Design, synthesis, docking and mechanistic studies of new thiazolyl/thiazolidinylpyrimidine-2,4-dione antiproliferative agents

Ashraf A. Aly; Mohammed B. Alshammari; Akil Ahmad; Hesham A. M. Gomaa; Bahaa G. M. Youssif; Stefan Bräse; Mahmoud A. A. Ibrahim; Asmaa H. Mohamed

doi:10.1016/j.arabjc.2023.104612

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Original article

04 2023

:16;

104612

doi:

10.1016/j.arabjc.2023.104612

Design, synthesis, docking and mechanistic studies of new thiazolyl/thiazolidinylpyrimidine-2,4-dione antiproliferative agents

Ashraf A. Aly^{^a,⁎}, Mohammed B. Alshammari^{^b}, Akil Ahmad^{^b}, Hesham A. M. Gomaa^{^c}, Bahaa G. M. Youssif^{^d,⁎}, Stefan Bräse^{^e,^f,⁎}, Mahmoud A. A. Ibrahim^{^g}, Asmaa H. Mohamed^{^a}

a Chemistry Department, Faculty of Science, Minia University, 61519 El-Minia, Egypt

b Chemistry Department, College of Sciences and Humanities, Prince Sattam Bin Abdulaziz University, Al-Kharij, Saudi Arabia

c Department of Pharmacology, College of Pharmacy, Jouf University, Sakaka 72341, Aljouf, Saudi Arabia

d Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Assiut University, 71526 Assiut, Egypt

e Institute of Organic Chemistry, Karlsruher Institut fur Technologie, 76131 Karlsruhe, Germany

f Institute of Biological and Chemical Systems (IBCS-FMS), Karlsruhe Institute of Technology, 76344 Eggenstein Leopoldshafen, Germany

g Computational Chemistry Laboratory, Chemistry Department, Faculty of Science, Minia University, Minia 61519, Egypt

⁎Corresponding authors at: Chemistry Department, Faculty of Science, Minia University, 61519, El-Minia, Egypt(A.A. Aly), Institute of Organic Chemistry, Karlsruher Institut fur Technologie, 76131 Karlsruhe, Germany(S. Bräse), Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Assiut University, Assiut 71526, Egypt(B.G.M. Youssif). ashraf.shehata@mu.edu.eg (Ashraf A. Aly), stefan.braese@kit.edu (Ashraf A. Aly), bgyoussif@ju.edu.sa (Bahaa G. M. Youssif), ashraf.shehata@mu.edu.eg (Stefan Bräse) stefan.braese@kit.edu (Stefan Bräse)

Received: 2022-11-20, Accepted: 2023-1-18,

Disclaimer:
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.

Peer review under responsibility of King Saud University.

Abstract

In this article, we display on the synthesis and biological evaluation of a new series of thiazolylpyrimidine 3a-l and thiazolidinylpyrimidine derivatives 5a-e. The structures of the new compounds were confirmed by using different spectral techniques including NMR, IR, mass spectroscopy in addition to elemental analyses. The cell viability of the new compounds was assessed against normal human mammary gland epithelial (MCF-10A) cell line. Data revealed that none of the compounds examined exhibited cytotoxic effects, and the cell viability for the compounds examined at 50 µM was greater than 87%. The antiproliferative activity of 3a-l and 5a-e was evaluated against four human cancer cell lines where the compounds showed promising activity. The most potent derivatives were compounds 3a, 3c, 3f, 3i, and 5b with GI₅₀ values ranging from 0.90 µM to 1.70 µM against the four cancer cell lines in comparison to doxorubicin (GI₅₀ = 1.10 µM). Compounds 3a, 3c and 3i showed potent antiproliferative activity with dual inhibitory action against EGFR and BRAF^V600E. Compounds 3a, 3c, and 3i demonstrated promising AutoDock scores towards EGFR and BRAF^V600E with values of − 9.1 and − 8.6, −9.0 and − 8.5, and − 8.4 and − 8.0 kcal/mol, respectively. The physicochemical and pharmacokinetic characteristics of 3a, 3c, and 3i were anticipated, demonstrating their oral bioavailability.

Keywords

Uracil

Thiazoles

Thiazolidines

Antiproliferative

Kinases

Docking

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1 Introduction

In anti-cancer research, a better understanding of druggable targets influences the development of new drugs. This strategy assumes that altering a specific cancer biomarker will result in a successful therapeutic outcome (Stanković et al., 2019). Selective anti-cancer drugs should be more effective at eliminating tumors while minimizing side effects on normal cells (Raghavendra et al., 2018). However, due to medication resistance, inhibiting just one target frequently has only a transient effect (Fu et al., 2017). Given the diversity of cancers, simultaneous intervention on multiple targets is required to achieve the best results (Zha et al., 2017). Combination chemotherapy is one method for simultaneously inhibiting two or more targets. However, differences in the pharmacokinetic profiles and metabolic stabilities of two or more medications are common.

Furthermore, combined chemotherapy may result in risky medication interactions (Palmeira et al., 2012). These problems could be solved by combining two medications with a single molecule that acts on several relevant targets (Zheng et al., 2017). In recent years, there has been much interest in multi-target medicines, also known as “hybrid” compounds, which have been developed by joining two or more separate pharmacophore moieties into a single molecule either directly through a covalent bond or indirectly through a linker (Mahboobi et al., 2010). Multi-target anti-cancer drugs have a wider range of activity than conventional medicines because they combine two different pharmacological features and target multiple signaling pathways (Alam et al., 2019, 2018; Banerji et al., 2018; Beckers et al., 2012; Cai et al., 2010 ).

The acquired BRAF^V600E mutation was suggested as a resistance mechanism following therapy with EGFR inhibitors (Ho et al., 2017). The feedback activation of EGFR signaling is also linked to the emergence of resistance in colorectal cancer (Hyman et al., 2015). Furthermore, BRAF inhibition may result in EGFR activation, which fuels tumor growth (Desai et al., 2017). To treat these issues, BRAF/EGFR were used in tandem. Several studies have shown that the BRAF-EGFR combination has a significant therapeutic effect in people with metastatic colorectal cancer who have the BRAF^V600E mutation (Ho et al., 2017). As a result, inhibiting the two kinases sequentially may address the issue of EGFR activation.

The pyrimidine ring is a popular heterocyclic scaffold in medicinal chemistry. Pyrimidine derivatives are widely used in medicine because of their therapeutic benefits (Bai et al., 2018; Borik et al., 2018; Jin et al., 2018; Madhu Sekhar et al., 2018; Rizk et al., 2018; Saleeb et al., 2018 ). The pyrimidine ring is also a component of DNA nucleic acid. Various drugs containing the pyrimidine nucleus are being used as effective anti-cancer agents via various mechanisms of action, i.e., 5-Fluorouracil (5-FU) I as thymidylate synthase inhibitor (Santi et al., 1974), Merbarone II as DNA topoisomerase II (topo II) inhibitor (Pastor et al., 2012), Nilotinib III and Imatinib IV as tyrosine kinase inhibitors (El-Mezayen et al., 2017; Shaker et al., 2013), and Ibrutinib (IBR) V as Bruton’s tyrosine kinase (BTK) inhibitor (Tang et al., 2018) (Fig. 1).

Some selected pyrimidine-based anti-cancer drugs I-V.

On the other hand, thiazole, a five-membered heterocyclic ligand containing sulfur and nitrogen, has recently piqued the interest of researchers due to its potent biological properties (Ayati et al., 2015; Sharma et al., 2017).

Thiazole and its derivatives are among the most active compounds with a wide range of activity, with anti-cancer activity topping the list (Lozynskyi Andrii et al., 2014). Furthermore, thiazole-containing compounds have been found in several clinically available anti-cancer drugs (Fig. 2), including tiazofurin (VI) (IMP dehydrogenase inhibitor) (Franchetti et al., 1995), dasatinib (VII) (Bcr-Abl tyrosine kinase inhibitor) (Li et al., 2009), and dabrafenib (VIII) (B-RAF inhibitor) (Hu-Lieskovan et al., 2015).

Some clinically used thiazole-containing anti-cancer drugs VI-VIII and reported thiazole-based kinase inhibitors IX and X.

Lv et al. developed two thiazolidinone derivatives to inhibit EGFR and HER-2 kinases. Some of the synthesized compounds exhibited significant EGFR and HER-2 inhibitory activity. Compound (IX) demonstrated the most potent EGFR and HER-2 inhibitory activities with IC₅₀ values of 0.09 µM and 0.42 µM, respectively,e that some of the thiazolidinone derivatives have Fig. 2 (Lv et al., 2010). In another study, Zhao et al. prepared a series of thiazole-based derivatives and evaluated them as potential inhibitors of BRAF^V600E. According to the biological activity data, compound (X, Fig. 2) exhibited the most potent antiproliferative activity with potent BRAF^V600E inhibitory activity with an IC₅₀ value of 0.05 µM. Furthermore, compound X induced remarkable apoptosis dose-dependent (Abdel-Maksoud et al., 2015).

Motivated by the findings above and as an extension of these studies (Alshammari et al., 2021; Aly et al., 2020, 2015; Mostafa et al., 2022 ), we present the synthesis of novel hybrid compounds based on pyrimidine and thiazole pharmacophores via the reaction of 3-substituted thiourea derivatives with 2-bromo-1-phenylethanone, 1-chloropropan-2-one, and ethyl bromoacetate. As a result, two new series of thiazolyl/pyrimidine-2,4-diones 3a-l (Scaffold A) and thiazolidinylpyrimidine-2,4-diones 5a-e (Scaffold B) were developed in the hope of obtaining new potent antiproliferative agents that can target EGFR and/or BRAF^V600E, as shown in Fig. 3.

All new compounds were tested for cell viability to see how they affected the viability of normal cell lines. The new compound will be tested as an antiproliferative agent against a panel of four cancer cell lines. Furthermore, the most active compounds will be tested against EGFR and BRAF^V600E as potential targets for antiproliferative activity. Ultimately, docking computations were executed to reveal the docking pose of the most active compounds toward EGFR and BRAF^V600E targets. The drug-likeness and ADMET characteristics were also anticipated for the investigated compounds.

2 Experimental (instruments in the Suppl. File)

2.1

2.1 Chemistry

General Details: Refer to Appendix A (Supplementary File).

Compounds 1a-e and α-bromoacetophenone (2a) were synthesized according to the literature (Alshammari et al., 2022; Nobuta et al., 2010) Chloroacetone (3b) and ethyl 2-bromoacetate (4) were obtained from Aldrich.

2.1.1

2.1.1 General procedure for the synthesis of compounds 3a-l

Compounds 3a-l were synthesized by refluxing thioureas 1a-e (1 mmol) with α-bromoacetophenone (2a) and/or chloroacetone (3b) (1 mmol) in 50 mL of absolute ethanol as a solvent and the presence of few drops (0.5 mL) of triethylamine as a catalyst for 4–7 h. The resulting solid was filtered and recrystallized from DMF/EtOH (1:10).

2.1.1.1

2.1.1.1 (Z)-5-((3,4-Diphenylthiazol-2(3H)-ylidene)amino)pyrimidine-2,4(1H,3H)-dione (3a)

Yield: 78 %; mp 314–316 °C, IR (KBr) υ_max/cm⁻¹ = 3135 (NH), 3000 (Ar-CH), 1688–1680 (C⚌O), 1597 (C⚌N), 1558 (C⚌C). ¹H NMR (400 MHz, DMSO‑d₆): δ_H = 7.17 (s, 1H, CH-5′), 7.35–7.36 (m, 2H, H- Ar-H), 7.46–7.49 (m, 6H, Ar-H), 7.60 (dd, 2H, J = 7.7, 7.6 Hz, Ar-H), 8.32 (d, 1H, J = 6.3 Hz, H-6), 11.62 (br,d, 1H, J = 5.1 Hz, NH-1), 11.73 ppm (br,s, 1H, NH-3). ¹³C NMR (100 MHz, DMSO‑d₆): δ_C = 104.7 (C-5′), 108.4 (C-5), 123.9, 128.3, 128.7, 129.2, 130.1 ppm (CH-Ar), 130.4, 142.0 (C-Ar), 143.1 (C-6), 146.0 (C-2′, 4′'), 150.6 (C-2), 160.2 ppm (C-4). MS (70 eV, %): m/z = 364 (M + 2, 15), 363 (M + 1, 62), 362 (M⁺, 8), 285 (5), 255 (3), 154 (20), 102 (1 0 0). Anal. Calcd for C₁₉H₁₄N₄O₂S (362.41): C, 62.97; H, 3.89; N, 15.46; S, 8.85. Found: C, 62.85; H, 3.92; N, 15.58; S, 8.94.

2.1.1.2

2.1.1.2 (Z)-5-((4-Phenyl-3-(p-tolyl)thiazol-2(3H)-ylidene)amino)pyrimidine-2,4(1H,3H)-dione (3b)

Yield: 74 %; mp 306–308 °C, IR (KBr): υ_max/cm⁻¹ = 3135 (NH), 3000 (Ar-CH), 1688–1670 (C⚌O), 1597 (C⚌N), 1558 (C⚌C). ¹H NMR (400 MHz, DMSO‑d₆): δ_H = 2.38 (s, 3H, CH₃), 7.18 (s, 1H, CH-5′), 7.34–7.36 (m, 4H, H-o, o'), 7.39–7.41 (m, 2H, H-m), 7.46–7.49 (m, 3H, H-m’, p’), 8.32 (d, 1H, J = 6.3 Hz, H-6), 11.62 (d, 1H, J = 5.7 Hz, NH-1), 11.73 ppm (br,s, 1H, NH-3). ¹³C NMR (100 MHz, DMSO‑d₆) δ_C = 20.66 (CH₃), 104.97 (C-5′), 108.20 (C-5), 123.91 (C-o), 128.25 (C-i'), 128.70 (C-m'), 129.22 (C-o'), 130.13 (C-p'), 130.86 (C-m), 138.20 (C-p), 142.01 (C-6, C-i), 146.07 (C-2′, C-4′), 150.58 (C-2), 160.16 ppm (C-4). MS (70 eV, %): m/z = 378 (M + 2, 27), 377 (M + 1, 100), 376 (M⁺, 15), 372 (10), 283 (5), 255 (5), 154 (25), 136 (17), 102 (83). Anal. Calcd for C₂₀H₁₆N₄O₂S (376.43): C, 63.81; H, 4.28; N, 14.88; S, 8.52. Found: C, 63.92; H, 4.31; N, 14.98; S, 8.67.

2.1.1.3

2.1.1.3 (Z)-5-((3-(3-Methoxyphenyl)-4-phenylthiazol-2(3H)-ylidene)amino)-pyrimidine-2,4(1H,3H)-dione (3c)

Yield: 76 %; mp 310–312 °C, IR (KBr) υ_max/cm⁻¹ = 3188 (NH), 3055 (Ar-CH), 1679–1670 (C⚌O), 1607 (C⚌N), 1555 (C⚌C). ¹H NMR (400 MHz, DMSO‑d₆): δ_H = 3.81 (s, 3H, OCH₃), 6.96–7.01 (m, 3H, Ar-H), 7.13 (s, 1H, C-5′), 7.35–7.36 (m, 2H, H-o, o'), 7.47–7.49 (m, 4H, Ar-H), 8.26 (d, 1H, J = 6.0 Hz, H-6), 11.60 (d, 1H, J = 4.0 Hz, NH-1), 11.70 ppm (br,s, 1H, NH-3). ¹³C NMR (100 MHz, DMSO‑d₆): δ_C = 55.5 (OCH₃), 108.6 (C-5′), 109.1 (C-5), 113.7 (C-o), 115.4 (C-o'), 128.7 (C-i'), 128.8 (C-m'), 129.2 (C-p'), 130.0 (C-m'), 131.3 (C-p), 138.2 (C-i), 141.8 (C-6), 145.9 (C-4′), 150.6 (C-2′), 160.2 (C-2), 160.5 ppm (C-4). MS (70 eV, %): m/z = 394 (M + 2, 26), 393 (M + 1, 100), 392 (M⁺, 10), 289 (5), 154 (17), 136 (13). Anal. Calcd for C₂₀H₁₆N₄O₃S (392.43): C, 61.21; H, 4.11; N, 14.28; S, 8.17. Found: C, 61.35; H, 4.15; N, 14.40; S, 8.28.

2.1.1.4

2.1.1.4 (Z)-5-((3-Benzyl-4-phenylthiazol-2(3H)-ylidene)amino)pyrimidine-2,4(1H,3H)-dione (3d)

Yield: 75 %; mp 312–314 °C, IR (KBr) υ_max/cm⁻¹ = 3135 (NH), 3000 (Ar-CH), 1678–1670 (C⚌O), 1600 (C⚌N), 1556 (C⚌C). ¹H NMR (400 MHz, DMSO‑d₆): δ_H = 5.28 (s, 2H, NCH₂), 6.95–7.00 (m, 3H, Ar-H), 7.25–7.30 (m, 5H, Ar-H + H-5′), 7.35–7.49 (m, 3H, Ar-H), 7.58 (s, 1H, H-6), 11.19 (br,s, 1H, NH-1), 11.56 ppm (br,s, 1H, NH-3). ¹³C NMR (100 MHz, DMSO‑d₆): δ_C = 45.7 (NCH₂), 104.2 (CH-5′), 108.6 (C-5), 126.6, 127.4, 127.6, 128.2, 128.5, 128.7, 129.3, 129.9 (CH-Ar), 132.2, 138.2 (C-Ar), 141.8 (C-6), 146.2 (C-4′), 148.2 (C-2′), 150.5 (C-2), 160.2 ppm (C-4). MS (70 eV, %): m/z = 376 (M⁺, 100), 283 (14), 255 (10), 154 (15), 136 (10), 102 (40). Anal. Calcd for C₂₀H₁₆N₄O₂S (376.43): C, 63.81; H, 4.28; N, 14.88; S, 8.52. Found: C, 63.94; H, 4.31; N, 14.98; S, 8.61.

2.1.1.5

2.1.1.5 (Z)-5-((3-Methyl-4-phenylthiazol-2(3H)-ylidene)amino)pyrimidine-2,4(1H,3H)-dione (3e)

Yield: 78 %; mp 308–310 °C, IR (KBr) υ_max/cm⁻¹ = 3142 (NH), 3055 (Ar-CH), 1668–1660 (C⚌O), 1598 (C⚌N), 1578 (C⚌C). ¹H NMR (400 MHz, DMSO‑d₆): δ_H = 3.32 (s, 3H, NCH₃), 6.51 (s, 1H, CH-5′), 7.27 (d, 1H, J = 4.0 Hz, H-6), 7.52–7.58 (m, 5H, Ar-H), 10.78 (br,s, 1H, NH-1), 11.29 ppm (br,s, 1H, NH-3). ¹³C NMR (100 MHz, DMSO‑d₆): δ_C = 33.6 (NCH₂), 99.1 (C-5′), 108.3 (C-5), 128.8, 128.9, 129.4 (CH-Ar), 138.3 (C-Ar), 140.6 (C-6), 142.3 (C-4′), 148.6 (C-2′), 150.5 (C-2), 160.4 ppm (C-4). MS (70 eV, %): m/z = 300 (M⁺, 13), 289 (10), 255 (10), 154 (48), 102 (1 0 0). Anal. Calcd for C₁₄H₁₂N₄O₂S (300.34): C, 55.99; H, 4.03; N, 18.65; S, 10.68. Found: C, 55.88; H, 4.07; N, 18.78; S, 10.77.

2.1.1.6

2.1.1.6 (Z)-5-((3-Allyl-4-phenylthiazol-2(3H)-ylidene)amino)pyrimidine-2,4(1H,3H)-dione (3f)

Yield: 74 %; mp 304–306 °C, IR (KBr) υ_max/cm⁻¹ = 3297 (NH), 3089 (Ar-CH), 1675–1665 (C⚌O), 1656 (C⚌N), 1582 (C⚌C). ¹H NMR (400 MHz, DMSO‑d₆): δ_H = 4.15 (d, 2H, J = 8 Hz, N-CH₂), 5.08 (d, 1H, J = 10.1 Hz, H-3′c), 5.18 (dd, 1H, J = 17.2, 1.1 Hz, H-3′c), 5.83 (ddt, 1H, J_d = 17.2, 10.4 Hz, J_t = 5.2 Hz, H-3b), 7.14–7.18 (m, 2H, Ar-H), 7.25–7.31 (m, 3H, Ar-H), 8.89 (s, 1H, H-6), 11.34 (br,s, 1H, NH-1), 11.66 ppm (br,s, 1H, NH-3). ¹³C NMR (100 MHz, DMSO‑d₆): δ_C = 45.7 (N-CH₂), 107.0 (C-5′), 110.0 (C-5), 116.5 (C-3′c), 131.7 (C-3′b), 127.1, 127.5, 128.7, 129.2, 129.9 (CH-Ar), 131.2 (C-Ar), 140.5 (C-6), 142.2 (C-4′), 148.1 (C-2′), 150.4 (C-2), 160.2 ppm (C-4). MS (70 eV, %): m/z = 326 (M⁺, 50), 283 (9), 255 (10), 154 (25), 136 (15), 102 (1 0 0). Anal. Calcd for C₁₆H₁₄N₄O₂S (326.37): C, 58.88; H, 4.32; N, 17.17; S, 9.82. Found: C, 55.96; H, 4.36; N, 17.28; S, 9.94.

2.1.1.7

2.1.1.7 (Z)-5-((4-Methyl-3-phenylthiazol-2(3H)-ylidene)amino)pyrimidine-2,4(1H,3H)-dione (3g)

Yield: 74 %; mp 302–304 °C, IR (KBr) υ_max/cm⁻¹ = 3172 (NH), 3017 (Ar-CH), 1676–1660 (CO), 1605 (C⚌N), 1597 (C⚌C). ¹H NMR (400 MHz, DMSO‑d₆): δ_H = 2.21 (s, 3H, CH₃), 6.10 (s, 1H, CH-5′), 7.15–7.21 (m, 5H, H- Ar-H), 8.05 (s, 1H, H-6), 10.86 (br,s, 1H, NH-1), 11.36 ppm (br,s, 1H, NH-3). ¹³C NMR (100 MHz, DMSO‑d₆) δ_C = 20.4 (CH₃), 104.1 (C-5′), 113.3 (C-5), 125.3, 128.2, 128.9 (CH-Ar), 137.3 (C-Ar), 140.2 (C-6), 143.7 (C-4′), 148.3 (C-2′), 150.0 (C-2), 161.4 ppm (C-4). MS (70 eV, %): m/z = 300 (M⁺, 85), 283 (10), 239 (40), 102 (1 0 0). Anal. Calcd for C₁₄H₁₂N₄O₂S (300.34): C, 55.99; H, 4.03; N, 18.65; S, 10.68. Found: C, 55.88; H, 4.07; N, 18.78; S, 10.77.

2.1.1.8

2.1.1.8 (Z)-5-((4-Methyl-3-(p-tolyl)thiazol-2(3H)-ylidene)amino)pyrimidine-2,4(1H,3H)-dione (3h)

Yield: 75 %; mp 312–314 °C, IR (KBr) υ_max/cm⁻¹ = 3145 (NH), 3000 (Ar-CH), 1668–1660 (C⚌O), 1600 (C⚌N), 1547 (C⚌N). ¹H NMR (400 MHz, DMSO‑d₆): δ_H = 1.88 (s, 3H, CH₃), 2.24 (s, 3H, CH₃), 5.92 (s, 1H, CH-5′), 6.75 (d, 2H, J = 8.0 Hz, Ar-H), 7.06 (d, 2H, J = 8.0 Hz, Ar-H), 7.90 (d, 1H, J = 7.6 Hz, H-6), 11.30 (d, 1H, J = 7.6 Hz, NH-1), 11.50 ppm (br,s, 1H, NH-3). ¹³C NMR (100 MHz, DMSO‑d₆): δ_C = 18.2 (CH₃), 20.4 (CH₃), 95.0 (C-5′), 108.9 (C-5), 120.7, 129.6, 129.8 (CH-Ar), 134.9, 138.5 (C-Ar), 142.6 (C-6), 148.2 (C-2′, C-4′), 150.9 (C-2), 160.2 ppm (C-4). MS: m/z = 314 (M⁺, 100), 295 (15), 273 (5), 242 (5), 167 (10). Anal. Calcd for C₁₅H₁₄N₄O₂S (314.36): C, 57.31; H, 4.49; N, 17.82; S, 10.20. Found: C, 57.42; H, 4.52; N, 17.85; S, 10.29.

2.1.1.9

2.1.1.9 (Z)-5-((3-(3-Methoxyphenyl)-4-methylthiazol-2(3H)-ylidene)amino)pyrimidine-2,4(1H,3H)-dione (3i)

Yield: 75 %; mp 308–310 °C, IR (KBr) υ_max/cm⁻¹ = 3168 (NH), 3015 (Ar-CH), 1691–1668 (C⚌O), 1601 (C⚌N), 1545 (C⚌C). ¹H NMR (400 MHz, DMSO‑d₆): δ_H = 1.92 (s, 3H, CH₃), 3.76 (s, 3H, OCH₃), 5.99 (s, 1H, CH-5′), 6.72 (d, 1H, J = 8.0 Hz, Ar-H), 7.02 (d, 1H, J = 8.0 Hz, Ar-H), 7.22–7.26 (m, 2H, Ar-H), 8.10 (d, 1H, J = 7.6 Hz, H-6), 11.45 (d, 1H, J = 7.6 Hz, NH-1), 11.62 ppm (br,s, 1H, NH-3). ¹³C NMR (100 MHz, DMSO‑d₆): δ_C = 19.1 (CH₃), 53.6 (CH₃), 95.1 (C-5′), 108.8 (C-5), 117.8, 125.1, 128.7, 129.4 (CH-Ar), 138.1, 139.6 (C-Ar), 142.5 (C-6), 146.2 (C-2′), 149.0 (C-4′), 151.0 (C-2), 161.2 ppm (C-4). MS (70 eV, %): m/z = 330 (M⁺, 32), 302 (10), 263 (5), 154 (1 0 0), 136 (68). Anal. Calcd for C₁₅H₁₄N₄O₃S (330.36): C, 54.53; H, 4.27; N, 16.96; S, 9.71. Found: C, 54.65; H, 4.30; N, 16.88; S, 9.82.

2.1.1.10

2.1.1.10 (Z)-5-((3-Benzyl-4-methylthiazol-2(3H)-ylidene)amino)pyrimidine-2,4(1H,3H)-dione (3j)

Yield: 76 %; mp 315–317 °C, IR (KBr) υ_max/cm⁻¹ = 3232 (NH), 3012 (Ar-CH), 1680–1663 (C⚌O), 1614 (C⚌N), 1568 (C⚌C). ¹H NMR (400 MHz, DMSO‑d₆): δ_H = 2.15 (s, 3H, CH₃), 5.02 (s, 2H, NCH₂), 5.88 (s, 1H, CH-5′), 7.27–7.45 (m, 5H, Ar-H), 7.57 (s, 1H, H-6), 11.40 (s, 1H, NH-1), 11.62 ppm (br,s, 1H, NH-3). ¹³C NMR (100 MHz, DMSO‑d₆): δ_C = 18.2 (CH₃), 52.6 (NCH₂), 99.2 (C-5′), 108.7 (C-5), 127.6, 128.0, 128.6 (CH-Ar), 132.0 (C-Ar), 142.6 (C-6), 146.4 (C-2′), 149.0 (C-4′), 151.0 (C-2), 161.2 ppm (C-4). MS (70 eV, %): m/z = 314 (M⁺, 100), 289 (15), 273 (5), 242 (5), 195 (10), 154 (40), 107 (23). Anal. Calcd for C₁₅H₁₄N₄O₂S (314.36): C, 57.31; H, 4.49; N, 17.82; S, 10.20. Found: C, 57.43; H, 4.52; N, 17.91; S, 10.31.

2.1.1.11

2.1.1.11 (Z)-5-((3,4-Dimethylthiazol-2(3H)-ylidene)amino)pyrimidine-2,4(1H,3H)-dione (3k)

Yield: 77 %; mp 306–308 °C, IR (KBr) υ_max/cm⁻¹ = 3210 (NH), 1672–1665 (CO), 1608 (C⚌N), 1576 (C⚌C). ¹H NMR (400 MHz, DMSO‑d₆): δ_H = 2.16 (s, 3H, CH₃), 3.47 (s, 3H, CH₃), 6.32 (s, 1H, CH-5′), 7.36 (s, 1H, H-6), 10.97 (br,s, 1H, NH-1), 11.32 ppm (br,s, 1H, NH-3). ¹³C NMR (100 MHz, DMSO‑d₆): δ_C = 13.9 (CH₃), 32.1 (NCH₃), 97.3 (C-5′), 119.3 (C-5), 134.6 (C-6), 137.3 (C-4′), 150.4 (C-2′), 160.4 (C-2), 166.2 ppm (C-4). MS (70 eV, %): m/z = 240 (M + 2, 7), 239 (M + 1, 56), 238 (M⁺, 10), 168 (5), 154 (30), 136 (20), 102 (1 0 0). Anal. Calcd for C₉H₁₀N₄O₂S (238.27): C, 45.37; H, 4.23; N, 23.51; S, 13.46. Found: C, 45.46; H, 4.27; N, 23.64; S, 13.55.

2.1.1.12

2.1.1.12 (Z)-5-((3-Allyl-4-methylthiazol-2(3H)-ylidene)amino)pyrimidine-2,4(1H,3H)-dione (3l)

Yield: 74 %; mp 298–300 °C, IR (KBr) υ_max/cm⁻¹ = 3211 (NH), 1677–1665 (C⚌O), 1648 (C⚌N), 1595 (C⚌C). ¹H NMR (400 MHz, DMSO‑d₆): δ_H = 2.18 (CH₃), 4.55 (d, 2H, J = 12.0 Hz, N-CH₂), 4.99 (d, 1H, J = 18.0 Hz, H-3′c), 5.10 (dd, 1H, J = 16.0, 1.1 Hz, H-3′c), 5.94 (ddt, 1H, Jd = 8.0 Hz, 12.0 Hz, Jt = 4.0 Hz, H-3b), 6.05 (s, 1H, CH-5′), 11.25 (br, s, 1H, NH-1), 11.37 ppm (br, s, 1H, NH-3). ¹³C NMR (100 MHz, DMSO‑d₆): δ_C = 13.6 (CH₃), 45.3 (N-CH₂), 110.1 (C-5′), 117.0 (C-5), 117.9 (C-3′c), 130.6 (C-3′b), 131.5 (C-6), 137.1 (C-4′), 148.8 (C-2′), 150.4 (C-2), 160.2 ppm (C-4). MS (70 eV, %): m/z = 266 (M + 2, 17), 265 (M + 1, 100), 264 (M⁺, 14), 224 (7), 165 (5), 154 (32), 136 (24), 102 (14). Anal. Calcd for C₁₁H₁₂N₄O₂S (264.30): C, 49.99; H, 4.58; N, 21.20; S, 12.13. Found: C, 49.89; H, 4.61; N, 21.28; S, 12.21.

2.1.2

2.1.2 General procedure for the synthesis of compounds 5a-e

Compounds 5a-e were synthesized by refluxing thioureas 1a-e (1 mmol) with ethyl 2-bromo-acetate (4) (0.167 g, 1 mmol) in 30 mL of absolute ethanol as a solvent and the presence of few drops (0.5 mL) of triethylamine (Et₃N) as a catalyst for 6–8 h. The resulting solid was filtered and recrystallized from DMF/EtOH.

2.1.2.1

2.1.2.1 (Z)-5-((4-Oxo-3-phenylthiazolidin-2-ylidene)amino)pyrimidine-2,4(1H,3H)-dione (5a)

Yield: 78 %; mp 298–300 °C, IR (KBr) υ_max/cm⁻¹ = 3205 (NH), 3005 (Ar-CH), 1690–1675 (C⚌O), 1648 (C⚌N). ¹H NMR (400 MHz, DMSO‑d₆): δ_H = 4.05 (s, 2H, CH₂), 7.17 (d, 1H, J = 5.5 Hz, H-6), 7.32–7.40 (m, 5H, Ar-H), 11.50 ppm (br,s, 1H, NH-1), 11.75 ppm (br,s, 1H, NH-3). ¹³C NMR (100 MHz, DMSO‑d₆): δ_C = 32.6 (CH₂), 106.6 (C-5), 127.4, 127.7, 128.0, 128.3 (CH-Ar), 135.7 (C-Ar), 144.4 (C-6), 150.5 (C-2), 159.8 (C-4), 170.5 ppm (C-4′). MS (70 eV, %): m/z = 302 (M⁺, 60), 288 (10), 154 (15), 102 (1 0 0). Anal. Calcd for C₁₃H₁₀N₄O₃S (302.31): C, 51.65; H, 3.33; N, 18.53; S, 10.61. Found: C, 51.77; H, 3.37; N, 18.62; S, 10.73.

2.1.2.2

2.1.2.2 (Z)-5-((3-(3-Methoxyphenyl)-4-oxothiazolidin-2-ylidene)amino)pyrimidine-2,4(1H,3H)-dione (5b)

Yield: 76 %; mp 306–308 °C, IR (KBr) υ_max/cm⁻¹ = 3204 (NH), 3002 (Ar-CH), 1690–1676 (C⚌O), 1640 (C⚌N). ¹H NMR (400 MHz, DMSO‑d₆): δ_H = 3.64 (s, 3H, CH₃), 4.10 (s, 2H, CH₂), 6.88–6.92 (m, 2H, Ar-H), 7.17–7.22 (m, 2H, Ar-H), 7.64 (d, 1H, J = 5.4 Hz, H-6), 11.52 (br,s, 1H, NH-1), 11.70 ppm (br,s, 1H, NH-3). ¹³C NMR (100 MHz, DMSO‑d₆): δ_C = 33.8 (CH₂), 108.1 (C-5), 125.2, 126.4, 128.1, 129.2 (CH-Ar), 131.3, 138.6 (C-Ar), 144.3 (C-6), 150.8 (C-2), 158.6 (C-4), 171.0 ppm (C-4′). MS (70 eV, %): m/z = 304 (M + 2, 17), 303 (M + 1, 80), 302 (M⁺, 13), 225 (15), 190 (12), 177 (100). Anal. Calcd for C₁₄H₁₂N₄O₄S (302.31): C, 50.60; H, 3.64; N, 16.86; S, 9.65. Found: C, 50.61; H, 3.67; N, 16.99; S, 9.73.

2.1.2.3

2.1.2.3 (Z)-5-((3-Benzyl-4-oxothiazolidin-2-ylidene)amino)pyrimidine-2,4(1H,3H)-dione (5c)

Yield: 79 %; mp 314–316 °C, IR (KBr) υ_max/cm⁻¹ = 3152 (NH), 2974 (Ar-CH), 1689–1670 (CO), 1646 (C⚌N). ¹H NMR (400 MHz, DMSO‑d₆): δ_H = 4.11 (s, 2H, CH₂), 4.88 (s, 2H, NCH₂), 7.30–7.39 (m, 5H, Ar-H), 7.94 (d,1H, J = 8.0 Hz, H-6), 11.25 (br,s, 1H, NH-1), 11.50 ppm (br,s, 1H, NH-3). ¹³C NMR (100 MHz, DMSO‑d₆): δ_C = 33.9 (CH₂), 45.4 (NCH₂), 106.6 (C-5), 127.8, 128.3, 128.6, 128.3 (CH-Ar), 136.0 (C-Ar), 138.1 (C-6), 144.7 (C-2′), 150.6 (C-2), 160.0 (C-4), 170.5 ppm (C-4′). MS (70 eV, %): m/z = 318 (M + 2, 20), 317 (M + 1, 100), 316 (M⁺, 15), 232 (5), 178 (7), 155 (10), 154 (35), 136 (30), 102 (37), 91 (42). Anal. Calcd for C₁₄H₁₂N₄O₃S (316.34): C, 53.16; H, 3.82; N, 17.71; S, 10.14. Found: C, 53.28; H, 3.85; N, 17.82; S, 10.22.

2.1.2.4

2.1.2.4 (Z)-5-((3-Methyl-4-oxothiazolidin-2-ylidene)amino)pyrimidine-2,4(1H,3H)-dione (5d)

Yield: 76 %; mp 294–296 °C, IR (KBr) υ_max/cm⁻¹ = 3168 (NH), 1687–1670 (C⚌O), 1650 (C⚌N), 1598 (C⚌C). ¹H NMR (400 MHz, DMSO‑d₆): δ_H = 4.04 (s, 2H, CH₂), 4.43 (d, 2H, J = 4.0 Hz, NCH₂), 5.19 (dd, 1H, J = 12.0 Hz, 1.0 Hz, ^'H-3c'), 5.22 (dd, 1H, J = 12.0 Hz, 1.1 Hz, H-3c'), 5.90 (ddt, 1H, J_d = 17.1, 10.4 Hz, J_t = 5.1 Hz, H-3b'), 7.30 (d, 1H, J = 6.4 Hz, H-6), 11.14 (bd, 1H, J = 4.0 Hz, NH-1), 11.56 ppm (br,s, 1H, NH-3). ¹³C NMR (100 MHz, DMSO‑d₆): δ_C = 35.14 (CH₂), 44.45 (NCH₂), 117.60 (C-3c'), 118.20 (C-5), 130.85 (C-3b'), 131.90 (C-6), 148.73 (C-2′), 150.60 (C-2), 160.05 (C-4), 172.10 ppm (C-4′). MS (70 eV, %): m/z = 240 (M⁺, 100), 229 (5), 188 (34), 154 (10), 102 (80). Anal. Calcd for C₈H₈N₄O₃S (240.24): C, 40.00; H, 3.36; N, 23.32; S, 13.35. Found: C, 40.09; H, 3.40; N, 23.43; S, 13.43.

2.1.2.5

2.1.2.5 (Z)-5-((3-Allyl-4-oxothiazolidin-2-ylidene)amino)pyrimidine-2,4(1H,3H)-dione (5e)

Yield: 76 %; mp 296–298 °C, IR (KBr) υ_max/cm⁻¹ = 3155 (NH), 1690–1668 (C⚌O), 1605 (C⚌N), 1545 (C⚌C). ¹H NMR (400 MHz, DMSO‑d₆): δ_H = 4.04 (s, 2H, CH₂), 4.43 (d, 2H, J = 4.0 Hz, NCH₂), 5.19 (dd, 1H, J = 12.0 Hz, 1.0 Hz, ^'H-3c'), 5.22 (dd, 1H, J = 12.0 Hz, 1.1 Hz, H-3c'), 5.90 (ddt, 1H, J_d = 17.1, 10.4 Hz, J_t = 5.1 Hz, H-3b'), 7.30 (d, 1H, J = 6.4 Hz, H-6), 11.14 (b,d; 1H, J = 4.0 Hz, NH-1), 11.56 ppm (br,s, 1H, NH-3). ¹³C NMR (100 MHz, DMSO‑d₆): δ_C = 35.1 (CH₂), 44.5 (NCH₂), 117.6 (C-3c'), 118.2 (C-5), 130.9 (C-3b'), 131.9 (C-6), 148.7 (C-2′), 150.6 (C-2), 160.1 (C-4), 172.1 ppm (C-4′). MS (70 eV, %): m/z = 268 (M + 2, 20), 267 (M + 1, 85), 266 (M⁺, 14), 111 (15), 225 (10), 195 (10), 154 (1 0 0), 137 (66), 102 (20). Anal. Calcd for C₁₀H₁₀N₄O₃S (266.28): C, 45.11; H, 3.79; N, 21.04; S, 12.04. Found: C, 45.23; H, 3.82; N, 21.11; S, 12.14.

2.2

2.2 Biology

2.2.1

2.2.1 Cell viability assay and evaluation of IC₅₀

2.2.1.1

2.2.1.1 MTT assay

The MTT assay was used to determine how the synthesized compounds affected the viability of the mammary epithelial normal cell line (MCF-10A) (Al-Wahaibi et al., 2020; Mohassab et al., 2021). See Appendix A (Supplementary file).

2.2.1.2

2.2.1.2 Antiproliferative test

To investigate the antiproliferative potential of compounds 3a-l and 5a-e, the MTT assay was carried out using various cancer cell lines following previously reported procedures (Al-Sanea et al., 2020). See Appendix A (Supplementary file).

2.2.1.3

2.2.1.3 EGFR inhibitory activity assay

2.2.1.4

2.2.1.4 BRAF kinase inhibitory assay

Compounds 3a, 3c, 3f, 3i, 5b and 5d were tested for their inhibitory activity against BRAF^V600E, and the results are shown as IC₅₀ values (Gomaa et al., 2022; El-Sherief et al., 2019). See Appendix A (Supplementary file).

3 Results and discussion

3.1

3.1 Chemistry

The synthetic sequence for the novel potentially biologically active molecules 3a-l and 5a-e are depicted in Scheme 1. Compounds 3a-l and 5a-e were synthesized from previously reported 1-(2,4-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl)thiourea 1a-e (Alshammari et al., 2022). The new compounds were obtained by heating the compounds 1a-e with α-bromo-acetophenone (2a), chloroacetone (2b), and ethyl bromoacetate (4) in refluxing ethanol and in the presence of triethylamine as a catalyst. IR, NMR, mass spectra, and elemental analyses confirmed the structure of the isolated compounds. COSY, ¹H–¹³C HSQC, and ¹H–¹³C HMBC experiments were used to assign signals from skeletal atoms unambiguously.

Synthesis of uracil-based derivatives 3a-l and 5a-e.

For example, the IR spectrum of 3b revealed absorption at υ_max = 3135 for NH, 1688–1670 for C⚌O, 1597 for C⚌N, and 1558 for C⚌C. ¹H NMR spectral data of the assigned compound 3b was identified as (Z)-5-((4-phenyl-3-(p-tolyl)thiazol-2(3H)-ylidene)amino)-pyrimidine-2,4(1H,3H)-dione (3b, Fig. 4) revealed singlet signals at δ_H = 11.73, 11.62, 7.18 and 2.38 ppm due to NH-3, NH-1, H-5′ and CH₃, respectively. H-6 appeared as a doublet at δ_H = 8.32 ppm (J = 6.3 Hz). The aromatic hydrogens system appeared between δ_H = 7.36–7.49 ppm. ¹³C NMR spectrum revealed represented signals at δ_C = 160.2, 150.2, 108.2, 105.0 and 20.7 ppm due to ⁴C = O, ²C = O, C-5, C-5′ and CH₃, respectively (Fig. 4). The p-tolyl methyl hydrogens (H-a) and carbon (C-a) are distinctive at δ_H = 2.38 and δ_C = 20.7 ppm. H-a gives COSY correlation with a 2H signal at δ_H = 7.39 ppm assigned as H-m; H-a also gives HMBC correlation with a tall, ¹³C-H signal at δ_C = 130.9 ppm, assigned as C-m, and an i-carbon-H at δ_C = 138.2 ppm, assigned as C-p. C-p gives HMBC correlation with a 4H signal at δ_H = 7.36 ppm. As the only signal with an odd number of hydrogens, H-p’ must appear at δ_H = 7.49 ppm. The hydrogens at δ_H = 7.36 ppm give HSQC correlation with carbons at δ_C = 129.2 and 124.0 ppm, and the hydrogens at δ_H = 7.49 give HSQC correlation with carbons at δ_C = 130.1 and 128.7 ppm. The line at δ_C = 130.1 is much smaller than that at δ_C = 128.7, so the line at δ_C = 130.1 ppm is assigned as C-p’; on chemical-shift grounds, the up-field line at δ_C = 124.0 ppm is assigned as C-o. (See Fig. 4).

Distinctive carbons and hydrogens for compound 3b.

On reacting compounds 1a-e with ethyl bromoacetate (4), a new series of 4-oxothiazolidin-2-ylidene)amino)pyrimidine-2,4(1H,3H)-diones 5a-e as more analogs for biological testing, was produced. The structure of 5a-e was determined using spectroscopic properties and elemental analyses. The Z-form of the obtained products was supported based on similar reactions between substituted thiosemicarbazides with α-bromoacetophenone and the X-ray structure of the obtained thiazole (Aly et al., 2019).

To rationalize our results, we choose compound 5d, which is assigned as (Z)-5-((3-methyl-4-oxothiazolidin-2-ylidene)amino)-pyrimidine-2,4(1H,3H)-dione (Fig. 5). Elemental analysis and mass spectrometry agreed with its general formula C₈H₈N₄O₃S and molecular weight (m/z = 240). The IR spectrum of compound 5d revealed absorptions of NH, C⚌O, C⚌N, and C⚌C at υ_max = 3168, 1687–1670, 1650, and 1598 cm⁻¹, respectively. The ¹H NMR spectrum for compound 5d showed three-singlet signals at δ_H = 11.26 (1H), 4.05 (2H), and 3.11 ppm (3H), which they are assigned as NH-3, CH_2, and methyl (CH₃) group, respectively. Further, the H-6 and NH-1 are distinctive at δ_H = 7.07 and 10.72 ppm. The ¹³C NMR spectrum of 5d clearly showed the presence of CH₃ and CH₂ carbons of the thiazole ring, which resonated at δ_C = 29.1 and 32.7 ppm. Furthermore, the ¹³C NMR spectrum revealed the presence of three carbonyl groups at δ_C = 172.1, 159.2, and 150.5 ppm for ^5′CO, ⁴CO, and ²CO, respectively. At the same time, the (C-6 and C-2′) carbon atoms resonated at δ = 122.0 and 159.9 ppm, respectively, as can be observed in Fig. 5.

Distinctive carbons and hydrogens for compound 5d.

The mechanism describes the formation of compounds 3a-l and 5a-e due to the attack of the thione-lone pair on the α-bromo-C in 2a,b, accompanied by elimination of a molecule of HX to give the intermediate 6 (Scheme 2). Subsequently, the cyclization process would occur via the internal nucleophilic attack of nitrogen lone-pair on the carbonyl carbon to give intermediates 7 (Scheme 2). Ultimately, a water molecule was eliminated from 7, resulting in thiazole 3 (Scheme 2). Similarly, intermediate 8 would be formed from the reaction of 1a-e with 4 and the elimination of the HX molecule. The cyclization step would be accompanied by the elimination of ethanol molecules to give compounds 5 (Scheme 2).

The suggested mechanism for the formation of 3a-l and 5a-e.

3.2

3.2 Biology

3.2.1

3.2.1 Cell viability assay

The human mammary gland epithelial (MCF-10A) cell line was used to evaluate the viability of new compounds. Compounds 3a-l and 5a-e were incubated on MCF-10A cells for four days before the viability of the cells was assessed using the MTT assay (-Al-Wahaibi et al., 2020; Mohassab et al., 2021). Table 1 demonstrates that none of the compounds examined exhibited cytotoxic effects, and the cell viability for the compounds examined at 50 µM was greater than 87 %.

Table 1 IC₅₀ of compounds 3a-l and 5a-e against four cancer cell lines.

Compound	Cell viability %	Antiproliferative activity IC₅₀ ± SEM (µM)
Compound	Cell viability %	A-549	MCF-7	Panc-1	HT-29	Average (GI₅₀)
3a	90	1.20 ± 0.20	1.10 ± 0.10	1.40 ± 0.20	1.30 ± 0.20	1.25
3b	89	2.70 ± 0.30	2.60 ± 0.30	3.10 ± 0.30	2.90 ± 0.30	2.80
3c	91	0.80 ± 0.10	0.90 ± 0.10	1.00 ± 0.10	0.90 ± 0.10	0.90
3d	91	2.00 ± 0.20	1.90 ± 0.20	2.30 ± 0.20	2.20 ± 0.20	2.10
3e	87	2.90 ± 0.30	2.80 ± 0.30	3.30 ± 0.30	3.20 ± 0.30	3.05
3f	89	1.80 ± 0.20	1.60 ± 0.10	2.10 ± 0.20	2.10 ± 0.20	1.90
3 g	91	2.30 ± 0.20	2.10 ± 0.20	2.50 ± 0.20	2.40 ± 0.20	2.30
3 h	92	3.30 ± 0.30	3.10 ± 0.30	3.50 ± 0.30	3.60 ± 0.30	3.40
3i	89	1.10 ± 0.10	1.00 ± 0.10	1.30 ± 0.10	1.20 ± 0.10	1.15
3j	87	6.20 ± 0.60	6.00 ± 0.60	6.50 ± 0.60	6.40 ± 0.60	6.30
3 k	90	4.80 ± 0.50	4.70 ± 0.40	4.90 ± 0.50	4.90 ± 0.50	4.80
3 l	87	5.50 ± 0.60	5.30 ± 0.50	5.80 ± 0.60	5.70 ± 0.60	5.60
5a	92	2.40 ± 0.20	2.30 ± 0.20	2.80 ± 0.20	2.70 ± 0.20	2.55
5b	90	1.50 ± 0.10	1.60 ± 0.10	1.80 ± 0.20	1.80 ± 0.20	1.70
5c	91	1.40 ± 0.10	1.30 ± 0.10	1.60 ± 0.10	1.50 ± 0.10	1.45
5d	87	7.70 ± 0.70	7.50 ± 0.70	7.80 ± 0.80	7.90 ± 0.80	7.70
5e	89	7.00 ± 0.60	6.80 ± 0.70	7.30 ± 0.70	7.20 ± 0.70	7.10
Doxorubicin	–	1.20 ± 0.10	0.90 ± 0.10	1.40 ± 0.10	1.00 ± 0.10	1.10

3.2.2

3.2.2 Antiproliferative assay

The antiproliferative activity of 3a-l and 5a-e was evaluated against the four human cancer cell lines Panc-1 (pancreatic cancer cell line), MCF-7 (breast cancer cell line), HT-29 (colon cancer cell line), and A-549 (lung cancer cell line) using the MTT assay (Al-Sanea et al., 2020) and doxorubicin as the reference drug. Table 1 displays the median inhibitory concentration (IC₅₀).

Overall, the antiproliferative activity of the compound examined was encouraging, with mean GI₅₀ values against the four cancer cell lines ranging from 0.90 µM to 7.70 µM. The most potent derivatives were compounds 3a, 3c, 3f, 3i, and 5b with GI₅₀ values ranging from 0.90 µM to 1.70 µM. With a mean GI₅₀ value of 0.90 µM against the four cancer cell lines, compound 3c (R = m-CH₃O-Ph, R¹ = Ph) was the most effective derivative of the synthetic compounds compared to doxorubicin (GI₅₀ = 1.10 µM). Compound 3c was found to be more potent than doxorubicin against three of the four cancer cell lines tested, namely A-549 (lung cancer cell line), Panc-1 (pancreatic cancer cell line), and HT-29 (colon cancer cell line) while being equipotent to doxorubicin against MCF-7 (breast cancer) cell line.

Compound 3i (R = m-CH₃O-Ph, R¹ = CH₃) ranks second in activity with a GI₅₀ of 1.15 µM, matching the potency of doxorubicin (GI₅₀ = 1.10 µM). Compound 3i has the same structural backbone as 3c except for the methyl group at position four of the thiazolidine ring moiety, indicating that the phenyl group, as in compound 3c, is more tolerated for antiproliferative activity than the methyl group.

Compounds 3a (R = Ph, R¹ = Ph) and 3b (R = p-CH₃-Ph, R¹ = CH₃) showed promising antiproliferative activity, with GI₅₀ values of 1.25 µM and 2.80 µM, respectively, being 1.4-fold and 3-fold less potent than 3c. These findings show that the substitution pattern of the phenyl moiety in the third position of the thiazolidine ring is essential for antiproliferative action, with activity increasing in the order m-CH₃O- > H > p-CH₃-.

Compounds 3e (R = CH₃, R¹ = Ph) and 3f (R = CH₂ = CH-CH₂–, R¹ = Ph) had GI₅₀ values of 3.05 µM and 1.90 µM, respectively, and were less potent than 3a (R = Ph, R¹ = Ph) indicating that un(substituted) phenyl ring is more tolerated for the antiproliferative action in the third position of thiazolidine moiety than allyl group and finally the methyl group. Compounds 3c (R = m-CH₃O-Ph) and 3d (R = CH₂-Ph) demonstrated great activity, with GI₅₀ values of 1.70 µM and 1.45 µM, respectively, whereas compounds 3e (R = CH₃) and 3f (R = CH₂ = CH-CH₂–) were the least potent derivatives, with GI₅₀ values of 7.70 µM and 7.10 µM, respectively.

3.2.3

3.2.3 EGFR inhibitory assay

The most effective antiproliferative derivatives (3a, 3c, 3f, 3i, 5b and 5c) were investigated for their inhibitory activity against EGFR as a possible target for their antiproliferative activity (Abdel-Aziz et al., 2021; Mohamed et al., 2021). The findings are presented as IC₅₀ values in Table 2. The results of this test are in line with those of antiproliferative assay, with compound 3c (R = m-CH₃O-Ph, R¹ = Ph), the most potent antiproliferative derivative, demonstrating the highest inhibitory activity against EGFR with an IC₅₀ value of 74 ± 07 nM, which is more potent than the reference erlotinib (IC₅₀ = 80 ± 05 nM). Compound 3i (R = m-CH₃O-Ph, R¹ = CH₃) is the second most active compound, with an IC₅₀ value of 89 ± 08 nM, followed by compound 3a (R = Ph, R¹ = Ph), which has an IC₅₀ value of 97 ± 08 nM. The remaining compounds tested had lower activity, with an IC₅₀ greater than 100 nM. After optimization, the results of this inhibitory assay test demonstrated that compounds 3c and 3i could be considered potent antiproliferative agents targeting EGFR.

Table 2 IC₅₀ of compounds 3a, 3c, 3f, 3i, 5b, and 5d against EGFR and BRAF^V600E.

Compound	EGFR inhibition IC₅₀ ± SEM (nM)	BRAF^V600E inhibition IC₅₀ ± SEM (nM)
3a	97 ± 08	138 ± 14
3c	74 ± 07	107 ± 10
3f	133 ± 13	167 ± 16
3i	89 ± 08	123 ± 12
5b	127 ± 13	159 ± 16
5d	115 ± 11	149 ± 15
Erlotinib	80 ± 05	60 ± 05

3.2.4

3.2.4 BRAF^V600E inhibitory assay

Compounds 3a, 3c, 3f, 3i, 5b, and 5d were tested further for inhibitory activity against BRAF^V600E, and the results are shown in Table 2 as IC₅₀ values (El-Sherief et al., 2019; Gomaa et al., 2022). Again, compound 3c was found to be the most potent BRAF^V600E inhibitor, with an IC₅₀ value of 107 ± 10 nM, 1.8-fold less potent than the reference drug erlotinib (IC₅₀ = 60 ± 05 nM).

Compounds 3i and 3a ranked second and third in activity with IC₅₀ values of 123 ± 12 nM and 138 ± 14 nM, respectively. Considering the previous, we can be concluded that compounds 3c and 3i could be regarded as potent antiproliferative agents with dual inhibitory activity against EGFR and BRAF^V600E, though further structural modifications to develop more potent derivatives are required.

3.3

3.3 Molecular docking

The 3D structures of EGFR and BRAF^V600E with PDB codes 1 M17 (Stamos et al., 2002) and 3OG7 (Bollag et al., 2010), respectively, were retrieved and utilized as templates for all docking predictions. All heteroatoms, ions, inhibitors, and water molecules were extracted to prepare the PDB files. All missing residues were built using Modeller software (Martí-Renom et al., 2000). The empirical program PropKa was utilized to assign the protonation state of residues of the investigated target (Olsson et al., 2011). The 3D structures of compounds 3a, 3c and 3i were modeled using Omega2 software (Hawkins et al., 2010; OMEGA 2013). The generated structures were energetically minimized using the MMFF94S force field within SZYBKI software (Halgren, 1999; SZYBKI 2016).

AutoDock Vina1.1.2 (Trott and Olson, 2010) and AutoDock4.2.6 (Morris et al., 2009) software were used to execute all docking computations. The investigated targets were prepared as described elsewhere (Ibrahim et al., 2022, 2021b, 2021a ). The docking parameters of AutoDock Vina1.1.2 and AutoDock4.2.6 software were generally kept at their default. For AutoDock Vina1.1.2 software, the exhaustiveness number was adjusted to 200. The grid box size was located at 19 Å × 19 Å × 19 Å. Besides, the grid spacing value was set to 1.0 Å. For AutoDock4.2.6 software, A maximum number of 25,000,000 energy evaluations and 250 independent docking runs were utilized. The grid box was tailored to fit the active site of EGFR and BRAF^V600E proteins, with a grid size of 50 Å × 50 Å × 50 Å, with a spacing value of 0.375 Å. The grid center was positioned at the center of the active sites of EGFR and BRAF^V600E proteins. The Gasteiger-Marsili method was employed to assign the atomic charges of these compounds (Gasteiger and Marsili, 1980). All molecular interactions were visualized using BIOVIA Discovery Studio Visualizer 2020 (Dassault Systèmes BIOVIA).

The binding features and scores of compounds 3a, 3c, and 3i against EGFR and BRAF^V600E were predicted using AutoDock Vina1.1.2 and AutoDock4.2.6 software. The anticipated binding scores and features of these compounds are summarized in Table 3. From data in Table 3, all investigated compounds demonstrated promising AutoDock scores against EGFR and BRAFV600E with values of − 8.9 to − 9.1 kcal/mol and − 8.4 to − 8.6 kcal/mol, respectively.

Table 3 Computed Vina and AutoDock scores (kcal/mol) and binding features for compounds 3a, 3c, 3i, and erlotinib towards EGFR and BRAF^V600E.

Compound	EGFR			BRAF^V600E
Compound	Vina Score (kcal/mol)	AutoDock Score (kcal/mol)	Binding features^a,b	Vina Score (kcal/mol)	AutoDock Score (kcal/mol)	Binding features^a,b
3a	−8.5	−8.9	CYS773 (2.31 Å), ARG817 (2.11 Å)	−7.2	−8.4	THR529 (3.17 Å), ALA481 (2.71 Å), ILE527 (2.84 Å)
3c	−8.7	−9.1	MET769 (1.96 Å), ARG817 (2.16 Å), CYS773 (2.35 Å)	−7.4	−8.6	THR529 (3.30 Å), ILE527 (3.08 Å), LYS483 (3.26 Å)
3i	−8.6	−9.0	MET769 (1.96 Å), ARG817 (2.17 Å), CYS773 (2.28 Å)	−7.3	−8.5	CYS532 (1.92, 2.07 Å)
Erlotinib	−7.9	−8.6	CYS773 (1.91 Å), MET769 (1.62 Å)	−7.0	−8.4	CYS532 (2.02 Å), THR529 (2.07 Å)

a Only conventional hydrogen bonds are listed.

b Binding features predicted by AutoDock4.2.6 software are listed.

Compared to AutoDock scores, all investigated compounds demonstrated plausible Vina scores towards EGFR and BRAF^V600E with values ranging from − 8.5 to − 8.7 kcal/mol and from − 7.2 to − 7.4 kcal/mol, respectively (Table 3). Notably, the binding modes predicted by AutoDock Vina1.1.2 software of the investigated compounds with the targets were very similar to those anticipated by AutoDock4.2.6 software. The good binding affinities of investigated compounds against EGFR and BRAF^V600E may be attributed to their ability to exhibit H-bonds, pi-based, hydrophobic, and van der Waals interactions with the proximal amino acids inside the active sites of EGFR and BRAF^V600E. Compound 3c revealed eminent AutoDock scores of − 9.1 and − 8.6 kcal/mol towards EGFR and BRAF^V600E, respectively (Table 3). More exactly, compound 3c exhibited three hydrogen bonds with MET769 (1.96 Å), ARG817 (2.16 Å), and CYS773 (2.35 Å) and three hydrogen bonds with THR529 (3.30 Å), ILE527 (3.08 Å) and LYS483 (3.26 Å) inside the binding pockets of EGFR and BRAF^V600E, respectively (Figs. 6 and 7). Compound 3i unveiled the second-lowest AutoDock score with values of − 9.0 and − 8.5 kcal/mol with EGFR and BRAF^V600E, respectively (Table 3). Inspecting the binding modes revealed that compound 3i formed three and two hydrogen bonds with MET769 (1.96 Å), ARG817 (2.17 Å), and CYS773 (2.28 Å) and CYS532 (1.92, 2.07 Å) inside the binding pockets of EGFR and BRAF^V600E, respectively (Figs. 6 and 7).

3D and 2D molecular interactions of compounds (a) 3a, (b) 3c, and (c) 3i with key residues of EGFR predicted by AutoDock4.2.6 software.

3D and 2D molecular interactions of compounds (a) 3a, (b) 3c, and (c) 3i with key residues of BRAFV600E predicted by AutoDock4.2.6 software.

As listed in Table 3, compound 3a manifested good docking scores with values of − 8.9 and − 8.4 kcal/mol towards EGFR and BRAF^V600E, respectively. Notably, compound 3a exhibited two and three hydrogen bonds with CYS773 (2.31 Å) and ARG817 (2.11 Å) and THR529 (3.17 Å), ALA481 (2.71 Å), and ILE527 (2.84 Å) inside the active sites of EGFR and BRAF^V600E, respectively (Table 3 and Fig. 6).

It is worth mentioning that compounds 3a, 3c and 3i demonstrated pi-sulfur interaction with MET742 residue inside the active site of EGFR (Fig. 6). Besides, compound 3a interacted with CYS773 by pi-sigma interaction within the binding site of EGFR (Fig. 6). On the other hand, compounds 3a, 3c and 3i pi-lone pair with CYS532, pi-pi stacking with TRP531, and pi-pi T-shaped were noted with PHE583 amino acid within the binding pocket of BRAF^V600E (Fig. 7).

Erlotinib, a positive control, exposed good docking scores against EGFR and BRAF^V600E with values of − 8.6 and − 8.4 kcal/mol, respectively (Table 3). Erlotinib formed two hydrogen bonds with CYS773 (1.91 Å) and MET769 (1.62 Å) and two hydrogen bonds with CYS532 (2.02 Å) and THR529 (2.07 Å) within the binding pockets of EGFR and BRAF^V600E, respectively (Table 3). The current outcomes highlighted the importance of compounds 3a, 3c, and 3i as potential antiproliferative agents.

3.4

3.4 ADMET study

Swiss ADME server was employed to predict the drug-likeness characteristics of compounds 3a, 3c, and 3i (Daina et al., 2017). Under the framework of the Lipinski rule, five properties were deemed, involving the hydrogen bond acceptors (HBA ≤ 10), hydrogen bond donors (HBD ≤ 5), log p-value (log p0/w ≤ 5), molecular weight (MWt ≤ 500), and topological polar surface area (TPSA ≤ 140). Crossing these properties points out that the investigated compounds are orally bioavailable.

The absorption, distribution, metabolism, excretion, and toxicity (ADMET) properties for compounds 3a, 3c, and 3i were estimated using a freely accessible web server pkCSM tool (Pires et al., 2015). Absorption (A) includes HIA (human intestinal absorption) and Caco-2 (human colorectal carcinoma) permeability. Distribution (D) is predicted by BBB (blood–brain barrier) permeability. The metabolism (M) is anticipated by CYP3A4 substrate/inhibitor. The excretion (E) and toxicity (T) were estimated by inhibitor total clearance and hepatotoxicity.

SwissADME server was utilized to predict the prospective character of compounds 3a, 3c, 3i and erlotinib as drugs via evaluating the drug-likeness properties. The drug-likeness properties included MWt (g/mol), HBA, HBD, log p0/w, and TPSA (Å²) (Table 4). As enrolled in Table 4, the MWts were 362.4, 392.4, 330.4, and 393.4 g/mol for compounds 3a, 3c, 3i and erlotinib, respectively, allowing skin absorption. The number of HBA was < 10, and the number of HBA was < 5 (Table 4). Additionally, the Log P0/w values of compounds 3a, 3c, 3i, and erlotinib were promising, with values < 5 (Table 4). The current results demonstrated that compounds 3a, 3c, 3i, and erlotinib are promising inhibitors against EGFR and BRAF^V600E.

Table 4 Predicted physiochemical and pharmacokinetic characteristics of compounds 3a, 3c, 3i, and erlotinib as EGFR and BRAF^V600E inhibitors.

Characteristics	3a	3c	3i	Erlotinib
Drug Likeness Properties
MWt	362.4	392.4	330.4	393.4
HBD	2	2	2	1
HBA	3	4	4	6
log p0/w	2.6	1.9	0.9	1.9
TPSA	111.3	120.5	120.5	74.7
ADMET Properties
Absorption (A)
HIA	97.9 %	89.1 %	82.0 %	95.4 %
Caco2 Permeability	0.8	0.5	0.1	1.0
Distribution (D)
BBB Permeability	−0.5	−0.9	−0.9	−0.6
Metabolism (M)
CYP1A2 Inhibitor	Yes	Yes	No	No
Excretion (E)
Total Clearance	−0.07	0.2	0.03	0.5
Toxicity (T)
AMES toxicity	No	No	Yes	No

The knowledge of ADMET characteristics presents significant guidelines for starting stage drug discovery. HIA and Caco2 permeability must be deemed in any drug discovery process to anticipate the absorption property (Pires et al., 2015). The inspected compounds manifested good absorption with HIA values of 97.9 %, 89.1 %, 82.0 %, and 95.4 % for compounds 3a, 3c, 3i and erlotinib, respectively (Table 4). Compounds 3a, 3c, and 3i, exposed perfect Caco2 permeability with a value of < 0.9 cm/s. However, erlotinib showed poor Caco2 permeability with a value of 1.0 cm/s (Table 4). To examine drug distribution, the BBB membrane permeability was evaluated. Remarkable distribution volumes were observed for compounds 3a, 3c, 3i, and erlotinib with log BB values of − 0.5, −0.9, −0.9 and − 0.6, respectively, demonstrating these compounds can cross BBB facilely (Table 4). CYP450 has an essential role in drug metabolism. The metabolism anticipations disclosed that compound 3i and erlotinib could not inhibit CYP1A2 (Table 4). The total drug clearance was − 0.07, 0.2, 0.03, and 0.5 mL/min/kg for compounds 3a, 3c, 3i, and erlotinib, respectively (Table 4). Toxicity plays a vital role in selecting drugs. Compounds 3a, 3c, and erlotinib did not demonstrate AMES toxicity; however, compound 3i showed AMES toxicity (Table 4). These findings proved that compounds 3a, 3c and 3i might be utilized as putative antiproliferative agents.

3.5

3.5 Structural activity relationship (SAR) analysis

The structural activity relationship of compounds 3a-l and 5a-e can be summarized in Fig. 8.

SAR analysis of compounds 3a-l and 5a-e.

4 Conclusion

Due to the importance of thiazolyl pyrimidine derivatives, we direct for the synthesis of (Z)-5-((thiazol-2(3H)-ylidene)amino)pyrimidine-2,4(1H,3H)-diones 3a-l through the reaction of thioureas 1a-e with α-bromoacetophenone (2a) and/or chloroacetone (3b). Similarly, thiazolidinones 5a-e derived by uracil molecule were obtained via the reaction of 1a-e with ethyl 2-bromoacetate. The structure of compounds was examined by ¹H, ¹³C NMR, 2D NMR, and ¹⁵N NMR spectroscopy and elemental analyses. The tested compounds displayed promising antiproliferative action, encouraging inhibitory activities against EGFR and/or BRAF^V600E. Moreover, the docking poses and scores of compounds 3a, 3c, and 3i towards EGFR and BRAF^V600E were revealed using AutoDock4.2.6 software. Based on the docking scores, compounds 3a, 3c and 3i displayed good docking scores towards EGFR and BRAF^V600E. Notably, the computed docking scores were in line with the IC₅₀ values. Compounds 3a, 3c and 3i also demonstrated excellent physicochemical and pharmacokinetic characteristics.

CRediT authorship contribution statement

Ashraf A. Aly: Conceptualization, Writing – original draft, Writing – review & editing. M.B. Alshammari: Editing and revision. A. Ahmad: Editing. H.A.M. Gomaa: Biology, Editing. Bahaa G.M. Youssif: Biology, Methodology, Writing – review & editing. S. Bräse: Editing and revision. Mahmoud A.A. Ibrahim: Writing – review & editing. Asmaa H. Mohamed: Conceptualization, Methodology, Writing – original draft.

Acknowledgments

The authors thank the Deanship of Scientific Research at Prince Sattam bin Abdulaziz University under research project No 2021/01/18104. We also acknowledge support from the KIT-Publication Fund of the Karlsruhe Institute of Technology. Resources department at KIT for providing Prof Ashraf A. Aly with a two-month contract at the Karlsruhe Institute of Technology, Karlsruhe, Germany, from 2 August to 26 September 2022

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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Appendix A

Supplementary material

Supplementary data to this article can be found online at https://doi.org/10.1016/j.arabjc.2023.104612.

Appendix A

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ADMET study
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Design, synthesis, docking and mechanistic studies of new thiazolyl/thiazolidinylpyrimidine-2,4-dione antiproliferative agents

Abstract

Keywords

Uracil

Thiazoles

Thiazolidines

Antiproliferative

Kinases

Docking

Abbreviations

1 Introduction

2 Experimental (instruments in the Suppl. File)

2.1 Chemistry

2.1.1 General procedure for the synthesis of compounds 3a-l

2.1.1.1 (Z)-5-((3,4-Diphenylthiazol-2(3H)-ylidene)amino)pyrimidine-2,4(1H,3H)-dione (3a)

2.1.1.2 (Z)-5-((4-Phenyl-3-(p-tolyl)thiazol-2(3H)-ylidene)amino)pyrimidine-2,4(1H,3H)-dione (3b)

2.1.1.3 (Z)-5-((3-(3-Methoxyphenyl)-4-phenylthiazol-2(3H)-ylidene)amino)-pyrimidine-2,4(1H,3H)-dione (3c)

2.1.1.4 (Z)-5-((3-Benzyl-4-phenylthiazol-2(3H)-ylidene)amino)pyrimidine-2,4(1H,3H)-dione (3d)

2.1.1.5 (Z)-5-((3-Methyl-4-phenylthiazol-2(3H)-ylidene)amino)pyrimidine-2,4(1H,3H)-dione (3e)

2.1.1.6 (Z)-5-((3-Allyl-4-phenylthiazol-2(3H)-ylidene)amino)pyrimidine-2,4(1H,3H)-dione (3f)

2.1.1.7 (Z)-5-((4-Methyl-3-phenylthiazol-2(3H)-ylidene)amino)pyrimidine-2,4(1H,3H)-dione (3g)

2.1.1.8 (Z)-5-((4-Methyl-3-(p-tolyl)thiazol-2(3H)-ylidene)amino)pyrimidine-2,4(1H,3H)-dione (3h)

2.1.1.9 (Z)-5-((3-(3-Methoxyphenyl)-4-methylthiazol-2(3H)-ylidene)amino)pyrimidine-2,4(1H,3H)-dione (3i)

2.1.1.10 (Z)-5-((3-Benzyl-4-methylthiazol-2(3H)-ylidene)amino)pyrimidine-2,4(1H,3H)-dione (3j)

2.1.1.11 (Z)-5-((3,4-Dimethylthiazol-2(3H)-ylidene)amino)pyrimidine-2,4(1H,3H)-dione (3k)

2.1.1.12 (Z)-5-((3-Allyl-4-methylthiazol-2(3H)-ylidene)amino)pyrimidine-2,4(1H,3H)-dione (3l)

2.1.2 General procedure for the synthesis of compounds 5a-e

2.1.2.1 (Z)-5-((4-Oxo-3-phenylthiazolidin-2-ylidene)amino)pyrimidine-2,4(1H,3H)-dione (5a)

2.1.2.2 (Z)-5-((3-(3-Methoxyphenyl)-4-oxothiazolidin-2-ylidene)amino)pyrimidine-2,4(1H,3H)-dione (5b)

2.1.2.3 (Z)-5-((3-Benzyl-4-oxothiazolidin-2-ylidene)amino)pyrimidine-2,4(1H,3H)-dione (5c)

2.1.2.4 (Z)-5-((3-Methyl-4-oxothiazolidin-2-ylidene)amino)pyrimidine-2,4(1H,3H)-dione (5d)

2.1.2.5 (Z)-5-((3-Allyl-4-oxothiazolidin-2-ylidene)amino)pyrimidine-2,4(1H,3H)-dione (5e)

2.2 Biology

2.2.1 Cell viability assay and evaluation of IC50

2.2.1.1 MTT assay

2.2.1.2 Antiproliferative test

2.2.1.3 EGFR inhibitory activity assay

2.2.1.4 BRAF kinase inhibitory assay

3 Results and discussion

3.1 Chemistry

3.2 Biology

3.2.1 Cell viability assay

3.2.2 Antiproliferative assay

3.2.3 EGFR inhibitory assay

3.2.4 BRAFV600E inhibitory assay

3.3 Molecular docking

3.4 ADMET study

3.5 Structural activity relationship (SAR) analysis

4 Conclusion

CRediT authorship contribution statement

Acknowledgments

Declaration of Competing Interest

References

Appendix A

Supplementary material

Appendix A

Supplementary material

Supplementary data 1

Suggested read for related articles:

2.2.1 Cell viability assay and evaluation of IC₅₀

3.2.4 BRAF^V600E inhibitory assay