Novel Sulfonamide Derivatives Carrying a Biologically Active 3,4-Dimethoxyphenyl Moiety as VEGFR-2 Inhibitors

Abstract

Novel sulfonamides 3–19 with a biologically active 3,4-dimethoxyphenyl moiety were designed and synthesized. The structures of the synthesized compounds were established using elemental analyses, IR, ¹H-NMR, ¹³C-NMR spectral data and mass spectroscopy. All the synthesized compounds were evaluated for their in vitro anticancer activity against four cancer cell lines, namely human hepatocellular carcinoma (HepG2), human medulloblastoma (Daoy), human cervical cancer (HeLa), and human colon cancer (HT-29), by using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and dasatinib as the reference drug. Among the tested derivatives, compounds 4, 10, 16, and 19 showed good activity as cytotoxic agents. The most active derivatives were evaluated for their ability to inhibit vascular endothelial growth factor receptor (VEGFR)-2. Compounds Z-4-(3-(3,4-dimethoxyphenyl)-3-oxoprop-1-enylamino)-N-(5-methyl-1,3,4-thiadiazol-2-yl)-benzenesulfonamide 10 and Z-4-(3-(3,4-dimethoxyphenyl)-3-oxoprop-1-enylamino)-N-(1H-indazol-6-yl)-benzenesulfonamide 19 were more active as VEGFR-2 inhibitors than dasatinib. Molecular docking of the most active derivatives on the active site of VEGFR-2 revealed that compound 19 exhibited favorable and promising results.

Cancer is considered the second common cause of death after cardiovascular diseases, and is expected to become first in the next few years.^1,2) Chemotherapeutic agents used for therapy have two drawbacks: lack of selectivity as most used drugs act on normal as well as tumor cells, leading to serious side effects³⁾ and refractoriness of tumor cells to traditional anticancer agents. Although the latter may be overcome by using several drug combinations, it will however lead to higher cost and longer treatment durations.⁴⁾ Vascular endothelial growth factor receptor (VEGFR) inhibitors have received great attention over the last decade as novel anticancer agents.^5–12) VEGF is a key angiogenic stimulator secreted by tumor cells to switch on the angiogenic phenotype.¹³⁾ Moreover, through circulation, angiogenic capillaries stimulate tumor cells to migrate to other sites (metastasis), leading to an increased mortality rate.¹³⁾ Through binding to a number of receptors, VEGF has become one of the most important regulators of angiogenesis.^14,15) This process is mainly facilitated through a specific VEGF receptor, the kinase insert domain-containing receptor (KDR or VEGFR-2),¹⁶⁾ which is mostly expressed on vascular endothelial cells, and is up-regulated in angiogenic blood vessels.¹⁷⁾ Inhibition of angiogenesis may result in the arrest of tumor growth, a theory that was first proposed by Folkman in 1971,¹⁸⁾ and has now become a reality, since several VEGFR-2 inhibitors are currently used in clinics as anticancer agents.^19–21) Pazopanib I is a VEGFR-2 inhibitor that has been recently approved for the treatment of advanced metastatic renal carcinoma and soft tissue sarcoma, and contains an active benzenesulfonamide moiety in its chemical structure.^22–24) Many sulfonamides exhibited favorable anticancer activity through numerous mechanisms including cell cycle arrest in the G1 phase²⁵⁾ and inhibition of carbonic anhydrase (CA),²⁶⁾ histone deacetylases (HDACs),²⁷⁾ methionine aminopeptidases (MetAPs),²⁸⁾ matrix metalloproteinase (MMPs),²⁹⁾ nicotinamide adenine dinucleotide (NADH) oxidase,³⁰⁾ cyclin-dependent kinase (CDK).³¹⁾ Moreover, by binding to β-tubulin, they can disrupt the microtubule assembly.³²⁾ A different agent, KRN 633 II, is under clinical trials as a VEGFR-2 inhibitor.³³⁾ It contains the active moiety 3,4-dimethoxyphenyl. Based on the aforementioned facts and as a continuation of our research on novel anticancer agents,^34–40) the current study focuses on the synthesis of novel benzenesulfonamide derivatives bearing the 3,4-dimethoxyphenyl moiety, as VEGFR-2 inhibitors. The most active cytotoxic compounds were evaluated for their VEGFR-2 inhibition activity; moreover, docking of the VEGFR-2 active site was performed to explore the binding mode of the inhibitors. It’s worth mentioning that similar work was performed on 4′,7-dicholorquinoline moiety with the substitution of different sulfonamides and good cytotoxic activity was observed to several derivatives against four different cell lines.⁴¹⁾

Results and Discussion

Chemistry

The goal of this study was to synthesize a novel series of benzenesulfonamides with a biologically active 3,4-dimethoxyphenyl moiety and to assess their anticancer activity. Thus, reaction of 3,4-dimethoxyacetophenone 1 with dimethylformamide-dimethylacetal in dry xylene yielded the starting material E-1-(3,4-dimethoxyphenyl)-3-(dimethylamino)prop-2-en-1-one 2. Enaminone 2 was allocated an E-configuration based on its ¹H-NMR spectrum, which indicated that the coupling constant of the doublet signals for olefinic protons was equivalent to 12.3 Hz and associated to E-isomers. Reaction of enaminone 2 with sulfa drugs in absolute ethanol and glacial acetic acid (2 : 1) resulted in benzenesulfonamide derivatives 3–19 at a reasonable yield (Chart 1). The structures of the obtained products were established using microanalysis, IR, ¹H-NMR, and ¹³C-NMR spectral data. The ¹H-NMR spectra for compounds 3–19 revealed that these structures were in the Z-, and not in the E-form, while the coupling constant of the doublet signals for olefinic protons was 8.5–8.6 Hz. Moreover, the Z-form is stabilized by intramolecular hydrogen bonds (Chart 1). Similar reaction of 2-enaminone derivative in E-configuration with several primary amines revealed Z-configuration products were reported in literature.⁴²⁾ The ¹H-NMR spectra of 3 shows a singlet at 10.03 ppm for SO₂NH₂ that was exchangeable with D₂O. The ¹H-NMR spectra of 4 revealed a singlet at 1.92 ppm for the COCH₃ group whereas that of 6 showed a singlet at 2.30 ppm for the CH₃ group and a singlet at 6.25 ppm for the isoxazole CH. The ¹H-NMR spectra of 7 revealed two singlets at 2.07 and 2.09 ppm for the 2CH₃ groups whereas that of 8 showed a doublet signal at 5.90 ppm for the 2CH groups of pyrazole. The ¹H-NMR spectra of 10 exhibited a singlet at 2.44 ppm for the CH₃ group whereas that of 13 presented a singlet at 2.30 ppm for the CH₃ group and that of 14 revealed a singlet at 2.24 ppm for 2CH₃ groups and a singlet at 5.96 ppm for the pyrimidine CH. The ¹H-NMR spectra of 15 displayed a singlet at 2.51 ppm for 2CH₃ groups whereas that of 17 indicated the presence of two singlets at 3.78 and 3.79 ppm for 2OCH₃ groups, and a singlet at 5.96 ppm for the pyrimidine CH. The ¹H-NMR spectra of 18 showed two singlets at 3.70 and 3.90 ppm for 2OCH₃ groups, and a singlet at 8.12 ppm for the pyrimidine CH whereas that of 19 showed a singlet signal at 8.13 ppm for the pyrazole CH.

Chart 1. Synthetic Pathways for Compounds 3–19

In Vitro Anticancer Activity

The synthesized compounds were evaluated for their cytotoxic activity against the human cervical cancer (HeLa), human hepatocellular carcinoma (HepG2), human medulloblastoma (Daoy), and human colon cancer (HT-29) cells by using an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and dasatinib as the reference drug. Percentages of growth inhibition for all compounds as well as the IC₅₀ for the most active compounds are listed in Table 1. Compound 1 inhibited HepG2 cell growth by 58.36% with an IC₅₀ of 14.17 µg/mL. However, the percentage of inhibition for the remaining cell lines was below 50. In contrast, compounds 4, 10 and 19 showed over 50% growth inhibition of HeLa, HepG2, and Daoy cells, but below 50% for HT-29 cells. Compound 16 was the only compound that showed over 50% growth inhibition of all the cancer cell lines tested. Compound 4 was the most active against HeLa cells with a remarkable IC₅₀ of 1.78 µg/mL, an order of magnitude more potent than dasatinib (IC₅₀: 16.22 µg/mL). Compound 19 was the most active against HepG2 cells, with an IC₅₀ of 1.6 µg/mL, and was more potent than dasatinib (IC₅₀: 6.91 µg/mL). The same compound was also the most active against Daoy cells with an IC₅₀ of 5.58 µg/mL, almost twice as potent as dasatinib (IC₅₀: 9.2 µg/mL).

Table 1. In Vitro Cytotoxicity of New Compounds against Cancer Cell Lines

Compd.	Hela		HepG-2		Daoy		HT-29
	% Inhibition*	IC50**	% Inhibition*	IC50**	% Inhibition*	IC50**	% Inhibition*	IC50**
1	45.9	nt#	58.36	14.17±0.83	41.37	nt#	34.83	nt#
2	32.53	nt#	33.76	nt#	29.78	nt#	35.82	nt#
3	30.91	nt#	34.36	nt#	3.34	nt#	0	nt#
4	58.26	1.78±0.08	63.41	2.63±0.13	55.3	7.74±0.26	46.05	nt#
5	37.48	nt#	31.32	nt#	23.67	nt#	28.2	nt#
6	9.36	nt#	5.91	nt#	41.49	nt#	0	nt#
7	36.72	nt#	36.04	nt#	30.3	nt#	0	nt#
8	46.1	nt#	46.69	nt#	35.59	nt#	36.39	nt#
9	29.32	nt#	30.21	nt#	36.82	nt#	19.44	nt#
10	57.51	20.45±0.15	56.5	20.78±0.68	51.32	19.9±1.6	33.7	nt#
11	26.72	nt#	21.65	nt#	24.86	nt#	0	nt#
12	17.12	nt#	33.03	nt#	17.12	nt#	3.7	nt#
13	22.6	nt#	5.5	nt#	22.6	nt#	0	nt#
14	6.04	nt#	0	nt#	6.04	nt#	0	nt#
15	44.07	nt#	44.66	nt#	44.7	nt#	3.65	nt#
16	57.27	22.5±0.9	61	20.74±0.48	53.02	18.44±0.84	52.03	24.46±1.46
17	39.24	nt#	41.17	nt#	35.1	nt#	15.26	nt#
18	24.79	nt#	24.45	nt#	17.59	nt#	0	nt#
19	60.6	2.69±0.19	68.49	1.6±0.2	56.12	5.58±0.42	39.21	nt#
Dasatinib	56.4	16.22±0.78	73.98	6.91±0.31	90.36	9.2±0.2	39.48	25.56±1.44

* Percent of growth inhibition of cells at a concentration of 25 µg/mL, relative to control. ** Dose–response curves were used to calculate IC₅₀ values (µg/mL), expressed as the mean±S.D. ^# nt: Not tested.

Structure–Activity Relationship

It appeared obvious that the substitution of sulfonamide derivatives used in the synthesis of compounds 3–19 has a remarkable effect on their cytotoxic activity. The acetyl derivative 4 showed favorable activity against HepG2 cells, whereas both the unsubstituted sulfonamide derivative 3 and the guanido derivative 5 exhibited low activity. Among the various heterocyclic sulfonamide derivatives 6–19, the methyl thidiazole derivative 10, the 5-methoxy pyrimidine derivative 16, and the benzopyrazole derivative 19 displayed an interesting activity against HepG2 and Daoy cells.

VEGFR-2 Inhibition Activity

The most active compounds (4, 10, 16, 19) were evaluated for their in vitro VEGFR-2 inhibitory activity in the HeLa cell line, using dasatinib as the reference drug. All four compounds showed a good inhibition (72.8–84.2% of control). Compounds 10 and 19 were more active than the reference drug (79.5%) with an inhibition of 80.6 and 84.2%, respectively. VEGFR-2 residual concentrations as well as VEGFR-2 inhibition percentages for the tested compounds are listed in Table 2. These interesting results urge to synthesize and evaluate VEGFR-2 inhibition activity of more derivatives using other sulfonamide moieties in the future.

Table 2. In Vitro Inhibition of VEGFR-2 for the Most Active Compounds

Compounds	Mw (g/mol)	IC50 values (µg/mL)	VEGFR residual (conc. pg/mL)	VEGFR % inhibition
4	404	1.78	686.491	72.8
10	460	20.45	740.914	80.6
16	470	22.5	775.862	74.7
19	478	2.69	590.541	84.2
Dasatinib	488	0.8	978.443	79.5

Molecular Docking

In order to explore the potential binding mode of the most active compounds to VEGFR-2, compounds 4, 10, 16, and 19 were docked on the receptor active site. The protein databank file (PDB ID) 4AGD was selected for this purpose. The file contains data on VEGFR-2 co-crystallized with sunitinib, a type I VEGFR-2 inhibitor. Type I inhibitors bind only to the ATP binding site of kinases with the hinge region with no interactions with allosteric pocket. All docking procedures were achieved by MOE (Molecular Operating Environment) software 10.2008 provided by the chemical computing group, Canada. Docking on the VEGFR-2 active site was performed for the sulfonamide derivatives 4, 10, 16, and 19. The docking protocol was verified by re-docking the co-crystallized ligand near the active site with an energy score (S) of −22.9143 kcal/mol and a root mean standard deviation (RMSD) of 1.5923. The co-crystalized inhibitor interacts with the active site of VEGFR-2 via two hydrogen bonds: the first, between Glu917 and the nitrogen of the pyrrole moiety, and the second, between Cys919 and the carbonyl group (Fig. 1).

Fig. 1. Co-crystallized Ligand on the Active Site of VEGFR-2

The four compounds, 4, 10, 16, and 19 were fit in the active site of VEGFR-2 with energy scores ranging between −11.9827 to −19.6112 kcal/mol. Molecular docking for pazopanib, KNR 633 and dasatinib was also performed. The energy scores of the docked compounds and the amino acid interactions are listed in Table 3.

Table 3. Docking Score (S) and Amino Acid Interactions for the Most Active Compounds on the VEGFR-2 Active Site

Compounds	(S) (kcal/mol)	Amino acid	Interacting group	Type of interaction	H-Bond length (Å)
4	−13.5853	Lys838	C=O	H bond (acceptor)	2.49
		Cys919	C=O	H bond (acceptor)	2.70
10	−11.9827	Cys919	SO2	H bond (acceptor)	2.99
16	−13.9947	Cys919	OCH3	H bond (acceptor)	2.99
19	−19.6112	Lys838	C=O	H bond (acceptor)	2.48
		Glu917	NH pyrazole	H bond (donor)	2.02
		Cys919	SO2	H bond (acceptor)	3.03
Pazopanib	−20.6243	Cys919	N pyrazole	H bond (acceptor)	3.18
KRN 633	−17.8653	Cys919	N pyrimidin	H bond (acceptor)	2.89
Dasatinib	−8.2171	Cys919	C=O	H bond (acceptor)	3.05
Sunitinib	−22.9143	Glu917	N pyrrole	H bond (donor)	2.93
		Cys919	C=O	H bond (acceptor)	2.92

The optimal docking energy score among the synthesized compounds was displayed by compound 19 with a value of −19.6112 kcal/mol supporting its excellent activity as a VEGFR-2 inhibitor (84.2% growth inhibition rate). Compound 19 interacted with the same amino acids (Glu917 and Cys919) in a comparable manner to the co-crystallized ligand did, and had an additional hydrogen bond with Lys838. The amino acid interactions exhibited by compound 19 are illustrated in Figs. 2 and 3. Amino acid interactions of pazopanib, KRN 633 and dasatinib are illustrated in Figs. 4–6, respectively.

Fig. 2. 2D Image of Compound 19 Docked onto the VEGFR-2 Active Site

Fig. 3. 3D Image of Compound 19 Docked to the VEGFR-2 Active Site

Fig. 4. 2D Image of Pazopanib Docked onto the VEGFR-2 Active Site

Fig. 5. 2D Image of KRN 633 Docked onto the VEGFR-2 Active Site

Fig. 6. 2D Image of Dasatinib Docked onto the VEGFR-2 Active Site

Experimental

Chemistry

Elemental analyses were performed using a model 2400 CHNS/O analyzer (PerkinElmer, Inc., U.S.A.). All the values were within ±0.4% of the theoretical values; melting points (mp; uncorrected) were determined using an open capillary on a Gallenkamp melting point apparatus (Sanyo Gallenkamp, U.K.). Precoated silica gel plates (Kieselgel 0.25 mm, 60 F254, Merck, Germany) were used for thin layer chromatography. A developing solvent system of chloroform–methanol (8 : 2) was used, and the spots were detected by ultraviolet light. IR spectra (KBr disc) were recorded using a Fourier transform infrared (FT-IR) spectrophotometer (PerkinElmer, Inc., U.S.A.). ¹H-NMR spectra were scanned on a NMR spectrophotometer (Bruker AXS Inc., Switzerland), operating at 500 MHz for ¹H, and 125.76 MHz for ¹³C. Chemical shifts are expressed as δ-values (ppm) relative to tetramethylsilane (TMS) as an internal standard, and dimethyl sulfoxide (DMSO)-d₆ as a solvent. All reagents used were of analytical grade. The starting material 3,4-dimethoxyacetophenone 1 was purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.), and was directly used for the preparation of the target compounds.

Z-1-(3,4-Dimethxoyphenyl)-3-(dimethylamino)prop-2-en-1-one (2)

A solution of 3,4-dimethoxyacetophenone (1.8 g, 0.01 mol) in dimethylformamide-dimethylacetal (1.19 g, 0.01 mol) was refluxed in dry xylene (20 mL) for 24 h. The obtained solid was collected by filtration, while still hot, and recrystallized from dioxane to yield compound 2.

Yield, 66%; mp 123.7°C. IR: 3080 (arom.), 2997, 2931, 2839 (aliph.), 1635 (CO). ¹H-NMR: 3.11 (s, 6H, 2NCH₃), 3.80 (s, 6H, 2OCH₃), 5.82, 6.98 (2d, 2H, CH=CH, J=12.3 Hz), 7.47–7.69 (m, 3H, Ar-H). ¹³C-NMR: 44.8(2), 56.0(2), 91.1, 110.8, 111.1, 121.2, 133.4, 148.7, 151.7, 154.1, 185.3. MS m/z (%): 235 (M⁺) (12.73), 136 (100). Anal. Calcd for C₁₃H₁₇NO₃ (235): C, 66.36; H, 7.28; N, 5.95. Found: C, 66.07; H, 7.54; N, 6.23.

Synthesis of Benzenesulfonamide Derivatives (3–19)

General Procedure

A mixture of 2 (2.35 g, 0.01 mol) and sulfa drugs (0.012 mol) in absolute ethanol (10 mL) and glacial acetic acid (5 mL) was refluxed for 22h, and subsequently left to cool. The solid product formed was collected by filtration and recrystallized from acetic acid to give compounds 3–19.

Z-4-(3-(3,4-Dimethoxyphenyl)-3-oxoprop-1-enylamino)benzenesulfonamide (3)

Yield, 89%; mp 276.4°C. IR: 3329, 3236 (NH₂, NH), 3005 (arom.), 2974, 2939, 2839 (aliph.), 1635 (CO), 1375, 1188 (SO₂). ¹H-NMR: 3.83 (s, 6H, 2OCH₃), 6.23, 6.60 (2d, 2H, CH=CH, J=8.5 Hz), 7.03–8.15 (m, 7H, Ar-H), 10.30 (s, 2H, SO₂NH₂), 12.04 (s, 1H, NH). ¹³C-NMR: 55.9, 56.1, 95.2, 110.5, 111.3, 111.4, 115.2, 121.7, 127.9, 128.1, 131.5, 132.2, 142.8, 144.5, 149.0, 152.7, 189.6. MS m/z (%): 362 (M⁺) (6.48), 155 (100). Anal. Calcd for C₁₇H₁₈N₂O₅S (362): C, 56.34; H, 5.01; N, 7.73. Found: C, 56.62; H, 5.29; N, 7.46.

Z-N-(4-(3-(3,4-Dimethoxyphenyl)-3-oxoprop-1-enylamino)phenylsulfonyl)acetamide (4)

Yield, 87%; mp 215.5°C. IR: 3329, 3236 (NH), 3078 (arom.), 2974, 2939, 2841 (aliph.), 1718, 1635 (2CO), 1373, 1155 (SO₂). ¹H-NMR: 1.92 [s, 3H, COCH₃], 3.83 [s, 6H, 2OCH₃], 6.22, 6.61 [2d, 2H, CH=CH, J=8.6 Hz]. 7.02–8.17 [m, 7H, Ar-H], 10.40 [s, 1H, SO₂NH], 12.03 [s, 1H, NH]. ¹³C-NMR: 23.6, 55.9, 56.0, 95.2, 110.5, 111.3, 115.1, 115.2, 121.7, 122.0, 130.1(2), 131.4, 143.8, 145.1, 152.7, 152.8, 169.2, 189.6. MS m/z (%): 404 (M⁺) (2.65), 93 (100). Anal. Calcd for C₁₉H₂₀N₂O₆S (404): C, 56.42; H, 4.98; N, 6.93. Found: C, 56.16; H, 5.35; N, 7.26.

Z-N-Carbamimidoyl-4-(3-(3,4-dimethoxyphenyl)-3-oxoprop-1-enylamino)benzenesulfonamide (5)

Yield, 91%; mp 260.1°C. IR: 3435, 3336, 3228 (NH₂, NH), 3100 (arom.), 2976, 2939, 2843 (aliph.), 1635 (CO), 1593 (CN), 1375, 1166 (SO₂). ¹H-NMR: 3.83 (s, 6H, 2OCH₃), 6.20, 6.57 (2d, 2H, CH=CH, J=8.6 Hz), 7.03–7.73 (m, 9H, Ar-H+NH₂), 8.13 (s, 1H, SO₂NH), 10.15 (s, 1H, NH), 12.03 (s, 1H, NH, imino).¹³C-NMR: 55.9, 56.1, 94.9, 110.4, 111.3, 115.1, 115.2, 121.7, 127.8, 131.6(2), 133.3, 142.9, 144.5, 152.5, 158.4, 158.5, 189.5. MS m/z (%): 404 (M⁺) (17.22), 77 (100). Anal. Calcd for C₁₈H₂₀N₄O₅S (404): C, 53.45; H, 4.98; N, 13.85. Found: C, 53.16; H, 5.31; N, 13.56.

Z-4-(3-(3,4-Dimethoxyphenyl)-3-oxoprop-1-enylamino)-N-(3-methylisoxaz-ol-5-yl)benzenesulfonamide (6)

Yield, 88%; mp 160.3°C. IR: 3161(NH), 3066 (arom.), 2978, 2889, 2839 (aliph.), 1637 (CO), 1589 (CN), 1375, 1161 (SO₂). ¹H-NMR: 2.30 (s, 3H, CH₃), 3.83 (s, 6H, 2OCH₃), 6.14, 6.61 (2d, 2H, CH=CH, J=8.5 Hz), 6.25 (s, 1H, CH isoxazole), 7.03–8.15 (m, 7H, Ar-H), 10.40 (s, 1H, SO₂NH), 12.00 (s, 1H, NH). ¹³C-NMR: 12.4, 56.1(2), 95.8, 100.7, 110.5, 111.3, 113.0(2), 121.8, 122.0, 129.2, 131.8(2), 142.4, 145.9, 149.1, 152.8, 158.0, 170.8, 189.7. MS m/z (%): 443 (M⁺) (37.18), 96 (100). Anal. Calcd for C₂₁H₂₁N₃O₆S (443): C, 56.87; H, 4.77; N, 9.48. Found: C, 56.52; H, 4.40; N, 9.11.

Z-4-(3-(3,4-Dimethoxyphenyl)-3-oxoprop-1-enylamino)-N-(3,4-dimethylisoxazol-5-yl)benzenesulfonamide (7)

Yield, 86%; mp 238.2°C. IR: 3334 (NH), 3089 (arom.), 2960, 2835 (aliph.), 1635 (CO), 1589 (CN), 1346, 1159 (SO₂). ¹H-NMR: 2.07, 2.09 (2s, 3H, 2CH₃), 3.83 (s, 6H, 2OCH₃), 6.26, 6.63 (2d, 2H, CH=CH, J=8.5 Hz), 7.03–8.16 (m, 7H, Ar-H), 10.42 (s, 1H, SO₂NH)], 12.03 (s, 1H, NH). ¹³C-NMR: 6.3, 10.7, 56.10, 56.12, 95.8, 100.7, 105.5, 110.6, 111.4(2), 121.8, 122.0, 129.1, 129.2(2), 143.8, 145.9, 149.1, 152.8, 156.1, 161.9, 189.7. MS m/z (%): 457 (M⁺) (31.52), 135 (100). Anal. Calcd for C₂₂H₂₃N₃O₆S (457): C, 57.76; H, 5.07; N, 9.18. Found: C, 57.98; H, 5.36; N, 9.39.

Z-4-(3-(3,4-Dimethoxyphenyl)-3-oxoprop-1-enylamino)-N-(1-phenyl-1H-pyrazol-5-yl)-benzenesulfonamide (8)

Yield, 80%; mp 236.1°C. IR: 3415(NH), 3049 (arom.), 2966, 2835, 2802 (aliph.), 1653 (CO), 1570 (CN), 1338, 1159 (SO₂). ¹H-NMR: 3.84 (s, 6H, 2OCH₃), 5.90 (d, 2H, 2CH pyrazole, J=7.1 Hz), 6.28, 6.63 (2d, 2H, CH=CH, J=8.5 Hz), 7.05–8.13 (m, 12H, Ar-H), 10.41 (s, 1H, SO₂NH), 12.00 (s, 1H, NH). ¹³C-NMR: 56.13, 56.16, 95.7, 104.3, 110.5, 110.6, 111.4(2), 121.8, 122.0, 124.7(2), 127.9, 129.3(2), 131.5, 132.1(2), 135.0, 138.7, 142.5, 144.6, 145.7, 149.1, 152.8, 189.7. MS m/z (%): 504 (M⁺) (16.38), 169 (100). Anal. Calcd for C₂₆H₂₄N₄O₅S (504): C, 61.89; H, 4.79; N, 11.10. Found: C, 61.56; H, 5.28; N, 11.41.

Z-4-(3-(3,4-Dimethoxyphenyl)-3-oxoprop-1-enylamino)-N-(thiazol-2-yl)-benzenesulfonamide (9)

Yield, 83%; mp 260.3°C. IR: 3151 (NH), 3101 (arom.), 2971, 2929, 2860 (aliph.), 1635 (CO), 1589 (CN), 1375, 1168 (SO₂). ¹H-NMR: 3.83 (s, 6H, 2OCH₃), 6.22, 6.58 (2d, 2H, CH=CH, J=8.6 Hz), 6.80–8.14 (m, 9H, Ar-H+2CH thiazole), 10.30 (s, 1H, SO₂NH), 12.02 (s, 1H, NH). ¹³C-NMR: 56.11, 56.13, 95.2, 108.5, 110.5, 110.6, 111.3, 111.4, 121.9, 124.9, 128.2, 131.5(2), 136.4, 142.7, 144.7, 149.0, 152.7, 169.1, 189.5. MS m/z (%): 445 (M⁺) (29.11), 93 (100). Anal. Calcd for C₂₀H₁₉N₃O₅S₂ (445): C, 53.92; H, 4.30; N, 9.43. Found: C, 53.59; H, 3.95; N, 9.14.

Z-4-(3-(3,4-Dimethoxyphenyl)-3-oxoprop-1-enylamino)-N-(5-methyl-1,3,4-thiadiazol-2-yl)benzenesulfonamide (10)

Yield, 80%; mp 186.9°C. IR: 3153 (NH), 3055 (arom.), 2926, 2839, (aliph.), 1635 (CO), 1589 (CN), 1375, 1141 (SO₂). ¹H-NMR: 2.44 (s, 3H, CH₃), 3.83 (s, 6H, 2OCH₃), 6.24, 6.59 (2d, 2H, CH=CH, J=8.5 Hz), 7.04–8.13 (m, 7H, Ar-H), 10.31 (s, 1H, SO₂NH), 12.02 (s, 1H, NH). ¹³C-NMR: 16.5, 55.1(2), 95.4, 110.5, 110.6, 111.3, 111.4, 121.7, 121.9, 128.1, 131.5(2), 144.1, 145.1, 149.1, 152.5, 154.8, 168.2, 186.7. MS m/z (%): 460 (M⁺) (8.75), 124 (100). Anal. Calcd for C₂₀H₂₀N₄O₅S₂ (460.53): C, 52.16; H, 4.38; N, 12.17. Found: C, 52.42; H, 4.09; N, 12.39.

Z-4-(3-(3,4-Dimethoxyphenyl)-3-oxoprop-1-enylamino)-N-(pyridin-2-yl)benzenesulfonamide (11)

Yield, 79%; mp 240.4°C. IR: 3431 (NH), 3101 (arom.), 2931, 2835 (aliph.), 1635 (CO), 1589 (CN), 1394, 1151 (SO₂). ¹H-NMR: 3.83 (s, 6H, 2OCH₃), 6.22, 6.58 (2d, 2H, CH=CH, J=8.5 Hz), 6.87–8.13 (m, 11H, Ar-H), 10.30 (s, 1H, SO₂NH), 12.00 (s, 1H, NH). ¹³C-NMR: 56.1(2), 95.4, 110.5, 110.6, 111.3, 113.8, 113.9, 115.2, 121.8, 121.9, 129.2, 129.3(2), 135.6, 140.6, 144.1, 149.0, 152.5, 152.7, 152.7, 189.6. MS m/z (%): 439 (M⁺) (23.19), 77 (100). Anal. Calcd for C₂₂H₂₁N₃O₅S (439): C, 60.12; H, 4.82; N, 9.56. Found: C, 60.39; H, 4.49; N, 9.21.

Z-4-(3-(3,4-Dimethoxyphenyl)-3-oxoprop-1-enylamino)-N-(pyrimidin-2-yl)benzenesulfonamide (12)

Yield, 83%; mp 272.5°C. IR: 3221 (NH), 3088 (arom.), 2939, 2875, 2823 (aliph.), 1653 (CO), 1577 (CN), 1375, 1184 (SO₂). ¹H-NMR: 3.83 (s, 6H, 2OCH₃), 6.24, 6.59 (2d, 2H, CH=CH, J=8.5 Hz), 7.04–8.51 (m, 10H, Ar-H), 10.34 (s, 1H, SO₂NH), 12.00 (s, 1H, NH). ¹³C-NMR: 56.5(2), 95.6, 110.4, 110.6, 111.3, 111.4, 115.0, 121.8, 121.9, 130.0, 131.4(2), 142.5, 144.5, 149.0, 152.5, 157.4, 158.8, 172.5, 189.6. MS m/z (%): 440 (M⁺) (1.85), 79 (100). Anal. Calcd for C₂₁H₂₀N₄O₅S (440): C, 57.26; H, 4.58; N, 12.72. Found: C, 56.88; H, 4.22; N, 12.44.

Z-4-(3-(3,4-Dimethoxyphenyl)-3-oxoprop-1-enylamino)-N-(4-methylpyrimidin-2-yl)benzenesulfonamide (13)

Yield, 90%; mp 245.2°C. IR: 3410 (NH), 3100 (arom.), 2958, 2923, 2836 (aliph.), 1635 (CO), 1589 (CN), 1338, 1151 (SO₂). ¹H-NMR: 2.30 (s, 3H, CH₃), 3.83 (s, 6H, 2OCH₃), 6.23, 6.59 (2d, 2H, CH=CH, J=8.6 Hz), 6.89–8.32 (m, 9H, Ar-H), 10.33 (s, 1H, SO₂NH), 12.01 (s, 1H, NH). ¹³C-NMR: 23.7, 56.11, 56.12, 95.5, 110.5, 110.6, 111.3, 112.5(2), 121.8, 121.9, 130.3, 131.5(2), 142.5, 145.4, 149.0, 152.8, 157.0, 157.9, 168.7, 189.6. MS m/z (%): 454 (M⁺) (24.28), 93 (100). Anal. Calcd for C₂₂H₂₂N₄O₅S (454): C, 58.14; H, 4.88; N, 12.33. Found: C, 58.48; H, 4.56; N, 12.03.

Z-4-(3-(3,4-Dimethoxyphenyl)-3-oxoprop-1-enylamino)-N-(4,6-dimethylpyrimidin-2-yl)benzenesulfonamide (14)

Yield, 92%; mp 276.5°C. IR: 3220 (NH), 3056 (arom.), 2939, 2843 (aliph.), 1635 (CO), 1587 (CN), 1340, 1149 (SO₂). ¹H-NMR: 2.24 (s, 6H, 2CH₃), 3.83 (s, 6H, 2OCH₃), 5.96 (s, 1H, CH pyrimidine), 6.55, 6.76 (2d, 2H, CH=CH, J=8.5 Hz), 7.04–8.11 (m, 7H, Ar-H), 10.31 (s, 1H, SO₂NH), 12.01 (s, 1H, NH). ¹³C-NMR: 23.5(2), 56.1(2), 96.4, 108.2, 110.6, 111.4, 112.3(2), 114.6, 115.5, 130.5, 130.7(2), 142.8, 146.7, 149.1, 152.6, 163.8(2), 167.7, 184.2. MS m/z (%): 468 (M⁺) (22.17), 164 (100). Anal. Calcd for C₂₃H₂₄N₄O₅S (468): C, 58.96; H, 5.16; N, 11.96. Found: C, 58.67; H, 5.44; N, 12.33.

Z-4-(3-(3,4-Dimethoxyphenyl)-3-oxoprop-1-enylamino)-N-(2,6-dimethylpyrimidin-4-yl)benzenesulfonamide (15)

Yield, 87%; mp 243.3°C. IR: 3415 (NH), 3072 (arom.), 2935, 2839 (aliph.), 1637 (CO), 1589 (CN), 1348, 1149 (SO₂). ¹H-NMR: 2.51 (s, 6H, 2CH₃), 3.82 (s, 6H, 2OCH₃), 6.22, 6.59 (2d, 2H, CH=CH, J=8.5 Hz), 7.01–8.14 (m, 8H, Ar-H), 10.34 (s, 1H, SO₂NH), 12.00 (s, 1H, NH). ¹³C-NMR: 19.0, 22.3, 56.1(2), 95.7, 100.6, 110.5, 110.6, 111.3, 111.4, 121.8, 121.9, 127.7, 129.2, 130.3, 142.3, 146.5, 149.0, 152.5, 152.8, 162.6, 163.0, 186.8. MS m/z (%): 468 (M⁺) (5.63), 89 (100). Anal. Calcd for C₂₃H₂₄N₄O₅S (468): C, 58.96; H, 5.16; N, 11.96. Found: C, 59.32; H, 4.87; N, 11.63.

Z-4-(3-(3,4-Dimethoxyphenyl)-3-oxoprop-1-enylamino)-N-(5-methoxylpyrimidin-2-yl)benzenesulfonamide (16)

Yield, 81%; mp 216.7°C. IR: 3373 (NH), 3101 (arom.), 2837, 2735 (aliph.), 1635 (CO), 1591 (CN), 1375, 1153 (SO₂). ¹H-NMR: 3.78 (s, 3H, OCH₃), 3.83 (s, 6H, 2OCH₃), 6.23, 6.59 (2d, 2H, CH=CH, J=8.5 Hz), 7.02–8.30 (m, 9H, Ar-H), 10.35 (s, 1H, SO₂NH), 12.02 (s, 1H, NH). ¹³C-NMR: 55.9, 56.6, 56.7, 95.6, 110.5, 110.6, 111.3, 111.4, 121.8, 125.7, 129.9, 130.1(2), 142.5(2), 144.0, 145.0, 145.4, 149.8, 152.5, 172.5, 186.8. MS m/z (%): 470 (M⁺) (33.82), 123 (100). Anal. Calcd for C₂₂H₂₂N₄O₆S (470): C, 56.16; H, 4.71; N, 11.91. Found: C, 55.79; H, 4.47; N, 12.25.

Z-4-(3-(3,4-Dimethoxyphenyl)-3-oxoprop-1-enylamino)-N-(2,6-dimethoxylpyrimidin-4-yl)benzenesulfonamide (17)

Yield, 86%; mp 201.3°C. IR: 3134 (NH), 3061 (arom.), 2902, 2835 (aliph.), 1637 (CO), 1589 (CN), 1363, 1151 (SO₂). ¹H-NMR: 3.78, 3.79 (2s, 6H, 2OCH₃ pyrimidine), 3.83 (s, 6H, 2OCH₃), 5.96 (s, 1H, CH pyrimidine), 6.24, 6.58 (2d, 2H, CH=CH, J=8.6 Hz), 7.02–8.12 (m, 7H, Ar-H), 10.37 (s, 1H, SO₂NH), 12.01 (s, 1H, NH). ¹³C-NMR: 54.2, 54.9, 55.8, 55.9, 84.9, 95.8, 110.5, 110.6, 111.4(2), 121.8, 121.9, 129.6, 129.8(2), 142.4, 145.8, 149.1, 152.8, 160.3, 164.7, 172.5, 189.6. MS m/z (%): 500 (M⁺) (19.34), 152 (100). Anal. Calcd for C₂₃H₂₄N₄O₇S (500): C, 55.19; H, 4.83; N, 11.19. Found: C, 55.52; H, 4.57; N, 10.90.

Z-4-(3-(3,4-Dimethoxyphenyl)-3-oxoprop-1-enylamino)-N-(5,6-dimethoxylpyrimidin-4-yl)benzenesulfonamide (18)

Yield, 80%; mp 244.1°C. IR: 3189 (NH), 3082 (arom.), 2941, 2839 (aliph.), 1631 (CO), 1589 (CN), 1375, 1161 (SO₂). ¹H-NMR: 3.70, 3. 90 (2s, 6H, 2OCH₃ pyrimidine), 3.83 (s, 6H, 2OCH₃), 6.20, 6.60 (2d, 2H, CH=CH, J=8.5 Hz), 7.06–7.94 (m, 7H, Ar-H), 8.12 (s, 1H, CH pyrimidine), 10.30 (s, 1H, SO₂NH), 12.00 (s, 1H, NH). ¹³C-NMR: 54.5, 55.9, 56.1, 56.5, 95.6, 110.5, 110.6, 111.4(2), 121.8, 121.9, 127.6, 130.2(2), 131.5, 142.5, 144.0, 149.1, 150.9, 152.6(2), 162.0, 189.6. MS m/z (%): 500 (M⁺) (5.84), 151 (100). Anal. Calcd for C₂₃H₂₄N₄O₇S (500): C, 55.19; H, 4.83; N, 11.19. Found: C, 54.92; H, 4.50; N, 11.48.

Z-4-(3-(3,4-Dimethoxyphenyl)-3-oxoprop-1-enylamino)-N-(1H-indazol-6-yl)benzenesulfonamide (19)

Yield, 89%; mp 252.4°C. IR: 3346 (NH), 3100 (arom.), 2966, 2933, 2839 (aliph.), 1627 (CO), 1589 (CN), 1350, 1157 (SO₂). ¹H-NMR: 3.82 (s, 6H, 2OCH₃), 6.18, 6.60 (2d, 2H, CH=CH, J=8.6 Hz), 6.95–8.08 (m, 10H, Ar-H), 8.13 (s, 1H, CH pyrazole), 10.32 (s, 1H, SO₂NH), 12.00 (s, 1H, NH), 12.8 (s, 1H, NH pyrazole). ¹³C-NMR: 56.5(2), 95.6, 100.3, 110.4, 110.6, 111.3, 115.3(2), 116.2, 120.2, 121.7, 129.3, 131.4(2), 132.2, 140.7, 142.4, 143.8, 144.4, 149.0, 152.8, 186.8. MS m/z (%): 478(M⁺) (14.84), 116 (100). Anal. Calcd for C₂₄H₂₂N₄O₅S (478): C, 60.24; H, 4.63; N, 11.71. Found: C, 60.53; H, 4.31; N, 11.98.

In Vitro Anticancer Evaluation

Chemicals and Supplies

MTT and DMSO were purchased from Sigma-Aldrich. Dulbecco’s modified Eagle’s medium (DMEM)/high glucose, DMEM/F12, fetal bovine serum (FBS), L-glutamine, and penicillin–streptomycin were obtained from Gibco Inc. (NY, U.S.A.).

Cell Lines

Four tumor cell lines were utilized in this study, namely the HeLa, HepG2, Daoy, and the HT-29 cells. These cell lines were obtained from American Type Culture Collection (Manassas, VA, U.S.A.). HeLa, HepG2, and HT-29 cells were cultured in DMEM/high glucose supplemented with 10% FBS, 2 mM L-glutamine, and 1% penicillin–streptomycin. Daoy cells were cultured in DMEM/F12 supplemented with 10% FBS, 2 mM L-glutamine, and 1% penicillin–streptomycin.

Methods

Growth Inhibitory Activity of the New Compounds Screened by MTT Assay

The antiproliferative activity of the new compounds was assessed at the Cell Culture Laboratory, College of Pharmacy, King Saud University, in a primary four cell lines-one concentration (25 µg/mL) anticancer assay by using the aforementioned cell lines. The cytotoxicity of the newly synthesized compounds was determined by testing the capacity of the reducing enzymes present in the viable cells to convert MTT to formazan crystals as previously reported.⁴³⁾ Briefly, cells were seeded in complete medium, at a density of 2 ×10⁴ cells/well, into 96-well microtiter plates and incubated at 37°C in a humidified atmosphere with 5% CO₂, for 24 h. The complete medium was then changed to serum-free medium (SFM), containing the test compounds (25 µg/mL), or an equivalent volume of solvent (DMSO), as control. After incubation, the SFM in the control and test wells was replaced by MTT (100 µL/well; 0.5 mg/mL) in phosphate-buffered saline (PBS) and incubated at 37°C for an additional 3 h. The MTT solution was removed and the purple formazan crystals formed at the bottom of the wells were dissolved using isopropyl alcohol (100 µL/well), and the plate was further incubated for 2 h at room temperature, with constant agitation. The plate was read in a microplate reader (ELX 800; Bio-Tek Instruments, Winooski, VT, U.S.A.), and the absorbance at 549 nm recorded.

The dose response curves of the compounds exhibiting ≥50% inhibition in the aforementioned one-dose prescreening assay for each cell line were established using the following concentrations: 1.56, 3.125, 6.25, 12.5, 25, and 50 µg/mL. The concentration effecting IC₅₀ was calculated. The cytotoxic activity of the well-known antitumor drug dasatinib, a potent multi-targeted inhibitor of BCR-ABL and SRC family kinases,⁴⁴⁾ was determined at the same concentrations of the tested compounds against the four cell lines, and utilized as a standard for comparative purposes.

In Vitro VEGFR-2 Inhibition Assay

HeLa cells (1.2–1.8×10⁴ cells/well) were cultured in a volume of 200 µL/well (100 µL of complete growth medium +100 µL of the tested compounds) in a 96-well plate for 18–24 h before the VEGFR-2 enzyme assay was performed.⁴⁵⁾

Molecular Docking

Molecular docking studies were performed on an Intel Pentium 1.6 GHz processor, 512 MB memory with Windows XP operating system using Molecular Operating Environment (MOE, 10.2008) software. All the minimizations were performed with MOE until an RMSD gradient of 0.05 kcal mol⁻¹ Å⁻¹ with MMFF94X force field, and the partial charges were automatically calculated. The X-ray crystallographic structure of VEGFR-2 complexed with sunitinib was obtained from the protein data bank (PDB ID: 4AGD). The protein was prepared for docking studies in the following way: (i) the co-crystallized ligand molecule was removed from the enzyme active site; (ii) hydrogen atoms were added to the structure with their standard geometry; (iii) MOE Alpha Site Finder was used to search for the active site in the protein structure and to generate dummy atoms from the obtained alpha spheres; (iv) the obtained model was then used in predicting ligand–protein interactions at the active site.

Conclusion

A novel series of benzenesulfonamide derivatives bearing the 3,4-dimethoxyphenyl moiety were synthesized and evaluated for their cytotoxic activity against four different cell lines. Four compounds (4, 10, 16, 19) showed good cytotoxic activity when compared to dasatinib (reference drug). The same compounds were evaluated for their in vitro ability to inhibit VEGFR-2. Compounds 10 and 19 were more active than dasatinib as VEGFR-2 inhibitors. Structure–activity relationship favors heterocyclic sulfonamide derivatives and, in particular, methyl thiadiazole derivative 10 and benzopyrazole derivative 19. Molecular docking on the active site of VEGFR-2 for compound 19 exhibited good fitting and comparable amino acid interactions to those displayed by sunitinib.

Acknowledgment

This project was funded by the National Plan for Science, Technology and Innovation (MAARIFAH), King Abdulaziz City for Science and Technology, Kingdom of Saudi Arabia, Award Number (13-MED 997-02).

Conflict of Interest

The authors declare no conflict of interest

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