Diversely substituted sulfamides for fragment-based drug discovery of carbonic anhydrase inhibitors: synthesis and inhibitory profile

Abstract

A series of sulfamide fragments has been synthesised and investigated for human carbonic anhydrase inhibition. One of the fragments showing greater selectivity for cancer-related isoforms hCA IX and XII was co-crystalized with hCA II showing significant potential for fragment periphery evolution via fragment growth and linking. These opportunities will be identified in the future via the screening of this fragment structure for co-operative carbonic anhydrase binding with other structurally diverse fragments.

Graphical Abstract

Keywords:

• Carbonic anhydrase
• zinc-binding groups
• sulfamides
• co-operative fragment screening
• solubility

1. Introduction

The carbonic anhydrase (CA) family of Zn(II) metalloenzymes (EC 4.2.1.1) catalyses the reversible hydration of carbon dioxide to bicarbonate anion, a fundamental reaction that controls physiological processes requiring pH control as well as ion transport and fluid secretion¹. Hyperactivity of specific CA isoforms² in various disease states makes these enzymes potential (and sometimes already validated) targets for therapeutic intervention with small molecule carbonic anhydrase inhibitors (CAIs)³.

Clinically validated applications of CAIs currently include, among other diseases, the treatment of glaucoma⁴, idiopathic intracranial hypertension⁵, high-altitude sickness⁶, congestive heart failure⁷, peptic ulcers⁸ and epilepsy⁹. Another important potential application of CAIs (specifically, of related human (h) CA IX and XII isoforms¹⁰) is in neoplastic therapy¹¹. The current state of development in this field is underscored by the hCA IX-selective drug SLC-0111¹² which is undergoing phase 1 b clinical study for tumours overexpressing hCA IX¹³ and non-selective inhibitor E7070 (indisulam) developed by Eisai Co., Ltd. which successfully completed phase II clinical study¹⁴. The highly promising application of CAIs as antibacterials is based on the premise of selective inhibition of microbial CAs (crucial for the survival of bacteria) without affecting the CAs in the same concentration range¹⁵. Thus, CA inhibition from such microorganisms as Vibrio cholerae¹⁶, Burkholderia pseudomallei¹⁷, Mycobacterium tuberculosis¹⁸, Salmonella enterica¹⁹, Helicobacter pylori²⁰, Escherichia coli²¹ and many others¹⁵.

Considering the plethora of validated and potential therapeutic applications of CAIs, the discovery of new chemotypes endowed with CA inhibitory activity will continue to be a significant aim. CA contains a Zn²⁺ ion in its active site which mandates the zinc-binding nature of active site targeting pharmacophoric groups which can be employed in the design of new CAIs. Indeed, most of the clinically investigated (SLC-0111 and E7070) and used (e.g. acetazolamide, methazolamide, dorzolamide, brinzolamide and zonisamide) are primary sulphonamides (CSO₂NH₂) in which the sulphonamide group anchors to the prosthetic zinc ion and the molecular periphery defines the potency and isoform selectivity of these CAIs² (Figure 1).

Figure 1. CAIs in clinical development and clinical use.

Many other zinc-binding motifs have been implicated as warheads in the CAI design²². Among them, sulfamides (NSO₂NH₂) appears as an attractive alternative to the frequently studied sulphonamides. Indeed, sulphonamides are expected to have greater polarity and solubility compared to sulphonamides, due to the presence of an additional nitrogen atom. Moreover, primary sulphonamide group is found in such drugs as anticancer epacadostat²³ and gastric ulcer medication famotidine²⁴, both of which (epacadostat²⁵ and famotidine²⁶) were also found to inhibit various CA isoforms (Figure 2).

Figure 2. Examples of clinically used sulfamide drugs.

The discovery of novel sulfamide CAIs would traditionally entail synthesis of structurally diverse libraries of compounds and their screening against an isoform panel of CAs. We have recently validated an approach²⁷ to the discovery of new sulphonamide CAIs based on the simultaneous screening of a diverse set of chemical fragments (i.e. small, Mw <∼250 and polar, cLopP< 3.0²⁸) along with sulphonamide zink-binding wasread (in particular, benzenesulfonamide or BSA). This led not only to the discovery of over 100 fragment hits which potentiated the binding of BSA but also to rediscovery of BSA-based CAIs with the molecular periphery replicating the fragment co-binders. In our intent aimed at the discovery of novel sulfamide CAIs, we decided to take a similar approach. Realisation of such an approach would require synthesis of a library of fragment-like sulfamides and profiling them against a panel of human CAs (in this case, anti-glaucoma target hCA II, two membrane-associated cancer-related targets hCA IX and XII and the usual cytosolic off-target hCA I). In this work, we aimed at the realisation of this approach and selection of a suitable sulfamide zinc-binding warhead for fragment-based discovery of novel sulfamide-type CAIs, a chemotype much less studied in the context of CA inhibition compared to sulphonamides²⁹. Moreover, in this study, we were looking to identify: fragments that do not display apparently high intrinsic selectivity towards specific CA isoforms (mindful that such a selectivity will be gained in the future from co-operative screening of specific “tail” fragments³⁰) and yet would show a tendency to inhibit cancer-related isoforms hCA IX and XII over cytosolic hCA I and II. Of particular interest would be fragments that do not display a pronounce potency against CA isoforms of interest, ideally in the 10⁻⁷M range of K_i values (so that the future contribution from co-operative fragment binding would be more pronounced). Specific emphasis was put on conformationally constrained fragments which would be structurally close to the classical BSA zinc-binding motif and would co-crystallize with any of the isoforms (e.g. the most readily available hCA II) to further guide further fragment evolution via growing, linking and merging³¹. Herein, we report on the successful realisation of this strategy.

2. Results and discussion

Seventeen non-symmetrically substituted primary sulfamides were synthesised from inorganic sulfamide 1 via direct nucleophilic substitution at the sulphur atom, via the thermally promoted reaction in dioxane with a four-fold excess of 1, conducted at 110 °C over 48 h³². The yields of the resulting compounds 2a–q were generally modest to good (Scheme 1).

Scheme 1. Synthesis of unsymmetrically substituted primary sulfamides 2a–q.

The same thermally promoted protocol, not unexpectedly, did not work for less reactive (hetero)aromatic amines. Thus, an alternative approach was taken³³. Instead of sulfamide, commercially available chlorosulfonyl isocyanate 3 dissolved in dichloromethane at 0 °C was reacted with 1 equiv of tert-butanol to give the Boc-protected amino-sulfonyl-chloride (4), which was subsequently added slowly to a solution of 1 equiv of the respective hetero(aromatic) amine in the presence of 2 equiv of triethylamine in dichloromethane at 0 °C. In this case, again, the yields of unsymmetrically substituted primary sulfamides 2r–w were modest to good over two steps (Scheme 2).

Scheme 2. Synthesis of unsymmetrically (hetero)aromatic amine-substituted sulfamides 2r–w.

From the physicochemical data summarised for fragments 2a–w in Table 1, one can appreciate their being distinctly fragment-like (M_w = 165.2 … 257.4, cLogP = −1.87 … 1.71). Furthermore, the inhibitory data reveal the absence of apparent isoform selectivity displayed by these fragments which is perfectly in line with the limited size of the molecular periphery (typically responsible for making additional contacts with the protein and ensuring higher potency and isoform selectivity). With our initial focus on the cancer-related, membrane-bound CA isoforms hCA IX and XII compounds displaying greater selectivity towards these isoforms against cytosolic hCA I and II (structural homologs of each other 2r and 2v) received our priority attention.

Table 1. Calculated physicochemical properties and hCA I, II, IX and XII inhibitory profile of compounds 2a–w.

Compound	Mw	cLogP^a	Ki (nM)^b
			hCA I	hCA II	hCA IX	hCA XII
2a	180.2	−1.87	75.6	42.7	54.2	25.3
2b	179.2	−1.01	64.2	32.1	58.6	52.5
2c	204.2	0.42	126.2	70.5	30.7	51.7
2d	226.3	1.22	120.7	42.6	36.8	59.7
2e	180.2	−0.77	38.4	63.4	92.7	98.0
2f	208.2	−0.65	52.1	69.8	78.2	83.6
2g	193.3	−0.72	89.7	52.1	63.2	38.4
2h	240.3	1.57	359.5	120.4	58.2	96.7
2i	257.4	0.53	28.4	39.8	65.1	19.7
2j	187.2	−0.98	98.5	90.2	29.5	16.5
2k	192.3	0.15	22.6	48.2	73.2	66.8
2l	209.3	−0.12	59.6	92.3	126.5	72.5
2m	236.3	0.11	62.8	42.8	58.1	25.9
2n	247.4	0.43	497.3	99.3	72.5	35.1
2o	166.2	−1.05	59.2	66.9	82.1	100.9
2p	207.3	−1.39	88.2	59.4	46.2	40.2
2q	165.2	−1.60	68.9	52.9	78.2	100.2
2r	212.3	1.12	867.9	315.7	94.7	116.2
2s	174.2	−1.27	29.6	59.4	68.0	39.3
2t	212.3	1.31	231.4	89.5	34.2	114.2
2u	222.3	1.71	160.1	56.7	25.4	72.3
2v	198.2	0.79	723.5	472.2	102.5	95.7
2w	208.2	0.81	45.7	76.3	38.5	224.0
Acetazolamide			250	125	25	5.7

^aCaclculated using Molinspiration Chemoinformatics [34].

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

After much experimentation, compound 2v was co-crystallized with recombinant hCA II and its structure was resolved (Figure 3).

Figure 3. Co-crystal structure of fragment sulfamide 2v with hCA II (PDB code 7QSI).

As one can see from the crystal structure of 2v with hCA II isoform, the small (Mw = 198.2) N-(aminosulfonyl)indoline fragment displayed two finding poses within the hCA II active site. In both poses, the sulfonylamino groups are anchored to the prosthetic zinc ion (displayed as a grey sphere). The validity of the two binding poses signifies the fact that the binding of 2v leaves a significant room for the periphery growth around this fragment and makes it a highly suitable candidate for co-operative screening with other structurally diverse fragments in order to identify the starting points for the structural evolution of this fragment.

3. Materials and methods

3.1. Chemical synthesis – general

NMR spectra were recorded on a Bruker Avance III 400 spectrometer (¹H: 400.13 MHz; ¹³С: 100.61 MHz; chemical shifts are reported as parts per million (δ, ppm); the residual solvent peaks were used as internal standards: δ 7.28 ¹H in CDCl₃, δ 77.02 ppm for ¹³C in CDCl₃; multiplicities are abbreviated as follows: s = singlet, d = doublet, t = triplet, q = quartette, m = multiplet, br = broad; coupling constants, J, are reported in Hz. Mass spectra were recorded on a Bruker microTOF spectrometer (ESI ionization).

3.1.1. General procedure for the synthesis of compounds 2a–q

A mixture of corresponding amine (2 mmol) and sulphuric diamide (8 mmol, 768 mg) in dry 1.4-dioxane (4 mL) was stirred at 110 ^oC for 48 h. If amine was in the form of hydrochloride salt, an additional equivalent of triethylamine was added. CH₂Cl₂ (5 mL) was added and the resulting precipitate was filtered off, washed with ethyl acetate (5 mL). The filtrate and the washings were combined and solvent was evaporated under reduced pressure. The crude product was purified by column chromatography on silica gel using CH₂Cl₂:2-propanol 20:1–10:1 gradient as eluent (R_f values are given for solvent system indicated).

3.1.2. 4-Hydroxypiperidine-1-sulphonamide (2a)

R_f = 0.30 (EtOAc); ¹H NMR (400 MHz, DMSO-d₆) δ = 6.66 (s, 2H), 4.69 (d, J = 3.9 Hz, 1H), 3.58 (dq, J = 7.8, 4.0 Hz, 1H), 3.22 (ddd, J = 11.0, 6.5, 3.8 Hz, 2H), 2.74 (ddd, J = 12.0, 9.0, 3.3 Hz, 2H), 1.77 (ddt, J = 13.7, 7.2, 3.6 Hz, 2H), 1.46 (dtd, J = 12.5, 8.6, 3.7 Hz, 2H) ppm; ¹³C NMR (101 MHz, DMSO-d₆, Dept) δ = 65.049 (C-OH), 43.80 and 33.47 (2 CH₂) ppm; HRMS (ESI) C₅H₁₁N₂O₃S⁺ m/z: [M – H]⁻ 179.0492 (calc 179.0485).

3.1.3. 4-Methylpiperazine-1-sulphonamide (2 b)

R_f = 0.61 (MeOH); ¹H NMR (400 MHz, DMSO-d₆) δ = 6.76 (s, 2H), 2.95 (t, J = 4.9 Hz, 4H), 2.38 (t, J = 5.0 Hz, 4H), 2.19 (s, 3H) ppm; ¹³C NMR (101 MHz, DMSO-d₆, DEPT) δ = 54.08 (CH₂), 46.14 (CH₂), 45.89 (CH₃) ppm; HRMS (ESI) C₅H₁₂N₃O₂S+ m/z: [M − H]⁻ 178.0644 (calc 178.0645).

3.1.4. N-(4-Fluorobenzyl)sulfamide (2c)

¹H NMR (400 MHz, DMSO-d₆) δ = 7.39 (dd, J = 8.5, 5.7 Hz, 2H), 7.15 (t, J = 8.9 Hz, 2H), 7.07 (t, J = 6.5 Hz, 1H), 6.63 (s, 2H), 4.07 (d, J = 6.5 Hz, 2H) ppm; ¹³C NMR (101 MHz, DMSO-d₆, DEPT) δ = 161.70 (d, J = 242.2 Hz), 135.39 (d, J = 2.9 Hz), 130.05 (d, J = 8.1 Hz, CH), 115.29 (d, J = 21.3 Hz, CH), 45.77 (s, CH₂) ppm; ¹⁹F NMR (470 MHz, DMSO-d₆) δ = −116.15 ppm; HRMS (ESI) C₇H₉FN₂NaO₂S⁺ m/z: [M + Na]⁺ 227.0272 (calc 227.0261).

3.1.5. N-(1,2,3,4-Tetrahydronaphthalen-1-yl)sulfamide (2d)

R_f = 0.80 (CH₂Cl₂-CH₃OH 4:1); ¹H NMR (400 MHz, DMSO-d₆) δ = 7.57 − 7.50 (m, 1H), 7.19 − 7.11 (m, 2H), 7.08 − 7.02 (m, 1H), 6.96 (d, J = 8.9 Hz, 1H), 6.65 (s, 2H), 4.39 (td, J = 8.0, 5.2 Hz, 1H), 2.79 − 2.57 (m, 2H), 2.11 − 2.00 (m, 1H), 1.96 − 1.80 (m, 2H), 1.74 − 1.60 (m, 1H) ppm;

¹³C NMR (101 MHz, DMSO-d₆, DEPT) δ = 138.15, 137.41, 129.43, 128.94, 127.07, 126.02, 51.32 (CH), 30.89, 29.23 and 20.39 (3CH₂) ppm; HRMS (ESI) C₁₀H₁₄N₂NaOS⁺ m/z: [M + Na]⁺ 249.0671 (calc 249.0668).

3.1.6. N-((tetrahydrofuran-2-yl)methyl)sulfamide (2e)

R_f = 0.59 (DCM:i-PrOH = 9:1); ¹H NMR (400 MHz, CDCl₃) δ = 4.94 (br. t, J = 6.0 Hz, 1H), 4.88 (br. s, 2H), 4.08 (ddt, J = 10.5, 7.3, 3.4 Hz, 1H), 3.88 (dt, J = 8.3, 6.7 Hz, 1H), 3.82 − 3.73 (m, 1H), 3.28 (ddd, J = 13.3, 6.4, 3.2 Hz, 1H), 3.14 (ddd, J = 13.4, 7.8, 5.8 Hz, 1H), 2.05 − 1.85 (m, 3H), 1.68 − 1.58 (m, 1H) ppm; ¹³C NMR (101 MHz, CDCl₃, DEPT) δ = 77.65 (CH), 68.21, 47.52, 28.61 and 25.76 (4 CH₂) ppm; HRMS (ESI) C₅H₁₂N₂NaO₃S+ m/z: [M + Na]⁺ 203.0464 (calc 203.0461).

3.1.7. Methyl sulfamoylprolinate (2f)

R_f = 0.75 (EtOAc); ¹H NMR (400 MHz, DMSO-d₆) = 6.82 (s, 2H), 4.18 (dd, J = 8.9, 4.1 Hz, 1H), 3.27 (dq, J = 6.2, 3.5, 2.9 Hz, 2H), 2.18 − 2.07 (m, 1H), 1.92 − 1.80 (m, 3H) ppm; ¹³C NMR (101 MHz, DMSO-d₆, DEPT) δ = 173.24, 60.47 and 52.28 (CH and CH₃), 49.11, 31.06 and 24.92 (3 CH₂) ppm; HRMS (ESI) C₆H₁₃N₂O₄S⁺m/z: [M + H]⁺ 209.0599 (calc 209.0591).

3.1.8. (1-Methylpiperidin-4-yl)sulfamide (2g)

R_f = 0,13 (MeOH); ¹H NMR (400 MHz, DMSO-d₆) δ = 6.47 (s, 3H), 3.01 (tt, J = 10.6, 4.2 Hz, 1H), 2.68 (d, J = 11.9 Hz, 2H), 2.12 (s, 3H), 1.85 (qd, 11.9, 2.0 Hz, 4H), 1.42 (qd, J = 12.5, 4.2 Hz, 2H) ppm; ¹³C NMR (101 MHz, DMSO-d₆) δ = 54.78, 50.36, 46.41, 32.86 ppm; HRMS (ESI) C₆H₁₆N₃O₂S+ m/z: [M + H]⁺ 194.0955 (calc 194.0958).

3.1.9. 4-Phenylpiperidine-1-sulphonamide (2 h)

R_f = 0.86 (EtOAc); ¹H NMR (400 MHz, DMSO-d₆) δ = 7.34 − 7.25 (m, 4H), 7.21 (td, J = 6.8, 1.7 Hz, 1H), 6.75 (br.s, 2H), 3.59 (br.d, J = 12.1 Hz, 2H), 2.63 (td, J = 12.2, 2.7 Hz, 2H), 2.59 − 2.53 (m, 1H), 1.86 (br.d, J = 12.2 Hz, 2H), 1.69 (qd, J = 12.5, 4.0 Hz, 2H) ppm;¹³C NMR (101 MHz, DMSO-d₆, DEPT) δ = 145.97, 128.84 (C_ArH), 127.20 (C_ArH), 126.67 (C_ArH), 46.96 (CH₂), 41.45 (CH), 32.45 (CH₂) ppm; HRMS (ESI) C₁₁H₁₆N₂NaO₂S⁺ m/z: [M + Na]⁺ 263.0830 (calc 263.0825).

3.1.10. Benzyl (2-(dimethylamino)ethyl)sulfamide (2i)

R_f = 0.58 (MeOH); ¹H NMR (400 MHz, DMSO-d₆) δ = 7.40 − 7.33 (m, 4H), 7.31 − 7.26 (m, 1H), 6.91 (s, 2H), 4.26 (s, 2H), 3.09 (t, J = 6.8 Hz, 2H), 2.30 (t, J = 6.8 Hz, 2H), 2.07 (s, 6H) ppm; ¹³C NMR (101 MHz, DMSO-d₆, DEPT) δ = 138.06, 128.76, 128.56 and 127.74 (3 C_ArH), 57.05, 51.41 and 45.72 (3 CH₂), 45.52 (CH₃) ppm; HRMS (ESI) C₁₁H₂₀N₃O₂S⁺ m/z: [M + H]⁺ 258.1276 (calc 258.1271).

3.1.11. (Pyridin-3-ylmethyl)sulfamide (2j)

R_f = 0.90 (MeOH); ¹H NMR (400 MHz, CD₃CN) δ = 8.55 (d, J = 2.2 Hz, 1H), 8.48 (dd, J = 4.8, 1.6 Hz, 1H), 7.76 (dt, J = 7.9, 1.9 Hz, 1H), 7.33 (dd, J = 7.8, 4.8 Hz, 1H), 5.62 (br. s, 1H), 5.31 (br. s, 2H), 4.20 (d, J = 6.3 Hz, 2H) ppm; ¹³C NMR (101 MHz, CD₃CN) δ = 149.23, 148.59, 135.72, 133.71, 123.46, 44.32 ppm; HRMS (ESI) C₆H₉N₃NaO₂S⁺ m/z: [M + Na]⁺ 210.0308 (calc 210.0308).

3.1.12. (Thiophen-2-ylmethyl) sulfamide (2k)

R_f = 0.49 (DCM:i-PrOH = 20:1); ¹H NMR (400 MHz, Acetone-d₆) = 7.37 (dd, J = 5.2, 1.2 Hz, 1H), 7.07 (dq, J = 3.3, 1.1 Hz, 1H), 6.97 (dd, J = 5.1, 3.5 Hz, 1H), 6.20 (s, 1H), 5.99 (s, 2H), 4.45 (dd, J = 6.4, 1.0 Hz, 2H) ppm; ¹³C NMR (101 MHz, Acetone-d₆, DEPT) δ = 141.21 (>C=), 126.62, 125.80, and 125.08 (3 C_ArH), 41.97 (CH₂) ppm; HRMS (ESI) C₅H₈N₂NaO₂S₂⁺ m/z: [M + Na]⁺ 214.9919 and 216.9877 (calc 214.9919 and 216.9878).

3.1.13. (2-(Diethylamino)ethyl)(methyl)sulfamide (2 l)

R_f = 0.58 (MeOH); ¹H NMR (400 MHz, DMSO-d₆) = 6.73 (s, 2H), 3.05 (t, J = 6.8 Hz, 2H), 2.68 (s, 2H), 2.55 (t, J = 6.8 Hz, 2H), 2.49 (q, J = 7.1 Hz, 4H), 0.96 (t, J = 7.1 Hz, 6H) ppm; ¹³C NMR (101 MHz, DMSO-d₆, DEPT) δ = 50.53, 48.57 and 46.95(3 CH₂), 35.57 and 12.01 (2 CH₃) ppm; HRMS (ESI) C₇H₂₀N₃O₂S⁺ m/z: [M + H]⁺ 210.1274 (calc 210.1271).

3.1.14. Ethyl 1-sulfamoylpiperidine-4-carboxylate (2 m)

R_f = 0.81 (EtOAc); ¹H NMR (400 MHz, DMSO-d₆) δ = 6.71 (s, 2H), 4.07 (q, J = 7.1 Hz, 2H), 3.36 (dt, J = 12.3, 3.8 Hz, 2H), 2.62 (td, J = 11.6, 2.8 Hz, 2H), 2.42 (tt, J = 10.7, 4.0 Hz, 1H), 1.90 (br. dd, J = 13.5, 3.8 Hz, 2H), 1.60 (qd, J = 11.1, 3.8 Hz, 2H), 1.18 (t, J = 7.1 Hz, 3H) ppm; ¹³C NMR (101 MHz, DMSO-d₆) δ = 174.25, 60.44, 45.55, 27.47, 14.54 ppm; HRMS (ESI) C₈H₁₆N₂NaO₄S⁺ m/z: [M + Na]⁺ 259.0722 (calc 259.0723).

3.1.15. [1,4'-Bipiperidine]-1'-sulphonamide (2n)

R_f = 0.24 (MeOH); ¹H NMR (400 MHz, DMSO-d₆) δ = 6.68 (br.s, 2H), 3.48 (br.d, J = 12.4 Hz, 2H), 2.53 − 2.41 (m, 3H), 2.44 (t, J = 5.1 Hz, 3H), 2.24 (tt, J = 11.3, 3.6 Hz, 1H), 1.76 (br.d, J = 11.7 Hz, 2H), 1.52 − 1.43 (m, 6H), 1.38 (q, J = 5.9 Hz, 2H) ppm; ¹³C NMR (101 MHz, DMSO-d₆, DEPT) δ = 61.56, 50.16, 46.22, 27.21, 26.52, 25.01; HRMS (ESI) C₁₀H₂₂N₃O₂S⁺ m/z: [M + H]⁺ 248.1431 (calc 248.1428).

3.1.16. Morpholine-4-sulphonamide (2o)

R_f = 0.56 (EtOAc); ¹H NMR (400 MHz, DMSO-d₆) δ = 6.82 (s, 2H), 3.86 − 3.50 (m, 4H), 3.02 − 2.77 (m, 4H) ppm; ¹³C NMR (101 MHz, DMSO-d₆) δ = 65.73, 46.43 ppm; HRMS (ESI) C₄H₉N₂O₃S⁺ m/z: [M − H]⁻ 165.0326 (calc 165.0339)

3.1.17. 1-Sulfamoylpiperidine-4-carboxamide (2p)

R_f = 0.61 (EtOAc:MeOH 3:1); ¹H NMR (400 MHz, DMSO-d₆) δ = 7.27 (s, 1H), 6.82 (s, 1H), 6.79 − 6.44 (m, 2H), 3.52 − 3.39 (m, 2H), 2.21 − 2.05 (m, 1H), 1.78 (d, J = 13.1 Hz, 2H), 1.57 (t, J = 12.7 Hz, 2H) ppm; ¹³C NMR (101 MHz, DMSO-d₆) δ = 176.30, 45.96, 41.12, 28.07 ppm; HRMS (ESI) C₆H₁₃N₃NaO₃S⁺ m/z: [M + Na]⁺ 230.0565 (calc 230.0570)

3.1.18. Piperazine-1-sulphonamide hydrochloride (2q)

¹H NMR (400 MHz, DMSO-d₆) δ = 9.60 (s, 2H), 7.08 (s, 2H), 3.21 (dd, J = 7.1, 3.6 Hz, 4H), 3.14 (dd, J = 7.0, 3.7 Hz, 4H) ppm; ¹³C NMR (101 MHz, DMSO-d₆) δ = 43.27, 42.37 ppm; HRMS (ESI) C₄H₁₂N₃O₂S⁺ m/z: [M + H]⁻ 166.0649 (calc 166.0645).

3.1.19. 2-Methylindoline-1-sulphonamide (2r)

R_f = 0.81 (n-Hexane:EtOAc 1:1); ¹H NMR (400 MHz, CDCl₃) = 7.40 (d, J = 8.2 Hz, 1H), 7.19 (dt, J = 7.7, 3.7 Hz, 2H), 7.04 (t, J = 7.4 Hz, 1H), 4.46 (s br., 1H), 4.07 (s br., 2H), 3.52 − 3.43 (m, 2H), 2.67 (dd, J = 15.9, 3.5 Hz, 1H), 1.43 (d, J = 6.1 Hz, 3H) ppm; ¹³C NMR (101 MHz, CDCl₃) δ = 141.32, 131.15, 127.79, 125.40, 124.10, 115.77, 59.19, 36.64, 22.63 ppm; HRMS (ESI) C₉H₁₂N₂NaO₂S⁺ m/z: [M + Na]⁺ 235.0515 (calc 235.0512).

3.1.20. N-(Pyrimidin-2-yl)sulfamide (2s)

¹H NMR (400 MHz, DMSO-d₆) = 8.56 (d, J = 4.8 Hz, 4H), 7.06 (t, J = 4.9 Hz, 2H) ppm; ¹³C NMR (101 MHz, DMSO-d₆) δ = 158.73, 158.26, 115.28 ppm; HRMS (ESI) C₄H₆N₄NaO₂S⁺ m/z: [M + Na]⁺ 197.1668 (calc 197.1672)

3.1.21. 3,4-Dihydroquinoline-1(2H)-sulphonamide (2t)

R_f = 0.55 (n-Hexane:EtOAc 3:1); ¹H NMR (400 MHz, CDCl₃) δ = 7.67 (d, J = 8.2 Hz, 1H), 7.17 (t, J = 7.6 Hz, 1H), 7.14 − 7.02 (m, 2H), 4.56 (s, 2H), 3.87 − 3.53 (m, 2H), 2.86 (t, J = 6.6 Hz, 2H), 2.06 (p, J = 6.5 Hz, 2H) ppm; ¹³C NMR (400 MHz, CDCl₃, DEPT) δ = 137.20, 129.92, 129.53, 126.60, 124.65 and 123.61 (4 C_ArH), 47.28, 26.94 and 21.87 (3 CH₂) ppm; HRMS (ESI) C₉H₁₂N₂NaO₂S⁺ m/z: [M + Na]⁺ 235.05110 (calc 235.0511)

3.1.22. N-(Naphthalen-1-yl)sulfamide (2 u)

R_f = 0.44 (n-Hexane:EtOAc 1:1); ¹H NMR (400 MHz, DMSO-d₆) δ = 9.27 (s, 1H), 8.35 − 8.25 (m, 1H), 7.96 − 7.87 (m, 1H), 7.75 (d, J = 8.1 Hz, 1H), 7.59 (dd, J = 7.6, 1.2 Hz, 1H), 7.55 − 7.47 (m, 3H), 7.00 (s, 2H) ppm; ¹³C NMR (101 MHz, DMSO-d₆, Dept) δ = 134.74, 134.36, 129.17, 128.26, 126.43, 126.10, 125.49, 124.10, 121.57 ppm; HRMS (ESI) C₁₀H₁₀N₂NaO2S⁺ m/z: [M + Na]⁺ 245.0359 (calc 245.0356)

3.1.23. Indoline-1-sulphonamide (2v)

R_f = 0.77 (EtOAc); ¹H NMR (400 MHz, DMSO-d₆) = 7.28 (d, J = 8.0 Hz, 1H), 7.25 − 7.20 (m, 1H), 7.23 (s, 2H), 7.16 (t, J = 7.7 Hz, 1H), 6.96 (t, J = 7.4 Hz, 1H), 3.80 (t, J = 8.4 Hz, 2H), 3.05 (t, J = 8.4 Hz, 2H) ppm; ¹³C NMR (101 MHz, DMSO-d₆, DEPT) δ = 143.51 and 131.99 (2 >C_Ar=), 127.63, 125.40, 122.83 and 114.21 (4 C_ArH), 50.39 (CH₂), 27.76 (CH₂) ppm; HRMS (ESI) C₈H₁₀N₂NaO₂S+ m/z: [M + Na]⁺ 221.0357 (calc 221.0355)

3.1.24. N-(2,4-Difluorophenyl)sulfamide (2w)

¹H NMR (400 MHz, CDCl₃) δ = 7.40 (d, J = 8.2 Hz, 1H), 7.19 (dt, J = 7.7, 3.7 Hz, 2H), 7.04 (t, J = 7.4 Hz, 1H), 4.46 (s br., 1H), 4.07 (s br., 2H), 3.52 − 3.43 (m, 2H), 2.67 (dd, J = 15.9, 3.5 Hz, 1H), 1.43 (d, J = 6.1 Hz, 3H) ppm; ¹³C NMR (101 MHz, CDCl₃) δ = 160.29 (dd, J = 248.7, 11.3 Hz), 154.88 (dd, J = 247.7, 12.1 Hz), 125.60 (dd, J = 9.6, 1.4 Hz, C_ArH), 121.02 (dd, J = 12.5, 3.7 Hz), 111.97 (dd, J = 22.3, 3.8 Hz), 104.40 (dd, J = 26.7, 23.7 Hz) ppm; ¹⁹F NMR (376 MHz, CDCl₃) δ = −111.98, −123.53 ppm; HRMS (ESI) C₆H₆F₂N₂NaO₂S+ m/z: [M + Na]⁺ 231.0010 (calc 231.0011)

3.2. Carbonic anhydrase inhibition testing

An Applied Photophysics stopped-flow instrument has been used for assaying the CA catalysed CO₂ hydration activity³⁵. Phenol red (at a concentration of 0.2 mM) has been used as indicator, working at the absorbance maximum of 557 nm, with 20 mM Hepes (pH 7.5) as buffer, and 20 mM Na₂SO₄ (for maintaining constant the ionic strength), following the initial rates of the CA-catalysed CO₂ hydration reaction for a period of 10–100 s. The CO₂ concentrations ranged from 1.7 to 17 mM for the determination of the kinetic parameters and inhibition constants. For each inhibitor at least six traces of the initial 5–10% of the reaction have been used for determining the initial velocity. The uncatalyzed rates were determined in the same manner and subtracted from the total observed rates. Stock solutions of inhibitor (0.1 mM) were prepared in distilled deionised water and dilutions up to 0.01 nM were done thereafter with the assay buffer. Inhibitor and enzyme solutions were preincubated together for 15 min at room temperature prior to assay, in order to allow for the formation of the EI complex. The inhibition constants were subsequently obtained by nonlinear least-squares methods using PRISM 3 and the ChengPrusoff equation, as reported earlier, and represent the mean from at least three different determinations. All CA isoforms were recombinant ones obtained in-house as reported earlier^36–39.

3.3. Co-crystallization and X-ray data collection

Crystals of hCA II complexed with compound 2v were obtained using the sitting drop vapour diffusion method. An equal volume of 0.8 mM solution of hCA II in Tris pH = 8.0 and 1.6 mM of the inhibitors in Hepes 20 mM pH = 7.4 was mixed and incubated for 15 min. 2 mL of the complex solution were mixed with 2 mL of a solution of 1.6 M sodium citrate, 50 mM Tris pH 8.0 and were equilibrated against the same solution at 296 K. Crystals of the complex grew in a few days. The crystals were flash-frozen at 100 K using a solution obtained by adding 25% (v/v) glycerol to the mother liquor solution as cryoprotectant. A data set on a crystal of the complex with the inhibitor 2v was collected at the Centro di Cristallografia Strutturale (CRIST) in Florence using an Oxford Diffraction instrument equipped with a sealed tube Enhance Ultra (Cu) and a Onyx CCD detector. Data were integrated and scaled using the program XDS.24 Data processing statistics are showed in Table 2.

Table 2. Summary of data collection and atomic model refinement statistics for hCAII.^a

	hCAII + 2v
PDB ID	7QSI
Wavelength (Å)	0.999900
Space Group	P21
Unit cell (a, b, c, α, β, γ) (Å,°)	42.363, 41.557, 72.068 90.000, 104.495, 90.000
Limiting resolution (Å)	41.02–1.30 (1.34–1.30)
Unique reflections	52,866 (2056)
Rmerge (%)	6.4 (73.2)
Rmeas (%)	7.0 (84.4)
Redundancy	6.11 (3.98)
Completeness overall (%)	88.9 (46.7)
<I/σ(I)>	19.54 (2.1)
CC (1/2)	99.9 (63.8)
Refinement statistics
Resolution range (Å)	41.02–1.30
Rfactor (%)	16.27
Rfree(%)	18.45
r.m.s.d. bonds(Å)	0.0135
r.m.s.d. angles (°)	1.8542
Ramachandran statistics (%)
Most favoured	96.9
additionally allowed	3.1
outlier regions	0.0
Average B factor (Å^2)
All atoms	15.116
inhibitors	11.348
Solvent	24.123

^aValues in parentheses are for the highest resolution shell.

3.4. Structure determination

The crystal structure of hCA II (PDB accession code: 7QSI) without solvent molecules and other heteroatoms was used to obtain initial phases of the structures using Refmac5⁴⁰. 5% of the unique reflections were selected randomly and excluded from the refinement data set for the purpose of Rfree calculations. The initial |Fo ̶ Fc| difference electron density maps unambiguously showed the inhibitor molecules. An electron density, which could be interpreted as a second molecule of inhibitor 2v, was present near the N-terminus of the protein. Thus, a second 2v molecule was introduced in the model with 0.75 occupancy. Atomic model for the inhibitor was calculated and energy minimised using the program JLigand 1.0.39. Refinements proceeded using normal protocols of positional, isotropic atomic displacement parameters alternating with manual building of the models using COOT⁴¹. Solvent molecules were introduced automatically using the program ARP⁴². The quality of the final model was assessed with COOT and Rampage⁴³. Crystal parameters and refinement data are summarised in Table 2. Atomic coordinates were deposited in the Protein Data Bank (PDB accession code: 7QSI). Graphical representations were generated with Chimaera⁴⁴.

4. Conclusions

We have described the synthesis and testing against a panel of human carbonic anhydrases (hCA I, II, IX and XII) of a series of hydrophilic, fragment sulfamides intended for fragment-based drug discovery of isoform-selective carbonic anhydrase inhibitors via cooperative screening with other, non-zinc-binding fragments. As expected from the minimal-periphery zinc-binding moieties, these fragment sulfamides demonstrated little selectivity across the panel of hCAs. However, for one of the fragment inhibitors (2v) which showed higher selectivity towards the cancer-related hCA isoforms (IX and CII), we obtained a crystal structure with the most abundant cytosolic isoform hCA II which showed two possible binding modes and thus significant room for cooperative fragment biding and subsequent periphery evolution.

Supplementary materials

Copies of ¹H and ¹³C NMR spectra of compounds 2a–w.

Acknowledgements

We are grateful to the Research Centre for Magnetic Resonance and the Centre for Chemical Analysis and Materials Research of Saint Petersburg State University Research Park for the analytical data.

Disclosure statement

No potential conflict of interest was reported by all author(s) except CTS. CT Supuran is Editor-in-Chief of the Journal of Enzyme Inhibition and Medicinal Chemistry. He was not involved in the assessment, peer review, or decision-making process of this paper. The authors have no relevant affiliations of financial involvement with any organisation or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. This article was submitted before the start of the Russia-Ukraine military conflict on February 24, 2022.

Correction Statement

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

Additional information

Funding

This research was supported by the Ministry of Education of the Russian Federation [government contract 073–00077-21–02 “Development of an innovative glaucoma drug based on selective inhibition of carbonic anhydrase II,” registry number 730000Ф.99.1.БВ10АА00006].