Journal of Chemistry
Volume 2016 (2016), Article ID 2135893, 12 pages
http://dx.doi.org/10.1155/2016/2135893

Research Article

Synthesis of Some Polysubstituted Nicotinonitriles and Derived Pyrido[2,3-d]pyrimidines as In Vitro Cytotoxic and Antimicrobial Candidates

Hassan M. Faidallah,1,2 Sherif A. F. Rostom,3,4 and Khalid A. Khan¹

¹Department of Chemistry, Faculty of Science, King Abdulaziz University, P.O. Box 80203, Jeddah 21589, Saudi Arabia
²Department of Chemistry, Faculty of Science, University of Alexandria, Alexandria, Egypt
³Department of Pharmaceutical Chemistry, Faculty of Pharmacy, King Abdulaziz University, P.O. Box 80260, Jeddah 21589, Saudi Arabia
⁴Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Alexandria, Alexandria 21521, Egypt

Received 8 February 2016; Revised 20 April 2016; Accepted 21 April 2016

Academic Editor: Artur M. S. Silva

Copyright © 2016 Hassan M. Faidallah et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

The synthesis of polysubstituted pyridines, in addition to some derived pyrido[2,3-d]pyrimidine ring systems supported with chemotherapeutically active functionalities, is described. They were evaluated for their in vitro cytotoxic effects against three different human tumor cell lines (human colon carcinoma HT29, hepatocellular carcinoma Hep-G2, and Caucasian breast adenocarcinoma MCF7). Nine compounds displayed variable cytotoxic potential, among which alkylthio analogs 33, 34, and 37 emerged as the most active members, being almost twice as active as doxorubicin against the colon carcinoma HT29 cell line. In addition, the same three analogs showed a clear differential cytotoxic profile as they exhibited a marginal inhibitory effect on the growth of the normal nontransformed human foreskin fibroblast Hs27 cell line. Meanwhile, nineteen compounds were able to exhibit significant antibacterial activity against both Gram-positive and Gram-negative bacteria, together with moderate antifungal activities. The pyrido[2,3-d]pyrimidine-2(1H)-thione 30 together with its alkylthio derivatives 33 and 34 stemmed as the most active antimicrobial members being equipotent to ampicillin against S. aureus, E. coli, and P. aeruginosa, together with a noticeable antifungal activity against C. albicans. Compounds 33 and 34 could be considered as a promising template for possible dual antimicrobial-anticancer candidates.

1. Introduction

Over the past two decades, several reports have discussed the growing number of cancerous diseases and the prominent role displayed by chemotherapy as the most fruitful treatment for many disseminated types of tumors. This has resulted in the activation of much research targeting the discovery of prominent lead structures that would be beneficial in styling novel antitumor chemotherapeutic agents [1–3]. Additionally, the dilemma of multi-drug resistant (MDR) bacteria arising from the misuse of traditional antimicrobial agents has been extensively studied [4]. In addition, mycotic invasions became a complicated problem especially in patients who experience immunotherapy, organ transplantation, carcinomas, or AIDS [5]. Therefore, there is a genuine demand for finding out new agents endowed with antimicrobial activity that acts with nonconventional mechanisms. Patients who undergo cancer chemotherapy are clearly liable to microbial infections owing to the subsidiary reduction of immunity. Concomitant use of several drugs for treating cancer joined with microbial invasions might cause additional complications in patients with malfunctioned kidneys. Consequently, the idea of administering one drug with dual action could be beneficial from both curative and economic viewpoints. In recent years, cyanopyridines and some of their fused-ring systems such as quinolines and pyridopyrimidines have been under focus as potential chemotherapeutic candidates because of their reported distinctive antimicrobial [6–8], antitubercular [9], and antiviral [10] activities, in addition to the well documented antineoplastic [11], antiproliferative [12], and cytotoxic [13] potentials. Furthermore, several pyridine-containing compounds were reported to possess dual anticancer and antimicrobial activities [14–17].

During our current interest in exploring new lead structures that possess chemotherapeutic potential, much attention was focused on the antimicrobial and anticancer activities of some pyridine derivatives [18–24], particularly those possessing the 4,6-disubstituted-2-aminopyridin-3-carbonitrile (nicotinonitriles) scaffold which revealed favourable broad spectrum anticancer and/or antimicrobial efficacy. The collected results persuaded the design and synthesis of novel polysubstituted nicotinonitriles and some derived pyrido[2,3-d]pyrimidines incorporating different functional groups that are claimed to account for the bioactivity of related antitumor and antimicrobial candidates. Additionally, the substituents encountered in these derivatives were chosen so as to affect the electronic environment of the compounds, which would synergistically influence the targeted biological activities, in an attempt to obtain novel candidates with both anticancer and antimicrobial potentials.

2. Experimental

2.1. Chemistry

Melting points were determined on a Gallenkamp melting point apparatus and are uncorrected. The infrared (IR) spectra were recorded on Shimadzu FT-IR 8400S infrared spectrophotometer using the KBr pellet technique. ¹H and ¹³C NMR spectra were recorded on a Bruker DPX-400 FT NMR spectrometer using Tetramethylsilane (TMS) as the internal standard and DMSO- as a solvent (Chemical shifts in δ, ppm). Splitting patterns were designated as follows: s: singlet; t: triplet; q: quartet; m: multiplet. The Electron Impact Ionization Mass Spectra were recorded on a Nermag R10-10C at 70 ev. 1000 Ex spectrometer. Elemental analyses were performed on a 2400 Perkin Elmer Series 2 analyzer and the values found were within ±0.4% of the theoretical values. Follow-up of the reactions and checking the homogeneity of the compounds were made by TLC on silica gel-protected aluminum sheets (Type 60 F254, Merck) and the spots were detected by exposure to UV-lamp at 254.

2.1.1. 2-Amino-4,6-disubstituted Nicotinonitriles (1–5)

A one-pot mixture of the appropriate aromatic aldehyde (10 mmol), the ketone (10 mmol), malononitrile (0.66 g, 10 mmol), and ammonium acetate (6.2 g, 80 mmol) in absolute ethanol (50 mL) was refluxed for 4-5 h. The reaction mixture was allowed to attain room temperature and the resulting precipitate was filtered, washed with water, dried, and recrystallized from ethanol.

( 1) 2-Amino-4-(benzo[d][1,3]dioxol-5-yl)-6-phenylnicotinonitrile ( 1). Yield: (1.7 g, 67%); m.p.: 162–164°C; IR (cm⁻¹, KBr): 3149, 3354 (NH₂), 2218 (CN). ¹H NMR (δ, ppm): 5.98 (s, 2H, CH₂), 6.17 (s, 2H, NH₂), 6.74–7.88 (m, 9H, 8 Ar-H + pyridine C₅-H). ¹³C NMR (δ, ppm): 91.4 (CH₂), 113.4, 115.6, 117.5 (CN), 120.3, 124.8, 126.6, 127.1, 128.4, 129.5, 139.9, 142.5, 148.1, 150.6, 161.5, 165.7 (Ar-C). MS: m/z 315 (M⁺, 100), 303 (21), 289 (82), 277 (14), 273 (76), 272 (42), 227 (55), 84 (58), 75 (28). Anal.% Calcd for C₁₉H₁₃N₃O₂ (315.33): C, 72.37; H, 4.16; N, 13.33. Found: C, 72.25; H, 4.20; N, 13.21.

( 2) 2-Amino-6-phenyl-4-(2-thienyl)nicotinonitrile (2). Yield: (1.8 g, 54%); m.p.: 190–192°C; IR (cm⁻¹, KBr): 3167, 3387 (NH₂), 2228 (CN). ¹H NMR (δ, ppm): 6.10 (s, 2H, NH₂), 7.02–8.11 (m, 9H, 8 Ar-H + pyridine C₅-H). ¹³C NMR (δ, ppm): 90.6, 110.0, 118.1 (CN), 122.3, 125.2, 126.8, 127.1, 128.3, 129.6, 139.7, 142.6, 150.7, 162.2, 165.4 (Ar-C). Anal.% Calcd for C₁₆H₁₁N₃S (277.35): C, 69.29; H, 4.00; N, 15.15. Found: C, 69.21; H, 4.40; N, 15.21.

( 3) 2-Amino-4-(benzo[d][1,3]dioxol-5-yl)-6-(4-bromophenyl)nicotinonitrile ( 3). Yield: (1.9 g, 68%); m.p.: 198–200°C; IR (cm⁻¹, KBr): 3175, 3358 (NH₂), 2223 (CN). ¹H NMR (δ, ppm): 6.03 (s, 2H, CH₂), 6.21 (s, 2H, NH₂), 7.68–7.87 (m, 8H, 7 Ar-H + pyridine C₅-H). ¹³C NMR (δ, ppm): 91.2 (CH₂), 113.6, 115.4, 117.8 (CN), 121.6, 125.5, 126.3, 127.0, 128.2, 129.2, 139.4, 142.2, 148.0, 150.5, 162.5, 164.9 (Ar-C). Anal.% Calcd for C₁₉H₁₂BrN₃O₂ (394.23): C, 57.89; H, 3.07; N, 10.66. Found: C, 57.82; H, 3.03; N, 10.61.

( 4) 2-Amino-6-(4-bromophenyl)-4-(2-thienyl)nicotinonitrile (4). Yield: (2.02 g, 73%); m.p.: 200–202°C; IR (cm⁻¹, KBr): 3416, 3198 (NH₂), 2220 (CN). ¹H NMR (δ, ppm): 6.18 (s, 2H, NH₂), 7.05–8.01 (m, 8H, 7 Ar-H + pyridine C₅-H). ¹³C NMR (δ, ppm): 91.4, 110.9, 118.0 (CN), 122.2, 125.3, 126.4, 127.2, 128.4, 129.3, 139.8, 142.7, 151.2, 162.8, 165.5 (Ar-C). Anal.% Calcd for C₁₆H₁₀BrN₃S (356.24): C, 53.95; H, 2.83; N, 11.80. Found: C, 53.88; H, 2.79; N, 11.85.

( 5) 2-Amino-6-(4-methoxyphenyl)-4-(2-thienyl)nicotinonitrile (5). Yield: (1.63 g, 62%); m.p.: 160–162°C; IR (cm⁻¹, KBr): 3172, 3382 (NH₂), 2228 (CN). ¹H NMR (δ, ppm): 3.75 (s, 3H, OCH₃), 6.18 (s, 2H, NH₂), 7.10–8.12 (m, 8H, 7 Ar-H + pyridine C₅-H). ¹³C NMR (δ, ppm): 56.1 (OCH₃), 97.9, 110.0, 117.8 (CN), 122.1, 125.4, 126.4, 127.2, 128.1, 129.2, 138.9, 142.2, 151.8, 161.2, 165.4 (Ar-C). MS: m/z 307 (M⁺, 100), 225 (18), 281 (85), 265 (78), 182 (63), 84 (15), 83 (12), 75 (34). Anal.% Calcd for C₁₇H₁₃N₃OS (307.37): C, 66.43; H, 4.26; N, 13.67. Found: C, 66.08; H, 3.90; N, 13.34.

2.1.2. 5,7-Disubstituted Pyrido[2,3-d]pyrimidine-4(3H)-ones (6–10)

A mixture of the appropriate nicotinonitriles 1–5 (10 mmol) and formic acid (5 mL) was heated in a boiling water bath for 30 min. After being cooled to room temperature, the reaction mixture was poured onto ice-cold water; the precipitated solid product was filtered, washed with water, dried, and recrystallized from the appropriate solvent.

( 1) 5-(Benzo[d][1,3]dioxol-5-yl)-7-phenylpyrido[2,3-d]pyrimidine-4(3H)-one ( 6). Yield: (1.8 g, 59%; methanol); m.p.: 168–170°C; IR (cm⁻¹, KBr): 3285 (NH), 1648 (C=O). ¹H NMR (δ, ppm): 6.18 (s, 2H, CH₂); 6.84–7.95 (m, 10 H, Ar-H); 8.16 (s, 1H, NH). ¹³C NMR (δ, ppm): 91.8 (CH₂), 113.5, 116.5, 118.2, 120.1, 120.2, 122.8, 125.3, 127.5, 131.4, 142.6, 144.2, 148.1, 151.9, 157.6, 164.3, 173.7 (Ar-C), 170.5 (CO). MS: m/z 343 (M⁺, 90), 342 (100), 331 (32), 326 (20), 314 (18), 297 (44), 284 (53), 206 (10), 83 (12), 75 (22). Anal.% Calcd for C₂₀H₁₃N₃O₃ (343.34): C, 69.97; H, 3.82; N, 12.24. Found: C, 70.02; H, 3.77; N, 12.31.

( 2) 7-Phenyl-5-(2-thienyl)pyrido[2,3-d]pyrimidine-4(3H)-one (7). Yield: (1.9 g, 62%, ethanol); m.p.: 160–162°C; IR (cm⁻¹, KBr): 3292 (NH), 1656 (C=O). ¹H NMR (δ, ppm): 6.76–8.02 (m, 10H, Ar-H); 8.16 (s, 1H, NH). ¹³C NMR (δ, ppm): 117.2, 121.6, 122.2, 122.8, 125.3, 125.4, 127.5, 138.4, 142.6, 144.3, 151.9, 157.8, 164.5, 173.4 (Ar-C), 170.2 (CO). Anal.% Calcd for C₁₇H₁₁N₃OS (305.36): C, 66.87; H, 3.63; N, 13.76. Found: C, 66.80; H, 3.65; N, 13.81.

( 3) 5-(Benzo[d][1,3]dioxol-5-yl)-7-(4-bromophenyl)pyrido[2,3-d]pyrimidine-4(3H)-one ( 8). Yield: (2.3 g, 72%, ethanol); m.p.: 110–112°C; IR (cm⁻¹, KBr): 3320 (NH), 1652 (C=O). ¹H NMR (δ, ppm): 6.11 (s, 2H, CH₂); 6.80–7.98 (m, 9H, Ar-H); 8.21 (s, 1H, NH). ¹³C NMR (δ, ppm): 91.5 (CH₂), 113.2, 116.7, 119.3, 120.1, 121.7, 122.8, 125.5, 127.2, 131.6, 142.3, 144.0, 148.2, 151.7, 157.3, 164.0, 173.6 (Ar-C), 170.2 (CO). Anal.% Calcd for C₂₀H₁₂BrN₃O₃ (422.24): C, 56.89; H, 2.86; N, 9.95. Found: C, 56.82; H, 2.79; N, 9.91.

( 4) 7-(4-Bromophenyl)-5-(2-thienyl)pyrido[2,3-d]pyrimidine-4(3H)-one (9). Yield: (2.0 g, 70%, methanol); m.p.: 128–130°C; IR (cm⁻¹, KBr): 3298 (NH), 1650 (C=O). ¹H NMR (δ, ppm): 6.72–7.69 (m, 9H, Ar-H); 8.22 (s, 1H, NH). ¹³C NMR (δ, ppm): 118.5, 120.6, 121.7, 122.8, 125.3, 127.5, 129.4, 132.3, 138.4, 142.3, 147.3, 157.8, 162.5, 173.1 (Ar-C), 170.3 (CO). Anal.% Calcd for C₁₇H₁₀BrN₃OS (384.25): C, 53.14; H, 2.62; N, 10.94. Found: C, 53.03; H, 2.60; N, 11.03.

( 5) 7-(4-Methoxyphenyl)-5-(2-thienyl)pyrido[2,3-d]pyrimidine-4(3H)-one (10). Yield: (1.8 g, 66%, ethanol); m.p.: 166–168°C; IR (cm⁻¹, KBr): 3304 (NH), 1654 (C=O). ¹H NMR (δ, ppm): 3.82 (s, 3H, OCH₃); 6.92–8.01 (m, 9H, Ar-H); 8.02 (s, 1H, NH). ¹³C NMR (δ, ppm): 56.1 (OCH₃), 114.5, 118.5, 120.7, 122.8, 125.5, 127.4, 128.1, 132.2, 142.4, 147.5, 157.7, 160.6, 163.8, 173.6 (Ar-C), 170.2 (CO). MS: m/z 335 (M⁺, 89), 334 (100), 318 (62), 304 (48), 252 (31), 206 (14), 132 (22), 83 (16), 75 (21). Anal.% Calcd for C₁₈H₁₃N₃O₂S (335.38): C, 64.46; H, 3.91; N, 12.53. Found: C, 64.61; H, 3.59; N, 12.44.

2.1.3. 2-Methyl-5,7-disubstituted-pyrido[2,3-d]pyrimidine-4(3H)-ones (11–15)

A mixture of the starting nicotinonitriles 1–5 (10 mmol), acetic anhydride (5 mL), and conc. H₂SO₄ (0.5 mL) was heated in a boiling water bath for 10 min and then cooled. The reaction mixture was poured onto ice-cold water, treated with 20% NaOH solution until it acquires pH of 11-12. The crude solid product was filtered, dried, and recrystallized from the appropriate solvent.

( 1) 5-(Benzo[d][1,3]dioxol-5-yl)-2-methyl-7-phenylpyrido[2,3-d]pyrimidine-4(3H)-one ( 11). Yield: (2.42 g, 60%, methanol); m.p.: 238–240°C; IR (cm⁻¹, KBr): 3277 (NH), 1656 (C=O). ¹H NMR (δ, ppm): 2.35 (s, 3H, CH₃), 6.18 (s, 2H, CH₂); 6.74–7.88 (m, 9H, Ar-H); 8.16 (s, 1H, NH). ¹³C NMR (δ, ppm): 20.9 (CH₃), 90.9 (CH₂), 113.4, 116.2, 118.1, 120.0, 121.2, 122.7, 125.4, 127.3, 131.2, 142.5, 144.1, 148.1, 151.8, 157.3, 164.1, 173.0 (Ar-C), 170.1 (CO). Anal.% Calcd for C₂₁H₁₅N₃O₃ (357.37): C, 70.58; H, 4.23; N, 11.76. Found: C, 70.47; H, 4.19; N, 11.85.

( 2) 2-Methyl-7-phenyl-5-(2-thienyl)pyrido[2,3-d]pyrimidine-4(3H)-one (12). Yield: (2.11 g, 62%, ethanol); m.p.: 228–230°C; IR (cm⁻¹, KBr): 3270 (NH), 1654 (C=O). ¹H NMR (δ; ppm): 2.28 (s, 3H, CH₃), 7.25–8.09 (m, 9H, Ar-H); 8.14 (s, 1H, NH). ¹³C NMR (δ, ppm): 21.1 (CH₃), 117.5, 121.4, 122.2, 122.8, 125.5, 125.6, 127.1, 138.5, 142.4, 144.5, 151.8, 157.7, 164.2, 173.4 (Ar-C), 168.8 (CO). MS: m/z 319 (M⁺, 82), 318 (100), 304 (78), 302 (35), 236 (12), 206 (14), 132 (28), 84 (43), 83 (12), 75 (21). Anal.% Calcd for C₁₈H₁₃N₃OS (319.38): C, 67.69; H, 4.10; N, 13.16. Found: C, 67.54; H, 4.02; N, 13.11.

( 3) 5-(Benzo[d][1,3]dioxol-5-yl)-7-(4-bromophenyl)-2-methylpyrido[2,3-d]pyrimidine-4(3H)-one ( 13). Yield: (2.10 g, 64%, ethanol); m.p.: 220–222°C; IR (cm⁻¹, KBr): 3265 (NH), 1651 (C=O). ¹H NMR (δ, ppm): 2.32 (s, 3H, CH₃), 6.16 (s, 2H, CH₂); 6.82–7.96 (m, 8H, Ar-H); 8.23 (s, 1H, NH). ¹³C NMR (δ, ppm): 20.2 (CH₃), 91.3 (CH₂), 113.5, 116.3, 118.2, 119.9, 120.0, 122.1, 125.4, 127.6, 131.4, 142.6, 144.2, 152.8, 148.1, 157.9, 163.4, 171.7 (Ar-C), 173.2 (CO). Anal.% Calcd for C₂₁H₁₄BrN₃O₃ (436.27): C, 57.82; H, 3.23; N, 9.63. Found: C, 57.91; H, 3.17; N, 9.75.

( 4) 7-(4-Bromophenyl)-2-methyl-5-(2-thienyl)pyrido[2,3-d]pyrimidine-4(3H)-one (14). Yield: (2.0 g, 63%, ethanol); m.p.: 248–250°C; IR (cm⁻¹, KBr): 3270 (NH), 1652 (C=O). ¹H NMR (δ, ppm): 2.35 (s, 3H, CH₃), 6.68–7.77 (m, 8H, Ar-H); 8.25 (s, 1H, NH). ¹³C NMR (δ, ppm): 21.0 (CH₃), 118.8, 120.5, 121.8, 122.9, 125.2, 127.4, 129.3, 132.2, 138.3, 142.2, 147.1, 157.7, 163.1, 172.9 (Ar-C), 170.2 (CO). Anal.% Calcd for C₁₈H₁₂BrN₃OS (398.28): C, 54.28; H, 3.04; N, 10.55. Found: C, 54.17; H, 3.00; N, 10.61.

( 5) 2-Methyl-7-(4-methoxyphenyl)-5-(2-thienyl)pyrido[2,3-d]pyrimidine-4(3H)-one (15). Yield: (2.2 g, 65%, ethanol); m.p.: 200–202°C; IR (cm⁻¹, KBr): 3259 (NH), 1653 (C=O). ¹H NMR (δ, ppm): 2.36 (s, 3H, CH₃), 3.81 (s, 3H, OCH₃); 6.81–8.00 (m, 8H, Ar-H); 8.22 (s, 1H, NH). ¹³C NMR (δ, ppm): 20.8 (CH₃), 56.1 (OCH₃), 91.9 (CH₂), 113.5, 116.6, 118.8, 120.41, 120.6, 122.7, 125.5, 127.3, 131.3, 142.2, 144.7, 148.4, 160.1, 157.3, 164.3, 173.3 (Ar-C), 170.2 (CO). Anal.% Calcd for C₁₉H₁₅N₃O₂S (349.41): C, 65.31; H, 4.33; N, 12.03. Found: C, 65.25; H, 4.29; N, 12.13.

2.1.4. 4-Imino-3-phenyl-5,7-disubstituted-dihydropyrido[2,3-d]pyrimidine-2(1H)-thiones (16–20)

To a solution of the appropriate nicotinonitriles 1–5 (10 mmol) in pyridine (15 mL), phenyl isothiocyanate (0.15 g, 15 mmol) was added, and the mixture was refluxed for 4 h. After cooling, the solid product thus formed was filtered, washed thoroughly with water, dried, and recrystallized from the appropriate solvent.

( 1) 5-(Benzo[d][1,3]dioxol-5-yl)-4-imino-3,7-diphenyl-3,4-dihydropyrido[2,3-d]pyrimidine-2(1H)-thione ( 16). Yield: (3.2 g, 82%, methanol); m.p.: 178–180°C; IR (cm⁻¹, KBr): 3328 (NH), 1635 (C=N), 1204 (C=S). ¹H NMR (δ, ppm): 6.12 (s, 2H, CH₂), 6.75 (s, 1H, C=NH); 6.78–7.99 (m, 15H, 14 Ar-H + NH). ¹³C NMR (δ, ppm): 90.9 (CH₂), 108.1, 109.8, 113.5, 115.6, 120.2, 121.3, 124.5, 125.3, 127.1, 127.5, 128.8, 129.0, 131.4, 139.5, 139.7, 148.3, 149.0, 160.0 (Ar-C), 163.6 (C=NH), 179.8 (CS). Anal.% Calcd for C₂₆H₁₈N₄O₂S (450.52): C, 69.32; H, 4.03; N, 12.44. Found: C, 69.24; H, 3.99; N, 12.40.

( 2) 4-Imino-3,7-diphenyl-5-(2-thienyl)-3,4-dihydropyrido[2,3-d]pyrimidine-2(1H)-thione (17). Yield: (3.89 g, 82%, methanol); m.p.: 168–170°C; IR (cm⁻¹, KBr): 3287 (NH), 1639 (C=N), 1198 (C=S). ¹H NMR (δ, ppm): 6.86 (s, 1H, C=NH); 6.78–7.80 (m, 15H, 14 Ar-H + NH). ¹³C NMR (δ, ppm): 109.2, 110.6, 121.5, 122.8, 124.3, 125.9, 127.1, 127.3, 127.6, 129.1, 132.3, 139.9, 142.4, 144.9, 149.7, 158.1, 162.1 (Ar-C), 164.7 (C=NH), 180.3 (C=S). Anal.% Calcd for C₂₃H₁₆N₄S₂ (412.53): C, 66.97; H, 3.91; N, 13.58. Found: C, 66.90; H, 3.88; N, 13.65.

( 3) 5-(Benzo[d][1,3]dioxol-5-yl)-7-(4-bromophenyl)-4-imino-3-phenyl-3,4-dihydropyrido[2,3-d]pyrimidine-2(1H)-thione ( 18). Yield: (3.3 g, 78%, ethanol); m.p.: 198–200°C; IR (cm⁻¹, KBr): 3321 (NH), 1637 (C=N), 1203 (C=S). ¹H NMR (δ, ppm): 5.98 (s, 2H, CH₂), 6.78 (s, 1H, C=NH); 6.88–8.12 (m, 14H, 13 Ar-H + NH). ¹³C NMR (δ, ppm): 91.3 (CH₂), 108.6, 109.8, 113.8, 116.1, 120.4, 124.8, 125.5, 127.1, 127.4, 128.8, 129.3, 131.6, 139.5, 140.0, 148.1, 149.2, 158.2, 160.8 (Ar-C), 164.4 (C=NH), 181.2 (C=S). MS: m/z 530 (M⁺, 74), 529 (100), 449 (80), 369 (29), 361 (17), 330 (25), 132 (26), 84 (30), 75 (22). Anal.% Calcd for C₂₆H₁₇BrN₄O₂S (529.41): C, 58.99; H, 3.24; N, 10.58. Found: C, 58.91; H, 3.26; N, 10.45.

( 4) 7-(4-Bromophenyl)-4-imino-3-phenyl-5-(2-thienyl)-3,4-dihydropyrido[2,3-d]pyrimidine-2(1H)-thione (19). Yield: (3.4 g, 76%, ethanol); m.p.: 180–182°C; IR (cm⁻¹, KBr): 3284 (NH), 1642 (C=N), 1195 (C=S). ¹H NMR (δ, ppm): 6.81 (s, 1H, C=NH); 7.34–7.82 (m, 14H, 13 Ar-H + NH). ¹³C NMR (δ, ppm): 108.4, 110.9, 121.6, 122.8, 124.5, 125.5, 126.8, 127.2, 127.4, 128.9, 132.4, 139.8, 142.5, 144.4, 149.0, 158.3, 160.0 (Ar-C), 164.6 (C=NH), 179.3 (C=S). Anal.% Calcd for C₂₃H₁₅BrN₄S₂ (491.43): C, 56.21; H, 3.08; N, 11.40. Found: C, 56.12; H, 3.06; N, 11.51.

( 5) 4-Imino-7-(4-methoxyphenyl)-3-phenyl-5-(2-thienyl)-3,4-dihydropyrido[2,3-d]pyrimidine-2(1H)-thione (20). Yield: (2.3 g, 73%, DMF); m.p.: 190–192°C; IR (cm⁻¹, KBr): 3312 (NH), 1639 (C=N), 1188 (C=S). ¹H NMR (δ, ppm): 3.73 (s, 3H, OCH₃), 6.83 (s, 1H, C=NH); 6.79–7.94 (m, 13H, 12 Ar-H + NH). ¹³C NMR (δ, ppm): 56.0 (OCH₃), 106.1, 114.6, 120.2, 121.6, 122.8, 124.5, 125.5, 126.8, 127.2, 127.4, 128.9, 132.4, 142.5, 144.4, 149.0, 158.3, 160.0 (Ar-C), 164.8 (C=NH), 179.6 (C=S). Anal.% Calcd for C₂₄H₁₈N₄OS₂ (422.56): C, 65.14; H, 4.10; N, 12.66. Found: C, 65.09; H, 4.17; N, 12.56.

2.1.5. N-((3-Cyano-4,6-disubstituted-pyridin-2-yl)thiocarbamoyl)benzamides (21–23)

To a solution of the appropriate nicotinonitriles 1–5 (20 mmol) in dry acetone (30 mL), a solution of benzoyl isothiocyanate (1.65 g, 20 mmol) in dry acetone (10 mL) was added. The resulting mixture was heated under reflux for 10 h. The reaction mixture was concentrated and left overnight at room temperature. The separated crystalline product was filtered, washed with Et₂O, and recrystallized from the appropriate solvent.

( 1) N-((3-Cyano-6-phenyl-4-(2-thienyl)pyridin-2-yl)carbamothioyl)benzamide (21). Yield: (3.7 g, 85%, methanol); m.p.: 126–128°C; IR (cm⁻¹, KBr): 3433 (NH), 2215 (CN), 1661 (C=O), 1166 (C=S). ¹H NMR (δ, ppm): 6.39 (s, 1H, CSNH); 7.10–8.14 (m, 14H, Ar-H); 8.43 (s, 1H, CONH). ¹³C NMR (δ, ppm): 96.4, 111.2, 118.0 (CN), 122.4, 125.5, 126.3, 127.2, 127.4, 128.7, 129.0, 132.0, 133.3, 139.9, 142.3, 151.7, 161.8, 165.2 (Ar-C), 169.2 (CO), 180.1 (CS). MS: m/z 440 (M⁺, 100), 414 (76), 333 (42), 311 (19), 252 (31), 206 (14), 105 (66), 83 (18), 75 (27). Anal.% Calcd for C₂₄H₁₆N₄OS₂ (440.54): C, 65.43; H, 3.66; N, 12.72. Found: C, 65.23; H, 3.96; N, 12.99.

( 2) N-((4-(Benzo[d][1,3]dioxol-5-yl)-6-(4-bromophenyl)-3-cyanopyridin-2-yl)carbamothioyl)benzamide ( 22). Yield: (4.5 g, 82%, acetic acid); m.p.: 198–200°C; IR (cm⁻¹, KBr): 3425 (NH), 2226 (CN), 1665 (C=O), 1174 (C=S). ¹H NMR (δ, ppm,): 6.02 (s, 2H, CH₂), 6.35 (s, 1H, CSNH); 6.63–7.89 (m, 13H, Ar-H); 8.48 (s, 1H, CONH). ¹³C NMR (δ, ppm): 91.7 (CH₂), 95.4, 113.0, 115.5, 117.7 (CN), 120.2, 122.3, 125.5, 126.6, 127.1, 127.3, 128.8, 129.2, 131.6, 133.4, 139.9, 142.4, 148.0, 151.7, 162.7, 165.2 (Ar-C), 168.1 (CO), 179.9 (CS). Anal.% Calcd for C₂₇H₁₇BrN₄O₃S (557.42): C, 58.18; H, 3.07; N, 10.05. Found: C, 58.11; H, 3.09; N, 10.15.

( 3) N-((6-(4-Bromophenyl)-3-cyano-4-(2-thienyl)pyridin-2-yl)carbamothioyl)benzamide (23). Yield: (4.1 g, 79%, acetic acid); m.p.: 210–212°C; IR (cm⁻¹, KBr): 3422 (NH), 2218 (CN), 1662 (C=O), 1170 (C=S). ¹H NMR (δ, ppm): 6.28 (s, 1H, CSNH); 7.02–8.32 (m, 12H, Ar-H); 8.52 (s, 1H, CONH). ¹³C NMR (δ, ppm): 94.4, 111.0, 118.1 (CN), 122.8, 125.3, 126.5, 127.3, 127.5, 128.6, 129.1, 131.9, 133.5, 139.8, 142.6, 151.5, 161.9, 165.4 (Ar-C), 169.3 (CO), 180.4 (CS). Anal.% Calcd for C₂₄H₁₅BrN₄OS₂ (519.44): C, 55.50; H, 2.91; N, 10.79. Found: C, 55.14; H, 3.19; N, 11.01.

2.1.6. 4-Amino-5,7-disubstituted-pyrido[2,3-d]pyrimidine-2(1H)-ones (24–27)

A mixture of the appropriate nicotinonitriles 1–5 (10 mmol) and urea (0.6 g, 10 mmol) was fused at 260–300°C using a sand bath for 1 h. The reaction mixture was allowed to attain room temperature; the crude solid product was treated with water, then rubbed with ethanol, filtered, and recrystallized from DMF/H₂O.

( 1) 4-Amino-5-(benzo[d][1,3]dioxol-5-yl)-7-phenylpyrido[2,3-d]pyrimidine-2(1H)-one ( 24). Yield: (2.4 g, 71%); m.p.: 278–280°C; IR (cm⁻¹, KBr): 3280, 3323, 3435 (NH & NH₂), 1660 (C=O). ¹H NMR (δ, ppm): 5.98 (s, 2H, CH₂), 6.52 (s, 2H, NH₂), 6.74–8.13 (m, 10H, 9 Ar-H + NH). ¹³C NMR (δ, ppm): 93.5 (CH₂), 113.7, 115.8, 120.6, 124.4, 126.5, 127.2, 128.8, 129.0, 129.3, 139.8, 142.4, 148.1, 150.8, 158.5, 160.4, 164.4 (Ar-C), 166.2 (CO). Anal.% Calcd for C₂₀H₁₄N₄O₃ (358.36): C, 67.03; H, 3.94; N, 15.63. Found: C, 67.10; H, 3.89; N, 15.49.

( 2) 4-Amino-7-phenyl-5-(2-thienyl)pyrido[2,3-d]pyrimidine-2(1H)-one (25). Yield: (2.1 g, 75%); m.p.: 280–282°C; IR (cm⁻¹, KBr): 3276, 3336, 34659 (NH & NH₂), 1665 (C=O). ¹H NMR (δ, ppm): 6.44 (s, 2H, NH₂), 6.82–8.15 (m, 10H, 9 Ar-H + NH). ¹³C NMR (δ, ppm): 109.6, 110.0, 122.2, 125.5, 126.5, 127.1, 128.1, 129.4, 139.7, 142.3, 151.2, 159.1, 162.5, 165.1 (Ar-C), 166.4 (CO). Anal.% Calcd for C₁₇H₁₂N₄OS (320.37): C, 63.73; H, 3.78; N, 17.49. Found: C, 63.81; H, 3.67; N, 17.57.

( 3) 4-Amino-5-(benzo[d][1,3]dioxol-5-yl)-7-(4-bromophenyl)pyrido[2,3-d]pyrimidine-2(1H)-one ( 26). Yield: (2.2 g, 75%); m.p.: 260–262°C; IR (cm⁻¹, KBr): 3271, 3326, 3460 (NH & NH₂), 1667 (C=O). ¹H NMR (δ, ppm): 6.10 (s, 2H, CH₂), 6.48 (s, 2H, NH₂), 6.84–8.13 (m, 9H, 8 Ar-H + NH). ¹³C NMR (δ, ppm): 92.6 (CH₂), 113.5, 115.6, 122.1, 125.5, 126.4, 127.2, 128.8, 129.0, 129.4, 139.6, 142.6, 148.1, 150.9, 157.8, 160.2, 164.8 (Ar-C). 166.2 (CO). Anal.% Calcd for C₂₀H₁₃BrN₄O₃ (437.25): C, 54.94; H, 3.00; N, 12.81. Found: C, 54.91; H, 2.97; N, 12.89.

( 4) 4-Amino-7-(4-bromophenyl)-5-(2-thienyl)pyrido[2,3-d]pyrimidine-2(1H)-one (27). Yield: (2.1 g, 73%); m.p.: 250–252°C; IR (cm⁻¹, KBr): 3284, 3346, 3455 (NH & NH₂), 1664 (C=O). ¹H NMR (δ, ppm): 6.41 (s, 2H, NH₂); 6.97–8.01 (m, 8H, Ar-H); 8.02 (s, 1H, NH). ¹³C NMR (δ, ppm): 108.1, 110.3, 122.7, 125.2, 126.7, 127.3, 127.4, 129.0, 139.3, 142.6, 144.5, 158.4, 159.3, 166.1 (Ar-C), 162.3 (CO). Anal.% Calcd for C₁₇H₁₁BrN₄OS (399.27): C, 51.14; H, 2.78; N, 14.03. Found: C, 51.05; H, 2.74; N, 14.07.

2.1.7. 4-Amino-5,7-disubstituted-pyrido[2,3-d]pyrimidine-2(1H)-thiones (28–32)

A mixture of the appropriate nicotinonitriles 1–5 (10 mmol) and thiourea (0.8 g, 10 mmol) was fused at 260–300°C using a sand bath for 1.5 h. The reaction mixture was worked up as described under compounds 24–27 and recrystallized from the appropriate solvent.

( 1) 4-Amino-5-(benzo[d][1,3]dioxol-5-yl)-7-phenylpyrido[2,3-d]pyrimidine-2(1H)-thione ( 28). Yield: (2.1 g, 67%, ethanol); m.p.: 220–222°C; IR (cm⁻¹, KBr): 3272, 3345, 3462 (NH & NH₂), 1179 (C=S). ¹H NMR (δ, ppm): 5.98 (s, 2H, CH₂), 6.52 (s, 2H, NH₂), 6.74–8.13 (m, 10H, 9 Ar-H + NH). ¹³C NMR (δ, ppm): 91.4 (CH₂), 113.4, 115.6, 120.3, 124.8, 126.6, 127.1, 128.4, 129.0, 129.5, 139.9, 142.5, 148.1, 150.6, 158.3, 160.5, 164.7 (Ar-C), 182.4 (CS). Anal.% Calcd for C₂₀H₁₄N₄O₂S (374.42): C, 64.16; H, 3.77; N, 14.96. Found: C, 64.09; H, 3.76; N, 14.90.

( 2) 4-Amino-7-phenyl-5-(2-thienyl)pyrido[2,3-d]pyrimidine-2(1H)-thione (29). Yield: (2.2 g, 52%, methanol); m.p.: 198–200°C; IR (cm⁻¹, KBr): 3277, 3325, 3438 (NH & NH₂), 1166 (C=S). ¹H NMR (δ, ppm): 6.44 (s, 2H, NH₂), 6.82–8.15 (m, 10H, 9 Ar-H + NH). ¹³C NMR (δ, ppm): 108.6, 110.0, 122.2, 125.5, 126.8, 127.1, 128.3, 129.6, 139.7, 142.6, 150.7, 158.2, 162.2, 165.4 (Ar-C), 182.3 (CS). MS: m/z 336 (M⁺, 74), 320 (100), 303 (72), 220 (31), 132 (15), 83 (11), 75 (25). Anal.% Calcd for C₁₇H₁₂N₄S₂ (336.43): C, 60.69; H, 3.60; N, 16.65. Found: C, 60.62; H, 3.57; N, 16.78.

( 3) 4-Amino-5-(benzo[d][1,3]dioxol-5-yl)-7-(4-bromophenyl)pyrido[2,3-d]pyrimidine-2(1H)-thione ( 30). Yield: (2.6 g, 73%, ethanol); m.p.: 230–232°C; IR (cm⁻¹, KBr): 3288, 3346, 3438 (NH & NH₂), 1173 (C=S). ¹H NMR (δ, ppm): 5.96 (s, 2H, CH₂), 6.59 (s, 2H, NH₂), 6.84–8.03 (m, 9H, 8 Ar-H + NH). ¹³C NMR (δ, ppm): 92.1 (CH₂), 113.5, 115.4, 120.1, 124.6, 126.4, 127.1, 128.8, 129.1, 129.5, 139.7, 142.7, 148.0, 151.0, 159.3, 160.5, 164.9 (Ar-C), 181.4 (CS). Anal.% Calcd for C₂₀H₁₃BrN₄O₂S (453.31): C, 52.99; H, 2.89; N, 12.36. Found: C, 52.93; H, 2.87; N, 12.47.

( 4) 4-Amino-7-(4-bromophenyl)-5-(2-thienyl)pyrido[2,3-d]pyrimidine-2(1H)-thione (31). Yield: (1.7 g, 52%, ethanol); m.p.: 243–245°C; IR (cm⁻¹, KBr): 3279, 3329, 3460 (NH & NH₂), 1177 (C=S). ¹H NMR (δ, ppm): 6.49 (s, 2H, NH₂), 6.67–8.11 (m, 9H, 8 Ar-H + NH). ¹³C NMR (δ, ppm): 109.3, 110.7, 122.1, 125.5, 127.1, 127.6, 127.9, 129.4, 139.6, 143.2, 150.7, 158.1, 162.0, 165.5 (Ar-C), 182.7 (CS). Anal.% Calcd for C₁₇H₁₁BrN₄S₂ (415.33): C, 49.16; H, 2.67; N, 13.49. Found: C, 49.12; H, 2.64; N, 13.38.

( 5) 4-Amino-7-(4-methoxyphenyl)-5-(2-thienyl)pyrido[2,3-d]pyrimidine-2(1H)-thione (32). Yield: (2.6 g, 71%, ethanol); m.p.: 298–300°C; IR (cm⁻¹, KBr): 3277, 3344, 3431 (NH & NH₂), 1168 (C=S). ¹H NMR (δ, ppm): 2.89 (s, 3H, OCH₃), 6.75 (s, 2H, NH₂); 6.94–8.27 (m, 9H, 8 Ar-H + NH). ¹³C NMR (δ, ppm): 56.1 (OCH₃) 108.8, 110.6, 114.6, 122.9, 125.3, 127.1, 128.2, 129.0, 139.7, 142.2, 144.6, 157.8, 158.7, 164.5 (Ar-C), 183.3 (CS). Anal.% Calcd for C₁₈H₁₄N₄OS₂ (366.46): C, 59.00; H, 3.85; N, 15.29. Found: C, 58.86; H, 3.83; N, 15.17.

2.1.8. 2-Alkylthio-4-amino-5,7-disubstituted-pyrido[2,3-d]pyrimidines (33–37)

To a stirred solution of proper thiones 30–32 (10 mmol) in 1N NaOH (5 mL) and ethanol (2 mL), the selected alkyl iodide (12 mmol) was added. The reaction mixture was stirred at room temperature for 2-3 h and the precipitated product was filtered, washed with cold ethanol, dried, and recrystallized from proper solvent.

( 1) 4-Amino-5-(benzo[d][1,3]dioxol-5-yl)-7-(4-bromophenyl)-2-(methylthio)pyrido[2,3-d]pyrimidine ( 33). Yield: (3.5 g, 76%, methanol); m.p. 240–242°C; IR (cm⁻¹, KBr): 3365, 3435 (NH₂). ¹H NMR (δ, ppm): 2.49 (s, 3H, SCH₃); 6.41 (s, 2H, CH₂); 6.92–8.06 (m, 10H, 8 Ar-H + NH₂). ¹³C NMR (δ, ppm): 19.4 (SCH₃), 91.4 (CH₂), 102.1, 113.8, 115.7, 120.1, 120.2, 121.7, 127.4, 129.1, 131.2, 132.1, 138.9, 148.1, 149.3, 157.8, 158.2, 167.1, 167.7 (Ar-C). Anal.% Calcd for C₂₁H₁₅BrN₄O₂S (467.34): C, 53.97; H, 3.24; N, 11.99. Found: C, 53.87; H, 3.19; N, 12.14.

( 2) 4-Amino-5-(benzo[d][1,3]dioxol-5-yl)-7-(4-bromophenyl)-2-(ethylthio)pyrido[2,3-d]pyrimidine ( 34). Yield: (2.3 g, 59%, ethanol); m.p.: 176–178°C; IR (cm⁻¹, KBr): 3288, 3429 (NH₂). ¹H NMR (δ, ppm): 1.24 (t, J = 6 Hz, 3H, CH₃); 2.88 (q, J = 6 Hz, SCH₂), 6.41 (s, 2H, CH₂); 6.92–8.06 (m, 10H, 8 Ar-H + NH₂). ¹³C NMR (δ, ppm): 16.1 (CH₃), 28.1 (SCH₂), 91.4 (CH₂), 102.1, 113.8, 115.7, 120.1, 120.2, 121.7, 127.4, 129.1, 131.2, 132.1, 138.9, 148.1, 149.3, 157.8, 158.2, 167.1, 167.7 (Ar-C). Anal.% Calcd for C₂₂H₁₇BrN₄O₂S (481.36): C, 54.89; H, 3.56; N, 11.64. Found: C, 54.79; H, 3.53; N, 11.72.

( 3) 4-Amino-7-(4-bromophenyl)-2-(methylthio)-5-(2-thienyl)pyrido[2,3-d]pyrimidine (35). Yield: (2.3 g, 66%, ethanol); m.p.: 185–187°C; IR (cm⁻¹, KBr): 3391, 3452 (NH₂). ¹H NMR (δ, ppm): 2.42 (s, 3H, SCH₃); 6.55 (s, 2H, NH₂) 7.10–7.89 (m, 8H, Ar-H). ¹³C NMR (δ, ppm): 19.1 (SCH₃), 100.6, 120.9, 121.7, 122.8, 125.3, 127.4, 129.2, 129.5, 132.4, 138.6, 142.7, 145.0, 159.6, 158.7, 167.1, 167.6 (Ar-C). Anal.% Calcd for C₁₈H₁₃BrN₄S₂ (429.36): C, 50.35; H, 3.05; N, 13.05. Found: C, 50.41; H, 3.00; N, 13.18.

( 4) 4-Amino-7-(4-methoxyphenyl)-2-(methylthio)-5-(2-thienyl)pyrido[2,3-d]pyrimidine (36). Yield: (2.6 g, 61%, DMF/H₂O); m.p.: 238–240°C; IR (cm⁻¹, KBr): 3385, 3447 (NH₂). ¹H NMR (δ, ppm): 2.45 (s, 3H, SCH₃), 3.82 (s, 3H, OCH₃), 6.62 (s, 2H, NH₂); 6.74–8.12 (m, 8H, Ar-H). ¹³C NMR (δ, ppm): 56.1 (OCH₃), 108.7, 110.4, 114.4, 122.3, 125.5, 127.1, 128.3, 129.2, 139.6, 142.2, 144.8, 157.8, 158.3, 167.3, 167.8 (Ar-C). MS: m/z 380 (M⁺, 100), 364 (70), 333 (82), 317 (54), 286 (22), 234 (30), 132 (26), 83 (16), 75 (24). Anal.% Calcd for C₁₉H₁₆N₄OS₂ (380.49): C, 59.98; H, 4.24; N, 14.73. Found: C, 59.91; H, 4.20; N, 14.84.

( 5) 4-Amino-2-(ethylthio)-7-(4-methoxyphenyl)-5-(2-thienyl)pyrido[2,3-d]pyrimidine (37). Yield: (2.1 g, 61%, DMF/H₂O); m.p.: 238–240°C; IR (cm⁻¹, KBr): 3378, 3425 (NH₂). ¹H NMR (δ, ppm): 1.28 (t, J = 6 Hz, 3H, CH₃); 2.85 (q, J = 6 Hz, 2H, SCH₂), 3.81 (s, 3H, OCH₃), 6.60 (s, 2H, NH₂); 6.75–8.04 (m, 8H, Ar-H). ¹³C NMR (δ, ppm): 15.7 (CH₃), 28.0 (SCH₂), 56.0 (OCH₃), 108.4, 110.1, 114.5, 122.2, 125.5, 127.0, 128.2, 129.1, 139.9, 142.5, 144.7, 157.7, 158.2, 167.0, 167.6 (Ar-C). Anal.% Calcd for C₂₀H₁₈N₄OS₂ (394.51): C, 60.89; H, 4.60; N, 14.20. Found: C, 60.79; H, 4.57; N, 14.34.

2.2. Biological Activity

2.2.1. In Vitro MTT Cytotoxicity Assay

The synthesized compounds were investigated for their in vitro cytotoxic effect via the standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method [25, 26] against a panel of three human tumor cell lines, namely, Caucasian breast adenocarcinoma MCF7, hepatocellular carcinoma HepG2, and colon carcinoma HT29 and a normal nontransformed human foreskin fibroblast Hs27 cell line. The procedures were done in a sterile area using a laminar flow cabinet biosafety class II level (Baker, SG403INT, Stanford, ME, USA). Cells were batch-cultured for 10 days and then seeded at concentration of 10 × 10³ cells/well in fresh complete growth medium in 96-well microtiter plastic plates at 37°C for 24 h under 5% CO₂ using a water jacketed carbon dioxide incubator (Sheldon, TC2323, Cornelius, OR, USA). Media was aspirated, fresh medium (without serum) was added, and cells were incubated either alone (negative control) or with different concentrations of the test compounds to give a final concentration of 100-50-25-12.5-6.25-3.125-1.56-0.78 μg/mL. DMSO was employed as a vehicle for dissolution of the tested compounds and its final concentration on the cells was less than 0.2%. Cells were suspended in RPMI 1640 medium (for HepG2 and HT29 cell lines) and DMEM (for MCF 7 cell line), 1% antibiotic-antimycotic mixture (10,000 IU/mL Penicillin Potassium, 10,000 μg/mL Streptomycin Sulphate, and 25 μg/mL Amphotericin B), and 1% L-Glutamine in 96-well flat bottom microplate at 37°C under 5% CO₂. After 24 h of incubation, the medium was aspirated and 40 μL of MTT salt (2.5 μg/mL) was added to each well and incubated for further 4 h at 37°C under 5% CO₂. To stop the reaction and dissolve the formed crystals, 200 μL of 10% sodium dodecyl sulphate (SDS) in deionized water was added to each well and incubated overnight at 37°C. The absorbance was then measured using a microplate multiwell reader (Bio-Rad Laboratories Inc., model 3350, Hercules, California, USA) at 595 nm and a reference wavelength of 620 nm. A statistical significance was tested between samples and negative control (cells with vehicle) using independent t-test by SPSS 11 program. The results are presented in Table 1 as LC₅₀ (μM) which is the lethal concentration of the compound which causes death of 50% of the cells in 24 h.

Table 1: Cytotoxic effects LC₅₀ (μM) of the active compounds on three human tumor and nontransformed cell lines and using the MTT assay.

Compound number
Human tumor cell lines
Nontransformed normal cell line

Colon carcinoma (HT29)
Hepatocellular carcinoma (HePG2)
Breast cancer (MCF 7)
Foreskin fibroblast (Hs27)

21
70.5
—
—
NT

22
46.7
98.9
97.3
NT

23
62.2
—
—
NT

30
113.6
107
—
NT

32
153.4
111.3
123.0
NT

33
25.2
64.6
6.4
>200

34
28.8
70.1
7.9
>200

36
40.4
97.7
22.8
>200

37
26.9
71.2
8.91
>200

D oxo rubicin
40.0
3.0
4.0
NT

LC₅₀: lethal concentration of the compound which causes death of 50% of cells in 24 h; totally inactive against this cell line; NT: not tested; D oxo rubicin: positive control cytotoxic agent.

2.2.2. In Vitro Antibacterial and Antifungal Activities

All of the newly synthesized compounds were evaluated for their in vitro antibacterial activity against Staphylococcus aureus (ATCC 6538), Bacillus subtilis (ATCC 6633), and Micrococcus luteus (ATCC 21881) as examples of Gram-positive bacteria and Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), and Klebsiella pneumonia (clinical isolate) as examples of Gram-negative bacteria. They were also evaluated for their in vitro antifungal potential against Candida albicans (ATCC 10231) and Aspergillus niger (recultured) fungal strains. Each 100 mL of sterile molten agar (at 45°C) received 1 mL of 6 h-broth culture and then the seeded agar was poured into sterile Petri dishes. Cups (8 mm in diameter) were cut in the agar. Each cup received 0.1 mL of the 1 mg/mL solution of the test compounds. The plates were then incubated at 37°C for 24 h or, in case of C. albicans, for 48 h. A control using DMSO without the test compound was included for each organism. Ampicillin trihydrate and Clotrimazole were used as reference drugs. The results were recorded for each tested compound as the average diameter of inhibition zones (IZ) of bacterial or fungal growth around the discs in mm. The minimal inhibitory concentrations (MIC) of the most active compounds were measured using the twofold serial broth dilution method [27]. The test organisms were grown in their suitable broth: 24 h for bacteria and 48 h for fungi at 37°C. Twofold serial dilutions of solutions of the test compounds were prepared [28] using 200, 100, 50, 25, 12.5, and 6.25 μg/mL. The tubes were then inoculated with the test organisms; each 5 mL received 0.1 mL of the above inoculum and was incubated at 37°C for 48 h. Then, the tubes were observed for the presence or absence of microbial growth.

3. Results and Discussion

3.1. Chemistry

The pathways adopted for the preparation of the intermediate and target compounds are illustrated in Scheme 1. Preparation of the key intermediates 2-amino-4,6-disubstituted nicotinonitriles 1–5 was performed according to Hantzsch-type synthesis via multicomponent one-pot cyclocondensation of the appropriate aromatic aldehyde (piperonal or thiophene-2-carbaldehyde) with the substituted acetophenone, malononitrile, and ammonium acetate as a nitrogen source. The reaction passes through a two-step procedure that involved the formation of chalcones via Claisen-Schmidt condensation, followed by cyclocondensation with malononitrile and ammonium acetate. When compounds 1–5 were heated with either formic acid or acetic anhydride, the targeted 5,7-disubstituted pyrido[2,3-d]pyrimidine-4(3H)-ones 6–10 and their 2-methyl analogs 11–15, respectively, were successfully obtained. The IR spectra of the latter compounds showed the absence of the CN group absorption and the appearance of new sharp absorption bands at 1656–1648 cm⁻¹ attributed to the newly formed C=O groups at position 4, beside the NH absorption bands at 3320–3259 cm⁻¹. Meanwhile, the ¹H-NMR spectra of compounds 11–15 showed new singlets at δ 2.28–2.36 ppm due to the newly introduced CH₃ group, whereas their ¹³C NMR spectral data exhibited new singlets at δ 20.2–21.1 ppm due to the new CH₃ group and the CO signals at δ 168.8–173.2 ppm.

Scheme 1: Synthesis of the target compounds 1–37. Reagents and reaction conditions: i: malononitrile, NH₄OAc, EtOH, reflux, 4-5 h; ii: HCOOH, heating, 30 min.; iii: Ac₂O, heating, 10 min.; iv: PhNCS, pyridine, reflux, 4 h; v: PhCONCS, acetone, reflux, 10 h; vi: urea, 260–300°C, 1 h; vii: thiourea, 260–300°C, 1.5 h; viii: R₂X, 1N NaOH, ethanol, r.t., 2-3 h.

Reacting compounds 1–5 with the phenyl isothiocyanate in pyridine medium led to the formation of the substituted dihydropyrido[2,3-d]pyrimidine-2(1H)-thiones 16–20. Their IR spectra showed new bands at 1642–1635 cm⁻¹ and at 1204–1188 cm⁻¹ corresponding to the C=N moiety and to the C=S group, respectively. Their ¹³C NMR spectral data were characterized by the presence of two signals at δ 179.3–181.2 and δ 164.4–164.8 ppm for the C=S and C=NH groups, respectively. However, condensing the starting compounds 2–4 with benzoyl isothiocyanate in acetone afforded the corresponding thioureido derivatives 21–23. Their IR spectra revealed the CN absorption bands at 2226–2215 cm⁻¹, in addition to the C=O and C=S bands at 1665–1661 and 1174–1166 cm⁻¹, respectively, whereas their ¹³C NMR spectra exhibited two characteristic signals at δ 168.1–169.3 and 179.9–180.4 ppm for the CO and CS carbons, respectively.

Furthermore, direct condensation of the starting compounds 1–4 with urea at 260–300°C was attempted to produce the target 4-amino-5,7-disubstituted-pyrido[2,3-d]pyrimidine-2(1H)-ones 24–27. The newly introduced C=O function was obvious both in the IR at 1667–1660 cm⁻¹ (beside the lack of CN absorption) and in the ¹³C NMR spectra at δ 166.1–166.4 ppm. Analogously, when compounds 1–5 were directly fused with thiourea at 260–300°C, the corresponding 4-amino-5,7-disubstituted-pyrido[2,3-d]pyrimidine-2(1H)-thiones 28–32 were obtained. Their ¹³C NMR spectra clearly revealed the signals attributed to the C=S carbons at δ 181.4–183.3 ppm. Finally, the synthesis of the targeted alkylthio derivatives 33–37 was successfully achieved via thioalkylation of the 2-thione derivatives 30–32 with either methyl or ethyl iodide in 1N NaOH medium. The ¹H-NMR spectra of 33, 35, and 36 showed the methylthio singlets at δ 2.42–2.49 ppm, whereas the ethylthio derivatives 34 and 37 exhibited triplets at δ 1.24–1.28 ppm and quartets at δ 2.85–2.88 ppm due to the ethyl CH₃ and CH₂ protons, respectively.

3.2. Biological Evaluation

3.2.1. In Vitro MTT Cytotoxicity Assay

All the newly synthesized compounds were evaluated for their in vitro cytotoxic effect via the standard MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] method [25, 26] against a panel of three human tumor cell lines, namely, colon carcinoma HT29, hepatocellular carcinoma HePG2, and Caucasian breast adenocarcinoma MCF7. The results are presented in Table 1 as LC₅₀ (μM) which is the lethal concentration of the compound that causes death of 50% of the cells in 24 h.

The obtained data revealed that the three tested human tumor cell lines exhibited variable degree of sensitivity profiles towards nine of the tested compounds, namely, 21, 22, 23, 30, 32, 33, 34, 36, and 37, whereas the other twenty-six compounds were either marginally active or even totally inactive. Regarding the activity against the human colon carcinoma HT29, this cell line proved to be very sensitive to all the nine active compounds. In particular, it revealed distinctive sensitivity towards compounds 33, 34, and 37 (LC₅₀ 25.2, 28.8, and 26.9 μM, resp.) even higher than doxorubicin (LC₅₀ 40.0 μM), the reference standard cytotoxic agent utilized in this assay. Meanwhile, compounds 22 and 36 (LC₅₀ 46.7 and 40.4 μM, resp.) were nearly equipotent to doxorubicin (LC₅₀ 40.0 μM), whereas compounds 21 and 23 (LC₅₀ 70.5 and 62.2 μM, resp.) showed moderate cytotoxic potential against the same cell line. Shifting to the hepatocellular carcinoma HepG2, this cell line showed mild to weak sensitivity towards seven of the tested analogs with LC₅₀ range 64.6–111.3 μM, when compared to doxorubicin (LC₅₀ 3.0 μM). Among these, the highest activity was displayed by compounds 33, 34, and 37 (LC₅₀ 64.4, 70.1, and 71.2 μM, resp.). On the other hand, the human breast cancer MCF 7 emerged as the least sensitive among the cell lines tested as its growth was affected by the presence of only six test compounds. However, a remarkable growth inhibition potential was shown by analogs 33, 34, and 37 as evidenced from their LC₅₀ values (LC₅₀ 6.4, 7.9, and 8.91 μM, resp.), which represents about 40–60% of the activity of doxorubicin (LC₅₀ 4.0 μM).

Further interpretation of the results revealed that compounds 33, 34, 36, and 37 showed considerable broad spectrum cytotoxic activity against the three tested human tumor cell lines. In particular, compounds 33, 34, and 37 proved to be the most active members in this study with special effectiveness against both the colon carcinoma HT29 (almost twice as active as doxorubicin; LC₅₀ 25.2, 28.8, and 26.9 versus 40 μM, resp.) and human breast cancer MCF 7 (about 40–60% of the activity of doxorubicin; LC₅₀ 6.4, 7.9, and 8.91 versus 4.0 μM, resp.).

A close examination of the structures of the active compounds showed that the nature of substituents (X and/or R), together with ring entity (mono- or bicyclic), seemed to influence the cytotoxic activity. In this context, compounds substituted with the 4-bromo- or 4-methoxyphenyl moities ( = Br and OCH₃) together with the benzo[d][1,3]dioxol-5-yl counterpart (22, 30, 33, and 34) were in favour of better cytotoxic activity, when compared with their 2-thienyl congeners (23, 32, 36, and 37), as revealed from their LC₅₀ values in Table 1. Moreover, the bicyclic pyrido[2,3-d]pyrimidines proved to be more active than the monocyclic nicotinonitriles. In this view, although the starting nicotinonitriles 1–5 lacked cytotoxic efficacy, yet the thiocarbamoyl benzamide derivatives 21–23 showed overall mild to moderate activity, among which analog 22 (X = Br; R = 3,4-(OCH₂O)C₆H₃) was relatively the most active regarding both potency and spectrum. Cyclization of the nicotinonitriles 1–5 with different reagents yielded variably substituted bicyclic pyrido[2,3-d]pyrimidines 6–10, 11–15, 16–20, and 24–27, which were all inactive against the three tested cell lines. However, isosteric replacement of 2-one functionality in pyrido[2,3-d]pyrimidine-2(1H)-ones 24–27, with a 2-thione group, yielded two weakly active analogs, namely, 30 ( = Br; = 3,4-(OCH₂O)C₆H₃) and 32 ( = OCH₃; = 2-thienyl). On the other hand, a remarkable improvement in both cytotoxic spectrum and potency was observed by thioalkylation of the 2-thione function of compounds 28–32 either with a methyl or ethyl group, where four potentially active compounds 33, 34, 36, and 37 were obtained. Among these, the analogs compounds 33 ( = Br; = 3,4-(OCH₂O)C₆H₃; = CH₃), 34 ( = Br; = 3,4-(OCH₂O)C₆H₃; R₂ = C₂H₅), and 37 ( = OCH₃; = 2-thienyl; = C₂H₅) stemmed as the most active members in this study.

3.2.2. In Vitro Differential Cytotoxicity Effect

Owing to the known common side effects often induced by chemotherapeutic agents exemplified by the possible destruction of normal cells, it was thought worthwhile to evaluate the in vitro differential cytotoxic effect of the most active analogs 33, 34, 36, and 37 on the nontransformed human foreskin fibroblast Hs27 cell line, under the same previously mentioned MTT assay experimental conditions.

Considering the obvious selective inhibitory potential displayed by the analogs (33, 34, 36, and 37) on the growth of the human breast MCF7 and colon HT29 cancer cell lines, the results revealed that the potential cytotoxic activity exhibited by 33, 34, and 37 (LC₅₀ values range 6.4–28.8 μM) was confirmed by their clear differential cytotoxic behavior, showing a marginal inhibitory effect on the growth of the nontransformed human foreskin fibroblast Hs27 cell line (LC₅₀ values > 200 μM), whereas a lower differential cytotoxic profile was shown by analog 36 against the tested normal cell line (LC₅₀ > 200 μM), when compared with its LC₅₀ values against the human breast MCF7 and colon HT29 cancer cell lines (22.8 and 40.4 μM, resp.) (Table 1).

3.2.3. In Vitro Antibacterial and Antifungal Activities

All compounds were evaluated for their in vitro antibacterial activity against Staphylococcus aureus (ATCC 6538), Bacillus subtilis (ATCC 6633), and Micrococcus luteus (ATCC 21881) as examples of Gram positive bacteria and Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), and Klebsiella pneumonia (clinical isolate) as examples of Gram-negative bacteria. They were also evaluated for their in vitro antifungal potential against Candida albicans (ATCC 10231) and Aspergillus niger (recultured) fungal strains. Agar-diffusion method was used for determination of preliminary antibacterial and antifungal activity. Ampicillin trihydrate (antibiotic) and Clotrimazole (antifungal) were used as reference drugs [27]. Dimethylsulfoxide (DMSO) was used as a blank and showed no antimicrobial activity. The results were recorded for each tested compound as the average diameter of inhibition zones (IZ) of bacterial or fungal growth around the discs in mm. The minimum inhibitory concentration (MIC, μg/mL) was determined for compounds that showed significant growth inhibition zones (≥13 mm) using the twofold serial dilution method [29].

As revealed from MIC data recorded in Table 2, nineteen of the newly synthesized compounds displayed variable inhibitory effects on the growth of the tested Gram-positive and Gram-negative microorganisms with pronounced activity against S. aureus and E. coli bacterial strains. In addition, some members exhibited moderate antifungal activity against C. albicans, whereas all the tested compounds lacked antifungal activity against Aspergillus niger.

Table 2: Minimal inhibitory concentrations (MIC; µg/mL) of the tested compounds.

Compound number
S. aureus
B. subtilis
M. luteus
E. coli
P. aeruginosa
K. pneumonia
C. albicans

3
50
50
—
25
100
—
—

4
50
100
—
50
—
—
—

5
50
100
200
100
200
—
—

13
100
100
100
50
100
—
—

15
100
200
—
100
100
200
—

18
25
100
50
25
50
100
100

19
100
100
—
50
50
—
—

20
25
100
—
25
50
200
100

21
100
100
50
100
200
50
—

22
50
100
100
50
100
50
—

23
50
100
200
100
50
100
—

29
100
100
50
50
100
200
100

30
12.5
50
50
6.25
25
100
50

31
50
100
—
12.5
200
100
50

33
6.25
25
25
6.25
12.5
50
12.5

34
12.5
25
100
6.25
25
100
25

35
25
100
200
25
50
—
100

36
12.5
50
100
12.5
25
—
25

37
25
50
50
12.5
100
—
50

Ampicillin
6.25
12.5
12.5
6.25
12.5
12.5
—

Clotrimazole
—
—
—
—
—
—
6.25

Inactive: MIC > 200 µg/mL.

Among the tested Gram-positive bacterial strains, two organisms, namely, S. aureus and B. subtilis, showed relatively high sensitivity towards the tested compounds. Concerning the activity against Gram-positive bacteria, compound 33 (MIC 6.25 μg/mL) was equipotent to ampicillin against S. aureus; meanwhile analogs 30, 34, and 36 (MIC 12.5 μg/mL) and 18, 20, 35, and 37 (MIC 25 μg/mL) showed 50% and 25% of the activity of ampicillin, respectively. With regard to the activity against B. subtilis, compounds 33 and 34 (MIC 25 μg/mL) exhibited half the activity of ampicillin (MIC 12.5 μg/mL). M. luteus was the least sensitive Gram-positive microorganism towards the majority of the tested compounds, except analog 33 which showed 50% of the activity of ampicillin (MIC 25 versus 12.5 μg/mL). On the other hand, investigation of anti-Gram-negative efficacy of the active compounds revealed that three analogs 30, 33, and 34 were able to produce a distinctive growth inhibitory profile against E. coli being equipotent to ampicillin (MIC 6.25 μg/mL), whereas compounds 31, 36, and 37 (MIC 12.5 μg/mL) were 50% less active than ampicillin against the same organism. Moreover, the tested P. aeruginosa strain showed high sensitivity towards compound 33 (MIC 12.5 μg/mL) which was equiactive with ampicillin, whereas the rest of the active compounds revealed moderate to weak activity against the same strain (MIC range 25–200 μg/mL). Shifting to the antifungal potential, ten compounds 18, 20, 29–31, and 33–37 were able to display appreciable growth inhibitory activity against C. albicans (MIC values 12.5–100 μg/mL, resp.) when compared with Clotrimazole (MIC 6.25 μg/mL), the standard antifungal agent utilized in this assay. Among these, analog 33 exhibited the best antifungal activity (MIC 12.5 μg/mL) that represents 50% of the standard. It is to be noted that all the tested compounds lacked any antifungal activity against Aspergillus niger.

Structurally, the nineteen active compounds represent two variations: the substituted nicotinonitriles and the bicyclic pyrido[2,3-d]pyrimidines. In general, relatively better antibacterial potential and spectrum were mostly confined to the derivatives comprising the bromine ( = Br) and the benzo[d][1,3]dioxol-5-yl groups, when compared with their methoxylated (X = OCH₃) and 2-thienyl congeners. Besides, better antimicrobial activity was associated with the bicyclic pyrido[2,3-d]pyrimidines when compared with the monocyclic nicotinonitrile precursors (Table 2). In this context, while the key nicotinonitrile precursors 3–5 showed mild antimicrobial activity, yet condensation of the 2-amino group with benzoyl isothiocyanate afforded three thiocarbamoyl benzamide derivatives 21–23 which revealed a slight improvement in the antimicrobial spectrum although not in efficacy. Annelation of nicotinonitriles 1–5 into the pyrido[2,3-d]pyrimidine-4(3H)-ones 6–10 resulted in complete abolishment of activity, whereas introducing a methyl group at C₂ to the latter ring system furnished two 2-methyl-pyrido[2,3-d]pyrimidine-4(3H)-one derivatives 13 and 15 which were noticeably less active than their parent compounds 3 and 5. Furthermore, reaction of starting nicotinonitriles 1–5 with phenyl isothiocyanate gave rise to three active compounds 18–20, among which analog 18 ( =Br; = 3,4-(OCH₂O)C₆H₃) showed relatively better antimicrobial spectrum and potential. Although cyclization of nicotinonitriles 1–5 with urea into the corresponding pyrido[2,3-d]pyrimidine-2(1H)-ones 24–27 led to total loss of activity, yet isosteric replacement of the 2-one with a 2-thione functionality yielded three significantly active broad spectrum compounds 29–31, among which analog 30 ( = Br; = 3,4-(OCH₂O)C₆H₃) was the most active being equipotent to ampicillin against E. coli, together with a mild antifungal activity. Finally, thioalkylation of the 2-thione function of 28–32 afforded five methyl- or ethylthio derivatives 33–37 with a great enhancement in both antimicrobial spectrum and potential. In particular, compound 33 ( = Br; = 3,4-(OCH₂O)C₆H₃; R₂ = CH₃) proved to be equipotent to ampicillin against S. aureus, E. coli, and P. aeruginosa, together with a noticeable antifungal activity against C. albicans, whereas analog 34 ( = Br; = 3,4-(OCH₂O)C₆H₃; R₂ = C₂H₅) was equiactive with ampicillin against E. coli.

4. Conclusions

Thirty-seven polysubstituted pyridines and some derived bicyclic pyrido[2,3-d]pyrimidine ring systems supported with various chemotherapeutically active functionalities were successfully synthesized, characterized, and evaluated for their biological activity as cytotoxic and/or antimicrobial agents. Globally, the data obtained from both the cytotoxic and antimicrobial assays suggested that higher biological activities were confined mainly to compounds comprising the 4-bromo, 4-methoxy, benzo[d][1,3]dioxol-5-yl substituents, and the bicyclic pyrido[2,3-d]pyrimidines rather than the monocyclic nicotinonitriles.

Regarding the cytotoxic activity, nine compounds were able to show variable cytotoxic efficiency, among which alkylthio analogs 33, 34, and 37 proved to be the most active members with considerable broad spectrum cytotoxic activity against the three tested human tumor cell lines and particular effectiveness against both the colon carcinoma HT29 (almost twice as active as doxorubicin; LC₅₀ 25.2, 28.8, and 26.9 versus 40 μM, resp.) and human breast cancer MCF 7 (about 40–60% of the activity of doxorubicin; LC₅₀ 6.4, 7.9, and 8.91 versus 4.0 μM, resp.). In addition, the same three analogs showed a clear differential cytotoxic profile as they exhibited a marginal inhibitory effect on the growth of the normal nontransformed human foreskin fibroblast Hs27 cell line (LC₅₀ values > 200 μM).

On the other hand, the results of the in vitro antimicrobial screening revealed that nineteen compounds were able to exhibit significant antibacterial potential against both Gram-positive and Gram-negative bacteria, together with mild to moderate antifungal activities. The pyrido[2,3-d]pyrimidine-2(1H)-thione 30 in connection with its alkylthio derivatives 33 and 34 stemmed as the most active antimicrobial members being equipotent to ampicillin against S. aureus, E. coli, and P. aeruginosa, together with a noticeable antifungal activity against C. albicans. Globally, the distinctive cytotoxic and antimicrobial behavior displayed by alkylthio derivatives 33 and 34 are concordant with several literature findings that emphasized the role of thioethers in enhancing the antimicrobial and antitumor activities [28, 30, 31]. This makes such type of compounds a fruitful matrix for further development of more potent and selective anticancer and/or antimicrobial agents. In particular, compounds 33 and 34 could be considered as possible dual antimicrobial-anticancer candidates that deserve further investigation and derivatization in order to explore the scope and limitation of their biological activities.

Competing Interests

The authors declare that there is no conflict of interests regarding the publication of this paper.

Acknowledgments

This project was funded by Saudi Basic Industries Corporation (SABIC) and the Deanship of Scientific Research (DSR), King Abdulaziz University, Jeddah, under Grant no. S-1436-130-3. The authors therefore acknowledge with thanks SABIC and DSR for technical and financial support. Extendable thanks are due to the staff members of the Bioassay-Cell Culture Laboratory, National Research Centre (NRC), Cairo, Egypt, for their efforts in performing the MTT cytotoxicity assay.

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