A new class of quinazoline-sulfonamides acting as efficient inhibitors against the α-carbonic anhydrase from Trypanosoma cruzi

Abstract

The protozoan parasite Trypanosoma cruzi is the agent responsible for trypanosomiasis (Chagas disease) in humans and other animals. It has been recently reported that this pathogen encodes for an α-class carbonic anhydrase (CA, EC 4.2.1.1), denominated TcCA, which was shown to be crucial for its life cycle. Inhibition studies of a class of 4-oxoquinazoline containing a benzensulfonamide moiety and their 4-thioxo bioisosteres against the protozoan enzyme TcCA are described here. Most of 4-oxoquinazoline sulfonamides showed nanomolar TcCA inhibition activity with K_Is in the same order of magnitude of acetazolamide (AAZ), whereas their thioxo bioisosters showed moderate anti-Trypanosoma CA potency with K_Is in the micromolar range. The discovery of compounds incorporating a 4-oxoquinazoline ring as a low-nanomolar TcCA inhibitor is quite promising and it may be useful for developing anti-Trypanosoma agents with a novel mechanism of action compared to the clinically used drugs (such as benznidazole, nifurtimox) for which significant resistance and serious adverse effects due to their high-toxicity appeared.

• α-Carbonic anhydrase
• enzyme inhibitor
• sulfonamide
• Trypanosoma cruzi
• quinazoline

Introduction

American trypanosomiasis or Chagas disease affects millions of people mostly in South and Central Americas, but nowadays it spreads also in Europe and North America1, being caused by the protozoan pathogen Trypanosoma cruzi. The only two clinically used drugs for its treatment are nitroazoles, such as benznidazole and nifurtimox. Their mechanism of action most probably involves the formation of free radicals from the nitro groups of the drugs towards which the parasite results to be particularly sensitive and consequently reduction of detoxification capabilities of T. cruzi2. However, since these two drugs are not always effective to abolish the chronic infection3, new anti-infective drugs started to be explored ultimately1.

Our groups recently reported that an α-CA with very high-catalytic activity is present in this pathogenic protozoan2. This enzyme, denominated TcCA, was cloned and characterized2, and further investigations showed that this enzyme may be inhibited by sulfonamides and thiols2^,4, two of the main classes of CA inhibitors (CAIs)5–10 as well as a wide range of inorganic anions6. Interestingly, most aromatic/heterocyclic sulfonamides as well as some 5-mercapto-1,3,4-thiadiazoles investigated earlier were not highly effective as TcCA inhibitors in vitro2^,11. However, some of these thiols, incorporating heterocyclic scaffolds, were among the most potent in vitro TcCA inhibitors (K_Is of 21.1–79.0 nM) and some of them also showed anti-trypanosomal activity in vivo, inhibiting the epimastigotes growth of two T. cruzi strains2.

The inhibition studies of TcCA by inorganic and complex anions and other molecules interacting with zinc proteins, such as sulfamide, sulfamic acid, phenylboronic/arsonic acids, have also been reported6. It showed that the protozoan enzyme TcCA was inhibited in the low-micromolar range by iodide, cyanate, thiocyanate, hydrogensulfide and trithiocarbonate as well as by diethyldithiocarbamate (K_I = 5 μM). Therefore, protozoan CA inhibition may be a valid strategy to control infection such as Chagas disease mainly caused by a pathogen agent like the protozoa T. cruzi.

In this study, we reported the inhibition studies of new classes of quinazoline derivatives which were designed and then synthesised as potential selective TcCA inhibitors.

Materials and methods

Chemistry

Melting points (°C) were determined in open capillaries on a Gallenkamp melting point apparatus (Sanyo Gallenkamp, Southborough, UK) and were uncorrected. Precoated silica gel plates (silica gel 0.25 mm, 60G F254; Merck, Germany) were used for thin layer chromatography, and the dichloromethane/methanol (9.5:0.5) mixture was used as a developing solvent system. Iodine was used for staining together with ultraviolet light. Infrared spectra were recorded in KBr discs using IR-470 Shimadzu spectrometer (Shimadzu, Tokyo, Japan). ¹H-NMR spectra were recorded on Bruker AC-400 Ultra Shield NMR spectrometer (Bruker, Flawil, Switzerland, δ ppm) at 300 MHz for ¹H and 75 MHz for ¹³C, using TMS as internal standard, and peak multiplicities are designed as follows: s, singlet; d, doublet; t, triplet; m, multiplet. Electron impact mass spectra were recorded on a Shimadzu GC-MS-QP 5000 instrument (Shimadzu, Tokyo, Japan). Elemental analyses were performed, on Carlo Erba 1108 Elemental Analyzer (Heraeus, Hanau, Germany), at the Micro-analytical Unit, Faculty of Science, Cairo University, Cairo, Egypt, and the results found were within ±0.4% of the theoretical values.

The synthesis and the full characterization of the compounds investigated here (8a–h and 9a–e) as CA inhibitors are reported elsewhere12.

CA inhibition studies

An applied photophysics stopped-flow instrument has been used for assaying the CA-catalyzed CO₂ hydration activity13. 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 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 room temperature prior to assay, in order to allow for the formation of the E-I complex. The inhibition constants were obtained by non-linear least-squares methods using PRISM 3, whereas the kinetic parameters for the uninhibited enzymes from Lineweaver-Burk plots, as reported earlier14–16 represent the mean from at least three different determinations. All CAs were recombinant proteins obtained as reported earlier by these groups2^,14–16.

Results and discussion

Chemistry

The present findings extend our previous studies which reported the synthesis of a new series of 2,3,5,6- and/or 8-substituted-4-oxoquinazoline derivatives and their bioisosters 4-thioxoquinazoline12, presenting here the inhibition studies of these compounds against the α-CA from T. cruzi (TcCA). The approach to prepare the target potentially bioactive quinazoline derivatives 8a–h and thioxoquinazolin-4-one analogues 9a–e was via a generalized route described in Scheme 1 The starting materials, thioxoquinazoline derivatives 3a–h and their 2,4-dithioxo isosteres 4a–e, were prepared according to reported literature procedure17^,18. The reaction of the anthranilic acid derivatives appropriate 3,5 or 6 substituted with the selected isothiocyanates in boiling ethanol containing catalytic amount of triethylamine afforded the key intermediates 3,5,6 and/or 8 substituted-2-thio-4-oxoquinazoline and 2-thio-4-thioxoquinazoline derivatives 3a–h and 4a–e, which were reacted with 2-chloro-N-(4-sulfamoyl-phenyl)-acetamide to give the new sulfonamide derivatives containing quinazoline ring 8a–h and their isosteres 9a–e, respectively (Scheme 1). These isothiocyanates were chosen in such a way as to contain moieties which were shown earlier to lead to interesting CA inhibitory compounds in the quinazoline-containing sulfonamide derivatives19^,20.

Scheme 1. Synthesis of the 2,3,5,6 and/or 8-substituted-4-oxoquinazoline derivatives 8a–h and their bioisosters 4-thioxoquinazoline derivatives 9a–e.

In order to obtain compounds with higher biological activity21–29, it has been thought that the oxoquinazoline derivatives could be converted to their thioxo isosteres. Therefore, the conversion of the thioxoquinazolin-4-one 3a–e derivatives to their corresponding di-thioxo 4a–e isosteres was obtained by the treatment of compounds 3a–e with phosphorus pentasulfide in dry pyridine. 2-Chloro-N-(4-sulfamoylphenyl)-acetamide was prepared by acylation of sulfanilamide at room temperature under basic condition.

The new compounds and their intermediates were characterized by elemental analyses, IR, ¹H- and ¹³C-NMR as well as by mass spectroscopy (see Experimental Protocols for details12).

CA inhibition

The inhibition studies of a new quinazolin-4-one sulfonamide series (8a–h) and their 4-thioxo bioisosteres (9a–e), as well as acetazolamide (AAZ, 5-acetamido-1,3,4-thiadiazole-2-sulfonamide, a clinically used drug) against the human (h) CA isoforms hCA I, II, and the protozoan enzyme TcCA are reported in Table 1. Data for the selectivity ratios of the dominant and physiologically relevant human carbonic anhydrase isoforms (hCA I and II) over the TcCA enzyme with these compounds are also included in Table 1, as they may be the main off-targets in case they are very efficient TcCA inhibitors. The following structure–activity relationship (SAR) for TcCA inhibition with the new group of quinazoline-sulfonamide derivatives 8a–h and 9a–e reported here can be observed from Table 1.

• (i) The cytosolic slow isoform hCA I was generally poorly inhibited by the new quinazoline derivatives and their bioisosteres reported here with inhibition constants in the range of 674–5432 nM. However, some derivatives belonging to the 4-oxoquinazoline series such as 8a–c showed to be high/medium potent inhibitors against this human isoform, with K_Is of 86.5–119 nM (Table 1).

• (ii) Against the physiologically dominant human isoform hCA II, the derivatives 8a–c were very potent inhibitors, with K_Is in the range of 1.3–1.7 nM. Indeed, they showed to have better inhibition activity than the clinically used acetazolamide AAZ (K_I of 12 nM). The derivative 8g showed K_I against this cytosolic human isoform of the same order of magnitude as acetazolamide (12.1 nM). Most of the 4-thioxoquinazoline derivatives, such as 9a–b, 9d and 9e, were medium potency hCA II inhibitors (K_Is in the range of 44.0–91.9 nM). The remaining compounds reported here such as 8d–8f, 8h and 9c showed ineffective hCA II inhibitory activity, with K_Is in the range of 114.2–233.8 nM (Table 1).

• (iii) As reported in Table 1, acetazolamide (AAZ) acts as a medium-potency CA inhibitor against the protozoan enzyme TcCA (K_I of 61.6 nM)2. In the same way, the new sulphonamides belonging to the 4-oxoquinazoline series 8 reported here generally acted as medium potent inhibitors of TcCA, with K_Is of the same order of magnitude as acetazolamide in the range of 43.3–68.4 nM (except for 8f with K_I equal to 272.7 nM and thus 4.4 times higher than AAZ, and 8c with K_I of 6.6 nM about 10 times lower than AAZ). Only one 4-thioxoquinazoline derivative 9d with K_I of 56.7 nM showed similar inhibition potency of acetazolamide.

• (iv) Although most of the 4-oxoquinazoline sulphonamides reported here inhibited medium efficiently TcCA, no significant change of K_I values has been observed. Therefore, SAR is almost impossible to delineate as all substitution patterns lead to highly effective inhibitors of this protozoan CA, TcCA.

• (v) However, it is also interesting to notice that the best TcCA inhibitor was derivative 8c (K_I of 6.6 nM), which was also a very strong inhibitor of the cytosolic isoform hCA II with K_I of 1.7 nM and a medium potency inhibitor of the other cytosolic human isoform CA I with K_I of 86.5 nM.

• (vi) Generally, the derivatives 8a–h belonging to the 4-oxoquinazoline series are more potent TcCA inhibitors than their bioisosters 4-thioxoquinazoline derivatives 9a–9e (except for compounds 8f and 9d which showed K_I of 272.7 and 56.7 nM, respectively). Indeed, the least effective TcCA inhibitors were mostly the 4-thioxoquinazoline derivatives, such as 9a–c and 9e, which showed K_Is in the range of 349.2–763.5 nM being thus low-potency inhibitors of this protozoan enzyme together with the only 4-oxoquinazoline derivatives 8f with K_I of 272.7 nM.

• (vii) Furthermore, the 8-methyl-3-benzyl (8d) and the 6-nitro-3-allyl 4-oxoquinazoline (8g) derivatives with K_I of 34.9 and 43.3 nM, respectively, were slightly more active compounds compared to the standard drug AAZ. However, keeping the nitro side chain in the same 6th position and just changing the substitution group in 3rd position from allyl to benzyl (such as in the 8h derivative) bring a small increase of K_I from 43.3 to 68.4 nM (Table 1). However, due to the presence of the nitro group, these two derivatives most probably possess the same mechanism of action as TcCA inhibitors of the only two clinically available drugs, benznidazole and nifurtimox. They are indeed nitroazoles whose mechanism of action is based on the production of free radicals involving the NO₂ groups of the drugs, to which T. cruzi is particularly sensitive given its reduced detoxification capabilities2.

• (viii) The remaining oxoquinazoline derivatives with either 3-allyl or 3-benzyl side chain mostly possessed very similar inhibition activity against the protozoan enzyme TcCA. However, changing position of the methoxy group from 6 to 8 in the quinazoline ring lead an enhancement of the inhibition constant by a factor of 4.5 as observed for derivative 8f (K_I 272.7 nM).

• (ix) Some of the TcCA inhibitors reported here also showed a high-selective inhibition of the protozoan CA over the human isoforms. Indeed, compound 8d which shows to be one of the most potent TcCA inhibitor with K_I of 34.9 nM represents also the most selective inhibitor against the protozoan enzyme over the human CA isoforms reported here with selectivity ratios as high as 34.9 against hCA I and 5.98 against hCA II (Table 1). Other derivatives of the same quinazolin-4-one series possessing the inhibition activity against TcCA of the same order of magnitude as AAZ showed good selectivity ratios for inhibiting the bacterial over the human enzymes, such as 8e and 8h (Table 1).

Table 1. Inhibition data of quinazoline-sulfonamides 8a–h, 9a–e and acetazolamide AAZ (as standard inhibitor) against human (h) isoforms hCA I, II (cytosolic) and the protozoan enzyme TcCA by a stopped-flow CO₂ hydrase assay (13). The selectivity ratios are also reported.

			KI (nM)*			Selectivity ratios
n	R	R1	hCA I	hCA II	TcCA	hCA I/TcCA	hCAII/TcCA
8-a	5-CH3	CH2CH=CH2	105	1.3	54.0	2.0	0.02
8-b	6-CH3	CH2CH=CH2	119	1.8	55.0		2.2
8-c	6-CH3	CH2C6H5	86.5	1.7	6.6	13.1	0.26
8-d	8-CH3	CH2C6H5	2078	208.4	34.9	59.6	5.98
8-e	6-OCH3	CH2C6H5	4761	233.8	60.0	79.3	3.90
8-f	8-OCH3	CH2C6H5	5432	208.9	272.7	19.9	0.77
8-g	6-NO2	CH2CH=CH2	674	12.1	43.3	15.6	0.28
8-h	6-NO2	CH2C6H5	3371	122.9	68.4	49.3	1.80
9-a	5-CH3	−	4168	67.5	349.2	11.9	0.19
9-b	6-CH3	−	793	78.3	531.9	1.5	0.15
9-c	8-CH3	−	2162	114.2	666.4	3.2	0.17
9-d	8-OCH3	−	803	65.3	56.7	14.2	1.15
9-e	6-NO2	−	4549	96.0	763.5	6.0	0.13
AAZ	−	−	250	12	61.6	4.06	0.19

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

Although, most of the compounds reported here acted as medium selective inhibitors for the protozoan isoform over the human ones, some of them presented better selectivity inhibition ratios against hCA I and hCA II than the drug clinically used AAZ which shows a selectivity ratios for inhibiting TcCA against hCA I and II equal to 4.06 and 0.19, respectively. However, it is interesting to notice how acetazolamide acts as an efficient TcCA inhibitor even showing low-selectivity ratios against hCA I and II.

Conclusions

We evaluated a new series of quinazolin-4-one and quinazolin-4-thione derivatives containing a benzenesulfonamide moiety for the inhibition of TcCA, an α-class CA recently cloned from Trypanosoma cruzi, the causative agent of Chagas disease, which was recently shown to be crucial for its life cycle. The inhibition studies of the protozoan enzyme TcCA with the reported compounds revealed that the 4-oxoquinazoline derivatives were generally more efficient than their 4-thioxoquinazoline bioisosters 4. However, most of the novel tested 4-oxoquinazoline sulfonamides, showing moderate/high inhibition against TcCA, were as potent as acetazolamide AAZ (as standard inhibitor) and one of them high-effectively inhibited TcCA at a subnanomolar concentration. In order to determine the selectivity ratios for inhibiting the protozoan over the human enzymes, the CA inhibitory activity of these compounds was also determined against the human cytosolic isoforms hCA I and II. According to the results obtained in the present study, many of the potent TcCA inhibitors reported here also possessed moderate selectivity ratios for inhibiting the protozoan over the human enzymes. The discovery of the 6-methyl-3-allyl 4-oxoquinazoline derivative as a low-nanomolar TcCA inhibitor may be useful to detect lead molecules for developing anti-Trypanosoma agents with a different mechanism of action compared to clinically used drugs (benznidazole, nifurtimox) for which significant resistance appeared. Indeed, the quinazoline-sulfonamides may represent a new class of compounds with potent inhibitory activity against protozoan enzymes, adding thus a relevant biological profile to the sulfonamides with CA inhibitory action30–33.

Declaration of interest

Most of the authors declare no financial interest. CTS reports conflict of interest as author of many patents on CA inhibitors. This research was funded by National Plan of Science, Technology and Innovation (Grant No. 11-MED1874-02), King Saud University, Riyadh (to AF), Coordenação de Aperfeiçoamento Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (MCT/CNPq) and Fundação Carlos Chagas Filho de Amparo à Pesquisa do Rio de Janeiro (FAPERJ).