Synthesis and Biological Evaluation of Imidazo[2,1-b]Thiazole based Sulfonyl Piperazines as Novel Carbonic Anhydrase II Inhibitors

A novel series of imidazo[2,1-b]thiazole-sulfonyl piperazine conjugates (9aa-ee) has been synthesized and evaluated for carbonic anhydrase (CA, EC 4.2.1.1) inhibitory potency against four isoforms: The cytosolic isozyme hCA I, II and trans-membrane tumor-associated isoform hCA IX and hCA XII, taking acetazolamide (AAZ) as standard drug, using a stopped flow CO2 hydrase assay. The results revealed that most of the compounds showed selective activity against hCA II whereas none of them were active against hCA I, IX, XII (Ki > 100 µM). The physiologically dominant cytosolic isoform hCA II was inhibited by these molecules with inhibition constants in the range of 57.7–98.2 µM. This new derivative, thus, selectively inhibits hCA II over the hCA I, IX, XII isoforms, which may be used for further understanding the physiological roles of some of these isoforms in various pathologies.


Introduction
Carbonic anhydrases (CAs, EC 4.2.1.1) are a group of zinc containing metalloenzymes that effectively catalyze the reversible hydration of CO 2 to bicarbonate and proton, an important reaction for many physiological processes. The human carbonic anhydrases (hCAs) belong to theα-class of carbonic anhydrases having sixteen different form of isoforms. CA isoenzymes play an important role in various cellular processes, such as tumorigenicity, respiration, regulation of pH, electrolytes secretion and many other processes. hCA I and hCA II act as targets for diseases, such as glaucoma and epilepsy, while hCA IX and hCA XII are targets for imaging of tumors and are found to over-express in many cancers. [1][2][3]. Therefore, it is important to develop an effective approach that can selectively inhibit these isoforms involved in different diseases. Nowadays, the major concern of the researchers working in this field is to develop new concept for designing the compounds that are capable of blocking specifically the process catalyzed by one or two isoform ms of these enzymes.
N and S containing fused heterocyclic systems have gained considerable attraction in the field of medicinal chemistry due to their broad spectrum of pharmacological activities [4].
Metabolites 2020, 10, 136 2 of 13 N and S containing fused heterocyclic systems have gained considerable attraction in the field of medicinal chemistry due to their broad spectrum of pharmacological activities [4]. Imidazo [2,1-b]thiazole-guanylhydrazone is a promising lead with potent anticancer activity against a number of cancer cell lines [5]. These fused heterocycles exhibit potent anticancer activity through multiple mechanisms [6]. However, a number of imidazo [2,1-b][1, 3,4]thiadiazole derivatives have been reported to exhibit antitumor activity [7]. Sharma and Supuran et al. have reported a series of benzenesulfonamide bearing imidazothiadiazole and thiazolotriazole hybrids as carbonic anhydrases IX and XII inhibitors [8]. The amide derivatives of probenecid have been reported as selective inhibitors of carbonic anhydrase IX and XII by Carradori et al. [9,10] (Figure 1). Based on the literature, sulfonamides were the most investigated class of CA inhibitors [11,12]. Acetazolamide (AZA), methazolamide (MZA), ethoxzolamide (EZA), dorzolamide (DZA) (Figure 1) are few of the many sulfonamide-based carbonic anhydrase inhibitors in clinical use [13][14][15]. Pazopanib has been clinically used for several years now whereas indisulam and celecoxib are currently under clinical investigations in various types of tumors ( Figure 1) [16]. Supuran et al. have reported various classes of piperazine derivatives as activators of human carbonic anhydrase I, II, IV and VII [17]. Thus, design of novel heterocyclic/aromatic scaffolds with sulfonamide moiety is crucial in developing selective CA inhibitors (CAIs) with potent activity.
Propelled by these findings and continuing our interest in the design of diversified classes of heterocyclic based CAIs, we endeavor to synthesize a new series of imidazo[2,1-b]thiazole-sulfonyl piperazine conjugates (Figure 1), which have not been reported earlier. Thus, a novel imidazo[2,1-b]thiazole-sulfonyl piperazine hybrids (9aa-ee) were synthesized and demonstrated CA inhibition potential against I, II, IX and XII isoforms of CA using acetazolamide (AAZ) as standard.
Propelled by these findings and continuing our interest in the design of diversified classes of heterocyclic based CAIs, we endeavor to synthesize a new series of imidazo[2,1-b]thiazole-sulfonyl piperazine conjugates (Figure 1), which have not been reported earlier.
All synthesized compounds (9aa-ee) were characterized by spectral techniques viz. 1 H NMR, 13 C NMR and high resolution mass spectrometry (HRMS). The 1 H NMR spectrum of 9ab displayed characteristic protons of piperazine around δ2. 8-3.8 ppm. Methyl and Methoxy group of phenyl ring was found to be around δ 2.40 ppm and 3.5-4.0 ppm. All the remaining protons appeared in the range of δ 6.8-8.20 ppm. 13 C NMR spectrum of 9ab showed the characteristic carbonyl and methyl group's at δ 158.61, and δ 21.02 ppm, respectively and all the remaining carbons appeared in the range of δ 108.97-158.56 ppm. The similar pattern was observed for the remaining compounds (9aa-ee). Scheme 1. Synthesis of imidazo[2,1-b]thiazole-sulfonyl piperazine hybrids (9aa-ee).

Carbonic Anhydrase Inhibition
The newly synthesized imidazo[2,1-b]thiazole-sulfonyl piperazines (9aa-ee) were screened for their CA inhibition activity against cytosolic (hCA I, hCA II) and the tumor associated (hCA IX, hCAXII) isoforms by a stopped flow CO2 hydrase assay [20]. Acetazolamide (AAZ), a clinically used reference standard and the obtained results are represented as Ki (µM) are summarized in Table 1. The following structure-activity relationship (SAR) was figured out from the inhibition data of 9aa-ee as shown in Table 1: i.
The cytosolic isoforms hCA I and tumor associated isoform hCA IX, hCAXII were not inhibited by the compounds 9aa-ee (Ki > 100 µM).
All synthesized compounds (9aa-ee) were characterized by spectral techniques viz. 1 H NMR, 13 C NMR and high resolution mass spectrometry (HRMS). The 1 H NMR spectrum of 9ab displayed characteristic protons of piperazine around δ2.8-3.8 ppm. Methyl and Methoxy group of phenyl ring was found to be around δ 2.40 ppm and 3.5-4.0 ppm. All the remaining protons appeared in the range of δ 6.8-8.20 ppm. 13 C NMR spectrum of 9ab showed the characteristic carbonyl and methyl group's at δ 158.61, and δ 21.02 ppm, respectively and all the remaining carbons appeared in the range of δ 108.97-158.56 ppm. The similar pattern was observed for the remaining compounds (9aa-ee).

Carbonic Anhydrase Inhibition
The newly synthesized imidazo[2,1-b]thiazole-sulfonyl piperazines (9aa-ee) were screened for their CA inhibition activity against cytosolic (hCA I, hCA II) and the tumor associated (hCA IX, hCAXII) isoforms by a stopped flow CO 2 hydrase assay [20]. Acetazolamide (AAZ), a clinically used reference standard and the obtained results are represented as K i (µM) are summarized in Table 1. The following structure-activity relationship (SAR) was figured out from the inhibition data of 9aa-ee as shown in Table 1: i.
The cytosolic isoforms hCA I and tumor associated isoform hCA IX, hCAXII were not inhibited by the compounds 9aa-ee (K i > 100 µM). ii. The compounds 9aa-ee showed varied inhibitory profiles against cytosolic isoform hCA II.
iv. Among all the congeners, some of the potent compounds exhibited selective inhibition of hCA II as compared to hCA IX and hCAXII in micromolar range. iv. Among all the congeners, some of the potent compounds exhibited selective inhibition of hCA II as compared to hCA IX and hCAXII in micromolar range. iv. Among all the congeners, some of the potent compounds exhibited selective inhibition of hCA II as compared to hCA IX and hCAXII in micromolar range. iv. Among all the congeners, some of the potent compounds exhibited selective inhibition of hCA II as compared to hCA IX and hCAXII in micromolar range. iv. Among all the congeners, some of the potent compounds exhibited selective inhibition of hCA II as compared to hCA IX and hCAXII in micromolar range. iv. Among all the congeners, some of the potent compounds exhibited selective inhibition of hCA II as compared to hCA IX and hCAXII in micromolar range. iv. Among all the congeners, some of the potent compounds exhibited selective inhibition of hCA II as compared to hCA IX and hCAXII in micromolar range. iv. Among all the congeners, some of the potent compounds exhibited selective inhibition of hCA II as compared to hCA IX and hCAXII in micromolar range. iv. Among all the congeners, some of the potent compounds exhibited selective inhibition of hCA II as compared to hCA IX and hCAXII in micromolar range.

Conclusions
In conclusion, we have reported the synthesis of imidazo[2,1-b]thiazole-sulfonyl piperazine conjugates (9aa-ee), which are designed to target CA isoforms. The synthesized compounds (9aa-ee) were screened against the cytosolic isoforms hCA I and II, as well as the transmembrane tumor associated isoforms, hCA IX and XII. Few of the compounds (9ae, 9bb, 9ca, 9cc-ce, 9da and 9dc) exhibited acceptable inhibitory activity against cytosolic isoform hCA II, Ki value in the range of >100 >100 >100 >100

Conclusions
In conclusion, we have reported the synthesis of imidazo[2,1-b]thiazole-sulfonyl piperazine conjugates (9aa-ee), which are designed to target CA isoforms. The synthesized compounds (9aa-ee) were screened against the cytosolic isoforms hCA I and II, as well as the transmembrane tumor associated isoforms, hCA IX and XII. Few of the compounds (9ae, 9bb, 9ca, 9cc-ce, 9da and 9dc) exhibited acceptable inhibitory activity against cytosolic isoform hCA II, Ki value in the range of >100 >100 >100 >100

Conclusions
In conclusion, we have reported the synthesis of imidazo[2,1-b]thiazole-sulfonyl piperazine conjugates (9aa-ee), which are designed to target CA isoforms. The synthesized compounds (9aa-ee) were screened against the cytosolic isoforms hCA I and II, as well as the transmembrane tumor associated isoforms, hCA IX and XII. Few of the compounds (9ae, 9bb, 9ca, 9cc-ce, 9da and 9dc) exhibited acceptable inhibitory activity against cytosolic isoform hCA II, Ki value in the range of >100 >100 >100 >100

Conclusions
In conclusion, we have reported the synthesis of imidazo[2,1-b]thiazole-sulfonyl piperazine conjugates (9aa-ee), which are designed to target CA isoforms. The synthesized compounds (9aa-ee) were screened against the cytosolic isoforms hCA I and II, as well as the transmembrane tumor associated isoforms, hCA IX and XII. Few of the compounds (9ae, 9bb, 9ca, 9cc-ce, 9da and 9dc) exhibited acceptable inhibitory activity against cytosolic isoform hCA II, Ki value in the range of >100 >100 >100 >100

Conclusions
In conclusion, we have reported the synthesis of imidazo[2,1-b]thiazole-sulfonyl piperazine conjugates (9aa-ee), which are designed to target CA isoforms. The synthesized compounds (9aa-ee) were screened against the cytosolic isoforms hCA I and II, as well as the transmembrane tumor associated isoforms, hCA IX and XII. Few of the compounds (9ae, 9bb, 9ca, 9cc-ce, 9da and 9dc) exhibited acceptable inhibitory activity against cytosolic isoform hCA II, Ki value in the range of >100 >100 >100 >100

Conclusions
In conclusion, we have reported the synthesis of imidazo[2,1-b]thiazole-sulfonyl piperazine conjugates (9aa-ee), which are designed to target CA isoforms. The synthesized compounds (9aa-ee) were screened against the cytosolic isoforms hCA I and II, as well as the transmembrane tumor associated isoforms, hCA IX and XII. Few of the compounds (9ae, 9bb, 9ca, 9cc-ce, 9da and 9dc) exhibited acceptable inhibitory activity against cytosolic isoform hCA II, Ki value in the range of >100 >100 >100 >100

Conclusions
In conclusion, we have reported the synthesis of imidazo[2,1-b]thiazole-sulfonyl piperazine conjugates (9aa-ee), which are designed to target CA isoforms. The synthesized compounds (9aa-ee) were screened against the cytosolic isoforms hCA I and II, as well as the transmembrane tumor associated isoforms, hCA IX and XII. Few of the compounds (9ae, 9bb, 9ca, 9cc-ce, 9da and 9dc) exhibited acceptable inhibitory activity against cytosolic isoform hCA II, Ki value in the range of >100 >100 >100 >100

Conclusions
In conclusion, we have reported the synthesis of imidazo[2,1-b]thiazole-sulfonyl piperazine conjugates (9aa-ee), which are designed to target CA isoforms. The synthesized compounds (9aa-ee) were screened against the cytosolic isoforms hCA I and II, as well as the transmembrane tumor associated isoforms, hCA IX and XII. Few of the compounds (9ae, 9bb, 9ca, 9cc-ce, 9da and 9dc) exhibited acceptable inhibitory activity against cytosolic isoform hCA II, Ki value in the range of

Conclusions
In conclusion, we have reported the synthesis of imidazo[2,1-b]thiazole-sulfonyl piperazine conjugates (9aa-ee), which are designed to target CA isoforms. The synthesized compounds (9aa-ee) were screened against the cytosolic isoforms hCA I and II, as well as the transmembrane tumor associated isoforms, hCA IX and XII. Few of the compounds (9ae, 9bb, 9ca, 9cc-ce, 9da and 9dc) exhibited acceptable inhibitory activity against cytosolic isoform hCA II, K i value in the range of 57.7-67.9 µM, while some compounds were found to be weaker inhibitors of tumor associated isoform hCA IX and hCA XII. Hence, further modifications of these newly designed imidazo[2,1-b]thiazole-sulfonyl piperazine derivatives (9aa-ee) have the potential to emerge as isoform II selective CAIs. Notably, as for all secondary/tertiary sulfonamides reported to date as CAIs, the inhibition mechanism with these derivatives reported here is for the moment unknown [21][22][23].

Materials and Methods
All Chemicals and reagents were purchased from the commercial suppliers Alfa Aesar, Sigma Aldrich and used without further purification. The reaction progress was monitored by Thin layer chromatography (TLC) was performed using pre-coated silica gel 60 F 254 MERCK. TLC plates were visualized and analyzed by exposure to UV light or iodine vapors and aqueous solution of ninhydrin. Column chromatography was performed with Merck flash silica gel with 60-120 mesh size. Melting points were determined on an Electro thermal melting point apparatus and are uncorrected. Nuclear magnetic resonance spectra for 1 H NMR were obtained on Avance 300, 400 and 500 MHz and analyzed using Mestrenova software and the chemical shifts are reported in ppm from tetramethylsilane (0 ppm) or the solvent resonance as the internal standard (CDCl 3 7.26 ppm, DMSO-d 6 2.49 ppm) and for 13  Substituted phenyl sulfonyl piperazines 8a-e was synthesized by adding triethylamine (31.5 mmol) slowly to a solution of piperazine in CH 2 Cl 2 at 0 • C, and then commercially available benzenesulfonyl chloride (7a-e, 11.4 mmol) was added and stirred for 30 min. After the completion of reaction, which was confirmed by TLC (petroleum ether and ethyl acetate (2:1)), the reaction was quenched with water and extracted with CH 2 Cl 2 . The organic layer was dried over anhydrous Na 2 SO 4 and concentrated on reduced pressure to give 8a-e [19]. 4.1.5. General Synthetic Procedure for the Preparation of (5-(aryl)imidazo[2,1-b]thiazol-2-yl)(4-((aryl)sulfonyl)piperazin-1-yl)methanone (9aa-ee) Above obtained carboxylic acid (6a-e, 1.0 mmol) was dissolved in dry dimethyl formamide (10 mL), cooled to 0 • C (ice bath). EDCI (1.2 mmol), HOBt (1.2 mmol), amine intermediate (8a-e, 1.0 mmol) and triethylamine (3 mmol) were added slowly. After 10 min removed the ice bath and the mixture was stirred at room temperature for 12 h until the starting materials were consumed. Then ice cold water was added to the reaction mixture and extracted with dichloro methane (DCM). The combined organic extracts were washed with aq NaHCO 3 and dried with Na 2 SO 4 and concentrated in vacuo. The residue was purified by column chromatography by using ethyl acetate and hexane solvent system to afford the title compounds (9aa-ee).   as reported earlier, [24,25] and represent the mean from at least three different determinations. All CA isozymes used here were recombinant proteins obtained as reported earlier by our group [26][27][28][29].