Previously, we have confirmed that the antiviral activities of the chromone derivatives were controlled by the type as well as the position of the substituents attached to the chromone core structure. In the course of our ongoing efforts to optimize the antiviral activity of the chromone derivatives, we have been attempting to derivatize the chromone scaffold introduction of various substituents. In this proof-of-concept study, we introduced a 3-amino-4-piperazinylphenyl functionality to the chromone scaffold and evaluated the antiviral activities of the resulting chromone derivatives. The synthesized 2-(3-amino-4-piperazinylphenyl)-chromones showed severe acute respiratory syndrome-corona virus (SARS-CoV)-specific antiviral activity. In particular, the 2-pyridinylpiperazinylphenyl substituents provided the resulting chromone derivatives with selective antiviral activity. Taken together, this result indicates the possible pharmacophoric role of the 2-pyridinylpiperazine functionality attached to the chromone scaffold, which warrants further in-depth structure–activity relationship study.
The appropriate aldehyde (1.1 equiv)and DIPA (1.1 equiv) were added to a 0.4 M solution of the appropriate2′-hydroxyacetophenone in EtOH. The mixture was heated by microwaveirradiation at 160–170 °C for 1 h (fixed hold time, normalabsorption), diluted with CH2Cl2, and washedwith NaOH (aq, 10%), HCl (aq, 1 M), water, and finally brine. Theorganic phase was dried over MgSO4, filtered, and concentratedunder reduced pressure. Purification by flash column chromatographygave chroman-4-ones 1b–n.
In vitro antifungal activity of various synthesized compounds (3a–j) was carried out against three fungal strains, that is, Saccharomyces cerevisiae (MTCC 172), Candida albicans (MTCC 3018), and Cryptococcus gastricus (MTCC 1715). Fluconazole was used as a positive control. Inhibitory potential of the synthesized compounds was determined in terms of minimum inhibitory concentration (MIC) in μg/mL using turbidity method ().
Recently, we have reported the synthesis of 3-(5-phenyl-3H-[1,2,4]-dithiazol-3-yl) chromen-4-ones with significant inhibitory potential against microbial strains; particularly, compound having both electron withdrawing groups such as chloro and fluoro linked with chromone ring showed more inhibitory potential against fungal strains than standard drug . Therefore, it was decided in the present study to incorporate chloro group at the phenyl ring of the 1,2,4-dithiazolylchromones in order to observe the effect of novel substitution on antimicrobial activity.
In the case of fungal strain S. cerevisiae, compound 3h displayed very high inhibitory activity with MIC 0.78, which is more than that of fluconazole used as positive control under similar conditions, followed by compound 3g with MIC 3.12 and compound 3a with MIC 6.25. Compound 3c exhibited promising inhibitory activity against C. albicans with MIC 3.12, which is higher than standard positive control, followed by compounds 3a, 3e, and 3h with MIC 6.25. Compounds 3h showed comparable inhibitory activity against C. gastricus (MIC 6.25) with standard drug fluconazole, followed by 3c with MIC 12.5, respectively. The structure activity relationship of all the synthesized compounds was developed on the basis of obtained in vitro antimicrobial results. The substitution of -chloro group at para position of phenyl ring of the 1,2,4-dithiazole enhanced the inhibitory activity against various bacterial and fungal strains comparison to our previously synthesized compounds . It was observed that substitution of electron withdrawing groups at C6 and C7 positions of chromone ring leads to an increase in both antifungal and antibacterial activities. Therefore, efforts are made to perform substitution with different electron withdrawing groups at C6, C7, and C8 positions of the chromone ring. C6 position of chromone moiety also has been substituted with electron donating substitution that is, -methyl, to compare the antimicrobial activity with electron withdrawing substitutions and results revealing that electron donating substitution decreases both antifungal and antibacterial activities. Substitution at C7 with electron withdrawing groups fluoro and bromo leads to an increase in both antifungal and antibacterial activities, especially, against B. subtilis, E. coli, and S. cerevisiae.
For the synthesis of the 2-pentylchromone 3a, chroman-4-one 1a was converted to the corresponding3-bromo-2-pentylchroman-4-one 2 using Py·Br3 as the brominating agent. Interestingly, 2 was isolated asa diastereomeric mixture of 80:20 according to 1H NMR spectroscopywith the cis-isomer as the major product. Computational studies confirmedthe higher stability of the cis-product. Hydrobromide elimination of 2 by CaCO3 inDMF in a microwave-assisted reaction provided the desired chromone 3a in 84% yield. Flavone 3b was prepared from3′-bromo-5′-chloro-2′-hydroxyacetophenone viaesterification with benzoyl chloride followed by a Baker–Venkataramanrearrangement yielding a diketo intermediate that was cyclized inan acid-catalyzed reaction.30 To obtain compounds 5 and 6, the carbonyl group in 1a was reduced by NaBH4 in MeOH, providing 8-bromo-6-chloro-2-pentylchroman-4-ol 4 in almost quantitative yield and in 96:4 diastereomericratio. With the chroman-4-ol 4 in hand, the hydroxylgroup could be removed either by dehydroxylation or dehydration. Dehydroxylationwas carried out using triethylsilane as hydrogen source in the presenceof BF3·Et2O yielding 8-bromo-6-chloro-2-pentylchroman 5 in moderate yield (44%). Dehydration was performed usinga catalytic amount of p-toluenesulfonic acid withMgSO4 present as drying agent. The corresponding 2H-chromene 6 could be isolated in a yield of63%.
The synthetic pathways toward the test compounds 1a–p, 3a,b,and 4–6 are presented in Scheme . The chroman-4-ones 1a–p were synthesized according to a procedure previously reportedby our group. Commercially available2′-hydroxyacetophenones were reacted with appropriate aldehydesin a base-promoted crossed aldol condensation followed by an intramolecularoxa-Michael addition. The reactions were conducted by heating ethanolicmixtures to 160–170 °C using microwave (MW) irradiationfor 1 h in the presence of DIPA as base. The desired 2-alkyl-chroman-4-ones(1a–p) were isolated in low to highyields (17–88%). The outcome of the reaction was strongly dependedon the substitution pattern of the acetophenones. In general, electron-deficient2′-hydroxyacetophenones gave high yields of the desired chroman-4-oneswhereas electron-donating groups led to higher amounts of byproductoriginating from self-condensation of the used aldehyde. This causedpurification problems, lowering the yields; for example, the 6,8-dimethyl-and 6-methoxy-substituted 2-pentylchroman-4-one derivatives 1d and 1h were obtained in only 17% yield. Theenantiomers of 1a were separated from the racemic mixtureby preparative HPLC on a chiral stationary phase.
The set of synthesized substituted chroman-4-one and chromone derivativeswas used to explore the SAR (Table ). Theinhibitory activities of the synthesized compounds 1a–p, 3a,b, and 4–6 were determined using in vitro SIRT1,SIRT2, and SIRT3 assays. It is noteworthythat the most potent inhibitors (over 70% inhibition at 200 μM)are completely SIRT2 selective. They show less than 10% inhibitionof SIRT1 and SIRT3 at 200 μM, the only exception being 16% inhibitionof SIRT3 by compound 1m. The results from the SIRT2 assayare summarized in Table (see for results from the SIRT1 and SIRT3assays).
To confirm that the detected SIRT2 inhibitionby 1a was not caused by interaction with an artificialfluorophore, wefurther verified SIRT2 inhibition with two different methods. First,a Western blot analysis of the SIRT2-mediated deacetylation of acetylatedα-tubulin was carried out and inhibition of the SIRT2-catalyzedreaction by 1a was observed (Figure A). Second, a SIRT2 activity assay based on the release ofradioactive 14C-nicotinamide was performed in the presenceof an acetylated peptidic substrate (RSTGGK(Ac)APRKQ) without a fluorophore(Figure B). In this assay 1a gave66% inhibition. Taken together, 1a was able to inhibitthe deacetylation of three different substrates: an artificial substratewith a fluorophore and a peptide and a protein substrate without afluorophore. On the basis of these results, a series of analoguesof 1a was synthesized and evaluated as SIRT2 inhibitors.
Chromones and chroman-4-ones constitute a naturallyoccurring classof substances which are classified asprivileged structures, as compounds basedon these scaffolds display a wide range of biological activities definedby the substitution pattern of the scaffold. We have put considerable effort into the development of syntheticstrategies for this class of compounds resulting, for example, inan efficient synthetic route to 2-alkyl-substituted chroman-4-ones. The incorporation of various functional groupsto furnish highly substituted structures have successfully been conductedthrough different Pd-mediated cross-coupling reactions,, through Mannich reactions, and viaa SmI2–KHMDS-mediated Reformatsky type reaction. Recently, we have also developed chromone/chroman-4-one-basedβ-turn peptidomimetics., In the present studywe report substituted chromone and chroman-4-one derivatives as potentand highly selective SIRT2 inhibitors.