Convenient synthesis and X-ray determination of 2-amino-6H-1,3,4-thiadiazin-3-ium bromides endowed with antiproliferative activity
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This is a self-archived version of an original article. This version may differ from the original in pagination and typographic details. Author(s): Title: Year: Version: Copyright: Rights: Rights url: Please cite the original version: CC BY-NC 4.0 https://creativecommons.org/licenses/by-nc/4.0/ Convenient synthesis and X-ray determination of 2-amino-6H-1,3,4-thiadiazin-3-ium bromides endowed with antiproliferative activity © 2024 the Authors Published version Tawfeek, Hendawy N.; Abdelmoez, Alshaimaa; Dahlous, Kholood A.; Youssif, Bahaa G. M.; Bräse, Stefan; Rissanen, Kari; Nieger, Martin; El-Sheref, Essmat M. Tawfeek, H. N., Abdelmoez, A., Dahlous, K. A., Youssif, B. G. M., Bräse, S., Rissanen, K., Nieger, M., & El-Sheref, E. M. (2024). Convenient synthesis and X-ray determination of 2-amino-6H1,3,4-thiadiazin-3-ium bromides endowed with antiproliferative activity. RSC Advances, 14(25), 17866-17876. https://doi.org/10.1039/D4RA02531H 2024
Convenient synthesis and X-ray determination of 2amino-6H-1,3,4-thiadiazin-3-ium bromides endowed with antiproliferative activity† Hendawy N. Tawfeek, ab Alshaimaa Abdelmoez, cd Kholood A. Dahlous, e Bahaa G. M. Youssif, * c Stefan Br¨ ase, * f Kari Rissanen, g Martin Nieger h and Essmat M. El-Sheref a A new series of 1,3,4-thiadiazin-3-ium bromide derivatives 9a–gwere prepared as a six-member ring by interactions between 4-substituted thiosemicarbazides 8a–eand a-halo ketones 2a,b. The reaction was conducted using hydrazine-NH 2 and yielded a hexagonal shape. The structures of all obtained compounds have been verified using IR, NMR spectra, mass spectrometry, elemental analysis, and X-ray crystallography. The X-ray crystallographic analysis of compounds 9a and 9b has revealed that the salt is formed with the nitrogen atom N3 when the aromatic substituents 9a and 9d are present, but in the case of compounds 9b,9c,9e,9f, and 9g with the aliphatic substituent, the salt is formed outside the ring. Compounds 9a–gwere evaluated for antiproliferative activity as multitargeted inhibitors. Results revealed that targets 9a–gdisplayed good antiproliferative activity, with GI 50 ranging from 38 nM to 66 nM against a panel of four cancer cell lines compared to the reference Erlotinib (GI 50 =33 nM). Compounds 9a,9c, and 9d were the most potent antiproliferative derivatives, with GI 50 values of 43, 38, and 47 nM, respectively. Compounds 9a,9c, and 9d were evaluated for their inhibitory activity against EGFR, BRAF V600E , and VEGFR-2. The in vitro experiments demonstrated that the compounds being examined exhibit potent antiproliferative properties and have the potential to function as multitargeted inhibitors. In addition, the western blotting investigation demonstrated the inhibitory effects of 9c on EGFR, BRAF V600E , and VEGFR-2. 1. Introduction Cancer remains a signicant global problem. It is the second leading cause of death aer heart problems. 1 There are currently more than 100 different forms of cancer, each necessitating a distinct diagnosis and therapy. 2 Many effective anticancer medicines, including standard chemotherapy drugs, suppress cell division and DNA replication. Nevertheless, many medications on the market are generic and oen have common adverse effects. 3 Thus, searching for new lead structures and chemical components to develop effective, innovative anticancer drugs has become a top priority in medicinal chemistry, considering current cancer treatments' limitations and adverse effects. 4–6 Anti-cancer medication discovery has heavily focused on developing treatments targeting a specic site with good efficacy and specicity. Clinical observations, such as identifying drug resistance in cancer treatment, have shown that focusing on one target may not always provide the intended biological outcome, even if the target is deactivated or suppressed. 7–9 The resistance develops due to the target's self-modication through mutation or the cancer cell's adoption of new routes for growth and multiplication. 10 Targeting a single oncoprotein has not led to successful treatment and may not be enough to establish long-lasting remission in patients. 11 Thus, adjusting the biological network is acknowledged to be advantageous. There are currently two opposing methodologies for designing multi-targeting medicines. Combination medication therapy involves using numerous drugs that act on different a Chemistry Department, Faculty of Science, Minia University, El Minia, 61519 Egypt b Unit of Occupational of Safety and Health, Administration Office of Minia University, El-Minia 61519, Egypt c Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Assiut University, Assiut 71526, Egypt. E-mail: [email protected]; bahaa.youssif@ pharm.aun.edu.eg; Tel: +20-1098294419 d Department of Neurology, Ulm University, Ulm, Germany e Department of Chemistry, College of Science, King Saud University, Riyadh 11451, Saudi Arabia f Institute of Biological and Chemical Systems, IBCS-FMS, Karlsruhe Institute of Technology, 76131 Karlsruhe, Germany. E-mail: [email protected] g Department of Chemistry, University of Jyv¨ askyl¨ a, PO Box 35, 40014 Jyv¨ askyl¨ a, Finland h Department of Chemistry, University of Helsinki, PO Box 55, A. I. Virtasen Aukio 1, 00014 Helsinki, Finland †Electronic supplementary information (ESI) available. CCDC 2329624 (9a)and 2329625 (9b). For ESI and crystallographic data in CIF or other electronic format see DOI: https://doi.org/10.1039/d4ra02531h Cite this: RSC Adv.,2024,14, 17866 Received 3rd April 2024 Accepted 28th May 2024 DOI: 10.1039/d4ra02531h rsc.li/rsc-advances 17866 |RSC Adv.,2024,14,17866–17876 © 2024 The Author(s). Published by the Royal Society of Chemistry RSC Advances PAPER Open Access Article. Published on 27 June 2024. Downloaded on 7/18/2024 9:06:31 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online View Journal | View Issue
targets to provide an additive or synergistic impact. Combination therapies have shown success in treating metastatic melanoma with BRAF mutations. 12,13 The FDA has approved the combination of dabrafenib (BRAF inhibitor) and trametinib (MEK inhibitor) based on preclinical evidence of increased apoptosis and delayed resistance to serine/threonine-protein kinase B-Raf. 14,15 Phase III clinical trials have shown good results for combination therapy using RAF inhibitor (vemurafenib) and MEK inhibitor (cobimetinib) in treating BRAF mutant melanoma. 16 Another successful example of combination therapy involves utilizing palbociclib and letrozole to treat advanced breast cancer. 17 The second approach is to identify and develop medicines that target numerous oncogenic pathways simultaneously. Multi-targeting therapies entail the discovery of a single agent capable of simultaneously acting on two or more targets. 18–20 The US Food and Drug Administration (FDA) has approved Lenvima (Lenvatinib) as a receptor tyrosine kinase inhibitor that blocks the kinase activities of vascular endothelial growth factor (VEGF) receptors VEGFR1, VEGFR2, and VEGFR3. 21 Cabozantinib, known as cabometyx, is an FDA-approved small molecule inhibitor targeting the tyrosine kinases c-Met and VEGFR-2. It has effectively reduced tumor growth, metastasis, and angiogenesis. 21 Nitrogen and sulfur-containing compounds are commonly used as the basis for various biological components in medicinal chemistry. 22–24 Thiadiazine-based compounds have garnered considerable attention for their broad applicability as physiologically active molecules. 25,26 Thiadiazine derivatives have shown signicant cytotoxic effects on cancer cell lines by disrupting DNA synthesis, causing cell cycle arrest, inhibiting tumor cell invasion and migration, triggering apoptosis through the mitochondrial pathway, and blocking various nuclear enzymes. 27–31 Ragab et al. 27 described the design, synthesis, and biological testing of novel 1,3,4-thiadiazine-based derivatives as potential anti-cancer agents against non-small cell lung cancer (NSCLC) cells. The most effective compounds were chosen for further study as multitargeted inhibitors of VEGFR-2, BRAF V600E , and matrix metalloproteinase 9 (MMP9). Compound I(Fig. 1) showed the most promising results among the compounds tested. It demonstrated inhibitory activity against VEGFR-2, similar to Sorafenib, with an IC 50 value of 0.11 ±0.01 mM. It also exhibited the most potent suppression of BRAF V600E activity, with an IC 50 value of 0.178 ±0.004 mM, and MMP9 inhibition, with an IC 50 value of 0.08 ±0.004 mM. Another study explored the synthesis of some novel anticancer agents derived from triazolothiadiazine. When tested against a panel of NCI-60 cancer cell lines. 29 Compound II (Fig. 1) showed a potential antiproliferative effect. Compound II was evaluated against 16 kinases to determine its antiproliferative mechanism. The study signicantly suppressed EGFR, VEGFR-2, CDK-2, and GSK-3b. Furthermore, compound II increased the amount of active caspase-3, causing cell cycle arrest at the G2-M phase with apoptotic action. On the other hand, phenacyl bromides, also known as a-halo ketones and primary alkyl halides, play a crucial role in chemical synthesis. They serve as crucial building blocks in heterocyclic constructions as a vital intermediate for synthesizing various bioactive chemicals and natural products. 32 Also, thiosemicarbazides are a vital class of organic compounds that operate as the backbone of a range of heterocyclic compounds of considerable interest in medical and industrial uses. 33,34 Additionally, interactions with phenacyl bromides revealed that thiosemicarbazides have several active centers. 35 Busby and Domincy were the rst to investigate the behavior of 4substituted thiosemicarbazides toward haloketones, obtaining 2-amino-1,3,4-thiadiazines. 36 Pfeiffer et al. synthesized 1,3,4thiadiazines by combining a-halo ketones and 1,2,4-trialkyl thiosemicarbazides. The structure of the thiadiazines was conrmed through desulfurization with acetic acid. 37 Several methods synthesized 1,3,4-thiadiazine, including interactions Fig. 1 Structures of some reported 1,3,4-thiadiazine-based multitargeted anti-cancer agents and target compounds 9a–g. © 2024 The Author(s). Published by the Royal Society of Chemistry RSC Adv.,2024,14,17866–17876 | 17867 Paper RSC Advances Open Access Article. Published on 27 June 2024. Downloaded on 7/18/2024 9:06:31 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online
between thiosemicarbazides and a-halo ketones in different solvents. 38,39 However, Aly et al. 40 demonstrated the one-pot synthesis of 2-ylidenehydrazonothiazoles from the reaction of 4-substituted thiosemicarbazides, ketones, and phenacyl bromide, and they proposed that the product was obtained through the formation of the hydrazinylthiazole intermediate, which contradicted the ndings of Pfeiffer et al. 41 In light of the previous data on the multitargeted inhibitory action of 1,3,4-thiadiazine derivatives and our ongoing efforts to develop multitargeted anti-cancer agents, 6,7,19,42–44 this investigation focuses on the synthesis of new 1,3,4-thiadiazine derivatives (9a–g, Fig. 1) as multitargeted antiproliferative agents. 2. Experiments 2.1. Chemistry 2.1.1 General details: see Appendix A. 4-Substituted thiosemicarbazides 8a–e 45 were prepared according to published literature, and 2-bromo-1-phenylethanone (2a), 2-bromo-1-(4bromophenyl)ethanone (2b) were used as purchased from Merck. 2.1.2. General procedure for the synthesis of 2-substituted amino-1,3,4-thiadiazin-3-ium bromide derivatives (9a–g). In a conical ask, mix 1 mmol of 4-substituted thiosemicarbazides 8a–ewith 199 mg of 2-bromo-1-phenylethanone (1 mmol, 2a)or 278 mg of 2-bromo-1-(4-bromophenyl)ethanone (1 mmol, 2b)in 30 ml ethanol at low temperature. The reaction mixtures were stirred magnetically at room temperature for 1–2 hours. Then, the resulting solid was ltered offand washed with ethanol three times, dried well, and recrystallized from hot ethanol to afford the thiadiazinium bromides 9a–gin excellent yields. 2.1.3. 5-Phenyl-2-(phenylamino)-6H-1,3,4-thiadiazin-3-ium bromide (9a). Colorless crystals (95%, 0.329 g), m.p. =197– 198 °C. IR (KBr, cm −1 ): 3164 (NH), 3015 (Ar-CH), 2988 (ali-CH), 1617 (C]N), 1594 (C]C) cm −1 . 1 H NMR (300 MHz, CDCl 3 ): d H =3.91 (s, 2H, CH 2 ), 7.35–7.48 (m, 6H, Ar-H), 7.88–7.94 (m, 2H, Ar-H), 8.01–8.04 (m, 2H, Ar-H), 9.45 (bs, 1H, exocyclic-NH), 12.85 (bs, 1H, cyclic-NH + ) ppm. 13 C NMR (75 MHz, DMSO-d 6 ): d c =23.26 (CH 2 ), 123.84, 126.01, 127.67, 128.80, 129.46, 130.03 (Ar-CH), 132.94, 139.15 (Ar–C), 144.32 (thiadiazine-C5), 150.71 (thiadiazine-C2) ppm. MS (70 eV) m/z(%): 347 (M + , 9), 267 (100), 155 (10), 119 (5). Anal. calcd for C 15 H 14 BrN 3 S (348.26) calcd: C, 51.73; H, 4.05; N, 12.07; S, 9.21. Found: C, 51.81; H, 3.99; N, 12.13; S, 9.28. 2.1.4. (Z)-N-(5-Phenyl-3,6-dihydro-2H-1,3,4-thiadiazin-2ylidene)prop-2-en-1-aminium bromide (9b). Yellow crystals (91%, 0.287 g), m.p. =224–226 °C. IR (KBr, cm −1 ). 1 H NMR (300 MHz, CDCl 3 ): d H =4.01–4.04 (m, 2H, Allyl-CH 2 ), 4.10 (s, 2H, CH 2 ), 5.23–5.34 (m, 2H, Allyl-CH 2 ]), 5.72–5.83 (m, 1H, AllylCH]), 7.33–7.44 (m, 3H, Ar-H), 7.72–7.79 (m, 2H, Ar-H), 10.62 (bs, 1H, exocyclic-NH + ), 12.98 (bs, 1H, cyclic-NH) ppm. 13 C NMR (75 MHz, CDCl 3 ): d c =22.99 (CH 2 ), 48.01 (CH 2 -Allyl), 119.91 (Allyl-CH 2 ]), 127.01, 129.02, 130.17, 131.71 (Ar-CH), 132.18 (ArC), 149.36 (C5), 166.75 (C2) ppm. MS (70 eV) m/z(%): 311 (M + , 15), 283 (28), 199 (54), 189 (10), 119 (33), 102 (100). Anal. calcd for C 12 H 14 BrN 3 S (311.23) calcd: C, 46.16; H, 4.52; N, 13.46; S, 10.27. Found: C, 46.23; H, 4.59; N, 13.39; S, 10.30. 2.1.5. (Z)-N-(5-Phenyl-3,6-dihydro-2H-1,3,4-thiadiazin-2ylidene) cyclohexanaminium bromide (9c). Yellow crystals (86%, 0.300 g), m.p. =217–219 °C. IR (KBr, cm −1 ): 3158 (NH), 3106, 3027 (Ar-CH), 2992, 2917 (ali-CH), 1613, 1581 (C]N), 1538, 1445 (C]C) cm −1 . 1 H NMR (300 MHz, CDCl 3 ): d H =125– 1.33, 1.36–1.69, 2.01–2.04 (m, 10H, cyclohexyl-CH 2 ), 3.02–3.09 (m, 1H, cyclohexyl-CH), 3.55 (s, 2H, CH 2 ), 7.33–7.37 (m, 3H, ArH), 7.80–7.82 (m, 2H, Ar-H), 10.20 (bs, 1H, exocyclic-NH + ), 11.84 (bs, 1H, cyclic-NH) ppm. 13 C NMR (75 MHz, CDCl 3 ): d c =22.46 (CH 2 ), 24.54, 25.23, 33.30 (cyclohexyl-CH 2 ), 52.98 (cyclohexylCH), 126.34, 128.42, 129.50 (Ar-CH), 135.31 (Ar-C), 147.38 (C5), 166.85 (C2) ppm. MS (70 eV) m/z(%): 353 (M + , 3), 274 (M + -HBr, 100), 192 (7), 133 (3), 104 ([PhCN] + , 4). Anal. calcd for C 15 H 20 BrN 3 S (354.31) calcd: C, 50.85; H, 5.69; N, 11.86; S, 9.05. Found: C, 50.78; H, 5.63; N, 11.90; S, 9.11. 2.1.6. 5-(4-Bromophenyl)-2-(phenylamino)-6H-1,3,4thiadiazin-3-ium bromide (9d). Yellow crystals (87%, 0.371 g), m.p. =185–186 °C. 1 H NMR (300 MHz, DMSO-d 6 ): d H =3.96 (s, 2H, CH 2 ), 7.19–7.39 (m, 3H, Ar-H), 7.61–7.68 (m, 2H, Ar-H), 7.71–7.76 (m, 2H, Ar-H), 7.78–7.89 (m, 2H, Ar-H), 10.48 (bs, 1H, exocyclic-NH), 12.64 (bs, 1H, cyclic-NH + ) ppm. 13 C NMR (75 MHz, DMSO-d 6 ): d c =40.33 (CH 2 ), 124.39, 125.81, 128.27, 129.30, 131.47 (Ar-CH), 123.64, 135.13, 138.55 (Ar-C), 146.12 (C5), 158.63 (C2) ppm. MS (70 eV) m/z(%): 347 (M + , 9), 267 (100), 155 (10), 119 (5). Anal. calcd for C 15 H 13 Br 2 N 3 S (427.16) calcd: C, 42.81; H, 3.07; N, 9.84; S, 7.51. Found: C, 42.87; H, 3.00; N, 9.78; S, 7.47. 2.1.7. (Z)-N-(5-(4-Bromophenyl)-3,6-dihydro-2H-1,3,4thiadiazin-2-ylidene) methanaminium bromide (9e). Yellow crystals (78%, 0.295 g), m.p. =211–213 °C, 1 H NMR (300 MHz, DMSO-d 6 ): d H =2.69 (s, 2H, CH 3 ), 4.09 (s, 3H, CH 2 ), 7.62–7.65 (dd, 2H, J=6.6, Ar-H), 7.70–7.73 (dd, 2H, J=6.6 Ar-H), 11.78 (bs, 1H, exocyclic-NH + ), 12.23 (bs, 1H, cyclic-NH) ppm. 13 C NMR (75 MHz, DMSO-d 6 ): d c =30.18 (CH 3 ), 48.66 (CH 2 ), 129.17, 131.40 (Ar-CH), 123.33, 137.17 (Ar-C), 145.66 (C5), 172.76 (C2) ppm. Anal. calcd for C 10 H 11 Br 2 N 3 S (365.09) calcd: C, 32.90; H, 3.04; N, 11.51; S, 8.78. Found: C, 32.83; H, 3.08; N, 11.46; S, 8.80. 2.1.8. (Z)-N-(5-(4-Bromophenyl)-3,6-dihydro-2H-1,3,4thiadiazin-2-ylidene)ethan aminium bromide (9f). Yellow crystals (78%, 0.295 g), m.p. =190–192 °C, 1 H NMR (300 MHz, DMSO-d 6 ): d H =1.38–1.79 (t, 3H, J=7.5, CH 3 –CH 2 ), 3.55–3.60 (q, 2H, J=7.5, CH 2 –CH 3 ) 3.65 (s, 1H, cyclic-CH 2 ), 7.62–7.65 (dd, 2H, J=8.1, Ar-H), 7.71–7.74 (dd, 2H, J=8.4 Ar-H), 11.73 (bs, 1H, exocyclic-NH + ), 12.31 (bs, 1H, cyclic-NH) ppm. 13 C NMR (75 MHz, CDCl 3 ): d c =14.29 (CH 3 CH 2 –), 38.66 (CH 2 –CH 3 ), 40.33 (CH 2 ), 129.00, 131.14 (Ar-CH), 122.74, 135.79 (Ar-C), 147.24 (C5), 165.64 (C2) ppm. Anal. calcd for C 11 H 13 Br 2 N 3 S (379.11) calcd: C, 34.85; H, 3.46; N, 11.08; S, 8.46. Found: C, 34.90; H, 3.41; N, 11.12; S, 8.43. 2.1.9. (Z)-N-(5-(4-Bromophenyl)-3,6-dihydro-2H-1,3,4thiadiazin-2-ylidene) cyclohexanaminium bromide (9g). Yellow crystals (81%, 0.350 g), m.p. =203–205 °C 1 H NMR (300 MHz, CDCl 3 ): d H =1.17–1.30, 1.38–1.79 (m, 8H, cyclohexyl-CH 2 ), 2.04– 2.14 (m, 3H, cyclohexyl-CH 2 and cyclohexyl-CH), 4.29 (s, 2H, CH 2 ), 7.45–7.48 (dd, 2H, J=8.4, Ar-H), 7.49–7.58 (dd, 2H, J= 17868 |RSC Adv.,2024,14,17866–17876 © 2024 The Author(s). Published by the Royal Society of Chemistry RSC Advances Paper Open Access Article. Published on 27 June 2024. Downloaded on 7/18/2024 9:06:31 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online
8.4, Ar–H), 10.89 (bs, 1H, exocyclic-NH + ), 12.65 (bs, 1H, cyclicNH) ppm. 13 C NMR (75 MHz, CDCl 3 ): d c =24.74 (CH 2 ), 25.03, 25.22, 32.51 (cyclohexyl-CH 2 ), 52.40 (cyclohexyl-CH), 128.05, 130.24 (Ar-CH), 132.05, 135.62 (Ar-C), 158.20 (C5), 174.57 (C2) ppm. Anal. calcd for C 15 H 19 Br 2 N 3 S (433.21) calcd: C, 41.59; H, 4.42; N, 9.70; S, 7.40. Found: C, 41.63; H, 4.43; N, 9.62; S, 7.51. 2.2. Crystal X-ray structure determination of 9a and 9b The single-crystal X-ray diffraction studies were carried out on a Rigaku XtaLAB Synergy R diffractometer with HyPix-Arc 100 detector at 133(2) K using Cu-Karadiation (9a,l=1.54178 Å, PhotonJet R rotating anode generator) or a Bruker D8 Venture diffractometer with a PhotonII detector at 173(2) K using Cu-Ka radiation (9b,l=1.54178 Å). Dual space methods (SHELXT) [G. M. Sheldrick, Acta Crystallogr., 2015, A71,3–8] were used for structure solution, and renement was carried out using SHELXL-2014 (full-matrix least-squares on F 2 ) [G. M. Sheldrick, Acta Crystallogr., 2015, C71,3–8]. Hydrogen atoms were localized by difference electron density determination and rened using a riding model. An extinction correction and a semiempirical absorption correction were applied. 9a: colourless crystals, C 15 H 14 N 3 S/Br, M r =348.26, crystal size 0.18 ×0.14 ×0.13 mm, monoclinic, space group P2 1 /n(no. 14), a=8.9077(1) Å, b=9.1847(1) Å, c=18.1367(2) Å, b= 93.364(1)°, V=1481.29(3) Å 3 ,Z=4, r=1.562 mg m 3 ,m(Cu-K a ) =5.04 mm −1 ,F(000) =704, T=133 K, 2q max =158.6°, 16 484 Scheme 1 Reactivity of substituted thiosemicarbazides toward phenacyl bromide. © 2024 The Author(s). Published by the Royal Society of Chemistry RSC Adv.,2024,14,17866–17876 | 17869 Paper RSC Advances Open Access Article. Published on 27 June 2024. Downloaded on 7/18/2024 9:06:31 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online
reections, of which 3177 were independent (R int =0.016), 188 parameters, 2 restraints, R 1 =0.020 (for 3161 I>2s(I)), wR 2 = 0.051 (all data), S=1.09, largest diff. peak/hole =0.33/−0.31 e Å −3 . 9b: yellow crystals, C 12 H 14 N 3 S/Br, M r =312.23, crystal size 0.14 ×0.25 ×0.20 mm, orthorhombic, space group Pbca (no. 61), a=7.4414(3) Å, b=18.2272(7) Å, c=19.6573(7) Å, V= 2666.24(18) Å 3 ,Z=8, r=1.556 mg m 3 ,m(Cu-K a )=5.51 mm −1 , F(000) =1264, T=173 K, 2q max =144.2°, 19 426 reections, of which 2613 were independent (R int =0.033), 161 parameters, 2 restraints, R 1 =0.022 (for 2558 I>2s(I)), wR 2 =0.056 (all data), S =1.05, largest diff. peak/hole =0.28/−0.25 e Å −3 . CCDC 2329624 (9a) and 2329625 (9b) contain the supplementary crystallographic data for this paper. 2.3. Biology 2.3.1. Cell viability assay. The human mammary gland epithelial (MCF-10A) normal cell line was used to investigate the viability effect of new targets 9a–gon viability. 46,47 Refer to Appendix A for more details. 2.3.2. Antiproliferative assay. The MTT assay 5,48 was used to investigate the antiproliferative efficacy of targets 9a–gagainst four human cancer cell lines: lung cancer (A-549) cell line, colon cancer (HT-29), pancreatic cancer (Panc-1) cell line, and breast cancer (MCF-7) cell line. Erlotinib was used as a reference. See Appendix A for more details. 2.3.3. EGFR inhibitory assay. The EGFR-TK test 49,50 was performed to assess the inhibitory strength of the most potent antiproliferative derivatives 9a,9c, and 9d against EGFR. See Appendix A for more information. 2.3.4. BRAF V600E inhibitory assay. In vitro testing assessed the anti-BRAF V600E activity of compounds 9a,9c, and 9d. 51,52 Refer to Appendix A. 2.3.5. VEGFR-2 assay. The inhibitory potency of compounds 9a,9c, and 9d against VEGFR-2 was assessed using kinase-glo-luminescent kinase assays, 53 with Sorafenib as the reference drug. See Appendix A for more details. 2.4. Western blotting assay The western blotting analysis 54 aids in elucidating the probable mechanisms behind the observed antiproliferative activity of 9c. Appendix A contain all details related to the assay methodology. 3. Results and discussion 3.1. Chemistry According to prior research and our own, 35,41,55 the interaction between thiosemicarbazides and phenacyl bromides is principally determined by the reaction conditions, substituted position, and solvent employed, as seen in Scheme 1. All obtained compounds were conrmed by X-ray crystallographic determination. Based on previous research and our interest in phenacyl bromide reactions, we found that the reaction between phenacyl bromide and 1/or 2-substituted thiosemicarbazides results in the formation of a ve-membered ring (thiazole-ring). In the current study, the reaction of phenacyl bromides (2a,b) with 4-substituted thiosemicarbazides (8a–e) in absolute ethanol with no catalyst resulted in the synthesis of 2substituted amino-1,3,4-thiadiazinium bromide derivatives (9a– g). This reaction formed six-membered rings, as shown in Scheme 2. In this context, the differential behavior of 4substituted thiosemicarbazides 8a–eversus 1/or 2-substituted thiosemicarbazides inuences hexagonal ring formation. This mismatch explains the observed differential in nucleophilicity of the NH 2 group, which rst reacts with the carbonyl group before cycling through the SH group. This effect compelled the reaction to yield thiadiazine as six-membered ring, rather than the ve-membered ring characteristic of thiazole. All obtained compounds are recrystallized from ethanol, and their structures were conrmed with advanced spectroscopic analysis tools such as IR, mass, and NMR spectrum ( 1 H and 13 C). Also, the structures are unambiguously conrmed with Xray crystallographic analysis. Scheme 2 Synthesis of 1,3,4-thiadiazin-3-ium bromide derivatives 9a–g. 17870 |RSC Adv.,2024,14,17866–17876 © 2024 The Author(s). Published by the Royal Society of Chemistry RSC Advances Paper Open Access Article. Published on 27 June 2024. Downloaded on 7/18/2024 9:06:31 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online
To illustrate our obtained products, we chose compound 9a (Fig. 2) as an example, which was assigned as 5-phenyl-2-(phenylamino)-6H-1,3,4-thiadiazin-3-ium bromide. In the IR spectrum of compound 9a, broad bands were observed at 3164 cm −1 due to NH 3015 cm −1 because of aromatic-CH—the other two peaks at 1617 and 1594 cm −1 due to C]N and C]C, respectively. Also, the 1 H NMR spectra of 9a showed three broad singlet signals at d H =3.91, 9.45, and 12.85 ppm, which were assigned as 1,3,4-thiadizinium-CH 2 , 56 exocyclic-NH and 1,3,4thiadizinium-NH + , respectively. The two NH-protons show a downeld shi, attributed to the high deshielding caused by the positive charge on 1,3,4-thiadizinium-ringas observed from the X-ray structure analysis of 9a (Fig. 2); furthermore, in 13 C NMR spectrum of 9a signals at 23.26, 144.32, and 150.71 ppm, which were assigned as CH 2 , thiadiazinium-C5 and thiadiazinium-C2, respectively, in addition to aromatic carbons. Through the preceding data and X-rays displayed in Fig. 2 and 3, we do not doubt that the structures for our obtained products are conrmed. Furthermore, the NMR of compound 9b, also known as (Z)- N-(5-phenyl-3,6-dihydro-2H-1,3,4-thiadiazin-2-ylidene)prop-2en-1-aminium bromide. The 1 H NMR spectrum revealed two downeld shisatd12.98 and 10.62 ppm, corresponding to cyclic-NH and exocyclic-NH + . The allylic protons appeared at d H =4.01–4.04 ppm as triplet with two protons for allyl-CH 2 ,at 5.23–5.34 ppm, as doublet of doublet (dd) with two protons for allyl-CH 2 ]and multiplet signal with one proton at d H =5.72– 5.83 ppm, for the allylic-CH], in addition to singlet signal with upeld chemical shiat d H =4.10 ppm, which assigned as CH 2 - group. The inverted values of the cyclic and exocyclic-NH's in both compounds 9a and 9b are owing to the delocalization of the positive charge over the cyclic-N and amino-nitrogen (exocyclic-N), as well as the hydrogen bond between the nitrogen atoms-H and bromine. The mass spectra of both 9a and 9b compounds revealed molecular ion peaks (M + ) at 347 and 311 m/z, conrming that the products are formed as a result of the reaction between phenacyl bromide and 4-phenyl/allyl thiosemicarbazide, with the elimination of an H 2 O molecule and another HBr molecule. Moreover, the structure of 9b was conrmed by X-ray crystallography, as illustrated in Fig. 3. The X-ray structure of compound 9b conrms the loss of one molecule of H 2 O, and the HBr molecule adheres to the crystallite, as indicated in the mass fragmentation pattern of compound 9b in Fig. 4. Furthermore, X-ray crystallographic studies validated the structure of all obtained products, as claried by the crystallographic data of compounds 9a and 9b (note that the Fig. 3 X-ray crystal structure of compound 9b ((Z)-N-(5-phenyl-3,6-dihydro-2H-1,3,4-thiadiazin-2-ylidene)prop-2-en-1-aminium bromide) (displacement parameters are drawn at 50% probability level). Fig. 2 X-ray crystal structure of compound 9a (5-phenyl-2-(phenylamino)-6H-1,3,4-thiadiazin-3-ium bromide) (displacement parameters are drawn at 50% probability level). © 2024 The Author(s). Published by the Royal Society of Chemistry RSC Adv.,2024,14,17866–17876 | 17871 Paper RSC Advances Open Access Article. Published on 27 June 2024. Downloaded on 7/18/2024 9:06:31 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online
crystallographic numbering does not mirror the systematic IUPAC numbering) (Fig. 2 and 3). The X-ray data of 9a revealed that its molecular formula is C 15 H 14 N 3 S$Br with a weight of m/z =348.26, a colorless crystal, and a monoclinic shape with space group P2 1 /n. The measured bond lengths of S1–C2 and S1–C6 bond length are 1.7304 (14) and 1.8148 (19) Å, respectively, are slightly different from the S–C single bond of 1.737 Å, and this is attributed to the sp 2 character of C2 and the sp 3 character of C6. The bond lengths of C2–N3 and C2–N7 are equal to 1.3346 (18) Å, and 1.3259 (19) Å showed some double bond character as they are comparable to the C–Ns-bond length of 1.47 Å and this conrmed the delocalization of the double bond as well as the positive charge. In contrast, the X-ray data of compound 9b showed that the crystal has an orthorhombic shape with space group pbca. It is observed from the bond length table of compound 9b that the S1–C2 and S1–C6 bond lengths are 1.7290 (15) and 1.8034 (16) Å, respectively, are slightly different from the S–C single bond of 1.737 Å and this is attributed to the sp 2 character of C2 and the sp 3 character of C6. The bond lengths of C2–N3 (1.331 (2)) Å and C2–N7 (1.316 (2)) Å suggest that these bonds have some double bond character as they are comparable to the C–Ns-bond length of 1.47 Å, the N3–N4 bond length is 1.3954(18) Å is closed to the N–N single bond 1.401 Å. Observing the mentioned values of bond lengths conrms the presence of a positive charge, which makes the bond length longer. The suggested mechanism for forming thiadiazin-3-ium bromides 9a–gderivatives is as depicted in Scheme 3. Initially, a nucleophilic attack from the NH 2 group to the carbonyl group and condensation occurs with a loss of H 2 O molecule. Aer that, inter-nucleophilic occurs via S-atom (S N 2 reaction S-alkylation step) on the methylene group, and cyclization takes place, resulting in the formation of the salt 12 which undergoes rearrangement and forming 2-amino-1,3,4thiadiazinium bromide derivatives 8a–g. 3.2. Biology 3.2.1. Cell viability assay. To test the viability impact of new targets 9a–g, the human mammary gland epithelial (MCF-10A) normal cell line was used. 46,47 The cell viability of 9a–gwas determined using the MTT test aer four days of incubation on MCF-10A cells. Table 1 indicates that none of the compounds examined were cytotoxic, and all derivatives showed more than 88% cell viability at 50 mM. 3.2.2. Antiproliferative activity. The MTT assay 5,48 was used to investigate the antiproliferative efficacy of targets 9a–g against four human cancer cell lines: lung cancer (A-549) cell line, colon cancer (HT-29), pancreatic cancer (Panc-1) cell line, and breast cancer (MCF-7) cell line. Erlotinib was used as a reference. Table 1 shows each compound's median inhibitory concentration (IC50) and average inhibitory concentration (GI50) against the four cancer cell lines. In general, targets 9a–gdisplayed good antiproliferative activity, with GI 50 ranging from 38 nM to 66 nM against the four cancer cell lines, when compared to the reference Erlotinib (GI 50 =33 nM), and all examined compounds demonstrated less potent efficacy than Erlotinib. Compounds 9a,9c, and 9d Fig. 4 Fragmentation pattern of compound 9b. 17872 |RSC Adv.,2024,14,17866–17876 © 2024 The Author(s). Published by the Royal Society of Chemistry RSC Advances Paper Open Access Article. Published on 27 June 2024. Downloaded on 7/18/2024 9:06:31 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online
were the most potent antiproliferative derivatives, with GI 50 values of 43, 38, and 47 nM, respectively. Compound 9c (Ar =cyclohexyl, Y =H) was the most potent derivative of all developed compounds, with a GI 50 value of 38 nM, and was more effective than Erlotinib against the MCF-7 cancer cell line, with an IC 50 value of 35 nM versus Erlotinib's IC 50 value of 40 nM. The replacement of the cyclohexyl group in the thiadiazine moiety with a phenyl group, as in compound 9a (Ar =Ph, Y =H), or with an allyl group, as in compound 9b (Ar =Allyl, Y =H), resulting in a reported decrease in antiproliferative activity. Compounds 9a and 9b had GI 50 values of 43 and 56 nM, respectively, which were 1.2and 1.5-fold lower than 9c, suggesting that the cyclohexyl group at position 2 of the thiadiazine moiety is better tolerated for antiproliferative action than the phenyl and allyl groups. Moreover, the substitution pattern of the phenyl group at position 5 of the thiadiazine moiety might greatly inuence the antiproliferative activity of these molecules. For instance, the 4bromo derivative 9g (Ar =cyclohexyl, Y =Br) was less effective than the unsubstituted derivative 9c (Ar =cyclohexyl, Y =H). Compound 9g exhibited a GI 50 value of 54 nM, suggesting that substituting a bromine atom in the para-position of the phenyl group is not benecial for its activity. The same holds when comparing compound 9a (Ar =Ph, Y =H) with compound 9d (Ar =Ph, Y =Br). It was found that compound 9a had a GI 50 value of 43 nM, but compound 9d had a GI 50 value of 47 nM. Finally, compounds 9e (Ar =Me, Y =Br) and 9f (Ar =Et, Y = Br) were the least effective, with GI 50 values of 62 and 66 nM, respectively. This suggests that having methyl and ethyl groups at position 2 of the thiadiazine moiety is not benecial for activity. 3.2.3. EGFR inhibitory assay. The EGFR-TK test 49,50 was conducted to assess the inhibitory strength of the most potent antiproliferative derivatives 9a,9c, and 9d against EGFR, and the results are detailed in Table 2. The assay results align with the antiproliferative assay, showing that the most effective derivatives of EGFR inhibitors were compounds 9a (Ar =Ph, Y = H), 9c (Ar =cyclohexyl, Y =H), and 9d (Ar =Ph, Y =Br) with IC 50 values of 92 ±7, 86 ±5 nM, and 96 ±7, respectively. The studied compounds were consistently less effective as EGFR inhibitors than the reference Erlotinib, which has an IC 50 value of 80 nM, Table 2. Compound 9c (Ar =cyclohexyl, Y =H) exhibited notable anti-EGFR effects, with an IC 50 value of 86 ± 6 nM, comparable to Erlotinib. On the other hand, compounds 9a (Ar =Ph, Y =H) and 9d (Ar =Ph, Y =Br) demonstrated moderate EGFR inhibition, with IC 50 values of 92 ±7 and 96 ± 7 nM, respectively. The data suggest that compound 9c has potential as an EGFR inhibitor and could be used as a candidate for antiproliferative purposes. 3.2.4. BRAF V600E inhibitory assay. In vitro testing assessed the anti-BRAF V600E activity of compounds 9a,9c, and 9d. 51,52 The enzyme testing showed that the examined derivatives moderately hindered BRAF V600E , with IC 50 values varying from 74 to 88 nM, as indicated in Table 2. The IC 50 values of the compounds studied are higher than those of the reference drugs Erlotinib (IC 50 =60 nM) and Vemurafenib (IC 50 =30 nM). Compound 9c had the highest inhibitory action against BRAF V600E with an IC 50 of 74 nM and was identied as a potential inhibitor of cancer cell proliferation with a GI 50 of 38 nM. Consequently, compound 9c becomes a potential antiproliferative agent that functions as a dual inhibitor of EGFR and BRAF V600E . On the other hand, additional structural Scheme 3 Suggested mechanism for the formation of 2-amino-1,3,4-thiadiazinium bromide derivatives 9a–g. Table 1 IC 50 values of compounds 9a–gagainst four cancer cell lines Comp. Cell viability% Antiproliferative activity IC 50 SEM (nM) A-549 MCF-7 Panc-1 HT-29 Average (GI 50 ) 9a 91 43 440444444443 9b 90 56 552558558556 9c 92 37 335338340438 9d 89 48 444448447447 9e 92 62 658564664662 9f 89 65 664666666666 9g 88 54 551556556554 Erlotinib ND 30 340330330333 © 2024 The Author(s). Published by the Royal Society of Chemistry RSC Adv.,2024,14,17866–17876 | 17873 Paper RSC Advances Open Access Article. Published on 27 June 2024. Downloaded on 7/18/2024 9:06:31 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online