CCR5 antagonists as anti-HIV-1 agents. 1. Synthesis and biological evaluation of 5-oxopyrrolidine-3-carboxamide derivatives

A novel lead compound, N-(3-[4-(4-fluorobenzoyl)piperidin-1-yl]propyl)-1-methyl-5-oxo-N-phenylpyrrolidine-3-carboxamide (1), was identified as a CCR5 antagonist by high-throughput screening using [(125)I]RANTES and CCR5-expressing CHO cells. The IC(50) value of 1 was 1.9 microM. In an effort to improve the binding affinity of 1, a series of 5-oxopyrrolidine-3-carboxamides was synthesized. Introduction of 3,4-dichloro substituents to the central phenyl ring (10i, IC(50)=0.057 microM; 11b, IC(50)=0.050 microM) or replacing the 1-methyl group of the 5-oxopyrrolidine moiety with a 1-benzyl group (12e, IC(50)=0.038 microM) was found to be effective for improving CCR5 affinity. Compound 10i, 11b, and 12e also inhibited CCR5-using HIV-1 envelope-mediated membrane fusion with IC(50) values of 0.44, 0.19, and 0.49 microM, respectively.     

Pharmacophore Model Generation Based on Pyrrolidine- and Butane-derived CCR5 Antagonists

A three-dimensional pharmacophore model was developed for a considerable number of pyrrolidine-based and butane-based chemokine (C-C motif) receptor 5 (CCR5) antagonists, which can block the entry of human immunodeficiency virus type 1 (HIV-1) by inhibiting the interaction of HIV-1 envelope protein and CCR5. The pharmacophore model was generated using a training set consisting of 25 carefully selected antagonists with the diverse molecular architecture and bioactivity, as required by the Catalyst/HypoGen program. The activity of the training set molecules expressed in IC₅₀ (half-inhibitory concentration) covered from 0.06 to 10000 nmol·L^–1. The most predictive pharmacophore model (Hypo 1), consisting of two positive ionizable points and three hydrophobic groups, had a correlation of 0.924 and a root mean square of 1.068, and a cost difference of 63.67 bits between the null cost and the total cost. The model was applied in predicting the activity of 74 compounds as a test set. The results indicated that the model was able to provide clear guidelines and accurate activity prediction for novel antagonist design.     

基于吡咯烷与正丁烷类衍生物CCR5拮抗剂的药效团模型构建

吡咯烷与正丁烷类CCR5(化学趋化因子受体5)拮抗剂可通过抑制人类免疫缺陷病毒(HIV-1)包膜蛋白与CCR5的相互作用而阻断病毒进入细胞. 本文使用已知拮抗剂结构和活性信息构建了一个三维药效团模型. 按照Catalyst/HypoGen模块的要求, 选择了25个结构和活性均具备差异性的分子作为药效团产生的训练集. 其中训练集分子以IC50值表示的生物活性值跨度为0.06到10000 nmol·L-1. 最好的药效团模型(Hypo 1)由两个正离子化特征以及三个疏水特征组成, 训练集预测相关系数为0.924, 均方根偏差为1.068. 模型用于预测由74个分子组成的测试集化合物活性, 结果表明模型可以提供较好的活性预测结果并用于新的拮抗剂的设计.     

Retinoid X receptor-antagonistic diazepinylbenzoic acids

Several dibenzodiazepine derivatives were identified as novel retinoid X receptor (RXR) antagonists on the basis of inhibitory activity on retinoid-induced cell differentiation of human promyelocytic leukemia cells HL-60 and transactivation assay using retinoic acid receptors (RARs) and RXRs in COS-1 cells. 4-(5H-2,3-(2,5-Dimethyl-2,5-hexano)-5-n- propyldibenzo[b,e][1,4]diazepin-11-yl)benzoic acid (HX603, 6c) is an N-n-propyl derivative of an RXR pan-agonist HX600 (6a), and exhibited RXR-selective antagonistic activity. Similar RXR-antagonistic activities were observed with 4-(5H-2,3-(2,5-dimethyl-2,5-hexano)-5-methyl- 8-nitrodibenzo[b,e][1,4]diazepin-11-yl)benzoic acid (HX531, 7a) and 4-(5H-10,11-dihydro-5,10-dimethyl-2,3-(2,5-dimethyl- 2,5-hexano)-dibenzo[b,e][1,4]diazepin-11-yl)benzoic acid (HX711, 8b), which also inhibited transactivation of RARs induced by an RAR agonist, Am80. These compounds inhibited HL-60 cell differentiation induced by the combination of a low concentration of the retinoid agonist Am80 with an RXR agonist (a retinoid synergist, HX600). These results indicated that HX603 (6c), and the related RXR antagonists inhibit the activation of RAR-RXR heterodimers as well as RXR homodimers, which is a distinct characteristic different from that of the known RXR antagonist, LG100754 (9).     

Heterocyclic effect for optical properties of naphthoquinone-based pigment: 2-methyl-3-heteroarylthio-1,4-naphthalenedione

Three novel naphthoquinone-based heterocyclic pigments, 2-methyl-3-[(1-methyl-1H-imidazol-2-yl)thio-1,4-naphthalenedione, (4-methyl-4H-1,2,4-triazol-3-yl)thio-1,4-naphthalenedione, and (1-methyl-1H-tetrazol-5-yl)thio]-1,4-naphthalenedione, are synthesized, and their optical properties in both solution and solid states are investigated. Depending on the heteroarylthio ring in the pigment, variation in optical properties is observed, e.g. characteristic colours for each pigment in the solution and solid states. The achiral pigment containing the 1-methyl-1H-tetrazol-5-yl ring exhibits a chiral space group and a CD signal in the solid state.     

Crystal structure of a 1,4-dihydropyridine with enantiomers showing opposite effects on calcium channels: structural features of calcium channel agonists and antagonists

The structure of the calcium channel modulator isopropyl 4-(2,1,3-benzoxadiazol-4-yl)-1,4-dihydro-2,6-dimethyl-5-nitro- 3-pyridinecarboxylate has been determined by X-ray analysis. Structural and stereochemical features are discussed in relation to previously determined structures of calcium agonists and antagonists of the 1,4-dihydropyridine type, and in relation to a newly proposed model for the dihydropyridine binding site.     

Orally active CCR5 antagonists as anti-HIV-1 agents 2: synthesis and biological activities of anilide derivatives containing a pyridine N-oxide moiety

In order to develop orally active CCR5 antagonists, we investigated 1-benzoxepine derivatives containing new polar substituents, such as phosphonate, phosphine oxide or pyridine N-oxide moieties, as replacements for the previously reported quaternary ammonium moiety. Among these compounds, the 2-(alpha-hydroxybenzyl)pyridine N-oxide 5e exhibited moderate CCR5 antagonistic activity and had an acceptable pharmacokinetic profile in rats. Subsequent chemical modification was performed and compound (S)-5f possessing the (S)-configuration hydroxy group was found to be more active than the (R)-isomer. Replacement of the 1-benzoxepine ring with a 4-methylphenyl group by a 1-benzazepine ring with a 4-[2-(butoxy)ethoxy]phenyl group enhanced the activity in the binding assay. In addition, introduction of a 3-trifluoromethyl group on the phenyl group of the anilide moiety led to greatly increased activity in the HIV-1 envelope-mediated membrane fusion assay. In particular, compound (S)-5s showed the most potent CCR5 antagonistic activity (IC(50)=7.2 nM) and inhibitory effect (IC(50)=5.4 nM) in the fusion assay, together with good pharmacokinetic properties in rats.     

Orally active CCR5 antagonists as anti-HIV-1 agents: synthesis and biological activity of 1-benzothiepine 1,1-dioxide and 1-benzazepine derivatives containing a tertiary amine moiety

The search for orally active CCR5 antagonists was performed by chemical modification of the 1-benzothiepine 1,1-dioxide 3 and 1-benzazepine 4 lead compounds containing a tertiary amine moiety. Replacement of methyl group with a 2-(C(2-4) alkoxy)ethoxy group at the 4-position on the 7-phenyl group of the 1-benzothiepine ring resulted in both enhanced activity and significant improvement in the pharmacokinetic properties upon oral administration in rats. Introduction of C(2-4) alkyl, phenyl or (hetero)arylmethyl groups as the 1-substituent on the 1-benzazepine ring together with the 2-(butoxy)ethoxy group led to further increase of activity. Among the 1-benzazepine derivatives, the isobutyl (6i), benzyl (6o) or 1-methylpyrazol-4-ylmethyl (6s) compounds were found to exhibit highly potent inhibitory effects, equivalent to the injectable CCR5 antagonist 1, in the HIV-1 envelope-mediated membrane fusion assay. In particular, compound 6s showed the most potent CCR5 antagonistic activity (IC(50)=2.7 nM) and inhibitory effect (IC(50)=1.2 nM) on membrane fusion, together with good pharmacokinetic properties in rats. The synthesis of 1-benzothiepine 1,1-dioxide and 1-benzazepine derivatives and their biological activity are described.     

Synthesis of Fluorine-Containing 2-(1-Aryl-4-oxo-1,4-dihydrocinnolin-3-yl)-2-oxoacetic Acids

Acid hydrolysis of ethyl 2-(1-aryl-5,6,7,8-tetrafluoro-4-oxo-1,4-dihydrocinnolin-3-yl)-2-oxoacetates gives 2-(1-aryl-5,6,7,8-tetrafluoro-4-oxo-1,4-dihydrocinnolin-3-yl)-2,2-dihydroxyacetic acids which undergo dehydration on heating in toluene to afford 2-(1-aryl-5,6,7,8-tetrafluoro-4-oxo-1,4-dihydrocinnolin-3-yl)-2-oxoacetic acids. Reactions of the latter with excess morpholine result in replacement of two fluorine atoms in positions 5 and 7 by the amine residues.     

Targeted disruption of the CCR5 gene in human hematopoietic stem cells stimulated by peptide nucleic acids

Peptide nucleic acids (PNAs) bind duplex DNA in a sequence-specific manner, creating triplex structures that can provoke DNA repair and produce genome modification. CCR5 encodes a chemokine receptor required for HIV-1 entry into human cells, and individuals carrying mutations in this gene are resistant to HIV-1 infection. Transfection of human cells with PNAs targeted to the CCR5 gene, plus donor DNAs designed to introduce stop codons mimicking the naturally occurring CCR5-delta32 mutation, produced 2.46% targeted gene modification. CCR5 modification was confirmed at the DNA, RNA, and protein levels and was shown to confer resistance to infection with HIV-1. Targeting of CCR5 was achieved in human CD34(+) hematopoietic stem cells (HSCs) with subsequent engraftment into mice and persistence of the gene modification more than four months posttransplantation. This work suggests a therapeutic strategy for CCR5 knockout in HSCs from HIV-1-infected individuals, rendering cells resistant to HIV-1 and preserving immune system function.     

新型α1受体拮抗剂先导化合物的寻找(I): 5-氯-2-(4-(芳氧烷基)哌嗪-1-基)苯并噁唑类化合物的设计、合成及生物活性研究

为寻找用于治疗良性前列腺增生的新型α1受体拮抗剂, 以本研究组发现的2-[(4-(2-(2-氯苯氧基)乙基)哌嗪-1-基)甲基]-5-甲基苯并噁唑(wb5c)为先导化合物, 结合已构建的α1-AR拮抗剂药效团模型, 通过骨架改造, 设计出以苯并噁唑-2-基哌嗪为母核的目标物, 然后以5-氯-2-氨基酚和取代苯酚为原料, 经缩合、卤代、氨化、Williamson醚合成、取代等反应共合成11个新目标化合物, 结构经ESI-MS, 1H NMR, IR及HRMS确证. 初步药理活性实验表明, 目标物具有中等强度α1受体拮抗活性, 符合我们提出的三元素药效团模型. 5-氯-2-[4-(芳氧烷基)哌嗪-1-基]类化合物是一类新的具有潜在开发价值的α1受体拮抗剂.     

Penicillixanthone A,a marine-derived dual-coreceptor antagonist as anti-HIV-1 agent

Marine micro-organisms have been proven to be excellent sources of bioactive compounds against HIV-1. Several natural products obtained from marine-derived Aspergillus fungi were screened for their activities to inhibit HIV-1 infection. Penicillixanthone A (PXA), a natural xanthone dimer from jellyfish-derived fungus Aspergillus fumigates, displayed potent anti-HIV-1 activity by inhibiting infection against CCR5-tropic HIV-1 SF162 and CXCR4-tropic HIV-1 NL4-3, with IC₅₀ of 0.36 and 0.26 μM, respectively. Molecular docking study was conducted to understand the possible binding mode of PXA with the CCR5/CXCR4. The results revealed that, the marine-derived PXA, as a CCR5/CXCR4 dual-coreceptor antagonist, presents a new type of potential lead product for the development of anti-HIV therapeutics.     

Studies on Thiazolopyridines. Part 4: Synthesis of Hitherto Unknown 1,4-Bis(thiazolopyridine)benzene Derivatives

2-Cyanomethyl-4-thiazolinone ( 1 ) was condensed with terephthalaldehyde ( 2 ) (2:1 molar ratio) and produced 1,4-bis(2-cyanomethyl-4,5-dihydro-5-methylidene-4-thiazolinone-5-yl) benzene ( 3 ). Treatment of compound ( 3 ) with benzylidenemalononitrile ( 4 ) (1:2 molar ratio) furnished the novel bisthiazolopyridines ( 6a-e ). Condensation of compound ( 1 ) with aromatic aldehyde yielded the benzylidene derivatives ( 7a-f ), which on treatment with compound ( 8 ) (2:1 molar ratio) afforded the novel bisthiazolopyridines ( 10a-f ). Structures of the synthesized compounds have been established by elemental analyses and spectral data.     

A synthetic heparan sulfate-mimetic peptide conjugated to a mini CD4 displays very high anti- HIV-1 activity independently of coreceptor usage

The HIV-1 envelope gp120, which features both the virus receptor (CD4) and coreceptor (CCR5/CXCR4) binding sites, offers multiple sites for therapeutic intervention. However, the latter becomes exposed, thus vulnerable to inhibition, only transiently when the virus has already bound cellular CD4. To pierce this defense mechanism, we engineered a series of heparan sulfate mimicking tridecapeptides and showed that one of them target the gp120 coreceptor binding site with μM affinity. Covalently linked to a CD4-mimetic that binds to gp120 and renders the coreceptor binding domain available to be targeted, the conjugated tridecapeptide now displays nanomolar affinity for its target. Using solubilized coreceptors captured on top of sensorchip we show that it inhibits gp120 binding to both CCR5 and CXCR4 and in peripheral blood mononuclear cells broadly inhibits HIV-1 replication with an IC(50) of 1 nM.     

Synthesis of novel unsymmetrical coumarinyl-1,4-dihydropyridines

The novel unsymmetrical 3,5-dialkoxycarbonyl-2,6-dimethyl-4-(7′,8′-dimethoxycoumarin-4′-yl)-1,4-dihydropyridines and 5-acetyl-3-alkoxycarbonyl-2,6-dimethyl-4-(7′,8′-dimethoxycoumarin-4′-yl)-1,4-dihydropyridines (coumarinyl-1,4-dihydropyridines) have been synthesized by Knoevenagel condensation of 4-formyl-7,8-dimethoxycoumarin with alkyl acetoacetates in the presence of AlCl₃ followed by cyclization of the resulted Knoevenagel product with other alkyl acetoacetate or acetyl acetone and ammonium acetate. The structure of the intermediate Knoevenagel product and the cyclized unsymmetrical coumarinyl-1,4-dihydropyridines has been established on the basis of their spectral data analysis and single-crystal X-ray diffraction analysis. The observed conformation of the coumarinyl-1,4-dihydropyridines holds the key to promising calcium antagonistic activity of the synthesized coumarinyl-1,4-dihydropyridines.     

Theoretical studies on the interactions and interferences of HIV-1 glycoprotein gp120 and its coreceptor CCR5

The interaction between the HIV gp120 protein and coreceptor CCR5 or CXCR4 of the host cell is critical in mediating the HIV entry process. A model for the CCR5-gp120 complex has been developed. In the model, the N-terminus of CCR5 binds to three discontinuous domains of gp120, including the fourth conserved (C4) region, β19/β20 connecting loop, and V3 loop. The second extra-cellular loop (ECL2) of CCR5 also interacts with the crown part of the gp120 V3 loop. The bindings of the three CCR5 antagonists, maraviroc, aplaviroc, and vicriviroc, to the trans-membrane domain of CCR5 have been modeled. The bindings are found to affect the conformation of the ECL2 domain, which in turn drives the N-terminus of CCR5 to an altered state. Aplaviroc is more hydrophilic than maraviroc and vicriviroc, and its binding is more interfered by solvent, resulting in a quite different effect to the structure of CCR5 compared with those of the other two molecules. The above results are in accord with experimental observations and provide a structural basis for further design of CCR5 antagonists.     

Condensed pyridazines. Part III. Rearrangement and ring cyclization during the thermolysis of (z)-methyl 3-(6-azido-3-chloro-1-methyl-4-oxo-1,4-dihydropyridazin-5-yl)-2-methylacrylate

The thermolysis of (Z)-methyl 3-(6-azido-3-chloro-1-methyl-4-oxo-1,4-dihydropyridazin-5-yl)-2-methylacrylate ( II ) provides a new synthetic route to pyrrolo[2,3-c-]pyridazines, specifically, methyl 3-chloro-1,6-dimethyl-4-oxo-1,4-dihydro-7H-pyrrolo[2,3-c]pyridazine-5-carboxylate ( III ) in 91% yield. Treatment of III with ozone provides an entry into the novel pyridazino[3,4-d][1,3]oxazine ring system, specifically, 3-chloro-1,7-dimethylpyridazino[3,4-d][1,3]oxazine-4,5-dione ( IV ) in 73% yield. Compound IV is smoothly hydrolyzed into 6-acetylamino-3-chloro-1-methyl-4-oxo-1,4-dihydropyridazine-5-carboxylic acid ( V ) which is readily recyclized into IV by dehydration with acetic anhydride. Furthermore, IV undergoes a facile reductive ring opening reaction with sodium borohydride to give 3-chloro-6-ethylamino-1-methyl-4-oxo-1,4-dihydropyridazine-5-carboxylic acid ( VI ) in 95% yield.     

Synthesis of nucleoside analogs of 1,4-oxathiane, 1,4-dithiane,and 1,4-dioxane

The two regioisomers 6-chloro-9-(1, 4-oxathian-3-yl)-9H-purine ( 5 ) and 6-chloro-9-(1,4-oxathian-2-yl)-9H-purine ( 6 ) were obtained when 3-acetoxy-1,4-oxathiane ( 3 ) was subjected to the acid-catalyzed fusion procedure; compound 3 was prepared by a Pummerer reaction with 1,4-oxathiane 4-oxide ( 2 ). The nucleoside analog 6 could he converted into the adenine derivative 7 and 9-(1,4-oxathian-2-yl)-9H-purine-6(1H)thione ( 8 ). The following nucleoside analogs have also been synthesized: 6-chloro-9-(1,4-dithian-2-yl)-9H-purine ( 13 ), 9-(1,4-dithian-2-yl)adenine ( 14 ), 9-(1,4-dithian-2-yl)-9H-purine-6(1H)thione ( 15 ), and 6-chloro-9-(1,4-dioxan-2-yl)-9H-purine ( 18 ).     

Synthesis and biological evaluation of some new substituted benzoxazepine and benzothiazepine as antipsychotic as well as anticonvulsant agents

Various 2-((2-((5-benzylideneamino)-1,3,4-oxa/thiadiazol-2-yl)methyl)hydrazinyl) methyl)benzo[b][1,4]oxa/thiazepin-4(5H)-ones (4a–4l), 2-((2-((5-(4-oxo-2-substitutedphenyl thiazolidin-3-yl)-1,3,4-oxa/thiadiazol-2-yl)methyl)hydrazinyl)methyl)benzo [b] [1,4]oxa/thiazepin-4(5H)-ones (5a–5l) and 2-((2-((5-(3-chloro-2-(substitutedphenyl)-4-oxoazetidin-1-yl)-1,3,4-oxa/thia diazol-2-yl)methyl)hydrazinyl)methyl)benzo[b][1,4]oxa/thiazepin-4(5H)-ones (6a–6l) have been synthesized. The structures of these compounds have been established by elemental (C, H, N) and spectral (IR, ¹H-NMR and Mass) analysis. The synthesized compounds were screened for their antipsychotic and anticonvulsant activities. Compound 5l was found to be the most active compound of this series.     

Reactions of 1,4-bis(tetrazole)benzenes: formation of long chain alkyl halides

The reactions of 1,4-bis[2-(tributylstannyl)tetrazol-5-yl]benzene with α,ω-dibromoalkanes were carried out in order to synthesise pendant alkyl halide derivatives of the parent bis-tetrazole. This led to the formation of several alkyl halide derivatives, substituted variously at N1 or N2 on the tetrazole ring. The crystal structures of 1,4-bis[(2-(4-bromobutyl)tetrazol-5-yl)]benzene (2-N,2-N′), 1,4-bis[(2-(4-bromobutyl)tetrazol-5-yl)]benzene (1-N,2-N′) and 1,4-bis[(2-(8-bromooctyl)tetrazol-5-yl)]benzene (2-N,2-N′) are reported. Further discussion involves the structure of 1,4-bis[2-(6-bromohexyl)-2H-tetrazol-5-yl]benzene (2-N,2-N′) previously reported.