The Uses of 2-Amino-4-Phenylthiazole in the Synthesis of Coumarin, Pyran, Pyridine and Thiazole Derivatives with Antitumor Activities

The thiazole derivative 3 was used for a series of heterocyclization reaction to produce pyran, pyridine and thiazole derivatives. The cytotoxicity of the newly synthesized compounds was studied against the six cancer cell lin es namely NUGC, HR, DLD1, HA22T, HEPG2, MCF, HONE1 and normal fibroblast cells (WI38). The results showed that most of the synthesized compounds were of high potency. Among the tested compounds, 2-Amino-4-(4-chlorophenyl)-6-(4-phenylthiazol-2-yl)-4H-pyran-3,5-dicarbonitrile 17b showed the highest potency among the tested compounds.

• Thiazole
• Pyran
• Pyridine
• Antitumor

1. Introduction

Thiazole is a core structural motif present in a variety of natural products, such as vitamin B1 (thiamine) and penicillin. Thiazole derivatives also exhibit a broad spectrum of medicinal and biological properties, such as antibacterial, antifungal [1] , anti-inflammatory [2] , antiviral [3] , antimalarial [4] and anti-HIV activities [5] . Thiazole analogs have also been reported as ligands at estrogen receptors [6] , neuropeptide Y5 [7] , adenosine receptors [8] , and act as inhibitors of human platelet aggregation factor [9] , urokinase [10] and poly (ADP-Ribose) polymerase-1 [11] . Selenazoles have been reported to possess antibacterial [12] , and superoxide anion scavenging activity [13] , and exhibit cytotoxicity and DNA fragmentation effects in human HT-1080 fibrosarcoma cells [14] . The structures of sulfathiazole, meloxicam, and selenazofurin and their pharmacological activities are given in Figure 1.

2. Results and Discussion

The reaction of ɷ-bromoacetophenone (1) with thiourea (2) in ethanol gave the thiazole derivative (3) [15] .

The latter compound underwent acetylation when reacted with acetic anhydride to give the N-acetyl derivative 5. The structure of compound 5 was confirmed on the basis of analytical and spectral data. The reaction of compound 3 with phenylisothiocyanate gave the N-phenylthiourea derivative 7. On the other hand, the reaction of compound 3 with ethyl cyanoacetate in dimethylformamide gave N-cyanoacetamide derivative 9. The reaction of compound 9 with any of the aromatic aldehydes namely benzaldehyde (10a), 4-chlorobenzaldehyde (10b) or 4-methoxybenzaldehyde (10c) gave benzylidene derivatives 11a-c, respectively. In addition, the reaction of compound 9 with salicylaldehyde (12) gave the coumarin derivative 13 (Figure 2).

The structure of compound 13 was established on the basis of analytical and spectral data. Thus, the ¹H NMR spectrum showed δ = 6.13 (s, 1H, thiazole H-5), 6.29 (s, 1H, coumarin H-4), 7.21 - 7.43 (m, 9H, C₆H₅, C₆H₄), 8.30 (s, 1H, D₂O exchangeable, NH). The reaction of compound 9 with any of the benzenediazonium chloride derivatives 14a-c gave aryl hydrazone derivatives 15a-c, respectively. Moreover, the multi-component reaction of compound 9 with any of the aromatic aldehydes 10a, 10b or 10c and malononitrile (16) gave the pyran derivatives 17a-c, respectively (Figure 3).

The analytical and spectral data of 17a-c were the basis of their structural identification. Thus, the ¹H NMR spectrum of compound 17a (as an example) showed δ = 4.82 (s, 2H, D₂O exchangeable, NH₂), 6.14 (s, 1H, thiazole H-5), 6.28 (s, 1H, D₂O exchangeable, NH), 6.49 (s, 1H, pyran H-4), 7.28 - 7.42 (m, 10H, 2C₆H₅). Similarly, the multi-component reaction of compound 9 with any of the aromatic aldehydes 10a, 10b or 10c and ethyl cyanoacetate (8) gave the pyran derivatives 18a-c, respectively (Figure 4).

The analytical and spectral data of 18a-c were the basis of their structural identification. In addition, the multi-component reaction of compound 9 with any of the aromatic aldehydes 10a, 10b or 10c and thiourea (2) gave the pyrimidine derivatives 19a-c, respectively. The analytical and spectral data of 19a-c

Figure 1

Biologically active thiazole and selenazole derivatives

Figure 2

Compounds 3, 5, 7, 9,11a-c, 13

were the basis of their structural identification. Thus, the ¹H NMR spectra of compound 19a (as an example) showed δ = 6.18 (s, 1H, thiazole H-5), 6.28 (s, 1H, D₂O exchangeable, NH), 7.29 - 7.36 (m, 10H, 2C₆H₅), 8.24 (s, 1H, D₂O exchangeable, NH). Compound 9 was capable for thiazole synthesis, thus the reaction of compound 9 with elemental sulfur and phenylisothiocyanate (6) gave the thiazole derivative 20.

3. Experiment

General

All melting points were determined on an electrothermal digital melting point apparatus and are uncorrected. IR spectra (KBr discs) were recorded on a FTIR plus 460 or PyeUnicam SP-1000 spectrophotometer. ¹H NMR spectra were recorded with Varian Gemini-200 (200 MHz) and Jeol AS 500 MHz instruments spectra were performed in DMSO-d6 as solvent using TMS as internal standard-

Figure 3

Compounds 15a-c, 17a-c

and chemical shifts are expressed as δ ppm. MS (EI) spectra were recorded with Hewlett Packard 5988 A GC/MS system and GCMS-QP 1000 Ex Shimadzu instruments. Analytical data were obtained from the Micro-analytical Data Unit at Cairo University and were performed on Vario EL III Elemental analyzer. Compound 3 was synthesized according to method reported in literature [15] . All synthesized compounds are filtered using Whatman filter paper 42 Ashless.

1) N-(4-phenylthiazol-2-yl)acetamide (5)

To a solution of compound 3 (1.76 g, 0.01 mol) in acetic acid (40 mL) acetic anhydride (10 mL) was added. The reaction mixture was heated under reflux (118˚C) for 2 h then poured onto ice/water and left to room temperature for 4 h. The formed solid product was collected by filtration.

Yellow crystals from ethanol, yield 70% (1.52 g), m.p. 206˚C - 208˚C. Anal. Calculated for C₁₁H₁₀N₂OS (218.27): C, 60.53; H, 4.62; N, 12.83; S, 14.69. Found: C, 60.74; H, 4.59; N, 12.93; S, 14.80. MS: m/e 218 (M⁺, 28%). IR, υ: 3492 - 3330 (NH), 3056 (CH, aromatic), 2970 (CH₃), 1688 (CO), 1638 (C=C). ¹H NMR (DMSO-d₆, 200 MHz): δ = 2.80 (s, 3H, CH₃), 6.12 (s, 1H, thiazole H-5), 7.26 -

Figure 4

Compounds 18a-c, 19a-c, 20

7.39 (m, 5H, C₆H₅), 8.30 (s, 1H, D₂O exchangeable, NH).

2) 1-phenyl-3-(4-phenylthiazol-2-yl)thiourea (7)

To a solution of compound 3 (1.76 g, 0.01 mol) in 1,4-dioxane (20 mL) phenylisothiocyanate (1.35 g, 0.01 mol) was added. The reaction mixture was heated under reflux (101˚C) for 3 h then poured onto ice/water and the formed solid product was collected by filtration.

Orange crystals from ethanol, yield 78% (2.42 g), m.p. 130˚C - 132˚C. Anal. Calculated for C₁₆H₁₃N₂S₂ (311.42): C, 61.71; H, 4.21; N, 13.49; S, 20.59. Found: C, 61.95; H, 4.31; N, 14.22; S, 20.72. MS: m/e 311 (M⁺, 22%). IR, υ: 3468 - 3324 (2NH), 3054 (CH, aromatic), 1638 (C=C). ¹H NMR (DMSO-d₆, 200 MHz): δ = 6.14 (s, 1H, thiazole H-5), 7.23 - 7.42 (m, 10H, 2C₆H₅), 8.26, 8.32 (2s, 2H, D₂O exchangeable, 2NH).

3) 2-Cyano-N-(4-phenylthiazol-2-yl)acetamide (9)

To a solution of compound 3 (1.76 g, 0.01 mol) in dimethylformamide (20 mL) ethyl cyanoacetate (1.13 g, 0.01 mol) was added. The reaction mixture was heated under reflux (153˚C) for 2 h then poured onto ice/water and the formed solid product was collected by filtration.

Orange crystals from ethanol, yield 67% (1.63 g), m.p. 154˚C - 157˚C. Anal. Calculated for C₁₂H₉N₃OS (243.28): C, 59.24; H, 3.73; N, 17.27; S, 13.18. Found: C, 59.36; H, 4.01; N, 16.96; S, 13.47. MS: m/e 243 (M⁺, 36%). IR, υ: 3452-3328 (NH), 3057 (CH, aromatic), 2220 (CN), 1678 (CO), 1632 (C=C),. ¹H NMR (DMSO-d₆, 200 MHz): δ = 3.84 (s, 2H, CH₂), 6.13 (s, 1H, thiazole H-5), 7.28 - 7.39 (m, 5H, C₆H₅), 8.30 (s, 1H, D₂O exchangeable, NH).

4) General procedure for the synthesis of the benzylidine derivatives 11a-c

To a solution of compound 9 (2.43 g, 0.01 mol) in 1,4-dioxane (40 mL) containing piperidine (0.50 mL), any of benzaldehyde (1.08 g, 0.01 mol), 4-chloro- benzaldehyde (1.40 g, 0.01 mol) or 4-methoxybenzaldehyde (1.36 g, 0.01 mol) was added. The reaction mixture was heated under reflux (101˚C) for 3 h then poured onto ice/water containing few drops of hydrochloric acid. The formed solid product, formed in each case was collected by filtration.

5) 2-Cyano-3-phenyl-N-(4-phenylthiazol-2-yl)acrylamide (11a)

Pale brown crystals from ethanol, yield 70% (2.32 g), m.p. 139˚C - 141˚C. Anal. Calculated for C₁₉H₁₃N₃OS (331.39): C, 68.86; H, 3.95; N, 12.68; S, 9.68. Found: C, 68.47; H, 4.16; N, 12.51; S, 9.38. MS: m/e 331 (M⁺, 28%). IR, υ: 3462 - 3341 (NH), 3053 (CH, aromatic), 2220 (CN), 1682 (CO), 1635 (C=C). ¹H NMR (DMSO-d₆, 200 MHz): δ = 6.09 (s, 1H, CH), 6.14 (s, 1H, thiazole H-5), 7.25-7.37 (m, 10H, 2C₆H₅), 8.32 (s, 1H, D₂O exchangeable, NH).

6) 3-(4-chlorophenyl)-2-cyano-N-(4-phenylthiazol-2-yl)acrylamide (11b)

Pale brown crystals from ethanol, yield 66% (2.41 g), m.p. 188˚C - 191˚C. Anal. Calculated for C₁₉H₁₂ClN₃OS (365.84): C, 62.38; H, 3.31; N, 11.49; S, 8.76. Found: C, 62.19; H, 3.53; N, 11.60; S, 8.57. MS: m/e 365 (M⁺, 40%). IR, υ: 3472 - 3329 (NH), 3056 (CH, aromatic), 2222 (CN), 1680 (CO), 1632 (C=C). ¹H NMR (DMSO-d₆, 200 MHz): δ = 6.06 (s, 1H, CH), 6.12 (s, 1H, thiazole H-5), 7.23-7.42 (m, 9H, C₆H_5, C₆H₄), 8.34 (s, 1H, D₂O exchangeable, NH).

7) 2-cyano-3-(4-methoxyphenyl)-N-(4-phenylthiazol-2-yl)acrylamide (11c)

Yellow crystals from ethanol, yield 78% (2.83 g), m.p. 166-169˚C. Anal. Calculated for C₂₀H₁₅N₃O₂S (361.42): C, 66.46; H, 4.18; N, 11.63; S, 8.87. Found: C, 66.37; H, 3.86; N, 11.41; S, 8.72. MS: m/e 361 (M⁺, 22%). IR, υ: 3463 - 3342 (NH), 3053 (CH, aromatic), 2220 (CN), 1682 (CO), 1631 (C=C). ¹H NMR (DMSO-d₆, 200 MHz): δ = 3.68 (s, 3H, OCH₃), 6.08 (s, 1H, CH), 6.14 (s, 1H, thiazole H-5), 7.24-7.38 (m, 9H, C₆H_5, C₆H₄), 8.32 (s, 1H, D₂O exchangeable, NH).

8) 2-Oxo-N-(4-phenylthiazol-2-yl)-2H-chromene-3-carboxamide (13)

To a solution of compound 9 (2.43 g, 0.01 mol) in 1,4-dioxane (40 mL) containing piperidine (0.50 mL) salicylaldehyde (1.22 g, 0.01 mol) was added. The reaction mixture was heated under reflux (101˚C) for 2 h then poured onto ice/water containing few drops of hydrochloric acid. The formed solid product was collected by filtration.

Yellow crystals from ethanol, yield 62% (2.16 g), m.p. 122˚C - 124˚C. Anal. Calculated for C₁₉H₁₂N₂O₃S (348.38): C, 65.51; H, 3.47; N, 8.04; S, 9.20. Found: C, 65.44; H, 3.59; N, 7.94; S, 9.38. MS: m/e 348 (M⁺, 18%). IR, υ: 3439-3312 (NH), 3056 (CH, aromatic), 1690, 1682 (2CO), 1665 (C=N), 1628 (C=C). ¹H NMR (DMSO-d₆, 200 MHz): δ = 6.13 (s, 1H, thiazole H-5), 6.29 (s, 1H, coumarin H-4), 7.21 - 7.43 (m, 9H, C₆H_5, C₆H₄), 8.30 (s, 1H, D₂O exchangeable, NH).

9) General procedure for the synthesis of the aryl hydrazone derivatives 15a-c

To a cold solution (0˚C - 5˚C) of compound 9 (2.43 g, 0.01 mol) in ethanol (40 mL) containing sodium acetate (2.50 g) any of the diazonium salts namely benzenediazonium chloride (14a) (0.01 mol) 4-chlorobenzene diazonium chloride (14b) (0.01 mol) or 4-methylbenzenediazonium chloride (14c) (0.01 mol) [prepared by the addition of sodium nitrite (0.70 g, 0.01 mol) to a cold solution (0-5 ^˚C) of any of aniline (0.93 g, 0.01 mol), 4-chloroaniline (1.27 g, 0.01 mol) or 4-methylaniline (1.14 g, 0.01 mol) in concentrated hydrochloric acid (16 mL)] was added. The whole reaction mixture, in each case, was stirred at room temperature for 2 h and the formed solid product was collected by filtration.

10) 2-Oxo-N’-phenyl-2-((4-phenylthiazol-2-yl)amino)acetohydrazonoyl cyanide (15a)

Orange crystals from ethanol, yield 65% (2.24 g), m.p. 153˚C - 156˚C. Anal. Calculated for C₁₈H₁₃N₅OS (347.39): C, 62.23; H, 3.77; N, 20.16; S, 9.23. Found: C, 62.41; H, 3.54; N, 20.08; S, 9.52. MS: m/e 347 (M⁺, 36%). IR, υ: 3452-3316 (2NH), 3053 (CH, aromatic), 2220 (CN), 1686 (CO), 1660 (C=N), 1626 (C=C). ¹H NMR (DMSO-d₆, 200 MHz): δ = 6.12 (s, 1H, thiazole H-5), 7.24-7.38 (m, 10H, 2C₆H₅), 8.26, 8.30 (2s, 2H, D₂O exchangeable, 2NH).

11) N’-(4-Chlorophenyl)-2-oxo-2-((4-phenylthiazol-2-yl)amino)acetohydra- zonoyl cyanide (15b)

Orange crystals from ethanol, yield 74% (2.82 g), m.p. 177˚C - 179˚C. Anal. Calculated for C₁₈H₁₂ClN₅OS (381.84): C, 56.62; H, 3.17; N, 18.34; S, 8.40. Found: C, 56.82; H, 3.38; N, 18.51; S, 8.29. MS: m/e 381 (M⁺, 28%). IR, υ: 3458 - 3331 (2NH), 3056 (CH, aromatic), 2222 (CN), 1688 (CO), 1653 (C=N), 1628 (C=C). ¹H NMR (DMSO-d₆, 200 MHz): δ = 6.18 (s, 1H, thiazole H-5), 7.22-7.41 (m, 9H, C₆H₅, C₆H₄), 8.28, 8.31 (2s, 2H, D₂O exchangeable, 2NH).

12) 2-Oxo-2-((4-phenylthiazol-2-yl)amino)-N’-(p-tolyl)acetohydrazonoylcya- nide (15c)

Orange crystals from ethanol, yield 72% (2.59 g), m.p. 203˚C - 206˚C. Anal. Calculated for C₁₉H₁₅N₅OS (361.42): C, 63.14; H, 4.18; N, 19.38; S, 8.87. Found: C, 62.97; H, 3.92; N, 19.26; S, 8.65. MS: m/e 361 (M⁺, 38%). IR, υ: 3471 - 3369 (2NH), 3055 (CH, aromatic), 2220 (CN), 1687 (CO), 1656 (C=N), 1629 (C=C). ¹H NMR (DMSO-d₆, 200 MHz): δ = 2.69 (s, 3H, CH₃), 6.17 (s, 1H, thiazole H-5), 7.24 - 7.45 (m, 9H, C₆H₅, C₆H₄), 8.26, 8.33 (2s, 2H, D₂O exchangeable, 2NH).

13) General procedure for the synthesis of the pyran derivatives 17a-c

To a solution of compound 9 (2.43 g, 0.01 mol) in ethanol (30 mL) containing triethylamine (0.50 mL) malononitrile (0.66 g, 0.01 mol) and any of benzaldehyde (1.06 g, 0.01 mol), 4-chlorobenzaldehyde (1.40 g, 0.01 mol) or 4-methox- ybenzaldehyde (1.36 g, 0.01 mol) was added. The reaction mixture, in each case, was heated under reflux (78˚C) for 3 h then left to cool and the formed solid product was collected by filtration.

14) 2-Amino-4-phenyl-6-(4-phenylthiazol-2-yl)amino)-4H-pyran-3,5-dicar- bonitrile (17a)

Orange crystals from ethanol, yield 83% (3.29 g), m.p. 213˚C - 215˚C. Anal. Calculated for C₂₂H₁₅N₅OS (397.45): C, 66.48; H, 3.80; N, 17.62; S, 8.07. Found: C, 66.73; H, 3.92; N, 17.94; S, 8.19. MS: m/e 397 (M⁺, 26%). IR, υ: 3462 - 3338 (NH, NH₂), 3056 (CH, aromatic), 2221 (CN), 1662 (C=N), 1621 (C=C). ¹H NMR (DMSO-d₆, 200 MHz): δ = 4.82 (s, 2H, D₂O exchangeable, NH₂), 6.14 (s, 1H, thiazole H-5), 6.28 (s, 1H, D₂O exchangeable, NH), 6.49 (s, 1H, pyran H-4), 7.28-7.42 (m, 10H, 2C₆H₅).

15) 2-Amino-4-(4-chlorophenyl)-6-(4-phenylthiazol-2-yl)amino)-4H-pyran- 3,5-dicarbonitrile (17b)

Orange crystals from ethanol, yield 76% (3.27 g), m.p. 183˚C - 185˚C. Anal. Calculated for C₂₂H₁₄ClN₅OS (431.90): C, 61.18; H, 3.27; N, 16.22; S, 7.42. Found: C, 61.28; H, 3.37; N, 16.49; S, 7.80. MS: m/e 431 (M⁺, 18%). IR, υ: 3462 - 3338 (NH, NH₂), 3056 (CH, aromatic), 2222 (CN), 1659 (C=N), 1626 (C=C). ¹H NMR (DMSO-d₆, 200 MHz): δ = 4.80 (s, 2H, D₂O exchangeable, NH₂), 6.17 (s, 1H, thiazole H-5), 6.26 (s, 1H, D₂O exchangeable, NH), 6.49 (s, 1H, pyran H-4), 7.22 - 7.48 (m, 9H, C₆H₅, C₆H₄).

16) 2-Amino-6-(4-phenylthiazol-2-yl)amino)-4-(p-tolyl)-4H-pyran-3,5-dicar- bonitrile (17c)

Orange crystals from ethanol, yield 68% (2.90 g), m.p. 97˚C - 99˚C. Anal. Calculated for C₂₃H₁₇N₅O₂S (427.48): C, 64.62; H, 4.01; N, 16.38; S, 7.50. Found: C, 64.80; H, 4.26; N, 16.60; S, 7.44. MS: m/e 427 (M⁺, 33%). IR, υ: 3462 - 3338 (NH, NH₂), 3055 (CH, aromatic), 2220 (CN), 1655 (C=N), 1628 (C=C). ¹H NMR (DMSO-d₆, 200 MHz): δ = 3.77 (s, 3H, CH₃), 4.81 (s, 2H, D₂O exchangeable, NH₂), 6.19 (s, 1H, thiazole H-5), 6.28 (s, 1H, D2O exchangeable, NH), 6.46 (s, 1H, pyran H-4), 7.24 - 7.45 (m, 9H, C₆H₅, C₆H₄).

17) General procedure for the synthesis of the pyran derivatives 18a-c

To a solution of compound 9 (2.43 g, 0.01 mol) in ethanol (30 mL) containing triethylamine (0.50 mL) ethyl cyanoacetate (1.13 g, 0.01 mol) and any of benzaldehyde (1.06 g, 0.01 mol), 4-chlorobenzaldehyde (1.40 g, 0.01 mol) or 4-me- thoxybenzaldehyde (1.36 g, 0.01 mol) was added. The reaction mixture, in each case, was heated under reflux (78˚C) for 6 h then left to cool and the formed solid product was collected by filtration.

18) 2-Hydroxy-4-phenyl-6-(4-phenylthiazol-2-yl)amino)-4H-pyran-3,5-dicar- bonitrile (18a)

Orange crystals from ethanol, yield 70% (2.79 g), m.p. 228˚C - 230˚C. Anal. Calculated for C₂₂H₁₄N₄O₂S (398.44): C, 66.32; H, 3.54; N, 14.06; S, 8.05. Found: C, 66.88; H, 3.51; N, 14.14; S, 8.40. MS: m/e 398 (M⁺, 19%). IR, υ: 3560 - 3328 (NH, OH), 3056 (CH, aromatic), 2223 (CN), 1660 (C=N), 1626 (C=C). ¹H NMR (DMSO-d₆, 200 MHz): δ = 6.16 (s, 1H, thiazole H-5), 6.46 (s, 1H, pyran H-4), 6.49 (s, 1H, D₂O exchangeable, NH),7.29 - 7.36 (m, 10H, 2C₆H₅), 10.20 (s, 1H, D₂O exchangeable, OH).

19) 4-(4-Chlorophenyl)-2-hydroxy-6-(4-phenylthiazol-2-yl)amino)-4H-py- ran-3,5-dicarbonitrile (18b)

Yellow crystals from ethanol, yield 80% (3.46 g), m.p. 220˚C - 223˚C. Anal. Calculated for C₂₂H₁₃ClN₄O₂S (432.88): C, 61.04; H, 3.03; N, 12.94; S, 7.41. Found: C, 61.42; H, 3.02; N, 12.83; S, 7.83. MS: m/e 432 (M⁺, 20%). IR, υ: 3560 - 3328 (NH, OH), 3054 (CH, aromatic), 2220 (CN), 1659 (C=N), 1624 (C=C). ¹H NMR (DMSO-d₆, 200 MHz): δ = 6.13 (s, 1H, thiazole H-5), 6.47 (s, 1H, pyran H-4), 6.6 (s, 1H, D₂O exchangeable, NH), 7.23 - 7.47 (m, 9H, C₆H₅, C₆H₄), 10.22 (s, 1H, D₂O exchangeable, OH).

20) 2-Hydroxy-6-(4-phenylthiazol-2-yl)amino)-4-(p-tolyl)-4H-pyran-3,5-di- carbonitrile (18c)

Orange crystals from ethanol, yield 72% (3.08 g), m.p. 234˚C - 237˚C. Anal. Calculated for C₂₃H₁₆N₄O₃S (428.46): C, 64.47; H, 3.76; N, 13.08; S, 7.48. Found: C, 64.43; H, 3.91; N; 13.22; S, 7.86. MS: m/e 428 (M⁺, 20%). IR, υ: 3560 - 3328 (NH, OH), 3056 (CH, aromatic), 2223 (CN), 1652 (C=N), 1623 (C=C). ¹H NMR (DMSO-d₆, 200 MHz): δ = 3.79 (s, 3H, CH₃), 6.16 (s, 1H, thiazole H-5), 6.27 (s, 1H, D₂O exchangeable NH), 6.48 (s, 1H, pyran H-4), 7. 28 - 7.41 (m, 9H, C₆H₅, C₆H₄), 10.18 (s, 1H, D₂O exchan- geable, OH).

21) General procedure for the synthesis of the pyrimidine derivatives 19a-c

To a solution of compound 9 (2.43 g, 0.01 mol) in ethanol (30 mL) containing triethylamine (0.50 mL) thiourea (0.76 g, 0.01 mol) and any of benzaldehyde (1.06 g, 0.01 mol), 4-chlorobenzaldehyde (1.40 g, 0.01 mol) or 4-methoxybenzal- dehyde (1.36 g, 0.01 mol) was added. The reaction mixture, in each case, was heated under reflux (78˚C) for 6 h then left to cool and the formed solid product was collected by filtration.

22) 6-Phenyl-4-(4-phenylthiazol-2-yl)amino)-2-thioxo-1,2-dihydropyrimi- dine-5-carbonitrile (19a)

Orange crystals from ethanol, yield 66% (2.55 g), m.p. 162˚C - 165˚C. Anal. Calculated for C₂₀H₁₃N₅S₂ (387.48): C, 61.99; H, 3.38; N, 18.07; S, 16.55. Found: C, 62.32; H, 3.49; N; 18.33; S, 16.19. MS: m/e 387 (M⁺, 25%). IR, υ: 3480 - 3337 (2NH), 3054 (CH, aromatic), 2220 (CN), 1663 (C=N), 1629 (C=C), 1205 (C=S). ¹H NMR (DMSO-d₆, 200 MHz): δ = 6.18 (s, 1H, thiazole H-5), 6.28 (s, 1H, D₂O exchangeable, NH), 7.29 - 7.36 (m, 10H, 2C₆H₅), 8.24 (s, 1H, D₂O exchangeable, NH).

23) 6-(4-Chlorophenyl)-4-(4-phenylthiazol-2-yl)amino)-2-thioxo-1,2-dihy- dropyrimidine-5-carbonitrile (19b)

Yellow crystals from ethanol, yield 75% (3.15 g), m.p. 133˚C - 135˚C. Anal. Calculated for C₂₀H₁₂ClN₅S₂ (421.93): C, 56.93; H, 2.87; N, 16.60; S, 15.20. Found: C, 56.73; H, 2.99; N; 16.83; S, 15.69. MS: m/e 421 (M⁺, 28%). IR, υ: 3487 - 3346 (2NH), 3056 (CH, aromatic), 2223 (CN), 1656 (C=N), 1628 (C=C), 1221 (C=S). ¹H NMR (DMSO-d₆, 200 MHz): δ = 6.14 (s, 1H, thiazole H-5), 6.6 (s, 1H, D₂O exchangeable, NH), 7.21 - 7.49 (m, 9H, C₆H₅, C₆H₄), 8.25 (s, 1H, D₂O exchangeable, NH).

24) 6-(4-Methoxyphenyl)-4-(4-phenylthiazol-2-yl)amino)-2-thioxo-1,2-dihy- dropyrimidine-5-carbonitrile (19c)

Orange crystals from ethanol, yield 68% (2.83 g), m.p. 177˚C - 179˚C. Anal. Calculated for C₂₁H₁₅N₅OS₂ (417.51): C, 60.41; H, 3.62; N, 16.77; S, 15.36. Found: C, 60.52; H, 3.79; N; 16.55; S, 15.26. MS: m/e 417 (M⁺, 15%). IR, υ: 3480 - 3329 (2NH), 3054 (CH, aromatic), 2221 (CN), 1658 (C=N), 1622 (C=C), 1205 (C=S). ¹H NMR (DMSO-d₆, 200 MHz): δ = 3.74 (s, 3H, CH₃), 6.16 (s, 1H, thiazole H-5), 6.36 (s, 1H, D₂O exchangeable, NH), 7.23 - 7.46 (m, 9H, C₆H₅, C₆H₄), 8.23 (s, 1H, D₂O exchangeable, NH).

25) 4-Amino-3-phenyl-N-(4-phenylthiazol-2-yl)amino)-2-thioxo-2,3-dihy- drothiazole-5-carboxamide (20)

To a solution of compound 9 (2.43 g, 0.01 mol) in ethanol (30 mL) containing triethylamine (0.50 mL), elemental sulfur (0.32 g, 0.01 mol) and phenylisothiocyanate (1.35 g, 0.01 mol) were added. The reaction mixture, in each case, was heated under reflux (78˚C) for 6 h then left to cool then poured onto ice/water containing few drops of hydrochloric acid and the formed solid product was collected by filtration.

Orange crystals from ethanol, yield 50% (2.05 g), m.p. 164˚C - 167˚C. Anal. Calculated for C₁₉H₁₄N₄OS₃ (410.54): C, 55.59; H, 3.44; N, 13.65; S, 23.43. Found: C, 55.73; H, 3.83; N; 13.83; S, 23.44. MS: m/e 410 (M⁺, 35%). IR, υ: 3475 - 3342 (NH, NH₂), 3056 (CH, aromatic), 1688 (CO), 1655 (C=N), 1623 (C=C), 1230 (C=S). ¹H NMR (DMSO-d₆, 200 MHz): δ = 4.34 (s, 2H, D₂O exchangeable NH₂), 6.19 (s, 1H, thiazole H-5), 7. 23 - 7.46 (m, 10H, 2C₆H₅), 8.39 (s, 1H, D₂O exchan- geable, NH).

4. Biological Activity

4.1. In Vitro Cytotoxic Assay

4.1.1. Chemicals

Fetal bovine serum (FBS) and L-glutamine, were purchased from Gibco Invitrogen Co. (Scotland, UK). RPMI-1640 medium was purchased from Cambrex (New Jersey, USA). Dimethyl sulfoxide (DMSO), doxorubicin, penicillin, streptomycin and sulforhodamine B (SRB) were purchased from Sigma Chemical Co. (Saint Louis, USA).

4.1.2. Cell cultures

Were obtained from the European Collection of cell Cultures (ECACC, Salisbury, UK) and human gastric cancer (NUGC and HR), human colon cancer (DLD1), human liver cancer (HA22T and HEPG2), human breast cancer (MCF), nasopharyngeal carcinoma (HONE1) and normal fibroblast cells (WI38) were kindly provided by the National Cancer Institute (NCI, Cairo, Egypt). They grow as monolayer and routinely maintained in RPMI-1640 medium supplemented with 5% heat inactivated FBS, 2 mM glutamine and antibiotics (penicillin 100 U/mL, streptomycin 100 lg/mL), at 37˚C in a humidified atmosphere containing 5% CO₂. Exponentially growing cells were obtained by plating 1.5 ´ 10⁵ cells/mL for the seven human cancer cell lines including cells derived from 0.75 ´ 10⁴ cells/mL followed by 24 h of incubation. The effect of the vehicle solvent (DMSO) on the growth of these cell lines was evaluated in all the experiments by exposing untreated control cells to the maximum concentration (0.5%) of DMSO used in each assay.

The heterocyclic compounds, prepared in this study, were evaluated according to standard protocols for their in vitro cytotoxicity against six human cancer cell lines including cells derived from human gastric cancer (NUGC), human colon cancer (DLD1), human liver cancer (HA22T and HEPG2), human breast cancer (MCF), nasopharyngeal carcinoma (HONE1) and a normal fibroblast cells (WI38). All of IC₅₀ values were listed in Table 1. Some heterocyclic compounds was observed with significant cytotoxicity against most of the cancer cell lines tested (IC₅₀ = 10 - 1000 nM). Normal fibroblasts cells (WI38) were affected to a much lesser extent (IC50 > 10,000 nM). The reference compound used is the CHS-828 which is a pyridyl cyanoguanidine anti-tumor agent.

4.2. Structure Activity Relationship

It is clear from Table 1 that most of the tested compounds showed high cytotoxicity against the six cancer cell line. The thiazole derivative 3 showed moderate cytotoxicity against the six cancer cell lines. Acetylation of compound 3 to give the N-acetyl derivative 3 did not give a remarkable difference in activity compared with the original thiazole 3. Reaction of compound 3 with phenylisothiocyanate to give the N-phenylthiourea derivative 7 showed also moderate potency. Similarly, the N-cyanoacetyl derivative 9 showed moderate potency against the six cancer cell lines. However, the reaction of compound 9 with any of the aromatic aldehydes gave the arylidene derivatives 11a-c. It is obvious that compound 11b (X = Cl) showed the highest potency among such series of compounds. On the other hand, 11c (X = OCH₃) showed high potency against only NUGC cell lines and moderate potency against the other cell lines. The reaction of compound 9 with any of the diazonium salts 14a-c gave the aryl hydrazone derivatives 15a-c. Compound 15b with the electronegative Cl group showed the highest cytotoxicity among the three compounds. The multicomponent reactions of compound 9 with any of the aromatic aldehydes 10a-c and malononitrile gave the pyran derivatives 17a-c. It is obvious from Table 1 that compounds 17b and 17c showed high potency against the six cancer cell lines. However compound 17a showed high potency against NUGC, DLDI, HA22T, HEPG2and HONE1 cell lines and low potency against MCF cell lines. On the other hand, for the pyran derivatives 18a-c it is clear from Table 1 that compound 18a (X = H) showed high potency against DLDI, HEPG2 and MCF cell lines with IC₅₀’s 368, 224 and 310 nM and 18b showed high potency against HONE1 cell line with IC₅₀ 666 nM. For the pyrimidine derivatives 19a-c, compound 19c with X = OCH₃ showed high potency against NUGC, DLDI, HA22,

Table 1

Cytotoxicity of novel derivatives against a variety of cancer cell lines [IC 50 b (nM)]

Compound					Cytotoxocity (IC50 in nM)
	NUGC	DLDI	HA22T	HEPG2	HONE1	MCF	WI38
3 5 7 9 11a 11b 11c 13 15a 15b 15c 17a 17b 17c 18a 18b 18c 19a 19b 19c 20 CHS 828	1220 1329 3228 1487 2760 49 462 2366 1180 182 1280 270 22 39 1750 2210 2130 2080 1026 880 220 25	2068 3260 1248 3210 1185 36 2102 1089 1500 1062 2872 348 39 682 368 1011 1148 2188 2180 488 328 2315	1028 1120 2077 3240 2688 66 1058 260 1981 560 1329 163 160 211 1270 1140 2013 3840 2138 150 214 2067	1432 1446 1686 2336 3311 320 1260 82 1371 299 1258 331 82 202 225 2134 2176 2060 2132 1040 223 1245	2019 1240 3120 3382 2276 39 1160 71 1289 3254 2107 178 49 130 1140 666 1670 3160 2470 1188 329 15	1880 1430 1133 2322 2355 286 3320 559 1133 210 1169 2071 122 52 310 1280 1742 2246 2140 504 128 18	na na na 665 na na na na na na na na na na na na na na na na na na

^aNUGC, gastric cancer, DLDI, colon cancer, HA22T, liver cancer, HEPG2, liver cancer; HONEI, nasopharyngeal carcinoma; HR, gastric cancer; MCF, breast cancer; WI38, normal fibroblast cells.

and MCF cell lines. Finally, the thiazole derivative 20 showed high potency against the six cancer cell lines. Its high potency is attributed to the presence of high content of N and S together with the phenyl moiety through the molecule.

5. Conclusions

A series of new heterocyclic compounds with the thiazole nucleus were synthesized and characterized. Their cytotoxicity against six cancer cell lines was measured and the results showed that compounds 11b, 11c, 15b, 17a, 17b, 17c, 19c and 20 were the most potent compounds among the synthesized compounds. The 2-amino-4-(4-chlorophenyl)-6-(4-phenylthiazol-2-yl)-4H-pyran-3, 5-dicarbonitrile (17b) showed the maximum cytotoxicity among the synthesized compounds towards the six cancer cell lines.

References

1. Bharti, S.K., Nath, G., Tilak, R. and Singh, S.K. (2010) Synthesis, Anti-Bacterial and Anti-Fungal Activities of Some Novel Schiff Bases Containing 2,4-Disubstituted Thiazole Ring. European Journal of Medicinal Chemistry, 45, 651–660. https://doi.org/10.1016/j.ejmech.2009.11.008

2. Yang, B.V., Weinstein, D.S., Doweyko, L.M., Gong, H., Vaccaro, W., Huynh, T., Xiao, H.-Y., Doweyko, A.M., Mckay, L., Holloway, D.A., Somerville, J.E., Habte, S., Cunningham, M., McMahon, M., Townsend, R.,, Shuster, D., Dodd, J.H., Nadler, S.G. and Barrish, J.C. (2010) Dimethyl-Diphenyl-Propanamide Derivatives as Nonsteroidal Dissociated Glucocorticoid Receptor Agonists. Journal of Medicinal Chemistry, 53, 8241-8251. https://doi.org/10.1021/jm100957a

3. Spector, F.C., Liang, L., Giordano, H., Sivaraja, M. and Peterson, M.G. (1998) Inhibition of Herpes Simplex Virus Replication by a 2-Amino Thiazole via Interactions with the Helicase Component of the UL5-UL8-UL52 Complex. Journal of Virology, 72, 6979-6987.

4. Diego, G.C., Frederic, D., Feng, T.-S., Nchinda, A., Younis, Y., White, K.L., Wu, Q., Ryan, E., Burrows, J., Waterson, D., Witty, J.M., Wittlin, S., Charman, S.A. and Chibale, K. (2011) Novel Orally Active Antimalarial Thiazoles. Journal of Medicinal Chemistry, 54, 7713-7719. https://doi.org/10.1021/jm201108k

5. Bell, F.W., Cantrell, A.S., Högberg, M., Jaskunas, S.R., Johansson, N.G., Jordan, C.L., Kinnick, M.D., Lind, P., Morin Jr, J.M., Noréen, R., Oberg, B., Palkoeitz, J.A., Parrish, C.A., Pranc, P., Sahiberg, C., Ternansky, R.J., Vasileff, R.T., Vrang, L., West, S.J., Zhang, H. and Zhou, X.-X. (1995) Phenethylthiazolethiourea (PETT) Compounds, a New Class of HIV-1 Reverse Transcriptase Inhibitors. 1. Synthesis and Basic Structure-Activity Relationship Studies of PETT Analogs. Journal of Medicinal Chemistry, 38, 4929-4936. https://doi.org/10.1021/jm00025a010

6. Fink, B.E., Mortensen, D.S., Stauffer, S.R., Aron, Z.D. and Katzenellenbogen, J.A. (1999) Novel Structural Templates for Estrogen-Receptor Ligands and Prospects for Combinatorial Synthesis of Estrogens. Cell Chemical Biology, 6, 205-219. https://doi.org/10.1016/s1074-5521(99)80037-4

7. Matteo, B., Colin, P.L., Angelica, M. and Caberlotto, L. (2010) Synthesis and Structure-Activity Relationship of N-(3-Azabicyclo[3.1.0]Hex-6-ylMethyl)-5-(2-Pyridinyl)-1,3-Tthiazol-2-Amines Derivatives as NPY Y5 Antagonists. Bioorganic and Medicinal Chemistry Letters, 20, 4741-4744. https://doi.org/10.1016/j.bmcl.2010.06.140

8. Tilburg, E.W.V., Van der Klein, P.A.M., Groote, M.D., Beukers, M.W. and Jzerman, A.P.I. (2001) Substituted 4-Phenyl-2-(Phenylcarboxamido)-1,3-Thiazole Derivatives as Antagonists for the AdenosineA1 Receptor. Bioorganic and Medicinal Chemistry Letters, 11, 2017-2019. https://doi.org/10.1016/S0960-894X(01)00356-0

9. Bhoga, U. (2007) Novel Synthetic Approach to N-aryl-4-(3-Pyridyl)Thiazol-2-Amine and Analogues Using HMCM-41 as Catalyst, and Their Biological Evaluation as Human Platelet Aggregation Inhibitors. European Journal of Medicinal Chemistry, 42, 1144-1150. https://doi.org/10.1016/j.ejmech.2007.01.016

10. Wilson, K.J., Illig, C.R., Subasinghe, N. and Spurlino, J. (2001) Synthesis of Thiophene-2 -Carboxamidines Containing 2-Aminothiazoles and Their Biological Evaluation as Urokinase Inhibitors. Bioorganic and Medicinal Chemistry Letters, 11, 915-918. https://doi.org/10.1016/S0960-894X(01)00102-0

11. Zhang, W.T., Ruan, J.L., Wu, P.F., Jiang, F.-C., Zhang, L.-N., Fang, W., Chen, X.-L., Wang, Y., Cao, B.-S., Chen, G.-Y., Zhu, Y.-J., Gu, J. and Chen, J.-G. (2009) Design, Synthesis, and Cytoprotective Effect of 2-Aminothiazole Analogues as Potent Poly (ADP-Ribose) Polymerase-1 Inhibitors. Journal of Medicinal Chemistry, 52, 718-725. https://doi.org/10.1021/jm800902t

12. Gebeyehu, G., Marquez, V.E., Cott, A.V., Cooney, D.A., Kelley, J.A., Jayaram, H.N., Ahluwalia, G.S., Dion, R.L., Wilson, Y.A. and Johns, D.G. (1985) Ribavirin, Tiazofurin, and Selenazofurin: Mononucleotides and Nicotinamide Adenine Dinucleotide Analogues. Synthesis, Structure, and Interactions with IMP Dehydrogenase. Journal of Medicinal Chemistry, 28, 99-105. https://doi.org/10.1021/jm00379a018

13. Sekhiguchi, A., Nishina, A., Kimura, H., Fukumoto, R.-H., Kanoh, K., Ishihara, H. and Koketsu, M. (2005) Superoxide Anion-Scavenging Effect of 2-Amino-1, 3-Selenazoles. Chemical and Pharmaceutical Bulletin, 53, 1439-1442. https://doi.org/10.1248/cpb.53.1439

14. Koketsua, M., Ishihara, H., Wu, W., Murakami, K. and Saiki, I. (1999) 1,3-Selenazine Derivatives Induce Cytotoxicity and DNA Fragmentation in Human HT-1080 Fibrosarcoma Cells. European Journal of Pharmaceutical Sciences, 9, 157-161. https://doi.org/10.1016/S0928-0987(99)00058-5

15. Hantzsch, A. and Weber, J.H. (1887) Ueber Verbindungen des Thiazols (Pyridins der Thiophenreihe). Berichte der deutschen chemischen Gesellschaft, 20, 3118-3132. https://doi.org/10.1002/cber.188702002200