Synthesis, Molecular Docking Analysis, and Biological Evaluations of Saccharide-Modified Sulfonamides as Carbonic Anhydrase IX Inhibitors

Based on the strategy of the “tail approach”, 15 novel saccharide-modified sulfonamides were designed and synthesised. The novel compounds were evaluated as inhibitors of three human carbonic anhydrase (CA) isoforms, namely cytoplasmic CA II, transmembrane CA IX, and XII. Most of these compounds showed good activity against CAs and high topological polar surface area (TPSA) values, which had a positive effect on the selective inhibition of transmembrane isoforms CA IX and XII. In the in vitro activity studies, compounds 16a, 16b, and 16e reduced the viability of HT-29 and MDA-MB-231 cells with a high expression of CA IX under hypoxia. The inhibitory activity of compound 16e on the human osteosarcoma cell line MG-63 with a high expression of CA IX and XII was better than that of AZM. Moreover, high concentrations of compounds 16a and 16b reversed the acidification of the tumour microenvironment. In addition, compound 16a had a certain inhibitory effect on the migration of MDA-MB-231 cells. All the above results indicate that the saccharide-modified sulfonamide has further research value for the development of CA IX inhibitors.


Introduction
Hypoxia and acidification are salient features of many tumour tissues [1]. Such a microenvironment is of great significance to tumour cell survival, proliferation, invasion, and the development of drug resistance [1][2][3]. Hypoxia in the microenvironment is caused by an imbalance between the rapid growth of tumour cells and the oxygen supply capacity, while acidification is caused by a variety of proteins including carbonic anhydrase (CAs, EC 4.2.1.1) [1,4,5]. In the CA family, CA IX and XII are isoforms related to acidification of the tumour microenvironment [6]. CA IX is not significantly expressed in most normal tissues but is overexpressed in many aggressive tumours including lung, breast, colorectal, head and neck, lung, and kidney cancers. CA IX is of great significance for maintaining the tumour microenvironment and is closely related to the proliferation, migration, and invasion of tumour cells [7,8]. Therefore, CA IX has been validated as a promising target for cancer diagnostics and treatments. Compared with CA IX, CA XII is distributed in a variety of normal tissues. CA XII is minimally expressed in various normal tissues (including in the kidney, colon, prostate, pancreas, ovary, testis, lung, and brain), but its expression is upregulated in cancer cells derived from these tissues [9]. Several recent studies have shown that CA XII is an attractive and potential target for tumour treatment and diagnostic intervention, especially for hypoxic tumours that are resistant to traditional radiotherapy and chemotherapy [9][10][11]. Both CA IX and CA XII play an important role in maintaining the acidic microenvironment of tumours in a variety of tumour tissues and have been proven to be vital to tumour growth and survival [12,13].

hCAs Inhibitory Activity
All the synthesised compounds and key intermediates were assayed as inhibitors of two physiologically relevant CA isoforms, namely transmembrane tumour-associated hCA IX and off-target cytosolic hCA II, and some of the compounds with significant

hCAs Inhibitory Activity
All the synthesised compounds and key intermediates were assayed as inhibitors of two physiologically relevant CA isoforms, namely transmembrane tumour-associated hCA IX and off-target cytosolic hCA II, and some of the compounds with significant activity were supplemented with the hCA XII inhibitory activity test. The test used a method of monitoring the hydrolysis of 4-nitrophenylacetate (4-NPA.) The control compound was acetazolamide (AZM), which is clinically used as a carbonic anhydrase inhibitor. The in vitro enzyme inhibition test results of sulfatriazole derivatives 14a-h and carbohydratebased compounds 16a-o are listed in Table 1. Based on the data in Table 1, the structure-activity relationship (SAR) is summarised as follows: (i). The inhibitory activities of sulfatriazole derivatives 14a-h against hCA II and IX were 12.3-102.0 and 40.4-120.2 nM, respectively. Among compounds 14a-h, 14a-c showed comparable inhibitory activities to hCAII and IX as AZM, and the inhibitory activities of other compounds against hCAII and IX were weaker. The SAR of compounds 14a-h for hCA II and IX was as follows: when the aromatic amine ring was unsubstituted or substituted by a fluorine atom with a small atomic volume, the inhibitory activities for hCAII and IX were good, but when the volume of substituents on aniline increased, both the electron-donating and electron-withdrawing groups caused the inhibitory activity against hCA II to decrease.
(ii). The inhibitory activities of saccharide-modified compounds 16a-o against hCA II and IX were 10.1-177.2 and 51.6-543.9 nM, respectively. Compared with compounds 14a-h without saccharide, the inhibitory activities of compounds 16a-o on hCA II did not change significantly, but the activity of hCA IX decreased noticeably. At the same time, although the introduction of saccharide fragments did not significantly improve the inhibitory activities on the enzyme, the TPSA of the saccharide derivatives increased significantly, which reduced the membrane permeability of compounds, and provided the possibility of isoform selectivity based on subcellular localisation.
(iii). Compounds 16a-b and 16h-i with unsubstituted benzene rings showed the best inhibitory activity against hCA II, ranging from 10.1 to 27.7 nM. When the benzene ring was substituted by halogen (F or Cl) and electron-withdrawing substituents (nitro or difluoro), the activity against hCA II decreased. When the benzene ring was substituted by electron-donating substituents, methyl substitution led to a slight decrease or maintenance of the inhibitory activity against hCA II compared with unsubstituted compounds, while methoxy caused a significant decrease. The effect of aliphatic chain length on hCA II inhibitory activities was not obvious.
(iv). The inhibitory effect of compounds 16a-o on hCA IX was not as clear as that on hCA II. The compounds with the best inhibitory activity against hCA IX were 16a, 16b, 16e and 16m, ranging from 51.6 to 99.6 nM. When the aliphatic chain was succinamide (n = 2), the most active compound was 16a, and replacement with meta-aniline or substitution with different groups both caused a decrease in activity. The decrease in activity caused by difluoro substitution was the most significant, resulting in a 10-fold decrease in activity. When the aliphatic chain was glutamide (n = 3), the most active compound was 16m. Compared with succinamide, the methoxy group played a positive role in increasing the activity.
(v). Compounds 16a, 16b, 16e, and 16m were selected to test the inhibitory activity against hCA XII, and the values ranged from 135.3 to 255.9 nM. Their inhibitory effect on hCA XII was weaker than that on hCA II and IX.

Docking Studies
We re-docked the native ligand (9FK) into CA IX (PDB ID: 5FL4) with optimised docking parameters and successfully produced the binding modes observed in the crystal structures. The ligand formed hydrogen bonding with Gln92, His119, and Thr200 in the active site, and hydrophobic interaction with His94, His96, Val130, and Leu134. The RMSD values of docking and experimental posture is 0.699 Å (detailed data is provided in the Table S1 and Figure S1 of supplementary information). Then, we conducted docking studies on the saccharide-modified compounds 16a and 16g to explain why they exhibit different inhibitory activities. The results are shown in Figure 1.
As shown in Figure 1, the modes of action of compounds 16a and 16g at the active site were quite different. Compound 16a formed strong interactions with Gln92, His94, and Thr200 at the active site, while 16g only interacted with Thr200 through the sulfonamide group. In addition, the mode of binding of the two glycosyl fragments at the entrance of the active site was different. Compound 16a formed hydrogen bonds with Arg129 and Asp131 through the glycosyl hydroxyl group and the amide in the structure, while the glycosyl in 16g only had a hydrogen bond with Val20. Moreover, the docking scores of 16a and 16g were −8.37 kcal/mol and −6.49 kcal/mol, respectively. From the docking scores, it was apparent that compound 16a was better than 16g. The fewer interactions and lower binding stability might be responsible for the lower activity of 16g.

In Vitro Cytotoxicity Studies on Cancer Cells
The CCK-8 assay was used to evaluate the cytotoxicity of compounds 16a, 16b, and 16e on the breast cancer cell line MDA-MB-231 and colon cancer cell line HT-29. MDA-MB-231 and HT-29 are tumour cells that have been reported to express CA IX. We cultivated them under normoxia and hypoxia respectively and then evaluated the effects of the compounds on tumour cell viability at different concentrations. The clinical drug AZM was selected as the positive cytotoxic reference compound at the same concentrations (detailed data is provided in the Tables S2 and S3 of supplementary information). As shown in Figure 1, the modes of action of compounds 16a and 16g at the active site were quite different. Compound 16a formed strong interactions with Gln92, His94, and Thr200 at the active site, while 16g only interacted with Thr200 through the sulfonamide group. In addition, the mode of binding of the two glycosyl fragments at the entrance of the active site was different. Compound 16a formed hydrogen bonds with Arg129 and Asp131 through the glycosyl hydroxyl group and the amide in the structure, while the glycosyl in 16g only had a hydrogen bond with Val20. Moreover, the docking scores of 16a and 16g were −8.37 kcal/mol and −6.49 kcal/mol, respectively. From the docking scores, it was apparent that compound 16a was better than 16g. The fewer interactions and lower binding stability might be responsible for the lower activity of 16g.

In Vitro Cytotoxicity Studies on Cancer Cells
The CCK-8 assay was used to evaluate the cytotoxicity of compounds 16a, 16b, and 16e on the breast cancer cell line MDA-MB-231 and colon cancer cell line HT-29. MDA-MB-231 and HT-29 are tumour cells that have been reported to express CA IX. We cultivated them under normoxia and hypoxia respectively and then evaluated the effects of the compounds on tumour cell viability at different concentrations. The clinical drug AZM was selected as the positive cytotoxic reference compound at the same concentrations (detailed data is provided in the Table S1-2 of supplementary information).
According to the results of the cell viability test, we summarised the following conclusions: (i). Data from Figure 2 show that both AZM and the tested compounds had a very limited effect on the growth of the two tumour cell lines under normoxic conditions. When the cell lines were cultured under hypoxia, the inhibitory effect of the compounds was According to the results of the cell viability test, we summarised the following conclusions: (i). Data from Figure 2 show that both AZM and the tested compounds had a very limited effect on the growth of the two tumour cell lines under normoxic conditions. When the cell lines were cultured under hypoxia, the inhibitory effect of the compounds was improved expect for that of 16e; the inhibitory effect of 16e on MDA-MB-231 under hypoxia is slightly weaker than that under normoxic condition.
(ii). The results showed that HT-29 colon cancer cells were more susceptible to inhibition by the tested compounds, and a 20% decrease in cell viability was observed in compounds 16a and 16b at the highest concentration of 400 µM.
(iii). Compared to AZM, compounds 16a, 16b, and 16e exhibited higher inhibitory activity against HT-29 and MDA-MB-231 cells, which was particularly evident in the hypoxic HT-29 cell line.
The human osteosarcoma cell line MG-63 is a high-expressing cell line of CA IX and XII [24]. We selected compound 16b with the best activity in the above studies to study the cell viability inhibitory activity of MG-63 (detailed data is provided in the Table S4 of supplementary information). The results are shown in Figure 3.
The test results showed that the inhibition of MG-63 by 16b was concentrationdependent, and its inhibitory activity was better than that of the positive control AZM. Moreover, under a hypoxic environment, the inhibitory effect of 16b on MG-63 cells was slightly higher than that of normoxia.
(ii). The results showed that HT-29 colon cancer cells were more susceptible to inhibition by the tested compounds, and a 20% decrease in cell viability was observed in compounds 16a and 16b at the highest concentration of 400 μM.
(iii). Compared to AZM, compounds 16a, 16b, and 16e exhibited higher inhibitory activity against HT-29 and MDA-MB-231 cells, which was particularly evident in the hypoxic HT-29 cell line. The human osteosarcoma cell line MG-63 is a high-expressing cell line of CA IX and XII [24]. We selected compound 16b with the best activity in the above studies to study the cell viability inhibitory activity of MG-63 (detailed data is provided in the Table S3 o supplementary information). The results are shown in Figure 3.  The human osteosarcoma cell line MG-63 is a high-expressing cell line o XII [24]. We selected compound 16b with the best activity in the above stud the cell viability inhibitory activity of MG-63 (detailed data is provided in th supplementary information). The results are shown in Figure 3.

Measurement of Extracellular pH
The main role of CA IX and XII in tumour development is to regulate the pH of the tumour growth microenvironment so that tumour cells are in a weakly acidic environment. Such an acidic environment is conducive to the growth of tumour cells and protects tumour cells from being killed by chemotherapeutics. We evaluated the effects of 0.1 mM and 1.0 mM of compounds 16a and 16b on the pH of the tumour cell growth environment under normoxia or hypoxia (detailed data is provided in the Tables S5 and S6 of supplementary  information). The results are shown in Figure 4.
The main role of CA IX and XII in tumour development is to re tumour growth microenvironment so that tumour cells are environment. Such an acidic environment is conducive to the growth protects tumour cells from being killed by chemotherapeutics. We ev 0.1 mM and 1.0 mM of compounds 16a and 16b on the pH of the environment under normoxia or hypoxia (detailed data is provided supplementary information). The results are shown in Figure 4. The results showed that both 16a and 16b could improve the MDA-MB-231 and HT-29 cells at concentrations of 0.1 mM and 1.0 m concentration-dependent. The pH change of the extracellular space w in hypoxic culture. After the tumour cells were cultured with the 1.0 extracellular pH increased significantly so that the environment ch alkaline.

Cell Migration Assay
Tumour cell migration is one of the basic biological characteri We carried out a study on the antitumour migration activity of co The results showed that both 16a and 16b could improve the extracellular pH of MDA-MB-231 and HT-29 cells at concentrations of 0.1 mM and 1.0 mM, and the effect was concentration-dependent. The pH change of the extracellular space was especially distinct in hypoxic culture. After the tumour cells were cultured with the 1.0 mM compounds, the extracellular pH increased significantly so that the environment changed from acidic to alkaline.

Cell Migration Assay
Tumour cell migration is one of the basic biological characteristics of tumour cells. We carried out a study on the antitumour migration activity of compound 16a, which showed high CA IX inhibitory activity, to investigate the effect of a CA IX inhibitor on tumour cell migration. MDA-MB-231 was selected as the experimental cell line to explore the ability of 16a to reduce tumour cells migration under hypoxia. As shown in Figure 5, after the positive control AZM and 16a at a concentration of 400 µM were incubated with MDA-MB-231 hypoxia for 72 h. Compared with the blank group, 16a had a slight inhibitory effect on tumour cell migration in the first 24 h, and this inhibitory effect could hardly be detected at 72 h. Compared with AZM, 16a improved the inhibition of tumour cell migration, but it was not obvious.
showed high CA IX inhibitory activity, to investigate the effect of a CA IX inh tumour cell migration. MDA-MB-231 was selected as the experimental cell line to the ability of 16a to reduce tumour cells migration under hypoxia. As shown in after the positive control AZM and 16a at a concentration of 400 μM were incuba MDA-MB-231 hypoxia for 72 h. Compared with the blank group, 16a had inhibitory effect on tumour cell migration in the first 24 h, and this inhibitory effe hardly be detected at 72 h. Compared with AZM, 16a improved the inhibition o cell migration, but it was not obvious.

Chemistry
All the reagents were obtained from commercial sources and used withou purification unless otherwise specified. Solvents were dried and redistilled befo the usual manner. All chemicals and solvents were analytical grades. Analytical performed using silica gel HF254 (Qingdao Haiyang Chemical, Qingdao, Preparative column chromatography was performed with silica gel H. Meltin were obtained on a Büchi melting point B-540 apparatus (Büchi Labortechnik Switzerland). 1 H and 13 C NMR spectra (details of raw data for compounds, see S1-S82) were recorded on Bruker ARX 600 MHz or 400 MHz spectrometers Zurich, Switzerland). HRMS was obtained on an Agilent ESI-QTOF instrument Santa Clara, CA, USA).

General Procedure of Synthesis of N-(4-Ethynylphenyl)acetamide Derivatives (12a-h)
Intermediates 12a-h were synthesized from compounds 11a-h by deprotection of trimethylsilyl using TBAF [28] and extraction after the reaction and used without further purification.

Measurement of Extracellular pH
The changes in extracellular pH were evaluated according to previously published procedures [31]. Cancer cells were grown in the medium containing 10% FBS and incubated in 6-well plates for 12 h. After removing the medium, we added fresh medium to the 6-well plates and incubated them for 48 h under hypoxia or normoxia. Then, we measured the pH of the culture medium immediately after collection. The tested compounds were dissolved in DMSO and diluted to the desired concentration. After the addition of tested compounds, the same procedure was carried out.

Cell Migration Assay
MDA-MB-231 cells (1 × 10 5 cells/well) were cultured in 6-well plates and wounded by scratching with a pipette tip. The wounded cell monolayers were washed three times with PBS and incubated with serum-free medium. Then, cells were treated with AZM and 16a respectively followed by a 72 h incubation under hypoxic condition, and they were photographed at 0, 24, 48, and 72 h with an inverted microscope.

Molecular Docking Studies
The CA IX crystal structure (PDB code: 5FL4) was downloaded from the protein data bank (https://www.rcsb.org/, accessed on 9 November 2021) and prepared with the Protein Preparation Wizard in the Schrödinger suite. The preparation of protein structure included adding bond orders, adding hydrogen atoms, deleting water molecules, and producing appropriate protonation states. The protonation and tautomeric states of Asp, Lys, and His were assigned at pH 7.4 state. The co-crystal ligand of 5FL4 was separated from the crystal structure. The selected inhibitors were prepared by using LigPrep from the Schrödinger suite with the OPLS_2005 force field. The structure of the inhibitor has also been modified by adding all hydrogen atoms, checking the bond sequence and atom type.
Receptor grids were generated before docking with the activity site determined by the literature. The prepared protein-ligand complex was imported into Glide 9.7, which defined it as the receptor structure with a size box (20 Å × 20 Å × 20 Å) [32,33]. The grid of the CA IX crystal structure was generated based on the OPLS_2005 force field. The standard precision (SP) mode was set for docking studies [34].
The standard descriptors protocol in the DS (version 3.0, Accelrys Inc., San Diego, CA, USA) was used to assess the ADME/T profile of the selected compounds. With the help of the ADME/T module, various parameters including TPSA are used to predict the ADME/T characteristics of specified compounds [35].

Conclusions
In this work, 15 novel saccharide-modified carbonic anhydrase inhibitors, 16a-o, were designed and synthesised via the "tail approach". Saccharide-modified compounds 16a-o and key intermediates 14a-h were evaluated for their enzymatic activity against tumour-associated isoforms CA IX and XII and the common off-target isoform CA II. The results showed that all the saccharide-modified compounds had inhibitory activity against hCA II, IX, and XII, with IC 50 values ranging from 13.3 to 177.2 nM, 51.6 to 543.9.6 nM, and 135.3 to 255.9 nM, respectively. Afterwards, the series of compounds were tested at the cellular level to investigate their effects on the cell viabilities of the MDA-MB-231, HT-29, and MG-63 cell lines. Compounds 16a, 16b, and 16e could reduce cell viability at high concentrations, and this effect was particularly obvious under hypoxic conditions. Moreover, we found that compounds 16a and 16b could significantly increase the extracellular pH of tumour cells and transform the microenvironment from acidic to alkaline. In addition, in the cell migration experiment, 16a exhibited a more apparent antimigration effect than the positive control AZM, and we predicted the pharmacokinetic properties of the synthesised compounds using the SwissADME website to find compounds that may have good pharmacokinetic properties (detailed results are provided in the Tables S7-S13 of supplementary information) [36]. Based on the above test results, we believe that modification of the glycosyl tail of targeted CA IX inhibitors is an effective strategy worthy of further study.