Dependence on linkers’ flexibility designed for benzenesulfonamides targeting discovery of novel hCA IX inhibitors as potent anticancer agents

Figures & data

Figure 1. Structures of SLC-0111, some reported CAIs I–VI and design of target sulphonamides, Series I (3a–c, 5a–d, and 7a–e) and Series II (9, 11a,b, 13a–e, and 15a,b).

Figure 2. The E-isomer of compound 3a and Z-isomer of compound 3b with the pseudo-six-membered ring (in blue color).

Scheme 1. Synthetic pathway of new sulphonamides analogues 3a–c, 5a–d, and 7a–e.

Scheme 2. Synthetic pathway of target sulphonamides 9, 11a,b, 13a–e, and 15a,b.

Scheme 3. The two proposed isomers of compound 15b when refluxed with CS₂ in pyridine.

Scheme 4. A plausible mechanism for the formation of one of the two isomers regarding analogue 15b.^31,32

Table 1. Inhibition profile concerning human CA isoforms, off-target isoforms (hCA I and II), and the tumour-associated isoforms (hCA IX and XII) with triazine-based benzenesulfonamides 3a–c, 5a–d, 7a–e, 9, 11a,b, 13a–e, 15a,b, besides the standard inhibitors acetazolamide (AAZ) and SLC-0111 by a stopped-flow CO₂ hydrase assay.

	KI ^a (nM)
Compounds	hCA I	hCA II	hCA IX	hCA XII
3a	8164	98.2	818.4	59.2
3b	8223	399.3	95.9	82.2
3c	3924	1516	43.8	24.4
5a	5111	175.4	575.5	64.1
5b	3523	668	33.4	14.9
5c	5309	2352	75.4	36.7
5d	4083	4291	28.6	31.3
7a	5811	46.4	68.6	85.5
7b	156.3	74.4	36.6	78.1
7c	8896	3147	47.3	439.0
7d	980.4	68.4	56.9	2185
7e	3720	2224	52.8	888.3
9	195.9	4585	443.0	27.3
11a	<50 000	4192	227	47.7
11b	<50 000	2131	31.9	8.29
13a	583.1	645.8	91.9	82.4
13b	368.9	114.3	94.7	77.8
13c	4710	2253	92.9	82.4
13d	92.6	30.8	91.8	89.9
13e	476.8	96.0	70.0	751.3
15a	728.0	704.7	871.4	40.6
15b	26.6	46.2	40.7	194.8
AAZ^b	250.0	12.1	25.8	5.7
SLC-0111^b	5080	960	45.0	4.5

^aMean from three different tests by a stopped-flow technique (approximately 5:10% of the reported numbers were erroneous).

^bAAZ, a standard sulphonamide carbonic anhydrase inhibitor. SLC-0111is also provided for comparison.

Table 2. Selectivity ratios for the inhibition of hCA IX and XII over hCA I and II for target compounds 3a–c, 5a–d, 7a–e, 9, 11a,b, 13a–e, 15a,b and the standard inhibitors acetazolamide (AAZ) and SLC-0111.

Compounds	Selectivity ratio (SR)^a,b (KI off-target CA/KI target CA)
	I/IX	II/IX	I/XII	II/XII
3a	9.97	0.12	137.91	1.66
3b	85.75	4.16	100.3	4.86
3c	89.59	34.61	160.82	62.13
5a	8.88	0.30	79.73	2.73
5b	105.48	20	236.44	44.83
5c	70.41	31.19	144.66	64.09
5d	142.76	150.03	130.44	137.09
7a	84.71	0.68	67.96	0.54
7b	4.27	2.03	2.00	0.95
7c	188.08	66.53	20.26	7.17
7d	17.23	1.20	0.45	0.03
7e	70.45	42.12	4.19	2.5
9	0.44	10.35	7.18	167.95
11a	220.26	18.47	1048.22	87.88
11b	1567.40	66.80	6031.36	257.06
13a	6.34	7.03	7.08	7.84
13b	3.89	1.21	4.7	1.47
13c	50.70	24.25	57.16	1.13
13d	1.01	0.34	1.03	0.34
13e	6.81	1.37	0.63	0.13
15a	0.84	0.81	17.93	17.36
15b	0.65	1.14	0.14	0.24
AAZ	10	0.48	43.86	2.10
SLC-0111	112.9	21.3	1128.9	213.3

^aThe K_I ratios indicate isozyme selectivity: A low-value ratio indicates the presence of a weak selective inhibitor.

^bSelectivity as measured by the hCA I and II KI ratio in comparison to hCA IX and hCA XII.

Table 3. Sixty human tumour cell lines in vitro subpanel at a concentration of 10 µM for the presence of compounds 3a, 5 b–d, 7c,d, 11a,b, and 13 b–e.

Subpanel cell lines	Growth inhibition percentage (GI%)
	3a	5b	5c	5d	7c	7d	11a	11b	13b	13c	13d	13e
Leukaemia
CCRF-CEM	73	56	89	72	51	43	85	63	45	89	62	–
HL-60(TB)	35	34	52	59	–	–	74	26	51	95	53	NT
K-562	56	33	85	66	41	28	67	32	48	88	43	NT
MOLT-4	57	59	118	81	89	68	82	51	55	95	64	NT
RPMI-8226	56	32	20	67	38	32	62	34	32	93	61	–
SR	66	38	112	80	67	51	94	59	31	99	44	NT
NSC lung cancer
A549/ATCC	–	14	–	41	–	–	33	21	39	74	17	17
EKVX	–	18	13	44	22	15	43	21	30	59	21	15
HOP-62	11	–	73	–	–	–	24	–	–	36	17	–
HOP-92	44	–	104	24	–	–	25	16	10	73	52	–
NCI-H226	24	19	45	53	–	–	44	13	35	59	34	27
NCI-H23	31	25	72	49	14	–	45	–	–	76	21	16
NCI-H322M	26	–	62	–	–	–	35	15	–	42	26	36
NCI-H460	12	24	–	76	34	25	79	12	27	94	30	34
NCI-H522	42	–	50	22	–	–	16	12	29	41	18	41
Colon cancer
COLO 205	17	23	11	39	–	–	91	29	17	90	12	22
HCC-2998	15	–	94	48	20	–	86	13	–	56	–	28
HCT-116	49	28	71	67	50	–	85	32	41	89	42	36
HCT-15	–	–	19	70	31	42	63	23	42	81	22	43
HT29	40	–	67	73	10	–	90	17	29	95	12	10
KM 12	27	18	38	64	40	34	94	40	27	90	26	43
SW-620	–	–	58	26	–	–	45	–	28	61	14	10
CNS cancer
SF-268	32	13	78	40	24	18	40	17	30	62	23	20
SF-295	26	18	56	40	24	14	43	18	10	65	27	17
SF-539	14	15	125	35	11	17	41	18	19	62	25	20
SNB-19	15	10	85	26	20	13	38	13	19	49	18	10
SNB-75	–	–	74	35	–	13	32	–	–	43	17	31
U251	47	21	72	46	26	13	38	25	45	66	19	28
Melanoma
LOX IMVI	53	NT	123	NT	33	18	87	27	27	NT	NT	32
MALME-3M	21	13	58	12	16	11	57	–	–	39	–	–
M14	NT	23	40	44	26	15	65	25	20	73	36	24
MDA-MB-435	29	33	43	50	28	16	60	23	30	73	28	14
SK-MEL-2	13	–	105	19	21	14	34	–	10	43	–	29
SK-MEL-28	20	20	65	24	19	13	52	23	18	45	17	–
SK-MEL-5	44	12	126	34	25	25	48	20	19	60	43	14
UACC-257	–	–	–	13	–	–	34	–	–	40	–	–
UACC-62	14	–	126	20	–	–	–	–	38	45	28	35
Ovarian cancer
IGROV1	33	14	40	39	12	–	65	23	15	74	41	41
OVCAR-3	39	–	79	14	17	13	69	–	10	68	22	32
OVCAR-4	27	16	28	38	32	26	50	–	29	62	28	–
OVCAR-5	–	14	–	–	–	–	56	18	–	21	–	–
OVCAR-8	43	12	42	54	19	10	31	21	27	60	33	33
NCI/ADR-RES	–	–	–	34	–	–	37	–	–	55	–	13
SK-OV-3	–	–	–	11	–	–	35	–	–	34	–	–
Renal cancer
786-0	23	21	74	46	23	15	34	15	16	79	40	21
A498	27	NT	–	NT	–	–	44	22	NT	NT	NT	22
ACHN	–	13	–	49	11	–	67	25	18	84	33	54
CAKI-1	33	25	–	50	–	–	57	13	27	79	43	65
RXF 393	27	42	45	74	31	30	103	39	29	77	41	36
SN 12 C	34	–	53	28	12	–	59	19	19	58	25	44
TK-10	–	–	–	–	–	–	10	–	–	–	–	19
UO-31	–	27	14	36	22	21	51	29	19	70	33	42
Prostate cancer
PC-3	29	34	42	76	51	45	57	39	48	73	46	30
DU-145	24	15	49	32	12	–	49	11	25	67	25	24
Breast cancer
MCF7	51	56	62	82	66	51	87	53	48	93	60	27
MDA-MB-231	12	13	101	28	–	–	45	26	23	62	41	32
HS 578 T	–	–	108	23	–	–	21	–	13	35	–	23
BT-549	19	–	114	32	12	–	26	–	17	47	31	37
T-47D	53	26	74	54	32	27	48	33	56	63	29	15
MDA-MB-468	45	22	157	29	19	14	73	22	30	88	42	29
GI% Mean	25	16	58	41	17	12	53	19	23	65	27	22

NT: not tested. “–” indicates GI% less than 10. GI% up to 60 indicates moderate activity, GI% up to 100 indicates strong activity, and GI% exceeds 100 shows lethal activity.

Table 4. Five doses of in vitro anticancer activity results ^aagainst all sixty cancer cell lines expressed as GI₅₀^b (μM), TGI^c (μM), and LC₅₀^d (μM) for compounds 5c and 13c.

Subpanel/cell lines	5c			13c
	GI50	TGI	LC50	GI50	TGI	LC50
Leukaemia
CCRF-CEM	11.1	41.6	>100	2.94	>100	>100
HL-60(TB)	22.0	60.9	>100	2.24	8.26	>100
K-562	23.4	>100	>100	2.62	10.8	>100
MOLT-4	19.7	52.3	>100	1.51	6.47	>100
RPMI-8226	22.8	93.5	>100	2.49	8.69	>100
SR	15.9	>100	>100	1.94	5.94	91.4
NSC lung cancer
A549/ATCC	21.8	67.5	>100	3.18	17.9	>100
EKVX	21.4	69.2	>100	2.96	17.4	>100
HOP-62	21.3	58.9	>100	3.29	12.4	47.1
HOP-92	26.7	81.2	>100	2.16	6.18	41.1
NCI-H226	26.5	98.2	>100	2.52	11.0	>100
NCI-H23	20.9	50.1	>100	2.84	9.12	30.5
NCI-H322M	14.2	27.3	52.4	2.78	29.9	>100
NCI-H460	19.6	60.1	>100	2.07	4.66	11.7
NCI-H522	16.8	42.4	>100	2.29	7.39	>100
Colon cancer
COLO 205	16.0	29.8	55.5	1.78	3.27	6.01
HCC-2998	14.8	29.2	57.8	2.98	6.84	33.4
HCT-116	17.7	38.9	85.5	1.74	3.39	6.58
HCT-15	32.3	>100	>100	3.08	11.2	>100
HT29	19.6	49.1	>100	2.38	5.48	16.5
KM 12	17.5	37.1	78.9	2.77	8.20	>100
SW-620	17.2	42.4	>100	3.52	32.1	>100
CNS cancer
SF-268	19.9	58.6	>100	3.97	>100	>100
SF-295	22.2	60.2	>100	3.02	10.5	>100
SF-539	17.9	41.3	95.0	2.66	10.3	44.5
SNB-19	14.2	27.7	53.9	3.44	20.9	>100
SNB-75	18.6	61.7	>100	4.61	>100	>100
U251	20.0	71.0	>100	2.91	10.3	39.6
Melanoma
LOX IMVI	15.8	30.4	58.6	NT	NT	NT
MALME-3M	14.7	34.8	82.0	2.43	7.09	45.2
M14	18.8	45.7	>100	3.22	12.5	55.7
MDA-MB-435	17.8	40.6	93.0	3.32	12.9	>100
SK-MEL-2	15.7	38.1	92.3	2.39	6.95	30.1
SK-MEL-28	18.1	40.6	91.2	3.87	16.9	>100
SK-MEL-5	14.9	28.6	54.8	2.33	5.54	17.8
UACC-257	20.6	47.9	>100	3.26	23.3	>100
UACC-62	14.5	28.3	55.3	1.81	3.76	7.80
Ovarian cancer
IGROV1	18.5	42.2	96.4	2.72	11.4	81.5
OVCAR-3	21.4	63.1	>100	2.24	4.97	19.2
OVCAR-4	23.2	81.4	>100	2.77	9.32	>100
OVCAR-5	22.7	62.6	>100	3.34	14.3	72.7
OVCAR-8	26.9	>100	>100	3.22	16.0	>100
NCI/ADR-RES	>100	>100	>100	2.93	11.0	65.5
SK-OV-3	21.3	61.9	>100	5.37	>100	>100
Renal cancer
786-0	15.1	44.3	>100	3.09	10.6	76.4
A498	13.3	36.6	>100	3.33	8.08	27.0
ACHN	24.8	93.8	>100	2.59	9.95	88.5
CAKI-1	24.0	85.7	>100	2.45	9.55	>100
RXF 393	14.7	32.8	73.3	1.99	5.23	22.8
SN 12 C	15.6	32.1	66.0	1.47	2.88	5.63
TK-10	27.7	87.0	>100	3.87	8.93	>100
UO-31	26.7	>100	>100	2.38	10.0	49.4
Prostate cancer
PC-3	19.5	55.2	>100	2.37	7.89	>100
DU-145	16.1	30.2	56.5	3.16	13.0	>100
Breast cancer
MCF7	16.9	45.4	>100	2.27	6.61	51.6
MDA-MB-231	17.2	47.4	>100	1.81	3.85	8.18
HS 578 T	77.6	81.3	>100	4.31	62.7	>100
BT-549	16.8	42.7	>100	2.24	5.54	26.7
T-47D	7.39	27.3	85.6	2.96	13.2	>100
MDA-MB-468	2.94	15.5	>100	2.31	6.95	92.3

^aIn vitro human cancer cell lines screen data from NCI. ^bMolar concentration necessary to inhibit 50% of growth of cancer cell line. ^cMolar concentration necessary to inhibit 100% of growth of cancer cell line. ^dMolar concentration necessary to kill 50% of cancer cell line.

NT: not tested.

Figure 3. GI₅₀-MID and average sensitivity of all cell lines (µM) of 5c (blue), and 13c (orange), in comparison to 5-FU (grey).

Table 5. Selectivity ratios of the analouges 5c and 13c in comparison to 5-FU towards nine tumours.

Panel	5c			13c			5-FU
	MID^a	MID^b	Selectivity^c	MID^a	MID^b	Selectivity^c	MID^a	MID^b	Selectivity^c
Leukaemia	19.15	21.06	1.09	2.29	2.78	1.21	3.24	7.24	2.23
NSC lung cancer	21.02		1.00	2.67		1.04	20.11		0.36
Colon cancer	19.30		1.09	2.60		1.06	1.83		3.95
CNS cancer	18.80		1.12	3.43		0.81	17.02		0.42
Melanoma	16.76		1.25	2.82		0.98	7.92		0.91
Ovarian cancer	33.42		0.63	3.22		0.86	5.77		1.25
Renal cancer	20.23		1.04	2.64		1.05	0.88		8.22
Prostate cancer	17.80		1.18	2.76		1.00	1.36		5.32
Breast cancer	23.13		0.91	2.65		1.04	7.03		1.02
GI50-MID	189.01			25.08			65.16

MID^a: Average sensitivity of all cell lines of a particular subpanel in µM; MID^b: Average sensitivity of all cell lines (µM); Selectivity^c: ratio MID^a: MID^b.

Table 6. Cytotoxic activities of compounds 5d and 13c against MCF7 (breast cancer), and NCI-H460 (lung cancer) under hypoxia compared to 5-FU.

Compound No.	IC50 ± SD (μM)^a
	MCF7 (Breast cancer)	NCI-H460 (Lung cancer)
5d	15.02 ± 0.02	10.12 ± 0.03
13c	3.03 ± 0.01	4.62 ± 0.02
5-FU	4.10 ± 0.02	6.77 ± 0.02

^aIC₅₀ values are the mean ± SD of three experiments.

Table 7. Anti-proliferative activity of compound 13c and 5-FU against normal human cells.

Compound No.	IC50 ± SD (µM)^a
	LO2	HK2
13c	30.88 ± 0.98	53.39 ± 1.58
5-FU	18.71 ± 0.48	34.01 ± 0.98

^aIC₅₀ values are the mean ± SD of three experiments.

Figure 4. Compound 13c reduces the migratory capacities of NCI-H460 cells versus control. (A) Effect of negative control on wound healing, (B) Effect of compound 13c on wound healing, and (C) Quantitative analysis of the percentage of mobility inhibition rate. The values are the mean ± SD of three experiments.

Figure 5. The influence of compound 13c on the clonogenicity of NCI-H460. (A) Effect of negative control on clonogenicity of NCI-H460 cells, (B) Effect of compound 13c on clonogenicity of NCI-H460 cells, and (C) Quantitative analysis of the colony number. The values are the mean ± SD of three experiments.

Figure 6. Effect of compound 13c (right panel) and DMSO (left panel) on the proportion of annexin V-FITC-positive staining in NCI-H460 cells during an apoptosis experiment. The four quarters were designated as LL for viable, LR for early apoptosis, UR for late apoptosis, and UL for necrotic.

Figure 7. The percentage of NCI-H460 cells stained positively for annexin V-FITC in the apoptosis assay is affected by compound 13c and DMSO. The values are the mean ± SD of three experiments.

Figure 8. Cell cycle analysis of compound 13c (left panel) and DMSO (right panel)-treated NCI-H460 cells (right panel).

Figure 9. Docking of compounds 5d and 13c within the active site of hCA IX (PDB, ID: 3IAI). (A and C) The 3D binding mode of compounds 5d and 13c with hCA IX. The analogues 5d and 13c are coloured in cyan. The surrounding residues in the binding pocket are coloured in yellow. The hydrogen bond is depicted as a magenta dashed line. (B and D) The surface binding mode of hCA IX with compounds 5d and 13c viewing large cavity size in the active site filled with two bulky phenyl rings attached to hydrazone linker in 5d and pyrazole linker in 13c.

Table 8. Outcomes for docking of target compounds, 5d and 13c in the hCA IX active site (PDB, ID: 3IAI).

Compound	Docking Score (kcal/mol)	Ionic interaction	H-bond interaction	Hydrophobic interaction
5d	−9.97	Zn^2+	Thr199 and Thr200	Arg60, Leu91, His94, His96, His119, Val121, Val131, Leu141.
13c	−8.92	Zn^2+	Thr199 and Thr200	His94, Val131, Pro202

Table 9. Calculated binding free energy of the compound 5d (kJ/mol).

Complex 5d-hCA IX; ΔG = −118.76 ± 30.58
Van der Waal energy	−154.67 ± 17.16	Polar solvation energy	192.19 ± 25.08
Electrostatic energy	−133.32 ± 27.50	SASA energy	−22.96 ± 1.55

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