Synthesis and exploratory biological evaluation of 3-[( N -4- benzyloxyphenyl)iminoethyl]- and 3-(1-hydrazonoethyl)-4-hydroxycoumarins

Three series of 4-hydroxycoumarin derivatives, comprising a total of 20 novel compounds have been prepared from 2-hydroxyacetophenones. These include a set of 3-[( N -4-benzyloxyphenyl)iminoethyl]-4-hydroxycoum- arins


Introduction
Naturally occurring and synthetic chromone (2H-1-benzopyran-2-one) derivatives comprise an important and extensive class of heterocyclic compounds. Reviewed by Sethna and Shah 1 more than 75 years ago, these compounds continue to enjoy the attention of synthetic and medicinal chemists and remain the subject of ongoing reviews. 2,3 Coumarin derivatives are known to exhibit a broad range of medicinal properties, including anti-coagulant, anti-HIV, anti-cancer and anti-inflammatory activities, and inhibitory activity against important enzymes, such as monoamine oxidase and cyclooxygenase. 4 Synthetic N-aryl coumarin-3carboxamides have been evaluated 5,6 for activity against human lung fibroblasts and breast cancer cell lines, and the most potent compound (with an IC50 of 0.166 μM against ErbB-2 kinase) was the 6-chlorocoumarin derivative 1 (Figure 1). Küçükbay et al. 7 have reported carbonic anhydrase inhibition by amino acid-coumarin conjugates at sub-micromolar concentrations. A common feature in many biologically active coumarin derivatives is the presence of a 4-hydroxy group, as in the anti-coagulant phenprocoumon 2; 8 in fact, this appears to be one of the minimum structural requirements for anti-coagulant activity in analogous systems. 9 A series of hybrid coumarin derivatives, including the hydrazine derivative 3, were shown to exhibit promising anti-HIV activity. 10 Compounds containing an imino group have exhibited a range of biological activities, including anticancer, 11 anti-viral 12 and anti-β-secretase 13 properties, while there appears to be growing interest in the medicinal potential of hydrazone derivatives. 14,15 The alkyne moiety is present in a number of pharmaceuticals, including Efavirenz, 14 an antiretroviral, and the antifungal agent Terbanafine. 16  18 We have a close and ongoing interest in applications of Morita-Baylis-Hillman methodology in the synthesis of benzannulated heterocyclic systems in general 17 and coumarin derivatives 18 in particular. Attention has been given to the synthesis of novel furocoumarins 4 -designed to serve as HIV-1 Integrase inhibitors 19 -and coumarin-AZT conjugates as portmanteau HIV-1 protease and reverse transcriptase inhibitors. 20 Access to 4-hydroxycoumarins, however, has required the application of a different methodology and, in a recent study, 21 we made use of a procedure developed by Zhao et al. 22 to access these systems. We now report the preparation and exploratory biological evaluation of three series of compounds containing 4hydroxycoumarin, iminoethyl , hydrazonoethyl and alkynyl moieties.

Synthesis of 4-hydroxycoumarin derivatives
With minor modifications of the method reported by Zhao et al., 22 the 2-hydroxyacetophenones 5a-f were reacted with diethyl or dimethyl carbonate in the presence of sodium hydride to afford the 4hydroxycoumarins 6a-f in yields of 60-87% (Scheme 1). Adapting a procedure described by Sukdolak et al., 23 the 4-hydroxycoumarins 6a-f were reacted with POCl3 in acetic acid to give the 3-acetylated derivatives 7a-f in yields ranging from 41% to 90%. Increasing the reaction time to at least 1 hour improved the yield, while washing the resulting precipitates with methanol gave the respective products in high purity thus obviating the need for recrystallisation. Each of the 3-acetyl-4-hydroxycoumarins 7a-f was condensed with 4benzyloxyaniline 8 (obtained by prior neutralisation of the hydrochloride salt) in an acetic acid-catalysed reaction in refluxing methanol for at least an hour. The resulting precipitates were filtered off, washed with methanol and dried to give the targeted products 9a-f in high yield (82 -94%) and high purity -as confirmed by NMR analysis.

Scheme 1. Synthesis of 3-[(N-4-benzyloxyphenyl)iminoethyl]-4-hydroxycoumarins 9a-f.
The 3-acetyl-4-hydroxycoumarin 7a was reacted with hydrazine hydrate under reflux for 1 hour using ethanol as the solvent and acetic acid as a catalyst to afford the hydrazone derivative 12 in 80% yield (Scheme 2). The deshielding of the OH signal (at 15.53 ppm) in the 1 H NMR spectrum of this compound is consistent with involvement of the hydrazone N 1 atom and the phenolic proton in a six-membered hydrogen-bonded chelate. The hydrazone 12 then provided access to two series of dihydrazone derivatives 13a-g and 15a-g. For the first series, the hydrazone 12 was reacted with each of seven disubstituted benzaldehydes 11a-g in refluxing ethanol (to which a catalytic quantity of 2N-HCl had been added) to yield the novel 3-[1-(benzylidenehydrazono)ethyl]-4-hydroxycoumarins 13a-g in moderate to good yields (51-76%). Four of the benzaldehyde precursors 11a-d had been obtained in 65-75% yields by prior O-benzylation of their phenolic precursors 10a-d; the other three benzaldehyde precursors (11e-g) were commercially available. For the second series, the hydrazone 12 was similarly reacted with each of the propargyloxy-substituted benzaldehydes 14a-g to afford the corresponding 3-[1-(benzylidenehydrazono)ethyl]-4-hydroxycoumarins 15a-g in good yields (65-80%).  (13) and (15).
All of the novel compounds were fully characterised using HRMS, IR and 1-D and, where appropriate, 2-D NMR analytical data. Use of COSY and HSQC data, as illustrated in Figures 2 and 3, respectively, facilitated the assignment of 1 H-and 13 C-NMR signals to the corresponding aromatic nuclei, with different and distinctive proton-proton coupling patterns, in the two benzenoid rings in compound 13f. Interestingly, Milenko et al. 24 have reported the preparation of analogous compounds with very encouraging antimicrobial properties and have used NOE and HMBC experiments to confirm their structural identity. In vitro bioassay and molecular docking studies of closely related 3-hydroxycoumarin derivatives have been undertaken by Batran et al. 25 to investigate antifungal properties and by Abdel Latif et al. 26 to explore their anti-cancer potential.

Biological evaluation
The novel, synthetic products were screened for cytotoxicity against HeLa cells and, variously, for HIV-1 IN and PR enzyme inhibitory potential and for antimalarial, trypanocidal and anti-mycobacterial activity. The bioassay protocols and results for each series of products are provided in the Supporting Information file. The 3-[(N-4benzyloxyphenyl)iminoethyl]-4-hydroxycoumarins 9a-f, at 20 μM concentration, exhibited little if any significant cytotoxicity (< 50% viability of Hela cells) nor activity against the malaria parasite (pLDH) -patterns generally common to the analogous benzylidenehydrazono series (13) and (15). Compounds 9a-d, however, decreased the viability of the T.b. brucei parasite to 23.3 -37.9% with the most active compound 9d having an IC50 value of 27.88 μM. While some of the 3-[1-(benzylidenehydrazono)ethyl]-4-hydroxycoumarins (13) exhibited a measure of trypanocidal activity, compound 13g was notable, decreasing T.b. brucei viability to 1.53% and exhibiting an IC50 value of 0.90 μM. Compound 13g was also the only compound observed to exhibit, albeit weakly, anti-mycobacterial (anti-TB) activity with MIC90 values of 62.50 μM (visual) and 62.44 μM (calculated).

Conclusions
Convenient synthetic access has been successfully established to three series of novel 4-hydroxycoumarin derivatives from substituted acetophenone and benzaldehyde precursors. The products contain several biologically important motifs, viz., 4-hydroxycoumarin, imino, hydrazono and alkynyl moieties. Exploratory bioactivity studies have revealed that some of the products exhibit anti-trypanosomal activity, with one of them having an IC50 value as low as of 0.90 μM against the T.b. brucei parasite.

Experimental Section
General. Chemicals were purchased from Sigma-Aldrich Chemical Co. and used without further purification. NMR spectra were recorded on Bruker Biospin 600 MHz NMR and AMX 400 MHz spectrometers and chemical shifts were calibrated relative to the residual proton signal in DMSO-d6 (2.5 ppm) and in CDCl3 (7.26 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.