Ultrasound Mediated One-Pot, Three Component Synthesis, Docking and ADME Prediction of Novel 5-Amino-2-(4-chlorophenyl)-7-Substituted Phenyl-8,8a-dihydro-7H-(1,3,4)thiadiazolo(3,2-α)pyrimidine-6-carbonitrile Derivatives as Anticancer Agents

molecules Article Ultrasound Mediated One-Pot, Three Component Synthesis, Docking and ADME Prediction of Novel 5-Amino-2-(4-chlorophenyl)-7-Substituted Phenyl-8,8a-dihydro-7H-(1,3,4)thiadiazolo(3,2-α) pyrimidine-6-carbonitrile Derivatives as Anticancer Agents Shailee V. Tiwari 1, Julio A. Seijas 2, M. Pilar Vazquez-Tato 2, Aniket P. Sarkate 3, Deepak K. Lokwani 3and Anna Pratima G. Nikalje 1,* 1Y.B. Chavan College of Pharmacy, Dr. Rafiq Zakaria Campus, Rauza Baug, Aurangabad, Maharashtra 431001, India; [email protected] 2Departamento de Química Orgánica, Facultad de Ciencias, Universidad of Santiago De Compostela, Alfonso X el Sabio, Lugo 27002, Spain; [email protected] (J.A.S.); pilar[email protected] (M.P.V.-T.) 3Department of Chemical Technology, Dr. Babasaheb Ambedkar Marathwada University, Aurangabad, Maharashtra 431004, India; [email protected] (A.P.S.); [email protected] (D.K.L.) *Correspondence: [email protected]; Tel.: +91-916-892-9111 Academic Editor: Derek J. McPhee Received: 28 May 2016; Accepted: 5 July 2016; Published: 29 July 2016 Abstract: Herein, we report an environmentally friendly, rapid, and convenient one-pot ultrasoundpromoted synthesis of 5-amino-2-(4-chlorophenyl)-7-substituted phenyl-8,8a-dihydro-7H-(1,3,4) thiadiazolo(3,2α )pyrimidine-6-carbonitrile derivatives. The in-vitro anticancer activities of these compounds were evaluated against four human tumor cell lines. Among all the synthesized derivatives, compound 4i , which has substituent 3-hydroxy-4-methoxyphenyl is found to have the highest GI 50 value of 32.7 µ M, 55.3 µ M, 34.3 µ M, 28.9 µ M for MCF-7, K562, HeLa and PC-3 cancer cell lines respectively. A docking study of the newly synthesized compounds were performed, and the results showed good binding mode in the active site of thymidylate synthase enzyme. ADME properties of synthesized compounds were also studied and showed good drug like properties. Keywords: 1,3,4-thiadiazolo(3,2-α)pyrimidine; ultrasound-promoted synthesis; ADME; docking 1. Introduction Cancer is a disease in which cells grow and proliferate in an uncontrolled manner. Cancer disease evokes a high level of mortality regardless of recent advances in the development of clinically authorized anticancer agents [ 1 ]. On the basis of source and action mechanisms, the anticancer drugs are classified as alkylating agents, antimetabolites, natural products, hormones and antagonistic, miscellaneous agents. Many scientists are intensively engaged in the development of new anticancer active agents that reveal a selective cytotoxicity for cancer cells over normal cells which is undoubtedly needed to treat the severe cancer disease more efficiently and is also less toxic, since many of the marketed anticancer drugs are toxic in nature [2–4]. Folate metabolism is considered as an important target for the development of new anticancer agents due to its role in the biosynthesis of nucleic acid precursors [ 5 , 6 ]. The inhibition of folate dependent enzymes such as thymidylate synthase (TS), which catalyzes the reductive methylation of deoxyuridylate (dUMP) to thymidylate (dTMP) has also been recognized as an interesting target for drug discovery [ 7 , 8 ]. Molecules 2016,21, 894; doi:10.3390/molecules21080894 www.mdpi.com/journal/molecules Molecules 2016,21, 894 2 of 13 Inhibition of this enzyme leads to thymineless state, rendering cells incapable of undergoing accurate DNA replication, ultimately resulting in cell death. Classical, antifolate inhibitors of thymidylate synthase often suffer from a number of potential disadvantages when used as anticancer agents. These include impaired uptake due to an alteration of the active transport system required for cellular uptake, as well as the formation of long acting, non-effluxing polyglutamates via folypolyglutamate synthetase, which are responsible for toxicity to normal cells. Nonclassical antifolates such as nolatrexed (AG337), AG331, pyrimethamine, trimethoprim (TMP), piritrexim (PTX) and trimetrexate (TMQ) do not require folate transport systems but enter cells via passive diffusion. Recent advances in non-classical antifolates have been documentedin theliterature[ 9 – 11 ]. To overcome some of the disadvantages of classical thymidylate synthase inhibitors, our team has tried to synthesize novel 5-amino-2-(4-chlorophenyl)-7-substituted phenyl-8,8a-dihydro-7H-(1,3,4)thiadiazolo(3,2-α)pyrimidine-6-carbonitrile derivatives. Pyrimidine and its derivatives have been recognized as important heterocyclic compounds due to their variety of chemical and biological significance to medicinal chemistry [ 12 – 15 ]. Hybridization of two different bioactive molecules with complementary pharmacophoric functions often showed synergistic effects [ 16 , 17 ]. During recent years, there have been intense investigations of fused thiadiazole and pyrimidine systems. Literature survey revealed that (1,3,4)thiadiazolo(3,2α )pyrimidine nucleus is associated with diverse pharmacodynamic and chemotherapeutic activities [ 18 , 19 ], including antimicrobial [ 19 – 21 ] and antitumor activities [ 18 , 20 ], herbicidal, antifungal, neuramidase inhibitors. 1,3,4-thiadiazolo(3,2α )pyrimidines have been used as key building blocks for the preparation of a variety of novel bioactive agents [ 22 ], therefore, we thought it worthwhile to explore this coupled heterocyclic system for evaluation of anticancer activity. The designing protocol for a targeted compound is as shown in Figure 1. Molecules 2016, 21, 894 2 of 13 of undergoing accurate DNA replication, ultimately resulting in cell death. Classical, antifolate inhibitors of thymidylate synthase often suffer from a number of potential disadvantages when used as anticancer agents. These include impaired uptake due to an alteration of the active transport system required for cellular uptake, as well as the formation of long acting, non-effluxing polyglutamates via folypolyglutamate synthetase, which are responsible for toxicity to normal cells. Nonclassical antifolates such as nolatrexed (AG337), AG331, pyrimethamine, trimethoprim (TMP), piritrexim (PTX) and trimetrexate (TMQ) do not require folate transport systems but enter cells via passive diffusion. Recent advances in non-classical antifolates have been documented in the literature [9–11]. To overcome some of the disadvantages of classical thymidylate synthase inhibitors, our team has tried to synthesize novel 5-amino-2-(4-chlorophenyl)-7-substituted phenyl-8,8a-dihydro-7H-(1,3,4)thiadiazolo (3,2-α)pyrimidine-6-carbonitrile derivatives. Pyrimidine and its derivatives have been recognized as important heterocyclic compounds due to their variety of chemical and biological significance to medicinal chemistry [12–15]. Hybridization of two different bioactive molecules with complementary pharmacophoric functions often showed synergistic effects [16,17]. During recent years, there have been intense investigations of fused thiadiazole and pyrimidine systems. Literature survey revealed that (1,3,4)thiadiazolo(3,2-α)pyrimidine nucleus is associated with diverse pharmacodynamic and chemotherapeutic activities [18,19], including antimicrobial [19–21] and antitumor activities [18,20], herbicidal, antifungal, neuramidase inhibitors. 1,3,4-thiadiazolo(3,2-α)pyrimidines have been used as key building blocks for the preparation of a variety of novel bioactive agents [22], therefore, we thought it worthwhile to explore this coupled heterocyclic system for evaluation of anticancer activity. The designing protocol for a targeted compound is as shown in Figure 1. N N NH O O F O OOH OH HN N H F O O Capecitibine 5-Flurouracil (5FU) HN NN H O H2N ONH COOH HOOC HN N ON S HN O COOH COOH Raltitrexed Pemetrexed HN N NH2 N N H2N N NH O COOH COOH Methotrexate HN N H2N O NH O COOH HOOC PDDF N N N S NH2 CN R Cl Synthesized molecule Figure 1. Designing protocol for targeted molecule. The conventional multistep methods for the preparation of complex molecules involve large synthetic operations, including extraction and purification processes for each individual step, that lead to synthetic inefficiency and the generation of large amounts of waste. Therefore, designing multicomponent reactions (MCRs) in one pot and the creation of several bonds in a single operation are the major challenges for modern organic chemistry. The synthesis of heterocycles using toxic and hazardous chemicals which cause pollution has given birth to “Green Chemistry,” coined by Paul Anastas in 1998 [23]. Ultrasound-promoted synthesis is one of the green methods of synthesis which we have used in our present research work in order to prevent pollution. Ultrasound-promoted synthesis has various advantages over conventional synthetic techniques such as highly accelerated reaction rate, reasonably good yields, simple open systems, very Figure 1. Designing protocol for targeted molecule. The conventional multistep methods for the preparation of complex molecules involve large synthetic operations, including extraction and purification processes for each individual step, that lead to synthetic inefficiency and the generation of large amounts of waste. Therefore, designing multicomponent reactions (MCRs) in one pot and the creation of several bonds in a single operation are the major challenges for modern organic chemistry. The synthesis of heterocycles using toxic and hazardous chemicals which cause pollution has given birth to “Green Chemistry,” coined by Paul Anastas in 1998 [ 23 ]. Ultrasound-promoted synthesis is one of the green methods of synthesis which we have used in our present research work in order to prevent pollution. Ultrasound-promoted synthesis has various advantages over conventional synthetic Molecules 2016,21, 894 3 of 13 techniques such as highly accelerated reaction rate, reasonably good yields, simple open systems, very low amount of solvents required, eco friendly method, clean heating system, neat and clean synthetic protocol, cheaper reagents and less extreme physical conditions, control on reaction parameters, milder reaction conditions. The existing synthetic methodologies for (1,3,4)thiadiazolo(3,2α )pyrimidine nucleus in a modular fashion are not straightforward and the synthetic routes involve multiple steps. For example, 1,3,4-thiadiazolo(3,2α )pyrimidine-7-sulfonamide derivatives were synthesized from 5-aminol-3,4thiadiazole-2-sulfonamide via a two steps approach [ 24 ]. Salimov et al. [ 25 ] prepared 2-bromo-7-methyl5-oxo-5H-1,3,4-thiadiazolo(3,2α )pyrimidine by two steps involving the addition of 2-aminothiadiazole derivatives to ethyl acetoacetate, tandem hydrolysis of the ester to the acid, and cyclization to give the ring-fused thiadiazolo(3,2α )pyrimidines in PPA. Most of these are multistep protocols, which suffer from generation of by-products, low yields, and use of metal-containing reagents. Therefore, it is quite significant to develop the direct, efficient, and green alternative approaches to get the functionalized thiadiazolo(3,2α )pyrimidine derivatives from viewpoint of green chemistry. Herein we reported a new, simple protocol for an environment friendly, rapid and convenient synthesis, and antitumor activity against MCF-7, K562, HeLa, PC-3 cancer cell lines, of novel 5-amino-2-(4-chlorophenyl)-7-substituted phenyl-8,8a-dihydro-7H-(1,3,4)thiadiazolo(3,2α )pyrimidine6-carbonitrile derivatives obtained in excellent yield through a one-pot three component condensation reaction of 5-(4-chlorophenyl)-1,3,4-thiadiazol-2 amine, aromatic aldehyde and malononitrile using sodium hydroxide as catalyst in ultrasound. Moreover, docking studies using thymidylate synthase (TS) enzyme are presented in this paper as well. 2. Result and Discussion 2.1. Chemistry Herein we report the one-pot synthesis of novel 5-amino-2-(4-chlorophenyl)-7-substituted phenyl-8,8a-dihydro-7H-(1,3,4)thiadiazolo(3,2α )pyrimidine-6-carbonitrile derivatives from three component reactions of an 5-(4-chlorophenyl)-1,3,4-thiadiazol-2 amine ( 1 ), aromatic aldehydes ( 3 ) and malononitrile ( 2 ) in presence of NaOH under reflux and ultrasonic irradiation as shown in Scheme 1. To determine the optimal reaction conditions, the one pot reactions between 5-(4-chlorophenyl)- 1,3,4-thiadiazol-2 amine ( 1 ), suitable aldehyde ( 3 ), malononitrile ( 2 ) were carried out using different solvents in the presence of NaOH as a catalyst at different mole percentage as shown in Table 1, the desired product was not formed when H 2 O was chosen as solvent and instead acetonitrile, methanol and dimethylformaide was chosen as solvent. The desired product was formed in low yield under reflux and ultrasonic irradiation as shown in Table 1. The optimization of reaction conditions for 1,3,4-thiadiazolo(3,2α )pyrimidine skeleton is as shown in Table 2. All the synthesized compounds were characterized by 1H-NMR, 13C-NMR, mass spectroscopy and IR. Molecules 2016, 21, 894 3 of 13 low amount of solvents required, eco friendly method, clean heating system, neat and clean synthetic protocol, cheaper reagents and less extreme physical conditions, control on reaction parameters, milder reaction conditions. The existing synthetic methodologies for (1,3,4)thiadiazolo(3,2-α)pyrimidine nucleus in a modular fashion are not straightforward and the synthetic routes involve multiple steps. For example, 1,3,4-thiadiazolo(3,2-α)pyrimidine-7-sulfonamide derivatives were synthesized from 5-aminol-3,4thiadiazole-2-sulfonamide via a two steps approach [24]. Salimov et al. [25] prepared 2-bromo-7-methyl5-oxo-5H-1,3,4-thiadiazolo(3,2-α)pyrimidine by two steps involving the addition of 2-aminothiadiazole derivatives to ethyl acetoacetate, tandem hydrolysis of the ester to the acid, and cyclization to give the ring-fused thiadiazolo(3,2-α)pyrimidines in PPA. Most of these are multistep protocols, which suffer from generation of by-products, low yields, and use of metal-containing reagents. Therefore, it is quite significant to develop the direct, efficient, and green alternative approaches to get the functionalized thiadiazolo(3,2-α)pyrimidine derivatives from viewpoint of green chemistry. Herein we reported a new, simple protocol for an environment friendly, rapid and convenient synthesis, and antitumor activity against MCF-7, K562, HeLa, PC-3 cancer cell lines, of novel 5-amino-2-(4-chlorophenyl)-7-substituted phenyl-8,8a-dihydro-7H-(1,3,4)thiadiazolo(3,2-α)pyrimidine-6-carbonitrile derivatives obtained in excellent yield through a one-pot three component condensation reaction of 5-(4-chlorophenyl)- 1,3,4-thiadiazol-2 amine, aromatic aldehyde and malononitrile using sodium hydroxide as catalyst in ultrasound. Moreover, docking studies using thymidylate synthase (TS) enzyme are presented in this paper as well. 2. Result and Discussion 2.1. Chemistry Herein we report the one-pot synthesis of novel 5-amino-2-(4-chlorophenyl)-7-substituted phenyl-8,8a-dihydro-7H-(1,3,4)thiadiazolo(3,2-α)pyrimidine-6-carbonitrile derivatives from three component reactions of an 5-(4-chlorophenyl)-1,3,4-thiadiazol-2 amine (1), aromatic aldehydes (3) and malononitrile (2) in presence of NaOH under reflux and ultrasonic irradiation as shown in Scheme 1. To determine the optimal reaction conditions, the one pot reactions between 5-(4-chlorophenyl)- 1,3,4-thiadiazol-2 amine (1), suitable aldehyde (3), malononitrile (2) were carried out using different solvents in the presence of NaOH as a catalyst at different mole percentage as shown in Table 1, the desired product was not formed when H2O was chosen as solvent and instead acetonitrile, methanol and dimethylformaide was chosen as solvent. The desired product was formed in low yield under reflux and ultrasonic irradiation as shown in Table 1. The optimization of reaction conditions for 1,3,4-thiadiazolo(3,2-α)pyrimidine skeleton is as shown in Table 2. All the synthesized compounds were characterized by 1H-NMR, 13C-NMR, mass spectroscopy and IR. Scheme 1. One-Pot, three component synthesis of novel 5-amino-2-(4-chlorophenyl)-7-substituted phenyl-8,8a-dihydro-7H-(1,3,4)thiadiazolo(3,2-α)pyrimidine-6-carbonitrile derivatives. Scheme 1. One-Pot, three component synthesis of novel 5-amino-2-(4-chlorophenyl)-7-substituted phenyl-8,8a-dihydro-7H-(1,3,4)thiadiazolo(3,2-α)pyrimidine-6-carbonitrile derivatives. Molecules 2016,21, 894 4 of 13 Table 1. Optimization of reaction conditions for novel 5-amino-2-(4-chlorophenyl)-7-substituted phenyl-8,8a-dihydro-7H-(1,3,4)thiadiazolo(3,2α )pyrimidine-6-carbonitrile derivatives using various solvent and different mole percentage of NaOH. Entry Catalyst Amount (% mol) Solvent Method A Conventional Method B Ultrasound Time (h) Yield (%) Time (h) Yield (%) 1 No catalyst - EtOH 9 - 2 - 2 NaOH 30 EtOH 9 70 2 89 3 NaOH 20 EtOH 9 70 2 89 4 NaOH 20 H2O 9 - 2 - 5 NaOH 20 MeOH 11 55 2.30 60 6 NaOH 20 CH3CN 11 40 2.30 50 7 NaOH 20 DMF 12 40 2.30 55 8 NaOH 10 EtOH 13 60 2.45 65 9 NaOH 5 EtOH 15 50 3.30 60 Table 2. Optimization of reaction conditions for 1,3,4-thiadiazolo(3,2-α)pyrimidine skeleton. Compound RConventional Ultrasound Time (h) Yield (%) Time (h) Yield (%) 4a 4-chlorophenyl 7 70 1 89 4b 2-chlorophenyl 7.30 68 1.30 85 4c 3-chlorophenyl 7.30 65 1.30 85 4d 4-flurophenyl 7 62 1.30 80 4e 4-methoxyphenyl 8 58 2 75 4f 3,4,5-methoxyphenyl 9 58 2 78 4g 3,4-methoxyphenyl 9 55 2 75 4h Phenyl 7 60 1 80 4i 3-hydroxy-4 methoxyphenyl 9 60 2 82 4j furan-2-yl 8 45 1.30 65 2.2. In Vitro Anticancer Activity The target compounds ( 4a – j ) were evaluated for their anticancer activity against MCF-7, K562, HeLa and PC-3 cancer cell lines. The GI 50 values (concentration required to Growth inhibition of 50%) for the synthesized compounds were determined using SRB assays method. The anticancer evaluation results and GI 50 values were listed in Table 3and the well-known anticancer drug 5-FU was used as positive control. Table 3. In-vitro anticancer activity. Compound GI50 µM MCF-7 K-562 HeLa PC-3 4a 88.5 47.9 56.2 38.9 4b 43.8 57.1 54.3 37.9 4c 55.0 60.1 55.7 38.4 4d 80.6 >100 58.1 30.2 4e 38.9 54.2 43.8 26.7 4f 38.3 58.1 48.6 25.4 4g 34.8 54.3 47.9 25.3 4h 38.9 58.3 38.7 34.7 4i 32.7 55.3 34.3 28.9 4j 82.5 >100 60.9 55.3 5-FU 32.18 47.03 43.71 12.00 5-Flurouracil (5-FU) was the standard drug used. GI 50 is the concentration exhibiting 50% inhibition of the growth as compared to the growth of control. MCF-7 Human breast cancer, K-562 Human Leukemia cancer, HeLa Human cervical cancer, PC-3 Human prostate cancer. Molecules 2016,21, 894 5 of 13 The results indicated that the compounds, 4e , 4f , 4g and 4i exhibited significant cell growth inhibition compared to reference standard 5-fluorouracil against MCF-7, K562, HeLa and PC-3 cancer cell lines. From the anticancer activity results, it was observed that compound 4i , which has substituent 3-hydroxy-4-methoxyphenyl, is found to have the highest GI 50 values of 32.7 µ M, 34.3 µ M, 55.3 µ M, 28.9 µ M for MCF-7, HeLa, K562 and PC-3 cancer cell lines respectively. Compound 4g , which has 3,4-dimethoxyphenyl, is found to have GI 50 values of 34.8 µ M, 54.3 µ M, 47.9 µ M, 25.3 µ M for MCF-7, K562, HeLa, and PC-3 cancer cell lines respectively. All the synthesized compounds were less active towards PC-3 in comparison to standard drug 5-FU. Structural activity relationship (SAR) studies for these compounds demonstrated that electron withdrawing groups such as fluoro ( 4d ), chloro ( 4a , 4b , 4c ) exhibited less activity compared to electron donating, polar groups. The anticancer activity for derivatives bearing an electron withdrawing group such as chloro group at ortho position ( 4b ) (43.8 µ M, 57.1 µ M, 54.3 µ M, 37.9 µ M for MCF-7, K-562, HeLa and PC-3 cancer cell lines respectively) exhibited more activity than compound 4c (chloro group at meta position) (55.0 µ M, 60.1 µ M, 55.7 µ M, 38.4 µ M for MCF-7, K-562, HeLa and PC-3 cancer cell lines respectively) and compound 4a (chloro group at para position) (88.5 µ M, 47.9 µ M, 56.2 µ M, 38.9 µ M for MCF-7, K-562, HeLa and PC-3 cancer cell lines respectively). Replacement of the phenyl group in the parent compound by furan ring in 4j has shown decreased activity in comparison to the standard drug 5-FU (82.5 µ M, >100 µ M, 60.9 µ M, 55.30 µ M, for MCF-7, K-562, HeLa and PC-3 cancer cell lines, respectively). From SAR it can be considered that compounds containing electron donating, polar groups such as 4e , 4f , 4g , 4i have good anticancer activity in comparison to electron withdrawing groups such as 4a , 4b , 4c , 4d . It is also clear that the replacement of the phenyl ring with the furan ring decreases anticancer activity. 4i and 4g are equipotent with the clinically used anticancer drug 5-FU against MCF-7, HeLa cell lines, hence these compounds can be developed as anticancer agents in the future. Compound 4a is equipotent with the clinically used anticancer drug 5-FU against K-562. 2.3. Molecular Docking 5-Fluorouracil derivatives and their structurally related compounds like pemetrexed, Capecitibine, Raltitrexed are well known to inhibit thymidylate synthase. We decided to carry out a molecular docking study on our newly synthesized 1,3,4-thiadiazolo(3,2α )pyrimidine skeleton derivatives into the binding site of thymidylate synthase (PDB ID: 1JU6) using 5-FU as a reference for docking results. The docking results indicated that compounds were held in the active pocket by combination of various hydrogen and hydrophobic interactions with TS. The docking results revealed that the highest binding compound to TS was 4i with a G-Score of ´ 7.17. The compound 4g shows hydrogen bonding with ASN 112, ILE 108 and ARG 50 with a G-Score of ´ 5.59 as shown in Figure 2. Similarly, the compound 4i shows hydrogen bonding with GLN 214, SER 214 and ASN 226 with a highest G-Score of ´ 7.17 as shown in Figure 3. The compounds 4b ( ´ 5.59), 4c ( ´ 5.24), 4d ( ´ 5.14), 4f ( ´ 5.14), 4h ( ´ 5.38) and 4j (´4.98), all showed G-Score greater than 5-FU (´4.75). On the basis of the anticancer activity and docking results, it was found that compounds 4g , 4h and 4i had potential to inhibit thymidylate synthase. G-Score of all the synthesized derivatives is as shown in Table 4. Table 4. G-Score of all the synthesized derivatives. Compound G-Score Compound G-Score 4a ´4.65 4g ´5.59 4b ´5.59 4h ´5.38 4c ´5.24 4i ´7.17 4d ´5.14 4j ´4.98 4e ´4.74 5-FU ´4.75 4f ´5.14 Molecules 2016,21, 894 6 of 13 Molecules 2016, 21, 894 6 of 13 Figure 2. Docking pose of compound 4g in the active site of thymidylate synthase enzyme. Figure 3. Docking pose of compound 4i in the active site of thymidylate synthase enzyme. 2.4. In Silico ADME Prediction The prediction of the ADME parameters prior to the experimental studies is one of the most important aspects of drug discovery and development of the drug molecule. ADME studies have always played a critical role in helping to optimize the pharmacokinetic properties of new drugs, thereby increasing their success rate. The analysis of Lipinski’s rule of five was performed to indicate whether a chemical compound could be an orally active drug in humans. It was observed that the compounds exhibited a good % absorption (% ABS) ranging from 87.50% to 100% (Table 5). The results of the prediction of ADME properties are depicted in Table 5. All synthesized compounds 4 a–j had good absorption and were found to be nontoxic. Figure 2. Docking pose of compound 4g in the active site of thymidylate synthase enzyme. Molecules 2016, 21, 894 6 of 13 Figure 2. Docking pose of compound 4g in the active site of thymidylate synthase enzyme. Figure 3. Docking pose of compound 4i in the active site of thymidylate synthase enzyme. 2.4. In Silico ADME Prediction The prediction of the ADME parameters prior to the experimental studies is one of the most important aspects of drug discovery and development of the drug molecule. ADME studies have always played a critical role in helping to optimize the pharmacokinetic properties of new drugs, thereby increasing their success rate. The analysis of Lipinski’s rule of five was performed to indicate whether a chemical compound could be an orally active drug in humans. It was observed that the compounds exhibited a good % absorption (% ABS) ranging from 87.50% to 100% (Table 5). The results of the prediction of ADME properties are depicted in Table 5. All synthesized compounds 4 a–j had good absorption and were found to be nontoxic. Figure 3. Docking pose of compound 4i in the active site of thymidylate synthase enzyme. 2.4. In Silico ADME Prediction The prediction of the ADME parameters prior to the experimental studies is one of the most important aspects of drug discovery and development of the drug molecule. ADME studies have always played a critical role in helping to optimize the pharmacokinetic properties of new drugs, thereby increasing their success rate. The analysis of Lipinski’s rule of five was performed to indicate whether a chemical compound could be an orally active drug in humans. It was observed that the compounds exhibited a good % absorption (% ABS) ranging from 87.50% to 100% (Table 5). The results of the prediction of ADME properties are depicted in Table 5. All synthesized compounds 4a–jhad good absorption and were found to be nontoxic. Molecules 2016,21, 894 7 of 13 Table 5. Absorption, distribution, metabolism and excretion (ADME) results of synthesized compounds. Compound MW a Percentage of Absorption (% ABS) b logP o/w c (–2.0 to 6.5) n-ON d(<10) n-OHNH e(<5) Polar Surface Area (PSA) f(7–200.0) Number of Violations of Rule of 5 (<5) Toxicity 4a 400 100 4.57 4 2 74.81 0 N 4b 400 100 4.42 4 2 74.87 0 N 4c 400 100 4.57 4 2 74.81 0 N 4d 383.8 100 4.30 4 2 74.81 0 N 4e 395.8 100 4.19 4.75 2 83.10 0 N 4f 455.9 100 4.47 6.25 2 95.95 0 N 4g 425.8 100 4.36 5.5 2 88.02 0 N 4h 365.8 100 4.01 4 2 74.81 0 N 4i 411.8 87.50 3.46 5.5 3 104.7 0 N 4j 355.8 94.19 3.12 4.5 2 83.51 0 N 5-FU 130.0 66.30 ´0.89 3.5 2 87.56 0 N a Molecular weight of the molecule; b Percentage human oral absorption (% ABS) (>80% is high, <25% is poor); c Predicted octanol-water partition coefficient (logP o/w) (–2.0 to 6.5); dn-ON number of hydrogen bond acceptors <10; en-OHNH number of hydrogen bonds donors <5; fPolar surface area (PSA) (7.0–200.0); N: non-toxic. Molecules 2016,21, 894 8 of 13 3. Materials and Methods 3.1. General All the reactions were performed in oven-dried glasswares. All reagents and solvents were used as obtained from the supplier or recrystallized/redistilled unless otherwise noted. The ultrasound sonicator (Sonics Vibra-cell, Modelno. VCX 500, Newtown, CT, USA) equipped with solid synthetic probe, 13 mm in tip diameter, operating at 20 kHz with a maximum power output of 500 W, was used for synthesis of final title compounds. The purity of the synthesized compounds was monitored by ascending thin layer chromatography (TLC) on silica gel-G (Merck, Darmstadt, Germany) coated aluminum plates, visualized by iodine vapor and melting points were determined in open capillary tubes. Infrared (IR) spectra were recorded on a PS 4000 FTIR (JASCO, Tokyo, Japan) using KBr pellets. Elemental analyses (C, H, and N) were done with a FLASHEA 112 Shimadzu’ analyzer (Mumbai, Maharashtra, India) and all analyses were consistent (within 0.4%) with theoretical values. The 1 H-NMR and 13 C-NMR spectra of synthesized compounds were recorded on Bruker Avance II 400 NMR Spectrometer (Billerica, MA, USA) at 400 MHz Frequency in deuterated DMSO and CDCl 3 and using TMS as internal standard (chemical shift δ in ppm). Mass spectra of some compounds were scanned on FTMS + p ESI full mass (100.00–1500.00). 3.2. General Procedure for the Synthesis of 5-Amino-2-(4-chlorophenyl)-7-Substituted Phenyl-8,8a-dihydro-7H-(1,3,4)thiadiazolo(3,2-α)pyrimidine-6-carbonitrile Derivatives Method A: A 25 mL round bottom flask was charged with a mixture of an 5-(4-chlorophenyl)- 1,3,4-thiadiazol-2 amine (0.01 mol) ( 1 ), malononitrile (0.01 mol) ( 2 ), suitable aldehyde (0.01 mol) ( 3 ) in ethanol (10–12 mL) and the catalyst NaOH (20% mmol) and the reaction mixture was refluxed . After completion of the reaction (monitored by TLC), the mixture was poured into ice cold water. The product obtained, was filtered and dried. The corresponding product was obtained in high purity after recrystallization of the crude product from ethanol. The authenticity of compounds was established by 1H-NMR, 13C-NMR, IR and HRMS. Method B: A 25 mL a beaker was charged with a mixture of an 5-(4-chlorophenyl)-1,3,4thiadiazol-2 amine (0.01 mol) ( 1 ), malononitrile (0.01 mol) ( 2 ), suitable aldehyde (0.01 mol) ( 3 ) in ethanol (10–12 mL) and the catalyst NaOH (20% mmol) and the reaction mixture was kept inside an Ultrasonicator acoustic chamber at 80 ˝ C at 20%. After completion of the reaction (monitored by TLC), the mixture was poured into ice cold water. The product obtained, was filtered and dried. The corresponding product was obtained in high purity after recrystallization of the crude product from ethanol. The authenticity of compounds was established by 1H-NMR, 13C-NMR, IR and HRMS. 5-Amino-2,7-bis(4-chlorophenyl)-7H-(1,3,4)thiadiazolo(3,2α )pyrimidine-6-carbonitrile 4a . M.P: 237–240 ˝ C. Rfvalue: 0.28. IR (KBr) υ max cm ´1 : 3400 (C-NH 2 ), 3100 (Aromatic C-H stretching), 1623 (C=N), 740.55 (C-Cl of aromatic rings), 1 H-NMR δ ppm: 10.00 (s, 2H, NH 2 ), 8.00–7.02 (m, 8H two aromatic rings), 3.35 (s, 1H, ArC, of pyrimidine ring), 13 C-NMR δ : 172.11 (C), 158.31 (C), 143.21 (C), 139.50 (C), 136.51 (C), 131.00 (C), 130.52 (CH), 129.87 (CH), 128.53 (CH), 128.23 (C), 127.93 (CH), 118.27 (C), 60.22 (C), 54.53 (C), m/z399 (100.0%), 401.01 (68.9%), 400.01 (22.7%) Molecular Formula: C 18 H 11 Cl 2 N 5 S. Elemental Analysis: Calculated: (C, H, Cl, N, S) 54.01, 2.77, 17.71, 17.50, 8.01 Found: 55.04, 2.74, 17.68, 17.51, 8.00. 5-Amino-7-(2-chlorophenyl)-2-(4-chlorophenyl)7H-(1,3,4)thiadiazolo(3,2α )pyrimidine-6-carbonitrile 4b . M.P: 240–242 ˝ C. Rfvalue: 0.24. IR (KBr) υ max cm ´1 : 3400 (C-NH 2 ), 3000 (Aromatic C-H stretching), 1623 (C=N), 740.55 (C-Cl of aromatic rings). 1H-NMR δ ppm: 9.98 (s, 2H, NH 2 ), 8.00–7.09 (m, 8H two aromatic rings), 3.35 (s, 1H, ArC, of pyrimidine ring), 13 C-NMR δ ppm: 170.24 (C), 158.37 (C), 143.77 (C), 138.10 (C), 134.26 (C), 129.52 (CH), 129.00 (CH), 128.51 (CH), 128.03 (C), 127.99 (CH), 126.11 (CH), 117.59 (C), 60.00 (C), 55.02 (C), m/z399 (100.0%), 401.01 (68.9%), 400.01 (22.7%) Molecular Formula: C 18 H 11 C l2 N 5 S. Elemental Analysis: Calculated: (C, H, Cl, N, S) 54.01, 2.77, 17.71, 17.50, 8.01 Found: 55.02, 2.76, 17.70, 17.52, 8.02. Molecules 2016,21, 894 9 of 13 5-Amino-7-(3-chlorophenyl)-2-(4-chlorophenyl)7H-(1,3,4)thiadiazolo(3,2α )pyrimidine-6-carbonitrile 4c . M.P: 235–238 ˝ C. Rfvalue: 0.20. IR (KBr) υ max cm ´1 : 3400 (C-NH 2 ), 3100 (Aromatic C-H stretching), 162.3 (C=N), 740.55 (C-Cl of aromatic rings), 1H-NMR δ ppm: 10.01 (s, 2H, NH 2 ), 8.00–7.05 (m, 8H two aromatic rings), 3.32 (s, 1H, ArC, of pyrimidine ring), 13 C-NMR δ ppm: 172.21 (C), 158.20 (C), 155.00 (C), 142.50 (C), 137.69 (C), 136.17 (C), 134.09 (C), 130.03 (CH), 129.99 (CH), 129.02 (CH), 128.93 (CH), 128.37 (CH), 125.59 (CH), 123.95 (CH), 119.11 (CH), 117.57 (C), 59.97 (C), 52.00 (C), m/z399 (100.0%), 401.01 (68.9%), 400.01 (22.7%), Molecular Formula: C 18 H 11 C l2 N 5 S. Elemental Analysis: Calculated: (C, H, Cl, N, S) 54.01, 2.77, 17.71, 17.50, 8.01 Found: 55.01, 2.77, 17.67, 17.49, 8.03. 5-Amino-2-(4-chlorophenyl)-7-(4-fluorophenyl)-7H-(1,3,4)thiadiazolo(3,2α )pyrimidine-6-carbonitrile 4d . M.P: 239–240 ˝ C. Rfvalue: 0.37. IR (KBr) υ max cm ´1 : 3400 (C-NH 2 ), 3000 (Aromatic C-H stretching), 1623 (C=N), 740.55 (C-Cl of aromatic ring), 1053 (C-F of aromatic rings), 1 H-NMR δ ppm: 9.79 (s, 2H, NH 2 ), 8.02–7.06 (m, 8H two aromatic rings), 3.33 (s, 1H, ArC, of pyrimidine ring), 13 C-NMR δ ppm: 172.22 (C), 159.07 (C), 158.31 (C), 143.77 (C), 136.62 (C), 136.99 (C), 130.67 (CH), 129.52 (CH), 128.99 (C), 128.77 (CH), 117.54 (C), 115.34 (CH), 60.07 (C), 52.97 (C), m/z383.04 (100%), 385.04 (37%), 384.04 (22.7%), Molecular Formula: C 18 H 11 ClFN 5 S. Elemental Analysis: Calculated: (C, H, Cl, F, N, S) 56.33, 2.89, 9.24, 4.95, 18.25, 8.35 Found: 56.37, 2.90, 9.20, 4.91, 18.23, 8.36. 5-Amino-2-(4-chlorophenyl)-7-(4-methoxyphenyl)-7H-(1,3,4)thiadiazolo(3,2α )pyrimidine-6-carbonitrile 4e . M.P: 210–212 ˝ C. Rfvalue: 0.36. IR (KBr) υ max cm ´1 : 3400 (C-NH 2 ); 3000 (Aromatic C-H stretching), 1623 (C=N), 1055 (C-OCH 3 ), 740.55 (C-Cl of aromatic ring), 1 H-NMR δ ppm: 9.99 (s, 2H, NH 2 ), 8.02–6.79 (m, 8H two aromatic rings), 3.36 (s, 1H, ArC, of pyrimidine ring), 3.56 (s, 3H, OCH 3 ), 13 C-NMR δ ppm: 172.26 (C), 158.99 (C), 157.51 (C), 143.70 (C), 136.99 (C), 133.04 (C), 130.07 (CH), 129.55 (CH), 128.52 (CH), 128.00 (C), 117.37 (C), 114.26 (CH), 60.00 (C), 55.99 (CH 3 ), 53.00 (C), m/z395.06 (100%), 397.06 (37.0%), 396.06 (23.8%), Molecular Formula: C 19 H 14 ClN 5 OS. Elemental Analysis: Calculated: (C, H, Cl, N, O, S) 57.65, 3.56, 8.96, 17.69, 4.04, 8.10 Found: 57.68, 3.59, 8.93, 17.67, 4.03, 8.11. 5-Amino-2-(4-chlorophenyl)-7-(3,4,5-trimethoxyphenyl)-7H-(1,3,4)thiadiazolo(3,2α )pyrimidine-6-carbonitrile 4f . M.P: 220–222 ˝ C. Rfvalue: 0.26. IR (KBr) υ max cm ´1 : 3400 (C-NH 2 ), 3100 (Aromatic C-H stretching), 1623 (C=N), 1059 (C-OCH 3 ), 740.55 (C-Cl of aromatic ring), 1 H-NMR δ ppm: 10.00 (s, 2H, NH 2 ), 8.02–6.79 (m, 6H two aromatic rings), 3.36 (s, 1H, ArC, of pyrimidine ring), 3.56 (s, 9H, OCH 3 ), 13 C-NMR δ ppm: 172.21 (C), 158.89 (C), 152.81 (C), 143.32 (C), 136.52 (C), 136.00 (C), 135.01 (C), 129.51 (CH), 128.52 (CH), 128.77 (C), 117.51 (C), 106.50 (CH), 61.00 (CH 3 ), 60.52 (C), 56.00 (CH 3 ), 53.00 (C), m/z455.08 (100%), 457.08 (37.1%), 456.09 (23.7%), Molecular Formula: C 21 H 18 ClN 5 O 3 S. Elemental Analysis: Calculated: (C, H, Cl, N, O, S) 55.32, 3.98, 7.78, 15.36, 10.53, 7.03 Found: 55.37, 3.99, 7.77, 15.33, 10.52, 7.00. 5-Amino-2-(4-chlorophenyl)-7-(3,4-dimethoxyphenyl)-7H-(1,3,4)thiadiazolo(3,2α )pyrimidine-6-carbonitrile 4g . M.P: 225–228 ˝ C. Rfvalue: 0.50. IR (KBr) υ max cm ´1 : 3400 (C-NH 2 ), 3100 (Aromatic C-H stretching), 1623 (C=N), 1623 (C=N), 1052 (C-OCH 3 ), 740.55 (C-Cl of aromatic ring), 1 H-NMR δ ppm: 10.02 (s, 2H, NH 2 ), 8.02–6.79 (m, 7H two aromatic rings), 3.36 (s, 1H, ArC, of pyrimidine ring), 3.56 (s, 6H, OCH 3 ), 13 C-NMR δ ppm: 172.07 (C), 158.00 (C), 149.07 (C), 146.72 (C), 143.54 (C), 136.58 (C), 134.51 (C), 129.59 (CH), 128.55 (CH), 128.70 (C), 122.01 (C), 117.56 (C), 114.99 (CH), 112.53 (CH), 59.11 (C), 56.09 (CH 3 ), 53.00 (C), m/z425.07 (100.0%), 427.07 (37.0%), 426.07 (24.09%), Molecular Formula: C 20 H 16 ClN 5 O 2 S. Elemental Analysis: Calculated: (C, H, Cl, N, O, S) 56.40, 3.79, 8.32, 16.44, 7.51, 7.53 Found: 56.44, 3.82, 8.30, 16.00, 7.50, 7.52. 5-Amino-2-(4-chlorophenyl)-7-phenyl-7H-(1,3,4)thiadiazolo(3,2α )pyrimidine-6-carbonitrile 4h . M.P: 218–220 ˝ C. Rfvalue: 0.43. IR (KBr) υ max cm ´1 : 3400 (C-NH 2 ), 3000 (Aromatic C-H stretching), 1623 (C=N), 740.55 (C-Cl of aromatic ring), 1 H-NMR δ ppm: 10.00 (s, 2H, NH 2 ), 8.02–7.27 (m, 9H two aromatic rings), 3.34 (s, 1H, ArC, of pyrimidine ring), 13 C-NMR δ ppm: 172.12 (C), 158.37 (C), 143.77 (C), 141.00 (C), 136.50 (C), 129.55 (CH), 129.08 (CH), 128.61 (C), 128.52 (CH), 125.79 (CH), 125.60 (CH), 117.51 (C), 60.00 (C), 53.12 (C), m/z365.05 (100.0%), 367.05 (37.0%), 366.05 (22.7%), Molecular Formula: C 18 H 12 ClN 5 S.