Synthesis and in vitro Bioactivity Evaluation of N 4-benzyl Substituted 5-Chloroisatin-3-thiosemicarbazones as Urease and Glycation Inhibitors

A series of fifteen N4-benzyl substituted 5-chloroisatin-3-thiosemicarbazones 5a–o were synthesized and screened mainly for their antiurease and antiglycation effects. Lemna aequinocitalis growth and Artemia salina assays were carried out to determine their phytotoxicity and cytotoxicity potential. All the compounds proved to be extremely effective urease inhibitors, demonstrating enzyme inhibition much better than the reference inhibitor, thiourea (IC50 values 1.31 ± 0.06 to 3.24 ± 0.15 vs. 22.3 ± 1.12 μM). On the other hand, eight out of fifteen compounds tested, i.e. 5b, 5c, 5h–k, 5m and 5n were found to be potent glycation inhibitors. Of these, five viz. 5c, 5h–j and 5n proved to be exceedingly efficient, displaying glycation inhibition greater than the reference inhibitor, rutin (IC50 values 114.51 ± 1.08 to 229.94 ± 3.40 vs. 294.5 ± 1.5 μM).


Introduction
Isatin and its derivatives are known to have a diversity of chemotherapeutic activities. 1-35Of the isatin derivatives, isatin-thiosemicarbazones have been found to exhibit a range of biological properties, including antiulcer, 1  anticancer, 1,4,7,12,21-23 antimicrobial, 1-3,5,7,22,24,25 antituberculosis, 26-28 antiviral 1,2,5,7,12,28 and enzyme inhibitory activities. 1,4,11,17Incited by these findings and as a part of our synthetic work on bioactive isatin derivatives, we have recently reported the synthesis of numerous N 4 -aryl substituted isatin-3-thiosemicarbazones (thiourea derivatives) as antimicrobial, 36-39 cytotoxic, 38-42 phytotoxic, 38-41,43 antileishmanial 44 and more importantly antiurease 36-40,43,45 compounds.SAR studies in the synthesized thiosemicarbazones disclosed that in some cases the nature and location of the substituent on the phenyl ring attached to thiosemicarbazone N 4 and/or the existence of lipophilic/inductively electron-withdrawing functions (Cl, F, F 3 CO, NO 2 ) at po-sition 5 of the isatin moiety played a significant role in inducing and/or increasing certain activities, including urease inhibition.In view of this and as an extension of our earlier studies 36-47 aiming to the synthesis of novel isatin derivatives with enhanced or diverse biological activities, we very recently reported the synthesis of a series of N 4 -benzyl substituted 5-nitroisatin-3-thiosemicarbazones (derivatives of thiourea, a substrate-like urease inhibitor) as highly effective urease inhibitors. 48Interestingly, all these compounds demonstrated either induced or increased urease inhibitory activity in comparison to the respective N 4 -phenyl substituted derivatives tested in our earlier assays. 38,45Furthermore, a number of other derivatives of thiourea are reported to show promising glycation inhibitory activity. 49-53Also, some isatin-derived imines (Schiff bases) have been recognized as potent inhibitors of glycation. 54,55Prompted by these observations and as a continuation of our previous studies on bioactive isatin derivatives, we have synthesized a series of fifteen title thiosemicarbazones (a class of Schiff bases) and tested them principally for their antiurease and antiglycation influences.L. aequinocitalis growth and A. salina assays were carried out to determine their phytotoxic and cytotoxic effects, respectively.This paper is one of the first to report on N 4 -benzyl substituted isatin-thiosemicarbazones possessing urease and glycation inhibitory potential.In it we limit our discussion to those compounds which have concurrently attached lipophilic/inductively electron-withdrawing chloro substituent at position 5 of the isatin scaffold and the benzyl functions (modified by placing one inductively electron-donating and one or two electron-attracting groups around the phenyl ring) to thiosemicarbazone N 4 .

Results and Discussion
This study describes the preparation and in vitro testing of antiurease, antiglycation, phytotoxic and cytotoxic potentiality of fourteen presently and one formerly reported 22 N 4 -benzyl substituted 5-chloroisatin-thiosemicarbazones 5b-o and 5a, respectively.

1. Chemistry of Compounds 5a-o
Appropriate N-substituted thiosemicarbazides 3 were reacted with 5-chloroisatin 4 in 50% ethanol (aq.) to furnish the respective thiosemicarbazones 5a-o (Scheme 1).The structures of 5a-o were established by elemental and spectral analyses.Acceptable elemental data were obtained in all the cases.The infrared (IR) spectra of 5a-o showed absorption bands in the 3375-3051, 1737-1684, 1620-1599 and 1193-1149 cm -1 regions attributed to NH, C=O, C=N and C=S functions, respectively. 22,56,57The proton nuclear magnetic resonance ( 1 H NMR) spectra displayed two separate singlets at δ 7.50-11.36and 10.84-12.75 for indole NH and thiosemicarbazone N 2 -H.The thiosemicarbazone N 4 -H appeared as a triplet at δ 8.03-10.91 in all the cases, 22,27 except 5e, 5g and 5h, wherein it resonated as a singlet at δ 8.26, 10.91 and 10.85, respectively.Similarly, the benzyl CH 2 protons were observed as a doublet at δ 4. 40-5.02 in all the cases, 22,27 except 5b, 5g and 5h; in the case of 5h they appeared as a singlet at δ 3.79, whilst in 5b and 5g they resonated together with the DMSO and DMSO, CH 3 group protons, respectively.The carbon nuclear magnetic resonance ( 13 C NMR) spectra of compounds 5a-o upheld the IR and 1 H NMR conclusions.Also, the EI (electron impact) mass spectra of all the compounds displayed molecular ions with mixed intensity and the fragments characteristic of isatin and thiosemicarbazone moieties.The main fragmentation path engaged the split of endocyclic NH-CO and exocyclic NH-CS, N-N bonds. 57The proposed fragmentation pattern of 5i is depicted in Figure S1.
Pervez et al.: Synthesis and in vitro Bio-activity Evaluation ... inhibitory effects and found to be extremely effective inhibitors of the enzyme, displaying superb inhibition (IC 50 = 1.31 ± 0.06 to 4.07 ± 0.11 μM) in contrast to the reference inhibitor, thiourea, which showed inhibitory activity with IC 50 value 22.3 ± 1.12 μM in this assay (Table 1).
SAR studies in the N 4 -benzyl substituted isatin-thiosemicarbazones 5a-o divulged that in comparison to compound 5a having no functional group on the phenyl ring of the benzyl group attached to thiosemicarbazone N 4 , all the rest compounds except 5d and 5k, regardless of the number, type and place of the functionalities present on the phenyl ring, displayed enhanced enzymatic inhibition (IC 50 values 1.31 ± 0.06 to 3.05 ± 0.15 vs. 3.24 ± 0.15 μM).Compound 5l bearing chloro substituent at position 3 of the phenyl ring was the most effective urease inhibitor of the series in the present assay, demonstrating two and half, and seventeen times larger activity than compound 5a and the reference inhibitor, thiourea, respectively (IC 50 = 1.31 ± 0.06 vs. 3.24 ± 0.15 and 22.3 ± 1.12 μM).The next most effective enzyme inhibitor was 5e possessing methoxy group at position 2 of the phenyl ring.This compound showed a somewhat lesser inhibitory activity than 5l but is still about two and fifteen times more active than compound 5a and thiourea (IC 50 value 1.45 ± 0.23 vs. 1.31 ± 0.06, 3.24 ± 0.15 and 22.3 ± 1.12 μM, respectively).The third most effective inhibitor of the enzyme was compound 5j having fluoro group at position 4 of the phenyl ring.This compound like 5e appeared to be a bit less active than 5l (IC 50 value 1.77 ± 0.24 vs. 1.31 ± 0.06 μM) but about two and twelve times more active than compound 5a and thiourea (IC 50 value 1.77 ± 0.24 vs. 3.24 ± 0.15 and 22.3 ± 1.12 μM, respectively).The isomeric compounds 5h and 5i possessing fluoro functionalities at positions 2 and 3 of the phenyl ring, however, demonstrated comparatively inferior but comparable enzymatic inhibition (IC 50 = 2.03 ± 0.14 and 2.07 ± 0.09 μM, respectively).The remaining relatively more effective inhibitors of the enzyme were the dichloro-substituted derivatives 5n and 5o.Among these, 5n with the substituents at positions 2 and 4 of the phenyl ring displayed about two fold larger enzymatic inhibition in comparison to the monochloro-substituted compound 5k having the substituent at position 2 of the phenyl ring (IC 50 value 2.36 ± 0.16 vs. 4.07 ± 0.11 μM).On the other hand, 5o possessing the substituents at positions 3 and 4 of the phenyl ring exhibited distinctly decreased inhibitory activity in contrast to monochloro-substituted compound 5l (the most potent inhibitor of the series) with the substituent at position 3 of the phenyl ring (IC 50 value 1.97 ± 0.16 vs. 1.31 ± 0.06 μM).
The above results revealed that the attachment of varied benzyl groups (possessing one inductively electron-donating and one or two electron-withdrawing functional groups on the phenyl ring) to N 4 of the thiosemicarbazone moiety caused the molecules to intermingle with the enzyme differently and in some cases more competently.Moreover, exhibition of enhanced enzymatic inhibition by N 4 -benzyl substituted isatin-thiosemicarbazones in contrast to the parallel N 4 -aryl substituted ones may be explained on the basis of their better hydrophobic character, which may play a role in their indiscriminate walk process to the active site, and in the hydrophobic binding near the active site of the enzyme.
The limited structure-activity relationship (SAR) studies in the synthetic thiosemicarbazones 5a-o indicated that compound 5c possessing methyl substituent at position 3 of the phenyl ring attached to N 4 of the thiosemicarbazone moiety was the most potent antiglycating agent of the series in the present assay, exhibiting antiglycation activity with IC 50 value 114.51 ± 1.08 μM.The next most potent antiglycating derivative was 5j bearing the fluoro substituent at position 4 of the phenyl ring.This compound displayed a bit lesser inhibition of glycation than the most potent derivative 5c but much larger than the reference inhibitor, rutin (IC 50 value 168.71 ± 8.58 vs. 114.51± 1.08 and 294.5 ± 1.50 μM, respectively).The third most active antiglycating agent was found to be compound 5i bearing the fluoro substituent at position 3 of the phenyl ring, demonstrating a somewhat lower activity than 5j but still considerably higher than the reference inhibitor, rutin (IC 50 = 202.21± 0.55 vs. 168.71± 8.58 and 294.5 ± 1.5 μM, respectively).The remaining relatively more potent glyca-tion inhibitors 5h and 5n possessing fluoro and chloro substituents at positions 2 and 2,4 (ortho and para) of the phenyl ring, respectively, showed inhibitory activity with IC 50 values 229.94 ± 3.40 and 217.71 ± 1.65 μM.The other potent compounds viz.5b and 5k, 5m having methyl and chloro substituents at positions 2 and 2,4 of the phenyl ring, respectively, demonstrated moderate antiglycation activity with IC 50 values 333.06 ± 9.80 and 343.75 ± 2.76, 433.88 ± 2.66 μM.The rest of the compounds viz.5d-g and 5l displayed less than 50% inhibition of glycation and were, therefore, not assessed further for their IC 50 values.Compound 5a showed an anomalous behaviour, displaying enhancement in glycation.
The above results showed that the CH 3 , F and Cl groups worked as active moieties in causing effective inhibition of glycation.Further, the location and/or the number of the substituents on the phenyl ring played a key role in improving the glycation inhibitory potency of the compounds.

4. Cytotoxicity (in vitro)
The synthesized thiosemicarbazones 5a-o were further screened for their cytotoxicity potential by a brine shrimp (Artemia salina) bioassay.All the compounds gave LD 50 values larger than 2.43 × 10 -4 M to 2.91 × 10 -4 M in this assay and were thus considered to be almost inactive.

5. Molecular Docking Studies of Compounds 5a-o
All the compounds showed interactions with His409, Ala436, His492, Asp494, Arg609, His519, Arg639, His593, Ala636 and His594.Figure 1 represents the putative bind-ing mode of 5l, the most potent inhibitor of the enzyme Jack bean urease.Hydrogen-bonding interactions were observed between the oxygen and hydrogen atoms of the inhibitors and the amino acid residues Ala436, Ala636, Arg639, Arg609 and His593.The 3-chlorobenzyl group was directed towards amino acids Ala436, Asp494 and His492, whereas the remaining part of the compound was directed towards Arg639 and Ala636.
All the synthetic derivatives 5a-o were docked inside the active site of the enzyme.No specific interactions were noticed with Ni atoms by any compound; however, aromatic interactions were observed in the active pocket of the enzyme, as shown in Figure 2.This suggests that all the compounds exhibit inhibitory activity by blocking the entrance of the catalytic domain in the enzyme.The docking scores were obtained in the range of -19.57 to -16.27 and are given in Table 4.   2. 2. 5. 1. ADME Profile of N 4 -Benzyl Substituted 5-Chloroisatin-3-thiosemicarbazones 5a-o Drug-likeness is a qualitative property of chemicals evaluated with respect to factors like bioavailability.In order to evaluate drug-likeness of the synthetic derivatives 5a-o, ADME (Absorption, Distribution, Metabolism and Excretion) properties were taken into account.The parameters selected for the purpose were octanol-water distribution coefficients (Slog P and log P), the log solubility in water (log S), the number of hydrogen bond acceptors (HBAH), the number of hydrogen bond donors (HBDH) and the topological polar surface area (TPSA).Along with in silico evaluation of ADME profile, free binding energies were also calculated for all the derivatives.The TPSA values indicated that all the compounds 5a-o can be easily penetrable in the form of drugs, as they showed polar surface area in the range of 95-107 Å 2 .Typically, for molecules to penetrate the blood-brain barrier and, therefore, to act as receptors, an area less than 120 Å 2 is usually needed and molecules with a polar surface area of greater than 120-140 Å 2 tend to be poor in permeating cell membranes. 58In general, compounds with molecular weight less than 500, HBAH less than 10, HBDH less than 5 and a logP value of less than 5 are treated as orally bioavailable with favorable ADME profile. 59,60The determined ADME properties of compounds 5a-o are given in Table 4.All the trial compounds exhibited favorable ADME properties and no violation of Lipinski's rule was observed.According to Lipinski's rule of 5, only two violations are allowed in the molecular docking studies. 61-63

1. General
Melting points (uncorrected) were measured on a Fisher-Johns melting point apparatus.Elemental analyses were carried out using a Leco CHNS-9320 (USA) instrument.IR (KBr discs) spectra were recorded with a Shimadzu Prestige-21 or Shimadzu 8400 spectrophotometer. 1 H NMR spectra were obtained in CDCl 3 /DMSO-d 6 with Bruker Avance/Spectrospin 300 and Bruker Avance 400 spectrometers, using tetramethylsilane (TMS) as the internal standard.

13
C NMR spectra were determined in DMSO-d 6 with a Bruker Avance 300 spectrometer, operating at 75 MHz.EI (electron impact) mass spectra were obtained on MAT-312 and JEOL MSRoute mass spectrometers.All the chemicals and reagents used in the present study were purchased from Merck-Schuchardt, Fluka and Sigma-Aldrich.

2. Synthesis of N-Substituted
Thiosemicarbazides 3 The N-substituted thiosemicarbazides used in this study were synthesized according to the synthetic route reported in the literature. 63

Synthesis of Isatin-thiosemicarbazones 5a-o
Appropriate thiosemicarbazides 3 (5 mmol) were treated with 5-chloroisatin 4 (5 mmol) in 50% aqueous ethanol (25 mL) under reflux for 2 h.The solid formed during heating in each case was filtered and washed thoroughly with hot aqueous ethanol, affording the required compounds 5a-o in pure form.

5. Molecular Docking Studies
Molecular docking studies were performed in accordance with the protocol used in our recently published article. 65

Conclusions
In this study, a series of fifteen N 4 -benzyl substituted 5-chloroisatin-3-thiosemicarbazones 5a-o were synthesized and screened primarily for urease and glycation inhibitory potential.All the synthetic thiosemicarbazones emerged as highly effective urease inhibitors, demonstrating enzymatic inhibition even better than the reference inhibitor, thiourea, used in the assay.These compounds, by displaying non-significant phytotoxic influences, draw a great deal of consideration towards their effectiveness as important inhibitors of soil ureases because they might be combined with fertilizers in minute amounts to greatly improve the usefulness of nitrogen consumption.Also, being non-toxic, they could prove to be promissing candidates for orally effective remedial agents used for the treatment of certain clinical conditions induced by microbial ureases.In antiglycation assay, five out of fifteen compounds tested, i.e. 5c, 5h-j and 5n were recognized as extremely effective inhibitors of glycation, showing inhibitory activity much larger than the reference inhibitor, rutin, and may thus act as credible leads for further studies.
This study presents the first example of exhibiting glycation inhibition by N 4 -benzyl substituted isatin-thiosemicarbazones and offers a firm base for additional studies on such compounds to build up more persuasive and safer glycation inhibitors of pharmaceutical interest.

Acknowledgements
H. Pervez acknowledges the Higher Education Commission (HEC), Pakistan for funding under National Re-

Figure 1 .
Figure 1.The putative binding mode of 5l (the most active inhibitor; light green colored) in the active site pocket of Jack bean urease.

Figure 2 .
Figure 2. Common binding modes of all the inhibitors inside the active site pocket of the urease enzyme.

Table 1 .
Inhibition of Jack bean urease by compounds

Table 2 .
Antiglycation activity of compounds

Table 3 .
Growth inhibition of Lemna aequinocitalis by compounds 5a-o at different concentrations

Table 4 .
Free binding energy and ADME profile of compounds 5a-o Pervez et al.: Synthesis and in vitro Bio-activity Evaluation ...
Pervez et al.: Synthesis and in vitro Bio-activity Evaluation ...
Pervez et al.: Synthesis and in vitro Bio-activity Evaluation ...