Design, Synthesis, and Biological Evaluation of 1,2,3-Triazole-linked triazino[5,6-b]indole-benzene sulfonamide Conjugates as Potent Carbonic Anhydrase I, II, IX, and XIII Inhibitors

Abstract

A series of 1,2,3-triazole-linked triazino[5,6-b]indole-benzene sulfonamide hybrids (6a–6o) was synthesized and evaluated for carbonic anhydrase (CA, EC 4.2.1.1) inhibitory activity against the human (h) isoforms hCA I, II, XIII (cytosolic isoforms), and hCA IX (transmembrane tumor-associated isoform). The results revealed that the compounds 6a–6o exhibited K_i values in the low to medium nanomolar range against hCA II and hCA IX (K_is ranging from 7.7 nM to 41.3 nM) and higher K_i values against hCA I and hCA XIII. Compound 6i showed potent inhibition of hCA II (K_i = 7.7nM), being more effective compared to the standard inhibitor acetazolamide (AAZ) (K_i = 12.1 nM). Compounds 6b and 6d showed moderate activity against hCA XIII (K_i = 69.8 and 65.8 nM). Hence, compound 6i could be consider as potential lead candidate for the design of potent and selective hCA II inhibitors.

1. Introduction

Carbonic anhydrases (CAs, EC 4.2.1.1) are omnipresent metalloenzymes that they play a pivotal catalytic role in the hydration of carbon dioxide to bicarbonate and protons by means of a ping pong mechanism, which is a slow process under non-catalytic conditions [1,2,3,4].

$$C{O}_2+H_{2}O\underset{}{\iff }HC{O}_3{}^{-}+H^{+}$$

These enzymes are encoded by eight genetically unrelated gene families, namely, α, β, γ, δ, ζ, η, θ, and the recently reported ι class [5]. Among these, the α family is predominantly present in mammals, and 16 isoforms have been reported with different catalytic activity, subcellular localization, and tissue distribution [1,2,3,4]. There are five cytosolic forms (CA I, CA II, CA III, CA VII, and CA XIII), five membrane-bound isozymes (CA IV, CA IX, CA XII, CA XIV, and CA XV), two mitochondrial forms (CA VA and CA VB), and a secreted isozyme (CA VI) [1,2,3,4]. These isoforms are implicated in different diseases, as shown in

Table 1. Carbonic anhydrase (CA) isoforms and associated diseases.

Disease	Isoform Target
Glaucoma	CA II, CA IV, CA XII
Cancer	CA IX, CA XII
Epilepsy	CA VII
Antineuropathic pain	CA VII
Obesity	CA VA

. Therefore, selective inhibition of a particular isoform redresses the particular disease in which it plays a major role.

To date, the sulfonamide group is considered as main zinc-binding group for the design of carbonic anhydrase inhibitors. Sulfonamides and their bio-isosteres such as sulfamides / sulfamates are known to elicit potent carbonic anhydrase inhibition and hence they are present in drugs, which are prescribed for the treatment of glaucoma, epilepsy, obesity, and cancer. The diuretic drugs mainly target CA II, CA IV, CA XII, and CA XIV [6,7], the anti-glaucoma drugs target CA II, CA IV, and CA XII [8,9], while the anti-epileptics target CA VII and CAXIV [10,11,12]. CA IX and CA XII specifically expressed in tumor cells, and their inhibition results in anti-metastatic effects [13,14,15]. However, the main drawback of all these drugs is the lack of selectivity, which results in serious side effects. Therefore, there is an urgent need to design and develop selective isoform inhibitors. The tail approach has been very successful in addressing this issue, and many novel scaffolds have been developed [16,17]. In this approach, the attached tails bind to the active site cavity, preferably the middle and the rim part, which shows variation in different CA isoforms. Some clinically/pre-clinically used sulfonamides are illustrated in the Figure 1:

Owing to the development of novel carbonic anhydrase inhibitors with better isoform selectivity, our group designed some novel hybrids in which the triazino[5,6-b]indole tail was conjugated to benzene sulfonamide via a 1,2,3-triazole linker. Triazino[5,6-b]indole is a flexible tail with diverse pharmacological activities, like anti-fungal/anti-bacterial [18], anti-diabetic [19], anti-depressant [20], anti-hypertensive [21], anti-inflammatory [22], and anti-hypoxic activities [23].

The design of this new series of compounds was based on the tail approach via the fusion of indole and 1,2,4-triazine, which were reported for high interactions with carbonic anhydrase (Figure 2) [24,25,26,27,28]. The present design mainly is mainly involved two strategies. The first one was to fuse the two CA-binding scaffolds i.e., Indole and 1,2,4-triazine in order to develop a flexible tail with better interactions in the enzymatic site and the second one was to incorporate different N-alkyl substituents in the indole tail in a systematic fashion to define optimal length (methyl, ethyl, propyl), bulkiness (isopropyl) and un-saturation (allyl), which would confer the best CA inhibitory activity. It is reported in the literature that 1,2,3-triazole is an efficient linker, useful in the design of potent CA inhibitors, as it is an amide bioisostere and maintains high stability under basic as well as acidic hydrolysis conditions. It also has high dipole moment and capability of H bonding in vivo. Due to its aromatic character, it shows some π-stacking interactions with relevant amino acid residues [29].

2. Results and Discussion

2.1. Synthesis of the Target Molecules

The current design of experiment (DOE) was based on the molecular hybridization approach. We synthesized molecular hybrids of a bulky triazino[5,6-b]indole, used as a tail, conjugated to benzene sulfonamide through a flexible 1,2,3-triazole linker.

The synthesis of 1,2,3-triazole-linked triazino[5,6-b]indole-benzene sulfonamide hybrids (6a–6o) was performed according to the general synthetic scheme illustrated in Scheme 1. The intermediate compounds (4a–o) were synthesized according to previously reported methods [30,31]. The N-alkylated isatins (3a–o) were synthesized from the simple five-substituted isatins (1a–c) by nucleophilic substitution of different alkyl halides (2a–e). The 3a–o were condensed with thiosemicarbazide in aqueous 1,4-dioxane under reflux conditions by using cesium carbonate as a base followed by propargylation to generate intermediates (5a–o). Finally the 5a–o were subjected to click reaction with 4-azido benzene sulfonamide to generate 1,2,3-triazole-linked triazino[5,6-b]indole-benzene sulfonamide hybrids (6a–o).

2.2. Carbonic Anhydrase Inhibition

The newly synthesized 1,2,3-triazole linked triazino[5,6-b]indole-benzene sulfonamide hybrids (6a–6o) were evaluated for their carbonic anhydrase inhibitory activity against a panel of carbonic anhydrases, i.e., hCA I, hCA II, hCA IX, and hCA XIII, by the stopped-flow CO₂ hydrase assay method. Highly purified CA isoforms were employed, for which the kinetic parameters for the physiologic reaction (CO₂ hydration) were measured (see the Experimental section for details), monitoring the color change produced by the formation of H⁺ ions (and bicarbonate). For all the pure enzymes, the kinetic parameters (k_cat and k_cat/K_M) are measured and these values are given in the

Table 2. Kinetic parameters of the pure CA isoforms employed in this work and inhibition constants for acetazolamide (AAZ), a standard sulfonamide drug.

Organisms	CA Class	Acronym	Kcat (s−1)	kcat/KM (M−1 × s−1)	KI (Acetazolamide) (nM)
Homo sapiens	α	hCA I	2.0 × 105	5.0 × 107	250
	α	hCA II	1.4 × 106	1.5 × 108	12.1
	α	hCA IX a	3.8 × 105	5.5 × 107	25.8
	α	hCA_XIII	1.5 × 105	1.1 × 107	17.0

^a Catalytic domain.

. These activities were highly inhibited by the clinically used sulfonamide inhibitor acetazolamide (AAZ), as shown in Table 2. It was observe that all these enzymes are highly efficient catalysts with k_cat/K_M > 10⁷ M⁻¹x s⁻¹.

The following structure-activity relationship can be inferred from the inhibition data of compounds 6a–o (

Table 3. Inhibition of hCA isoforms I, II, IX, and XIII with compounds 6a–o and AAZ as a standard inhibitor.

		KI (nM)
Compound	Structure	hCA I	hCA II	hCA IX	hCA XIII
6a		910.1	65.5	285.6	77.8
6b		642.2	72.0	172.5	69.8
6c		3960	88.7	219.4	364.8
6d		314.7	20.9	37.8	65.8
6e		535.8	41.7	41.1	626.7
6f		766.2	59.6	41.3	834.8
6g		698.5	63.9	193.1	675.0
6h		764.2	682.7	118.6	1815
6i		379.2	7.7	34.9	736.2
6j		4592	73.7	401.7	793.6
6k		5140	252.7	330.7	867.0
6l		2837	184.6	150.4	3980
6m		6513	61.7	204.5	823.8
6n		571.9	179.2	320.8	91.3
6o		694.4	97.1	324.6	2300
AAZ		250.0	12.1	25.8	17.0

).

I

Tthe cytosolic hCA II isoform was strongly inhibited by all the synthesized compounds 6a–o, in a low to medium nanomolar range (K_is = 7.7 nM to 0.2527 µM). The best activity against hCA II was shown by compound 6i (K_i = 7.7 nM), possessing a fluoro group attached at the 5th position of the indole ring and an isopropyl group anchored to the nitrogen of indole. It was almost twofold more active than the standard AAZ (K_i = 12.1 nM). Compounds 6d–6g, were found to have potent activity at the nanomolar concentration against hCA II, with K_i ranging from 20.9 to 63.9 nM. Compounds 6k–6o, containing a chloro group at the 5th position of indole, showed lower activity in the range of 61.7 to 252.7 nM, compared to compounds containing a fluoro group and unsubstituted indole.

II

The transmembrane hCA IX isoform, which is expressed exclusively in tumors, was also strongly inhibited by the synthesized compounds in the medium nanomolar range (K_is = 34.9 nM to 0.3246 µM). Compounds 6d, 6e, 6f, and 6i showed equipotent nanomolar activity with AAZ, with K_is ranging from 34.9 nM to 41.3 nM. Among these compounds, 6i showed the best activity (K_i = 34.9 nM) against hCA IX isoform.

III

The cytosolic hCA I and hCA XIII isoforms were inhibited by all synthesized compounds in the high nanomolar range (K_is > 500 nM). However, compounds 6b and 6d showed moderate activity with K_is of 69.8 nM and 65.8 nM respectively against hCA XIII isoform.

From the above structure–activity relationship, it was found that compound 6i was the most potent compound with a K_i values of 7.7 nM against hCAII and 34.9 nM against hCA IX.

3. Conclusions

In conclusion, we report here the synthesis of a series of 1,2,3-triazole-linked triazino[5,6-b]indole-benzene sulfonamide hybrids (6a–6o). The structures of these compounds where confirmed by different spectral and elemental analyses methods (Supplemental Data S1). The Biological evaluation of sulfonamides was performed against hCA I, hCA II, hCA IX, and hCA XIII. All compounds showed low to moderate inhibitory activity against hCA II and hCA IX isoforms, at concentrations in the range between 7.7 nM and 0.3246 µM. Compound 6i emerged as a potent hCA II and hCA IX inhibitor (K_i = 7.7 nM against hCA II and 34.9 nM against hCA IX). The compounds 6b and 6d was showed activity at medium nanomolar concentrations, with K_i of 69.8 nM and 65.8 nM, respectively, against hCA XIII isoform. Thus, the compound 6i can be emerged as a novel potential lead compound to develop selective carbonic anhydrase inhibitors against the hCA II isoform.

4. Experimental Section

4.1. General Experimental Conditions

All the chemicals and solvents utilized as obtained from the suppliers. Wherever necessary, anhydrous solvents are used. Thin-layer chromatography analysis (TLC), was carried-out by utilizing Merck silica gel 60 F₂₅₄ aluminum plates. A Stuart Digital Melting point Apparatus (SMP 30) was used to determining the melting point of the compounds, which were uncorrected. The ¹H and ¹³C NMR spectra were recorded on Bruker Avance 500 MHz and 125 MHz respectively, with DMSO-d₆ as the solvent. The chemical shift values were calculated in ppm using TMS as the standard reference. The HRMS were recorded on Agilent QTOF mass spectrometer 6540 series and were performed using ESI techniques at 70 eV. All the newly synthesized analogs were evaluated in vitro for their inhibitory activity against a panel of recombinant CA isoforms, i.e., hCA I, hCA II, hCA IX, and hCA XIII, obtained inhouse, using the stopped-flow CO₂ hydrase assay.

4.1.1. Synthesis of N-Alkylated isatins (3a–o)

To a stirred solution of isatin (0.5 g, 0.00398 mole) in DMF (10 mL) we added potassium carbonate (0.939 g, 0.006796 mole) and potassium iodide (0.05 mole%), and the resulting solution was stirred for about 30 min. After the specified time interval, the respective alkyl halides (2a–e) was added and the resulting solution was allowed to reflux. The progress of the reaction was monitored by using TLC. Upon the completion of the reaction as assessed by TLC, the reaction mixture was poured into ice water, and the precipitated solid was collected, washed with water, and recrystallized from ethanol to yield compounds (3a–o) (yield 72–75%) [31,32].

4.1.2. Synthesis of N-Alkylated triazino[5,6-b]indolethioether derivatives (4a–o)

To a stirred solution of N-alkylated isatin (0.450 g, 0.002378 mol) in 40% aqueous 1,4-dioxane (5 mL), thiosemicarbazide (0.260 g, 0.00285 mol) and Cs₂CO₃ (0.720 g, 0.00285 mol) were added. The resulting solution was refluxed overnight. Upon completion of the reaction (as determined by TLC), the reaction mixture was cooled to rt. The solid byproducts were filtered off and the filtrate was acidified with conc. HCl to pH 1–3. The obtained solids were collected washed with water and dried to give yellow-colored solids which were used without any further purification (yield 68–70%) [31,32].

4.1.3. Synthesis of N-Alkyl-3-prop-2-yn-1-ylthio)-5H-[1,2,4]triazino[5,6b]indole derivatives (5a–o)

To a stirred solution of compounds 4a–o (0.140 g, 0.00607 mole) in DMF (3 mL), K₂CO₃ (0.101 g, 0.000729 mol) and propargyl bromide (0.087 g, 0.000729 mol) were added. The resulting reaction mixture was stirred at rt overnight. Upon completion of the reaction (monitored by TLC), the reaction mixture was poured in ice-cold water, and the formed solid was collected, washed with water, and dried to give brown-colored solids that were used without any further purification (yield 86–90%).

4.1.4. Synthesis of 4-(4-(N-Alkyl-5H-[1,2,4]triazino[5,6b]indol-3-yl)thio)methyl)- 1H-1,2,3-triazol-1-yl)benzenesulfonamide derivatives (6a–o)

The compounds 5a–o (0.04 g, 0.0001 mol) and 4-azido benzene sulfonamide (0.024 g, 0.000 mol) were suspended in 2 mL of a 1:1 water/tert-butanol mixture. Sodium ascorbate (0.048 g, 0.0002 mol) was added, followed by copper (II) sulfate pentahydrate (0.031 g, 0.0001 mol). The heterogeneous mixture was stirred vigorously overnight at which point it cleared and TLC analysis indicated complete consumption of the starting materials. The reaction mixture were diluted with water and cooled in ice to obtained the brown precipitate, which were collected by filtration. After washing with cold water the precipitate were dried under vacuum to afford a pure product as a brown amorphous solid (6a–o).

4-(4-(((5-methyl-5H-[1,2,4]triazino[5,6b]indol-3-yl)thio)methyl-1H-1,2,3-triazol-1-yl)benzene sulfonamide (6a) Yield: 57%; Color: Brown solid; mp: 235–240 °C; ¹H NMR (500 MHz, DMSO-d₆) δ 8.87 (s, 1H), 8.34 (s, 1H), 8.10 (s, 2H), 7.99 (s, 2H), 7.80 (s, 2H), 7.51 (s, 3H), 4.77 (s, 2H), 3.86 (s, 3H);13C NMR (125 MHz, DMSO) δ 167.1, 159.9, 158.0, 147.2, 145.7, 144.2, 141.3, 139.0, 138.5, 127.9, 122.5, 120.8, 113.2, 108.1, 107.9, 28.1, 25.29. HR-MS (ESI-QTOF): m/z calculated for [M + H]⁺ C₁₉H₁₆N₈O₂S₂; 453.0916; found 453.0965.

4-(4-(((5-ethyl-5H-[1,2,4]triazino[5,6b]indol-3-yl)thio)methyl-1H-1,2,3-triazol-1-yl)benzene sulfonamide (6b) Yield: 59%; Color: Brown solid; mp: 202–204 °C; ¹H NMR (500 MHz, DMSO-d₆) δ8.85 (s, 10H), 8.38 (dd, J = 17.8, 4.8 Hz, 11H), 8.10 (d, J = 8.6 Hz, 21H), 7.99 (d, J = 8.6 Hz, 20H), 7.85 (d, J = 8.2 Hz, 8H), 7.83–7.75 (m, 12H), 7.50 (s, 20H), 4.75 (s, 19H), 4.44 (q, J = 7.0 Hz, 21H), 1.35 (t, J = 7.2 Hz, 28H); ¹³C NMR (125 MHz, DMSO-d₆) δ 166.58, 146.11, 145.83, 144.23, 141.55, 141.02, 139.02, 131.43, 127.91, 123.28, 122.37, 122.06, 120.72, 117.94, 111.68, 36.45, 25.31, 13.73; HR-MS (ESI-QTOF): m/z calculated for [M + H]⁺ C₂₀H₁₈N₈O₂S₂; 467.1072; found 467.1120.

4-(4-(((5-propyl-5H-[1,2,4]triazino[5,6b]indol-3-yl)thio)methyl-1H-1,2,3-triazol-1-yl)benzene sulfonamide (6c) Yield: 61%; Color: Brown solid; mp: 261–263 °C; ¹H NMR (500 MHz, DMSO) δ 8.62 (s, 1H), 8.13 (s, 1H), 7.81 (d, J = 56.5 Hz, 5H), 7.57 (d, J = 35.0 Hz, 3H), 7.27 (s, 3H), 4.51 (s, 2H), 4.12 (s, 2H), 1.57 (s, 3H); ¹³C NMR (125 MHz, DMSO) δ 166.7, 146.6, 144.2, 141.4, 139.0, 131.4, 127.9, 123.2, 122.3, 121.9, 120.7, 117.9, 111.8, 43.0, 25.35, 21.62, 11.6.HR-MS (ESI-QTOF): m/z calculated for [M + H]⁺ C₂₁H₂₀N₈O₂S₂; 481.1229; found 481.1268.

4-(4-(((5-isopropyl-5H-[1,2,4]triazino[5,6b]indol-3-yl)thio)methyl-1H-1,2,3-triazol-1-yl)benzene sulfonamide (6d) Yield: 53%; Color: Brown solid; mp: 213–215 °C; ¹H NMR (500 MHz, DMSO-d₆) δ 8.84 (s, 1H), 8.19 (d, J = 7.5 Hz, 1H), 8.09 (d, J = 8.4 Hz, 2H), 8.04–7.93 (m, 4H), 7.62 (t, J = 8.1 Hz, 1H), 7.49 (s, 2H), 5.21–5.13 (m, 1H), 4.73 (s, 2H), 1.61 (d, J = 6.7 Hz, 6H). ¹³C NMR (125 MHz, DMSO) δ 166.2, 146.2, 145.9, 144.2, 141.5, 140.6, 139.0, 131.3, 127.9, 123.1, 122.3, 122.0, 120.7, 118.2, 112.6, 46.7, 25.3, 20.3. HR-MS (ESI-QTOF): m/z calculated for [M + H]⁺ C₂₁H₂₀N₈O₂S₂; 481.1229; found 481.1294.

4-(4-(((5-allyl-5H-[1,2,4]triazino[5,6b]indol-3-yl)thio)methyl-1H-1,2,3-triazol-1-yl)benzene sulfonamide (6e) Yield: 61%; Color: Brown solid; mp: 230–232 °C; ¹H NMR (500 MHz, DMSO-d₆) δ8.84 (s, 1H), 8.39 (s, 1H), 8.05 (d, J = 45.9 Hz, 5H), 7.78 (s, 2H), 7.52 (s, 2H), 6.02 (s, 1H), 5.08 (s, 4H), 4.77 (s, 2H); ¹³C NMR (125 MHz, DMSO) δ 166.4, 147.0, 146.5, 144.3, 141.5, 141.3, 139.0, 131.4, 123.5, 122.5, 122.0, 120.8, 117.9, 112.0, 43.5, 25.3. HR-MS (ESI-QTOF): m/z calculated for [M + H]⁺ C₂₁H₁₀N₈O₂S₂; 479.1072; found 479.1087.

4-(4-(((8-fluoro-5-methyl-5H-[1,2,4]triazino[5,6b]indol-3-yl)thio)methyl-1H-1,2,3-triazol-1-yl)benzene sulfonamide (6f) Yield: 59%; Color: Brown solid; mp: 244–246 °C; ¹H NMR (500 MHz, DMSO-d₆) δ 8.86 (s, 1H), 8.20–8.16 (m, 1H), 8.10 (d, J = 8.6 Hz, 2H), 7.99 (d, J = 8.6 Hz, 2H), 7.84 (dd, J = 8.8, 3.9 Hz, 1H), 7.70–7.63 (m, 3H), 7.51 (s, 1H), 4.76 (s, 2H), 3.86 (s, 3H); ¹³C NMR (125 MHz, DMSO-d₆) δ 167.15, 159.89, 147.16, 144.25, 139.02, 138.48, 127.90, 122.49, 120.78, 118.93, 118.73, 118.67, 118.59, 113.14, 107.91, 28.04, 25.29; HR-MS (ESI-QTOF): m/z calculated for [M + H]⁺ C₁₉H₁₅FN₈O₂S₂;471.0822; found 471.0824.

4-(4-(((8-fluoro-5-ethyl-5H-[1,2,4]triazino[5,6b]indol-3-yl)thio)methyl-1H-1,2,3-triazol-1-yl)benzene sulfonamide (6g) Yield: 53%; Color: Brown solid; mp: 246–248 °C; ¹H NMR (500 MHz, DMSO-d₆) δ 8.85 (s, 1H), 8.19 (dd, J = 8.1, 2.3 Hz, 1H), 8.10 (d, J = 8.7 Hz, 2H), 7.99 (d, J = 8.7 Hz, 2H), 7.91 (dd, J = 8.9, 4.0 Hz, 1H), 7.66 (td, J = 9.2, 2.4 Hz, 1H), 7.50 (s, 2H), 4.75 (s, 2H), 4.45 (q, J = 7.0 Hz, 2H), 1.34 (t, J = 7.1 Hz, 3H); ¹³C NMR (125 MHz, DMSO-d₆) δ 167.16, 159.85, 146.63, 145.75, 144.26, 139.03, 137.40, 127.91, 122.39, 120.73, 118.97, 118.87, 118.77, 113.24, 108.09, 36.63, 25.33, 13.73; HR-MS (ESI-QTOF): m/z calculated for [M + H]⁺ C₂₀H₁₇FN₈O₂S₂; 485.0978; found 485.0979.

4-(4-(((8-fluoro-5-propyl-5H-[1,2,4]triazino[5,6b]indol-3-yl)thio)methyl-1H-1,2,3-triazol-1-yl)benzene sulfonamide (6h) Yield: 63%; Color: Brown solid; mp: 240–242 °C; ¹H NMR (500 MHz, DMSO) δ 8.84 (s, 1H), 8.18 (dd, J = 8.2, 2.5 Hz, 1H), 8.09 (d, J = 8.8 Hz, 2H), 7.98 (d, J = 8.8 Hz, 2H), 7.89 (dd, J = 9.0, 4.0 Hz, 1H), 7.64 (td, J = 9.2, 2.6 Hz, 1H), 7.50 (s, 2H), 4.74 (s, 2H), 4.35 (t, J = 6.9 Hz, 2H), 1.83–1.74 (m, 2H), 0.78 (t, J = 7.4 Hz, 3H); ¹³C NMR (125 MHz, DMSO) δ 167.3, 159.8, 157.9, 147.1, 145.8, 144.3, 141.1, 139.0, 137.8, 127.9, 122.3, 120.7, 113.4, 108.2, 107.9, 43.2, 25.4, 21.6, 11.5. HR-MS (ESI-QTOF): m/z calculated for [M + H]⁺ C₂₁H₁₉ FN₈O₂S₂ 499.1135; found 499.1153.

4-(4-(((8-fluoro-5-isopropyl-5H-[1,2,4]triazino[5,6b]indol-3-yl)thio)methyl-1H-1,2,3-triazol-1-yl)benzene sulfonamide (6i) Yield: 55%; Color: Brown solid; mp: 235–237 °C; ¹H NMR (500 MHz, DMSO-d₆) δ 8.84 (s, 1H), 8.20 (d, J = 7.8 Hz, 1H), 8.10 (d, J = 8.0 Hz, 2H), 7.98 (d, J = 8.3 Hz, 3H), 7.63 (s, 1H), 7.50 (s, 2H), 5.17 (s, 1H), 4.73 (s, 2H), 1.61 (d, J = 6.1 Hz, 6H); ¹³C NMR (125 MHz, DMSO) δ 166.8, 159.7, 157.8, 146.7, 145.8, 144.2, 141.1, 139.0, 137.0, 127.9, 122.3, 120.7, 119.2, 118.8, 46.9, 25.3, 20.3. HR-MS (ESI-QTOF): m/z calculated for [M + H]⁺ C₂₁H₁₉ FN₈O₂S₂499.1135; found 499.1174.

4-(4-(((8-fluoro-5-allyl-5H-[1,2,4]triazino[5,6b]indol-3-yl)thio)methyl-1H-1,2,3-triazol-1-yl)benzene sulfonamide (6j) Yield: 51%; Color: Brown solid; mp: 245–247 °C; ¹H NMR (500 MHz, DMSO-d₆) δ 8.81 (s, 1H), 8.21 (d, J = 6.7 Hz, 1H), 8.09 (d, J = 8.3 Hz, 2H), 7.99 (d, J = 8.3 Hz, 2H), 7.78 (d, J = 5.0 Hz, 1H), 7.65 (t, J = 8.2 Hz, 1H), 7.50 (s, 2H), 6.06-5.96 (m, 1H), 5.13–5.05 (m, 4H), 4.74 (s, 2H); ¹³C NMR (125 MHz, DMSO) δ 167.4, 159.9, 158.0, 146.9, 145.7, 144.3, 141.3, 139.0, 137.6, 132.1, 127.9, 122.4, 120.8, 119.0, 118.7, 113.6, 108.3, 43.8, 25.3. HR-MS (ESI-QTOF): m/z calculated for [M + H]⁺ C₂₁H₁₇FN₈O₂S₂ 497.0978; found 497.1027.

4-(4-(((8-chloro-5-methyl-5H-[1,2,4]triazino[5,6b]indol-3-yl)thio)methyl-1H-1,2,3-triazol-1-yl)benzene sulfonamide (6k) Yield: 62%; Color: Brown solid; mp: 243–248 °C; ¹H NMR (500 MHz, DMSO) δ 8.87 (s, 1H), 8.39 (s, 1H), 8.10 (d, J = 8.1 Hz, 1H), 7.99 (d, J = 8.1 Hz, 1H), 7.84 (d, J = 5.5 Hz, 1H), 7.51 (s, 1H), 4.77 (s, 1H), 3.86 (s, 1H);¹³C NMR (126 MHz, CDCl₃) δ 172.04, 151.12, 150.44, 148.97, 145.57, 144.27, 143.71, 135.73, 132.74, 127.25, 126.31, 125.47, 124.14, 118.20, 109.43, 41.18, 29.99. HR-MS (ESI-QTOF): m/z calculated for [M + H]⁺ C₁₉H₁₅ClN₈O₂S₂ 487.0526; found 487.0530.

4-(4-(((8-chloro-5-ethyl-5H-[1,2,4]triazino[5,6b]indol-3-yl)thio)methyl-1H-1,2,3-triazol-1-yl)benzene sulfonamide (6l) Yield: 57%; color: Brown solid; mp: 252–256 °C; ¹H NMR (500 MHz, DMSO) δ 8.86 (s, 1H), 8.40 (s, 1H), 8.10 (d, J = 7.3 Hz, 1H), 7.99 (d, J = 7.0 Hz, 1H), 7.91 (d, J = 7.6 Hz, 1H), 7.82 (s, 1H), 7.51 (s, 1H), 4.75 (s, 1H), 4.44 (d, J = 5.7 Hz, 1H), 1.29 (d, J = 47.6 Hz, 1H); ¹³C NMR (126 MHz, CDCl₃) δ 172.04, 151.80, 150.30, 149.06, 145.56, 145.35, 143.81, 132.66, 132.51, 127.17, 126.07, 125.52, 123.87, 122.79, 118.21, 53.69, 32.67, 29.99.HR-MS (ESI-QTOF): m/z calculated for [M + H]⁺ C₂₀H₁₈ClN₈O₂S₂ 501.0682; found 501.0694.

4-(4-(((8-chloro-5-propyl-5H-[1,2,4]triazino[5,6b]indol-3-yl)thio)methyl-1H-1,2,3-triazol-1-yl)benzene sulfonamide (6m) Yield: 63%; Color: Brown solid; mp: 257–262 °C; ¹H NMR (500 MHz, DMSO) δ 8.85 (s, 1H), 8.39 (s, 1H), 8.10 (d, J = 7.1 Hz, 1H), 7.99 (d, J = 7.0 Hz, 1H), 7.90 (d, J = 7.9 Hz, 1H), 7.80 (d, J = 7.3 Hz, 1H), 7.51 (s, 1H), 4.74 (s, 1H), 4.35 (s, 1H), 1.78 (d, J = 6.1 Hz, 1H), 0.79 (s, 2H); ¹³C NMR (126 MHz, DMSO) δ 167.46, 146.95, 145.67, 144.25, 140.63, 139.99, 138.86, 131.10, 127.90, 127.75, 122.34, 121.44, 120.70, 119.24, 113.68, 43.18, 25.42, 21.48, 11.56.HR-MS (ESI-QTOF): m/z calculated for [M + H]⁺ C₂₁H₂₀ClN₈O₂S₂ 515.0839; found 515.0839.

4-(4-(((8-chloro-5-isopropyl-5H-[1,2,4]triazino[5,6b]indol-3-yl)thio)methyl-1H-1,2,3-triazol-1-yl)benzene sulfonamide (6n) Yield: 56%; Color: Brown solid; mp: 263–265 °C; ¹H NMR (500 MHz, DMSO) δ 8.85 (s, 1H), 8.40 (s, 1H), 8.10 (d, J = 8.6 Hz, 1H), 7.99 (d, J = 8.6 Hz, 1H), 7.95 (d, J = 8.9 Hz, 1H), 7.80–7.75 (m, 1H), 7.50 (s, 1H), 5.16 (dt, J = 13.6, 6.7 Hz, 1H), 4.73 (s, 1H), 1.61 (d, J = 6.8 Hz, 1H);¹³C NMR (126 MHz, DMSO) δ 166.92, 146.52, 145.82, 144.26, 140.54, 139.11, 139.01, 130.89, 127.92, 127.53, 122.36, 121.33, 120.73, 119.71, 114.31, 47.03, 30.95, 25.38, 20.33.HR-MS (ESI-QTOF): m/z calculated for [M + H]⁺ C₂₁H₂₀ClN₈O₂S₂ 515.0839; found 515.0856.

4-(4-(((8-chloro-5-allyl-5H-[1,2,4]triazino[5,6b]indol-3-yl)thio)methyl-1H-1,2,3-triazol-1-yl)benzene sulfonamide (6o) Yield: 67%; Color: Brown solid; mp: 261–264 °C; ¹H NMR (500 MHz, TFE) δ 8.81 (s, 1H), 8.40 (s, 1H), 8.08 (d, J = 8.7 Hz, 1H), 7.98 (d, J = 8.7 Hz, 1H), 7.81–7.75 (m, 1H), 7.50 (s, 1H), 5.99 (ddd, J = 22.0, 10.3, 5.0 Hz, 1H), 5.13–5.02 (m, 2H), 4.73 (s, 1H);¹³C NMR (126 MHz, DMSO) δ 167.54, 146.67, 145.70, 144.27, 140.68, 139.65, 138.99, 131.82, 131.04, 127.89, 122.51, 121.44, 120.76, 119.42, 117.79, 113.91, 43.48, 31.33, 25.06.HR-MS (ESI-QTOF): m/z calculated for [M + H]⁺ C₂₁H₁₇ClN₈O₂S₂ 513.0682; found 513.069.

4.2. CA Inhibition

An SX.18 V-R Applied Photophysics (Oxford, UK) stopped-flow instrument was used to assay the catalytic inhibition of various CA enzymes [32]. Phenol Red (at a concentration of 0.2 mM) was used as an indicator, working at maximum absorbance of 557 nm, with 10 mM Hepes (pH 7.4) buffer, 0.1 M Na₂SO₄ (for maintaining constant ionic strength; these anions are not inhibitory in the used concentration), following the CA-catalyzed CO₂ hydration reaction for a period of 5–10 s. Saturated CO₂ solutions in water at 25 °C were used as a substrate. Stock solutions of the inhibitors were prepared at a concentration of 10 mM (in DMSO/Water 1:1, v/v), and dilutions up to 0.01 nM were prepared using the assay buffer mentioned above. At least 7 different inhibitor concentrations were used for measuring the inhibition constant. Inhibitor (I) and enzyme (E) solutions were pre-incubated together for 10 min at room temperature prior to the assay, in order to allow for the formation of the E–I complex. IC₅₀-s values were calculated from the enzyme activity (as k_cat, see Table 2) with respect to the inhibitor concentration. Triplicate experiments were done for each inhibitor concentration, and the values reported in this paper are the mean of such results. The inhibition constants were obtained by non-linear least-square methods, using the Cheng–Prusoff equation, as reported earlier, and represent the mean from at least three different determinations: K_I = IC₅₀/[(1 + [S]/K_M)]. K_M values for all enzymes were reported earlier (see Table 2) by us [26,27,28,29,30,31]; [S] is the CO₂ concentration at which the experiments were performed. All CA isozymes used here were recombinant proteins obtained as reported earlier by our group [33,34,35,36,37,38].