Synthesis and carbonic anhydrase activating properties of a series of 2-amino-imidazolines structurally related to clonidine¹

Abstract

The Carbonic Anhydrase (CA, EC 4.2.1.1) activating properties of histamine have been known for a long time. This compound has been extensively modified but only in few instances the imidazole ring has been replaced with other heterocycles. It was envisaged that the imidazoline ring could be a bioisoster of the imidazole moiety. Indeed, we report that clonidine, a 2-aminoimidazoline derivative, was found able to activate several human CA isoforms (hCA I, IV, VA, VII, IX, XII and XIII), with potency in the micromolar range, while it was inactive on hCA II. A series of 2-aminoimidazoline, structurally related to clonidine, was then synthesised and tested on selected hCA isoforms. The compounds were inactive on hCA II while displayed activating properties on hCA I, VA, VII and XIII, with K_A values in the micromolar range. Two compounds (10 and 11) showed some preference for the hCA VA or VII isoforms.

Keywords:

• Carbonic anhydrase
• activator
• clonidine
• histamine
• imidazoline

1. Introduction

Carbonic anhydrases (CAs, EC 4.2.1.1) are metallo-enzymes widespread in all life kingdoms. These enzymes catalyse a plethora of reactions, among which the reversible hydration of CO₂ is the most important one¹. The active site contains the cofactor, a metal ion (usually Zn²⁺) which coordinates a water molecule responsible, once activated as hydroxide ion, of the nucleophilic attack onto carbon dioxide. Eight genetically different families have been found (α-ι); 15 isoforms belonging to the α class have been characterised in humans^1,2.

CAs have been drug targets since more than 70 years; inhibitors of these enzyme are used for the treatment of oedema, glaucoma and epilepsy but several new therapeutic applications are under study³. In recent years, the attention has been focussed also on activators of these enzymes, despite the fact that CA are among the most efficient enzymes known. In fact, genetic deficiencies of several CA isoforms were reported in the last decades (reviewed in Refs. [3,4]), and in principle a loss of function of these enzymes could be treated with CA selective activators (CAAs). In addition, there is evidence that CA activation improves cognitive performance^5–10. However, the influence of CA on these processes is complex since also inhibitors have been found to improve memory deficits in animal models (reviewed in Ref. [11]); these findings point out the need for isoform selective inhibitors or activators to elucidate the role of CA isoforms in cognitive processes. Other possible applications of CAAs could be in the formation of artificial tissues¹² and in CO₂ capture and sequestration processes¹³.

Histamine (HST, Chart 1) was among the first reported activators, whose interaction mode was elucidated by means of X-ray crystallography¹⁴. The adduct with hCA II revealed a complex network of H-bonds involving the Zn-bound water molecule, His64 and the imidazole ring of the activator, which is located far away from the metal ion, in a region approaching the edge of the active site cavity. X-ray crystallographic studies have later shown that also other activators bind in this area⁴.

Chart 1. Chemical structure of CA activators.

As common structural feature, CAAs possess flexible tails decorated with protonable moieties, with pK_a values spanning between 6 and 8. The molecule of histamine has been extensively modified, placing substituents on the imidazole C atoms and on the NH₂ group, showing that the latter is not essential, since it can be largely modified to keep or improve potency (reviewed in Ref. [4]). Only in few instances the imidazole ring has been replaced by another heterocycle, such as a thiadiazole ring¹⁵.

In search for bioisosters of the imidazole moiety, our attention was attracted by the imidazoline ring. This feature is present in a well-known drug, Clonidine (CLO, Chart 1), which is clinically used as an antihypertensive agent being an agonist at the central α2-adrenergic receptor, but it is able to interact with other targets, such as the imidazoline binding sites and the hyperpolarization-activated cyclic nucleotide gated channels^16,17. Therefore, we decided to measure the potential CA activating properties of this compound, finding that CLO behaves as CAA on several CA isoforms (Table 1). Encouraged by this positive outcome, we synthesised a series of 2-substituted imidazolines (compounds 1–24, Chart 1) and tested their activity on five different hCA isoforms. The ubiquitous cytosolic enzymes CA I and II, the mitochondrial CA VA, which is associated with the glucose homeostasis,¹⁸ the cytosolic CA VII which is particularly abundant in the CNS and has been recently demonstrated to have a protective role against oxidative damage,¹⁹ and the cytosolic CA XIII, which is particularly expressed in the reproductive organs^20,21 were selected.

Table 1. Activation constants of Clonidine (CLO) and histamine (HST) on selected human CA isoforms, measured by means of a stopped-flow, CO₂ hydrase assay.^a

Compound	hCA KA (µM)
	I	II	IV	VA	VII	IX	XII	XIII
CLO	73.6	>200	132	42.6	8.4	54.1	126	7.8
HST^b	2.1	125	25.3	0.01	37.5	35.1	27.9	4.6

^aErrors (data not shown) are in the range of ±5–10% of the reported values from three different assays.

^bFrom Ref. [4].

2. Materials and methods

2.1. Chemistry

All melting points were taken on a Büchi apparatus and are uncorrected. NMR spectra were recorded on a Brucker Avance 400 spectrometer (400 MHz for ¹H NMR, 100 MHz for ¹³C). Chromatographic separations were performed on a silica gel column by gravity chromatography (Kieselgel 40, 0.063–0.200 mm; Merck) or flash chromatography (Kieselgel 40, 0.040–0.063 mm; Merck). Yields are given after purification, unless differently stated. When reactions were performed under anhydrous conditions, the mixtures were maintained under nitrogen. High-resolution mass spectrometry (HR-MS) analyses were performed with a Thermo Finnigan LTQ Orbitrap mass spectrometer equipped with an electrospray ionisation source (ESI). Analyses were carried out in positive ion mode monitoring protonated molecules, [M + H]⁺ species, and a proper dwell time acquisition was used to achieve 60,000 units of resolution at Full Width at Half Maximum (FWHM). Elemental composition of compounds were calculated on the basis of their measured accurate masses, accepting only results with an attribution error less than 5 ppm and a not integer RDB (double bond/ring equivalents) value, in order to consider only the protonated species²². Compounds were named following IUPAC rules by means of ChemDraw 14.0.

2.1.1. General procedure for the preparation of 2-amino-imidazoline 2–20

A mixture of the appropriate intermediate (1a²³ or 1b²⁴, 0.05 g) and the amine (1 eq) was suspended in THF (5 ml) and the mixture was heated at 70 °C until reaction completion (TLC monitoring, eluent CH₂Cl₂/CH₃OH/NH₄OH 90:10:1); alternatively, an excess of amine (5 eq) was used as solvent. Volatiles were evaporated under vacuum and the residue was triturated with Et₂O to remove the unreacted amine, affording the desired imidazoline derivative. In some instances, additional purification by means of flash chromatography was necessary. The conditions for each compound are reported in Table 2. With this method, the following compounds were prepared.

Table 2. Synthetic details for the preparation of compounds 2–20 (see general formula in Scheme 1) starting from 1a (R₁ = H) or 1 b (R₁ = Me) and amines R₂R₃NH.

N	Starting material	R2	R3	Solvent	Reaction time (h)^a	Yields (%)^b
2	1a	H	–CH2Ph	THF	7	94
3	1a	H	–(CH2)2Ph	THF	16	90
(S)-4	1a	H	(S)–CH(Me)Ph	Amine^c	2	36^d
(R)-4	1a	H	(R)–CH(Me)Ph	Amine^c	2	74^d
5	1a	H	–(CH2)3Ph	THF	3	88
6	1a	Me	–CH2Ph	THF	22	32^d
7	1a	Me	–(CH2)2Ph	THF	2	88
8	1b	H	–(CH2)2Ph	Amine^c	2	86
9	1b	Me	–(CH2)2Ph	Amine^c	2	97
10	1b	Me	–CH2Ph	Amine^c	3	70^d
11	1b	H	–CH2Ph	Amine^c	2	81
12	1b	H	–(CH2)3Ph	Amine^c	2	45^d
13	1b	H	–CH2C6H4Cl(4)	THF	16	97
14	1b	H	–CH2C6H4OCH3(4)	THF	4	89^d
15	1b	H	–CH2C6H4F(4)	Amine^c	6	89
16	1b	H	–CH2C6H4Cl(3)	Amine^c	6	88^d
17	1b	H	–CH2C6H4OCH3(3)	Amine^c	24	92
18	1b	H	–CH2C6H4 F(3)	Amine^c	16	87
19	1a	H	–(CH2)2NHCH2Ph	Amine^c	3	56^d
20	1b	H	–(CH2)2NHCH2Ph	Amine^c	4	57^d

^a Heating at 70 °C; all amines were commercially available.

^b Unless otherwise stated, yields are given after trituration with diethyl ether.

^c The amine was used in excess (5 eq).

^d After chromatographic separation.

N-benzyl-4,5-dihydro-1H-imidazol-2-amine hydroiodide 2²³. White solid; m.p. 143–146 °C ESI-LC-MS (m/z) 176.0 [M + H]⁺ [¹H]-NMR (D₂O) δ: 3.56 (s, 4H, CH₂CH₂); 4.31 (s, 2H, CH₂); 7.24–7.35 (m, 5H, Ar) ppm. [¹³C]-NMR (D₂O) δ: 42.70 (CH₂Ph); 45.65 (2CH₂); 126.93 (CH_Ar); 128.00 (CH_Ar); 128.95 (CH_Ar); 136.25 (C_Ar); 159.91 (C=N) ppm.

N-Phenethyl-4,5-dihydro-1H-imidazol-2-amine hydroiodide 3²⁵. White solid; m.p 82–83 °C; ESI-LCMS (m/z) 189.7 [M + H]⁺. [¹H]-NMR (D₂O) δ: 2.75 (t, J = 6.6 Hz, 2H, CH₂); 3.33 (t, J = 6.6 Hz, 2H, CH₂); 3.43 (s, 4H, CH₂CH₂); 7.14–7.31 (m, 5H, Ar) ppm. [¹³C]-NMR (D₂O) δ: 34.76 (CH₂); 42.63 (2CH₂); 43.78 (CH₂); 126.94 (CH_Ar); 128.84 (CH_Ar); 129.13 (CH_Ar); 138.50 (C_Ar); 159.83 (C=N) ppm.

(S) N-(1-Phenylethyl)-4,5-dihydro-1H-imidazol-2-amine hydroiodide (S)-4²⁶. Purification by flash chromatography (CH₂Cl₂/CH₃OH/NH₃ 87:13:1.3 as eluent); m.p. 103–107 °C; ESI-LCMS (m/z) 190.2 [M + H]⁺. [¹H]-NMR (D₂O) δ: 1.41 (d, J = 6.8 Hz, 3H, CH₃); 3.49–3.53 (m, 4H, 2CH₂); 4.55 (q, J = 6.8 Hz, 1H, CH); 7.23–7.35 (m, 5H, Ar) ppm. [¹³C]-NMR (D₂O) δ: 22.52 (CH₃); 42.62 (2CH₂); 52.79 (CH); 125.59 (CH_Ar); 128.03 (CH_Ar); 129.08 (CH_Ar); 142.03 (C_Ar); 158.98 (C=N) ppm.

(R) N-(1-Phenylethyl)-4,5-dihydro-1H-imidazol-2-amine hydroiodide (R)-4²⁶. Purification by flash chromatography (CH₂Cl₂/CH₃OH/NH₃ 87:13:1.3 as eluent); m.p. 105–109 °C; ESI-LCMS (m/z) 190.2 [M + H]⁺. [¹H]-NMR (D₂O) δ: 1.40 (d, J = 6.8 Hz, 3H, CH₃); 3.47–3.52 (m, 4H, 2CH₂); 4.54 (q, J = 6.8 Hz, 1H, CH); 7.21–7.40 (m, 5H, Ar) ppm. [¹³C]-NMR (D₂O) δ: 22.54 (CH₃); 42.67 (2CH₂); 52.84 (CH); 125.63 (CH_Ar); 128.07 (CH_Ar); 129.13 (CH_Ar); 142.06 (C_Ar); 159.03 (C=N) ppm.

N-(3-Phenylpropyl)-4,5-dihydro-1H-imidazol-2-amine hydroiodide 5. White solid; m.p. 85–87 °C. [¹H]-NMR (CDCl₃) δ: 1.90 (p, J = 7.2 Hz, 2H, CH₂); 2.70 (t, 2H, J = 7.9 Hz, PhCH₂); 3.34 (apparent q, J = 6.4 Hz, 2H, NCH₂); 3.60 (s, 4H, CH₂CH₂); 7.07 (bs, 1H, NH), 7.12–7.26 (m, 5H, Ar); 7.63 (s, 1H, NH) ppm. [¹³C]-NMR (CDCl₃) δ: 30.68 (CH_2); 32.63 (CH₂); 42.57 (CH₂); 43.39 (CH₂); 126.22 (CH_Ar); 128.54 (CH_Ar); 140.62 (C_Ar); 159.67 (C=N) ppm. ESI-HRMS (m/z) [M + H]⁺: calculated for C₁₂H₁₈N₃⁺ 204.1495; found 204.1498.

N-Benzyl-N-methyl-4,5-dihydro-1H-imidazol-2-amine hydroiodide 6. Purification by flash chromatography (CH₂Cl₂/CH₃OH/NH₃ 80:20:1 as eluent). White solid; m.p. 150–153 °C. [¹H]-NMR (D₂O) δ: 2.89 (s, 3H, NCH₃); 3.61 (s, 4H, CH₂CH₂); 4.42 (s, 2H, CH₂Ph); 7.15–7.40 (m, 5H, Ar) ppm. [¹³C]-NMR (D₂O) δ: 36.38 (CH₃); 43.14 (2CH₂); 54.29 (CH₂Ph); 127.05 (CH_Ar); 128.25 (CH_Ar); 129.11 (CH_Ar); 134.71 (C_Ar); 160.548 (C=N) ppm. ESI-HRMS (m/z) [M + H]⁺: calculated for C₁₁H₁₆N₃⁺ 190.1339; found 190.1342.

N-Methyl-N-phenethyl-4,5-dihydro-1H-imidazol-2-amine hydroiodide 7. White solid; m.p. 200–204 °C. [¹H]-NMR (D₂O) δ: 2.80 – 2.89 (m, 5H, NCH₃ + _PhCH₂); 3.42 (s, 4H, 2CH₂); 3.46 (t, J = 6.6 Hz, 2H, NCH₂); 7.19–7.33 (m, 5H, Ar) ppm. [¹³C]-NMR (D₂O) δ: 32.62 (CH₂Ph); 36.19 (NCH₃); 42.83 (N-CH₂); 52.53 (CH₂CH₂); 126.98 (CH_Ar); 128.82 (CH_Ar); 129.05 (CH_Ar); 138.13 (C_Ar); 159.89 (C=N) ppm. ESI-HRMS (m/z) [M + H]⁺: calculated for C₁₂H₁₈N₃⁺ 204.1495; found 204.1499.

1-Methyl-N-phenethyl-4,5-dihydro-1H-imidazol-2-amine hydroiodide 8. White solid; m.p. 118–220 °C. [¹H]-NMR (CDCl₃) δ: 3.00–3.08 (m, 5H, PhCH₂ + NCH₃); 3.48 (s, 4H, 2CH₂); 3.65 (apparent q, J = 6.8 Hz, 2H, NCH₂); 6.96 (s, 1H, NH), 7.12–7.35 (m, 5H, Ar), 7.49 (t, J = 5.6, 1H, NH) ppm. [¹³C]-NMR (CDCl₃) δ: 33.78 (CH₃); ₃₅.49 (CH₂); 41.04 (CH_2); 45.22 (CH₂); 50.11 (CH₂); 126.79 (CH_Ar); 128.67 (CH_Ar); 129.32 (CH_Ar); 137.87 (C_Ar); 158.22 (C=N) ppm. ESI-HRMS (m/z) [M + H]⁺: calculated for C₁₂H₁₈N₃⁺ 204.1495; found 204.1494.

N,1-Dimethyl-N-phenethyl-4,5-dihydro-1H-imidazol-2-amine hydroiodide 9. White solid; m.p. 109–110 °C. [¹H]-NMR (CDCl₃) δ: 2.82 (s, 3H, CH₃); 2.97 (t, J = 6.8 Hz, 2H, PhCH₂); 3.13 (s, 3H, CH₃); 3.55–3.62 (m, 4H, CH₂CH₂); 3.77 (t, J = 6.9 Hz, 2H, CH₂N); 7.32 (t, J = 7.5 Hz, 5H, Ar), 8.34 (bs, 1H, NH) ppm. [¹³C]-NMR (CDCl₃) δ: 33.66 (CH_2); 36.97 (CH₃); 39.70 (CH₃); 40.33 (CH₂); 52.89 (CH₂); 54.71 (CH₂); 127.23 (CH-Ar); 128.92 (CH-Ar); 136.96 (C-Ar); 162.47 (C=N) ppm. ESI-HRMS (m/z) [M + H]⁺: calculated for C₁₃H₂₀N₃⁺ 218.1652; found 218.1649.

N-Benzyl-N,1-dimethyl-4,5-dihydro-1H-imidazol-2-amine hydroiodide 10. Purification by flash chromatography (CH₂Cl₂/CH₃OH/NH₃ 90:10:1 as eluent). Oil. [¹H]-NMR (CDCl₃) δ: 3.01 (s, 3H, CH₃); 3.07 (s, 3H, CH₃); 3.70–3.87 (m, 4H, CH₂CH₂); 4.63 (s, 2H, CH₂Ph); 7.22–7.35 (m, 5H, Ar), 8.10 (s, 1H, NH) ppm. [¹³C]-NMR (CDCl₃) δ: 37.03 (CH₃); 39.72 (CH₃); 40.53 (CH₂); 53.14 (CH₂); 56.60 (CH₂); 127.20 (CH_Ar); 128.44 (CH_Ar); 129.25 (CH_Ar); 133.94 (C_Ar); 162.63 (C=N) ppm. ESI-HRMS (m/z) [M + H]⁺: calculated for C₁₂H₁₈N₃⁺ 204.1495; found 204.1497.

N-Benzyl-1-methyl-4,5-dihydro-1H-imidazol-2-amine hydroiodide 11. White solid; m.p. 130–133 °C. [¹H]-NMR (CDCl₃) δ: 3.16 (s, 3H, CH₃); 3.58–3.66 (m, 4H, CH₂CH₂); 4.67 (d, J = 5.6 Hz, 2H, CH₂Ph); 6.87 (s, 1H, NH); 7.25–7.38 (m, 3H, Ar); 7.49 (d, J = 7.2 Hz, 2H, Ar); 8.21 (s, 1H, NH) ppm. [¹³C]-NMR (CDCl₃) δ: 33.76 (CH₃); 41.31 (CH₂); 46.38 (CH₂); 50.22 (CH₂); 128.13 (CH_Ar); 128.34 (CH_Ar); 129.04 (CH_Ar); 135.63 (C_Ar); 158.35 (C=N) ppm. ESI-HRMS (m/z) [M + H]⁺: calculated for C₁₁H₁₆N₃⁺ 190.1339; found 190.1338.

1-Methyl-N-(3-phenylpropyl)-4,5-dihydro-1H-imidazol-2-amine hydroiodide 12. Purification by flash chromatography (CH₂Cl₂/CH₃OH/NH₃ 90:10:1 as eluent). Gum. [¹H]-NMR (CDCl₃) δ: 2.02 (p, J = 7.6 Hz, 2H); 2.69 (t, J = 8.0 Hz, 2H, CH₂); 2.97 (s, 3H, CH₃); 3.41–3.54 (m, 4H, CH₂CH₂); 3.60 (t, J = 8.0 Hz, 2H, CH₂); 7.10–7.25 (m, 5H, Ar) ppm. [¹³C]-NMR (CDCl₃) δ: 30.62 (CH₂), 32.93 (CH₂), 33.77 (NCH₃), 40.97 (CH₂), 43.83 (CH₂), 50.11 (CH₂), 126.01 (CH_Ar), 128.44 (CH_Ar), 128.58 (CH_Ar), 141.25 (C_Ar), 157.91 (C=N) ppm. ESI-HRMS (m/z) [M + H]⁺: calculated for C₁₃H₂₀N₃⁺ 218.1652; found 218.1654.

N-(4-Chlorobenzyl)-1-methyl-4,5-dihydro-1H-imidazol-2-amine hydroiodide 13. White solid, m.p. 84 °C. [¹H]-NMR (MeOD) δ: 2.96 (s, 3H, CH₃), 3.61–3.76 (m, 4H, CH₂CH₂), 4.41 (s, 2H, CH₂), 7.31 (d, J = 8.4 Hz, 2H, Ar), 7.35 (d, J = 8.4 Hz, 2H, Ar), 7.42 (s, 2H, NH) ppm. [¹³C]-NMR (MeOD) δ: 30.68 (CH₃), 40.95 (CH₂), 45.41 (CH₂), 50.09 (CH₂), 128.92 (CH_Ar), 130.37 (CH_Ar), 133.46 (CCl), 135.00 (C_Ar), 158.68 (C=N), ppm. ESI-HRMS (m/z) [M + H]⁺: calculated for C₁₁H₁₅ClN₃⁺ 224.0949; found 224.0946.

N-(4-Methoxybenzyl)-1-methyl-4,5-dihydro-1H-imidazol-2-amine hydroiodide 14. White solid, m.p. 167 °C. [¹H]-NMR (CDCl₃) δ: 3.18 (s, 3H, CH₃), 3.64 (s, 4H, CH₂CH₂), 3.76 (s, 3H, OCH₃), 4.58 (d, J = 5.7 Hz, 2H, CH₂), 6.38 (s, 1H, NH), 6.85 (d, J = 8.6 Hz, 2H, Ar), 7.39 (d, J = 8.6 Hz, 2H, Ar), 8.10 (s, 1H, NH), ppm. [¹³C]-NMR (CDCl₃) δ: 33.77 (NCH₃), 41.29 (CH₂), 45.86 (CH₂), 50.14 (CH₂), 55.36 (OCH₃), 114.38 (CH_Ar), 127.62 (C_Ar), 129.69 (CH_Ar), 158.19 (C_Ar), 159.59 (C=N) ppm. ESI-HRMS (m/z) [M + H]⁺: calculated for C₁₂H₁₈N₃O⁺ 220.1444; found 220.1443.

N-(4-Fluorobenzyl)-1-methyl-4,5-dihydro-1H-imidazol-2-amine hydroiodide 15. White solid, m.p. 157 °C. [¹H]-NMR (MeOD) δ: 2.97 (s, 3H, CH₃), 3.61 – 3.75 (m, 4H, CH₂CH₂), 4.42 (s, 2H, CH₂), 7.08 (t, J = 8.5 Hz, 2H, Ar), 7.42 (dd, J = 8.5, 5.4 Hz, 2H, Ar), ppm. [¹³C]-NMR (MeOD) δ: 30.81 (CH₃), 40.98 (CH₂), 45.44 (CH₂), 50.11 (CH₂), 115.26 (d, J_C-F = 22 Hz, CH_Ar), 129.12 (d, J_C-F = 8 Hz, CH_Ar), 132.25 (C_Ar), 158.62 (C=N), 162.48 (d, J_C-F = 244 Hz, CF), ppm. ESI-HRMS (m/z) [M + H]⁺: calculated for C₁₁H₁₅FN₃⁺ 208.1245; found 208.1245.

N-(3-Chlorobenzyl)-1-methyl-4,5-dihydro-1H-imidazol-2-amine hydroiodide 16. White solid, m.p. 190 °C. [¹H]-NMR (MeOD) δ: 2.96 (s, 3H, CH₃), 3.58–3.71 (m, 4H), 4.41 (s, 2H, CH₂), 7.20–7.25 (m, 1H, Ar), 7.25–7.37 (m, 3H, Ar) ppm. [¹³C]-NMR (MeOD) δ: 30.56 (CH₃), 40.93 (CH₂), 45.45 (CH₂), 50.06 (CH₂), 125.25 (CH_Ar), 126.91 (CH_Ar), 127.74 (CH_Ar), 130.07 (CH_Ar), 134.35 (C-Cl), 138.59 (C_Ar), 154.10 (C=N) ppm. ESI-HRMS (m/z) [M + H]⁺: calculated for C₁₁H₁₅ClN₃⁺ 224.0949; found 224.0949.

N-(3-Methoxybenzyl)-1-methyl-4,5-dihydro-1H-imidazol-2-amine hydroiodide 17. White solid, m.p. 147 °C. [¹H]-NMR (CDCl₃) δ: 2.95 (s, 3H, CH₃), 3.61 (s, 4H, CH₂CH₂), 3.76 (s, 3H, OCH₃), 4.59 (s, 2H, CH₂), 6.77 (d, J = 6.4 Hz, 1H, NH), 7.06–7.66 (m, 4H, Ar), 8.31 (s, 1H, NH) ppm. [¹³C]-NMR (CDCl₃) δ: 33.70 (CH₃), 41.32 (CH₂), 46.20 (CH₂), 50.22 (CH₂), 55.68 (OCH₃), 113.67 (CH_Ar), 114.00 (CH_Ar), 120.27 (CH_Ar), 129.96 (CH_Ar), 137.43 (C_Ar), 158.22 (C_Ar), 159.94 (C=N), ppm. ESI-HRMS (m/z) [M + H]⁺: calculated for C₁₂H₁₈N₃O⁺ 220.1444; found 220.1447.

N-(3-Fluorobenzyl)-1-methyl-4,5-dihydro-1H-imidazol-2-amine hydroiodide 18. White solid, m.p. 195 °C. [¹H]-NMR (CDCl₃) δ: 3.19 (s, 3H, CH₃), 3.61–3.71 (m, 4H, CH₂CH₂), 4.68 (s, 2H, CH₂), 6.95 (t, J = 7.7 Hz, 1H, NH), 7.09 – 7.41 (m, 4H, Ar), 8.27 (bs, 1H, NH) ppm. [¹³C]-NMR (CDCl₃) δ: 33.86 (CH₃), 41.19 (CH₂), 45.42 (CH₂), 50.20 (CH₂), 115.05 (d, J_CF= 25 Hz, CH_Ar), 123.94 (CH_Ar), 130.45 (d, J = 8 Hz, CH_Ar), 138.48 (C_Ar), 158.11 (C=N), 162.78 (d, J = 246 Hz, CF) ppm.

N¹-Benzyl-N²-(4,5-dihydro-1H-imidazol-2-yl)ethane-1,2-diamine hydroiodide 19. White solid, m.p. 230 °C (dec). [¹H]-NMR (MeOD) δ: 3.37 (t, J = 6.2 Hz, 2H, CH₂), 3.64 (t, J = 6.2 Hz, 2H, CH₂), 3.70 (s, 4H, CH₂CH₂), 4.34 (s, 2H, PhCH₂), 7.40–7.47 (m, 3H, Ar), 7.59–7.64 (m, 2H, Ar) ppm. [¹³C]-NMR (MeOD) δ: 38.87 (CH₃), 42.82 (CH₂CH₂), 45.73 (CH₂), 51.20 (PhCH₂), 128.92 (CH_Ar), 129.49 (CH_Ar), 129.99 (CH_Ar), 130.69 (C_Ar), 159.88 (C=N) ppm. ESI-HRMS (m/z) [M + H]⁺: calculated for C₁₂H₁₉N₄⁺ 219.1604; found 219.1606.

N¹-Benzyl-N²-(1-methyl-4,5-dihydro-1H-imidazol-2-yl)ethane-1,2-diamine hydroiodide 20. White solid, m.p. 235 °C (dec). [¹H]-NMR (MeOD) δ: 2.93 (s, 3H, CH₃), 3.33 (t, J = 6.0 Hz, 2H, CH₂), 3.63–3.72 (m, 6H, 3CH₂), 4.28 (s, 2H, PhCH₂), 7.38–7.44 (m, 3H, Ar), 7.48 – 7.54 (m, 2H, Ar) ppm. [¹³C]-NMR (MeOD) δ: 30.59 (CH₃), 39.07 (CH₃), 40.94 (CH₂), 45.61 (CH₂), 50.04 (PhCH₂), 51.17 (CH₂), 128.56 (CH_Ar), 129.37 (CH_Ar), 129.80 (CH_Ar), 130.81 (C_Ar), 156.18 (C=N) ppm. ESI-HRMS (m/z) [M + H]⁺: calculated for C₁₃H₂₁N₄⁺ 233.1761; found 233.1760.

2.1.2. N1-(4,5-Dihydro-1H-imidazol-2-yl)ethane-1,2-diamine 21²⁷

The compound was prepared following the procedure reported by A. Bucio-Cano et al.²⁷ Yellow oil, isolated then as oxalate salt. Yield: 61%; m.p. (oxalate salt): 153–154 °C. [¹H] NMR (D₂O) δ: 3.10 (t, J = 6 Hz, 2H, CH₂); 3.44 (t, J = 6 Hz, 2H, CH₂); 3.59 (s, 4H, NHCH₂CH₂NH₂) ppm. [¹³C] NMR (D₂O) δ: 38.28 (CH₂); 39.70 (CH₂); 42.82 (2CH₂); 159.80 (C); 165.0 (CO Oxalate) ppm.

2.1.3. N-Phenyl-4,5-dihydro-1H-imidazol-2-amine 22²⁸

1-(2-Aminoethyl)-3-phenylthiourea²⁹ (110 mg, 0.56 mmol) was dissolved in THF (5 ml) and a 1 M sodium hydroxide/water solution (1 eq) was added while stirring. After 0.5 h, a solution of TsCl (1–1.1 equiv) in THF (5 ml) was slowly added and the mixture was stirred for 5 h at room T. The solvent was evaporated and diethyl ether and brine were added to the residue. The layers were separated and the aqueous layer was extracted three times with diethyl ether. The combined organic layers were dried (Na₂SO₄), filtered and evaporated, leaving a residue which was purified by flash chromatography (CH₂Cl₂: MeOH: NH₃ 80:20:1 as eluent). Compound 22 was obtained as a white solid, m.p. 137–140 °C, in 50% yields. [¹H]-NMR (D₂O) δ: 3.40 (s, 4H, CH₂CH₂), 6.94 (d, J = 7.2 Hz, 2H), 7.03 (t, J = 7.2 Hz, 1H), 7.27 (t, J = 8.0 Hz, 2H) ppm. [¹³C]-NMR (D₂O) δ: 42.37 (CH₂), 123.34 (CH_Ar), 123.40 (CH_Ar), 129.52 (CH_Ar), 146.90 (C_Ar), 161.03 (C=N) ppm.

2.1.4. 1–(2-(Methylthio)-4,5-dihydro-1H-imidazol-1-yl) ethanone 23³⁰

The imidazoline 1a was acylated on one imidazoline-nitrogen atom to obtain 23 as reported by Gomez-San Juan et al.³⁰ Yellow solid, yield: 100%; m.p.= 107–110 °C. [¹H]-NMR (D₂O) δ: 2.07 (s, 3H, SCH₃); 2.23 (s, 3H, CH₃); 3.76 (t, J = 8.8 Hz, 2H, CH₂); 3.92 (t, J = 8.8 Hz, 2H, CH₂) ppm. [¹³C]-NMR (D₂O) δ: 14.5 (SCH₃); 23.5 (CH₃); 48.0 (2CH₂); 161.5 (C=N); 171.9 (CO) ppm.

2.1.5. 2-(Benzylthio)-4,5-dihydro-1H-imidazole 24 hydrobromide³¹

A mixture of commercially-available 2-imidazolidinethione (0.1 g, 0.98 mmol) and benzyl bromide (1.1 eq) in anhydrous methanol (5 ml) was heated at 65 °C for 2 h. After cooling, the solvent was removed under vacuum and the residue was treated with Et₂O until it became solid; filtration and drying under vacuum gave the desired compound as a white solid, m.p. 181–183 °C. ESI-LC-MS (m/z) 193.1 [M + H]⁺ [¹H]-NMR (D₂O) δ: 3.81 (s, 4H, CH₂CH₂); 4.35 (s, 2H, CH₂); 7.31– 7.42 (m, 5H, Ar) ppm. [¹³C]-NMR (D₂O) δ: 35.15 (CH₂Ph); 45.24 (2CH₂); 128.31 (CH_Ar); 128.72 (CH_Ar); 129.10 (CH_Ar); 134.12 (C_Ar); 169.69 (C=N) ppm.

2.2. CA activation

An Sx.18Mv-R Applied Photophysics (Oxford, UK) stopped-flow instrument has been used to assay the catalytic activity of various CA isozymes for CO₂ hydration reaction³². Phenol red (at a concentration of 0.2 mM) was used as indicator, working at the absorbance maximum of 557 nm, with 10 mM Hepes (pH 7.5, for α-CAs)^33–37 as buffers, 0.1 M NaClO₄ (for maintaining constant ionic strength), following the CA-catalysed CO₂ hydration reaction for a period of 10 s at 25 °C. The CO₂ concentrations ranged from 1.7 to 17 mM for the determination of the kinetic parameters and inhibition constants. For each activator at least six traces of the initial 5–10% of the reaction have been used for determining the initial velocity. The uncatalysed rates were determined in the same manner and subtracted from the total observed rates. Stock solutions of activators (at 0.1 mM) were prepared in distilled-deionised water and dilutions up to 1 nM were made thereafter with the assay buffer. Enzyme and activator solutions were pre-incubated together for 15 min prior to assay, in order to allow for the formation of the enzyme–activator complexes. The activation constant (K_A), defined similarly with the inhibition constant K_I, can be obtained by considering the classical Michaelis–Menten equation (Equation (1)(1) $$\mathrm{v}\mathrm{ }={\mathrm{v}}_{\text{max}}/\left\{1+\left({\mathrm{K}}_{\mathrm{M}}/\left[\mathrm{S}\right]\right)\left(1+{\left[\mathrm{A}\right]}_{\mathrm{f}}/{\mathrm{K}}_{\mathrm{A}}\right)\right\}$$(1) , which has been fitted by non-linear least squares by using PRISM 3: (1) $$\mathrm{v}\mathrm{ }={\mathrm{v}}_{\text{max}}/\left\{1+\left({\mathrm{K}}_{\mathrm{M}}/\left[\mathrm{S}\right]\right)\left(1+{\left[\mathrm{A}\right]}_{\mathrm{f}}/{\mathrm{K}}_{\mathrm{A}}\right)\right\}$$(1) where [A]_f is the free concentration of activator.

Working at substrate concentrations considerably lower than K_M ([S] ≪K_M), and considering that [A]_f can be represented in the form of the total concentration of the enzyme ([E]_t) and activator ([A]_t), the obtained competitive steady-state equation for determining the activation constant is given by Equation (2)(2) $$\mathrm{v}={\mathrm{v}}_0.{\mathrm{K}}_{\mathrm{A}}/\{{\mathrm{K}}_{\mathrm{A}}+{\left({\left[\mathrm{A}\right]}_{\mathrm{t}}-0.5\{\left({\left[\mathrm{A}\right]}_{\mathrm{t}}+{\left[\mathrm{E}\right]}_{\mathrm{t}}+{\mathrm{K}}_{\mathrm{A}}\right)-{\left({\left[\mathrm{A}\right]}_{\mathrm{t}}+{\left[\mathrm{E}\right]}_{\mathrm{t}}+{\mathrm{K}}_{\mathrm{A}}\right)}^2-4{\left[\mathrm{A}\right]}_{\mathrm{t}}.{\left[\mathrm{E}\right]}_{\mathrm{t}}\right)}^{1/2}\}\}$$(2) : (2) $$\mathrm{v}={\mathrm{v}}_0.{\mathrm{K}}_{\mathrm{A}}/\{{\mathrm{K}}_{\mathrm{A}}+{\left({\left[\mathrm{A}\right]}_{\mathrm{t}}-0.5\{\left({\left[\mathrm{A}\right]}_{\mathrm{t}}+{\left[\mathrm{E}\right]}_{\mathrm{t}}+{\mathrm{K}}_{\mathrm{A}}\right)-{\left({\left[\mathrm{A}\right]}_{\mathrm{t}}+{\left[\mathrm{E}\right]}_{\mathrm{t}}+{\mathrm{K}}_{\mathrm{A}}\right)}^2-4{\left[\mathrm{A}\right]}_{\mathrm{t}}.{\left[\mathrm{E}\right]}_{\mathrm{t}}\right)}^{1/2}\}\}$$(2) where v₀ represents the initial velocity of the enzyme-catalysed reaction in the absence of activator^33–39. This type of approach to measure enzyme–ligand interactions is in excellent agreement with recent results from native mass spectrometry measurements⁴⁰.

3. Results and discussion

3.1. Chemistry

2-Amino imidazolines 2–20 were prepared by reacting primary and secondary amines with 2-(methylthio)-4,5-dihydro-1H-imidazole hydroiodide 1a²³ or its N¹-methyl derivative 1b²⁴ using tetrahydrofuran or an excess of amine as solvents (Scheme 1). The final compounds were obtained as hydroiodide salts in different yields. Synthetic details are reported in the experimental section. Reactions with 3- or 4-nitrobenzyl amine or with dibenzyl amine were unsuccessful.

Scheme 1. Synthesis of compounds 2-20.

This method failed also to give compound 22 (step b, Scheme 2), whose synthesis was therefore attempted according to the procedure of Gomez Saint-Juan (step c, Scheme 2);³⁰ however, also this method was abandoned since the reaction of 1-acetyl-2-(methylthio)-4,5-dihydro-1H-imidazole 23 with aniline was not successful. Finally, 22 was prepared from 1–(2-aminoethyl)-3-phenylthiourea²⁹ according to Heinelt et al.²⁸ Compound 21²⁷ was synthesised through condensation of guanidine and ethylenediamine while 24³¹ was prepared by reaction of imidazoline-2-thione with benzyl bromide.

Scheme 2. Synthesis of compounds 22 and 23.

3.2. CA activating profile

The stopped-flow method³² has been used for assaying the CO₂ hydration activity catalysed by different CA isoforms; the results are expressed as K_A (activation constant, µM). The activating profile of CLO is reported in Table 1, in comparison with HST. CLO behaved as an activator on several CA isoforms (I, IV, VA, VII, IX, XII and XIII), with K_A values in the range 7.8–136 µM, while on CA II it was inactive up to a dose of 200 µM. In particular, the isoforms most sensitive to CLO were CA VII and CA XIII.

The K_A values of the synthesised compounds are reported in Table 3. All compounds have been tested as hydroiodide salts, with the exception of 21 (oxalate), 22 and 23 (free bases), and 24 (as HBr salt). As CLO, none of the compounds was active on hCA II at the highest tested concentration, while on the other isoforms the compounds showed K_A values mainly in the low-medium range, allowing to derive the following structure–activity relationships.

Table 3. Activation constant (K_A) of the synthesised compounds and Clonidine (CLO) for human I, II, VA, VII and XIII Carbonic Anhydrase isoforms.

N	R1	X	R2	KA (μM)^b
				hCA I	hCA II	hCA VA	hCA VII	hCA XIII
1a	H	S	–CH3	9.61	>150	38.3	41.9	>100
2	H	NH	–CH2Ph	4.18	>150	45.7	35.2	>100
3	H	NH	–(CH2)2Ph	>150	>150	16.7	18.9	>100
S-4	H	NH	(S)–CH(Me)Ph	>150	>150	12.4	31.5	>100
R-4	H	NH	(R)–CH(Me)Ph	>150	>150	4.92	24.2	>100
5	H	NH	–(CH2)3Ph	36.7	>100	9.9	11.4	24.3
6	H	NMe	–CH2Ph	>150	>150	40.5	11.0	>100
7	H	NMe	–(CH2)2Ph	68.6	>100	14.9	2.4	6.5
8	CH3	NH	–(CH2)2Ph	95.4	>100	14.6	16.2	31.0
9	CH3	NMe	–(CH2)2Ph	20.2	>100	14.9	2.6	36.9
10	CH3	NMe	–CH2Ph	30.2	>100	0.9	6.5	17.4
11	CH3	NH	–CH2Ph	16.9	>100	3.7	0.9	19.1
12	CH3	NH	–(CH2)3Ph	10.9	>100	17.2	3.1	10.9
13	CH3	NH	–CH2C6H4Cl(4)	17.7	>100	10.5	22.7	22.9
14	CH3	NH	–CH2C6H4OCH3(4)	37.6	>100	15.3	30.2	19.3
15	CH3	NH	–CH2C6H4F(4)	59.7	>100	9.3	17.4	24.4
16	CH3	NH	–CH2C6H4Cl(3)	99.2	>100	10.4	24.0	37.0
17	CH3	NH	–CH2C6H4OCH3(3)	>100	>100	13.1	29.4	14.7
18	CH3	NH	–CH2C6H4 F(3)	>100	>100	14.9	19.4	23.3
19	H	NH	–(CH2)2NHCH2Ph	>100	>100	11.0	41.7	20.1
20	CH3	NH	–(CH2)2NHCH2Ph	>100	>100	11.9	29.0	16.3
21	H	NH	–CH2CH2NH2	3.87	>150	31.2	91.6	>100
22	H	NH	–Ph	>150	>150	52.7	32.6	>100
23	COCH3	S	–CH3	12.7	>150	15.0	30.9	>100
24	H	S	–CH2Ph	>150	>150	11.1	46.7	>100
CLO	H	NH	(2,6-dichloro)Ph	76.3	>200	42.6	8.4	7.8

^a All compounds have been tested as HI salts, with the exception of 21 (oxalate), 22 and 23 (free bases), 24 (HBr), and CLO (HCl).

^b Mean from 3 different determinations (errors in the range of 5–10% of the reported values, data not shown).

3.2.1. hCA I

The K_A value of CLO on this isoform was 76.3 µM. Removal of both chlorine atoms abolished activity, since 22 (R₂ = Ph) was inactive when tested up to a 150 µM concentration. On the contrary, inserting a CH₂ unit between the exocyclic N atom and the Ph ring of 22 improved the activity: in fact, 2 (K_A 4.18 µM), 18 times more potent than CLO, was one of the most potent compounds on this isoform among the newly synthesised analogues. The elongation of the methylene chain gave compounds less active than 2; interestingly, a chain formed by 3 CH₂ units was tolerated (5, K_A 36.7 µM) while a CH₂CH₂ chain was not (3, K_A >150 µM). Side-chain branching abolished activity (S-4, R-4: K_A >150 µM). Methylation of the exocyclic N_α atom was tolerated when R₂ was a phenethyl group (7: K_A 68.6 µM) but not when R₂ was a benzyl moiety (6: K_A >150 µM). Also the methylation of the endocyclic N₁ atom gave contrasting results, since compounds 8 and 12 were more active than their non-methylated analogues 3 and 5, but 11 (R₂ = benzyl) showed a fourfold decrease of activity with respect to 2 (11: K_A 16.9; 2: 4.18 µM). Double methylation was productive for the phenethyl analogues (compare 9 with 3, 7 and 8); for the benzyl analogue 10 this modification improved the activity only with respect to 6. The aminoethyl derivative 21 (K_A 3.87 µM) was the most potent compound on the hCAI isoform; the addition of a benzyl moiety on the primary amine group abolished activity (19, K_A > 100 µM), and also the N¹-methyl analogue 20 was inactive.

Aromatic substitution on the benzyl moiety did not improve the potency: in fact, while the 4-Cl derivative 13 was equiactive with 11, a 4-OMe (14) or 4-F substituent (15) increased from 2 to 3.5 times the K_A values. The same substituents in the meta position reduced to a higher extent or abolished the activity. As far as the sulphur analogues 1a, 23 and 24 are concerned, the small methyl group seemed tolerated, not the bulkier benzyl moiety (24, K_A >150 µM). The basicity of the amidine group appeared to be not crucial, since the NH and the N-acetyl derivatives (1a and 23, respectively) were equipotent.

3.2.2. hCA VA

The K_A value of CLO on this isoform was 42.6 µM. The removal of both chlorine atoms did not affect activity, since 22 (R₂ = Ph, K_A 52.7 µM) was almost equipotent with CLO. Also the insertion of a CH₂ unit between the exocyclic N atom and the Ph ring of 22 did not substantially modified potency (2, K_A 45.7 µM). On this isoform, the majority of the compounds showed good activating properties, with potency higher than CLO: the K_A values of 24, 23, 3–5, 7–9, 11 and 12 were in the range 3.7–17.2 µM.

The most potent compound was 10 (K_A 0.9 µM), a benzyl derivative carrying a methyl group on both N₁ and N_α atoms; this compound was 47 times more active than CLO. The removal of the exocyclic N_α-Me group decreased 4 times the activity (11, K_A 3.7 µM), while the removal of the N₁-methyl group was more detrimental: as a matter of fact, compounds 6 (K_A 40.5 µM) and 2 (K_A 45.5 µM) were about 40 times less potent than 10 (K_A 0.9 µM). On the contrary, the degree of methylation did not substantially affect the potency of the phenylethyl and phenyl propyl derivatives, since compounds 3, 5, 7, 8 and 12 had K_A values in the range 9.9–17.2 µM. Similarly, aromatic substitution on the benzyl moiety slightly decreased the potency without substantial modulation, the K_A values of compounds 13–18 being 2–4 times higher than 11. Side-chain branching (compounds S-4 and R-4) improved the activity on this isoform, and a small enantioselectivity was observed: the R-enantiomer was twice more potent as the S-isomer. A benzyl moiety on the terminal amino group of 21 (K_A 31.2 µM) increased the activity, as analogue 19 and its N¹-methyl derivative 20 were about 3 times more potent than the parent compound.

As far as the sulphur derivatives are concerned, the replacement of the N_αH moiety of 2 (K_A 45.5 µM) with S (24, K_A 11.1 µM) brought a fourfold improvement in activity. Acetylation of the N¹ nitrogen was also favourable, as 23 was twice more potent than 1a.

3.2.3. hCA VII

The K_A value of CLO on this isoform was 8.4 µM. All the tested compounds showed activation properties on this isoform, with K_A values between 0.9 and 91.6 µM. The least potent compound was the primary amine 21 (K_A 91.6 µM), whose activity was however improved by adding a benzyl group on the terminal NH₂ moiety (19, K_A 41.7 µM) and a methyl group on the endocyclic N atom (20, K_A 29.0 µM). The removal of chlorine atoms of CLO reduced 4 times the activity (22, R₂ = Ph, K_A 32.6 µM) while the separation of the phenyl and N_αH moieties by means of a CH₂ unit did not substantially modified potency (3, K_A 35.2 µM). On the contrary, the potency increased by elongating the chain from 1 to 3 CH₂ units (3, K_A 35.2 µM; 5, K_A 11.4 µM) and by adding a methyl group on the N_αH moiety: with the latter modification the potency of 3 (K_A 18.9 µM) and of 2 (K_A 35.2 µM) were increased 3 (6, K_A 11.0 µM) and 8 times (7, K_A 2.4 µM), respectively. Side-chain branching did not substantially affect activity, since S-4 and R-4 were equipotent with 2. Methylation on the endocyclic N atom was the most effective modification in this set of molecules: as a matter of fact, with this structural change the K_A value of 2 (K_A 35.2 µM) was reduced 39 times, and 11 (K_A 0.9 µM) resulted in the most potent compound on this isoform. The same modification was also effective on the phenylpropyl derivative 5 (K_A 11.4 µM), whose activity was increased 4 times (12, K_A 3.1 µM). The double methylation on the N₁ and N_α atoms gave potent compounds (10, K_A 6.5 µM and 9, K_A 2.6 µM) even if the K_A values are, respectively, 7 and 3 times lower than that of 11. Aromatic substitution on the benzyl moiety of 11 was detrimental for activity, as compounds 13–18 were 19–33 times less potent than 11. As far as the sulphur analogues 1a, 23 and 24 are concerned, their K_A values were in the range 30.9–46.7 µM, not better that the other tested 2-aminoimidazoline derivatives. Attempts to crystallise adducts of 7, 11 and 12 with hCA VII are ongoing.

3.2.4. hCA XIII

This is the isoform most sensitive to CLO among those studied (K_A 7.8 µM). As it happened on the hCA I isoform, the removal of both chlorine atoms, to give 22, abolished activity. Several other compounds resulted inactive when tested at concentrations up to 100 µM, i.e. the sulphur derivatives, the polar aminoethyl derivative 21, and all the compounds having both the N₁ and N_α atoms as secondary amines, with the exception of the lipophilic phenylpropyl derivative 5 (K_A 24.3 µM). The activity of 21 could be restored by adding a benzyl moiety on the primary amino-group (19, K_A 20.1 µM) and a methyl group on the endocyclic N atom (20, K_A 16.3 µM). Also the methylation of the phenethyl analogue 3 on the N_α atom re-established activity, giving 7 (K_A 6.5 µM) which resulted the most potent compound of the series on this isoform. Methylation on the endocyclic N₁ atom gave compounds 8, 11 and 12 whose potency ranged from 10.9 to 31.0 µM, the most potent being the derivative carrying a phenylpropylamino side chain (12). Methylation on both N₁ and N_α atoms or aromatic substitution on the benzyl moiety did not improve activity.

As far as selectivity is concerned, the two compounds showing submicromolar K_A values displayed also interesting selectivity profiles: 10 was more active on hCA VA with respect to hCA I (33 times), II (>100 times), VII (7 times), and XIII (19 times), while 11 showed a preference for hCA VII over hCA I (19 times), II (>100 times), VA (4 times), and XII (21 times).

4. Conclusions

We have synthesised a series of 2-aminoimidazolines, structurally related to Clonidine, and tested them on five different hCA isoforms (I, II, VA, VII and XIII). As the lead compound, none of the newly synthesised molecules was active on the ubiquitously expressed CA II; on the contrary, the compounds showed activity in the micromolar range on the other tested CA isoforms. Structure–activity relationships were derived, which were different on the various isoforms, suggesting that it could be possible, in this class of compounds, to find molecules, selective for a particular CA isoform. Indeed, from these preliminary modifications it has been possible to find two compounds, 10 and 11, with a promising preference towards, respectively, CA VA and VII. Work is underway to improve both potency and selectivity, in order to find new pharmacological tool to activate specific CA isoforms in pathologies characterised by their loss of functionality.

Disclosure statement

The authors report no conflict of interest.

Additional information

Funding

This work was supported by grants from the University of Florence [Fondo Ricerca Ateneo RICATEN18].