Chapter INTRODUCTION Nitrogen containing heterocycles are of immense importance biologically and industrially1 The triazines are among the oldest known organic nitrogen-containing heterocycles that possess three ‘N’ atoms in a six membered ring They may exist in three possible isomeric forms namely 1,2,3-triazine, 1,2,4-triazine and 1,3,5-triazine (Fig 1) N N N 1,2,3 -triazine N N N N 1,2,4 -triazine N N 1,3,5 -triazine Fig Isomeric forms of triazine (C3H3N3) In the literature, there are many anticancer agents that possess the 1,3,5-triazine heterocycle Many of them are being used in the clinic and some are undergoing clinical trials (Fig 2) HMM (Hexamethylmelamine) is a substituted 1,3,5-triazine derivative which is used clinically as an alkylating anti-neoplastic agent for the treatment of ovarian cancer2 A190 is a dihydrotriazine derivative with anti-tumor activity against lung cancer The mechanism of A190 is known to be associated with blocking the G1 phase of the cell cycle and death by apoptosis3 ZSTK 474, a morpholinyl analogue, inhibits an ATP-competitive pan-class I phosphatidylinositol kinase which plays a fundamental role in cellular responses such as proliferation, survival, motility and metabolism It has demonstrated antitumor activity against human cancer xenografts without showing toxic effects in critical organs4,5 Irsogladine [2,4-diamino-6-(2,5-dichlorophenyl)-1,3,5-triazine], an anti-gastric ulcer agent that is commonly used in Japan, has been shown to possess anti-angiogenic properties which contribute to the anti-cancer effect of the drug6 Dioxadet, a cytostatic agent, is used for the treatment of primary hepatic tumors and multiple intrahepatic metastases of colorectal carcinoma7 5-Aza-2'-deoxycytidine (decitabine) is a DNA methyltransferase -I and -3B inhibitor It also stops silencing of the proapoptotic BIK8 N Me N N Me N Me Me F2HC Cl N N N NH2 N Me N Me N H Hexamethylmelamine N N NH2 Cl- N N O ZSTK 474 NH2 N H2N N N Cl N N N N O A190 NH2 N N O HN N N N HO N O HO O O Irsogladine Cl OH 5-aza-2'deoxycytidine Dioxadet Fig Selected examples of existing anti-cancer agents that possess the 1,3,5-trazine heterocycle From the examples listed above, it is evident that the monocyclic 1,3,5-triazine heterocycle is an excellent scaffold for anti-cancer drug design and variation of substituents around this 1,3,5-triazine heterocycle may alter the mechanism of the anti-cancer action (ranging from alkylating RNA/DNA to anti-metabolite) Besides the monocyclic triazines, intensive research has been carried out on fused 1,3,5triazines9-12 in the search for antiproliferative lead compounds Recent reports have shown that a number of polycyclic fused 1,3,5-triazines also possess antiproliferative activity Some of these compounds include derivatives of pyrazolo[1,5-a] [1,3,5]triazine (e.g with IC50 (HCT 116) value = 0.99µM)9, 1,2,4-triazolo[1,5-a] [1,3,5]triazine (e.g with IC50 (MDA-MB-231) value = 28μM)10,11 and 1,3,5triazino[1,2-a]benzimidazole12 (e.g with IC50(DHFR) value =10.9μM) (Fig 3) 2 Fig Examples of polycyclic fused 1,3,5-triazines that show antiproliferative activity Hence, it would be rational to explore the fusion of 1,3,5-triazine ring with other heterocycles in an attempt to discover new fused 1,3,5-triazine scaffolds which may possess antiproliferative property With this as the aim, and in continuation of the efforts of our research group to find potent anti-cancer leads, the interest was extended to explore the synthesis of pyrimido fused 1,3,5-triazine system and its benzofused analogues In the following section, a comprehensive literature review of the approaches for the synthesis of pyrimido[1,2-a][1,3,5]triazines is presented It should be noted that the latest review of this scaffold presented by Mahajan and coworkers13 in 2008 contained only 16 references and have missed out many valuable references 1.1 Literature review on the synthesis of pyrimido[1,2-a][1,3,5]-triazines and its fused analogues Ziegler and Noelken14 were the first to synthesize the pyrimido[1,2-a][1,3,5]triazine system in 1961 (vide infra Scheme 1) The aim of this review is to summarize the methods developed for the preparation of compounds with the pyrimido[1,2a][1,3,5]triazine scaffold (Fig 4) and polyfused system bearing this heterocyclic core Information on the biological activity, if any, of pyrimido[1,2-a][1,3,5]triazine derivatives is also included N3 N N N Fig Pyrimido[1,2-a][1,3,5]triazine scaffold The review is collectively categorized according to the common strategies in the synthesis of pyrimido[1,2-a][1,3,5]triazines: (A) annulation of pyrimidine onto a 1,3,5-triazine scaffold; (B) annulation of the 1,3,5-triazine ring onto a pyrimidine scaffold; (C) concurrent formation of both the 1,3,5-triazine and pyrimidine ring The information on the biological activity of pyrimido[1,2-a][1,3,5]triazine derivatives is also included 1.1.1 Synthesis by annulation of pyrimidine ring onto a 1,3,5-triazine scaffold This synthetic strategy can be further classified into three methods as described below Annulation of pyrimidine to amino[1,3,5]triazine with three carbon synthons The reaction of 2-amino-1,3,5-triazines with synthons (such as the malonates 5) that contribute three atoms to pyrimidine annulation would yield pyrimido[1,2a][1,3,5]triazines The regioselectivity of the ring closure in the formation of the product was reported to be “questionable” by Ziegler and Noelken14 as equal probability existed for the formation of or 6’ However, the spectral support could not be obtained Similarly, various 2,4-substituted pyrimido[1,2-a][1,3,5]triazin-6,8diones were synthesized and patented as insecticidal agents15 There is a limitation using these malonates as only 6,8-dioxopyrimido[1,2-a][1,3,5]triazines can be synthesized Much later, Katritzky and Yousaf16 studied the mechanism of 1,3dicarbonyl compounds with bis nucleophiles and concluded that the ring closure was faster than the initial nucleophilic attack without the isolation of the intermediate (Scheme 1) R2 N N OR3 200°C, + R R1 N OR3 NH2 R2 R2 O N R O O N R4 N or N N R1 N N N O O O R4 R = Ph (5a), Et (5b), C6H3-2,4-Cl2 (5c) 6' R1 = H, R2 = H, R4 = Bn (6f) (ref 14) R1 = NH2, R2 = morpholinyl, R4 = H (6a); R1 = NH2, R2 = NMe2, R4 = H (6b); R1 = NH2, R2 = NEt2, R4 = H (6c); R1 = NH2, R2 = NH2, R4 = H (6d); R1 = NMe2, R2 = NMe2, R4 = H (6e) (ref 15) Scheme 1: Reaction of 2-amino-1,3,5-triazines with malonates to give 2,4,7 substituted pyrimido[1,2-a][1,3,5]triazines (6a-f) On the other hand, 1,4-dipolar cycloaddition of substituted bis-(2,4,6- trichlorophenyl)malonates 5d17 to N-alkyl substituted amino 1,3,5-triazines resulted in the formation of mesoionic 6-oxo pyrimido[1,2-a]-1,3,5-triazin-9-ium-8-olates in high yields (Scheme 2) According to IUPAC Compendium of Chemical Terminology, mesoionic compounds are defined as “dipolar five or six membered heterocyclic compounds in which both the negative and the positive charge are delocalized and which cannot be represented satisfactorily by any one polar structure” 18 R HN N N O NHR N SCH3 OC6H2Cl3 fusion, 180°C, 10-20 + R OC6H2Cl3 N N 62-92% O R1HN N O R2 SMe O 5d R1 N  2 R = R = C2H5, 92% (8a); R = C2H5, R = C4H9, 95% (8b); R = C2H5, R = Bn, 76% (8c); R1= i-Pr, R2 = C2H5, 62% (8d); R1= i-Pr, R2 = Bn, 68% (8e) Scheme 2: Reaction of 2-amino-1,3,5-triazines with substituted bis-(2,4,6-trichloro phenyl)malonates Cyclocondensation of pyrimidine ring to give 6-oxo-7-carboxylic acid ester derivative 11 of pyrimido[1,2-a][1,3,5]triazine scaffold can be easily done using ethoxymethylene malonate 10 as triatomic synthon Al-Shaar and co-workers19 synthesized this fused tricyclic 1,3,5-triazine derivative 11 by adding imidazo[1,5a][1,3,5]triazin-4-amine to boiling ethoxymethylene malonate (Scheme 3) EtO2C NH2 N EtO2C N OEt N + O N 55% OEt O N 10 N N N 10 N 11 Scheme 3: Reaction of imidazo[1,5-a][1,3,5]triazin-4-amine with ethoxymethylene malonate 10 fused pyrimido[1,2-a][1,3,5]-triazine 11 Annulation of pyrimidine to 1,3,5-triazine via base catalyzed rearrangement of the isoxazolone substituted 1,3,5-triazine20 In this method, acetamide derivative of 4,6-diamino-2-chloro-1,3,5-triazine 12 was reacted with isoxazolone ester 13 in ethanol Both N-acetyl groups were hydrolysed during the reaction period (10h, 80°C) to give 14 Compound 14 underwent ready rearrangement to 7-carboxyethyl-8-hydroxy-6-oxo derivative (15) on warming with 2.5 M NaOH (Scheme 4) NH2 NHAc N Cl + O N N 12 NHAc O N NH EtOH, 80°C, overnight 78% O O N N N NH2 EtO2C 13 EtO2C 14 NaOH/40°C 82% O EtO2C HO NH2 N N N N NH2 15 Scheme 4: Base catalyzed rearrangement of ethyl 2-(4,6-diamino-1,3,5-triazin-2-yl)5-oxo-2,5-dihydroisoxazole-4-carboxylate 14 to give pyrimido[1,2-a][1,3,5]-triazine 15 In this reaction, two different regioisomeric products might result via oxazolone ring opening and subsequent pyrimidine ring annulation of 14 However, since the chosen substrate 12 for the reaction had a plane of symmetry, there was no regioselectivity issue Therefore, regiochemistry of heterocyclization when unsymmetrically substituted substrates are used remains to be explored Annulation of pyrimidine via intramolecular ring closure21-24 In an attempt to obtain 18 via EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide) catalyzed amide coupling of N-triazino anthranillic acid 16 with alanine, the formation of 15% of fused pyrimido[1,2-a][1,3,5]-triazine derivative 17 was also reported This formation of 17 was a result of intramolecular ring closure leading to pyrimidine ring annulation which happened prior to coupling (Scheme 5) This kind of intramolecular ring closure was reported by many authors for the synthesis of various benzofused pyrimido[1,2-a][1,3,5]triazines Polyfused pyrimido[1,2-a][1,3,5]triazine 20 was also synthesized by similar intramolecular ring closure of N-triazino anthranillic acid substrate 19 21 (Scheme 6) OMe O OMe N N H OH O N Alanine Bz, EDC.HCl, HOBt, TEA, DMF N N OMe 0°C, 24h OMe N N H 16 Me N N N H + CO2Bn HN Me 17 C O N H N N CO2Bn N H Me CO2Bn 18 Scheme 5: EDC catalyzed intramolecular ring closure of 2-(4,6-dimethoxy-1,3,5triazin-2-ylamino)benzoic acid 16 O HN N O OR N N 19 Ac2O, reflux, 20h 76% N N CH3 N N N N N N CH3 20 R = H, Me Scheme 6: Intra-molecular ring closure of pentazacycl[3.3.3]azine 19 Refluxing 2,4-bis(o-carbomethoxyanilino)-6-dimethylamino-1,3,5-triazine 21 in acidic condition for 16h yielded 2(4)-anilino-4(2)hydroxy-6H-1,3,5-triazino[2,1b]quinazolin-6-one (22) Cyclization-deamination and decarboxylation were believed to have happened in the same step These compounds were patented as peripheral vascular dilating agents as well as for the preparation of dyestuff material22 (Scheme 7) NMe2 N O N H OMe O GAA/propionic acid, reflux, 16h N N N H N N O O OH N N N HN N N H N OH N OMe 22' 22 21 Scheme 7: Intra-molecular ring closure of 2,4-bis(o-carbomethoxyanilino)-6-dimethyl amino-1,3,5-triazine 21 N,N-diallylmelamine or N-alkyl-N-allylmelamine 23 would undergo (6+0) intramolecular heterocyclization to give 24 (Scheme 8) These compounds were reported to depress CNS and were patented for tranquilizing property23,24 NH NH2 N H2N N N TFA, CF3CO)2O, H2O2, 6°C to 72°C, 5h N 23 R R = CH3, -(CH2)nCH3, n= 1-7, -CH2CH=CH2 43% N H2N OH N N N R 24 Scheme 8: (6+0) intramolecular heterocyclization of N-alkyl-N’-allylmelamine 23 1.1.2 Synthesis by annulation of 1,3,5-triazine ring onto a pyrimidine scaffold Appropriately substituted pyrimidines may also serve as starting material for the synthesis of fused 1,3,5-triazine heterocycles The desired fused 1,3,5-triazine scaffold can be formed by annulation reaction on pyrimidine This approach has been adopted extensively for the preparation of pyrimido[1,2-a][1,3,5]triazines Suitably functionalized 2-amino pyrimidines can be cyclized to generate pyrimido[1,2a][1,3,5]triazines according to the strategies described below (Scheme 9) C N A) + N N N C N N N B) N N N + N + C C) N C N N N + N N N N N N N N F) N N N N + N N + C N N N + C + C N E) N N N N N N D) C N + C N N N N N +C N N N Scheme 9: Schematic strategies plan for the annulation reactions that generate pyrimido[1,2-a][1,3,5]triazines A) Two-bond formation (3+3) through cyclization of 2-aminopyrimidines with reagents introducing C-N-C fragment This synthetic approach provides an excellent opportunity for the preparation of a variety of functionalized pyrimido[1,2-a][1,3,5]triazines using 2-aminopyrimidines The triatomic C-N-C synthons used in the cyclization determine the substitution pattern at the positions and of the formed 1,3,5-triazine ring N-cyano-imidates 25, isocyanates 38/46 or isothiocyanates 50 are the examples of this type of synthons found in the literature for the construction of the desired scaffold (Fig 5) It should be noted that the regiochemistry of the ring closure was not always unambiguous and the structure assignments were often inadequate and therefore would require further verification O N N RO 25 R = Me, Et X N C Y X = Cl, Y = O, Chlorocarbonyl isocyanate 38 X = OPh, Y = O, Phenoxycarbonyl isocyanate 46 X = Ar, Y = S, Benzoyl isothiocyanate 50 Fig Examples of C-N-C synthons used in the annulation of 1,3,5-triazine onto a pyrimidine Annulation of 1,3,5-triazine ring with methyl N-cyanoformimidate 25a as C-N-C fragment introducing reagent The reaction of substituted 2-amino pyrimidin-4-ones 26 with methyl Ncyanoformimidate 25a in the presence of sodium methoxide could proceed via two possible modes of condensation-cyclization (N2-1 vs N2-3) with 27 being the possible intermediate 4-amino-8-oxo-8H-pyrimido[1,2-a][1,3,5]triazine (28) was formed via N2-1 by reaction in HMPA (hexamethyl phosphoramide) at 50-55°C25 The basic hydrolysis of 28 using 5% aqueous NaHCO3 yielded 29 whose crystal structure (as triflate (CF3SO3-) salt) was also reported (Scheme 10) The salt formation occurred, surprisingly, at the bridgehead nitrogen26 At low temperature (20°C), 2-amino-6methyl- 4-oxo-pyrimidine gave N2-3 cyclized product 30 regioselectively in anhydrous methanol The structure of this regioisomer 30 could be easily confirmed as two clearly differentiated N-H signals were observed in 1H NMR analysis because of the non-equivalence of the hydrogens of NH2 due to intra-molecular H-bonding Conversion of regioisomer 30 to 31 was also achieved by heating in dry dimethyl formamide (DMF) at 120°C for 2h It is important to note here that the 1,3,5-triazine ring undergoes a hydrolytic ring opening step in preference to the pyrimidine ring in dimethyl formamide (DMF) Isomers 32 and 33 were not observed This is consistent with the mechanism and is expected due to higher nucleophilicity of exocyclic nitrogen atom of 2-amino pyrimidin-4-one The authors demonstrated that regioisomeric 4-aminopyrimido[1,2-a][1,3,5]triazines can be exclusively isolated by controlling the reaction conditions (viz lowering temperature, solvent change) This opened up the possibility of interconversion among the regioisomers of pyrimido[1,2-a][1,3,5]triazinone 10 C-5’), 130.0 (C-2’ and C-4’), 131.1 (C-1’), 137.7 (C-4’), 157.5 (C-2), 159.7 (C-4), 161.0, 162.6 1-(4-methoxybenzyl)-3-(4-methyl-6-oxo-1,6-dihydropyrimidin-2-yl)guanidine (204) Yield 59%; mp 227-228°C; TLC (silica gel, MeOH:CH2Cl2, 1:6): Rf 0.57 1H NMR (300 MHz, Me2SO-d6): δ 2.03 (3H, s, Me), 3.71 (3H, s, OMe), 5.21 (2H, s, CH2), 5.45 (1H, s, H-5), 8.46 (2H, br s, NH-C(=NH)N), 10.58 (1H, br s, NH); N-(4-methyl-6-oxo-1,6-dihydropyrimidin-2-yl)indoline-1-carboxamidine (205) Yield 61%; mp 268-269°C; 1H NMR (300 MHz, Me2SO-d6): δ 2.10 (3H, s, Me), 3.15 (2H, t, 3J = 8.5 Hz, CH2), 3.97 (2H, t, 3J = 8.5 Hz, CH2), 6.93 (1H, t, 3J = 7.2 Hz), 7.09 (1H, t, 3J = 7.7 Hz), 7.17 (1H, d, 3J = 7.2 Hz), 8.64 (1H, d, 3J = 8.3 Hz), 11.24 (1H, s, NH) 13 C NMR (75 MHz, Me2SO-d6): δ 23.5 (Me), 26.4 (3’-CH2), 47.3 (2’-CH2), 103.5 (C-5), 118.1, 122.1, 124.1, 126.9, 131.6, 142.4, 157.9 (C-2), 155.3 (C-4), 162.7 (C=NH), 163.7 (C=O) 1-(3-bromophenyl)-3-(6-oxo-4-(trifluoromethyl)-1,6-dihydropyrimidin-2yl)guanidine (206) Yield 70%; mp 161-162°C (EtOH); 1H NMR (300 MHz, Me2SO-d6): δ 6.18 (1H, s, H7), 7.12-7.28 (2H, m, HAr), 7.74-7.89 (2H, m, HAr), 8.32 (1H, br s., NH), 10.19 (1H, br s., NH), 12.05 (1H, br s., NH); 13C NMR (75 MHz, Me2SO-d6): δ 103.4 (q, 3JC-F = 3.1 Hz, C-7), 119.1, 120.9 (q,1JC-F = 274.4 Hz, CF3), 121.4, 121.9, 122.8, 125.0, 130.4, 140.8, 150.9 (q, 2JC-F = 33.3 Hz, C-8), 156.2, 159.5, 163.4 General procedure for the synthesis of 197 and 199 Appropriate guanidine, 0.25 ml acetic acid and excess triethylorthoacetate were refluxed under nitrogen atmosphere Solvent was evaporated to dryness on rotary evaporator, purified using column chromatography and finally recrystallised using suitable solvent 4-amino-2,8-dimethyl-6H-pyrimido[1,2-a][1,3,5]triazin-6-one (199a) mp 264-265°C; TLC (silica gel, CH2Cl2): Rf 0.55; LC–MS (ESI) m/z 192.0841 (MH+); Anal Calcd for C8H9N5O: C, 50.26; H, 4.74; N, 36.63; found: C, 50.35, H, 4.95, N, 36.03.1H NMR (300 MHz, Me2SO-d6): δ 2.18 (1H, s, 2-Me), 2.24 (1H, s, 8-Me), 6.04 (1H, s, H-7), 9.26 (1H, s, NH), 10.18 (1H, s, NH); 13 C NMR (75 MHz, Me2SO-d6): 23.8 (8-Me), 129 25.2 (2-Me), 104.6 (C-7), 152.9, 156.8, 162.7, 167.6, 172.7; IR (KBr); v 3294 NH, 3116 br, 1695 C=O, 1647, 1575, 1400, 1197, 1170, 1060, 821, 792, 748, 702 4-amino-2-methyl-8-phenyl-6H-pyrimido[1,2-a][1,3,5]triazin-6-one (199b) Yield: 58%; mp 258-259°C (80AcOEt:20Hex); TLC (silica gel, 9:1 AcOEt:Hex): Rf 0.45; 1H NMR (300 MHz, Me2SO-d6): δ 2.29 (1H, s, 2-CH3), 6.77 (1H, s, H-7), 7.447.61 (3H, m, H-3’, H-4’, H-5’), 8.12 (2H, d, J = 8.1 Hz, H-2’ H-6’), 9.32 (1H, s, NH), 10.22 (1H, s, NH); 13C NMR (75 MHz, Me2SO-d6): 25.2 (2-CH3), 101.2 (C-7), 127.1, 128.6, 131.1, 135.6 (C-1’), 153.4, 156.8, 162.3, 163.6, 172.9; IR (KBr); v 3344 NH, 3213, 1674 C=O, 1624, 1570, 1544, 1448, 1382, 1220, 1174, 908, 778 purity 99.4%; tR 8.9 (MeOH:H2O) 2,8-dimethyl-4-(methylamino)-6H-pyrimido[1,2-a][1,3,5]triazin-6-one (199c) mp 166-167°C; TLC (silica gel, 9:1 AcOEt:Hex): Rf 0.16; LC–MS (ESI) m/z 206.0946 (MH+); Anal Calcd for C9H11N5O: C, 52.67; H, 5.40; N, 34.13; found: C, 52.49, H, 5.69, N, 32.83 1H NMR (300 MHz, Me2SO-d6): δ 2.18 (3H, s, 2-Me), 2.28 (3H, s, 8Me), 2.98 (3H, d, J = 4.9 Hz, NMe), 6.07 (1H, s, H-7), 10.90 (1H, d, J = 4.5 Hz, NH); 13 C NMR (75 MHz, Me2SO-d6): 23.7 (8-Me), 25.6 (2-Me), 28.4 (NMe), 104.8 (C-7), 152.6, 155.5, 163.0, 167.5, 172.3; IR (KBr); v 3324 NH, 3201, 1690 C=O, 1642, 1574, 1440, 1187, 1165, 1065, 793, 774 4-(4-chlorobenzylamino)-2,8-dimethyl-6H-pyrimido[1,2-a][1,3,5]triazin-6-one (199g) mp 133-134°C; TLC (silica gel, 9:1 AcOEt:Hex): Rf 0.8; LC–MS (ESI) m/z 316.0817 (MH+); Anal Calcd for C15H14ClN5O: C, 57.06; H, 4.47; Cl, 11.23; N, 22.18; found C, 56.99; H, 4.51; Cl 11.44; N, 22.05 1H NMR (300 MHz, Me2SO-d6): δ 2.19 (3H, s , 8-Me), 2.25 (3H, s, 2-Me), 4.69 (2H, s, CH2), 6.08 (1H, s, H-7), 7.39 (2H, d, J = 8.7 Hz, H-3’ and H-5’), 7.43 (2H, d, J = 8.7 Hz, H-2’ and H-6’), 11.43 (1H, br s., NH); 13C NMR (75 MHz, Me2SO-d6): 23.8 (2-Me), 25.6 (8-Me), 43.9 (CH2), 105.0 (C-7), 128.3 (C-3’and C-5’), 129.5 (C-2’ and C-6’), 131.8 (C-4’), 136.4 (C-1’), 152.7, 155.2, 163.2, 167.6, 172.3 ; IR (KBr); v 3170 br NH, 1689 C=O, 1618, 1411, 1377, 1344, 827, 794, 711 4-(4-methoxybenzylamino)-2,8-dimethyl-6H-pyrimido[1,2-a][1,3,5]triazin-6-one (199h) mp 138-139°C; TLC (silica gel, 9:1 AcOEt:Hex): Rf 0.26; LC–MS (ESI) m/z 312.1409 (MH+); Anal Calcd for C16H17N5O2: C, 61.72; H, 5.50; N, 22.49; found: C, 130 61.78; H, 5.42; N, 22.52 1H NMR (300 MHz, Me2SO-d6): δ 2.19 (3H, s, 2-Me), 2.28 (3H, s, 8-Me), 3.74 (3H, s, OMe), 4.62 (2H, d, J = 5.7 Hz, CH2), 6.07 (1H, s, H-3), 6.91 (1H, d, J = 8.7 Hz, H-3’ and H-5’), 7.34 (1H, d, J = 8.3 Hz, H-2’ and H-6’), 11.36 (1H, t, J = 5.7 Hz, NH); 13 C NMR (75 MHz, Me2SO-d6): 23.8 (8-Me), 25.6 (2-Me), 44.1 (CH2), 55.0 (OMe), 104.9 (C-7), 113.8 (C-2’ and C-6’), 128.9 (C-1’), 129.2 (C-3’ and C-5’), 152.6 (C-4’), 155.1, 158.6, 163.3, 167.7, 172.4; IR (KBr); v 3109 br NH, 2950 (CH), 1683 C=O, 1629, 1516, 1458, 1379, 1342, 1172, 1114, 1026, 821, 792 4,8-dimethyl-2-(phenylamino)-6H-pyrimido[1,2-a][1,3,5]triazin-6-one (197d) mp 220-221°C; TLC (silica gel, 9:1 AcOEt:Hex): Rf 0.53; LC–MS (ESI) m/z 268.1141 (MH+); Anal Calcd for C14H13N5O: C, 62.91; H, 4.90; N, 26.20; Found 62.61, 4.95, 26.03 1H NMR (300 MHz, Me2SO-d6): δ 2.18 (3H, s, 8-Me), 2.90 (3H, s, 4-Me), 5.92 (1H, s, H-7), 7.13 (1H, t, 3J = 7.4 Hz, H-5’), 7.38 (2H, t, 3J = 7.7 Hz, H-3’ and H-5’), 7.86 (2H, d, 3J = 7.5 Hz, H-2’ and H-6’), 10.63 (1H, s, NH); 13 C NMR (75 MHz, Me2SO-d6): δ 23.7 (2-Me), 26.2 (8-Me), 103.6 (C-5), 120.4 (C-2’ and C-6’), 123.9 (C4’), 128.6 (C-3’ and C-5’), 138.0 (C-1’), 152.9, 156.8 (C-2), 160.0, 163.8, 166.7; IR (KBr); v 3109 br NH, 1685 C=O, 1627, 1535, 1244, 1036, 821, 752 2-(3-chlorophenylamino)-4,8-dimethyl-6H-pyrimido[1,2-a][1,3,5]triazin-6-one (197e) mp 239-240°C; TLC (silica gel, 9:1 AcOEt:Hex): Rf 0.60; LC–MS (ESI) m/z 302.0708 (MH+); Anal Calcd for C14H12ClN5O: C, 55.73; H, 4.01; Cl, 11.75; N, 23.21; Found: C, 55.49; H, 4.05; Cl 11.50; N, 22.98 1H NMR (300 MHz, Me2SO-d6): δ 2.19 (1H, s, 8-Me), 2.91 (1H, s, 4-Me), 5.96 (1H, s, H-7), 7.18 (1H, d, J = 8.3 Hz, H4’), 7.40 (1H, t, J = 8.1 Hz, H-5’), 7.76 (1H, d, J = 7.9 Hz, H-6’), 8.06 (1H, s, H-2’), 10.79 (1H, s, NH); 13 C NMR (75 MHz, Me2SO-d6): 23.8 (8-Me), 26.2 (4-Me), 104.0 (C-7), 118.7, 119.5, 123.5, 130.3, 133.0, 139.7 (C-1’), 152.7, 156.9, 160.0, 164.3, 166.7; IR (KBr); v 3273 br NH, 3103 (CH), 3082, 1678 C=O, 1636, 1095, 866, 788, 717 purity 95.0%; tR 25.1 (MeOH:H2O) 2-(4-methoxyphenylamino)-4,8-dimethyl-6H-pyrimido[1,2-a][1,3,5]triazin-6-one (197f) mp 177-178°C; TLC (silica gel, 9:1 Hex:AcOEt): Rf 0.25; LC–MS (ESI) m/z 298.1296 (MH+); Anal Calcd for C15H15N5O2: C, 60.60; H, 5.09; N, 23.56 Found: C, 60.47; H, 5.10; N, 23.29 1H NMR (300 MHz, Me2SO-d6): δ 2.16 (3H, s, 8-Me), 2.88 (3H, s, 4-Me), 3.75 (3H, s, OMe), 5.89 (1H, s, H-7), 6.96 (2H, d, J = 8.8 Hz, H-3’ 5’), 7.74 (2H, d, J = 8.8 Hz, H-2’ 6’), 10.52 (1H, s, NH); 13C NMR (75 MHz, Me2SO-d6): 131 23.7 (4-Me), 26.2 (8-Me), 55.2 (OMe), 103.2 (C-7), 113.8 (C-2’ and C-6’), 122.1 (C3’ and C-5’), 130.9 (C-1’), 153.1 (C-4’), 155.8, 156.5, 160.1, 163.5, 166.7; IR (KBr); v 3109, 2920 (CH), 2850, 1670 C=O, 1627, 1541, 1419, 1236, 1174, 1028, 831, 788 2-(dimethylamino)-4,8-dimethyl-6H-pyrimido[1,2-a][1,3,5]triazin-6-one (197i) mp 190-191°C; TLC (silica gel, CH2Cl2): Rf 0.30; LC–MS (ESI) m/z 220.1198 (MH+); Anal Calcd for C10H13N5O: C, 54.78; H, 5.98; N, 31.94; found: C, 54.42; H, 5.87; N, 31.69 1H NMR (300 MHz, Me2SO-d6): δ 2.12 (3H, s, 8-Me), 2.85 (3H, s, 4-Me), 3.14 (3H, s, N(Me)2), 3.25 (3H, s, N(Me)2), 5.78 (1H, s, H-7); 13C NMR (75 MHz, Me2SOd6): δ 23.8 (8-Me), 26.6 (4-Me), 36.3 (N(Me)2, 36.4 (N(Me)2, 101.8 (C-7), 153.1, 158.0, 160.1, 163.5, 167.2 (C=O) ; IR (KBr); v 3420 br NH, 3034 (CH), 2978, 1714, 1670 C=O, 1620, 1516, 1317, 1238, 1192, 1078, 1033, 966, 825, 794, 717 purity 95.0%; tR 15.7 (MeOH:H2O) 4,8-dimethyl-2-morpholino-6H-pyrimido[1,2-a][1,3,5]triazin-6-one (197j) mp 191192°C; TLC (silica gel, 9:1 CH2Cl2): Rf 0.40; LC–MS (ESI) m/z 262 (MH+); Anal Calcd for C12H15N5O2: C, 55.16; H, 5.79; N, 26.80; found C, 55.22; H, 5.77; N, 26.86 H NMR (300 MHz, Me2SO-d6): δ 2.13 (3H, s, 8-Me), 2.86 (3H, s, 4-Me), 3.67 (t, J = 4.5 Hz, 4H, (CH2)2O), 3.78 (t, J = 4.3 Hz, 2H, N(CH2), 3.89 (t, J = 4.3 Hz, 2H, N(CH2), 5.81 (s, 1H, H-7); 13 C NMR (75 MHz, Me2SO-d6): δ 25.0 (8-Me), 27.8 (4- Me), 44.8 (CH2), 45.3 (CH2), 66.7 (CH2), 67.1 (CH2), 103.3 (C-7), 154.4, 158.3, 161.2, 165.5, 168.3 (C=O); IR (KBr); v 3388 br NH, 3076 (CH), 2950, 1627 C=O, 1543, 1508, 1406, 1352, 1246, 1181, 1028, 966, 834 2-(indolin-1-yl)-4,8-dimethyl-6H-pyrimido[1,2-a][1,3,5]triazin-6-one (197k) mp 212213°C (AcOEt); TLC (silica gel, 9:1 AcOEt:Hex): Rf 0.32; Anal Calcd for C16H15N5O: C, 65.52; H, 5.15; N, 23.88; found C, 65.27; H, 5.14; N, 23.74 1H NMR (300 MHz, Me2SO-d6): δ 2.18 (3H, s, 8-Me) and 2.21 (3H, s, 8-Me), 2.94 (3H, s, 4Me) and 3.00 (3H, s, 4-Me), 3.20 (2H, t, 3J = 8.5 Hz, 3’CH2), 4.19 (1H, t, 3J = 8.3 Hz, CH2) and 4.32 (1H, t, 3J = 8.9 Hz, CH2), 5.92 (2H, s, CH), 6.98-7.16 (2H, m, H-4’), 7.21-7.40 (4H, m, H-5’ and H-6’), 8.29 (1H, d, 3J = 8.2 Hz, H-7’), 8.49 (1H, d, 3J = 8.1 Hz, H-7’); 13C NMR (75 MHz, Me2SO-d6): δ 23.5 (Me), 26.4 (3’-CH2), 47.3 (2’-CH2), 103.5 (C-5), 118.1, 122.1, 124.1, 126.9, 131.6, 142.4, 157.9 (C-2), 155.3 (C-4), 162.7 (C=NH), 163.7 (C=O) Two rotamers in the ratio of 1:0.8 were observed for 197k; IR 132 (KBr); v 3446 br NH, 2935 (CH), 2918, 2854, 1707 C=O, 1624, 1576, 1481, 1456, 1249, 1195, 785 2-(3-bromophenylamino)-4-methyl-8-(trifluoromethyl)-6H-pyrimido[1,2-a][1,3,5] triazin-6-one (197l) Yield: 70%; mp 216-217°C (EtOH); TLC (silica gel, 9:1 AcOEt:Hex): Rf 0.90 1H NMR (300 MHz, Me2SO-d6): δ 2.91 (1H, s, Me), 6.54 (1H, s, H-7), 7.32-7.42 (2H, m, HAr), 7.86 (1H, d, J = 6.8 Hz), 8.10 (1H, s, HAr), 11.08 (1H, s, NH); 26.07 (CH3), (C4), 103.3 (q, 3JC-F = 2.6 Hz, C-7), 119.5, 120.6 (q,1JC-F = 275.4 Hz, CF3), 121.5, 122.8, 127.1, 130.8, 139.2, 152.6 (q, 2JC-F = 34.0 Hz, C-8), 155.1, 157.2, 160.2, 164.4; IR (KBr); v 3282 NH, 3081, 2945, 1699 C=O, 1631, 1608, 1587, 1552, 1465, 1375, 1340, 1278, 1192, 1155, 1101, 1083, 925, 875, 788, 707 purity 94.8% tR = 15.7 (MeOH:H2O); purity 96.7% tR = 7.1 (CH3CN:H2O) 3-(6-methyl-4-(phenylamino)-1,3,5-triazin-2(1H)-ylideneamino)but-2-enoic acid (208d) LC–MS (ESI) m/z 285.1293 (MH+); TLC (silica gel, 9:1 AcOEt:Hex): Rf 0.16; Anal Calcd for C14H15N5O2: C, 58.94; H, 5.30; N, 24.55; found 56.18, H 4.89, N 23.58; 1H NMR (300 MHz, Me2SO-d6): δ 2.17 (3H, s, 8-Me), 2.25 (3H, s, 4-Me), 5.77 (1H, s, CH), 7.13 (1H, t, J = 7.3 Hz, H-4’), 7.33 (2H, t, J = 7.7 Hz, H-3’ and H-5’), 7.84 (2H, d, J = 7.9 Hz, H-2’ and H-6’), 10.94 (1H, s, NHPh), 12.06 (1H, br s, NH), 13.75 (1H, br s, COOH) 3-(4-(4-methoxyphenylamino)-6-methyl-1,3,5-triazin-2(1H)-ylideneamino) but-2- enoic acid (208f) LC–MS (ESI) m/z 316.1255 (MH+); TLC (silica gel, 9:1 Hex:AcOEt): Rf 0.11; Anal Calcd for C15H17N5O3: C, 57.13; H, 5.43; N, 22.21; found C, 57.44; H, 5.21; N, 22.45 1H NMR (300 MHz, Me2SO-d6): δ 2.17 (3H, s, 8-Me), 2.24 (3H, s, 4-Me), 3.75 (3H, s, OMe), 5.74 (1H, s, CH), 6.87 (2H, d, J = 8.3 Hz, H2’and H-6’), 7.74 (2H, d, J = 9.0 Hz, H-3’and H-5’), 10.82 (1H, s, NHPh), 11.97 (1H, s, NH), 13.72 (1H, s, COOH) 3-(4-(4-methoxybenzylamino)-6-methyl-1,3,5-triazin-2(1H)-ylideneamino) but-2- enoic acid (208h) 1H NMR (300 MHz, Me2SO-d6): δ 2.12 (3H, s, 4-Me), 2.17 (3H, s, Me), 3.74 (3H, s, OMe), 4.57 (2H, d, J = 5.7 Hz, CH2), 5.66 (1H, s, H-3), 7.37 (2H, d, 133 J = 8.7 Hz, H-3’ and H-5’), 7.48 (1H, d, J = 8.3 Hz, H-2’ and H-6’), 9.30 (1H, t, J = 5.7 Hz, NH), 11.63 (1H, br s, NH), 13.72 (1H, s, COOH) 3-(4-(4-chlorobenzylamino)-6-methyl-1,3,5-triazin-2(1H)-ylideneamino) but-2-enoic acid (208g) H NMR (300 MHz, Me2SO-d6): δ 2.12 (3H, s , 4-Me), 2.17 (3H, s, Me), 4.56 (2H, d, J = 6.0 Hz, CH2), 5.66 (1H, s, CH), 7.37 (2H, d, J = 8.7 Hz, H-3’ and H-5’), 7.48 (2H, d, J = 8.7 Hz, H-2’ and H-6’), 9.30 (1H, t, J = 5.9 Hz, NH), 11.48 (1H, br s., NH), 13.72 (1H, s, COOH); 13 C NMR (75 MHz, CDCl3): 23.7 (4-Me), 24.8 (Me), 44.0 (CH2), 105.7 (CH), 128.9 (C-3’and C-5’), 129.1 (C-2’ and C-6’), 133.5 (C-4’), 135.9 (C-1’), 153.5, 156.7, 162.9, 163.6, 172.0 General procedure for the synthesis of 213 Appropriate guanidine and excess equivalent of DMA-DMA (with/without toluene) were refluxed under nitrogen atmosphere The reaction was monitored using TLC The solvent was evaporated to dryness on rotary evaporator, purified using column chromatography and finally recrystallised using suitable solvent (E)-2-(dimethylamino)-4-(2-(dimethylamino)prop-1-enyl)-8-methyl-6Hpyrimido[1,2-a][1,3,5]triazin-6-one (213a) Yield: 73%; physical appearance: orange; mp 212-213°C (MeOH:AcOEt); MS (ESI) m/z: 289 (MH+); Anal calc for C14H20N6O: C, 58.31; H, 6.99; N, 29.15; found: 58.54; H, 7.17; N, 28.89 1H NMR (300 MHz, Me2SO-d6): δ 2.06 (3H, s , 9-Me), 2.64 (3H, s, 4-Me), 3.05 (6H, s, (N(Me)2), 3.05 (6H, s, (N(Me)2), 3.10 (6H, s, (NMe)2), 5.63 (1H, s, 8-CH), 6.13 (1H, s, =CH-N); 13 C NMR (75 MHz, Me2SO-d6): 18.2 (4-Me), 23.6 (9- Me), 36.2 (N(Me)2), 36.4 (N(Me)2), 90.9 (=CH-N), 101.1 (C-8), 154.7, 157.5, 158.4 (C-5), 157.5, 158.4, 161.3, 164.9, 166.1 (E)-4-(2-(dimethylamino)prop-1-enyl)-8-methyl-2-morpholino-6H-pyrimido[1,2a][1,3,5]triazin-6-one (213b) Yield: 69%; physical appearance: yellow; mp 217-218°C (MeOH:AcOEt); MS (ESI) m/z: 331 (MH+); Anal calc for C16H22N6O2: C, 58.17; H, 6.71; N, 25.44; 1H NMR (300 MHz, Me2SO-d6): δ 2.06 (3H, s , 9-Me), 2.61 (3H, s, 4-Me), 3.11 (6H, s, (N(Me)2), 3.65 (4H, m, (CH2)2, 3.72 (4H, m, (CH2)2), 5.67 (1H, s, 8-CH), 6.17 (1H, s, 134 =CH-N); 13 C NMR (75 MHz, Me2SO-d6): 18.5 (4-Me), 23.5 (9-Me), 43.8, 65.8, (N(Me)2), 91.2 (=CH-N), 101.5 (C-8), 154.8, 157.6, 158.0 (C-5), 161.2, 165.4, 165.9; IR (KBr); v 3352 br NH, 1681 C=O, 1616, 1565, 1171, 979 (E)-2-(3-chlorophenylamino)-4-(2-(dimethylamino)prop-1-enyl)-8-methyl-6Hpyrimido[1,2-a][1,3,5]triazin-6-one (213c) Yield: 59%; 1H NMR (300 MHz, CDCl3): δ 2.28 (3H, s , 9-Me), 2.66 (3H, s, 4-Me), 3.09 (6H, s, (N(Me)2), 5.21 (1H, s, 8-CH), 5.87 (1H, s, =CH-N), 7.14 (1H, d, J = 8.3 Hz, H-4’), 7.53 (1H, m, H-5’), 7.74 (1H, d, J = 7.9 Hz, H-6’), 7.85 (1H, s, H-2’), 13.32 (1H, s, NH) (E)-4-(2-(dimethylamino)prop-1-enyl)-8-methyl-2-(methylamino)-6H-pyrimido[1,2a][1,3,5]triazin-6-one (213d) Yield: 52%; 1H NMR (300 MHz, Me2SO): δ 2.07 (3H, s, 4-Me), 2.28 (3H, s, 9-Me), 2.97 (6H, s, (N(Me)2), 3.52 (3H, s, NMe), 5.21 (1H, s, 8-CH), 5.87 (1H, s, =CH-N), 7.14 (1H, d, J = 8.3 Hz, H-4’), 7.53 (1H, m, H-5’), 7.74 (1H, d, J = 7.9 Hz, H-6’), 7.85 (1H, s, H-2’), 13.32 (1H, s, NH) (E)-2-amino-4-(2-(dimethylamino)prop-1-enyl)-8-phenyl-6H-pyrimido[1,2a][1,3,5]triazin-6-one (213e) mp 232-233°C (MeOH:AcOEt); MS (ESI) m/z: 323 (MH+) 1H NMR (300 MHz, Me2SO): δ 2.30 (3H, s, 3-Me), 3.11 (6H, s, (N(Me)2), 6.34 (1H, s, =CH-N), 6.45 (1H, s, 8-CH), 7.49 (3H, m, H-3’, H-4’ and H-5’), 8.04 (2H, m, H-2’ and H-6’), 10.65 (1H, br s, NH), 13.66 (1H, s, NH); Anal calc for C16H22N6O2: C17H18N6O C, 63.34; H, 5.63; N, 26.07; found C, 63.04; H, 5.41; N, 25.95; IR (KBr); v 3045, 2922 (CH), 1678 C=O, 1645, 1471, 1398, 910, 819, 785, 711 135 CHAPTER POTENTIAL MECHANISM OF ACTION AND TARGET IDENTIFICATION OF LEAD PYRIMIDO[1,2-a][1,3,5]TRIAZINONES Understanding the relationship between a molecule and its biological activity are a central theme in medicinal chemistry In previous chapters, six compounds viz 149b and 149f from the 8-methyl pyrimido[1,2-a][1,3,5]triazin-6-one series and 173b, 173c, 173f and 173h from the benzo fused pyrimido[1,2-a][1,3,5]triazin-6-one series were identified as bioactive leads having IC50 value in the range of 4.9-7.4µM based on MTT assays Since the MTT assay only measures cell viability, information on the mechanism of action and target identification are not provided This often is among the most difficult processes in chemical biology research144 Therefore, the objective of this chapter was to investigate the potential mechanism of cell death brought about by the exposure to the bioactive leads as observed in the MTT assay In addition, an attempt to identify putative targets of the synthesized compounds using “reverse/inverse" virtual screening approaches was carried out 4.1 Modes of cell death Cell death and cell proliferation are tightly controlled in their activation and execution Both these processes co-ordinate to keep the cell numbers in the body at appropriate levels Cell death is classified into types: Type cell death (Apoptosis): Apoptosis is an evolutionarily conserved form of cellular suicide and requires a specialized proteolytic system that involves a family of proteases called caspases145 Caspases are synthesized in cells as inactive zymogens, which upon stimulation by apoptotic signals, are processed into mature tetrameric forms with two large and two small subunits146 No matter whether the activation is initiated by the activation of cell-surface death receptors, (such as Fas and TNF) or by cytochrome c released from the mitochondria (mitochondria-independent apoptosis pathway/ extrinsic apoptosis), these activated caspases participate in the cleavage of a set of proteins, resulting in the disassembly of the cell One such protein is nuclear poly (ADP-ribose) polymerase (PARP) This 116kD protein is cleaved to give cPARP 89kD147, which can be detected by techniques like immune-blotting Defects in apoptosis may contribute to tumor progression and resistance to treatment148 It has 136 been documented that elucidating the molecular mechanisms associated with specific apoptotic processes, such as identifying means to trigger effective apoptosis, is an attractive therapeutic strategy in discovering potential cure for human cancers149 Type cell death (Autophagy): is a physiological process which involves the degradation of a cell's own components through the lysosomal machinery Portions of cytosol and organelles are sequestered into a double-membrane vesicle (autophagosome) and delivered into a degradative organelle (the vacuole or lysosome) for breakdown of the resulting macromolecules150 Type cell death (Necrosis) is characterized by general disintegration and deletion of organelles Two subtypes of necrosis were reported In type 3A, lysosome maintains its integrity whereas in type 3B, lysosomal membrane damage leads to cytoplasmic cell death151 Using the immunoblotting experimental approach (Western blot analysis), observed cytotoxicity of the six lead compounds in MTT assay was semi-quantified based on chemically induced stimulation of caspase dependent apoptotic signal 4.2 Computer-aided Virtual Screening Approaches Virtual screening (VS) approaches for druggable target identification generally involve molecular docking of chemical libraries into the 3D structure of a protein target or performing ligand similarity search using pharmacophore/fragment fingerprint In VS, the aim is to identify compounds that will fit a specific protein (i.e when target is known) On the other hand, term “reverse” or “inverse” VS is used when the aim is to identify protein(s) that will fit a given test compound By screening a test compound against a protein target database in-silico, it is possible to identify proteins as potential targets which can be experimentally validated subsequently To date, only a few reverse VS methods152-156 are available, most of which involve a docking algorithm based approach for target identification However, two new reverse VS approaches based on pharmacophore mapping (PharmMapper) and ligand similarity with bioactive conformation (Reverse Screen 3D) were introduced recently and the webservers of these methods were available for query submission 137 Fig 26 Schematic flowsheet of the reverse/inverse VS approach used Despite having their own limitations, it was hypothesized that the new combined approach towards target identification could give us more realistic list of targets for experimental validation Therefore, the aim of the present study was to identify the potential targets for the six selected compounds having IC50 values below 10µM Step 1: Finding the common targets (proteins/receptors) among the target hits obtained from two different VS approaches viz pharmacophore mapping (pharmMapper) and active ligand conformation matching approach (Reverse Screen 3D) This narrows down the number of target hits Step 2: Re-ranking of the predicted target hits using docking approach (INVDOCK) and evaluating the role of each predicted target that is implicated in cancer development This further narrows down the number of predicted targets Step 3: Target validation using substrate/ligand similarity with the six selected compounds as the criteria Tanimoto or Jaccard Coefficient157 was used as an index of similarity among molecules 138 According to Tanimoto (or Jaccard) Coefficient, if Na is the number of features in A, Nb is the number of features in B, and Nab is the number of features common to both A and B, then the Tanimoto coefficient is derived as: Tab = Nab / Na + Nb - Nab Tab >0.7 is considered to be good cut off for likely similar molecules having a high chance of interacting with the same target biologically158 Molecules with similar structure are likely to have similar biological activity159 4.3 Results and Discussion Western blot results Fig 27 Western blot analysis of cell lysates probed with PARP antibody As observed from the Figure 26, no band was observed for cleaved PARP in vehicle controls whereas the strong band corresponding to the positive control was observed The cleaved PARP bands of very low intensity were observed for 149f, 173c and 173h The selected compounds elicited only faint PARP cleavage, suggesting that these compounds might induce modes of cell death other than caspase dependent apoptosis The appearance of only faint bands suggested that other mechanisms of cell death might be synergistic with caspase dependent apoptosis It is known that mitochondria, as the central integrator of programmed cell death signaling pathways, is able to release multiple factors that may trigger a caspase-independent cell death Caspase independent apoptosis or other modes of cell death as cellular response to anticancer therapy were highlighted by Brown et al.160 139 Due to the involvement of an enormous number of targets (proteins/receptors/genes) in various tumorigenic pathways, in-silico target profiling methods are emerging as efficient alternatives to the expensive experimental high-throughput in-vitro target profiling of compounds161-163 Therefore, it was worthwhile to explore in-silico approaches to identify the possible druggable targets involved in cell death observed in MTT assay in response to the pyrimido[1,2-a][1,3,5]triazine exposure The list of targets found common in different VS approaches viz Pharm mapper and Reverse Screen 3D are presented in Tables XVIII-XXIII In Table XVIII, 44 common targets were identified for 149b Careful evaluation of retrieved targets suggest many entries to be false hits and not implicated in cancer These include purine nucleoside phosphorylase (S/N 4), HCV RNA polymerase (S/N 16), 2-C-methyl-D-erythritol 2,4cyclodiphosphate synthase (S/N 19), 3-oxoacyl-[acyl-carrier-protein] synthase (S/N 29), scytalone dehydratase (S/N 33), pantothenate synthetase (S/N 34), UDP-glucose 4-epimerase (S/N 35), transthyretin (S/N 40) and 1-aminocyclopropane-1-carboxylate deaminase (S/N 43) EGFR and HSP90α were predicted to be the topmost targets based on the fit score obtained from pharmMapper for 149b whereas purine nucleoside phosphorylase and nicotinate-nucleotide- dimethylbenzimidazolephosphoribosyltransferase were predicted to be topmost targets by Reverse screen 3D In Table XIX, 22 targets were found to be common among the targets predicted for 149f by PharmMapper and Reverse screen 3D Purine nucleoside phosphorylase (S/N 2), UDP-glucose 4-epimerase (S/N 4), 6,7-dimethyl-8-ribityllumazine synthase (S/N 6), imidazole glycerol phosphate synthase (S/N 9), enoyl-[acyl-carrier-protein] reductase [NADH] (S/N 13), 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (S/N 15), nicotinate-nucleotide-dimethylbenzimidazole phosphoribosyltransferase (S/N 20), phosphopantetheine adenylyltransferase (S/N 21) appeared to be false positives Nucleoside diphosphate kinase and purine nucleoside phosphorylase were among the top targets predicted by PharmMapper whereas imidazole glycerol phosphate synthase and thymidylate synthase were among the top targets based on Reverse Screen 3D results 31 targets were found to be common among the targets predicted for 173b by PharmMapper and Reverse Screen 3D (Table XX) 2-C-methyl-D-erythritol 2,4140 cyclodiphosphate synthase (S/N 6), enoyl-[acyl-carrier-protein] reductase [NADH] (S/N 9), 2-amino-4-hydroxy-6-hydroxymethyl dihydropteridine pyrophosphokinase (S/N 10), purine nucleoside phosphorylase (S/N 11), scytalone dehydratase (S/N 20) seems to be false positives Adenylate cyclase was among the top targets predicted by PharmMapper whereas androgen receptor and DHFR were among the top targets based on Reverse Screen 3D results 51 targets were found to be common among the targets predicted for 173c by PharmMapper and Reverse Screen 3D (Table XXI) Phosphoenolpyruvate carboxykinase (S/N 3), scytalone dehydratase (S/N 5), streptogramin A acetyltransferase (S/N 8), mast/stem cell growth factor receptor (S/N 11), Neprilysin (S/N20), pyruvate oxidase (S/N 21), renin (S/N 23), biotin carboxylase (S/N 27), trypanothione reductase (S/N 33), oxygen-insensitive NAD(P)H nitroreductase (S/N 35), purine nucleoside phosphorylase (S/N 44) and nicotinate-nucleotide-dimethylbenzimidazole phosphoribosyltransferase (S/N 51) seems to be false positives Phosphatidylinositol-4,5-bisphosphate-3-kinase were among the top targets predicted by both PharmMapper and Reverse Screen 3D 73 targets were found to be common among the targets predicted for 173f by PharmMapper and Reverse Screen 3D (Table XXII) Dihydrodipicolinate reductase (S/N 5), chorismate synthase (S/N 11), phosphopantetheine adenylyltransferase (S/N 13), purine nucleoside phosphorylase (S/N 19), 2-amino-4-hydroxy-6- hydroxymethyldihydro pteridine pyrophospho kinase (S/N 20), 6,7-dimethyl-8ribityllumazine synthase (S/N 22), scytalone dehydratase (S/N 28), sepiapterin reductase (S/N 29), renin (S/N 31), branched-chain-amino-acid aminotransferase (S/N 34), protease (S/N 37), 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (S/N 45), UDP-glucose 4-epimerase (S/N 50), 1-aminocyclopropane-1-carboxylate deaminase (S/N 52), Homoserine dehydrogenase (S/N 65) seems to be false positives RXR retinoid x receptor and EGFR were among the top targets predicted by pharmMapper whereas RAC-α serine/threonine-protein kinase were among the top targets based on Reverse screen 3D results 52 targets were found to be common among the targets predicted for 173h by pharmMapper and Reverse screen 3D (Table XXIII) Chorismate synthase (S/N 5), dihydrodipicolinate reductase (S/N 7), nicotinate-nucleotide-dimethylbenzimidazole 141 phosphoribosyltransferase (S/N 13), scytalone dehydratase (S/N 40), transthyretin (S/N 48) seems to be false positives Type PI3K were among the top targets predicted by PharmMapper whereas phospholipase A2 were among the top targets based on Reverse Screen 3D results After removal of the false hits, each of the six selected compounds were docked to the list of predicted target hits (found common in both Pharmmapper and Reverse Screen 3D) using INVDOCK The list of targets which were implicated in cancer were obtained and presented in the Table XXIV with the function of each target and their scores ELP (Ligand Protein interaction energies in Kcal/mol) The known ligands (substrates/inhibitors/drugs) for these targets and their similarity with the synthesized compounds (based on Tanimoto coefficient) are presented in Table XXV Ligands of four targets can be considered biologically similar to the synthesized compounds as TC was found to be greater than 0.7 Ligands known to bind to the enzyme phosphatidyl Inositol-3-Kinase (PI3K) were found to be similar to 173b, 173c and 173f (TC = 0.72-0.74) whereas ligands known to bind to the enzyme mitogenactivated protein kinase (MAP kinase) were found to be similar to all four benzo fused pyrimido[1,2-a][1,3,5]triazin-6-ones (173b, 173c, 173f and 173h) As can be seen from Table XXV, ligands known to bind to the enzyme DHFR were found to be similar to only 173b and 173c (TC = 0.66-0.70) whereas ligands known to bind to the enzyme Bcr-Abl tyrosine kinase were found to be similar to 149b, 173b, 173c and 173f (TC = 0.67-0.73) Based on Tanimoto similarity scores between lead compounds and known ligands/inhibitors of these proteins- PI3K, MAP kinase, dihydrofolate reductase (DHFR) and Bcr-Abl tyrosine kinase could be the potential target(s) for benzofused pyrimido[1,2-a][1,3,5]triazinones, and Bcr-Abl tyrosine kinase could be the potential target for bicyclic pyrimido[1,2-a][1,3,5]triazinone (149b) Moreover, a chemist describes “similar” compounds in terms of approximately similar backbone and almost the same functional groups So, the scaffolds like- pyrido[2,3-d]pyrimidinone, quinazolinone, dihydropyrimido[4,5d]pyrimidinone can be considered approximately similar to the synthesized pyrimido[1,2-a][1,3,5]triazinones and its analogues Therefore, 6-6 fused ring systempyrimido[1,2-a][1,3,5]triazinone and its benzofused analogues have a good likelihood of binding to these four target(s) However, experimental validation will be required 142 to corroborate these findings Still these enzymes can be reasonably good starting points for experimental validation to be done in future 143 ... derivatives Anti-fungal activity was stated for derivatives 10 1a against Microsporum canis and average affinity of 10 1b for serotoninergic 5-HT 1A and 5-HT2B receptors was also published by Lucry and. .. cyclocondensation of biguanide and its analogues 13 2 with ethyl acetoacetate gave 12 8 -13 1 as reported by Curd and Rose92 (Scheme 39, Method A) There was a need to develop an alternative method of obtaining... catalyzed cyclization of potassium N,N-dicyanobenzamidine 11 2 with methyl anthranilate 11 1 in methanol gave triazino[2 ,1- b]quinazolone 11 4 instead of triazino [1, 2 -a] quinazolone 11 4’ In order to confirm