Dck Class Extension Study Of The Anti-hiv Compounds

Suksdorfin (1), a natural product isolated from the fruit of Lomatium Suksdorfii, has an angular pyranocoumarin skeleton with interesting biological properties, especially its anti-HIV activity (EC₅₀ = 2.6±2.1μM, TI = 30.6±22.4, TI = therapeutic index IC₅₀/EC₅₀). Structural modification of 1 yielded DCK (2) and 4-methyl-DCK (11), which demonstrated extremely potent inhibitory activity against HIV-1 replication in H9 lymphocytic cells (EC₅₀= 4×10^-4μM, TI = 136719 and EC₅₀ = 1.57×10^-7μM, TI > 10⁹, respectively).The recent mechanistic studies of pharmacological action indicated that HIV-1 RT is possibly the target of DCK and DCK is a special HIV-1 RT inhibitor that inhibits the DNA-dependent DNA polymerase activity.¹⁷ In contrast, DCK did not significantly affect the RNA-dependent DNA polymerase activity. Due to its unique mode of action, DCK and its derivatives could be used to functionally dissect HIV-1 RT and might have the potential to be clinically useful. Accordingly, selected modifications on DCK skeleton are highly desirable in order to identify the pharmacophores in this class of potent anti-HIV agents and explore the structural aspect of the target bio-molecule(s). To this end, a variety of novel DCK analogs containing N,N-, O,N-, N,O-, O,S- or S,O- by displacing the O,O atoms in the DCK skeleton were synthesized previously in our group. The structural modifications of 4-methyl-DCK by replacing the ring oxygen atom of DCK with a sulfur or a nitrogen atom (21a, 22b and 17a) demonstrated that these analogs also exhibited potent inhibitory effects on HIV-1 replication in H9 lymphocytes. In the previous SAR study of our group on DCK derivatives, the size of alkyl substituents on 8-position was sensitive to the antiviral activity in this series of compounds. Gem-dimethyl substitution was preferable; on the other hand, either a larger alkyl substituent or smaller hydrogen atoms dramatically decreased the activity. The rank order of activity was two hydrogen atoms (unsubstituted)≈methyl+propyl < gem-diethyl < gem-dimethyl.As a continuous effort towards the development of this type of potential anti-HIV drugs, a new series of DCK analogs, 4-methyl-7-thio-DCK 26, 8-Monomethyl-4-methyl-DCK (27-28), 8-Monomethyl-7-thio-4-methyl-DCK (29-30), 8-Monomethyl-7-sulfoxide-4-methyl-DCK (31-32) and 8-Monomethyl-7-sulfone-4-methyl-DCK (33-34), were designed. The studies on the SAR of these compounds might provide the useful information for understanding the impact of 7-hetero atom, the spatial orientation of 8,8-dimethyl groups on the anti-HIV activity. Structural modification of A ring of DCK by modifying the coumarin skeleton of DCK analogues to isoflavone skeleton, Professor Lee synthesized a series of new DCP analogues which exhibited extremely high anti-HIV activity in the non-drug-resistant strain assay. More promising was that 39b also showed excellent activity against multi-RT inhibitor-resistant HIV-1 strains. This result demonstrated that the activity against drug-resistant strains could be improved by modifying the chromene skeleton of DCK analogues.Combined the research results of DCP analogues with the SAR knowledge of DCK accumulated through the former structural modification, a series of novel DCK analogues 35, 36, 37, 38 were designed in order to explore the impact of changing C ring skeleton and A ring skeleton on the anti-HIV activity, especially on the activity against multi-RT inhibitor-resistant HIV-1 strains.Moreover, all of the target molecules are new heterocyclic compounds and their preparation remains a challenge from the viewpoint of synthetic chemistry. This study has potential scientific significance by accumulating new knowledge and providing a synthetic method of such novel heterocyclic skeletons.The syntheses of all designed compounds were accomplished in seven synthetic routes, respectively, starting from 7-hydroxy-4-methylcoumarin, naphthalene-1,7-diol, 3,5-dihydroxy-2-naphthoic acid and resorcinol. The structures of all these target analogs were confirmed by various spectral analyses, including ¹H NMR, ¹³C NMR and mass spectra. The anti-HIV activities of these target compounds were tested in H9 lymphocytes or TZM-bl cells with AZT as a reference compound. The preliminary bioassay data indicated that compounds 26, 27a and 29a demonstrated significantly potent anti-HIV activity in H9 lymphocytes, with ^aIC₅₀ = 38.3μM, ^aEC₅₀ < 0.0383μM, ^aTI > 1000; ^aIC₅₀ = 28.4μM, ^aEC₅₀ < 0.0402μM, ^aTI > 705.6; ^aIC₅₀ = 39.1μM, ^aEC₅₀ < 0.0391μM, ^aTI > 1000, respectively. Compounds 26, 35a, 100 and 118c also showed activity against a multi-RT inhibitor-resistant strain, HIV-1 RTMDR1, with ^cIC₅₀ = 38.3μM, ^cEC₅₀ < 0.0383μM, ^cTI > 1000; ^cIC₅₀ > 33.19μM, ^cEC₅₀ = 2.32μM, ^cTI > 14.33; ^cIC₅₀ > 61.29μM, ^cEC₅₀ = 0.899μM, ^cTI > 68.14; ^cIC₅₀ > 50.58μM, ^cEC₅₀ = 11.27μM, ^cTI > 4.49, respectively. The further study on the end points of anti-HIV activities of these compounds is undergoing. At this stage, the following new concepts can be gained on the basis of the information attained from these preliminary bioassay results:(1) The preliminary bioassay results suggested that interchange of 7-oxygen atom with 7-sulfur atom in DCK series derivatives apparently enhances the activity against both a non-drug-resistant strain in H9 lymphocytes and a multiple reverse transcriptase (RT) inhibitor-resistant strain in the TZM-bl cells.(2) The biological result elucidated 8S configuration methyl substitution was much more favorable than 8R configuration methyl substitution for anti-HIV activity. This study firstly revealed the configuration of 8-substituents exerted a significant impact on the anti-HIV activity, which would have a profound influence on directing the future structural modification. These results implied that in the receptor at the site fitting 8-positions of DCK analogs, there is probably only a small space at a orientation.(3) Converting 7-thia-4-methyl DCK analogs to the corresponding 7-sulfoxide- and 7-sulfone-4-methyl DCK analogs resulted in decreased or completely abolished activity. The increasing spatial bulkiness or change of electrical effect on the 7-position may lead to the decreased activity.(4) The low potency and inactivity of analogs 83, 27b and 29b with 9α, 10α-dicamphanoyl groups were in accord with the preferred configuration (β) and spatial demand for the two camphanoyls found previously with the DCK series.(5) Compound 35a with C benzene ring also showed good anti-HIV activity both in non-drug-resistant strain and a multiple reverse transcriptase (RT) inhibitor-resistant strain, HIV-1 RTMDR1. However, compounds 35b and 35c with larger steric bulkiness of 9,10-substituents completely lost activity. The data showed that too large 9,10-substituents are not favorable against the resistant strain.(6) Compound 100 with 9,10-diacetoxy showed potent activities against non-drug-resistant HIV strain and HIV-1 RTMDR1, which were much better than those of compound 35a with 9,10-dicamphanoyl. These results implied that the interchange of chromene skeleton of DCK analogues (Ring B and C) with naphthalene skeleton will change the spatial orientation of two substituents at the corresponding 9 and 10 positions. Therefore, the previous SAR is not suitable for this novel class of compounds. Discovery of compounds without 9,10-dicamphanoyl which showed potent anti-HIV activity is a new breakthrough. Because the pharmacophores of two bulky camphanoyls decrease the water solubility and stability in vivo, which presented the main obstacle to the development of DCK-related drug candidates.(7) Compound 118c with 7-morpholinyl also showed significant anti-HIV activity, but which is much less that compound 100. The data showed that substitution on the 7-position is not favorable for anti-HIV activity.(8) All of 10-monosubstituent analogs with C benzene ring displayed no anti-HIV activity, which demonstrated the importance of the substituents at 9,10-positions for the activity.(9) Compounds 76a and 132 with very simple structures showed certain anti-HIV activity with ^aIC₅₀ = 90.6μM, ^aEC₅₀ = 42.7μM,^aTI = 2.12; ^aIC₅₀ = 58.1μM, ^aEC₅₀ = 52.8μM, ^aTI = 1.10, respectively. It may represent a novel structural class of inhibitors.The SAR results mentioned above will undoubtedly be helpful in further optimization of anti-HIV activities of this class of compounds, and may provide clues for exploring the structure of receptor biomolecule.During the synthesis of the target molecules, we also disclosed a series ofinteresting chemical reaction features, which are summarized as follows:(1) An unexpected rearrangement reaction was discovered in the reaction of7-mercapto-4-methyl-2H-chromen-2-one 43 with 2-methylbut-3-yn-2-ol in H₃PO₄.The structure of the rearrangement product was elucidated by the combination of¹N NMR, ¹³C NMR, DEPT, EIMS and HMBC spectrum. Similar rearrangement was also found in other substrate and a possible mechanism of this rearrangement was proposed.(2) The reaction of 7-mercapto-4-methyl-2H-chromen-2-one 43 with 2-methylbut-3-yn-2-ol and PPTS in toluene was found to give an unexpected product 4-methyl-9-(prop-1-en-2-yl)-2H-mieno[2,3-h]chromen-2-one 80. The structure of abnormal product was elucidated by X-ray diffraction analysis. The possible mechanism for the formation of 80 was discussed.(3) The reactions of 7-mercapto-4-methyl-2H-chromen-2-one 43 with different propargyl halides had different reaction behaviors. A reasonable explanation was discussed based on experiment.(4) Attempts to follow the literature procedure⁶⁹ for the preparation of 6-hydroxybenzofuran-3(2H)-one 66 only led to a very low yield of the desired product 66. Instead, 2-chloro-1-(2,4-dihydroxyphenyl)ethanone 67 and 2-(2-chloroacetyl)-5-hydroxyphenyl 2-chloroacetate 133 were obtained as the major products. The desired compound 66 was obtained via a one pot synthesis by selective hydrolyzation of 133 followed by cyclization.(5) During the Claisen Rearrangement of 6-(2-methylbut-3-yn-2-yloxy)benzofuran-3(2H)-one 65 in N,N-diethylaniline, an abnormal product (Z)-2-ethylidene-7,7-dimethyl-2H-furo[2,3-f]chromen-3(7H)-one 138 was yielded. In addition to normal rearrangement of propargyl ether, an ethylene group turned out to be added to the 2 position of benzofuranone. Similar reactions were also observed other substrates with benzofuran-3(2H)-one skeleton. The pathway for the formation of products via free redical was proposed based on experiment.(6) The dihydroxylation reactions of 64 and 144 showed the quite different reaction behaviors with other coumarin substrates in our previous study. The products were separated and purified (The structure are not determined).The study mentioned above provided valuable information on the synthesis and chemical properties of these new types of heterocyclic compounds.