Novel chalcone 2-thiopyrimidine conjugates as dual VEGFR-2/BRAF inhibitors: design, synthesis, in vitro cytotoxicity, and molecular docking study

Abstract

Chalcone-based derivatives have shown potential anticancer activity via multiple mechanisms including protein kinase inhibition. In the current study, two series of chalcone/2-thiopyrimidine conjugates 4a–4d and 6a–6i were designed, synthesized and screened for their antiproliferative activity in a single-dose assay against NCI-60 cancer cell lines. Ten compounds, 4a–4d, 6a–6c, 6f, 6h, and 6i, were selected for a five-dose assay and their GI₅₀ values were determined. Compound 4c showed potent anticancer activity against LOX IMVI melanoma cell line with a GI₅₀ value of 0.0128 μM. Seven compounds, 4a, 4c, 4d, 6c, 6f, 6h, and 6i, were found to be non-cytotoxic against fibroblast (hFB) normal cell line. Additionally, investigation of the VEGFR-2 inhibitory activity of the ten promising compounds revealed that 4c, 4d and 6i displayed promising VEGFR-2 inhibition (IC₅₀ = 0.144, 0.105, and 0.072 μM, respectively) compared to sorafenib (IC₅₀ = 0.081 μM). Moreover, 4c inhibited BRAF_WT and BRAF_V600E kinases (IC₅₀ = 0.201 and 0.101 μM, respectively) relative to vemurafenib (IC₅₀ = 0.156 and 0.063 μM, respectively). Furthermore, 4c arrested the cell cycle progression at the G₁ phase and induced late apoptosis in LOX IMVI cells. Moreover, evaluation of the effect of 4c on apoptotic markers in the mentioned cells indicated an increase in the Bax/Bcl-2 ratio by 28.12-fold along with upregulation of caspases-3 and -9 by 7.40- and 5.63-fold, respectively, in addition to anti-migratory effect. Molecular docking study of the most promising derivatives revealed a common binding pattern in the binding site of the target kinases that extends from the hinge region through the gate area towards the allosteric back pocket interacting with the key amino acids in a type II inhibitor-like binding pattern.

A chalcone-based thiopyrimidine (4c) has displayed a potent anticancer activity via VEGFR-2 and BRAF inhibition.

Introduction

Cancer is a massive health burden with 35 million new cancer cases expected in 2050 with a 77% increase in cancer incidence from the number of cases in 2022 according to the World Health Organization (WHO).^1,2 Targeting angiogenesis is one of the most promising strategies in cancer therapy as it inhibits the continuous increase in tumor vascularization required for its rising demand for oxygen and nutrient supply.³ Sprouting angiogenesis is the common mode of angiogenesis where angiogenic growth factors initiate cellular changes in endothelial cells such as proliferation, differentiation and migration of tip cells which are pivotal for the production of new functional blood vessels.^4,5 Several angiogenic growth factors and their receptors are involved in transmitting signals that regulate various cellular functions including cellular growth, differentiation, migration, and further angiogenesis such as vascular endothelial growth factor receptor (VEGFR), fibroblast growth factor receptor (FGFR), epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGFR), and insulin-like growth factor receptor (IGFR).⁶

VEGFR-2 is a key receptor tyrosine kinase (RTK) which upon binding to its ligand, VEGF, initiates angiogenesis as well as the signaling pathways controlling cellular differentiation, proliferation, migration, and survival.^7,8 BRAF is a serine/threonine kinase in the mitogen-activated protein kinase (MAPK) signaling pathway, which is a vital regulator involved in cellular division and differentiation.⁹ BRAF mutations are frequently linked to various types of melanoma, with BRAF V600E as the most common BRAF mutation where the valine residue is replaced with glutamate at position 600.¹⁰ Several small molecules with diverse scaffolds targeting VEGFR-2 and BRAF kinases among other kinases (multi-kinase inhibitors) are clinically approved by the FDA for targeted cancer therapy.¹¹

Sorafenib is a multi-kinase inhibitor targeting VEGFR-2, BRAF, and PDGFR kinases.¹² It blocks tumor angiogenesis as well as cancer cell proliferation and it is approved for the treatment of hepatocellular carcinoma (HCC), renal cell carcinoma (RCC), and differentiated thyroid carcinoma (DTC).¹³ Several pyrimidine-based derivatives acting as VEGFR-2 inhibitors have been approved for cancer treatment. Pazopanib, an indazole–pyrimidine sulfonamide hybrid, is an orally active angiogenesis inhibitor targeting VEGFR-1/2/3, PDGFRα/β, and c-KIT kinases for the management of advanced RCC.^14,15 Sulfatinib, an indole–pyrimidine sulfonamide derivative, is another multi-kinase inhibitor targeting VEGFR-1/2/3 and FGFR-1 kinases that is used for the treatment of patients with advanced solid tumors through tumor angiogenesis inhibition and immune modulation.¹⁶ Fruquintinib, a quinazoline–benzofuran hybrid, is a VEGFR-1/2/3 inhibitor that is used for the treatment of metastatic colorectal cancer (Fig. 1).^17,18

Fig. 1. Molecular structures of FDA-approved drugs targeting VEGFR-2 and/or BRAF kinases.

Chalcones are privileged scaffolds which show promising anticancer activity through various mechanisms including microtubule inhibition, topoisomerase inhibition, angiogenesis inhibition, histone deacetylase inhibition, EGFR inhibition, and apoptosis induction.¹⁹ Ahmed et al.²⁰ reported that the N-aryl piperazine–chalcone derivatives Ia and Ib displayed a significant anticancer activity against NCI-60 cancer cell lines at 10 μM, showing growth inhibition % (GI%) ranges of 16.55–114.19% and 12.37–121.73%, respectively.²⁰ Furthermore, compounds Ia and Ib showed a potent VEGFR-2 inhibitory activity with IC₅₀ values of 0.80 and 0.57 μM, respectively.²⁰ Hafezz and co-workers²¹ reported the chalcones IIa and IIb as promising VEGFR-2 inhibitors with IC₅₀ values of 0.37 and 0.31 μM, respectively.²¹ The N-acetylchalcone IIb exhibited a potent antiproliferative activity against NCI-60 cancer cell lines with a full panel MG-MID value of 4.48 μM (Fig. 2).²¹ The pyrimidine nucleus is another core framework which displays a promising VEGFR-2 inhibitory activity.^11,22–24 Abdel-Mohsen et al.²⁵ reported that the pyrimidine-2-thioacetamide ethyl carboxylate derivative III acted as a dual VEGFR-2 and BRAF inhibitor with submicromolar IC₅₀ values of 0.17 and 0.15 μM, respectively.²⁵ Moreover, compound III disclosed a potent anticancer activity against the T-47D breast cancer cell line with an IC₅₀ value of 2.18 μM.²⁵ Marzouk et al.²⁶ cited the 1,6-dihydropyrimidine-2-thioacetamide derivative IV as a potent VEGFR-2 inhibitor with an IC₅₀ value of 0.199 μM, which showed anticancer activity against SR leukemia cell line with a GI₅₀ value of 19.0 μM.²⁶ The study of Lamie et al.²⁷ revealed that the chalcone 4,6-diphenyl-2-thiopyrimdine hybrids Va–Vc showed a potent anticancer activity against K-562 myelogenous leukemia cell line (IC₅₀ = 0.77–1.37 μM) in comparison to cisplatin and erlotinib (IC₅₀ = 2.31 and 9.85 μM, respectively).²⁷ Altwaijry and co-workers²⁸ demonstrated that some chalcone 1,6-dihydro-2-thiopyrimidine conjugates VIa–VIe exhibited a potent anti-proliferative activity against HuH-7 liver cancer cell line (IC₅₀ values = 0.55–8.32 μM) relative to doxorubicin (IC₅₀ = 5.70 μM) (Fig. 2).²⁸

Fig. 2. Molecular structures of reported chalcones and 2-thiopyrimidines I–VI as promising VEGFR-2 and/or BRAF inhibitors and anticancer agents.

Consequently, guided by the reported cytotoxicity and VEGFR-2 inhibitory activity of the piperazine–chalcone hybrids Ia and Ib and 2-thiopyrimidine derivative IV along with the dual VEGFR-2/BRAF activity of pyrimidine-2-thioacetamide III, and based on the concept of using molecular hybridization approach to obtain new conjugates with enhanced VEGFR-2/BRAF inhibition and potent anticancer activity, the authors designed and synthesized some chalcone 2-thiopyrimidine hybrids 4a–4d and 6a–6i as promising cytotoxic agents with potential VEGFR-2/BRAF inhibition (Fig. 3). The designed compounds were evaluated for their antiproliferative activity at 10 μM against NCI-60 cancer cell lines and their GI% were determined. Promising compounds were further tested by the NCI five-dose assay to determine their GI₅₀ and MG-MID values. Furthermore, their VEGFR-2 inhibitory activity was evaluated, whereas BRAF inhibition was appraised on the most potent cytotoxic derivative in addition to determination of its effect on cell cycle progression and apoptotic induction. Molecular docking study was also carried out to explore the binding characteristics of the designed compounds in VEGFR-2 and BRAF kinase domains.

Fig. 3. Design strategy of the target chalcone 2-thiopyrimidine hybrids 4a–4d and 6a–6i as cytotoxic agents with anticipated VEGFR-2 and BRAF inhibition.

Discussion

Chemistry

Synthesis of chalcones 1a–1e was performed according to reported procedures^29–32via the reaction of p-aminoacetophenone with the appropriate benzaldehyde derivative in absolute ethanol in the presence of 20–50% aqueous sodium hydroxide solution; the mixture was stirred for 1–2 h at room temperature. The corresponding 2-chloro-N-(4-((E)-3-((substituted)phenyl)acryloyl)phenyl)acetamides 2a–2e were synthesized according to reported procedures²⁹ through the reaction of chalcones 1a–1e with chloroacetyl chloride in dry DMF and in the presence of anhydrous potassium carbonate for 0.5–2 h at room temperature. 4,6-Dimethyl-2-thioxopyrimidine 3 and 2-thioxo-2,3-dihydropyrimidinones 5a and 5b were synthesized according to reported procedures.^11,33 The synthesis of the chalcone/pyrimidine thioacetamide derivatives 4a–4d and 6a–6i was achieved by the reaction of the appropriate 2-chloro-N-(4-((E)-3-((substituted)phenyl)acryloyl)phenyl)acetamides 2a–2e and 4,6-dimethyl-2-thioxopyrimidine 3 or 2-thioxo-2,3-dihydropyrimidinones 5a and 5b in dry DMF and in the presence of anhydrous potassium carbonate under reflux for 1–2 h as outlined in Scheme 1.

Scheme 1. Synthesis of the chalcone 2-thiopyrimidine conjugates 4a–4d and 6a–6i.

The IR spectrum of 4a demonstrated two C O and one C N absorption bands at 1693, 1655, and 1593 cm⁻¹, respectively. The ¹H NMR spectrum of 4a revealed that the compound is in (E) configuration, where two chalcone protons were observed at 7.46 and 8.15 ppm along with a coupling constant (J = 16.0 Hz). In addition, two methyl, pyrimidine protons and one NH proton appeared as a singlet at 2.51, 6.86, and 9.86 ppm, respectively. The ¹³C NMR spectrum of 4a illustrated three signals attributed to the methyl carbons, the amide C O, and the chalcone C O at 24.09, 170.19, and 188.82 ppm, respectively. For 6c, the IR spectrum showed two C O and one C N absorption bands at 1710, 1669, and 1601 cm⁻¹, respectively. ¹H NMR of 6c revealed chalcone protons at 7.74 and 8.12 ppm with a coupling constant (J = 15.0 Hz) that confirms the (E) configuration, whereas pyrimidine protons and two NH protons appeared at 6.13, 8.17, 10.67, and 12.79 ppm, respectively. The ¹³C NMR spectrum of 6c displayed two signals for the amide C O and chalcone C O at 166.46 and 187.16 ppm, respectively. The ¹H and ¹³C NMR and HRMS data for the synthesized compounds 2c, 4a–4d and 6a–6i are included in the SI (Fig. S.1–S.41).

Biology

NCI-60 anticancer screening

The newly synthesized chalcone/pyrimidine thioacetamide derivatives 4a–4d and 6a–6i were selected by the Developmental Therapeutic Program (DTP) at the National Cancer Institute (NCI) to be screened for their in vitro anticancer activity following the protocols established in the NCI's Drug Evaluation Branch in Bethesda, MD, USA.

Preliminary cytotoxicity screening at 10 μM concentration

The tested compounds were evaluated in a single-dose anticancer assay against a panel of NCI-60 cancer cell lines at 10 μM concentration. The most active compounds 4a–4c, 6c, 6f, and 6h demonstrated growth inhibition against the most sensitive cancer cell lines with mean GI% values of more than 100% as presented in Fig. 4. The obtained data revealed that the chalcone/4,6-dimethylpyrimidine-2-yl thioacetamide conjugates 4a–4d displayed prominent cytotoxicity against the entire tumor cell panel. Compounds 4a–4c bearing electron-withdrawing group (2-Cl, 4-Cl, and 3-NO₂-4-Cl, respectively) at the chalcone moiety exhibited potent anticancer activity with mean GI% values of 115.88%, 142.86%, and 192.40%, respectively, relative to 4d having a 3,4,5-trimethoxy group with a mean GI% value of 74.57%.

Fig. 4. Growth inhibition % (GI%) of compounds 4a–4c, 6c, 6f, and 6h in a one-dose assay at 10 μM.

Regarding the chalcone/6-oxo-1,6-dihydropyrimidin-2-yl thioacetamide hybrids 6a–6i, they showed significant cytotoxicity towards the majority of the tested sub-panels. Also, compounds 6a–6c, 6f and 6h featuring electron-withdrawing group (2-Cl, 4-Cl, and 3-NO₂-4-Cl) substituents at the chalcone moiety elicited the most potent cytotoxic activity among this series. Compounds 6c (R₁ = 3-NO₂-4-Cl, R₂ = H), 6f (R₁ = 2-Cl, R₂ = CH₃) and 6h (R₁ = 3-NO₂-4-Cl, R₂ = CH₃) showed growth inhibition with mean GI% values of 140.13%, 156.79%, and 134.30%, respectively. However, compounds 6a (R₁ = 2-Cl, R₂ = H), 6b (R₁ = 4-Cl, R₂ = H) and 6i (R₁ = 3,4,5-tri-OCH₃, R₂ = CH₃) revealed lower cytotoxicity with mean GI% values of 39.34%, 63.85%, and 68.64%, respectively. The results representing the growth inhibition percentage (GI%) of the tested compounds are shown in Table S.1.

In vitro cytotoxic evaluation against NCI-60 cancer cell lines at a five-dose level

Based on the outstanding results of the single-dose screening, ten compounds, 4a–4d, 6a–6c, 6f, 6h and 6i, were chosen to be evaluated in a five-dose assay. The preliminary results of the screened compounds are illustrated in Table 1 to present the response parameters, particularly GI₅₀, across various cell lines.

Table 1. GI₅₀ (μM) values of selected compounds 4a–4d, 6a–6c, 6f, 6h and 6i against a panel of 60 cancer cell lines in a five-dose assay.

Subpanel		Compound ID
		4a	4b	4c	4d	6a	6b	6c	6f	6h	6i
	R 1	2-Cl	4-Cl	3-NO2-4-Cl	3,4,5-tri-OCH3	2-Cl	4-Cl	3-NO2-4-Cl	2-Cl	3-NO2-4-Cl	3,4,5-tri-OCH3
	R 2	—	—	—	—	H	H	H	Me	Me	Me
		GI 50 (μM)
Leukemia
CCRF-CEM		1.90	3.77	1.50	2.80	4.57	4.31	1.45	5.73	1.91	2.26
HL-60(TB)		2.24	3.37	0.235	3.97	18.10	14.40	1.56	0.844	2.26	7.71
K-52		1.74	2.81	1.21	2.16	3.83	5.40	1.47	1.34	1.89	3.00
MOLT-4		2.03	2.43	1.51	1.80	11.60	14.70	1.40	3.58	2.11	6.28
RPMI-8226		1.53	0.517	0.343	1.34	7.53	3.64	1.77	0.90	2.14	11.80
SR		0.504	0.422	1.18	nd	1.30	2.22	nd	0.0055	1.15	1.44
Non-small cell lung cancer
A549/ATTC		5.80	4.51	3.79	5.51	16.80	60.90	12.50	6.44	16.20	10.80
EKVX		6.44	3.61	3.55	9.59	17.20	—	12.10	6.49	5.78	10.70
HOP-62		2.19	3.26	1.65	7.60	15.80	12.60	9.25	5.42	1.90	9.17
HOP-92		2.96	3.57	2.88	—	14.50	13.60	8.60	5.55	4.87	6.28
NCI-H226		4.60	2.98	2.07	8.61	11.40	19.70	3.09	4.70	4.50	6.73
NCI-H23		3.45	3.53	1.78	7.42	15.70	24.60	3.94	4.50	2.31	7.17
NCI-H322M		12.10	3.40	3.22	8.80	16.90	28.90	3.36	1.46	2.35	5.55
NCI-H460		4.26	3.49	1.86	4.34	17.20	42.50	12.50	7.03	11.40	10.80
NCI-H522		5.36	3.00	1.87	3.65	17.20	23.40	10.90	5.81	4.21	5.72
Colon cancer
COLO 205		11.50	3.66	2.03	5.69	20.50	26.20	9.34	6.20	7.03	6.72
HCC-2998		1.74	3.17	0.515	7.29	11.00	1.82	1.73	1.21	1.72	5.17
HCT-116		1.84	1.22	1.14	1.99	3.29	3.14	1.67	1.14	1.86	4.40
HCT-15		1.99	3.24	1.01	1.77	16.10	23.30	9.47	6.25	3.01	8.59
HT29		3.91	4.80	1.81	2.17	15.70	32.50	9.18	5.23	3.80	8.52
KM12		1.73	1.90	1.66	2.56	4.26	3.39	1.44	0.710	2.03	6.48
SW-620		2.05	3.18	1.67	2.94	17.40	12.80	1.60	2.61	2.16	11.90
CNS cancer
SF-268		4.08	3.66	1.77	10.60	12.10	15.80	2.66	2.20	1.93	7.01
SF-295		11.90	5.33	6.45	7.37	15.90	33.00	11.70	6.05	12.20	10.40
SF-539		2.13	3.39	1.79	nd	13.40	9.74	1.40	1.95	1.98	nd
SNB-19		5.36	5.82	2.96	6.00	6.11	19.40	1.39	1.45	4.43	5.37
SNB-75		4.63	3.96	2.03	2.86	16.10	14.70	1.90	4.87	2.63	1.16
U251		2.06	3.10	1.64	1.66	4.58	8.55	1.40	1.12	2.01	2.38
Melanoma
LOX IMVI		2.15	2.68	0.0128	1.99	5.21	5.34	1.21	1.35	2.00	5.05
MALME-3 M		—a	3.37	2.25	7.37	13.80	27.20	2.64	6.31	5.72	3.35
M14		4.11	3.51	2.07	6.06	12.70	12.70	3.37	5.35	3.40	6.13
MDA-MB-435		5.41	3.21	2.56	5.35	17.20	17.30	2.78	6.17	4.15	7.64
SK-MEL-2		4.63	3.58	1.76	8.83	10.10	21.20	1.88	2.11	2.33	6.41
SK-MEL-28		6.77	7.92	1.78	6.05	18.70	18.90	8.79	8.39	5.65	5.70
SK-MEL-5		3.32	2.60	1.75	6.80	14.00	28.70	2.91	4.25	3.60	6.13
UACC-257		2.91	3.36	1.77	5.99	14.70	16.30	5.47	4.93	2.70	5.11
UACC-62		3.41	2.93	1.54	5.01	11.50	13.40	3.03	3.26	3.45	5.27
Ovarian cancer
IGROV1		3.63	2.98	1.92	3.04	15.90	23.20	8.95	5.78	4.23	6.77
OVCAR-3		2.83	2.86	1.51	2.98	13.10	13.50	1.97	3.20	1.99	4.54
OVCAR-4		7.36	2.71	1.94	9.24	13.20	48.90	9.14	5.29	7.91	5.11
OVCAR-5		15.70	3.72	12.80	2.61	17.50	—	14.10	7.09	17.40	7.87
OVCAR-8		3.93	3.50	1.76	2.92	2.56	21.40	2.00	3.94	2.57	7.11
NCI/ADR-RES		3.89	3.34	2.26	5.09	18.90	—	18.00	14.10	32.70	—
SK-OV-3		2.95	3.79	1.63	18.70	14.70	11.60	7.83	5.04	2.27	19.80
Renal cancer
786–0		3.64	2.47	nd	7.30	11.00	nd	1.22	5.19	2.16	9.15
A498		2.60	3.77	1.90	—	12.80	25.70	8.54	4.58	3.82	35.80
ACHN		ndb	4.13	1.94	5.55	17.20	—	12.60	6.34	nd	12.00
CAKI-1		7.64	3.79	1.56	2.99	17.00	47.40	8.58	5.57	6.35	6.37
RXF 393		2.11	nd	1.77	2.16	nd	19.30	nd	nd	1.85	1.30
SN 12C		3.83	3.67	1.79	4.07	11.40	18.80	9.13	5.22	2.43	5.68
TK-10		16.10	3.53	3.12	37.10	15.60	59.80	10.00	6.16	10.50	18.00
UO-31		8.44	nd	1.80	3.18	nd	31.70	nd	nd	7.02	8.71
Prostate cancer
PC-3		4.75	nd	1.97	4.26	nd	34.80	nd	nd	2.67	13.50
DU-145		3.37	6.90	1.91	2.88	17.40	87.80	12.4	6.20	2.16	7.87
Breast cancer
MCF7		2.35	1.12	1.28	1.52	4.29	4.03	1.80	0.955	1.34	3.66
MDA-MB-231/ATTC		7.28	3.53	2.60	8.44	16.90	42.00	2.36	1.98	9.03	7.27
HS 578 T		1.12	3.15	1.68	—	12.90	17.60	1.53	1.38	1.21	11.00
BT-549		1.96	3.15	1.87	2.77	12.10	8.84	1.60	4.68	2.00	3.84
T-47D		5.75	2.63	1.87	7.65	14.50	19.90	1.80	3.27	1.88	5.94
MDA-MB-468		2.41	0.685	0.425	1.91	1.87	2.28	1.10	0.734	1.67	1.14

^aGI₅₀ >100 μM.

^bNot determined.

It was noticed that the majority of the tested compounds showed significant GI₅₀ in a low micromolar concentration, specifically against leukemia, colon, melanoma and breast cancer cell lines. In this context, compound 4c showed a potent anticancer activity in a submicromolar concentration against leukemia (HL-60(TB), RPMI-8226), colon (HCC-2998), melanoma (LOX IMVI), and breast (MDA-MB-468) cancer cell lines (GI₅₀ values 0.0128–0.515 μM) (Table 1). Additionally, the most potent compounds 4a–4d, 6c, 6f, 6h, and 6i are outlined in Fig. 5. The mean graph midpoints (MG-MIDs) for the GI₅₀ parameter of each of the tested compounds against sub-panel and full panel cell lines were calculated and presented in Table 2.

Fig. 5. Heat map representing the GI₅₀ of compounds 4a–4d, 6c, 6f, 6h, and 6i on different cancer cell lines.

Table 2. MG-MID (μM) values of compounds 4a–4d, 6a–6c, 6f, 6h, and 6i on a subpanel of cancer cell lines.

Subpanel	Compound ID
	4a	4b	4c	4d	6a	6b	6c	6f	6h	6i
	MG-MIDa (μM)
Leukemia	1.657	2.220	0.996	2.414	7.822	7.445	1.530	2.067	1.910	5.415
Non-small cell lung cancer	5.240	3.483	2.519	6.940	15.856	28.275	8.471	5.267	5.947	8.102
Colon cancer	3.537	3.024	1.405	3.487	12.607	14.736	4.919	3.336	3.087	7.397
CNS cancer	5.027	4.210	2.773	5.698	11.365	16.865	3.408	2.940	4.197	5.698
Melanoma	4.089	3.684	1.721	5.939	13.101	17.893	3.564	4.680	3.667	5.643
Ovarian cancer	5.756	3.271	3.403	6.369	13.694	23.720	8.856	6.349	9.867	8.533
Renal cancer	5.966	3.560	1.983	8.907	14.167	33.783	8.345	5.510	4.876	12.126
Prostate cancer	4.060	6.900	1.940	3.570	17.400	61.300	12.400	6.200	2.415	10.685
Breast cancer	3.478	2.378	1.621	4.458	10.427	15.775	1.698	2.167	2.855	5.475
Full panel MG-MIDb	4.312	3.637	2.040	5.309	12.938	24.421	5.910	4.279	4.313	7.675

^aMedian value of GI₅₀ (μM) calculated according to the data obtained from NCI's tumor cell screen.

^bFull panel mean graph midpoint (MG-MID) is the average sensitivity of all cell lines towards the tested compounds.

The low MG-MID values of the tested compounds in a micromolar concentration demonstrated a remarkable anticancer activity. Interestingly, five compounds, namely, 4a–4c, 6f, and 6h, out of ten displayed a potent anticancer activity across the full panel with MG-MID values of 2.040–4.313 μM. The MG-MID values of 4c (0.996–3.403 μM) highlighted its potential as a broad-spectrum anticancer agent. The one-dose mean graphs, dose–response curves for the five-dose assay, and GI₅₀, TGI, and LC₅₀ values are included in the SI (Fig. S.42–S.74 and Tables S.2 and S.3).

In vitro cytotoxicity against human primary skin fibroblast (hFB) normal cell line

Based on the results outlined in Table 2, the highly active compounds 4a–4d, 6c, 6f, 6h, and 6i were further tested against skin fibroblast (hFB) normal cell line to determine their cytotoxicity on normal cells (tolerability) and their IC₅₀ values were calculated and presented in Table 3. It was found that compounds 4a, 4c, 4d, 6c, 6f, 6h, and 6i were non-cytotoxic against the hFB cell line, with IC₅₀ values >100 μM; however, compound 4b was moderately toxic (IC₅₀ = 25.26 μM). The IC₅₀ graphs for the compounds are illustrated in Fig. S.75 in the SI.

Table 3. Cytotoxic effect of compounds 4a–4d, 6c, 6f, 6h, and 6i against human fibroblast (hFB) normal cell line.

Compound ID	IC50 (μM)	Compound ID	IC50 (μM)
4a	>100	6c	>100
4b	25.26	6f	>100
4c	>100	6h	>100
4d	>100	6i	>100

Kinase inhibitory assays

VEGFR-2 kinase inhibitory activity

Compounds 4a–4d, 6a–6c, 6f, 6h, and 6i which were screened in the five-dose assay were further assessed for their VEGFR-2 inhibitory activity relative to sorafenib and the results are presented in Table 4. Regarding the 4,6-dimethyl-2-thiopyrimidines 4a–4d, they exhibited significant VEGFR-2 inhibition (IC₅₀ = 0.105–1.176 μM) relative to sorafenib (IC₅₀ = 0.081 μM). The monosubstituted derivatives 4a (R₁ = 2-Cl) and 4b (R₁ 4-Cl) showed moderate VEGFR-2 inhibition (IC₅₀ = 1.176 and 0.319 μM, respectively), whereas the disubstituted congener 4c (R₁ = 3-NO₂-4-Cl) and the trisubstituted one 4d (R₁ = 3,4,5-tri-OCH₃) displayed the most potent VEGFR-2 inhibition in this series (IC₅₀ = 0.144 and 0.105 μM, respectively). Concerning 1,6-dihydro-2-thioxopyrimidines 6a–6i, they showed potent VEGFR-2 inhibition in the submicromolar level (IC₅₀ = 0.072–0.561 μM) more than that expressed by the 4,6-dimethyl-2-thiopyrimidines 4a–4d. Compounds 6a (R₁ = 2-Cl, R₂ = H) and 6f (R₁ = 2-Cl, R₂ = CH₃), bearing a 2-Cl substituent at R₁ and regardless of the substitution on R₂ (H or CH₃), demonstrated remarkable VEGFR-2 inhibition (IC₅₀ = 0.189 and 0.172 μM, respectively). With regard to 6b (R₁ = 4-Cl, R₂ = H), 6c (R₁ = 3-NO₂-4-Cl, R₂ = H), and 6h (R₁ = 3-NO₂-4-Cl, R₂ = CH₃), they displayed moderate VEGFR-2 inhibition (IC₅₀ = 0.371, 0.406, and 0.561 μM, respectively). The most potent VEGFR-2 inhibition was observed in 6i (R₁ = 3,4,5-tri-OCH₃, R₂ = CH₃) with IC₅₀ = 0.072 μM, surpassing that of sorafenib (IC₅₀ = 0.081 μM). The structure–activity relationship of the tested compounds on VEGFR-2 is illustrated in Fig. 6. The IC₅₀ graphs of compounds 4a–4d, 6a–6c, 6f, 6h, 6i and sorafenib on VEGFR-2 kinase are presented in the SI (Fig. S.76 and S.77).

Table 4. VEGFR-2 kinase inhibitory activity of compounds 4a–4d, 6a–6c, 6f, 6h, 6i and sorafenib.

Compound ID	VEGFR-2 kinase	Compound ID	VEGFR-2 kinase
	IC50 (μM)		IC50 (μM)
4a	1.176	6a	0.189
4b	0.319	6b	0.371
4c	0.144	6c	0.406
4d	0.105	6f	0.172
Sorafenib	0.081	6h	0.561
		6i	0.072

Fig. 6. Structure–activity relationship of target compounds on VEGFR-2 kinase.

BRAF kinase inhibitory activity

Compound 4c that displayed a potent anticancer activity as shown by its GI₅₀ or MG-MID value was evaluated for its inhibitory activity versus wild-type BRAF and mutant BRAF V600E kinases relative to vemurafenib and the results are depicted in Table 5. It was observed that 4c exhibited significant inhibition towards wild-type and mutant kinases (IC₅₀ = 0.201 and 0.101 μM, respectively) compared to vemurafenib (IC₅₀ = 0.156 and 0.063 μM, respectively). The IC₅₀ graphs of 4c and vemurafenib on BRAF_WT and BRAF_V600E kinases are presented in the SI (Fig. S.78 and S.79).

Table 5. BRAF_WT/BRAF_V600E kinases inhibitory activity of compound 4c and vemurafenib.

Compound ID	BRAFWT kinase	BRAFV600E kinase
	IC50 (μM)
4c	0.201 ± 0.006	0.101 ± 0.003
Vemurafenib	0.156 ± 0.005	0.063 ± 0.002

Cell cycle analysis

Compound 4c, which showed the most potent anticancer activity against the LOX IMVI melanoma cell line, was screened for its impact on cell cycle progression at half of its GI₅₀ concentration in the LOX IMVI melanoma cell line. The cells were stained with propidium iodide (PI) to appraise their DNA content using flow cytometry. The results are shown in Fig. 7A. Compound 4c was found to halt the cell cycle at the G₁ phase via increasing the population of LOX IMVI cells to 79.62% in 4c-treated LOX IMVI cells relative to control untreated LOX IMVI cells (58.26%) after 48 h (Fig. 7B).

Fig. 7. (A) Effect of 4c on cell cycle analysis in LOX IMVI melanoma cells after 48 h. (B) Graphical representation of the cells' population percentage in cell cycle phases in the untreated and 4c-treated LOX IMVI cells.

Effect on cell apoptosis

The apoptotic induction of 4c was determined via distinguishing between early and late apoptosis in LOX IMVI melanoma treated and untreated cells using annexin V–FITC labelling. The results are presented as early, late and total apoptosis and necrosis in Fig. 8A. The results revealed that 4c induced 27.77% total apoptosis in 4c-treated LOX IMVI cells in comparison to the control untreated ones 0.57%, and necrosis of 4.64% in 4c-treated LOX IMVI cells relative to 2.09% of the control untreated cells. This finding shows that 4c induced cell death and reduced cell proliferation (Fig. 8B).

Fig. 8. (A) Effect of 4c on apoptosis in LOX IMVI melanoma cells after 48 h. (B) Graphical representation of the cells' population percentage on apoptosis in the untreated and 4c-treated LOX IMVI cells.

Effect of compound 4c on the apoptotic markers Bax, Bcl-2, and caspases-3 and -9 in LOX IMVI cells

Evaluation of the apoptotic markers of 4c was carried out in LOX IMVI cells to assess these apoptotic indicators, which highlighted the potential of 4c as an anticancer agent. The results are shown in Table 6. It was noticed that 4c significantly modulated the Bax/Bcl-2 apoptotic pathway via upregulation of the Bax level by 9.60-fold while decreasing the anti-apoptotic protein Bcl-2 by 0.34-fold, resulting in a Bax/Bcl-2 ratio of 28.12-fold. This elevated ratio indicates the magnified apoptotic induction effect of 4c. Moreover, 4c activated caspase-3 and caspase-9 levels by 7.40- and 5.63-fold, respectively, which further supported its role in inducing apoptosis via the intrinsic mitochondrial route.

Table 6. Effects of compound 4c on fold change of Bax, Bcl-2, and caspases-3 and -9 in LOX IMVI cells.

Compound ID	Bax	Bcl-2	Bax/Bcl-2	Caspase-3	Caspase-9
	Fold change (RT-PCR)
4c/LOX IMVI	9.6042	0.3415	28.1235	7.4035	5.6314
Control LOX IMVI	1	1	1	1	1

Cell migration assay

Cell migration is one of the crucial stages of angiogenesis and metastasis in the development of tumor cells.³⁴ Cell migration assay was conducted to demonstrate the effect of 4c on the angiogenesis process in LOX IMVI melanoma cells at half of its GI₅₀ value. After wound formation on a monolayer of LOX IMVI melanoma cells, the migration of epidermal cells is induced for wound closure with loosely connected cell populations.³⁵ The wound closure % observed in LOX IMVI cells after 72 h declined from 97.037% in untreated control LOX IMVI cells to 63.704% in 4c-treated cells (Table 7, Fig. 9). This confirms the proposed anti-migratory effect of 4c in LOX IMVI cells.

Table 7. Effect of 4c on migration of LOX IMVI cells after 72 h.

Compound ID	Wound closure%
Control LOX IMVI	97.037 ± 3.12a
4c	63.704 ± 2.05

^aData are presented as mean ± SD.

Fig. 9. Anti-migratory effect of 4c in LOX IMVI cells after 72 h.

Molecular docking study

To study the binding features of the most promising compounds 4a–4d, 6a–6c, 6f, 6h, and 6i in the kinase domain of the target kinases (VEGFR-2, BRAF_WT, BRAF_V600E), molecular docking simulations were performed using Molecular Operating Environment (MOE, 2024.06) software. The X-ray crystal structures of VEGFR-2, BRAF_WT, and BRAF_V600E in their inactive conformation (DFG-out) co-crystallized with sorafenib; PDB ID 4ASD, PDB ID 1UWH, and PDB ID 1UWJ, respectively, were utilized for the planned molecular docking study.^36–38

Self-docking of the co-crystallized ligand, sorafenib, in the kinase domain of the target kinases was initially carried out to check the validity of the adopted molecular docking setup. Self-docking simulations efficaciously duplicated the binding mode of sorafenib in the target kinases VEGFR-2, BRAF_WT, and BRAF_V600E with low RMSD values of 0.319, 0.194, and 0.732 Å, respectively, between the docked poses and the co-crystallized ligand. Furthermore, it replicated all the key interactions attained by sorafenib with the kinase domain hot spots in VEGFR-2 (Glu885, Cys919 and Asp1046), and in BRAF_WT/V600E (Glu500, Cys531, and Asp593) (see the SI for further details). Self-docking validation suggested the aptness of the adopted molecular docking protocols for the planned molecular docking study of the tested compounds in the kinase domain of the target kinases.

Generally, the target compounds 4a–4d, 6a–6c, 6f, 6h, and 6i showed a comparable binding pattern in the target kinase domains with docking energy score ranges of −16.28 to −13.41 kcal mol⁻¹ (in VEGFR-2), −15.17 to −13.12 kcal mol⁻¹ (in BRAF_WT), and −15.56 to −13.06 kcal mol⁻¹ (in BRAF_V600E) in comparison to the native ligand, sorafenib, binding scores of −15.05, −14.96, and −15.48 kcal mol⁻¹, respectively (Table S.4, see the SI for further details).

The tested compounds exhibited favorable binding patterns in the kinase domains of the target kinases, interacting with the key amino acids in a type II inhibitor-like binding mode. Generally, the amide moiety is located at the boundary between the gate area and the hydrophobic allosteric back pocket interacting through hydrogen bonding with the side chain carboxylate of Glu885 and Glu500 and with backbone NH of Asp1046 and Asp593 in VEGFR-2 and BRAF_WT/V600E, respectively. From one side, this binding mode fits the thiopyrimidine moiety into the hydrophobic allosteric back pocket interacting through hydrophobic interactions with the surrounding amino acids lining the allosteric back pocket, Ile888, Leu889, Ile892, Val898, Val899, Leu1019 and Ile1044 (in VEGFR-2) and Val503, Ile512, Leu566, Ile571, and Ile591 (in BRAF_WT/V600E). On the other side, the phenyl spacer fits in the gate area interacting through hydrophobic interactions with the surrounding amino acids Lys868, Leu513, Cys1045, and Phe1047 (in VEGFR-2) and Lys482, Leu513, and Phe594 (in BRAF_WT/V600E). This binding pattern directs the peripheral substituted phenyl ring into the hinge region to interact through hydrophobic interactions with the surrounding amino acids Leu840, Val848, Ala866, Val899, Phe918, Leu1035 and Phe1047 (in VEGFR-2) and residues Ile462, Val470, Trp530, Cys531, Phe582, and Phe594 (in BRAF_WT/V600E). Furthermore, additional π–π stacking interaction takes place at the hinge region between this phenyl moiety and the side chains of Phe918 (in VEGFR-2) and Trp530 (in BRAF_WT/V600E). Additionally, compounds containing 2-chloro substitution at the peripheral phenyl ring 4a, 6a, and 6f show halogen bond interactions with Glu917 (in VEGFR-2) and Gln529 (in BRAF_WT/V600E) (Fig. 10–12) (see the SI for further details).

Fig. 10. 2D (A) and 3D (B) representation of compound 4c interactions within the VEGFR-2 active site.

Fig. 11. 2D (A) and 3D (B) representation of compound 4c interactions within the BRAF_WT active site.

Fig. 12. 2D (A) and 3D (B) representation of compound 4c interactions within the BRAF_V600E active site.

Conclusion

In the present study, two series of chalcone 2-thiopyrimdine conjugates 4a–4d and 6a–6i were designed, synthesized, and assessed for their anticancer activity against a panel of 60 cancer cell lines (NCI-60) at a single concentration (10 μM). Ten compounds (4a–4d, 6a–6c, 6f, 6h, and 6i) were further selected and tested by the NCI five-dose assay where they showed a GI₅₀ value range of 0.0055–87.80 μM. Seven compounds (4a, 4c, 4d, 6c, 6f, 6h, and 6i) revealed a safety profile towards normal cells. Furthermore, the VEGFR-2 inhibitory activity for the promising compounds 4a–4d, 6a–6c, 6f, 6h and 6i elicited IC₅₀ values of 0.072–1.176 μM in comparison to sorafenib (IC₅₀ = 0.081 μM). Moreover, compound 4c showed potent BRAF_WT and BRAF_V600E inhibitory activities relative to vemurafenib. In LOX IMVI cells, 4c engendered cell cycle arrest at the G₁ phase and induced late apoptosis. Furthermore, apoptotic marker investigation in LOX IMVI cells showed that compound 4c increased Bax and caspases-3 and -9 expression levels while decreasing the expression level of the anti-apoptotic protein Bcl-2. Molecular docking study revealed that the designed compounds showed a common favorable interaction pattern in the binding site of the target kinases that extends from the hinge region through the gate area towards the allosteric back pocket interacting with the key amino acids in a type II inhibitor-like binding pattern.

Experimental

Chemistry

General remarks

Organic solvents and chemicals were purchased from commercial suppliers: Sigma Aldrich, ACROS, Fischer, and Alfa Aesar. Using pre-coated silica gel 60 F245 aluminum sheets (Merck), thin layer chromatography (TLC) was performed with UV light viewing. Melting points were measured using a melting point analyzer (Daihan Scientific, MP360D, South Korea) and were uncorrected. IR spectra were recorded via a Fourier-transform infrared (FTIR) spectrophotometer (8400 Shimadzu, Kyoto, Japan) utilizing KBr pellets. ¹H and ¹³C NMR spectra were recorded on a 500 (125) MHz JEOL spectrometer (Japan), a 400 (100) MHz Bruker spectrometer, and a 300 (75) MHz Varian spectrometer. DMSO-d₆ and CDCl₃ were utilized as NMR solvents. The unit of measurement for coupling constants was hertz (Hz). Chemical shifts (δ) were measured in relation to tetramethylsilane (TMS) as an internal standard and were recorded in parts per million. IR and NMR spectra were recorded at the Microanalytical Unit, Faculty of Pharmacy, and Faculty of Science, Cairo University, Egypt. HRMS was reported using a Thermo Exactive Plus Orbitrap mass spectrometer at Joseph Banks Laboratories, University of Lincoln, UK.

Synthesis of the chalcones 1a–1e, key intermediates 2a–2e, and 2-thioxopyrimidine 3 and 5a and 5b

The synthesis of the reported chalcones 1a–1e,²⁹ the 2-chloro-N-(4-((E)-3-((substituted)phenyl)acryloyl)phenyl)acetamides 2a, 2b, 2d, and 2e,²⁹ 4,6-dimethyl-2-thioxopyrimidine 3,¹¹ and 6-methyl-2-thioxo-2,3-dihydropyrimidin-4(1H)-one 5b,³³ is outlined in the SI.

2-Chloro-N-(4-((E)-3-(4-chloro-3-nitrophenyl)acryloyl)phenyl)acetamide (2c)

A mixture of (E)-1-(4-aminophenyl)-3-(4-chloro-3-nitrophenyl)prop-2-en-1-one 1c (1.241 g, 4.1 mmol) and chloroacetyl chloride (0.463 g, 4.1 mmol) in DMF (5 mL) in the presence of anhydrous potassium carbonate (0.622 g, 4.51 mmol) was stirred for 1.5 h at room temperature and then poured onto ice-cold water. The formed precipitate was filtered, washed with water, and dried to afford 2c as a yellowish orange precipitate which was filtered, washed with water, and dried. Yield: 80%; mp: 166–168 °C; IR (KBr) υ_max: 3399, 3071, 2920, 2851, 1702, 1693, 1667, 1601, 1531 cm⁻¹; ¹H NMR (500 MHz; DMSO-d₆, δ): 4.29 (s, 2H, CH₂), 7.73 (d, J = 15.3 Hz, 1H, CH–CO), 7.76 (d, J = 8.6 Hz, 2H, CH_ar), 7.84 (d, J = 8.1 Hz, 1H, CH_ar), 8.10 (d, J = 15.5 Hz, 1H, CH CH), 8.15–8.18 (m, 3H, CH_ar), 8.64 (s, 1H, CH_ar), 10.68 (s, 1H, NH) ppm; ¹³C NMR (125 MHz; DMSO-d₆, δ): 44.02, 119.31, 125.18, 125.74, 130.70, 132.55, 132.98, 134.25, 135.38, 136.13, 140.29, 143.71, 148.80, 165.82, 187.80 ppm.

General procedure for the synthesis of chalcone/2-thiopyrimidines 4a–4d

A mixture of the appropriate 2-chloro-N-(4-((E)-3-((substituted)phenyl)acryloyl)phenyl)acetamides 2a–2c and 2e (1 mmol) and 4,6-dimethyl-2-thioxopyrimidine 3 (0.14 g, 1 mmol) in dry DMF (5 mL) and in the presence of anhydrous potassium carbonate (0.25 g, 1.1 mmol) was stirred under reflux for 1–2 h and then poured onto ice-cold water. The formed precipitate was filtered, washed with water, dried, and recrystallized from an appropriate solvent to afford the target compounds 4a–4d.

2-(1,6-Dihydro-4,6-dimethylpyrimidin-2-ylthio)-N-(4-((E)-3-(2-chlorophenyl)acryloyl)phenyl)acetamide (4a)

Yellowish white powder; yield: 80%; solvent of crystallization: THF; mp: 188–190 °C; IR (KBr) υ_max: 3248, 3178, 3066, 2916, 2851, 1693, 1655, 1593, 1550 cm⁻¹; ¹H NMR (400 MHz; CDCl₃, δ): 2.51 (s, 6H, 2CH₃), 3.91 (s, 2H, CH₂), 6.86 (s, 1H, CH_pyrimid), 7.30–7.32 (m, 2H, CH_ar), 7.42 (d, J = 8.0 Hz, 1H, CH_ar), 7.46 (d, J = 16.0 Hz, 1H, CH–CO), 7.60 (d, J = 8.0 Hz, 2H, CH_ar), 7.73 (d, J = 8.0 Hz, 1H, CH_ar), 7.99 (d, J = 8.0 Hz, 2H, CH_ar), 8.15 (d, J = 16.0 Hz, 1H, CH CH), 9.86 (s, 1H, NH) ppm; ¹³C NMR (100 MHz; CDCl₃, δ): 24.09, 35.84, 117.2, 119.12, 124.60, 127.19, 127.90, 130.19, 130.38, 131.23, 133.39, 133.49, 135.54, 140.35, 142.57, 167.82, 168.29, 170.19, 188.82 ppm; HRMS (−) ESI m/z calculated for C₂₃H₂₀ClN₃O₂S [M − H]⁻: 436.0897, found: 436.0959.

2-(1,6-Dihydro-4,6-dimethylpyrimidin-2-ylthio)-N-(4-((E)-3-(4-chlorophenyl)acryloyl)phenyl)acetamide (4b)

Dark yellow powder; yield: 66%; solvent of crystallization: absolute ethanol; mp: 262–263 °C; IR (KBr) υ_max: 3237, 3082, 2924, 2855, 1701, 1669, 1608, 1589 cm⁻¹; ¹H NMR (300 MHz; CDCl₃, δ): 2.51 (s, 6H, 2CH₃), 3.91 (s, 2H CH₂), 6.86 (s, 1H, CH_pyrimid), 7.39 (d, J = 9.0 Hz, 2H, CH_ar), 7.48 (d, J = 15.0 Hz, 1H, CH CH), 7.55–7.61 (m, 4H, CH_ar),7.73 (d, J = 15.0 Hz, 1H, CH–CO), 8.00 (d, J = 9.0 Hz, 2H, CH_ar), 9.82 (s, 1H, NH) ppm; ¹³C NMR (75 MHz; CDCl₃, δ): 24.12, 29.84, 35.80, 117.01, 119.13, 120.93, 122.29, 129.37, 129.70, 130.11, 133.57, 142.58, 143.11, 167.84, 168.36 ppm; HRMS (−) ESI m/z calculated for C₂₃H₂₀ClN₃O₂S [M − H]⁻: 436.0913, found: 436.0959.

2-(1,6-Dihydro-4,6-dimethylpyrimidin-2-ylthio)-N-(4-((E)-3-(4-chloro-3-nitrophenyl)acryloyl)phenyl)acetamide (4c)

Dark yellow powder; yield: 70%; solvent of crystallization: petroleum ether (60–80 °C) : ethyl acetate (5 : 1); mp: 166–167 °C; IR (KBr) υ_max: 3387, 3163, 2920, 2851, 1718, 1694, 1663, 1585, 1535 cm⁻¹; ¹H NMR (400 MHz; CDCl₃, δ): 2.91 (s, 6H, 2CH₃), 4.34 (s, 2H, CH₂), 7.63 (s, 1H, CH_pyrimid), 7.94 (d, J = 16.0 Hz, 1H, CH–CO), 7.98–8.02 (m, 3H, CH_ar), 8.07–8.11 (m, 2H, CH_ar, CH CH), 8.37 (d, J = 8.0 Hz, 2H, CH_ar), 8.50 (s, 1H, CH_ar), 10.31 (s, 1H, NH) ppm; ¹³C NMR (100 MHz; CDCl₃, δ): 24.03, 29.82, 35.80, 117.07, 119.22, 124.60, 124.79, 128.40, 130.23, 132.56, 132.64, 133.02, 135.25, 140.17, 143.00, 148.45, 167.89, 168.29, 170.12, 187.79 ppm; HRMS (−) ESI m/z calculated for C₂₃H₁₉ClN₄O₄S [M − H]⁻: 481.0750, found: 481.0810.

2-(4,6-Dimethylpyrimidin-2-ylthio)-N-(4-(E)-3-(3,4,5-trimethoxyphenyl)acryloyl)acetamide (4d)

Yellow powder; yield: 77%; solvent of crystallization: petroleum ether (60–80 °C) : ethyl acetate (1 : 1); mp: 100–101 °C; IR (KBr) υ_max: 3318, 3186, 2924, 2851, 1705, 1694, 1655, 1582, 1528, 1501 cm⁻¹; ¹H NMR (300 MHz; CDCl₃, δ): 2.52 (s, 6H, 2CH₃), 3.91 (s, 2H, CH₂), 3.93 (s, 9H, 3OCH₃), 6.86 (s, 3H, CH_ar, CH_pyrimid), 7.38 (d, J = 15.0 Hz, 1H, CH–CO), 7.61 (d, J = 9.0 Hz, 2H, CH_ar), 7.71 (d, J = 15.0 Hz, 1H, CH CH), 8.00 (d, J = 9.0 Hz, 2H, CH_ar), 9.82 (s, 1H, NH) ppm; ¹³C NMR (75 MHz; CDCl₃, δ): 24.05, 35.73, 56.30, 61.09, 105.67, 116.94, 119.06, 121.21, 130.02, 130.48, 133.76, 142.35, 144.80, 153.53, 167.76, 168.33, 170.22, 189.00 ppm; HRMS (−) ESI m/z calculated for C₂₆H₂₇N₃O₅S [M − H]⁻: 492.1607, found: 492.1666.

General procedure for the synthesis of chalcone/2-thiopyrimidines 6a–6i

A mixture of the appropriate 2-chloro-N-(4-((E)-3-((substituted)phenyl)acryloyl)phenyl)acetamides 2a–2e (1 mmol) and the commercially available 2-thiouracil 5a (0.13 g, 1 mmol) or 6-methyl-2-thiouracil 5b (0.14 g, 1 mmol) in dry DMF (10 mL) in the presence of anhydrous potassium carbonate (0.25 g, 1.1 mmol) was stirred under reflux for 1–2 h and then poured onto ice-cold water. The formed precipitate was filtered, washed with water, dried, and purified either by recrystallization from appropriate solvents or by column chromatography to obtain the target compounds 6a–6i.

2-(1,6-Dihydro-6-oxopyrimidin-2-ylthio)-N-(4-((E)-3-(2-chlorophenyl)acryloyl)phenyl)acetamide (6a)

Yellowish white powder; yield: 62%; solvent of crystallization: THF; mp: 229–230 °C; IR (KBr) υ_max: 3298, 3186, 3047, 2920, 2847, 1701, 1659, 1609, 1595, 1524 cm⁻¹; ¹H NMR (300 MHz; DMSO-d₆, δ): 4.16 (s, 2H, CH₂), 6.13 (d, J = 6.0 Hz, 1H, CH_pyrimid), 7.40–7.47 (m, 2H, CH_ar), 7.52–7.55 (m, 1H, CH_ar), 7.78 (d, J = 9.0 Hz, 2H, CH_ar), 7.86 (d, J = 6.0 Hz, 1H, CH_ar), 7.89 (d, J = 12.0 Hz, 1H, CH–CO), 7.98 (d, J = 6.0 Hz, 1H, CH_ar), 8.02 (d, J = 12.0 Hz, 1H, CH CH), 8.16 (d, J = 6.0 Hz, 2H, CH_ar, CH_pyrimid), 10.61 (br. s, 1H, NH), 10.67 (s, 1H, NH) ppm; ¹³C NMR (75 MHz; DMSO-d₆, δ): 35.23, 109.67, 118.59, 124.70, 127.71, 128.57, 130.06, 130.20, 131.92, 132.23, 132.42, 134.38, 138.06143.55, 166.49, 187.36 ppm; HRMS (−) ESI m/z calculated for C₂₁H₁₆ClN₃O₃S [M − H]⁻: 424.0540, found: 424.0595.

2-(1,6-Dihydro-6-oxopyrimidin-2-ylthio)-N-(4-((E)-3-(4-chlorophenyl)acryloyl)phenyl)acetamide (6b)

Yellowish white powder; yield: 70%; eluent of column chromatography: ethyl acetate : petroleum ether (60–80 °C): (2 : 1); mp: 230–232 °C; IR (KBr) υ_max: 3379, 2920, 2851, 1724, 1678, 1655, 1585, 1535 cm⁻¹; ¹H NMR (400 MHz; DMSO-d₆, δ): 3.68 (s, 2H, CH₂), 5.70 (d, J = 4.0 Hz, 1H, CH_pyrimid), 7.51 (d, J = 8.0 Hz, 2H, CH_ar), 7.55 (d, J = 8.0 Hz, 1H, CH_ar), 7.69 (d, J = 16.0 Hz, 1H, CH–CO), 7.85 (d, J = 12.0 Hz, 2H, CH_ar), 7.92 (d, J = 8.0 Hz, 2H, CH_ar, CH_pyrimid), 7.96 (d, J = 16.0 Hz, 1H, CH CH), 8.13 (d, J = 8.0 Hz, 2H, CH_ar), 10.37 (br. s, 1H, NH), 12.62 (s, 1H, NH) ppm; ¹³C NMR (100 MHz; DMSO-d₆, δ): 23.26, 108.60, 118.50, 122.88, 129.04, 130.13, 130.61, 132.04, 133.87, 135.02, 141.92, 144.01, 153.50, 167.82, 169.60, 171.80, 174.48, 187.55 ppm; HRMS (−) ESI m/z calculated for C₂₁H₁₆ClN₃O₃S [M − H]⁻: 424.0540, found: 424.0595.

2-(1,6-Dihydro-6-oxopyrimidin-2-ylthio)-N-(4-((E)-3-(4-chloro-3-nitrophenyl)acryloyl)phenyl)acetamide (6c)

Yellow powder; yield: 77%; solvent of crystallization: THF; mp: 221–222 °C; IR (KBr) υ_max: 3318, 3036, 2920, 1710, 1669, 1601, 1581, 1531 cm⁻¹; ¹H NMR (300 MHz; DMSO-d₆, δ): 4.16 (s, 2H, CH₂), 6.13 (s, 1H, CH_pyrimid), 7.74 (d, J = 15.0 Hz, 1H, CH–CO), 7.77 (d, J = 6.0 Hz, 2H, CH_ar), 7.86 (d, J = 9.0 Hz, 2H, CH_ar), 8.12 (d, J = 15.0 Hz, 1H, CH CH), 8.17 (d, J = 9.0 Hz, 3H, CH_ar, CH_pyrimid), 8.65 (s, 1H, CH_ar), 10.67 (s, 1H, NH), 12.79 (br. s, 1H, NH) ppm; ¹³C NMR (75 MHz; DMSO-d₆, δ): 35.21, 118.52, 124.70, 125.15, 125.82, 130.23, 132.03, 132.11, 133.75, 135.60, 139.70, 143.62, 148.22, 166.46, 187.16 ppm; HRMS (−) ESI m/z calculated for C₂₁H₁₅ClN₄O₅S [M − H]⁻: 469.0390, found: 469.0446.

2-(1,6-Dihydro-6-oxopyrimidin-2-ylthio)-N-(4-((E)-3-p-tolylacryloyl)phenyl)acetamide (6d)

Off-white powder; yield: 74%; solvent of crystallization: ethyl acetate; mp: 221–223 °C; IR (KBr) υ_max: 3267, 3283, 2982, 2835, 1686, 1659, 1593, 1527, 1504 cm⁻¹; ¹H NMR (400 MHz; DMSO-d₆, δ): 2.35 (s, 3H, CH₃), 4.16 (s, 2H CH₂), 6.14 (s, 1H, CH_pyrimid), 7.27 (d, J = 8.0 Hz, 2H, CH_ar), 7.69 (d, 1H, 16.0 Hz, CH CH), 7.75–7.78 (m, 4H, CH_ar), 7.88 (d, 2H, 16.0 Hz, CH–CO, CH_pyrimid), 8.15 (d, J = 8.0 Hz, 2H, CH_ar), 10.66 (s, 1H, NH), 12.78 (br. s, 1H, NH) ppm; ¹³C NMR (100 MHz; DMSO-d₆, δ): 21.09, 35.15, 118.49, 120.88, 128.85, 129.54, 129.91, 132.06, 132.59, 140.57, 143.19, 143.50, 166.36, 187.50 ppm; HRMS (−) ESI m/z calculated for C₂₂H₁₉N₃O₃S [M − H]⁻: 404.1082, found: 404.1142.

2-(1,6-Dihydro-6-oxopyrimidin-2-ylthio)-N-(4-((E)-3-(3,4,5-trimethoxyphenyl)acryloyl)phenyl)acetamide (6e)

Dark yellow powder; yield: 72%; solvent of crystallization: petroleum ether (60–80 °C) : ethyl acetate (2 : 1); mp: 158–160 °C; IR (KBr) υ_max: 3379, 3086, 2924, 2855, 1724, 1678, 1655, 1597, 1525 cm⁻¹; ¹H NMR (400 MHz; DMSO-d₆, δ): 3.71 (s, 3H, CH₃), 3.86 (s, 6H, 2CH₃), 4.10 (s, 2H, CH₂), 6.09 (s, 1H, CH_pyrimid), 7.22 (s, 2H, CH_ar), 7.67 (d, J = 16.0 Hz, 1H, HC CH), 7.79 (d, J = 8.0 Hz, 2H, CH_ar), 7.83 (s, 1H, CH_pyrimid), 7.89 (d, J = 16.0 Hz, 1H, CH–CO), 8.17 (d, J = 8.0 Hz, 2H, CH_ar), 10.82 (br. s, 1H, NH), 10.93 (s, 1H, NH) ppm; ¹³C NMR (100 MHz; DMSO-d₆, δ): 35.18, 56.18, 60.19, 106.51, 109.44, 118.51, 121.15, 130.02, 130.37, 132.58, 139.71, 143.34, 143.99, 153.16, 153.88, 166.86, 187.53 ppm; HRMS (−) ESI m/z calculated for C₂₄H₂₃N₃O₆S [M − H]⁻: 480.1248, found: 480.1302.

2-(1,6-Dihydro-4-methyl-6-oxopyrimidin-2-ylthio)-N-(4-((E)-3-(2-chlorophenyl)acryloyl)phenyl)acetamide (6f)

Light yellow powder; yield: 76%; solvent of crystallization: ethyl acetate; mp: 224–225 °C; IR (KBr) υ_max: 3294, 3186, 3040, 2905, 2851, 1693, 1667, 1601, 1535 cm⁻¹; ¹H NMR (300 MHz; DMSO-d₆, δ): 2.11 (s, 3H, CH₃), 4.11 (s, 2H, CH₂), 5.99 (s, 1H, CH_pyrimid), 7.74 (d, J = 15.0 Hz, 1H, CH–CO), 7.78 (d, J = 9.0 Hz, 2H, CH_ar), 7.85 (d, J = 9.0 Hz, 2H, CH_ar), 8.11 (d, J = 15.0 Hz, 1H, CH CH), 8.18 (d, J = 9.0 Hz, 3H, CH_ar), 8.64 (s, 1H, CH_ar), 10.69 (s, 2H, NH) ppm; ¹³C NMR (75 MHz; DMSO-d₆, δ): 35.31118.56, 124.72, 125.20, 125.83, 130.25, 132.09, 133.78, 135.63, 139.72, 143.71, 148.26, 166.82, 187.22 ppm; HRMS (−) ESI m/z calculated for C₂₂H₁₈ClN₃O₃S [M − H]⁻: 438.0694, found: 438.0752.

2-(1,6-Dihydro-4-methyl-6-oxopyrimidin-2-ylthio)-N-(4-((E)-3-(4-chlorophenyl)acryloyl)phenyl)acetamide (6g)

Off-white powder; yield: 68%; solvent of crystallization: THF; mp: 236–238 °C; IR (KBr) υ_max: 3302, 3186, 2974, 2908, 2851, 1691, 1658, 1605, 1593, 1519 cm⁻¹; ¹H NMR (400 MHz; DMSO-d₆, δ): 2.11 (s, 3H, CH₃), 4.12 (s, 2H, CH₂), 5.99 (s, 1H, CH_pyrimid), 7.51 (s, 2H, CH_ar), 7.72–7.92 (m, 6H, CH_ar), 8.16 (s, 2H, CH_ar), 10.66 (s, 1H, NH), 12.51 (br. s, 1H, NH) ppm; ¹³C NMR (100 MHz; DMSO-d₆, δ): 23.25, 35.28, 118.52, 122.73, 128.95, 130.04, 130.51, 132.37, 133.77, 134.99, 141.94, 143.41, 166.70, 187.44 ppm; HRMS (−) ESI m/z calculated for C₂₂H₁₈ClN₃O₃S [M − H]⁻: 438.0693, found: 438.0752.

2-(1,6-Dihydro-4-methyl-6-oxopyrimidin-2-ylthio)-N-(4-((E)-3-(4-chloro-3-nitrophenyl)acryloyl)phenyl)acetamide (6h)

Yellowish white powder; yield: 69%; solvent of crystallization: THF; mp: 221–222 °C; IR (KBr) υ_max: 3298, 3040, 2920, 2851, 1694, 1667, 1610, 1589, 1535 cm⁻¹; ¹H NMR (400 MHz; DMSO-d₆, δ): 2.11 (s, 3H, CH₃), 4.13 (s, 2H, CH₂), 6.00 (s, 1H, CH_pyrimid), 7.74 (d, J = 16.0 Hz, 1H, CH–CO), 7.81 (d, J = 8.0 Hz, 2H, CH_ar), 8.86 (d, J = 8.0 Hz, 1H, CH_ar), 8.13 (d, J = 16.0 Hz, 1H, CH CH), 8.19 (d, J = 8.0 Hz, 3H, CH_ar), 8.67 (s, 1H, CH_ar), 10.86 (s, 1H, NH), 12.78 (br. s, 1H, NH) ppm; ¹³C NMR (100 MHz; DMSO-d₆, δ): 35.23, 118.52, 124.67, 125.22, 125.74, 130.18, 132.03, 133.73, 135.62, 139.64, 143.73, 148.25, 166.83, 187.20 ppm; HRMS (−) ESI m/z calculated for C₂₂H₁₇ClN₄O₅S [M − H]⁻: 483.0546, found: 483.0603.

2-(1,6-Dihydro-4-methyl-6-oxopyrimidin-2-ylthio)-N-(4-((E)-3-(3,4,5-trimethoxyphenyl)acryloyl)phenyl)acetamide (6i)

Yellow powder; yield: 82%; solvent of crystallization: petroleum ether (60–80 °C) : ethyl acetate (1 : 1); mp: 155–157 °C; IR (KBr) υ_max: 3264, 3040, 2924, 2855, 1724, 1678, 1655, 1603, 1597, 1535 cm⁻¹; ¹H NMR (400 MHz; DMSO-d₆, δ): 2.12 (s, 3H, CH₃), 3.72 (s, 3H, OCH₃), 3.86 (s, 6H, OCH₃), 4.09 (s, 2H, CH₂), 5.97 (s, 1H, CH_pyrimid), 7.22 (s, 2H, CH_ar), 7.68 (d, J = 12.0 Hz, 1H, CH–CO), 7.78 (d, J = 8.0 Hz, 2H, CH_ar), 7.89 (d, J = 16.0 Hz, 1H, CH CH), 8.17 (d, J = 8.0 Hz, 2H, CH_ar), 10.72 (br. s, 1H, NH), 10.80 (s, 1H, NH) ppm; ¹³C NMR (100 MHz; DMSO-d₆, δ): 23.17, 35.32, 56.16, 60.18, 106.48, 118.51, 121.12, 130.02, 130.39, 132.60, 143.33, 143.98, 153.14, 166.76, 187.48 ppm; HRMS (−) ESI m/z calculated for C₂₅H₂₅N₃O₆S [M − H]⁻: 494.1405, found: 494.1459.

Biology

NCI-60 anticancer screening

All the newly synthesized chalcone/2-thiopyrimidine hybrids 4a–4d and 6a–6i were selected and evaluated by the Developmental Therapeutic Program (DTP) for the one-dose assay (National Cancer Institute (NCI), Bethesda, Maryland, USA),^39–43 and the screening results were reported as growth inhibition (GI) % values at a single concentration. Compounds 4a–4d, 6a–6c, 6f, 6h and 6i were selected for the five-dose assay. The mean graphs, dose–response curves, GI₅₀, TGI, and LC₅₀ values, and the detailed assay protocol are reported in the SI.

In vitro cytotoxicity on human primary skin fibroblast normal cell line

Cytotoxicity evaluation was performed on compounds 4a–4d, 6c, 6f, 6h, and 6i against skin fibroblast (hFB) cell line, supplied from the Brazilian cell bank (BCRJ, Rio de Janeiro, Brazil), through MTT assay according to the reported procedures^11,35 and is reported in the SI.

Kinase inhibition assays

The detailed VEGFR-2 and BRAF kinase assay protocol is presented in the SI according to reported procedures.^11,44 Compounds 4a–4d, 6a–6c, 6f, 6h and 6i were selected and tested for VEGFR-2 kinase assay relative to sorafenib, and the IC₅₀ values were calculated. Compound 4c was further selected and tested on BRAF_WT and BRAF_V600E kinases in comparison to vemurafenib, and IC₅₀ values were also calculated.

Cell cycle analysis

The cell cycle analysis performed by a flow cytometry protocol is illustrated in the SI according to reported procedures.^45–47 Cell cycle distribution was performed for compound 4c at half of its GI₅₀ value in LOX IMVI melanoma cells obtained from ATCC, USA.

Apoptosis study

Apoptosis assay

The detailed apoptotic study protocol is outlined in the SI according to reported procedures.^45–47 Apoptosis was performed via flow cytometry for compound 4c at half of its GI₅₀ value in LOX IMVI cells.

Apoptosis marker determination

Bax, Bcl-2, and caspase-3 and -9 expression levels and Bax/Bcl-2 ratio were determined via RT-PCR for compound 4c at half of its GI₅₀ value in LOX IMVI cells via a Qiagen RNA extraction kit and a BioRad iScript One-Step RT-PCR Kit.

Cell migration assay

Cell migration assay was carried out for compound 4c in LOX IMVI cells after 72 h, and the cell migration assay protocol is presented in the SI according to reported procedures.^34,48

Molecular docking study

Molecular Operating Environment (MOE, 2024.06) software was utilized to perform the molecular docking simulation of the most promising compounds 4a–4d, 6a–6c, 6f, 6h, and 6i in the kinase domain of the target kinases (VEGFR-2, BRAF_WT, BRAF_V600E). The X-ray crystal structures of VEGFR-2, BRAF_WT, and BRAF_V600E in their inactive conformation (DFG-out) co-crystallized with sorafenib; PDB ID 4ASD, PDB ID 1UWH, and PDB ID 1UWJ, respectively, were utilized for the planned molecular docking study.^36–38 The detailed docking protocol is included in the SI.

Author contributions

Conceptualization: I. A. Y. G.; funding acquisition: R. A. I. A.; supervision and project adminstration: H. A. A., S. E. A., I. A. Y. G.; methodology: R. A. I. A., A. M. E., M. T. A.; data curation and validation: S. E. A., I. A. Y. G.; formal analysis: A. M. E., M. T. A., I. A. Y. G.; investigation: I. A. Y. G.; writing – original draft: all co-authors, writing – reviewing and editing: H. A. A., A. M. E., S. E. A., I. A. Y. G.

Conflicts of interest

The authors declare no conflict of interest to disclose.

Data availability

The data supporting this article (NMR and HRMS spectra) have been included as part of the supporting information (SI). Supplementary information is available. See DOI: https://doi.org/10.1039/d5md00787a.