全碳C环DCK类似物的设计、合成及生物活性研究

【作者】 王洋；

【导师】 夏鹏；

【作者基本信息】 复旦大学 ， 药物化学， 2005， 博士

【摘要】 Suksdorfin（1）是美国北卡大学药学院K. H. Lee课题组从植物Lomatium Suksdorfii的果实中分离而得的双氧杂三环稠杂环化合物,具有明显抗HIV活性(EC₅₀=2.6±2.1 μM,TI=30.6±22.4,TI=therapeutic index IC₅₀/EC₅₀)。Lee小组以此化合物为先导,进行了一系列的结构修饰和生物活性观察,先后获得DCK（2）(EC₅₀=4×10^-4μM,TI=136719)和4-甲基-DCK（11）(EC₅₀=1.57×10^-7μM,TI>10⁹)等化合物,其极低的EC₅₀值和极高的TI值引起了极为广泛的关注。 最近的药理作用机理研究表明,HIV-1逆转录酶可能是DCK类化合物的作用靶点,DCK类化合物不明显抑制依赖RNA的DNA合成,而对依赖DNA的DNA聚合酶的活性具有强的抑制活性,这一独特的作用位点使得DCK类化合物极具成为临床用药的潜力。因此,对DCK类化合物进一步的化学修饰和结构改造研究,进而揭示其结构与活性之间的关系,一方面可以为寻求活性更好的先导化合物提供信息和线索,同时还可通过化合物的活性及化合物上各基团的特性,探测与之相结合的作用靶点生物大分子的模型和结构状况,对研究这类化合物的分子药理作用机制具有重要的学术意义。 近年来,本课题组相继完成了用N,N-,O,N-,N,O-,O,S-及S,O-分别代替DCK类化合物中A环或C环原有的氧原子而形成的三环稠杂环衍生物的合成及生物活性测试,其中1-thio-DCK（23a）、7-thio-DCK（22a）和4-甲基-DCK的内酰胺类似物（17a）（N,O-杂）均显示出优异的抗HIV活性。更多还原

【Abstract】 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 ± 2.1 uM, 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= 4×10-4 μM, TI = 136719 and EC50 = 1.57×10-7 μM, TI > 109, respectively）.Suksdorfin （1）4-Methyl-DCK （11）The recent mechanistic studies indicated that HIV-1 RT is possibly the target ofDCK and DCK is a unique HIV-1 RT inhibitor that inhibits the DNA-dependent DNApolymerase activity.47 In contrast, DCK did not significantly affect theRNA-dependent DNA polymerase activity. Due to its unique mode of action, DCKand its derivatives could be used to functionally dissect HIV-1 RT and might have thepotential to be clinically useful. Accordingly, selected modifications on DCK skeletonare highly desirable in order to identify the pharmacophores in this class of potentanti-HIV agents and explore the structural aspect of the target bio-molecule（s）. To thisend, a variety of novel DCK analogs containing N,N-, O,N-, N,O-, O,S- or S,O- bydisplacing the O,O atoms in the DCK skeleton were synthesized previously in ourgroup. The structural modifications of 4-methyl-DCK by replacing the ring oxygenatom of DCK with a sulfur or a nitrogen atom （23a, 22a and 17a） demonstrated thatthese analogs also exhibited potent inhibitory effects on HIV-1 replication in H9lymphocytes.o22a!C50= 155 [JM EC50 = 0.141|jM Tl = 111023aIC50 = 169 uM EC50 = 0.00012 MM Tl = 1408000As a continuous effort towards the development of this type of potential anti-HIV drugs, a new series of DCK analogs, namely 7-carbon- and 7-carbonyl-DCK derivatives （25-30, 73） and five-membered-C-ring DCK analogs （109 and 117）, were designed based on the principle of bioisomerism and synthesized via four different routes in this dissertation. The studies on the S AR of these compounds might provide the useful information for understanding the impact of 7-oxygen atom, 4,8,8-trimethyl groups, as well as the location and the number of the ester groups on the anti-HIV activity. Moreover, all of the target molecules are new heterocyclic compounds and their preparation remains a challenge from the viewpoint of synthetic chemistry.ROR1OR1OR’OR’OR125a-b2627R’O30a-bR’Oa: R = CH3, b: R = H; R’ = CamphanoylThe syntheses of designed all-carbon-C-ring-DCK analogs 25a-b and their corresponding regio-isomers 29a-b and 30a-b were accomplished in seven, four and seven steps, respectively, starting from 1-naphthol. The target compounds 26, 27 and 28 were synthesized via nine-step, nine-step and seven-step transformations, respectively, by using 3,4-dihydro-5-methoxy-2//-l-tetralone as the starting material. The target compounds 109 and 117 were synthesized by using phenol as the starting material. The structures of all these target DCK analogs were confirmed by various spectral analyses, including *H NMR, 13C NMR and mass spectra.The anti-HIV activities of these compounds and some of their synthetic intermediates were tested in H9 lymphocytes with AZT as a reference compound. The preliminary bioassay data indicated that both compounds 26 and 25b demonstrated obvious anti-HIV activity, with IC50 > 25 ]ug/mL（39.39 uM）, EC50 < 0.25 jag/mL（0.39 uM）, TI > 100 and IC50 > 25 ug/mL（42.18 uM）, EC50 < 0.25 \igiml（0A2 uM）, TI > 100, respectively. Compound 25a showed anti-HIV activity with IC50 = 22.92 \xg/mL （37.78 pM）, EC50 < 0.25 ug/mL（0.41 uM） and TI > 88. Compound 27 exhibited anti-HIV activity with IC50 = 20.71 ug/mL（31.93 uM）, EC50 = 1.428 ^g/mL（2.20 uM） and TI = 14.5. The further studies 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 the DCK analogs （25a-b and 26） with replacement of 7-oxygen atom by carbon atom or carbonyl group demonstrated obvious anti-HIV activities, which suggests that 7-oxygen in DCK analogs is not the necessity for anti-HIV effect. Thus, the 7-oxygen atom of DCK should not be the key pharmacophore responsible for the activity and 7-carbon-DCK analogs also merit attention as potential HIV-1 inhibitors.（2） We have demonstrated that the positional effect of ester groups in DCK analogs on their anti-HIV activities. It was found that all of 9,10-diester-DCK analogs showed more potent activity than their corresponding regio-isomers（8,9-diester-DCK or 7,8-diester-DCK analogs）. These results implied that in the receptor at the site fitting 7- and 8-positions of DCK analogs, there is probably only a small space. On the contrary, the binding site corresponding to 9- and 10-posiitons of DCK probably provides a large enough space to allow the incorporation of two big camphanoyl ester groups at 9-and 10-positions of DCK analogs.（3） All of 9,10-diester-DCK analogs （26 and 25a-b） showed more potent activity than their corresponding 10-monoester-DCK analogs （28 and 73a-b）. These results further demonstrated the importance of the steric bulkiness of 9,10-diester groups for the activity.（4） In contrast to the 9,10-diester-DCK analogs （26 and 25a-b）, 9,10-dihydroxy derivatives （42 and 45） displayed no anti-HIV activity, which demonstrated the importance of large camphanoyl ester groups at 9,10-positions of DCK-analogs once again. This observation is consistent with the aforementioned conclusions in （2） and （3）.（5） The etherized product 3-（4-chloro-2,3-dihydro-7-hydroxy-lH-inden-l-yloxy） -7-chloro-2,3-dihydro-lH-inden-4-ol （91a） showed considerable potent anti-HIV activity （IC50 = 15.24 u.g/mL（43.39 uM）, EC50 = 2.126 ug/mL（6.05 uM）, TI = 7.2）. The poor IC50 value might be due to the presence of two Cl atoms in the molecules. The bioassay test of the analogs in which the Cl atoms were substituted by CH3 groups is undergoing.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） The condensation reaction of 5,8-dihydro-l-naphthol （40） with ethyl acetoacetateor malic acid was found to give the expected coumarin derivative together with anC-ring aromatized coumarin derivative as byproduct. The formation of thisbyproduct could be significantly reduced through addition of some anti-oxidationreagent into the reaction mixture and under Ar atmosphere. Moreover, in thecondensation reaction of 5,8-dihydro-l-naphthol （40） with ethyl acetoacetate, aself-condensation product of ethyl acetoacetate, 3-acetyl-2-hydroxy-6-methyl-pyran-4-one （63）, was also isolated and its structure was elucidated by X-raydiffraction analysis.（2） The regioselectivities for the oxidation reactions of 7,8,9,10-tetrahydrobenzo|7z] coumarins （67a-b and 50） were studied and rationalized with quantum chemical calculations and further confirmed by the X-ray crystallographic analysis of the derivatives of the oxidation products. It was found that the oxidation occurs preferentially at 7-position of the substrate, leading to 7-carbonyl products as the major products and their regioisomers 10-carbonyl products as the minors.（3） In the dihydroxylation reactions of 37a-b, the over-oxidation products, i.e. 7-carbonyl-8-hydroxy-substituted products （74a-b）, could be obtained beside the expected dihydoxylation products 36a-b when an excess amount of AD-mix-a was employed or when the reaction temperature was raised. These results further demonstrated 7-position of this coumarin derivative was easier to be oxidized than its 10-position, which was similar to the results of the oxidation reactions of 7,8,9,10-tetrahydrobenzo|7z]coumarms （67a-b and 50） with CrC^. The structures of 74a and its esterification product （75a） were confirmed by X-ray diffraction analysis.（4） In the bromination of 4,8,8-trimethyl-7,8,9,10-tetrahydrobenzo|7z] coumarin （50）, an unexpected rearrangement product, 4,7,8-trimethyl-2#-benzo [/j]coumarin （82）, was obtained in 34% yield together with the expected brominated product （37%）, 7-bromo-substituted coumarin （81）. The possible mechanism for the formation of 82 was discussed.（5） The unexpected etherification reactions of indan-l,7-diols （57a-b） were discovered and three ethers （91a-b and 99） were isolated for the bioassay screening. The self-catalyzed pathway for the formation of ethers was proposed based on experiment.（6） Attempts to follow the literature procedure for the preparation of 2,3-dihydro-4-methoxy-l,l-dimethyl-li/-indene （103b） by an acid-catalyzed cyclization of l-methoxy-2-（3-methyl-2-butenyl）benzene （102b） only led to a low yield of the desired product 103b. Instead, 1 ^{4-（2-methoxyphenyl）-2 -methylbutan-2-yl}benzene （112b）, formed by the reaction of 102b with the solvent benzene, was obtained as the major product （yield 50%）. Similar phenomenon has also been observed in the reaction of 4-chloro-l-methoxy-2-（3-methyl-2-butenyl）benzene （102a） to form 7-chloro-2,3-dihydro-4-methoxy-l,l-dimethyl-l//-indene（103a）.更多还原