Carbonic anhydrase inhibitors: synthesis and inhibition of cytosolic/tumor-associated carbonic anhydrase isozymes I, II, and IX with sulfonamides incorporating thioureido-sulfanilyl scaffolds

doi:10.1016/j.bmcl.2005.02.087

Bioorganic & Medicinal Chemistry Letters

Volume 15, Issue 9, 2 May 2005, Pages 2359-2364

https://doi.org/10.1016/j.bmcl.2005.02.087 Get rights and content

Abstract

The tumor-associated transmembrane carbonic anhydrase (CA, EC 4.2.1.1) isozyme IX (CA IX) is overexpressed in hypoxic tumors and appears to be involved in acidification of the tumor microenvironment, a process correlated with cancer progression and bad prognosis. The acidification may be reduced by inhibiting the enzyme with potent sulfonamide/sulfamate CA inhibitors. A series of such aromatic sulfonamides incorporating thioureido-sulfanilyl moieties has been prepared and investigated for its interaction with the catalytic domain of the human isozyme hCA IX. The key intermediates in the synthesis were obtained by reacting sulfanilamide, homosulfanilamide, or 4-aminoethylbenzenesulfonamide with 4-acetamido-benzenesulfonyl chloride followed by deacetylation and reaction with thiophosgene. The obtained isothiocyanato sulfonamides were reacted with aliphatic or aromatic primary amines or hydrazines, leading to the corresponding thioureas. Some of these compounds showed excellent inhibitory properties against isozymes I, II, and IX, with several inhibitors also presenting selectivity for the inhibition of CA IX over that of the ubiquitous isozyme CA II. Such sulfonamides may constitute interesting candidates for the development of novel antitumor therapies based on the inhibition of the CA isozymes overexpressed in hypoxic tumors. Due to the highest expression of CA IX in clear renal cell carcinoma and its chemo/radioresistance, our efforts are first of all directed to generate effective therapeutic strategies for the cure of this malignancy.

Graphical abstract

Introduction

We have recently shown¹ that the tumor-associated carbonic anhydrase (CA, EC 4.2.1.1) isozyme IX, hCA IX, is involved in acidification of hypoxic tumors, and that this process may be diminished (and probably abolished) by inhibiting the enzyme with potent sulfonamide inhibitors. This constitutes the proof-of-concept demonstration that inhibiting the tumor-associated CAs (two such isozymes are presently known, CA IX and CA XII)2, 3, 4, 5, 6 may be relevant for the design of novel antitumor therapies. Thus, much work is currently being done in this and other laboratories for discovering either small molecule, iRNA-s or immunologic-based agents targeting these proteins present in a multitude of hypoxic tumors.7, 8, 9, 10

Sulfonamide CA inhibitors (CAIs), such as acetazolamide AZA, methazolamide MZA, ethoxzolamide EZA, or dichlorophenamide DCP among others, initially played a crucial role in the understanding of renal physiology and pharmacology, leading then to the development of widely used drugs such as the benzothiadiazine and high ceiling diuretics.1, 2, 3, 4, 5 More recently, this class of drugs showed important applications for the development of antiglaucoma agents,² being also useful for the design of other types of pharmacological agents, as it will be shown here shortly. Thus, a recent and new field in CAI research has been opened by the report of the potent antitumor properties of a rather large number of sulfonamide CAIs, as well as by the isolation, purification, and characterization of the isozymes predominantly present in tumor cells, such as CA IX and CA XII.1, 2, 3, 4, 5, 6 The mechanisms by which these isozymes participate in the tumor cell growth and differentiation, as well as those by which their inhibitors interfere with the tumor growth only now begin to be understood,¹ but important advances in these directions have recently been achieved.¹ Indeed, several laboratories are involved in the synthesis, evaluation, and in vitro/in vivo antitumor testing of novel types of CAIs with potential application as anticancer therapeutic agents.6, 7, 8, 9, 10, 11, 12 Furthermore, a compound of this type—indisulam IND—has progressed to Phase II clinical trials for the treatment of solid tumors.6, 9, 10

CA IX gene regulation is tightly dependent on the HIF-1α gene expression, which is evoked by environmental hypoxic stimuli. Initially, the strategy to counteract hypoxic tumor growth was by targeting of the HIF-1α protein, but its expression is usually transient and its half-life is very short.5, 6, 7 On the contrary, CA IX is a very stable protein. The activity of CA IX, whose half-life is of at least 96 h, is long lasting, even in the case of hypoxia abrogation. Finally, CA IX is anchored to the outermost layer of the cell and not placed in the inner nuclear compartment (as HIF-1α is), making this enzyme a druggable interesting target.1, 5, 6, 7

In previous work from this laboratory,¹³ we have developed thioureido-containing sulfonamide CAIs derived from simple aromatic sulfonamides, such as sulfanilamide 1 and homosulfanilamide 2, which showed excellent CA I, II, and IV inhibitory properties and were effective topically acting antiglaucoma agents in an animal model of this disease (some of the above-mentioned isozymes are involved in aqueous humor secretion within the eye, being the targets of antiglaucoma sulfonamides).1, 2, 3, 4 Continuing this work and the same type of chemistry,¹³ but using as lead molecule benzolamide BZA (an orphan drug belonging to the family of CAIs),² we then reported novel thioureido-containing benzolamide-like compounds, which were also shown to act as very potent inhibitors of the transmembrane, tumor-associated isozyme CA IX (in addition to the strong inhibition of the ubiquitous, cytosolic isozymes I and II).¹⁴ Using these last compounds as lead molecules,¹⁴ we report this time novel derivatives incorporating thioureido-sulfanilyl scaffolds. Thus, the thiadiazole scaffold of the above mentioned lead compounds has been changed to an 1,4-phenylene moiety, and the spacer between this structural element and the thioureido-sulfanilyl scaffold has been also varied, with 0, 1, and 2 CH₂ moieties present in the new compounds reported in this paper. Some of these compounds showed excellent inhibitory properties against isozymes CA I, II, and IX, as well as some specificity for inhibiting the last isozyme over the ubiquitous cytosolic isoform CA II.

Section snippets

Chemistry

The reaction of amino-substituted sulfonamides such as sulfanilamide 1, homosulfanilamide 2, and 4-aminoethylbenzenesulfonamide 3 with alkyl/arylsulfonyl halides has been investigated in detail by our group.¹⁵ During the previous studies, we also reported the sulfanylated derivatives 5–7,^15a which were obtained by treating the parent amino sulfonamides 1–3 with 4-acetamidobenzenesulfonyl chloride 4, followed by deacetylation with concentrated hydrochloric acid for removal of the protecting

Carbonic anhydrase inhibition

Inhibition data against three physiologically relevant isozymes, that is, the cytosolic isoforms hCA I and II and the membrane-bound, tumor-associated isozyme hCA IX (all of them of human origin) with the new compounds 11–13 as well as the standard, clinically used CA inhibitors acetazolamide AZA, methazolamide MZA, ethoxzolamide EZA, dichlorophenamide DCP, and indisulam IND, are shown in Table 1. Inhibition data for the parent sulfonamides from which the new derivatives were obtained (such as 1

Conclusions

A series of aromatic sulfonamides incorporating thioureido-sulfanilyl moieties have been prepared and investigated for its interaction with the catalytic domain of the human isozyme hCA IX as well as with the cytosolic isozymes I and II. The key intermediates in the synthesis were obtained by reacting sulfanilamide, homosulfanilamide, or 4-aminoethylbenzenesulfonamide with 4-acetamido-benzenesulfonyl chloride followed by deacetylation and reaction with thiophosgene. The obtained isothiocyanato

Acknowledgments

This research was financed in part by a grant of the 6th Framework Programme of the European Union (EUROXY project) and by a grant from Miroglio Vestebene S.p.A Via Santa Barbara 11 12051 (Alba, Cuneo, Italy). J.Y.W. is grateful to CSGI, University of Florence and University of Montpellier II for a travel grant to Florence.

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Carbonic anhydrase inhibitors: synthesis and inhibition of cytosolic/tumor-associated carbonic anhydrase isozymes I, II, and IX with sulfonamides incorporating thioureido-sulfanilyl scaffolds

Abstract

Graphical abstract

Introduction

Section snippets

Chemistry

Carbonic anhydrase inhibition

Conclusions

Acknowledgments

FEBS Lett.

Expert Opin. Invest. Drugs

Bioorg. Med. Chem. Lett.

Expert Opin. Ther. Pat.

Expert Opin. Ther. Pat.

Med. Res. Rev.

Expert Opin. Ther. Pat.

J. Enzyme Inhib. Med. Chem.

Curr. Med. Chem.—Cardiovasc. Hematol. Agents

Cancer-related carbonic anhydrase isozymes

Curr. Med. Chem.

Curr. Cancer Drug Targets

Bioorg. Med. Chem. Lett.

Cancer Res.