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Article

Design, Synthesis and Molecular Docking Study of Novel 3-Phenyl-β-Alanine-Based Oxadiazole Analogues as Potent Carbonic Anhydrase II Inhibitors

by
Kashif Rafiq
1,2,
Najeeb Ur Rehman
1,*,
Sobia Ahsan Halim
1,
Majid Khan
1,3,
Ajmal Khan
1 and
Ahmed Al-Harrasi
1,*
1
Natural and Medical Sciences Research Center, University of Nizwa, P.O Box 33, Birkat Al Mauz, Nizwa 616, Oman
2
Department of Chemistry, Abdul Wali Khan University Mardan, Mardan 23200, Pakistan
3
H. E. J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistan
*
Authors to whom correspondence should be addressed.
Molecules 2022, 27(3), 816; https://doi.org/10.3390/molecules27030816
Submission received: 26 December 2021 / Revised: 22 January 2022 / Accepted: 24 January 2022 / Published: 26 January 2022
(This article belongs to the Special Issue Designing Next-Generation Drug-Like Molecules for Medicinal Applications)
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Abstract

:
Carbonic anhydrase-II (CA-II) is strongly related with gastric, glaucoma, tumors, malignant brain, renal and pancreatic carcinomas and is mainly involved in the regulation of the bicarbonate concentration in the eyes. With an aim to develop novel heterocyclic hybrids as potent enzyme inhibitors, we synthesized a series of twelve novel 3-phenyl-β-alanine 1,3,4-oxadiazole hybrids (4a–l), characterized by 1H- and 13C-NMR with the support of HRESIMS, and evaluated for their inhibitory activity against CA-II. The CA-II inhibition results clearly indicated that the 3-phenyl-β-alanine 1,3,4-oxadiazole derivatives 4a–l exhibited selective inhibition against CA-II. All the compounds (except 4d) exhibited good to moderate CA-II inhibitory activities with IC50 value in range of 12.1 to 53.6 µM. Among all the compounds, 4a (12.1 ± 0.86 µM), 4c (13.8 ± 0.64 µM), 4b (19.1 ± 0.88 µM) and 4h (20.7 ± 1.13 µM) are the most active hybrids against carbonic CA-II. Moreover, molecular docking was performed to understand the putative binding mode of the active compounds. The docking results indicates that these compounds block the biological activity of CA-II by nicely fitting at the entrance of the active site of CA-II. These compounds specifically mediating hydrogen bonding with Thr199, Thr200, Gln92 of CA-II.

1. Introduction

Carbonic anhydrases (CAs, EC 4.2.1.1) are a class of well-studied metalloenzymes widely distributed in living organisms [1]. These are strongly involved in regulating cell homeostasis, intracellular pH, fluid secretion, ion transport and biosynthetic reactions by catalyzing the reversible hydration of carbon dioxide (CO2) to bicarbonate ion (HCO3−) and proton (H+) [2,3,4,5,6,7]. This simple reaction is crucial for many physiological mechanisms including electrolyte secretion, acid-base tuning, tumorigenesis, respiration, bone resorption, calcification and biosynthesis of important molecules such as glucose, urea, and lipids [8,9]. These enzymes are common in almost all organisms from simple to complex [10,11]. The extracellular pH in tumors is more acidic than intracellular pH [11]. To generate the pH gradient between the extracellular and intracellular compartments, tumor cells express ion transport proteins and CA enzymes [12,13,14]. The CA-II is expressed in malignant brain tumors [15,16,17], renal cancer cell lines, and gastric and pancreatic carcinomas [15,17]. CA-II has also been used since long time for the treatment of glaucoma, epilepsy, leukemia, and cystic fibrosis [18,19]. It is remarkable that ubiquitous hCA-I and II are the main off-target isoforms because these are involved in many physiological and biochemical processes [20]. Due to the key role of this enzyme in several diseases, its inhibition is considered therapeutically important.
Oxadiazoles are heterocyclic compounds which have a diversity of useful biological effects including antibacterial [21], antifungal [22], analgesic [23], anti-inflammatory [24], antiviral [25], anticancer [26], antihypertensive [27], anticonvulsant [28] and anti-diabetic [29]. 1,3,4-Oxadiazole nucleus is present in the molecules of drugs such as: furamizole (antibacterial), tiodazosin (an α-1 adrenergic antagonist), nesapidil (antihypertensive), raltegravir (antiretroviral), and zibotentan (anticancer) (Figure 1) [30]. The most commonly used synthetic route for the synthesis of 1,3,4-oxadiazoles includes the reactions of acid hydrazides with acid chlorides, carboxaldehyde, carboxylic acids and cyclization of the formed acylhydrazines using a dehydrating agent, such as phosphorous pentaoxide, thionyl chloride, or phosphorous oxychloride [31]. On the other hand, 1,3,4-oxadiazoles are thermally stable and neutral heteroaromatic compounds containing two nitrogens and one oxygen atom, affects the pharmacokinetic and physicochemical properties of the compounds in which it is present [32]. It makes diverse noncovalent interactions with various active sites of enzymes and receptors in biological systems and, thus display versatile pharmacological activities like anti-inflammatory [33], antidepressant [34], anti-proliferative [35], analgesic [36] and antiviral effect [32,37,38].
Previously, Vats et al. have synthesized novel 4-functionalized 1,5-diaryl-1,2,3-triazoles containing benzenesulfonamide moiety as carbonic anhydrase I, II, IV and IX inhibitors [39], while novel benzenesulfonamides bearing 1,2,3-triazole linked hydroxy trifluoromethylpyrazolines and hydrazones as selective carbonic anhydrase isoforms IX and XII inhibitors were reported by Sharma et al. [40]. Similarly, Kumar et al. described that the synthesis of novel benzenesulfonamide containing 1,2,3-triazoles [41] and benzenesulphonamide-bearing 1,4,5-trisubstituted-1,2,3-triazoles [42] showed potent inhibition against human carbonic anhydrase isoforms I, II, IV and IX inhibitors. Bianco et al. have successfully been synthesized N-acylbenzenesulphonamide dihydro-1,3,4-oxadiazole hybrids against hCA IX and XII [43]. Recently, Sharma et al. have reported the novel benzenesulfonamides incorporating 1,3,4-oxadiazole hybrids as potent inhibitor of carbonic anhydrase I, II, IX, and XII isoenzymes [44], while Swain et al., have efficiently been synthesized have efficiently been synthesized benzenesulphonamide based 1,3,4-oxadiazoles as selective carbonic anhydrase XIII inhibitors [45]. Our group recently reported a novel class of CAs inhibitors belonging to the 1H-1,2,3-triazole derivatives [46,47].
Our focus was on the identification of novel drug-like compounds against CA-II enzyme to combat CA-II related disorders. Keeping in mind the importance of these scaffolds in the present work, we designed and synthesized a series of novel 3-phenyl-β-alanine 1,3,4-oxadiazole hybrids 4a–l with the oxadiazole ring offering an important pharmacophore. With the hope to obtain an effective carbonic anhydrase II enzyme inhibitors, we planned to synthesize new compounds, characterized by spectroscopic techniques including 1H-, 13C-NMR and HRMS, and to explore their carbonic anhydrase enzyme inhibition. Later, computational docking method was applied to investigate the mode of binding of these compounds in the active site of CA-II.

2. Results and Discussion

2.1. Chemistry

The interest in finding an effective carbonic anhydrase II enzyme inhibitor has been increased in recent decades, especially with the exploring of possible relationships between carbonic anhydrase II and cancer [48,49,50]. CA is present in human (h) with sixteen (16) different isoforms identified from hCA I-hCA XV. All these isoforms are widely dispersed in different tissues/organs and are associated with a range of pivotal physiological activities. Due to their involvement in various physiological roles, inhibitors of different human isoforms of CA have found clinical applications for the treatment of various diseases including glaucoma, retinopathy, epilepsy, hemolytic anemia, and obesity [51]. However, clinically used inhibitors of CA (acetazolamide, brinzolamide, dorzolamide, etc.) are not selective causing the undesirable side effects. Recently we have investigated the interaction of CA-II isozymes with several types of natural and synthetic compounds [46,47,52,53]. Furthermore, 1,3,4-oxadiazole derivatives were found to be strong inhibitors against carbonic anhydrase II enzyme [39,40,41,42,44]. Inspired by these advances, we sought to investigate the application of this reagent in oxadiazole synthesis.
Molecular iodine plays an important role in organic synthesis, owing to its commercial availability, low cost, and low toxicity [54,55]. Recently, it has been successfully employed to synthesize indole derivatives [56,57] and oxazoles [58,59,60]. Compound 1 was synthesized through esterification of 3-phenyl-β-alanine in methanol (MeOH) by adding thionyl chloride, followed by protection of the amino group as tert-butyloxycarbonyl (Boc), which is then treated with hydrazine in the presence of MeOH at room temperature Scheme 1).
Our investigation started with the cyclization of 3-phenyl-β-alanine hydrazones 3a–l to the corresponding 1,3,4-oxadiazole 4a–l. The synthesis and NMR data of the substrates 3a–l were already reported by our group. These compounds were prepared via the condensation of different benzaldehyde moieties 2a–l (0.6 mmol, 1.2 equiv.) with phenylalanine hydrazide (1, 0.5 mmol, 1 equiv.) in ethanol at refluxing temperature in 90% yield [52] (Scheme 2). The oxidative cyclization of 3a–l to 4a–l was achieved by utilizing molecular iodine in the presence of potassium carbonate (1.8 mmol, 3 equiv.) and DMSO (2 mL) which is the most effective media (solvent) for this conversion at 100 °C. The structures of the resulting compounds were confirmed by 1H-, IR, and 13C-NMR spectroscopy as well as mass spectrometry (HRESIMS).

2.2. Carbonic Anhydrase-II Enzyme Inhibition and Structural-Activity Relationship

All the synthetic compounds reported herein, and standard carbonic anhydrase inhibitor acetazolamide were assessed for their inhibition properties towards the relevant recombinant human carbonic anhydrase-II using the colorimetric method. Most of the synthesized compounds strongly inhibited CA-II enzyme ranging from 12.1 to 53.6 μM. Among all, compounds 4a (12.1 ± 0.86 μM), 4c (13.8 ± 0.64 μM), 4i (18.1 ± 1.31 μM), 4b (19.1 ± 0.88 μM) and 4h (20.7 ± 1.13 μM) were found to be the best CA-II inhibitors, while compounds 4g (21.5 ± 0.99 μM), 4k (22.4 ± 1.32 μM), 4f (25.1 ± 1.04 μM), and 4e (26.6 ± 0.80 μM) demonstrated moderate activity (Table 1). Compared to standard (acetazolamide), compound 4l (53.6 ± 0.96 μM) was found to be weak inhibitor against CA-II among all derivatives.
Comparing compound 4i with 4b, the higher activity of the 4i may be possible due to the presence of -OH group at para position of phenyl ring. A comparison between 4g with 4h revealed a slight decrease in inhibition of 4g, which is likely due to the presence of nitro group (electron-withdrawing substituent) at ortho position of the phenyl ring. This suggests that the inhibitory activity of CA-II enzyme could be possibly increased with an electron-withdrawing substituent at para position of the phenyl ring. A slight change in activities between 4j and 4k as well as 4e and 4f was observed. The higher inhibition of 4k and 4f could be accounted for the arrangements of functional groups attached to the phenyl ring. Compound 4a (IC50 = 12.1 μM) exerted a stronger inhibition than other compounds in the series, which is possibly due to the presence of pyridine ring instead of phenyl. Similarly, a higher inhibition of compound 4c is observed when compared with compounds 4d and 4l which is probably due to the absence of electron withdrawing groups at para position of the phenyl ring.

2.3. Molecular Docking Results

All the active compounds were docked at the active site of CA-II (PDB code: 3HS4) to predict the best possible binding modes of each active compound through molecular docking. The standard drug, acetazolamide binds at the active site and mediates several interactions including ionic interactions with Zn ion, hydrogen bonding with the side chains of His94, His96, Thr199 and Thr200 within the active pocket of CA-II. The binding mode of acetazolamide is shown in Figure 2. Several active compounds, including 4a, 4c and 4i, exhibited CA-II inhibitory activity (in range of 12.1 to 18.1 µM) higher than the standard drug. The binding mode of the most active compound 4a depict that the carbamate moiety of the compound is involved in binding with the active site residues. Whereas oxadiazol ring and its substituted R group (pyridinyl ring) resided at the entrance of the active site. Moreover, the carbamate substituted phenyl ring was also fitted neatly in the groove near the active site entrance. The carbamate moiety of the compound mediated multiple H-bonding interaction with the amino group of Thr199 and Thr200, and side chain of Thr200. Moreover, the side chain of Gln92 also provides H-bond to the oxadiazole ring of the compound. We observed that the compound does not interact with the Zn ion in the active site, however, by interacting with several active site residues through H-bonds, and complete blockage of active site entrance, 4a inhibits the function of CA-II. Similar orientation was observed for compound 4c (the second highest active inhibitor), 4i and 4b, however, carbamate moiety of these compounds interacted with the amino group and the side chain of Thr200. Additionally, -OH group at the substituted hydroxyl phenyl ring of 4b mediated H-bonding with the side chain of Gln92. The docked conformation of 4h was completely like the binding mode of 4b, the carbonyl oxygen of 4h binds with the amino group of Thr200, however, the oxadiazol and the substituted nitrophenyl rings did not interact with the surrounding residues. Interestingly, the carbamate and the oxadiazol ring of 4g do not interact with the surrounding residues, while its methoxy oxygen interacted with the amino group of Thr199 through H-bond. The substituted nitrophenyl rings of compounds 4h and 4g were found to be surface exposed. Similarly, the carbamate oxygen of 4k and 4f and oxadiazol ring of 4f formed H-bonds with the amino nitrogen of Thr200, and side chain of Gln92, respectively. The binding mode of compounds 4e and 4j demonstrated that the carbamate nitrogen of 4e interacted with a water molecule in the vicinity of active site, while the substituted R group mediated π-π interaction with the phenyl ring of Phe131, whereas the carbonyl oxygen of 4j accepted H-bond with the amino group of Thr199. The least active compound of this series, 4l only mediates bidentate interaction with the amino nitrogen and side chain -OH of Thr200 through its carbamate moiety. The binding interactions of each compound within the active site of CA-II are tabulated in Table 2. The docked view of the most active compound 4a is presented in Figure 2.
The pharmacokinetic properties of the active compounds were predicted through BOILED- Egg model of SwissADME [61]. In the BOILED-Egg analysis, all the compounds showed high gastro-intestinal absorption, while none of the compounds exhibited blood brain barrier permeability. Moreover, all the compounds followed Lipinski rule of five drug-likeness criteria, and no PAIN alerts. In addition, accept 4a, all the compounds were found as non-substrate for P-glycoprotein (Table 3). The predicted ADMET profile of the compounds suggest that these compounds could act as beneficial inhibitor of CA-II.

3. Material and Methods

3.1. General Instrumentation

All reagents were purchased from Sigma-Aldrich Chemical Company (St. Louis, MO, USA). Solvents used for chemical reactions were purified and dried by standard procedures. Melting point was determined using digital melting point apparatus SMP10 (StuartTM, Cole-Parmer, Beacon Rd, Stone, Staffordshire, ST15 OSA, UK). Infrared (IR) spectra were recorded on an ATR-Tensor 37 attenuated total reflectance spectrometer (Bruker, Ettlingen, Baden-Württemberg, Germany) in the range from 400 to 4000 cm−1. High-resolution electrospray ionization mass spectrometry (HR-ESI-MS, Agilent technologies, 6530, Q-TOF LC/MS, Agilent, country of origin USA/EU, made in Singapore) was used for the determination of compound masses. The 1H- (600 MHz) and 13C- (150 MHz) NMR spectra were recorded on Bruker (Zürich, Switzerland) nuclear magnetic resonance spectrometers using the solvent peak as internal reference (CDCl3, δH: 7.26; δC: 77.2–76.8; DMSO, δH: 2.49; δC: 40.0–39.1). The following abbreviations were used to explain for NMR signals as s = singlet, d = doublet, dd = doublet of doublet, t = triplet, m = multiplet, J = coupling constant. Chemical shifts are expressed in parts per million (δ) values and coupling constants (J) are given in Hertz (Hz). All reactions were monitored by Thin-Layer-Chromatography (TLC, Merck, Darmstadt, Germany) using pre-coated aluminum sheets (silica gel 60 F254). TLC plates were visualized under the UV light at 254 and 366 nm and by spraying with the ninhydrin reagent. Solvents for chromatography were of technical grade and distilled prior to use.

3.2. General Procedure for the Synthesis 3-phenyl-β-alanine-1,3,4-Oxadiazoles Derivatives

3-Phenyl-β-alanine (10 mmol, 1.65 g) was used as a starting material for the synthesis of Boc-3-phenyl-β-alanine hydrazide (1). Esterification of the carboxylate group was done by the slow addition of thionyl chloride (15 mmol, 2.1 mL) to a solution of methanol (20 mL) containing 3-phenyl-β-alanine at 0 °C. The reaction solution was stirred for overnight (24 h.) at room temperature, while the completion of the reaction was carefully monitored by TLC for ensuring that all amount of amino acid changed to ester. After reaction completion, the solvent (MeOH) was evaporated under vacuum and the resultant product was filtered.
In the second step, amino acid methyl ester (9.8 mmol, 1.75 g) was added to a solution of anhydrous Na₂CO₃ (1.2 g, 12 mmol) and H2O (40 mL) in a 100 mL RB flask. Then, di-tert-butyl dicarbonate (Boc anhydride, 10 mmol, 2.1 g) was dissolved in EtoAc (20 mL), added to that solution and the reaction was allowed to stir for 24 h at room temperature. The progress of the reaction was continuously observed by TLC. When the reaction was completed, the pH was adjusted to 5.5 with oxalic acid and then the desired product was extracted with an organic solvent (EtOAc). The solvent was dried over anhydrous MgSO4 and evaporated under reduced pressure to obtain the corresponding Boc-phenylalanine methyl ester (91%).
In the third step, hydrazine monohydrate (2 mL, 32 mmol) was added to a solution of methanol (20 mL) containing Boc-phenylalanine methyl ester (9.1 mmol, 2.5 g) and stirred under room temperature for 24 h to afford phenylalanine hydrazide (1). The reaction mixture was thoroughly checked by TLC. After reaction completion, the solvent was evaporated under reduced pressure and the product tert-butyl (S)-(3-hydrazinyl-3-oxo-1-phenylpropyl) carbamate (1, yield 89%) was washed with methanol. White powder; Yield: 86%; m-p. 187–190 °C; FT-IR (solid, cm−1): 3318, 1694, 1664, and 1580, 1362, 1260, 1160, and 1092; 1H-NMR (CDCl3): δ 8.54 (1H, s, NH), 7.30 (1H, C-NH), 4.88 (2H, NH-NH2), 7.11–7.28 (5H, m), 4.97 (CH-NH), 2.93–3.14 (2H, CH2), 1.36 (9H, s); 13C-NMR (CDCl3): δ 28.3, 38.5, 55.2, 79.7, 126.0, 126.6, 126.7, 128.0, 128.5, 155.3, 143.2, 155.9, 177.3; HRMS (ESI+) m/z: 302.1477 [M + Na]+.
In the fourth step, a solution of aldehyde (0.6 mmol) added to a stirred solution of 1 (0.5 mmol) in EtOH (5 mL) at room temperature for 24 h. The progress of the reaction was monitored with TLC system of EtOAc/n-hexane (3:7). After completion of the reaction, the product (3) was filtered, washed with n-hexane for removing any excess of aldehyde. The resulting residue was redissolved in DMSO (2 mL), followed by addition of potassium carbonate (3 mmol) and iodine (1.2 mmol) in sequence. The reaction mixture was stirred at 100 °C until the conversion was complete (1−4 h, monitored by TLC,). After being cooled to room temperature, it was treated with 5% Na2S2O3 (20 mL), extracted with EtOAc (10 mL, three times). The combined organic layer was washed with brine (10 mL × 1), dried over anhydrous sodium sulfate, and concentrated. The given residue was purified through preparative thin layer chromatography (TLC) using a mobile phase of EtOAc and n-hexane (3:7) to afford the desired oxadiazoles. All compounds were obtained in fare yields ranging from 57 to 64%. The structures of all compounds were established by HRMS, 1H- and 13C-NMR and same procedure was use for the synthesis of other compounds.

3.2.1. Tert-Butyl (S)-(1-phenyl-2-(5-(pyridin-2-yl)-1,3,4-oxadiazol-2-yl) ethyl) carbamate (4a)

White solid powder; Yield: 57%; FTIR (solid, cm−1): 3320, 1690, 1666, and 1585, 1365, 1264, 1160, and 1094; 1H-NMR (CDCl3): δ 8.76 (1H), 8.18 (1H), 7.86 (1H), 7.45 (1H), 7.25–7.10 (5H, m), 5.40 (1H, NH), 5.19 (1H, CH), 3.22, 3.35 (2H), 1.37 (9H, s); 13C-NMR (CDCl3): δ 28.2, 39.9, 48.5, 80.4, 123.2, 126.0, 127.2, 128.7, 129.4, 135.2, 137.3, 143.3, 150.3, 164.2, 167.1; HRMS (ESI+) m/z: 366.1756 [M + H]+.

3.2.2. Tert-Butyl (S)-(2-(5-(2-hydroxyphenyl)-1,3,4-oxadiazol-2-yl)-1-phenylethyl) carbamate (4b)

Solid powder; Yield: 56%; FTIR (solid, cm−1): 3318, 1689, 1670, and 1594, 1445, 1375, 1340, 1302 and 1160; 1H-NMR (CDCl3): δ 10.86 (1H, OH, s), 8.15 (1H), 7.65 (1H), 7.42 (1H), 7.30 (1H), 7.25–6.72 (5H, m), 5.36 (1H, NH), 5.09 (1H, CH), 3.29, 3.28 (2H), 1.41 (9H, s); 13C-NMR (CDCl3): δ 28.2, 39.8, 48.6, 81.2, 86.8, 109.5, 119.9, 121.5, 124.9, 126.8, 127.5, 128.7, 128.9, 129.3, 133.8, 134.7, 143.3, 154.8, 157.7, 165.8; HRMS (ESI+) m/z: 382.1814 [M + H]+.

3.2.3. Tert-Butyl(S)-(1-phenyl-2-(5-phenyl-1,3,4-oxadiazol-2-yl)ethyl) carbamate (4c)

Pale yellow Color; Yield: 58%; FTIR (solid, cm−1): 3336, 1710, 1668, 1608, 1375, 1335, 1290 and 1080; 1H-NMR (CDCl3, 600 MHz): δ 7.88–7.39 (5H, m), 7.22–7.07 (5H, m), 5.47 (1H, NH), 5.25 (1H, CH), 3.23, 3.20 (2H), 1.33 (9H, s); 13C-NMR (150 MHz, CDCl3): δ 28.2, 29.6, 48.5, 80.3, 123.6, 126.8, 127.1, 128.6, 129.0, 132.9, 131.7, 135.6, 154.9, 164.9, 166.2; HRMS (ESI+) m/z: 366.1948 [M + H]+.

3.2.4. Tert-Butyl(S)-(2-(5-(4-methoxyphenyl)-1,3,4-oxadiazol-2-yl)-1-phenylethyl) carbamate (4d)

White solid powder; Yield: 57%; FTIR (solid, cm−1): 3324, 1704, 1672, 1602, 1452, 1370, 1268, 1160, and 1020; 1H-NMR (CDCl3): δ 7.81 (1H), 7.80 (1H), 6.90 (1H), 6.89 (1H), 7.24–7.07 (5H, m), 5.47 (1H, NH), 5.22 (1H, CH), 3.78 (3H, CH3), 3.23,3.17 (2H), 1.32 (9H, s); 13C-NMR (CDCl3): δ 28.2, 29.6, 48.5, 55.4, 114.4, 116.0, 127.1, 128.6, 129.3, 135.6, 154.9, 162.3, 164.8, 165.6; HRMS (ESI+) m/z: 396.1960 [M + H]+.

3.2.5. Tert-Butyl(S)-(2-(5-(4-hydroxy-3-methoxyphenyl)-1,3,4-oxadiazol-2-yl)-1-phenylethyl) carbamate (4e)

Yellow powder; Yield: 56%; FTIR (solid, cm−1): 3333, 1696, 1668, 1590, 1450, 1370, 1252, 1160, 1030; 1H-NMR (CDCl3): δ 9.80 (1H, OH, s), 7.63 (1H), 7.51 (1H), 7.25–7.11 (5H, m), 6.87 (1H), 5.43 (1H, NH), 5.31 (1H, CH), 4.44 (3H, OCH3), 3.94,3.86 (2H), 1.38 (9H, s); 13C-NMR (CDCl3): δ 28.2, 34.4, 38.5, 48.6, 40.9, 52.5, 56.1, 107.7, 109.5, 119.9, 121.5, 124.9, 126.8, 128.7, 129.3, 136.5, 145.0, 155.3, 167.8, 190.8; HRMS (ESI+) m/z: 436.2279 [M + H]+.

3.2.6. Tert-Butyl(S)-(2-(5-(2-hydroxy-3-methoxyphenyl)-1,3,4-oxadiazol-2-yl)-1-phenylethyl) carbamate (4f)

Crystalline powder; Yield: 61%; FTIR (solid, cm−1): 3320, 1690, 1664, 1594, 1450, 1405, 1358, 1257, 1164, and 1090; 1H-NMR (CDCl3, 600 MHz): δ 9.90 (1H, OH, s), 8.17 (1H), 7.93 (1H), 7.29–7.10 (5H, m), 6.85 (1H), 5.27 (1H, NH), 4.45 (1H, CH), 3.91 (3H, OCH3), 3.91,3.87 (2H), 1.37 (9H, s); 13C-NMR (150 MHz, CDCl3): δ 28.2, 34.4, 38.1, 56.3, 113.7, 114.2, 117.5, 119.5, 121.4, 124.5, 126.9, 128.4, 129.5, 136.3, 145.6, 151.1, 155.9, 167.4, 172.8, 196.6; HRMS (ESI+) m/z: 436.1883 [M + H]+.

3.2.7. Tert-Butyl(S)-(2-(5-(2-nitrophenyl)-1,3,4-oxadiazol-2-yl)-1-phenylethyl) carbamate (4g)

Yellow powder; Yield: 56%; FTIR (solid, cm−1): 3310, 1703, 1666, 1614, 1455, 1346, 1324, 1236, 1059; 1H-NMR (CDCl3): δ 8.05 (1H), 7.87 (1H), 7.67 (1H), 7.71 (1H), 7.29–7.13 (5H, m), 5.34 (1H, NH), 5.13 (1H, CH), 3.30,3.18 (2H), 1.38 (9H, s); 13C-NMR (CDCl3): δ 28.2, 39.8, 48.4, 118.7, 124.8, 127.3, 128.7, 129.4, 131.9, 132.6, 133.3, 135.0, 148.1, 154.8, 161.8, 167.3; HRMS (ESI+) m/z: 411.1694 [M + H]+.

3.2.8. Tert-Butyl(S)-(2-(5-(4-nitrophenyl)-1,3,4-oxadiazol-2-yl)-1-phenylethyl) carbamate (4h)

Light yellow powder; Yield: 61%; FTIR (solid, cm−1): 3322, 1701, 1664, 1610, 1430, 1365, 1345, 1208, and 1163; 1H-NMR (CDCl3): δ 8.38 (1H), 8.37 (1H), 8.17 (1H), 8.16 (1H), 7.32–7.17 (5H, m), 5.41 (1H, NH), 5.20 (1H, CH), 3.34,3.33 (2H), 1.45 (9H, s); 13C-NMR (CDCl3): δ 28.2, 29.7, 40.0, 48.6, 124.3, 124.5, 126.9, 127.4, 127.8, 128.8, 129.0, 129.1, 129.3, 135.1, 149.7, 154.8, 163.2, 167.4; HRMS (ESI+) m/z: 411.1705 [M + H]+.

3.2.9. Tert-Butyl (S)-(2-(5-(4-hydroxyphenyl)-1,3,4-oxadiazol-2-yl)-1-phenylethyl) carbamate (4i)

White solid powder; Yield: 59%; FTIR (solid, cm−1): 3330, 1705, 1668, and 1608; 1H-NMR (CDCl3): δ 7.78 (1H), 7.77 (1H), 7.26 (1H), 7.20 (1H), 7.12–6.86 (5H, m), 5.30 (1H, NH), 5.16 (1H, CH), 3.29,3.24 (2H), 1.38 (9H, s); 13C-NMR (CDCl3): δ 28.2, 29.3, 29.7, 29.7, 31.9, 40.0, 48.5, 80.7, 116.0, 116.1, 127.2, 128.7, 128.9, 129.4, 135.4, 158.9, 164.9, 165.5; HRMS (ESI+) m/z: 382.1463 [M + H]+.

3.2.10. Tert-Butyl (S)-(2-(5-(3,4-dimethoxyphenyl)-1,3,4-oxadiazol-2-yl)-1-phenylethyl) carbamate (4j)

White amorphous powder; Yield: 64%; FTIR (solid, cm−1): 3326, 1701, 1670, 1595, 1420, 1370, 1336, 1252, 1208, and 1143; 1H-NMR (CDCl3): δ 7.50 (1H), 7.49 (1H), 7.45 (1H), 7.27–7.12 (5H, m), 6.90 (1H), 5.32 (1H, NH), 5.18 (1H, CH), 3.92 (6H, OCH3), 3.27,3.26 (2H), 1.40 (9H, s); 13C-NMR (CDCl3): δ 28.3, 40.1, 48.5, 56.0, 56.1, 80.5, 109.5, 111.1, 116.2, 120.4, 127.2, 128.6, 129.4, 135.5, 149.3, 152.1, 164.9, 165.7; HRMS (ESI+) m/z: 426.2066 [M + H]+.

3.2.11. Tert-Butyl (S)-(2-(5-(2,5-dimethoxyphenyl)-1,3,4-oxadiazol-2-yl)-1-phenylethyl) carbamate (4k)

Colorless solid; Yield: 61%; FTIR (solid, cm−1): 3328, 1711, 1667, 1602, 1524, 1342, 1272, 1228, 1155, and 1066; 1H-NMR (CDCl3): δ 7.30 (1H), 7.24–7.11 (5H, m), 7.03 (1H), 6.96 (1H), 5.36 (1H, NH), 5.22 (1H, CH), 3.86,3.78 (6H, OCH3), 3.30,3.27 (2H), 1.40 (9H, s); 13C-NMR (CDCl3): δ 28.3, 40.0, 48.5, 56.0, 56.6, 60.4, 80.3, 113.1, 113.7, 114.5, 119.4, 127.1, 128.6, 129.4, 135.5, 152.3, 153.4, 163.6, 165.8; HRMS (ESI+) m/z: 426.2065 [M + H]+.

3.2.12. Tert-Butyl (S)-(2-(5-(4-chlorophenyl)-1,3,4-oxadiazol-2-yl)-1-phenylethyl) carbamate (4l)

White powder; Yield: 57%; FTIR (solid, cm−1): 3350, 1698, 1663, and 1603, 1532, 1336, 1266, 1224, 1172, and 1066; 1H-NMR (CDCl3): δ 7.88 (1H), 7.87 (1H), 7.45 (1H), 7.44 (1H), 7.27–7.12 (5H, m), 5.32 (1H, NH), 5.16 (1H, CH), 3.28,3.27 (2H), 1.40 (9H, s); 13C-NMR (CDCl3): δ 28.2, 29.7, 39.9, 48.6, 80.6, 122.2, 127.3, 128.2, 128.7, 129.3, 129.4, 135.3, 138.1, 154.8; HRMS (ESI+) m/z: 400.1467 [M + H]+.

3.3. Carbonic Anhydrase II Inhibition Assay

A total reaction volume of 200 µL containing 20 µL of the synthetic compounds 4a–l prepared in DMSO, followed by the addition of 140 µL of the HEPES-tris buffer, 20 µL of purified bovine erythrocyte CA-II (0.15 mg/mL) prepared in buffer, and 20 µL of a solution of 4-nitrophenyl acetate [36,44]. 20 µL of tested compounds were incubated with the enzyme carbonic anhydrase II (EC 4.2.1.1) for 15 min in 96-well flat bottom plate. The rate of product formation was monitored with the addition of 20 µL of 4-NPA as substrate, prepared in ethanol at the final concentration of 0.7 mM at 25 °C for 30 min with regular intervals of 1 min, by using spectrophotometer (xMarkTM Microplate, Bio-Rad, Hercules, CA, USA). HEPES-tris was used as a buffer for the reaction at the final concentration of 20 mM at pH 7.4.

3.4. Molecular Docking

Docking was conducted on Molecular Operating Environment [45] using X-ray crystal structure of human carbonic anhydrase II complexed with acetazolamide (PDB code: 3HS4, resolution: 1.10 Å) [62]. The docking performance of MOE was tested previously [36,44] by re-docking experiment which shows that MOE is efficient in docking of CA-II inhibitors. In the re-docking, the X-ray conformation of acetazolamide was re-docked at its cognate binding site, where it was docked with RMSD = 0.61 Å. The re-docked conformation is shown in Figure S37. The protein file was prepared for docking by QuickPrep module of MOE which adds missing hydrogen atoms on residues and calculate partial charges using pre-defined force field (we applied Amber10:EHT force field). The two-dimensional (2D-) structures of ligands were prepared by ChemDraw, later converted into 3D-form by MOE using WASH module of MOE which adds hydrogen atoms and partial charges on ligands. Subsequently, the 3D-structure of each compound was minimized until the gradient was reached to 0.1RMS kcal/mol/Å. After the preparation of protein and ligand files, docking was performed by Triangle Matcher docking algorithm and London dG scoring function. Thirty docked conformation of each ligand was saved, and the best docked orientation was selected based on the docking score and binding interactions. The pharmacokinetic profile of the active hits was predicted through SwissADME server using BOILED-Egg model [61,63].

4. Conclusions

In summary, a series of twelve new 3-phenyl-β-alanine 1,3,4-oxadiazoles 4a–l have been synthesized and CA-II were performed in vitro. From all derivatives, compounds 4a (12.1 µM) and 4c (13.8 µM) exhibited the most potent activity against CA-II enzyme. In addition, structure-activity relationship of the active compounds has been established. When the results were compared, it was observed that these molecules show more effective inhibition than the standard inhibitor. Based on molecular docking strategy, we observed the mode of binding interaction of the active hits with the active site residues of CA-II, which suggests that the compounds mainly interact with the Thr199 and Thr200, thus inhibit the activity of CA-II. Based on these results, we suggest that these molecules can be used against CA-II related diseases.

Supplementary Materials

The following supporting information can be downloaded, Figures S1–S36: 1H- (CDCl3, 600 MHz), 13C-NMR and HRESIMS of the compounds 4a–l, Figure S37: The re-docked orientation of acetazolamide.

Author Contributions

K.R. synthesized the compounds. N.U.R. performed structural elucidation and wrote original draft of the manuscript. A.K. and M.K., performed carbonic anhydrase II inhibition. S.A.H. conducted in silico studies. A.A.-H. supervised the project and assisted in reviewing and editing the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

The authors are thankful to The Oman Research Council (TRC) through the funded Research Grant Program (BFP/RGP/CBS/21/002).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The spectroscopic data is available in the supporting information to the researchers.

Acknowledgments

The authors are thankful to the University of Nizwa for the generous support to this project. We are also grateful to the analytical and technical staffs of the University for assistance.

Conflicts of Interest

The authors declare no conflict of interest.

Sample Availability

The data presented in this study is available in the Supplementary Material.

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Molecules 27 00816 g001 550
Figure 1. Some reported 1,3,4-oxadiazole derivative drugs.
Figure 1. Some reported 1,3,4-oxadiazole derivative drugs.
Molecules 27 00816 g001
Molecules 27 00816 sch001 550
Scheme 1. Reagents and conditions: (a) SOCl2, MeOH, 0 °C, overnight; (b) NaCO3, H2O, Boc, EtOAc (c) NH2NH2.H2O, MeOH, rt, 24 h (1).
Scheme 1. Reagents and conditions: (a) SOCl2, MeOH, 0 °C, overnight; (b) NaCO3, H2O, Boc, EtOAc (c) NH2NH2.H2O, MeOH, rt, 24 h (1).
Molecules 27 00816 sch001
Molecules 27 00816 sch002 550
Scheme 2. Reagents and conditions: (a) EtOH, 80 °C, Reflux overnight, (3a–l, 90%); (b) K2CO3, DMSO, 100 °C, Reflux overnight (4a–l).
Scheme 2. Reagents and conditions: (a) EtOH, 80 °C, Reflux overnight, (3a–l, 90%); (b) K2CO3, DMSO, 100 °C, Reflux overnight (4a–l).
Molecules 27 00816 sch002
Molecules 27 00816 g002 550
Figure 2. The binding mode of most active compound 4a is shown in the active site of CA-II. The ligand is presented in magenta stick model, 3D-structure of CA-II is shown in surface model, interacting residues are depicted in coral stick model and H-bonds are presented in green lines.
Figure 2. The binding mode of most active compound 4a is shown in the active site of CA-II. The ligand is presented in magenta stick model, 3D-structure of CA-II is shown in surface model, interacting residues are depicted in coral stick model and H-bonds are presented in green lines.
Molecules 27 00816 g002
Table
Table 1. Carbonic anhydrase-II activity of the compounds 4a–l.
Table 1. Carbonic anhydrase-II activity of the compounds 4a–l.
CompoundsR% InhibitionIC50 ± SEM (µM)
4a2a Molecules 27 00816 i00196.212.1 ± 0.86
4b2b Molecules 27 00816 i00293.519.1 ± 0.88
4c2c Molecules 27 00816 i00395.513.8 ± 0.64
4d2d Molecules 27 00816 i00429.0N/A
4e2e Molecules 27 00816 i00597.326.6 ± 0.80
4f2f Molecules 27 00816 i00693.825.1 ± 1.04
4g2g Molecules 27 00816 i00779.521.5 ± 0.99
4h2h Molecules 27 00816 i00894.620.7 ± 1.13
4i2i Molecules 27 00816 i00958.018.1 ± 1.31
4j2j Molecules 27 00816 i01096.726.6 ± 1.47
4k2k Molecules 27 00816 i01187.822.4 ± 1.43
4l2l Molecules 27 00816 i01267.653.6 ± 0.96
StandardAcetazolamide84.618.2 ± 1.43
N/A = Not active, SEM = Standard error mean.
Table
Table 2. Molecular docking results of active inhibitors.
Table 2. Molecular docking results of active inhibitors.
CompoundsScore (kcal/mol)Interactions
Ligand AtomReceptor AtomBond TypeDistance (Å)
4a−8.96O25N-THR199HBA1.87
O25N-THR200HBA2.37
6-ringHOH-1247π-H3.53
4c−8.2N22OG1-THR200HBD1.97
O25N-THR200HBA2.39
4i−8.05N22OG1-THR200HBD2.95
O25N-THR200HBA2.61
4b−7.88O25N-THR200HBA2.26
O25OG1-THR 200HBA1.80
6-ringO-HOH1247π-H3.46
4h−7.63O25OG1-THR200HBA2.23
4g−7.68O26N-THR199HBA1.87
4k−7.80O25OG1-THR200HBA2.11
4f−7.10O21NE2-GLN92HBA2.97
O25N-THR200HBA2.21
4e−7.09N22O-HOH1191HBD1.99
6-ring6-ring-PHE131π-π3.01
4j−7.10O25N-THR199HBA2.20
4l−6.23N22OG1-THR200HBD1.82
O25N-THR200HBA2.08
HBA = Hydrogen bond acceptor, HBD = Hydrogen bond donor.
Table
Table 3. The pharmacokinetic profile of active inhibitors.
Table 3. The pharmacokinetic profile of active inhibitors.
MoleculeNRBHBAHBDTPSAiLOGPGIABBBPPgpSLipinski VPAINS Alerts
4a86190.143HighNoYes00
4b86297.483.34HighNoNo00
4c85177.253.54HighNoNo00
4e972106.713.56HighNoNo00
4f972106.713.45HighNoNo00
4g971123.072.96HighNoNo00
4h971123.073.11HighNoNo00
4i86297.483.15HighNoNo00
4j107195.713.81HighNoNo00
4k107195.713.64HighNoNo00
4l85177.253.75HighNoNo00
AZM361178.330LowNoNo00
NRB = Number of rotatable bonds, HBA = H-bond acceptors, HBD = H-bond donors, TPSA = Topological polar surface area, GIA = Gastrointestinal absorption, BBBP = Blood brain barrier permeant, PgpS = P glycoprotein substrate, Lipinski V = Violation of lipinski rule of 5.
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Rafiq, K.; Ur Rehman, N.; Halim, S.A.; Khan, M.; Khan, A.; Al-Harrasi, A. Design, Synthesis and Molecular Docking Study of Novel 3-Phenyl-β-Alanine-Based Oxadiazole Analogues as Potent Carbonic Anhydrase II Inhibitors. Molecules 2022, 27, 816. https://doi.org/10.3390/molecules27030816

AMA Style

Rafiq K, Ur Rehman N, Halim SA, Khan M, Khan A, Al-Harrasi A. Design, Synthesis and Molecular Docking Study of Novel 3-Phenyl-β-Alanine-Based Oxadiazole Analogues as Potent Carbonic Anhydrase II Inhibitors. Molecules. 2022; 27(3):816. https://doi.org/10.3390/molecules27030816

Chicago/Turabian Style

Rafiq, Kashif, Najeeb Ur Rehman, Sobia Ahsan Halim, Majid Khan, Ajmal Khan, and Ahmed Al-Harrasi. 2022. "Design, Synthesis and Molecular Docking Study of Novel 3-Phenyl-β-Alanine-Based Oxadiazole Analogues as Potent Carbonic Anhydrase II Inhibitors" Molecules 27, no. 3: 816. https://doi.org/10.3390/molecules27030816

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