Bio-Oriented Synthesis of Novel (S)-Flurbiprofen Clubbed Hydrazone Schiff’s Bases for Diabetic Management: In Vitro and In Silico Studies

A new series of (S)-flurbiprofen derivatives 4a–4p and 5a–5n were synthesized with different aromatic or aliphatic aldehydes and ketones to produce Schiff’s bases and their structures were confirmed through HR-ESI-MS, 1H, and 13C-NMR spectroscopy. The α-glucosidase inhibitory activities of the newly synthesized compounds were scrutinized, in which six compounds 5k, 4h, 5h, 4d, 4b, and 5i showed potent inhibition in the range of 0.93 to 10.26 µM, respectively, whereas fifteen compounds 4c, 4g, 4i, 4j, 4l, 4m, 4o, 4p, 5c, 5d, 5j, 5l, 5m, 5n and 1 exhibited significant inhibitory activity with IC50 in range of = 11.42 to 48.39 µM. In addition, compounds 5g, 5f, 4k, 4n, and 4f displayed moderate-to-low activities. The modes of binding of all the active compounds were determined through the molecular docking approach, which revealed that two residues, specifically Glu277 and His351 are important in the stabilization of the active compounds in the active site of α-glucosidase. Furthermore, these compounds block the active site with high binding energies (−7.51 to −3.36 kcal/mol) thereby inhibiting the function of the enzyme.


Chemistry
In continuation of our efforts to discover pharmacologically potent α-glucosidase inhibitors, thirty Schiff base derivatives of flurbiprofen were successfully synthesized through multi-step reactions. Flurbiprofen was reacted with hydrazine hydrate in the presence of CDI (1, 1 -carbonyldiimidazole) and triethylamine (Et 3 N) in THF solvent. The hydrazide was further reacted with various substituted aliphatic/aromatic aldehydes or ketones in the presence of acid using ethanol solvent to give the desired Schiff's base derivatives of flurbiprofen (Scheme 1). Scheme 1. Schiff's base derivatives of flurbiprofen.

In Vitro α-Glucosidase Inhibitory Activity
The synthesized compounds were tested for in vitro α-glucosidase inhibitory potential and compared with standard drug acarbose. All the compounds showed α-glucosidase inhibitory activity except five compounds (4a, 4e, 5a, 5b, and 5e). In this series, compounds 4b, 4d, 4h, 5k, 5h and 5i were found potent against α-glucosidase as compared to the standard inhibitor, acarbose (IC 50 = 875.75 ± 1.24 µM). Compound 5k (IC 50 = 0.93 µM), was the most potent inhibitor followed by 4h (IC 50 = 1.52 µM), 5h (IC 50 = 3.41 µM), 4d (IC 50 = 4.77 µM), 4b (IC 50 = 7.61 µM), and 5i (IC 50 = 10.26 µM), (Figure 1, Table 1). Furthermore, compounds 4g, 5m, 4i, 5n, 5l, 4l, 4p, 4j, 4m, 4o, 5j, 1, 5c, 5d, and 4c also demonstrated significant inhibitory activity (IC 50 value ranging from 11.42 to 48.39 µM) against α-glucosidase, while five compounds [5g, (IC 50 = 68.75 µM), 5f (IC 50 = 136.36 µM), 4k (IC 50 = 146.78 µM), 4n (IC 50 = 147.26 µM), and 4f (IC 50 = 426.82 µM)] showed moderate to least inhibitory activities (Table 1). The structure-activity relationship (SAR), for better understanding, was discussed by analyzing the changes in position and nature of attached substituents (R) on the benzene ring. In the case of the aldehydes series, compound 4h was the most active inhibitor of α-glucosidase having IC 50 value of 1.52 ± 0.07 µM (Figure 2),~580 times stronger than acarbose as the standard inhibitor ( Figure 2). The most potent activity of 4h could be due to the presence of bromine substituent at the meta position of the benzene ring. The decrease in α-glucosidase inhibition of 4g (IC 50 = 11.42 ± 1.09 µM) may be likely due to the presence of bromine at the para substitution and additional substituent (fluorine) at the ortho position of the benzene ring. Similarly, a further slight decline in the inhibition of 4i (IC 50 = 13.20 ± 2.47 µM) could be due to the presence of additional bromine at the ortho substituent and hydroxyl group at the para position of the benzene ring. Comparing 4d (IC 50 = 4.77 ± 2.03 µM) with 4j (IC 50 = 18.71 ± 0.25 µM) and 4m (IC 50 = 19.84 ± 1.89), the higher activity of the 4d might be due to the presence of hydroxyl and methoxy groups at ortho and meta positions, respectively, attached to the benzene ring. The compounds (4j and 4m) attributed almost the same inhibition, while the lower activity of the compound 4f (IC 50 = 426.82 ± 4.63 µM) suggested that the -OH group at ortho position and -OCH 3 at ortho and meta substitution on the benzene ring may interact well with the α-glucosidase enzyme compared to para ( Table 1). The slight decline in activity of 4c (IC 50 = 48.39 ± 3.75 µM) is likely due to the substitution of second -OH at para position, indicating the interaction of -OH at para substitution with the screen enzyme as well. Comparing straight-chain alkanes, compounds 4l (IC 50 = 17.36 ± 1.68 µM), 4o (IC 50 = 22.72 ± 1.46 µM), displayed outstanding inhibition than 4k (IC 50 =146.78 ± 0.86 µM), indicating that n-hexyl and octyl groups might have better interaction with the enzyme compared to butyl in the aldehyde series. Compound 4p revealed promising activity with IC 50 value of 18.61 ± 0.14 µM having a carboxylic group at the para substitution of the benzene ring, while 4n (IC 50 = 147.26 ± 2.78 µM, naphthalene substituent), determined good inhibition compared to the standard. Similarly, in the case of ketone series, 5k attributed excellent inhibitory activity ((IC 50 = 0.93 ± 0.06 µM),~940 times stronger) compared to the standard, which could be attributed due to the presence of benzene ring on one side and propyl group on the other side of the attached moiety.
Comparing the most active compound 5k with 5h and 5i, a decrease in inhibition of the can be attributed to the replacement of propyl with isopropyl (5h, IC 50 = 3.41 ± 0.05 µM) and butyl (5i, IC 50 = 10.26 ± 0.13 µM) on one side of the attached moiety. On the other side, comparing compounds 5h and 5i, the higher inhibition of 5i is likely due to the isomerization of the propyl group ( Figure 2). Comparing compounds 5f and 5g with the most active compound (5k), a further decrease in inhibition of the 5g (IC 50 = 68.75 ± 1.42 µM) and 5f (IC 50 = 136.36 ± 2.57 µM) may be possible due to the replacement of ethyl and methyl groups on one side of the attached moiety, respectively. The lower inhibition activity of 5f than 5g could be possibly due to the replacing of ethyl with a methyl group on one side of the attached substituent. Overall, it can be concluded that the propyl group on one side of the substituent, among the ketone series having straight-chain alkanes on one side and benzene ring on the other side, may have a beneficial effect on the interaction with the α-glucosidase enzyme. Compound 5a and 5b (having methyl group on one side and electron-withdrawing substituent on the para position of a benzene ring on the other side) was found inactive, indicating that methyl with para hydroxyl and methyl with para nitro benzene groups had no beneficial effect on the interaction with the α-glucosidase enzyme. Similarly, compound 5e having cyclohexane on one side and benzene on the other side was found to be inactive. The higher inhibition of 5m (IC 50 = 11.83 ± 0.18 µM) than 5n (IC 50 = 14.24 ± 0.16 µM) might be possibly due to the presence of methoxy group at ortho substation on benzene ring on one side in addition to having the same functional group on the other side. Compounds 5j (IC 50 = 29.31 ± 0.60 µM), 5c (IC 50 = 35.52 ± 2.04 µM), and 5d (IC 50 = 42.03 ± 3.51 µM) displayed promising inhibition in the series as well (Table 1).

Molecular Docking Studies
The mode of binding of all the active compounds was studied in the active site of the α-glucosidase enzyme by molecular docking. The binding orientation of the parent compound (1) indicates that the 2-fluoro-substituted-biphenyl moiety of the compound lies near the entrance of the active site where Tyr158 provides hydrophobic interaction to the biphenyl ring of the compound 1, while its hydrazide moiety mediated H-bonding (H-bond) with the side chains of Asp69 and Arg442. However, the substitution of the R group at the terminal amino group of 1 was responsible to tilt the other compounds towards the hydrophobic pocket in the active site constituted by Trp58, Phe301, Phe303, and Tyr347 and the R group resided in that pocket.
Among compounds 4a-4p, 4h was found most active inhibitor with an IC 50 value of 1.52 µM, followed by 4d and 4b which exhibited an IC 50 value of 4.77 µM and 7.16 µM, respectively. The binding orientation of compound 4h, which is the second most active compound in this series, was like the docked conformation of 5k. The addition of isobutylbenzene at the R position of compound 4h made this molecule tilt more towards the Phe303 which stabilized the 4h by π-π interaction. Due to the tilted position, the hydrazide moiety of 4h got far away from Glu277 and His351. A similar trend of binding was followed by compounds 4d and 4b. The hydrazide moiety of 4d mediated H-bonding with the side chains of Asp215 and His351, while the hydrazide group of 4b did not form any significant interaction; however, its nitro group interacted with the side chain of Arg213 through H-bond.
The compounds 4g, 4i, 4l, 4p, 4j, 4m, and 4o showed moderate inhibitory activities with IC 50 in the range of 11.42 to 22.72 µM. The docked orientation of 4g reflects that the substituted bromo-flouro-benzene ring is responsible to decrease the activity of this compound since the bulky group is not favorable for binding at this position. The addition of dibromophenol ring at the R position in 4i further reduced the inhibitory activity of 4i. The compound 4i further slipped towards the entrance of the active site, thereby its carbonyl only interacted with the side chain of Arg213. The docked orientations of compounds 4g, 4i, 4j, 4l, 4m, and 4p are quite similar; however, the hydrazide moiety of 4g, 4l, and 4m interacted with the Glu277 and His351 like compound 4h, while the hydrazide group of 4p mediated H-bonding with Asp215 and His351. The hydrazide carbonyl group of 4j was stabilized by His351, whereas the ethoxy group of 4j and hydrazide carbonyl of 4o accepted H-bond with the side chain of Tyr347. One of the −OH groups of 4c (IC 50 = 48.39 µM) exhibited H-bonding with Asp352, while its hydrazide group interacted with Asp215 and His351. The compounds 4k, 4n, and 4f exhibited the least activity with IC 50 values of 146.78, 147.26, and 426.82 µM, respectively. The compound 4k slipped more towards the outside of the active site while 4n adopt a bent position, the carbonyl of 4k was stabilized by the side chain of Arg442 and the hydrazide of 4n mediated H-bonding with Asp215 and Arg213. The docked orientation of 4f was twisted and its biphenyl ring acquired a position in opposite direction, due to the reason this compound did not form any significant interaction in the active site, which may be the reason for the least activity in this compound.
In this series, compound 5k was identified as the most active inhibitor against αglucosidase. Like 4h, the hydrazide group of 5k mediated H-bonds with the side chains of Glu277 and His351, and the side chains of Trp58, and Phe301 provide hydrophobic interaction to the R group of 5k, and the biphenyl ring of the compound formed hydrophobic interaction with the side chain of Phe178 at the entrance of the active site of the enzyme. The docked mode of 5h was found more twisted, therefore the carbonyl group of 5h interacted with the side chain of Arg213. The addition of pentyl-benzene moiety at the R position of 5i made the compound more flexible. Due to high flexibility, 5i moved more towards the entrance of the active site, however, the hydrazide group of 5i retained its H-bonding with Glu277 and His351, and π-π interaction with Phe303. The hydrazide moiety of 5l, 5n, 5j, 5c, and 5f interacted with the Glu277 and His351-like compounds 5k and 4h. The biphenyl ring of 5m was tilted towards Arg69, His112, and Phe178, and its hydrazide group mediated H-bond with the Asn350 and Glu277. Like 4f, the biphenyl ring of 5d was tilted in opposite direction, therefore 5d could only make hydrophobic interaction with Glu277 and Phe303, as a result, its activity was decreased. Similarly, the biphenyl ring of 5g slipped more outside the active site, while its R-group acquired the position near the biphenyl ring of other compounds, instead of residing in the hydrophobic groove. Due to this conformational change, the compound showed the least inhibitory activity. The docking results are tabulated in Table 2. In docking studies, it was observed that Glu277 and HIS351 play a crucial role in the stabilization of binding of those compounds in the active site of the enzyme ( Figure 3). The docking scores of the compounds are in the range of −7.51 to −3.36 kcal/mol, which indicates that these compounds are good binders of protein, therefore exhibit excellent inhibitory potential for α-glucosidase.

General Instrumentation
All the chemicals, solvents, and reagents used in this research work were analytical grade and purchased from Sigma-Aldrich, MO, USA. For thin-layer chromatography (TLC), pre-coated aluminum sheets (silica gel 60F-254, Merck, Darmstadt, Hesse, Germany) were used and visualized with the help of UV light at 254 nm. LC-HRESIMS (liquid chromatography high-resolution electrospray ionization mass spectrometry) spectra were recorded on a mass spectrometer (Waters Quattro Premier XE, Waters, Milford, MA, USA). Melting point apparatus Stuart ® SMP10 was used for the determination of melting points of the synthesized derivatives. The 1 H-and 13 C NMR spectra were recorded on a nuclear magnetic resonance (NMR) spectrometer (Bruker, Zürich, Switzerland) operating at 600 MHz (150 MHz for 13 C; 600 MHz for 1 H; chemical shift (δ) = ppm; coupling constants (J) = Hz) using the solvent peaks as internal references (CDCl 3 , δ H : 7.26; δ C : 77.0). Column chromatography was carried out by using silica gel of the selected particle size of 100-200 mesh. (1) Coupling reagent CDI (1,1 -carbonyldiimidazole) was used for the synthesis of hydrazide by reacting an appropriate amount of flurbiprofen with hydrazine hydrate in the presence of triethyl amine in THF solvent. In this reaction, flurbiprofen (0.01 M) and CDI (0.012 M) were taken in a round bottom flask and dissolved in minimum amount of tetrahydrofuran (THF). The reaction mixture was stirred for 30-40 min to activate the carboxylic group of flurbiprofen. Finally, hydrazine hydrates (4 mL) were added and reflux it for 5 h, with continuous stirring. The completion of the reaction was checked through TLC (4:6 = ethyl acetate: hexane) from time to time. After completion the reaction mixture was cooled to room temperature (RT) and poured onto crushed ice (100 mL), precipitates were formed, filtered, and washed with excess distilled water to obtain pure hydrazide for further reactions, and the ppt. were dried, weighed and recrystallize with ethanol earlier reported by Momin khan et al. [15].

General Procedure for the Synthesis of Substituted Schiff's Bases of Flurbiprofen (4a-p and 5a-n)
Acid hydrazide of flurbiprofen (0.0005 mole) and corresponding substituted aromatic/aliphatic aldehydes and ketones (0.0005 mole) were added to a 100 mL RB flask and dissolved in 15 mL ethanol in the presence of 4-5 drops glacial acetic acid as a catalyst. The reaction mixture was refluxed for 3-4 h with constant stirring. The reaction progress was checked through TLC (3:7 = ethyl acetate:hexane). After completion of reaction, it was poured into ice-cold distilled water, precipitates were formed, filtered, and washed with distilled water and hot hexane to remove unreacted aldehydes/ketones. The ppt. was dried under air, recrystallized with ethanol, and weighed. The desired products were further confirmed by different spectroscopic techniques like HRMS, and NMR ( Figures S1-S30, Supplementary Materials).

Molecular Docking
The docking study of the active compounds was conducted in molecular operating environment [42] on the X-ray crystal structure of isomaltase from Saccharomyces cerevisiae in complex with alpha-D-glucopyranose (PDB code: 3A4A, resolution: 1.60 Å) [43]. In our previous studies [11,40,44], we have thoroughly checked the docking performance of applied docking protocol via re-docking of acarbose in the active site of α-glucosidase where MOE was found efficient with an RMSD value of 0.36 Å. The enzyme file was handled through QuickPrep module of MOE to add missing hydrogen atoms on each residue and to calculate partial charges using pre-defined force field (Amber10:EHT force field). The structures of active compounds were drawn by ChemDraw and converted into three-dimensional (3D)-format by MOE-WASH module after importing all the prepared structures in the MOE database where hydrogen atoms and partial charges were added to all the compounds simultaneously and the structure of each ligand was minimized until RMS gradient of 0.1 RMS kcal/mol/Å was obtained. In subsequent step, triangle matcher docking algorithm in combination with London dG scoring function of MOE was applied during docking. In the end, the best-docked conformation of each ligand was selected based on the good docking score and appropriate binding interaction.

Conclusions
Novel acyl hydrazones of flurbiprofen (4a-4p) and (5a-5n) were synthesized and evaluated for in vitro inhibition against the α-glucosidase enzyme. Among the synthesized compounds, 5k, 4h, 5h, 4d, 4b, and 5i showed excellent inhibitory potential. The structureactivity relationship (SAR) advocated that the deviation in the activity of the synthesized compounds may be due to the attachment and nature of substituents. Likewise, in silico studies have shown that the synthesized hydrazones (ligands) have widespread binding interactions within the active site of the enzyme, and due to their different substituent, their conformation is different in the active site of the enzyme. This study recognized numerous lead candidates derived from flurbiprofen and further exploration of these molecules for coming research to obtain novel α-glucosidase inhibitors.