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. 2024 Mar 13;15(4):470–477. doi: 10.1021/acsmedchemlett.3c00484

Discovery of a New Class of 1-(4-Sulfamoylbenzoyl)piperidine-4-carboxamides as Human Carbonic Anhydrase Inhibitors

Davide Moi §,*, Serena Vittorio †, Andrea Angeli ‡, Claudiu T Supuran ‡, Valentina Onnis §
PMCID: PMC11017293  PMID: 38628786

Abstract

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A series of 1-(4-sulfamoylbenzoyl)piperidine-4-carboxamides deriving from substituted piperazines/benzylamines was designed, synthesized, and tested on human carbonic anhydrase (hCA). The inhibitory activity of the new sulfonamides was analyzed using acetazolamide (AAZ) as a standard inhibitor against hCA I, II, IX, and XII. Several sulfonamides showed both inhibitory activity at low nanomolar concentrations and selectivity against the cytosolic hCA II isoform, and the same trend was observed on the tumor-associated hCA IX and XII. The benzenesulfonamido carboxamides 11 and 15 were the most potent of the piperazino- and benzylamino-based series, respectively. Docking and molecular dynamics studies related the high selectivity of compound 11 toward the tumor-associated hCA isoforms to its capability to participate in favorable interactions within hCA IX and hCA XII active sites, whereas no such interactions were detected within both hCA I and hCA II isoforms.

Keywords: carbonic anhydrase enzyme inhibition, carboxamides, sulfonamides

Abnormal cell growth and spreading into neighboring tissues are common features of solid tumors, but this overgrowing is typically not followed by an adequate delivery of oxygen and nourishment due to the inadequate tumor vasculature.1 Hypoxia may activate angiogenesis, aggressiveness, and metastasis precesses, improving tumor survival and reducing anticancer drugs’ efficiency.2 This condition leads to significant changes in gene expressions, mediated by hypoxia-inducible transcription factor (HIF-1α), promoting glycolysis to enhance cell survival. The exploitation of the glycolysis metabolism resulted in a huge production of lactic acid with a consequent decrease of the cytosolic pH.3 In this context, pH regulatory systems are crucial for cancer cells’ survival and proliferation; thus, the development of molecules which alter this mechanism may be an effective strategy for developing anticancer drugs. Carbonic anhydrases (CAs) are ubiquitous metalloenzymes responsible for the reversible conversion of CO2 into hydrogen carbonate ions (HCO3–) and protons (H+), water-soluble products involved in pH regulation.4 Currently 15 CAs have been discovered and described in humans and are involved in several physiological and biochemical processes, such as pH homeostasis, gluconeogenesis, liquid secretion, and tumorgenicity. The active site of human CAs (hCAs) contains zinc ions in a tetrahedral geometric shape, coordinated by three amino acid residues, H94, H96, and H119, in addition to a H2O molecule or OH– ion.5−8 The hCA IX and hCA XII isoenzymes have been deeply studied due to their important role in cancer cell survival.9−11 Although small metabolic changes are tolerated in cancer cells, strong pH control is necessary for their survival and proliferation.12 Hydrogen carbonate ions, which are important pH buffers, are imported by transporters such as Cl–/HCO3– anion exchanger and Na+/HCO3– co-transporter, consuming cytosolic protons to give a new molecule of CO2 which leaves the cytoplasm to be hydrated by hCA IX and XII.13 These two CA isoforms control the intracellular pH and participate in extracellular acidification, which supports the tumor aggressiveness, being an extremely interesting biological target. Moreover, the development of selective CA IX and XII inhibition over off-target isoforms is crucial to avoid off-target effects.14,15 Generally, a Zn2+ binding group is required for the inhibition of CAs, and benzenesulfonamide is the most commonly used group due to its ability to coordinate the metal ion in a tetrahedral or bipyramidal way. Selective CA inhibitors can be obtained by maintaining the sulfonamide group and modifying the general scaffold of the molecule using the “tail approach”.16−18 It is possible to vary the tail functionality bound to a heterocyclic/aromatic portion, optimizing the interaction with the central and border parts of the active site, which are the most variable portions among the different CA isoforms.19

The structure of SLC-0111, a selective hCA IX inhibitor in phase I/II clinical trials for the treatment of advanced metastatic hypoxic solid tumors, was recently employed in our group for the preparation of a new class of 4-sulfamoylbenzoylpiperidine analogs with the aim of obtaining potent and selective hCA IX and XII molecules.20−22 With the intent to increase the knowledge about tail modification effects on the CA inhibitory activity and selectivity, in this study we present a small library of 1-(4-sulfamoylbenzoyl)piperidine-4-carboxamide derivatives (Figure 1). In these compounds, the benzenesulfonamide group is connected through a carbonyl group to a piperidine ring, facing the two tails with both the hydrophilic and hydrophobic regions of the active site. The flexibility and hydrophilicity were also improved by the piperazine and benzylamine amides, enhancing the hCA activity and selectivity on the cancer-related isoforms through advantageous interactions with restricted residues in the hydrophilic portion of the active site.

Figure 1.

Figure 1

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Drug design strategy for the generation of the new carbonic anhydrase inhibitors.

The new compounds 5–24 were prepared by following the synthetic pathway reported in Scheme 1. Using our previously reported synthetic procedure, the pathway started with the coupling of sulfanilamide 1 with ethylpiperidine-4-carboxylate 2 in the presence of 1-(3-(dimethylamino)propyl)-3-ethylcarbodiimide hydrochloride (EDCI) using dry acetonitrile (MeCN) to obtain the corresponding ethyl 1-(4-sulfamoylbenzoyl)piperidine-4-carboxylate 3. Intermediate 3 was than dissolved in ethanol and treated with aqueous sodium hydroxide to achieve the corresponding 1-(4-sulfamoylbenzoyl)piperidine-4-carboxylic acid 4. The carboxylic acid 4 was further treated with an equimolar amount of appropriately substituted benzylamine/piperazine in MeCN solution in the presence of EDCI, obtaining the desired amides 5–24 in 43–90% yields. 1H and 13C NMR and ESI-MS confirmed the structures of amides 5–24, while the purity grade was elucidated by elemental analysis.

Scheme 1. General Synthetic Procedure for 1-(4-Sulfamoylbenzoyl)piperidine-4-carboxamide Derivatives 5–24.

Scheme 1

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Reagents and conditions: (a) EDCI, HOBt, dry CH3CN, r.t. 12h, 77% yield; (b) LiOH, EtOH/H2O 3:1, r.t. 12h, 89% yield; (c) substituted piperazine, EDCI, HOBt, dry CH3CN, r.t. 12h, 43–64% yield; (d) substituted benzylamine, EDCI, HOBt, dry CH3CN, r.t. 12h, 46–90% yield.

The inhibitory activities on hCAs of benzenesulfonamides 5–13 (Table 1) and 14–24 (Table 2) have been explored by stopped-flow CO2 hydrase assay,23 using acetazolamide (AAZ) as reference compound. The cytosolic hCA I isoform has been taken as an off-target isoform, while both cytosolic hCA II and transmembrane hCA IX are outstanding targets for the treatment of glaucoma due to their role in maintaining intraocular pressure. On the other hand, hCA IX and hCA XII are already validated targets for cancer treatment due to their overexpression in solid tumors. To study the structure–activity relationship (SAR), the CA inhibitory activities against all four isoforms were examined in depth. All the piperazine-based compounds 5–13 inhibited hCA I in the nanomolar range. The best inhibitor compound was derivative 6, bearing a 4-methoxyphenyl group (Ki = 7.9 nM), while moving the methoxy group to the 3-position decreased the activity (compound 5, Ki = 38.6 nM). Compound 7, endowed with a 2-methylphenyl group, displayed a Ki of 11.8 nM, while the introduction of a second methyl group in the 2-position, as shown in compound 8, did not produce important changes in the inhibitory activity. The presence of a halogen atom on the phenyl ring, such as a 4-fluorine (compound 9) or a 4-chlorine (compound 10), generates compounds with Ki values of 8.9 and 9.7 nM, respectively. A decrease of the inhibitory activity is observed for compounds 11–13, substituting the piperazine ring with an aliphatic chain.

Table 1. Inhibition Activity of Compounds 5–13 on hCA I, hCA II, hCA IX, and hCA XII, Using AAZ as a Reference Compound.

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    Ki (nM)
Compd R hCA I hCA II hCA IX hCA XII
5 3-OCH3-C6H4 38.6 5.8 7.6 9.2
6 4-OCH3-C6H4 7.9 3.7 0.9 6.2
7 2-CH3-C6H4 11.8 5.7 3.9 9.4
8 2,3-(CH3)2-C6H3 16.8 7.1 7.4 3.9
9 4-F-C6H4 8.9 5.6 8.8 7.1
10 4-Cl-C6H4 9.7 4.4 5.2 7.6
11 CH2-C6H5 47.6 36.5 8.3 2.7
12 n-C7H15 68.5 37.7 5.6 18.7
13 n-C8H17 88.2 40.6 8.1 6.7
AAZ – 250 12.1 25.8 5.7
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Table 2. Inhibition Activity of Compounds 14–24 on hCA I, hCA II, hCA IX, and hCAX II, Using AAZ as Reference Compound.

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    Ki (nM)
Compd R hCA I hCA II hCA IX hCA XII
14 C6H5 9.0 3.9 4.7 3.6
15 2-CH3-C6H4 6.1 34.8 8.4 6.9
16 4-CH3-C6H4 5.6 3.8 0.8 80.1
17 3,5-(CH3)2-C6H3 5.6 4.4 4.2 94.6
18 2-OCH3-C6H4 8.7 2.9 6.1 33.4
19 2-Cl-C6H4 5.7 4.1 4.3 51.7
20 4-Cl-C6H4 6.8 3.8 0.9 75.7
21 2,4-Cl2-C6H3 8.8 3.9 4.8 65.3
22 3,4-Cl2-C6H3 8.4 4.2 2.1 75.3
23 3,5-Cl2-C6H3 6.5 3.2 3.2 6.6
24 4-F-C6H4 4.1 1.4 6.4 2.6
AAZ – 250 12.1 25.8 5.7
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All of the compounds of this series inhibited hCA II in the 3.7–40.6 nM range. Compound 6, containing the 4-methoxyphenyl group, was the compound with the best activity on this isoform (Ki = 3.7 nM). Moving the methoxy group to the 3-position slightly reduced the activity (compound 5, Ki = 5.8 nM), as did its substitution with a halogen, as in compounds 9 (4-fluorophenyl, Ki = 5.6 nM) and 10 (4-chlorophenyl, Ki = 4.4 nM). The introduction of a 2-methylphenyl group did not affect the inhibitory activity (compound 7, Ki = 11.8 nM), nor did the introduction of a second methyl to obtain the 2,3-dimethylphenyl compound 8 (Ki = 16.8 nM). Changing from an aromatic to an aliphatic substituent on the piperazine nitrogen resulted in a decrease of activity, maintaining the same trend observed for the hCA I inhibition.

All the newly synthesized compounds displayed Ki values in the low nanomolar range on the cancer-related isoform hCA IX. Compound 6 showed an inhibitory activity at sub-nanomolar levels (Ki = 0.9 nM), being the most potent compound so far. Compound 6 exhibited also good selectivity on hCA IX, being about 8-fold and 4-fold more selective on hCA IX as compared to hCA I and hCA II, respectively. Both moving and changing the methoxy group reduced the inhibitory activity from 4- to 10-fold. The introduction of an aliphatic chain in the “tail” portion resulted in a great selectivity on hCA IX, as we can see for compounds 11–13. The benzyl-substituted derivative 11, with a Ki of 8.3 nM, was about 6-fold and 4-fold more selective on hCA IX as compared to hCA I and hCA II, respectively, while the introduction of an aliphatic chain further improved the selectivity. The n-heptyl-substituted compound 12, with a Ki of 5.6 nM on hCA IX, was about 12-fold and 7-fold more selective against hCA I and hCA II, respectively. The n-octyl analog 13, with a Ki of 8.1 nM, was about 11-fold and 5-fold more selective on hCA IX than hCA I and hCA II, respectively.

The same trend was observed for the inhibition on hCA XII. Indeed, even though all the compounds 5–13 were active on the target isoform at low nanomolar levels, the “aromatic tails” are related with poor selectivity, while compounds 11–13, endowed with “aliphatic tails”, displayed good inhibitory activity and selectivity on hCA XII. The benzyl derivative 11 showed a Ki of 2.7 nM, making it the best inhibitor of the series on hCA XII. Compound 11 was about 17-fold more selective as compared to hCA I and about 13-fold more selective as compared to hCA II, being the most selective compound so far. Compound 12 inhibited the target isoform with a Ki of 18.7 nM, while moving from the n-heptyl to the n-octyl analog (compound 13) improved the activity and the selectivity. Compound 13 inhibited hCA XII with a Ki of 6.7 nM, being about 13-fold more selective as compared to hCA I and about 6-fold more selective as compared to hCA II. Overall, compounds 11–13 displayed the best activity/selectivity combinations for the inhibition of the cancer-related isoforms hCA IX and hCA XII.

The benzylamino-based analogs 14–24 displayed activity in the nanomolar range for all of the tested isoforms. Starting from the hCA I, compound 14, bearing a benzylamino group, inhibited the isoform with a Ki of 9.0 nM. The introduction of a 2-methylbenzyl (compound 15, Ki = 6.1 nM), a 4-methylbenzyl (compound 16, Ki = 5.6 nM), or two methyl groups (compound 17, Ki = 5.6 nM) did not affect the activity. The same trend was observed by introducing one or two chlorine atoms on the benzyl ring (compounds 19–23). The introduction of a 4-fluorine atom (compound 24, Ki = 4.1 nM) slightly improved the inhibitory activity. Compound 24 was also found to be the best hCA II inhibitor, with a Ki of 1.4 nM, while substitution of the fluorine atom with either a 4-chlorine atom (compound 20, Ki = 3.8 nM) or a 4-methyl group (compound 16, Ki = 3.8 nM) reduced the activity on hCA II. Moving the methyl group into the 2-position resulted in a 10-fold reduction in activity, as observed for compound 15 (Ki = 34.8 nM). Interestingly, replacing the 2-methyl group with a 2-methoxy group (compound 18) decreased the Ki value from 34.8 nM to 2.9 nM. The presence of two chlorine atoms at different position in the aromatic ring did not affect the inhibitory activity (compounds 21–23, Ki = 3.9, 4.2, and 3.2 nM, respectively).

Compounds 14–24 inhibited hCA IX at low nanomolar levels, and specifically compounds 16 and 20 inhibited this isoform at sub-nanomolar levels, with Ki values of 0.8 and 0.9 nM, respectively. Compound 16 showed about 7-fold and 5-fold more selectivity on hCA IX as compared to hCA I and hCA II, respectively. Moving the 4-methyl group into the 2-position resulted in about a 10-fold reduction in the activity on the target isoform (compound 15, Ki = 8.4 nM), as did the introduction of a second methyl group (compound 17, Ki = 4.2 nM).

Important differences in activities were also observed upon moving the chlorine atom on the benzyl ring. Starting from compound 20, moving the chlorine atom to the 2-position resulted in a 5-fold decrease of activity (compound 19, Ki = 4.2 nM). The same reduction in activity was observed upon introducing a second chlorine atom in the 2-position (compound 21, Ki = 4.8 nM), while the introduction of a second chlorine atom in the 3-position partially restored the activity (compound 22, Ki = 2.1 nM). Substitution of the 4-chlorine atom with a 4-fluorine atom decreased the activity by 7-fold (compound 24Ki = 6.4 nM). Moving on to the activity on hCA XII, the best compound of the series was found to be the 4-fluorine derivative 24, showing a Ki of 2.6 nM. Exchanging the fluorine with a 4-chlorine atom resulted in a 30-fold decrease in the activity. The same tendency was observed for the 2-chlorine analog (compound 19, Ki = 51.7 nM) as well as for the 2,4-dichlorophenyl analog (compound 21, Ki = 65.3 nM) and for the 3,4-dichlorophenyl analog (compound 22, Ki = 75.3 nM). Interestingly, the 3,5-dichlorophenyl analog partially recovered the activity (compound 23, Ki = 6.6 nM). The unsubstituted benzyl compound 14 displayed a Ki of 3.6 nM, making it one of the best hCA XII inhibitors in this series. The presence of a 2-methylbenzyl group slightly reduced the activity (compound 15, Ki = 6.9 nM), while moving the methyl group into the 4-position (compound 16, Ki = 80.1 nM) or introducing a second methyl group in the aromatic ring (compound 17, Ki = 94.6 nM) drastically decreased the activity.

Molecular docking was carried out to elucidate the binding mode in the four analyzed hCA isoforms. Derivatives 11 and 15 were selected as representative compounds of the piperazino- and benzylamino-based series, respectively, as they showed the best selectivity toward the tumor-associated isoforms, hCA IX and hCA XII. The benzenesulfonamide moiety is deeply inserted into the hCA’s active site, with the sulfonamide group coordinating the Zn2+ ion stabilized by hydrogen bonds with Thr199 and the benzene ring involved in hydrophobic contacts with L198 and V121 (A121 in hCA I). When bound to hCA I, the long tail of compound 11 is oriented toward A132, A135, and L131, involved in hydrophobic contacts with the inhibitor (Figure 2A), while a different orientation was observed for the 2-methylbenzylamine moiety of compound 15, which is located close to P201 and P202 and is implicated in hydrophobic interactions with the aromatic ring (Figure 3A). Derivative 15 could be further stabilized by an H-bond with Q92. Within the hCA II binding pocket, compound 11 might engage hydrophobic contacts with F131 and I91 through the piperidine ring, while no significant interactions were detected for the tail portion (Figure 2B). Derivative 15 could approach P201, forming a H-bond through the amino group of the tail, and W5, establishing aromatic and hydrophobic interactions through the 2-methylbenzyl ring (Figure 3B).

Figure 2.

Figure 2

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Binding modes of compound 11 (cyan sticks) into the active sites of A) hCA I, B) hCA II, C) hCA IX, and D) hCA XII. The residues involved in the interactions with the inhibitors are highlighted as sticks. H-bond interactions are displayed as blue dashed lines.

Figure 3.

Figure 3

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Binding modes of compound 15 (green sticks) into the active sites of A) hCA I, B) hCA II, C) hCA IX, and D) hCA XII. The residues involved in the interactions with the inhibitors are highlighted as sticks. H-bond interactions are displayed as blue dashed lines.

Concerning the hCA IX isoform, both compounds 11 and 15 could elicit hydrophobic contacts with V131 through the piperidine ring while their tails occupy a niche lined by S3, W5, V19, S20, P201, and P202 involved in hydrophobic contacts with the aromatic rings of the inhibitors’ tails. Derivative 15 could also establish two H-bonds between (i) the amino group of the tail and the backbone of P200 and (ii) the carbonyl group linked to the benzenesulfonamide portion and Q92. Additional hydrophobic contacts were detected between the piperidine ring of compound 11 and L135. Finally, when bound to the hCA XII active site, compound 11 might elicit aromatic interactions with H94 through the benzenesulfonamide ring and a H-bond with S132 through the carbonyl group linked to the molecule tail. Instead, derivative 15 might engage two H-bonds between (i) the amide carbonyl group linked to the benzenesulfonamide moiety and Q92 and (ii) the carbonyl group linked to the molecule’s tail and S135. Both derivatives can establish hydrophobic contacts with A131 through the piperidine ring.

In order to gain more insight into the hCA isoform selectivity of derivatives 11 and 15, their binding modes were further explored by performing 200 ns molecular dynamics (MD) simulations. MD has been widely recognized as a useful tool to investigate the dynamic behavior of macromolecules, which strongly influences the protein–ligand recognition process.24,25 The stability of the simulated systems was checked by root-mean-square deviation (RMSD) analysis, as shown in Figures S1 and S2. Both proteins and ligands exhibited a quite stable behavior throughout the simulation time, maintaining RMSD values lower than 3 Å. The distance between the NH– of the sulfonamide moiety and the Zn2+ ion was monitored along the trajectories yielding values of ∼2 Å, in good agreement with the literature.26 Clustering analysis was carried out to obtain representative conformations of the systems. The representative structures of the most populated clusters of each simulated system are displayed in Figures 4 and 5. In all of the simulated complexes, the benzenesulfonamide moiety retained the H-bond interactions with T199 and the hydrophobic contacts with L198. The results obtained from the MD simulations involving hCA I revealed that the tail of compound 11 shifts toward V62, forming hydrophobic contacts through its phenyl moiety (Figure 4A). Concerning compound 15, during the trajectory the methylbenzylamine portion moves toward L131, A132, and A135, which establish hydrophobic interactions with the 2-methylbenzyl ring of the inhibitor (Figure 5A). The piperazine ring elicits hydrophobic contacts with P202, A121, and F91. The most persistent conformer of 15 maintains the H-bond with Q92 detected in the initial docking pose. These outcomes might explain the higher affinity toward hCA I observed for derivative 15 if compared to compound 11. Inside the hCA II binding pocket, the most prevalent conformation of compound 11 did not exhibit relevant interactions with the surrounding residues except for hydrophobic contacts between the piperidine ring and V135 and L141 (Figure 4B). Regarding derivative 15, during the MD simulation the carbonyl group linked to the piperidine ring is able to reach Q92, forming a H-bond. (Figure 5B) The 2-methylbenzylamine moiety moves toward V135, which establishes hydrophobic contacts with the inhibitor, causing the loss of the H-bond with P201 observed in the starting complex (Figure 3B).27

Figure 4.

Figure 4

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Most persistent conformers obtained from the MD simulation of compound 11 in complex with A) hCA I, B) hCA II, C) hCA IX, and D) hCA XII. H-bond interactions are displayed as blue dashed lines.

Figure 5.

Figure 5

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Most persistent conformers obtained from the MD simulation of compound 15 in complex with A) hCA I, B) hCA II, C) hCA IX, and D) hCA XII. H-bond interactions are displayed as blue dashed lines.

Within the hCA IX active site, the most persistent conformer of inhibitor 11 (Figure 4C) conveniently approaches Q92 and Q67, forming H-bonds with their side chains while retaining the hydrophobic contacts with V131 and L135 detected in the initial docking pose (Figure 2C). During the MD simulation, the tail of compound 11 shifts toward D132, which establishes a salt bridge with the positively charged nitrogen of the piperazine ring, while the phenyl ring is stabilized by pi-cation interactions with R130. The most prevalent conformer of 15 adopts a binding orientation within the hCA IX active site similar to the starting pose, with the 2-methylbenzylamine moiety inserted into a sub-pocket lined by S3, W5, V19, S20, P201, and P202 (Figure 5C). However, due to the rotation of the amide bond linked to the molecule’s tail, the H-bond with P202 observed in the starting complex is not retained during the simulation. Conversely, the H-bond involving Q92 is maintained, while an additional H-bond between Q67 and the carbonyl group linked to the benzenesulfonamide moiety was detected during the MD trajectory. The MD simulation of compound 11 in complex with hCA XII revealed that the inhibitor conveniently approaches Q92, engaging a H-bond with its side chain, while the piperazine-based tail shifts toward N136, forming a H-bond through the protonated nitrogen of the piperazine ring (Figure 4D). The hydrophobic contacts observed in the starting pose (Figure 2D) are retained during the trajectory. The most persistent conformer of compound 15 (Figure 5D) maintains the H-bonds involving Q92 and S135 detected in the docking outcomes and establishes additional hydrophobic contacts with L141.

The results of our computational studies suggested that the higher selectivity of compound 11 toward the tumor-associated hCA isoforms is ascribable to its capability to elicit profitable contacts within hCA IX and hCA XII active sites, while no significant interactions were detected within both hCA I and hCA II isoforms. Concerning derivative 15, the isoform selectivity on hCA I, hCA IX, and hCA XII is mainly due to the engagement of peculiar residues of the above-mentioned isoenzymes, which enhances the stability of the inhibitors within the pocket. In more details, within hCA I the inhibitor favorably contacts L131 and A132, while the presence of the more hindered F131 in hCA II exposes the inhibitor’s tail toward the solvent. When bound to hCA IX, the binding of 15 is stabilized by an additional H-bond with Q67, which is replaced by N67 in hCA II. Due to its shorter side chain, N67 did not provide any contact with the inhibitor in the hCA II binding pocket. Finally, the more polar hCA XII active site allows the formation of a H-bond with the peculiar S135 (V135 in hCA II) which contributes to the stabilization of the molecule’s tail.

The predicted drug-like properties of compounds 11 and 15 were examinated using SwissADME software,28 showing significant oral bioavailability scores, noticeable pharmacokinetic profiles, and drug-like properties (Table 3). The predicted analysis showed that these compounds exhibited no Lipinski violation and high gastrointestinal absorption, comparable with the predicted results for AAZ.

Table 3. Predicted Drug-like Properties of Compounds 11, 15, and Acetazolamide.

Compd MW MlogP < 5 H-bond acceptors H-bond donors Lipinski violations GI absorption Bioavailability score PAINS
11 470.58 1.43 6 1 No High 0.55 0
15 415.51 1.87 5 2 No High 0.55 0
AAZ 221.24 –2.34 6 2 No High 0.55 0
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In conclusion, this study led to the discovery of 1-(4-sulfamoylbenzoyl)piperidine-4-carboxamides as new human carbonic anhydrase inhibitors, with the purpose of investigating the impact of piperazine and benzylamine amides as “tails” of the inhibitors. All the compounds inhibited the activity of the human carbonic anhydrases hCA I, hCA II, hCA IX, and hCA XII at nanomolar concentrations, while compounds 6, 16, and 20 inhibited the cancer-related isoform hCA IX at sub-nanomolar concentrations (Ki = 0.9, 0.8, and 0.9 nM, respectively). Compounds 11 and 15 displayed high activity and selectivity on cancer-related isoforms hCA IX and hCA XII, confirmed by the interactions with specific residues of the isoenzymes, which improves the stability of the inhibitors, endorsed by both docking analysis and molecular dynamics studies. These preliminary results demonstrated the efficacy of 1-(4-sulfamoylbenzoyl)piperidine-4-carboxamides as a starting point for the further treatment of CA-expressing cancer cell lines.

Glossary

Abbreviations

CA/CAs

carbonic anhydrase/s

EDCI

1-(3-(dimethylamino)propyl)-3-ethylcarbodiimide hydrochloride

OHBt

1-hydroxybenzotriazole

AAZ

acetazolamide

MD

molecular dynamics

RMSD

root-mean-square deviation

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmedchemlett.3c00484.

  • Materials and methods; procedures for the synthesis of intermediate 4; general procedure for the synthesis and characterization data of final compounds 5–24; carbonic anhydrase inhibition assay protocol; computational studies; 1H and 13C NMR spectra of final compounds 5–24; additional references (PDF)

This research was funded by the Italian Ministero dell’Istruzione, Università e della Ricerca, Italy; grant PRIN 2017, Prot. No. 2010E84AA4_002.

The authors declare no competing financial interest.

Supplementary Material

ml3c00484_si_001.pdf (2.2MB, pdf)

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