2-Arylnaphthoquinone analogues: potential anti-TB and pro-apoptotic agents

A useful library of substituted 2-arylnaphthoquinones prepared by reaction between the corresponding bromonaphthoquinones and arylboronic acids via Suzuki-Miyaura protocols has been established. Conversion of some of the products into new analogues was effected.


Introduction
The bisnaphthoquinone disospyrin 1 comprises two 7-methyljuglone units linked between C2 and C6. 1,2Antimycobacterial activity studies performed on diospyrin 1 alerted the scientific community to the potential importance of this natural product 3 which was soon followed by its first published synthesis by Yoshida and Mori in the same year. 4][7] In order to gain a better understanding of the structural features and functional parameters that are necessary for such systems to demonstrate activity, we synthesized a number of biquinone molecules in which the two quinone moieties were directly linked viz., 2 and evaluated their pro-apoptotic activities against three human cancer cells lines.In addition the demethylated analogues viz., 3 were also evaluated and demonstrated that the molecules were specific for the available cancer cell lines evaluated. 8,9(Figure 1).All the former studies involved use of the 7-methyljuglone moiety as one component in the quinone-quinone linked systems.It was thus considered necessary to investigate a) whether two quinone-quinone linked moieties are necessary for biological activity, b) whether the 7-methyl group was necessary to enhance any biological activity, c) what, if any, relative reactivities between the peri OMe vs the peri OH groups are detectable and d) what the nature of the substituent attached at C2 of the final juglone moiety should be viz., either phenyl or naphthyl.The latter investigation was prompted by numerous examples reported in the literature. 10his paper describes the syntheses of a number of families of substituted 2-phenyl-and 2naphthyl-1,4-naphthoquinones related to the diospyrin type molecules together with a very brief mention of the biological activities of a few of the more active molecules selected to emphasize the importance of this work.

Results and Discussion
In the first series of substituted 2-arylnapthoquinoidal compounds synthesized, 2-bromo-7methylnaphthoquinone 4 8,12 was selected since this moiety represented the 5-deoxy analogue of the 'left hand' juglone moiety in diospyrin 1.Thus treatment of bromoquinone 4 with boronic acid 5 under Suzuki-Miyaura conditions 13a-c afforded the expected naphthoquinone product 6 (90%) while a similar synthetic protocol between bromoquinone 4 and boronic acid 7 afforded the naphthoquinone product 8 (86%).Since one of our major future goals involved the biological evaluation of analogues containing a disulfoxide nucleus to mimic a 9,10-anthraquinoidal moiety, bromoquinone 4 was coupled to the thianthrenyl boronic acid 9 to afford the desired thianthrenyl naphthoquinone 10 (82%).This was in turn was oxidized using metachloroperbenzoic acid (m-CPBA) in dichloromethane under stirring to the corresponding disulfoxide 11 (58%) according to the protocol of Nakayama et al. 14 The IR spectrum of the product 11 demonstrated clear S=O stretching vibrations at 1328 and 1164 cm -1 .Apart from some reasonable variations between the quite complex NMR spectra of the thianthrenyl naphthoquinone 10 and the corresponding naphthoquinone sulfoxide 11, the molecular formula of 11 viz., C23H14O4S2 requires M + 418.0334 amu (found M + 418.0330) proved to be the major factor establishing the correct structure (Scheme 1).Scheme 1. Products from 2-bromo-7-methylnaphthoquinone and naphthyl and thianthrenyl boronic acids.
For the second series of 2-arylnaphthoquinoidal compounds related to the basic skeleton of diospyrin 1, 2-bromo-5-methoxynaphthoquinone 12 was prepared 15,16 which represented the 7demethyl analogue of the 'left hand' ring.Treatment of quinone 12 with a range of naphthyl boronic acids under Suzuki-Miyaura conditons 13 afforded the expected coupled products in yields ranging from 79-94% (Scheme 2).Chemoselective peri demethylation was effected in the transformation of quinone 18 to 19 (60%) by treatment with AlCl3 in dichloromethane. 8xidation of the thianthrenyl naphthoquinone 20 to the corresponding disulfoxide 21 was achieved in 68% yield using the protocol described earlier.This was done to investigate the relative activities between disulfoxides 11 and 21 among others.
Literature indicates that introduction of a fluorine atom into a biologically active molecule will lead to an expectation of an increase in its biological activity profile. 17,18Treatment of Scheme 3. Products from 2-bromo-5-methoxynaphthoquinone and fluorinated and aminated phenylboronic acids and naphthyl boronic acids.Demethylation of the 5-methoxynaphthoquinone 26 to the corresponding 5-hydroxy analogue 27 was effected using AlCl3 as described earlier and would serve as a comparative model for peri hydrogen bonding influences.An example of a powerful electron-donating substituent in the 2-phenyl analogues was obtained in the form of the dimethylaminophenyl naphthoquinone 28 (78%) derived from the coupling between quinone 12 and boronic acid 24.Scheme 4. Products from 2-bromo-8-methoxynaphthoquinoneand naphthyl and thianthrenyl boronic acids.
The two acetoxy analogues 30 and 31 were prepared from the corresponding acetoxybromonaphthoquinone 29 15 and boronic acids 7 and 5 respectively (Scheme 3) since we wished to evaluated what comparative electronic effects might operate in these molecules compared to their MeO isomers 16 and 13 respectively and how these might be manifested in their biological activities.
Studies by Lall et al. 5 and Mahapatra et al. 19 indicated that the peri alkoxy derivatives of diospyrin 1 and 7-methyl-5-hydroxynaphthoquinone exhibited a somewhat reduced activity against the susceptible strain of M. tuberculosis, viz., H37Rv.On the other hand, Chakrabarty et al. 20 reported that the peri alkoxy derivatives were more active compared to their peri hydroxyl analogues when evaluated against four human cancer cell lines.
It is for these reasons that a number of the peri alkoxy compounds were demethylated to serve as comparable test molecules for evaluations.
The isomeric peri methoxynaphthoquinone of 12, viz., 32 21 was then employed to synthesize the next series of naphthylnaphthoquinones and is shown in Scheme 4. Of note in this series, is the finding that reaction between naphthoquinone 32 and boronic acid 15 produced two products viz., the Suzuki product 34 in addition to a 2,3-disubstituted product 35.
In order to evaluate any variations in activity between the isomeric quinones shown in Schemes 2 and 4 due to the electronic effects of the MeO group of the quinone, the isomeric 2bromo-7-methoxynaphthoquinone 41 was prepared by reaction between the commercially available diene 39 and 2,5-dibromobenzoquinone 40. 22Passage of the crude adduct through a silica gel column produced a mixture of the expected product 42 (18%) together with the desired naphthoquinone 41 (50%).Subsequently, the crude adduct obtained from the reaction described above, was methylated by treatment with methyl iodide and Ag(II) oxide in benzene at 24 o C in which case the desired quinone 41 was isolated in 80% yield (Scheme 5).Treatment of naphthoquinone 41 with boronic acid 15 also produced two products viz., the expected naphthoquinone 43 in addition to the 2,3-disubstitued product 44 in yields of 61 and 28% respectively similar to that found for reaction between quinone 32 and boronic acid 15 (Scheme 4).The coupled thianthrenyl naphthoquinone adduct 45, derived between reaction of naphthoquinone 41 and boronic acid 9, could not be oxidized to a stable product in our hands.
Bromoquinone 46 23 was selected for the final synthesis of a new series of 2arylnaphthoquinones since it represented the left hand ring of diospyrin 1.A brief series of 2naphthylnaphthoquinones having the best evaluated activity are illustrated in Scheme 6. Demethylations and oxidation of the thianthrenyl naphthoquinone 54 into the corresponding disulfoxide 55 with m-CPBA are also shown.Scheme 5. Synthesis of 2-bromo-7-methoxynaphthoquinone and its products with arylboronic acids.
A series of biologically active 2-aryl and 2-naphthylnaphthoquinones has been prepared via Suzuki Miyaura methodology and subjected to evaluations for their anti-TB and behaviour.A comprehensive article on the biological activities will be published elsewhere.

Experimental Section
General.All melting points were obtained on a Fischer Johns melting point stage and are uncorrected. 1H NMR/ 13 C NMR spectra were obtained on a 200 MHz / 50 MHz Varian Gemini 2000 spectrometer in CDCl3 with  7.26 for 1 H NMR and  77.11 for 13 C NMR spectra as internal reference.Mass spectra were performed on a Waters GCT Premier 70 eV High Resolution Mass Spectrometer and elemental analyses were performed on a Carlo Erba Strumentazione 1106 apparatus.IR spectra were recorded as nujol mulls for solids and thin films for oils on a Perkin Elmer FT-IR spectrometer Pragon 2000.Column chromatography was carried out on dry-packed columns using Merck silica gel 60 (0.063-0.2 mm) as stationary phase.The term "residue obtained upon work-up" refers to the residue obtained when the organic layer was separated, dried over MgSO4, filtered and the filtrate evaporated under rotatory evaporation.All the boronic acids were purchased from Sigma Aldrich and used without further purification.Dichloromethane (DCM) was dried by distillation from calcium chloride and stored over sodium wire while benzene was dried by distillation and stored over sodium wire.