Convergent synthesis and biological evaluation of 3-[2-(benzylidenehydrazinyl)thiazol-4-yl]-4-hydroxycoumarins

A convenient synthesis of a small library of 4-hydroxycoumarin derivatives, containing thiazole and benzylidenehydrazine moieties has been achieved, using 2-hydroxyacetophenone and aryl-and heteroaryl aldehydes as starting materials. In vit ro biological studies revealed that some of the compounds exhibited modest activities, viz.; anti-malarial (65% PLDH viability), anti-trypanosomal (52% T.b.brucei viability), anti-mycobacterial (Visual MIC90 5.63  M) and anti-bacterial (restricting S.aureus metabolic activity after 6 h)


Introduction
2][3][4] Examples of naturally occurring coumarin derivatives include the antibiotic, novobiocin 1, 5 and the highly toxic aflatoxins produced as fungal metabolites and illustrated by the fused-ring derivative, aflatoxin B1 2. 6 Recently reported synthetic coumarin derivatives of medicinal interest include inter alia: antimycobacterial hydrazinyl thiazolyl derivatives, such as compound 3; 7 non-steroidal anti-inflammatories, such as cloricromine 4; 8 and anti-bacterial coumarin-triazole conjugates, such as compound 5 which exhibited a minimum MRSA inhibitory concentration of 4 μg/mL.Classic approaches to coumarin synthesis have included the Perkin, Knoevenagel and Pechmann and Wittig reactions. 10In our group, we have pioneered applications of Morita-Baylis-Hillman (MBH) methodology in the construction of coumarin derivatives 11,12 and have reported the synthesis and in vitro biological evaluation of: novel furocoumarins 6 as potential HIV-1 IN inhibitors; 13 and coumarin-AZT conjugates. 14,15as potential HIV-1 PR and dual-action HIV-1 PR/RT inhibitors.The 4-hydroxycoumarin motif is present in many biologically active compounds and, in this communication, we report on the preparation and bioassay of a series of 4-hydroxy analogues of the hydrazinyl thiazolyl derivative 3 reported by Arshad et al. 7

Synthesis of 3-[2-(3-[2-(benzylidinehydrazinyl)thiazol-4-yl]-yl]-4-hydroxycoumarins 14a-g
The title compounds were designed to incorporate, within a single hybrid structure, 4-hydroxycoumarin, thiazole and hydrazinyl moieties.It was hoped that MBH methodology, which we have successfully developed to access various benzannulated heterocyclic systems, 16 could be extended to generate the critical 4hydroxycoumarin platform.Unfortunately, our efforts to access 4-hydroxycoumarins using MBH methodology were unsuccessful, and attention was turned to the procedure reported by Zhao et al. 17 Thus, in the first arm of the convergent synthesis, summarised in Scheme Following the approach used by Arshad et al., 7 the second arm of the convergent synthesis (Scheme 1) of the 3-[2-(benzylidenehydrazinyl)thiazol-4-yl]-4-hydroxycoumarins 14a-g involved preparation of the series of critical thiosemicarbazone derivatives 13a-g by refluxing ethanolic solutions of the substituted benzaldehydes 12a-g and thiosemicarbazide to which a trace of 2N-HCl had been added.Most of the substituted benzaldehydes (12) were commercially available, but compounds 12f and 12g were specially prepared by benzylating the phenolic groups of the benzaldehyde precursors 11f and 11g.The thiosemicarbazones 13a-g were isolated in satisfactory yields (65 -76%).

Biological activity and computer docking studies
The 3-[2-(benzylidenehydrazinyl)thiazol-4-yl]-4-hydroxycoumarins 14a-g were subjected to anti-malarial, trypanocidal, cytotoxicity, anti-mycobacterial and anti-bacterial studies.The bioassay protocols are detailed and the results are tabulated in the Supporting Material file.The compounds generally exhibited weak antimalarial and trypanocidal activity with the most active exhibiting, at best, only modest activity ( 14a: 65.3% PLDH viability: and 14b: 52.0%T.b.brucei viability).Cytotoxicity, as determined at 20 μM against HeLa cells, ranged from 74.9 to 100% cell viability.Two of the compounds showed interesting anti-mycobacterial potential with respective visual and calculated MIC90 values of 15.63 and 31.25 μM for 14a and and 31.25 and 15.63 μM for 14c.In preliminary anti-bacterial assays conducted at Rhodes (CCBR), compounds 14a,c,d,e,g proved inactive against E.coli cells but consistently active against S.aureus, with compound 14d restricting metabolic activity to as little as 31% after 6h.Interestingly, parallel cell growth assays conducted at the University of Cape Town (H3-D) revealed no activity for these ligands against S.aureus after 16 h -raising the possibilities of rapid ligand metabolism, followed by pathogen recovery, or an initial delay in the implementation of the pathogen resistance mechanisms.
In silico docking studies of the synthetic ligands 14a-14g in relevant enzyme structures were conducted using Schrӧdinger software. 19Protein structures were obtained from the Protein Data Bank (5FRN 20 for HIV-1 integrase; 1YT9 21 and 1ZP8 22 for HIV-1 protease; 1T2C 23 and 1V0O 24 for P.falciparum; 3FWN 25 for T.b.brucei; and 4BFW 26 for M.tuberculosis).The binding affinities of ligands 14a-g docked into the T.b.brucei enzyme structure 4FWN 25 ranged from -3.240 kcal/mol (14d) to -5.337 kcal/mol (14b).Compounds 14a and 14b, which exhibited significant activity against T.b. brucei in vitro, are among the three best ligands in silico.Interestingly, compound 14b exhibited the best activity in both the in vitro T.b.brucei enzyme bioassay and the in silico 4FWN 25 docking analysis -results indicative of its potential as an anti-trypanosomal agent.In general, the binding affinities exhibited by the ligands (14) in their docking in the M.tb 4BFW 26 enzyme structure suggest their potential as lead compounds in the development of novel anti-mycobacterial agents.Only one of the ligands (14d) had a binding affinity weaker than -5.7 kcal/mol.Compound 14a, on the other hand, exhibited the strongest binding affinity (-7.479 kcal/mol) -consistent with having the highest in vitro Mtb activity.

Conclusions
A small library of novel 3-[2-(benzylidenehydrazinyl)thiazol-4-yl]-4-hydroxycoumarins, which contain medicinally significant 4-hydroxycoumarin, thiazole and hydrazinyl motifs, has been successfully prepared.The compounds have been fully characterised, and in silico docking studies and/or in vitro biological evaluation have suggested the trypanocidal, anti-mycobacterial and antibacterial (against S.aureus) activity of some of these compounds.

Experimental Section
General.Chemicals were purchased from Sigma-Aldrich and used without further purification.NMR spectra were recorded on a Bruker Biospin 600 MHz NMR spectrometer and chemical shifts were calibrated relative to the residual proton signal in DMSO-d6 (2.5 ppm).IR Spectra were recorded on a Perkin Elmer Spectrum 100 FTIR spectrometer with a diamond window.Melting points were determined using a Stuart SMP30 apparatus apparatus and are uncorrected.HRMS analyses were conducted at Rhodes University and by the Central Analytical Facilities Unit at the University of Stellenbosch.
The Supporting Information File contains: experimental methods and characterisation data for synthesised compounds; NMR spectra for new compounds; in vitro bioassay and in silico docking protocols; and in silico docking data.8][29] Benzylation of the phenolic substrates 11f,g to access compounds 12f,g was effected following the method reported by Olomola et al. 30 N 1 -[2-(benzyloxy)-5-bromobenzylidene]-N 2 -(thiocarbamoyl)hydrazine (13g).A mixture of 2-(benzyloxy)-5bromobenzaldehyde 0.58 g, 2 mmol), thiosemicarbazide (0.19, 2.1 mmol) and one or two drops of 2N-HCl was boiled under reflux in ethanol for at least 1.5 h.after which the resulting precipitate was filtered off and washed with several small volumes of methanol, filtered and air dried to afford N 1

Figure 1 .
Figure 1.Structures of biologically active naturally occurring and synthetic coumarin derivatives.
POCl3 and acetic acid; increasing the reported duration of the reaction from 30 min.to 1 hour gave compound 9 in excellent yield (90%).Treatment of 2-acetyl-4-hydroxycoumarin 9 with bromine in acetic acid at 100 o C afforded the  -brominated derivative 10.