Synthesis of 2-{ [ 5-( aralkyl / aryl )-1 , 3 , 4-oxadiazol-2-yl ] sulfanyl }-N-( 4-methyl-1 , 3-thiazol-2-yl ) acetamides : Novel bi-heterocycles as potential therapeutic agents

Purpose: To evaluate the therapeutic potential of new bi-heterocycles containing a 1,3-thiazole and 1,3,4-oxadiazole in the skeleton against Alzheimer's disease and diabetes, supported by in-silico study. Methods: The synthesis was initiated by the reaction of 4-methyl-1,3-thiazol-2-amine (1) with bromoacetyl bromide (2) in aqueous basic medium to obtain an electrophile,2-bromo-N-(4-methyl-1,3thiazol-2-yl)acetamide (3). In parallel reactions, a series of carboxylic acids, 4a-r, were converted through a sequence of three steps, into respective 1,3,4-oxadiazole heterocyclic cores, 7a-r, to utilize as nucleophiles. Finally, the designed molecules, 8a-r, were synthesized by coupling 7a-r individually with 3 in an aprotic polar solvent. The structures of these bi-heterocycles were elucidated by infrared (IR), electron ionization-mass spectrometry (EI-MS), proton nuclear magnetic resonance (H-NMR) and carbon nuclear magnetic resonance (C-NMR). To evaluate their enzyme inhibitory potential, 8a-r were screened against acetylcholinesterase (AChE), but brine shrimp lethality bioassay. Results: The most active compound against AChE was 8l with half-maximal inhibitory concentration (IC50) of 17.25 ± 0.07 μM. Against BChE, the highest inhibitory effect was shown by 8k (56.23 ± 0.09 μM). Compound 8f (161.26 ± 0.23μM) was recognized as a fairly good inhibitor of urease. In view of its inhibition of α-glucosidase, 8o (57.35 ± 0.17μM) was considered a potential therapeutic agent. Conclusion: The results indicate that some of the synthesized products with low toxicity exhibit notable enzyme inhibitory activity against selected enzymes compared with the reference drug, and therefore, are of potential therapeutic interest


INTRODUCTION
The chemistry and biological study of heterocyclic compounds has been an interesting field for a long time in medicinal chemistry.Thiazoles play a crucial role in the activity of biological compounds.For example, the thiazole ring is a component of vitamin B1 (thiamine), an important coenzyme of carboxylases.This heterocycle is also present in penicillins, which serve as antibiotics [1].In addition, high antioxidant, anti-inflammatory and inhibitory effects of thiazoles have been observed in vitro in their action against parasites (Plasmodium and Trypanosoma) and fungi (Candida albicans) [2].Similarly, 1,3,4-oxadiazoles have demonstrated a range of bioactivities [3][4][5].Molecules bearing this moiety are known to possess anti-parasitic, hypoglycemic, anti-inflammatory, antifungal and antibacterial activities [6][7][8].
Cholinesterases, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), belong to the class of serine hydrolases.Acetylcholine activity is terminated at cholinergic synapses by these enzymes, and these enzymes are also found at neuromuscular junctions and cholinergic brain synapses [9].It is known that BChE is associated with Alzheimer's plaque in notably elevated quantities.The inhibitors of these enzymes may have future prospects as curative agents for Alzheimer's disease [10].α-Glucosidase inhibitors (AGIs) such as acarbose and miglitol have been approved for clinical use in controlling the digestion of complex carbohydrates in the gut and may be used in the treatment of patients with type 2 diabetes or impaired glucose tolerance [11].Urease is involved in different pathogenic processes such as pyelonephritis, peptic ulceration, hepatic encephalopathy, urolithiasis and urinary catheter encrustation [12].Molecular docking analysis approximates ligands regarding their orientation and conformation at the binding site of target proteins.The precise prediction of activity and structural modeling can be achieved by docking studies.Furthermore, it elucidates the active site of proteins and interactions by inhibitors [13].
New drugs are continually being synthesized, aimed at treating and/or preventing various human diseases.The documented bioactivities of heterocyclic compounds prompted us to synthesize molecules having an amalgamation of two heterocyclic cores, thiazole and 1,3,4oxadiazole, linked through an acetamide.As it is known that structural modifications have an effect on the therapeutic behavior of drugs [14], the molecules were synthesized with variation of the groups attached to the 5-position of the 1,3,4-oxadiazole core.The whole series of compounds was tested against the four aforementioned enzymes and also cytotoxicity.

EXPERIMENTAL General
All the chemicals and analytical grade solvents were purchased from Sigma Aldrich, Alfa Aesar (Germany) and Merck through local suppliers.Pre-coated silica gel Al-plates were used for TLC with ethyl acetate and n-hexane as solvent system.Spots were detected under UV light at 254 nm.With open capillary tubes, a Gallonkamp apparatus was used to determine the melting point.IR spectra were recorded using the KBr pellet method on a Jasco-320-A spectrometer. 1 H-NMR spectra were recorded at 600 MHz in DMSO with a Bruker spectrometer.Mass spectra (EIMS) were obtained on a JEOL JMS-600H instrument with data system.The coupling constant (J) is given in Hz and chemical shift ( ) in ppm.The abbreviations used in the interpretation of 1 H NMR spectra are as follows: s, singlet; d, doublet; dd, doublet of doublets; t, triplet; br.t, broad triplet; q, quartet; quin, quintet; sex, sextet; sep, septet; m, multiplet.

General procedure for synthesis of ethyl aralkyl/arylcarboxylates (5a-r)
Aralkyl/arylcarboxylic acids (4a-r; 2.5 g each) were refluxed, individually, with 60 mL of EtOH for 4-5 h in the presence of conc.H 2 SO 4 (1.25 mL) in a 250-mL round-bottom flask.TLC plates were used to round-bottom monitor the reactions.Excess distilled water (150 mL) was added after maximum completion of the reaction, and the pH was adjusted to 8-10 with 20 % aq.Na 2 CO 3 .The product was collected through sequential extraction with CHCl 3 (50 mL × 3).CHCl 3 was distilled off to collect the products.In some cases, the products were collected by filtration.
The esters obtained, 5a-r, were then used further.

General
procedure for synthesis of aralkyl/arylcarbohydrazides (6a-r) Ethyl esters (5a-r; 4.5 mL) were refluxed with 80% N 2 H 4 .H 2 O (7.2 mL) for 3-4 h in 20 mL of EtOH in a round-bottom flask (100 mL).The reaction was monitored by TLC.At completion, excess ice-cold distilled H 2 O (60 mL) was added to obtain the precipitate, which was filtered off, washed with distilled H 2 O and dried to acquire the compounds, 6a-r.

Cholinesterase inhibition assay
AChE and BChE inhibitory activity was determined in 96-well microplates according to a reported method [15] with slight modifications.The reaction mixture totaled 100 µL and contained the following: 60µL of 50 mM Na 2 HPO 4 buffer, pH 7.7; 10 µL of acetylthiocholine iodide for AChE and butyrylthiocholine bromide for BChE, at final concentrations of acetylthiocholine iodide;10 µL of test compound at 0.5 mM, and 10 µL of enzyme (0.005 U/well AChE or BChE).The microplates were mixed, pre-read, and incubated for 10 min at 37 ºC.The enzyme reaction product was determined by the addition of 0.5 mM Ellman's reagent (5,5'-dithio-bis-[2-nitrobenzoic acid)and further incubation for 15 min at 37ºC, followed by reading absorbance at 405 nm.All experiments were performed with controls and in triplicate.Eserine (0.5 mM) served as the positive control.Inhibition (H) was calculated using Eq 1.
where Ac = absorbance of control and At = absorbance of test compound.IC 50 values were calculated using EZ-Fit Enzyme kinetics software (Perrella Scientific Inc. Amherst, NH, USA).
The reaction mixture was mixed, incubated for 10 min at 37 ºC and pre-read at 400nm.The reaction was initiated by the addition of 10 µL of 0.5 mM p-nitrophenyl-alpha-D-glucopyranoside.Acarbose was used as positive control.Microplates were incubated for 30 min at37 ºC and absorbance read at 400 nm using a Synergy HT microplate reader.All experiments were performed in duplicate.The equation discussed for cholinesterase enzymes was used for the determination of % inhibition and IC 50 values.

Urease inhibition assay
This enzyme inhibition assay is the customized form of the commonly known Berthelot assay [17].To each well of a 96-well microplate, 45 µL of reaction mixture containing 10μL of phosphate buffer, pH 7.0, 10μL of test sample and 25μL of urease solution (0.135 U) were added.Contents were pre-incubated at 37 ºC for 5 min.A volume (40μL) of urea stock solution (20 mM) was added to each well, and the microplate incubated for 10 min at 37ºC.This was followed by the addition of 115μL of phenol-hypochlorite reagent (freshly prepared by mixing 45μL phenol with 70 μL of alkali) per well.For color development, the microplate was incubated for another 10 min at 37 ºC, and absorbance measured at 625 nm.The percentage enzyme inhibition and IC 50 values were determined as mentioned above.

Cytotoxicity assay
Cytotoxicity was evaluated by the brine shrimp lethality bioassay [18].Artificial sea water was prepared with sea salt at 34g/L.A shallow rectangular dish (22×32 cm) was used for the hatching of brine shrimp (Artemia salina) eggs (Sera, Heidelberg, Germany) under constant aeration for 48 h at room temperature.After hatching, active shrimp were collected from the brighter portion of the hatching chamber and used for the assay.Vials containing 5 mL of artificial sea water (with different concentrations of test compounds from the stock) were used.Ten shrimp were transferred to each vial.The temperature of the vials was maintained at 26 °C.The number of surviving shrimp were calculated after one day.The experiment was performed in triplicate and data analyzed with the Finney computer program to determine the LD 50 (lethal dose that killed 50 % of shrimp).

Molecular docking studies
The reported MOE-Dock method of MOE 2009-2010 was utilized to study molecular recognition [19].The Protein Data Bank site was used to retrieve protein molecules of acetylcholinesterase (PDB code: 1GQR), butyrylcholinesterase (PDB code: 1POP), αglucosidase (PDB ID: 3NO 4 ) and urease (PDB ID: 4UBP).After removing water molecules, MOE applications were used for performing 3D protonation of the protein molecule.The energy minimization algorithm of the MOE tool was used to minimize the energy of protein molecules using the following parameters; gradient of 0.05, Force Field of MMFF94X & Solvation and Chiral Constraint of Current Geometry.However, minimization of energy was ended for the gradient below 0.05.The templates for docking were the energy minimized protonated structures that were saved in a separate database (using a mdb file format).Finally, all compounds were docked into the binding pockets of enzymes.For the confirmation of validity, a re-docking procedure was applied.After docking analysis of each compound with 30 conformations, the best 2D images were selected for their specific types of interactions and their 3D images were drawn along with their bond lengths.

Statistical analysis
All measurements were carried out in triplicate and statistical analysis was performed by Microsoft Excel 2010.The results are presented as mean ± SEM with 90 % CL.P < 0.05 was considered statistically significant.

Chemistry
The protocol for the synthesis of the new molecules 1, and the various groups are given in Table 1.The first step in the synthesis was the reaction of 4methyl-1,3-thiazol-2-amine (1) with 2bromoethanoyl bromide (2) in a basic aqueous medium to synthesize 2-bromo-N-(4-methyl-1,3thiazol-2-yl)acetamide ( 3), an electrophile.Various nucleophiles were then synthesized in a parallel sequence of reactions, starting from a series of aralkyl/arylcarboxylic acids, 4a-r, which were converted to respective esters, 5a-r; acid hydrazides, 6a-r, which were cyclized to the respective 5-aralkyl/aryl-1,3,4-oxadiazole-2-thiols (7a-r).These thiols, serving as nucleophiles, were finally coupled one by one with 3in an aprotic polar solvent, i.e., DMF, using LiH as a base to obtain the target heterocyclic derivatives, 8a-r.The structural verification was performed through spectral data analysis.All compounds were tested for enzyme inhibitory activity against cholinesterases, urease, and α-glucosidase, and the results are as shown in Table 2.The brine shrimp lethality bioassay was used to evaluate the cytotoxicity of the synthesized compounds and these results are given in Table 3.

Enzyme inhibition
In the search for new more effective therapeutic agents, the synthesized bi-heterocycles 8a-p were screened for their inhibitory activity against four pharmacologically relevant enzymes: acetylcholinesterase, butyrylcholinesterase, urease and α-glucosidase.The results of enzyme inhibition are represented as % inhibition and IC 50 for cholinesterases, urease, glucosidase (Table 2) and LD 50 values for brine shrimps (Table 3).

Enzyme inhibition, molecular docking and structure-activity relationship
To ascertain the therapeutic potential of 8a-r, these compounds were tested for inhibitory activity against four pharmacologically relevant enzymes, namely, AChE, BChE, α-glucosidase and urease.The results are given as % inhibition and IC 50 (50% inhibitory concentration) values in Table 2.The modes of interactions of the synthesized molecules (8a-r) with these enzymes were also described with in silico (molecular docking) study.All compounds except two, 8a and 8r, exhibited an inhibitory potential against AChE.The most promising compounds against this enzyme were 8l and 8q with IC 50 values of 17.25 ± 0.07 and 18.36 ± 0.08 µM, respectively, which might have been due to the presence of a substituted nitro group at the three position of the phenyl ring.
Eserine was used as a positive control against this enzyme, having an IC 50 of 0.04 ± 0.0001 µM.The better activity of 8l and 8q, relative to other derivatives in the series, might have been due to the presence of a 3-nitrophenyl group and benzyl group, respectively, in these molecules.
Varying trends were observed in other derivatives of the series.All derivatives, 8a-r, were docked into the active pocket of this enzyme.
All synthesized derivatives were computationally docked against α -glucosidase, AChE and BChE to explore the binding modes of the ligands.Four interactions were shown by the most active compound 8l in this series.Ser122 makes an acidic interaction with carbonyl oxygen of the ligand, with 8l giving a bond length of 3.46 Å. Trp84 gave the strongest backbone donor interaction with -HNof thiazol and two arenearene interactions with oxadiazol ring, having bond distances of 2.12, 3.75 and 3.93 Å , respectively, as identified from the 2D and 3D images of Figure 4.
Against BChE, weak activity was shown by the synthesized compounds, and almost half of the synthesized compound were inactive, where a moderate inhibition potential was shown by the compound 8k, with an IC 50 of 56.23±0.09µM.This might have been attributed to the substitution of a 2-nitrophenyl group in the skeleton.The docking results revealed that the 8k showed three strong interactions, an acidic interaction between Ser122 and acetyl moiety, a backbone donor and an arene-arene interaction with Trp84, having bond lengths of 3.48, 2.07 and 3.86 Å, respectively, as indicated in Figure 5 (2D & 3D).Similarly, a very moderate inhibitory potential was demonstrated by most of the compounds against α-glucosidase.Acarbose was used as the reference standard (IC 50 = 37.38 ± 0.12 μM).The molecule 8o (57.35 ± 0.17μM) exhibited relatively better inhibitory activity in the series studied against this enzyme.This improved activity might have been due to the incorporation of a 2-methyl-3,5-dinitrophenyl ring in the molecule.
The 1,4 glycosidic linkage is hydrolyzed by αglucosidase.Postprandial hyperglycemia is caused by the inhibition of α-glucosidase [20].Therefore, inhibition of α-glucosidase is considered important in managing type-2 diabetes whose blood sugar is highest after eating complex carbohydrates.From the in silico study, (Figure 6) the hydrogen attached to nitrogen of the acetamoyl group in 8o was involved in strong polar bonding with Asp349 giving a bond length of 2.12Å.In addition, 1,3,4oxadiazole and 4-methyl-1,3-thiazol-2-yl groups were also found to show arene-cation interactions with Arg312, Arg212 and Arg439, respectively.Some compounds were found to be active against urease but displayed feeble inhibition relative to the reference, thiourea (IC 50 = 21.11±0.12μM).In comparison with the other synthesized derivatives, 8f possessed relatively considerable inhibitory activity, indicating that the incorporation of 3-chlorophenyl moiety was adequate for the inhibition of this enzyme.Against urease, the molecular docking study (Figure 7) of 8f revealed π-π and arene-cation interactions for the 4-methyl-1,3-thiazol-2-yl and 3-chlorophenyl groups with His323 and His324, respectively.The hydrogen attached to nitrogen of the acetamoyl group was involved in polar bonding with Asp224, giving a bond length of 2.16Å.
The cytotoxicity of the synthesized compounds was evaluated by brine shrimp lethality.The higher LD 50 values of compounds 8d (498.7 mM), 8l (466.7 mM) and 8h (395.4 mM) of brine shrimp lethality analysis demonstrated the lowest toxicity of these compounds.Thus the synthesized molecules might be used as new drug candidates for the related diseases shown by their enzyme inhibition behavior.

CONCLUSION
Most of the derivatives among the synthesized molecules displayed notable inhibitory activity against the selected enzymes, along with very low cytotoxicity.Therefore, these synthesized compounds may have important roles in drug design and development.