Synthesis, Biological Evaluation, and In Silico Studies of Novel Coumarin-Based 4H,5H-pyrano[3,2-c]chromenes as Potent β-Glucuronidase and Carbonic Anhydrase Inhibitors

The search for novel heterocyclic compounds with a natural product skeleton as potent enzyme inhibitors against clinical hits is our prime concern in this study. Here, a simple and facile two-step strategy has been designed to synthesize a series of novel coumarin-based dihydropyranochromenes (12a–12m) in a basic moiety. The synthesized compounds were thus characterized through spectroscopic techniques and screened for inhibition potency against the cytosolic hCA II isoform and β-glucuronidase. Few of these compounds were potent inhibitors of hCA II and β-glucuronidase with varying IC50 values ranging from 4.55 ± 0.22 to 21.77 ± 3.32 μM and 440.1 ± 1.17 to 971.3 ± 0.05 μM, respectively. Among the stream of synthesized compounds, 12e and 12i were the most potent inhibitors of β-glucuronidase, while 12h, 12i, and 12j showed greater potency against hCA II. In silico docking studies illustrated the significance of substituted groups on the pyranochromene skeleton and binding pattern of these highly potent compounds inside enzyme pockets.


■ INTRODUCTION
β-Glucuronidase belongs to the family of lysosomal enzymes, which brings about the cleavage of glucuronosyl-O-bond, thus removing individual sugars from glycosaminoglycans (GAGs). 1 The shortage of β-glucuronidase in body tissues consequently results in proliferation of GAGs inside the lysosomes, leading to the enlargement of their size. 2 As a consequence of overexpression of this enzyme in various cancers like the neoplasm of bladder, 3 liver cancer, 4 and colon carcinoma, 5,6 the quantitative measurement of β-glucuronidase has been proved as a detection tool at the initial stage diagnosis of cancer and the assessment of therapeutic treatments. 7,8 Moreover, it is evident from the literature that an elevated level of βglucuronidase in blood serum is related to various physiological disorders such as urinary tract infection, 9 rejection of transplantation, 10 epilepsy, 11 renal diseases, 12 rheumatoid arthritis, 13 and larynx 14 and breast cancer. 15 Besides these, acquired immune deficiency syndrome (AIDs) and hepatic disorders have also been reported as a consequence of a high level of this enzyme. 16,17 Carbonic anhydrase (CA) isoenzymes are pervasive members of metalloenzymes with a Zn(II) ion metallic core at the active center of the enzyme. 18,19 They are prevalent in all organisms where they are engaged in the reversible catalysis of hydration of CO 2 to (HCO 3 −1 ) and (H + ). 20 This reaction is associated with various physiological processes in humans such as electrolytic secretion, pH regulation, gluconeogenesis, lipogenesis, bone resorption, calcification, tumorigenicity, and various other pathological processes. 21 CAs are committed as attractive targets for drug design to cure therapeutic diseases like acid−base disequilibria, epilepsy, glaucoma, cancer, osteoporosis and other neuromuscular disorders, edema, altitude sickness, and obesity. 22,23 Recent studies have shown the role of CA inhibitors (CAIs) as a therapeutic hit for other diseases like neuropathic pain, 24 cerebral ischemia, 25 and tumor. 26 The unexpected and novel application of CAIs in synaptic transformation and attentional gating of memory is quite appreciating in the treatment of Alzheimer's disease. 27 The synthesis of efficient heterocyclic molecules with a natural product core and their exploration as therapeutic agents against human diseases have become an indispensable paradigm of clinical trials in modern drug development. 28 Chromenes and coumarins 29 are among the O-ring heterocycles with a benzopyrone skeleton and have gained the attention of pharmaceutical chemists for a number of years because of their therapeutic potential and biological significance. 30,31 The ubiquitous chromene framework is well known for several pharmacological activities like antifungal, 32 antimicrobial, 33 antiviral, 34 anti-allergic, 35 antiulcer, 36 and antiinflammatory activities. 37 Furthermore, fused-chromene compounds are renowned to exhibit a wide range of therapeutic applications starting with antitumor, 38 antileishmanial, 39 antiproliferation, 40 anti-HIV agent, 41 and antioxidative applications. 42 The substituted side chains in the structural framework of benzopyran exhibited the pharmaceutical potential of coumarin-containing drugs, thus exploring their prominence in synthetic and clinical perspective. 43 Coumarin-based compounds are reported as a class of CAIs possessing unique features of their skeletal structure for binding with carbonic anhydrase enzyme. 44 Some of the firstand second-generation pharmacophores like sulfamides, sulfamates, and sulfonamides, still utilized in medical treatment as CAIs, are zinc binders exhibiting various complications. 45−48 Contrary to the above-mentioned compounds, coumarins and thiocoumarins are contrived as CAIs 49 for selective inhibition of hCA isoforms, exploring an inhibition pathway not vulnerable to Zn 2+ ( Figure S1). 50,51 Coumarin compounds are hydrolyzed by the esterase activity of CA and proposed to bind with the entrance of the active site of hCA, the region that is quite variable in various isoforms, where the interaction of coumarin substrates with amino acids leads to higher selectivity toward various isoforms. 52,53 Earlier studies from the literature revealed that some of the coumarin derivatives could not access the enzyme cavity of CA due to bulkiness, which therefore could not be hydrolyzed by the esterase activity of the enzyme. 54−57 Some of the selected compounds from the literature 58−63 are reported here in Figure 1 as hCA II and β-glucuronidase inhibitors, which are rationalized with current work to disclose their metabolic liabilities and/or biological potencies.
Encouraged by above findings about coumarins and chromenes in enzyme inhibition and their role in drug development, we have decided to employ this privileged core with multifarious medicinal properties in synthesizing a series of novel fused ring dihydropyranochromenes (12a−12m). Furthermore, these synthesized compounds were subjected to enzyme inhibition studies to find new inhibitors. β-Glucuronidase and carbonic anhydrase are among the important enzyme targets discussed in the present study for inhibitory properties of synthesized biomolecules against the diseased state. The molecular docking 64−68 study of synthesized compounds is also reported here to compare the results of biological potential of molecules with theoretical calculations.  ■ RESULTS AND DISCUSSION Chemistry. In our targeted approach toward seeking O-ring heterocycles (chromenes) as potent enzyme inhibitors, a series of novel fused ring coumarin-based dihydropyranochromenes (12a−12m) were synthesized in two steps (Scheme 1). In the first step, coumarin-based aldehydes (9a−9b) were synthesized by utilizing the Baylis−Hillman reaction protocol as depicted in the literature. 69 Compound 9a showed physical parameters (i.e., color, m.p., etc.) and spectroscopic data precisely unified with literature values. 69 The structure of the other novel bromo-substituted coumarin-based aldehyde (9b) was precisely verified from its spectroscopic data that was in good agreement. The presence of aldehydic and ester moieties in these precursors could be easily depicted on the basis of IR stretching frequency bands in the regions of 1670−1695 and 1700−1730 cm −1 , and the 1 H NMR spectra also unveiled the singlet of 1H at δ 10.48−10.53 ppm for the presence of a CHO group. The 13 C NMR spectra of compound 9a also displayed required chemical shifts, i.e., δ 125−150 ppm (aromatic region) and δ 190.17 ppm (aldehydic-C). The collected experimental data from elemental analysis was also in close agreement with calculated % ages of elements in targeted heterocyclic compounds. In the second step (Scheme 1), arylidenes of respective aldehydes (9a and 9b) with different nitriles (10a−10c) were further treated with different derivatives of 4-hydroxycoumarin (11a−11d) in the presence of Et 3 N through the Knoevenagel−Michael cyclization path in a one-pot strategy to get targeted dihydropyranochromenes (12a−12m) with excellent yield ( Table 1). The enhancement in the regioselectivity of reaction as well as yield of product was obtained by monitoring various parameters during the course of reaction (i.e., temperature, solvent, and catalyst). The optimization of various reaction parameters for the preparation of model compound 12a is illustrated in Table S1. Thus, ethyl acetate as a solvent, Et 3 N or Me 3 N as a base, and 60°C temperature were found to be the suitable parameters for this reaction. Spectroscopic techniques like IR, NMR, etc., were utilized to elaborate the structure of a synthesized series of targeted heterocycles. The absorption peaks in the ranges of 1680−1735 and 2190−2197 cm −1 and two peaks at 3150− 3300 and 3300−3450 cm −1 were used to identify the desired functionalities like carbonyl, cyano, and NH 2 groups in the relevant product. The evidence for other substituents such as the OH group on the coumarin skeleton was also comprehended on the basis of the absorption band at 3400− 3650 cm −1 . The 1 H NMR spectra of 12a−12m gave the confirmation of cyclized products as there was no downfield signal of CHO proton at δ 10.48−10.53 ppm; instead, a singlet of two protons for the NH 2 group was observed at δ 7.10−7.70 ppm, and this peak was merged in the aromatic range in accordance with shielding or deshielding. Further evidence for the cyclized product came from the fact that the signal for a singlet of the OH group of 4-hydroxylated coumarins disappeared as well. The appearance of two signals (i.e., triplet of 3H at δ 0.80−1.10 ppm and quartet of 2H at δ 3.95−3.96 ppm) and one signal for the singlet of 3H at δ 3.50−3.52 ppm illustrated the ethyl ester in 12c, 12f, 12g, and 12j and methylester in compounds 12b, 12e, and 12i, respectively. Total proton counts in the aromatic region also fulfill the structural requirement of the compound. Their chemical shifts were allocated on behalf of spin multiplicity as well as coupling constant. The 13 C NMR spectra of 12a−12m unveiled the accordance of chemical shifts of aliphatic, aromatic, and carbonyl moieties with desired structures. Further confirmation of representative compound 12a came from absorption peaks of aliphatic carbons at 32.63 and 57.25 ppm, aromatic carbons at 125−150 ppm, and carbonyl at 159.88 and 160.4 ppm. CHN analyses satisfactorily supported the desired structure of targeted dihydropyranochromenes, as experimentally collected data was in close agreement with calculated % ages.
Biological Evaluation. In Vitro hCA II Inhibition. Literature findings enlighten the scope of CA inhibition study in the clinical treatment of various therapeutic diseases like acid−base disequilibria, cancer, epilepsy, glaucoma, osteoporosis and neuromuscular disorders, edema, altitude sickness, obesity, etc. 22,23 In this perspective, the role of coumarin-based compounds as CAIs cannot be ignored. 44 In the present study, a series of synthesized compounds (12a− 12m) were tested against cytosolic hCA II. The inhibition results of the stream of synthesized compounds are presented in Table 2a, which explore the broad spectrum of inhibitory potential against cytosolic hCA II using zonisamide as a reference. IC 50 values thus obtained were found to be staggered between 4.55 ± 0.22 and 21.77 ± 3.32 μM. Few of these compounds like 12h, 12i, and 12j with IC 50 = 4.55 ± 0.22, 4.91 ± 1.13, and 7.78 ± 0.08 μM, respectively, unveiled excellent inhibitory power to block the enzyme. An innovative  inhibition mechanism has been proposed based on the activity profile of the series of synthesized coumarin derivatives. Our reported compounds in this study could not enter deep inside the cavity of the enzyme due to their massive size, therefore attaching at the mouth of the cavity of hCA II via substituents present at the phenyl ring of the coumarin skeleton. This inhibition mode followed by these coumarin derivatives because of their bulkiness is called the allosteric inhibition pathway. Thus, the high-rank inhibitory potential of compounds 12h, 12i, and 12j against hCA II is attributed to the presence of the electron-donating OH group at C-7 of the coumarin motif. Compound 12e also exhibits moderate inhibitory power, bearing a CH 3 group at C-6 of coumarin and COOCH 3 and NH 2 on the pyran ring. Compounds 12a− 12m showed the following descending order of inhibitory activity illustrated on behalf of the SAR study of compounds: In Vitro β-Glucuronidase Inhibition. Owing to the great therapeutic potential of synthetic coumarins as enzyme inhibitory molecules and important drug candidates, 30,31,43 the prepared coumarin-based pyranochromenes captivated our attention to bring about this study. Thus, the β-glucuronidase inhibitory power of synthesized derivatives (12a−12m) was checked by utilizing the literature protocol 74 and IC 50 values calculated were compared with std. silymarin. The results are illustrated in Table 2b, which showed inhibition of IC 50 ± SEM in the range from 440.1 ± 1.17 to 971.3 ± 0.05 μM. Compounds 12e with IC 50 = 670.7 ± 1.18 μM and 12i with IC 50 = 440.1 ± 1.17 μM were found to be more active against this enzyme when compared with std. silymarin with IC 50 = 731.9 ± 3.34 μM, while other compounds with mild inhibitory power are 12d (IC 50 = 789.5 ± 0.75 μM), 12g (IC 50 = 971.3 ± 0.05 μM), and 12j (IC 50 = 773.9 ± 2.22 μM). The SAR studies on these O-heterocycles reveal that the presence of the COOCH 3 group on the pyran ring of 12e and 12i may be responsible for this high-rank inhibitory potential against βglucuronidase. Furthermore, the presence of the electrondonating OH group at C-7 of the coumarin nucleus enhanced the activity in compound 12i. Compounds 12a−12m showed the following descending order of inhibitory activity illustrated on behalf of the SAR study of compounds: 12i > 12e > 12j > 12d > 12g. Molecular Docking Studies. Molecular docking studies enabled scientists in recent decades to unveil the interaction and binding tendency of a given molecule inside the active pocket of protein. The use of molecular docking studies to rationalize the mechanism behind various fundamental biochemical processes is well documented. 70−72 Keeping in view the good enzyme inhibitory potential of synthesized compounds 12a−12m, we performed molecular docking studies against carbonic anhydrase II and β-glucoronidase enzyme. The compounds having a good binding score presented in Tables 3a and 3b and an interaction pattern inside the active pocket of given enzymes gave insight into their inhibitory potential and suggested them as potent inhibitors. The binding interaction of potent inhibitors with active site residues of hCA II and β-glucoronidase is mentioned in Table S2.
Carbonic Anhydrase II. On account of results obtained from in vitro CA II studies, compounds 12h, 12i, and 12j exhibited potent inhibition against the hCA II isoform. Thus, the inhibition mechanism of these highly potent compounds     has been evaluated via molecular docking studies. The accuracy of the docking methodology was further tested with redocking phenomena, and RMSD values were evaluated and compared with docking scores. The best docked conformation was observed for compounds 12h, 12i, and 12j having binding scores of −13.627, −11.252, and −9.886 kcal/mol, respectively. The molecular docking studies are facilitated to understand the mechanism behind the selective potency and inhibition tendency of these three compounds against hCA II. Previously reported coumarin derivatives as hCA II inhibitors revealed the interaction of substituents with active site residues and non-involvement of the coumarin ring due to steric hindrance, 54−57 thus exhibiting an innovative mechanistic approach where the coumarin ring could not enter deep into the active pocket of the enzyme due to bulkiness and rigidity, which therefore could not be hydrolyzed due to the esterase activity of CA II. The attachment of the molecule at the entrance of the cavity of the enzyme blocks and deshapes the active site, so the natural substrate could not enter into the enzyme pocket. This inhibition mode is illustrated as allosteric inhibition. For compound 12h, the OH group at 7-position showed H-bonding interaction with Thr199 (1.79 Å) alongside pi−alkyl interaction with Val121 and the coumarin ring did not form any H-bonded interaction with active site residues. Similar interaction patterns were observed in the case of 12j where coumarin rings did not interact through H-bond and the only H-bond observed was between the OH functional group and Thr199 (1.77 Å), although pi−pi stacking was revealed with His94 as shown in Figure 2. Steric hindrance prevents the coumarin ring from entering deep into the active pocket and interacting with residues. The inhibition activity of these selective compounds is attributed to allosteric inhibition due to substituents attached at the phenyl ring of coumarin rings. In the case of 12i, we observed two H-bonding interactions with Thr199 and Gln92 alongside some pi−pi stacking and pi−alkyl interactions as depicted in Figure 2. The 2D interaction tool of Discovery Studio Visualizer was used for clear depiction of binding interactions of all these compounds inside the active pocket of hCA II (Figure 2).
β-Glucoronidase Enzyme. The compounds of current series were also evaluated through molecular docking studies for their binding tendency inside the active pocket of the βglucoronidase enzyme. The best docked conformation was observed for compound 12i with a binding score of −7.894 kcal/mol showing H-bonding interactions with Val410 (2.76 Å) and Asp207 (2.65 Å) alongside pi−alkyl interaction with Ala413 as shown in Figure 3. The second best binding score (−6.749 kcal/mol) was observed for compound 12e having Hbonding interaction with Asp207 (3.09 Å), pi−pi stacking with Tyr508, and pi−anion interaction with Glu451.
2D views for the binding interaction pattern of compounds 12e and 12i with active site residues of β-glucoronidase are shown in Figure S1. In silico studies are in good agreement with in vitro enzyme inhibition studies and suggest some of the compounds of current series as potential inhibitors of carbonic anhydrase II and β-glucoronidase, evident from their binding scores, IC 50 values, and binding interaction pattern inside the active pocket.

■ CONCLUSIONS
A series of novel coumarin-based 4H,5H-pyrano[3,2-c]chromenes (12a−12m) were synthesized via a two-step simple, facile route and evaluated for their inhibition potency against hCA II and β-glucuronidase. Few of these compounds like 12h, 12i, and 12j with IC 50 = 4.55 ± 0.22, 4.91 ± 1.13, and 7.78 ± 0.08 μM, respectively, unveiled excellent inhibitory power against hCA II as compared to the reference compound zonisamide (IC 50 = 12.02 ± 0.33 μM). Compounds 12e (IC 50 = 670.7 ± 1.18 μM) and 12i (IC 50 = 440.1 ± 1.17 μM) are found to be more potent against β-glucuronidase when compared with std. silymarin (IC 50 = 731.9 ± 3.34 μM). The results from molecular docking studies showed good agreement between theoretical and experimental findings. In the future, our results would help find new CA and βglucuronidase inhibitors and pave the direction in designing new drugs for the treatment of multifactorial clinical hits including cancer, epilepsy, Alzheimer's disease, glaucoma, and obesity. The required chemicals in synthetic work were purchased from a supplier and redistilled or recrystallized on need. The melting points of synthesized compounds were measured using a Fisher-Johns melting point apparatus (48061, Cole-Parmer). The reaction processing and product purity were examined utilizing TLC plates coated with Merck silica gel 60 F 254 , while spots were envisioned in UV light (λ = 254 and 366 nm). The IR absorption spectra were taken on an FTIR instrument, while the 1 H NMR and 13 C NMR spectra of final products were done in deuterated (CH 3 ) 2 SO utilizing (CH 3 ) 4 Si as ref. std. at 300 and 400 MHz, respectively, on a Bruker AV-500 (Bruker, Rheinstetten, Forchheim, Germany). Elemental (N, C, and H) analysis was accomplished on a Leco-CHNS-9320, and results collected were in good agreement with those of calculated ones. Synthesis.
Step 1: General Protocol for the Synthesis of Coumarin-Based Aromatic Aldehydes (9a and 9b). A standard literature methodology 69 with little amendment was adapted to prepare coumarin-based aldehydes (9a and 9b). An equimolar mixture of appropriate salicylaldehyde (7a and 7b) and triethylamine (5 mmol each) was stirred with methylacrylate (8) (15 mmol) in methanol or ethylacetate (5 mL) at room temperature. White precipitates appeared after 24 h of stirring, which were filtered and purified by subsequent washing and recrystallization from hot ethanol or ethylacetate. Compound 9b was recrystallized from hot chloroform.
Step 2: General Methodology for the Synthesis of Coumarin- Based Heterocycles (12a−12m). An equimolar mixture of coumarin-based aldehyde (9a and 9b) (1 mmol) and appropriate nitrile (10a−10c) (1 mmol) in EtOAc was initially stirred for half an hour at room temperature by employing two to three drops of Et 3 N as a catalyst, and then 4hydroxy coumarin or its substituted analog (11a−11d) (1 mmol) was added. The consumption of reactants was explored by TLC. White precipitates appeared during the course of reaction; otherwise, on cooling the reaction mixture to environmental parameters, they were filtered and washed two to three times with hot ethylacetate. Further purification was done by recrystallization using a mixture of CHCl 3 (4 parts) and MeOH (1 part) to furnish a pure, newly synthesized series of heterocyclic coumarin-based compounds (12a−12m) with excellent yield.

2-Amino-9-methyl-5-oxo-4-[2-(2-oxo-2H-chromen-3-ylmethoxy)-phenyl]-4H,5H-pyrano[3,2-c]chromene-3-carboxylic Acid
The reaction was initialized by adding 4-NPA to the previously incubated test sample for 15 min to 6 h and carried out utilizing 96-well plates. The data reported in Table 2a shows the inhibition after 15 min of incubation as there is no difference of inhibitory power when the enzyme and inhibitor are kept for a long period in incubation. The amount of product obtained during the reaction was recorded for 30 min in a spectrophotometer with an interval of 1 min at 400 nm. 73 β-Glucuronidase Inhibition Assay. The assay for inhibition of β-glucuronidase was performed utilizing a conventionally reported methodology with slight amendments. 74 In short, 100 μL of β-glucuronidase (0.1 M of 986.4 units/mL with phosphate-buffered solution at pH 7) and 340 μL of test sample solution/reference standard of varying concentrations with the same buffer were pre-incubated for 15 min at 37°C. Sixty microliters of p-nitrophenyl-(β)-D-glucuronide (3.15 mg/ mL buffered at pH 7 in 0.1 M phosphate buffer) was put onto the aforementioned solution and further incubated for 50 min under the same parameters. The developed color was perceived at 405 nm in absorbance spectrum. Control assay was performed without test samples, and finally, % age inhibition was premediated.
Molecular Docking Studies (Methodology). Molecular docking studies were performed via ICM v3.8-7d (Molsoft L.L.C., La Jolla, CA) 75 to evaluate the binding tendency of test compounds into the active pocket of carbonic anhydrase and β-glucuronidase. The 3D structures of carbonic anhydrase and β-glucuronidase were retrieved from Protein Data Bank with accession codes 1BN1 (resolution: 1.66 Å) 76 and 5CZK (resolution: 2.39 Å) 77 (Figure 4). The receptor preparation tool of ICM v3.8-7d (Molsoft L.L.C., La Jolla, CA) was used to prepare protein for docking that involved (i) optimization of hydrogen atoms, (ii) addition of Gasteiger charges, and (iii) removal of water molecules and reference ligand. The energy optimization was carried out using the default force field. The three-dimensional (3D) structures of compounds were modeled using the ligedit tool of ICM and later optimized. 75 The active pocket of protein was specified through a grid box, and compounds were docked into the pocket to evaluate their binding interaction with pocket residues. The 10 best docked conformations were generated for each compound.
The view of the docking results and the analysis of their surface with graphical representations were done using ICM, while the ligand interaction module of Discovery Studio Visualizer was used to calculate the 2D ligand−enzyme interactions.
General mechanism of carbonic anhydrase inhibition by coumarin analogs; optimization of reaction parameters for the preparation of model compound 12a; binding interaction of potent inhibitors with active site residues of hCA II and β-glucoronidase; 2D view for the binding interaction pattern of compounds 12d, 12e, 12g, and 12i with active site residues of β-glucoronidase; 1 H NMR and 13 C NMR spectra of 9a, 9b, and 12a−12m (PDF)