Design, synthesis, and biological investigation of selective human carbonic anhydrase II, IX, and XII inhibitors using 7-aryl/heteroaryl triazolopyrimidines bearing a sulfanilamide scaffold

Figures & data

Figure 1. Structure of SLC-0111 and molecular structures of 2-sulphanilamide-7-substituted [1,2,4]triazolo [1,5-a]pyrimidines 1a–ac reported in this article.

Scheme 1. Reagents. (a) THF, sulphanilamide, reflux, 18 h; (b) NH₂NH₂·H₂O, THF, rx, 18 h; (c) DMF-DMA, DMF, reflux, 4 h; (d) AcOH, 80 °C, 4 h.

Table 1. Inhibition data of human CA isoforms I, II, IV, IX, and XII with novel synthesised triazolopyrimidine derivatives 1a–ac using acetazolamide (AAZ) and SLC-0111 as standard inhibitors.

Compound	R	KI (nM)^a
		hCA I	hCA II	hCA IV	hCA IX	hCA XII
1a	C6H5	841.0	75.3	1326	33.2	71.7
1b	2′-Thienyl	500.8	98.2	2659	49.5	66.9
1c	4′-Pyridinyl	725.1	43.7	726.5	41.6	15.3
1d	3′-Pyridinyl	601.9	24.8	332.9	5.1	8.8
1e	2′-Naphthyl	5874	326.4	8201	57.7	62.5
1f	4′-F-C6H4	763.1	82.7	992.7	28.8	62.4
1g	3′-F-C6H4	551.2	45.9	853.4	22.4	42.6
1h	2′-F-C6H4	682.1	35.9	920.1	19.5	25.8
1i	2′,6′-2F-C6H3	742.9	17.6	702.6	21.9	22.5
1j	2′,3′,6′-3F-C6H2	496.3	13.9	662.1	8.6	5.4
1k	4′-Cl-C6H4	2253	62.4	3997	74.1	29.2
1l	3′-Cl-C6H4	6579	123.7	5983	30.4	46.4
1m	2′-Cl-C6H4	9150	154.9	6482	14.8	16.9
1n	4′-Br-C6H4	3365	200.4	5212	52.7	58.2
1o	3′-Br-C6H4	9201	133.0	4329	38.1	78.6
1p	4′-I-C6H4	7632	559.4	8854	95.7	39.0
1q	4′-NO2-C6H4	4287	86.3	3257	69.4	42.1
1r	3′-NO2-C6H4	3468	48.0	689.5	49.9	4.3
1s	2′-NO2-C6H4	8455	77.1	3982	20.4	48.9
1t	4′-CH3-C6H4	1472	64.0	2289	41.5	61.2
1u	3′-CH3-C6H4	1402	55.2	4847	26.2	70.2
1v	2′-CH3-C6H4	2678	42.8	8821	4.7	30.4
1w	4′-OCH3-C6H4	1628	89.6	2004	50.8	39.8
1x	3′-OCH3-C6H4	1056	33.8	559.5	5.1	12.5
1y	2′-OCH3-C6H4	2198	72.9	6324	11.0	31.6
1z	4′-OCH2CH3-C6H4	2258	69.3	3652	43.5	55.2
1aa	3′,5′-(OCH3)2C6H3	4528	110.4	1008	46.8	27.1
1ab	3′,4′-(OCH3)2C6H3	992.7	95.2	1520	35.8	9.0
1ac	3′,4′,5′-(OCH3)3C6H3	1230	169.8	1598	73.4	31.9
SLC-0111	/	5080	960.0	286.0	45.0	4.5
AAZ	/	250	12	74	25	5.7

^a Mean from three different assays, by a stopped flow technique (errors were in the range of ±5–10% of the reported values).

Table 2. Selectivity indexes (SIs) for the inhibition of transmembrane human CA isoforms hCA IX and XII over off-targets isoforms hCA I, IV, and II for target 2-sulfanilamido triazolopyrimidines 1a–ac, acetazolamide (AAZ) and SLC-0111.

	Selectivity indexes^a
Compd	I/IX	II/IX	IV/IX	I/XII	II/XII	IV/XII
1a	25.3	2.3	39.9	11.7	1.0	18.5
1b	10.1	2.0	53.7	7.5	1.5	39.7
1c	17.4	1.0	17.5	47.4	2.9	47.5
1d	118	4.9	65.3	68.4	2.8	37.8
1e	102	5.7	142	94	5.2	131
1f	26.5	2.9	34.5	12.2	1.3	15.9
1g	24.6	2.0	38.1	12.9	1.1	20.0
1h	35.0	1.8	47.2	26.4	1.4	35.7
1i	33.9	0.8	32.1	33	0.8	31.2
1j	57.7	1.6	77.0	91.9	2.6	123
1k	30.4	0.8	53.9	77.1	2.1	137
1l	216	4.06	197	142	2.7	129
1m	618	10.5	438	541	9.2	384
1n	63.8	3.8	98.9	57.8	3.4	89.6
1o	241	3.5	114	117	1.7	55.1
1p	79.7	5.8	92.5	196	14.3	227
1q	61.8	1.2	46.9	102	2.0	77.4
1r	69.5	1.0	13.8	806	11.2	160
1s	414	3.8	195	173	1.6	81.4
1t	35.5	1.5	55.1	24.0	1.0	37.4
1u	53.5	2.1	185	20.0	0.8	69.0
1v	570	9.1	1877	88.1	1.4	290
1w	32.0	1.8	39.4	40.9	2.2	50.3
1x	207	6.6	110	84.5	2.7	44.8
1y	200	6.6	575	69.5	2.3	200
1z	51.9	1.6	83.9	40.9	1.2	66.1
1aa	96.7	2.4	21.5	167	4.1	37.2
1ab	27.7	2.6	42.4	110	10.6	169
1ac	16.7	2.3	21.8	38.6	5.3	50.1
SLC-0111	113	21	6.3	1,129	213	63.5
AAZ	10	0.5	3.0	43.8	2.1	13

^a Selectivity index (SI) of inhibitors for transmembrane hCA IX, and XII over isoforms hCA I, II, and IV calculated as the ratio of K_I off-target hCA/K_I target hCA. A potent, selective inhibitor is characterised by a high value ratio.

Figure 2. Binding mode of compound 1a in the active site of CA isoforms I (A). Binding modes of compound 1j in the active site of CA isoforms II (B), IV(C), and XII (E). Binding mode of compound 1v in the active site of CA isoforms IX (D).

Figure 3. Binding modes of 1x (A), 1y (B), and 1w (C) in the active site of CA isoforms IX, and the superposition of the binding poses of the three analogues 1w–y (D).

Table 3. Cytotoxicity of selected compound in PBL.

Compounds	PBL resting^a GI50 (µM)c	PBL + PHA^b GI50 (µM)c
1d	>10	>10
1j	>10	>10
1m	>10	>10
1x	>10	6.0 ± 0.8
1y	>10	>10
SLC-0111	>10	>10

^a PBL not stimulated with PHA.

^b PBL stimulated with PHA.

^c Compound concentration required to reduce cell growth by 50%.

Figure 4. Antiproliferative activity of the indicated compounds in medulloblastoma cell lines (DAOY, upper panels; HDMB lower panels). Cells were treated with the compounds at the concentration of 10 µM alone or in combination with cis-Pt (1.8 µM) for 72 h. SLC-0111 was used as reference compounds at the concentration of 10 µM. Data are expressed as mean ± SE of three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 5. Antiproliferative activity of the indicated compounds in glioblastoma primary cell lines (HuTuP192, upper panels; HuTuP108 lower panels). Cells were treated with the compounds at the concentration of 10 µM alone or in combination with TMZ (500 µM) for 72 h. SLC-0111 was used as reference compounds at the concentration of 10 µM. Data are expressed as mean ± SE of three independent experiments. *p < 0.05; ****p < 0.0001.

Figure 6. Induction of apoptosis in medulloblastoma (HDMB, panels A and C) and GBM cells (HuTu-108, panels B and D) by 1j and SLC-0111 as reference compounds. Cells were treated with 1j (10 µM) or SLC-0111 (10 µM) both alone or in combination with cis-Pt (1.8 µM) or TMZ (500 µM) for 72 h. The cells were then labelled with Annexin-V and PI as described in “Materials and methods” section and analysed by flow cytometry. Data are expressed as mean ± SE of three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001.

Data availability statement

¹H NMR, ¹³C NMR, ESI-mass, and UV–vis spectra of compounds 1a–ac. Preparation and purification CA isoforms. Supplementary data associated with this article can be found in the online version.