Synthesis of New 1H-1,2,3-Triazole Analogs in Aqueous Medium via “Click” Chemistry: A Novel Class of Potential Carbonic Anhydrase-II Inhibitors

A series of novel 1H-1,2,3-triazole analogs (9a–j) were synthesized via “Click” chemistry and Suzuki–Miyaura cross-coupling reaction in aqueous medium. The compounds were evaluated for their carbonic anhydrase-II enzyme inhibitory activity in vitro. The synthesis of triazole 7a was accomplished using (S)-(-) ethyl lactate as a starting material. This compound (7a) underwent Suzuki–Miyaura cross-coupling reaction with different arylboronic acids in aqueous medium to afford the target molecules, 9a–j in good yields. All newly synthesized compounds were characterized by 1H NMR, 13C NMR, FT-IR, HRMS, and where applicable 19F NMR spectroscopy (9b, 9e, 9h, and 9j). The new compounds have shown moderate inhibition potential against carbonic anhydrase-II enzyme. A preliminary structure-activity relationship suggested that the presence of polar group at the 1H-1,2,3-triazole substituted phenyl ring in these derivatives (9a–j) has contributed to the overall activity of these compounds. Furthermore, via molecular docking, it was deduced that the compounds exhibit inhibitory potential through direct binding with the active site residues of carbonic anhydrase-II enzyme. This study has unraveled a new series of triazole derivatives as good inhibitors against carbonic anhydrase-II.


INTRODUCTION
1H-1,2,3-Triazole molecules play a vital role in pharmaceuticals and agrochemicals (Abdel-Wahab et al., 2012). The triazole moiety is very important in organic chemistry due to its broad range of applications in biomedicinal, biochemical, and material sciences (Singh et al., 2010). The chemistry of the compounds containing this moiety underwent substantial growth over the past decades (Thirumurugan et al., 2013). These compounds are widely used in industrial applications such as dyes, photographic materials, photostabilizers, agrochemicals, and corrosion inhibitors (copper alloys) (Fan et al., 1996).
Carbonic anhydrase (CAs, EC 4.2.1.1), a Zn +2 containing metallic enzyme, catalyzes the reversible reaction of carbon dioxide into bicarbonate ions (Supuran and De Simone, 2015;Ozensoy and Guler et al., 2016). There are fifteen isoforms of CA which have been identified so far (Lindskog, 1997;Aggarwal et al., 2013). They possess a difference in their organ distributions, levels of gene expression, molecular sequence features, and kinetic parameters (Krishnamurthy et al., 2008). CAs are key contributors to various physiological and pathological processes. Thus, they are considered the prime therapeutic target for the treatment of several chronic diseases.
In continuation of our research work on 1H-1,2,3-triazole derivatives (Avula et al., 2018;Avula et al., 2019), we herein report a new series of 1H-1,2,3-triazole analogs (9a-j) as carbonic anhydrase-II inhibitors (Huisgen, 1963;Pham Thi et al., 2016). We have selected the 8th triazole structure ( Figure 1A) for our present synthesis because it contains a chiral dioxyaryl moiety which is present in some bioactive natural products such as surinamensinols A and B ( Figure 1B) (add two references). A structural-activity relationship is discussed to demonstrate the influence of structural moieties on the triazole derivatives.

RESULTS AND DISCUSSION
Chemistry: Synthesis of 1H-1,2,3-Triazole Analogs (9a-j) The first step employed Mitsunobu reaction between (S)-(-) ethyl lactate 1 and 4-bromo-2-methoxy phenol using diisopropylazodicarboxylate (DIAD). The reaction afforded the expected compound 2 in 86% yield. In this step, a chiral center has been successfully introduced. Reduction of compound 2 to compound 3 was achieved by using DIBAL-H which furnished compound 3 in 90% yield. The hydroxyl group of 3 was converted into a tosyl moiety to provide compounds 4 in high yield (95%). The latter was treated with NaN 3 and afforded the corresponding azide derivative 5 in 78% yield.
Azide 5 underwent 1,3-dipolar cycloaddition with the alkyne derivative 6a in the presence of CuI and Hunig's base. The reaction furnished the desired product 1H-1,2,3-triazole derivative 7a as a colorless amorphous solid in 76% yield. The 1 H NMR spectrum of compound 7a showed singlet at δ 8.05 for triazole proton (-CH-N 3 ). The eight aromatic protons appeared in the region of δ 7.78-6.63 ppm. One multiplet and doublet of doublet signals at δ 4. 61-4.56 and δ 4.48 correspond to the-CH-O-and -CH-N-, respectively. A singlet peak at δ 3.69 is due to methoxy protons on the phenyl ring and a doublet at δ 1.24 is attributed to methyl protons of -CH-O-Ph. The high-resolution mass spectrometric data at 388.0661 (M + ) support the structure of compound 7a. Similarly, using the same reaction conditions described for the synthesis of compound 7a, compound 7b was obtained in 82% yield using different alkyne derivative (6b). The synthesis of compounds 7a and 7b is summarized in Scheme 1.

Molecular Docking Studies and Predicted Structure-Activity Relationship
Molecular docking studies of all active triazole derivatives were performed using molecular operating software (MOE) (Chemical Computing Group, 2014), in order to determine the best plausible binding modes of the ligands in the active site of the enzyme. The active site of CA-II is depicted in Figure 3A. Compound 7b (IC 50 13.8 ± 0.63 µM) was found to be the most active compound of the series and its best predicted binding pose is presented in Figure 3B.
The binding interaction of 7b demonstrated that its aniline moiety formed a metallic bond with Zn +2 ion and hydrogen bonding with the amino and -OH groups of Thr199. Furthermore, the dioxyaryl group of 7b exhibited H-bonding with the amide group of Asn62. Additionally, a water molecule (HOH270) also offered an H-bond to the triazole moiety of 7b. This multiple bonding of the compound with the active site residues is responsible for the enhanced biological activity of 7b as compared to the rest of the compounds. Similarly, the triazole nitrogen and methoxy oxygen of the 9e (IC 50 18.1 ± 1.31 µM) interacted with the side chains of Gln92 and Asn62, respectively. The loss of hydrogen bond donor/acceptor group at triazole substituted aryl group of 9e makes the molecule less active than 7b; moreover, water molecule does not contribute to protein-ligand bridging for 9e. The nitro group of 9d mediated H-bonding and metallic interaction with the amide group of Thr199 and Zn ion, respectively. Additionally, the side chain of His64 provided π-cation interaction to the triazole ring of 9d. The dioxyaryl group does not interact with the surrounding residues including Asn62, Asn67, Gln92, and water molecules; this may be the reason for lower activity of 9d than 7b and 9e. The docked view of 9c showed that the carbonyl oxygen interacted with the Zn ion via metallic bond; however, the other polar groups do not interact with the active site residues and the docked orientation of 9c is surface exposed. This is the reason for the further reduced activity of 9c. Similarly, the triazole substituted aryl group of 9b does not possess any polar moiety to interact with Zn ion or Thr199 and His94. However, the triazole nitrogen of 9b formed H-bond with the side chain of Gln92. The dioxyaryl moiety and its substituted R group of 9b remained surface exposed, which further decreased the inhibitory activity of 9b. Likewise, the triazole substituted aryl group and dioxyaryl substituted aryl group of 9f mediated hydrophobic interactions with the side chains of Thr199 and His64, respectively, while the triazole ring and the dioxyaryl group lost interaction with Gln92 and Asn62, respectively. Due to the loss of these interactions, the compound exhibited less biological activity than 9b. The binding mode of 9h was similar to the docked view of 9f; however, the triazole nitrogen and the dioxyaryl substituted aryl group of 9h formed H-bonding and hydrophobic interaction with Asn62 and Trp5, respectively. Similarly, the least active compound, 9j, exhibited only π-cation interaction with the side chain of His64, while its triazole ring and the dioxyaryl group do not interact with the surrounding residues; due to the loss of major H-bonding or metallic interactions, the compound exhibited the least inhibitory activity against CA-II. The best-docked poses of the active compounds are depicted in Figure 3C. The docking scores and the binding interactions are tabulated in Table 2. Acetazolamide was used as a positive control in docking which exhibited biological activity with IC 50 value of 18.2 µM. The sulfate group of acetazolamide interacted with the Zn ion and the side chain of Thr198 through metallic interaction and H-bonding, respectively. The docking score of acetazolamide is −5.40, which is lesser than the docking scores of 7b and 9e, while being greater than the docking scores of 9d, 9c, 9b, 9f, 9h, and 9j. The docked view of acetazolamide is shown in Figure 4 in both 3D and 2D format. The docking scores and binding interactions of the compounds are well correlated with the inhibitory activities of the compounds. The docked orientation of compounds showed that Thr199 and Zn ions play important role in the stabilization of the triazole substituted aryl group, while the dioxyaryl group interacts with Asn62 or Gln92. Additionally, His64 provides hydrophobic interactions to the compounds.

CONCLUSION
In summary, a series of novel 1H-1,2,3-triazole analogs were synthesized (9a-j) and evaluated for their carbonic anhydrase-II inhibitory activity in vitro. (S)-(-) Ethyl lactate was used as a starting material to introduce the chirality of the target molecules. The triazole moiety was prepared via "Click" chemistry and the aryl derivatives by utilizing Suzuki-Miyaura cross-coupling reaction in aqueous medium. All the compounds have shown moderate inhibition potential against carbonic anhydrase-II enzyme reported for the first time. The molecular docking studies showed that all the active compounds well accommodate to the active site of the CA enzyme.

EXPERIMENTAL SECTION General
Reagents were obtained from Sigma-Aldrich, Germany. Silica gel for column chromatography was of 100-200 mesh. Solvents were purified by following standard procedures. Thin-layer chromatography (TLC) was carried using silica gel F 254 precoated plates. UV-light and I 2 stain were used to visualize the spots. The 1 H and 13 C NMR spectra were recorded on NMR spectrometer (Bruker: 600 MHz for 1 H, 150 MHz for 13 C, and
General Procedure for Synthesis of 1H-1,2,3-Triazole Derivatives (7a and 7b) CuI (2.0 equiv) and triethylamine (3.0 equiv) were added to a solution of azide compound 5 (1.0 equiv) and alkyne derivatives 6a-b (1.2 equiv) in acetonitrile (10 ml) at room temperature, and the mixture was stirred for 3 h. The reaction mixture was diluted with EtOAc (20 ml), 10 ml of aqueous NH 4 Cl was added, the aqueous layer was extracted with EtOAc (3 × 15 ml), and the combined organic layer was washed with brine solution, dried over anhydrous Na 2 SO 4 , and concentrated in vacuo to obtain a crude residue that was purified by flash chromatography to obtain desired 1H-1,2,3-triazole derivatives (7a) 76% and (7b) 82%.

In vitro Carbonic Anhydrase-II Inhibition Assay
In vitro experiment of bovine erythrocyte CA-II was conducted in HEPES-Tris buffer (20 mM) to maintain the pH 7.4. The purified was dissolved in HEPES-Tris buffer (0.1 mg/ml), and the reaction mixture was comprised of 140 μL of the HEPES-Tris buffer, 20 μL of the enzyme, and 20 μL of test compound (prepared in DMSO) and was incubated for 15 min at 25°C. After completion of the preincubation, the reaction was started by adding instantly the substrate p-nitrophenylacetate (P-NPA) at a concentration of 0.7 mM and prepared in methanol. To initiate the reaction, the 96-well plate was placed in a microplate reader and continuous product formation was monitored with a one-minute time interval for 30 min at 400 nm. The assay temperature was strictly controlled and kept at 25°C. All the reaction was conducted in triplicate, and the results are presented as mean.

Molecular Docking Protocol
The molecular docking experiment was performed on Molecular Operating Environment (MOE, 2014.14). The structures of ligands were prepared by MOE and minimized with MMFF94x force field until an RMSD gradient of 0.1 kcal mol −1 Å −1 was attained, and partial charges were automatically calculated. The crystal structure of bovine carbonic anhydrase-II (PDB ID: 1V9E) was downloaded from the Protein Data Bank (https://www.rcsb.org/). Water molecules at an active site within the vicinity of 3 Å were retained and the rest of them were removed. The enzyme structure was then prepared for docking simulation using Protonate 3D option in MOE. Triangle Matcher placement method and London dG scoring function were used for docking using Zn 2+ metal ion as a constrain for molecular docking. The bestdocked pose of each compound was selected based on the binding interactions and docking score. The docking protocol was first validated by redocking the standard acetazolamide in the active site of the enzyme. The best-docked pose of the known inhibitor showed a highly negative docking score (S −5.40 kcal/mol). The calculated RMSD between the docked and the native confirmation of sulfonamide was 2.31 Å. The docked view and binding interactions of the known active inhibitor are shown in Figure 4. The validated molecular docking protocol was then used to predict the binding pattern of the newly synthesized triazole derivatives in the active site of CA-II and to elucidate their structure-activity relationship.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material; further inquiries can be directed to the corresponding author.

AUTHOR CONTRIBUTIONS
SA designed the study and wrote the manuscript; MK and AK were involved in biological activity; SH and SA-R performed molecular docking studies; RC and BD were involved in manuscript corrections and writing part.