One pot Synthesis, DNA binding and fragmentation in vitro of new fused uracil derivatives for anticancer properties Bothaina A. Mousaa, Ashraf H. Bayoumi*b, Makarem M. Korraaa, Mohamed G. Assyc, and Samar A. El-kalyoubid aDepartment of Organic Chemistry, Faculty of Pharmacy, Cairo University, Egypt bDepartment of Organic Chemistry, Faculty of Pharmacy (boys), Al-Azhar University, Egypt; cDepartment of Organic Chemistry, Faculty of Science, Zagazig University, Egypt; dDepartment of Organic Chemistry, Faculty of Pharmacy (girls), Al-Azhar University, Egypt Síntesis en un solo recipiente, combinación ADN y fragmentación in vitro de derivados condensados de uracilo con propiedades anticancerígenas Síntesi en un sol recipient, combinació ADN i fragmentació in vitro de derivats condensats d’uracil amb propietats anticancerígenes Recibido: 9 de junio de 2012; aceptado: 28 de junio de 2012 RESUMEN RESUM Se ha sintetizado una nueva serie de pirimidopirimidinas S’ha sintetitzat una nova sèrie de pirimidopirimidines partiendo de 6-amino-1-benzyl [o 1-(2-chlorobenzyl)] ura- partint de 6-amino-1-benzyl [o 1-(2-chlorobenzyl)] uracil cilo a través de la formación de un aducto de Michael no a través de la formació d’un aducte de Michael no aïllat aislado seguido de adición nucleofílica y aromatización fi- seguit d’addició nucleofílica i aromatització final. El ma- nal. El mismo material de partida puede ser utilizado para teix material de partida pot ser utilitzat per a la prepara- la preparación de algunas nuevas pirimidodipirimidinas ció d’algunes noves pirimidodipirimidines per reflux amb por reflujo con diferentes aldehídos en medio ligeramente diferents aldehids en medi lleugerament bàsic. Finalment básico. Finalmente se han sintetizado diferentes lumazinas s’han sintetitzat diferents lumazines via reacció de tipus 2 vía reacción de tipo SN entre clorhidrato de diaminouracilo SN2 entre clorhidrat de diaminouracil amb bromur de fe- con bromuro de fenacilo seguida de ciclodeshidratación nacil seguida de ciclodeshidratació intramolecular i aro- intramolecular y aromatización. Estos nuevos compuestos matització. Aquests nous compostos de síntesis van mos- de síntesis mostraron propiedades de combinación, que- trar propietats de combinació, quelació i fragmentació de lación y fragmentación del ácido nucleico ADN. l’àcid nucleic ADN. Palabras clave: 6-Amino-1-bencil [o 1-(2-clorobenzil)] Paraules clau: 6-Amino-1-bencil [o 1-(2-clorobenzil)] ura- uracilo, clorhidrato de diaminouracilo, pirimidopirimidinas, cil, clorhidrato de diaminouracil, pirimidopirimidines, piri- pirimidodipirimidinas y lumazinas. midodipirimidines i lumazines. SUMMARY INTRODUCTION A new series of pyrimidopyrimidines are synthesized start- Resistance of tumor cells to chemotherapeutic agents is ing from 6-amino-1-benzyl[or1-(2-chlorobenzyl)]uracil via a major problem in the treatment of human malignancy.1-3 the formation of nonisolable acyclic Michael type adduct The causes of drug resistance fall into two groups; firstly, followed by nucleophilic addition and finally aromatization. those leading to inadequate drug exposure and, secondly, The same starting material can be used for the preparation alterations in the cancer cell itself that affect drug sensitiv- of some new pyrimidodipyrimidines by refluxing with dif- ity.4 Differences in the metabolic and structural properties ferent aldehydes in slightly basic medium. Finally different of cells may lead to drug-resistant phenotypes.5 The anti- 2 lumazines are synthesized via SN type reaction of diami- metabolite group of drugs blocks the de novo synthesis of nouracil hydrochloride with phenacyl bromides followed the purines and pyrimidines required for DNA synthesis.5-7 by intramolecular cyclodehydration then aromatization. Resistance has been reported due to the increased ac- The newly synthesized compounds showed binding, che- tivities of the enzymes in alternative pathways for purine lation and fragmentation of the nucleic acid DNA. and pyrimidine biosynthesis, the salvage pathways.5,7,8 The inhibitory activity of an antimetabolite depends on its Keywords: 6-Amino-1-benzyl[or1-(2-chlorobenzyl)]uracil, successful competition with the corresponding metabolite diaminouracil hydrochloride, pyrimidopyrimidines, pyrimi- for the enzymes of which the metabolite is the natural sub- dodipyrimidines and lumazines. 224 AFINIDAD LXIX, 559, Julio - Septiembre 2012 strate or cofactor. A series of nonnucleoside drugs as fla- (DMSO-d6) δ 11.34 (bs, 1H, NH), 7.70 (bs, 2H, NH2), 7.51- vin analogues9-11 e.g. alloxazine9 have exhibited antitumor 7.48 (m, 2H, arom.), 7.34-7.25 (m, 4H, arom.), 7.06-7.04 activity against different tumor cell lines. 5-Fluorouracil12,13, (m, 2H, arom.), 5.35 (s, 2H, NCH2). MS: m/z 439 (M++2, methotrexate (MTX)14,15 is the oldest antifolate anticancer 0.9), 437 (M+, 1.73), 404 (38), 402 (100). Anal. Calcd for 16 drugs, which is widely used as chemotherapeutic drug. C21H13Cl2N5O2 (438.26), Calcd.: C, 57.55, H, 2.99, N, 15.98, It competes with the normal substrates, folic acid and di- Found: C, 57.25, H, 3.45, N, 15.79. hydrofolate, for the active site on the enzyme dihydrofolate 7-Amino-1-(2-chlorobenzyl)-5-(4-fluorophenyl)-6- reductase (DHFR).2,17,18 Mild reduction in reduced folates cyanopyrido[2,3-d]pyrimidine-2,4(1H,3H)-dione (2b) at low serum concentrations of MTX results in inhibition of Yield: A 81%, m.p.= 312-314° C. IR= 3311, 3226 (NH2& pyrimidine (i.e. thymidine) synthesis.19,20 Thus, by inhibit- NH), 3072 (CH arom.), 2923, 2844 (CH aliph), 2219 (CN), ing DHFR, methotrexate prevents DNA synthesis and kills 1701, 1665 (2 C = O), 1637 (C = N), 1560 (C = C), 803 cells by depleting thymidylic acid.21-23 We rationalized to (p-substituted phenyl), 754 (o-substituted phenyl). Anal. synthesize new series of bicyclic fused uracil of pyridopy- Calcd for C21H13ClFN5O2 (421.81), Calcd.: C, 59.80, H, rimidines and lumazines as well as tricyclic derivatives of 3.11, N, 16.60, Found: C, 59.37, H, 3.10, N, 16.51. pyridodipyrimidines. 7-Amino-1-(2-chlorobenzyl)-5-(2-methoxyphenyl)-6- cyanopyrido[2,3-d]pyrimidine-2,4(1H, 3H)-dione (2c) Yield: A 89%, m.p.= 316-318° C. IR= 3310, 3227 (NH2& Materials AND METHODS NH), 3074 (CH arom.), 2953, 2838 (CH aliph), 2217 (CN), 1702, 1665 (2 C = O), 1635 (C = N), 1557 (C = C), 755 1 Chemistry (o-substituted phenyl). H-NMR (DMSO-d6) δ 11.32 (s, 1H, All melting points were determined with an Electrothermal NH, exchangeable), 7.70 (bs, 2H, NH2, exchangeable), Mel.-Temp. II apparatus and were uncorrected. Element 7.51-7.48 (m, 2H, arom.), 7.48-7.40 (m, 2H, arom.), 7.40- analyses were performed at the Micro Analytical Unit, 6.98 (m, 4H, arom.), 5.43-5.29 (two d, 2H, NCH2), 3.72 (s, Chemistry Department, Mansoura University. The infra- 3H, OCH3). Anal. Calcd for C22H16ClN5O3 (433.84), Calcd.: red (IR) spectra were recorded using potassium bromide C, 60.91, H, 3.72, N, 16.14, Found: C, 61.22, H, 4.21, N, disc technique on Nikolet IR 200 FT IR at Pharmaceutical 15.87. Analytical Unit, Faculty of Pharmacy, Al-Azhar University. 7-Amino-1-(2-chlorobenzyl)-5-(4-hydroxyphenyl)-6- The proton nuclear magnetic resonance (1H-NMR) spectra cyanopyrido[2,3-d]pyrimidine-2,4(1H,3H)-dione (2d) were recorded on Varian Gemini 300 MHz Spectrometer Yield: A 63%, m.p.= 293-295° C. IR= 3441 (OH), 3336, using DMSO-d6 as a solvent and tetramethylsilane (TMS) 3227 (NH2& NH), 3070 (CH arom.), 2980 (CH aliph), 2224 as an internal standard (Chemical shift in δ, ppm), Faculty (CN), 1690-1641 (br, C = O), 1569 (C = C &/or C = N), 821 of Science, Chemistry Department, Cairo University. Mass (p-substituted phenyl), 754 (o-substituted phenyl). Anal. spectra were recorded on DI-50 unit of Shimadzu GC/ Calcd for C21H14ClN5O3 (419.82), Calcd.: C, 60.08, H, 3.36, MS-QP 5050A at the Regional Center for Mycology and N, 16.68, Found: C, 60.15, H, 3.81, N, 17.00. Biotechnology at Al-Azhar University. All reactions were 5-Aryl-1-benzyl[or 1-(2-chlorobenzyl)]pyrido[2,3-d] py- monitored by TLC using precoted plastic sheets silica gel rimidine-2,4,7(1H,3H,8H)-triones (3a-d) (Merck 60 F254) and spots were visualized by irradiation A mixture of the appropriate 6-amino-1-benzyluracil 1a or with UV light (254 nm). The used solvent system was chlo- b (1.2 mmol) and ethyl arylidenacetoacetate (1.2 mmol) in roform: methanol (9:1) & ethyl acetate:toluene (1:1). DMF (3 ml) in the presence of TEA (0.3 ml) was heated un- 7-Amino-5-aryl-1-(2-chlorobenzyl)-6-cyanopyrido[2,3- der reflux for 6-8 hours. The reaction mixture was evapo- d]pyrimidine-2,4(1H,3H)-diones (2a-d) rated under diminished pressure. The residue was treated with ethanol (10 ml), the formed precipitate was filtered, Two methods were used to synthesize the target com- washed with ethanol and crystallized from DMF/ethanol pounds: (3:1) to afford the target pyridopyrimidines 3a-d. A- A mixture of 6-amino-1-(2-chlorobenzyl)uracil (1b) 1-Benzyl-5-(4-chlorophenyl)pyrido[2,3-d]pyrimidine- (0.3 g, 1.2 mmol) and the appropriate benzylidene- 2,4,7(1H,3H,8H)-trione (3a) malononitrile (1.2 mmol) in DMF (3 ml) in the presence Yield: 58%, m.p.= 250-252 °C. IR= 3160 (NH), 3028 (CH of TEA (0.3 ml) was heated under reflux for 6-8 hours. arom.), 2920, 2848 (CH aliph.), 1693 (C = O), 1596 (C = C), 1 The reaction mixture was evaporated under reduced 829 (p-substituted phenyl). H-NMR (DMSO-d6) δ 11.79 pressure. The residue was treated with ethanol (10 (bs, 1H, NH), 8.40-8.37 (d, 1H, arom.), 8.11-8.08 (d, 2H, ml), the formed precipitate was filtered, washed with arom.), 7.87-7.85 (d, 1H, arom.), 7.56-7.53 (d, 2H, arom.), ethanol and crystallized from DMF/ethanol (2:1) to af- 7.38- 7.22 (m, 4H, arom.), 7.20 (bs, 1H, NH &/or OH), 5.45 + ford 2a-d.