S-substituted 2-mercaptoquinazolin-4(3H)-one and 4-ethylbenzensulfonamides act as potent and selective human carbonic anhydrase IX and XII inhibitors

Abstract

We evaluated the hCA (CA, EC 4.2.1.1) inhibitory activity of novel 4-(2-(2-substituted-thio-4-oxoquinazolin-3(4H)-yl)ethyl)benzenesulfonamides (compounds 2–20) towards the isoforms I, II, IX, and XII. hCA Isoforms were effectively inhibited by most of new compounds comparable to those of AAZ. Compounds 2 and 4 showed interestingly efficient and selective antitumor (hCA IX and hCA XII) inhibitor activities (K_Is; 40.7, 13.0, and 8.0, 10.8 nM, respectively). Compounds 4 and 5 showed selective hCA IX inhibitory activity over hCA I (SI; 95 and 24), hCA IX/hCA II (SI; 23 and 5.8) and selective hCA XII inhibitory activity over hCA I (SI; 70 and 44), hCA XII/hCA II, (SI; 17 and 10) respectively compared to AAZ. Compounds 12–17, and 19–20 showed selective inhibitory activity towards hCA IX over hCA I and hCA II, with selectivity ranges of 27–195 and 3.2–19, respectively, while compounds 12, 14–17, and 19 exhibited selective inhibition towards hCA XII over hCA I and hCA II, with selectivity ratios of 48–158 and 5.4–31 respectively, compared to AAZ. Molecular docking analysis was carried out to investigate the selective interactions among the most active derivatives, 17 and 20 and hCAs isoenzymes. Compounds 17 and 20, which are highly selective CA IX and XII inhibitors, exhibited excellent interaction within the putative binding site of both enzymes, comparable to the co-crystallized inhibitors.

Highlights

• Quinazoline-linked ethylbenzenesulfonamides inhibiting CA were synthesised.

• The new molecules potently inhibited the hCA isoforms I, II, IV, and IX.

• Compounds 4 and 5 were found to be selective hCA IX/hCA I and hCA IX/hCA II inhibitors.

• Compounds 4 and 5 were found to be selective hCA XII/hCA I and hCA XII/hCA II inhibitors.

• Compounds 12–17, 19, and 20 were found to be selective hCA IX/hCA I and hCA IX/hCA II inhibitors.

• Compounds 12, 14–17, 19 were found to be selective hCA XII/hCA I and hCA XII/hCA II inhibitors.

Graphical Abstract

• Compounds 4 and 5 are selective hCA IX and XII inhibitors over hCA I (selectivity ratios of 95, 23, and 24, 5.8, respectively) and hCA II (selectivity ratios of 70, 17, and 44, 10 respectively). Compounds 12–17, and 19–20 are selective hCA IX inhibitors over hCA I (selectivity ratios of 27-195) and hCA II (selectivity ratios of 3.2-19). Compounds 12, 14–17 and 19 are also selective hCA XII inhibitors over hCA I (selectivity ratios of 48-158) and hCA II (selectivity ratios of 5.4-31).

1. Introduction

Carbonic anhydrases (CAs; EC 4.2.1.1) constitute the superfamily of metalloenzymes that catalyse the CO₂ hydration and dehydration reactions. CAs are classified into eight genetically distinct families, named α-, β-, γ-, δ-, ζ-, η-, ɵ- and ι-CAs^1,2. 15 α-class CA isozymes have been detected in humans, which are further classified into four different subsets on the basis of their subcellular localisation—CA I, II, III, VII, VIII, X, XI, XIIII are cytosolic proteins, CA IV is a glycosylphosphatidylinositol (GPI)-anchored protein, CA VA and VB are located in the mitochondrial matrix, CA VI is secreted, and CA IX, XII and XIV are trans-membrane isoforms^1–3. Human CAs (hCAs) are spread in the human body, and are implicated in a plethora of essential physiological processes. Therefore, the dysregulated expression and/or activity of the CAs can lead to various pathological conditions². CA II is the most physiologically relevant CA isoform, implicated in various disorders including cerebral oedema, glaucoma (such as CA XII), and epilepsy. It is conversely off-target, as CA I, when targeting tumours where CA IX and XII are overexpressed and represent validated targets to combat the growth of both primary tumours and metastasis^4,5. The high structural similarities between various CA isoforms necessitate high selectivity in the design of small-molecule anti-CA drugs for the treatment of diseases associated with CA dysregulation, to minimise the side effects³. Benzene sulphonamides are one of the best-known molecules clinically used as CA inhibitors. Additionally, “SLC-011 (Figure 1), a benzenesulfonamide, is a selective CA IX/XII inhibitor currently being evaluated in a Phase I trial for the treatment of solid, metastatic tumors”^6–10. Sulphonamide derivatives are not only one of the most preferred CA inhibitor classes^9,11–23, but also important COX-2 inhibitors and antitumor agents^{17,19,24–26}. The quinazolinone scaffold is also used widely across medicinal chemistry^27–43. (6-Iodo or 7-flouro-2-merqapto-4-(3H)-quinazolinone3-yl)-benzenesulfonamides (A, Figure 1) have been shown to potently inhibit CA I, II, IX, and XII^44,45. A number of 2-((3-benzyl-4-oxo-3,4-dihydroquinazolin-2-yl)thio)-N-(4-sulfamoylphenethyl)anildes (B, Figure 1) also showed potent inhibitory activity against different hCA isoforms³⁸. The 2-mercapto-4(3H)-quinazolinone derivatives containing ethylsulfonamide tail (C, Figure 1) showed strong inhibitory activity against different hCA isoforms with low-concentration inhibition constants

Figure 1.

Structures of AAZ, SLC-0111, A–C, and the designed quinazoline derivatives (2–20) as CAIs.

Here, we studied 2-mercaptoquinazolinone, (C, Figure 1) a slightly polar and non-selective hCA inhibitor. Because the sulfhydryl group has been reported to be associated with various metabolic and pharmacological problems^46–49, we used a 2-mercaptoquinazolinone scaffold bearing an ethylsulfonamide head with alkylation of the thione group with a terminal lipophilic moiety, so that it can interact selectively with CA through both, hydrogen and hydrophobic interactions. Here, we synthesised various derivatives of 2-mercaptoquinazolinone (2–20, Figure 1) with different selectivity criteria for the hCA inhibitors, particularly for the tumor-associated hCA IX and hCA XII. The role of alkyl substituent in 2-mercaptoquinazolinone was computationally analysed and the conserved residues responsible for the target selectivity were identified.

2. Materials and methods

2.1. Chemistry

Melting points were recorded on a Barnstead 9100 electrothermal melting point apparatus (UK). IR spectra (KBr) were recorded on a FT-IR Perkin-Elmer spectrometer (Perkin Elmer Inc., MA). NMR (¹H and ¹³C NMR) spectra were recorded with Bruker 700 MHz spectrometers (Zurich, Switzerland). Micro-analytical data (C, H, and N) were obtained using a Perkin-Elmer 240 analyser (Perkin Elmer Inc., MA) and agreed with the proposed structures within ±0.4% of the theoretical values. Mass spectra were recorded on a Varian TQ 320 GC/MS/MS mass spectrometer (Varian, Palo Alto, CA). Thione 1 and compounds 8–20 were prepared as described earlier^50,51.

2.1.1. General procedure for synthesis of 4-(2-(2-((2-(4-substituted-phenyl)-2-oxoethyl)thio)-4-oxoquinazolin-3(4H)-yl)ethyl)benzenesulfonamide (2–7)

A mixture of thione 1 (1 mmol, 361 mg) and potassium carbonate (3 mmol, 415 mg) in 6 ml acetone were stirred at room temperature for one hour. Appropriate phenacyl bromide (1 mmol) was added and the reaction mixture was stirred at room temperature for 9–12 h, filtered, and the crude solid was washed with water, dried and recrystallized from ethanol (¹H & ¹³C NMR supplementary material).

4-(2-(4-Oxo-2-((2-oxo-2-phenylethyl)thio)quinazolin-3(4H)-yl)ethyl)benzenesulfonamide (2): m.p 246–247°; 94% yield; IR (KBr, cm⁻¹) ν: 3284, 3237 (NH), 1665 (C=O), 1342, 1151 (O=S=O); ¹H NMR (700 MHz, DMSO-d₆): δ 8.14 (t, 2H, J = 7.14 and 1.26 Hz), 8.04 (dd, 1H, J = 7.91 and 1.26 Hz), 7.82 (d, 2H, J = 8.26 Hz), 7.74 (t, 1H, J = 7.49 Hz), 7.66 (t, 1H, J = 16.71 and 6.96 Hz), 7.62 (t, 2H, J = 7.80 and 7.77 Hz), 7.52 (d, 2H, J = 8.26 Hz), 7.41 (t, 1H, J = 7.17 Hz), 7.37 (s, 2H), 6.98 (d, 1H, J = 8.12 Hz), 4.92 (s, 2H), 4.33 (t, 2H, J = 16.25 Hz), 3.14 (t, 2H, J = 16.20 Hz); ¹³C NMR (176 MHz, DMSO-d₆): δ 194.04, 160.76, 156.10, 146.92, 143.11, 142.29, 136.90, 135.19, 134.01, 129.67, 129.29, 128.79, 126.92, 126.45, 125.87, 119.08, 45.69, 39.38, 33.67; Ms; m/z (479).

4-(2-(2-((2-(4-Bromophenyl)-2-oxoethyl)thio)-4-oxoquinazolin-3(4H)-yl)ethyl)benzenesulfonamide (3): m.p 248–248°; 95% yield; IR (KBr, cm⁻¹) ν: 3280, 3236 (NH), 1686 (C=O), 1340, 1153 (O=S=O); ¹H NMR (700 MHz, DMSO-d₆): δ 8.07 (d, 2H, J = 8.26 Hz), 8.04 (d, 1H, J = 7.84 Hz), 8.85 (d, 2H, J = 8.19 Hz), 7.81 (d, 2H, J = 7.98 Hz), 7.68 (t, 1H, J = 7.63 Hz), 7.52 (d, 2H, J = 8.05 Hz), 7.41 (d, 1H, J = 7.45 Hz), 7.37 (s, 2H), 7.00 (d, 1H, J = 8.19 Hz), 4.89 (s, 2H), 4.32 (t, 2H, J = 16.05 Hz), 3.13 (t, 2H, J = 16.04 Hz); ¹³C NMR (176 MHz, DMSO-d₆): δ 193.41, 160.74, 156.02, 146.88, 143.12, 142.27, 135.9266, 135.26, 132.38, 130.81, 129.67, 128.10, 126.93, 126.55, 126.52, 126.45, 125.87, 119.08, 45.72, 39.27, 33.67; Ms; 558.0; Ms; (m/z; 557, M + 2; 559).

4-(2-(2-((2-(4-Chlorophenyl)-2-oxoethyl)thio)-4-oxoquinazolin-3(4H)-yl)ethyl)benzenesulfonamide (4): m.p 250–251°; 93% yield; IR (KBr, cm⁻¹) ν: 3281, 3239 (NH), 1684 (C=O), 1345, 1159 (O=S=O); ¹H NMR (700 MHz, DMSO-d₆): δ 8.15 (d, 2H, J = 8.43 Hz), 8.04 (d, 1H, J = 7.85 Hz), 7.82 (d, 2H, J = 8.05 Hz), 7.70 (d, 2H, J = 8.40 Hz), 7.67 (d, 1H, J = 7.05 Hz), 7.52 (d, 2H, J = 8.05 Hz), 7.41 (t, 1H, J = 7.45 Hz), 7.37 (s, 2H), 6.99 (d, 1H, J = 8.19 Hz), 4.89 (s, 2H), 4.32 (t, 2H, J = 16.09 Hz), 3.13 (t, 2H, J = 16.06 Hz); ¹³C NMR (176 MHz, DMSO-d₆): δ 193.20, 160.74, 156.03, 146.89, 143.12, 142.28, 138.89, 135.60, 135.25, 130.73, 129.67, 129.43, 126.93, 126.53, 125.86, 119.08, 45.72, 39.28, 33.67; Ms; 514; Ms; (m/z; 513, M + 1; 514).

4-(2-(2-((2-(4-Fluorophenyl)-2-oxoethyl)thio)-4-oxoquinazolin-3(4H)-yl)ethyl)benzenesulfonamide (5): m.p 249–250°; 92% yield; IR (KBr, cm⁻¹) ν: 3278, 3238(NH), 1666 (C=O), 1342, 1152 (O=S=O); ¹H NMR (700 MHz, DMSO-d₆): δ 8.23 (dd, 2H, J = 13.95 and 2.66 Hz), 8.04 (d, 1H, J = 7.84 Hz), 7 82 (d, 2H, J = 8.05 Hz), 7.68 (t, 1H, J = 7.63 Hz), 7.52 (d, 2H, J = 7.98 Hz), 7.46 (t, 2H, J = 8.71 Hz), 7.41 (t, 1H, J = 7.49 Hz), 7.37 (s, 2H), 6.99 (d, 1H, J = 8.12 Hz), 4.90 (s, 2H), 4.33 (t, 2H, J = 16.09 Hz), 3.14 (t, 2H, J = 16.06 Hz); ¹³C NMR (176 MHz, DMSO-d₆): δ 192.73, 166.36, 164.93, 160.7571, 156.07, 146.89, 143.12, 142.28, 135.24, 133.65, 133.64, 131.88, 131.83, 129.67, 126.92, 126.52, 125.87, 119.08, 116.41, 116.29, 45.70, 39.26, 33.67; Ms; m/z (497).

4-(2-(4-Oxo-2-((2-oxo-2-(p-tolyl)ethyl)thio)quinazolin-3(4H)-yl)ethyl)benzenesulfonamide (6): m.p 257–258°; 92% yield; IR (KBr, cm⁻¹) ν: 3281, 3237 (NH), 1665 (C=O), 1339, 1150 (O=S=O); ¹H NMR (700 MHz, DMSO-d₆): δ 8.04 (t, 3H, J = 7.12 and 4.90 Hz), 7.82 (d, 2H, J = 7.75 Hz), 7.68 (t, 1H, J = 7.59 Hz), 7.52 (d, 2H, J = 7.84 Hz), 7.42 (t, 3H, J = 7.84 and 10.71 Hz), 7.37 (s, 2H), 7.04 (d, 1H, J = 8.19 Hz), 4.90 (s, 2H), 4.33 (t, 2H, J = 15.79 Hz), 3.14 (t, 2H, J = 15.79 Hz), 2.44 (s, 3H); ¹³C NMR (176 MHz, DMSO-d₆): δ 193.40, 160.78, 156.12, 146.94, 144.46, 143.12, 142.30, 135.21, 134.33, 129.83, 129.67, 128.93, 126.91, 126.52, 125.95, 119.09, 45.65, 39.41, 33.67, 21.73; Ms; m/z (493).

4-(2-(4-Oxo-2-((1-oxo-1-phenylpropan-2-yl)thio)quinazolin-3(4H)-yl)ethyl)benzenesulfonamide (7): m.p 245–246°; 90% yield; IR (KBr, cm⁻¹) ν: 3279, 3237 (NH), 1668 (C=O), 1347, 1154 (O=S=O); ¹H NMR (700 MHz, DMSO-d₆): δ 8.16 (d, 2H, J = 7.76 Hz), 8.29 (d, 1H, J = 7.85 Hz), 7.80 (d, 2H, J = 7.84 Hz), 7.73 (t, 1H, J = 7.31 Hz), 7.65–7.61 (m, 3H), 7.84 (d, 2H, J = 7.84 Hz), 7.39 (t, 1H, J = 7.52 Hz), 7.37 (s, 2H), 6.78 (d, 1H, J = 8.12 Hz), 5.75 (q, 1H, J = 7.16 Hz), 4.29–4.19 (m, 2H), 3.07 (t, 2H, t, J = 12.58 Hz), 1.57 (d, 3H, J = 7.19 Hz); ¹³C NMR (176 MHz, DMSO-d₆): δ 198.20, 160.67, 155.81, 146.87, 143.12, 142.23, 135.98, 135.15, 134.01, 129.69, 129.34, 128.95, 126.92, 126.57, 126.48, 125.46, 119.13, 46.23, 45.76, 33.60, 16.44; Ms; 493.00; Ms; m/z (493).

2.2. CA inhibition

The hCA I, II, IX, and XII isoenzyme inhibition assays were performed according to the reported method using the SX.18 MV-R stopped-flow instrument (Applied Photophysics, Oxford, UK)^52–54. All CA isoforms were recombinant isoforms obtained in-house, as reported earlier^55,56.

2.3. Molecular docking method

The molecular docking protocol was conducted according to the reported methods^{28,32,33,41–43,57–64} using MOE 2008.10 from the Chemical Computing Group Inc⁶⁵. The crystal structures of CA-IX (PDB ID: 5FL4) and CA-XII (PDB ID: 1JCZ) were obtained from the protein data bank^66,67.

3. Results and discussion

3.1. Chemistry

4-(2-(4-Oxo-2-thioxo-1,4-dihydroquinazolin-3(2H)-yl)ethyl)benzenesulfonamide (1) was obtained via the reaction of 4-(2-isothiocyanatoethyl)benzenesulfonamide, triethylamine and 2-aminobenzoic acid in boiling ethanol^50,51 (Scheme 1). Stirring of compound 1 with potassium carbonate in acetone and different phenacyl bromides produced the corresponding 4-(2-(2-((2-(4-substituted-phenyl)-2-oxoethyl)thio)-4-oxoquinazolin-3(4H)-yl)ethyl)benzenesulfonamides 2–7 with 90–95% yield. Various spectroscopic studies were conducted to validate the structures of the newly synthesised compounds, 2–7. The target compounds, 2–6, were validated by the diminishing of the thioamidic proton (NH–C=S) at 13.03 ppm and that of the thione moiety (NH–C=S) at 175.29 ppm, as well as by the presence of the phenacyl carbonyl group (SCH₂COAr) at 194.04–192.73 ppm, with singlet peaks at 4.92–4.89 ppm and 39.41–39.26 ppm due to the phenacyl (SCH₂COAr) moiety, in the ¹H and ¹³C NMR spectra, respectively. Additionally, 4-(2-(4-oxo-2-((1-oxo-1-phenylpropan-2-yl)thio)quinazolin-3(4H)-yl)ethyl)benzenesulfonamide (7) was confirmed by presence of the carbonyl group of (S(CH)CH₃COAr) at 198.20 ppm in the ¹³C NMR spectrum, as well as by the quartette (S(CH)CH₃COAr) and doublet (S(CH)CH₃COAr) peaks at 5.75 and 1.57 ppm respectively in the ¹H NMR spectrum, together with the characteristic peaks (S(CH)CH₃COAr) at 46.23 and (S(CH)CH₃COAr) at 16.44 ppm in the ¹³C NMR spectrum. The ethylbenzenesulfonamide amino group (NH₂) (in compounds 2–7) was long-established by the presence of a typical singlet peak at 7.37 ppm in the ¹H NMR spectrum. The tails of aliphatic ethylbenzenesulfonamide moiety were fixed by triplet peaks at 4.33–4.32 and 3.15–3.12 ppm in the ¹H NMR spectrum and distinctive peaks at 45.72–45.65 and 33.67 ppm in ¹³C NMR spectrum, respectively. 2-Substituted mercapto-4(3H)-quinazolinones (8–20) were prepared in 90–96% yield by mixing compound 1 and 2-chloro-N-substitutedamide in acetone at room temperature in the presence of potassium carbonate⁵¹.

Scheme 1.

Synthesis of the designed quinazoline derivatives (2–20).

3.2. CA inhibitory activity

The CA inhibitory activity of 4-(2-(2-(substituted-thio)-4(3H)-quinazolinon-3-yl)ethyl)benzenesulfonamides (compounds 2–20) towards hCA I, II, IV, and IX isoforms was measured and compared to acetazolamide (AAZ), a typical sulphonamide inhibitor. hCA I was effectively inhibited by compounds 2 and 4–13 with the inhibition-constant (K_I) values ranging from 114.5–938.3 nM (AAZ: K_I = 250.0 nM). Compounds 3 and 16 showed moderate activity with K_I values of 1447.0 and 1697.0 nM, respectively, while compounds 14–15 and 17–20 showed weak activity with K_I values ranging from 2048–5467 nM. Compounds 5, 8, 9, 11, 12, and 20 were verified to be effective hCA II inhibitors, with K_I values of 25.4–95.4 nM (AAZ: K_I = 12.0 nM). Compounds 2, 3, 4, 6, 7, 10, 14, and 16 showed modest hCA II inhibitory activity with K_I values ranging between 116.2 and 266.1 nM, whereas compounds 13 and 15 showed a weak inhibitory activity with K_I values of 304.6 and 1099.0 nM, respectively. Compounds 2–17 and 20 displayed potent hCA IX inhibitory activity with K_I values ranging from 8.0 to 100.4 nM, which were greater than or nearly identical to that of AAZ (K_I = 25.0 nM), whereas compounds 18 and 19 showed modest hCA IX inhibitory activity with K_I values ranging between 256.4 and 145.1 nM, respectively. 4-(2-(2-(Substituted-thio)-4(3H)-quinazolinon-3-yl)ethyl)benzenesulfonamide derivatives 2, 4, 5, 8, 9, 11, 12, 13, 14, 16 and 17 showed potent hCA XII inhibitory activity with K_I values of 2.4–49.1 nM compared to AAZ (K_I = 5.7 nM), whereas compounds 3, 6, 7, 10, 15, 18, 19, and 20 exerted moderate hCA XII inhibitory activities with K_I values of 59.7–113.4 nM (Table 1). On the other hand, the selectivity factor is critical goal to increase the value of the new synthesised compounds. New compounds, such as 2 and 4 showed characteristic effective and selective antitumor (hCA IX and hCA XII) carbonic anhydrase inhibitory activity with K_I values (compound 2; 40.7 and 13.0 nM) and K_I values (compound 4; 8.0, and 10.8 nM) compared with AAZ (K_I values of 25 and 5.7 respectively). 4-(2-(2-((2-(4-Substituted-phenyl)-2-oxoethyl)thio)-4-oxoquinazolin-3(4H)-yl)ethyl)benzenesulfonamides (compounds 2–7) showed high selectivity in the inhibition of hCA IX over hCA I and hCA II (in the range of 15.0–95.0 and 2.3–23.0, respectively), as well as selectivity in the inhibition of hCA XII over hCA I and hCA II (in the range of 5.5–70.0 and 2.5–17.0, respectively) (Table 1).

Table 1.

Inhibition data of hCA isoforms hCA I, II, IX and XII for sulphonamides using AAZ as standard drug.

Comps	R	R1	KI (nM)a				Selectivity analysis
			hCA I	hCA II	hCA IX	hCA XII	hCA I/IX	hCA I/XII	hCA II/IX	hCA II/XII
1	–	–	31.5	0.62	–	0.59	–	53.12	–	1.05
2	COPh	H	592.7	140.8	40.7	13.0	15	46	3.5	11
3	CO(4-Br-Ph)	H	1447	174.7	75.2	69.6	19	21	2.3	2.5
4	CO(4-Cl-Ph)	H	758.7	186.6	8.0	10.8	95	70	23	17
5	CO(4-F-Ph)	H	399.5	95.4	16.5	9.1	24	44	5.8	10
6	CO(4-CH3-Ph)	H	471.0	116.2	25.1	85.1	19	5.5	4.6	1.4
7	CO(4-Br-Ph)	CH3	978.3	202.6	63.2	76.8	15	13	3.2	2.6
8	CONH2	H	114.5	25.4	34.5	2.4	3.3	48	0.7	11
9	CONHPh	H	459.7	69.7	27.3	38.4	17	12	2.6	1.8
10	CONH(4-Br-Ph)	H	697.1	119.3	64.9	61.0	11	11	1.8	2
11	CONH(4-Cl-Ph)	H	726.4	92.0	66.8	31.6	11	23	1.4	2.9
12	CONH(4-F-Ph)	H	548.6	87.6	12.7	8.7	43	63	6.9	10
13	CONH(4-CH3-Ph)	H	878.1	304.6	37.4	45.2	23	19	8.1	6.7
14	CONH(4-OCH3-Ph)	H	2567	266.1	84.0	49.1	31	52	3.2	5.4
15	CONH(4-OC2H5-Ph)	H	3654	684.2	35.9	59.7	102	61	19	11
16	CONH(4-COCH3-Ph)	H	1697	200.1	24.1	22.5	70	75	8.3	8.9
17	CONH(3,4,5-tri-OCH3-Ph)	H	2672	519.4	100.4	16.9	27	158	5.2	31
18	CONH(4-F-Bn)	H	2048	975.4	256.4	113.4	8	18	3.8	8.6
19	CONH(3,4-diOCH3-Bn)	H	5467	1099	145.1	97.3	38	56	7.6	11
20	CONH(4-Cl-Ph)	CH3	3628	75.4	18.6	66.7	195	54	4.1	1.1
AAZ	–		250.0	12.0	25.0	5.7	10	44	0.5	2.1

^aMean from 3 different assays, obtained using a stopped flow technique (errors were in the range of ±5–10% of the reported values).

Compounds 4 and 5 showed high selectivity in the inhibition of hCA IX over hCA I and hCA II, with selectivity ratios of 95.0 and 23.0, respectively for compound 4, and those of 24.0 and 5.8, respectively, for compound 5, compared with AAZ selectivity ratios of 10.0 and 0.5, respectively. Additionally, compounds 4 and 5 showed selective inhibition of hCA XII over hCA I and hCA II with selectivity ratios of 70.0 and 17.0, respectively, for compound 4, and 44.0 and 10.0, respectively, for compound 5, compared with AAZ selectivity ratios of 44.0 and 2.1, respectively. N-(substituted)-2-((4-oxo-3-(4-sulfamoylphenethyl)-3,4-dihydroquinazolin-2-yl)thio)amides (compounds 8–20) showed high selectivity in the inhibition of hCA IX over hCA I and hCA II, with selectivity ratios in the range 3.3–195.0 and 0.7–8.3 respectively, compared with AAZ selectivity ratios of 10.0 and 0.5 respectively, and that of hCA XII over hCA I and hCA II, respectively with selectivity ratios in the range 11.0–158.0 and 1.8–31.0, compared with AAZ selectivity ratios of 44.0 and 2.1 respectively. Compounds 12–17, and 19–20 showed selective inhibition of hCA IX over hCA I and hCA II, with selectivity ratios of 23.0–195.0 and 3.2–19.0, respectively, compared with AAZ selectivity ratios of 10.0 and 0.5. Additionally, Compounds 12, 14–17, and 19 showed selective inhibition of hCA XII over hCA I and hCA II, with selectivity ratios of 48.0–158.0 and 5.4–31.0, respectively, compared with AAZ selectivity ratios of 44.0 and 2.1 respectively.

3.3. Structure-activity relationship (SAR) analysis

Several synthesised quinazolinone derivatives (compounds 2–20) were potent inhibitors of the hCA isoforms.

3.3.1. SAR analysis of hCA I inhibition

SAR analysis of hCA I inhibition indicated revealed several key features. (1) 4–(2-(4-Oxo-2-((2-oxo-2-phenylethyl)thio)quinazolin-3(4H)-yl)ethyl)benzenesulfonamide (2), with a K_I value of 592.7 nM, was more potent than 4-(2-(2-((1-(4-substituted-phenyl)-1-oxopropan-2-yl)thio)-4-oxoquinazolin-3(4H)-yl)ethyl)benzenesulfonamides 3–4 and 4-(2-(4-oxo-2-((1-oxo-1-phenylpropan-2-yl)thio)quinazolin-3(4H)-yl)ethyl)benzenesulfonamide 7, with K_I values of 758.7–1447 nM, but less potent than 4-(2-(2-((1-(4-flouro/4-methyl-phenyl)-1-oxopropan-2-yl)thio)-4-oxoquinazolin-3(4H)-yl)ethyl)benzenesulfonamide 5 and 6 with K_I values of 399.5–471.0 nM. (2) Unsubstituted-N-acetamide 8 (K_I value = 114.5 nM) was more active than the corresponding N-phenylacetamide 9 (K_I value = 459.7 nM. (3) Substitution of the phenyl ring of N-phenylacetamide 9 (K_I value = 459.7 nM) resulted in substituted-N-phenylacetamides 10–17 and N-phenylpropanamide 20 with significantly decreased CA inhibitory activity (K_I values = 548.6–3654 nM). (4) The hCA I inhibitory activity of N-(4-fluorophenyl)-2-((4-oxo-3-(4-sulfamoylphenethyl)-3,4-dihydroquinazolin-2-yl)thio)acetamide (12), with a K_I value of 548.6 nM, was more stronger than the corresponding N-(4-fluorobenzyl)-2-((4-oxo-3-(4-sulfamoylphenethyl)-3,4-dihydroquinazolin-2-yl)thio)acetamide (18) K_I of 2048 nM. (5) hCA I inhibition of N-acetamide 11, with a K_I value of 726.4 nM, was more powerful than the corresponding N-propanamide 20 with a K_I value of 3628 nM.

3.3.2. SAR analysis for hCA II inhibition

The SAR analysis for hCA II inhibition revealed several key features. (1) 4-(2-(2-((1-(4-Fluorophenyl/4-methylphenyl)-1-oxopropan-2-yl)thio)-4-oxoquinazolin-3(4H)-yl)ethyl)benzenesulfonamides 5 and 6 with K_I values of 95.4–116.2 nM were more effective than unsubstituted phenyl and other substituted phenyl derivatives, such as compounds 2–4 and 7 with K_I values of 140.8–202.6 nM. (2) hCA II inhibition of 2-((2-oxo-2-phenylethyl)thio)quinazolinone 2, with a K_I value of 140.8 µM, was stronger than the corresponding 2-((1-oxo-1-phenylpropan-2-yl)thio)quinazolinone 7 with a K_I value of 202.6 nM. (3) N-Phenylacetamide 9 with a K_I value of 69.7 nM was less potent than unsubstituted-N-acetamide 8 (K_I value = 25.4 nM). (4) Substitution of the phenyl ring of N-phenylacetamide 9 (K_I; 69.7 nM) resulted in substituted-N-phenylacetamides 10–17 and N-phenylpropanamide 20 with considerably diminished CA II inhibitory activity (K_I values of 75.4–684.2 nM); (5) The hCA II inhibitory effect of N-acetamide 11 (K_I value = 92 nM) was less potent than the corresponding N-propanamide 20 (K_I value = 75.4 nM). (6) The hCA II inhibitory activity of N-(4-fluorophenyl)acetamide 12, with a K_I value of 87.6 nM, was stronger than the corresponding N-(4-fluorobenzyl)acetamide 18 (K_I of 2048 nM).

3.3.3. SAR analysis of hCA IX inhibition

SAR analysis of hCA IX inhibition revealed several key factors. (1) The 2-((2-oxo-2-phenylethyl)thio)quinazolinone 2, with a K_I value of 40.7 nM, was more potent than 2-((1-oxo-1-phenylpropan-2-yl)thio)quinazolinone 7 with K_I value of 63.2 nM. (2) The induction of the activating group, such as the 4-methyl group on the phenyl ring of compound 2 (K_I value = 40.7 nM) led to compound 6, with an increased hCA IX inhibitory activity (K_I value = 25.1 nM). (3) The introduction of the deactivating group on phenyl ring of compound 2, such as the 4-bromo group, resulted in compound 3 with diminished hCA IX inhibition activity (K_I value of 75.2 nM); in contrast, the introduction of 4-fluoro/4-chloro groups produced compounds 4–5 with boosted the inhibitory potency of the hCA IX (K_I values of 8.0–16.5 nM). (4) N-propanamide 20, with a K_I value of 18.6 nM, was powerful than the corresponding N-acetamide 11 with a K_I value of 66.8 nM. (5) The introduction of activating/deactivating groups on the phenyl ring of compound 2 (K_I value = 27.3 nM) yielded compounds 10–17 with reduced inhibitory activity (K_I values = 35.9–100.4 nM), except for compounds 12 and 16, which had improved hCA IX inhibitory potency (K_I values = 12.7–24.1 nM). (6) Substitution of the phenyl group of compound 12 (K_I value = 12.7 nM) with a benzyl moiety resulted in compound 18, which had significantly reduced hCA IX inhibitory activity (K_I value = 256.4 nM).

3.3.4. SAR analysis for hCA XII inhibition

SAR analysis for hCA XII inhibition revealed several key factors. (1) 2-((2-Oxo-2-phenylethyl)thio)quinazolinone 2, with a K_I value of 13.0 nM, was more potent than 2-((1-oxo-1-phenylpropan-2-yl)thio)quinazolinone 7 with a K_I value of 76.8 nM. (2) The introduction of a chloro/fluoro group at the phenyl ring, such as in compounds 4 and 5 (K_I values = 9.1–10.8 nM), improved the hCA XII inhibition activity and was similar to that of compound 2 (K_I value = 13.0 nM). (3) The unsubstituted N-acetamide, compound 8, (K_I value = 2.4 nM) resulted in more powerful hCA XII inhibition than N-substituted amides, compounds 9–20, (K_I values = 8.7–113.4 nM). (4) hCA XII inhibition of N-acetamide 11, with a K_I value of 31.6 nM, was more powerful than that of the corresponding N-propanamide 20 with a K_I value of 66.7 nM. (5) The substitution of the phenyl group of N-(4-fluorophenyl)acetamide 12 (K_I value of 8.7 nM) with a benzyl moiety resulted in the N-(4-fluorobenzyl)acetamide 18, with sharply reduced CA inhibitory activity (K_I value = 113.4 nM).

3.4. Molecular docking

3.4.1. Molecular docking of compounds 17 and 20 with CA IX and CA XII isoenzymes

To further investigate the interactions between the selected active compounds 17 and 20 with the hCAs targets, we performed docking simulations into the binding pockets of the hCA isoforms, IX and XII, using the MOE Suite⁶⁵ (data are summarised in Figures 2 and 3).

Figure 2.

Docking modes of active compounds 17 and 20 in the binding pockets of CA isoenzyme IX (PDB 5FL4). Predicted binding mode of compounds 17 (2D and 3D in upper panel), and 20 (2D and 3D in lower panel) with the hCA-IX target.

Figure 3.

Docking modes of the active compounds 17 and 20 in the binding pockets of CA isoenzyme XII (PDB 1JCZ). Predicted binding mode of compounds 17 (2D and 3D in upper panel) and 20 (2D and 3D in lower panel) with hCA-XII target.

Both the compounds 17 and 20 were shown to directly interact with the zinc ion of CA IX and CA XII isoenzymes, via the sulphonamide anion of the active sites of both enzymes. However, the contributions of the quinazoline scaffold and the terminal bulky thioether fragments interaction are different, based on the CA isoform. In CA IX, the quinazoline ring of compound 20 interacts with the Gln71 residue through a stable hydrogen bond, and gets accommodated in the hydrophobic pocket lined by the Val121, Val130, Leu134, and Leu91 residues, thereby stabilising the binding (Figure 2, lower panel). In addition, the terminal p-chlorobenzamide fragment formed a hydrophobic interaction with the Leu91 residue (Figure 2, lower panel). In contrast, compound 17 was shown to bind similarly to the pocket of CA IX, except the unfavourable orientation of the quinazoline carbonyl moiety of compound 17 towards the hydrophobic pocket formed by Leu91 residue in CA IX (Figure 2, upper panel). Also, the benzamide core showed a polar-nonpolar interaction with the Leu91 and Thr73 residues, as the bulky side chain causes steric hindrance, inducing conformational changes in the bulky thioether tail and the quinazoline groups (Figure 2, upper panel). These differences in the binding of compounds 17 and 20 could be responsible for the observed differences in the K_I values of the two compounds for CA IX.

Results also showed different interactions between CA XII and compounds 17 and 20 (Figure 3). The carbonyl group on the quinazoline ring in compound 17 was stabilised by direct hydrogen bonding with the target residue Ser132 of CA XII (Figure 3, upper panel). In addition, the Lys67 residue showed favourable hydrophobic binding to the quinazoline core of compound 17. The trimethoxybenzamide group of compound 17 was accommodated in the polar pocket of CA XII that included Ser132 and Thr133 residues (Figure 3, upper panel). The placement of compound 20 within the CA XII pocket was not favoured, particularly because the quinazoline ring of compound 20 was trapped between the polar pocket of CA lined by the Ser135, Gln92, and Ser132 residues (Figure 3, lower panel). Therefore, this interaction causes an energetically unfavourable change in the terminal benzamide and quinazoline scaffold of compound 20, which could be responsible for the decreased inhibitory activity of compound 20 (Figure 3, lower panel).

3.4.2. Molecular orbital analyses

According to the frontier molecular orbital theory, HOMO and LUMO are the most important orbitals found in a molecule, as they can affect its biological activity, the molecular reactivity, the ionisation and the electron affinity^68–70. The molecular orbital analysis of the representative compounds 4, 17, and 20 (Figure 4) as an active and selective derivatives was done by exploring their structure-selectivity relationship. The electron transition from HOMO to LUMO occurs freely when the energy gap is small. The HOMO-LUMO energy gap for the compounds 4, 17, and 20 was calculated to be −0.3125, −0.2834, and −0.28949 eV, respectively. The negative energy values are indicative of a stable structure and confirm the eventual charge transfer interactions. The distributions and energy levels of the HOMO-LUMO orbitals computed for the above-mentioned compounds are represented in Figure 4. HOMO and LUMO orbitals are mainly delocalised in the carbon and nitrogen of the quinazoline scaffolds and the sulphur ether atoms in the active compound 4. While they are mainly delocalised in the S-linker of the benzamide moiety, ring substituents in the compounds 17 and 20 reverse their interactions with the enzyme isoforms. These results indicate that the affinity of the selective compounds for the CA IX and CA XII binding sites could be because of the involvement of the thioether moiety, and that the quinazoline moiety could mostly provide the structural basis and the lipophilic function, contributing strongly to their selectivity. In addition, the low HOMO-LOMO energy gap suggests that the molecules have high stability and are in their lowest energy conformation.

Figure 4.

Molecular orbital spatial distribution and localisation for the HOMO and LUMO of three representative compounds, 4 (left panel), 17 (middle panel), and 20 (right panel).

4. Conclusion

The CA inhibitory activity of 4-(2-(2-(substituted-thio)-4(3H)-quinazolinon-3-yl)ethyl)benzenesulfonamides (compounds 2–20) towards the hCA I, II, IV, and IX isoforms was assessed and compared with acetazolamide (AAZ), a typical sulphonamide inhibitor. Of the different hCA isoforms, hCA I was effectively inhibited by the compounds 2 and 4–13, with inhibition constant (K_I) values in the range of 114.5–938.3 nM (AAZ: K_I value of 250.0 nM), while compounds 3 and 14–20 showed moderate to weak CA inhibitory activity with K_I values of 1447.0–5467 nM. Compounds 5, 8, 9, 11, 12, and 20 were revealed to be effective hCA II inhibitors, with K_I values of 25.4–95.4 nM (AAZ: K_I value of 12.0 nM). Compounds 2, 3, 4, 6, 7, 10, 13, 14, 15, and 16 showed modest to weak hCA II inhibitory activity with K_I values ranging between 116.2 and 1099.0 nM. Compounds 2–17 and 20 displayed potent hCA IX inhibitory activity with K_I values ranging from 8.0 to 100.4 nM compared to AAZ (K_I value of 25.0 nM), whereas compounds 18 and 19 showed modest hCA IX inhibitory activity with K_I values ranging between 256.4 and 145.1 nM, respectively. Ethylbenzenesulfonamide derivatives, 2, 4, 5, 8, 9, 11, 12, 13, 14, 16, and 17 showed potent hCA XII inhibitory activities with K_I values of 2.4–49.1 nM compared to AAZ (K_I value of 5.7 nM), whereas compounds 3, 6, 7, 10, 15, 18, 19, and 20 showed moderate hCA XII inhibitory activities with K_I values of 59.7–113.4 nM. Compounds 2 and 4 showed characteristic effective and selective antitumor (hCA IX and hCA XII) carbonic anhydrase inhibitory activity with K_I values (compound 2; 40.7 and 13.0 nM) and K_I values (compound 4; 8.0, 10.8 nM). Compounds 2–7 showed high selectivity ratios for the inhibition of hCA IX over hCA I (15.0–95.0) and hCA IX over hCA II (2.3–23.0), while selectivity ratios of hCA XII over hCA I (5.5–70.0) and hCA XII over hCA II (1.4–17.0). Compounds 4 and 5 displayed selective inhibitory activity towards hCA IX over hCA I with selectivity ratios of 95.0 and 24.0 respectively, and hCA IX over hCA II with selectivity ratios of 23.0 and 5.8 respectively, as well as, selective inhibitory activity for hCA XII over hCA I and hCA XII over hCA II (selectivity ratios of 70.0, 44.0 and 17.0, 10.0, respectively). Compounds 12–17, and 19–20 exhibited selective inhibitory activities towards hCA IX over hCA I and hCA IX over hCA II (selectivity ratios of 23.0–195.0 and 3.2–19.0, respectively). In addition, compounds 8, 12, 14–17, and 19 showed selective inhibitory activity towards hCA XII over hCA I and hCA XII over hCA II (selectivity ratios of 48.0–158.0 and 5.4–31.0, respectively). Docking study of the selective derivatives, compounds 17 and 20, with the hCAs revealed consistent interactions, particularly selectivity-oriented hydrophobic and aromatic interactions through the S-alkyl substituent.

Correction Statement

This article has been republished with minor changes. These changes do not impact the academic content of the article.

Funding Statement

The authors extend their appreciation to the Deanship of Scientific Research at King Saud University for funding the work through the research group project No. RGP-163.

Disclosure statement

No potential conflict of interest was reported by the author(s).