Synthesis, antifungal activity, and QSAR studies of 1,6-dihydropyrimidine derivatives.

INTRODUCTION: A practical synthesis of pyrimidinone would be very helpful for chemists because pyrimidinone is found in many bioactive natural products and exhibits a wide range of biological properties. The biological significance of pyrimidine derivatives has led us to the synthesis of substituted pyrimidine.
MATERIALS AND METHODS: With the aim of developing potential antimicrobials, new series of 5-cyano-6-oxo-1,6-dihydro-pyrimidine derivatives namely 2-(5-cyano-6-oxo-4-substituted (aryl)-1,6-dihydropyrimidin-2-ylthio)-N-substituted (phenyl) acetamide (C1-C41) were synthesized and characterized by Fourier transform infrared spectroscopy (FTIR), mass analysis, and proton nuclear magnetic resonance ((1)H NMR). All the compounds were screened for their antifungal activity against Candida albicans (MTCC, 227). RESULTS AND DISCUSSION: Quantitative structure activity relationship (QSAR) studies of a series of 1,6-dihydro-pyrimidine were carried out to study various structural requirements for fungal inhibition. Various lipophilic, electronic, geometric, and spatial descriptors were correlated with antifungal activity using genetic function approximation. Developed models were found predictive as indicated by their square of predictive regression values (r(2pred)) and their internal and external cross-validation. Study reveals that CHI_3_C, Molecular_SurfaceArea, and Jurs_DPSA_1 contributed significantly to the activity along with some electronic, geometric, and quantum mechanical descriptors.
CONCLUSION: A careful analysis of the antifungal activity data of synthesized compounds revealed that electron withdrawing substitution on N-phenyl acetamide ring of 1,6-dihydropyrimidine moiety possess good activity.

Over the past two decades, health benefits ascribed to commercially available antimicrobials became doubtful, since many commonly used antibiotics have become less effective against certain bacterial infections; not only because of the toxic reactions they produce, but also due to emergence of drug resistant bacteria like methicillin resistant Staphylococcus aureus (MRSA) and vancomycin resistant Enterococcus faecium (VRE).[12] Resistance to a number of antimicrobial agents (β-lactam antibiotics, macrolides, quinolones, and vancomycin) among a variety of clinically significant species of bacteria is becoming increasingly major global problem. These pose a serious challenge to the scientific community, hence emphasis has been laid on development of new antimicrobial agents.[34] Moreover, there has been a rapid spread in primary and opportunistic fungal infections, particularly C. albicans, because of the increased number of immunocompromised patients suffering from AIDS, cancer, and organ transplantation.[56] Consequently, such types of infections continue to provide impetus for the search and discovery of novel, more potent, and selective nontraditional antimicrobial agents so that no cross-resistance with the present therapeuticals can take place. A practical synthesis of pyrimidinone would be very helpful for chemists because pyrimidinone is found in many bioactive natural products and exhibits a wide range of biological properties. The biological significance of pyrimidine derivatives has led us to the synthesis of substituted pyrimidine. As pyrimidine is a basic nucleus in DNA and RNA, it has been found to be associated with diverse biological activities. Pyrimidines occupy a distinct and unique place in medicine. The chemotherapeutic efficacy of pyrimidine derivatives is related to their ability to inhibit vital enzymes responsible for DNA biosynthesis as dihydrofolate reductase (DHFR), thymidylate synthetase (TSase), thymidine phosphorylase (TPase), and reverse transcriptase (RTase). Pyrimidine derivatives were found to be associated with a variety of chemotherapeutic effects including anticancer,[7] antiviral,[891011] antibacterial,[1213] antifungal,[1415] antiprotozoal,[161718192021] antihypertensive,[22] antihistaminic,[2324] anti-inflammatory,[252627] and central nervous activities.[282930] Moreover, several pyrimidine carbonitrile derivatives were reported to possess antiviral and antimicrobial activities.[31323334] Interest in the chemotherapeutic activity of pyrimidines stemmed from the early success of some pyrimidine-based antimetabolites such as 5-flurouracil, carmofur, 5-azauracil, cytarabine, and gemcitabine as potential antineoplastic agents. Diverse mechanisms of actions were reported to be encountered with the chemotherapeutic bioactivity of pyrimidines including inhibition of kinases, inhibition of enzymes involved in pyrimidine biosynthesis, incorporation into RNA and DNA which subsequently cause misreading, and inhibition of DNA polymerase.[35] A literature survey revealed that many pyrimidine-5-carbonitriles[3637] and pyrimidinethione derivatives proved to exhibit potent anticancer as well as antimicrobial activities. Moreover, it has been well documented that incorporation of alkoxy substituents (methoxy and/or benzyloxy moieties) results in significant enhancement of several biological activities due to the magnification of compounds lipophilicity.[383940] Consequently, the newly synthesized compounds were designed so as to constitute essentially a pyrimidinethione ring system substituted basically with a cyano group and substituted thione functional group. The thrust in derivatization of such scaffold was focused on the thione function that was linked to a variety of pharmacologically active substituents and functionalities through -CH2 CONH- bridges, to the sulfur atom to produce the target compounds. In continuation to our interest in the chemical and pharmacological properties of pyrimidine derivatives, we report herein the synthesis of new series of 5-cyano-6-oxo-1,6-dihydro-pyrimidine derivatives as potential antifungal agents.

Quantitative structure activity relationship (QSAR) study remains as a very useful tool in the era of modern drug discovery to get better insights into structure activity relationships.[41424344] The behavior of QSAR models developed is examined with a variety of statistical parameters and the contribution of various descriptors is analyzed. In this communication, we describe the results of QSAR studies carried out on a series of 1,6-dihydro-pyrimidine derivatives as potential antifungal agents using genetic function approximation (GFA) technique.[45]

GFA algorithm offers a new approach to build structure activity models. It automates the search for QSAR models by combining a genetic algorithm with statistical modeling tools. Thousands of candidate models are created and tested during evolution; only the superior models survive, and are then used as ‘parents’ for the creation of next generation of candidate models. GFA has been successfully applied for the generation of variety of QSAR models.[464748] Such model provides structure-activity insights, which can be used for designing of new compounds and activity prediction prior to synthesis.

Materials and Methods

All the solvents were of laboratory reagent (LR) grade and were obtained from Merck, SD Fine, and Finar Chemicals. The melting points were determined in open capillary tubes and were uncorrected. The purity of synthesized compound was confirmed by thin layer chromatography (TLC) using silica gel G (Merck) in a developing solvent system of chloroform and methanol (9:1) and spot were visualized with ultraviolet (UV) light and by exposure to iodine vapors or sulfuric acid (30%). Infrared (IR) spectra of all compounds were recorded in Fourier transform infrared spectroscopy (FTIR)-8400S Shimadzu spectrophotometer using KBr pellets in the range of 4000-500 cm-1. Mass spectra were obtained using 2010EV LCMS Shimadzu instrument. The proton nuclear magnetic resonance (1H NMR) spectra was recorded on Bruker advanced-II NMR-400MHz model spectrophotometer in DMSO-d6 as solvent and tetramethylchlorosilane (TMSi) as internal standard with 1H resonant frequency of 400 MHz. The chemical shifts were measured in δ ppm downfield from internal standard TMSi at δ = 0.

Chemistry

Synthesis of the intermediate and target compounds was accomplished according to the steps depicted in Scheme 1. The present synthetic strategy begins with the synthesis of 6-substituted aryl-2-thiouracil-5-carbonitrile (A1–A7) by the prolonged heating of aromatic aldehyde (1), ethyl cyanoacetate (2), and thiourea (3) in ethanol; in the presence of potassium carbonate. Compounds (A1–A7) were allowed to react with the appropriate 2-chloro-N-substituted-phenylacetamide (B1–B9) in the presence of potassium carbonate, in dimethylformamide (DMF) at room temperature for 8-10 h to yield the target derivatives (C1–C41) in 51.38-73.49% yields [Table 1]. 1H NMR spectra of compounds (C1–C41) revealed that singlet was observed at δ 7.85-9.42 ppm due to N-H proton, δ 4.06-4.21 ppm due to presence of S-CH2 group, and aromatic protons resonated in the range of δ 6.18-8.02 ppm. The structures of all the newly synthesized compounds were confirmed by the IR, 1H NMR, and electron spray ionization-mass spectra (ESI-MS) spectral data, which were in full agreement with their structures.

Scheme 1

Schematic representation for synthesis of pyrimidine derivatives (C1-C41)

Table 1

Physicochemical characteristics of the novel pyrimidine derivatives (C1-C41)

General synthetic procedures used for the preparation of target compounds (C1-C41)

Preparation of 6-substituted aryl-2-thiouracil-5-carbonitrile (A1-A7)

A mixture of ethylcyanoacetate (5.7 g, 0.01 mol), thiourea (3.8 g, 0.01 mol), aromatic aldehyde (0.01 mol), and potassium carbonate (6.9 g, 0.01 mol) in absolute ethanol was refluxed for 5-8 h. The completion of reaction was monitored by TLC; the product was obtained in form of potassium salt which was dissolved in warm water and acidifies by acetic acid to precipitate pure nucleobase; filtered and dried (in vacuo). The crude product was recrystallized from acetic acid.[4950]

Preparation of 2-chloro-N-substituted-phenylacetamide (B1-B9)

Chloroacetyl chloride (0.06 mol) was added dropwise to a mixture of the appropriate amine (0.05 mol) and K2CO3 (0.06 mol) in acetone (50 ml) at room temperature. The reaction mixture was refluxed for 4-5 h, then, after cooling to room temperature, it was slowly poured into 100 ml of ice water. A solid was formed thereafter. The precipitate was separated by filtration and washed successively with water. The product was dried under vacuum to obtain (B1-B9). The progress of the reaction was monitored by TLC using toluene: acetone (8:2) solvent system.[51]

Preparation of 2-(5-cyano-6-oxo-4-substituted (aryl)-1,6- dihydropyrimidin-2-ylthio)-N-substituted (phenyl) acetamide (C1-C41)

To a suspension of (A1–A7) (0.01 mol) in dry DMF (20 ml) containing anhydrous K2CO3 (0.01 mol), and 2-chloro-N-substituted-phenylacetamide (0.01 mol) (B1-B9) was added. The reaction mixture was stirred at room temperature for 8-10 h, poured onto crushed ice, and then acidified with dilute acetic acid. The obtained precipitate was filtered, washed with H2O, dried, and washed with n-hexane. Purification of all the compounds were done by repeated recrystallization by methanol. The progress of the reaction was monitored by TLC using chloroform: methanol (90:10) solvent system.

2-(5-cyano-6-oxo-4-phenyl-1,6-dihydropyrimidin-2-ylthio)- N-phenylacetamide (C1)

IR (KBr, υ cm-1): 3290.33 (-NH), 2221.84 (-CN), 1662.52 (-C = O); 1H NMR (400MHz, δ ppm, DMSO-d6): 4.12 (s, 2H, S-CH2), 7.05-7.72 (m, 10H, Ar-H), 9.72 (s, 1H, NH), 13.10 (s, 1H, NH-Pyrimidine); ESI-MS: 362.9 (M+).

2-(5-cyano-6-oxo-4-phenyl-1,6-dihydropyrimidin-2-ylthio)- N-(4-methylphenyl) acetamide (C2)

IR (KBr, υ cm-1): 3290.33 (-NH), 2221.28 (-CN), 1658.62 (-C = O); 1H NMR (400MHz, δ ppm, DMSO-d6): 2.29 (s, CH3, 3H), 4.18 (s, 2H, S-CH2), 7.08-7.78 (m, 9H, Ar-H), 9.32 (s, 1H, NH), 13.08 (s, 1H, NH-Pyrimidine); ESI-MS: 376.9 (M+).

2-(4-(4-chlorophenyl)-5-cyano-6-oxo-1,6-dihydropyrimidin- 2-ylthio)-N-phenyl acetamide (C3)

IR (KBr, υ cm-1): 3263.33 (N-H Str.), 2225.84 (C ≡ N Str.), 1666.38 (C = O Str.), 833.19 (C-Cl Str.); 1H NMR (400MHz, δ ppm, DMSO-d6): 4.15 (s, 2H, S-CH2), 7.15-7.75 (m, 9H, Ar-H), 9.37 (s, 1H, NH), 13.12 (s, 1H, NH-Pyrimidine); ESI-MS: 396.7 (M+), 398.7(M+ + 2).

2-(5-cyano-4-(4-chlorophenyl)-6-oxo-1,6-dihydropyrimidin- 2-ylthio)-N-(3-chloro-4-fluorophenyl) acetamide (C4)

IR (KBr, υ cm-1): 3290.33 (N-H Str.), 2221.44 (C ≡ N Str.), 1674.10 (C = O Str.), 852.43 (C-F Str.), 817.62 (C-Cl Str.); 1H NMR (400MHz, δ ppm, DMSO-d6): 4.15 (s, 2H, S-CH2), 7.20-7.70 (m, 7H, Ar-H), 9.25 (s, 1H, NH), 13.16 (s, 1H, NH-Pyrimidine); ESI-MS: 451.1(M+), 453.1(M+ + 2) 455.1(M+ + 4).

2-(5-cyano-4-(4-chlorophenyl)-6-oxo-1,6-dihydropyrimidin- 2-ylthio)-N-(4-fluorophenyl) acetamide (C5)

IR (KBr, υ cm-1): 3282.62 (N-H Str.), 2229.56 (C ≡ N Str.), 1654.81 (C = O Str.), 894.91 (C-F Str.), 821.62 (C-Cl Str.); 1H NMR (400MHz, δ ppm, DMSO-d6): 4.18 (s, 2H, S-CH2), 7.12-7.69 (m, 8H, Ar-H), 9.32 (s, 1H, NH), 13.15 (s, 1H, NH-Pyrimidine); ESI-MS: 415.1(M+), 417.1(M+ + 2).

2-(5-cyano-4-(4-chlorophenyl)-6-oxo-1,6-dihydropyrimidin - 2-ylthio)-N-(4-chloro phenyl) acetamide (C6)

IR (KBr, υ cm-1): 3282.62 (N-H Str.), 2229.56 (C ≡ N Str.), 1654.81 (C = O Str.), 821.62 (C-Cl Str.); 1H NMR (400MHz, δ ppm, DMSO-d6): 4.13 (s, 2H, S-CH2), 7.09-7.72 (m, 8H, Ar-H), 9.29 (s, 1H, NH), 13.19 (s, 1H, NH-Pyrimidine); ESI-MS: 431.9 (M+), 433.9 (M+ + 2), 435.9 (M+ + 4).

2-(5-cyano-4-(4-nitrophenyl)-6-oxo-1,6-dihydropyrimidin - 2-ylthio)-N-phenylacetamide (C7)

IR (KBr, υ cm-1): 3271.05 (N-H Str.), 2225.70 (C ≡ N Str.), 1662.52 (C = O Str.), 1531.37 (NO2 Str.); 1H NMR (400MHz, δ ppm, DMSO-d6): 4.18 (s, 2H, S-CH2), 7.25-7.80 (m, 9H, Ar-H), 9.22 (s, 1H, NH), 13.42 (s, 1H, NH-Pyrimidine); ESI-MS: 408.0 (M+).

2-(5-cyano-4-(4-nitrophenyl)-6-oxo-1,6-dihydropyrimidin - 2-ylthio)-N-(4-chlorophenyl) acetamide (C8)

IR (KBr, υ cm-1): 3263.33 (N-H Str.), 2225.70 (C ≡ N Str.), 1662.52 (C = O Str.), 1531.37 (NO2 Str.), 867.91 (C-Cl Str.); 1H NMR (400MHz, δ ppm, DMSO-d6): 4.15 (s, 2H, S-CH2), 7.22-7.78 (m, 8H, Ar-H), 9.39 (s, 1H, NH), 13.35 (s, 1H, NH-Pyrimidine); ESI-MS: 442.1(M+), 443.9(M+ + 2).

2-(5-cyano-4-(4-nitrophenyl)-6-oxo-1,6-dihydropyrimidin - 2-ylthio)-N-(4-methyl phenyl) acetamide (C9)

IR (KBr, υ cm-1): 3259.47 (N-H Str.), 2225.70 (C ≡ N Str.), 1662.52 (C = O Str.), 1531.37 (NO2 Str.); 1H NMR (400MHz, δ ppm, DMSO-d6): 2.24 (s, CH3, 3H), 4.19 (s, 2H, S-CH2), 7.18-7.72 (m, 7H, Ar-H), 9.43 (s, 1H, NH), 13.28 (s, 1H, NH-Pyrimidine); ESI-MS: 422.0 (M+).

2-(5-cyano-4-(4-nitrophenyl)-6-oxo-1,6-dihydropyrimidin-2- ylthio)-N-(4-methoxy phenyl) acetamide (C10)

IR (KBr, υ cm-1): 3256.62 (N-H Str.), 2225.70 (C ≡ N Str.), 1666.38 (C = O Str.), 1527.52 (NO2 Str.); 1H NMR (400MHz, δ ppm, DMSO-d6): 3.67 (s, 3H, OCH3), 4.21 (s, 2H, S-CH2), 7.20-7.71 (m, 8H, Ar-H), 9.30 (s, 1H, NH), 13.28 (s, 1H, NH-Pyrimidine); ESI-MS: 438.1 (M+).

2-(5-cyano-4-(4-nitrophenyl)-6-oxo-1,6-dihydropyrimidin- 2-ylthio)-N-(3-chloro-4-fluoro phenyl) acetamide (C11)

IR (KBr, υ cm-1): 3259.47 (N-H Str.), 2229.56 (C ≡ N Str.), 1670.24 (C = O Str.) 1512.09 (NO2 Str.), 833.19 (C-F Str.), 867.91(C-Cl Str.); 1H NMR (400MHz, δ ppm, DMSO-d6): 4.18 (s, 2H, S-CH2), 7.15-7.80 (m, 7H, Ar-H), 9.22 (s, 1H, NH), 13.35 (s, 1H, NH-Pyrimidine); ESI-MS: 459.9 (M+), 461.9 (M+ + 2).

2-(5-cyano-4-(4-nitrophenyl)-6-oxo-1,6-dihydropyrimidin- 2-ylthio)-N-(4-fluoro phenyl) acetamide (C12)

IR (KBr, υ cm-1): 3263.33 (N-H Str.), 2225.84 (C ≡ N Str.), 1666.38 (C = O Str.), 1512.09 (NO2 Str.), 833.19 (C-F Str.); 1H NMR (400MHz, δ ppm, DMSO-d6): 4.12 (s, 2H, S-CH2), 7.19-7.75 (m, 7H, Ar-H), 9.30 (s, 1H, NH), 13.32 (s, 1H, NH-Pyrimidine); ESI-MS: 426.1(M+).

2-(5-cyano-4-(4-nitrophenyl)-6-oxo-1,6-dihydropyrimidin- 2-ylthio)-N-(4-bromo phenyl) acetamide (C13)

IR (KBr, υ cm-1): 3263.93 (N-H Str.), 2229.56 (C ≡ N Str.), 1674.10 (C = O Str.), 1512.09 (NO2 Str.), 833.19 (C-Br Str.); 1H NMR (400MHz, δ ppm, DMSO-d6): 4.15 (s, 2H, S-CH2), 7.20-7.85 (m, 8H, Ar-H), 9.40 (s, 1H, NH), 13.29 (s, 1H, NH-Pyrimidine); ESI-MS: 486.8(M + 1), 488.7(M+ + 2).

2-(5-cyano-4-(3,4-dimethoxyphenyl)-6-oxo-1,6- dihydropyrimidin-2-ylthio)-N-phenyl acetamide (C14)

IR (KBr, υ cm-1): 3128.32 (N-H Str.), 2241.13 (C ≡ N Str.), 1681.81 (C = O Str.); 1H NMR (400MHz, δ ppm, DMSO-d6): 3.69-3.77 (2 × OCH3 6H), 4.18 (s, 2H, S-CH2), 6.67-7.72 (m, 8H, Ar-H), 9.32 (s, 1H, NH), 13.10 (s, 1H, NH-Pyrimidine); ESI-MS: 423.6 (M+).

2-(5-cyano-4-(3,4-dimethoxyphenyl)-6-oxo-1,6-dihydropyrimidin -2-ylthio)-N-(4-chloro phenyl) acetamide (C15)

IR (KBr, υ cm-1): 3290.33 (N-H Str.), 2229.56 (C ≡ N Str.), 1689.53 (C = O Str.), 824.42 (C-Cl Str.); 1H NMR (400MHz, δ ppm, DMSO-d6): 3.67-3.79 (2 × OCH3 6H), 4.12 (s, 2H, S-CH2), 6.65-7.72 (m, 7H, Ar-H), 9.37 (s, 1H, NH), 13.12 (s, 1H, NH-Pyrimidine); ESI-MS: 457.0 (M+), 459.0 (M+ + 2).

2-(5-cyano-4-(3,4-dimethoxyphenyl)-6-oxo-1,6- dihydropyrimidin-2-ylthio)-N-(4-methyl phenyl) acetamide (C16)

IR (KBr, υ cm-1): 3292.59 (-NH), 2221.18 (-CN), 1683.58 (-C = O); 1H NMR (400MHz, δ ppm, DMSO-d6): 2.24 (s, CH3, 3H) 3.67-3.79 (2 × OCH3 6H), 4.10(s, 2H, S-CH2), 6.67-7.75 (m, 7H, Ar-H), 9.37 (s, 1H, NH), 13.12 (s, 1H, NH-Pyrimidine); ESI-MS: 437.1 (M+).

2-(5-cyano-4-(3,4-dimethoxyphenyl)-6-oxo-1,6-dihydropyrimidin- 2-ylthio)-N-(4-methoxyphenyl) acetamide (C17)

IR (KBr, υ cm-1): 3291.19 (N-H Str.), 2217.99 (C ≡ N Str.),1686.18 (C = O Str.); 1H NMR (400MHz, δ ppm, DMSO-d6): 3.67-3.80 (m, 9H,3 × OCH3), 4.18 (s, 1H, S-CH2), 6.86-7.78 (m, 7H, Aromatic H), 9.38 (s, 1H, NH), 13.18 (s, 1H, NH-Pyrimidine); ESI-MS: 452.9 (M+).

2-(5-cyano-4-(3,4-dimethoxyphenyl)-6-oxo-1, 6- dihydropyrimidin-2-ylthio)-N-(3-chloro-4-fluoro phenyl) acetamide (C18)

IR (KBr, υ cm-1): 3267.19 (N-H Str.), 2217.99 (C ≡ N Str.), 1647.10 (C = O Str.), 864.05 (C-Cl Str.), 825.48 (C-F Str.); 1H NMR (400MHz, δ ppm, DMSO-d6): 3.69-3.77 (2 × OCH3 6H), 4.15 (s, 2H, S-CH2), 6.68-7.78 (m, 6H, Ar-H), 9.42 (s, 1H, NH), 13.19 (s, 1H, NH-Pyrimidine); ESI-MS: 475.0 (M+), 477.0 (M+ + 2).

2-(5-cyano-4-(3,4-dimethoxyphenyl)-6-oxo-1,6-dihydropyrimidin- 2-ylthio)-N-(4-fluoro phenyl) acetamide (C19)

IR (KBr, υ cm-1): 3282.62 (N-H Str.), 2217.99 (C ≡ N Str.), 1662.52 (C = O Str.), 821.62 (C-F Str.); 1H NMR (400MHz, δ ppm, DMSO-d6): 3.68-3.92 (m, 6H,2 × OCH3), 4.06 (s, 1H, S-CH2), 7.55-7.96 (m, 7H, Ar-H), 8.04 (s, 1H, NH), 10.51 (s, 1H, NH-Pyrimidine); ESI-MS: 441.2 (M+).

2-(5-cyano-4-(3,4-dimethoxyphenyl)-6-oxo-1,6-dihydropyrimidin- 2-ylthio)-N-(2,4-di chlorophenyl) acetamide (C20)

IR (KBr, υ cm-1): 3240.19 (N-H Str.), 2217.99 (C ≡ N Str.), 1674.10 (C = O Str.), 864.05 (C-Cl Str.); 1H NMR (400MHz, δ ppm, DMSO-d6): 3.69-3.81 (m, 6H,2 × OCH3), 4.29 (s, 1H, S-CH2), 6.99-7.65 (m, 6H, Ar-H), 9.87 (s, 1H, NH), 13.18 (s, 1H, NH-Pyrimidine); ESI-MS: 491.0 (M+), 493.0 (M+ + 2), 495.0 (M+ + 4).

2-(5-cyano-6-oxo-4-(3,4,5-trimethoxyphenyl)-1, 6-dihydropyrimidin-2-ylthio)-N-phenyl acetamide (C21)

IR (KBr, υ cm-1): 3271.05 (N-H Str.), 2225.70 (C ≡ N Str.), 1662.52 (C = O Str.); 1H NMR (400MHz, δ ppm, DMSO-d6): 3.50-3.89 (m, 9H,3 × OCH3), 4.24 (s, 1H, S-CH2), 7.03-7.52 (m, 6H, Ar-H), 10.28 (s, 1H, NH), 13.20 (s, 1H, NH-Pyrimidine); ESI-MS: 453.2 (M+).

2-(5-cyano-6-oxo-4-(3,4,5-trimethoxyphenyl)-1,6-dihydropyrimidin- 2-ylthio)-N-(4-chlorophenyl) acetamide (C22)

IR (KBr, υ cm-1): 3301.91 (N-H Str.), 2225.70 (C ≡ N Str.), 1666.38 (C = O Str.), 824.42 (C-Cl Str.); 1H NMR (400MHz, δ ppm, DMSO-d6): 3.52-3.92 (2 × OCH3 6H), 4.13 (s, 2H, S-CH2), 7.09-7.63 (m, 7H, Ar-H), 9.33 (s, 1H, NH), 13.22 (s, 1H, NH-Pyrimidine); ESI-MS: 486.7(M+), 488.7(M+ + 2).

2-(5-cyano-6-oxo-4-(3,4,5-trimethoxyphenyl)-1, 6-dihydropyrimidin-2-ylthio)-N-(4-methyl phenyl) acetamide (C23)

IR (KBr, υ cm-1): 3247.05 (N-H Str.), 2206.70 (C ≡ N Str.), 1662.52 (C = O Str.); 1H NMR (400MHz, δ ppm, DMSO-d6): 2.32 (s, 3H, CH3), 3.64-3.89 (m, 9H, 3 × OCH3), 4.23 (s, 1H, S-CH2), 7.07-7.49 (m, 6H, Ar-H), 7.95 (s, 1H, NH), 13.8 (s, 1H, NH-Pyrimidine); ESI-MS: 466.9 (M+).

2-(5-cyano-6-oxo-4-(3,4,5-trimethoxyphenyl)-1,6- dihydropyrimidin-2-ylthio)-N-(4-methoxy phenyl) acetamide (C24)

IR (KBr, υ cm-1): 3263.33 (N-H Str.), 2214.13 (C ≡ N Str.), 1677.95 (C = O Str.); 1H NMR (400MHz, δ ppm, DMSO-d6): 3.67-3.79 (4 × OCH3 12H), 4.18 (s, 2H, S-CH2), 7.09-7.85 (m, 6H, Ar-H), 9.02 (s, 1H, NH), 13.36 (s, 1H, NH-Pyrimidine); ESI-MS: 483.7 (M+).

2-(5-cyano-6-oxo-4-(3,4,5-trimethoxyphenyl)-1,6- dihydropyrimidin-2-ylthio)-N- (4-fluoro phenyl) acetamide (C25)

IR (KBr, υ cm-1): 3301.91 (N-H Str.), 2214.13 (C ≡ N Str.), 1666.38 (C = O Str.),833.19 (C-F Str.); 1H NMR (400MHz, δ ppm, DMSO-d6): 3.64-3.89 (m, 9H, 3 × OCH3), 4.20 (s, 1H, S-CH2), 7.09-7.54 (m, 6H, Ar-H), 7.85 (s, 1H, NH),13.82 (s, 1H, NH-Pyrimidine); ESI-MS: 471.1(M+).

2-(5-cyano-4-(naphthalen-1-yl)-6-oxo-1,6-dihydropyrimidin-2-ylthio)-N-phenyl acetamide (C26)

IR (KBr, υ cm-1): 3236.33 (N-H Str.), 2225.70 (C ≡ N Str.), 1681.81 (C = O Str.); 1H NMR (400MHz, δ ppm, DMSO-d6): 4.21 (s, 1H, S-CH2), 7.03-8.08 (m, 12H, Ar-H), 8.41 (s, 1H, NH), 13.86 (s, 1H, NH-Pyrimidine); ESI-MS: 413.0 (M+).

2-(5-cyano-4-(naphthalen-1-yl)-6-oxo-1,6-dihydropyrimidin- 2-ylthio)-N-(4-chloro phenyl) acetamide (C27)

IR (KBr, υ cm-1): 3220.90 (N-H Str.), 2225.70 (C ≡ N Str.), 1681.81 (C = O Str.), 879.48 (C-Cl Str.); 1H NMR (400MHz, δ ppm, DMSO-d6): 4.10 (s, 1H, S-CH2), 7.24-8.08 (m, 11H, Ar-H), 8.39 (s, 1H, NH), 13.91 (s, 1H, NH-Pyrimidine); ESI-MS: 447.1(M+), 449.3(M+ + 2).

2-(5-cyano-4-(naphthalen-1-yl)-6-oxo-1,6-dihydropyrimidin- 2-ylthio)-N-(4-methyl phenyl) acetamide (C28)

IR (KBr, υ cm-1): 3292.59 (-NH), 2221.18 (-CN), 1683.58 (-C = O); 1H NMR (400MHz, δ ppm, DMSO-d6) 2.32 (s, 3H, -CH3), 4.09 (s, 1H, S-CH2), 7.06-8.09 (m, 11H, Ar-H), 8.41 (s, 1H, NH), 10.91 (s, 1H, NH-Pyrimidine); ESI-MS: 427.2 (M+).

2-(5-cyano-4-(naphthalen-1-yl)-6-oxo-1,6-dihydropyrimidin- 2-ylthio)-N-(4-methoxy phenyl) acetamide (C29)

IR (KBr, υ cm-1): 3245.45 (N-H Str.), 2229.56 (C ≡ N Str.), 1689.53 (C = O Str.); 1H NMR (400MHz, δ ppm, DMSO-d6):) 3.74 (s, 3H, -OCH3), 4.12 (s, 1H, S-CH2), 7.06-8.12 (m, 11H, Ar-H), 8.56 (s, 1H, NH), 10.96 (s, 1H, NH-Pyrimidine); ESI-MS: 443.2 (M+).

2-(5-cyano-4-(naphthalen-1-yl)-6-oxo-1,6-dihydropyrimidin- 2-ylthio)-N-(3-chloro-4-fluorophenyl) acetamide (C30)

IR (KBr, υ cm-1): 3228.62 (N-H Str.), 2225.70 (C ≡ N Str.), 1681.81 (C = O Str.) 879.48(C-Cl Str.), 824.82(C-F Str.); 1H NMR (400MHz, δ ppm, DMSO-d6): 4.07 (s, 1H, S-CH2), 7.16-8.07 (m, 10H, Aromatic H), 8.12 (s, 1H, NH), 13.86 (s, 1H, NH-Pyrimidine); ESI-MS: 465.1(M+), 467.1(M+ + 2).

2-(5-cyano-4-(naphthalen-1-yl)-6-oxo-1,6- dihydropyrimidin- 2-ylthio)-N-(4-fluoro phenyl) acetamide (C31)

IR (KBr, υ cm-1): 3244.05 (N-H Str.), 2221.84 (C ≡ N Str.), 1681.81 (C = O Str.), 833.19 (C-F Str.); 1H NMR (400MHz, δ ppm, DMSO-d6): 4.15 (s, 1H, S-CH2), 7.12-8.12 (m, 11H, Ar-H), 8.23 (s, 1H, NH), 13.76 (s, 1H, NH-Pyrimidine); ESI-MS: 431.0 (M+).

2-(5-cyano-4-(naphthalen-1-yl)-6-oxo-1,6-dihydropyrimidin- 2-ylthio)-N-(4-bromo phenyl) acetamide (C32)

IR (KBr, υ cm-1): 3224.76 (N-H Str.), 2221.84 (C ≡ N Str.), 1681.81 (C = O Str.), 880.19 (C-Br Str.); 1H NMR (400MHz, δ ppm, DMSO-d6): 4.18 (s, 1H, S-CH2), 7.09-8.02 (m, 11H, Ar-H), 8.83 (s, 1H, NH), 13.56 (s, 1H, NH-Pyrimidine); ESI-MS: 492.2 (M+), 494.2(M+ + 2).

2-(5-cyano-4-(furan-2-yl)-6-oxo-1,6-dihydropyrimidin- 2-ylthio)-N-phenylacetamide (C33)

IR (KBr, υ cm-1): 3217.04 (N-H Str.), 2229.56 (C ≡ N Str.), 1689.53 (C = O Str.); 1H NMR (400MHz, δ ppm, DMSO-d6): 4.08 (s, 1H, S-CH2), 6.18-7.82 (m, 8H, Ar-H), 9.32 (s, 1H, NH), 10.79 (s, 1H, NH-Pyrimidine); ESI-MS: 353.2 (M+).

2-(5-cyano-4-(furan-2-yl)-6-oxo-1,6-dihydropyrimidin- 2-ylthio)-N-(4-chlorophenyl) acetamide (C34)

IR (KBr, υ cm-1): 3224.76 (N-H Str.), 2229.56 (C ≡ N Str.), 1683.59 (C = O Str.), 867.91 (C-Cl Str.); 1H NMR (400MHz, δ ppm, DMSO-d6): 4.12 (s, 1H, S-CH2), 6.30-7.79 (m, 7H, Ar-H), 9.40 (s, 1H, NH), 10.78 (s, 1H, NH-Pyrimidine); ESI-MS: 386.81(M+), 388.8 (M+ + 2).

2-(5-cyano-4-(furan-2-yl)-6-oxo-1,6- dihydropyrimidin- 2-ylthio)-N-(4-methylphenyl) acetamide (C35)

IR (KBr, υ cm-1): 3238.25 (N-H Str.), 2229.53 (C ≡ N Str.), 1689.69 (C = O Str.); 1H NMR (400MHz, δ ppm, DMSO-d6): 4.11 (s, 1H, S-CH2), 6.19-7.71 (m, 7H, Ar-H), 9.32 (s, 1H, NH), 10.79 (s, 1H, NH-Pyrimidine); ESI-MS: 367.3(M+).

2-(5-cyano-4-(furan-2-yl)-6-oxo-1,6- dihydropyrimidin- 2-ylthio)-N-(3,4-dichloro phenyl) acetamide (C36)

IR (KBr, υ cm-1): 3205.47 (N-H Str.), 2229.56 (C ≡ N Str.), 1658.67 (C = O Str.), 867.91 (C-Cl Str.); 1H NMR (400MHz, δ ppm, DMSO-d6): 4.15 (s, 1H, S-CH2), 6.21-7.75 (m, 6H, Ar-H), 9.39 (s, 1H, NH), 10.85 (s, 1H, NH-Pyrimidine); ESI-MS: 422.9 (M+), 424.9 (M+ + 2), 426.9 (M+ + 4).

2-(5-cyano-4-(furan-2-yl)-6-oxo-1,6- dihydropyrimidin- 2-ylthio)-N-(4-methoxyphenyl) acetamide (C37)

IR (KBr, υ cm-1): 3120.16 (N-H Str.), 2229.56 (C ≡ N Str.), 1689.53 (C = O Str.); 1H NMR (400MHz, δ ppm, DMSO-d6): 3.79 (s, 3H, OCH3), 4.12 (s, 1H, S-CH2), 6.69-7.65 (m, 6H, Ar-H), 9.40 (s, 1H, NH), 13.09 (s, 1H, NH-Pyrimidine); ESI-MS: 383.2 (M+).

2-(5-cyano-4-(furan-2-yl)-6-oxo-1,6-dihydropyrimidin- 2-ylthio)-N-(3-chloro-4-fluoro phenyl) acetamide (C38)

IR (KBr, υ cm-1): 3205.47 (N-H Str.), 2229.56 (C ≡ N Str.), 1658.67 (C = O Str.), 867.91 (C-Cl Str.), 813.90 (C-F Str.); 1H NMR (400MHz, δ ppm, DMSO-d6) 4.11 (s, 1H, S-CH2), 6.32-7.02 (m, 6H, Ar-H), 9.25 (s, 1H, NH), 10.99 (s, 1H, NH-Pyrimidine); ESI-MS: 404.7(M+), 406.7(M+ + 2).

2-(5-cyano-4-(furan-2-yl)-6-oxo-1,6-dihydropyrimidin- 2-ylthio)-N-(4-fluorophenyl) acetamide (C39)

IR (KBr, υ cm-1): 3274.90 (N-H Str.), 2237.27 (C ≡ N Str.), 1666.38 (C = O Str.), 813.90 (C-F Str.); 1H NMR (400MHz, δ ppm, DMSO-d6): 4.09 (s, 1H, S-CH2), 6.50-7.09 (m, 7H, Ar-H), 9.33 (s, 1H, NH), 10.97 (s, 1H, NH-Pyrimidine); ESI-MS: 371.4 (M+).

2-(5-cyano-4-(furan-2-yl)-6-oxo-1,6-dihydropyrimidin-2-ylthio)-N-(4-bromophenyl) acetamide (C40)

IR (KBr, υ cm-1): 3217.04 (N-H Str.), 2217.99 (C ≡ N Str.), 1658.67 (C = O Str.), 821.62 (C-Br Str.); 1H NMR (400MHz, δ ppm, DMSO-d6): 4.12 (s, 1H, S-CH2), 6.53-7.19 (m, 7H, Ar-H), 9.36 (s, 1H, NH), 10.43 (s, 1H, NH-Pyrimidine); ESI-MS: 432.9 (M+), 434.9 (M+ + 2).

2-(5-cyano-4-(furan-2-yl)-6-oxo-1,6-dihydropyrimidin-2-ylthio)-N-(2,4-dichloro phenyl) acetamide (C41)

IR (KBr, υ cm-1): 3224.76 (N-H Str.), 2221.84 (C ≡ N Str.), 1662.52 (C = O Str.), 867.91 (C-Cl Str.); 1H NMR (400MHz, δ ppm, DMSO-d6): 4.12 (s, 1H, S-CH2), 6.22-7.78 (m, 6H, Ar-H), 9.25 (s, 1H, NH), 10.58 (s, 1H, NH-Pyrimidine); ESI-MS: 422.9 (M+ + 1), 424.9(M+ + 2).

Antifungal activity

All the synthesized compounds were evaluated for their in vitro antifungal activities against C. albicans by two-fold tube dilution method. Dimethyl sulfoxide (DMSO) was used as negative control while fluconazole was used as a positive control showing inhibition of growth of microbes. According to the values of control, the results were evaluated.

Minimum inhibitory concentration measurement

Two-fold dilution techniques were followed to determine the minimum inhibitory concentration of synthesized compounds.[52] The microdilution susceptibility test in Sabouraud liquid medium was used for determination of antifungal activity. Stock solutions of the tested compounds, and fluconazole were prepared in DMSO at concentration of 500 μg/ml followed by two-fold dilution at concentrations of (250, 200,…, 6.25 μg/ml). The microorganism suspensions at 106 colony forming units (CFU)/ml concentrations were inoculated to the corresponding wells. Plates were incubated at 36°C for 24-48 h and the minimal inhibitory concentrations (MIC) were determined. The lowest concentration of test substance that completely inhibited the growth of microorganism was reported as pMIC which is the negative logarithm of molar minimum inhibitory concentration. Fluconazole was used as a positive control. All experiments were performed in triplicate.

Results and Discussion

Antifungal evaluation

All the synthesized compounds were screened for antifungal activity by two-fold dilution method.[53] The antifungal screening was done on C. albicans (MTCC 227) fungal strain. The strain used in this study was maintained at the Department of Microbiology, Shri Sarvajanik Pharmacy College, Mehsana, Gujarat, India. Fluconazole was used as a standard drug for antifungal activity. The test compounds were prepared with different concentrations using DMSO and their MICs were determined. The MICs were defined as the lowest concentration of the compounds that prevented visible growth. The results of antifungal activity are presented in Table 2. The compounds C4, C15, C20, C26, and C37 were found to be the most effective compounds with MIC value of 6.25 μg/ml against C. albicans.

Table 2

Antifungal activity of the synthesized compounds (C1-C41)

From the analysis of structures and the activity displayed, some structure-activity relationships can be extracted. (a) The structural requirements for antifungal activity are different for substituted phenylacetamide at the C2 position of the dihydropyrimidine moiety. This is evidenced by the fact that the most active antifungal compounds C4, C15, C20, C26, and C37 showed potent antifungal activity. The presence of electron-withdrawing groups (-F, -Cl, -Br) on aromatic ring improved the antifungal activity of compounds C4, C15, C20, C26, and C37. (b) The introduction of an additional electron-withdrawing halo group (-Cl or -F) to the compounds C11, C18, C30, and C38 did not appreciably improve antifungal activity in comparison to compound C4 which contains -Cl group as an electron-withdrawing group at C4 aryl ring of the dihydropyrimidine moiety. (c) The introduction of an electron-donating group (3,4,-(OCH3)2 or 3,4,5-(OCH3)3) at C4 aryl ring and phenyl substitution on C2 position of the dihydropyrimidine moiety showed poor antifungal activity in compounds C14 and C24.

QSAR analysis

Data set

In present studies, the data set of the structures and their quantitative values of antifungal activities were used for QSAR study. Forty-one compounds were randomly divided into training and test sets, the former set consisting of 30 compounds and the remaining 11 compounds were taken in the test set. Structures of all the compounds used for QSAR analysis and their antifungal activity (MIC, micromolar concentrations, μg/ml) are given in Table 2. For every compound of the series, the experimental values of biological activity (pMIC) are used in the negative logarithmic scale. The structures of all compounds used in this study were sketched by using Visualizer module of Discovery Studio 2.1 software (Accelrys Inc, USA). CHARMM force field was used for the calculation of potential energy. An energy minimization of all the compounds was done by using Smart Minimizer method until the root mean square gradient value becomes smaller than 0.001 kcal/mol Å and followed by geometry optimization by semi empirical MOPAC-AM1 method (Astin Method-1). Further, optimized structures for all compounds were aligned with compound 1, and these structures were used for calculation of various descriptors.

Descriptor calculation

Various physicochemical descriptors like structural, thermodynamic, steric and electronic, and quantum mechanical descriptors were calculated using calculate molecular properties protocol of the Discovery Studio 2.1. A correlation matrix of the molecular descriptors was prepared and highly correlated descriptors with a correlation value of 0.9 or above were removed from the study. Remaining descriptors were used to develop QSAR models. Descriptors included to develop QSAR models are listed and described in Table 3.

Table 3

List of descriptors used in the study

Regression analysis

The data set was modeled using the GFA technique to generate a population of equations rather than one single equation for correlation between the binding affinity and descriptors. GFA is genetics based method of variable selection, which combines Holland's genetic algorithm with Friedman's multivariate adaptive regression splines to evolve the population of equations that best fit the training set data.

The GFA method works in the following way: First of all a particular number of equations (set at 100 by default in the Discovery Studio 2.1 software) are generated randomly. Then pairs of “parent” equations are chosen randomly from this set of 100 equations and “crossover” operations were performed at random. The number of crossing over was set by default at 5,000. The goodness of each progeny equation is assessed by Friedman's lack of fit (LOF) score, which is given by following formula:

LOF = LSE/[1 – (c + dp)/m]2 (1)

Where; LSE is the least-squares error, c is the number of basic functions in the model, d is smoothing parameter, P is the number of descriptors, and m is the number of observations in the training set. The smoothing parameter, which controls the scoring bias between equations of different sizes, was set at default value of 0.5 and GFA crossover of 5,000 were set to give reasonable convergence. The length of equation was fixed to seven terms, the population size was established as 100, the equation term was set to linear polynomial with spline functionality and the mutation probability was specified as 0.1. The best equations, out of the 100 equations, were chosen based on the statistical parameters such as regression coefficient (r), adjusted regression coefficient (radj), regression coefficient cross validation (rcv), and F-test values.

Validation test

To check the intercorrelation of descriptors, variance inflation factor (VIF) analysis was performed. VIF value is calculated from 1/1- r2, where r2 is the multiple correlation coefficient of one descriptor's effect regressed on the remaining molecular descriptors. VIF value greater than 10 signals towards chance correlation and hide the information of descriptors by intercorrelation of descriptors.[54]

It has been shown that a high value of statistical characteristics (r, s, F, LOF) need not be the proof of a highly predictive model.[55] Hence, in order to evaluate the predictive ability of the QSAR model, we used the method described by Roy et al. External predictability of the model was determined by calculating the value of predictive r2 (r2pred) using the following equation:

Where; YPred (test) and YObs (test) are the predicted and observed activity values, respectively, of the test set compounds and Ytraining is the mean activity value of the training set.

Results and discussion of QSAR study

In the present study, we screened 21 preselected descriptors for 41 antifungal activity using GFA method. Initially, 100 QSAR equations were generated that consist of five descriptors among QSAR random models. However, finally the results of the best four models are given in Table 4 along with their regression statistics.

Table 4

Selected QSAR equations and their regression statistics

For a statistically significant model, it is necessary that the descriptors evolved in the equation should not be intercorrelated with each other. The intercorrelation of the descriptors used in the selected models [Table 4] was very low. The correlation matrix for the used descriptors is shown in Table 5. To further check the intercorrelation of descriptors, variance inflation factor (VIF) analysis was performed (as described in Section 2.4). The VIF values of these descriptors were found to be 1.195 (PMI_Y), 1.140 (Shadow_X length), 6.369 (CHI_3_C), 4.050 (Jurs_DPSA_1), and 7.062 (Molecular_SurfaceArea). All the VIF values were found to be less than ten. Thus, from the VIF analysis, it is clear that the descriptors used in the final models have very low intercorrelation.

Table 5

Correlation matrix of the descriptors used in the equations

The models were also evaluated for their capacity to predict the activity of training set and test set compounds, that is, internal and external cross-validation, respectively. The results for the Equation 1 are summarized in Tables 6 and 7. Figures 1 and 2 depicts the plot of observed vs predicted activity for training and test set compounds, respectively. The models displayed satisfactory r2pred. For all the models, r2pred was found to be in the accepted range.[55]

Table 6

Observed and predicted pMIC values for training set compounds (as per Equation 1)

Table 7

Observed and predicted pMIC values for test set compounds (as per Equation 1)

Figure 1

Plot of observed vs predicted negative logarithm of molar minimum inhibitory concentration (pMIC) values for training set compounds (as per Equation 1)

Figure 2

Plot of observed vs predicted pMIC values for test set compounds (as per Equation 1)

Molecular_SurfaceArea calculates the total surface area for each molecule using a two-dimensional (2D) approximation. Molecular_SurfaceArea was a very useful parameter for prediction of drug transport properties. They were significantly positively correlated with the biological activity. Positive coefficient of this term in the equation means that the higher the value, the better the activity. This set of geometric descriptors helps to characterize the shape of the molecules. The descriptors were calculated by projecting the model surface on three mutually perpendicular planes: xy, yz, and xz. These descriptors depend not only on conformation, but also on the orientation of the model. To calculate them, the models were first rotated to align the principal moments of inertia with the X, Y, and Z axes. Shadow_Xlength means length of molecule in the X dimension. This was significantly negatively correlated with the biological activity. Jurs descriptors were combination of shape and electronic information to characterize molecules.[56] These descriptors were calculated by mapping atomic partial charges on solvent-accessible surface areas of individual atoms. Jurs_DPSA_1 represents difference in charged partial surface areas and can be calculated by Partial positive solvent accessible surface area minus partial negative solvent-accessible surface area. Jurs_DPSA_1 shows positive contribution towards biological activity. Topological descriptors are a special class of descriptors that do not rely on a three-dimensional (3D) model. CHI_3_C was a molecular connectivity index 3 (cluster) which shows positive contribution towards biological activity, which indicates that molecules with bulkier substituents are more likely to show activity. PMI_Y is the principal moments of inertia about the principal axes of a molecule. The moments of inertia are computed for a series of straight lines through the center of mass. The value of PMI depends on the total mass of the molecule, the distribution within the molecule, and position of axis rotation of the molecule. Principal moment of inertia (PMI_Y) is a spatial descriptor which explains the significance of orientation and conformational rigidity of biological activity. The negative coefficient of PMI_Y suggests that the presence of bulky substituents oriented towards Z-axis of the molecule will give better activity.

Conclusion

Summarizing, a series of 1,6-dihydro-pyrimidines derivatives have been synthesized successfully in appreciable yields and screened for their in vitro antifungal activity against C. albicans. QSAR analysis carried out to investigate the role of molecular descriptors in attributing the antifungal activity of synthesized derivatives indicated the importance of CHI_3_C, Molecular_SurfaceArea, and Jurs_DPSA_1 contributed significantly to explain the activity along with some other topological, electronic, geometric, and quantum mechanical descriptors. These important parameters can be taken into consideration while designing new antifungal agents belonging to the above said class of compounds in predicting the antifungal activity. Developed QSAR models are statistically significant and have excellent predictive power. Our results may provide a preliminary valuable guidance for improving the biological activity of analogs and continuing search for potent antifungal agents prior to synthesis.