Synthesis of novel (E)-1-(2-(2-(4(dimethylamino) benzylidene) hydrazinyl)-4-methylthiazol-5-yl)ethanone derivatives as ecto-5′-nucleotidase inhibitors

Ecto-5′-nucleotidase (e5′NT), a membrane-bound enzyme and an essential member of ecto-nucleotidases which regulates extracellular purinergic signalling. Their upregulation results in various disease conditions, for example, inflammation, hypoxia and cancer. Therefore, efforts have been made to synthesize potent and selective inhibitors of e5′NT. Here we have synthesized, characterized and evaluated six thiazole derivatives (3a–3f) as potent e5′NT inhibitors. Among all derivatives, the compound (E)-1-(4-methyl-2-(2-(pyridin-3-ylmethylene)hydrazinyl) thiazol-5-yl)ethanone (3a) exhibited maximum inhibition towards both human and rat enzymes. However, their potency against h-e5′NT was 24-fold higher than r-e5′NT. Only two compounds exhibited inhibitory behaviour towards r-e5′NT. The molecular structures of these derivatives were confirmed with the help of solid-state characterization through NMR (1H and 13C), FTIR and elemental analysis. Additionally, molecular docking was also implemented to explain putative bonding interaction between the active site of an enzyme and potent inhibitors.


Background
As a regulator of adenosine signalling pathway, the membrane-bound ecto-5 0 -nucleotidase (e5 0 NT, CD73) speeds up the final reaction step that involves the hydrolysis of extracellular nucleotides and their conversion from adenosine monophosphate (AMP) to adenosine [1]. These enzymes belong to metallophosphoesterase superfamily which contains divalent cation in its active site [2]. The end product of this catalytic activity is adenosine molecule which further activates P1 receptor and accelerates a number of physiological and pathophysiological processes like anti-inflammatory, immunosuppressive, tranquillizing, vasodilatation and antidiuretic effects [3]. It is evident from previous reports that numerous cancer cells display high levels of e5 0 NT, which ultimately increases the intensity of adenosine production to promote angiogenesis and T-cells death [4]. An e5 0 NT has been involved in cancer progression by performing several functions; e.g. enzymatic and nonenzymatic functions. An uncontrolled enzymatic activity results in stimulation of adenosine receptors that reflects its role in breast cancer cells migration as well as invasion and adhesion to the extracellular membrane (ECM). Irrespective of its catalytic activity, an e5 0 NT functions as co-receptor in T-cell activation or promotes cell interaction with ECM component as well as migration and regulates cell-cell adhesion [5,6]. An e5 0 NT has been supposed to be a motility factor in the development and progression of cancers. Hence several studies have suggested the correlation between e5 0 NT expression and tumour progression [7]. Therefore, the e5 0 NT enzyme is considered a significant target in cancer therapy. Eukaryotic e5 0 NTs are competitively inhibited by ADP and ATP, regarded as physiological inhibitors. Previously, small inhibitory molecules or antibodies have been investigated that resulted into reduction of metastasis and tumour growth [8]. It has been found that ADP analogues such as AMPCP resulted in inhibition of all forms of e5 0 NT. In addition, several other molecules like anthraquinone, sulfonamide and flavonoid-based compounds were found to be active against e5 0 NT [9,10]. All these known molecules lack specificity and selectivity towards their target. Hence the therapeutic importance of e5 0 NT plus need of potent and selective moieties encourages us to synthesize a series of compounds as a potential therapeutic agent in various disease conditions.
Azomethines are widely known as Schiff bases; which represent a pharmacologically active class of organic compounds. Schiff bases have grabbed immense importance in medicinal chemistry research due to their widespread therapeutic activities including anti-microbial [11,12], anti-tumour [13], antimycobacterial [14], trypanocidal [15], anti-inflammatory [16], anti-HIV [17], anti-diabetic [18] and anti-malarial activities [19]. Apart from medicinal significance, Schiff bases elicit applications in dyes and pigments, catalysts, polymer stabilizers and serve as intermediates in organic synthesis. Azomethines can be easily synthesized by the condensation reaction of amines with suitable aldehydes or ketones.

Synthesis of 1-(2-hydrazinyl-4-methylthiazol-5-yl)ethanone(2)
A 0.02 M of 3-chloropentane-2,4-dione (1) in dry distilled methanol (50 ml) was refluxed for 7 h in the presence of thiosemicarbazide (0.02 M). The intermediates formed during each step of reaction were evaluated with the help of thin layer chromatograph (TLC). When the reaction was completed the mixture was decanted in ice cold water. The acquired precipitates (86% yield) were filtered and recrystallized in ethanol.

Biochemical assays
The plasmids expressing e5 0 NT, either rat or human, were used to transfect the COS-7 cells through lipofectamine [30]. The confluent cells were allowed to incubate in DMEM/F-12 having plasmid DNA (6 mg) and transfecting reagent (24 ml) for 5 h at ambient temperature (378C). To discontinue transfection, a specified volume of DMEM/F-12 was added, containing FBS (20%). These cells were then harvested after 2 -3 days.

Production of membrane fractions
The transfected cells were separated from harvesting buffer (45 mM Tris buffer, 95 mM NaCl, and 0.1 mM PMSF, pH 7.5) through scraping, followed by washing with Tris buffer and then allowed to centrifuge by spinning at 300 r.p.m. at 48C for 5 min [30]. Finally, the cells remained suspended in harvesting buffer solution containing aprotinin (10 mg ml 21 ). As a result of sonication, the produced cellular debris was removed by cold centrifugation operated at 300 r.p.m. for 10 min. Glycerol (7.5%) rsos.royalsocietypublishing.org R. Soc. open sci. 5: 180837 was mixed with the resultant supernatant. The Bradford microplate assay was employed for estimation of protein concentration where albumin was used as a standard [31].

Ecto-5 0 -nucleotidase inhibition assay
The e5 0 NT inhibition assay was performed with respect to our previously described procedure [32] on P/ACE MDQ capillary electrophoresis system (Beckman Instruments, Fullerton, CA, USA) provided with UV detection system. The samples were prepared in the assay buffer (1 mM CaCl 2 , 10 mM Tris HCl and 2 mM MgCl 2 , pH 7.4) and analysed at 0.1 mM concentration. The total assay volume (100 ml) comprises sample (10 ml), 10 ml of h-e5 0 NT (6.94 mg ml 21 ) protein extract or r-e5 0 NT (7.17 mg ml 21 ) and 70 ml of assay buffer. The above mixture was allowed to incubate at 378C for 10 min. Subsequently, biochemical reaction was started with the addition of substrate (10 ml) and AMP (500 mM). The mixture was incubated again for 30 min at 378C. The enzyme-substrate reaction was stopped by thermal denaturation for the duration of 20 min by placing the mixture in a water bath at 998C. Then, 50 ml of obtained mixture was filled into CE mini vials and injected with 0.5 psi into the capillary in 5 s, whereas 15 kV was set to split-up substrate and product peaks. The demonstration of greater than 50% inhibitory activity by compounds on either human or rat enzyme was further investigated for estimation of IC 50 values. Therefore, successive dilution of each active compound was made and different inhibitor concentrations were assayed to obtain the dose -response curve against both enzymes. All experiments were conducted thrice. The nonlinear regression analysis program (PRISM 5.0) was employed to calculate IC 50 value.

Molecular docking studies
Molecular docking analysis of every compound was performed to explore binding mode with the active site of h-e5 0 NT as well as r-e5 0 NT target enzymes. X-ray crystallographic structures were present in RCSB Protein Data Bank which was downloaded in the form of PDB ID: 4H2I [33], while X-ray crystallographic structure of r-e5 0 NT was not reported till now, so a previous homology model was selected for docking studies [34].

Structure-activity relationship
A series of (E)-1-(2-(2-ethylidenehydrazinyl)-4-methylthiazol-5-yl)ethanones was synthesized containing Schiff bases along with thiazole ring. These six derivatives possessing different substitution were tested against e5 0 NT to evaluate their inhibitory potential. From table 1 data, it was evident that these derivatives had showed higher inhibitory effects towards human enzyme as compared to rat. Except two derivatives, all four derivatives showed inhibition in the range of 0.32 + 0.03-6.19 + 0.32 (mM). After initial screening, compounds 3c and 3d showed no inhibitory potential toward e5 0 NT (h-, r-) enzymes (table 1) due to presence of furan and 2-methyl furan ring, respectively. Two derivatives (3e and 3f ) displayed selective inhibitory behaviour against h-e5 0 NT while these were inactive against r-e5 0 NT with per cent values 10.8 + 4.25 and 32.7 + 1.62 (%), respectively. The compound 3e possessed pyrrole ring substitution at Schiff base that favours its selective reactivity towards h-e5 0 NT. In the case of compound 3f, the presence of indole ring resulted in selective and significant activity. With regard to r-e5 0 NT, only two derivatives (3a and 3b) displayed some inhibitory activity but were less reactive when compared with their activity against h-e5 0 NT. Both compounds 3a and 3b showed their inhibition with IC 50 + s.e.m. values 7.81 + 0.89 and 10.1 + 0.58 (mM), respectively.

Molecular docking analysis
Docking analysis of 3a -3f was executed to investigate putative binding mode with h-e5 0 NT and r-e5 0 NT enzymes. Figure 2a  interactions. Contrary to the binding interface of 3a inside active site of h-e5 0 NT, three hydrogen bonds formed by 3a with amino acid residue of r-e5 0 NT involved were Arg356, Asn392 and Arg397 while three p-p stacked interactions were formed by Phe419, Try502 and Gly394. The compound 3a, the most potent compound against h-e5 0 NT possesses the pyridine-3-ylmethylene and therefore was found most active. However, the second active compound, 3b, did not possess the pyrimidin, and instead have dimethylamino group attached to the benzylidene hydrazinyl. The compound showed significant inhibitory potential against both the enzymes, and especially, against h-e5 0 NT.

Conclusion
In conclusion, a novel series of thiazole derivatives was produced and evaluated for their anti-cancer potential, i.e. e5 0 NT. The derivatives possessed significant inhibition potential against h-e5 0 NT as compared to r-e5 0 NT. The compound 3a was found to exhibit maximum inhibition against h-e5 0 NT with IC 50 value 0.32 + 0.03 mM that is 24-fold higher than its activity towards r-e5 0 NT. Moreover, molecular docking was also performed to determine their putative binding sites. Hence, these derivatives can be further evaluated for their therapeutic importance in the management of various diseases.
Data accessibility. All data are included within the article. Authors' contributions. All the authors contributed in drafting the article and revising it critically for important intellectual content and towards conception and design, or acquisition of data, or analysis and interpretation of data. Moreover, all the authors gave final approval for publication and agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. More specifically, P. Acknowledgements. We acknowledge Shafi Ullah Khan for assisting in docking studies.