Seminaris de Recerca de la Facultat de Farmàcia 27 de setembre de 2016

RECERCA EN PRODUCTES NATURALS

Plantes ornamentals de la família com a font de compostos bioactius Dr. Laura Torras Claveria; Luciana Tallini, Dr. Francesc Viladomat, Dr. Jaume Bastida littoralis Phaedranasa dubia Crinum x powellii nivalis Lycoris radiata

Narcissus cv. Pinza AMARYLLIDACEAE Natural Products 1981-2010

All new approved drugs Natural product and/or natural product structures play a highly significant role in the drug discovery and development

Newman & Cragg, J Nat Prod 2007, 70, 461-477 Newman & Cragg, J Nat Prod 2012, 75, 311-335 Research and development of new products

FDA Production/ New drugs Pre-clinic Clinic assays (Phases I-III) Revision Phase IV

10.000 1 approved

compounds 250 compunds 5 compounds drug Presentación NDA Presentación Presentación IND Presentación FDA

5 Years 1,5 Years 6 Years 2 Years 2 Years

Average time in each step

IND: Investigational New Drug NDA: New Drug Application Amaryllidaceae family • Bulbous family with ornamental and medical value

• 3 subfamilies: , , and (previously 3 families)

• Term “Amaryllidaceae” corresponds with subfamily Amaryllidoideae Amaryllidaceae medical uses through History

Hippocrates of Kos Dioscorides (40-90 AD) Pliny the Elder (23-79 AD) (460-370 BC). Soranus of Ephesus (98-138 AD) Topic use oil Same therapy Narcissus extracts Uterus tumors

Bible Middle Ages European doctors Narcissus tazetta Chinese medicine, N Anti-tumor therapy Narcissus oil tumors – inflammation Amaryllidaceae geographical distribution Amaryllidaceae: Africa Amaryllidaceae: America () Amaryllidaceae: Asia Amaryllidaceae: Europe Mediterranean area

Caucasus Narcissus poeticus (Vall d’Aran, Catalonia) Amaryllidaceae alkaloids

12 11 OH 3 2 4 HO 4 2 HO 1 MeN 3 3 4a 5' 10 10b H 4 10 H 10b HO 4' 6' 9 10a MeO 9 10a 1 2 5 1  O 11 4a 6 H H H NH  N 12 O HO 3' 1' O 8 6a MeO 8 6a 6 2' ' 7 6 7 norbelladine lycorine homolycorine O OH 2 2 2 1 3 OH 1 3 OMe 1 OH 11 11 3 10 10 OH 10 H 4 O 9 10a 4a 4 O 9 10a 4a 4 O 9 10a 10b 10b 10b OH H 12 H 12 4a N N NH O 8 6a O 8 6a O 8 6a 6 7 6 7 6 7 crinine haemanthamine OH O narciclasine OMe 1 OMe 3 10 11 11a 2 2 2 4 3 OH O 9 10a OH 1 H 4a 1 4a 3 O 10 NMe 12 10 10b 9 10a 4 MeO 9 10a O 11 4 10b 12 O 8 6a H N 4a OH 7 6 11 12 O O 8 6a montanine 8 6a NMe 7 6 7 6 tazettine galanthamine Other Amaryllidaceae alkaloids

OMe

H NMe 10b O O R NMe R 4a O N H O O O O Graciline Plicamine OH Graciline PlicNonami ndimerice dinitrogenated compound DibenzoEsquele[tob,d di]b piranenzo[skeletonb,d]pirano Compuesto dinitrogenado no dimérico AbsoluteConfigur aconfigurationción absoluta 4aR/10bR 4aR/10bR CenSpirotro s pcenteriro = T =a Tazettinezettina [O[O/N]/N]

GalanthusGalanthusgracilis gracilis GaGalanthuslanthus plicplicatusatus sspssp. by zByzantinusantinus GalanthusGalanthusplicatus plicatusssp. ssp Byzantinusbyzantinus CyrCyrthantusthantus oblobliquusiquus maritimum Narcissus broussonetii Exceptions

NMe O 2 OMe OMe OMe OMe

NMe2 O N OH N O Me Me amisine mesembrenol mesembrenone Aizoaceae Aizoaceae Aizoaceae Hymenocallis arenicola Crinum oliganthum Narcissus pallidulus Z Chem 1980, 20: 298 Z Chem 1981, 21: 358 J Nat Prod 1989, 52: 478

O O HO NMe NMe O O MeO NH H O O H HO Me H salsoline O MeO Chenopodiaceae (Salsola richteri) O Galanthus woronowii (-)-capnoidine (+)-bulbocapnine Fumariaceae Papaveraceae, Fumariaceae y Biochem Syst Ecol 2013, 51, 276 Lauraceae Galanthus nivalis ssp cilicicus Biochem Syst Ecol 2004, 32, 1059 Chemotaxonomic value

Behria tenuiflora Greene

NMe NMe O COO O Shihunine

NMe H COOH

Dehidroshihunine Amaryllidaceae Alliaceae Bastida, Sellés, Codina, Viladomat, León de la Luz Planta Medica 1996, 62, 575-577 Amaryllidaceae alkaloids biosynthesis 1.- Enzimatic preparation of precursors

HO NH2 L-Phe L-Tyr COOH

H2N COOH PAL

hydroxylasesPAL TTyryr-decarboxylasedecarboxylase

R1 -oxidation HO HO

reduction R2 COOH HO CHO tyramine trans-cinnamic acid, R1=R2=H protocatechuic aldehyde H2N para-coumaric acid, R1=OH, R2=H caffeic acid, R1=R2=OH Amaryllidaceae alkaloids biosynthesis 2.- Primary ciclation HO HO

HO CHO protocatechuic aldehyde tyramine H2N

HO HO HO

HO HO

N NH H N HO O H

Schiff's base HO (isomeric structures in solution) HO OH

HO

NH HO

norbelladine Amaryllidaceae alkaloids biosynthesis 3.- Enzimatic preparation of intermediary agents

HO HO

HO 4' MeO 4'

NH NH HO HO norbelladine O-methylnorbelladine Amaryllidaceae alkaloids biosynthesis 4.- Secondary ciclation. Diversification HO

MeO 4'

NH HO

O-methylnorbelladine

ortho-para' para-para' para-ortho'

types types types lycorine crinine galanthamine homolycorine haemanthamine tazettine narciclasine montanine Amaryllidaceae alkaloids biosynthesis 4.- Secondary ciclation. Diversification OH

MeO

NH HO O-methylnorbelladine

ortho-para' para-para' para-ortho'

types types types lycorine crinine galanthamine homolycorine haemanthamine tazettine narciclasine montanine Amaryllidaceae alkaloids biosynthesis 4.- Secondary ciclation. Diversification OH OH MeO

NH

O-methylnorbelladine

ortho-para' para-para' para-ortho'

types types types lycorine crinine galanthamine homolycorine haemanthamine tazettine narciclasine montanine Natural Products Research

SCREENING STRUCTURAL CHARACTERIZATION

1980 1983 I 1993 II 2005 III 2011

Study of Study of Study of Catalan flora Amaryllidaceae galanthamine Narcissus spp. used in producer [Spain-] TradMed [Chemotaxonomy] [SA/Ibero- America] Phytochemical screening

SCREENING • Collection, Drying, Storage • Taxonomic Determination 1980 1983 • Obtaining genus information • Simple method  rapid selection

Catalan flora > 1000 samples Green & Fungus  different habitats from Catalonia

Narcissus assoanus Duf. =N. requienii Roem. =N. juncifolius Lag. (Amaryllidaceae) Natural Products Research

SCREENING STRUCTURAL CHARACTERIZATION

1980 1983 I 1993 II 2005 III 2011

Study of Study of Study of genus Catalan flora Amaryllidaceae galanthamine Narcissus spp. used in producer species [Spain-Portugal] TradMed [Chemotaxonomy] [SA/Ibero- America] Selection of genus Narcissus

In base of:

• High content in exclusive Amaryllidaceae alkaloids • Biological and pharmacological activity of extracts and alkaloids previously isolated from this family species • Poor studied genus • Influence area: Mediterranean (easy to work) Selection of genus Narcissus

• 80-100 species • Narkào (=narcotic) penetrating smell of flowers from some species • Growing from lowlands to highlands • Flowering at the end of winter - spring

• Distribution South Europe (Iberian Peninsula and Italy) (also in North Africa and East Mediterranean)

* * *

* Methodology

Spectroscopy Plant Pure UV, IR, MS, Extracts Fractions material compounds NMR (1D/2D), X Ray

Structure elucidation Alkaloids from genus Narcissus OH 2 HO 1 3

10 10b H 4 9 10a O 11 H 4a N 12 O 8 6a 7 6 Lycorine, Narcissus pseudonarcissus Gerrad, 1877: isolation Nagakawa et al., 1956: characterization New alkaloids from Narcissus

R

OMe MeO O MeO N NMe N MeO O MeO roserine bicolorine vasconine R= H tortuosine R= OMe OR2 R1O H HO MeO H N N MeO MeO O 1-O-acetylpseudolycorine R1= Ac, R2= H 2-O-acetylpseudolycorine R1= H, R2= Ac oxoassoanine

MeN MeN H H H H R O 9 R O 9 1 R 1 R H 3 H 3 O O R2O R2O O OMe

9-O-demethylhomolycorine R1=H, R2= Me, R3= H O-methyllycorenine R1=R2= Me, R3=H dubiusine R1= COCH2CHOHMe, R2= Me, R3= OAc 2-hydroxy-6-O-methyloduline R1+R2= CH2, R3=OH 9-O-demethyl-2-hydroxyhomolycorine R1= H, R2= Me, R3= OH 8-O-acetylhomolycorine R1= Me, R2= Ac, R3= H New alkaloids from Narcissus

OAc OH OMe OMe 11 OAc MeCOOCH2 HO 9 HO 9 O O O H H H H N N N N MeO MeO O O cantabricine 9-O-demethylmartidine 11-O-acetylhaemanthamine bujeine OMe

HO H NMe O NH O O O OH O O N O N H OH O nangustine obesine pallidiflorine OMe O HO

OH OH OH O O O MeO MeO MeO

NH NH N O epinorgalanthamine epinorlycoramine H N-formylnorgalanthamine

Bastida, Lavilla, Viladomat. The Alkaloids 2006, 63, 87-179 Natural Products Research

SCREENING STRUCTURAL CHARACTERIZATION

1980 1983 I 1993 II 2005 III 2011

Study of Study of Study of genus Catalan flora Amaryllidaceae galanthamine Narcissus spp. used in producer species [Spain-Portugal] TradMed [Chemotaxonomy] [SA/Ibero- America] Amaryllidaceae in traditional medicine

KwaZulu-Natal 200.000 Traditional healers (Year 2004 – licences authorization) South Africa Amaryllidaceae in traditional medicine

Brunsvigia Crinum Nerine B. josephinae C. sthulmanii C. elatus N. filifolia B. orientalis C. kirkii C. sanguineus B. radulosa C. delagoense C. obliquus B. littoralis C. macowanii pregnancy B. bosmeniae (facilitate birth) sexually transmitted stomach pain urinary diseases (STD) Leprosy affected areas lactation problems South Africa Amaryllidaceae in traditional medicine

Ammocharis Boophane A. tinneana B. flava

Used by indigenous population (Xhosa, Shoto, San, Zulu ...) Infusions of [> 200 years]. A lot of species from genus Boophane, Brunsvigia, Crinum and Nerine are used to treat ♀ infertility

South Africa Methodology

Mass spectrometry GC-MS, LC-MS Spectroscopy Plant Pure UV, IR, MS, Extracts Fractions material compounds NMR (1D/2D), X Ray Biological assays

Structure elucidation Amaryllidaceae alkaloids OAc OR2 OAc HO R1O 1 R1 O O H OH H H N O N N R2 O R delagoensine R1+R2= OCH2O josephinine 3 delagoenine R1= R2= OMe 1-epibowdensine R1= R2= Ac, R3= OMe 1-epidemethoxybowdensine R1= R2= Ac, R3= H 1-epideacetylbowdensine R1= R2 =H, R3= OMe O O OMe N OMe OR1 AcO O O O O H H H N N O O O N OMe OMe OMe OH 11-O-acetylambelline filifoline 6-hydroxycrinamidine R= H 6-hydroxyundulatine R= Me

OH OMe OMe OH OH MeO O O H H H N N N HO O O OMe OMe OH macowiine brunsbelline 6-hydroxybuphanidrine South Africa Amaryllidaceae alkaloids OAc HO AcO AcO H H H MeO MeO O H H H N N N HO HO O kirkine 1-O-acetylnorpluviine 1,2-O-diacetylzephyranthine

OMe

H MeO NMe O MeO MeO H O O N NH RO 8 NMe MeO

obliquine buflavine R= Me N-demethylbelladine OH 8-O-demethylbuflavine R= H

H OH N N

O OMe O O O O O O O N O O montabuphine noraugustamine 4a-dedihydronoraugustamine South Africa Amaryllidaceae in Ibero-America Amaryllidaceae alkaloids

O O O O O MeO H H N N N O O MeO ungeremine apohaemanthamine oxomaritidine OMe OH

H O NMe MeO O MeO OH H N O MeO O NMe OMe O OMe 7-methoxyassoanine 6-O-methylpretazettine 11-hydroxygalanthamine

Andrade, Berkov, Viladomat, Codina, Zuanazzi, Bastida Molecules 2011,16, 7097-7104 (11β-hydroxygalanthamine) Ibero-America Amaryllidaceae micropropagation

• Study of species in danger of extinction • Study of insufficient samples

Ibero-America Natural Products Research

SCREENING STRUCTURAL CHARACTERIZATION

1980 1983 I 1993 II 2005 III 2011

Study of Study of Study of genus Catalan flora Amaryllidaceae galanthamine Narcissus spp. used in producer species [Spain-Portugal] TradMed [Chemotaxonomy] [SA/Ibero- America] Galanthamine Galanthamine

OH • Acetylcholinesterase inhibitory activity O

H3CO • Interaction with nicotinic receptors NCH3

Commercialized for the treatment of mild to moderate states of Alzheimer’s disease Geographic study Leucojum aestivum

Distribution of different alkaloid types in Leucojum aestivum populations in

Berkov, Georgieva, Kondakova, Viladomat, Bastida, Atanassov, Codina Biochemical Systematics in Ecology 2013, 46, 152-161 Methodology: GC-MS software AMDIS Automatic Mass Spectral Deconvolution and Identification System

Separation and identification of Amaryllidaceae alkaloids in complex mixtures and extracts without derivatisating

Differentiation of coeluting compounds

Structural information of new compounds Methodology: GC-MS

software AMDIS Automatic Mass Spectral Deconvolution and Identification System

Identification of Amaryllidaceae alkaloids Amaryllidaceae • Kovats Retention Index alkaloids library • Mass spectra NIST database Literature Methodology: GC-MS software AMDIS GAL in ornamental Narcissus

105 ornamental Narcissus were evaluated in terms of GAL content and AChE inhibitory activity, as a new source of GAL for pharmaceutical industry

Torras-Claveria, Berkov, Codina, Viladomat, Bastida Industrial Crops and Products, 2013, 43, 237-244 GAL in ornamental Narcissus Narcissus hispanicus ‘Rip Van Winkle’

‘Pumilus’ GAL in ornamental Narcissus ‘Pinza’ ‘Rijnveld’s Early Sensation’

‘Ara’

‘February Gold’ GAL in ornamental Narcissus

Bulbs and leaves were evaluated separately

GAL content GC-MS

AChE inhibitory activity In vitro test

Torras-Claveria, Berkov, Codina, Viladomat, Bastida Industrial Crops and Products, 2013, 43, 237-244 GAL content 8000 7000 Galanthamine 6000 content in 5000 leaves 4000

3000 µg Gal /g DW /g Gal µg 2000

1000

0 N. confusus N. hispanicus Yellow Wings Rijnveld Early Rip Van Sensation Winkle

16000

14000

12000 Galanthamine 10000 content in 8000 bulbs

6000 µg Gal/g DW Gal/g µg

4000

2000

0 N. confusus Yellow Wings Bella Estrella Enkele Jonquilla N. hispanicus Simplex Torras-Claveria, Berkov, Codina, Viladomat, Bastida Industrial Crops and Products, 2013, 43, 237-244 AchE inhibitory activity 1,200

1,000

0,800

.(IC50) IC 50 Act

. . 0,600 AChE inhib

0,400 inhibition AChE 0,200 in leaves

0,000 Snowball Green N. confusus Hawera Albus Plenus Pink N. hispanicus Howard Odoratus Paradise 1,200

1,000

0,800

. (IC50) . Act

. . 0,600 IC 50

inhib AChE 0,400

AChE inhibition 0,200 in bulbs

0,000 N. confusus Hawera Little Spell

Torras-Claveria, Berkov, Codina, Viladomat, Bastida Industrial Crops and Products, 2013, 43, 237-244 AchE inhibitory activity

Galanthamine AChE activity content inhibition

N. hispanicus ‘Snowball’

Torras-Claveria, Berkov, Codina, Viladomat, Bastida Industrial Crops and Products, 2013, 43, 237-244 Clustering , the Cluster analysis organization of a collection of patterns based on similarity. Its aim to find structure in ALKALOID PROFILE data

LEAF samples 5 clusters

BULB samples 5 clusters

Torras-Claveria, Berkov, Codina, Viladomat, Bastida Industrial Crops and Products, 2014, 56, 211-222 Hierarchical cluster LEAF samples

Rup Sud Vel

BULB samples

Torras-Claveria, Berkov, Codina, Viladomat, Bastida Industrial Crops and Products, 2014, 56, 211-222 LEAF samples Horticultural divisions

Lycorine type

3000,00

2500,00

2000,00 Galanthamine type Haemanthamine type

1500,00 Homolycorine type Lycorine type Narciclasine type 1000,00

Tazettine type µg GAL/DW µg Other 500,00 Protoalkaloids

0,00 D1 D2 D3 D4 D5 D6 D7 D8 D9 D10 D11 D12 D13 Horticultural divisions BULB samples Horticultural divisions

Galanthamine type

3000,00

2500,00

2000,00 Galanthamine type Haemanthamine type

1500,00 Homolycorine type Lycorine type Narciclasine type 1000,00

Tazettine type µg GAL/DWµg Other 500,00 Protoalkaloids

0,00 D1 D2 D3 D4 D5 D6 D7 D8 D9 D10 D11 D12 D13 Horticultural divisions Statistical analysis C1 C2 C3 C4 C5 All C1 C2 C3 C4 C5 All

D1 0 7 1 8 0 16 D1 4 2 3 7 0 16

D2 0 4 0 9 0 13 D2 2 3 3 5 0 13

D3 0 3 0 9 0 12 D3 2 5 3 2 0 12 Cross D4 0 4 0 8 0 12 D4 7 1 3 1 0 12 D5 0 2 0 5 0 7 D5 1 0 6 0 0 7 tabulation D6 0 7 2 6 0 15 D6 6 6 2 1 0 15

D7 0 2 0 6 0 8 D7 2 1 3 2 0 8

D8 0 0 1 0 0 1 D8 0 0 1 0 0 1

D9 0 2 0 0 0 2 D9 0 1 0 1 0 2

D10 0 1 0 2 0 3 D10 0 0 3 0 0 3

D11 0 3 0 3 0 6 D11 1 0 1 4 0 6

D12 0 2 0 0 0 2 D12 0 1 0 1 0 2

D13 1 1 2 4 1 9 D13 2 0 2 4 1 9

All 1 38 6 60 1 106 All 1 38 6 60 1 106 Correspondence analysis

Torras-Claveria, Berkov, Codina, Viladomat, Bastida Industrial Crops and Products, 2014, 56, 211-222 Natural Products Research

2011 - today

Metabolomics

Ibero-America

Lycoris Metabolomics

Pancratium canariense Torras-Claveria, Berkov, Jáuregui, Caujapé, Viladomat, Codina, Bastida Phytochemical analysis, 2010, 21, 80-88

Narcissus Section Ganymedes Pigni, Berkov, Ríos-Ruiz, Luque, Viladomat, Codina, Bastida Phytochemistry, 2013, 95, 384-393

Ornamental Narcissus varieties and cultivars Torras-Claveria, Berkov, Codina, Viladomat, Bastida Industrial Crops and Products, 2014, 56, 211-222 Metabolomics

Polyphenols – LC-MS/MS and LC high resolution MS 2000 - today Sánchez-Rabaneda, Jáuregui, Lamuela-Raventós, Bastida, Viladomat, Codina. Journal of Chromatography A, 2003, 1008, 57-72 Artichoke waste Sánchez-Rabaneda, Jáuregui, Lamuela-Raventós, Viladomat, Bastida, Codina. Rapid Communications in Mass Spectrometry, 2004, 18, 553-563 Apple pomace

Parejo, Jáuregui, Viladomat, Bastida, Codina. Rapid

Communications in Mass Spectrometry, 2004, 18, 2801-2810 Tagetes maxima

Torras-Claveria, Jáuregui, Bastida, Codina, Viladomat. Journal of Agricultural and Food Chemistry, 2007, 55, 8436-8443 Lavandin Metabolomics

Polyphenols – LC-MS/MS and LC high resolution MS 2000 - today

Torras-Claveria, Jáuregui, Codina, Tiburcio, Bastida, Viladomat. Plant Science, 2012, 182, 71-78 Tobacco

Gasca, Cabezas, Torras, Bastida, Codina. Free Radicals and Antioxidants, 2013, 3, S55-S61 Pavonia sepioides

Luna, Pigni, Torras-Claveria, Monferran, Maestri, Wunderlin, Ferensin, Bastida, Tapia. Journal of Food Composition and Analysis, 2013, 31, 120-128 Ramorinoa girolae Genus Hippeastrum De Andrade, Pigni, Torras-Claveria, Guo, Berkov, Reyes-Chilpa, El Amrani, Zuanazzi, Codina, Viladomat, Bastida. Revista Latinoamericana de Química, 2012, 40, 83-98 Hippeastrum papilio De Andrade, Berkov, Viladomat, Codina, Zuanazzi, Bastida. Molecules, 2011, 16, 7097-7104 Guo, de Andrade, Pigni, Torras-Claveria, Tallini, Borges, Viladomat, Nair, Zuanazzi, Bastida. Helvetica Chimica Acta, 2016, 99, 143-147 Hippeastrum aulicum and calyptratum De Andrade, Guo, Font-Bardia, Clavet, Dutilh, Viladomat, Codina, Nair, Zuanazzi, Bastida. Phytochemistry, 2014, 56, 188-195 Hippeastrum breviflorum Sebben, Giordani, de Andrade, Berkov, Osorio, Sobral, de Almeida, Henriques, Bastida, Zuanazzi. Revista Brasileira de Farmacognosia, 2015, 25, 353-355 Phaedranassa dubia Osorio, Berkov, Brun, Guo, Codina, Viladomat, Cabezas, Bastida. Phytochemistry Letters, 2010, 3, 161-163 Zephyranthes concolor Reyes-Chilpa, Berkov, Hernández-Ortega, Jankowski, Arseneau, Clotet-Codina, Esté, Codina, Viladomat, Bastida. Molecules, 2011, 16, 9250-9533 Brazilian Amaryllidaceae as a source of acetylcholinesterase inhibitory alakaloids De Andrade, Giordani, Torras-Claveria, Pigni, Berkov, Font-Bardia, Calvet, Konrath, Bueno, Sachett, Dutilh, Borges, Viladomat, Henriques, Nair, Zuanazzin, Bastida Phytochemistry Reviews, 2016, 15, 147-160 Bioactive alkaloids in Lycoris species

Guo, Pigni, Zheng, de Andrade, Torras-Claveria, Borges, Viladomat, Codina, Bastida Natural Product Communication, 2014, 9, 1081-1086 Pharmacological activity

Increase of interest in pharmacological activity of Amaryllidaceae alkaloids since GAL approval by FDA

Anti-tumoral Anti-parasitic Acetylcholinesterase inhibition Docking studies Pharmacological activity

Citotoxicity test in anti-tumor compounds

Some Amaryllidaceae have been historically used in symptomatologies identified as cancer

• Previous evaluation of Toxicity test in Artemia salina Amaryllidaceae compounds • Reproducible and comparable values Pharmacological activity Citotoxicity test in anti-tumor compounds

Evaluation in 60 cell lines Pharmacological activity Citotoxicity test in anti-tumor compounds Pharmacological activity

Apoptosis – Selectivity OH • Selective apoptosis of HO OH tumoral cells • Minimal effects in normal O cells OH • Early activation of Caspasa-3 NH • Fast apoptosis in Human O Linfoma cells OH O

Pancratistatin

McLachlan, Kekre, McNulty, Pandey. Apoptosis 2005, 10, 619-630 Kekre, Griffin, McNulty, Pandey. Cancer Chemother. & Pharmacol. 2005, 56, 29-38 Pharmacological activity

Apoptosis – SAR studies Selectivity Hepatoma (5123tc)

OH OAc HO HO OH OMe HO O O O O H H H H N O N O N O O N OMe amabiline josephinine crinine ambelline (3) (4) (5) (6) OMe CRININE-HAEMANTHAMINE OMe OH OH O 100 O

H 90 H N N O 80 O

crinamine 70 haemanthamine

(7) 60 (8)

50

O Apoptotic % O OMe 40 OMe

30 HO % Apoptosis

O 20 O H H N 10 O O N 0 cC A1 3 4A2 5A3 A47 A5 8 A7 6 A8 9 10A10 OMe C 3 4 5 7 8 6 9 10 MeO OH epoxyambelline 25mM – 48h treatment 6-hydroxyundulatine (9) (10)

McNulty, Nair, Codina, Bastida, Pandey, Gerasimoff, Griffin. Phytochemistry 2007, 68, 1068-1074 Pharmacological activity

Apoptosis – SAR studies Selectivity Hepatoma (5123tc)

Pharmacophoric requirements

• Presence of ethano bridge α-5,10b • Methoxyl group tolerance in α or β in C-3 • Hydroxyl group in position 11

3 OMe 11 OH O H O N

McNulty, Nair, Codina, Bastida, Pandey, Gerasimoff, Griffin. Phytochemistry 2007, 68, 1068-1074 Pharmacological activity

Anti-parasitic

Parasite Disease Infectected people Detection Plasmodium Malaria 300 millions Hypoxanthine H3 falciparum (2 millions death/year) incorporation Leishmania sp. Leishmaniasis 12 millions Microscopy

Trypanosoma Sleeping sickness 300.000 Alamar blue assay brucei-rhodesiense (100% mortality) (colorimetry) Trypanosoma cruzi Chagas disease 18 milions Colorimetry

Spencer et al., Lloydia 1947, 10, 145-174 Pharmacological activity Anti-parasitic

Parasite: strain: stage: reference drug: T. b. rhodesiense STIB 900 trypomastigotes Melarsoprol T. cruzi Tulahuen C4 amastigotes Benznidazole L. donovani MHOM-ET-67/L82 amastigotes Miltefosine P. falciparum K1 IEF Chloroquine Cytotoxicity L6 Podophyllotoxin All values as: mg / mL

T.b.rhod. T. cruzi L.don. axen. P. falc. K1 Cytotox. L6 ID No IC-50 ref drug IC-50 ref drug IC-50 ref drug IC-50 ref drug IC-50 ref drug Ungeremine 0,979 0,003 0,85 0,22 >30 0,17 0,0933 0,0321 17,3 0,008 Buphanamine 28,75 0,003 >30 0,22 >30 0,17 4,31 0,046 >90 0,008 Zefbetaine 39,22 0,003 >30 0,22 >30 0,17 >5 0,046 >90 0,008 Galanthamine 37,92 0,003 >30 0,22 >30 0,17 >5 0,046 >90 0,008 Pdub Extract 27,43 0,003 >30 0,22 >30 0,17 >5 0,046 >90 0,008 O HO OH O R1O O MeO H N O N NMe R2O OMe

Ungeremine: R1+R2=CH2 Buphanamine Galanthamine Zefbetaine: R1=R2=Me

Osorio, Robledo, Bastida. The Alkaloids 2008, 66, 113-190 Pharmacological activity

Acetylcholinesterase activity inhibition

Strategy to palliate Alzheimer Drugs with cholinergic activity symptomatology

OH Galanthamine O

H3CO

NCH3

Ellman in vitro assay to test Amaryllidaceae alkaloids and plant extracts Pharmacological activity

Acetylcholinesterase activity inhibition

2 OH 1 3 López, Bastida, Viladomat, Codina O 10b 4 10 Life Sciences, 2002, 71, 2521-2529 MeO 10a 4a 11 Narcissus confusus 9 12 8 6a NR 7 6

1: N-allylnorgalanthamine, Berkov, Codina, HA Viladomat, Bastida. R= Bioorg. Med. Chem. Lett. HB 2008, 18, 2263-2266 2: N-(14-methylallyl) Leucojum aestivum norgalanthamine,

Me HA 16 15 R= 14 HB 13

Torras-Claveria, Berkov, Codina, Viladomat, Bastida Industrial Crops and Products, 2013, 43, 237-244 Ornamental Narcissus varieties Pharmacological activity Docking – Molecular Modeling Prediction of the preferred orientation of a molecule to another one when they form a stable complex

4-O-methylnangustine

Ortiz, Pigni, Andujar, Roitman, Suvire, Enriz, Tapia, Bastida Feresin Journal of Natural Products 2016, 00, 000-000 In press Pharmacological activity Docking butyrylcholinesterase Prediction of the preferred orientation of a molecule to another one when they form a stable complex

Simulate molecular interactions of Amaryllidaceae compounds in the active site of butyrylcholinesterase

Molecular Dynamics Simulations Estimation of binding energies

4-O-methylangustine Which aminoacids are involved in the interaction in the binding site

Ortiz, Pigni, Andujar, Roitman, Suvire, Enriz, Tapia, Bastida Feresin. Journal of Natural Products 2016, 00, 000-000 In press Pharmacological activity Docking GSK-3β

Glycogen Synthase Kinase 3

Proteins involved in Kinases of unicellular Alzheimer Disease parasites

Increased GSK-3 activity in AD frontal cortex Plasmodium falciparum

Alzheimer Disease Anti-parasitic Pharmacological activity Docking GSK-3β Comparison of the best “X-ray like” Putative binding mode poses for narciclasine in each for narciclasine system

Hydrogen Bond interactions are marked Red: 1Q3D_NOWAT; as black dashed lines. green: 1Q3D_WAT; Red circles highlight other functional blue: 3F88_NOWAT; groups that could contribute to binding orange: 3F88_WAT. through water-mediated interactions. Ornamental value Biodiversification

Amaryllidaceae plants

Amaryllidaceae Pharmacological OH alkaloids activity HO OH

O OH OH O NH OH O O pancratistatine H3CO

galanthamine NCH3 Team

Dr. C. Codina / Dr. F. Viladomat / Dr J. Bastida

Dra. M. Galobardes (†), Dr. J.M. LLabrés, Dr. J.M. Fernández, Dr. S. Bergoñón, Dra. M. Sellés, Dra. R. Riera, Dra. G. Almanza, Dr. E. Ellington, Dra. S. López, Dr. R. Colque, Dr. J.C. Alarcón, Dr. A.K. Machocho, Dr. J.J. Nair, Dra. M. Herrera, Dra. I. Parejo, Dr. F. Sánchez, Dr. S. Berkov, Dr. R. Reyes, Dra. L. Torras-Claveria, Dr. E.J. Osorio, Dra. M. Nappo, Dr. J.P. Andrade, Dra. N. Pigni, Dra. Y. Guo, Msc. L. Tallini

Prof. O. de Bolós (†), Dr. M. Rubiralta, Dr. J. Fdez-Casas, Dr. A. Susanna, Dr. J. Molero Societat Catalana de Micologia

Dr. M. Feliz / Dra. M.A. Molins (RMN-PC) Dra. A. Linares (RMN) / Dra. A. Marín (GC-MS) / Dra. O. Jáuregui (LC-MS) Collaborations

Alemania – Prof. I. Merfort Argentina – Dr. A.A. Tapia, Dra. G.E. Feresin Bolivia – Dra. G. Almanza, Dr. R. Colque Brasil – Dr. J.A.S. Zuanazzi, Dra. R. Giordani, Dr. J.P. Andrade Bulgaria – Dr. S. Berkov, Dr. A. Paulov, Dra. Z. Yordanova Canada – Prof. J. McNulty Chile – Dr. G. Schmeda-Hirshmann China – Prof. G.E. Ma – Dr. F. Cabezas, Dr. J.C. Alarcón, Dr. E. Osorio Costa Rica – Dr. R. Murillo, Dra. R. Romero, Dr. F. Binns, Dr. B. Lomonte – Dra. M.R. Herrera Equador – Dra. N. Oleas, Msc. K. Acosta Francia – Prof. H.P. Husson, Prof. J.C. Quirion, Dr. B. Weniger – Dr. E. Ellington Kenia – Prof. S. Chaabra, Dr. A. Machocho Marruecos –Dr. Elamrami México – Dr. R. Reyes-Chilpa, Dr. J.L. León, Dra. E. López Sudáfrica – Prof. W.C. Campbell (†), Dr. J.J. Nair, Dr. N. Koorbanally Suiza – Prof. R. Brun, Dr. Marcel (STI) Turquía – Dra. N. Unver, Dra. B. Sarikaya UK – Prof. J.D. Phillipson, Dr. C.W. Wright, Dra. D. Mulholland USA – Dr. A. Meerow, Dr. V.L. Narayanan & E. Sausville (NCI) Gràcies per la vostra atenció

Dra. Laura Torras Claveria 27 setembre 2016

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