New series of sulfonamides containing amino acid moiety act as effective and selective inhibitors of tumor-associated carbonic anhydrase XII

Abstract

New benzenesulfonamides incorporating water solubilizing moieties were synthesized using N-α-acetyl-l-lysine or γ-aminobutyric acid as scaffolds followed by the conversion of their terminal amino group to the guanidine one. Their inhibition activity was assessed by determining their K_Is values against the human (h) carbonic anhydrase (CA, EC 4.2.1.1) isoforms hCA I, II, IX and XII. Some of these compounds were medium potency inhibitors of the cytosolic (CA I, II) and transmembrane (CA IX) isoforms and highly effective, nanomolar inhibitors of the second transmembrane isoform hCA XII. Some of these sulfonamides possessing good selectivity inhibition for the tumor-associated CA XII isoform over the cytosolic and physiologically dominant isoforms CA I and II may be used as tools to develop new anticancer agents.

• Amino acid
• carbonic anhydrase inhibitor
• sulfonamide
• tumor-associated CA XII

Introduction

Carbonic anhydrases (CAs, EC 4.2.1.1) are metalloenzymes that catalyze a simple and physiologically relevant reaction in all life kingdoms: the carbon dioxide hydration to bicarbonate and protons1–7. In humans, 15 CA isoforms belonging to the α-CA family are known to be involved in many physiologic processes. Due to the fact that different isoforms are involved in diverse physiologic or pathologic processes1^,2, this class of enzyme constitutes drug targets for diuretics, antiglaucoma agents, antiepileptics, antiobesity and antitumor drugs1–7.

It has been found that isoforms CA IX and XII are transmembrane proteins characterized by an N-terminal extracellular domain, a putative transmembrane α-helix and an intramolecular C-terminal domain1^,8. Both of them are overexpressed in a large number of human cancers, such as glioblastoma9, colorectal10 and breast cancer11 as markers of hypoxia and thus used in clinical settings1^,2^,8.

Although it has been demonstrated that the overexpression of CA IX can be initiated by the activation of the hypoxia-inducible factor-1 (HIF-1) cascade6, the molecular mechanisms at the basis of the induction of CA XII are not so well understood yet4. However, CA XII is known to be involved in breast cancer and recently it has been shown to be regulated by the estrogen receptor α (ERα), a hormone-regulated transcription factor belonging to the superfamily of nuclear receptors, which is expressed in about 70% of breast cancers12. CA XII has also been identified in brain tumors, such as astrocytoma and glioblastoma13–16.

The main consequence of an overexpression of hypoxia-induced CA IX and CA XII isoforms is a pH imbalance, with most of hypoxic tumors having lower pH value (about 6–6.5) compared with that characteristic of normal tissue equal to 7.417–19.

Therefore, hypoxia-induced CA IX and CA XII overexpression can be considered as the major tumor pH-regulating enzymes, and their inhibition may represent a promising target for antitumor therapy1^,8^,13–16.

Although all α-CAs, including CA IX and XII, are inhibited by several main classes of inhibitors, such as the inorganic anions20, the phenols21 and the coumarins22 the sulfonamides and their bioisosteres (sulfamates, sulfamides, etc.) are still the most investigated CAIs18^,19^,23. Many low nanomolar sulfonamide CA IX17^,18 or CA XII24 inhibitors have been identified in the last few years. It has been recently shown that in vitro, in cell cultures and in animals with transplanted tumors, CA IX inhibition with sulfonamides led to a return to more normal extracellular pH values, with consequential delay of tumor growth5^,6. Furthermore, in mice suffering from T lymphoma, it was observed that the inhibition of the overexpressed CA XII by sulfonamides decreased cell proliferation and induced apoptosis16.

In this article we continue our interest in sulfonamide CAIs as potential anticancer agents, and report the synthesis of benzenesulfonamides incorporating amino acid scaffolds and guanidine moieties. These new compounds were investigated for the inhibition of the overexpressed in hypoxic tumors human CA (hCA) IX and XII isoforms over the physiologically dominant, cytosolic ones hCA I and II, in order to develop possibly isoform-selective CA IX/XII inhibitors as anticancer agents5.

Materials and methods

Chemistry

Anhydrous solvents and all reagents were purchased from Aldrich, Merck or Carlo Erba. All reactions involving air- or moisture-sensitive compounds were performed under nitrogen atmosphere using oven-dried glassware and syringes to transfer solutions. Nuclear magnetic resonance (¹H-NMR and NOE-difference) spectra were determined in CDCl₃ and DMSO-d₆ on a Varian XL-400 (400 MHz) spectrometer (Germany). Chemical shifts (δ scale) are reported in parts per million downfield from tetramethylsilane used as an internal standard. The assignment of exchangeable protons (OH and NH) was confirmed by the addition of D₂O. Electron ionization mass spectra (70 eV) were recorded on a Hewlett-Packard 5989 Mass Engine Spectrometer (Germany). Analytical thin-layer chromatography was performed on Merck silica gel F-254 plates (Darmstadt, Germany). For flash chromatography Merck Silica gel 60 was used with a particle size 0.040–0.063 mm (230–400 mesh ASTM). The synthesis and the full characterization of the compounds investigated here (4–25) as hCA inhibitors are reported elsewhere25.

CA inhibition studies

An applied photophysics stopped-flow instrument has been used for assaying the CA catalyzed CO₂ hydration activity26. 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 7.4) and 20 mM NaBF₄ (for maintaining constant the ionic strength), following the initial rates of the CA-catalyzed 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 (10 mM) were prepared in distilled–deionized water and dilutions up to 0.01 nM were done thereafter with distilled–deionized water. Inhibitor and enzyme solutions were preincubated together for 15 min at RT prior to assay, in order to allow for the formation of the E-I complex. The inhibition constants were obtained by nonlinear least-squares method using PRISM 3, and the Cheng–Prusoff equation, as reported earlier27^,28, and represent the mean from at least three different determinations. All CAs were recombinant proteins obtained as reported earlier by these groups27^,28.

Results and discussion

Chemistry

Sulfonamides derivates are known to be highly effective CAIs1–3. However, their low water solubility represents one of the main weak points of this class of pharmacological agents29. This is probably due to the fact that the highly polar sulfamoyl moiety generally is attached to aromatic/heterocyclic rings and no water hydrophilic moieties are present in the molecule. Therefore, in this work we considered to add amino acyl moieties as tails to the scaffolds of aromatic sulfonamides, in order to increase the water solubility of such compounds30. We then used the tail approach for our drug design employing N-α-acetyl-l-lysine or γ-aminobutyric acid (GABA) as scaffolds, and the conversion of their terminal amino group to the guanidine one, for synthesizing compounds incorporating 4-aminoethyl/methyl-benzenesulfonamide as well as metanilamide/sulfanilamide as heads for binding to the zinc ion from the enzyme active site (Schemes 1 and 2, and Chart 1).

Scheme 1. Synthesis of N-α-acetyl-l-lysine sulfonamides 4–12.

Scheme 2. Synthesis of GABA-containing sulfonamides 14–21.

Chart 1. Structure of compounds 22–25 and the clinically used sulfonamide acetazolamide AAZ.

The first group of compounds were obtained starting from N-α-acetyl-l-lysine 1, followed by the protection of the ɛ-amino group with the Boc moiety, and the coupling with benzenesulfonamides 2 or 3 to give the ɛ-Boc-α-acetyl-protected-lysyl sulfonamides 4 and 5 (Scheme 1). After removing of the Boc (tert-butyloxycarbonyl) protecting group in 3 M HCL, the hydrochlorides 6, 7 were treated with N,N′-di-Boc-N″-trifluoromethane-sulfonylguanidine 8 for converting the terminal amino moiety to the di-Boc-protected guanidines 9 and 10. Deprotection of the guanidine moiety in the presence of acid led to the guanidine-substituted sulfonamides 11 and 12 (Scheme 1).

The remaining derivatives were synthesized using a similar approach. In this case GABA 13 was used as starting material following by similar derivatization reactions, compounds 14–21 incorporating guanidine–GABA moieties were thus obtained (Scheme 2). Furthermore, several metanilamide (22, 23), sulfanilamide (24) and 4-carboxy-benzenesulfonamide (25) derivatives incorporating lysyl- or GABA moieties were also obtained by following the same synthetic procedure (Chart 1).

CA inhibition

Inhibition data with the new group of sulfonamides 4–25 reported here, against hCA isoforms hCA I, II (cytosolic) and hCA IX, XII (transmembrane and tumor associated) are shown in Table 1. Therefore, the following structure–activity relationship can be observed from data reported in mentioned table:

1. The new sulfonamides reported here were generally ineffective or medium potency inhibitors against the cytosolic slow isoform hCA I, with K_Is values ranging between 88.9 and 6030 nM. However, only two sulfonamides, 14 and 23, both containing Boc–GABA moieties, showed to be effective inhibitors against this isoform, with K_Is of 19.8–22.1 nM. For all substitution patterns, generally the 4-aminoethyl-benzenesulfonamides were more effective hCA I inhibitors compared with the corresponding 4-aminomethylbenzenesulfonamide derivates (Table 1).

2. The physiologically dominant human isoform hCA II was effectively inhibited by derivatives 14 and 23–25. Indeed, these compounds showed K_Is in the range of 4.4–29.4 nM, of the same order of magnitude as acetazolamide (AAZ; K_Is of 12 nM). These compounds again incorporate the Boc-GABA moieties, except 25 which is a derivative of 4-carboxybenzenesulfonamide to which the derivatization was achieved at the α-amino moiety, whereas the ɛ-amino one was protected by the benzyloxycarbonyl group. Some of the derivatives reported here, such as 4, 5, 12, 15, 16, 18 and 19, were medium potency hCA II inhibitors (K_Is in the range of 44.0–91.9 nM). The remaining compounds showed to be ineffective inhibitors against hCA II, with K_Is in the range of 384–5100 nM (Table 1).

3. The transmembrane isoform, hCA IX was poorly inhibited by most of the derivatives reported here. Generally the GABA derivatives were better hCA IX inhibitors compared with the N-α-acetyl-l-lysines possessing similar sulfonamide and protecting group scaffolds (Table 1). Indeed, the compounds 4, 5, 7, 9–12 and 22 have shown high K_Is values in the range of 1373–2474 nM (except for the compound 16 with K_I of 1015 nM, which contains a GABA moiety). Although this tumor-associated isoform, hCA IX, was slightly better inhibited by the derivatives 14, 17, 18, 20, 21 and 23 containing a GABA moiety (K_Is of 517.8–862.2 nM) and derivatives 6 which contain N-α-acetyl-l-Lysine (K_I of 876.4 nM), the most efficient hCA IX inhibitors were 15, 19 and 24, which showed inhibition constants between 65.7 and 95.7 nM. The best activity is correlated with the presence of derivatized GABA moieties and 4-amino-ethylbenzenesulfonamide (15 and 19) and 4-aminobenzenesulfonamide (24) head group. However, the best hCA IX inhibitor was compound 25, (containing a different scaffold, as mentioned above) with K_I value of 47.7 nM, which was anyhow a weaker inhibitor compared with the clinically used inhibitor AAZ (K_I of 25 nM).

4. The second transmembrane isoform associated with tumors, hCA XII, was much better inhibited by the new sulfonamides investigated here, compared with hCA IX. Several low nanomolar hCA XII inhibitors are reported here, such as 14, 18, 20, 24 and 25 derivatives with K_Is in the range of 4.8–12.3 nM. Although many of the highly effective hCA XII inhibitors reported here have not possessed excellent selectivity ratios for inhibiting this transmenbrane isoform over the cytosolic ones, the most interesting CA XII inhibitor listed here was derivate 20 which has shown selectivity ratios as high as 139 against hCA I and 67 against hCA II (Table 1). The remaining derivatives 10, 15, 16, 19 and 23 have showed moderate inhibitory potency against this isoform (K_Is in the range of 26.5–69 nM).

Table 1. Inhibition data against human isoforms hCA I, II (cytosolic) and IX, XII (transmembrane and tumor-associated) of sulfonamides 4–25 reported here and AAZ (as standard inhibitor) by a stopped-flow CO₂ hydrase assay26.

		KI (nM)*
Compound	hCA I	hCA II	hCA IX	CA XII
4	484	71.2	1718.2	150.7
5	430	75.9	2130	157.7
6	2330	563	876.4	1356
7	626	473	2242	201.2
9	2310	384	2474	126.2
10	519	452	1373	31.6
11	3150	824	2216	849.7
12	289	44.0	1795	142.2
14	19.8	9.8	517.8	12.1
15	253	91.9	65.7	69.0
16	2270	83.5	1015	59.0
17	517	197	751.4	145.6
18	193	83.1	540.9	12.3
19	267	71.3	95.7	53.6
20	972	470	862.2	7.0
21	2120	847	737.3	246.8
22	6030	5100	1825	413.7
23	22.1	5.9	648.6	26.5
24	151.3	29.4	67.5	6.5
25	88.9	4.4	47.7	4.8
AAZ	250	12	25	5.7

*Errors in the range of ± 10% of the reported value (from three different assays).

The least effective hCA XII inhibitors were derivatives 4, 5, 7, 9, 12, 17, 21 and 23, which showed K_Is in the range of 126.2–413.7 nM, being thus low potency inhibitors of this isoform compared with the clinically used inhibitor AAZ.

Conclusions

Two series of benzenesulfonamides incorporating amino acid scaffolds (GABA or the N-α-acetyl-l-lysine) were explored here as a new generation of selective tumor-associated hCA XII inhibitors. The compounds investigated contain guanidine groups at the terminal amino moiety which confer them higher water solubility. Most of the new compounds were medium potency or ineffective as inhibitors of the human CAs, hCA I, II and IX, but they showed significant inhibition potency against the transmembrane isoform hCA XII, with efficacy in nanomolar range.

Therefore, considering the selectivity ratios of some of these derivatives for inhibiting the tumor-associated hCA XII over the physiologically dominant hCA I and II isoforms, these compounds may constitute interesting tools for the development of new anticancer agents.

Declaration of interest

C.T.S. reports conflict of interest as author of many patents on CA inhibitors. The authors alone are responsible for the content and writing of this article. This project was funded by a 7th FP EU grant (METOXIA).