Synthesis and Biological Evaluation of Coumarin-Linked 4-Anilinomethyl-1,2,3-Triazoles as Potent Inhibitors of Carbonic Anhydrases IX and XIII Involved in Tumorigenesis

A series of coumarin-linked 4-anilinomethyl-1,2,3-triazoles (6a–t) was synthesized via a molecular hybridization approach, through carbon C-6 of the coumarin moiety. The synthesized compounds were evaluated for their inhibition of carbonic anhydrase (CA) isoforms I, II, IX and XIII. CAs IX and XIII were selectively inhibited over the off-target isoforms I and II. The best inhibitory profiles against CA IX were shown by compounds 6a, 6e and 6f (Ki < 50 nM), with compound 6e displaying the best inhibition with a Ki value of 36.3 nM. Compounds 6a, 6b, 6j, 6o and 6q exhibited the best inhibitory profiles against CA XIII (Ki < 100 nM). These compounds can be further explored for the discovery of potent and effective CA IX and CA XIII inhibitors.


Introduction
Metalloenzymes and metalloproteins are two key players in cellular regulation, which act by maintenance of homoeostasis, control of metal ion concentrations, buffering and activation of signal pathways. Carbonic anhydrases (CAs, EC 4.2.1.1) are a super family of metalloenzymes, which play a fundamental role in the inter conversion of CO 2 to bicarbonate and proton. Thereby, they perform the constant physiological demand of converting CO 2 generated during metabolic oxidative processes into ionic, soluble species [1,2].
These enzymes have evolved convergently over the eons into eight families, which are genetically distinct. The families are designated as α, β, γ, δ, ζ, η, θ and ι CAs. All CAs exhibit catalytic activity, on account of a metal ion present within their active sites. Most of them contain a Zn(II) ion in their active sites, but in ζ CAs the Zn(II) ion is interchangeable with Cd(II). Similarly, Co(II) may substitute Zn(II) in α CAs and Fe(II) might be present in γ CAs, especially under anaerobic conditions. The human CAs belong to α class (denoted as human carbonic anhydrases (hCAs)), consisting of sixteen isozymes differing in various aspects like structure, cellular localization, organ and tissue distribution, catalytic properties and response to various inhibitors. The hCAs I, II, III, VII, VIII, X and XIII are cytosolic, hCAs IV, IX, XII, XIV and XV are membrane-bound, hCAs VA and VB are mitochondrial and hCA VI is secretory in nature [3,4].
Hypoxia has been determined to be one of the key features of several solid tumors, due to incomplete vascularization and overexpression of proteins which dramatically change the metabolism. Hypoxia promotes tumor-adaptive processes such as acidosis, tumor

Chemistry
The synthesis of the coumarin-linked 4-anilinomethyl-1,2,3-triazoles (6a-t) was performed according to the general synthetic scheme as illustrated in Scheme 1. The reduction of 6-nitrocoumarin to 6-aminocoumarin (2) and the conversion of 6-aminocoumarin to 6-azidocoumarin (3) was carried out according to the reported

Carbonic Anhydrase Inhibition
The coumarin-linked 4-anilinomethyl-1,2,3-triazoles, 6a-t, were tested for their inhibition of three cytosolic isoforms, namely hCAs I, II and XIII, as well as the transmembrane tumor-associated isoform, hCA IX. Acetazolamide (AAZ) was used as the standard drug. The following conclusions can be drawn from the results shown in Table 1: The newly synthesized compounds, 6a-twere found to be ineffective against the cytosolic isoforms hCAs I and II (Ki> 50,000 nM).

Carbonic Anhydrase Inhibition
The coumarin-linked 4-anilinomethyl-1,2,3-triazoles, 6a-t, were tested for their inhibition of three cytosolic isoforms, namely hCAs I, II and XIII, as well as the transmembrane tumor-associated isoform, hCA IX. Acetazolamide (AAZ) was used as the standard drug. The following conclusions can be drawn from the results shown in Table 1: The newly synthesized compounds, 6a-t were found to be ineffective against the cytosolic isoforms hCAs I and II (K i > 50,000 nM). II.
The transmembrane tumor-associated isoform hCA IX was inhibited by the compounds, 6a-t in a low to moderate nanomolar range with the K i values ranging from 36.3 to 642.8 nM. Compound 6e possessing a 4-bromo substitution on the aniline moiety exhibited the most potent inhibition of hCA IX with a K i value of 36.3 nM. Compound 6f is possessing a 4-isopropyl substitution on the aniline moiety and compound 6a possessing a 4-methyl substitution on the aniline moiety exhibited K i values of 45.0 and 48.4 nM, respectively. Barring compounds 6p, 6r and 6t, all the compounds displayed K i values < 100 nM against hCA IX. Additionally, the compounds containing electron-donating substituents on the phenyl ring of aniline displayed better inhibitory profiles over the electron-withdrawing substituents. Thus, it is implied from the overall results that the compounds 6a-t are highly selective inhibitors of hCAs IX and XIII over the off-target isoforms, hCAs I and II. Thus, it is implied from the overall results that the compounds 6a-t are highly selective inhibitors of hCAs IX and XIII over the off-target isoforms, hCAs I and II. pounds, 6a-t in a low to moderate nanomolar range with the Ki values ranging from 36.3 to 642.8 nM. Compound 6e possessing a 4-bromo substitution on the aniline moiety exhibited the most potent inhibition of hCA IX with a Ki value of 36.3 nM. Compound 6f is possessing a 4-isopropyl substitution on the aniline moiety and compound 6a possessing a 4-methyl substitution on the aniline moiety exhibited Ki values of 45.0 and 48.4 nM, respectively. Barring compounds 6p, 6r and 6t, all the compounds displayed Ki values <100 nM against hCA IX. Additionally, the compounds containing electron-donating substituents on the phenyl ring of aniline displayed better inhibitory profiles over the electron-withdrawing substituents. III.
The cytosolic isoform hCA XIII was inhibited by the compounds 6a-t in a low to high nanomolar range with the Ki values ranging from 90. Thus, it is implied from the overall results that the compounds 6a-t are highly selective inhibitors of hCAs IX and XIII over the off-target isoforms, hCAs I and II. Thus, it is implied from the overall results that the compounds 6a-t are highly selective inhibitors of hCAs IX and XIII over the off-target isoforms, hCAs I and II. Thus, it is implied from the overall results that the compounds 6a-t are highly selective inhibitors of hCAs IX and XIII over the off-target isoforms, hCAs I and II. Thus, it is implied from the overall results that the compounds 6a-t are highly selective inhibitors of hCAs IX and XIII over the off-target isoforms, hCAs I and II.

Conclusions
In the present study, two potent CA pharmacophores, coumarin and substituted 4-anilinomethyl-1,2,3-triazoles, were combined using a molecular hybridization approach and a series of new 6-substituted-coumarin derivatives 6a-t was synthesized. The 1,2,3-triazole moiety was tethered to the C-6 position of the coumarin moiety. All the synthesized compounds were screened against four CA isoforms, I, II, IX and XIII. None of the compounds showed significant inhibition of the off-target CA isoforms, I and II. Most compounds showed good inhibition of CA IX (Ki< 100 nM)

Conclusions
In the present study, two potent CA pharmacophores, coumarin and substituted 4-anilinomethyl-1,2,3-triazoles, were combined using a molecular hybridization approach and a series of new 6-substituted-coumarin derivatives 6a-t was synthesized. The 1,2,3-triazole moiety was tethered to the C-6 position of the coumarin moiety. All the synthesized compounds were screened against four CA isoforms, I, II, IX and XIII. None of the compounds showed significant inhibition of the off-target CA isoforms, I and II. Most compounds showed good inhibition of CA IX (Ki< 100 nM)

Conclusions
In the present study, two potent CA pharmacophores, coumarin and substituted 4-anilinomethyl-1,2,3-triazoles, were combined using a molecular hybridization approach and a series of new 6-substituted-coumarin derivatives 6a-t was synthesized. The 1,2,3-triazole moiety was tethered to the C-6 position of the coumarin moiety. All the synthesized compounds were screened against four CA isoforms, I, II, IX and XIII. None of the compounds showed significant inhibition of the off-target CA isoforms, I and II. Most compounds showed good inhibition of CA IX (Ki< 100 nM)

Conclusions
In the present study, two potent CA pharmacophores, coumarin and substituted 4-anilinomethyl-1,2,3-triazoles, were combined using a molecular hybridization approach and a series of new 6-substituted-coumarin derivatives 6a-t was synthesized. The 1,2,3-triazole moiety was tethered to the C-6 position of the coumarin moiety. All the synthesized compounds were screened against four CA isoforms, I, II, IX and XIII. None of the compounds showed significant inhibition of the off-target CA isoforms, I and II.

General
All the chemicals and solvents were procured and utilized as such from the suppliers. Wherever necessary, anhydrous solvents were used. Thin Layer Chromatography (TLC) analysis was done by utilizing Merck silica gel 60 F 254 aluminum plates. Stuart Digital Melting Point Apparatus (SMP 30) was used in determining the melting points of the compounds, which are uncorrected. 1 H and 13 C NMR spectra were recorded using Bruker Avance 500 MHz and 125 MHz, respectively, using DMSO-d 6 as the solvent. Chemical Shift values are recorded in ppm using TMS as the internal standard. HRMS were determined by Agilent QTOF mass spectrometer 6540 series instrument and were performed using ESI techniques at 70 eV.
3.1.1. Synthesis of 6-Amino-2H-Chromen-2-One (2) To a reaction mixture of Fe powder (1.7 g, 31 mmol) and NH 4 Cl (0.8 g, 15 mmol) in EtOH (30 mL) and water (10 mL) was added 6-nitro-2H-chromen-2-one (1.5 g, 7.8 mmol). The reaction mixture was stirred at 80 • C for 4-5 h. Upon completion of the reaction as monitored by TLC, the reaction mixture was cooled to room temperature and it was extracted with ethyl acetate (3 × 100 mL). The organic layer was washed with brine and dried over Na 2 SO 4 . The solid was filtered off and the filtrate was concentrated under reduced pressure. The resulting crude product was purified by column chromatography using silica gel (60-120 mesh) as the stationary phase and EtOAc in hexane (0 percent to 20 percent) as the mobile phase to afford the entitled compound 2 as a yellow-colored solid (Yield: 85%).

Synthesis of 6-Azido-2H-Chromen-2-One (3)
To a mixture of 2.5 mL of conc. H 2 SO 4 and 7 mL of water was dissolved 1.6 g (10 mmol) of intermediate 2 and the solution was cooled to 0 • C. To this solution, a saturated solution of 0.83 g (12 mmol) of NaNO 2 was added maintaining the temperature < 5 • C. After stirring for 5 min, a solution of 0.65 g (10 mmol) of NaN 3 in 5 mL of water was added. The reaction mixture was allowed to stir at room temperature and it was monitored by TLC. Upon completion of the reaction as monitored by TLC, the solid obtained was filtered, dried and weighed to afford the entitled compound 3 as a cream-colored solid (Yield: 90%).

Synthesis of N-Propargylated Anilines (5a-t)
To a solution of substituted anilines 4a-t (4.0 equivalent) in 5-10 mL DMF, potassium carbonate (2.0 equivalent) was added. The mixture was stirred for 5 min at room temperature. Thereafter, a solution of propargyl bromide (1.0 equivalent) was added to the mixture drop-wise. The resulting mixture was stirred at room temperature for 6 h. After completion of reaction as indicated by TLC, crushed ice was added to the reaction mixture and the solid obtained was filtered. The resulting crude solid was purified by column chromatography using silica gel (60-120 mesh) as the stationary phase and EtOAc in hexane (0 percent to 20 percent) as the mobile phase to afford the title compounds 5a-t.

Synthesis of Coumarin-Triazole Hybrids (6a-t)
To a mixture of 6-azidocoumarin (1 equivalent) and intermediates 5a-t (1 equivalent) in 6 mL of water/tert-butanol (1:1), was added sodium ascorbate (0.3 equivalent), followed by copper (II) sulfate pentahydrate (0.03 equivalent). The heterogeneous mixture was stirred overnight at room temperature, at which point it cleared and TLC analysis indicated complete consumption of the reactants. To the reaction mixture was added crushed ice and the solid obtained was collected by filtration. Thereafter, the solid was washed with cold water and dried in oven. It was purified by silica gel chromatography using silica gel (60-120 mesh) as the stationary phase and EtOAc:Hexane:1:1 as mobile phase to afford the final compounds 6a-t.

CA Inhibition
An SX.18V-R Applied Photophysics (Oxford, UK) stopped flow instrument has been used to assay the CA catalyzed CO 2 hydration activity [26]. Phenol Red (at a concentration of 0.2 mM) has been used as an indicator, working at an absorbance maximum of 557 nm, with 10 mM Hepes (pH 7.4) as a buffer, 0.1 M Na 2 SO 4 or NaClO 4 (for maintaining constant the ionic strength; these anions are not inhibitory in the used concentration), following the CA-catalyzed CO 2 hydration reaction for a period of 5-10 s. Saturated CO 2 solutions in water at 25 • C were used as substrate. Stock solutions of inhibitors were prepared at a concentration of 10 mM (in DMSO-water 1:1, v/v) and dilutions up to 0.01 nM done with the assay buffer mentioned above. At least 7 different inhibitor concentrations have been used for measuring the inhibition constant. Inhibitor and enzyme solutions were pre-incubated together for 10 min at room temperature prior to assay, in order to allow for the formation of the E-I complex. Triplicate experiments were done for each inhibitor concentration, and the values reported throughout the paper are the mean of such results. The inhibition constants were obtained by non-linear least squares methods using the Cheng-Prusoff equation, as reported earlier, 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 and their concentration was of 2.5-12 nM in the assay system [27][28][29][30].

Conclusions
In the present study, two potent CA pharmacophores, coumarin and substituted 4-anilinomethyl-1,2,3-triazoles, were combined using a molecular hybridization approach and a series of new 6-substituted-coumarin derivatives 6a-t was synthesized. The 1,2,3triazole moiety was tethered to the C-6 position of the coumarin moiety. All the synthesized compounds were screened against four CA isoforms, I, II, IX and XIII. None of the compounds showed significant inhibition of the off-target CA isoforms, I and II. Most compounds showed good inhibition of CA IX (K i < 100 nM), with compound 6e showing the most effective inhibition (K i = 36.3 nM). Against CA XIII, compounds 6a, 6b, 6j, 6o and 6q exhibited good inhibitory profiles (K i < 100 nM). Thus, these molecules can be further explored as a starting point for the design of potent and effective inhibitors against CA IX and CA XIII isoforms. The structure activity relationship SAR of the synthesized compounds has been depicted in Figure 2.

Conclusions
In the present study, two potent CA pharmacophores, coumarin and substituted 4-anilinomethyl-1,2,3-triazoles, were combined using a molecular hybridization approach and a series of new 6-substituted-coumarin derivatives 6a-t was synthesized. The 1,2,3-triazole moiety was tethered to the C-6 position of the coumarin moiety. All the synthesized compounds were screened against four CA isoforms, I, II, IX and XIII. None of the compounds showed significant inhibition of the off-target CA isoforms, I and II Most compounds showed good inhibition of CA IX (Ki< 100 nM), with compound 6e showing the most effective inhibition (Ki= 36.3 nM). Against CA XIII, compounds 6a, 6b 6j, 6o and 6q exhibited good inhibitory profiles (Ki< 100 nM). Thus, these molecules can be further explored as a starting point for the design of potent and effective inhibitors against CA IX and CA XIII isoforms. The structure activity relationship SAR of the synthesized compounds has been depicted in Figure 2.