Development of novel anilinoquinazoline-based carboxylic acids as non-classical carbonic anhydrase IX and XII inhibitors

Figures & data

Figure 1. Structure of acetazolamide, dorzolamide, SLC-0111, non-classical CAIs (I–III), and the target inhibitors (6, 7, and 8).

Scheme 1. Synthesis of chloroquinazolines (4a–c): Reaction conditions (i) FeCl₃/H₂O/heating 80 °C/3h, (ii) POCl₃/N,N-dimethylformamide (cat.)/heating 90 °C/4h.

Scheme 2. Synthesis of 2-aryl-quinazolin-4-yl aminobenzoic acids (6a–c, 7a–c and 8a–c): Reaction conditions (i) Isopropanol/HCl (cat.)/reflux/2h.

Table 1. Inhibition data of hCA isoforms I, II, IX, XII, for carboxylic acids (6a–c, 7a–c, and 8a–c) by a stopped flow CO₂ hydrase assay.

			KI (µM)^a
Cmpd	COOH	R	hCA I	hCA II	hCA IX	hCA XII
6a	2-COOH	3-CH3	>100	85.2	42.5	9.0
6b	2-COOH	4-CH3	>100	48.2	34.4	7.1
6c	2-COOH	4-OCH3	>100	83.1	46.4	8.9
7a	3-COOH	3-CH3	>100	26.0	29.3	4.8
7b	3-COOH	4-CH3	>100	41.7	24.2	0.91
7c	3-COOH	4-OCH3	>100	85.8	31.6	0.48
8a	4-COOH	3-CH3	87.7	9.3	4.3	3.8
8b	4-COOH	4-CH3	73.2	3.9	1.6	0.42
8c	4-COOH	4-OCH3	66.3	4.6	4.5	0.25
AAZ	–	–	0.25	0.01	0.02	0.006

^aMean from 3 different assays (errors were in the range of ± 5–10% of the reported values).

Figure 2. SARs summary for the hCA inhibitory activities of target quinazolines.

Table 2. Selectivity ratios for the inhibition of CA IX and XII isoforms over CA I and II isoforms for carboxylic acids (6a–c, 7a–c, and 8a–c) and acetazolamide.

Compound	I/IX	II/IX	I/XII	II/XII
6a	2.4	2.00	11.1	9.47
6b	2.9	1.40	14.1	6.79
6c	2.2	1.79	11.2	9.34
7a	3.4	0.89	20.8	5.42
7b	4.1	1.72	109.9	45.82
7c	3.2	2.72	208.3	178.75
8a	20.4	2.16	23.1	2.45
8b	45.8	2.44	174.3	9.29
8c	14.7	1.02	265.2	18.40
AAZ	10.0	0.5	43.9	2.2

Table 3. Percentage growth inhibition (GI%) of subpanel tumour cell lines at 10 μM concentration of the quinazoline based-carboxylic acids (6a–c, 7b, and 8a–b).

Subpanel/cell line		Compound^a
		6a	6b	6c	7b	8a	8b
Leukaemia	CCRF-CEM	82	87	58	21	25	28
	HL-60(TB)	60	82	67	–	–	24
	K-562	71	82	69	20	–	23
	MOLT-4	65	89	64	–	22	24
	RPMI-8226	71	83	80	29	30	45
	SR	77	81	NT	41	32	38
Non-Small Cell Lung Cancer	A549/ATCC	56	70	60	–	22	–
	EKVX	79	87	66	41	–	28
	HOP-62	54	72	49	–	–	–
	HOP-92	105	120	78	29	29	25
	NCI-H226	64	85	47	25	52	62
	NCI-H23	79	104	38	–	52	–
	NCI-H322M	52	66	24	–	–	–
	NCI-H460	81	109	89	–	22	–
	NCI-H522	40	67	63	–	–	–
Colon cancer	COLO 205	–	–	–	–	–	–
	HCC-2998	73	95	38	–	24	28
	HCT-116	78	93	62	26	–	22
	HCT-15	86	95	61	55	29	49
	HT29	60	82	37	–	–	–
	KM12	70	92	64	32	23	42
	SW-620	65	86	61	–	–	–
CNS cancer	SF-268	42	89	43	31	37	30
	SF-295	73	94	60	34	36	42
	SF-539	52	79	37	27	–	21
	SNB-19	79	91	55	28	33	29
	SNB-75	45	84	68	–	–	–
	U251	73	82	66	21	27	–
Melanoma	LOX IMVI	64	91	50	–	29	43
	MALME-3M	63	80	31	–	–	–
	M14	75	87	48	–	–	–
	MDA-MB-435	55	77	44	–	–	–
	SK-MEL-2	34	94	57	–	–	–
	SK-MEL-28	50	71	35	–	–	–
	SK-MEL-5	94	124	70	–	29	28
	UACC-257	51	71	28	–	–	–
	UACC-62	72	89	44	–	–	–
Ovarian cancer	IGROV1	41	69	21	–	–	–
	OVCAR-3	66	94	54	–	–	20
	OVCAR-4	58	78	66	23	–	26
	OVCAR-5	23	34	27	–	–	–
	OVCAR-8	40	69	40	–	–	–
	NCI/ADR-RES	75	95	46	–	–	24
	SK-OV-3	24	69	53	–	–	–
Renal cancer	786-0	75	81	48	24	29	–
	A498	52	78	41	–	54	–
	ACHN	63	92	54	24	–	24
	CAKI-1	48	79	53	–	–	–
	SN12C	59	78	32	23	42	51
	TK-10	–	41	52	–	–	–
	UO-31	69	87	25	51	54	58
Prostate	PC-3	71	84	60	–	–	23
	DU-145	50	81	43	–	–	–
Breast cancer	MCF7	76	92	49	–	–	37
	MDA-MB-231	34	65	–	–	–	–
	HS 578 T	84	109	72	34	48	32
	BT-549	100	123	62	–	34	41
	T-47D	73	89	71	–	22	32
	MDA-MB-468	115	126	57	–	–	–
Mean GI %		63	84	52	14	16	20
Number of sensitive cell lines		56	58	56	21	25	30

^aOnly GI% higher than 20% are shown.

Table 4. GI₅₀, TGI, and LC₅₀ values of NCI five doses anticancer assay for 6b (NSC: 835857).

Cancer type/cells	Compound 6b (NSC: 835857)
	GI50 (µM)	TGI (µM)	LC50 (µM)
Leukaemia
MOLT-4	11.7	<100	<100
RPMI-8226	4.4	<100	<100
K-562	7.05	<100	<100
SR	10.3	<100	<100
CCRF-CEM	5.7	<100	<100
HL-60(TB)	12.9	75.4	<100
Non-small cell lung cancer
HOP-92	2.9	29.3	<100
NCI-H226	10.2	54.2	<100
NCI-H522	4.4	27.1	<100
NCI-H322M	13.6	<100	<100
NCI-H460	8.5	27.7	84.0
NCI-H23	15.7	44.4	<100
EKVX	8.9	56.2	<100
HOP-62	10.8	33.7	<100
A549/ATCC	8.31	<100	<100
Colon cancer
KM 12	13.7	81.2	<100
SW-620	18.2	<100	<100
HT29	17.9	<100	<100
HCT-15	5.8	28.7	<100
COLO 205	17.8	41.0	94.1
HCC-2998	12.4	36.0	<100
HCT-116	10.1	36.1	<100
CNS cancer
SNB-75	1.4	80.3	<100
U251	5.6	35.0	<100
SF-539	11.9	28.3	67.2
SNB-19	8.1	81.7	<100
SF-295	7.04	27.0	86.0
SF-268	14.3	<100	<100
Melanoma
MDA-MB-435	13.9	87.2	<100
UACC-62	10.2	27.0	71.6
M14	15.5	77.4	<100
UACC-257	15.3	90.3	<100
SK-MEL-5	5.3	20.3	57.5
SK-MEL-28	15.9	97.4	<100
LOX IMVI	10.2	31.6	97.7
MALME-3M	15.6	64.5	<100
SK-MEL-2	3.7	15.6	83.5
Ovarian cancer
IGROV1	19.1	87.1	<100
OVCAR-4	8.8	<100	<100
OVCAR-5	21.8	68.9	<100
OVCAR-8	18.2	<100	<100
NCI/ADR-RES	16.8	98.5	<100
SK-OV-3	11.9	55.3	<100
Renal cancer
786-0	17.1	<100	<100
A498	19.9	58.1	<100
ACHN	6.4	91.1	<100
CAKI-1	13.8	<100	<100
RXF 393	13.1	65.9	<100
SN 12 C	11.0	60.4	<100
TK-10	29.3	<100	<100
UO-31	7.3	77.9	<100
Prostate cancer
PC-3	12.7	<100	<100
DU-145	17.7	<100	<100
Breast cancer
MCF7	9.4	89.9	<100
MDA-MB-231	17.2	59.7	<100
HS 578 T	10.2	67.0	<100
BT-549	14.6	33.8	78.1
T-47D	6.3	70.0	<100
MDA-MB-468	8.3	42.0	<100

Table 5. Median GI₅₀ values (µM) for compound 6b on subpanel tumour cell lines.

Subpanel tumour cell line	6b
	MG-MID	Selectivity index
Leukaemia	8.68	1.38
Non-small cell lung cancer	9.24	1.29
Colon cancer	13.70	0.87
CNS cancer	8.04	1.49
Melanoma	11.73	1.02
Ovarian cancer	15.66	0.76
Renal cancer	14.74	0.81
Prostate cancer	15.2	0.78
Breast cancer	10.99	1.09
Full panel MG-MID	11.99	–