“From Chemistry to Medicine”

INTERNATIONAL SYMPOSIUM on DRUG RESEARCH and DEVELOPMENT “From Chemistry to Medicine”

Kervansaray Convention Center & Hotel, LARA, ANTALYA / TÜRKİYE May 17-20, 2007

Hacettepe University Medicinal Chemistry Research, Development and Application Center (MAGUM)

“From Chemistry to Medicine” DRD 2007

A big THANK YOU Supporting Organizations

ASSOCIATION OF RESEARCH - BASED PHARMACEUTICAL THE SOCIETY OF PHARMACEUTICAL SCIENCES OF COMPANIES ANKARA

TURKISH PHARMACEUTICAL INDUSTRIES TURKISH PHARMACEUTICAL TECHNOLOGY SCIENTISTS ASSOCIATION ASSOCIATION

TURKISH PHARMACEUTICAL MANUFACTURERS TURKISH ASSOCIATION OF PHARMACEUTICAL AND ASSOCIATION MEDICINAL CHEMISTRY

Sponsoring Organizations

THE SCIENTIFIC & TECHNOLOGICAL RESEARCH COUNCIL OF TÜRKİYE

Pharmaceutical Companies

ADEKA İLAÇ SAN. A.Ş. ABBOTT LABORATUVARLARI İTHALAT İHRACAT ve TİC.LTD.ŞTİ.

ABDİ İBRAHİM İLAÇ SANAYİ ve TİC. A.Ş. BİEM TIBBİ CİHAZ ve İLAÇ SANAYİ LTD. ŞTİ.

Kervansaray Convention Center & Hotel, LARA, ANTALYA / TÜRKİYE iii International Symposium on Drug Research and Development

BİLİM İLAÇ SANAYİ ve TİC. A.Ş. PFIZER İLAÇLARI LTD. ŞTİ.

FAKO İLAÇLARI A.Ş. ROCHE MÜSTAHZARLARI SAN. A.Ş.

GLAXOSMITHKLINE İLAÇLARI A.Ş. SANOFİ-AVENTİS İLAÇLARI LTD.ŞTİ.

JOHNSON & JOHNSON SIHHİ MALZEME SANOVEL İLAÇ SAN. VE TİC. A.Ş. SAN. ve TİC. LTD. ŞTİ.

SANTA FARMA İLAÇ SANAYİ A.Ş. LİLLY İLAÇ TİCARET LİMİTED ŞTİ.

SCHERING PLOUGH TIBBİ ÜRÜNLER TİC. A.Ş. MUSTAFA NEVZAT İLAÇ SANAYİ A.Ş.

SERVIER İLAÇ ve ARAŞTIRMA A.Ş. NOVAGENIX BİO ANALİTİK İLAÇ AR-GE MERKEZİ SAN. TİC. A.Ş.

NOVARTIS SAĞLIK GIDA ve TARIM ÜRÜNLERİ ULKAR KİMYA SAN. VE TİC. A.Ş. SAN. VE TİC. A.Ş.

iv May 17-20, 2007 “From Chemistry to Medicine” DRD 2007

Institute

TURKISH PATENT INSTITUTE

Manufacturers/Distributors of Analysis Products and Equipments

ALBİO KİMYEVİ MADDELER İTHALAT ve TİC. A.Ş. HACETTEPE TEKNOKENT A.Ş.

ANAMED ANALİTİK ve MEDİKAL SİSTEMLER A.Ş. IŞIN TIP ARAŞTIRMA ÜRÜNLERİ TİC. LTD. ŞTİ.

ANT TEKNİK CİHAZLAR PAZ. ve DIŞ TİC. LTD.ŞTİ. İNCEKARA TEKNİK CİHAZLAR ENDÜSTRİSİ ve TİC. A.Ş.

ATOMİKA MAKİNA TİC. LTD. ŞTİ. İNFO KİMYA LABORATUVAR CİH. TİC. LTD. ŞTİ.

BETA LABORATUVAR CİHAZLARI LTD. ŞTİ. İNTERLAB LABORATUVAR ÜRÜNLERİ SANAYİ ve TİCARET A.Ş.

FARGEM, FARMASÖTİK ARAŞTIRMA GELİŞTİRME MERKEZİ SAN. ve TİC.A.Ş. KUTAY LABORATUVAR CİHAZLARI TİCARET A.Ş.

Kervansaray Convention Center & Hotel, LARA, ANTALYA / TÜRKİYE v International Symposium on Drug Research and Development

REFERANS KİMYA LTD. ŞTİ.

LABOR İLDAM LAB. MALZ. TİC. LTD. ŞTİ

SARTONET SEPERASYON TEKNOLOJİLERİ LTD. ŞTİ.

MEDSANTEK LABORATUVAR MALZEMELERİ SANAYİ ve TİCARET LTD.ŞTİ. SESA ELEKTRONİK SAN. TİC. A.Ş.

TETRA ENDÜSTRİYEL ve TEKNİK SİSTEMLER NÜVE SANAYİ MALZEMELERİ İMALAT ve TİC. A.Ş. DIŞ TİCARET A.Ş.

REDOKS KİMYASAL BİYOLOJİK MAD. ve LAB. CİH. PAZ. İTH. İHR. SAN. ve TİC. LTD. ŞTİ.

Others

BİOSTAR SOYDANLAR KIRTASİYE

LEDA PASTAHANESİ

ZEKİ KIRTASİYE SAYDAN ÜÇ AS TEKSTİL

SAYDAN ÜÇ AS TEKSTİL vi May 17-20, 2007 “From Chemistry to Medicine” DRD 2007

Honorary Chairman Prof. Tunçalp ÖZGEN, MD President of Hacettepe University

Organizing Committees Chairman : Prof. Ünsal ÇALIŞ, Ph. D. Scientific Secretary : Prof. Selma SARAÇ, Ph. D. Treasurer : Assoc. Prof. Kezban ULUBAYRAM, Ph. D Members : Prof. Sedef KIR, Ph. D. Prof. Gülberk UÇAR, Ph. D. S. Kutay DEMİRKAN, Pharm. D.

Scientific Committee

Analytical Chemistry Marianne FILLET University of Liege, Belgium Sacide ALTINÖZ Hacettepe University, Türkiye Alexander V. KABANOV University of Nebraska, USA José BARBOSA University of Barcelona , Spain Ningur NOYANALPAN Gazi University, Türkiye Nursabah BAŞCI Hacettepe University, Türkiye Seçkin ÖZDEN Ankara University, Türkiye Nuran ÖZALTIN Hacettepe University, Türkiye Wolfgang SIPPL Martin Luther University, Germany Ljubica SUTURKOVA Ss. Cyril and Methodius University Biochemistry Skopje, R. Macedonia Fethi ŞAHİN Gazi University, Türkiye Hamdi ÖĞÜŞ Hacettepe University, Türkiye İnci ÖZER Hacettepe University, Türkiye Pharmaceutical Technology Nazmi ÖZER Hacettepe University,Türkiye Sema ÇALIŞ Hacettepe University, Türkiye Angelino PARINI Rangueil Institute of Molecular Medicine, France Nevin ÇELEBİ Gazi University, Türkiye Kevser PİŞKİN Hacettepe University, Türkiye Ruxandra GREF University of Paris-Sud, France Mercedes UNZETA Barcelona Autonoma University, Spain Süeda HEKİMOĞLU Hacettepe University, Türkiye A. Atilla HINCAL Hacettepe University, Türkiye Biology Filiz ÖNER Hacettepe University, Türkiye Nilüfer AKSÖZ Hacettepe University, Türkiye Levent ÖNER Hacettepe University, Türkiye Vasıf HASIRCI Middle East Technical University, Türkiye A. Yekta ÖZER Hacettepe University, Türkiye Aşkın TÜMER Hacettepe University, Türkiye Nilüfer TARIMCI Ankara University, Türkiye

Chemistry and Chemical Engineering Pharmaceutical Microbiology Ayhan S. DEMİR Middle East Technical University, Türkiye Ufuk ABBASOĞLU Gazi University, Türkiye Adil DENİZLİ Hacettepe University, Türkiye Sulhiye YILDIZ Ankara University, Türkiye Emir Baki DENKBAŞ Hacettepe University, Türkiye Pharmacognosy and Pharmaceutical Botany Metin BALCI Middle East Technical University, Türkiye Ahmet BAŞARAN Hacettepe University, Türkiye Menemşe GÜMÜŞDERELİOĞLU Hacettepe University, Türkiye Ömür DEMİREZER Hacettepe University, Türkiye Olgun GÜVEN Hacettepe University, Türkiye Nurten EZER Hacettepe University, Türkiye Nesrin HASIRCI Middle East Technical University, Türkiye Erdem YEŞİLADA Yeditepe University, Türkiye Yunus KARA Atatürk University, Türkiye

Erhan PİŞKİN Hacettepe University, Türkiye Pharmacology Bekir SALİH Hacettepe University, Türkiye İsmail Hakkı AYHAN Ankara University, Türkiye Muzaffer TALU Gazi University, Türkiye E. Rüştü ONUR Hacettepe University, Türkiye

İnci ŞAHİN Hacettepe University, Türkiye Pharmaceutical and Medicinal Chemistry Serdar UMA Hacettepe University, Türkiye Peter CROOKS University of Kentucky, USA

Sevim DALKARA Hacettepe University, Türkiye Toxicology Şeref DEMİRAYAK Anadolu University, Türkiye Sema BURGAZ Gazi University, Türkiye Erçin ERCİYES Ege University, Türkiye Filiz HINCAL Hacettepe University, Türkiye Dilek EROL Yeditepe University, Türkiye Gönül ŞAHİN Hacettepe University, Türkiye Mevlüt ERTAN Hacettepe University, Türkiye

Kervansaray Convention Center & Hotel, LARA, ANTALYA / TÜRKİYE vii International Symposium on Drug Research and Development

viii May 17-20, 2007 “From Chemistry to Medicine” DRD 2007

Contents

• Exhibitor A - Z ...... x • Scientific Program ...... xi • Lectures ...... 1 SMART MOLECULES IN TARGETED THERAPIES ...... 3 PROPARGYLAMINES AS NEUROPROTECTIVE AGENTS IN NEURODEGENERATIVE DISEASES ...... 4 OXIDATIVE STRESS AND MONOAMINE OXIDASES: FROM BASIC STUDIES TO NOVEL THERAPEUTICAL INTERVENTIONS ...... 7 CHARACTERIZATION OF GLYCOPROTEINS BY CAPILLARY ELECTROPHORESIS ELECTROSPRAY MASS SPECTROMETRY (CE-ES-MS). APPLICATIONS TO DIAGNOSIS IN BIOMEDICINE ...... 8 BIOMARKER DISCOVERY FOR CHRONIC INFLAMMATORY DISEASES USING PROTEOMIC SERUM PROFILING ...... 14 IN SILICO MEDICINAL CHEMISTRY –

UNDERSTANDING BIOLOGICAL EFFECTS THROUGH MOLECULAR DOCKING AND MOLECULAR DYNAMICS SIMULATIONS ...... 17 SURFACE-MODIFIED NANOPARTICLES FOR DRUG ENTRAPMENT AND TARGETING ...... 18 CHALLENGES IN THE TREATMENT OF GRAM-POSITIVE INFECTIONS ...... 20 CHALLENGES IN TREATMENT OF GRAM-NEGATIVE BACTERIA ...... 22 CHALLENGES IN ANTIFUNGAL THERAPY ...... 24 NATIONAL AND INTERNATIONAL PATENT PROTECTION, FREE PATENT SEARCH TOOLS (ESPACENET) AND STRATEGIES ...... 25 PATENTABILITY OF MEDICAL AND PHARMACEUTICAL INVENTIONS ...... 27 IMPORTANCE OF POLYMORPHISM IN DRUG RESEARCH AND DEVELOPMENT ...... 28 A VAST SOURCE FOR THE DISCOVERY OF NOVEL DRUG LEADS: TRADITIONAL MEDICINES ...... 29 PARTHENOLIDE ANALOGS AS ANTILEUKEMIC AGENTS WITH CLINICAL POTENTIAL ...... 35 SYNTHESIS AND BIOLOGICAL EVALUATION OF NOVEL CAMPTOTHECIN CLASS TOPOISOMERASE I INHIBITORS ...... 36 NANOMATERIALS IN MEDICINE ...... 38 NANOMEDICINE FOR CENTRAL NERVOUS SYSTEM (CNS) DRUG DELIVERY ...... 39 • Oral Presentations ...... 41 • Poster Presentations ...... 49 • Author Index ...... 91

Kervansaray Convention Center & Hotel, LARA, ANTALYA / TÜRKİYE ix International Symposium on Drug Research and Development

Exhibitor A - Z

ABDİ İBRAHİM İLAÇ SANAYİ VE TİC. A.Ş...... A – 3 MEDSANTEK LABORATUVAR MALZEMELERİ SANAYİ VE TİCARET LTD.ŞTİ...... A – 19 ALBİO KİMYEVİ MADDELER İTHALAT VE TİC. A.Ş...... C – 11 NOVAGENIX BİO ANALİTİK İLAÇ AR-GE MERKEZİ SAN. TİC. A.Ş...... A – 17 ANAMED ANALİTİK VE MEDİKAL SİSTEMLER A.Ş...... C – 10 NÜVE SANAYİ MALZEMELERİ İMALAT VE TİC. A.Ş...... C – 9 ANT TEKNİK CİHAZLAR PAZ. VE DIŞ TİC. LTD.ŞTİ...... A – 4 ONDÖRT MAYIS DERGİSİ ...... A – 14 ATOMİKA TEKNİK CIH. TİC. LTD. ŞTİ...... A – 10 PATENT ENSTİTÜSÜ ...... A – 15,16 BİEM TIBBİ CİHAZ VE İLAÇ SANAYİ LTD. ŞTİ...... B – 9 PFIZER İLAÇLARI LTD. ŞTİ...... A – 5 BİLİM İLAÇ SANAYİ VE TİC. A.Ş...... B – 6 REDOKS KİMYASAL BİYOLOJİK MAD. VE FABAD ...... A – 8 LAB. CİH. PAZ. İTH. İHR. SAN. VE TİC.LTD.ŞTİ ...... A – 1 FARGEM, FARMASÖTİK ARAŞTIRMA GELİŞTİRME MERKEZİ SAN. VE TİC.A.Ş...... B – 5 REFERANS KİMYA LTD. ŞTİ...... C – 7 HACETTEPE TEKNOKENT A. Ş...... A – 7 SESA ELEKTRONİK SAN. TİC. A.Ş...... A – 20 IŞIN TIP ARAŞTIRMA ÜRÜNLERİ TİC. LTD. ŞTİ...... B – 7 TERRALAB LABORATUVAR MALZEMELERİ SANAYİ VE TİCARET A:Ş...... C – 8 İNCEKARA TEKNİK CİHAZLAR ENDÜSTRİSİ VE TİC. A.Ş...... C – 12 TUBİTAK ...... A – 6 İNFO KİMYA LABORATUVAR CİH. TİC. LTD. ŞTİ...... A – 12 TÜFTAD ...... A – 9 İNTERLAB LABORATUVAR ÜRÜNLERİ SANAYİ VE TİCARET A.Ş...... A – 18 TÜRK KİMYA DERNEĞİ ...... A – 13 LABOR İLDAM LAB. MALZ. TİC. LTD. ŞTİ...... B – 8 ULKAR KİMYA SAN. VE TİC. A.Ş...... B – 4

x May 17-20, 2007 “From Chemistry to Medicine” DRD 2007

www.drd2007.org PROGRAMME

MAY 17, 2007 – THURSDAY

16.16-16.0 Registration

Hall A 16.00-17.30 OPENING CEREMONY Plenary Lecture Smart molecules in targeted therapies Emin Kansu (Hacettepe University, Türkiye)

19.19-19.0 WELCOME COCKTAIL

MAY 18, 2007 - FRIDAY

Hall A 09.00-10.20 SESSION I Simultaneous Turkish translation will be provided. (New Approaches to Neurodegenerative Diseases) Chairpersons: Peter Crooks (University of Kentucky, USA) Emin Kansu (Hacettepe University, Türkiye) 09.00-09.40 Propargylamines as neuroprotective agents in neurodegenerative diseases Mercedes Unzeta (Universitat Autonoma de Barcelona, Spain) 09.40-10.20 Oxidative stress and monoamine oxidases: From basic studies to novel therapeutical interventions Angelo Parini (Rangueil Institute of Molecular Medicine, France) 10.20-10.50 Coffee Break Hall A 10.50-12.10 SESSION II Simultaneous Turkish translation will be provided. (Analytical Methods in Medicine) Chairpersons: Wolfgang Sippl (Martin Luther University, Germany) Nursabah Başçı (Hacettepe University, Türkiye) 10.50-11.30 Characterization of glycoproteins by capillary electrophoresis electrospray mass spectrometry (CE-ES-MS). Applications to diagnosis in biomedicine José Barbosa (University of Barcelona, Spain) 11.30–12.10 Biomarker discovery for chronic inflammatory diseases using proteomic serum profiling Marianne Fillet (University of Liege, Belgium) 12.10–12.35 Investigation of noncovalent protein-protein interactions by matrix- assisted llaser desorption/ ionization mass spectrometry Bekir Salih (Hacettepe University, Türkiye)

12.35-13.30 LUNCH

Kervansaray Convention Center & Hotel, LARA, ANTALYA / TÜRKİYE xi International Symposium on Drug Research and Development

Poster Hall 13.30-14.30 Poster Session Industry Hall All presentations will be in Turkish. (Mission and Vision of the Pharmaceutical Companies) Chairperson: Nilüfer Tarımcı (Ankara University, Türkiye) 13.30-14.00 Bilim İlaç Sanayii ve Ticaret A.Ş. (Türkiye) 14.00-14.30 Farmasötik Araştırma Geliştirme Merkezi Sanayii ve Ticaret A.Ş.- FARGEM (Türkiye) Satellite Hall All presentations will be in Turkish. Chairperson: Seçkin Özden (Ankara University, Türkiye) 13.30-14.00 New Technology: Near Infrared (NIR) Analysis Systems İncekara Holding/FOSS Analytical A.Ş. (Türkiye) 14.00-14.30 XRD Applications in Pharmaceutical Industry: Determination of Crystal Structure Ant Teknik Ltd. Şti./Shimadzu Corp. (Türkiye) Hall A 14.30-16.00 SESSION III Simultaneous Turkish translation will be provided (Molecular Modeling) Chairpersons: José Barbosa (University of Barcelona, Spain) Sevim Dalkara (Hacettepe University, Türkiye) 14.30-15.10 In silico medicinal chemistry – Understanding biological effects through molecular docking and molecular dynamics simulations Wolfgang Sippl (Martin Luther University, Germany) 15.10-15.35 Molecular dynamics simulation of thrombin: Target for anticoagulant drugs Özge Kül (Hacettepe University, Türkiye) 15.35-16.00 Molecular dynamics simulatıon of hirudins: Specific thrombin inhibitor isolated from medicinal leech Fulya Çağlar, (Hacettepe University, Türkiye) Hall B 14.30-16.00 SESSION IV (Current Approaches for Development of Novel Drug Delivery Systems) Chairpersons: Alexander V. Kabanov (University of Nebraska, USA) Nevin Çelebi (Gazi University, Türkiye) 14.30-15.10 Surface-modified nanoparticles for drug entrapment Ruxandra Gref (University of Paris-Sud, France) 15.10-15.35 Dendrimers as drug delivery agents to bone Rana Sanyal (Boğaziçi University, Türkiye) 15.35-16.00 Recent advances in brain drug delivery Yılmaz Çapan (Hacettepe University, Türkiye) 16.00-16.30 Coffee Break Anamed Hall Presentations will be in Turkish. 16.00-16.30 Coffee Break Session Chairperson: Ayhan S. Demir (Middle East Technical University, Türkiye) See the Future:”Microwave Synthesis from µL’s to L’s” Nejla Kılıç (ANAMED & ANALİTİK Grup, Türkiye) Hall A 16.30-18.15 SESSION V Simultaneous Turkish translation will be provided (Design and Synthesis of New Anticancer Drugs) Chairpersons: Fethi Şahin (Gazi University, Türkiye) Vildan Adar (Hacettepe University, Türkiye) 16.30-16.55 Rational design of novel photosensitizers as potential photodynamic therapy reagents Engin Umut Akkaya (Middle East Technical University, Türkiye) 16.55-17.20 A novel potential antitumor active drug: Platinum blue complex containing sulfur-donor ligand Şeniz Özalp-Yaman (Atılım University, Türkiye) 17.20-17.45 Synthesis of acetophenone and substituted acetophenone derived Mannich bases and their biological activities H. İnci Gül (Atatürk University, Türkiye) xii May 17-20, 2007 “From Chemistry to Medicine” DRD 2007

MAY 19, 2007 - SATURDAY

Hall A 09.00-10.30 SESSION VI Simultaneous translation will be provided (Experiences of Clinicians: New Horizons and Expectations From Research & Development in Antimicrobial Agents) Chairpersons: Angelo Parini (Rangueil Institute of Molecular Medicine, France) Ahmet Başaran (Hacettepe University, Türkiye) 09.00-09.30 Challenges in treatment of gram positive bacteria Serhat Ünal (Hacettepe University, Türkiye) 09.30-10.00 Challenges in treatment of gram negative bacteria Recep Öztürk (İstanbul University, Türkiye) 10.00-10.30 Challenges in antifungal agents Ömrüm Uzun (Hacettepe University, Türkiye)

10.30-11.00 Coffee Break

Info Kimya Hall Presentations will be in Turkish. 10.30-11.00 Coffee Break Session Chairperson: Filiz Öner (Hacettepe University, Türkiye) Technological Applications in Pharmaceutical QA/QC and R&D Oğuzhan Ay (Info Kimya Laboratuar Cihazları Tic. Ltd. Şti, Türkiye)

Hall A 11.00-12.10 SESSION VII Simultaneous translation will be provided (Patenting in Health and Pharmaceutics) Chairperson: Levent Öner (Hacettepe University, Türkiye) 11.00-11.40 National and international patent protection, free patent search tools (espacenet) and strategies Hakan Bayram (Turkish Patent Institute, Türkiye) 11.40-12.10 Patentability of health and pharmaceutical inventions Serkan Özkan (Turkish Patent Institute, Türkiye)

12.10-13.30 LUNCH

Poster Hall 13.30-14.30 Poster Session

Industry Hall All presentations will be in Turkish. (Mission and Vision of the Pharmaceutical Companies) Chairperson: H. İnci Gül (Atatürk University, Türkiye) 13.30-14.00 Novartis (Türkiye) 14.00-14.30 Abdi İbrahim İlaç Sanayii ve Ticaret A.Ş. (Türkiye)

Satellite Hall All presentations will be in Turkish. Chairperson: Yılmaz Çapan (Hacettepe University, Türkiye) 13.30-14.00 New Horizons in Drug Industry: Technological Platforms Ahu Yücesoy (The Scientific & Technological Research Council of Türkiye-TÜBİTAK) 14.00-14.30 Bioavailability and Bioequivalency Studies in Türkiye Tuncel Özden (Novagenics, Türkiye)

Kervansaray Convention Center & Hotel, LARA, ANTALYA / TÜRKİYE xiii International Symposium on Drug Research and Development

Hall A 14.30-16.25 SESSION VIII Simultaneous Turkish translation will be provided (Pharmacology and Molecular Pharmacology) Chairpersons: Mercedes Unzeta (Universitat Autonoma de Barcelona, Spain) İnci Erdemli (Hacettepe University, Türkiye) 14.30-15.10 Importance of polymorphism in drug research and development M. Fethi Şahin (Gazi University, Türkiye) 15.10-15.35 Studies on histone deacetylase inhibitory activity of some carboxylic acid derivatives and their structure-activity relationships Gamze Bora (Hacettepe University, Türkiye) 15.35-16.00 Free-radical scavenger activities of newly synthesized 2-benzoxazolinone derivatives containing thiosemicarbazide, triazole, thiadiazole and hydrazone Samiye Yabanoğlu (Hacettepe University, Türkiye) 16.00-16.25 UVB-Induced apoptotic effect of 11 P53 actinic keratosis mutations Ayşe Ercan (Hacettepe University, Türkiye) Hall B 14.30-16.00 SESSION IX (Natural Compounds as Drug Leads) Chairpersons: Engin Umut Akkaya (Middle East Technical University, Türkiye) Bekir Salih (Hacettepe University, Türkiye) 14.30-15.10 A vast source for the discovery of novel drug leads: Traditional medicines Erdem Yeşilada (Yeditepe University, Türkiye) 15.10-15.50 Parthenolide analogs as antileukemic agents with clinical potential Peter Crooks (University of Kentucky, USA) 15.50-16.30 Synthesis and biological evaluation of novel camptothecin class topo-isomerase inhibitors Ayhan S. Demir (Middle East Technical University, Türkiye) 16.30-17.00 Coffee Break Atomika Hall Presentations will be in Turkish. 16.30-17.00 Coffee Break Session Chairperson: Füsun Acartürk (Gazi University, Türkiye) Particle Characterization in the Pharmaceutical Industry: New Developments and Techniques for Size, Shape and Chemical Analysis in Laboratory and Process Environments Stuart Wakefield ( Malvern Instruments Ltd. England) Naci Saraçoğlu (Atomika Teknik Ltd., Türkiye) Hall A 17.00-18.30 PANEL Simultaneous English translation will be provided. (Expectations of Pharmaceutical Industry from Drug Research & Development Studies) Panelists: Altan Demirdere (Novartis, Türkiye) (Moderator) Erdal Akalın (Pfizer, Türkiye) Vedat Eğilmez (Abdi İbrahim İlaç Sanayi ve Ticaret A.Ş., Türkiye)

20.00-24.00 GALA DINNER

xiv May 17-20, 2007 “From Chemistry to Medicine” DRD 2007

MAY 20, 2007 - SUNDAY

Hall A 10.10-10.10 SESSION (Nanomedicine and Nanomaterials) Chairpersons: Ruxandra Gref (University of Paris-Sud, France) Sema Çalış (Hacettepe University, Türkiye) 09.30-10.20 Nanomaterials in medicine Erhan Pişkin (Hacettepe University, Türkiye) 10.20-11.00 Nanomedicine for central nervous system (CNS) drug delivery Alexander V. Kabanov (University of Nebraska, USA)

11.00-11.20 CLOSING REMARKS

MAY 18, 2007 - FRIDAY

TÜFTAD SATELLITE SYMPOSIUM organized by Turkish Pharmaceutical Technology Scientists’ Association (TÜFTAD)

Projects and Support Sources in the Development of Active Pharmaceutical Ingredients and Drug Formulations in University and Industry Cooperation

TÜFTAD Hall All presentations will be in Turkish. Simultaneous English translation will not be provided.

30.30-30.30 İlaç Etkin Maddesi ve Formulasyon Geliştirmede Üniversite Sanayi İşbirliği Projeleri ve Destek Kaynakları

TÜBİTAK Sanayi AR-GE Projeleri Destekleri Dr. Bülent İçgen (Türkiye Bilimsel ve Teknolojik Araştırma Kurumu -TEYDEB)

Eczacıbaşı Özgün Kimya’da Faydalanılan AR-GE Teşvikleri Dr. Mustafa Adıyaman (Eczacıbaşı Özgün Kimya)

Sanayide AR-GE’nin Rolü Doç.Dr. Tuncer Aslan (Ulkar İlaç Sanayi ve Tic. A.Ş.)

TÜBİTAK Sanayi AR-GE Destekleri ve Etkilerinin Değerlendirilmesi Uzm. Ecz. Ece Kut (Abdi İbrahim İlaç Sanayi)

TÜBİTAK-TEYDEB Projelerinde Hakemlik ve İzleyicilik Deneyimleri Prof. Dr. A.Atilla Hıncal (H.Ü. Eczacılık Fakültesi) Prof. Dr. Nilüfer Tarımcı (A.Ü. Eczacılık Fakültesi)

Kervansaray Convention Center & Hotel, LARA, ANTALYA / TÜRKİYE xv International Symposium on Drug Research and Development

Dear Participants, You can access to the “DRD 2007 Symposium Abstract e-Book” via internet using the following address:

http://www.magum.hacettepe.edu.tr/drd/drd2007.htm

DRD 2007 Organizing Committee

The authors are responsible for their presentations published in this Abstract Book. Lectures

“From Chemistry to Medicine” DRD 2007 S

L 01 SMART MOLECULES IN TARGETED THERAPIES LECTURE

Emin KANSU Hacettepe University, Institute of Oncology, Ankara, Turkey

n recent years there has been a significant progress spread. The p53 is an attractive therapeutic target in in the field of cancer cell biology and molecular oncology because its tumour-suppressor activity can oncology. Many researchers outlined very impor- be stimulated to eradicate tumour cells. Inhibiting the Itant processes of cancer genesis, growth, invasion and p53–MDM2 interaction is a promising approach for metastasis. As a result of this progress a large number activating p53, because this association is well charac- of molecular abnormalities that are “specific” to can- terized at the structural and biological levels. MDM2 cer cells and that are a critical feature of cancer phe- inhibits p53 transcriptional activity, favours its nucle- notype have been discovered. These advances in the ar export and stimulates its degradation, so inhibiting field of moleculer oncology over the past decade have the p53–MDM2 interaction with synthetic molecules led to a new era in cancer therapeutics and several should lead to p53-mediated cell-cycle arrest or apop- strategies directed to selective molecular targets. This tosis in p53-positive stressed cells. new class of anticancer agents has been named “targeted therapies”, because these structures target spe- Angiogenesis inhibitors are a new class of drugs, for cific cellular molecular and/or abnormalities. which the general rules involving conventional chemotherapy might not apply. The successful translation Tumour cells acquire the ability to proliferate uncon- of angiogenesis inhibitors to clinical application de- trollably, resist apoptosis, sustain angiogenesis and pends partly on the transfer of expertise from scien- evade immune surveillance. Different from conventi- tists who are familiar with the biology of angiogenesis onal chemotherapy agents, which mainly kill prolife- to clinicians. rating cells by interacting with general cellular processes, “targeted agents” are expected to affect only STAT proteins — especially STAT3 and STAT5 — reg- cells in which the specific molecular alteration is pre- ulate all of these processes and are persistently activat- sent, induce predominantly antiproliferative effects, ed in a surprisingly large number of human cancers. and be specific for cancer cells versus normal tissu- Consequently, STAT proteins are emerging — unex- es. In this context, there have been very significant pectedly — as ideal targets for cancer therapy. progress in the anti-cancer “targeted molecules” including tyrosine kinase inhibitors, angiogenesis inhi- Monoclonal antibodies have become the most bitors, modulators of cell matrix interactions, agents rapidly expanding class of pharmaceuticals for that interact with the cell cycle and cell death (apop- treating a wide variety of human diseases, in- tosis) and protein trafficking regulators. cluding cancer. Six antibodies including Trastu- zumab (Herceptin), Rituximab (Mabthera), Ale- Several of these new therapeutic agents are showing mtuzumab (Campath), Cetuximab, Gefinitib and promise in the clinic and many more are being de- Bevacizumab are now approved for cancer therapy. veloped. The RAS proteins control signalling path- Coupled antibodies to toxins or radionuclides is the ways that are key regulators of several aspects of nor- most widely investigated means for increasing their mal cell growth and malignant transformation. They antitumour activity. Two anti-CD20 radioimmu- are aberrant in most human tumours due to activat- noconjugates, Bexxar (tositumomab; 131iodine) and ing mutations in the RAS genes themselves or to al- Zevalin (ibritumomab tituxetan; 90yttrium), and My- terations in upstream or downstream signalling lotarg (anti-CD33-calicheamicin conjugate) are ap- components. Rational therapies that target the RAS proved for cancer therapy and currently in clinical pathways might inhibit tumour growth, survival and practice.

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L 02 LECTURE PROPARGYLAMINES AS NEUROPROTECTIVE AGENTS IN NEURODEGENERATIVE DISEASES

Elisenda SANZ, José Luis MARCO, Mercedes UNZETA Departament Bioquimica y Biologia Molecular, Facultad de Medicina-Instituto de Neurociencias, Universitat Autonoma de Barcelona, 8193 Barcelona, Spain

arkinson´s disease is a neurodegenerative dis- hibits it (bcl-2, bcl-xL, bcL-w, mcl-1). Defects in the order characterized by the progressive loss of physiological apoptotic pathway, leading to inappro- the dopaminergic neurons in the nigrostriatal priate cell death underlies in some neurodegenerative Psystem. Different factors have been suggested to in- diseases. duce Parkinsonism: environmental toxins, alteration of the intracellular calcium homeostasis, mitochon- The programmed cell death may contribute to human drial dysfunction, genetic factors, oxidative stress etc., neurodegeneration. It has been reported that apopto- [1]. All them are able by themselves to induce apop- sis is involved in the death of the dopaminergic neu- totic cell death [2]. rons in Parkinson´s disease [6, 7, 9].

Oxidative stress, arising from an imbalance between In humans, MPTP (1-methyl-4-phenyl-1, 2, 3, 6-tet- the production of reactive oxygen-containing free- rahydropyridine) produces a behavioural syndrome radical species (ROS) and antioxidant protective similar to the neuropathological features of idiopathic mechanisms, has been shown to induce apoptosis in Parkinson´s disease [10] and involves a selective de- dopaminergic cells. Dopaminergic neurones in the struction of nigrostriatal dopaminergic neurons. Al- substantia nigra (SN) are particularly vulnerable to though MPTP itself displays low chemical reactivity, oxidative stress because of relatively low antioxidant it is biotransformed by glial MAO-B, to form MPP+ levels. Dopamine itself can be readily autooxidised (1-methyl-4-phenylpyridinium) as the effective tox- at physiological pH, with generation of ROS and, fur- in [11]. MPP+ is able to inhibit the Complex-I of the thermore, it is metabolised by monoamine oxidase respiratory chain, inducing the free radical formation, in a reaction that forms H2O2. Furthermore, elevat- ATP depletion, and cellular death [12]. ed levels of MAO-B activity have been reported in the SN of patients with Parkinson´s disease [3]. Thus, The programmed cell death may contribute to the hu- the increase of the oxidation of dopamine by MAO- man neurodegeneration probably due to the free rad- B, might generate sufficient ROS to trigger the death icals formation as a consequence of the mitochon- of nigrostriatal neurones [4]. drial dysfunction [13]. The mitochondrial process implicated in the toxic and neurodegenerative condi- Besides the symptomatic therapeutically treatments tions is related with the opening of the mitochondrial based on L-Dopa administration, a precursor of do- pore in the inner membrane, that leads to the mem- pamine, at present there is a great interest on the use brane depolarization, release of small proteins such as of neuroprotective agents such as the antiapoptotic Cytochrome c, apoptosome complex formation, acti- factors [5]. Apoptosis has been reported to be present vation of the executer caspase 3, poly (ADP-ribose) in post-mortem human brain from Parkinson´s pa- polymerase (PARP) activation, DNA damage and cell tients [6, 7]. Apoptosis (Programmed cell death), death. is a highly regulated physiological process that occurs in all vertebrates and controls the cellular turn- Recent studies have demonstrated that ER stress in- over from fetal development to aging [8]. Apoptosis duced by protein missfolding as a consequence of the is morphologically characterized by chromatin con- inhibition of protein glycosylation, trafficking or al- densation, DNA fragmentation, cell shrinkage and teration of the Ca+ homeostasis in the endoplasmic plasma membrane blebing. The apoptotic pathway is reticulum (ER), may contribute to the pathogenesis induced by a cascade of events in which a family of and neurodegeneration in Parkinson and Alzheim- cysteine proteases named caspases, leads to the cleav- er diseases. It has been reported that ER stress can in- age and activation of different cellular substrates.The duce apoptotic cell death in mice trough another mo- apoptotic death is under genetic control and is char- lecular pathway different to mitochondrial one that acterized by the expression of some genes that en- involves the activation of an ER-specific Caspase 12 hance the apoptosis (bax, bad ) and others that in- [14]. Furthermore, pharmacological agents able to in-

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+ hibit the perturbation of the mitochondria and ER ment significantly reduced MPP induced cell death LECTURE function may represent a potential therapeutically and diminished the activation of one of the main ex- approach for the prevention of neurotoxin-induced ecutioner caspases, Caspase-3 and also inhibited the Parkinson´s disease. PARP activation. PF9601N showed the same neuroprotective behaviour when cells were treated with In this context, the design and synthesis of new an- ER-stress toxins. Further effects of PF9601N in the tiapoptotic molecules, able to interfere with different maintenance of mitochondrial membrane potential apoptotic molecular pathways, inhibiting the open- or changes in the expression of Bcl-2 family proteins ing of the mitochondrial pore, preserving the mem- were also examined by RPA analysis. brane potential and/or inducing the gene expression of some antiapoptotic proteins, is expected to protect MPP+ treatment also induced a prominent increase neurons from the cell death and to slow progression in p53 expression, nuclear translocation of this tran- of chronic neurodegenerative diseases [1, 15-17]. scription factor and transactivation of p53 response elements. Additionally, p53 inhibitor pifithrin-alpha It has been designed and synthesised, a novel series of partially prevented MPP+-induced apoptosis, suggest- acetylenic and allenic tryptamine derivatives as po- ing that activation of p53 contributes to cell death. tential MAO inhibitors [18]. Among them there is a new non-amphetamine molecule, the PF9601N [N- PF9601N pre-treatment was able to partially avoid (2-propynyl)-2-(5-benzyloxy-indolyl) ethylamine, MPP+ induced cell death through preventing an in- with antioxidant properties [19], that resulted to be a creasing p53 expression and thus reducing transcrip- MAO B inhibitor, more potent and selective than se- tional activity of p53. PF9601N was also able to show legyline (l-deprenyl), widely used as coadjuvant of l- the same degree of protection that pifithrin-alpha DOPA in PD therapy [20]. Moreover, PF 9601N did supporting that PF9601N would act inhibiting p53 not show the ‘amphetaminergic’ side-effects of l-de- pathway activation as well as Caspase 2 activation. prenyl [21] and was able to inhibit the dopamine uptake in human and rat striatum. PF 9601 N showed a These results allow us to conclude that the pharma- neuroprotective effect in vivo, using several dopamin- cological target of PF9601N is the p53 molecular ergic toxins in different experimental models such as pathway. In this context, this new non-amphetamine MPTP, [22] and 6-hydroxydopamine-striatal lesion MAO B inhibitor, could have a therapeutical potential [23]. Furthermore, PF 9601N also enhanced the du- use for those diseases where neurodegenation is de- ration of l-DOPA-induced contralateral turning in 6- pendent upon the p53 induced apoptosis. PF 9601N hydroxydopamine lesioned rats [24]. is a good candidate to be used in the therapy of the neurodegenerative diseases. A structurally different non-amphetamine compound, Rasagiline, has also been reported to be neuroprotective [25], but this compound also differs from References l-deprenyl in not being an inhibitor of presynaptic 1. Olanow CW, Tatton WG. Etiology and pathogenesis of dopamine uptake [26]. In that respect, it differs from Parkinson´s disease, Ann Rev Neurosci., 22, 123-144, PF9601N and thus comparison of its behaviour with 1999. that of Rasagiline should reveal whether this trans- 2. Simonian NA, Coyle JT. Oxidative stress in neurodegen- port inhibition is an important factor in the neuro- erative diseases, Annu Rev Pharmacol Toxicol, 36, 83-106, 1996. protective spectrum of activities. 3. Riederer P, Jellinger K. Neurochemical insights into monoamine oxidase inhibitors, with special reference to The present study was performed to determine the deprenyl (selegiline), Acta Neurol Scand Suppl , 95, 43-55, molecular mechanism involved in the neuroprotective 1983. effect of PF 9601N observed in vivo. In this context 4. Cohen G. Monoamine oxidase and oxidative stress at it has been studied the cytoprotective and antiapop- dopaminergic synapses, J Neural Transm 32 (Suppl), 229- totic effect of PF 9601N using human neuroblastoma 38, 1990. SHSY5Y cells lesioned with MPP+ as a toxin that in- 5. Kragten E, Lalande I, Zimmermann K, Roggo S, Schin- duces the apoptotic mitochondrial pathway, and le- dler P, Múller D, Oostrum J van, Waldemeir P, Fürst P. sioned as well with Brefeldin A, Tunicamycin and Glyceraldehide-3-phosphate deshydrogenase, the puta- Thapsigargin, as toxins inducers of the ER stress ap- tive target of the antiapoptotic compounds CGP3466 and l-deprenyl, J Biol Chem., 273, 5821-5828, 1998. optotic pathway. 6. Hartmann A, Hirsch EC. Parkinson’s disease. The apoptosis hipótesis revisited, Adv Neurol., 86, 143-153, 2001. The neuroprotective properties of PF9601N in these 7. Mochizuki H, Goto K, Mori H, Mizuno Y. Histochemi- different apoptotic cell death models, were assessed cal detection of apoptosis in Parkinson´s disease, J Neurol using several viability assays (Alamar Blue reduc- Sci., 137, 120-123, 1996. tion and Calcein-AM staining). PF9601N pretreat-

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LECTURE 8. Oppenheim RW. Cell death during development of the 19. Sanz E, Romrea M, Bellik L, Marco JI, Unzeta M. Indo- nervous system, Ann Rev Neurosci., 14, 453-501, 1991. lalkylamines derivatives as antioxidant and neuropro- 9. Tatton NA, Maclean-Fraser A, Tatton WG, Perl DP, tective agents,in a experimental model of Parkinson´s Olanow CW. (1998), A fluorescent double-labeling meth- disease, Med Sci Monit., 10, BR477-484, 2004. od to detect and confirm apoptotic nuclei in Parkinson´s 20. Perez V, Marco JL, Fernandez-Alvarez E, Unzeta M. Rel- disease, Ann Neurol., 44, S142-S148. evance of benzyloxy group in 2-indolyl methylamines in 10. Langston JW, Ballard PA, Tetrud JW, Irwin I. Chronic the selective MAO-B inhibition, Br J Pharmacol., 127, parkinsonism in humans due to a product of meperidine 869-76, 1999. analog synthesis, Science, 219, 979-980, 1983. 21. Lees AJ. Parkinson´s disease research group of the United 11. Chiba K, Trevor A, Castagnoli N. Metabolism of the Kingdom. Comparison of therapeutic effects and mor- neurotoxic tertiary amine MPTP, by brain monoamine tality data of levpdopa and levodopa combined with l- oxidase, J Biochem Biophys Res Commun., 120, 574-578, deprenyl in patienst with early, mild Parkinson´s disease, 1984. Br Med J., 311, 1602-1607, 1995. 12. Sanchez-Ramos J, Hefti F, Hollinden DE, Jasik T, Rosenthal 22. Perez V, Unzeta M. PF 9601N [N-(2-propynyl)-2-(5-ben- M. (1988) Mechanisms of neurotoxicity oxygen radicals zyloxy-indolyl) methylamine] a new MAO- B inhibitor, and mitochondrial inhibition hypothesis in Progress in attenuates MPTP-induced depletion of striatal dopamine Parkinson´s Research (Hefti F, eds), Plenum Press New levels in C57/Bl 6 mice, Neurochem Int., 42, 221-229, York pp 145-152. 2003. 13. Beal MF. Mitochondria, free radicals and neurodegenera- 23. Cutillas B, Ambrosio S, Unzeta M. Neuroprotective effect tion, Curr Opin Neurobiol., 6, 661-666, 1996. of the monoamine oxidase inhibitor PF9601N [N-(2- 14. Nakagawa T, Zhu H, Morishima N, Li E, Xu J, Yankner propynyl)-2-(5-benzyloxy-yndolyl) methylamine ] on rat BA, Yuan J. Caspase-12 mediates endoplasmic-reticu- nigral neurons after 6-hydroxydopamine-striatal lesion, lum-specific apoptosis and cytotoxicity by amyloid-beta, Neuroscience Lett., 329, 165-168, 2002. Nature, 403, 98-103, 2000. 24. Prat G, Perez V, Casas ARM, Unzeta M. The novel type 15. Djaldetti R, Melamed E. New drugs in the future treat- B-MAO inhibitor PF 9601N enhances the duration of ment of Parkinson´s disease, J Neurol., 249 (Suppl), II30- L-DOPA-induced contralateral turning in 6-hydroxy- II35, 2002. dopamine lesioned rats, J Neural Transm., 107, 409-417, 16. Wellington CL, Hayden MR. Caspases and neurodegen- 2000. eration: on the cutting edge of new therapeutic approach- 25. Sterling J, Veinberg A, Lerner D, Goldenberg W, Levy es, Clin Genet., 57, 1-10, 2000. R, Youdim M, Finberg JP. (R)(+)-N-propargyl-1-ami- 17. Tajrena A, Cesari V, Borlongan RLM, Faull CEW, Ros noindan (rasagiline) and derivatives: highly selective GC, Gluckman PD, Hughes PE. Neuroprotective strate- and potent inhibitors of monoamine oxidase B, J Neural gies for basal ganglia degeneration: Parkinson´s and Transm (Suppl), 52, 301-305, 1998. Huntington¨s diseases, Prog Neurobiol., 60:5, 409-470, 26. Lamensdorf I, Porat S, Simantov R, Finberg JP. Effect of 2000. low-dose treatment with selegiline on dopamine trans- 18. Cruces MA, Elorriaga C, Fernandez-Alvarez E. Acety- porter (DAT) expression and amphetamine-induced lenic and allenic derivatives of 2-(5-methoxyindolyl) and dopamine release in vivo, Br J Pharmacol., 126, 997-1002, 2-(5-hidroyindolyl) methylamines: synthesis and invitro 1999. evaluation as monoamine oxidase inhibitors, Eur Med Chem., 26, 33-41, 1991.

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L 03 OXIDATIVE STRESS AND MONOAMINE OXIDASES: FROM LECTURE BASIC STUDIES TO NOVEL THERAPEUTICAL INTERVENTIONS

Angelo PARINI INSERM U858, Institut de Médecine Moléculaire de Rangueil – I2MR, Toulouse, France

iogenic amines, including catecholamine and pargyline and the antioxidant N-acetylcysteine. These serotonin, regulate a variety of cell functions data show that dopamine and serotonin induces cell through the interaction of G-coupled mem- proliferation, hypertrophy and apoptosis by a recep- Bbrane receptors. During the last years, we described tor-independent mechanism requiring amine inter- a novel mechanism of action of dopamine and serot- nalisation into the cells, their degradation by MAOs onin that occurs independently of membrane recep- and hydrogen peroxide production. tor stimulation and requires hydrogen peroxide generation by monoamine oxidases (MAO). Based on these findings, we next investigated the potential role of hydrogen peroxide generated by MAOs Using different models of renal and cardiac cells we on cell death in vivo. Our results showed that MAO showed that, in addition to the classical receptor-de- inhibition largely reduced renal and myocardial dam- pendent effects, dopamine and serotonin induces cell age induced by ischemia/reperfusion in rat. The pro- proliferation and hypertrophy by a mechanism inde- tective effects of MAOs inhibitors were associated pendent of receptor activation. At higher concentra- with the prevention of post-ischemic oxidative stress, tions (up to 10 µM), dopamine and serotonin cause ceramide accumulation, neutrophil infiltration and cell apoptosis by sequential i) increase in the ratio mitochondrial-dependent cell death. of Bax/Bcl2 proteins, ii) mitochondrial cytochrome c release, iii) caspase 3 activation and iv) DNA frag- In conclusion, our results show the key role of H2O2 mentation. Both proliferative and apoptotic effects of produced by MAOs in mediating cell effects of bio- dopamine and serotonin were not inhibited by specif- genic amines and propose these enzymes as a phar- ic receptor antagonists but were prevented by amine macological target for prevention of organ damag- transporter inhibitors, the irreversible MAO inhibitor es.

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L 04 LECTURE CHARACTERIZATION OF GLYCOPROTEINS BY CAPILLARY ELECTROPHORESIS ELECTROSPRAY MASS SPECTROMETRY (CE-ES-MS). APPLICATIONS TO DIAGNOSIS IN BIOMEDICINE

J. BARBOSA, V. SANZ-NEBOT, F. BENAVENTE, E. GIMÉNEZ Departament de Química Analítica, Facultat de Química, C/Martí i Franquès 1, 08028 Barcelona, Spain

enomics and, even more important, the wide complex and time consuming procedure favoured field of proteomic and related clinical appli- the introduction of new alternative methods. Cap- cations like biomarkers, dramatically increase illary electrophoresis (CE), has become one of the Gthe demand of sensitive and selective analytical tools most important techniques for glycoform separa- for the analysis of biological samples. The sugar con- tion, combining high resolution power under non- tent of proteins has been demostrated to be critical denaturing conditions [7]. In previous studies Tf for its biological activity, and it is influenced during sialoforms were resolved by CE-UV (4). Also the its manufacturing process by the cell line and the in- two commercial ready-to-use pharmaceuticals of re- cubation culture conditions [1]. The polymorphism combinant human EPO (rHuEPO), epoetin-α and associated with the amount, the size, and the struc- epoetin-β, and the hyperglicosilated EPO derivated ture of the carbohydrate chains is known as micro- NESP were analysed [8, 9]. Separation and charac- heterogeneity, and the molecular species generated terization of the different glycoforms from each glyc- are termed glycoforms. In this context, investigations oprotein are presented. However endogenous EPO of glycoproteins has become increasing important, in are generally found at subnanomolar levels such as particular with respect to the variations in glycosyla- other endogenous hormones and the poor concen- tion patterns observed in sera from healthy individu- tration detection sensitivity of CE preclude the di- als and patiens. rect analysis of these hormones at the levels found in biological fluids. Glycosylation is the most common posttranslation- al modification in proteins carbohydrates partici- The high values of the concentration limits of de- pate in many biological processes and encode infor- tection in CE are closely related with the small vol- mation for molecular recognition, protein folding, ume capacity of the capillary columns. Several in- stability and pharmacokinetics [2]. The number and strumental, electrophoretic and chromatographic type of glycoforms for a certain glycoprotein may modifications have been described in order to over- change as a consequence of pathological process- come this limitation. In solid phase extraction cou- es [3]. For example, patients with Congenital Dis- pled on-line to capillary electrophoresis (SPE-CE), orders of Glycosylation (CDG) or chronic alcohol- a microcartridge or analyte concentrator is insert- ism present hypoglycosylation of several plasmatic ed near the inlet of the separation capillary [10]. The glycoproteins as transferrin (Tf), analysis of which analyte concentrator contains a solid phase extrac- is used as a model glycoprotein for CDG diagnosis tion sorbent which retains the target analyte, ena- [4]. Tf is one of the twenty high abundance human bling large volumes of sample to be intruduced. The plasma proteins. Other different analytical problem captured analyte is eluted in a small volume of an ap- is analysis of erythropoietin which is found as very propiate solution, resulting in sample clean-up and low abundance protein. Erythropoietin (EPO) is a concentration enhancement, with minimum sam- glycoprotein hormone, which regulates erythropoi- ple handling. Several researchers have perceived the esis and has been used extensively for the treatment suitability of SPE-CE to perform selective and sensi- of several anemias associated with acute and chronic tive analysis of proteins and peptides in complex di- diseases [5]. Despite the many benefits of EPO in the luted samples. In addition, the use of solid-phase ex- clinic, they have been most widely publicized on ac- traction coupled on-line to capillary electrophoresis count of their extensive misuse as performance en- electrospray mass spectrometry (SPE-CE-ESI-MS) hancing agents in endurance sports [6]. has demonstrated improved capabilities for characterization of compounds found at low concentration Until recently, isoelectric focuosing electrophore- in complex matrix [10]. sis was the reference method for glycoforms analysis due to its high selectivity allowing an easy detec- Mass spectrometry (MS) has emerged as a powerful tion of genetic glycoprotein variants. However, this tool for the analysis of large biomolecules. Howev-

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er, direct analysis of intact glycoproteins by MALDI- IU from Janssen-Cilag (Neuss, Germany) and epoe- LECTURE TOF and conventional electrospray ionization mass tin- (NeoRecormon) 4000 IU from Roche (Man- spectrometry (ESI-MS) has presented some problems nheim, Germany) were obtained as ready-to-use in order to resolve microheterogenous structures. drugs. Deionised and organic-eliminated water was Thus, a previous glycoform separation is mandato- obtained using a Milli-Q water purification system ry to obtain valuable information about the carbohy- (Millipore, Schwalbach, Germany). All solutions and drate heterogeneity of glycoproteins. CE-ESI-MS has background electrolytes were degassed by ultrasoni- been successfully used for this purpose [11-13]. How- cation before use. ever, volatile background electrolytes (BGE) are necessary to provide suitable electrospray ionization and Instrumental therefore, obtain good sensitivity. The non-volatile CE-ESI buffer used in the current CE-UV methods for gly- CE was performed on a Hewlett Packard CE (Agi- coform separation preclude the CE-ESI-MS coupling lent Technologies, Waldbronn, Germany). For CE- [14-16]. MS coupling, a coaxial sheath-liquid sprayer was used (Agilent Tecnologies). For intact EPO glycopro- In our work, CE-UV methods for the separation tein analysis, separation was performed in capillar- of glycoforms in volatile BGE have been devel- ies coated with polybren (PB) or ultra Tof Dynam- oped. The CE-ESI-MS separation method for in- ic Pre-Coat LN (LN). For Tf analysis the separation tact rHuEPO has been improved as a consequence capillary is coated with a polybren-dextran (PB-DE) of the use of a novel acrylamide-based coating double layer couting. that provides a stable suppression of EOF and allows a succesful glycoform separation [7, 17]. MS Also a method for the separations of Tf sialofor- Mass spectrometric Tf analysis are carried out in a ms has been developed, that permits the diagnostic Marimer TOF mass spectrometer (Perseptive Bio- of Congenital Disorders Glycosylation and chron- systems, Framingham, MA, USA) coupled to CE sys- ic alcoholism. A stable negative modified capillary tems whereas the hormones spectrometric investiga- is performed by a first amino quaternary coating tions were performed with the CE system compled to (Polybrene) attached to the capillary wall, followed a MSD Ion Trap mass spectrometer (Agilent Tech- by a second anionic coating (Dextran) obtaining a nologies) and characterization of glycoprotein were negative coated capillary [6, 7]. In order to improve performed using a CE-microTOF (Bruker Daltonik), de detection limits of CE-ESI-MS methodologies, an orthogonal accelerated TOF mass spectrometer the use of solid-phase extractions coupled on-line (oaTOF-MS). to capillary electrophoresis using electrospray mass spectrometry detection (SPE-CE-ESI-MS) is stud- RESULTS AND DISCUSSION ied for the analysis of peptide hormones in dilute Transferring glycoform analysis by CE-UV and CE-ESI-MS solution [10]. This resulted in sample clean-up and Application to CDG chronic alcoholism diagnosis concentration enhancement, with minimum sam- A CE-UV separation method has been developed us- ple handling. The CE-ESI-MS developed methods ing a MS compatible buffer with 25 mM NH4Ac at pH are applied for the characterization of rHuEPO gly- 8.5. Best separation conditions have been obtained in coforms. The achieved separation and the highly a coated capillary based on a Successive Multiple Ion- mass-resolving time of fligh (TOF) mass detection ic Layer (SMIL) performed by a first layer of PB and a allows to establish the most probable rHuEPO gly- second layer of dextran (DS). This coating provides a coforms. constant and positive EOF, and minimizes the interactions between Tf and capillary walls improving sep- EXPERIMENTAL PART aration reproducibility. Chemicals All chemicals used in the preparation of buffers and In order to deplete albumin and the most abundant solutions are analytical reagent grade. Standard hu- immunoglobulins from sera, a commercial kit based man transferrin (partially saturated, min 98%), insu- on dyes and immunoaffinity capture has been used lin, sodium dextran sulphate (M=500000), and hex- prior to electrophoretic analysis. Figure 1 shows the adimethrin bromide (Ploybrene, PB, M=15000) are obtained electropherograms in two different sera, one purchased from Sigma. Ultra TolDynamic PreCoat from a healthy individual and the other from a CDG LN was provided by Target Discovery (Palo Alto, CA, patient. USA). Trypsin Gold, Mass Spectrometry Grade, was obtained by Promega (Madison, WI, USA). Stand- A clear difference on the electrophoretic profiles is ard rHuEPO was obtained as BRP from Pharmaco- observed. The CE-UV developed method is now ap- poeia (EDQM, European Pharmacopoeia, Council of plied in clinical diagnosis. Europe, Strasbourg, France). Epoetin-α (Eprex) 6000

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LECTURE Using CE-ESI-MS methodologies the best sensitivity is obtained with a sheath liquid containing 90% of methanol and 0.5 % formic acid. Other experimental parameters as sheath liquid flow rate, nebulizer gas and ionization potentials have been optimized in order to achieve good sensitivity and separation .Sepa- ration of different proteins present in serum has been achieved and mass spectra can be deconvoluted. A glycoform of 77387 Da is obtained in a serum from a CDG patient that is not observed for a control serum (Figure 2). Analysis of hormones by on line SPE-CE-ESI-MS Endogenous hormones are generally found at subnanomolar levels, e.g. between 100 and 1 ng L depending on the hormone, in biological samples. In our studies, solid-phase extraction coupled on-line to capillary electrophoresis electrospray mass spectrometry (SPE-CE-ESI-MS) is explored for the preconcentration and separation of dilute solutions of peptide Figure 1. CE-UV electropherograms obtained for different serum samples. hormones. First, a CE-ESI-MS methodology was de- a) Non-treated healthy, b) healthy serum and c) CDG serum both passed veloped and validated. Limits of detection (LOD) of through the albumin depletion kit. around 1 g mL were obtained for all the studied hormones. For SPE-CE-ESI-MS experiments, a home-

Figure 2. a) Total Ion Electropherogram obtained for a serum from a healthy individual in the CE-ESI-MS optimized conditions. b) and c) deconvoluted mass spectra obtained from the beginning and the end of the Tf peak respectively. d) Total Ion Electropherogram obtained for a serum from a CDG patient. e) and f) deconvoluted mass spectra obtained from the two partial resolved glycoforms of Tf. The most probable glycan composition is displayed below the deconvoluted mass spectra.

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made SPE microcartridge containing a C18 sorbent LECTURE was constructed near the inlet of the separation capillary. (Figure 3). After optimizing the on-line preconcentration methodology, LOD between 10 and 0.1 ng mL-1 were achieved. The preconcentration methodologies have been applied to rHuEPO analysis using an in-line inmunoaffinity solid phase extraction(IA- CE-ESI-MS).The preliminary results obtained using a custom-made inmunoaffinity sorbent prepared from an anti-human EPO polyclonal antibody and glutaraldehyde-glass beads show the potential of this novel approach. Characterization of rHuEPO glycoforms by CE-ESI-MS

Several recombinant human erythropoietins (rHuE- Figure 3. Scheme of the manufacturing procedure for the C18 analyte PO) from different origen have been analysed. Coat- concentrator (C18AC). ed capillaries are mandatory in order to decrease or suppress the adsorption of glycoproteins to the silica capillary wall. In our works two different capillary coatings have been used: (1) polybrene (PB), from spectra is not as obvious as in more straight- an amino quaternary polymer with positive charges forward samples. Figure 4 shows the separation and that reverses the EOF, and (2) UltraTolDynamic mass spectra obtained from rHuEPO in a LN coated Pre-Coat Low Normal (LN), an acrylamide polymer capillary, summarizing the procedure performed in that suppresses the EOF at low pH. The best sensi- the data analysis. tivity has been obtained in sheath liquids containing 1% acetic acid and high resolution TOF-MS has In order to obtain more complete information about been found to be the most suitable mass analyzer the carbohydrate moiety of glycoproteins, a study of for detect intact glycoproteins differing in few Da. the composition and structure of the glycan is nec- In CE-ESI-MS analyses of intact glycoproteins, nu- essary. In our work a CE-ESI-MS separation meth- merous and complex data are obtained and there- od has been developed for the N-glycans obtained fore, extracting useful and valuable information by PNGase F release from the glycoproteins Fig-

Figure 4. Schematic view for data processing of intact CE-ESI-MS glycoprotein analysis in a LN coated capillary, BGE: 2M acetic acid, separation voltage + 30kV. Sample: Pharmacopoeia rHuEPO 2.5 µg/µL injected for 15s, at 50 mbar. a) Base Peak Electropherogram (BPE) obtained, b) mass spectrum obtained in 25.0-25.2 min, c) deconvoluted mass spectrum, d) ion identification for 29888.2 Da glycoform, e) Extracted Ion Electropherogram (EIE) obtained for 29888.2 Da glycoform (in purple) and for some of the different sialoforms present in rHuEPO (in grey).

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LECTURE ure 5. Also the sialic acid content of O-glycosylation site has been characterized based on the glycopeptides obtained by trypsin digest and CE-ESI-MS analysis. Therefore, the probability of every intact sialoform has been calculated taking into account the number of glycosylation sites, and the percent- ages of the N-glycans and the O-glycans depending on their sialic acid content. The data have been compared with the normalized areas obtained from the EIE of the intact sialoforms of Pharmacopoeia rHuEPO, and therefore the sialic acid assignment Figure 5. Typical tetra-antennary N-glycan of rHuEPO. has been performed. Once sialic acid number has been assigned, a global carbohydrate composition is deduced in high confidence. Thus, a main molecular mass of 29888.2 Da, consist of the protein backbone (165 amino acids, 18235.8 Da), 22 hexoses, 19 N-acetylhexosamines, 3 fucoses and 13 sialic acids (Figure 6). Table 1 shows the main observed molecular masses and the respective carbohydrate composition.

References 1. Skibeli, V, Nissen-Lie G, Torjesen P. Blood, 98, 3626-3634, 2001. 2. Mechref Y, Novotny V. Chem. Rev., 102, 321-369, 2002. Figure 6. One possible glycan composition for an intact rHuEPO glycoform 3. Balaguer E, Benavente F, NeusüB C, Sanz-Nebot V, Bar- of 29888.2 Da containing 22 hexoses (), 19 N-acetylhexosamines (), 3 bosa J. Electhrophoresis, (2006) in press. fucoses (◄) and 13 sialic acids (). 4. Sanz-Nebot V, González P, . Toro I, Ribes A, Barbosa J. J. Chromatogr.B, 798, 1-7, 2003.

Table 1. Main intact rHuEPO glycoforms and the respective carbohydrate composition obtained in a LN coated capillary.

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5. Fisher JW. Exp. Biol. Med., 228, 1-14, 2003. 11. Kelly JF, Locke SJ, Ramaley L, Thibault P. J. Chromatogr. LECTURE 6. Sanz-Nebot V, Benavente F, Vallverdú A, Guzmán NA, A, 720, 409-427, 1996. Barbosa J. Anal. Chem., 756, 5220-5229, 2003. 12. Demelbauer UM, Plematl A, Kremser L, Allmaier G, Josic 7. Balaguer E, Demelbauer U, Pelzing M, Sanz-Nebot V, D, Rizzi A. Electrophoresis 25, 2026-2032, 2001. Barbosa J, NeusüB C. Electrophoresis, 27, 2638-2350, 13. Neusüss C, Demelbauer U, Pelzing M. Electrophoresis, 2006. 26, 1442-1450, 2005. 8. Sanz-Nebot V, Benavente F, Giménez E, Barbosa J. Elec- 14. Klampfl CW. Electrophoresis, 27, 3-34, 2006. trophoresis 26, 1451-1456, 2005. 15. Stutz H. Electrophoresis, 26, 1254-1290, 2005. 9. Giménez E, Benavente F, Barbosa J, Sanz-Nebot V.(2006) 16. Hernández-Borges J, NeusüB C, Cifuentes A, Pelzing M. in press. Electrophoresis, 25, 2257-2281, 2004. 10. Benavente F, Vescina MC, Hernández E, Sanz-Nebot V, 17. Benavente F, Gimenéz E, Olivieri AC, Barbosa J, Sans- Barbosa J, Guzman N. J. Chromatogr. A, 1140, 205-212, Nebot V. Electrophoresis, 27, 4008-4015, 2006. 2007.

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L 05 LECTURE BIOMARKER DISCOVERY FOR CHRONIC INFLAMMATORY DISEASES USING PROTEOMIC SERUM PROFILING

M. FILLET1, D. de SENY2, M-A. MEUWIS1, P. GEURTS4, L. WEHENKEL4, Ed. LOUIS3, M. MALAISE2, M-P. MERVILLE1 1Laboratory of Clinical Chemistry, 2Rheumatology, 3Gastroenterology, 4Department of Electrical Engineering & Computer Science, GIGA Research, University of Liège, and CHU, 4000 Liège, Belgium

he development of new technologies in the last es, as it perfuses all the tissues of the body, sothat it decade in the field of genomic and proteom- carries not only specific blood proteins but also pro- ic analysis has brought novel optimism to the teins or fragments shed by diseased tissue. In order to Tdiscovery of new biomarkers. One of the novel strat- analyse circulating proteins and peptides, the cellular egies employed for the discovery of new biomarkers components of blood are removed, either in the pres- is the analysis of the peptides and proteins in plasma ence of anticoagulants, which yields plasma, or after or serum samples by mass spectrometry. Protein dif- blood coagulation, which yields serum. ferential display techniques such as two-dimensional gel electrophoresis (2-DE), one- or two dimensional Analysis of plasma or serum is a challenge because liquid chromatographic approaches (LC-MS) or sur- of the huge dynamic range of protein abundance and face-enhanced laser desorption and ionization time forms. It is well known that the concentration of the of flight (SELDI-TOF) mass spectrometry have be- proteins present in the blood covers at least 10 orders come increasingly useful to establish fingerprint pro- of magnitude, ranging from albumine (35-50mg/ml files of both disease and non-disease states from large in serum) to IL6 (0-5 pg/ml in serum). Ninety-sev- number of samples. en percent of the proteins found in plasma belong to one of the seven major groups of high-abundant plas- These methods are based on the assumption that the ma proteins: albumin, immunoglobulin, fibrinogen, pathology will affect the physiology of the organism alpha 1-antitrypsin, alpha 2-macroglobulin, transfer- and cause more or less severe changes in the expres- ring and lipoproteins. The remaining 3% is a com- sion level of proteins. Proteins common to both groups plex mixture of middle and low abundance proteins, are ignored when up- and downregulated proteins be- including proteins from the family of complements, come of the main interest and potential biomarkers. hormones, other proteins originated from normal tissue secretion or from tissue leakage upon cell death SELDI’s ProteinChip Arrays distinguish this tech- or damage. The dynamic range of protein mount that nology from other mass spectrometry-based analyt- can be detected in a single mass spectrum (2-3 or- ical systems. ProteinChip Arrays provide a variety ders of magnitude) is insufficient to cover the range of surface chemistries that allow researchers to opti- present in blood sample. To overcome this disadvan- mize protein capture and analysis. The surface chem- tage, two non-exclusive strategies have been devel- istries of the arrays include a series of classic chroma- oped. One consists of the selective depletion of the tographic chemistries and specialized affinity capture most abundant proteins in the plasma or the serum surfaces. Classic chromatographic surfaces include and the second one, on the fractionation of the sam- normal phase for generic protein binding; hydropho- ple. The use of antibodies for the albumin, immu- bic surfaces for reversed-phase capture; cation- and noglobulins and other high-abundance proteins is at anion-exchange surfaces; and immobilized metal af- present the most efficient and specific method. The finity capture (IMAC) for metal-binding proteins. major drawback is the high cost. These resins are ro- Specific proteins of interest can be covalently immo- bust, highly specific and yield to reproducible results. bilized on pre-activated surface arrays, enabling cus- However, it is known that albumin and other high- tomized experiments to investigate antibody-antigen, abundance proteins bind some less abundant pep- DNA-protein, receptor-ligand, and other molecular tides in the blood and act as their carriers. Moreover, interactions. Then, the unbound proteins are washed the depletion shows a certain degree of cross-reactiv- away, the chips are overlaid with an energy-absorbing ity with non-targeted proteins. matrix and finally spectra are acquired by using laser ionization and TOF separation mass spectrometry. In order to prevent the finding of artefactual biomarkers, it is important to eliminate as many of variables. Blood is an ideal source of markers because it is eas- The all procedure has to be standardized from the me- ily accessible and reflects secondary systemic chang- dical history of the blood donor to the acquisition of

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the spectra. When preparing the plasma, the nature of ibility and as a control protein profile for each SEL- LECTURE the anticoagulant added to the blood and the protocol DI experiment. employed to remove the cellular components influences the protein content of the sample. Similarly, when Several chip arrays such as strong anion-exchange preparing serum, the material of the tube for blood (SAX), weak cation-exchange (CM10) and hydro- collection (plastic or glass) and the conditions of the phobic (H4) were tested in order to determine which clotting affect the peak pattern. would provide the optimal profile for serum in terms of number and resolution of peaks. pH (from 4 to 9) This work describes how SELDI-TOF applied to and salt concentration (from 30 mM to 1M) in wash- blood samples may generate complex protein profile ing buffers were optimized using ion-exchange ar- for diagnostic and prognostic evaluation and become rays as well as the percentage of acetonitrile (ACN) a valuable tool to reliably predict arthritis and inflam- (from 10 to 60 %) in H4. CM10 and H4 were finally matory bowel diseases (IBD) outcome. The aim is to observed to give the best results. accurately distinguish patients with Rheumatoid Ar- thritis or Crohn disease from patients with other in- Mass accuracy was calibrated externally using peptide flammatory diseases and from healthy controls. molecular mass standards in order to cover a larger range of mass (0 to 20 000 Da). Chronic inflammatory diseases like Rheumatoid Ar- thritis (RA) and Crohn disease (CD) are autoimmune Several processing steps are required before data diseases of unknown etiology for which diagnosis are analysis such as baseline subtraction, normaliza- based on a pattern of clinical symptoms completed tion or peak detection. Baseline subtraction was by unspecific biological tests for RA and endoscop- achieved by employing a varying-width segment- ic tissue histology for CD. Biomarkers for early died convex hull algorithm that eliminates any base- agnosis or prognosis do not exist and clinicians of- line signal caused mostly by matrix distortions. Nor- ten manage the patients empirically and secondarily malization was essential to eliminate any systematic adapt the therapeutic strategy according to the clin- effects between samples due to varying amounts of ical evolution. protein or degradation over time in the sample or variation in the instrument detector sensitivity. All The project requires the establishment of a biobank data were normalized according to the total ion cur- including serum, tissue samples and epidemiologi- rent normalization function by following the soft- cal data, from large cohort of patients afflicted with ware instruction. Peak detection was performed us- RA, CD and related diseases (Psoriatic Arthritis - ing the ProteinChip Biomarker software version 3.0 PsA- and Ulcerative rectocolitis -UC-). The num- (Ciphergen Biosystems, Inc.). The part of the spec- ber of samples and types of samples is one of the trum m/z values < 1000 was not used for analysis, most important parameters that determine the suc- as the energy absorbing matrix signal generally in- cess of a project. Usually, we profile at least 30 sam- terfered with peak detection in this area. Peaks be- ples in each classification group (e.g., disease versus tween 1000 and 20 000 m/z ratio were autodetected healthy or treated versus untreated). This number of with a signal:noise ratio >3 and the peaks clustered samples is usually enough to give us > 90% statisti- using second-pass peak selection with signal:noise cal confidence in single marker with p values < .01 ratio >2 and a 0.3% mass window. and is also enough to use different forms of multivariate analysis. Standardization of all experimental conditions was drawn up to minimize the effects of irrelevant sourc- Control sera were selected on the basis of matching es of fluctuation and coefficient of variations (CVs) for age, sex and Caucasian race. All samples were al- were calculated to evaluate the reproducibility of ex- iquoted and frozen at -80°C until thawed specifical- periments using SELDI-TOF-MS approach. ly for SELDI analysis. We paid a careful attention thoughout all our experiments to avoid as much as Moreover, we chose to include patients with a vari- possible sources of variation in the procedure. For ex- ety of very close pathologies to RA (osteoarthritis, ample, the sera freeshly collected have been aliquot- PsA) in our control groups to mimic real life diagnos- ed, stored at –80°C and were only ones unfreezed. tic difficulties. We also wanted to include heterogene- Our quality control serum allowed us to detect any ous control groups, because we wanted to differenti- unusual features during the process. All those precau- ate markers that are inflammatory in nature from RA tions allowed us to obtained a very good repetability specific molecules. and a good reproducibity between the results. Data were analyzed with an original multivariate sta- A quality control serum sample was taking from a tistical method based on multiple decision trees al- healthy control. It was used to determine reproduc- gorithms. One of the challenges in the analysis of

Kervansaray Convention Center & Hotel, LARA, ANTALYA / TÜRKİYE 15 International Symposium on Drug Research and Development S

LECTURE SELDI mass spectrometry-generated data is avoid- In the second approach, RA spectra were compared ing the false discovery of proteins peaks, of which to PsA spectra. PsA is a chronic disease, whose clin- the discrimatory power is due to random variation. ical manifestations are very close to the ones of RA. The statistical approach we used to analyse the SEL- Sensitivity reaches 81% on CM10 and 90% on H4 DI profiles reduces this problem by ranking all of the arrays. Correlation between discriminant values detected protein peaks according to their relative con- obtained by boosting and p-values was also per- tribution to the separation of distinct data sets and formed. by using bootstrap cross-validation. As an additional safeguard against the identification of discriminating Finally, the multivariate analysis based on multiple peaks that are merely artefacts, we analysed all of the decision trees generated several models that could samples in duplicate, and only the peaks that exhibit- classify samples with good sensitivity and specificity ed a reproducibly high ranking in both sets of analy- (minimum 80%) discriminating inflammatory bowel sis were used. For most comparisons, bioinformatics diseases versus controls (inflammatory or healthy) or analysis yielded a panel of 10 or more distinguished Crohn versus ulcerative colitis. between patients groups. Once the optimum discrim- inatory decision process is created, blinded samples In conclusion we can say that Surface Enhanced La- can then be subjected to analysis to confirm the utili- ser Desorption Ionization-Time Of Flight-Mass Spec- ty of the algorithm. trometry technology combined with the use of the multiple decision trees method, as robust statistical In a first approach, we compared RA spectra versus tool, led to the selection of protein biomarker patterns control spectra (inflammatory controls and non-in- that may reveal to be helpful for diagnosis and under- flammatory controls). According to the boosting sta- standing of chronic inflammatory diseases. tistical analysis a sensitivity superior to 75% and 85% was obtained on CM10 and H4 arrays respectively.

16 May 17-20, 2007 “From Chemistry to Medicine” DRD 2007 S

L 06 IN SILICO MEDICINAL CHEMISTRY – UNDERSTANDING LECTURE BIOLOGICAL EFFECTS THROUGH MOLECULAR DOCKING AND MOLECULAR DYNAMICS SIMULATIONS

Wolfgang SIPPL Institute of Pharmaceutical Chemistry, Martin-Luther-University of Halle-Wittenberg, 06120 Halle/Saale, Germany

rotein homology models have been used in els can be used as structural basis for lead finding conjunction with structure-based approach- of yet not crystallized protein targets and are able to es to successfully identify novel inhibitors over provide important information concerning their bi- Pthe last few years. It is widely accepted that for exam- ological effects. ple docking to homology models is more challenging and less successful than docking to X-ray structures of proteins. To successfully apply structure-based References methods to homology models, in addition to accu- 1. Windshügel B, Jyrkkärinne J, Poso A, Honkakoski P, Sippl rate docking programs, high quality protein models W. Molecular dynamics simulations of the human CAR are needed. ligand binding domain: Deciphering the molecular basis for constitutive activity, J. Mol. Mod., 11, 69-79, 2005. The present talk will highlight the results obtained 2. Jyrkkärinne J, Windshügel B Mäkinen J, Ylisirniö M, Peräkylä M, Poso A, Sippl W, Honkakoski P. Amino acids for two protein targets where we used a combina- important for ligand specificity of the human constitution of comparative protein modelling and struc- tive androstane receptor, J. Biol. Chem., 280, 5960-5971, ture-based methods in order to find novel leads. For 2005. 1,2 both targets (the nuclear hormone receptor CAR 3. Spannhoff A, Heinke R, Bauer I, Trojer P, Metzger E, and the histone modifying enzymes PRMT1 and Gust R, Schüle R, Brosch G, Sippl W, Jung M. Target- SIRT2) we were able to identify new ligands based based approach to inhibitors of histone arginine methyl- on a structure-based virtual screening. Besides the transferases, J. Med. Chem., 2007, in press. successful identification of PRMT13 and SIRT24 in- 4. Trapp J, Jochum A, Meier R, Saunders L, Marshall hibitors as well as CAR agonists the validated ho- B, Kunick C, Verdin E, Goekjian P, Sippl W, Jung M. mology models were used to explain the structural Adenosine mimetics as inhibitors of NAD+-dependent basis for yet not understood biological effects. Our histone deacetylases - from kinase to sirtuin inhibition, J. Med. Chem., 49, 7307-16, 2006. findings suggest that high quality homology mod-

Kervansaray Convention Center & Hotel, LARA, ANTALYA / TÜRKİYE 17 International Symposium on Drug Research and Development S

L 07 LECTURE SURFACE-MODIFIED NANOPARTICLES FOR DRUG ENTRAPMENT AND TARGETING

Ruxandra GREF UMR CNRS 8612, Paris-Sud University, Châtenay-Malabry, France

ver the last decades, there has been a grow- col) (PEG) were the most commonly used polymers ing interest in the development of a drug car- for surface modification. rier that is small enough for intravenous ad- Oministration and has the ability to bypass the normal Core-shell (PEG) nanoparticles revealed to be of out- physiological defense processes of the organism. This most interest; the biodegradable core is able to entrap type of “stealth” carrier could therefore circulate for and release a variety of active molecules, whereas the prolonged times in the bloodstream, opening in this hydrophilic PEG surface governs the in vivo interac- way new therapeutic opportunities, such as control- tions and determines the fate after intravenous ad- led drug release into the vascular compartment, or ministration. targeted delivery. Examples of applications will be given, including the There are numerous potential applications for such entrapment of proteins and lipophilic drugs. The ac- a system, for example, the protection of highly sen- cent will be put on the challenge of busulfan entrap- sitive active molecules against in vivo degradations, ment. This small fragile molecule with a strong ten- the reduction of the toxic side effects that can occur dency to crystallize is indeed particularly difficult to when highly active drugs such as those used in cancer be encapsulated. therapy are administered as a solution, the increase of patient comfort by avoiding repetitive injections or Busulfan is a bifunctional alkylating agent, which is the use of perfusion pumps, and the achievement of widely used at high dose as a part of myeloablative more favorable drug pharmacokinetics. The success- regimen before both allogenic and autologous bone ful administration of protein drugs is also often prob- marrow transplantation for the treatment of haema- lematic. Hydrolyzed or denaturated in the gastroin- tological malignancies and non-malignant disorders testinal tract, most proteins cannot be administered such as immunodeficiency. For a long time, busulfan orally, and usually, they are quickly metabolized if in- has been available only in oral form and a wide in- travenously injected. Their typical half-lives,ranging tra-patient and inter-patient bioavailability variabili- from 2 to 30 min, could be improved by encapsulat- ty in both adult and children has been reported [1]. ing them in long-circulating drug delivery devices Moreover, severe side effects were reported such as acting as circulating microreservoirs. the veino-occlusive disease. This pathology has been correlated with a high systemic exposure to busulfan Many efforts have concentrated in recent years on the expressed as the area under the plasma concentra- design of submicronic long-circulating drug carri- tion-time curve. ers such as liposomes, lipid emulsions, micelles, solid lipid nanoparticles, and nanoparticles. Among all of In order to overcome these problems, intravenous these systems, we will focus on nanoparticles, which formulations of busulfan were developed, using co- were defined as solid colloidal particles ranging in solvent mixtures [2]. However, these organic solvents size from 10 to 1000 nm, consisting of macromo- have their own well-documented toxicity. Therefore, lecular materials in which the active principle is dis- to avoid the massive use of organic solvents, injecta- solved, entrapped, and encapsulated and/or to which ble colloidal carriers, such as conventional liposomes the drug is adsorbed or attached. [3] have been elaborated. However, these carriers had encapsulation efficiencies lower than 1% (w/w). The strategy of choice in the design of submicronic long-circulating drug carriers was to sterically ex- We aimed at designing composite core-shell nanopar- clude the cellular immune system from recognizing ticles able to combine the ability of the biodegradable and responding to the particles’ artificial surfaces. To polymeric cores to efficiently encapsulate busulfan, achieve this, polymers were covalently bound or ad- with the excellent steric repulsive properties of the di- sorbed on surfaces. Among these, poly (ethylene gly- block copolymer, poly (�-caprolactone)-poly (ethyl-

18 May 17-20, 2007 “From Chemistry to Medicine” DRD 2007 S

Figure 1. Typical transmission electron microscopy studies of PCL-PEG (left) LECTURE and composite PIBCA/ PCL-PEG (right) nanoparticles.

ene glycol) (PCL-PEG), which is expected to provide erential surface distribution of the PCL-PEG poly- increased blood half lives to the nanoparticles [4]. mer. Therefore, composite nanoparticles have a ‘core- shell’-type structure, where the “core” is essentially In a first step, we have chosen the biodegradable poly- formed by the PIBCA polymer and the “shell” by the mer able to encapsulate the highest amounts of busul- PCL-PEG copolymer. The use of PIBCA to form the fan. For this, busulfan was entrapment by nanoprecip- core of the nanoparticles leads to a 2-4 fold drug load- itation into polyesters and five different types of poly ing increase, in comparison to the single PCL-PEG (alkyl cyanoacrylate) polymers. The polymers leading nanoparticles. In addition, the complement activa- to the highest busulfan loading efficiencies were poly tion results showed a significant difference between (isobutyl cyanoacrylate) (PIBCA) and poly (ethyl cy- the composite nanoparticles and the single PIBCA anoacrylate) (PECA). Molecular modelling along nanoparticles, thus demonstrating that PEG at the with energy minimization process was employed to surface of the nanoparticles reduced the complement identify the nature of the interactions occurring be- consumption, one of the main opsonin responsible tween busulfan and PIBCA. Further, optimization for nanoparticle removal from the blood stream. studies enabled to obtain PIBCA nanoparticles displaying busulfan loading ratios equal to 5.9 % (w/ These data strongly suggest that PEG-coated nano- w) together with nanoparticle yields of 71 % (w/w). particles could serve as long-circulating busulfan car- Since busulfan is a highly reactive molecule, we per- riers. formed 1H-NMR spectroscopy experiments showing that chemical integrity of the drug was preserved after loading into nanoparticles. The in vitro release stud- References ies under ‘sink’ conditions, in water or in rat plasma 1. Hassan M, Oberg G, Bekassy AN, Aschan J, Ehrsson H, showed a fast release in the first minutes, followed by Ljungman P, Lonnerholm G, Smedmyr B, Taube A, Wal- a slower one over 6 hours. lin I, et al.. Pharmacokinetics of high-dose busulphan in relation to age and chronopharmacology, Cancer Chem- The composite busulfan-loaded core-shell nanopar- other. Pharmacol., 28(2), 130-134, 1991 2. Bhagwatwar HP, Phadungpojna S, Chow DSL, Andersson ticles were obtained by co-precipitation of mixtures BS. Formulation and stability of Busulfan for intrave- of PIBCA and of PCL-PEG, in different mass ratios nous administration in high-dose chemotherapy, Cancer [5]. The nanoparticle size, morphology and surface Chemother. Pharmacol., 37, 401-408, 1996. charge were assessed. For example, electron micros- 3. Hassan Z, Nilsson C, Hassan M. Liposomal Busulfan: copy studies revealed a particular core-shell structure bioavaibility and effect on bone marrow in mice, Bone typical of the composite nanoparticles (Fig. 1). Marrow Transplant., 22, 913-918, 1998. 4. Layre AM, Gref R, Richard J, Requier D, Chacun H, Appel The chemical composition of the top layers was deter- M, Domb AJ, Couvreur P. Nanoencapsulation of a crys- mined by X-ray photo-electron spectroscopy (XPS). talline drug, Int. J. Pharm., 298(2), 323-327, 2005. 3H-labelled busulfan was used in order to determine 5. Layre AM, Couvreur P, Chacun H, Richard J, Passirani the drug loading efficiency and the invitro drug re- C, Requier D, Benoit JP, Gref R. Novel composite core- shell nanoparticles as busulfan carriers, J. Contr. Release, lease by liquid scintillation counting. Physico-chem- 111(3), 271-80, 2006. ical techniques such as Zeta potential determination and XPS analysis provided evidence about a pref-

Kervansaray Convention Center & Hotel, LARA, ANTALYA / TÜRKİYE 19 International Symposium on Drug Research and Development S

L 08 LECTURE CHALLENGES IN THE TREATMENT OF GRAM-POSITIVE INFECTIONS

Serhat ÜNAL Hacettepe University, Faculty of Medicine, Department of Medicine, Section of Infectious Diseases, Ankara, Turkey

he introduction of benzylpenicillin in the dependent antimicrobial effect against Gram-posi- 1940s was the start of an era in the treatment of tive microorganisms, including MRSA. Its once–dai- bacterial infections but is soon became appar- ly dosing and and the favourable safety profile (except Tent that some strains of Staphylococcus aureus were some concerns about rhabdomyolysis and neuropa- resistant due to production of β-lactamase. Since the thy) are making daptomycin an attractive option for mid-1970s resistance to antimicrobials has become the treatment of Gram-positive infections. Succesful an escalating problem. A striking change over the past results had been reported for the treatment of bac- quarter-century has been the increasing role of Gram- teremia and right-sided infective endocarditis but not positive bacteria which might have resulted from the for the treatment of community acquired pneumo- emphasis placed on controlling Gram-negative infec- nia. tions. Today we have to deal with infections caused by multidrug-resistant organisms, particularly methicil- Tigecycline (Tygacil®) is a new, semisynthetic glycyl- lin resistant staphylococci, penicilin- and erythromy- cycline that was approved by US FDA for the treat- cin-resistant pneumococci and vancomycin-resistant ment of skin, soft tissue and intra-abdominal infec- enterococci. The emergence and rapid spread of me- tions It appears to be very active not only against thicillin- resistant S.aureus (MRSA), which were re- MRSA but also against glycopeptide-resistant staphy- sistant not only to all β-lactams but also to the main lococci and enterococci. Nausea and vomitting are antibiotic classes, resulted a an increase use of glyco- the most common side-effects. peptide antibiotics, namely vancomycin and teicho- planin. Unfortunately, the first glycopeptide-resistant Quinupristin-dalfopristin (Synercid®) is fixed mic- enterococci were described in late 1980s, they rapidly ture of semisynthetic streptogramin derivatives. It is spred in many different countries. bacteriostatic against Enterococcus faecium and bac- tericidal against methicillin-susceptible and methi- Against this background, there is a definitive need for cillin-resistant staphylococci. It is inactive against new antimicrobial agents, as well as for prudent use of Enterococcus faecalis. Pain and inflammation at the existing agents. infusion site, arthralgia, myalgia, drug interactions and liver function abnormalities have occured in pa- Linezolid (Zyvox®) is the first of a new class of anti- tients treated with quinupristin-dalfopristin. microbial agents, the oxazolidinones. It was approved by the U.S. Food and Drug Administration (FDA) in Oritavancin, telavancin, dalbavancin are the new 2000 for the treatment of uncomplicated and compli- glycopeptides in clinical development which appear cated skin and soft tissue infections, including diabet- as potent molecules wiht favourable pharmacokinetic ic foot infections without concomitant osteomyelitis, and pharmacodynamic properties. community acquired and nosocomial pneumonia and vancomycin-resistant Enterococcus faecium infec- Ceftobiprole is an investigational novel cephalosporin tions including cases with concurrent bacteremia. Al- that is active in vitro against streptococci and staphy- though clinical trials have shown linezolid to be as ef- lococci, including penicilin-resistant strains of pneu- fective as best established therapy, resistance has been mococci and MRSA, while maintaining the activity reported. In addition, high rates of adverse effects, in- of extended-spectrum cephalosporins against Gram- cluding thrombocytopenia and anemia, are seen with negative organisms. In vivo, screening models predict prolonged courses of therapy. Serotonin syndrome, good activity for ceftobiprole against Gram-positive potentially irrevesible peripheral neuropathy and lac- and Gram-negative bacteria. tic acidosis are also reported. Changing patterns of resistance have compound- Daptomycin (Cubicin®) is the first agent of a new class ed and exacerbated the need for new antimicrobi- of cyclic lipopeptides The drug exhibits a calcium– al agents. The appropriate indications and cost-effec-

20 May 17-20, 2007 “From Chemistry to Medicine” DRD 2007 S

ctiveness of these molecules will determine our future 2. Pace JL, Yang G. Glycopeptides: update on an old suc- LECTURE treatment options. Until then the prudent use of ex- cessful antibiotic class, Biochem Pharmacol., 71, 968-980, isting antibiotics with strict reinforcement of infec- 2006. tion control should be the rules of our practice in the 3. Eliopoulos GM. Current and new antimicrobial agents, treatment of gram-positive infections. Am Heart J, 147, 588-91, 2004. 4. Torres-Viera C, Dembry LM. Approaches to vancomycin-resistant enterococci, Curr Opin İnfec Dis., 17, 541-7, References 2004. 5. Bosso JA. The antimicrobial armamentaium: Evaluating 1. Appelbaum PC. MRSA-the tip of the iceberg, Clin Micro- current and future treatment options, Pharmacother., 25, biol Infec., 12 (Supp 2), 3-10, 2006. 55S-62S, 2005.

Kervansaray Convention Center & Hotel, LARA, ANTALYA / TÜRKİYE 21 International Symposium on Drug Research and Development S

L 09 LECTURE CHALLENGES IN TREATMENT OF GRAM-NEGATIVE BACTERIA

Recep ÖZTÜRK İstanbul University, Cerrahpaşa Medical Faculty, Infectious Diseases and Clinical Microbiology, İstanbul, Turkey

he issue of resistance among nosocomial or complicated skin, soft tissue and intra-abdominal in- even community acquired gram-negative mi- fections. Its activity in the treatment of community- coorganisms including primarily extend- and hospital-acquired pneumonias is under investi- Ted-spectrum beta-lactamase producing Enterobac- gation. It is approved by the Ministry of Health in our teriaceae (Escherichia coli, Klebsiella pneumoniae), country. Nausea, vomiting and diarrhea are among multidrug-resistan (MDR) Pseudomonas, and MDR the side effects [2, 5]. Acinetobacter produce a challenge for their tratement. Recently carbapenem-resistant, ESBL producing en- Ceftobiprole teric bacilli and panresistant Pseudomonas and Aci- It exhibits activity against resistant gram-posi- netobacter strains emerged [1]. tive bacteria such as MRSA and penicillin-resistant Streptococcus pneumoniae as well as gram-negative Developing new antimicrobials have considerable bacteria such as E. coli, K. Pneumoniae and Entero- problems since the resistance shortens market life bacter cloacae. It has oral and parenteral formula- and thus increases its cost. Pharmaceutical pipelines tions. Complicated skin and soft tissue infections, are less encouraged to maintain new agents. Discov- and community- and hospital-acquired pneumonias ery of novel antibiotics is not expected to give much are among the probable indications for its usage [2, alternatives in near future [2, 3]. On the other hand, 6]. a clear and urgent need for developing new anti-in- fectives is apparent. However especially develeop- Doripenem ing drugs for the agents especially for the multidrug- It is a carbapenem class antibiotic with activity against or pan-resistant ones is a social responsibility of the gram-positive, gram-negative and anaerobic bacte- companies [3, 4]. ria. It exhibits activity against ESBL-producing enter- ic rods similar to imipenem and meropenem. It has Antibiotics such as colistin that have been available partial activity against carbapenem-resistant P. aeru- but had limited use due to its toxicity gained a new ginosa. It is under investigation for the treatment of interest in this challange posed by resistant microor- nosocomial- and ventilator-associated pneumonias, ganisms. Recently available drugs against gram-nega- complicated urinary tract infections, pyelonephritis tives with ongoing studies are as follows: and complicated intra-abdominal infections. It has parenteral formulation [7, 8]. Tigecycline It is the first member of the glycylcycline class antibi- Faropenem otics, which are the synthetic analogues of classical tet- It is a carbapenem class antibiotic with oral formula- racyclines. The modification of tetracycline at its D-9 tion available. It has wide-spectrum of activity similar position of the central four-ring carbocyclic skeleton to other carbapenems [2]. enabled the molecule with much broader spectrum of antimicrobial activity as well as defense against tet- Garenoxacin racycline resistance. It exhibits activity against gram- It is a new quinolone antibiotic undergoing phase III positive bacteria including MRSA and VRE as well as clinical trials. It exhibits activity against gram-posi- gram-negative bacteria such as ESBL producing en- tive, gram-negative (M. catarrhalis and H. influenzae) teric rods and multidrug-resistant Acinetobacter spp. and anaerobic bacteria [3]. β-lactamase negative and positive strains of Haemo- philus influenzae and Moraxella catarrhalis are within Prulifloxacin its spectrum. Moreover, it is active against the anaer- This new quinolone is the prodrug of ulifloxacin that obic bacteria and MOTT (Mycobacterium other than exhibits activity against gram-positive, gram-negative tuberculosis) rods. It has limited activity against Pseu- (Enterobacteriaceae, H. influenzae and M. catarrhalis) domonas aeruginosa and Proteus spp. It is indicated in and anaerobic bacteria [3].

22 May 17-20, 2007 “From Chemistry to Medicine” DRD 2007 S

New ribosomal inhibitors

2. Bosso JA. The Antimicrobial Armamentarium: Evaluat- LECTURE There is ongoing research on these new class antibiot- ing Current and Future Treatment Options, Pharmaco- ics with wide spectrum (A-72310 and A-692345). This therapy, 25(10 Pt 2), 55S–62S, 2005. class exhibits activity against gram-positive, gram-neg- 3. Khardori N. Antibiotics--past, present, and future, Med ative and respiratory pathogens such as Streptococcus Clin North Am., 90(6), 1049-76, 2006. 4. Bush K. Antibacterial drug discovery in the 21st century, pneumoniae, Haemophilus influenzae, Staphylococcus Clin Microbiol Infect.., Nov;10 Suppl 4, 10-7, 2004. aureus and Moraxella catarrhalis [9]. Though similar 5. Stein GE, Craig WA. Tigecycline: a critical analysis, Clin to quinoles in spectrum of activity, they differ in struc- Infect Dis., 2006 Aug 15; 43 (4), 518-24. ture. As they have a different mechanism of action, 6. Ceftobiprole Medocaril: BAL5788, JNJ 30982081, they are not affected by cross-resistance to other ribos- JNJ30982081, RO 65-5788, RO 655788. Drugs R D. omal inhibitors such as macrolides, tetracyclines, chlo- 2006;7(5):305-11. ramphenicol, aminoglycosides and oxazolidinones. 7. Anderson DL. Doripenem, , 42(6), 399-404, 2006. 8. Jones RN, Huynh HK, Biedenbach DJ. Activities of dorip- Other developments enem (S-4661) against drug-resistant clinical pathogens, There is ongoing research on compounds and extracts Antimicrob Agents Chemother., 48, 3136–40, 2004. other than the ones mentioned above. In addition, 9. Dandliker PJ et al. Novel antibacterial class, Antimicrobial Agents and Chemotherapy; 47, 3831-9, 2003. there are studies to counteract resistance development 10. Mahady GB. Medicinal plants for the prevention and mechanisms using molecular methods [2, 3, 10, 11]. treatment of bacterial infections, Curr Pharm Des., 11, 2405-27, 2005. References 11. Wiart C, Hannah A, Yusof M, Hamimah H, Sulaiman M. Growth inhibition of foodborne and nosocomial 1. McGowan JE Jr. Resistance in nonfermenting gram-nega- pathogens by aqueous fraction of bearded Argostemma tive bacteria: multidrug resistance to the maximum, Am J (Argostemma involucratum Hemsl., Rubiaceae), J Herb Infect Control., 34(5 Suppl 1), S29-37, 2006. Pharmacother., 5, 97-102, 2005.

Kervansaray Convention Center & Hotel, LARA, ANTALYA / TÜRKİYE 23 International Symposium on Drug Research and Development S

L 10 LECTURE CHALLENGES IN ANTIFUNGAL THERAPY

Ömrüm UZUN Hacettepe University School of Medicine, Department of Medicine, Section of Infectious Diseases, Ankara, Turkey

he incidence of invasive fungal infections has sis have rompted a reappraisal of the concept of em- increased, and the spectrum of etiologic agents pirical antifungal therapy. The time period between has widened in recent years. Recent advanc- the biologicalstart of a fungal infection and the ap- Tes in newer classes of antifungals havepromised bet- pearance of clinicalsigns and symptoms represents ter outcome, especially in difficult-to-treat invasive a window of opportunity that, if identified through mould infections; however this has not proven to be prospective screening, may allow earlier therapeutic true. The empirical use of antifungal therapy in pa- intervention and may potentially improve outcome. tients with neutropenia who remain febrile in spite Such ‘preemptive’ strategy would not be triggered by of adequate antibacterial therapy has been a standard fever, but would rest on (1) a better identification of approach for almost two decades. However, thesta- those patients who are at the highest risk for fungal tistical weakness of data, the relatively low incidence infections so that they can be closely monitored;and of IFI in patients not undergoing allogeneic hemat- (2) the availability of sensitive techniques that facil- opoietic stem cell transplantation together with the itate rapid and early diagnosis of invasive fungal in- recent progress in the diagnostic accuracy of IFI has fections. On the other hand, the concept of combina- made the routine use of empirical antifungal therapy tion therapy has become appealing, but there is still a in all patients with fever and neutropenia question- long way to go. able. Recently, new developments in fungal diagno-

24 May 17-20, 2007 “From Chemistry to Medicine” DRD 2007 S

L 11 NATIONAL AND INTERNATIONAL PATENT PROTECTION, LECTURE FREE PATENT SEARCH TOOLS (ESPACENET) AND STRATEGIES

Hakan BAYRAM Patent Examiner, Turkish Patent Institute, Hipodrom Street 115, Yenimahalle, Ankara, Turkey

About PCT About EPC The PCT was concluded in 1970, amended in 1979, The Convention on the Grant of European Patents and modified in 1984 and 2001. of 5 October 1973, commonly known as the Europe- an Patent Convention (EPC), is a multilateral trea- It is open to States party to the Paris Convention for ty instituting the European Patent Organisation and the Protection of Industrial Property (1883). Instru- providing an autonomous legal system according to ments of ratification or accession must be deposit- which European patents are granted. Although the ed with the Director General of WIPO. The Treaty term European patent is used to refer to patents grant- makes it possible to seek patent protection for an in- ed by the EPO, after grant such a patent is not a uni- vention simultaneously in each of a large number of tary right, but a group of essentially independent na- countries by filing an “international” patent applica- tionally-enforceable, nationally-revocable patents, tion. Such an application may be filed by anyone who subject to revocation and/or narrowing as a group is a national or resident of a Contracting State. The pursuant a time-limited, unified, post-grant opposi- procedure under the PCT has great advantages for the tion procedure. applicant, the patent offices and the general public: The EPC provides a legal framework for the granting (i) the applicant has up to 18 months more than of European patents, via a single, harmonized pro- he has in a procedure outside the PCT to reflect cedure before the European Patent Office. A single on the desirability of seeking protection in for- patent application in one language, may be filed at eign countries, to appoint local patent agents in the European Patent Office at Munich, at its branch- each foreign country, to prepare the necessary es at The Hague or Berlin or at a national patent of- translations and to pay the national fees; he is fice of a Contracting State, if the national law of the assured that, if his international application is State so permits. This latter provision is important in the form prescribed by the PCT, it cannot be in countries such as the United Kingdom, in which rejected on formal grounds by any designated it is an offence for a UK resident to file a patent ap- Office during the national phase of the process- plication for inventions in certain sensitive areas ing of the application; on the basis of the inter- abroad without national search report or the written opinion, he can evaluate with reasonable probability the About Patent databases chances of his invention being patented; and the applicant has the possibility during the in- As any exclusive ownership rights, paatents must be ternational preliminary examination to amend accessible to the public if the owner wants to exert his the international application to put it in order right. Today, patents are usually published and acces- before processing by the designated Offices; sible through electronic means. (ii) the search and examination work of patent offices can be considerably reduced or virtually As any exclusive ownership rights, patents must be eliminated thanks to the international search accessible to the public if the owner wants to exert his report, the written opinion and, where applica- right. Today, patents are usually published and acces- ble, the international preliminary examination sible through electronic means. report that accompany the international application; In several technical fields, Patents are the most ef- (iii) since each international application is pub- ficient source of information for the following rea- lished together with an international search re- sons. port, third parties are in a better position to formulate a well-founded opinion about the patentability of the claimed invention.

Kervansaray Convention Center & Hotel, LARA, ANTALYA / TÜRKİYE 25 International Symposium on Drug Research and Development S

LECTURE • Accessibility: All patent documents have a universal format for the bibliographic data. More than 50 different fields, each representing valuable technical or strategic information, are accessible for each Patent. In addition to this, Patents are classified according to internationally agreed systems, which divides technical domains into more than 100,000 subdivisions.

• Content: A patent, to be valid, should enable a person skilled in that particular area to reproduce the invention. This strict requirement explains why 70% of the information contained in Patents is not available elsewhere. When a catalogue or an article describesa product in a few lines, the corresponding Patent often consists of 20 pages. Patentsrepresent about 350 million of A4 pages containing very relevant technical information.

• Concentration: Full patent collections are often present in national Patent Office archives. From a relevant list of patent documents you will need one or two hours in a patent libraryto collect all the data. In comparison, you will usually need several weeks to order andreceive the references quoted in a thesis.

• Up to date: A company is not inclined to make its inventions public. To get a legal protection for the invention, the company generally files the Patent Application at the earliest possible stage. Patent Applications are normally published 18 months after their first filing date and therefore very often represent the first published information available.In other words newly published Patents are the most up-to-date information available ina specific field.

About Coverage and Content About Classification Do not expect to cover the whole state-of-the-art by Patents are classified according to various classifica- searching in national Patents only. tion schemes covering all possible technical domains. When conducting a search, it is essential to systemat- Databases available on espa@cenet® have also some ically use this tool that offers an objective criteria to limitations in the number and the quality of fields ac- access relevant documents. Keywords are much more cessible that should be considered when drawing con- subjective as there are many words to describe a same clusions from the concept.

result of a search. Commercial patent databases usually offer extra value, by the number of fields you can access, and due to the content of the abstract that has been rewritten to facilitate easieraccess to the docu- ment by keyword searches.

To sum up, a patent database user should always be aware of the content of the database he is using and know the limitations of the fields.

26 May 17-20, 2007 “From Chemistry to Medicine” DRD 2007 S

L 12 PATENTABILITY OF MEDICAL AND PHARMACEUTICAL LECTURE INVENTIONS

Serkan ÖZKAN Patent Examiner, Turkish Patent Institute, Hipodrom Street 115, Yenimahalle, Ankara, Turkey

ntellectual property rights are the rights given to in respect of following subject matter: (a) Inventions persons over the creations of their minds. They whose subject matter is contrary to the public order usually give the creator an exclusive right over the or to morality as is generally accepted and (b) Plant Iuse of his/her creation for a certain period of time. In- and animal varieties/species or processes for breed- tellectual property rights are customarily divided into ing/plant or animal varieties/species, based mainly on two main areas: (i) Copyright and rights related to biological grounds. copyright; and (ii) Industrial property rights. In order to develop new drugs, mechanisms will have Industrial property rights are a result of intellectual to be put in place that foster innovation and the de- activities in the industrial spheres such as trademark velopment of new products, while at the same time laws, representing an indication of a business, or de- ensuring that patients have rapid access to the fruits sign rights, utility model rights, and patent rights. of such research. The pharmaceutical sector is a major user of the patent system. While only a small - and Patents are given for any inventions, whether prod- declining - number of new chemical entities are ap- ucts or processes, in all fields of technology without proved annually, thousands of patents are applied discrimination, subject to the normal tests of novel- for to protect variants of existing products, process- ty, inventiveness and industrial applicability. Patents es of manufacture or, where admitted, second uses of give the right holder exclusive right to make, use and known pharmaceutical products. sell the invention. The number of international patent applications con- Inventions, which are new, have an inventive step tinues to rise with impressive growth. The great- that is not obvious to someone with knowledge and est number of international applications (PCT) pub- experience in the subject, and capable of being made lished in 2006 were in telecommunications (10.5%), or used in some kind of industry, are protected by pharmaceuticals (10.4%), and information technolo- patents. gy (10.4%). The fastest growing technology areas are semiconductors (28% increase), information technol- Non-Patentable subject matters and inventions are ogy (22%) and pharmaceuticals (21%). mentioned in the article 6 of the Decree-Law No.551 Pertaining to The Protection of Patent Rights. Ac- There is a remarkable growth rate in the number of pat- cording to subparagraph (e) of this article 6, meth- ent applications received by the Turkish Patent Insti- ods of diagnosis, therapy and surgery applying to hu- tute (TPI) in last years. Number of patent applications man or animal body shall remain outside the scope increased from 1152 to 5165 between 2003 and 2006. the patent protection. The provision under subpara- Also, TPI experienced a %50 growth in the number of graph (e) of the Article 6, shall apply neither to the patent applications in 2006 as compared to 2005. Sim- products and compositions (per se) used in connec- ilar to the increase in the total number of patent ap- tion with these methods nor to their process of man- plications, number of pharmaceutical applications in- ufacturing. Patent shall not be granted for inventions creased from 320 to 467 between 2005 and 2006.

Kervansaray Convention Center & Hotel, LARA, ANTALYA / TÜRKİYE 27 International Symposium on Drug Research and Development S

L 13 LECTURE IMPORTANCE OF POLYMORPHISM IN DRUG RESEARCH AND DEVELOPMENT

Fethi ŞAHİN Gazi University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Ankara, Turkey

olymorphism is the property of a compound This circumstance cause the more intense studies on found in different crystal forms. Different the polymorphs and their patent protection. crystal forms exhibit different dissolution pro- Pfile and polymorphs of recently discovered drug sub- Importance of polymorphism in pharmaceutical instances show different lipophilicity. During the de- dustry can be classified as follows: velopment of an original active principal all the polimorphs should be determined before clinical in- • Thermodynamic stability vestigations. The more stable olymorph which is dis- • Chemical stability covered after clinical studies may cause repeating of • Mechanical features clinical studies or withdrawal of the medicine from • the market. (example Ritonavir). Solubility

28 May 17-20, 2007 “From Chemistry to Medicine” DRD 2007 S

L 14 A VAST SOURCE FOR THE DISCOVERY OF NOVEL DRUG LECTURE LEADS: TRADITIONAL MEDICINES

Erdem YEŞİLADA Yeditepe University, Faculty of Pharmacy, Kayışdağı 34755 İstanbul, Turkey

iscovery of new drug leads may be defined as ical targets. As the initial process the crude extract(s) a SACRED ART. In order to create MASTER- are investigated by in vitro and/or in vivo techniques to PIECES in this field, scientists should have to comb for the potential targets and in turn, to confirm Dembroider each detail obtained through recent com- or disapprove the applications from folk medicines. putational techniques, SAR studies, ethiological stud- This is the most critical step to select the convenient ies on the pathogenesis of diseases, etc. Once a viable pharmacological technique for the success of study. For structure is designed, further process is to obtain the example, during our field survey in Anatolia in a loca- compound in ample quantities by chemical synthesis tion was reported that the decoction of hawthorn (Cra- or biosynthesis for the succeeding procedures. taegus tanacetifolia) roots was used against rheumatic pain. Actually such utilization was interesting since Alternatively, during the photosynthesis hundreds was not previously reported for hawthorn. We pre- of primary or secondary metabolites as well as phy- pared aqueous and methanol extracts from the roots toallexins have been synthesized in each plant speci- of the plant and tested for their effects on carrageenan- men in the nature. Therefore, plants are well-known induced hind paw edema model in mice, a well-known as an important source of many biologically active technique for the assessment of anti-inflammatory ac- compounds. Since ancient times of civilizations, peo- tivity. However, we could not find any activity on this ple have been relying on plants as either prophylac- model and considered as possibly false information. In tic or therapeutically arsenal to restore and maintain a following attempt, we decided to test the in vitro ac- health. tivity of the extracts against phospholipase A2 enzyme and the methanol extract revealed a potent activity. However, random exploiting of these rich sources for Through activity-guided fractionation and isolation developing new drug leads is a long, tedious and ex- procedures we obtained a polyphenolic constituent pensive task and various tools are developed in order with a large molecular weight as the active ingredient. to increase success. Traditional medicine is among the As known, carrageenan-induced edema model is a con- most extensively implemented approach for this pur- venient technique for the investigation of nonsteroidal pose. Recently breeding sciences of Ethnobotany for anti-inflammatory agents which act through cycloox- documentation of traditional wealth of information ygenase pathway in the inflammatory process, while and Ethnopharmacology for the scientific evaluation phospholipase A2 is an enzyme catalyze the hydrolysis of this information to afford biologically active drug of membrane phospholipids to arachidonic acid and a molecules are useful tools for discovery of new drug convenient model for the corticosteroid type agents. In leads. Ethnopharmacology is defined as “the interdisci- order to emphasis the importance of “bioassay model plinary scientific exploration of biologically active agents selection”, I would like to give another example; the oily traditionally employed or observed by man“ (Bruhn and extract from the fruits of bitter cucumber (Momordica Holmstedt, 1981). The bio-rational approaches within charantia) has been used in Anatolia to treat wounds the drug discovery process are based on the ethnobo- and ulcers. In a previous study, targeting to evaluate the tanical knowledge. A careful evaluation of ethnobotan- antiulcer potential of this extract was employed water ical inquiries based on field surveys are compulsory to immersion-induced stress ulcer model in rats, howev- attain an idea about the selection of the correct materi- er was found totally ineffective. Water immersion-in- al, the way of preparation for the administration of the duced stress ulcer model is known as a representative remedy (ethnopharmaceutics), and the symptoms of model mainly for the determination of agents acting pathologies which the remedy claimed to be effective through inhibition of acid secretion. However, the oily by the lay people (ethnomedicine) may yield success- extract of the plant was also recommended as an effec- ful results. The latter issue is of special importance for tive remedy for wound healing, which suggests that the the elaboration of pharmacological studies. The diffus- activity of the extract might possibly, at least partial- er the descriptions about the diseases and troubles to ly, through a cytoprotective mechanism. Therefore we be cured with, the blurrier the focused pharmacolog- tested the effects of the extracts on indomethacin-in-

Kervansaray Convention Center & Hotel, LARA, ANTALYA / TÜRKİYE 29 International Symposium on Drug Research and Development S

LECTURE duced ulcer model as a representative model for assess- In particular, in the last two decades, extensive re- ing cytoprotective activity and a potent antiulcer activ- searches on BGFI procedures have been carried out ity was found (Gürbüz et al., 2000). for the identification and isolation of pharmacolog- ically active principles from the plants. In Turkey, I’m always enthusiastic that how the people had dis- we have conducted such studies for the evaluation of covered the biological effects of those remedies! How Turkish traditional medicines. many of them had sacrificed their life? For example, during our expeditions in south Anatolia (Isparta), Turkish folk medicine for the discovery of drug Spanish broom (Spartium junceum) flowers were men- molecules tioned to be effective in treating peptic ulcers. Due to Turkey has a rich flora as well as cultural diversi- the alkaloid content (spartein type), this plant is known ty which yielded the accumulation of notable tradi- as toxic and we deliberately recorded this information. tion originated remedic information. Through ex- However, in vivo experimental studies had shown that tensive field surveys between 1986 and 1994, we have the crude extract was highly effective in rat ulcer mod- documented this wealth of knowledge using scientif- els without inducing any apparent toxicity. In further ic ethnobotanical techniques. A database of Turkish bioassay-guided fractionation procedures, one fraction folk medicine was established (TUHIB) and 8000 folk which accumulates alkaloids induced sudden death of remedies have been accumulated so far. animals, while active fraction yielded a new triterpenoid saponin with potent antiulcer activity (Yeşilada et Several chemical and pharmacological studies have al., 2000a). Actually, because of the lowest alkaloid con- been conducted in order to evaluate this wealth of in- tent inhabitants have used the flowers of plant in thera- formation and through application of BGFI proce- py without any apparent toxicity. If leaves or other parts dures a number of molecules have been defined with of the plant were used, instead of flowers, might possi- anti-inflammatory, antinociceptive, antidiabetic, anti- bly cause death, due to the higher alkaloid content. Helicobacter, IL-2 inducing, antihepatotoxic, antiulcerogenic, antioxidant or antiviral activity so far. In Bioassay-guided fractionation and isolation order to demonstrate the different strategies for lead procedures discovery from folk remedies, we will focus on several The most effective way for the discovery of drug leads selected ethnopharmacological studies on TFM. from the natural sources in ethnopharmacology is the “Bioassay-guided Fractionation and Isolation” proce- Anti-inflammatory and antinociceptive agents from dures (BGFI). This process asks for a carefully selection Turkish folk medicines (TFM) of target, respectively bioassay. As soon as an activity is The upper ground part of water speedwell (Veronica manifested by the preliminary tests, the extract is sub- anagallis-aquatica), Scrophulariaceae, is used to treat jected to successive chemical fractionation and isola- rheumatic pain in the northeast part. Herbs boiled tion procedures and each fraction and/or components in a cauldron and then the patient takes bath inside is then submitted to bioassay model, which eventual- while still warm as well as the pulp applied externally yield the active ingredient(s). The chemical struc- ly on affected joints. Through BGFI techniques three ture of the isolated constituent is elucidated through catalpol derivative of iridoids were isolated. The anti- detailed spectroscopic analysis, i.e., mass spectrometry inflammatory (carrageeenan-induced hind paw ede- and 1H- and 13C-NMR techniques. Although, this pro- ma) and antinociceptive (p-benzoquinone-induced cedure sounds quite simple and logical, in compare to writing) activities were found to be shared by three dealing with a collection of pure natural compounds a compounds but, in particular, catalposide and ver- lot of experience is required for the successful chem- proside showed higher activity and were safe as re- ical and pharmacological processing, in particular to gard to gastric toxicity (Küpeli et al., 2005). exclude false positive results. One of the most common problems frequently encountered in BGFI procedures is the stated activity may be loss during chemical fractionation, or may be spread among many fractions and compounds. This may be, in particular, due to the syn- ergistic interaction where many constituents in the extract/fraction of sometimes even in different chemical classes contributing to a pharmacological effect. An- other problem is the possible decomposition of the active ingredient(s) during the chemical separation processing; due to the loss of co-metabolites responsible for the stability of the active ingredient or wrong extraction or chromatographic conditions employed for the isolation of constituents.

30 May 17-20, 2007 “From Chemistry to Medicine” DRD 2007 S

An isoflavone derivative, scandenone, was isolat- which is occasionally removed to drain the accumu- LECTURE ed from osage orange (Maclura pomifera) (Morace- lated inflammation out. On the other hand, for cur- ae) fruits by BGFI techniques showed comparable ing this open wound a fresh leaf of Plantago major anti-inflammatory (carrageenan-induced hid paw ssp. major L. is applied. Moreover, C. vitalba branch- edema, TPA-induced ear edema) and antinocicep- es are also used to stop toothache by smoking like a tive (PBQ-induced writhings) activity to indometh- cigarette in Northwestern Anatolia. acin, without inducing any gastric lesion (Küpeli et al., 2006a). Recently we have isolated a potent anti-inflammatory, antinociceptive and antipyretic principle from the dried herbs of Clematis vitalba, as a new C-glycosylflavon, vitalboside (4’-coumaroyl isovitexin). This compound was shown to possess a powerful and broad activity spectrum against a battery of tests; acute inflammation tests: carrageenan-, PGE2-, serotonin-induced paw edema, inhibitory activity on acetic acid-induced vascular permeability, castor oil-induced diarrhea; subacute inflammation test: Air-sac test; and chronic inflammation test:, Freund’s complete adjuvant (FCA)-induced chronic arthritis; antipyretic tests: yeast- and FCA-induced pyrexia; antinociceptive test: p-benzoquinone-induced writings without inducing any apparent gastric toxicity (Yes- ilada and Küpeli, 2007).

Another C-glycosylflavon with potent anti-inflammatory and antinociceptive activity isolated from several plants was isoorientin. Even in a very low dosage, isoorientin provided activity as potent as of indomethacin without inducing any apparent gastric toxicity (Küpeli et al., 2004). This compound was also found to possess a potent antihepatotoxic (Kü- peli et al., 2004) and antidiabetic (Sezik et al., 2005) activities.

In Anatolia, oil made from the flowers of mullein (Verbascum species) is used to help soothe earache Due to widespread distribution of Clematis spe- and can be applied externally for eczema and other cies at the northern sphere, have been reported to types of inflammatory skin conditions, wounds and be used in the traditional medicines worldwide. The rheumatic pain. Through BGFI procedures from the aerial parts of various Clematis species are used as flowers of Verbascum lasianthum (Scrophulariaceae) analgesic, antipyretic and diuretic, against rheumat- an iridoid glucoside, aucubin, and a triterpenoid sa- ic pain, eye infections, gonorrhoeal symptoms, bone ponin, ilwensisaponin A, were isolated as the potent illnesses, chronic skin disorders, gout and varicosity anti-inflammatory and antinociceptive components in particular in the Europe and Eastern Asia. In the without inducing any apparent acute toxicity or gas- field works, we have determined that different Turk- tric damage (Küpeli et al., 2007). ish species of Clematis are used as remedy among public of Anatolia. The most salient application related to folkloric usage is the one that is applied in the treatment of rheumatic ailments. It is stated that in Northern Anatolia, C. cirrhosa ve C. flammula leaves or the aerial parts are implemented to provide temporary relief in joint pains. Fresh herbs or leaves are applied on inflammatory joints kept for about 15-30 minutes after battering, as it irritates the skin opens a hole for draining the edema. In fact, in particular cases, in order to provide continuous draining of the edema for 20-25 days, the wound is plugged by inserting a grape dreg into the hole,

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LECTURE The roots of Astragalus membranaceus (Fabaceae), a Chinese species, has a reputation of immunomodula- tory and have been implemented against various diseases including cancers. In Turkey, ca.500 species have been recorded, however, almost none of these species are used in Turkish folk medicine. Fourteen triterpen saponins isolated from the roots of three Turkish species, A.oleifolius, A.cephalotes, A.trojanus, were studied using ELISA tests against cytokines induced by bacterial lipopolysaccharide stimulation; IL-1ß, IL-8, TNF- α, and phorbolacetate stimulation; IL-2, IL-4, INF-γ. Among the compounds studied only Astraga- Bath prepared with the whole plant (roots and aerial loside VII was isolated from Astragalus oleifolius sho- parts) of spruge olive (Daphne oleoides ssp.oleoides) wed a potent IL-2 stimulatory activity (142%). Since a (Thymelaeaceae) has been used to treat rheumatism, few local healers claimed that several Astragalus spe- lumbago and against fever in TFM. Through activ- cies (not revealed) from southeast Anatolia to possess ity guided fractionation and isolation techniques healing effect on cancers (personal note), it is suppo- 17 active constituents were isolated and their struc- sed that this result may partially support the above as- tures were chemically elucidated. Diterpenes [genk- sertions (Yesilada et al., 2005). wadaphnin, gnidilatin, gnidicin and 20-palmitates of these compounds and 1,2-dehydrodaphnetoxin], a coumarin derivative [daphnetin], Lignan derivatives [eudesmin, wikstromol, matairesinol]. Effects of these molecules were studied against a group of cytokines known to play key role in inflammatory disorders; interleukin-1α and –1β and tumor necrosis factor-α. Genkwadaphnin, 1,2-dehydrodaphnetoxin and daphnetin were found to possess potent inhibitory activity on these cytokines, but others showed a moderate activity (Yesilada et al., 2001).

Cistus laurifolius (Cistaceae), rockrose, is a widespread and perennial bush with charming flowers. The leaves are used externally as a remedy against rheumatic pain, urinary inflammations, high fever, peptic ulcers and diabetes in TFM. For this purpose, a warm decoction was used as a bath to relieve pain, or wilted leaves were externally applied to joints. The leaves were found to inhibit effectively IL-1α, an inflammatory cytokine (Yesilada et al., 1997).

32 May 17-20, 2007 “From Chemistry to Medicine” DRD 2007 S

Internal use of flowers for treatment of peptic ul- techniques yielded two sesquiterpenes as the active LECTURE cer was also reported. In a recent study, to evaluate components; Chlorojanerin (90%***), 13-acetyl sol- antirheumatic activity in TFM, we isolated 16 com- stitialin A (89%*), while Solstitialin A showed a weak pounds mainly flavonoids and lignans through ac- activity (31%) (Yesilada et al., 2004; Gürbüz and Yes- tivity-guided procedures and major component 3- ilada, 2007). O-methyl quercetin was found to possess potent prostaglandin inhibitory and antioxidant activity; inhibitory on PGE1 and PGE2-induced contractions in guinea pig ileum and DPPH (Sadhu et al., 2006). This compound was also found to possess potent anti-Heli- cobacter pylori activity (Üstün et al., 2006).

Other flavonoids, kaempferol-3,7-O-α-dirhamnoside and quercetin-3,7-O-α-dirhamnoside from the leaves of Tilia argentea showed antinociceptive (34% and 40% inhibition, respectively) and anti-inflammatory (36% for both) activities without inducing any apparent gastric lesions (Toker et al., 2004).

Ethnopharmacological studies have been conducted on TFM so far revealed that flavonoids are the most promising phytochemicals as drug leads. A wide activity spectrum for flavonoids has been reported since at least two or three decades and many studies have been Antiulcerogenic activity conducted on the semi- or full synthesis of various flavonoid type compounds. Among the isolated flavo- Tea prepared from the flowers of Spanish broom noids in the course of ethnopharmacological studies (Spartium junceum L.) (Fabaceae) are used in TFM from TFM; 3-O-methyl quercetin (isorhamnetin) de- to treat ulcers. Since Spanish broom is known to pos- serves further detailed attention. This compound sho- sess spartein type alkaloids, utilization of this plant as wed a potent anti-Helicobacter pylori activity (MIC 3.9 a folk remedy seemed quite risky. However, aqueous µg/ml; AMP 0.06 µg/ml) (Üstün et al., 2006), anti-inf- extract and polar fractions showed 100% inhibition lammatory and antinociceptive activities (Küpeli and against water immersion and restraint-induced stress Yesilada, 2007), prostaglandin inhibitory and antioxi- ulcer in rats. BGFI techniques yielded a new triter- dant activities (Sadhu et al., 2006) and antihepatotoxic penoid, spartitrioside. This compound showed po- activity (Küpeli et al., 2006b). Further studies may yield tent inhibitory activity against a wide range of ulcer successful results for the discovery of new drug leads. models; EtOH-ulcer (100%**), pyloric ligation-ulcer (84%*), gastric secretion (49%*), gastric pH (2.3 to 4.5**), peptic activity (61%*), gastric acidity (83%*) References (Yesilada et al., 2000a). Several flavonoids with potent 1. Bruhn, J.G., Holmstedt, B., 1981. Ethnopharmacology: antioxidant activity were also isolated from the flow- objectives, principles and perspectives. In: Natural Prod- ers (Yesilada et al., 2000b). ucts as Medicinal Agents. J.L. Beal, E. Reinhard (Eds). Hippokrates Verlag: Stuttgart, pp.18-19. Fresh flowers of yellow starthistle (Centaurea solstitia- 2. Gürbüz, İ., Akyüz, Ç., Yeşilada, E., Şener, B., 2000. Anti- ulcerogenic Effect of Momordica charantia L. fruits on lis ssp.solstitialis) (Asteraceae) are swallowed to treat Various Ulcer Models in Rats. Journal of Ethnopharmacol- ulcers in TFM. Methanol extract showed 100% in- ogy 71, 77-82. hibition against EtOH-induced lesions in rats. BGFI

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LECTURE 3. Gürbüz İ., Yeşilada, E., 2007. Evaluation of anti-ulcero- 12. Üstün O., Özçelik B., Akyön Y., Abbasoğlu U., Yeşilada E., genic effect of sesquiterpene lactones from Centaurea 2006. Flavonoids with anti-Helicobacter pylori activity solstitialis L. ssp.solstitialis by using various in vivo and from Cistus laurifolius leaves. Journal of Ethnopharmacol- biochemical techniques. Journal of Ethnopharmacology ogy 108, 457-61. (in press). 13. Yeşilada, E., Üstün, O., Sezik, E., Takaishi, Y., Ono, Honda, 4. Küpeli E., Aslan M., Gürbüz İ., Yeşilada E., 2004. Evalua- G., 1997. Inhibitory effects of Turkish folk remedies on tion of the in vivo biological activity profile of isoorientin. inflammatory cytokines: interleukin-1a, interleukin-1β Zeitschrift für Naturforshung C 59c, 787-89. and tumor necrosis factor α. Journal of Ethnopharmacol- 5. Küpeli E., Harput Ş., Varel M., Yeşilada, E., Saraçoğlu, İ.., ogy 58, 59-73. 2005. Bioassay-guided isolation of iridoid glucosides with 14. Yeşilada, E., Takaishi, Y., Fujita, T., Sezik, E., 2000a. Anti- antinociceptive and anti-inflammatory activities from ulcerogenic effects of Spartium junceum flowers on in Veronica anagallis-aquatica. Journal of Ethnopharmacol- vivo test models in rats. Journal of Ethnopharmacology ogy 102, 170-176. 70, 219-226. 6. Küpeli E., Orhan İ., Toker G., Yesilada E., 2006a. Anti- 15. Yeşilada, E., Tsuchiya, K., Takaishi, Y., Kawazoe, K., inflammatory and antinociceptive potential of Maclura 2000b. Isolation and characterization of free radical scav- pomifera (Rafin.) Schneider fruit extracts and its major enging flavonoid glycosides from the flowers of Spartium isoflavonoids, scandenone and auriculasin. Journal of junceum by activity-guided fractionation. Journal of Eth- Ethnopharmacology 107, 169-174. nopharmacology 73, 471-78. 7. Küpeli E., Orhan Deliorman D., Yeşilada E., 2006b. Effect 16. Yeşilada E., Taninaka H., Takaishi Y., Honda G., Sezik of Cistus laurifolius L. leaf extracts and isolated flavonoids E., Momota H., Ohmoto Y., Taki T., 2001. In vitro on acetaminophen-induced hepatotoxicity in mice. Jour- effects of Daphne oleoides ssp. oleoides on inflammatory nal of Ethnopharmacology 103, 455-60. cytokines and activity-guided isolation of active constitu- 8. Küpeli, E., Tatlı, İ.İ., Akdemir, Z.S., Yeşilada E., 2007. ents. Cytokine 13, 359-64. Bioassay-guided isolation of anti-inflammatory and anti- 17. Yeşilada E., Gürbüz İ., Bedir E., Tatlı İ., Khan I.A., 2004. nociceptive glycoproteins from the flowers of Verbascum Isolation of antiulcerogenic sesquiterpene lactones from lasiniathum Boiss. ex Bentham.. Journal of Ethnopharma- Centaurea solstitialis L. ssp.solstitialis through bioassay- cology 110, 444-450. guided fractionation procedures in rats. Journal of Ethno- 9. Sadhu S.K., Okuyama E., Fujimoto H., Ishibashi M., Yesi- pharmacology 95, 213-9. lada E., 2006. Prostaglandin Inhibitory and Antioxidant 18. Yeşilada, E., Bedir, E., Çalış, İ., Takaishi, T., Ohmoto, 2005. Components of Cistus laurifolius, a Turkish Medicinal Effects of triterpene saponins from Astragalus species on Plant. Journal of Ethnopharmacology 108, 371-78. in vitro cytokine release. Journal of Ethnopharmacology 10. Sezik E., Aslan M., Yeşilada E., Ito S., 2005. Hypogly- 96, 71-7. caemic activity of Gentiana olivieri and isolation of the 19. Yeşilada E., Küpeli E., 2007. Clematis vitalba L. aerial part active constituent through Bioassay-guided fractionation exhibits potent anti-inflammatory, antinociceptive and techniques. Life Sciences 76, 1223-38. antipyretic effects. Journal of Ethnopharmacology 110, 11. Toker G., Küpeli E., Temizer Memişoğlu M., Yesilada E., 504-515. 2004. Flavonoids with antinociceptive and anti-inflammatory activity from the leaves of Tilia argentea (Linden). Journal of Ethnopharmacology 95, 393-7.

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L 15 PARTHENOLIDE ANALOGS AS ANTILEUKEMIC AGENTS LECTURE WITH CLINICAL POTENTIAL

Peter A. CROOKS University of Kentucky, College of Pharmacy, 501A Pharmacy Building, Rose Street, Lexington, KY 40536-0082

he sesquiterpene lactone, parthenolide, the brary of chirally defined parthenolide analogs with principal constituent of the medicinal plant improved water-solubility and in vivo bioavailability. Feverfew (Tanacetum parthenum), has been From these studies we have identified a lead clinical Tshown to be capable of inducing human leukemia candidate, DMAPT fumarate, which induces selective stem cell (LSC) death in vitro, but is without effect and rapid death of primary human LSCs from both on hematopoietic stem cells. LSCs are believed to play myeloid and lymphoid leukemias. The mechanism of a central role in the pathogenesis of acute leukemia action of DMAPT fumarate includes NF-кB inhibi- (AML), and are known to be involved in both initi- tion, p53 activation, and induction of oxidative stress ation of AML and relapse. Thus, novel compounds responses. Pharmacokinetic and metabolic studies in such as parthenolide, that selectively target LSCs, rep- rodents indicate that DMAPT fumarate has ~70% bi- resent potential novel clinical agents for the treat- oavailability via the oral route, while studies in mouse ment of AML. Unfortunately, parthenolide has poor xenograft models and in dogs suffering from sponta- physicochemical properties that preclude its devel- neous acute leukemias indicate that DMAPT has in opment as an effective clinical agent. Utilizing clas- vivo bioactivity. Thus, based upon the above preclin- sical solution-phase Michael-addition chemistry, we ical data, DMAPT fumarate appears to be a potential have designed, synthesized and screened a small li- clinical candidate for the treatment of human LSCs.

Kervansaray Convention Center & Hotel, LARA, ANTALYA / TÜRKİYE 35 International Symposium on Drug Research and Development S

L 16 LECTURE SYNTHESIS AND BIOLOGICAL EVALUATION OF NOVEL CAMPTOTHECIN CLASS TOPOISOMERASE I INHIBITORS

Ayhan S. DEMİR Middle East Technical University, Department of Chemistry, 06531 Ankara, Turkey

amptothecin (CPT) is a pentacyclic alkaloid first isolated from extracts of the Chinese tree Camptotheca acuminata. Camptothecin Cis thought to inhibit the proliferation of cancer cells by interfering with the breakage/reunion reaction of the enzyme topoisomerase I, a nuclear enzyme implicated in DNA replication and RNA transcription. A camptothecin drug stabilizes and forms a reversible enzyme-camptothecin-DNA ternary complex, designated the cleavage complex. The formation of the cleavable complex specifically prevents the reunion step of the breakage/union cycle of the topoisomerase reaction.1

To date, some water-soluble camptothecin derivatives have been prepared by derivatizing the A and B rings and by opening the lactone E-ring. Water-soluble pro-drug type camptothecin compounds, in which the 20-position hydroxyl group is esterified. Enzymes present within the body can break the ester bond after injection to form the parent camptothecin compound. Camptothecin and its analogs are presently The natural fluorescent properties of CPT and its de- under study worldwide in research laboratories and rivatives have been exploited to monitor its concen- cancer clinics. In lab tests and in clinical trials, these tration in living cells. Significant attention has been camptothecin drugs have aroused considerable inter- paid to the equilibrium between the lactone and its est as a result their ability to halt the growth of a wide ring-opened carboxylate form, which influences its range of human tumors. For example, these drugs ex- antitumor ability.2 hibit unprecedented high levels of antitumor activities against human colon cancer.3 Camptothecin has also been shown to be effective against other experimental cancer types such as lung, breast, and malignant melanoma.

A need continues to exist for the development of new and better camptothecin compounds having still higher anti-tumor activity and still more improved water- solubility while exhibiting low levels of toxicity. Unfortunately, camptothecin and many structurally-related camptothecin analogs are water insolu- The primary object of the present work to provide ble. This water insolubility makes administration of new camptothecin analogs and derivatives displaying camptothecin compounds difficult. In an effort to cytotoxic activity and improved water-solubility for address this problem a number of synthetic efforts ease and efficiency of administration/delivery. have been directed to derivatizing the A-ring and/or B-ring to improve water-solubility while maintaining The compounds are prepared by substitution of OH cytotoxic activity. to form camptothecin prodrugs, which can be con- verted to active form by physical methods and sub-

36 May 17-20, 2007 “From Chemistry to Medicine” DRD 2007 S

stitution of the 7-position of camptothecin or ho- generated by camptothecin or 7-ethyl-10-hydroxyca- LECTURE mocamptothecin to form water-soluble derivatives mptothecin (SN-38). This slower reversal may corre- containing a 7- aminoalkyl moiety. Generally, the spond to more stable cleavable complexes. The gua- camptothecins serving as starting materials are sus- nadino amine compounds show cleavable complexes pended in organic solvents or water. To the mixture with intermediate stabilities between camptothecin is added the following compounds which give equiv- and SN-38. alent structure to Z: halo alcohols, amino alcohols, amino aldehydes, halo aldehydes, halo ketones, azido alcohols, azido aldehydes, sulfuric acid, iron sulfate References and oxidizing reagents such as per acids and hydro- 1. Camptothecins: New Anticancer Agents; Potmesil, M., gen peroxide. The mixture is then stirred to complete Pinedo, H., Eds.; CRC Press: Boca Raton, FL, 1995. (b) the reaction. Any precipitate which forms is removed Garcia-Carbonero, R.; Supko, J. G. Clin. Cancer Res. 2002, by filtration and the product is isolated after remov- 8, 641-661. (c) Croce, A. C.; Bottiroli, G.; Supino, R.; al of the solvent. Favini, E.; Zuco, V.; Zunino, F. Biochem. Pharm. 2004, 67, 1035-1045. 2. Burke, T. G.; Mi, Z. J. Med. Chem. 1994, 37, 40-46. (b) Mi, We have evaluated the abilities of the novel camp- Z.; Burke, T. G. Biochemistry 1994, 33, 10325-10336. (c) tothecins described here to inhibit the topoisomer- Mi, Z.; Burke, T. G. Biochemistry 1994, 33, 12540-12545. ase I enzyme. In these experiments the oligonu- (d) Burke, T. G.; Malak, H.; Gryczynski, I.; Mi, Z.; Lakow- cleotide was labeled at the 3 ′-end of the upper icz, J. R. Anal. Biochem. 1996, 242, 266-270. (scissile) strand with 32P-α-cordycepin and termi- 3. Giovanella B C; Stehlin J S; Wall M E; Wani M C; Nicholas nal transferase.4 Topoisomerase I-mediated cleav- A W; Liu L F; Silber R; Potmesil M. . Science 1989, 246: age generates DNA fragments that can be readily 1046-1048. detected by electrophoresis and quantitated by phos- 4. Pommier, Y., Kohlhagen, G., Kohn, F., Leteurtre, F., Wani, phorimager analysis.5 Several CPT analogs such M.C., and Wall, M.E. Proc. Natl. Acad. Sci. U.S.A. 1995, as 7-(1-Guanidinopropyl)-20(S)camptothecin and 92, 8861-8865. 5. Tanizawa, A., Kohn, K.W., Kohlhagen, G., Leteurtre, F., 7-(1-Guanadinoethyl)-20(S)camptothecin are good and Pommier, Y. Biochemistry 1995, 34(21), 7200-6. inducers of cleavable complex formation and the distribution of cleavage sites are comparable to those

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L 17 LECTURE NANOMATERIALS IN MEDICINE

Erhan PİŞKİN Hacettepe University, Chemical Engineering and Bioengineering; Biyomedtek: Society for Biomedical Technologies, Beytepe, Ankara, Turkey

anotechnology deals with materials and sys- polymers) have been utilized in controlled/targeted tems having at least one dimension of about delivery of several active agents including drugs, plas- 1-100 nm and aims to produce materials with mid DNA, etc., for effective therapy of a wide range of Nsuperior electrical, chemical, mechanical and optical diseases. Nanofibers can be electrospun and form po- properties. Nanotechnology has a great potential for rous nonwoven films as potential dressings and scaf- medical applications both in diagnosis and therapy. folds for tissue engineering. This presentation will Nanoparticles made of metallic, ceramic, polymer- cover some diverse medical applications of nano- ic or their composites have been proposed in many structured materials including author’s contributions applications in medical diagnosis, both in vitro and and ongoing studies. in vivo. Nanocarriers (particles, micelles and soluble

38 May 17-20, 2007 “From Chemistry to Medicine” DRD 2007 S

L 18 NANOMEDICINE FOR CENTRAL NERVOUS SYSTEM (CNS) LECTURE DRUG DELIVERY

A. V. KABANOV Department of Pharmaceutical Sciences, and Center for Drug Delivery and Nanomedicine, University of Nebraska Medical Center, 985830 Nebraska Medical Center, Omaha, Nebraska 68198-5830

eurodegenerative and infectious disorders in- be overcome by polymer science and nanotechnolo- cluding Alzheimer’s and Parkinson’s diseas- gy. Specific examples developed in our work include es, amyotrophic lateral sclerosis, and stroke 1) inhibition of drug efflux transport systems in the Nare rapidly increasing as population’s age. Alzheimer’s blood-brain barrier by amphiphilic block copolymers; disease alone currently affects 4.5 million Americans, 2) chemical modification of proteins with hydropho- and more than $100 billion is spent per year on med- bic anchors groups and amphiphilic polymers to en- ical and institutional care for affected people. Such hance their transport across the blood-brain barrier; numbers will double in the ensuing decades. Current- 3) development of nanogels capable of carrying anti- ly disease diagnosis for all disorders is made, in large sense oligonucleotides and siRNA across the blood- measure, on clinical grounds as laboratory and neu- brain barrier; 4) cell-mediated delivery of nanozymes roimaging tests confirm what is seen by more routine to the brain. Such approaches may improve diagnos- examination. Achieving early diagnosis would ena- tic and therapeutic outcomes. New developments ble improved disease outcomes. Drugs, vaccines or in polymer science coupled with cell based delivery regenerative proteins present “real” possibilities for strategies support the notion that diseases that now positively affecting disease outcomes, but are limit- have limited therapeutic options can show improved ed in that their entry into the brain is commonly re- outcomes by advances in nanomedicine. The work is stricted across the blood-brain barrier. This presen- supported by the United States National Institutes of tation will review highlights how these obstacles can Heath (RO1 NS36229 and RO1 NS051335).

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Oral Presentations

“From Chemistry to Medicine” DRD 2007

O 01 Ref: 0053 loss of blood. Thrombin inhibitors are very important in preventing the formation of blood clots inside veins and arteries. INVESTIGATION OF NONCOVALENT PROTEIN- Trombin consists of two polypeptide chains which are chain A and PROTEIN INTERACTIONS BY MATRIX-ASSISTED LASER chain B. Chain A consists of 36 aminoacid, Chain B consists of 259 DESORPTION/IONIZATION MASS SPECTROMETRY aminoacid. There are 492 X-ray structures which has been dissolved Bekir SALİH, Basri GÜLBAKAN, Özlem DEMİREL in the Protein databank (http://www.rcsb.org/pdb/home/home.do). Hacettepe University, Faculty of Science, Department of Chemistry, 06532 Beytepe- One of the principal tools in the theoretical studies of biological ORAL PRESENTATIONS Ankara, Turkey molecules is molecular dynamics simulations (MD). Md gives us dynamic picture of molecules and their motions. Matrix-Assisted Laser Desorption/Ionization-Mass spectrometry X-Ray structures of thrombin with bound different substrates of (MALDI-MS) is a very important and rapidly evolving analyti- 1qj6, 1qj7, 1qj1, 1qhr ve 1ppb [5] are taken from the protein da- cal tool that is used in many different areas, including protein in- tabank. teraction analysis. The work described herein was devised and implemented using MALDI-MS to investigate different types protein-protein interactions. Experimental conditions have been found in which specific noncovalent interactions in solution are maintained throughout the sample preparation and ionization process. Noncovalent interaction between Aβ 1-42 peptide and Aβ 1-42 antibody is studied. Aβ 1-42 peptide and Aβ 1-40 antibody was used as negative control to determine the specificity. Aβ 1-42 peptide was digested with trypsin and digestion products were incubated with Aβ 1-42 antibody and Aβ 1-40 antibody respectively. Fibril form of the Aβ 1-40 peptide was studied. The effect of TFA and Aβ 1-40 antibody onto disaggregation of Aβ 1-40 fibril peptide was followed by capillary electrophoresis (CE) and MALDI-MS. Interaction of bovine serum albumin (BSA) with Aβ 1- 42 was investigated with varying working concentrations by MALDI- MS and CE. Intensity fading MS was also applied to determine the Figure 1. X-Ray structure of thrombin (pdb code:1ppb). degree of interaction with varying working concentrations. Protein A-IgG interaction was further studied to show the applicability of Molecular dynamics simulations an energy minimizations were intensity fading approach in determination of protein-protein inter- carried out on Sun Fire x4600 x64 server: (8 Core AMD Opteron) actions. Finally, a very complex and high molecular weight enzyme, using the GROMACS 3.3.1 [1] package of programs. SYBYL 7.3 [2] cytochrome C oxidase, was studied by using CE and MALDI-MS was used to model of hirudins. For visualization of structures and to explore the capability of MALDI-MS in complex protein-protein trajocteries the VMD [3] 1.8 and PyMOL [4] respectively were used. interactions. After gained free forms of this structures (without water,ligands) by sybyl programme this structures are calculated by GROMACS 3.3.1. 1. Farmer TB, Caprioli RM. Determination of protein-protein interactions Some structural properties of the thrombin with different sub- by matrix-assisted laser desorption/ionization mass spectrometry, Jour- nal of Mass Spectrometry, 33(8), 697-704, 1998. strates including pdb code and number of residues are given in Table 2. Frenkel D, Solomon B, Benhar I. Modulation of Alzheimer’s amyloid 1. All water molecules and ligands from X-Ray structures are remo- neurotoxicity by site-directed single-chain antibody, Journal of Neu- ved and proteins were solvated with water. Trajectories were sampled roimmunology, 106, 23–31, 2000. at 2 ps. interval. 3. Heck AJ, Van Den Heuvel RH. Investigation of intact protein complexes by mass spectrometry, Mass Spectrometry Reviews, 23(5), 368-89, 2004. Table 1. MD simulation parameters of 1qj6,1qj7,1qhr,1ppb. 4. Solomon B, Koppel R, Hanan E. Katzav T. Monoclonal antibodies inhibit in vitro fibrillar aggregation of the Alzheimer β amyloid peptide, Pro- Start 1qj6 1qj7 1qhr 1ppb ceedings of National Academy of Science USA, 93, 452–455, 1996. structure

No.of atoms 287 287 287 287 (chain A) O 02 Ref: 0010 No.of atoms 2093 2093 2093 2093 MOLECULAR DYNAMICS SIMULATION OF (chain B) THROMBIN: TARGET FOR ANTICOAGULANT DRUGS No.of solvent 8559 8545 8501 8695 1Özge KÜL, 1Fulya ÇAĞLAR, 1Vildan ADAR, 2Timuçin YALÇINKAYA molecles 1Hacettepe University, Faculty of Science, Department of Chemistry, Computer-aided Total charge 3.000e 3.000e 3.000e 3.000e Drug Design Group, Ankara, Turkey in the system 2Forte Technology, Simulation Group, Ankara, Turkey Thrombin is a primary target for the development of novel anti- Box geometry octahedron octahedron octahedron octahedron coagulants, since it plays two important and opposite roles in he- Box volume 126.84 124.07 126.27 148.15 mostasis as procoagulant and anticoagulant. Two allosteric forms, (nm3) slow (S) and fast (F), are present that recognize natural substrates and inhibitors with significantly different affinities. Two forms are Temperature 300 K 300 K 300 K 300 K almost equally populated. THROMBIN is an enzyme that causes Simulation 20 ns. 20 ns. 20 ns. 20 ns. blood clotting when vascular system is injured, thus preventing the time

Kervansaray Convention Center & Hotel, LARA, ANTALYA / TÜRKİYE 43 International Symposium on Drug Research and Development

MD simulations have been performed for thrombins (1qj6, 1qj7, which is free form of HV1, whose first 49 residues’ structure has 1qj1, 1qhr ve 1ppb) to explore conformational transition from fast been determined in solution by nmr spectroscopy, and 1hrt which to slow forms.It was carry out with a time step of 20 ns. at 300K. is the structure of a complex of bovine alpha-thrombin and HV1, Analysis of MD simulations was examined by the root mean whose whole chain (65 residues) structure has been determined by square deviation(RMSD), the root mean square fluctation (RMSF). x-ray diffraction. X-Ray structure of hirudin variant 2 (HV2) with Active sides of thrombin has been determined. the pdb code 4htc, structure of the hirudin-thrombin complex, is modelled based on sequence of HV1, extracted from 1hrt, since ORAL PRESENTATIONS 1. Berendsen HJC, Van der Spoel D, van Drunen R. GROMACS: A mes- there is no coordinates for whole chain of HV2. Also hirudin variant sage-pssing parallel molecular dynamics implantation, Comp. Phys. 3 which has no x-ray structure is modelled based on sequence of Comm., 95, 43-56, 1995. http://www.gromacs.org/ HV1, extracted from 1hrt. 2. http://www.tripos.com, SYBYL 7.0, Tripos Inc., 1699 South Hanley Rd., Molecular dynamics simulations and energy minimizations were St. Louis, Missouri, 63144, USA. 3. Humphrey W, Dalke A, Schulten K. VMD - Visual Molecular Dynamics, carried out on Sun Fire x 4600 x 64 server: (8 Core AMD Opteron)

J. Molec. Graphics, 14, 33-38, 1996. using the GROMACS 3.3.1 [6] package of programs. SYBYL 7.3 [7] 4. DeLano WL. The PyMOL Molecular Graphics System, DeLano Scien- was used to model hirudins. For visualization of structures and tra- tific, Palo Alto, CA, USA, 2002. jocteries the VMD [8]1.8 and PyMOL [9] respectively were used. 5. Berman M, Westbrook J, Feng Z, Gilliland G, Bhat TNH, Weissig IN, MD simulations have been performed to determine time-depend- Shindyalov PE. Bourne: The Protein Data Bank, Nucleic Acids Research, ent characteristic of hirudin variants (HV1, HV2, HV3). All systems 28, 235-242, 2000. http://www.rcsb.org/ were minimized prior to simulation using the steepest descent method followed by the conjugate gradient method. MD simulations were Acknowledgement carried out with a time step of 20 ns temperatures over 300 K. We would like to thank Turkish Scientific and Technical Research Asso- ciation (TUBITAK) for the financial support (Grant no: TBAG-106T088) in Some structural properties of the hirudin variants, including pdb this research. code, the number of residues, resolution are given in Table 1. This work was carried out under the HPC-EUROPA project( RH3-CT- 2003-50607), with the support of the European Community’s Research Infra- Table 1. MD simulation parameters. structures action of the European Research Area Programme. 1hrt (HV1) 1hrt (HV1) 5hir (HV1) 1hrt (HV2) 4htc (HV2)

No. of residues 65 49 49 65 65 O 03 Ref: 0011 No. of atoms 483 347 348 478 479 MOLECULAR DYNAMICS SIMULATION OF HIRUDINS: No. of solvent 10404 2881 3183 10284 10751 SPECIFIC THROMBIN INHIBITOR ISOLATED FROM molecules MEDICINAL LEECH Total charge -9 -3 -3 -7 -6 1Fulya ÇAĞLAR, 1Özge KÜL, 1Vildan ADAR, 2Uğur BÜYÜKDEMİRCİ 1Hacettepe University, Faculty of Science, Department of Chemistry, Computer-aided Box geometry octahedron octahedron octahedron octahedron octahedron Drug Design Group, Ankara, Turkey 2Forte Technology, Simulation Group, Ankara, Turkey Box volume 326,893 93,46 104,041 321,3 341,39 (nm^3) Hirudin is a low molecular weight anticoagulant peptide (~7 kDa) excreted naturally from the salivary glands of the medicinal leech, Temperature (K) 300 300 300 300 300 Hirudo medicinalis [1]. Hirudin is the most powerful natural inhibi- Simulation time 20 20 20 20 20 tor of thrombin, due to its binding specificity. Native hirudin is not (ns) a single, homogeneous protein, but rather includes several isoforms. Three variants, designated HV-1, HV-2, and HV-3, have been iso- Methods X-Ray X-Ray NMR X-Ray X-Ray lated from Hirudo medicinalis [2-4]. In Figure 1 x-ray structure of Diffraction Diffraction Diffraction Diffraction HV1 obtained from 1hrt is given. A detailed comparison of simulations were examined by comput- ing the root mean square deviation (RMSD), the root mean square fluctuation (RMSF), the radius of gyration of a group of atoms and the radii of gyration. They are shown that hirudin is composed of a compact N-terminal region (residues 1-47, cross-linked by three di- sulfide bridges) and a flexible C-terminal tail (residues 48-65), and it has a disordered region made up of residues 31-36.

Acknowledgement We would like to thank Turkish Scientific and Technical Research Asso- ciation (TUBITAK) for the financial support (Grant no: TBAG-106T088) in this research. This work was carried out under the HPC-EUROPA Project (RH3-CT- 2003-50607), with the support of the European Community’s Research Infra- structures action of the European Research Area Programme.

1. Markwardt F. Untersuchungen uber Hirudin, Naturwissenschaften, 42, Figure 1. Structure of HV1 taken from complex, 1hrt. 537–538, 1955. 2. Dodt J, Muller HP, Seemuller U, Chang JY. The complete amino acid X-Ray structures of hirudin variant 1 (HV1) taken from the sequence of hirudin, a thrombin-specific inhibitor, Applied Microbiology Brook Protein Data Bank [5], with the pdb codes 5hir and 1hrt.5hir and Biotechnology, 165, 180–184, 1984.

44 May 17-20, 2007 “From Chemistry to Medicine” DRD 2007

3. Harvey RP, Degryse E, Stefani L, Schamber F, Cazenave JP,Courtney M, Mitoxantrone (MTZ) has potent in vitro activity against malignant Tolstoshev P, Lecocq JP. Cloning and expression of a cDNA coding for glioma cell lines, and MTZ-loaded poly(lactide-co-glycolide) (PLGA) the anticoagulant hirudin from the bloodsucking leech, Hirudo medici- microspheres may be injected into the peritumoral area and into tu- nalis. Proc Natl Acad Sci USA, 83, 1084–1088, 1986. mor tissue to provide effective and sustained local drug concentrations 4. Tripier D. Hirudin: a family of iso-proteins. Isolation and sequence determination of new hirudins, Folia Haematol (Leipzig), 115, 30–35, 1988. without causing systemic side effects. Thus, in rats treated with MTZ- 5. Berman M, J. Westbrook Z, Feng G, Gilliland TN, Bhat H, Weissig IN, loaded microspheres, tumor volumes were significantly reduced. No Shindyalov PE. Bourne: The Protein Data Bank. Nucleic Acids Research, tumor formation was observed when glioma cells and MTZ-load- 28, 235-242, 2000. http://www.rcsb.org/ ed PLGA microspheres were implanted concomitantly. Moreover, ORAL PRESENTATIONS 6. Berendsen HJC, Van der Spoel D, van Drunen R. GROMACS: A mes- no systemic side effects or parenchymal inflammatory infiltration sage-pssing parallel molecular dynamics implantation, Comp. Phys. were observed in either group of rats. Brain MTZ concentration Comm. 95, 43-56, 1995. http://www.gromacs.org/ was highest at the injection site and declined with time and dis- 7. http://www.tripos.com, SYBYL 7.0, Tripos Inc., 1699 South Hanley Rd., tance from the injection site. The data demonstrate that MTZ- St. Louis, Missouri, 63144, USA 8. Humphrey W, Dalke A, Schulten, K. VMD - Visual Molecular Dynamics, loaded PLGA microspheres can deliver therapeutic concentrations J. Molec. Graphics, 14, 33-38, 1996. of the drug to the tumor and prevent glioma growth without caus- 9. DeLano, WL. The PyMOL Molecular Graphics System (2002), DeLano ing side effects. Therefore, this treatment method may increase the Scientific, Palo Alto, CA, USA. efficiency of antineoplastic therapy and positively impact survival. The peptide Z-DEVD-FMK is a specific caspase inhibitor, which significantly reduces vulnerability to the neuronal cell death. The inhi- O 04 Ref: 0113 bition of the caspase-3 enzyme is reported to increase neuronal cell survival following cerebral ischemia. Using the avidin (SA)-biotin DENDRIMERS AS DRUG DELIVERY AGENTS TO BONE (BIO) technology, we have accomplished the design of chitosan (CS) Rana SANYAL nanoparticles conjugated with poly(ethylene glycol) (PEG) bearing Boğaziçi University, Faculty of Arts and Sciences, Department of Chemistry, 34342 the OX26 monoclonal antibody whose affinity for the transferrin Bebek, İstanbul, Turkey receptor (TfR) may trigger receptor-mediated transport across the BBB. Fluorescently labeled CS-PEG-BIO-SA/ OX26 nanoparticles Dendrimers have emerged as promising candidates for targeted were administered systemically to mice in order to evaluate their ef- drug carriers since they can be tailored to accommodate a high den- ficacy for brain translocation. sity and wide variety of functional groups on their surface 1]. Along The results showed that an important amount of nanoparticles with their well-defined molecular structure, segmented spherical were located in the brain, outside of the intravascular compartment. construction of dendrimers offers an interesting architecture. While These findings, which were also confirmed by electron microscopic one of these segments is ornamented with active drug molecules, the examination of the brain tissue, indicate that this novel targeted na- other one can be decorated with targeting groups. Since direct appli- noparticulate drug delivery system was able to translocate into the cation of drug molecules to the diseased tissue or organ increases the brain tissue after iv administration. Consequently, these novel nano- effect of the therapy and decreases the side effects, targeted drug de- particles are promising carriers for the transport of the anticaspase livery promises to solve the cytotoxicity issues associated with many peptide Z-DEVD-FMK into the brain. drug candidates. Presentation will provide a brief survey of some of the advances in this field as well as introduce some new dendritic carriers developed in our group aimed towards bone targeted delivery [2, 3]. Synthesis of different generations of biodegradable den- O 06 Ref: 0109 dritic compounds containing bone targeting molecules for targeting RATIONAL DESIGN OF NOVEL PHOTOSENSITIZERS AS chemotherapy agents to bone tissue will be presented. POTENTIAL PHOTODYNAMIC THERAPY REAGENTS 1. Lee CC, MacKay, JA, Frechet JMJ, Szoka FC. Designing dendrimers for Engin Umut AKKAYA biological applications, Nature Biotech., 23, 1517-1526, 2005. Middle East Technical University, Department of Chemistry, Ankara, Turkey 2. Wang D, Miller SC, Kopeckova P, Kopecek, J. Bone-targeting macromo- lecular therapeutics, Adv. Drug Delivery Rev., 57, 1049-1076, 2005. Photodynamic therapy (PDT) is a noninvasive method of treating 3. Gittens SA, Bansal G, Zernicke, RF, Uludağ, H. Designing proteins for malignant tumors and age-related macular degeneration, and par- bone targeting, Adv. Drug Delivery Rev., 57, 1011-1036, 2005. ticularly promising in the treatment of multidrug-resistant (MDR) tumors. The PDT strategy is based on the preferential localization of certain photosensitizers in tumor tissues upon systemic adminis- O 05 Ref: 0111 tration. The sensitizer is then excited with red or NIR light, gener- 1 ating reactive oxygen species (ROS) including singlet oxygen ( O2) RECENT ADVANCES IN BRAIN DRUG DELIVERY and thus irreversibly damaging tumor cells. Current practice of PDT Yilmaz ÇAPAN is limited to a few functionalized porphyrins, however these com- Hacettepe University, Faculty of Pharmacy, Department of Pharmaceutical Technology, pounds are not considered to be ideal drugs for use in PDT. Among 06100 Ankara, Turkey the limitations, the most prominent is the low extinction coefficient of porphyrins in the body’s therapeutic window (650-800 nm, low Drug delivery to the brain possess a major challenge due to the absorptivity region in typical mammalian tissues). Therefore, there blood brain barrier (BBB). Thus, several strategies have been devel- is a significant impetus to develop novel and better efficiency sensi- oped to overcome the BBB and to achieve successful brain drug de- tizers for use in PDT. Partially reduced porphyrins are an alternative. livery. Here, we describe two different approaches for drug delivery to As non-porphyrin photosensitizers, texaphyrins, phthalocyanines, the brain. In the first approach, we describe the formulation of mitox- squaraines, chalcogenopyrylium dyes, aza-boradiazaindacenes and antrone (MTZ) into poly (lactide-co-glycolide) (PLGA) microspheres perylenediimides have been suggested. There is also a recent report for local delivery. In the other approach, we describe the development of a diiodo-substituted boradiazaindacene (BODIPY) as a sensitizer, of a chitosan nanoparticle carrier system, functionalized with PEG- but it requires excitation outside of the therapeutic window. BIO-SA/ OX26 for the delivery of a caspase-3 inhibitor.

Kervansaray Convention Center & Hotel, LARA, ANTALYA / TÜRKİYE 45 International Symposium on Drug Research and Development

Boradiazaindacenes with methyl substituents on 3 or 5 positions the dose that can be administered to patients. The clinical incon- were previously shown to undergo condensation reactions with vencies associated with cisplatin therapy prompted the design and aldehydes to yield longer wavelength absorbing dyes (100 nm red synthesis of more effective and less toxic platinum analogs. shifted) of internal charge transfer (ICT) characteristics. The extend- A family of deeply colored platinum compounds, usually called ed conjugation in these dyes moves the absorption peak to 590-600 platinum blues, has attracted wide interest for years not only because nm. Incorporation of a second styryl group would result in further of their unusual color and intriguing chemistry but also for their red shifts in the absorption spectrum. We targeted dyes 1-3 in our high antitumor activities [2]. In contrast to the usual yellow, orange, ORAL PRESENTATIONS synthesis work, and demonstrated that these dyes have strong ab- red, or colorless platinum complexes, platinum blues are unusual sorptions in the 650-680 nm region. The singlet oxygen generation for their intense blue or purple colors [3]. The first blue platinum efficiency was studied using the singlet oxygen trap 1,3-diphenyl- compound was prepared by German chemists in 1908 [4]. ]. This

isobenzofuran (DPBF). In order to facilitate comparison to previ- unusual material was prepared by the reaction of Ag2SO4 with yellow II ously reported sensitizers, the activity was studied in isopropanol. cis- Pt Cl2(CH3CN)2 and was first proposed to have a mononuclear II Even at very low concentration levels of 9 nM dyes (1-3) and under composition of Pt (CH3CONH)2.H2O. However, the compound was relatively weak red LED irradiation at 625 nm, remarkable efficiency later proposed to be polymeric with bridging acetamidate linkages was observed. Encouraged by these observations, we tested the most [5].

promising sensitizer 3 on K562 human erythroleukemia cells. EC50 Moreover, special attention was paid to the platinum blues produ- value less than 200 nM was obtained [1]. ced from the reactions between the hydrolysis product of cis-DDP 2+ (i.e., cis-[Pt(NH3)2(OH2)2] ) and pyrimidine bases such as uracil, since these so-called “platinum-pyrimidine-blues” were found to have a high index of antitumor activity with a lower associated neph- rotoxicity than cis-DDP [6]. Various platinum blues complexes containing nitrogen and oxygen have been synthesized and reported in literature, however, no platinum blues compound containing sulphur and nitrogen has been reported. 2- The reaction of tetrachloroplatinate (II), [PtCl4] , with 2-, and 3-aminothiophenol first time have been investigated. The reaction with 2-aminothiophenol, first yielded a yellow solid, then a very dark blue colored product, which has a very distinct absorption band at 724 nm in acetonitrile. Elemental analysis, UV-Vis, IR, ESR, ESCA, SEM,1H-NMR, 13C-NMR , 195Pt-NMR and electrochemical measurements made on this dark blue colored compound suggest that it is a new “platinum blue” type complex.

Thus, we demonstrated that novel di-styrylborodiazaindacene dyes with bromo system are very efficient singlet oxygen-substituents on the fluorochrome pi- generators. In addition, these water soluble photosensitizers were shown to have spectacular photoinduced cytotoxicity at very low concentrations and even under low fluence rate LED irradiation. Dark toxicity was nil at the concentration range studied. Structure-activity fine tuning of the sensitizer with further in vitro and in vivo studies is likely to result in highly promising reagents for use in PDT. We are working on the design and synthesis of further red shifted photosensitizers and novel delivery systems including functionalized carbon nanotubes. DNA binding studies of the complex were carried out by voltammetric and UV titrations supporting the electrostatic binding of ct- III II 1. Atılgan S, Ekmekçi Z, Doğan AL, Güç, D, Akkaya EU. Water soluble DNA with suggesting preferential stabilization of Pt over Pt on distyryl-boradiazaindacenes as efficient photosensitizers for photodyna- binding to DNA. mic therapy. Chem. Commun., 4398-4400, 2006. The effect of platinum blue complex on GST, one of the most important enzymes involved in drug conjugation and biotransformation reactions ,were also investigated. O 07 Ref: 0115 1. Rosenberg B, VanCamp L., Nature, 205, 698, 1965. A NOVEL POTENTIAL ANTITUMOR ACTIVE DRUG: 2. Tejel C, Ciriano MA, Oro LA., Chem. Eur. J., 5, 1131, 1999. PLATINUM BLUE COMPLEX CONTAINING SULFUR- 3. Matsumoto K, Sakai, K. Adv. Inorg. Chem., 49, 375, 2000. 4. Hoffmann KA, Bugge G., Berichte, 41, 312, 1908. DONOR LIGAND 5. Gillard RD, Wilkinson G., J. Chem. Soc., 2835, 1964. 1Şeniz ÖZALP-YAMAN, 1İsmail ERİLHAN, 1S. Belgin İŞGÖR, 2Hüseyin İŞÇİ 6. Davidson JP, Faber PJ, Fisher RG., Cancer Chemother. Rep., 59, 287, 1Atılım University, Engineering Faculty Chemistry Group, 06836-İncek, Ankara, Turkey 1975. 2Middle East Technical University, Chemistry Department, Ankara, Turkey

Cisplatin (cis-diamminedichloroplatinum(II), Pt(NH3)2Cl2 (CDDP) was chemically described in 1845, but its antitumor properties were only found accidentally by Rosenberg in 1965 [1]. Decipite the success of cisplatin in chemotherapy, serious side effects limits

46 May 17-20, 2007 “From Chemistry to Medicine” DRD 2007

O 08 Ref: 0055 O 09 Ref: 0117 SYNTHESIS OF ACETOPHENONE AND SUBSTITUTED UVB-INDUCED APOPTOTIC EFFECT OF 11 P53 ACTINIC ACETOPHENONE DERIVED MANNICH BASES AND THEIR KERATOSIS MUTATIONS CYTOTOXIC ACTIVITIES 1Ayşe ERCAN, 2İ. Hamdi ÖĞÜŞ, 3Douglas BRASH H. İnci GÜL 1Hacettepe University, Faculty of Pharmacy, Department of Biochemistry, 06100 Ankara, Turkey Atatürk University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, ORAL PRESENTATIONS 25240 Erzurum, Turkey 2Hacettepe University, Faculty of Medicine, department of Biochemistry, 06100 Ankara, Turkey Mannich bases are generally formed by the reaction between a com- 3Yale University, Faculty of Medicine, Department of Therapeutic Radiology, New Haven, pound containing a reactive hydrogen atom, formaldehyde and a sec- USA ondary amine. The process whereby these compounds are formed is The incidence of skin cancer is increasing every year where 95 known as the Mannich reaction [1]. Their cytotoxic activities mostly % of them are non-melanoma skin cancers. Actinic Keratosis (AK) depend on the alkylation of cellular thiols. Since available alkylating is a non-melanoma skin cancer which is characterized by aberrant anticancer drugs in market have resistance problem or low selectivity proliferation and cell differentiation. UV contributes to the etiology against cancer cells [1], there is a need to find some new compounds of skin cancers by creating DNA photoproducts which generates with anticancer activity. Our laboratory has been focused on the syn- mutations, thus causing damage on the genes. The signature of UVB thesis of acetophenone derived Mannich bases and investigation of damage is C to T transitions which have been found on the p53 tu- their cytotoxic activities against some cancer cell lines. mor suppresor gene. The role of the P53 protein is to suppress tumor In this presentation, the synthesis of mono Mannich bases, 1-aryl- growth by arresting cell cycle and inducing apoptosis. P53 is mutated 3-amino-1-propanone hydrochlorides (series 1), bis Mannich bases, in more than 50 % of the cancers, where most of these are missen- bis(beta-aroylethyl) methyl/ethylamine hydrochlorides (series 2), se mutations. The aim of this work was to determine whether the 2-benzoyl-1,3-diaminopropan dihydrochlorides (series 3), piperidi- selected 11 p53 AK mutations have different abilities as a response nols, 3-aroyl-4-aryl-1-ethyl-4-piperidinol hydrochlorides (series 4), to UVB-induced apoptosis. For this purpose, after a bioinformatic which are structural non classical isomers of bis Mannich bases of study 11 mutations were selected and generated on an expression series 2, and azine derivatives of some mono Mannich bases, N, N’- vector by site-directed mutagenesis. The mutated and wild type bis(3-amino-1-aryl-propylidene)hydrazine dihydrochlorides (series vectors were transiently transfected into primary mouse fibroblasts 5) and evaluation of their cytotoxic activities against some cancer and the cells were irradiated with 1000 J/m2 UVB in order to induce cell lines such as Jurkat, Renca and Androgen-independent Prostate apoptosis. The apoptosis percentage was detected by flow cytometry. Cancer cell line, PC-3, will be mentioned [2-6]. Taken all the data together, the results show that the physical and Aryl part was changed as phenyl, substituted phenyl and 2-thienyl chemical change the mutation acquires, being on an evolutionary and amine part was methyl or ethylamine, dimethylamine, piperid- conserved domain, being close to the DNA binding region confers ine, morpholine in series depending on our design. Representative to the protein different properties on apoptosis. quaternary derivatives of series 1, 3 and 4 were also synthesized since the quaternary derivatives of these series were expected to deami- Keywords: Actinic keratosis, UV, P53, site-directed mutagenesis, apoptosis. nate faster comparing to their corresponding nonquaternary derivatives. Bis Mannich bases were also designed with an expectation that they may produce additional alkylating centers compared to their O 10 Ref: 0084 corresponding mono derivatives. The logic behind the synthesizing piperidinol compounds was to see alterations in cytotoxicity in iso- FREE-RADICAL SCAVENGER ACTIVITIES OF NEWLY mers. Azine derivatives were considered as bifunctional alkylating SYNTHESIZED 2-BENZOXAZOLINONE DERIVATIVES agents which may produce additonal alkylating centers compared to CONTAINING THIOSEMICARBAZIDE, TRIAZOLE, the Mannich bases they are derived from. Cytotoxic activities were THIADIAZOLE AND HYDRAZONE evaluated depending on chemical structure. The effects of the representative compounds on glutathione and related enzymes were test- 1Samiye YABANOĞLU, 2Umut SALGIN-GÖKŞEN, ed in order to determine the mechanism of thiol alkytion to confirm 2Nesrin GÖKHAN-KELEKÇİ, 1Gülberk UÇAR the stability study of some representative compounds [7, 8]. A pre- 1Hacettepe University, Faculty of Pharmacy, Department of Biochemistry, 06100 Ankara, liminary study has also been performed to determine the effects of Turkey 2Hacettepe University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, mono-Mannich bases and a cyclic Mannich base having piperidinol 06100 Ankara, Turkey sturucture on the expression of cytoprotective heat shock proteins (HSC70 and GRP75) and on the levels of thioredoxin (TRX) and It has been reported that some benzoxazolinone, triazole, thiadia- glutaredoxin (GRX) in Jurkat cells [9]. zole and hydrazone derivatives showed significant analgesic and antiinflammatory activities [1-3]. It has been speculated that non-ster- 1. Dimmock JR, Kumar P. Current. Med. Chem., 1, 1-22, 1997. oidal antiinflammatory agents (NSAIDs) can act as the free radical 2. Gül Hİ, Vepsalainen J, Gül M, Erciyas E, Hanninen O. Pharm. Acta Helv., 4, scavengers and show antioxidant activity. It is also well documented 393-8, 2000. that oxidative stress can play an important role in the side effects of 3. Gül Hİ, Gül M, Erciyas E. Arzneimittel Forschung, 52, 628-35, 2002. many xenobiotics including NSAIDs. Therefore, in the present study, 4. Gül Hİ, Gül M, Vepsalainen J, Erciyas E, Hanninen O. Biol. Pharm. Bull. 26, it was thought that the combination of different pharmacophores in 631-7, 2003. one frame may lead to compounds with interesting pharmacologi- 5. Gül Hİ, Das U, Pandit B, Li PK. Arzneimittel Forschung, 56, 850-4, 2006. cal profiles and the effects of thiosemicarbazide, triazole, thiadiazole 6. Gül M, Gül Hİ, Das U, Hanninen O. Arzneimittel Forschung, 55, 332-7, 2005. and hydrazone bearing 2-benzoxazolinone were investigated on free 7. Gül M, Atalay M, Gül Hİ, Nakao C, Lappalainen J, Hanninen O. Toxicol. In radical scavenging activity. Vitro, 19, 573-80, 2005. 8. Gül Hİ, Gül M, Erciyas E. Arzneimittel Forschung, 52, 628-35, 2002. 9. Gül M, Gül Hİ, Hanninen O. Toxicol. In Vitro, 16, 107-12, 2002.

Kervansaray Convention Center & Hotel, LARA, ANTALYA / TÜRKİYE 47 International Symposium on Drug Research and Development

O 11 Ref: 0108 STUDIES ON HISTONE DEACETYLASE INHIBITORY ACTIVITY OF SOME CARBOXYLIC ACID DERIVATIVES AND THEIR STRUCTURE-ACTIVITY RELATIONSHIPS 1Gamze BORA, 1Didem DAYANGAÇ-ERDEN, 2Peruze AYHAN, 3Kemal YELEKÇİ, 4Sevim DALKARA, 2Ayhan DEMİR, ORAL PRESENTATIONS 1Hayat ERDEM-YURTER 1Hacettepe University, Faculty of Medicine, Department of Medical Biology, Ankara, Turkey 2Middle East Technical University, Department of Chemistry, Ankara, Turkey 3Hadir Has University, Faculty of Arts and Sciences, İstanbul, Turkey 4Hacettepe University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Ankara, Turkey Histone deacetylation is responsible for transcriptional repres- sion whereas histone hyperacetylation facilitates gene expression. Therefore histon deacetylating enzymes (HDACs) are popular targets in drug development and HDAC inhibitors are potential drug Figure 1. candidates for cancer, inflammation and some single gene disorders caused by splicing defects of mRNA. Thiosemicarbazide, triazole, thiadiazole and hydrazone deriva- During the past 15 years, a number of structurally diverse HDAC tives (Figure 1) were synthesized according to a previous method inhibitors have been identified including short chain carboxylic ac- [4]. Antioxidant activities of the novel compounds were evaluated by ids. To date, a limited number of compounds such as butyric acid the determinations of 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical (BA), phenylbutyric acid (PBA) and valproic acid (VPA) are in phase scavenging activity, superoxide radical scavenging activity, hydrogen trails for the treatment. peroxide scavenging activity, nitric oxide scavenging activity and re- In this study, we designed eighteen BA, PBA and VPA analo- ducing power. The various antioxidant activities were compared to gous to understand the structural requirements for HDAC in- standard antioxidants such as butylated hydroxytoluen and ascorbic hibitory activity. To develop structure-activity relationships acid and the results were expressed as IC50 values [5,6]. the modifications listed below are realized in the molecules: All of the newly synthesized compounds exhibited varying degrees . increase or decrease the length of alkyl chain, of scavenging capacity on different active radicals; but compounds . branching in the alkyl chain, 13-18 (hydrazone derivatives) proved to be most active derivatives. . introduction of double bond and hydroxyl group into the chain, Among those, compound 17 and 18, having p-methoxy and p-hy- . replication of carboxylic group, droxyl groups on their phenyl rings, respectively showed the highest . amino acid analogy and scavenging capacity of active radical species. These preliminary in . isosteric replacements. vitro results may contribute to explain the potency of antiinflamma- HDAC inhibition activities of these molecules were investigated in tory activity of the compounds. vitro by using HeLa nuclear extract in a fluorimetric assay. Sodium 1. Köksal M, Gökhan N, Küpeli E, Yeşilada E, Erdoğan H. Synthesis, an- butyrate was used as reference compound to compare the effects of algesic and antiinflammatory properties of certain 5-/6-acyl-3-(4-sub- molecular modifications on HDAC inhibitory activities. Activity re- stituted-1-piperazinylmethyl)-2-benzoxazolinones derivatives, Arch. sults were evaluated to establish structure-activity relationship. Pharm., 338, 117-125, 2005. Acknowledgement 2. Labanauskas L, Kalcas E, Udrénaité E, Gaidelis P, Brukštus A, Daukšas, This project was supported by The Scientific & Technological Research V. Synthesis of 3-(3,4-dimethoxyphenyl)-1H-1,2,4-triazole-5-thiol and Council of Turkey, Project No: 105G014. 2-amino-5-(3,4-dimethoxyphenyl)-1,3,4-thiadiazole derivatives exhibiting anti-inflammatory activity, Pharmazie, 56, 617-619, 2001. 3. Sondhi SM, Dinodia M, Kumar A. Synthesis, anti-inflammatory and analgesic activity evaluation of some amidine and hydrazone derivatives, Bioorg. Med. Chem., 14, 4657-4663, 2006. 4. Salgın U et al. Unpublished data 5. Ferreira ICFR, Baptista P, Vilas-Boas M, Barros L. Free-radical scavenging capasity and reducing power of wild edible mushrooms from northeast Portugal: individual cap and stipe activity, Food Chem., 100, 1511- 1516, 2007. 6. Kumaran A, Karunakaran RJ. In vitro antioxidant activities of methanol extracts of five Phyllanthus species from India, LWT, 40, 344-352, 2007.

48 May 17-20, 2007 Poster Presentations

“From Chemistry to Medicine” DRD 2007

P 001 Ref: 0003 P 003 Ref: 0005 PRICEITE DOES NOT INDUCE GENOTOXICITY IN HUMAN IN VITRO EFFECTS OF ALUMINUM SULPHATE ON SISTER- LYMPHOCYTES IN VITRO CHROMATID EXCHANGES AND OXIDATIVE STRESS

Hasan TÜRKEZ, Fatime GEYİKOĞLU IN HUMAN BLOOD: PROTECTIVE ROLE OF BISMUTH PRESENTATIONS Atatürk University, Faculty of Arts and Sciences, Department of Biology, 25240 Erzurum, SUBNITRATE Turkey Fatime GEYİKOĞLU, Hasan TÜRKEZ Boron does not exist by itself in nature. This element combines with Atatürk University, Faculty of Arts and Sciences, Department of Biology, 25240 Erzurum, oxygen and other elements to form boric acid, or inorganic salts called Turkey POSTER borates. People need borates, too, as an important part of a healthy Aluminum, which is found in several different forms and oxida- diet and an essential ingredient in many products necessary for an tion states, causes acute and chronic adverse health effects. Medici- acceptable standard of living. And borates frequently used in indus- nally, the treatment methods with bismuth compounds especially trial, cosmetic, and medical settings. Priceite also (or pandermite = bismuth subnitrate (BSN) (as astringents, antacides, antiulcers and Ca4B10O19.7H2O) is one of the most important commercial boron antidiarrheals) have been increased. The goal of the present study compounds produced in large. The aim of the present study was to was to evaluate the genetic and oxidative effects of aluminum sul- investigate the ability of priceite to induce sister-chromatid exchange phate (Al2(SO4)3) and BSN in human blood in vitro. (SCE) and micronucleus (MN) in cultured human lymphocytes. The various concentrations of Al2(SO4)3 (10 and 20 µg/mL) and With this aim, whole heparinized blood samples were taken from BSN (0.75, 1.5, 3 and 5 µg/mL) were used. Evaluation of DNA dam- eight healthy non-smoking donors. Thirteen experimental concen- ages was carried out by using Sister-Chromatid Exchange (SCE) trations of priceite were used, ranging from 5 to 500 mg/L. method in blood lymphocytes. Oxidative status of erythrocytes was The peripheral blood cultures which were applied 400 and 500 assessed by measuring following oxidative stress markers: reduced mg/L of priceite was found to be sterile. Treatment with priceite did glutathione (GSH), superoxide dismutase (SOD), glucose-6- phos- not cause an increase in the frequency of SCE per cell at all other phate dehydrogenase (G6PDH) and catalase (CAT). concentrations. Moreover, there were no significant aneugenic and/ The SOD activity increased by Al2(SO4)3 (10 µg/mL) exposure but or clastogenic effects observed in the micronucleus assay. the ratio of SCEs didn’t change compared to the controls. On the Our results firstly indicated that priceite is not a genotoxic agent other hand, the high dose of Al2(SO4)3 (20 µg/mL) caused oxidative in human blood cultures and safe for use in medical and cosmetic stress and increased SCE frequency. When the concomitant treat- applications. ment with Al2(SO4)3 of BSN were investigated, BSN exerted an antioxidant action at low doses (0.75 and 1.5 µg/mL). It also reduced the formation of SCEs. P 002 Ref: 0004 This study suggests for the first time that BSN may be admin-

istered as a potential protective against the effects of Al2(SO4)3 in THE EFFECTS OF SOME LICHEN SPECIES AGAINST which oxidative and genetic damages are clearly involved. SISTER-CHROMATID EXCHANGE FREQUENCY INDUCED BY TITANIUM DIOXIDE IN HUMAN LYMPHOCYTES Fatime GEYİKOĞLU, Hasan TÜRKEZ P 004 Ref: 0014 Atatürk University, Faculty of Arts and Sciences, Department of Biology, 25240 Erzurum, Turkey GST-CDNB ACTIVITIES IN GILTHEAD SEABREAM (SPARUS Despite the increasing use of factory-made synthetic drugs, natu- AURATA) LIVER CYTOSOL OF DIFFERENT AGES ral healing materials have persisted as the treatment of choice for a 1Hatice ARDAĞ-AKDOĞAN, 2Alaattin ŞEN multitude of health problems in populations throughout the world. 1Pamukkale University, Science and Arts Faculty, Department of Chemistry, 20017 Investigations of genotoxicity and anti-genotoxicity can help evalu- Kınıklı, Denizli, Turkey ate the safety and effectiveness of herbal health products. Titanium 2Pamukkale University, Science and Arts Faculty, Department of Biology, 20017 Kınıklı, Denizli, Turkey dioxide (TiO2) is widely used in the pharmaceutical and cosmetic industries. It is also used for sterilization of waste water. The pur- Lipophilic compounds such as polycyclic aromatic hydrocarbons, pose of this work was to evaluate the effects of four lichen species polychlorinated biphenyls, nitroaromatics, dioxins, drugs, various (Dermotocopon intestiniformis, Pseudevernia furfuracea, Rhizoplaca pesticides and natural residual are readily taken up into the tissues melanophthalma and Xanthoparmelia pulla) against the genotoxicity of aquatic organisms where biotransformation via Phase I and Phase II metabolism can in part, determine the fate and toxicity of the induced by titanium dioxide (TiO2) for the first time. With this aim, whole heparinized blood samples were taken from xenobiotics. The glutathione S-transferases (GST) represent a major group of detoxification enzymes. GSTs are a family of phase II three healthy non-smoking donors. TiO2 was added to the cultures detoxification enzymes. It is known that the important changes in in concentrations of 5 and 10 µM. After the application of TiO2 and lichen extracts, seperate and together, the sister-chromatid exchange drug metabolism occur with ageing because the various factors that (SCE) test was used to assess DNA damage in lymphocytes. have significant influences on xenobiotic metabolizing enzymes are None of the lichen extracts showed a significant genotoxicity alone. altered by ageing and season. The extract of X. pulla did not show anti-genotoxic activity against In this study, gilthead seabream (Sparus aurata) fish liver cytosol of different ages were used as sample. The fish used in this study, the genotoxic effects induced by TiO2. However, D. intestiniformis, P. furfuracea, R. melanophthalma extracts caused significant decreases Gilthead Seabream (Sparus aurata), were bought from Pinar Fish in in titanium-induced SCE frequencies as dose dependent manner. İzmir, Aegean coast of Turkey. Glutathione S-transferase (GST) were The potency of anti-genotoxic activity was also in the following or- determined from this Gilthead Seabream (Sparus aurata) fish livers der: P. furfuracea > R. melanophthalma > D. intestiniformis. cytosoles. Our findings indicated that lichens can be a new resource of therapeu- Though various factors that may have a significant impact on drug tic potential as recognized here against to adverse effects of drugs used. metabolism are affected by ageing, our results suggest that some im-

Kervansaray Convention Center & Hotel, LARA, ANTALYA / TÜRKİYE 51 International Symposium on Drug Research and Development

portant age-related differences in xenobiotic metabolism do occur in Structurally, σ ligands are represented by wide variety of chemical Gilthead Seabream liver and are substrate specific, which might af- scaffolds, for example (+)-benzomorphans, phenylpiperidines, (+)- fect obtaining desired actions and/or responses to drugs, hormones pentazocine and haloperidol [2]. and dietary supplements used during breeding. Corbera et al. synthesized a series of novel tetrahydroindazole de-

PRESENTATIONS GST-CDNB activity increased in gilthead seabream (Sparus au- rivatives and tested for σ1 and σ2 receptor binding [3]. rata) liver cytosol of different ages (ranging from 1.5 to 20 months). Since sigma receptors are membrane-bound proteins, isolating 1.5 months of the fish activity is 17,7 ± 0,5 pmol/dakika/mg protein, and resolving their three-dimensional structure has proven to be dif- 20 months of the fish is 690,9 ± 32,0 pmol/dakika/mg protein. ficult. QSAR (quantitative structure-activity relationship) methods

POSTER assume that biological activity is correlated with chemical structure or properties and that as a consequence activity can be modeled as P 005 Ref: 0015 a function of computable physicochemical attributes. QSAR techniques are able to raise a predictive description of global structural THE PROTECTIVE ROLE OF PSEUDOVERNIA FURFURACEA requirements for interactions between substrates and receptor by us- AGAINST COLLOIDAL BISMUTH SUBCITRATE – INDUCED ing binding data. GENOTOXICITY IN HUMAN LYMPHOCYTE CULTURES We have described a detailed QSAR study on tetrahydroindazole series, in order to give better picture of action and to rationalize se- Hasan TÜRKEZ, Fatime GEYİKOĞLU lection of substituents. Atatürk University, Faculty of Arts and Sciences, Department of Biology, 25240 Erzurum, Turkey The QSAR functions in the Molecular Operating Environment (MOE) and SYBYL were used to compute theoretical molecular de- Bismuth compounds, especially, colloidal bismuth subcitrate scriptors related to physicochemical properties. (CBS) have been actively promoted for the treatment of diarrhoea, peptic and duedonal ulcer diseases. And the therapeutic bismuth 1. Lever JR, Gustafson JL, Xu R, Allmon RL, Lever SZ. Synapse, 59, 350- compounds are now being marred by episodes of serious adverse 358, 2006. reactions. On the other hand, the potential of lichens in cellular ac- 2. Vangveravong S, Xu J, Zeng C, Mach RH. Bioorg. Med. Chem., 14, 6988- tivities remains largely unexplored. The aim of this study was to as- 6997, 2006. sess the efficacy of the lichen Pseudovernia furfuracea (2.5, 5, 10 and 3. Corbera, J. et al. Chem. Med.Chem., 1, 140-154, 2006. 20 µg/mL) on the genotoxicity induced by CBS in the human blood cultures. With this aim, whole heparinized blood samples were taken from P 007 Ref: 0017 three healthy non-smoking donors. Sister-chromatid exchanges (SCE) and Micronuclei (MN) tests were used for evaluating the ge- SYNTHESIS AND DNA-CLEAVING ACTIVITY OF A SERIES notoxicity. OF SUBSTITUTED ARENEDIAZONIUM IONS SCEs and MN formations significantly increased by effect of 5 µg/ Murat KIZIL, Bircan ÇEKEN mL CBS when compared with the the control group. However, the Dicle University, Faculty of Science and Art, Department of Chemistry, 21280 Diyarbakır, decreased rates of such formations indicated that P. furfuracea was Turkey anti-genotoxic agent. Our results also showed that the protective role Aryl radicals and biradicals are generated in biological systems as of P. furfuracea was dose-related. intermediates of some drugs targeted DNA. Cleavage of DNA via On the basis of data, it is thought that this lichen species can pro- radical attack plays an important role in various biological processes, vide anti-genotoxic effects as due to their antioxidant defenses al- including chemoteraphy and carcinogenesis. Some antitumor drugs though there is no evidence for the content of the lichen species in generate aromatic biradicals that are belived to cleave double-strand- the present investigation. ed DNA via hydrogen atom abstraction from the sugar moiety. How- ever, the chemical behavior of substituted phenyl radicals toward DNA has not been extensively investigated. We have investigated the P 006 Ref: 0016 ability of causing the supercoiled DNA cleavage and the free radical formation of the substituted aryl radicals and aryl cations derived QSAR MODELING ON SIGMA RECEPTOR LIGANDS from arenediazonium ions. Mine YARIM, Ece GÜRDAL, Dilek EROL We prepared the substituted arenediazonium tetrafluoroborates in Yeditepe University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 22-74 % yields by reaction of the corresponding amine with isoamyl 34755 İstanbul, Türkiye nitrite and aqueous fluoroboric acid in ethanol. The plasmid pBlue- Sigma (σ) receptors are functional, membrane-bound proteins script M13+ DNA was prepared and isolated according to stand- distributed throughout the brain and peripheral organs. σ1 and σ2 ard protocols using Qiagene plasmid mini preparation kit. Gel was receptor types are clearly established, and further pharmacologi- scanned on Gel documentation system (Gel-Doc-XR, BioRad, Her- cal differentiation may be possible. σ1 receptors are implicated in cules, CA, USA). Bands on the gels were quantified using discovery central nervous system (CNS) disorders such as depression, schizo- series Quantity One programme (version 4.5.2, BioRad Co.). phrenia and dementia. Further, σ1 receptor agonists have value as Systematic studies indicate that both aryl radicals as well as aryl neuroactive agents, while antagonists may help alleviate cocaine ad- cation participates in the DNA cleavage pathway. diction. The significance of the σ receptor system to human health It has been found that the substituted arenediazonium salts have is augmented through the overexpression of σ sites by a number of capacity to cleave supercoiled DNA to form the open circular Form cancers. Thus, σ receptor ligands might be used for the detection II DNA and linear Form III DNA. and treatment of malignant neoplastic diseases. σ2 receptors may be of particular prognostic relevance, as the extent of their expression seems indicative of the proliferative status of tumors [1].

52 May 17-20, 2007 “From Chemistry to Medicine” DRD 2007

P 008 Ref: 0020 problem in many regions of the world. The need for new antimalar- ials comes from the widespread resistance to those in current use. In ANTIDEPRESSANT-LIKE EFFECT OF SOME NEW ARYL this study, the use of parasite’s lactate dehydrogenase as an antima- PROPANE DERIVATIVE COMPOUNDS larial drug target is evaluated. The cofactor binding site of Plasmo- 1Fulya MORAL, 1Meriç KÖKSAL, 1Mine YARIM, 2S. Sırrı BİLGE, 2S. Elif AKSÖZ dium vivax lactate dehydrogenase was compared to some other api- PRESENTATIONS 1Yeditepe University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, complexan counterparts by making a single residue change. Residue 34755 İstanbul, Turkey 163 is a leucine in Plasmodium, a serine in human and a methionine 2Ondokuz Mayıs University, Ondokuz Mayıs University, Faculty of Medicine, Department in other apicomplexans. Leucine 163 was replaced by methionine by of Pharmacology, 55139, Kurupelit, Samsun, Turkey site directed mutagenesis. It was observed that enzyme was tolerant POSTER In the early 1970s, evidence of the role of serotonin (5-hydroxytry- when leucine 163 residue in its cofactor binding site was replaced tamine or 5-HT) in depression began to emerge and the hypothesis with methionine like in its apicomplexan counterpart. This is a fea- that enhancing 5-HT neurotransmission would be available mecha- ture that distinguishes Plasmodium and other apicomplexan lactate nism to mediate antidepressant response was put forward. On the dehydrogenase enzymes from their human lactate dehydrogenase, basis of this hypothesis, efforts to develop agents that inhibit the up- supporting their suitability as targets in common drug design stud- take of 5-HT from the synaptic cleft were initiated [1]. ies. In recent years, selective serotonin reuptake inhibitors (SSRIs) have become a standard treatment because of their safety profile and fewer side effects than the other classes of antidepressants. Fluoxetine, P 010 Ref: 0024 (±)-N-methy-3-phenyl-3(α,α,α-trifluoro-p-tolyoxy)propylamine hydrochloride, is a potent selective serotonin reuptake inhibitor MUTAGENIC ANALYSIS OF ACTIVE SITE LOOP OF used for the treatment of major depression which marketed under LACTATE DEHYDROGENASE BY MIMICKING EIMERIA the well-known trade name Prozac® [2]. Furthermore, some of the TENELLA IN PLASMODIUM VIVAX undesired effects such as sexual dysfunction, gastrointestinal intol- 1Venhar ÇELİK, 2Dilek TURGUT-BALIK erance and activating effects such as nervousness, anxiety and im- 1University of Fırat, Faculty of Arts and Sciences, Department of Biology, 23169 Elazığ, sonia are showed with all available SSRIs [3, 4]. Therefore, one of Turkey the still therapeutic needs is the availability of antidepressants with a 2Yıldız Technical University, Faculty of Chemical and Metallurgical Engineering, more rapid onset of action and less side effects. Department of Bioengineering, Davutpaşa Campus, 34210 Esenler-İstanbul, Turkey In view of these literature results, we aimed modifications on Malaria, the most prevalent and most pernicious parasitic diseases Fluoxetine structure by changing the amine group with benzylpipe- of humans, is estimated to kill approximately three million people ridine group to reach new antidepressant compounds with a faster each year. The increasing occurence of drug resistance in many en- onset of action, a better efficacy and less side-effects. The antide- demic areas emphasizes the need for antimalarial drugs. The gene pressant-like effect of this synthesized compounds was studied in encoding Plasmodium vivax lactate dehydrogenase is a functinal comparison with other antidepressants (Fluoxetine, Sertraline, Imi- validation. We have isolated and cloned the lactate dehydrogenase, pramine) in forced swimming test (FST), a validated experimental and overexpressed the protein from Plasmodium vivax. The en- model of depression in mice. According to antidepressant profile zyme’s structure was also solved from P. vivax recently. Active site evaluation, three of six showed antidepressant-like effect some of loop of the enzyme has been determined by crystal structure studies which better than the standarts (fluoxetine, sertraline, imipramine) and shown to be an ideal site for the drug design studies. The active without changing any locomotor activity. site loop of this enzyme was found to have a pentapeptide insertion and this insertion differentiates this enzyme from its human coun- 1. Wong DT, Perry KW, Bymaster FP. Nature Reviews Drug Discovery, 4, terpart. Similar insertion sequence was also found in Eimeria tenella 764-774, 2005. 2. Takeuchi, K et al. Bioorg. Med. Chem. Lett., 13, 1903-1905, 2003. lactate dehydrogenase. It was observed in this study that replacing 3. Labbate A, Grimes JB, Arana GW. Biol. Psychiatry, 43, 904-907, 1998. the pentapeptide insertion sequence in Plasmodium vivax with the 4. Takeuchi K, Kohn TJ, Honigschmidt NA, Rocco VP, Spinazze PG, At- insertion sequence from Eimeria tenella has unaffected the enzyme kinson ST, Hertel W, Nelson DL, Wainscott DB, Ahmad LJ, Shaw J, activity. Threlkeld PG, Wong DT. Bioorg. Med. Chem. Lett., 13, 3939-3942, 2003.

P 011 Ref: 0025 P 009 Ref: 0023 SYNTHESIS OF AMINO ALCOHOL BASED COMPARATIVE ANALYSIS OF THE COFACTOR CHIRAL LIGANDS AND THEIR APPLICATIONS IN BINDING SITE OF PLASMODIUM VIVAX LACTATE ENANTIOSELECTIVE DIETHYLZINC ADDITION TO DEHYDROGENASE WITH SOME OTHER KNOWN BENZALDEHYDES APICOMPLEXAN COUNTERPARTS Dilek Işık TAŞGIN, Canan ÜNALEROĞLU 1 2 Venhar ÇELİK, Dilek TURGUT-BALIK Hacettepe University, Faculty of Science, Department of Chemistry, 06800 Beytepe- 1University of Fırat, Faculty of Arts And Sciences, Department of Biology, 23169 Elazığ, Ankara, Turkey Turkey Among asymmetric catalysis of C-C bond-formation, the enanti- 2Yıldız Technical University, Faculty of Chemical and Metallurgical Engineering, Department of Bioengineering, Davutpaşa Campus, 34210 Esenler-İstanbul, Turkey oselective addition of diorganozinc reagents to aldehydes in the presence of a catalytic amount of a chiral ligand is a convenient method Malaria is caused by the protozoan parasite Plasmodium from the for the preparation of optically active secondary alcohols [1]. These phylum Apicomplexa that also includes the important pathogens structural features are important building blocks for the synthesis of Toxoplasma, Eimeria, Theileria, Cryptosporidium and Babesia. Ma- many natural and biologically active compounds, and materials such laria is one of the greatest causes of human misery and death. Despite as liquid crystals [2]. For this purpose, a wide variety of chiral cata- continued efforts to control the disease, it remains a major health lysts, i.e. amino alcohols, amino thiols, diamines, disulfonamides,

Kervansaray Convention Center & Hotel, LARA, ANTALYA / TÜRKİYE 53 International Symposium on Drug Research and Development

and diols have been synthesized [3]. Among the chiral ligands reported, β-amino alcohols are the most often used chiral ligands [4]. Table 1. The addition of diethylzinc to benzaldehyde with ligands 1 and 2. In this study, norephedrine based chiral ligands were synthesized entry ligand solvent yield (%) ee (%) conf. PRESENTATIONS starting from pyrrole carbaldehydes and norephedrine. The synthesized (1S,2R)-2-((1H-pyrrol-2-yl)methylamino)-1-phenylpropan- 1 1 Toluene 36 71 S 1-ol (1) and (1R, 2S)-2-((1H-pyrrol-2-yl)methyl amino)-1-phenylpropan-1-ol (2) were used as chiral ligands in the enantioselective 2 1 Hexane 85 58 S

POSTER addition of diethylzinc to benzaldehyde. 3 1 Tol.+Hex. 67 66 S

4 2 Toluene 35 74 R

5 2 Hexane 77 64 R

6 2 Tol.+Hex. 70 78 R

7 2 Dichloromethane 17 27 R

1. Roudeau R, Pardo DG, Cossy J. Tetrahedron, 62, 2388, 2006. 2. Melo RPA, Vale JA, Zeni G, Menezes PH. Tetrahedron Lett., 47, 1829, 2006. 3. Xu Q, Zhu G, Pan X, Chan ASC. Chirality, 14, 716, 2002. 4. Cicchi S, Crea S, Goti A, Brandi A. Tetrahedron:Asymmetry, 8, 293, Synthesis of Amino Alcohols: Synthesized imines (1.22 mmol) 1997.

from pyrrolecarbaldehydes and norephedrine, and NaBH4 (1.35 mmol) in methanol was refluxed for 8 hours. When the reaction was completed, the mixture was cooled to room temperature and P 012 Ref: 0028 quenched with 15 mL water. It was extracted with ether (3x10 mL)

and dried over MgSO4. The mixture was filtered and the solvent was ELECTROCHEMICAL INVESTIGATION OF INTERACTIONS evaporated. Crude products 1 and 2 were dissolved in benzene and BETWEEN DNA AND SOME CHEMICALS filtered. The product was obtained after evaporation. 1Görkem YALÇIN, 2Murat ÇİZMECİOĞLU, 1Özlem SÖĞÜT, 1Mehmet ÖZSÖZ General Procedure for the Enantioselective Diethylzinc Addi- 1 tion to Benzaldehyde: System was dried under vacuum by applying Ege University, Faculty of Pharmacy, Department of Analytical Chemistry, İzmir, Turkey 2Ege University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, İzmir, the Schlenk technique for five times. 0.05 mmol chiral ligand was Turkey dissolved in dry solvent (5 mL) under the argon atmosphere. Af- Determinations of interactions between DNA and drugs are im- ter cooling to 0 oC, 2.2 mmol diethylzinc (1M in hexane) was added portant aspects of biological studies in drug discovery and pharma- with a syringe over a period of 5 min. The mixture was stirred for 5 ceutical development processes. hours 0 oC and 1 mmol aldehyde was added. The reaction mixture The interactions of some molecules with DNA have been inves- was stirred for 16 hours at room temperature and monitored by TLC. tigated by a variety of techniques. There has been growing interest The reaction was quenched with 5 mL of 1 N HCl solution and ex- to determine interacts between DNA and some molecules by using tracted with ether and dried over anhydrous MgSO and the solvent 4 electrochemical methods. was evaporated under reduced pressure. The crude product was pu- In this study, interactions of some chalcone derivatives with DNA rified by flash column chromatography ( EtOAc:hexane, 1:6). were investigated by using electrochemical methods. In recent years Amino alcohols were obtained by the reduction of imines with chalcone (1,3-diphenyl-2-propen-1-one) derivatives have been NaBH in methanol. Chiral ligands 1 and 2 were obtained in 65 % 4 synthesized in order to develop active compounds against cancer, and 70 % yields, respectively. Characterization of these compounds malaria, leishmaniase, tuberculosis and cardiovascular diseases. was carried out by 1H-NMR and 13C-NMR techniques. Therefore, determination of interactions between some chalcone The enantioselective addition of diethylzinc to benzaldehyde was derivatives and DNA will give some help to chalcone based drug de- carried out in different solvents in the presence of 5 mol % of ligands velopment studies. under argon atmosphere at 0oC to room temperature. Catalytic activity of 1 and 2 were examined in toluene, hexane, toluene-hexane mixture and dichloromethane (Table 1). The best result was obtained with ligand 2 (70 % yield and 78 % ee) in toluene-hexane solvent P 013 Ref: 0031 mixture. GLYCOSYLATION IN ROOM TEMPERATURE IONIC LIQUID (RTIL) USING UNPROTECTED AND UNACTIVATED DONORS 1Sultan N. BAYTAŞ, 2Tae-joon PARK, 1Robert J. LINHARDT 1Rensselaer Polytechnic Institute, Department of Chemistry and Chemical Biology, Troy, NY, USA 2Rensselaer Polytechnic Institute, Department of Chemical and Biological Engineering, Troy, NY, USA Glycosylation occurs between a donor and an acceptor in the presence of a promoter, which activates the donor. There are various factors that need to be considered when carrying out the synthesis of glycosides including the manipulation of protecting groups in both

54 May 17-20, 2007 “From Chemistry to Medicine” DRD 2007

donor and acceptor, the architecture of donor and acceptor and the P 015 Ref: 0034 solvent system [1]. These strategies involve often a large number of synthetic steps. The chemical synthesis of unprotected carbohydrates AMINO TERMINAL ANALYSIS OF THE ACTIVE SITE LOOP poses a number of challenges, including poor solubility in most con- OF LACTATE DEHYDROGENASE FROM THE MALARIA ventional organic solvents. Only very polar organic solvents, such as PARASITE PLASMODIUM VIVAX PRESENTATIONS formamide, dimethylformamide, dimethylsulfoxide, and pyridine, 1Bünyamin ATMIŞ, 1Dilek SADAK, 1Venhar ÇELİK, 2Dilek TURGUT-BALIK easily dissolve significant amounts of sugars. It is important to in- 1University of Fırat, Faculty of Arts And Sciences, Department of Biology, 23169 Elazığ- vestigate new solvent systems that dissolve carbohydrates, support Turkey glycosylation reactions of unprotected sugars, and facilitate product 2Yıldız Technical University, Faculty of Chemical and Metallurgical Engineering, POSTER recovery. Room temperature ionic liquids (RTILs) are becoming Department of Bioengineering, Davutpaşa Campus, 34210 Esenler-İstanbul,Turkey increasingly used as solvents for a wide variety of chemical reac- Malaria is parasitic disease that threatens nearly half the global tions [2]. RTILs also display desirable solvent properties and have population. It is caused by protozoan apicomplexan parasites of the the potential of replacing conventional volatile organic solvents in genus Plasmodium; four species cause malaria in man. In this study, carbohydrate chemistry. In this work, we report the glycosylation of amino acid exchanges were made in the amino terminal end of the various simple, unprotected monosaccharides in RTILs to give ben- active site loop of Plasmodium vivax lactate dehydrogenase (LDH) zyl glycosides, disaccharides and oligosaccharides. RTILs facilitate by mimicking Toxoplasma gondii I ve II, Eimeria acervulina, Eimeria the use of unactivated and unprotected donors in these reactions, re- tenella ve Theileria parva LDH’s using the site directed mutagenesis sulting in a simple synthetic strategy involving a single glycosylation method. Although enzymatic activity was decreased compared to step. The synthesis of galactose oligomers was also possible using that of the wild type protein, some enzymatic activity was present these novel solvents. meaning that enzyme was still in contact with its substrate. Decrease in the enzymatic activity indicates that this region is sensitive to 1. Sasaki K, Nagai H, Matsumura S, Toshima K. A novel greener glycosida- changes and this supports the idea that this site could be evaluated as tion using an acid-ionic liquid containing a protic acid, Tetrahedron Lett., an ideal target for the drug design studies for both Plasmodium and 44, 5605-5608, 2003. 2. Murugesan S, Linhardt RJ. Ionic liquids in carbohydrate chemistry - cur- some other apicomplexans. rent trends and future directions, Curr. Org. Synth., 2, 437-451, 2005.

P 016 Ref: 0035 P 014 Ref: 0033 AN ANALYSIS TO BE USED IN SCTRUCTURE-BASED DRUG DEVELOPMENT OF VALIDATED METHOD FOR DESIGN STUDIES: COMPARISON OF ACTIVE SITE LOOPS RISEDRONATE BY HPLC-MS MS FROM BIOLOGICAL OF ENZYMES, PLASMODIUM VIVAX AND TOXOPLASMA MATERIAL GONDII LACTATE DEHYDROGENASES 2Zeynep İrem DİLER, 2Gülay ŞAHİN-KOÇ, 2Hüseyin YALÇINKAYA, 1Venhar ÇELİK, 2Dilek TURGUT-BALIK 1Durişehvar ÖZER-ÜNAL, 1Dilek EROL 1University of Fırat, Faculty of Arts and Sciences, Department of Biology, 23169 Elazığ, 1Yeditepe University, Faculty of Pharmacy, Department of Analytical Chemistry, İstanbul, Turkey Turkey 2Yıldız Technical University, Faculty of Chemical and Metallurgical Engineering, 2Yeditepe University, Yeditepe Sağlık Hizmetleri A.Ş., GLP Laboratory, Acıbadem-İstanbul, Department of Bioengineering , Davutpaşa Campus, 34210 Esenler-İstanbul, Turkey Turkey Increasing resistance of Plasmodium to the antimalarial agents Risedronate (RS) is a third generation of bisphosphonate class of necessitates the development of new drugs which have different drugs. It is widely used for the treatment of bone disorders such as modes of action from the currently existing ones. Present study is osteopotrosis. Determination of RS from biological fluids have dif- targeted to lactate dehydrogenase (LDH) which is a key anaerobic ficulties because of its low level in urine and blood. A sentitive and metabolic pathway enzyme. LDH of Plasmodium has strikingly dif- reliable HPLC-MS MS method was developed and validated from ferent properties compared to its human counterparts. Inhibition of human urine. the activity of this enzyme has been shown to kill the parasites in the The extraction method was developed to analyse RS from bio- erythrocytes. Thus, we have isolated and cloned the LDH gene, and logical material. In this method TMS-diazomethan derivatization overexpressed the protein. The structure of the enzyme from P. vivax was used followed by solid phase extraction.The mobile phase was (PvLDH) recently reported. A five amino acids insertion in the ac-

MeOH:H20 (80:20; v/v) containing 0.1 % formic acid. The best reso- tive site formed a distinctive cleft on the surface of the PvLDH as did lution was obtained by using Agilent Zorbax Eclipse XDB reversed in P. falciparum LDH. This site has been identified as a potential tar- phase C18 (150x4.6mm, 5µm) and alendronate was used as an inter- get for the structure based drug design studies. The site is not unique nal standart. to Plasmodium. The five amino acid insertion was also observed in The developed method was applied succesfully to biological ma- some other apicomplexan parasites. This site was analysed by mak- terial. The limit of quantitation of RS from biological material was ing residue exchanges in the P. vivax LDH active site loop to mimick 5 ng/ml. The calibration curve was linear in between 5-400 ng/ml. the same region of Toxoplasma gondii LDH1. It was observed that Precision, recovery, accuracy and stability results were satisfactory making residue exchanges in the active site loop of PvLDH was pos- for the method developed. The method is suitable for routine analy- sible without losing enzymatic activity. This observation emphasizes sis of bioequivalence studies. that the active site loop is a crucial region accross the apicomplexan LDHs. 1. Zhu LS., Lapko VN., Lee JW et.al. Rapid Commun. Mass Spectrom., 20(22), 3421-6, 2006. 2. Vallano PT., Shugards SB., Kline WF. et. al. J. Chromatogr. B, 794, 23-33, 2003.

Kervansaray Convention Center & Hotel, LARA, ANTALYA / TÜRKİYE 55 International Symposium on Drug Research and Development

P 017 Ref: 0036 (Vit C) and tocopherol (Vit E) are the major antioxidant molecules combating the bed effects of free radicals. Catalase, one of the major INTERACTION OF SOME 3,4-DIHYDROQUINOLIN-(1H)- antioxidant enzyme, neutralize the hydrogen peroxide (H2O2) pro- 2-ONE DERIVATIVES WITH RAT LUNG SEMICARBAZIDE- duced form superoxide radical by dismutases.

PRESENTATIONS SENSITIVE AMINE OXIDASE (SSAO) In this study, effects of one water soluable antioxidant, ascorbic acid 1Samiye YABANOĞLU, 2Sevil Görkem SUNAL, 2Akgül YEŞİLADA, (Vit C), and one lipid soluable antioxidant, α-lipoic acid, on diabetic 1Gülberk UÇAR rat liver catalase activities were aimed to be studied. Furthermore, 1Hacettepe University, Faculty of Pharmacy, Department of Biochemistry, 16100 Ankara, effects of both antioxidants given together were also analyzed. To do

POSTER Turkey this, male Sprauge-Dawley rats were given streptozotocin (STZ) to 2Hacettepe University, Faculty of Pharmacy, Department of Basic Pharmaceutical induce diabetes, and groups were seperated as control (n=9), diabet- Sciences, 06100 Ankara, Turkey ic (n=9), diabetic+lipoic acid given (n=8), diabetic+vitamin C given Since semicarbazide-sensitive amine oxidase (SSAO) is shown to (n=12) and diabetic+vitC and lipoic acid given (n=7). Four weeks be involved in diabetes, Alzheimer’s and Parkinson diseases, heart after the development of diabetes, rats were decapitated and catalase and vascular diseases, the synthesis of new compounds as specific activites were measured. It has been observed that catalase activities SSAO inhibitors suggested to be useful developing novel therapeutic were significantly lowered in diabetic group (p<0.005) as compared agents. In view of the previous studies indicating that diazoheterocy- to controls. Application of lipoic acid were increased the diabetic clic compounds have been introduced as promising class of revers- CAT activities but not up to the control level. Similarly, vitamin C ible amine oxidase inhibitors, three compounds with 3,4-dihydro- rised the diabetic catalase activities and we observed that vitamin quinoline-(1H)-2-one structure and their open ring derivatives were C was better for the restoration of diabetic CAT activities. Moreo- synthesized and the interaction of these compounds with SSAO pu- ver, combined antioxidant given groups’ catalase activities were also rified from rat lung were evaluated. higher than those of the diabetics and this increament was as effec- The compounds of Q (N-amino-3,4-dihydroquinoline-(1H)-2- tive as the one of the vitamin C group. one) and QB (1-(Benzyliden-amino)-3,4-dihiydroquinoline-(1H)-2- In conclusion, due to the increased oxidative stress, catalases are one) were synthesized by the reduction and the ring closure reaction damaged or inhibited by the actions free radicals and administration of o-nitro-cinnamic acid whereas MBK (Tert-butyl-N-[cyclohexyl- of antioxidants help to reduce these side effects in streptozotocin in- carbamoyl-(3-hydroxyphenyl)methyl]-N-phenyl-carbamate), MG duced diabetes. (Tert-butyl-N-[cyclohexylcarbamoyl-(3-hidroxyphenyl)methyl]-N- (2-benzoylphenyl)-carbamate) and PCN (2-(3-cyano-2-oxo-4-phenyl-2H-quinolin-1-yl-N-cyclohexyl-2-(4’-chlorophenyl)acetamide) P 019 Ref: 0038 were synthesized by one pot Ugi-Knovenagel reaction. The structures of the novel compounds were evaluated using 1H NMR, 13C SYNTHESIS AND ANALGESIC ACTIVITY OF SOME NMR and MS techniques. BENZIMIDAZOLE DERIVATIVES Compound Q, which carries a free amine group appeared as a 1İlhan IŞIKDAĞ, 2Ümide DEMİR, 2Özgür Devrim CAN, 1Yusuf ÖZKAY, good substrate for rat liver SSAO suggesting that this relatively small 2Yusuf ÖZTÜRK compound may interact with the active site channel of the enzyme 1Anadolu University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, through its free amine group. QB, PCN and MG inhibited the en- 26470 Eskişehir, Turkey zyme non-competitively and irreversibly suggesting that these com- 2Anadolu University, Faculty of Pharmacy, Department of Pharmacology, 26470 pounds may interact with an another hydrophobic binding region Eskişehir, Turkey outside of the active site of the enzyme. It is concluded that these Heterocyclic compounds having two nitrogen atoms oriented in compounds may have promising features as novel anti-parkinson/ 1,3-positions in ring are endowed with broad spectrum of phar- anti-Alzheimer agents in case their SSAO inhibitory effects can be maceutical properties. Imidazole and benzimidazole, shown as the supported by in vivo studies. instances for these heterocyclics, drugs have broaden scope in rem- edying various dispositions in clinical medicine [1]. Pharmaceutical Keywords: 3,4-Dihydroquinoline-(1H)-2-ones, tissue-bound semicar- properties concern antifungal and antimycotic, antiprotozoal, anti- bazide-sensitive amine oxidase (SSAO), Ugi-Knovenagel condensation, neoplastic, antiulcer, antihistaminic and antiallergic, antihyperten- substrate, inhibition sive, anthelmintic, antioxidant, antiviral, antibacterial and antipara- sitic activities, all of which are unique characteristics known from imidazole and benzimidazole derivatives [2]. It is known well that P 018 Ref: 0037 synthetic chemical compounds especially lipophylic ones have vari- EFFECTS OF LIPOIC ACID AND VITAMIN C ous different effects on central nervous system. Benzimidazoles are examples of these derivatives with their reported pharmacological ADMINISTRATION ON STREPTOZOTOCIN INDUCED effects such as anesthetic and hypnotic[3], neuroleptic and antipsy- DIABETIC RAT LIVER CATALASE ACTIVITIES chotic [4], analgesic [5] and sedative [6] etc. These large research Gökhan SADİ, T ülin GÜRAY areas of benzimidazoles promted us to study with them. Middle East Technical University, Department of Biochemistry, Ankara, Turkey We synthesized five 2-aryl-4,5-dichloro-(1H)-benzimidazoles via the condensation of 4,5-dichloro-o-phenylenediamine and cor- Diabetes mellitus which is a glucose metabolism disorder is as- responding aldehyde derivatives in ethanol with the presence of sociated with consequences of oxidative stress due to non-enzymatic sodium bisulfite. Their structures were elucidated with 1H-NMR, protein glycation, glucose autoxidation and polyol pathway, which IR and MASS(Apci) spectral analysis. Tail-clip and tail-immersion augments the free radical production. In the tissues, cells evolve en- tests, were applied in order to investigate probable analgesic effects. zymatic and non-enzymatic antioxidants to defend themselves for Synthesized compounds were applied to mices at a dose of 100 mg/ the oxidation potential of radicals. Superoxide dismutase (SOD), kg (i.p). Morphine (1 mg/kg) was used as positive standart. Catalase (CAT) and Glutathione Peroxidase (GPx) are the major All of the synthesized compounds showed analgesic activities in antioxidant enzymes, and reduced glutathione (GSH), ascorbic acid tail-clip and tail-immersion tests. Compound 2 was found as the

56 May 17-20, 2007 “From Chemistry to Medicine” DRD 2007

most active derivative in the series. 1H-NMR and IR spectral datas were obtained as expected. Mass (Apci) spectras of the compounds were obtained as expected. Mass (Apci) spectras of the compounds were agreed well with their molecular weight. Our synthesis with were agreed well with their molecular weight. The compounds bisbenzimidazole compounds have recently begun. Further studies (compounds 1, 2 and 4) including metoxy groups at metha posi- are in progress to increase the number of compounds with smilar tions and/or hydroxyl group at para position of phenyl ring which structures. After reaching adequate number of the compounds, anti- PRESENTATIONS is substituted at second position of benzimidazole ring, have higher proliferative activity scaning will be started. analgesic activity. On the other hand, addition of nitro group at or- tho position of phenyl ring (compound 3) causes decrease in the an- 1. Townsend LB, Chem. Rev., 67, 533, 1976. algesic activity. These findings indicate the importance of chemical 2. Haugwitz RD, Angel RG, Jacobs GA, J. Med. Chem., 25, 969, 1982. POSTER sturucture and pharmacological activity relationships of the synthe- 3. Hisano T, Ichıkawa M, Tsumoto K, Tasaki M, Chem. Pharm. Bull., 30, 2996, 1982. sized compounds. In conclusion, it may be suggested that to obtain 4. Kamal A, Ramul P, Srinivas O, Ramesh G, Kumar PP, Bioorg. Med. Chem. more active derivatives, containing the same structure with the title Lett., 14, 4791, 2004. compounds, number of synthesis including substituted-p-hydroxy- 5. Alper S, Arpacı, ÖT, Akı, EŞ, Yalçın İ, Farmaco, 58, 497, 2003. benzaldehyde and substituted-m-methoxybenzaldehyde derivatives should be increased.

1. Kleeman A, Engel J, Kutscher B, Reichert D, Pharmaceutical Substances, P 021 Ref: 0041 3rd ed.; Stuttgart: New York, 1999. SYNTHESIS OF 3-PHENETHYLAMINO-1- 2. Nezhad AK, Rad MNS, Mohabatkar H, Asraria Z, Hemmateenejada B, Bioorg. Med. Chem., 13, 1931, 2005. PHENYL/SUBSTITUTED PHENYL-1-PROPANONE 3. Janssen PAJ, Niemegeers CJE, Schellekens KHL, Lenaerts FM, Arzneim. HYDROCHLORIDES Forsch., 21, 1234, 1971. 1Ebru METE, 2H. İnci GÜL, 1Cavit KAZAZ 4. Sato M, Arimoto M, Ueno K, Kojima H, Yamasaki T, Sakurai T, Kasahara 1 A, J. Med. Chem. 21, 1116, 1978. Atatürk University, Faculty of Arts and Sciences, Department of Chemistry, 25240 Erzurum, Turkey 5. Sondhi SM, Singh N, Kumar A, Lozach O, Meijer L, Bioorg. Med. Chem., 2 14, 3758, 2006. Atatürk University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 25240 Erzurum, Turkey 6. Seredenim SB, Eur. Neuropsychopharm., 6, 111, 1996. It has been reported that acetophenone derived several mono Mannich bases have cytotoxic, antifungal and anticonvulsant activi- P 020 Ref: 0039 ties [1-5]. In this study, synthesis of 1-aryl-3-phenethylamino-1-propanone hydrochlorides has been realised to investigate their biologi- STUDIES ON THE SYNTHESIS AND ANTIPROLIFERATIVE cal activities in future. ACTIVITY INVESTIGATION OF 2,2’-(3-METHOXYPHENYL) Chemical structure of the compounds were confirmed by 1H 13 AND 2,2’-(3-HYDROXYPHENYL)-1H,1H’-[5.5’]-BIS- NMR, C NMR, UV, IR and MS spectroscopies. Purity level of them BENZIMIDAZOLES was determined by elemental analyses. 1İlhan IŞIKDAĞ, 1Yusuf ÖZKAY, 2Zerrin İNCESU 1. Gül Hİ, Vepsalainen J, Gül M, Erciyas E, Hanninen O. Cytotoxic activi- 1Anadolu University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, ties of mono and bis Mannich bases derived from acetophenone against 26470 Eskişehir, Turkey Renca and Jurkat cells, Pharm. Acta Helv., 74, 393-8, 2000. 2Anadolu University, Faculty of Pharmacy, Department of Biochemistry, 26470 Eskişehir, 2. Gül Hİ, Gül M, Erciyas E. Syntheses and stability studies of some Man- Turkey nich bases of acetophenones and evaluation of their cytotoxicity against The incorporation of the imidazole and benzimidazole nuclei Jurkat cells, Arzneimittel Forschung, 52, 628-35, 2002. 3. Gül Hİ, Çalış Ü, Vepsalainen J. Synthesis of some mono-Mannich bases is an important synthetic strategy in drug discovery [1]. Previous and corresponding azine derivatives and evaluation of their anticonvul- observations suggest that substituted benzimidazoles and related sant activity, Arzneimittel Forschung, 54, 359-64, 2004. heterocycles, which are the structural isosters of nucleotides ow- 4. Gül Hİ, Şahin F, Gül M, Öztürk S, Yerdelen KO. Evaluation of antimi- ing fused heterocyclic nuclei in their structures that allow them crobial activities of several Mannich bases and their derivatives, Arch. to interact easily with the biopolymers, possess potential activity Pharm., 338, 335-8, 2005. with lower toxicities in the chemotherapeutic approach in man [2, 5. Gül Hİ, Das U, Pandit B, Li PK. Evaluation of the cytotoxicity of some 3]. The high therapeutic properties of the related drugs have en- mono-mannich bases and their corresponding azine derivatives against couraged the medicinal chemists to synthesize the large number of androgen-independent prostate cancer cells, Arzneimittel Forschung, 56, 850-4, 2006. novel chemotherapeutic agents. Antitumoral activities of benzimidazole and bisbenzimidazole compounds were reported in several studies. Furthermore, there are clinical anticancer drugs, known as Hoechst-33258 and Hoechst-33342 dyes including bisbenzimida- P 022 Ref: 0042 zole structure [4, 5]. Prompted above observations we synthesized SYNTHESIS OF 1-PHENETHYL-3-AROYL-4-ARYL-4- the title compounds to investigate their possible antiproliferative effects. PIPERIDINOLS We synthesized two bis-benzimidazoles via the condensation of 1Ebru METE, 2H. İnci GÜL, 1Cavit KAZAZ 3,3’-diaminobenzidine and corresponding aldehyde derivatives in 1Atatürk University, Faculty of Arts and Sciences, Department of Chemistry, 25240 ethanol with the presence of sodium bisulfite. Their structures were Erzurum, Turkey elucidated with 1H-NMR, IR and MASS(Apci) spectral analysis. 2Atatürk University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Their antiproliferative effects of the compounds will be determined 25240 Erzurum, Turkey on HDQ-P1 and HT-29 cancer cell lines by using MTT and BrdU It has been reported that piperidinol type of compounds have cy- assays. totoxic, antifungal and anticonvulsant activities [1-4]. In this study, Both of the bisbenzimidazole derivatives were synthesized and synthesis of 1-phenethyl-3-aroyl-4-aryl-4-piperidinols has been re- their spectral datas were recorded. 1H-NMR and IR spectral datas alised to investigate their biological activities in future.

Kervansaray Convention Center & Hotel, LARA, ANTALYA / TÜRKİYE 57 International Symposium on Drug Research and Development

Chemical structure of the compounds were confirmed by 1H 1. Stolfi RL, Martin DS. Therapeutic activity of maleic anhydride-vinyl NMR, 13C NMR, UV, IR and MS spectroscopies. Purity level of them ether copolymers against spontaneous, autochthonous murine mam- was determined by elemental analyses. mary tumors, Cancer Treat Rep., 62(11),1791-6,1978. 2. Ottenbrite, RM. The antitumor and antiviral effects of polycarboxylic acid polymers in biological activities of polymers, ACS Symposium Se- PRESENTATIONS 1. Gül Hİ, Çalış Ü, Vepsalainen J. Synthesis and evaluation of anticonvul- ries 186, Washington DC, 1982. sant activities of some bis Mannich bases and corresponding piperidi- 3. Kaplan Can, H., Doğan, AL., Rzaev, ZMO, Üner, AH, Güner, A. Syn- nols, Arzneimittel Forschung, 52, 863-9, 2002. thesis and Antitumor Activity of Poly(3,4-dihydro-2Hpyran-co-maleic 2. Gül M, Gül Hİ, Das U, Hanninen O. Biological evaluation and structure- anhydride-co-vinyl acetate), Journal Applied Polymer Science, 96, 2352- activity relationships of bis-(3-aryl-3-oxo-propyl)-methylamine hydro-

POSTER 2359, 2005. chlorides and 4-aryl-3-arylcarbonyl-1-methyl-4-piperidinol hydrochlorides as potential cytotoxic agents and their alkylating ability towards cellular glutathione in human leukemic T cells, Arzneimittel Forschung, 55, 332-7, 2005. P 024 Ref: 0044 3. Gül Hİ, Şahin F, Gül M, Öztürk S, Yerdelen KO. Evaluation of antimicrobial activities of several mannich bases and their derivatives, Arch. ANTIFUNGAL ACTIVITIES OF 3-PHENETHYLAMINO-1- Pharm., 338, 335-8, 2005. ARYL-1-PROPANONE HYDROCHLORIDES AND 3-AROYL- 4. Gül M, Atalay M, Gül Hİ, Nakao C, Lappalainen J, Hanninen O. The effects of some Mannich bases on heat shock proteins HSC70 and GRP75, 4-ARYL-1-PHENETHYL-4-PIPERIDINOLS AGAINST SOME and thioredoxin and glutaredoxin levels in Jurkat cells, Toxicol In Vitro, PLANT AND HUMAN PATHOGENIC FUNGI 19, 573-80, 2005. 1H. İnci GÜL, 1Canan ÖZELGÜL, 2Ebru METE, 3Fikrettin ŞAHİN, 3Dilşat YURDAKUL 1Atatürk University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, P 023 Ref: 0043 25240 Erzurum, Turkey 2Atatürk University, Faculty of Arts and Sciences, Department of Chemistry, 25240 SYNTHESIS AND ANTITUMOR ACTIVITY OF POLY(2,3- Erzurum, Turkey DIHYDROPYRAN-CO-MALEIC ANHYDRIDE-CO-VINYL 3Yeditepe University, Faculty of Engineering and Architecture, Department of Genetics and Bioengineering, İstanbul, Turkey ACETATE) 1-Aryl-3-phenethylamino-1-propanone hydrochlorides (1, 3, 5, 7, 1Hatice KAPLAN-CAN, 2A. Lale DOĞAN, 3Zakir M. O. RZAEV, 9, 11, 13, 15, 17, 19) and 3-aroyl-4-aryl-1-phenethyl-4-piperidinols 2Ayşegül HASEGELİ-ÜNER, 1Ali GÜNER (2, 4, 6, 8, 10, 12, 14, 16, 20) were synthesized. Aryl part was C6H5 1Hacettepe University, Faculty of Science, Department of Chemistry, Ankara, Turkey for 1, 2; p-CH C H for 3, 4; p-CH OC H for 5, 6; p-ClC H for 7, 2 3 6 4 3 6 4 6 4 Hacettepe University, Institute of Oncology, Department of Basic Oncology, Ankara, 8; p-FC H for 9, 10; p-BrC H for 11, 12; o, p-(Cl) C H for 13, 14; Turkey 6 4 6 4 2 6 3 p-NO C H for 15,16; p-HOC H for 17; C H S(2-yl) i.e. 2-Thienyl 3Hacettepe University, Faculty of Engineering, Department of Chemical Engineering, 2 6 4 6 4 4 3 Ankara, Turkey for 19, 20. The compounds synthesized were tested against 8 plant patho- It is known that the water soluble anhydride-containing copoly- genic fungi (Rhizoctonia soloni-EB-ML, Fusarium oxysporium CE1, mers as polyanions and their functional derivatives have high biologi- Sclerotinia sclerotiorum FD3, Aspergillus niger FS2, Aspergillus fla- cal and physiological activity, specially antimicrobial and antitumor vus Hak23, Alternaria alternata FS2002, Macrophamina phaseoli properties. Polyanions are also known for their potential to stimulate CE4, Botrytis cinerea MFD3) and 3 human pathogenic fungi (Mi- the immune system and invoke activities against tumors, viruses and crosporum canis-AO5, Candida albicans EA-07, Candida parapsilosis bacteria. Copolymers of dihydropyran and its derivatives with acrylic EA-08) using agar dilution assay in the concentration range of 6,25 acid, which contain tetrahydropyran rings and free carboxylic groups to 200 µg/ml [1]. Minimal Inhibition Concetrations (MIC) of the on the polymer backbone, as well as an alternating cyclocopolymer of compounds were determined. Amphotericin-B was used as a refer- divinyl ether (acyclic analogy of dihydropyran) with maleic anhydride ence compounds. Amphotericin-B was only effective against Mac- have exhibited antitumor activities in vitro [1, 2]. rophamina phaseoli CE4, Aspergillus niger FS2 and Aspergillus flavus Radical-initiated terpolymerization of 3,4-dihydro-2H-pyran Hak23 at 100 mg/ml. (DHP), maleic anhydride (MA) and vinyl acetate (VA) as a donor- None of the compounds showed antifungal activity at the concen- acceptor-donor systems was carried out MEK in the presence of tration range studied against Fusarium oxysporium-CE1, Aspergillus AIBN as initiator at 65oC in nitrogen atmosphere. Determination niger-FS7, Botrytis cinerea-MFD, Candida albicans-EA-07, which are of some kinetic parameters, constants of thermal−copolymerization plant pathogenic fungi. MIC values of the compounds (µg/ml) were of complexed comonomers, terpolymer composition behavior re- as follows: lationships of synthesized polymers with alternating structure are Compound (µg/ml): 13(25), 14(50) against Rhizoctonia soloni- described and discussed. Synthesized polymers were characterized 2001; 10, 13, 15 (12.5) , 2, 3, 8, 9, 11, 19, 20 (25), 1 (50), 7 (200) by analytical methods (acid number), viscometer, FTIR and thermal against Microsporum canis-AÖ5, 11 (100) against Sclerotinia sclero- (DSC and TGA) methods [3]. tiorum-FD3; 13(100), 15, 16 (200) against Aspergillus flavus Hak23; 8 In vitro cytotoxicities of synthesized poly(DHP-alt-MA) and (200), 14 (100) against Alternaria alternata-FS2002; 5, 7 (200) against poly(DHP-co-MA-co-VA) polymers were evaluated using Raji cells Macrophamina phaseoli-CE4; 9, 13, 15, 17, 19 (100) against Candida (human Burkitt lymphoma cell line). Antitumor activity of prepared parapisilosis EA-08. anion-active poly(DHP-alt-MA) and poly(DHP-co-MA-co-VA) polymers were studied by methyl-thiazol-tetrazolium (MTT) test- 1. Gül Hİ, Şahin F, Gül M, Öztürk S, Yerdelen KO. Evaluation of antimi- ing method using calorimetric measurements of chemotherapic ef- crobial activities of several mannich bases and their derivatives, Arch. fect and quantitative evaluation of LD50 dose for the total number of Pharm., 338, 335-8, 2005. tumor cells. Hydrolyzed copolymer has sufficiently high antitumor activity, which depends on the amount of hydrogen bonded carboxylic groups and on their regular distribution in side chain of the functional macromolecules [3].

58 May 17-20, 2007 “From Chemistry to Medicine” DRD 2007

P 025 Ref: 0045 been elucidated on the basis of 1H and 13C NMR spectral data (DEPT, COSY, HMQC, HMBC and NOE). 1H NMR spectra of the com- EVALUATION OF CYTOTOXICITY OF 1-ARYL-3- pound was well resolved and the unambiguous proton chemical-shift PHENETHYLAMINO-1-PROPANONE HYDROCHLORIDES assignments were based on the multiplicity pattern of proton reso-

AND 1-PHENETHYL-3-BENZOYL-4-ARYL-4-PIPERIDINOLS nances and also on the use of homonuclear 1H – 1H COSY spectra. PRESENTATIONS BY BRINE SHRIMP BIOASSAY The assignments of all carbon resonances of compound were based on the analysis of the HMQC and HMBC spectra. 1Murat ÇİZMECİOĞLU, 2H. İnci GÜL, 3Ebru METE X-Ray Study: For the crystal structure determination, the single– 1Ege University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 35100 İzmir, Turkey crystal of the molecule 3-(p-chlorobenzoyl)-4-(p-chlorophenyl)-1- POSTER 2Atatürk University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, phenethyl-4-piperidinol was used for data collection on a four–circle 25240 Erzurum, Turkey Rigaku R–AXIS RAPID–S diffractometer equipped with a two–di- 3Atatürk University, Faculty of Arts and Sciences, Department of Chemistry, 25240 mensional area IP detector. The graphite–monochromatized Mo Kα Erzurum, Turkey radiation (λ=0.71073 Å) and oscillation scans technique with ∆ω=5° Synthesis of the novel 1-aryl-3-phenethylamino-1-propanone for one image were used for data collection. The lattice parameters hydrochlorides (series 1) and 1-phenethyl-3-benzoyl-4-aryl-4-pip- were determined by the least–squares methods on the basis of all eridinols (series 2) were carried out. Evaluation of the cytotoxicity reflections with F2>2σ(F2). Integration of the intensities, correction of the compounds has been realised by brine shrimp bioassay as for Lorentz and polarization effects and cell refinement was per- previously described [1]. Chemical structure of the compounds were formed using CrystalClear software [1]. The structures were solved confirmed by 1H-NMR, 13C-NMR, UV, IR and MS spectroscopic by direct methods (SHELXS–97) [2] and non–H atoms were refined methods. Puririties of the synthesized compounds were determined by full–matrix least–squares method with anisotropic temperature by elemental analyses. factors (SHELXL-97) [2]. Of the compounds tested, compounds 13, 15, 17 (from the series Compound 1, 3-(p-chlorobenzoyl)-4-(p-chlorophenyl)-1-phene- 1), 4, 6, 8 (from the series 2) were found effective in brine shrimp bi- thyl-4-piperidinol, crystallizes in the monoclinic crystal system oassay (Table 1). Aryl part was (2,4-(Cl)2C6H3) for 13, (4-NO2C6H4) with space group P21/a and the following unit cell parameters: a= for 15, (4-HOC6H4) for 17, (4-CH3C6H4) for 4, (4-CH3OC6H4) for 10.792.(4) Å, b= 12.863 (5)Å, c= 16.950 (6) Å; α =90°, β= 97.77(4)°,

6, (4-ClC6H4) for 8. The most effective compound was detected as γ = 90°; V= 2331.3(16) Å3 , and Z=4. The compound 1 has intermo- compound 6 while the least effective one was found as compound lecular O1-H···N1 [O1-H; 0.820, H···N1; 2.11, O1···N1; 2.878(5) Å, 15. Cytotoxicities of these compounds were determined in the range O1···H-N1 156˚], C13···H-O2 [C13-H; 0.970, H···O2; 2.54, C13···O2; of 13.85 - 3467.52 µg/ml while reference compound 5-fluorouracil 3.373(6) Å, C13···H-O2 144˚] hydrogen bonds. exhibited 2.99 µg/ml of cytotoxicity. 1. Rigaku (2005), CrystalClear, Version 1.3.6. Rigaku American Corpora- tion, 9009 New Trails Drive, The woodlands, TX 77381–5209, USA. Table 1. Cytotoxicity of the compounds in brine shrimp bioassay. 2. Sheldrick GM. SHELXS97 and SHELXL97, University of Göttingen, Code Aryl Cytotoxicity (µg/ml) Germany, 1997.

Series 1 P 027 Ref: 0047 13 2,4-(Cl)2C6H3 65.79 ± 0.083

15 4-NO2C6H4 3467.52 ± 0.469 STRUCTURE ELUCIDATION OF MONO MANNICH BASE 1-(p-METHOXYPHENYL)-3-PHENETHYLAMINO-1- 17 4-HOC H 316.82 ± 0.122 6 4 PROPANONE HYDROCHLORIDE AND SEMICYCLIC Series 2 MONO MANNICH BASE 3-(p-METHOXYBENZOYL)-4-(p- 4 4-CH C H 165.70 ± 0.186 METHOXYPHENYL)-1-PHENETHYL-4-PIPERIDINOL USING 3 6 4 1D AND 2D NMR SPECTROSCOPY 6 4-CH OC H 13.85 ± 0.191 3 6 4 1Cavit KAZAZ, 1Ebru METE, 2H. İnci GÜL 1 8 4-ClC6H4 573.33 ± 0.156 Atatürk University, Faculty of Science & Arts, Department of Chemistry, 25240 Erzurum, Turkey 2Atatürk University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 1. Gül Hİ, Gül M, Hanninen O. Cytotoxic activities of some mono and bis 25240 Erzurum, Turkey Mannich bases derived from acetophenone in brine shrimp bioassay, Mono Mannich base 1-(p-methoxyphenyl)-3-phenethylamino-1- Arzneimittel Forschung, 52, 840-3, 2002. propanone hydrochloride and semicyclic mono Mannich base 3-(p- methoxybenzoyl)-4-(p-methoxyphenyl)-1-phenethyl-4-piperidinol have been synthesized according to the literature process [1]. P 026 Ref: 0046 The structural elucidations of the compound was accomplished using extensive 1D-NMR (1H, 13C, NOE-diff, DEPT) and 2D-NMR CRYSTAL STRUCTURE OF 3-(p-CHLOROBENZOYL)-4-(p- (COSY, NOESY, HMQC and HMBC) spectroscopic techniques. Pro- CHLOROPHENYL)-1-PHENETHYL-4-PIPERIDINOL ton and carbon-13 spectra were recorded at 27 °C in DMSO on a 1Ertan ŞAHİN, 1Cavit KAZAZ, 1Ebru METE, 2H. İnci GÜL Varian mercury-plus (Palo Alto, USA) instrument at a frequency of 1Atatürk University, Faculty of Science & Arts, Department of Chemistry, 25240 Erzurum, 400 and 100 MHz, respectively, using a 5 mm ASW PFG probe. Turkey 2Atatürk University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 1. Gül M, Gül Hİ, Das U, Hanninen O, Arzneimittel Forschung, 55(6), 332- 25240 Erzurum, Turkey 7, 2005. NMR Study: The characterisation of compound 1, 3-(p-chlorobenzoyl)-4-(p-chlorophenyl)-1-phenethyl-4-piperidinol, have

Kervansaray Convention Center & Hotel, LARA, ANTALYA / TÜRKİYE 59 International Symposium on Drug Research and Development

P 028 Ref: 0048 P 029 Ref: 0049 INVESTIGATION OF CASEIN KINASE 2 (CK2) ENZYME CYTOTOXICITIES OF N,N’-BIS(3-DIMETILAMINO-1-ARYL- AND ITS BINDING PROPERTIES WITH P53 PROTEIN, PROPYLIDENE)HYDRAZINE DIHYDROCHLORIDES

PRESENTATIONS POLYLYSINE AND HEPARIN BY MALDI-MS 1Kaan KÜÇÜKOĞLU, 2Mustafa GÜL, 1H. İnci GÜL, 3Osmo HANNINEN, 3 1Ömür ÇELİKBIÇAK, 1Cansel TAŞAĞIR, 2Wayne E. CRISS, 1Bekir SALİH Mustafa ATALAY 1 1Hacettepe University, Faculty of Science, Department of Chemistry, 06532 Beytepe- Atatürk University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Ankara, Turkey Erzurum, Turkey 2 POSTER 2Hacettepe Unıversity, Faculty of Medicine, Oncology Institute, Department of Atatürk University, Faculty of Medicine, Department of Physiology, Erzurum, Turkey Biochemıstry, 06100 Sıhhiye-Ankara, Turkey 3University of Kuopio, Department of Physiology, Finland Matrix-assisted Laser Desorption/Ionization, along with electro- Synthesis of N,N’-bis (3-dimethylamino-1-aryl-propylidene) hy- spray ionization, is now among the most important ionization meth- drazine dihydrochlorides were carried out according to the literature ods for nonvolatile, high molecular weight compounds, in particular procedure [1] by using acetophenone or substituted acetophenones as peptides, proteins, oligonucleotides, oligosaccharides and synthetic the ketone component of the reactions while dimethylamine hydro- polymers. MALDI has also been successfully used for the investiga- chloride was the amine component of the reactions. Substituents were tion of fullerenes, fullerene derivatives, and synthetically prepared methyl for 2, methoxy for 3, hydroxy for 4 and chloro for 5 at the para dendrimers. Under certain conditions, it has even been shown that position of the phenyl ring while compound 1 was nonsubstituted. MALDI can be used for the study of weakly bound noncovalent Chemical structures of the compounds were confirmed by 1H complexes [1]. Protein kinase CK2 (Casein Kinase 2) is a ubiquitous NMR. Cytotoxicity of the compounds was determined against Jur- serine/threonine protein kinase which is composed of two regulatory kat cells which is human T lymphocytes cells. Reference compounds β- and two catalytic α- or α’- subunits. Although its precise function were 5-fluorouracil and melphalane. in the cell is still unclear there is ample evidence that CK2 plays an All compounds have shown more powerful activity than both important role in the regulation of cell proliferation [2]. The activity references, cytotoxicity values of the compounds were in the range of CK2 is elevated in tissues with a high proliferation rate, such as of 9.75-13.27µM. The most effective compound was 4 in five com- tumors and embryonic tissue [2]. The p53 tumor suppressor protein pounds studied. is a potent transcription factor which is activated in response to a variety of DNA-damaging agents. Activation of p53 leads to cell growth 1. Gül Hİ, Das U, Pandit B, Li PK, Evaluation of the cytotoxicity of some arrest or the induction of apoptosis, thereby blocking the survival of mono-mannich bases and their corresponding azine derivatives against genetically damaged cells [2]. In the absence of functional p53 genes, androgen-independent prostate cancer cells, Arzneimittel Forschung, 56(12), 850-4, 2006. the cycle is not arrested and the apoptosis signal is not delivered, so a cell with abnormal DNA is allowed to replicate, thus increasing the chance of cancer developing [3]. P53 is phosphorylated at multiple sites in vivo and by several different protein kinases in vitro includ- P 030 Ref: 0050 ing the protein kinase CK2. In addition, it was shown previously by SYNTHESIS OF 1-[3-(PIPERIDINOMETHYL)-4- some researchers that mutation of p53 at the CK2 phosphorylation HYDROXYPHENYL]-3-ARYL-2-PROPEN-1-ONES AND site abolishes the growth suppressor activity of p53 [2]. Because of the importance of the binding properties of p53 to the CK2 enzyme, EVALUATION OF THEIR ANTICONVULSANT ACTIVITIES MALDI-MS analysis of CK2 and p53 proteins were performed and 1H. İnci GÜL, 1K. Özden YERDELEN, 2Ünsal ÇALIŞ interactions of p53 protein with α and β subunits of CK2 enzyme 1Atatürk University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, were investigated. Since CK2 was first discovered, many studies have 25240 Erzurum, Turkey showed us that the enzymatic activity of CK2 was stimulated by 2Hacettepe University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, polyamines and inhibited by heparin in vitro [2]. According to these 06100 Sıhhiye-Ankara, Turkey data, existence of interactions can be inferred between these mol- In this study, Mannich bases with piperidine, 1-[3-(piperidinome- ecules and CK2 and/or between these molecules and CK2 substrates, thyl)-4-hydroxyphenyl]-3-aryl-2-propen-1-one, B1-B5, were synthe- which probably improve the susceptibility of substrates to be phos- sized starting from the chalcones, 1,3-diaryl-2-propen-1-one, A1-A5. phorylated or not. In order to get information about the mechanism Chemical structures of the compounds have been confirmed by of noncovalent interactions between these biomolecules, MALDI- 1H-NMR, 13C-NMR, IR, and UV spectra and elemental analysis. MS was used by selecting suitable matrix, sample preparation and Anticonvulsant activities of the compounds were evaluated by MES, clean up procedures at appropriate pH and solvent conditions. scMet tests. Neurotoxicities of the compounds were also evaluated by rotorod test [1, 2]. 1. Zenobi R, Knochenmuss R. Ion Formation in MALDI Mass Spectrom- None of the compounds showed neurotoxicity at the screening of etry, Mass Spectrometry Reviews, 17, 337–366, 1998. anticonvulsant activity. Compounds B1-B5 at MES test, compounds 2. Schuster N, Götz C, Faust M, Schneider E, Prowald A, Jungbluth A, Mon- B1-B3 at scMet test have shown anticonvulsant activity at different tenarh M. Wild-Type p53 Inhibits Protein Kinase CK2 Activity, Journal dose levels (30-300 mg/kg) and time periods (1/2 h, 4 h). of Cellular Biochemistry, 81, 72-183, 2001. To conclude, of the compounds B4, B5 against grand-mal epilep- 3. Elliott, WH, Elliott, DC. Biochemistry and molecular biology, 3rd ed., Oxford University Press Inc., New York, 2001, p. 533-534. sia, B1, B2 and B3 against both types of epilepsia, can be choosen as candidate compounds to develop new anticonvulsant compounds for further studies.

1. Gül Hİ, Çalış Ü, Vepsalainen J. Synthesis of some mono-Mannich bases and corresponding azine derivatives and evaluation of their anticonvulsant activity, Arzneimittel Forschung, 54, 359-64 2004. 2. Gül Hİ, Çalış Ü, Vepsalainen J. Synthesis and evaluation of anticonvulsant activities of some bis Mannich bases and corresponding piperidinols, Arzneimittel Forschung, 52, 863-9 2002.

60 May 17-20, 2007 “From Chemistry to Medicine” DRD 2007

P 031 Ref: 0051 mentagrophytes (Hak-9), Microsporum canis (Hak-4)] and 13 fungi species pathogenic in plants [Sclerotinia sclerotiorum, Sclerotinia minor, DEVELOPMENT AND OPTIMIZATION OF A SURFACE Alternaria alternate (AA-1121), Aspergillus flavus (Hak-23), Aspergil- AIDED ENZYMATIC DIGESTION SYSTEM TO USE IN lus variecolor (IO-Balik), Fusarium acuminatum, Fusarium oxysporum

PROTEOMICS STUDIES (ED-10), Fusarium solani (ED-1S), Fusarium tabacinum (ED-1T), PRESENTATIONS 1Basri GÜLBAKAN, 1Aslı ÖZTÜRK-ÇAL, 2Talat YALÇIN, 1Bekir SALİH Moniliania fructicola (FS-M), Penicillium spp. (P-TY), Rhizopus spp. 1Hacettepe Unıversity, Faculty of Science, Department of Chemistry, 06532 Beytepe- (R-27), Rhizoctonia solani (EB-ML)] at the concetration range of 2-64 Ankara, Turkey µg/ml using amphotericin–B as the reference compound (1).

2İzmir Institute of Technology, Faculty of Science, Department of Chemistry, 35430 İzmir, Of the compounds, A1 against plant pathogens Sclerotinia scle- POSTER Turkey rotioruma and Rhizoctonia solani, while C5 against plant pathogen Proteomics is the study of all methods covering isolation, sepa- Fusarium oxysporum, have shown 2-4 times more powerful antifun- ration, identification, characterization of all proteins in living or- gal activity compared with amphotericin-B. Of the compounds syn- ganisms and finding their functional roles and expression of the thesized, A1 and C5, against plant pathogenic fungi can be choosen sequences of proteins in all tissues. The anomalies in the synthe- as candidate compounds for further studies to develop new antifun- sis and function of the proteins are interrelated with several lethal gal compounds. diseases. Understanding the cause and the therapy of a disease is related to determining and understanding the proteins that are in- 1. Gül Hİ, Şahin F, Gül M, Öztürk S, Yerdelen KO. Evaluation of antimi- volved. After completion of the Human Genome Project, proteom- crobial activities of several mannich bases and their derivatives, 1, Arch. Pharm., 338(7), 335-8, 2005. ics gained more importance. However, since proteins are more complex than that of genes, secondary cleavage reactions namely, enzymatic trypsin reactions are employed to obtain smaller peptide fragments and peptides are then analyzed to obtain structural in- P 033 Ref: 0054 formation about proteins. Thus the success of proteomics is closely SYNTHESIS OF 1-[3-(PIPERIDINOMETHYL)-4- related with the success of enzymatic trypsin cleavage. Isolation of HYDROXYPHENYL]-3-ARYL-2-PROPEN-1-ONES AND proteins from their natural environment, which is a very complex matrix, leads to protein loss and in many cases necessitates the ad- EVALUATION OF THEIR CYTOTOXIC ACTIVITIES AGAINST dition of contaminating agents like SDS, CHAPS and sucrose. This HUMAN T LYMPHOCYTES (JURKAT) CELLS AND RAT also limits the information that is obtained from proteomics studies. SKELETAL MUSCLE DERİVED MYOBLAST CELLS (L6) The presented study was carried out in order to eliminate the intrin- 1H. İnci GÜL, 2Mustafa GÜL, 1K. Özden YERDELEN, 3Osmo HANNINEN, sic difficulties of conventional in-solution and in-gel trypsin diges- 3Mustafa ATALAY tion protocols and to develop a new and effective digestion method. 1Atatürk University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Proteins samples having different molecular weights and different 25240 Erzurum, Turkey structural complexity was preconcentrated and purified by using 2Atatürk University, Faculty of Medicine, Department of Physiology, 25240 Erzurum, Turkey conventional reversed phase column packing materials that are C18 3University of Kuopio, Faculty of Medicine, Department of Physiology, 1627 Kuopio, bonded silica, polystyrene-divinylbenzene and modified derivatives Finland thereof and digested while bound to surface. The resulting peptides In this study, Mannich bases with piperidine, 1-[3-(piperidinome- were then analyzed by MALDI mass spectrometry. The effect of dif- thyl)-4-hydroxyphenyl]-3-aryl-2-propen-1-one, B1-B5 were synthe- ferent MALDI matrices, sample preparation methods, effect of func- sized starting from the chalcones, 1,3-diaryl-2-propen-1-one, A1- tional group loading onto poly (styrene-divinylbenzene) microbeads A5. Chemical structures of the compounds have been confirmed by was studied. The results were compared by conventional methods 1H-NMR, 13C-NMR, IR, and UV spectra and elemental analyses. using trypsin digestion to test the efficiency of the method. Cytotoxic activities of the compounds have been tested against rat skeletal muscle derived myoblast cells (L6) and transformed hu- 1. Gevaert K, Vandekerckhove J. Protein identification methods in pro- man T lymphocytes (Jurkat). Melphalan and 5-fluorouracil were also teomics, Electrophoresis, 21, 1145-1154, 2001. tested as reference drugs [1]. All compounds have shown 1.28-5.40 times more powerful cytotoxicity than 5-FU, and the compounds A3, B1, B2, B3 have shown P 032 Ref: 0052 1.03-2.76 times more powerful cytotoxicity than melphalan against L6 cells, respectively. Preparation of Mannich bases with piperidine from SYNTHESIS OF 1-[3-(DIBENZYLAMINOMETHYL)-4- the chalcones increased the cytotoxicity 1.55, 1.54, 1.33, 1.38 times at HYDROXYPHENYL]-3-ARYL-2-PROPEN-1-ONES AND the compounds B1, B2, B4, B5 respectively, compared with their corre- EVALUATION OF THEIR ANTIFUNGAL ACTIVITIES sponding chalcones. While all compounds synthesized had 3.20-9.43 1K. Özden YERDELEN, 1H. İnci GÜL, 2Fikrettin ŞAHİN times more powerful cytotoxicity than 5-FU against Jurkat cells, except B1, all other compounds showed 1.11-2.38 times more powerful 1Atatürk University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 25240 Erzurum, Turkey cytotoxicity than melphalan. Preparation of Mannich bases from the 2Yeditepe University, Faculty of Engineering and Architecture, Department of Genetics chalcones increased the cytotoxicity 1.42, 1.70, 1.43 times respectively and Bioengineering, 34755-Kayışdağı, İstanbul, Turkey at the compounds B2, B4 and B5 compared with the corresponding In this study, Mannich bases with dibenzylamine 1-[3-(diben- chalcones against Jurkat cells.The compounds synthesized have been zylaminomethyl)-4-hydroxyphenyl]-3-aryl-2-propen-1-one, (C1- found more selective against Jurkat cells compared with L6 cells. C5) were synthesized and the chemical structures of the compounds Of the compounds synthesized, B2, B4 and B5 can be choosen as have been confirmed by 1H-NMR, 13C-NMR, IR, and UV spectra candidate compounds for further cytotoxicity studies to develop new and elemental analysis. cytotoxic compounds. Antifungal activities of the compounds have been tested and compared with their precursor chalcones (A1-A5) against 3 fungi species 1. Gül M, Gül Hİ, Das U, Hanninen O, Arzneimittel Forschung, 55(6), 332- pathogenic in humans [Trichophyton rubrum (Hak-8), Trichophyton 7, 2005.

Kervansaray Convention Center & Hotel, LARA, ANTALYA / TÜRKİYE 61 International Symposium on Drug Research and Development

P 034 Ref: 0056 lar partition coefficient, Po, obtained in octanol-water systems by Countercurrent Chromatography (CCC) [4], and the correlated data DETERMINATION OF PHYSICOCHEMICAL fits perfectly. PROPERTIES OF SEVERAL SULFONAMIDES BY LIQUID

PRESENTATIONS CHROMATOGRAPHY IN ACETONITRILE-WATER BINARY 1. Mengelers MJB, Hougee PE, Janssen LHM, Van Miert AS. Structure-ac- MIXTURES tivity relationships between antibacterial activities and physicochemical properties of sulfonamides, J. Vet. Pharmacol. Therap., 20, 276-283, 1Nurullah ŞANLI, 1Güleren ALSANCAK, 2Adil DENİZLİ 1997. 1Süleyman Demirel University, Faculty of Science and Literature, Department of 2. Botsoglou NA, Fletouris DJ, Simeonidou EJ, Psomas IE. Retention be- POSTER Chemistry, Isparta, Turkey havior of multiple sulfonamides in various liquid chromatographic sys- 2Hacettepe University, Faculty of Science, Department of Chemistry, Ankara, Turkey tems, Chromatographia, 46, 9/10, 477-782, 1997. Sulfonamides are antibacterial compounds commonly used to 3. Qiang Z, Adams C. Potentiometric determination of acid dissociation constants (pKa) for human and veterinary antibiotics, Water Research, prevent and to treat diseases in medical and veterinary practice. A 38, 2874-2890, 2004. sulfonamide contains one basic amino group and one acidic amide 4. Carda-Broch S, Berthord A. Countercurrent chromatography for the group which correspond to pKa1 and pKa2 respectively. The degree measurement of the hydrophobicity of sulphonamide amphoteric com- of ionization of sulfonamides was strongly related with the in vitro pounds, Chromatographia, 59, 79-87, 2004. bacteriostatic activity [1]. The use of HPLC retention parameters to determine pKa values has been widely applied [1-2]. Literature studies of the chromato- P 035 Ref: 0057 graphic determination of pKa values of sulfonamides and the effect of the organic modifier content on the pKa values of these com- CONTROLLED RELEASE OF NAPROXEN FROM POLY pounds are scarce, but the investigation of Mengelers et al. should (VINYL ALCOHOL) / SODIUM ALGINATE MICROSPHERES be mentioned [1]. 1Oya ŞANLI, 2Ebru KONDOLOT-SOLAK In this study, the dissociation constants of related series of sulfon- 1 amides (sulfodiazine, sulfothiazole, sulfomerazine, sulfomethazine, Gazi University, Arts and Sciences Faculty, Department of Chemistry, 06500 Ankara, Turkey 2Gazi University, Atatürk Vocational College, Department of Chemistry, Ankara, Turkey sulfomonomethoxine, sulfodoxine, sulfomethoxazole) were determined by LC methodology and the effects of the ACN percentage In this study microspheres of poly(vinyl alcohol)/sodium algi- on the pKa values were investigated. The prodigy C-18 was used as nate (PVA/Na-Alg) and sodium alginate (Na-Alg) were prepared stationary phase. On changing the mobile phase of the system, the to encapsulate naproxen sodium drug. Microspheres were prepared column was thoroughly equilibrated with studied new mobile phase. by liquid curing method by crosslinking with glutaraldehyde, then The electrode system was calibrated with potassium acid phthalate characterized by fourier transform infrared (FTIR) spectroscopy, in organic mixture of the same composition as the mobile phase ac- differential scanning calorimetry (DSC) and scanning electron mi- cording to IUPAC rules. The pH of the mobile phase was measured croscopy (SEM). after the addition of the ACN to properly investigate the effect of Microspheres were also characterized by measuring the particle pH on the retention of ionizable compounds. The effect of pH on diameter, equilibrium swelling values and determining release pro- sulfonamides retention was investigated in the range of pH 1.7-9.7. files. Equilibrium swelling experiments indicated that the swelling The retention time was plotted against pH value of the mobile phase of the spheres decreased with an increase in crosslinking time and to calculate pKa2 values. The sigmoidal behaviors of studied sulfona- concentration however diameter of the spheres was not affected con- mides are shown in Figure 1. siderably. The release studies were carried out at three pH values 1.2, 6.8 and 7.4 respectively. The release of diclofenac from the micropheres increased as the drug/polymer ratio decreased. Optimum condition for the preparation and release of the spheres were determined as PVA/Na-Alg: 1/2, drug/polymer: 1/4 and pH: 7.8. The release of these conditions was found as 80.3 % at the end of 6th hour.

P 036 Ref: 0058 SPECTROPHOTOMETRIC ANALYSIS OF CABERGOLINE IN PHARMACEUTICAL PREPARATIONS 1Nursabah E. BAŞÇI, 2Demet SALMAN 1Hacettepe University, Faculty of Pharmacy, Department of Analytical Chemistry, Ankara, Turkey 2Turkish Republic Instutition of Social Security, Ankara, Turkey Plots of retention times of sulphonamides against Figure 1. Cabergoline is a synthetic dopamine agonist having high affinity mobile phase pH (4.5-9.0) at 30% (v/v) ACN. Symbols indicate: ■ sulfodiazine, ♦ sulfothiazole, ▲ +sulfomerazine, х sulfomethazine, ж to D2 receptors and used for early and advanced Parkinson’s patients

sulfomonomethoxine, • sulfodoxine, sulfomethoxazole). and hyperprolactinemy disorders [1]. Chromatographic and radio- immunoassay methods have been reported for quantification of ca-

Their pKa values in ACN – water mixture (50% v/v) were also bergoline in body fluids [2-5], but there was not any spectrophoto- potentiometricaly determined [3] and compared with the results ob- metric analysis of cabergoline in pharmaceutical preparations in the tained by LC methodology. literature. In this study, simple, fast, reliable and validated UV-VIS nd The retention data of sulfonamides obtained by Reversed Phase and 2 derivative spectroscopy methods were developed for deter- Liquid Chromatography (RPLC) were correlated with the molecu- mination of cabergoline in pharmaceutical preparations.

62 May 17-20, 2007 “From Chemistry to Medicine” DRD 2007

Determination of cabergoline was performed by UV-VIS spectro- Ethanesulfonic acid hydrazide, C2H5SO2NHNH2, (esh) ; 5-methyl- photometry at 280 nm wavelength and by 2nd derivative spectropho- 2-hydroxyacetophenoneethanesulfonylhydrazone (5mafesh) and its tometry at a range of 227-232 nm. HPLC (Shimadzu SCL-10AVP) Ni(II) ,Co(II) complexes containing sulfonamide and hydrazine connected to an electrochemical detector (Decade) was used in com- fragments were synthesized and their structures were determined parison analysis. Cabergoline substance was donated by Pharmacia. by using elemental analysis, NMR, FT-IR, LC-MS, magnetic and PRESENTATIONS Dostinex® (0.5 mg Cabergoline, Pfizer) and Cabaser® (1, 2 and 4 mg conductivity studies. Their antimicrobial activities were investigated Cabergoline, Pfizer) tablets were used for pharmaceutical applica- against to Escherichia coli ATCC 11230, Bacillus subtilis RSKK 244, tions. Developed methods were validated according to the regula- Bacillus cereus RSKK 863, Bacillus magaterium RSKK 5117, Salmo- tions and guidelines of ICH, EMEA and FDA. Validation results nella enteritidis ATCC 13076, Staphylococcus aureus ATCC 25923 by POSTER were statistically evaluated using related methods at a significant using MIC’s method. MIC’s was defined as the lowest concentrations level of 95%. of compounds which inhibit the growth of microorganisms. Developed UV-VIS and 2nd derivative spectrophotometry meth- The antimicrobial results evidently showed that the sulfonamide ods were linear over the range of 1-125 µg mL-1. The limit of detec- derivatives possessed a broad spectrum of activity against the tested tion for the methods was 0.5 µg mL-1 (RSD = 0.75%, Bias = 0.30) bacteria (MIC’s values g/mL). All compounds exhibited the most ac- and the limit of quantification was 1.0 µg mL-1 (RSD = 0.67%, Bias tivity against to Staphylococcus aureus between 60-672µg. The pres- = 0.33). The highest relative error and relative standard deviation ence of the NH group in the sulfonamides contributes positively to in inter-day and intra-day study for UV-VIS spectrophotometry the increase of the activity of compounds against bacteria. In addi- were 1.10% and 0.63%, respectively. The highest relative error and tion the negative charges on the other donor atoms show a tendency relative standard deviation in inter-day and intra-day study for 2nd to increase the activity [4]. derivative spectrophotometry were 1.10% and 0.70%, respectively. The methods were applied to the analysis of cabergoline in pharma- 1. Albert A. Selective Toxicity, Chapman and Hall, London, New York, ceutical preparations and there was no statistically significant differ- 1985. ence when the results were compared with the results of comparison 2. Topiol S, Sabio M, Erhardt PW, J. Chem. Soc.Perkin Trans., II, 437, 1988. 3. Dodoff NI, Özdemir Ü, Karacan N et al., Z. Naturforsch., 54 b , 1553, method (HPLC/ECD). In conclusion, the developed UV-VIS and 2nd 1999. derivative spectrophotometry methods were accurate, sensitive, pre- 4. Zanatta N, Alues SH, Coelho HS et al., Bioorganic & Medicinal Chemis- cise and repeatable and can be applied to the analysis of cabergoline try, 15, 1947, 2007. in pharmaceutical preparations as direct, fast and simple methods.

1. Gottwald MD, Bainbridge JL, Dowling GA, Aminoff MJ, Alldredge BK. New pharmacotherapy for parkinson’s disease, Annals of Pharmacothera- P 038 Ref: 0060 phy, 31(10), 1205-17, 1997. 2. Persiani S, Pianezzola E., Broutin F, Fonte G, Benedetti MS. Radioim- DETERMINATION OF DISSOCIATION CONSTANTS OF munoassay for the synthetic ergoline derivative cabergoline in biological SEVERAL SULFONAMIDES BY POTENTIOMETRY IN fluids, Journal of Immunoassay, 13(3), 457-76, 1992. METHANOL, 2-PROPANOL, TETRAHYDROFURAN-WATER 3. Pianezzola E, Bellotti V, Croix RL, Benedetti MS. Determination of ca- BINARY MIXTURES bergoline in plasma and urine by high-performance liquid chromatography with electrochemical detection, Journal of Chromatography, 574, 1Senem ŞANLI, 1Ebru ÇUBUK-DEMİRALAY, 2Hale SEÇİLMİŞ, 170-174, 1992. 3Jose Luis BELTRAN 4. Allievi C, Dostert P. Quantitative determination of cabergoline in human 1Süleyman Demirel University, Science & Literature Faculty, Department of Chemistry, plasma using liquid chromatography combined with tandem mass spec- 32260 Isparta, Turkey trometry, Rapid Communications in Mass Spectrometry, 12, 33-39, 1998. 2Süleyman Demirel University, Research Centre, 32260 Isparta, Turkey 5. Igarashi K, Hotta K, Kasuya F, Abe K, Sakoda S. Determination of caber- 3Barcelona University, Department of Analytical Chemistry, 08028 Barcelona, Spain. goline and L-dopa in human plasma using liquid chromatography-tan- The pK value is a main item in the biophysical characterization dem mass spectrometry, Journal of Chromatography B, 792, 55-61, 2003. a of a drug and may be helpful in predicting the behavior of a drug under in vivo conditions. Sulfonamides (SAs) are typical amphoteric compounds and dissociation pathways of sulfonamides are given in P 037 Ref: 0059 Figure 1. Ka1 and Ka2 are the dissociation constants of the aromatic INVESTIGATION OF ANTIMICROBIAL ACTIVITES OF amine and sulfonic groups respectively. NEW SULFONYLHYDRAZONE DERİVATİVES ON SOME MICROORGANISMS Ümmühan ÖZDEMİR-ÖZMEN, Fatma HAMURCU Gazi University, Arts and Sciences Faculty, Department of Chemistry, 06500 Ankara, Turkey The chemistry of hydrazones has been intensively investigated in recent years, owing to their coordinating capability, pharmacological activity, antibacterial and antifungal properties, Sulfonamide drugs are widely used chemotherapeutic agents with large spectrum of activity [1]. Methane sulfon amide residue has appeared as a suitable pharmacophoric equivalent to replace functional groups in drug design . Having hydrophilic character, like the sulfonyl group is con- Fig 1. Scheme of dissociation equilibrium of sulfonamides. sidered as a suitable pharmacophoric equivalent for replacing functional groups in drug design [2]. In previous paper, we reported the Literature studies of the potentiometric determination of pKa val- antibacterial and cytotoxic effect of methanesulfonic acid hydrazide ues of sulfonamides are scarce, but the investigation of Qiang and (msh) [3]. Adams should be mentioned [1]. In addition only a few pKa values

Kervansaray Convention Center & Hotel, LARA, ANTALYA / TÜRKİYE 63 International Symposium on Drug Research and Development

of sulfonamides in organic solvent-water binary mixtures can be ob- A simple, rapid, sensitive and selective method for the analysis tained from the literature [2]. of indapamide in human plasma, utilizing ultra performance liquid

Among the pKa determination techniques, the potentiometric chromatography (UPLC), has been developed and validated to fulfill titration is most economical of time and if due care is taken; this FDA guidelines for bioanalytical methods [3]. The analyte and the

PRESENTATIONS technique is accurate and has good reproducibility. This paper inves- internal standard, sulfamethazine, were isolated from plasma sam-

tigated the potential of potentiometric method to determine the pKa ples by liquid-liquid extraction with diethyl ether. The assay exhib- values of sulfonamides that show poor solubility. In this study the ited a linear dynamic range of 1 to 100 ngmL-1 for indapamide in

effect of the organic modifier type and content on the pKa values of human plasma. -1 POSTER these compounds were also investigated. These solvent mixtures can The limit of quantification (LOQ) was 1 ngmL with a relative dissolve drugs more effectively than water and in many cases they standard deviation of less than 12.2 %. Inter and intra-day preci- are more suitable solvent for the determination of the dissociation sion (CV %) and accuracy (%) for quality control samples (3, 50, 80 constants. Methanol and 2-propanol are closest to water in structure ngmL-1) ranged from 0.55 % to 8.48 % and from 94.62 % to 107.56 and properties. THF is one of the most widely used dipolar aprotic % respectively. Furthermore, this method was successfully applied to solvent and is much better differentiating solvent than water. the pharmacokinetic and bioequivalence study of indapamide tab-

The pKa1 and pKa2 values of sulfonamide were determined by up- lets in healthy male volunteers within 96 h period. ward titration using 0,025 M KOH. The typical titration curve for sulfametaxazole is shown in Fig 2. In this study PKPOT program 1. Caruso FS, Szabadi RR, Vukovich RA. Am. Heart, 106, 212-220, 1983. 2. Johnston MM, Rosenberg MJ, Yeung AK, Grebow PE. J. Pharm. Sci., 69, was used to correct the effect of ionic strength on pKa determination [3]. 1158-1160, 1980. 3. FDA Guidance for Industry, Bioanalytical Method Validation, May 2001.

P 040 Ref: 0062 THE EFFECT OF TAUROLIDINE ON THICKNESS OF SCAR TISSUE IN RABBIT MODEL 1Ali HAYAT, 2Füsun TEMAMOĞULLARI, 3Füsun BABA 1University of Harran, Faculty of Veterinary Medicine, Department of Surgery, Şanlıurfa, Turkey 2University of Harran, Faculty of Veterinary Medicine, Department of Pharmacology and Toxicology, Şanlıurfa, Turkey 3University of Harran, Faculty of Medicine, Department of Pathology, Şanlıurfa, Turkey Clinicans have used numerous strategies to combat wound infections, including topical and systemic administration of antibiotics and various antiseptic agents [1]. The antimicrobial properties of Fig 2. Potentiometric titration curve of sulfomethaxazole in MeOH- H O, 30% (v/v). taurolidine have been ascribed to the biological active methylol tau- 2 rinamide which reacts with cell wall constituents of microbial patho- 1. Qiang Z, Adams C. Potentiometric determination of acid dissociation gens via methylene iminium ions preventing bacterial adhesion to biological surfaces. Taurolidine has a short half-life and is metabo- constants (pKa) for human and veterinary antibiotics, Water Research, 38, 2874-2890, 2004. lised to taurine, carbon dioxide and water. It has been shown to be 2. Mengelers MJB, Hougee PE. Janssen LHM, Van Miert AS. Structure-ac- non-toxic to human and animals [4]. Povidone iodine is commonly tivity relationships between antibacterial activities and physicochemi- used in clinical practice but dermal hypersensitivity is associated cal properties of sulfonamides, J. Vet. Pharmacol. Therap., 20, 276-283, with the use of povidone iodine in humans and small animals [2]. 1997. In this study, we have investigated efficacy of 2% taurolidine solu- 3. Barbosa J, Barron D, Beltran JL, Sanz-Nebot V. PKPOT, a program for the potentiometric study of ionic equilibria in aqueous and non-aqueous tion on healing wound standing on the clinical and histopathologic media, Anal. Chim. Acta, 317, 75-81, 1995. parameters comparing with 10% povidine iodine solution and 0.9% sodium chloride. Six male and six female rabbits (mean weight: 2500±200) were P 039 Ref: 0061 taken for the study. All rabbits were anesthetized with intramuscu- lar administration of xylazine hydrochloride 10 mg/kg (Rompun, IMPROVED ULTRA-PERFORMANCE LIQUID Bayer) and ketamin hydrochloride 50 mg/kg (Ketanes, Alke) Right CHROMATOGRAPHIC DETERMINATION OF INDAPAMIDE and left costal regions of rabbits were clipped and the skin was pre- IN HUMAN PLASMA pared for aseptic surgery. Then full-thickness skin wounds (3 cm in diameter) as two cranial and one caudally located were created on Zeliha ATEŞ, Sami EREN, Selma ÖZİLHAN, Tuncel ÖZDEN each animal using a template prepared from x-ray film. Daily, 2% Novagenix Bioanalytical R&D Centre, Ankara, Turkey taurolidine, 10% povidine iodine solution and 0.9% sodium chloride Indapamide, 3-(aminosulfonyl)-4-chloro-N-(2,3-dihydro-2-me- were applied on wounds. Macroscopically wounds were examined thyl-1H-indol-1-yl)-benzamide, is an oral antihypertensive and from point of the exudation during the postoperative days. Biopsy diuretic agent [1]. Indapamide inhibits carbonic anhydrase en- specimens which were collected on the 4th, 8th, 12th and 16th PODs. zyme, which reduces the vascular response to noradrenalin and an- Specimens were evaluated according to several histopathologic pa- giotensin II by inhibiting the transportation of Ca²+ into vascular rameters, such as the thickness of scar tissue. smooth muscle. It shows diuretic effect by acting at the first parts of Macroscopically, the wounds treated with 10% povidine iodine so- distal tubules [2]. lution and 0.9% sodium chloride appeared to have marked fibrinous

64 May 17-20, 2007 “From Chemistry to Medicine” DRD 2007

exudate and crust formation which caused adherence to the gauze. P 042 Ref: 0064 On the other hand. 2% taurolidine gauze applied wounds showed lesser degree of exudate and lesser adherence. Daily applied 2% tau- COMPARISON OF THE ROYAL JELLY AND POVIDONE rolidine reduced the thickness of scar tissue when compared to other IODINE ON WOUND HEALING IN RABBITS two solution. The degree of attachment between dressing material 1Füsun TEMAMOĞULLARI, 2Ali HAYAT, 3Füsun BABA PRESENTATIONS and the wound surface is important [3]. The subjective examination 1University of Harran, Faculty of Veterinary Medicine, Department of Pharmacology and showed that the 2% taurolidine conformed well to the wound surface Toxicology, Şanlıurfa, Turkey and it was readily separeted from underlying wound. As a result, 2% 2University of Harran, Faculty of Veterinary Medicine, Department of Surgery, Şanlıurfa, taurolidine application to full thickness skin wounds in rabbits posi- Turkey POSTER tively effected wound healing but the mechanism underlies this fact 3University of Harran, Faculty of Medicine, Department of Pathology, Şanlıurfa, Turkey still needs furter investigations. Royal jelly (RJ) has been used worldwide for many years as medical products, health foods and cosmetics [1]. A number of biological 1. Burks, RI. Povidone-iodine solution in wound treatment, Phys. Ther., 78, and immuno-regulatory actions attributed to RJ have been report- 212-218, 1998. ed. In this study, we have investigated the efficacy of RJ on healing 2. Farstvedt, E, Stashak, TS., Othic, A. Update on Topical Wound Medica- wound standing on the clinically and histopathologically comparing tions, Clin. Tech. Equine Pract., 3, 164-172, 2004. with 10 % povidone iodine and 0,9% sodium chloride. 3. Kılıç, S, Timurkan, N, Ünsaldı, S, Günay, C, İstek, Ö, Yılmaz, B. Compari- son of the Effects of Some Wound Healing Materials on Full Thickness Six male and six female rabbits weighing about 2500 ± 200 g were Skin Wounds in Rabbits, Turk. J. Vet. Anim. Sci., 26, 263-272, 2002. anesthetized with i.m. administration of 10mg/kg xylazine hydro- 4. Koldehoff, M, Zakrzewski, JL. Taurolidine is effective in the treatment chloride (Rompun, Bayer) and 50mg/kg ketamine hydrochloride of central venous catheter-related bloodstream infections in cancer pa- (Ketanes, Albe). On dorsal aspect of each animal, two cranially and tients, International Journal of Antimicrobial Agents, 24, 491-495, 2004. one caudally located full-thickness skin wounds in 3,14 cm diameter were created using a template prepared from an X-ray film. Fol- lowing incision different wounds of each animal were treated with P 041 Ref: 0063 RJ (83 mg/ml Royal Jelly-Arıjel Co.,Ltd. ), 10% povidon iodine and 0,9% sodium chloride as the control respectively. Then the wounds THE QUANTITATIVE DETERMINATION OF FEXOFENADINE were closed with sterile gauze and fixed with circular adhesive bands. IN HUMAN PLASMA BY LC/MSD SYSTEM Wounds were examinated macroscopically and by exudation. The Müberra ŞEN, Evren İŞLEYEN, Selma ÖZİLHAN, Suna TOPTAN, beginning of the wound contraction as the indicator of the begin- Tuncel ÖZDEN ning of healing, granulation of the tissue and the first day of epithe- Novagenix Bioanalytical R&D Centre, Ankara, Turkey lization were noted regularly. SPSS 11.0 for Windows was used for statistical analyses. Fexofenadine hydrochloride, (±)-4-[1-hydroxy-4-[4-(hydroxy- Whole control wound surface were covered by a thin gelatinous diphenylmethyl)-1-piperidinyl]-butyl]-α, α-dimethyl benzeneacetic exudate (POD 4). After this exudate was removed, an ongoing acid hydrochloride has an empirical formula, C H NO .HCl with a 32 39 4 healthy granulation and epithelization tissues were determined. RJ molecular weight of 538.1 which is the active metabolite of terfena- gauze-applied wounds showed strong adherence and a lesser degree dine and is a second-generation histamine H1-receptor antagonist in exudate. Therefore, they required greater tearing force for removal piperidine-class drugs. Fexofenadine is a H1-receptor antagonist that of the dressing. According to other groups, the acceleration of epi- blocks peripheral histamine H1-receptors selectively [1, 2]. thelization in the RJ treated group appeared to occur between 7 and A simple, rapid, sensitive and selective LC-MS method was de- 9 days clinically as well as histologically. However, adhered strongly veloped and validated for quantification of fexofenadine in human and the frequent dressing may delay the healing. The epithelization plasma. The LC-MS system was operated under the positive elec- was completed on POD 16 on RJ and 10 % povidone iodine gauze- trospray ionisation mode (ESI). After liquid-liquid extraction, fex- applied wounds, whereas it wasn’t completed on 0.9 % sodium chlo- ofenadine analysis was performed through a C18 column with a mo- ride gauze-applied wounds and ulceration in central wounds was bile phase of acetonitrile: 10 mM ammonium acetate: formic acid, seen. The granulation tissues on all wounds were noted on PODs 70:30:0.1 (v/v/v) at a flow rate of 1 mLmin-1 by using loratadine as an 3-5, while epithelization was seen on PODs 6-8. The expansion proc- internal standard. The lower limit of quantitation was 3 ngmL-1 for ess (to POD 4) was followed by the contraction process (to PODs fexofenadine. 6-8). The contraction in RJ gauze-applied wounds (to PODs 16) be- Results of analysis showed after five days validation process, co- came more than other wounds. The thickness of scar tissue was sig- efficient correlation was 0.9993 – 0.9999. In quality control sam- nificantly different on PODs 4 and 12 between RJ groups (P< 0.05). ples, with-in-batch and batch-to batch accuracy ranges were 86.51 In our study, we did not observe any adverse effects of antiseptics – 113.50% and 97.92 – 106.06% respectively; precision ranges were on the thickness of scar tissue, the density of vascular proliferation 3.89 – 13.62% and 8.40 – 11.81% respectively. In calibration stand- and the degree of inflammatory cell infiltration in full thickness skin ard samples, batch-to batch accuracy ranges were 96.40 – 104.17%; wounds. According to current study, RJ gauze-applied wounds was batch-to batch precision ranges were 2.44 – 5.80%. Our whole study showed strong adherence, the dressing every day and required an was conducted according to FDA regulations about bioanalytical extra force to separate it from them. This force could cause epithelial method validation process [3]. The presented analytical method that damage and thus may increase the thickness of scar tissue. In conclu- is developed originally and validated in our laboratory was used to sion, RJ application in full-thickness skin defects in rabbits acceler- evaluate the bioequivalency of two different brand name fexofena- ated wound healing. dine products. 1. Hidaka S, Okamoto Y, Uchiyama S, Nakatsuma A, Hashimoto K, Ohni- 1. Simpson K, Jarvis B. Drugs, 59, 301-321, 2000. shi ST, Yamaguchi M. Royal jelly prevents osteoporosis in rats: Beneficial 2. Dollery C. Therapeutic Drugs 2nd edition Churchill Livingstone, United effects in ovariectomy model and in bone tissue culture model, Evid. Kingdom, A151-A154. 1999. Based Complement. Alternat. Med., Sep 3(3), 339-48, 2006. 3. FDA, Bioanalytical Method Validation, Guidance for Industry, May 2001.

Kervansaray Convention Center & Hotel, LARA, ANTALYA / TÜRKİYE 65 International Symposium on Drug Research and Development

P 043 Ref: 0065 P 044 Ref: 0066 INVESTIGATION OF CYTOTOXIC EFFECTS OF ANHYDRIDE TOPIC ADMINISTRATION OF YARROW EXTRACT ON CONTAINING WATER-SOLUBLE COPOLYMERS ON L929 WOUND IN RABBITS

PRESENTATIONS MOUSE FIBROBLASTS 1Füsun TEMAMOĞULLARI, 2Ali HAYAT, 3Füsun BABA 1Esin AKBAY, 1Handan SEVİM, 1Özer Aylin GÜRPINAR, 1University of Harran, Faculty of Veterinary Medicine, Department of Pharmacology and 2Hatice KAPLAN-CAN, 1Mehmet Ali ONUR, 3Zakİr M. O. RZAEV, 2Ali GÜNER Toxicology, Şanlıurfa, Turkey 2 1Hacettepe University, Faculty of Science, Department of Biology, 06800 Beytepe- University of Harran, Faculty of Veterinary Medicine, Department of Surgery, Şanlıurfa,

POSTER Ankara, Turkey Turkey 3 2Hacettepe University, Faculty of Science, Department of Chemistry, 06800 Beytepe- University of Harran, Faculty of Medicine, Department of Pathology, Şanlıurfa, Turkey Ankara, Turkey Many infectious diseases are known to be treated with herbal 3Hacettepe University, Department of Chemical Engineering, Faculty of Engineering, remedies throughout the history of mankind. Yarrow belongs to the 06800 Beytepe-Ankara, Turkey Asteraceae family and contains aquileic acid, essential oils, tannins, Synthetic polymers, water-soluble or in the form of hydrogels, na- flavonoids and acids. The application of infusions showed positive ef- noparticles, dendrimers or microspheres, are materials with which fects on wound healing and hemorrhages [1]. The aim of the present we are in daily contact or which are under development as materi- study was to investigate efficacy of yarrow extract on healing wound als for medical applications. At the end of the last century, synthetic standing on the clinical comparison with 10 % povidone iodine and polymers successfully replaced a number of natural materials, either 0.9 % sodium chloride. because the latter were in short supply or because the physicochemi- Six male and six female rabbits (2250 ± 100g) were taken for the cal characteristics of synthetic polymers exceeded those of materials study. All rabbits were anesthetized with i.m. administration of available from natural sources [1]. 10 mg/kg xylazine hydrochloride (Rompun, Bayer) and 50 mg/kg In this study, complex-radical copolymerization of maleic anhy- ketamine hydrochloride (Ketanes, Albe). Then full-thickness skin dride (MA), and acrylic acid (AA) and ternary polymerization of wounds were created (n: 3) on costal sides. Yarrow methanolic ex- maleic anhydride (MA), vinyl acceptor−donor−acetate (VA) and tract was prepared by infusion of the aerial parts of the plant (10 acrylic acid (AA) and, considered as acceptor systems, were carried days) in methanol at 1:5, w/v. The infusion filtered [2] with gauze out in 1,4-dioxane with benzoyl peroxide (BPO) as an initiator at 70 was applied to the defect on the right cranial side (yarrow ektract o C under a nitrogen atmosphere. The co- and terpolymer synthe- group), and 10 % povidone iodine was applied to the defect on the sized by the use of 1: 1 and 1: 2: 1 molar ratio of initial monomers, re- left cranial side, as for control 0.9 % sodium chloride was applied to spectively. Polymer samples were purified by several reprecipitating the defect on the left caudal side of the same animal. Wound surfaces from anhydrous acetone, n-hexane, diethyl ether and were dried in were examined macroscopically and microscopically from the points o vacuo at 60 C to a constant weight with quantitative yields [2]. The of exudation, bleeding, thickness of scar, contraction and epitheliza- cytotoxic effects of poly(MA-co-AA) and poly(MA-co-VA-co-AA) tion during the postoperative days (PODs). polymers samples were investigated in cell culture. The cytotoxicity Macroscopically, there was much less bleeding, and thicker scar and was observed on L929 mouse fibroblasts. In the first step, fibroblasts more contraction was observed in yarrow extract group compared to were cultured in DMEM at initial density of 50.000 cells/ml. Follow- others. The epithelization in this group was completed on PODs 12. ing a 24 hour of incubation, the cell culture medium was removed But the wounds treated with 0.9 % sodium chloride and 10 % povidon and fresh medium containing poly(MA-co-AA) and poly(MA-co- iodine was not completed on PODs 12. The density of vascular prolif- VA-co-AA) was added. Poly(MA-co-AA) and poly(MA-co-VA-co- eration progression was significantly different on PODs 4 and 16 with- AA) were prepared in five different dilutions (Dilution 1: 0.00114 in yarrow extract group. Such a relation was not found on PODs 8-12 g/mL; Dilution 2: 0.00057 g/mL; Dilution 3: 0.00028 g/mL; Dilution ((P> 0.05). The degree of inflammatory cell infiltration was signifi- 4: 0.00014 g/mL; Dilution 5: 0.00007 g/mL). Untreated cells served cantly different on PODs 8-12 within the yarrow extract group. Such a as controls. The cells were incubated during 5 days. Cell number and relation was not found on PODs 4,12 and 16 (P> 0.05). 10% povidone- cell morphology were investigated at the 1st and 5th days. Propidium iodine is a microbicidal, antiseptic agent. However, it is inactivated by iodide/acridine orange (PI/AO) staining was used to assess apopto- organic material and blood [3]. Candan et.al [4], observed that yarrow sis of treated cells and of the control group. possess strong antioxidative activity but low antimicrobial activitiy in The results showed that there were differences between poly(MA- vitro. In this study, the degree of inflammatory cell infiltration was co-AA) and poly(MA-co-VA-co-AA) in view of cell proliferation. more decreased in yarrow extract than 10 % povidone iodine applied In poly(MA-co-VA-co-AA) group cell number was higher than wounds on PODs 8-12. This might be a result of reduced bleeding due poly(MA-co-AA). A relatively few number of apoptotic cells were to yarrow extract Conclusively, we suggested that yarrow extract led observed in the Poly(MA-co-VA-co-AA) on day 5. Therefore it can limited bleeding, better contraction and decrease of inflammatory cell be said that toxicity of Poly(MA-co-AA) was related with the pro- infiltration in wound treatment process. liferation characteristics of cells. Cytotoxicity of results can be ex- plained polyanionic character of the co- and ternary polymers and 1. Teixeira RO, Camparoto ML, Mantoovani MS, Vicentini, VEP. Asses- also poly(MA-co-VA-co-AA) depicts the low cytotoxicity behavior. ment of two medicinal plants Psidium guajava L. and Achillea millefo- Vinyl acetate fragments in the terpolymer gives the immobility to the lium L. in vitro and in vivo assays, Genetics and Moleculer Biology, 26(4), polymer chains and lower polyanionic character [2]. 551-555, 2003. 2. Baytop, T. Türkiye’de Bitkiler ile Tedavi (Geçmişte ve Bugün), İstanbul 1. Ottenbrite RM, Kaplan AM. Some biologically active copolymers of Üniversitesi Eczacılık Fakültesi, İstanbul, s.166-167, 1999. maleic anhydride, macromolecules as drugs and as carriers for biologi- 3. Frastvedt E., Stashak TD, Othic A. Update on Topical wound medica- cally active materials, Annals of the New York Academy of Sciences, 446 tions, Clinical Techinique Practice, 3, 164-172, 2004. (1), 160–168, 1985. 4. Candan F, Ünlü M, Tepe B, Daferera D, Polissiu M, Sökmen A, Akpulat 2. Kaplan CH, Doğan AL, Rzaev ZMO, Uner AH, Güner A. Synthesis, HA. Antioxidant and antimicrobial activity of the essential oil and meth- characterization and antitumor activity of poly(maleic anhydride-co-vi- anol extracts of Achillea millefolium Subs. Millefolium Afan, Journal of nyl acetate-co-acrylic acid), Journal Applied Polymer Science, 100, 3425- Ethnopharmacology, 87, 215-220, 2003. 3432, 2006.

66 May 17-20, 2007 “From Chemistry to Medicine” DRD 2007

P 045 Ref: 0067 In this present study, poly(maleic anhydride-alt-acrylic acid) copolymer, poly(MA-alt-AA), and poly(N-vinyl-2-pyrrolidone), PVP, INVESTIGATION OF CELL PROLIFERATION OF are used in the preparation of blends. Poly(MA-alt-AA)/PVP blends, DEXAMETHASONE ON HUMAN PULP AND GINGIVAL covering a full range of compositions, were prepared by dissolution

FIBROBLASTS of both of the polymers in common solvent followed by the solvent PRESENTATIONS 1Handan SEVİM, 1Esin AKBAY, 1Özer Aylin GÜRPINAR, removal by drying at ambient temperature. Characterization of 2Zafer C. ÇEHRELİ, 1Mehmet Ali ONUR, 1Aşkın TÜMER blends was carried out by FTIR and Raman spectroscopy. 1Hacettepe University, Faculty of Science, Department of Biology, 06800 Beytepe- The FTIR and Raman measurements prooved the establishment

Ankara, Turkey of the interactions between copolymer and PVP. The significant POSTER 2Hacettepe University, Faculty of Dentistry, Department of Pediatric Dentistry, 06100 chemical shifts in the characteristic frequencies in FTIR and Raman Sıhhiye-Ankara, Turkey spectra support the strong hydrogen bond formation. Thus compat- Although previous studies have suggested that topical use of dex- ible blends were obtained due to this strong hydrogen bond forma- amethasone in replanted animal teeth enhances healing and results tion between copolymer and PVP. in fewer resorption complications, the effect of dexamethasone on the types of human cells involved in the periodontal healing process 1. Khutoryanskiy VV, Cascone MG, Lazzeri L, Nurkeeva ZS, Grigory MA, remains unknown [1, 2]. Mangazbaeva RA. Phase behaviour of methylcellulose-poly(acrylic acid) This study investigated the effects of dexamethasone on cultured blends and preparation of related films, Polym. Int., 52, 62-67, 2003. 2. Feldstein MM, Kuptsov SA, Shandryuk GA, Plate NA, Chalykh AE. human pulp fibroblasts (HPF) and gingival fibroblasts (HGF). HPF Coherence of thermal transitions in poly(N-vinyl pyrrolidone)- and HGF were cultured in DMEM at initial density of 20.000 cells/ poly(ethylene glycol) compatible blends 3. Impact of sorbed water upon ml and 30.000 cells/ml, respectively. Following 24h incubation, the phase behavior, Polymer, 41, 5349-5359, 2000. cell culture medium was removed and fresh medium containing 3. Abd El-Rehim HA, El-Hag Ali A, Mostafa TB, Farrag HA. Anti-micro- three different dilutions of dexamethasone (Dilution 1: 0.00001 mM bial activity of anhydride copolymers and their derivatives prepared by ; Dilution 2: 0.0001mM; Dilution 3: 0.05 mM) were added separate- ionizing radiation, Eur. Polym. J., 40, 2203-2210, 2004. ly. Untreated cells served as controls. The cells were incubated for 5 days. Cell number and cell morphology were investigated at the 1st, 2nd, 3rd, 4th and 5th days. Propidium iodide/acridine orange P 047 Ref: 0070 (PI/AO) staining was used to assess apoptosis of treated cells and of the control group at 1st and 5th days. In Dilution 1, cell number KINETIC EVALUATION OF THE SUBSTITUTIONS ON was significantly higher than those of Dilutions 2 and 3. Compared BISBENZIMIDAZOL DERIVATIVES ON THEIR INHIBITORY to other dilutions, the number of apoptotic cells observed in the 3rd ACTIVITIES OF MAMMALIAN DNA TOPOISOMERASE I dilution was relatively higher than those of other dilutions. 1Sevil ZENCİR, 2A. Selcen ALPAN, 2Pınar ALCIL, 2Güneş ÇOBAN, The proliferation of HPF was significantly lower than HGF. The 2H. Semih GÜNEŞ, 1Zeki TOPÇU results of this study indicate that the effect of topical administered 1Ege University, Faculty of Pharmacy, Department of Pharmaceutical Biotechnology, dexamethasone in an avulsion-type dental trauma varies for both 35100 İzmir, Turkey cell-type and concentration. 2Ege University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 35100 İzmir, Turkey 1. Cabral MC, Costa MA, Fernandes MH. In vitro models of periodontal 1H-Benzimidazole derivatives are known to have antibacterial, an- cells: a comparative study of long-term gingival, periodontal ligament tifungal, antimicrobial, antiprotozoal and antihelmintic activities [1]. and alveolar bone cell cultures in the presence of beta-glycerophosphate We have previously identified a considertable inhibition exerted by and dexamethasone, Journal of Materials Science, 2007 Feb 1, in press. 2. Soury B, Hentzen D, Vignal M, Christeff N, Doly J. Induction of inter- a number of 1H-benzimidazole derivatives on the mammalian type feron-beta gene expression by dexamethasone in murine L929 cells, Mo- I DNA topoisomerases [2]. Topoisomerases are ubiquitous enzymes lecular Endocrinology, 9, 199-207, 1995. that regulate the conformational changes in DNA topology by cata- lyzing the concerted breakage and rejoining of DNA strands during many genetic processes including DNA replication, transcription, re- P 046 Ref: 0069 combination and transposition [3]. Because of the increased aware- ness on the targetting of these enzymes as an effective approach for INVESTIGATION OF INTERACTIONS IN POLY(MA-ALT-AA)/ the development of chemotherapeutics, we extended our analysis on PVP BLENDS the 1H-benzimidazole-generated topoisomerase inhibition. In this study, we synthesized 1,2-bis(5,6-dimethyl-1H-benzo[d]imidazol-2- 1Hatice KAPLAN-CAN, 1Serap KAVLAK, 1Ali GÜNER, 2Zakir M. O. RZAEV yl)ethane and showed a significant interference of this compound on 1 Hacettepe University, Faculty of Science, Department of Chemistry, Ankara, Turkey the mammalian type I topoisomerase using in vitro plasmid super- 2Hacettepe University, Faculty of Engineering, Department of Chemical Engineering, Ankara, Turkey coil relaxation assays [4-6]. A known topoisomerase I poision, Camp- tothecin, was used as the reference compound in the evaluation of the In the last few decades the development of various hydrophilic inhibition. Our report also includes the effects of the substitutions of materials based on blends and interpolymer complexes of the hydrogen atom at the 5- and/or 6- position on 1H-benzimidazol poly(carboxylic acid)s and non-ionic water-soluble polymers is of ring with chloro, nitro and methyl hydrogen acceptor or donor atoms/ great importance because of their unique properties and possible groups on the inhibitory activity of the compound. applications in medicine [1]. Hydrophilic synthetic polymers have been widely investigated as carrier substances. PVP is an amorphous 1. Güneş HS, Coşar G. Arzneimittel-Forschung/ Drug Res., 42, 1045-1048, 1992. and biocompatible polymer with a high affinity for water and its in- 2. Alpan AS, Güneş HS, Topçu Z. 1H-Benzimidazole Derivatives As Mam- teraction with water has became the topic of a large body of research malian DNA Topoisomerase I Inhibitors, Amnusc., submitted 2007. [2]. It has been known that some maleic anhydride based polymers 3. Wang JC. Ann Rev Biochem, 65, 635-692, 1996. with high carboxylic acid content exhibit high biological activities 4. Shriner RL. Upson RW. J Am Chem Soc, 63, 2277-2278, 1941. such as inhibitory effect on viruses, bacteria and tumors [3]. 5. Topçu Z, Castora FJ. Biochim. Biophys. Acta, 1264, 377-387, 1995. 6. Topçu Z. J. Clin. Pharm. Ther., 26, 405-416, 2001.

Kervansaray Convention Center & Hotel, LARA, ANTALYA / TÜRKİYE 67 International Symposium on Drug Research and Development

P 048 Ref: 0071 P 049 Ref: 0072 LC-DAD METHOD FOR THE DETERMINATION OF SYNTHESIS AND CHARACTERZATION OF NOVEL

pKA VALUES OF SULFONAMIDES AND OPTIMIZING DENDRITIC POLYMERS FOR ANTI-CANCER DRUG

PRESENTATIONS CHROMATOGRAPHIC SEPARATION BY VARYING SOLVENT ENCAPSULATION AND RELEASE COMPOSITION 1Esra GÜÇ, 2Güngör GÜNDÜZ, 1Ufuk GÜNDÜZ 1Nurullah ŞANLI, 1Güleren ALSANCAK, 2Zerrin ERDEMGİL, 1Middle East Technical University, Department of Biological Sciences, Ankara, Turkey 3Jose Luis BELTRAN, 3Jose BARBOSA 2Middle East Technical University, Department of Chemical Engineering, Ankara, Turkey POSTER 1Süleyman Demirel University, Science & Literature Faculty, Departament of Chemistry, Controlled drug delivery by biocompatible artificial systems has be- 32260 Isparta, Turkey come one of the most interested research areas because of the ability to 2Anadolu Unıversity, BİBAM, 32260 Eskişehir, Turkey reduce the problems of conventional chemotherapy. By dendritic poly- 3Barcelona University, Department of Analytical Chemistry, 08028 Barcelona, Spain mer architectures, controlled drug delivery systems gain a new strategy. Systematic optimization of liquid chromatographic (LC) meth- Particularly dendritic polyesters are highly investigated due to their ods requires an accurate knowledgement of the parameters that unique properties including high degree of branching, nontoxicity and influence the separation of the compounds. The linear correlation water solubility [1]. Fatty acids are good candidates for polymeric sys- between the logarithm of retention factor and Reichardt’s polarity tems with their encapsulation stability for hydrophobic drugs and with parameter have been used to predict the chromatographic behavior their biosafety properties [2]. The aim of this study is to design hyper- of the compounds. Nowadays, acetonitrile-water mixtures are widely branched polyesters with fatty acids, to encapsulate anti-cancer drugs used in high performance liquid chromatography (HPLC) and LC and to study their release for chemotherapeutic purposes. retention parameters of the compounds strongly depend on the dis- Dendritic polyesters were based on dipentaerythritol (used as sociation behavior of the compounds and the pH of the acetonitrile- core molecule) and dimethylolpropionic acid (used as repeated end water mobile phases [1]. groups) and they were used in stoichiometrical ratios [3]. Ricinoleic The aim of this work is to analyze sulfodiazine, sulfothiazole, sul- acid, a C18 fatty acid was hydrolyzed from castor oil and was con- fomerazine, sulfomethazine, sulfodoxine, sulfomonometoxine, sulfo- jugated to the end groups of polyesters for effective encapsulation methoxazole like sulfonamides efficiently using HPLC method. The of hydrophobic drug. Dendritic polyesters were characterized by separation conditions have been optimized using the relationships fourier transform infrared spectroscopy (FTIR) and size exclusion between Reichardt’s polarity parameter and the capacity factors of the chromatography (SEC) analysis. For encapsulation of hydrophobic sulfonamides. The experimental region was selected in a such way that anti-cancer drug, idarubicin was used. Encapsulation of the drug the capacity factors of the sulfonamides would stay within the limits was followed with spectrophotometric measurements in the wave- 1< k < 10 [2]. These limits have been provided when the organic modi- lenght regions at 540 nm and 578 nm. Interactions of idarubicin and fier content of the mobile phase was in the range of 25 -16% (v/v). dendritic polyester were shown by FTIR analysis. For drug release The sulfonamides investigated are ampholytes with weakly basic profiles samples are placed into dialysis bags and drug delivery were and acidic characteristic. Because of this molecular structure the re- applied against phosphate buffer saline (PBS). Releasing medium tention of sulfonamides is expected to pass through a maximum at were analyzed spectrophotometrically to measure the released drug. an intermediate mobile phase pH. The pH dependent retention pro- For the next study, toxicity effect of unloaded and drug loaded den- files have been investigated and the results obtained demonstrated dritic polyesters on cell culture (MCF-7 breast cancer cell lines) will that the changes in retention are consistent with this expectation. be investigated by XTT tests and IC 50 values will be determined. The characterization by FTIR of the obtained hyperbranched pol- Before the optimization of chromatographic separation dissociation ymer have indicated the expected hydroxyl end groups. SEC studies constants of these compounds have been determined by LC-DAD have shown the molecular weight distribution of the dendritic poly- methodology [3]. The results obtained show that pKa1 and pKa2 val- mer. According to the molecular characterization results, various ues are between approximately 2 and 7. The pH of the mobile phase polymer to drug ratios were tested for encapsulation studies.The en- was kept constant at pH 4.50 where the neutral form is predominant. capsulation efficieny variations with respect to hyperbranched poly- Thus they will present a maximum hydrophobicity around this pH mer (HBR) to drug ratio is shown in the Figure 1. The results showed [4]. The greatest retention was obtained for sulfonamides bearing that encapsulation efficiency of the drug was increased by increasing additional methyl or methoxy groups on the R side chain. This sug- the polymer amount. Conversely efficiency of encapsulation was de- gests that the R side chain plays an important role in the hydropho- creased with increasing drug ratio. Sustained release profiles of den- bic interaction of the compounds with the reversed phase column. dritic nanoparticles were obtained and analyzed successfully. The next step in this study will be the determination of cytotoxicity of 1. Botsoglou NA, Fletouris DJ, Psomas IE. Retention behavior of multiple empty and drug loaded dendritic nanoparticles on MCF-7 cell line. sulfonamides in various liquid chromatographic systems, Chromato- These studies will bring new insights to successful cancer therapy by graphia, 46, 477-481, 1997. 2. Augiar de PF, Bourguignon B, Massart DL. Comparison of models and controlled delivery of anti-cancer drugs. designs for optimization of the pH and solvent strength in HPLC, Anal. Chim. Acta, 356, 7-18, 1997. 3. Jiménez-Lozano E, Marqués I, Barrón D, Beltrán JL, Barbosa J. Determi- nation of pKa values of quinolones from mobility and spectroscopic data obtained by capillary electrophoresis and diode array dedector, Anal. Chim. Acta, 464, 37-45, 2002. 4. Carda-Boch S, Berthod A. Countercurrent chromatography for the measurement of hydrophobicity of sulfonamide amphoteric compounds, Chromatographia, 59, 79-87, 2004.

68 May 17-20, 2007 “From Chemistry to Medicine” DRD 2007

when PANI and PVF+ClO4- were codeposited. The peaks belonging to both PVF and PANI were observed clearly from their cyclic vol- tammograms. It was also proven that film contains both PANI and PVF using FT-IR spectra. This PANI/PVF+ codeposited film on Pt

electrode were used for determination of catechol without using en- PRESENTATIONS zyme. The experimental results indicate that the anodic peak potential of catechol at the PANI/PVF+ modified electrode is lower than that at the Pt electrode in a solution consisting of catechol. The OH group

on the PANI chain in the composite film plays an important role in POSTER the electron transfer between PANI and catechol in the solution. Opti- mum conditions for the determination of the relationship between the response current and the concentration of catechol are that the poten- Figure 1. Encapsulation efficiency of idarubicin at 25mg, 50mg, 75mg of tial was set at 0.55 V and the pH of the solution controlled at 4.0. Fig. hyperbranched polymer (HBR). Encapsulation efficiency = Encapsulated 1 shows the change in the response current with the concentration of drug weight(mg)/Initial drug weight(mg)*100a. aMean ± SEM (n=2) catechol from 3,91 to 500 µM and from 3.91 to 64 000 µM, respectively. This modified electrode has a lower working potential and a good 1. Dhanikula RS, Hildgen P. Synthesis and evaluation of novel dendrimers operational stability due to reducing the electrode fouling, compared with a hydrophilic interior as nanocarriers for drug delivery, Bioconju- with the direct oxidation of catechol at the bare Pt electrode. gate Chem., 17 (1), 29, 2006. 2. Slivniak R, Ezra A, Domb AJ. Hydrolytic degredation and drug release of ricinoleic acid-lactic acid copolyesters, Pharmaceutical Research, 23, 6, 2006. 3. Bat E, Gündüz G, Kısakürek D, Akhmedov İ. M. Synthesis and characterization of hyperbranched and air drying fatty acid based resins, Prog. Org. Coat., 55, 330-336, 2006.

P 050 Ref: 0073 DETERMINATION OF CATECHOL USING MODIFIED ELECTRODE WITH A COMPOSITE OF POLY(VINYLFERROCENE) AND POLYANILINE Muammer KAVANOZ, Nuran ÖZÇİÇEK-PEKMEZ, Kadir PEKMEZ, Attila YILDIZ Hacettepe University, Faculty of Science, Department of Chemistry, Beytepe-Ankara, Turkey The determination of phenolic compounds has a great interest in many fields, such as neurochemistry, pharmaceutical and clinical chemistry. Some phenols as catechol, resorcinol and chlorogenic acid are found in plants, fruits and herbs. Among them, the catechol has acquired an increasing interest, not only to be a model molecule for bi-phenolic compounds such as dopamine, adrenaline, isopre- nalin, dobutamin and phenylethylamine but also have important pharmacological activities. Therefore, it is very important to develop a sensitive analytical method for the determination of catechol in biological studies. The interest in the determination of this phenolic compound has proportionated the development of several methods for their quantification, such as the chromatographic methods with different detection systems. These methods are very important, but time and reagent consuming are, generally, high. Thus, the development of new methods, that makes possible the minimal use of reagent and a lower time of analysis are very important. In this sense, electrochemical methods involving the development of chemical sensors have been utilized [1, 2]. Polyaniline (PANI) is one of the most promising conducting polymers due to its high conductivity, good redox reversibility and good Figure 1. a, b) Current time for different catechol concentrations. The stability in aqueous solutions and air. These properties provide favo- relationship between the response current and the concentration of rable conditions for its potential applications in super capacitor and catechol, c) from 3.91 µM to 500 µM, d) from 3.91 µM to 64000 µM. electrocatalysis. Poly(vinylferrocene) (PVF) is also an electroactive polymer. In this work, PVF in deposited composite film was used 1. Rita de Cassia Silva Luz, Flavio Santos Damos, Adriano Bof de Oliveira, Jo- as an electron transfer mediator in the electrochemical oxidation of hannes Beck, Lauro Tatsuo Kubota. Development of a voltammetric sensor for catechol in nanomolar levels using a modified electrode with Cu(phen)2 catechol due to its reversible redox. (TCNQ)2 and PLL, Sensors and Actuators, B 117, 274-281, 2006. In this study, electropolymerization of aniline in the presence of 2. Shaolin M. Catechol sensor poly (aniline-co-o- aminophenol) as an PVF was carried out by cyclic voltammetry in non-aqueous methyl- electron transfer mediator, Biosensors and Bioelectronics, 21, 1237-1243, ene chloride medium. Thin and more adhesive films were obtained 2006.

Kervansaray Convention Center & Hotel, LARA, ANTALYA / TÜRKİYE 69 International Symposium on Drug Research and Development

P 051 Ref: 0074 P 052 Ref: 0075 THE UV-SPECTROSCOPIC METHOD FOR DETERMINATION DETERMINATION OF PHYSICOCHEMICAL PARAMETERS OF DISSOCIATION CONSTANTS OF SEVERAL AND SAR STUDY ON THE SOME BENZIMIDAZO[1,2-

PRESENTATIONS SULFONAMIDES IN WATER AND ACETONITRILE-WATER a]PYRIMIDINE DERIVATIVES BINARY MIXTURES Asiye MERİÇ 1Senem ŞANLI, 1Güleren ALSANCAK, 2Jose Luis BELTRAN, 2Jose BARBOSA Anadolu University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 26470 Eskişehir, Turkey 1Süleyman Demirel University, Science and Literature Faculty, Department of Chemistry, POSTER 32260 Isparta, Turkey Condensed compounds bearing bridgehead nitrogen atom have 2Barcelona University, Department of Analytical Chemistry, 08028 Barcelona, Spaın. various activity, ranging from antihelmintic and anticonvulsant to Sulfonamides are anti-bacterial and anti-infective drugs com- antitumor and antiviral. Last study in our research group was re- monly used to treat in medicine and veterinary practice. The physi- vealed the cytotoxicities of some benzimidazo[1,2-a]pyrimidine cochemical profiling of drugs includes the determination of their compounds on non-cancer and cancer cell lines [1]. It is necessary dissociation constants. Data on proton dissociation of pharmacolog- to evaluate their SAR in order to understand the activity mechanism ically active substances are of utmost interest for the understanding of this type fused azole compounds, properly. of stability and solubility of drugs. A major factor of drug permea- Initially, physicochemical parameters of synthetic compounds

tion is their aqueous pKa. were calculated theoretically. Steric {molecular weight (MW), mo- Very often, the main difficulty in the determination of dissociation lecular refraction (MR) [2], molecular volume (MV) [3], molecular constants of drugs is their aqueous insolubility that forces the use connectivity index (MCI) [4], paracor (Par) [5-7]}, hydrophobic of spectroscopic method. UV spectroscopy is an excellent method {partition coefficient [8], hydrophobic substituent constant (π) [9]}

for pKa determination. This method requires very low analyte con- and electronic{electronic substituent constant [10]} parameters of centration and allows suitable absorbance measurement in aqueous 2,4-di- and 2,3,4-trisubstituted benzimidazo[1,2-a]pyrimidine de- solution even for products with low aqueous solubility. Acetonitrile rivatives were determined firstly. Then, correlations were constituted

(ACN)-water mixtures are usually employed for pKa determina- between cytotoxicities (IC50) and structural properties of compounds. tion of water insoluble drugs. Literature shows several extrapolation Some computerized programs were also used for further evaluation.

equations to estimate aqueous pKa from the pKa values determined in acetonitrile-water mixtures [1]. In this study, approximately 1.0 x 10-5 M solution of sulfonamides (sulfadiazine, sulfamethazine, sulfatiazole, sulfamethoxazole, sul- famonomethoxine, sulfamerazine, sulfadimethoxine) in water and ACN – water mixtures were titrated with NaOH in the range of pH 2-11 for determination of dissociation constants [2]. The data evaluation was performed by using STAR program [3]. The spectrum recorded for sulfadiazine at various pH values in Table . Training Set of Compounds ACN-water, 30 % (v/v) is shown in Fig 1. The aqueous pK obtained a Compound R R R Compound R R R from UV data and extrapolations of the data in ACN-water medium 1 2 3 1 2 3 is consistent between them and agree with those from literature [4]. 1 OH H Me 6 Ph H Me

2 OH H Ph 7 Ph H Ph

3 OH H n-Pr 8 Me H Me

4 OH Ph Me 9 Me Me Me

5 OH Me Me 10 Me Et Me

The SAR results of compounds can be summarized as follows:

At positions R1, R2 and R3; hydrophobic, electronic and steric properties were found important. The existence and abundance of methyl substituent on the structure increase the cytotoxic activity. It can be concluded that the substances abundantly bearing methyl subtituent may evaluate as promising compounds for potential antineoplastic activity. UV-spectrum of sulfadiazine obtained at various pH in ACN- Fig 1. 1. Meriç A, İncesu Z, Karayel A, Özbey S. Synthesis of some 2,4-di- and water mixture (30 % v/v). 2,3,4-trisubstituted benzimidazo[1,2-a]pyrimidines and evaluation of their cytotoxicities toward F2408 and 5RP7 cells, Revista de Chimie, 1. Ruiz R, Roses M, Rafols C, Bosch. Critical validation of a new simpler 57(11), 1090-1097, 2006. approach to estimate aqueous pKa of drugs sparingly soluble in water, 2. Dunn III WJ. Molar refractivity as an independent variable in quantita- Anal. Chim. Acta, 550, 210-221, 2005. tive structure-activity studies, Eur. J. Med. Chem.-Chim. Ther., 12, 109- 2. Polster J, Lachmann H. Spectrometric Titrations, VCH, 1989. 112, 1977. 3. Jiménez-Lozano E, Marqués I, Barrón D, Beltrán JL, Barbosa J. Determi- 3. Berkem AR, Baykut S., Fizikokimya Kitabı, İstanbul Üniversitesi nation of pKa values of quinolones from mobility and spectroscopic data Yayınları, Sayı: 2735, Kimya Fakültesi, No. 42, 1980. obtained by capillary electrophoresis and a diode array detector, Anal. 4. Kier LB, Hall LH. Derivation and significance of valence molecular con- Chim. Acta, 464, 37-45, 2002. nectivity, J. Pharm. Sci. 70, 583-9, 1981. 4. Lin CE, Lin WC, Chen YC, Wang SW. Migration behavior of sulfona- 5. Quayle QR. The parachors of organic compounds, Chem. Rev., 53, 439- mides in capillary electrophoresis, J. Chromatogr. A, 792, 37-47, 1997. 89, 1953.

70 May 17-20, 2007 “From Chemistry to Medicine” DRD 2007

6. Sugden S. A relation between surface tension, density, and chemical P 054 Ref: 0077 composition, J. Chem. Soc., 125, 1177, 1924. 7. Vogel AI. Physical properties and chemical constitution. Part IX. Aliphat- EFFECT OF USNIC ACID ON TISSUE NITRIC OXIDE ic hydrocarbons, J. Chem. Soc., 133, 1946. SYNTHASE ACTIVITY AND GLUTATHIONE LEVEL IN 8. Rekker RF, De Kort HM. The hydrophobic fragmental constant, an ex- PRESENTATIONS tension to a 1000 data point set, Eur. J. Med.-Chim. Ther., 14(6), 479-488, TITANIUM-IMPLANTED SUBJECTS 1979. 1Fehmi ODABAŞOĞLU, 2Hayati AYGÜN, 2Ömer Selim YILDIRIM, 9. Hansch C, Leo A, Unger SH, Kim KH, Nikaitanı D, Lien EJ. Aromatic 3Zekai HALICI, 1Mesut HALICI, 4Zafer OKUMUŞ, 5Ali ASLAN, substituent constant for structure activity correlations, J. Med. Chem., 16, 6Ahmet ÇAKIR, 7Cavit KAZAZ

1207-26, 1973. 1Atatürk University, Faculty of Pharmacy, Department of Biochemistry, 25240 Erzurum, POSTER 10. Akı-Şener E, Yalçın İ. Kantitatif Yapı-Etki İlişkileri Analizleri (QSAR), Turkey Ankara Üniversitesi Eczacılık Fakültesi Yayınları No: 86, 2003. (ISBN 2Atatürk University, Faculty of Medicine, Department of Orthopedics and Traumatology, 975-482-585-8) 25240 Erzurum, Turkey 3Atatürk University, Faculty of Medicine, Department of Pharmacology, 25240 Erzurum, Turkey 4Atatürk University, Faculty of Veterinary Medicine, Department of Surgery, 25240 P 053 Ref: 0076 Erzurum, Turkey ENHANCED ENZYME ACTIVITY OF IMMOBILIZED LIPASE 5Atatürk University, Kazım Karabekir Education Faculty, Department of Biology, 25240 Erzurum, Turkey AS BIOCATALYST FOR SYNTHESIS OF ESTERS IN ORGANIC 6Atatürk University, Kazım Karabekir Education Faculty, Department of Chemistry, MEDIA 25240 Erzurum, Turkey 7Atatürk University, Faculty of Science, Department of Chemistry, 25240 Erzurum, Turkey Taylan K. ÖZTÜRK, Funda KARTAL, Ali KILINÇ Ege University, Faculty of Science, Department of Biochemistry, İzmir, Turkey Debris due to the frictions as well as biochemical and magnetic reactions following orthopedic implantations may play a role in aseptic Lipases (triacylglycerol acylhydrolases, EC 3.1.1.3) catalyze the loosening through initiating a series of complex cellular reactions hydrolysis and the synthesis of esters of glycerol and long-chain between bone and implant. Loosening may be related to cytotoxic- fatty acids. The many applications of lipases include special organic ity [1, 2]. Usnic acid (UA) (Fig. 1) is a dibenzofuran derivative bio- syntheses, hydrolysis of fats and oils, modification of fats, flavor en- synthesised by lichens. Previously, it has been shown that UA has hancement in food processing, resolution of racemic mixtures and various biological activities [3]. The present study was conducted to chemical analyses [1]. evaluate the effect of UA on nitric oxide synthase (NOS) activity and Research on lipase catalyzed production of various kinds of ester glutathione level (GSH) in Ti-implanted tissues of rabbits. has increased tremendously in the recent past. Esters are present in fats and oils and in natural and synthetic polymers. They are useful intermediates or end products in the chemical industry. Enzymatic production of esters can be achieved either by reaction between free acid and hydroxyl groups of alcohol or by ester exchange or transes- terification (include alcoholysis, acidolysis and interesterification). Lipase catalized esterification reactions have been actively pur- sued to produce various kinds of commercially important esters. Esters of short chain fatty acids are extremely important aromatic compounds. Esters of short chain alcohols and long chain fatty acids are valuable oleochemicals that may be used as lubricants, diesel fuel and antistatic reagents. Esters of long chain fatty acids and polyhy- Fig. 1. Molecular structure of usnic acid (UA) dric alcohols like glycerol, sorbitol and other carbohydrates (called- emulsifiers/surfactants) find immense application in food and phar- UA was isolated from a lichen species, Usnea longissima [3]. Eight- maceutical industries [2]. een New Zealand rabbits divided into 6 groups, of which femurs of In this work the immobilized form of lipase was prepared by rabbits in 5 groups were subperiostally implanted with Ti. Then, they covalent attachment of enzyme on crosslinked polyvinyl alcohol [3]. received UA (30 mg/kg) and olive oil (OO) orally or locally every 3 Optimization of reaction conditions for ester synthesis was made by d for 21 d or received none. Rabbits from the other group served experimenting with different chain length acids and alcohols and ef- as control. Following euthanasia, tissues around the implant were fects of organic solvent type on esterification activity were studied. scrapped and then ground within liquid nitrogen for NOS activity The effects of reaction time and reaction temperature were also stud- and GSH level. ied. Esterification activity of immobilized lipase in organic solvents In the present study, 2.17, 1.6, and 1.7-fold increases were deter- was measured by GC-FID. mined in the activities of iNOS (inducible), cNOS (constitutive), and tNOS (total) respectively in Ti-implanted rabbits compared to con- 1. Hari Krishna S, Karanth N.G. Production, purification, characterization trol rabbits (Table 1). However, both local and oral administration of and applications of lipases. Catalysis Reviews, 44, 499-591, 2002. OO and oral administration of UA decreased NOS activities. Olive 2. Rohit S, Yusuf C, Uttam B. Lipases and lipase-catalyzed esterification oil was more effective than UA when administered locally, whereas reactions in nonaqueous media, Biotechnology Advances, 19,627-662, UA was more effective than OO when administered orally. Surgi- 2001. cal intervention was associated with a 31% reduction in GSH level. 3. Kılınç A, Önal S, Telefoncu A. Chemical attachment of porcine pancre- atic lipase to crosslinked poly(vinyl alcohol) by means of adipoyldichlo- Local and oral administration of UA and only local administration ride, Process Biochemistry, 38, 641-647, 2002. of OO alleviated this reduction. In conclusion, UA and OO administration may affect cytotoxicity via suppressing iNOS activity and increasing GSH level.

Keywords: Titanium implant, usnic acid, nitric oxide synthase, glutathione

Kervansaray Convention Center & Hotel, LARA, ANTALYA / TÜRKİYE 71 International Symposium on Drug Research and Development

Table 1. Effects of local and oral-administrated usnic acid (UA) and olive oil (OO) on the activities of nitric oxide synthase enzymes (tNOS, cNOS and iNOS) and amount of glutathione (GSH) in titanium-implanted subperiostal tissues of rabbits. Titanium group (TIT) was compared with healthy group. The 30 mg/kg dose of PRESENTATIONS UA and OO treated groups were compared with TIT group.

NITRIC OXIDE SYNTHASE Amount of (NOS) ACTIVITY GSH a

POSTER (µmol/min/mg tissue) (nmol/mg tissue)a

Treatments N tNOS cNOS iNOS The basic structures of these compounds were confirmed by IR, TIT+UA (local) 3 6.84±0.27** 3.07±0.17** 3.77±0.19** 3.44±0.02* 1H-NMR, mass spectral and elemental analyses data. The reaction TIT+UA (oral) 3 2.58±0.19** 2.47±0.18** 0.12±0.07** 3.96±0.04** products were assigned structure that were in accordance with their spectroscopic and chemical behaviors. Thus, compounds 1 and 2 TIT+OO (local) 3 3.65±0.27** 3.52±0.25* 0.13±0.02** 3.52±0.12* showed two strong band at 1771-1766 and 1745-1746 cm–1 in their IR spectra assignable to C=O groups. Also, compounds 3 and 4 have TIT+OO (oral) 3 3.40±0.37** 2.85±0.40** 0.55±0.05** 2.51±0.13* a one stretching band at 1783-1779 cm–1. The ethylene group protons 1 TIT (control) 3 5.43±0.21** 4.39±0.22** 1.04±0.03** 2.91±0.08** in the H-NMR spectra of all compounds appeared as a triplet at

3.31-4.07 ppm for -N-CH2-, a triplet signal at 4.23-4.14 ppm for -N- Healthy tissue 3 3.19±0.08 2.72±0.06 0.48±0.02 4.21±0.01 CH2-CH2-. Characteristic singlet peak of –NH for compounds 3 and a Means±SEM of tissues of six legs in each group. N: The number of rabbits. 4 were observed at 12.4 ppm. *Significant at p<0.05; **Significant at p<0.01. 1. Malawska B. New Anticonvulsant Agents, Curr Top Med Chem 5(1), 69- 85, 2005. 1. Stea S, Visentin M, Granchi D, Cenni E, Ciapetti G, Sudanese A, Toni A. 2. Popp FD. Potential anticonvulsants. IX. Some isatin hydrazones and re- Apoptosis in peri-implant, Biomaterials, 21, 1393-1398, 2000. lated compounds, J. Heteroc. Chem. 21, 1641-1645, 1984. 2. Raha S, Robinson BH. Mitochondria, Oxygen free radicals, and apopto- 3. Pandeya SN, Sriram D, Yogeeswari P, Stables JP. Anticonvulsant and sis, Am. J. Med. Gen., 106, 62-70, 2001. neurotoxicity evaluation of 5-(un)-substituted isatin-imino derivatives, 3. Odabaşoğlu F, Çakır A, Süleyman H, Aslan A, Bayır Y, Halıcı M, Kazaz, Pharmazie, 56, 875-876, 2001. C. Gastroprotective and antioxidant effects of usnic acid on indometha- 4. Pandeya SN, Smitha S, Stables JP. Anticonvulsant and sedative-hypnotic cine-induced gastric ulcer in rats, J. Ethnopharmacology, 103 (1), 59-65, activities of N-substituted isatin semicarbazones, Arch Pharm (Weinhe- 2006. im), 335(4), 129-134, 2002. 5. Tacconi G, Righetti PP, Desimoni G. Einfache Darstellung von N-substi- tuierten Isatinen, J. Prakt. Chem., 315(2), 339-344, 1973. P 055 Ref: 0078

SYNTHESIS AND CHARACTERIZATION OF SOME NOVEL P 056 Ref: 0079 ISATIN DERIVATIVES IN WHICH ANTICONVULSANT ACTIVITY IS PREDICTED PHENOLIC COMPOUNDS OF SIDERITIS OZTURKII AND THEIR IN VIVO ANTI-INFLAMMATORY AND Ebubekir SEPTİOĞLU, Mutlu DİLSİZ-AYTEMİR, Ünsal ÇALIŞ ANTINOCICEPTIVE ACTIVITIES Hacettepe University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 06100-Sıhhiye, Ankara, Turkey 1Pınar ŞAHİN, 2Esra KÜPELİ, 1İhsan ÇALIŞ, 1Nurten EZER, 3Erdem YEŞİLADA The restrictive treatment of epileptic seizures of the patients lets 1Hacettepe University, Faculty of Pharmacy, Department of Pharmaceutical Botany, the researches to find the new agents with more efficient activity Ankara, Turkey 2 and less toxicity [1]. Recently, the anticonvulsant activities of isatin Gazi University, Faculty of Pharmacy, Department of Pharmacognosy, Ankara, Turkey 3 derivatives were reported by various studies [2-4]. In this study, we Yeditepe University, Faculty of Pharmacy, Department of Pharmacognosy, İstanbul, Turkey have synthesized some new isatin derivatives as shown below and will evaluated the anticonvulsant activities of all compounds in fu- In Turkey, the genus Sideritis L. (Lamiaceae) is represented by 46 ture work. species [1] and some of which are used in traditional medicine for The synthesized compounds were prepared by Tacconi and col- their beneficial and curative effects [2]. Sideritis species growing in leagues’ method [5]. Treatment of isatin with sodium hydride in Turkey are known to be rich in essential oils, diterpenes, flavonoids N,N-dimethyl formamide (DMF) at room temperature gave isatin and phenylethanoid glycosides [3-5]. In a continuation of our phyto- sodium salt. This isatin salt was alkylated with 1,2-dibromoethane chemical studies on Turkish Sideritis species, we now report the iso- in DMF. Reaction of the alkylated isatin as named 1-(2-bromoe- lation of phenolic compounds from S. ozturkii Aytaç & Aksoy, which thyl)-1H-indole-2,3-dione with benzoxazol-2-one derivatives gave is endemic to Turkey, through in vivo bioassay-guided fractionation compounds 1 and 2. Final compounds 3 and 4 which have phenyl- procedures. hydrazone group were prepared by heating compounds 1 or 2 with Acetone extract from aerial parts of S. ozturkii and its fractions phenylhydrazine in ethanol. were investigated for its in vivo anti-inflammatory and antinociceptive activities. For the anti-inflammatory activity assessment, carrageenan-induced hind paw edema and for the antinociceptive activity, p-benzoquinone-induced abdominal constriction tests were used [6]. Acetone extract of the plant and its phenolic fraction were found to possess significant inhibitory activity on these models in mice. Ozturkosides A-C were isolated from the active phenolic fraction.

72 May 17-20, 2007 “From Chemistry to Medicine” DRD 2007

The structures of isolated compounds were elucidated by spectro- kg), indomethacine (IND, 25 mg/kg) or diclofenac (DIC, 25 mg/kg). scopic techniques (UV, IR, 1D- and 2D-NMR, MS). Ozturkoside In other series of experiments, the effect of ALA on the proliferative C showed notable antinociceptive and anti-inflammatory activities phase of inflammation was investigated using cotton pellet test [5]. without inducing any apparent acute toxicity or gastric damage. Al- In the present study, we found that 1) All doses of ALA, IND and though the activity of ozturkosides A and B were found insignifi- DIC have significantly decreasing effect on the mean weight of the PRESENTATIONS cant in statistical analysis, some inhibitory effects were observed. cotton pellets. The anti-proliferative effect of ALA was found as 67.7, Accordingly, it is suggested that these components in phenolic 68.9 and 69.9 %, of IND was 83.8 % and of DIC was 76.1 %; 2) ALA fraction might possibly share the antinociceptive and anti-inflam- reduced the development of CAR-induced paw edema, at a smaller matory activities together. magnitude for ALA than for IND and DIC; and 3) There were 2.57- POSTER fold increase in activity of inducible nitric oxide synthase (iNOS) in 1. Aytaç Z, Aksoy A. A new Sideritis species (Labiatae) from Turkey, Flora CAR-induced rats compared to control rats. However, oral adminis- Mediterranea, 10, 181-184, 2000. tration of ALA, IND and DIC significantly decreased iNOS activity. 2. Baytop T. Therapy with Medicinal Plants in Turkey (Past and Present), All doses of ALA were more effective than IND and DIC for de- Nobel Tıp Publications, İstanbul, p.375, 1999. creasing iNOS activity. These results suggest that the anti-inflamma- 3. Ezer N, Vila R, Cañigueral S, Adzet T. Essential oil composition of four Turkish species of Sideritis, Phytochem., 41, 203-205, 1996. tory effect of ALA on CAR-induced acute and cotton pellet-induced 4. Akcoş Y, Ezer N, Özçelik B, Abbasoğlu U. Iridoid glycosides from Sideri- chronic inflammations can be attributed to its decreasing effect on tis lycia Boiss & Heldr. and its antimicrobial activities, FABAD J. Pharm. activities of inducible nitric oxide synthase. Sci., 23, 99-103, 1998. 5. Şahin FP, Taşdemir D, Rüedi P, Ezer N, Çalış İ. Three new acylated flavon Table 1. Effects of ALA, indomethacine (IND) and diclofenac glycosides from Sideritis ozturkii Aytaç & Aksoy, Phytochem., 65, 2095- (DIC) on carrageenan (CAR)-induced paw edema and cotton 2099, 2004; 66, 125, 2005. pellet granuloma test in rats. 6. Küpeli E, Harput UŞ, Varel M, Yeşilada E, Saracoğlu İ. Bioassay-guided isolation of iridoid glucosides with antinociceptive and anti-inflammatory activities from Veronica anagallis-aquatica L., J. Ethnopharmacol., 102, 170–176, 2005.

P 057 Ref: 0080

ANTI-INFLAMMATORY EFFECTS OF THE ALPHA-LIPOIC Treatment Number of animals Dose mg/kg body wt Acute antiinflammatory effect (Inhibition %) Chronic antiinflammatory effect (Inhibition %) ACID ON CARRAGEENAN-INDUCED ACUTE AND COTTON ALA 2x6 50 18.5 67.7 PELLET-INDUCED CHRONIC INFLAMMATION MODELS IN RATS AND ITS RELATION WITH NITRIC OXIDE SYNTHASE - 2x6 100 29.6 68.9 ACTIVITY - 2x6 200 40.7 69.9 1Fehmi ODABAŞOĞLU, 2Zekai HALICI, 3Hayati AYGÜN, 1Mesut HALICI, 1Yasin BAYIR, 4Ahmet ÇAKIR, 5Elif ÇADIRCI, 1Fadime ATALAY DIC 2x6 25 44.4 76.1 1Atatürk University, Faculty of Pharmacy, Department of Biochemistry, IND 2x6 25 55.5 83.8 Erzurum, Turkey 2Atatürk University, Faculty of Medicine, Department of Pharmacology, Erzurum, Turkey CONTROLS 2x6 - - - 3Atatürk University, Faculty of Medicine, Department of Orthopedics and Traumatology, Erzurum, Turkey 4Atatürk University, Kazım Karabekir Education Faculty, Department of Biology, Table 2. Effects of ALA, IND and DIC on changes in activities of Erzurum, Turkey cNOS, iNOS and tNOS in CAR-induced paws (5th hour) of rats. 5Atatürk University, Faculty of Pharmacy, Department of Pharmacology, Erzurum, Turkey Alpha-lipoic acid (ALA) is a dithiol that is found naturally in mitochondria as the coenzyme for pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase. ALA has been shown to combat oxidative stress by quenching a variety of intracellular reactive oxy- Treatment Number of animals Dose mg/kg body wt. cNOS iNOS tNOS gen species (ROS) [1]. ALA has been demonstrated to be effective ALA 6 50 2.70±0.53* 0.17±0.02** 2.86±0.55 in preventing pathology in various experimental models in which ROS have been implicated [2]. Mediators such as free radicals, ni- - 6 100 3.10±0.25* 0.13±0.05** 3.23±0.29* tric oxide, prostaglandins, and cytokines play important roles in the - 6 200 3.23±0.37* 0.13±0.03** 3.37±0.37* development of acut or chronic inflammation [3]. The present study was conducted to evaluate the effect of ALA on acute and chronic CAR+DIC 6 25 2.28±0.07 0.18±0.03** 2.46±0.07 inflammation models in Dawley rats. Additionally, we have investigated the alterations in the activity of nitric oxide synthase following CAR+IND 6 25 2.04±0.05 0.22±0.01** 2.25±0.05 oral administration of ALA, indomethacine (IND) and diclofenac CAR 6 - 2.22±0.25 0.36±0.06** 2.58±0.24 (DIC) in CAR-induced paw edema tissues of rats. The present study was carried out in a total of 72 male Sprague- HEALTHY 6 - 2.51±0.37 0.14±0.03 2.65±0.37 Dawley rats, weighing 180-190 g. The animals were grouped before the experiments and kept under standard conditions [4]. Animals 1. Bilska A, Wlodex L. Lipoic acid- the drug of future?, Pharmacol Report, were assigned randomly for acute inflammation to the control 57, 570-577, 2005. groups receiving either CAR (0.1 ml of 1% per animal) or water and 2. Odabaşoğlu F. Alpha lipoic acid, Pharma Şark, 1 (4), 12-15, 2006. expamintal groups receiving CAR plus ALA (50, 100 and 200 mg/

Kervansaray Convention Center & Hotel, LARA, ANTALYA / TÜRKİYE 73 International Symposium on Drug Research and Development

3. Gualillo O, Eiras S, Lago F, Dieguez C, Casanueva FF. Evaluated serum leptin concentrations induced by experimental acute inflammation, J. Table 1. Effects of the MLK, FAM, RAN and LAN on IND-induced Ethnopharmacol., 75, 213-218, 2001. gastric damage in rats. IND was compared with healthy and 4. CCAC. 1993. Guide to the care and use of experimental animals, Vol I, treated groups with IND group. 2nd Ed. Canadian Council on Animal Care. Bradda Printing Services PRESENTATIONS Inc., Ottawa, ON, Canada. Treatment N Dose Ulcer index % 5. Süleyman H, Odabaşoğlu F, Aslan A, Çakır A, Karagöz Y, Göçer F, Halıcı mg/kg body wt. (UI) Inhibition M, Bayır Y. Antiinflammatory and antiulcer effects of aqueous extract of Lobaria pulmonaria, Phytomedicine, 10 (6-7), 552-557, 2003. IND+MLK 6 5 2.98±0.88** 59.1 POSTER IND+MLK 6 10 2.21±0.91** 69.7

P 058 Ref: 0081 IND+MLK 6 20 2.03±0.49** 72.2 GASTROPROTECTIVE EFFECT OF MONTELUKAST IND+FAM 6 25 0.16±0.08*** 97.8 (SINGULAIRE) ON INDOMETHACINE-INDUCED GASTRIC IND+RAN 6 25 2.54±0.76** 65.2 ULCER IN RATS AND ITS RELATION WITH SOME IND+LAN 6 30 0.0±0*** 100 GLUTATHIONE METABOLISM PARAMETERS 1Günnur ÖZBAKIŞ-DENGİZ, 2Zekai HALICI, 3Fehmi ODABAŞOĞLU, IND 6 25 7.29±0.43*** - 4 2 Elif ÇADIRCI, Halis SÜLEYMAN Healthy 6 - - - 1Karaelmas University, Faculty of Medicine, Department of Pharmacology, Zonguldak, Turkey 2Atatürk University, Faculty of Medicine, Department of Pharmacology, Erzurum, Turkey 3Atatürk University, Faculty of Pharmacy, Department of Biochemistry, Erzurum, Turkey Table 2. Effects of the MLK, RAN and LAN on GSH, GST and GR in rat’s IND-induced tissues. IND was compared with healthy and 4Atatürk University, Faculty of Pharmacy, Department of Pharmacology, Erzurum, Turkey treated groups with IND. Non-steroidal anti-inflammatory drugs (NSAID) are widely used in the treatment of pain, fever and inflammation. However, these Treatment N Dose GST GR GSH drugs have some side effects, especially on the gastrointestinal tract. (mg/kg) ACTIVITY ACTIVITY LEVEL Recently, reactive oxygen species (ROS) have also been shown to (EU) (EU) play a critical role in gastric ulceration process. The role of ROS in IND+MLK 6 5 26,29±0,5* 34.5±0.6* 3.31±0.06* the development of pathogenesis in acute experimental gastric lesions induced by stress, ethanol and NSAID’s is well known [1]. The IND+MLK 6 10 28,22±0,7* 29.6±0.8* 3.76±0.04* glutathione (GSH), glutathione S-transferase (GST) and glutathione IND+MLK 6 20 33,08±0,8** 27.6±0.7** 4.24±0.05** reductase (GR) play an important role in the prevention of the gastric damages [2]. On the other hand, montelukast (MLK), a selec- IND+LAN 6 30 33,65±0,4** 43.1±0.6* 4.85±0.03** tive reversible cysteinyl leukotriene-1 receptor (LTD4 receptor) an- IND+RAN 6 25 17,33±0,8* 25.2±0.2** 4.17±0.10** tagonist is used in the treatment of asthma [3] and presently nothing known about its effects on the gastro-intestinal system. We have in- IND 6 25 23,53±0,3* 37.7±0.9** 3.05±0.04** vestigated alterations in the GSH level and the activities of antioxidative enzymes (GST and GR), following oral administration of MLK, HEALTHY 6 - 28,60±0,4 25.6±0.5 4.12±0.04 lansoprazole (LAN), famotidine (FAM) and ranitidine (RAN) in rats with indomethacin (IND)-induced ulcer. 1. Das, D, Bandyopadhyay, D, Bhattacharjee, M, Banerjee, RK. Hydroxyl 48 male Sprague-Dawley rats, weighing 180–190 g were used in the radical is the major cousative factor in stress-induced gastric ulceration, present study. The animals were grouped before the experiments and Free Radical Biol. Med., 23, 8-18, 1997. kept under standard conditions [4]. MLK (5, 10 and 20 mg/kg), LAN 2. Odabaşoğlu F, Çakır A, Süleyman H, Aslan A, Bayır Y, Halıcı M, Kazaz, (30 mg/kg), FAM (25 mg/kg) and RAN (25 mg/kg) were administrated C. Gastroprotective and antioxidant effects of usnic acid on indometha- orally. 5 min after MLK, LAN, FAM and RAN administrations, IND cine-induced gastric ulcer in rats, J. Ethnopharmacology, 103 (1), 59-65, (25 mg/kg) was administrated to all animals orally. Control group re- 2006. ceived only water. After 6 h of all treatments, animals were sacrificed 3. Wenzel, SE. Leukotriene receptor antagonists and related compounds, Can. Respir. J., 6, 189-193, 1999. using sodium thiopental (50 mg/kg). The stomachs were removed and 4. CCAC. Guide to the care and use of experimental animals, Vol I, 2nd opened along the greater curvature and washed with saline [2]. The Ed. Canadian Council on Animal Care. Bradda Printing Services Inc., wideness of ulcer areas were determined using a magnifier and a mil- Ottawa, ON, Canada, 1993. limeter paper. Tissues were grounded under liquid nitrogen for the determinations of GSH level and GST and GR activities. In the present study, we found that 1) MLK, LAN, FAM and RAN reduced the development of IND-induced gastric damages, at a greater magnitude for MLK, FAM and LAN than for RAN; 2) MLK and RAN caused an increase in the activity of GST possibly resulted from gastric injury; and 3) MLK and RAN ameliorated depressions in the GSH levels and the GR activity possibly caused by IND administration. These results suggest that the gastroprotective effect of MLK on IND-induced ulceration can be attributed to its ameliorat- ing effect on the oxidative damage.

74 May 17-20, 2007 “From Chemistry to Medicine” DRD 2007

P 059 Ref: 0082 tric ulcers. These results suggest that the gastroprotective effect of AMD on IND-induced ulceration can be attributed to its ameliorat- GASTROPROTECTIVE EFFECT OF AMIODARONE ON ing effect on the oxidative damage, but can not be attributed to its INDOMETHACIN-INDUCED GASTRIC ULCER IN RATS AND effect on MPx activity, because of high iodine content, amiodarone

ITS RELATION WITH MYELOPEROXIDASE AND SOME can activate MPx activity in rat stomach [9]. PRESENTATIONS ANTIOXIDANT ENZYMES Keywords: Amiodarone; Indomethacin; Gastroprotective effect; 1 2 3 Günnur ÖZBAKIŞ-DENGİZ, Fehmi ODABAŞOĞLU, Zekai HALICI, Myeloperoxidase; Antioxidant enzymes 4Elif ÇADIRCI, 2Mesut HALICI, 3Halis SÜLEYMAN POSTER 1Karaelmas University., Faculty of Medicine, Department of Pharmacology, Zonguldak, Turkey Table 1. Effects of different doses of the amiodarone and single 2Atatürk University, Faculty of Pharmacy, Department of Biochemistry, Erzurum, Turkey dose of ranitidine and lansoprazole on indomethacin-induced 3Atatürk University, Faculty of Medicine, Department of Pharmacology, Erzurum, Turkey gastric damage in rats. Three doses of amiodarone, lansoprazole 4 Atatürk University, Faculty of Pharmacy, Department of Pharmacology, Erzurum, and ranitidine treated groups were compared with indomethacin Turkey group. Amiodarone (AMD){2-butyl-3-(3’;5’diiodo-4’α-diethyl-amino- ethoxy-benzoyl) -benzofuran} is a multiple ion (Ca++, Na+, K+) chan- a nel blocker drug and is also a non competitive α- and β-blocker in Treatment N Dose Ulcer index (UI) % [ b cardiac cell. It is an effective anti-arrhythmic and is used to treat a mg/kg Ulcerated area / Total Inhibition body stomach area] x 100 wide variety of ventricular and supraventicular tachyarrhytmias [1]. wt. Clinical use of AMD is limited because of its potential for developing numerous adverse side effects. Of greatest concern is AMD-in- 6 25 2.75 ±0.70 * 54.5 duced pulmonary toxicity (AIPT), due to the potential for mortality. Amiodarone 6 50 2.28 ±0.83 ** 62.3 However, hepatotoxicity and other adverse effects are also of clinical importance. This drug can also modulate thyroid function and phos- 6 100 0.89 ±0.46 *** 85.3 pholipid metabolism [1-3]. Non-steroidal anti-inflammatory drugs (NSAID) are widely used in the treatment of pain, fever and inflam- Ranitidine 6 25 1.64 ±0.85 ** 72.8 mation. However, these drugs have some side effects, especially on Lansoprazole 6 30 0 ±0 *** 100 the gastrointestinal tract. Recently, reactive oxygen species (ROS) have also been shown to play a critical role in gastric ulceration proc- Indomethacin 6 25 6.04 ±1.27 *** - ess. The role of ROS in the development of pathogenesis in acute Healthy group 6 - 0 ±0 - experimental gastric lesions induced by stress, ethanol and NSAID’s is well known [4]. ROS damage membrane proteins via causing lipid aMean damage index ±SEM of six animals in each group. N: The number of rats. b% Inhibition in ulcer index in relation to indomethacin group. *Significant at p<0.05; peroxidation in membranes by attacking to unsaturated fatty acids **Significant at p<0.01; *** Significant at p<0.005. as compared with indomethacin [5]. Oxygen-handling cells have antioxidant enzymes that are able group. to protect them against. The enzymatic antioxidant defenses include superoxide dismutase (SOD), catalase (CAT) and myeloperoxidase (MPx), as marker of ulceration process [6, 7]. These antioxidants Table 2. Effects of different doses of the amiodarone and single also play an important role in the prevention of the gastric damages. dose of ranitidine on the activity of superoxide dismutase (SOD), We have investigated alterations in the activities of SOD, CAT and catalase (CAT) and myeloperoxidase (MPx) enzymes in rat’s MPx, following oral administration of AMD, lansoprazole (LAN) indomethacin (IND)-induced gastric tissue. and ranitidine (RAN) in rats with indomethacin (IND)-induced ul- Treatment N Dose CAT SOD MPx Activity cer. mg/kg (mmol/min/ (mmol/min/mg (µmol/min/mg 42 male Sprague-Dawley rats, weighing 180–190 g were used in body mg tissue) tissue) tissue) this study. The animals were grouped before the experiments and wt kept under standard conditions [8]. AMD (25, 50 and 100 mg/kg 6 25 115,99±1,20 94,20±0.98* 10,22±0.08 body weight doses prepared as suspension in water) and positive controls [LAN (30 mg/kg body weight) and RAN (25 mg/kg body IND+AMD 6 50 90,17±1,20* 107,45±0.81** 11,47±0.26* weight)] were administrated orally to the assigned groups of rats. 5 min after AMD, LAN and RAN administrations, IND (25 mg/kg 6 100 74,05±1,10* 125,92±0.28** 13,51±0.14* body weight) was administrated to all animals orally. One group was IND+RAN 6 25 50,68±0,84* 116,08±0.06** 5,73±0.11* assigned as control group, which received only water. After 6 h of all treatments, animals were sacrificed using sodium thiopental (50 mg/ IND 6 25 117,89±0,36* 74,57±0.52** 9,75±0.07* kg). The rats’ stomachs were removed and opened along the greater Healthy rats 6 - 74,93±0,95 122,35±1.13 7,65±0.61 curvature and then washed with serum physiological solution. The wideness of ulcer areas was determined using a magnifier and a mil- N: The number of rats. Results are means ± SE of three measurements. IND group was compared with healthy group. Treated groups were compared with IND group. limeter paper. Then tissues grounded within liquid nitrogen for as- *Significant at p<0.05; **Significant at p<0.01 as compared with IND group. says of SOD, CAT and MPx activities. In the present study, we found that 1) AMD, LAN and RAN reduced the development of IND-induced gastric damages, at a greater 1. Mason JW. Amiodarone, New. Eng.. J. Med. 316, 455-466. 1987. magnitude for AMD and LAN than for RAN; 2) AMD and RAN al- 2. Vrobel TR, Miller PE, Mostow ND, Rakita, L. A general overview of leviated increase in the activity of CAT enzyme resulting from ulcer; amiodarone toxicity: its prevention, detection, and management, Prog. Cardiovasc. Dis. 31, 393-426, 1989. 3) AMD and RAN ameliorated depression in the activities of SOD 3. Figge HL, Figge J. The effects of amiodarone on thyroid hormone func- enzyme caused by IND administration; and 4) All doses of AMD tion: A review of the physiology and clinical manifestations, J. Clin. Phar- caused an amplification in MPx activity resulting from induced gas- macol., 30, 588-595, 1990.

Kervansaray Convention Center & Hotel, LARA, ANTALYA / TÜRKİYE 75 International Symposium on Drug Research and Development

4. Das D, Bandyopadhyay D, Bhattacharjee M, Banerjee RK. Hydroxyl radi- 2. Shin SS, Noh MS, Byun YJ, Choi JK, Kim JY, Lim KM, Ha JY, Kim JK, cal is the major cousative factor in stress-induced gastric ulceration, Free Lee CH, Chung S. 2,2-Dimethyl-4,5-diaryl-3(2H)furanone derivatives as Radical Biol. Med. 23, 8-18, 1997. selective cyclooxygenase-2 inhibitors, Bioorg. Med. Chem., 11, 165-168, 5. Ames BN, Shigenaga MK, Hagen TM. Oxidants, antioxidants, and the 2001. degenerative diseases of aging, Proc. Natl. Acad. Sci., 90, 7915-7922, 3. Dannhardt G, Laufer S. Structural approaches to explain the selectivity PRESENTATIONS 1993. of COX-2 inhibitors: Is there a common pharmacophore, Curr. Med. 6. Mates JM, Perez-Gomez C, Nudez de Castro I. Antioxidant enzymes and Chem., 7, 1101-1112, 2000. human diseases, Clinical Biochemistry, 32, 595-603, 1999. 4. Önkol T, Doğruer DS, Ito S, Şahin MF. Synthesis and antinociceptive 7. Elliot SN, Wallace JL.. Neutrophil-mediated gastrointestinal injury, Ca- activity of (5-chloro-2-benzothiazolinon-3-yl)acetamide derivatives, Ar-

POSTER nadian Journal of Gastroenterology, 12, 559-568, 1998. chiv der Pharmazie, 333(10), 337-340, 2000. 8. CCAC. 1993. Guide to the care and use of experimental animals, Vol I, 5. Kontogiorgis CA, Hadjipavlou-Litina DJ. Non steroidal anti-inflamma- 2nd Ed. Canadian Council on Animal Care. Bradda Printing Services tory and anti-allergy agents, Curr. Med. Chem., 9, 89-98, 2002. Inc., Ottawa, ON, Canada. 6. Kalgutkar AS, Zhao Z. Discovery and design of selective clooxygenase-2 9. Magnusson RP, Taurog A, Dorris ML. Mechanisms of thyroid peroxi- inhibitors as non-ulcerogenic, anti-inflammatory drugs with potential dase- and lactoperoxidase catalyzed reactions involving iodide, J. Biol. utility as anti-cancer agents. Curr. Drug Targets, 2, 79-106, 2001. Chem., 259, 13783-13790, 1984.

P 061 Ref: 0085 P 060 Ref: 0083 SYNTHESIS AND ANTIMICROBIAL EVALUATION SYNTHESIS OF SOME 5-CHLORO-6-(THIAZOL–4-YL)-2- OF 6-SUBSTITUTED-3(2H)-PYRIDAZINONE-2-YL OXO-3H-BENZOTHIAZOLE DERIVATIVES AS POTENTIAL ACETOHYDRAZİDE DERIVATIVES COX-2 INHIBITORS 1Mehtap GÖKÇE, 1Gülay YELKEN, 2Serpil POLAT, 3Seda TEZCAN, Selcen ADAK, Deniz S. DOĞRUER, Serdar ÜNLÜ 2Mehmet SERİN Gazi University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 1Gazi University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 06330 Ankara, Turkey Ankara, Turkey It is known that inhibition of the enzyme cyclooxygenase (COX) 2Mersin University, Faculty of Pharmacy, Department of Pharmaceutical Microbiology, Mersin, Turkey is the principal mechanism for the efficacy of NSAIDs. Two distinct 3Mersin University, Faculty of Medicine, Department of Medicinal Microbiology, Mersin, and independently COX isoforms have been identified COX-1 and Turkey COX-2 [1]. Interruption of COX-1 activity can lead to life-threating Several antibiotics have been prescribed and found to be effective gatro-intestinal (GI) toxicity of perforation, ulceration and bleeding. on various infectious disorders. However, the appearance of multi- In the meantime, COX-2 is induced upon inflammatory stimuli and drug resistant Gram-positive bacteria, in particular, methicillin-re- is responsible for progression of inflammation [2]. Therefore, many sistant Staphylococcus aureus (MRSA) and vancomycin-resistant studies have been focused on COX-2 inhibitors to reduce the risk of Enterococci (VRE) is causing a serious menace. Moreover, the emer- GI complications. The greatest research activity in the field of COX- gence of vancomycin resistant MRSA can be anticipated in foresee- 2 inhibitors has been made in the synthesis and pharmacological able future. For the treatment of these intractable infections, a new testing of the class of diaryl heterocyclics [3]. antibacterial agent is needed. Furthermore Synthetic antibiotics Some compounds bearing 5-chloro-2-oxo-3H-benzothiazole ring are widely prescribed drugs because of their safety, good tolerance, have been reported to have anti-inflammatory activity [4]. In addi- broad antibacterial spectrum and less resistance. tion, potent selective COX-2 inhibitors which bear thiazole struc- Due to favorable presence a pyridazinone moiety in known active ture as a central ring have been reported in the literature [5, 6]. In structures, pyridazinone derivatives provoked a special interest in the design of new compounds, development of the hybrid molecules the search for new antibacterial agents [1-4]. Meanwhile hyrazide- through the combination of different pharmacophores in one struc- hydrazones have been claimed to exhibit appreciable antimicrobial ture may lead to compounds with increased analgesic and anti-in- activity [5-9]. On the basis of these observations a new series of 6- flammatory activities. substituted-3(2H)-pyridazinone-2-yl acetohydrazide (I) was syn- Therefore, these observations prompted us to synthesize six new thesized using an appropriate synthetic route. The structure eluci- compounds which bear a central thiazole ring whose 2,4-positions dation of new compounds was based on the relevant 1H-NMR and are substituted with phenyl and 5-chlorobenzothiazolone moieties, IR spectral characteristics and purity of the products was confirmed respectively. Structures of the synthesized compounds have been both by TLC and elemental analyses. All the target compounds confirmed by IR, 1H-NMR and elemental analysis. Their COX-2 in- were evaluated for their in vitro antimicrobial activity against Sta- hibitory potency will be evaluated by using in vitro human whole phylococcus aureus, Bacillus subtilis as examples for Gram positive blood assay. bacteria, Escherichia coli, Pseudomonas aeruginosa as examples for gram negative bacteria and Candida albicans, Candida parapsilosis as representative of fungi. The minimum inhibitory concentration (MIC) was determined for test compounds as well as for reference standards.

1. Menozzi G, Merello L, Fossa P, Mosti L, Piana A, Mattioli F. 4-Substitut- ed 1,5-diarylpyrazole, analogues of celecoxib: synthesis and preliminary evaluation of biological properties, IL Farmaco, 58, 795-808, 2003.

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1. Sönmez M., Berber I., Akbaş E. Eur. J. Med. Chem., 41(1), 101-5, 2006. P 063 Ref: 0087 2. Fuks B, Talaga P, Huart C, Henichart JP, Bertrand K, Grimee R, Lorent G. Eur. J. Pharmacol., 519(1-2), 24-30, 2005. SYNTHESIS OF 6-(SUBSTITUE THIAZOL–4-YL)-2-OXO- 3. Akbaş E, Berber I, Şener A, Hasanov B. Farmaco, 60(1), 23-6, 2005. 3H-BENZOXAZOLE DERIVATIVES AS POTENTIAL COX-2 4. Takaya M. Yakugaku Zasshi., 113(9), 676-81, 1993. PRESENTATIONS 5. Koçyiğit-Kaymakçıoğlu B, Orçun E, Ünsalan S, Kandermirli F, Shvets N, INHIBITORS Rollas S, Antony D., Eur. J. Med. Chem., 41(11), 1253-61, 2006. Şeyma CANKARA, Serdar ÜNLÜ 6. Bijev A, Arzneimittel Forschung, 56(2), 96-103, 2006. Gazi University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 7. Küçükgüzel G, Kocatepe A, De Clercq E, Şahin F, Gulluce M. Eur. J. Med. Ankara, Turkey

Chem., 41(3), 353-9, 2006. POSTER 8. Sriram D, Yogeeswari P, Madhu K. Bioorg. Med. Chem. Lett., 16(4), 876- Selective inhibition of cyclooxygenase-2 (COX-2) which is being 8, 2006. induced during inflammation is off interest since the resulting drugs 9. Grover G, Kini SG. Eur. J. Med. Chem., 41(2), 256-62, 2006. lack the gastric ulceration side effects associated to classical NSAIDs. Since 2-oxo-3H-benzoxazole [1] and thiazole [2]ring system bear good anti-inflammatory property and the 2,4-diaryl/heteroaryl P 062 Ref: 0086 structures might also be important for selective COX-2 inhibitory activity, we hereby describe the biological consequences of incorpo- SYNTHESIS AND ANTIMICROBIAL EVALUATION OF ration of a 2-oxo-3H-benzoxazole ring as one of the aryl substituents 5-CHLORO-2(3H)-BENZOXAZOLINONE-3-ACETYL-2-(p-- and the effect of a 2,4-diarylsubstitution pattern around the thiazole SUBSTITUTED BENZAL)HYDRAZONE DERIVATIVES ring on the in vitro activity of the resulting derivatives towards COX- 2 inhibition. 1 1 1 2 Tijen ÖNKOL, Mehtap GÖKÇE, Ali Ulvi TOSUN, Serpil POLAT, Thus, we have designated a series of 6-(substitute thiazole–4-yl)- 2 3 Mehmet SERİN, Seda TEZCAN 2-oxo-3H-benzoxazole derivatives and these compounds were pre- 1 Gazi University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 06330 pared by the reaction of 6-bromoacetyl-2-oxo-3H-benzoxazole and Ankara, Turkey 2Mersin University, Faculty of Pharmacy, Department of Pharmaceutical Microbiology, substituted thiobenzamides. The result of inhibitory potency against

Mersin, Turkey. COX was evaluated using human whole blood assay [3]. 3Mersin University, Faculty of Medicine, Department of Clinical Microbiology, Mersin, Turkey Since it is a common agreement of multidrug-resistant bacteria are major cause of failure in the treatment of infectious diseases, the need for the synthesis novel antibiotics is a reality. The structural and therapeutic diversity coupled with commercial viability of small molecules has fascinated organic and medicinal chemists. It has been reported in the literature that hydrazide-hydrazones have been demonstrated to possess antibacterial, and antifungal activities [1-4]. Furthermore, it is reported in the literature that 2(3H)-benzoxazolinone derivatives can exhibit diverse activities. Particularly antibacterial [3] and antifungal [4] activities have been scrutinized intensively. In addition, it has been published that chlorinated 2(3H)-benzoxazolinone compounds have valuable fungicidal properties [5]. 1. Ünlü S, Önkol T, Dündar Y, Ökcelik B, Küpeli E, Yeşilada E, Noyanalpan These observations led us to synthesize a series of Schiff bases N, Şahin MF. Synthesis and Analgesic and Anti-inflammatory Activity of combining 5-chloro-2(3H)-benzoxazolinone, hydrazide and benzal- Some New (6-Acyl-2-benzoxazolinone and 6-Acyl-2-benzothiazolinone dehyde moieties in the same molecule. Synthesized 5-chloro-2(3H)- Derivatives with Acetic Acid and Propanoic Acid Residues, Arch. Pharm. benzoxazolinone-3-acetyl-2-(p-substituted benzal)hydrazone I de- Pharm. Med. Chem., 336, 353–361, 2003. rivatives were evaluated for screening antibacterial and antifungal 2. Narender M, Reddy MS, Sridhar R, Nageswar YVD, Rama Rao K. Aque- ous phase synthesis of thiazoles and aminothiazoles in the presence of activities on microorganisms respectively on four bacteria and two β-cyclodetrin, Tetrahedron Letters, 46, 5953-5955, 2005. Candida species. 3. Patrignani P, Panara Mr, Greco A, Fusco O, Natoli C, Iacobelli S, Cipol- lone F, Ganci A, Creminon C, Maclouf J, Patrono C. Biochemical and Pharmacological Characterization of the Cyclooxygenase Activity of Hu- man Blood Prostaglandin Endoperoxide Synthases, The Journal of Phar- macology and Experimental Therapeutics, 271, 1705-1712, 1994.

P 064 Ref: 0088

R1= H, F, Cl, Br, CH3, OCH3, OH INHIBITION EFFECT OF NEW SULFONAMIDE DERIVATIVES ON CARBONIC ANHYDRASE 1. Metwally KA, Abdel-Aziz LM, Lashine el-SM, Husseiny MI, Badawy RH., Bioorg. Med. Chem., 14(24), 8675-8682, 2006. Ümmühan ÖZDEMİR-ÖZMEN, Fatma ARSLAN, Fatma HAMURCU 2. Koçyiğit-Kaymakçıoğlu B, Orçun E, Ünsalan S, Kandemirli F, Shvets N, Gazi University, Arts and Sciences Faculty, Department of Chemistry, 06500 Ankara, Rollas S, Antony D., Eur. J. Med. Chem., 41(11), 1253-1261, 2006. Turkey 3. Mincheva ZP, Kalcheva VB, Golovinsky EG., Pharmazie, 48(11), 859- Sulfonamides have been known as powerful inhibitors of carbonic 860, 1993. anhydrase (CA) [1]. Since sulfonamides interact with the active site 4. Wang HX, Liu F, Ng TB., Comp. Biochem. Physiol., 30(3), 379-388, 2001. of CA, the active site charge requirements and the orientations of 5. Basel EM, Riehen JB., US Patent 2,922,794 (Cl. 260-304), January 26, sulfonamide groups are essential for the inhibitory powers of these 1960. drugs. Understanding the mechanism of the inhibition of CA isoen-

Kervansaray Convention Center & Hotel, LARA, ANTALYA / TÜRKİYE 77 International Symposium on Drug Research and Development

zymes with sulfonamide derivatives at the molecular level have been P 066 Ref: 0090 reported to be helpful for the rational design of novel derivatives with minimum side effects [2]. SYNTHESIS OF 5-CHLORO-2-OXO-6-(2-SUBSTITUTED In the present study, inhibitory effects of ethanesulfonic acid hy- PHENYL-1,3-THIAZOL-4-YL)-3H-BENZOXAZOLE

PRESENTATIONS drazide (esh), 5-methyl-2-hydroxyacetophenone ethanesulfonylhy- DERIVATIVES AND EVALUATION OF THEIR COX-1/COX-2 drazone (5mafesh) and its Ni(II) complex on CAII have been inves- INHIBITORY ACTIVITIES tigated. Enzyme activity was determined using p-nitrophenylacetate Şölen URLU, Serdar ÜNLÜ as substrate. According to the corresponding IC and K values, 50 i Gazi University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 06330 POSTER 5mafesh was found as the most potent inhibitor of CAII. Ankara, Turkey

1. Arslan O, Küfrevioğlu Öİ, Nalbantoğlu B., Bioorg. & Med. Chem., 5(3), The therapeutic efficacy of nonsteroidal anti-inflammatory drugs 515, 1997. (NSAIDs) results from the inhibition of cyclooxygenases (COX) 2. Chakravarty S , Kannan KK., J. of Mol. Biol., 243, 298, 1994. which were shown to exist as two distinct isoforms, namely, COX-1 and COX-2. COX-1 is constitutively expressed as a house keeping enzyme in nearly all tissues and mediates physiological responses. P 065 Ref: 0089 On the other hand, COX-2 is expressed by cells involved in inflammation and has emerged as the isoform that is primarily responsi- DESIGN AND SYNTHESIS OF NOVEL TIAZOLE DERIVATIVES ble for the synthesis of prostanoids involved in acute and chronic AS POTENTIAL CYCLOOXYGENASE- 2 (COX-2) INHIBITORS inflammatory states. It was hypotesised that selective inhibition of COX-2 might have therapeutic actions similar to those of NSAIDs, Leyla UZUN, Tijen ÖNKOL, Serdar ÜNLÜ but without causing gastrointestinal side effects [1]. Gazi University, Faculty of Pharmacy Department of Pharmaceutical Chemistry, 06330 Our ongoing studies towards the derivatives of chlorzoxazone Ankara, Turkey [2]and thiazole with anti-inflamatory activities [3] prompted us to The major side effects associated with the currently available design new compounds which could be selective COX-2 inhibitors. NSAIDs are gastrointestinal (GI) hemorrhagia and ulceration which For this purpose some 5-chloro-2-oxo-6-(2-substituted phenyl-1,3- result from the nonselective inhibition of COX enzymes, namely thiazol-4-yl)-3H-benzoxazole derivatives were synthesized by con- COX-1 and COX-2. After the discovery of the second isoform (COX- densation of 6-bromoacetyl-5-chloro-2-oxo-3H-benzoxazole and 2) that is expressed in inflammatory cells, but not in gastric mucosa, appropriate thiobenzamides derivatives in diethylenglicoldimethyl- novel NSAIDs with selective COX-2 inhibitory activity resulted to ether. The activities of the compounds for possible COX inhibitory

be more useful for the treatment of inflamatory diseases [1] without activity will be performed by enzyme immunoassay method. gastric side effects. For this reason, the series of 2,4-diarylthiazole derivatives were designed and synthesized for their evaluation as selective COX-2 inhibitors in an enzymatic assay using human whole blood. We selected thiazole ring to synthesize the title compounds since thiazole ring have been reported to have analgesic and anti-inflammatory activities [2]. The synthesis of 6-(substituted thiazol-4-yl)-2- oxo-3H-benzo- thiazoles were accomplished by the reaction of 6- bromoacetyl-2- oxo-3H-benzothiazole with o/p-substituted thiobenzamides using microwave-assisted synthesis. Physical and chemical properties of synthesized compounds have been confirmed by using their melting points, IR, 1H-NMR and elemental analysis.

1. Brune K, Hinz B. Selective cylooxygenase-2 inhibitors: similarities and differences, Scand. J. Rheumatol., 33, 1-6, 2004. 2. Park, JY, Kim KA, Park PW, Ha JM. Effect of high-dose aspirin on CYP2E1 activity in healty subjects measured using chlorzoxazone as a probe, Journal of Clinical Pharmacology, 46, 109-114, 2006. 3. Kazzouli Sl, Berteina-Raboin S, Mouaddib A, Guillaumet G. Solid support synthesis of 2,4-disubstituted thiazoles and aminothiazoles, Tet- rahedron Letters, 43, 3193-3196, 2002.

P 067 Ref: 0091 PHYSICOCHEMICAL CHARACTERIZATION OF BIODEGRADABLE CARDIOVASCULAR STENTS 1. Ulbrich H, Fiebich B, Dannhardt G. European Journal of Medicinal Chemistry, 37, 953-959, 2002. Can SARISÖZEN, Betül ARICA, Sema CALIŞ, A. Atilla HINCAL 2. Norender M, Reddy MS, Sridhar R, Nageswar YVD, Rao KR. Aqueous Hacettepe University, Faculty of Pharmacy, Department of Pharmaceutical Technology, phase Ssynthesis of thiazoles and aminothiazoles in the presence of β- 06100 Ankara, Turkey cyclodextrin, Tetrahedron Letters, 46, 5953-55, 2005. The aim of our study was to realize physicochemical characterization and in vitro evaluation of prepared biodegradable cardiovascular stents for preventing restenosis of blood vessels. Stents were prepared from solution-cast biodegradable formed polymeric films and a model drug prednisolone acetate (PA) was incorporated into the stents.

78 May 17-20, 2007 “From Chemistry to Medicine” DRD 2007

For this purpose, two types of synthetic biopolymer (PLGA 75:25 and PLGA 50:50) were dissolved in dichloromethane separately to Table 1. Characterization of biodegradable stent formulations. have a final concentration of 25% (w/v) in which PEG 4000 was added as a plasticizer. The polymer solutions cast onto the aluminum Polymer PA or Length Diameter Thickness Specific Microspheres (cm) (mm) (µm) Surface PRESENTATIONS pans (in 3x2 cm area). After evaporation of dichloromethane, the Area polymer films were cut into strips and coiled on the cylindrical rods to form a helical shaped stents. PLGA None (Empty 1.5 3 136.5±5 µm 1,32 m2/g Two types of stents were used in our study: stents formed by a 75:25 Stent) polymer film including PA and stents formed by a polymer film in- PLGA PA 1.5 3 140±3.6 µm 2,14 m2/g POSTER cluding PA containing spray-dried chitosan microspheres which has 75:25 Incorporated been developed by our group previously. The characteristics of these prepared stents such as wall thickness, diameter, length, morphology PLGA Microsphere 1.5 3 145±4 µm 0,44 m2/g were measured and evaluated. 75:25 Incorporated Differential Scanning Calorimetry (DSC) and Attenuated Total PLGA None (Empty 1.5 3 109±8 µm - Reflectance (ATR)-FTIR studies were also performed. Samples were 50:50 Stent) heated under nitrogen atmosphere in aluminum hermetic pans and thermograms recorded a range of (20°C to 300°C at a heating rate of PLGA PA 1.5 3 119.3±4.2 µm - 10°C/min). ATR-FTIR studies were performed to determine if there 50:50 Incorporated were drug present on the surface of the stents or not. Stents which PLGA Microsphere 1.5 3 118.7±3.2 µm - were prepared by PLGA 75:25 polymer were analyzed for specific 50:50 Incorporated surface area analyzes. In vitro drug release testing was performed to evaluate the drug release form the stents. The test was also performed in PBS solution containing 0.5%(w/v) sodium lauryl sulphate and 0.05%(w/v) sodium azide at pH 7.4. P 068 Ref: 0092 The prepared stents had outer diameters approximately 3mm and their length were 1.5cm. The polymer wall thicknesses of empty stents CO-ADMINISTRATION OF A NITRIC OXIDE SYNTHASE were measured using a micrometer and determined as 136µm±0.05. INHIBITOR AND MELATONIN EXERTS AN ADDITIVE Based on ease of handling for perivascular placement, this film thick- ANTIDEPRESSANT-LIKE EFFECT IN THE MOUSE FORCED ness was assessed to be optimum. Thin films (100µm) tended to fold SWIM TEST and 200µm films were not elastic and easy to crack. Incorporation of 1 2 3 4 drug or drug loaded microspheres did not interfere during the forma- Yusuf ERGÜN, Ufuk Güney ERGÜN, Özlem ORHAN, Ekrem KÜÇÜK 1 tion of homogenous films and helical stent structure. Prepared stents Kahramanmaraş Sütçü İmam University, School of Medicine, Department of Pharmacology, 46100 Kahramanmaraş, Turkey seemed to have smooth surface without any visible defects and cracks. 2Kahramanmaraş Sütçü İmam University, School of Medicine, Department of Family Incorporated drug or microspheres were homogenously distributed Medicine, 46100 Kahramanmaraş, Turkey in the polymer matrix. The ATR-FTIR spectrums showed that biode- 3Kahramanmaraş Sütçü İmam University, School of Medicine, Department of Psychiatry, gradable stent surfaces were free of drug and microspheres. 46100 Kahramanmaraş, Turkey Thermograms of the formulations are given in Figure 1. As it can 4Osmaniye High School of Science, Osmaniye, Turkey be seen from the graphics, melting peak of PA disappeared after it Previous studies have shown that nitric oxide synthase inhibi- was incorporated into the stents. The results for the specific surface tors and melatonin are as efficacious as conventional antidepres- area analyzes are given in Table 1. Although the release of PA from sant drugs in pre-clinical antidepressant screening procedures such the stents seemed to be very slow following the initial release pe- as Porsolt swim test. The aim of the present study was to assess the riod which was about 1-5 days in PBS buffer, a significant amount of combination therapy of these two distinct arrays of drugs. the drug was determined to be released at the second release period Porsolt swim test was conducted to resemble the symptomatology which started on the 5th day and on the 63th day. This might be ex- of major depressive disorder, and an open filed locomotor activity plained by the slow degradation of the PLGA polymer in the release was also used. medium. Nevertheless, approximately 5-62% of the PA from the PA- NG-nitro-L-arginine (L-NNA) at doses 3 to 30 mg/kg and me- loaded stents and 10-13% of the PA from the chitosan microspheres latonin at 3 and 10 mg/kg were examined in the forced swim test containing stents were released in 63 days. in mice. 3 mg/kg L-NNA slightly but not significantly reduced the duration of immobility, and increasing the dose to 10 mg/kg was sufficient to attain a significant reduction (Fig. 1). On the other hand, the maximal dose of L-NNA (30 mg/kg) had no effect although a non-significant small increase was observed (Fig. 1). The results obtained with L-NNA were in accordance with a U-shape effect. 3 mg/kg melatonin was found to be ineffective while a statistically significant decrease in the duration of immobility was found at the dose of 10 mg/kg (Fig. 1). The combination of ineffective doses (3 mg/kg, each) of L-NNA and melatonin revealed no further inhibition in the duration of immobility and the most effective doses (10 mg/kg, each) caused a more pronounced reduction when compared to those of each drug alone (Fig. 1). None of the drugs effected locomotor activity over the dose range applied (Fig. 2). In conclusion, the combined therapy with L-NNA and melatonin seems to have an additive effect and may be considered as a feasible candidate in attenuating the symptoms of major depressive disorder.

Kervansaray Convention Center & Hotel, LARA, ANTALYA / TÜRKİYE 79 International Symposium on Drug Research and Development

of natural products such as carbohydrates and prostaglandins [1]. A series of key precursors to the IMDA reaction of furane diene (2a–c) have been prepared via facile alkylation and protection. While the cycloaddition process for (3a–c) was afforded in hot toluene, a com-

PRESENTATIONS mercial microwave (2450 MHz) was used for the synthesis of (5a–b) (Figure 1). POSTER

Figure 1. The effects of saline, 1% alcohol, NG-nitro-L-arginine (L- NNA: 3, 10 and 30 mg/kg) and melatonin (3 and 10 mg/kg) on the duration of immobility (sec) in forced swim test. Values presented as mean ± SEM, n=10. * P<0.05 (versus control, saline or 1% alcohol), Figure 1. i. Br , Et N, DCM, 0°C, 98%; NaBH , CeCl .7H O, MeOH, ** P

2,3-dibromocyclohexene, K2CO3, THF, reflux, 3d; (Boc)2O, DMAP, DCM, 0°C, 2h.

Treatment of fused oxy- and thio-heterotricycles (5a–b) with bo- rontriluoride-etherate in dichloromethane at –78°C cleaved Epoxy- Bridge and concomitant aromatisation gave the isobenzo-furanol and thiophenol (6a–b) in 72–76% yields respectively (Figure 2) [2].

Figure 2. The effects of saline, 1% alcohol, NG-nitro-L-arginine (L-NNA: 3, 10 and 30 mg/kg) and melatonin (3 and 10 mg/kg) on the squares entered in locomotor activity test. Values presented as mean ± SEM, n=10. Figure 2. 1. Trullas R, Skolnick P. Functional antagonists at the NMDA receptor complex exhibit antidepressant actions, Eur. J. Pharmacol., 185, 1-10, 1. Lipshutz BH. Chem. Rev., 86, 795, 1986. Wang Q, Padwa A, Rh(I)-Cata- 1990. lyzed ring opening of an IMDAF-derived oxabicyclo cycloadduct as the 2. Harkin AJ, Bruce KH, Craft B, Paul IA. Nitric oxide synthase inhibitors key step in the synthesis of ±-epi-Zephyranthine, Org. Lett., 6, 2189, have antidepressant-like properties in mice. 1. Acute treatments are ac- 2004. tive in the forced swim test, Eur. J. Pharmacol., 372, 207-213, 1999. 2. Demircan A, Karaarslan M, Turac E.. A facile synthesis of heterotricycles 3. Karolewicz B, Paul IA, Antkiewicz-Michaluk L. Effect of NOS inhibitor from furfurylbromoalkenes using thermal IMDA cycloaddition. Hetero- on forced swim test and neurotransmitters turnover in the mouse brain, cyclic Commun., 3&4, 233, 2006. Karaarslan M, Göktürk E, Demircan A. Pol. J. Pharmacol., 53, 587-596, 2001. Thermal intramolecular Diels–Alder reaction of furan; Synthesis of ni- 4. Overstreet DH, Pucilowski O, Retton MC, Delangrange P, Guardiola-Le- trogen tetracycles, isobenzofuran and isobenzothiophene, J. Chem. Res., maitre B. Effects of melatonin receptor ligands on swim test immobility, 2, 117, 2007. Neuroreport, 9, 249-253, 1998. 5. Mantovani M, Pértile R, Calixto JB, Santos ARS., Rodrigues ALS. Me- latonin exerts an antidepressant-like effect in the tail suspension test in mice: evidence for involvement of N-methyl-D-aspartatereceptors and P 070 Ref: 0094 the L-arginine-nitric oxide pathway, Neurosci. Lett., 343, 1-4,2003. 6. Heiberg IL, Wegener G, Rosenberg R. Reduction of cGMP and nitric THE DETERMINATION OF ANTHOCYANINS oxide has antidepressant-like effects in the forced wimming test in rats, DELPHINIDIN AND PEONIDIN IN FRESH FRUITS BY HIGH Behav. Brain Res., 134, 479-484, 2002. PERFORMANCE LIQUID CHROMATOGRAPHY Nafiz Öncü CAN, Göksel ALTIOKKA Anadolu University, Faculty of Pharmacy, Department of Analytical Chemistry, Eskişehir, P 069 Ref: 0093 Turkey A UTILITY OF FURAN CORED COMPOUNDS; SYNTHESIS Anthocyanins constitute one of the major groups of natural pig- OF NITROGEN TETRACYCLES, ISOBENZOFURANOL AND ments and are responsible for many of the colours of fruits and ISOBENZOTHIOPHENOL vegetables [1]. It has been demonstrated that anthocyanins possess some positive therapeutic effects [2, 3] and are in use as nutritional Muhsin KARAARSLAN, Ersen GÖKTÜRK, Aydın DEMİRCAN supplements in many countries [4]. The aim of this study was to de- University of Niğde, Faculty of Sciences & Letters, Department of Chemistry, Kampus, velop a simple and precise procedure for the determination of two 51100 Niğde, Turkey anthocyanins, delphinidin and peonidin, and indicate their levels in Intramolecular Diels–Alder (IMDA) reactions involving furane as some fresh fruits consumed in Turkey. a diene form oxanorbornenes that have been used in the synthesis of 11 fruit samples, including pomegranate, cherry and grape, were numerous complex targets and as an intermediate in the synthesis examined using high performance liquid chromatography coupled

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to photodiode array detection. Two anthocyanins and cyanidin (in- thocyanins were resolved by reversed phase technique within about ternal standard) were separated using a gradient elution technique 16 minutes. Analytes were detected at 520 nm wavelength and mo- by a reversed phase column. Pumping the mobile phase at a flow bile phase was pumped through the column at a flow rate of 0.8 rate of 0.8 mL.min-1, analytes were detected at 520 nm wavelength mL.min-1. Samples were injected to the system directly, following a within an average time of 18 min. Samples were prepared from fresh simple filtration through 0.2 µm PVDF filters, without any extrac- PRESENTATIONS fruits by taking their juices with direct pressing which was followed tion or concentration steps. by filtration through 0.2 µm PVDF filters, and injected into the system with no further extraction or concentrating steps. POSTER

Figure 1. A typical chromatogram of grape juice sample (PG: Pelargonidin, MV: Malvidin, IS: Internal standard). Figure 1. A typical chromatogram of pomegranate fruit sample (DP: Delphinidin, PN: Peonidin, IS: Internal standard) Anthocyanins were detected in ppb levels with adequate chromatographic resolution. Linearities were obtained between the ranges The method was found to be linear between the ranges about 80 of 78.1 – 390.7 ppb for pelargonidin and 88.15 – 440.7 ppb for mal- – 400 ngmL-1, giving symmetrically sharp and repeatable analyte vidin. On the whole, malvidin and pelargonidin levels were found to peaks. The overall limits of quantitation (LOQ) were 58.4 and 50.1 be relatively high in grape products, such as wine and grape juice, in ngmL-1 for delphinidin (DP) and peonidin (PN), respectively. It was accordance with the previous studies on these compounds. observed that the amount of anthocyanins detected in fruits reach to the maximum due to freshness and exist especially in dark coloured Keywords: Pelargonidin, malvidin, food analysis, liquid chromatography fruits. 1. Bravo L. Polyphenols: chemistry, dietary sources, metabolism, and nutri- 1. Kong JM, Chia LS, Goh NK, Chia TF, Brouillard R. Analysis and biologi- tional significance. Nutr. Rev., 56, 317-333, 1998. cal activities of anthocyanins, Phytochemistry, 64, 923-933, 2003. 2. Dell’Agli M, Busciala A, Bosisio E. Vascular effects of wine polyphenols, 2. Dell’Agli M, Busciala A, Bosisio E. Vascular effects of wine polyphenols, Cardiovasc. Res., 63, 593-602, 2004. Cardiovasc. Res., 63, 593-602, 2004. 3. Kowalczyk E, Krzesinski P, Kura M, Szmigiel B, Blaszczyk J. Anthocy- anins in medicine, Pol. J. Pharmacol., 55, 699-702, 2003. P 072 Ref: 0097 4. Bravo L. Polyphenols: chemistry, dietary sources, metabolism, and nutritional significance, Nutr. Rev., 56, 317-333, 1998. RAPID DETERMINATION OF ACRYLAMIDE IN POTATO CHIPS BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY WITH DIODE ARRAY DETECTION P 071 Ref: 0096 Nafiz Öncü CAN, Göksel ALTIOKKA SCREENING OF ANTHOCYANINS PELARGONIDIN AND Anadolu University, Faculty of Pharmacy, Department of Analytical Chemistry, Eskişehir, MALVIDIN IN COMMERCIAL FRUIT JUICES USING LIQUID Turkey CHROMATOGRAPHY COUPLED TO DIODE ARRAY Acrylamide is one of the heat-generated food toxicants, which was DETECTION frequently found in various foodstuffs such as potato chips, biscuits and coffee [1, 2]. It has been demonstrated that acrylamide possesses Göksel ALTIOKKA, Nafiz Öncü CAN some neurotoxic and carcinogenic effects [3, 4] and is classified Anadolu University, Faculty of Pharmacy, Department of Analytical Chemistry, Eskişehir, in Group 2A as a possible carcinogen to humans by International Turkey Agency for Research on Cancer [5]. The aim of this study was to Commercial fruit juices are one of the most common consumed develop an inexpensive, simple and sensitive method for determi- food products that contain many naturally occurring compounds, nation of acrylamide and indicate acrylamide levels of some potato such as anthocyanins, which have therapeutic effects against cancer products produced in Turkey. and cardiovascular diseases [1, 2]. Development of a cheap and re- Acrylamide concentration of the samples was examined using producible method for the determination of two anthocyanins, pel- high performance liquid chromatography combined to diode ar- argonidin and malvidin, in fruit juices was introduced in this study. ray detection. Acrylamide and methacrylamide (internal standard) 8 samples of different fruit juices were examined using liquid were separated using isocratic elution technique by a reversed phase chromatography coupled to photodiode array detection. Utilizing column (GL Sciences, Inertsil® ODS-3). Mobile phase (acetonitrile: the sugar-free aglycone cyanidin as an internal standard, two an- water, 2:98, v/v) was pumped at 0.5 mL.min-1 flow rate and analytes

Kervansaray Convention Center & Hotel, LARA, ANTALYA / TÜRKİYE 81 International Symposium on Drug Research and Development

were detected at 200 nm wavelength within an average retention in many of the vegetables such as red cabbage and turnip [1]. An- time of 15 min. Samples were prepared by solid phase extraction and thocyanins, which act as colouring pigments in plants, have been injected into the system with no further extraction or pre-concentra- shown to be strong antioxidants with potential health benefits [2]. tion steps. These properties make these compounds preferable as nutritional

PRESENTATIONS supplements and possible therapeutic agents against cancer and cardiovascular diseases [3, 4]. The aim of this study was to investigate the levels of two major anthocyanins, cyanidin and petunidin, in vegetables using an improved liquid chromatographic method.

POSTER Anthocyanin content of some commonly consumed vegetables was determined using an ODS-3 reversed phase column (GL Sci- ences, Inertsil ODS-3, 3 µm, 150 x 4.6 mm) with excellent accuracy (recovery more than 99%) and precision (RSD% < 0.9). Both compounds were separated with adequate resolution factor which was higher than 3. Cyanidin and petunidin signals were found to be linear between the ranges of 80.8 – 404.0 ng/mL and 85.8 – 429.0 ng/mL, giving limit of detection levels of 7.9 and 9.1 ng/mL for cyanidin and petunidin, respectively. No intensive extraction steps were applied in order to keep the methodology as simple as possible; direct pressing was applied to the homogenized samples, and extracted juice was injected to the system after filtration. Figure 1. A typical chromatogram of homemade potato chip sample (AA: Acrylamide, MAA: Methacrylamide)

Acrylamide was detected in ppb levels with adequate chromatographic resolution. The developed method was validated according to the recommendations of International Conference of Harmoni- zation – Q2(R1) and system suitability parameters were tested according to United States Pharmacopeia. The method was found to be linear in the ranges of 2.9-14.2 ppb, giving LOD and LOQ concentrations of 0.93 and 2.82 ppb, respectively. As a result, it was observed that the amount of acrylamide in potato chips varies due to cooking conditions and was found to be relatively high in many of the samples.

Keywords: acrylamide, food analysis, liquid chromatography, Turkish foods

1. Tareke E, Rydberg P, Karlsson P, Eriksson S, Tornqvist M. Acrylamide: a Figure 1. A typical chromatogram of turnip sample (CY: Cyanidin, cooking carcinogen? Chem. Res. Toxicol., 13, 517-522, 2000. PT: Petunidin, IS: Internal standard) 2. Tareke E, Rydberg P, Karlsson P, Eriksson S, Tornqvist M. Analysis of acrylamide, a carcinogen formed in heated foodstuffs, J. Agric. Food Chem., Developed method was validated according to the recommenda- 50, 4998-5006, 2002. tions of International Conference of Harmonization. As a result, it 3. JECFA - Joint FAO/WHO Expert Committee on Food Additives, Sum- can be concluded that both anthocyanins exist at high levels espe- mary and conclusions of the sixty-fourth meeting, JECFA/64/SC, p. 7-17, World Health Organization WHO, Rome, Italy, 2005. cially in red cabbage and turnip, while there is no evidence was ob- 4. Shipp A, Lawrence G, Gentry R, McDonald T, Bartow H, Bounds J, Mac- served in tomato and red pepper. Further investigations are advised donald N, Clewell H, Allen B, van Landingham C. Acrylamide: Review to construct a general profile of anthocyanins in vegetables. of toxicity data and dose-response analyses for cancer and noncancer effects, Crit. Rev. Toxicol., 36, 481-608, 2006. 1. Bravo L. Polyphenols: chemistry, dietary sources, metabolism, and nutri- 5. IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, tional significance, Nutr. Rev., 56, 317-333, 1998. Volume 60: Some Industrial Chemicals, p. 389-433, International Agen- 2. Kong JM, Chia LS, Goh NK, Chia TF, Brouillard R. Analysis and biologi- cy for Research on Cancer (IARC),Geneva, Switzerland, 1997. cal activities of anthocyanins, Phytochemistry, 64, 923-933, 2003. 3. Kowalczyk E, Krzesinski P, Kura M, Szmigiel B, Blaszczyk J. Anthocy- anins in medicine, Pol. J. Pharmacol., 55, 699-702, 2003. 4. Dell’Agli M, Busciala A, Bosisio E. Vascular effects of wine polyphenols, P 073 Ref: 0098 Cardiovasc. Res., 63, 593-602, 2004. IMPROVED HIGH PERFORMANCE LIQUID CHROMATOGRAPHIC SEPARATION OF CYANIDIN AND PETUNIDIN IN FRESH VEGETABLES USING AN ODS-3 REVERSED-PHASE COLUMN Göksel ALTIOKKA, Nafiz Öncü CAN, Zeki ATKOŞAR Anadolu University, Faculty of Pharmacy, Department of Analytical Chemistry, Eskişehir, Turkey As a subgroup of the polyphenols family, anthocyanins are known as one of the best natural antioxidant compounds that can be found

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P 074 Ref: 0100 maintain normal concentrations of about 60 M in plasma It is the most common electroactive biological compound [1-4]. So, the elec- MOLECULAR MODELLING STUDIES OF trochemical oxidation of ascorbic acid has been carried out at glassy BENZO[α]CARBAZOLE DERIVATIVES AS ESTROGEN carbon electrode in various aqueous solution in the pH range of 0.64-

RECEPTOR INHIBITORS 10.15 (Britton-Robinson, acetat, phosphate buffers and 0.5 M sulfu- PRESENTATIONS Tu ğba TAŞKIN, Fatma SEVİN ric acide solution) by cyclic (CV) and differential pulse voltammetry Hacettepe University, Faculty of Science, Department of Cemistry, Ankara, Turkey (DPV). The best results were obtained for the quantitative determination of ascorbic acid by DPV technique in 0.2 M acetate buffer (

There have been some researches about a number of 5,6-dihydro- pH 3.49) at 0.342 V. The diffusion controlled nature of the peak was POSTER 11-alkylbenzo[α]carbazoles derivatives with one or two hydroxy established. This electroanalytical procedure enabled to determine groups in the aromatic rings for their binding affinities for the estro- ascorbic acid in the concentration range 8x10-6-8x10-4 M. The regres- gen receptor [1, 2]. According to analysis, the best conditions for the sion equation was obtained as Ip (µA) = 5.29 x10-3 C (M) + 0.035 (r receptor binding were provided by one hydroxy group at C-3 and a = 0.998). Limit of detection (LOD) and limit of quantitation (LOQ) second one at position 8 or 9. These results prompted us to elucidate were obtained as 5.16x10-7 M and 1.72x10-6 M, respectively. Based and visualize this experimental researches by using computational on this study, simple, rapid, selective, and sensitive DPV technique and molecular docking methods. was developed for the quantitative determination of ascorbic acid in Our conformatinal analysis consist of first generating conformers pharmaceutical dosage forms and some fruit juices. The proposed by using Gaussian 03 software and then optimizing with AM1, PM3, voltammetric technique was validated and good recoveries were ob- DFT-B3LYP/6-31G* methods. QSAR [3] is used to determine which tained in tablet dosage forms and some fruit juices. molecular descriptors preferences that can be rapidly computed and describe the structure, size, topology and other properties of a mol- 1. Erdurak-Kılıç CS, Uslu B, Doğan B, Özgen U, Özkan SA, Coşkun M. ecule. The most statistically significant descriptors like substituent Anodic voltammetric behaviour of ascorbic acid and its selective deter- hydrophobicity constant (π), electrophilicity index (ω) and heat of mination in pharmaceutical dosage forms and some rosa species of Tur- formation energy (HF) are selected using stepwise multiregression key, J. Analytical Chemistry, 61, 1113-1120, 2006. analysis (SMLR) to validate biochemical activity. According to the 2. Sweetman SC. in Martindale: The Complete Drug Reference, 33rd ed., results of analysis, the most fitting equation is determined to describe London: Pharmaceutical, 2002, p. 1389. the relationship between Log1/C and three independent variables for 3. Hardman JG, Limbird LE. Goodmann and Gilmann’s the pharmacological basis of therapeutics, 9th ed. [CD-ROM], New York: McGraw-Hill, each method. In addition, predicted biological activities have been 1996. compared with observed activities. 4. Pamuk F. Biochemistry (in Turkish), 1st ed. Ankara, Gazi press, 2000, Furthermore, to visualize the interactions with 5,6-dihydro-11- p.138-139. alkylbenzo[α]carbazole derivatives-binding sites on selected a-chain of estrogen receptor, ICM Pro.3.4. was performed. Results are used to compare physical and chemical properties in determining the P 076 Ref: 0102 conformational preference. Then the most stable conformers, 2 and 3 compounds were brought onto the ER and than the system are fully ONE-POT SYNTHESIS AND STRUCTURAL ELUCIDATION optimized allowing both systems interactions by using the potential OF SOME NEW BIGINELLI COMPOUNDS virtual screening of this method. The results are discussed in terms of the nature of the these compounds, ligands recognition on the İnci Selin DOĞAN, Selma SARAÇ estrogen receptor. Hacettepe University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 06100 Ankara, Turkey

1. Angerer E, Prekajac J. Journal of Medicinal Chemistry, 29, 3, 1986. 3,4-Dihydropyrimidin-2(1H)-ones(thiones) have been reported 2. Katzenellenbogen JA, Katzenellenbogen BS. Chem. Biol., 3, 529, 1996. to possess diverse pharmacological properties, such as antibacte- 3. Pahsa FA, Srıvastava HK, Singh PP. International Journal of Quantum rial, antiviral, antitumor, antiinflammatory, antihypertensive, cal- Chemistry, 104, 87–100, 2005. cium channel blocker, -1a-antagonist and neuropeptide Y (NPY) antagonist effects. The biological activity of some alkaloids isolated recently has also been attributed to the dihydropyrimidine moiety P 075 Ref: 0101 [1, 2]. Therefore, synthesis of these compounds is still of great interest. Earlier, we have described the synthesis and calcium antago- RAPID AND SENSITIVE VOLTAMMETRIC DETERMINATION nistic activity of some 5-acetyl-3,4-dihydro-6-methyl-4-(substituted OF ASCORBIC ACID IN TABLET DOSAGE FORMS AND phenyl)-2(1H)-pyrimidinone derivatives [3]. In continuation of our SOME FRUIT JUICES study, we aimed to report the synthesis of some new 5-acetyl–6-methyl-4-(substituted phenyl)-3,4-dihydropyrimidin-2(1H)-thiones 1Selehattin YILMAZ, 1Gökçe AŞKIN, 1Sultan YAĞMUR, 1Gülen TÜRKER, 1Gülşen SAĞLIKOĞLU, 1Hüseyin ERDUĞAN which are expected to have calcium channel blocker and antibacte- 1Çanakkale Onsekiz Mart University, Faculty of Arts and Sciences, Department of rial activities. Chemistry, 17020 Çanakkale, Turkey 2Çanakkale Onsekiz Mart University, Faculty of Arts and Sciences, Department of Biology, 17020 Çanakkale, Turkey Ascorbic acid, a water-soluble vitamin, is important in forming collagen, a protein that gives structure to bones, muscles, and blood vessels. Also it helps to take of ferrum into body. Ascorbic acid is one of the most ubiquitous vitamins ever discovered. Besides playing a paramount role as an antioxidant and free radical scavenger, it has been suggested to be an effective antiviral agent. Ascorbic acid is usually administered orally. Because of the notable loss of the in- fused ascorbic acid in the urine, daily doses of 200 mg are needed to

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The title compounds were prepared by using the Biginelli three- P 078 Ref: 0104 component cyclocondensation reaction of acetylacetone, substituted benzaldehyde and thiourea under strongly acidic conditions [4]. The STUDIES ON NEW OXIME ESTER DERIVATIVES reaction was carried out simply by stirring the mixture of the three OF NAFIMIDONE ALCOHOL AS POTENTIAL

PRESENTATIONS components dissolved in absolute ethanol with a catalytic amount ANTICONVULSANT COMPOUNDS of hydrochloric acid at room temperature. The solid products were 1Burcu SELİMOĞLU, 2Arzu KARAKURT, 1Sevim DALKARA isolated after cooling the reaction mixture. 1Hacettepe University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, The reactions were completed within 15-20 hours in 30%- 06100 Ankara, Turkey

POSTER 40% yields. The structures of the compounds were confirmed 2İnönü University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, by IR, 1H-NMR, 13C-NMR, mass spectra and elemental analysis. 44280 Malatya, Turkey The calcium channel blocker and antibacterial activities of the com- Nafimidone [1-(2-naphthyl)-2-(imidazol-1-yl)ethanone] is one of pounds are in progress. the two representatives of (arylalkyl)imidazole anticonvulsants [1] and nafimidone alcohol is a major and active metabolite of nafimidone. 1. Kappe CO. Biologically active dihydropyrimidones of the Biginelli-type Nafimidone oxime ethers also have anticonvulsant activities [2]. In – A literature survey, Eur. J. Med. Chem., 35, 1043-1052, 2000. this project we prepared ten new nafimidone oxime ester derivatives. 2. Zorkun İS, Saraç S, Çelebi S, Erol K. Synthesis of 4-aryl-3,4-dihydropyrimidine-2(1H)-thione derivatives as potential calcium channel blockers, These compounds (I-X) have been synthesized by esterification of Bioorg. Med.Chem., 14, 8582-8589, 2006. nafimidone alcohol with benzoyl and substituted benzoyl chlorides. 3. Yarım M, Saraç S, Ertan M, Batu ÖS, Erol K. Synthesis, structural elu- Total synthesis scheme starting from 2-acetylnaphthalene is given cidation and pharmacological properties of some 5-acetyl-3,4-dihydro- below: 6-methyl-4-(substituted phenyl)-2(1H)-pyrimidinones, Il Farmaco, 54, 359-363, 1999. 4. Biginelli P.Aldehyde-urea derivatives of aceto- and oxaloacetic acids. Gazz. Chim. Ital., 23, 360-416, 1893.

P 077 Ref: 0103 AN EXPERIMENTAL DESIGN APPROACH TO SELECTING THE OPTIMUM LIQUID CHROMATOGRAPHIC CONDITIONS FOR THE DETERMINATION OF LOCAL Their structural elucidation were realized by IR, 1H-NMR, Mass ANESTHETICS spectral data and elemental analysis.

1 2 Aysun DİNÇEL, Nursabah E. BAŞÇI 1. Walker KAM, Wallach BM, Hirschfeld RD. 1-(Naphthylalkyl)-1H-imi- 1Hacettepe University, Faculty of Medicine, Department of Pharmacology, Ankara, dazole Derivatives, a New Class of Anticonvulsant Agents. J. Med. Chem., Turkey 24, 67-74, 1981. 2Hacettepe University, Faculty of Pharmacy, Department of Analytical Chemistry, 2. Karakurt A, Dalkara S, Özalp M, Özbey S, Kendi E, Stables JP. Synthesis Ankara, Turkey of Some 1-(2-naphthyl)-2-(imidazole-1-yl)ethanone Oxime and Oxime A sensitive high performance liquid chromatographic (HPLC) Ether Derivatives and Their Anticonvulsant and Antimicrobial Activi- method has been developed and validated for the simultaneous ties, Eur. J. Med. Chem., 36, 421-433, 2001. determination of four local anesthetics: lidocaine, proparacaine, bupivacine and oxybuprocaine. A full factorial design was used. The Acknowledgement chromatographic separation was achieved using a Bondesil C (4.6 This project was supported by Hacettepe University Scientific Research 8 Fund, Project no: 06 D03 301 001. x 2.5 mm i.d., particle size 5 µm) analytical column. An optimised mobile phase consisted of acetonitrile and sodium dihydrogen phosphate (pH=3.0, 20 mM) (30:70, v/v) at a flow rate of 1.2 ml min-1. Local anesthetics detection was performed by UV/Vis detector at 220 nm. The retention times for lidocaine, proparacaine, bupivacine P 079 Ref: 0105 and oxybuprocaine 5.74, 9.28, 16.84 and 26.26 min, respectively. SEPARATION OF SEVEN QUINOLONES USING HPLC: -1 This method was linear in the range of 50-5000 ng ml for lidocaine DETERMINATION OF LEVOFLOXACIN IN PLASMA AND and proparacaine and 100-5000 ng ml-1 for bupivacine and oxybu- AMNIOTIC FLUID procaine. The limit of detection (LOD) was 25 ng ml-1 for lidocaine, proparacaine and 30 ng ml-1 for bupivacine and oxybuprocaine. The 1Emirhan NEMUTLU, 1Sedef KIR, 2Sinan BEKSAÇ limit of quantification (LOQ) was found to be 50 ng ml-1 for lido- 1Hacettepe University, Faculty of Pharmacy, Department of Analytical Chemistry, 06100 caine, proparacaine and 100 ng ml-1 for bupivacine, oxybuprocaine Ankara, Turkey (RSD ≤ 15 %, n=6). 2Hacettepe Unıversity, Faculty of Medicine, Department of Obstetrics and Gynecology, 06100 Ankara, Turkey Keywords: Local anesthetics, validation, experimental design and meth- A reversed-phase high-performance liquid chromatographic od optimization method is described for the separation and determination of seven quinolones (ciprofloxacin, levofloxacin, oxolinic acid, lomefloxacin moxifloxacin, enoxacin, and pefloxacin) in plasma and amniotic fluid. Chromatographic separation of the quinolones was performed on a Zorbax Eclipse XDB-C18 column (150 mm x 4.6 mm i.d.) in connec- tion to a Phenomenex C18 column (4 mm x 3.0 mm i.d.). The optimum assay conditions were found with experimental designs. Op- timum conditions were 15 mM citrate buffer, pH 3.2, 9 % MeCN, 5

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% MeOH, 5 mM TMAB, 1.5 mL min-1 flow rate and 40 °C column cies. The chloroform-soluble compounds extracted from Scor- temperature. Photodiode array detector was set to 280 nm. Marbo- zonera sandrasica were found to inhibit violacein production, a floxacin (MAR) was used as internal standard. The retention times QS-regulated behavior in C. violaceum which is used as a model of the quinolones in optimum conditions were 6.0 min for MAR, 6.7 system for QS inhibition studies. This extract was also able to in- min for enoxacin, 7.8 min for levofloxacin, 8.5 min for pefloxacin, 9.3 hibit QS-regulated carbapenem antibiotic production in an im- PRESENTATIONS min for ciprofloxacin, 11.2 min for lomefloxacin, 16.3 min for moxi- portant plant pathogen E. carotovora. In addition, some of these floxacin and 17.3 min for oxolonic acid. A simple and efficient solid plant materials such as Origanum onites L and Nigella sativa phase extraction was applied for sample preparation of plasma and have strong anti-bacterial effect against some bacterial species. amniotic fluid. The validation studies of the method for the analysis As the regulation of many bacterial processes is controlled by QS POSTER of levofloxacin was performed according to FDA guidelines and the systems, the finding of natural compounds acting as QS inhibitors linearity range was found as from LOQ to 30 µg mL-1. The LOD and suggests an attractive tool to control and handle detrimental infec- LOQ were 0.010 and 0.035 µg mL-1, respectively. The absolute recover- tions caused by human, animal and plant pathogens. ies from plasma and amniotic fluid were 98.0 and 95.9, respectively. In intra-day and inter-day studies, the developed method was found to 1. Hentzer M, Giskov M. Pharmacological inhibition of quorum sensing be accurate and precise with a bias value less than 2.6 % and the RSD for the treatment of chronical bacterial infections, J. Clin. Invest., 112, value less than 5 %. The robustness studies were performed with an 1300-1307, 2003. 2. Fuqua C, Parsek MR, Greenberg EP. Regulation of gene expression by experimental design. All validation data were showed that the method cell-to-cell communication: acyl-homoserine lactone quorum sensing, was accurate, precise, linear, robust, and specific. Finally, the method Annu. Rev. Genet., 35, 439-468, 2001. has been applied to quantification of levofloxacin in amniotic fluid 3. Finch RG, Pritchard DI, Bycroft BW, Stewart GSAB. Quorum sensing-a and plasma for investigation fetal transformation of levofloxacin. novel target for anti-infective therapy, J. Antimicrob. Chemother., 42:569- 571, 1998.

P 080 Ref: 0106 P 081 Ref: 0107 INVESTIGATION FOR NATURAL QUORUM SENSING INHIBITORS: ALTERNATIVE TO ANTIBIOTICS? DEVELOPMENT OF A CAPILLARY ZONE 1Gülgün BOŞGELMEZ-TINAZ, 1Seyhan ULUSOY, ELECTROPHORESIS METHOD FOR THE SIMULTANEOUS 1Fatma Filiz COŞKUN-ARI, 2Aysel UĞUR, 2Özgür CEYLAN ANALYSIS OF OLMESARTAN MEDOXOMIL AND 1Süleyman Demirel University, Faculty of Arts and Sciences, Department of Biology, HYDROCHLOROTHIAZIDE Isparta, Turkey Mustafa ÇELEBİER, Sacide ALTINÖZ 2Muğla University, Faculty of Arts and Sciences, Department of Biology, 480000 Muğla, Turkey Hacettepe University, Faculty of Pharmacy, Department of Analytical Chemistry, Ankara, Turkey As the bacterial resistance to multiple antibiotics increases, it is becoming more difficult to treat infections and, in many cases, the Olmesartan medoxomil is a selective AT1 subtype angiotensin II available therapeutic options are severely limited. Hence, there is a receptor antagonist [1]. Hydrochlorothiazide is a thiazide diuretic growing urgency to search for novel targets and the development of that is commonly used in combination with other antihypertensive new antimicrobials. Millions of dollars are devoted to antibiotics agents. It has been reported that the combination of olmesartan that almost immediately start to become less therapeutically useful medoxomil and hydrochlorothiazide is a safe, well tolerated, and ef- when they finally reach the marketplace. For this reason, pharma- fective option for antihypertensive therapy, demonstrating greater ceutical companies are now focusing on finding novel bacterial tar- blood pressure reduction than monotherapy [2]. In the literature, gets and new ways to control and eradicate bacterial infections [1]. there hasn’t been any method described for the simultaneous analy- To infect a host and cause a disease, bacteria produce an array of viru- sis of olmesartan medoxomil and hydrochlorothiazide. A capillary lence determinants that contribute to pathogenesis. It is now known that zone electrophoresis method with ultraviolet detection for the simul- many different Gram-negative pathogens communicate via production taneous analysis of olmesartan medoxomil and hydrochlorothiazide of small, diffusible N-acyl homoserine lactone (AHL) derivatives as in synthetic tablets was developed. A fused silica capillary (50 µm signaling molecules to coordinate virulence determinant production. internal diameter, 48.5 cm total length, 40 cm effective length) was This event is called “quorum sensing (QS)”. A variety of physiologi- used and the separation was obtained by 40 mM pH 9.5 borate buffer cal process in a range of bacterial species is regulated by QS, including followed by detection with an ultraviolet detector at 210 nm. The antibiotic biosynthesis, biofilm differentiation and the production of analysis were performed at 30 °C with the application of 3 seconds virulence determinants in animal and plant pathogens [2]. Since many hydrodynamic injection at 50 mbar pressure and a applied potential pathogens use QS to regulate virulence, strategies intended to interfere of 30 kV. Diflunisal was used as internal standard. The developed with signaling systems will likely have many potential applications. method was validated according to the literature [3]. Biotechnological research is now focused on the development of AHL antagonists. In medicine, usage of such molecules represents a novel therapeutic approach offering the opportunity to attenuate virulence, and thus infection, by blocking cell-to-cell communication [3]. This approach is highly attractive because it does not impose harsh selective pressure for the development of resistance as with antibiotics [1]. Various plant samples were screened for anti-QS activities using bioassays with Chromobacterium violaceum (ATCC 12472) and Erwinia carotovora. Plant materials were dried and extracted using chloroform, methanol and water. In this study, we tested 46 terrestrial plants materials for their ability to inhibit QS-regulated behaviors in different bacterial spe-

Kervansaray Convention Center & Hotel, LARA, ANTALYA / TÜRKİYE 85 International Symposium on Drug Research and Development

1. Mire DE, Silfani TN, Pugsley MK. A review of the structural and func- logical studies have been conducted on the roots of Paeonia species. tional features of olmesartan medoxomil, an angiotensin receptor block- Monoterpen glycosides are the active constituents for this plant spe- er, Journal of Cardiovascular Pharmacology, 46, 585-593, 2005. cies. Among the terpen glycosides, paeoniflorin (1) and oxypaeoni- 2. Steven GC, Michael AW, Antonia CW, Donald JH. Evaluation of antihy- florin (2) are the main compounds. pertensive therapy with the combination of olmesartan medoxomil and PRESENTATIONS hydrochlorothiazide, AJH, 17, 252–259, 2004. In the present study, reversed-phase high performance liquid 3. ICH Steering Committee. Text and Methodology Q2 (R1). Harmanized chromatographic (HPLC) method with diode array detection for the Tripartite Guideline, 2005. determination of the 1 and 2 has been developed. Compounds 1 and 2 were isolated from Paeonia species using chromatographic meth-

POSTER ods. The structure elucidations of 1 and 2 were achieved by combina- P 082 Ref: 0110 tion of 1D and 2D-NMR experiments and mass spectrometry. The HPLC separation was achieved on a Nucleosil 100-5 C18 (5 µm, 250 CYP1A2, CYP2A6, NAT2 AND XANTHINE OXIDASE x 4.6 mm) column using a mobile phase composed of acetonitrile:10 ACTIVITIES IN TURKISH VOLUNTEERS mM pH 3.5 phosphate buffer (20:80 v/v) at a flow rate of 1 mL min-1 . A wavelength of 230 nm for diode array detection was selected. The 1Aysun DİNÇEL, 2Nursabah E. BAŞÇI, 1Atila BOZKURT internal standard was metronidazol. 1Hacettepe University, Faculty of Medicine, Department of Pharmacology, 06100 Ankara, Turkey The retention times were 4.1 min for 1 and 7.8 min for 2. The 2Hacettepe University, Faculty of Pharmacy, Department of Analytical Chemistry, 06100 developed method was optimized by changing the chromatographic Ankara, Turkey parameters such as organic solvent ratio and pH of mobile phase. A RP-HPLC method has been developed for the determination Optimal conditions were selected by calculating capacity and tail- of caffeine metabolites in urine for the evaluation of the CYP1A2, ing factors of each peaks and by identifying the resolution for the CYP2A6, xanthine oxidase (XO) and N-acetyl-transferase-2 (NAT- separation. Low retention time, symmetric peak, high peak area and 2) activities in 101 Turkish volunteers (47 males and 54 females). high resolution were performed by optimized method. The validated Spot urine samples were analyzed 5 h after 100 mg caffeine con- method was found to be linear, accurate, precise, sensitive, rugged sumption. The urinary caffeine metabolites, 1,7-dimethylxanthine and robust. The linear ranges were 0.25-80 µg/ml for 1 and 0.25-60 (17X), 1,7-dimethyluric acid (17U), 1,3-dimethyluric acid (13U), µg/ml for 2. The developed method was found to be useful for de- 3-methylxanthine (3X), 1-methylxanthine (1X), 1-methyluric acid termination of compounds 1 and 2 in Paeonia species. By the help (1U), theobromine (37X), and 5-acethylamino-6-formylamino-3- this method, 3.47 %- 5.46 % paeoniflorin (1), and 0.049 % -0.422 % methyluracil (AFMU) were analysed. CYP1A2, CYP2A6, XO and oxypaeoniflorin (2) were found from 2 g of the crude samples of P. NAT-2 activities were estimated from the metabolic ratios (AFMU mascula, P. daurica, P. peregrina, P. tenuifolia, P. mascula subsp. bod- + 1U + 1X)/17U, 17U/17X, 1U/(1X + 1U) and AFMU/(AFMU + urii collected from the flora of Turkey. 1U + 1X), respectively. Urine samples were extracted with chloroform/isopropanol (85:15, v/v) and separated on a reversed phase P 084 Ref: 0114 C18 (4.6 x 2.5 mm i.d., particle size 5 µm) analytical column with acetic acid/tetrahydrofuran/acetonitrile/water (1:2.5:44:925, v/v/ STABILITY OF LIQUID AND SOLID FORMS OF v/v). Peaks were monitored with UV detection at 280 nm. The enzyme activities of CYP1A2, CYP2A6, NAT2 and XO were found as RECOMBINANT HUMAN INTERFERON 5.28±6.12, 0.22±0.11, 0.33±0.17, and 0.65±0.16 (mean±SD), respec- Bilgen ÖZBATIR, Filiz ÖNER tively. Smoking and gender were not affected CYP1A2, and CYP2A6 Hacettepe University, Faculty of Pharmacy, Department of Pharmaceutical activities. The developed RP-HPLC method was validated and suc- Biotechnology, 06100 Ankara, Turkey cessfully applied for the evaluation of CYP1A2, CYP2A6, XO and Due to the patient compliance problems of injectable formulations, NAT-2 activities. These results are comparable to those reported for alternative routes for peptide-protein drugs are under investigation Caucasian populations previously. in recent years. Mucosal route seems advantageous for administration of peptide drugs due to the direct systemic effect and avoidance Keywords: CYP1A2, CYP2A6, XO, NAT-2, HPLC, caffeine of first hepatic elimination [1]. The aim of this study was to develop a stable lyophilized solid lozenge form for mocosal application of rHuIFN-α2b which has a molecular mass of 19.269 kDa and has a P 083 Ref: 0112 significant therapeutic potential in treatment of some cancers and infectious diseases. DETERMINATION OF PAEONIFLORIN AND Studies on the therapeutic effects of low dose oral mucosal for- OXYPAEONIFLORIN IN PAEONIA SPECIES USING mulations of interferon present promising results in some references VALIDATED HPLC METHOD [2]. In this study we prepared a solid lozenge form and liquid form 1Semra KOYUNOĞLU, 2Sedef KIR, 3Ali A. DÖNMEZ, 4İhsan ÇALIŞ of rHuIFN-α2b (recombinant human interferon alfa 2b) and exa- 1Hacettepe University, Faculty of Pharmacy, Department of Basic Pharmaceutical mined the stability of the active substance in vitro by comparing with Sciences, 06100 Sıhhiye-Ankara, Turkey the commercial product. Composition of the formulated forms of 2Hacettepe University, Faculty of Pharmacy, Department of Analytical Chemistry, 06100 interferon alpha is shown in Table I. Sıhhiye-Ankara, Turkey Experiments were performed in an artificial saliva medium (pH 3Hacettepe University, Faculty of Science, Department of Biology, 06100 Sıhhiye-Ankara, 6.7) in order to simulate the medium in the oral cavity. Liquid and Turkey solid forms were kept at 4oC and 25oC for 1 and 4 months and the 4Hacettepe University, Faculty of Pharmacy, Department of Pharmacognosy, 06100 Sıhhiye-Ankara, Turkey amount of active substance was determined quantitatively by HPLC and qualitatively by SDS-PAGE. No degradation product was found Paeonia L., the largest genus in the family Paeoniacea, is repre- and rHuIFN-α2b was kept stable for 1 month at both 4oC and 25oC sented by 7 species in the flora of Turkey. Paeonia Radix is one of the temperatures. most important herbal drugs in traditional Chinese medicine, and as well as in different countries. Significant chemical and pharmaco-

86 May 17-20, 2007 “From Chemistry to Medicine” DRD 2007

P 086 Ref: 0118 Table I. Composition of the liquid and solid interferon formulations used in the study ANTIMICROBIAL ACTIVITY OF 3-HYDROXY-6-METHYL-2- SUBSTITUTED 4H-PYRAN-4-ONE DERIVATIVES Components 1Ekrem KILIÇ, 2Mutlu DİLSİZ-AYTEMİR PRESENTATIONS Liquid formulation Solid formulation 1Hacettepe University, Faculty of Pharmacy, Department of Pharmaceutical rHuIFN-α2b rHuIFN-α2b Microbiology, 06100 Sıhhiye-Ankara, Turkey 2 Sodium phosphate dibasic anhydrous Gelatin Hacettepe University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry,

06100 Sıhhiye-Ankara, Turkey POSTER Sodium phosphate monobasic monohydrate Glycerol Over the past several years the emergence of organisms resistant Disodium EDTA Sucrose to nearly all the class of antimicrobial agents has become a serious Polysorbate 80 (Tween 80) Water for injection public health concern. For these reasons the research of new antimicrobial agents with novel modes of action represents a main target in Phenol chemotherapy. It has been shown that hydroxypyran-4H-one deriva- Sodium chloride tives have antimicrobial activity [1, 2]. In our previous studies, we Water for injection. synthesized some Mannich bases of 3-hydroxy-6-methyl-4H-pyran- 4-one derivatives and investigated their antimicrobial activity [3].

1. Shojaei AH. Buccal mucosa as a route for systemic drug delivery: A review, J. Pharm. Pharmaceut. Sci.,1(1), 15-30, 1998. 2. Ship JA., Fox PC, Michalek JE, Cummins MJ, Richards AB. Treatment of pimary Sjögren’s syndrome with low-dose natural human interferon- α administered by the oral mucosal route: a phase II clinical trial, Journal of Interferon and Cytokine Research, 19, 943-951, 1999.

Antifungal activities of the compounds evaluated in vitro against P 085 Ref: 0116 fungi such as Candida albicans (ATCC 90028), C. krusei (ATCC 6258) and C. parapsilosis (ATCC 90018). They were also tested HPLC-ECD DETERMINATION OF EPINEPHRINE PLASMA against Gram-positive bacteria such as Staphylococcus aureus (ATCC CONCENTRATIONS IN VARIOUS DENTAL PATIENTS 29213), Enterococcus faecalis (ATCC 29212) and Gram-negative bacteria such as Escherichia coli (ATCC 25922), Pseudomonas aeruginosa 1Şeyda DEMİRCAN, 2Ayşe Gül AKALIN, 1Filiz SAYIN, 2Gökçe MERAL, 2Ferda TAŞAR, 1Sedef KIR, 1Nursabah E. BAŞÇI (ATCC 27853) by using microdilution broth method recommended by National Committee for Clinical Laboratory Standards (NCCLS) 1Hacettepe University, Faculty of Pharmacy, Department of Analytical Chemistry, 06100, Sıhhiye-Ankara, Turkey [4, 5]. The minimum inhibitory concentrations (MIC) were defined 2Hacettepe University, Faculty of Dentistry, Department of Oral Surgery, 06100-Sıhhiye- as the lowest concentrations of the compounds that prevented visible Ankara, Turkey growth. Fluconazole and Ampicillin were used as the standards for A sensitive and reliable reversed-phase high performance liquid antifungal and antibacterial tests, respectively. The screening data chromatographic (RP-HPLC) method with electrochemical detector indicates that compound 5 (MIC: 16 µg/ml), which was carrying (4-c (ECD) has been developed for the determination of local anesthetic hlorophenyl)phenylmethyl substituent showed antibacterial activity agent added epinephrine in plasma obtained during dental surgery. against S. aureus. The other compounds had no valuable inhibitory For this aim, the changes of plasma levels of epinephrine have been activity. Compound 5 (MIC: 16 µg/ml) was also the most effective measured in the blood samples taken at five different times (0, 3, 15, compound towards C. albicans than the others (MIC: 32-64 µg/ml). 30 and 60 minutes) after administration of the local anesthetic. The method was based on the use of a Nucleosil 100-5 C (5 µm, 250 x 1. Aytemir MD, Erol DD, Hider RC, Özalp M. Synthesis and evaluation 18 of antimicrobial activity of new 3-hydroxy-6-methyl-4-oxo-4H-pyran-2- 4,6 mm i.d.) as analytical column with a mobile phase containing -1 carboxamide derivatives, Turk J. Chem., 27(6), 757-764, 2003. methanol: 0.1 mol L citrate buffer (10:90, v/v, pH 2.5) with a flow 2. Kayahara H, Shibata N, Tadasa K, Kotani T. Amino acid and peptide de- -1 rate of 1.2 mL min . Electrochemical detector was set to 800 mV. rivatives of kojic acid and their antifungal properties Agric. Biol. Chem., Retention times of epinephrine and isoprotrenole (internal standard, 54(9), 2441-2442, 1990. IS) were 8.0 and 19.0 min, respectively. The plasma assay was vali- 3. Aytemir MD, Çalış Ü, Özalp M. Synthesis and evaluation of anticonvul- dated for the parameters such as specificity, accuracy and extraction sant and antimicrobial activities of 3-Hydroxy-6-methyl-2-substituted recovery and was applied to three different patient groups. The re- 4H-pyran-4-one derivatives. Arch. Pharm. Pharm. Med. Chem. 337, 281- covery of epinephrine after extraction from spiked plasma was 97 ± 288, 2004. 4. National Committee for Clinical Laboratory Standards (NCCLS), Meth- 0.03 % (mean±SD). The method was linear over the range of -1 2 ods for dilution antimicrobial susceptibility tests for bacteria that grow 15-200 ng mL (r = 0.9992). The detection limit (LOD) was found aerobically, Approved Standard M7-A, 37(2), Villanova, PA. (1997). -1 to be 5 ng mL (signal-to-noise ratio = 3) and the quantitation limit 5. National Committee for Clinical Laboratory Standards (NCCLS), Refer- (LOQ) was 15 ng mL-1 for epinephrine. The described HPLC-ECD ence method for broth dilution susceptibility testing of yeast, Approved method is simple, selective and can successfully be applied with high Standard M27-A, 17(9) Villanova, PA. (1997). degrees of precision and accuracy for the quantitative determination of epinephrine in plasma samples. The pharmacokinetic profiles for epinephrine in the healthy young, healthy older and ASA II CVS diseased older dental patients after the administration of local anesthe- sia with epinephrine were obtained.

Keywords: Epinephrine; HPLC; Electrochemical Detector (ECD); Vali- dation; Plasma; Dental Patients

Kervansaray Convention Center & Hotel, LARA, ANTALYA / TÜRKİYE 87 International Symposium on Drug Research and Development

P 087 Ref: 0119 4. Power MB, Hackett LP, Dusci LJ, Ilett KF. Antidepressant toxicity and the need for identification and concentration monitoring in overdose. Clin INHIBITION OF RAT LIVER MONOAMINE OXIDASE (MAO)- Pharmacokin 29: 154-171, 1995. A AND –B BY GENTIANELLA CAUCASEA (Loddiges ex Sms) 5. Haraguchi H, Tanaka Y, Kabbashi A, Fujioka T, Ishizu T, Yagi A. Mono- amine oxidase inhibitors from Gentiana lutea. Phytochemistry 65: 2255- PRESENTATIONS HOLUB EXTRACTS 2260, 2004. 1Tayfun ERSÖZ, 1Zeliha Ş. AKDEMİR, 2Samiye YABANOĞLU, 6. Pritchard NM, Gentianella “in Flora of Turkey and the East Aegean Is- 1, 3Funda Nuray YALÇIN, 1Duygu KAYA, 4İ. İrem ÇANKAYA, 2Gülberk UÇAR lands “(Ed. Davis PH) University Press, Vol 6 Edinburg, 1978. 1Hacettepe University, Faculty of Pharmacy, Department of Pharmacognosy, 06100 7. Suzuki O, Katsumata Y, Oya M, Chari VM, Vermes B, Wagner H, Hos-

POSTER Ankara, Turkey tettmann K. Inhibition of type A and B monoamine oxidases by naturally 2Hacettepe University, Faculty of Pharmacy, Department of Biochemistry, 06100 Ankara, occuring xanthones Planta Med 42: 17-21, 1981. Turkey 8. Holt A, Sharman DF, Baker GB, Pelcic MM. Continuous spectrophoto- 3Hacettepe University, Faculty of Pharmacy, Department of Pharmacy Management, metric assay for monoamine oxidase and related enzymes in tissue ho- 06100 Ankara, Turkey mogenates. Anal Biochem 244: 384-392, 1997. 4Hacettepe University, Faculty of Pharmacy, Department of Pharmaceutical Botany, 06100 Ankara, Turkey P 088 Ref: 0120 Monoamine oxidase (MAO), which is found in two forms designated as MAO-A and -B, plays an essential role in the metabolism of NEW MOLECULARLY IMPRINTED MICROSPHERES FOR the biogenic amines [1]. MAO-B inhibitors are shown to be useful COLONIC DELIVERY OF 5-AMINOSALICYLIC ACID in the treatment of Parkinson’s and Alzheimer’s diseases [2] while Yel iz TUNÇ, Ersin BAYKARA, Kezban ULUBAYRAM MAO-A inhibitors are known as antidepressant and antianxiety Hacettepe University, Faculty Pharmacy, Department of Basic Pharmaceutical Sciences, agents [3]. Since severe side-effects have been observed with some Ankara, Turkey classical MAO inhibitors [4], new inhibitors devoid of these adverse Molecular imprinting has become an increasingly active field of effects are needed. The presence of plant-derived MAO inhibitors study for the construction of new highly stable molecularly imprin- suggests that such plant extracts might be useful as potential neu- ted polymers (MIPs) that possess selective molecular recognition roprotectans in the treatment or prevention of depression, psychosis, properties [1]. MIPs have a large number of potential applications schizophrenia, Alzheimer’s and Parkinson’s diseases [5]. such as seperation process, immunoassays and antibody mimics, Gentianella caucasea is a biennial stem to 30 cm grown in North- biosensor recognition elements, and catalysis and artifical enzymes ern and Northeastern Anatolia [6]. Preliminary works on Gen- [2]. Recently, researchers have applied the molecular recognition tianella caucasea showed the presence of secoiridoid, flavonoid and properties of imprinted polymers for enhanced control in the release xanthone compounds in the chemical composition. Xanthones are of pharmaceutical compounds [3]. known to possess MAO inhibitor activity [7].The present study was In this study, feasibility of molecularly imprinted polymeric mi- designed to investigate the MAO inhibitory activities of the metha- crospheres (MIPs) has been investigated for colonic delivery of 5- nol, petrolum ether, chloroform and the remaining water extracts aminosalicylic acid (5-ASA), an active agent used in treatment of prepared from the aerial parts of Gentianella caucasea. ulcerative colitis and Crohn’s disease. For the first time, 5-ASA im- MAO was purified from the rat liver and MAO-A and –B activities printed microspheres were prepared by a single step precipitation were determined according to a previous method [8]. Kinetic data polymerization of 2-(diethylamino) ethyl methacrylate (DEAEMA; for interaction of the enzyme with the abstracts were determined us- functional monomer) and trimethylolpropane trimethacrylate ing Microsoft Excel package program. IC values were determined 50 (TRIM; crosslinker). The microspheres were prepared in the diame- from plots of inhibitory activity percentage, calculated in relation ter range between 1.2 and 2.7 by varying polymerization conditions. to a sample of the enzyme treated under the same conditions wit- The release characteristics of 5-ASA imprinted and non-imprinted hout extracts, versus extract concentration. All Gentianella extracts microspheres were determined and molecular imprinting effect tested inhibited rat liver MAO isoforms in a dose-and time-depen- on release behaviors was evaluated. We present a precipitation po- dent manner. Incubation of the enzyme with the extracts at 37 0C lymerization method for preparing uniform molecularly imprinted for 60 min. increased the inhibitory action of the extracts. Metha- microspheres in micron range, quickly and cleanly. Monodisperse nol extract inhibited MAO-B isoform selectively with IC value of 50 polymer particles with good spherical shapes and smooth surfaces 38.2±2.26 µg dry weight/mL whereas chloroform, petrolum ether, were obtained. Furthermore, the results show that the imprinted mi- and water extracts inhibited MAO-A selectively with IC values of 50 crospheres have a slower 5-ASA release in the initial stages than the 58.7±4.31, 64.33±5.06 and 102.13±7.55 µg dry weight/mL, respec- non-imprinted microspheres, because of the interaction of the drug tively. Xanthones of Gentianella caucasea may be a possible source molecules with the recognition sites in the imprinted microspheres. of pharmaceuticals for the treatment and prevention of depression, This result indicates that molecular imprinting may have a potential Parkinson’s and Alzheimer’s diseases. Phytochemical study on the for controlled delivery of drugs. title plant is carrying on. 1. Tunç Y, Hasırcı N, Yeşilada A, Ulubayram K. Comonomer effects on 1. Loscher W, Lehman H, Teschendorf H, Traut M, Gross, G. Inhibition of binding performances and morphology of acrylate-based imprinted monoamine oxidase type A, but not type B, is an effective means of in- polymers, Polymer, 47, 6931-6940, 2006. ducing anticonvulsant activity in the kindling model of epilepsy. J Phar- 2. Zhang H, Ye L, Mosbach K. Non-covalent molecular imprinting with macol Exp Ther 288: 984-992, 1999. emphasis on its application in separation and drug development, J. Mol. 2. Uçar G, Gökhan N, Yeşilada A, Bilgin A. 1-N-substituted thiocarbamoyl- Recognit., 19, 248-259, 2006. 3-phenyl-5-thienyl-2-pyrazolines: A novel cholinesterase and selective 3. Pepppas, N.A, Intelligent therapeutics: Biomimetic systems and nanote- monoamine oxidase-B inhibitors for the treatment of Parkinson’s and chnology in drug delivery, Adv. Drug Del. Rev., 56, 1529-1531, 2004. Alzheimer’s diseases. Neurosci Lett 382: 327-331, 2005. 3. Ertuğrul A, Uçar G, Başar K, Demir B, Yabanoglu S, Uluğ B. Influence of clozapine on platelet serotonin, monoamine oxidase and plasma serotonin levels. Pscyhiatr Res 149: 49-57, 2007.

88 May 17-20, 2007 “From Chemistry to Medicine” DRD 2007

P 089 Ref: 0121 tions on platelets aggregation were investigated in vitro using adenosine-5’-diphosphate (ADP) and arachidonic acid (AA) as agonists. BIOLOGICALLY ACTIVE COMPOSITE SCAFFOLD FOR The volatile oil of coriander seeds and the ethanolic extracts of TISSUE ENGINEERING APPLICATIONS leaves of coriander and parsley exhibited inhibitory effects on plate- 1Sinan GÜVEN, 2Nesrin HASIRCI, 3Sevda MÜFTÜOĞLU, let aggregation induced by ADP and AA in dose response manner. PRESENTATIONS 1Kezban ULUBAYRAM The effects of aspirin and dimethylsulfoxide (DMSO) on platelet 1Hacettepe University, Faculty of Pharmacy, Department of Basic Pharmaceutical aggregation induced by ADP and AA were also studied. It was found Sciences, Ankara, Turkey that Aspirin exhibited a strong inhibitory effect on platelet aggrega- 2 Middle East Technical University, Faculty of Arts and Sciences, Department of tion induced by ADP or AA, while DMSO exhibited an inhibitory POSTER Chemistry, Ankara, Turkey effect on platelets. 3 Hacettepe University, Faculty of Medicine, Department of Histology and Embrology, A combination of aspirin with coriander and parsley extracts was Ankara, Turkey studied in terms of its effect on platelet aggregation. İt was found that Tissue engineering plays a vital role in regenerative medicine in the combination of aspirin and coriander seed’s volatile oil extract order to create new tissues and organs from cultured cells for trans- were approximately additive effect on platelet aggregation. plantation [1]. In scaffold oriented tissue engineering, the scaffolds The effects of coriander and parsley extracts on blood coagulation should mimic the structure and biological function of native extra- were also investigated. The effects of plant preparations were inves- cellular matrix which provide mechanical support and regulate cell tigated by determination of their effects on activated partial thromo- activities [2]. plastine time (APTT). None of tested extracts affected prothrombine In the present study biologically active composite scaffolds were time (PT) values. developed from natural polymers by tissue engineering approach and As a result, the coriander’s seeds volatile oil and parsley leaves tested in vitro. Freeze-dried scaffolds composed of chitosan, gelatin extracts are of the interest for open new approaches towards expla- and dermatan sulfate were treated with different stirring rates, freez- nation of the beneficial effects on several manifestations of cardio- ing temperatures and molding. Among the prepared scaffolds at dif- vascular diseases like hypertension, arteriosclerosis and ischemic ferent conditions, scaffolds (SC) prepared at 500 rpm and frozen at - diseases and it could be assessed as useful natural source of pharma- 80°C were chosen for further studies. These scaffolds achieved 0.512 ceutical agents. MPa tensile strength, 9.165 MPa tension modulus and 3.428 MPa compression modulus. Besides in lysozyme containing degradation medium they conserved their integrity and lost about 30% of their initial weight in 30 days period. Mechanical and enzymatic degradation tests showed that scaffolds have physical integrity for the tissue engineering applications. To mimic the natural tissue and enhance cell growth, biologically active arginine-glycine-aspartic acid-serine (RGDS) peptides and platelet derived growth factor-BB (PDGF-BB) were immobilized on selected scaffolds. Fibroblast cells were seeded on the scaffolds containing RGDS, (SC-RGD), and PDGF-BB, (SC- RGD-GF), and incubated in media either free of serum or containing serum. Scaffolds immobilized with RGDS and PDGF-BB had the highest attached cell number by the day 15. According to scanning electron microscopy (SEM) results, cells on modified scaffolds demonstrated better cell morphology and attachment. Based on the obtained results, it can be concluded that RGDS-PDGF immobilized chitosan-gelatin-dermatan sulfate systems have a great potential to be used as a scaffold for soft tissue engineering applications.

1. Langer R, Vacanti JP, Tissue Engineering, Science, 260, 920-926, 1993. 2. Marler JJ, Upton J, Langer R, Vacanti JP. Transplantation of cells in matrices for tissue regeneration, Advanced Drug Delivery Reviews, 33, 65–182, 1998.

P 090 Ref: 0122 EFFECT OF CORIANDER (Coriander sativum L) AND PARSLEY (Petroseleinum sativum Hoffm.) EXTRACTS ON PLATELET AGGREGATION AND BLOOD COAGULATION Muhammad Jamal SHAMMOUT University of Jordan, Analytical Toxicology, Amman, Jordan Certain herbs may cause pharmacological effects on some cardiovascular aspects such as platelet aggregation and blood coagulation during homeostasis processes. In this study, the volatile oils and crude extracts of Coriander (Coriander sativum L) and Parsley (Pet- roseleinum sativum Hoffm.) were isolated using steam distillation and heating under reflux techniques. The effects of herb’s prepara-

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Author Index

“From Chemistry to Medicine” DRD 2007

A SARISÖZEN Can 78 GEYİKOĞLU, Fatime 51, 52 HASIRCI, Nesrin 89 KAZAZ Cavit 57, 59, 71 ARSLAN, Fatma 77 ARDAĞ-AKDOĞAN, Hatice 51 AKALIN, Ayşe Gül 87 CROOKS, Peter A. 35 HAMURCU Fatma 63, 77 KAPLAN-CAN, Hatice 58, AKDEMİR, Zeliha Ş. 88 AUTHOR INDEX ÇAKIR, Ahmet 71, 73 SEVİN Fatma 83 66, 67 YEŞİLADA, Akgül 56 ODABAŞOĞLU Fehmi 71, 73, AYGÜN, Hayati 71, 73 ŞEN, Alaattin 51 D 74, 75 ERDEM-YURTER, Hayat 48 ASLAN, Ali 71 SALMAN, Demet 62 ŞAHİN, Fikrettin 58, 61 ERDUĞAN, Hüseyin 83 GÜNER, Ali 58, 66, 67 DEMİR, Ayhan S. 36 COŞKUN-ARI, Fatma Filiz 85 YALÇINKAYA, Hüseyin 55 HAYAT, Ali 64, 65, 66 DEMİRCAN, Şeyda 87 FILLET, M. 14 KILINÇ, Ali 71 I DENİZLİ, Adil 62 ÇAĞLAR, Fulya 43, 44 ONUR, Mehmet Ali 66 CAN, Özgür Devrim 56 MORAL, Fulya 53 ÇALIŞ, İhsan 72, 86 KARAKURT, Arzu 84 DAYANGAÇ-ERDEN KARTAL, Funda 71 IŞIKDAĞ, İlhan 56, 57 MERİÇ, Asiye 70 Didem 48 BABA, Füsun 64, 65, 66 GÜL, H. İnci 47, 57, 58, 59, TÜMER, Aşkın 67 60, 61 EROL, Dilek 52, 55 TEMAMOĞULLARI, Füsun 64, ÖZTÜRK-ÇAL, Aslı 61 SADAK, Dilek 55 65, 66 DİLER, Zeynep İrem 55 BOZKURT, Atila 86 TURGUT-BALIK, Dilek 53, 55 İŞÇİ, Hüseyin 46 G HINCAL, A. Atilla 78 YURDAKUL, Dilşat 58 İŞGÖR, S. Belgin 46 YILDIZ, Attila 69 DİLSİZ-AYTEMİR, Mutlu 72, BORA, Gamze 48 DEMİRCAN, Aydın 80 87 GEURTS, P. 14 J DEMİR, Ayhan 48 DÖNMEZ, Ali A. 86 GIMÉNEZ, E. 8 J. LINHARDT, Robert 54 GÜRPINAR, Özer Aylin 66, 67 ÖZER-ÜNAL Durişehvar 55 AŞKIN, Gökçe 83 BARBOSA , Jose 68, 70 HASEGELİ-ÜNER, Ayşegül 58 SADİ, Gökhan 56 K UĞUR, Aysel 85 E ALTIOKKA, Göksel 80, 81, 82 DİNÇEL, Aysun 84, 86 BAŞÇI, Nursabah E. 62, 84 SUNAL, Sevil Görkem 56 ÖZTÜRK, Taylan K. 71 CRISS, Wayne E. 60 YALÇIN, Görkem 54 KÜÇÜKOĞLU, Kaan 60 B ÇUBUK-DEMİRALAY, Ebru 63 GREF, Ruxandra 18 KABANOV, A. V. 39 BARBOSA, J. 8 KONDOLOT-SOLAK, Ebru 62 ŞAHİN-KOÇ, Gülay 55 PEKMEZ, Kadir 69 BAŞÇI, Nursabah E. 86, 87 METE Ebru, 57, 58, 59 YELKEN, Gülay 76 KANSU, Emin 3 GÜLBAKAN, Basri 43, 61 GÜRDAL, Ece 52 UÇAR, Gülberk 47, 56 KAYA, Duygu 88 BAYKARA, Ersin 88 KÜÇÜK, Ekrem 79 TÜRKER, Gülen 83 YELEKÇİ, Kemal 48 BAYRAM, Hakan 25 AKSÖZ, S. Elif 53 ALSANCAK, Güleren 62, 68, KILIÇ, Ekrem 87 SALİH, Bekir 43, 60, 61 ÇADIRCI, Elif 73, 74, 75 70 KIR, Sedef 87 BENAVENTE, F. 8 NEMUTLU, Emirhan 84 BOŞGELMEZ-TINAZ, L ARICA, Betül 78 ERCAN, Ayşe 47 Gülgün 85 ÇEKEN, Bircan 52 YEŞİLADA, Erdem 72 SAĞLIKOĞLU, Gülşen 83 DOĞAN, A. Lale 58 BRASH, Douglas 47 ERİLHAN, İsmail 46 ÇOBAN, Güneş 67 UZUN, Leyla 78 ATMIŞ, Bünyamin 55 GÖKTÜRK, Ersen 80 ERGÜN, Ufuk Güney 79 LOUIS, Ed. 14 SELİMOĞLU, Burcu 84 ERSÖZ, Tayfun 88 GÜNDÜZ, Güngör 68 BELTRAN, Jose Luis 63, 68, 70 ŞAHİN, Ertan 59 ÖZBAKIŞ-DENGİZ, C Günnur 74, 75 M AKBAY, Esin 66, 67 GÜVEN, Sinan 89 MALAISE, M. 14 C. ÇEHRELİ, Zafer 67 GÜÇ, Esra 68 ÇALIŞ, Ünsal 72 MARCO, José Luis 4 KÜPELİ, Esra 72 H ÖZSÖZ, Mehmet 54 ÖZELGÜL, Canan 58 İŞLEYEN, Evren 65 SEÇİLMİŞ, Hale 63 ÜNALEROĞLU, Canan 53 SERİN, Mehmet 76, 77 SÜLEYMAN, Halis 74, 75 ÇANKAYA, İ. İrem 88 F GÖKÇE, Mehtap 76, 77 SEVİM, Handan 66, 67 TAŞAĞIR, Cansel 60 ATALAY, Fadime 73 MERAL, Gökçe 87 TÜRKEZ, Hasan 51, 52

Kervansaray Convention Center & Hotel, LARA, ANTALYA / TÜRKİYE 93 International Symposium on Drug Research and Development

KÖKSAL, Meriç 53 ŞAHİN, Pınar 72 YALÇIN, Talat 61 ATKOŞAR, Zeki 82 MERVILLE, M-P. 14 PİŞKİN, Erhan 38 TAŞAR, Ferda 87 TOPÇU, Zeki 67 HALICI, Mesut 71, 73, 75 TAŞGIN, Dilek Işık 53 ATEŞ, Zeliha 64 AUTHOR INDEX MEUWIS, M-A. 14 S ÖNKOL, Tijen 77, 78 ERDEMGİL, Zerrin 68 YARIM Mine 52, 53 DOĞRUER, Deniz S. 76 YALÇINKAYA, Timuçin 43 İNCESU, Zerrin 57 KAVANOZ, Muammer 69 ALTINÖZ, Sacide 85 TAŞKIN, Tuğba 83 ŞEN, Müberra 65 ŞAHİN, Fethi 28 GÜRAY, Tülin 56 MÜFTÜOĞLU, Sevda 89 YABANOĞLU, Samiye 47, 56 TUNÇ, Yeliz 88 KARAARSLAN, Muhsin 80 EREN, Sami 64 ÖZDEN, Tuncel 64, 65 ÇİZMECİOĞLU, Murat 54, 59 SANYAL, Rana 45 KIZIL, Murat 52 SANZ, Elisenda 4 U ATALAY, Mustafa 60, 61 SANZ-NEBOT, V. 8 UÇAR, Gülberk 88 ÇELEBİER, Mustafa 85 SAYIN, Filiz 87 GÜNDÜZ, Ufuk 68 GÜL, Mustafa 60, 61 TEZCAN, Seda 76, 77 BÜYÜKDEMİRCİ, Uğur 44 KIR, Sedef 84, 86 ULUBAYRAM, Kezban 88, 89 N ADAK, Selcen 76 TOSUN, Ali Ulvi 77 BAYTAŞ, Sultan N. 54 ALPAN, A. Selcen 67 DEMİR, Ümide 56 GÖKHAN-KELEKÇİ, Nesrin 47 YILMAZ, Selehattin 83 ÖZDEMİR-ÖZMEN, ÖZÇİÇEK-PEKMEZ, Nuran 69 YILDIRIM, Ömer Selim 71 Ümmühan 63, 77 EZER, Nurten 72 DOĞAN, İnci Selin 83 AKKAYA, Engin Umut 45 ŞANLI, Nurullah 62, 68 ÖZİLHAN, Selma 64, 65 SALGIN-GÖKŞEN, Umut 47 SARAÇ, Selma 83 ÜNAL, Serhat 20 O CALIŞ, Sema 78 ÇALIŞ, Ünsal 60 RZAEV, Zakir M. O. 58, 67, 66 GÜNEŞ, H. Semih 67 UNZETA, Mercedes 4 ÖĞÜŞ, İ. Hamdi 47 KOYUNOĞLU, Semra 86 UZUN, Ömrüm 24 ÇELİKBIÇAK, Ömür 60 ŞANLI, Senem 63, 70 V CAN, Nafiz Öncü 80, 81, 82 SENY, D. de 14 ÇELİK, Venhar 53, 55 ÖNER, Filiz 86 SEPTİOĞLU, Ebubekir 72 ADAR, Vildan 43, 44 ONUR, Mehmet Ali 67 KAVLAK, Serap 67 HANNINEN, Osmo 60, 61 ÜNLÜ, Serdar 76, 77, 78 W ŞANLI, Oya 62 POLAT, Serpil 76, 77 WEHENKEL, L. 14 ÖZALP-YAMAN, Şeniz 46 ZENCİR, Sevil 67 ÖZBATIR, Bilgen 86 DALKARA, Sevim 48, 84 Y YERDELEN, K. Özden 60, 61 ULUSOY, Seyhan 85 YABANOĞLU, Samiye 88 KÜL, Özge 43, 44 CANKARA, Şeyma 77 YALÇIN, Funda Nuray 88 CEYLAN, Özgür 85 SHAMMOUT, Muhammad BAYIR, Yasin 73 ÖZKAN, Serkan 27 Jamal 89 YEŞİLADA, Erdem 29 DEMİREL, Özlem 43 BEKSAÇ, Sinan 84 ÇAPAN, Yilmaz 45 ORHAN, Özlem 79 SIPPL, Wolfgang 17 ERGÜN, Yusuf 79 SÖĞÜT, Özlem 54 BİLGE, S. Sırrı 53 ÖZKAY, Yusuf 56, 57 ÖZTÜRK, Recep 22 URLU, Şölen 78 ÖZTÜRK, Yusuf 56 YAĞMUR, Sultan 83 P TOPTAN, Suna 65 Z PARINI, Angelo 7 OKUMUŞ, Zafer 71 AYHAN, Peruze 48 T HALICI, Zekai 71, 73, 74, 75 ALCIL,” Pınar 67 PARK, Tae-joon 54

94 May 17-20, 2007 Profesyonel Çözümler...

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Firmamz tümü belirli bir kalitenin üzerinde olan laboratuar malzemelerinin ve ara�trma kimyasallarnn 26 yllk deneyimle son kullanc sat�n yapmaktadr

Ürünlerle ilgili katalog, teknik bilgi, teklif iste�i ve tüm taleplerinize en etkin biçimde cevap almak için a�a�da belirtilen irtibat adreslerine ula�manz yeterlidir.

TÜRK�YE TEK D�STR�BÜTÖRÜ OLARAK SATI�INI YAPMAKTA OLDU�UMUZ ÜRÜNLER

Biyokimyasal ve “Life Science” ürünlerini;

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Ara�trma kimyasallar ve stoklarmzda devaml olarak bulundurdu�umuz rutin kullanm amaçl kimyasallarn;

Safla�trma ve kromotografi ürünlerini;

Laboratuarlarnzda kullanlmakta olan cam ve plastik tüm sarf malzemelerini;

Laboratuarlarnzda kullanlmakta olan cam ve plastik tüm sarf malzemelerini kapsamaktadr.

�NTERLAB LABORATUAR ÜRÜNLER� SAN.T�C.A.�.

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Web:www.interlab.com.tr [email protected] [email protected] [email protected] [email protected] [email protected] �NTERLAB LABORATUAR ÜRÜNLER� SAN. VE T�C. A.�.

Firmamz tümü belirli bir kalitenin üzerinde olan laboratuar malzemelerinin ve ara�trma kimyasallarnn 26 yllk deneyimle son kullanc sat�n yapmaktadr

Ürünlerle ilgili katalog, teknik bilgi, teklif iste�i ve tüm taleplerinize en etkin biçimde cevap almak için a�a�da belirtilen irtibat adreslerine ula�manz yeterlidir.

TÜRK�YE TEK D�STR�BÜTÖRÜ OLARAK SATI�INI YAPMAKTA OLDU�UMUZ ÜRÜNLER

Biyokimyasal ve “Life Science” ürünlerini;

Organik ve �norganik çal�malara yönelik kimyasallar ve sarflarn;

Ara�trma kimyasallar ve stoklarmzda devaml olarak bulundurdu�umuz rutin kullanm amaçl kimyasallarn;

Safla�trma ve kromotografi ürünlerini;

Laboratuarlarnzda kullanlmakta olan cam ve plastik tüm sarf malzemelerini;

Laboratuarlarnzda kullanlmakta olan cam ve plastik tüm sarf malzemelerini kapsamaktadr.

�NTERLAB LABORATUAR ÜRÜNLER� SAN.T�C.A.�.

Genel Müdürlük: Hadmköy Asfalt 4.Km. 34870 Hadmköy/�STANBUL Tel: 0212 798 21 68 Fax: 0212 798 21 52

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