WO2020007070A1 - 4'-取代核苷的晶型、制备和应用 - Google Patents

4'-取代核苷的晶型、制备和应用 Download PDF

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WO2020007070A1
WO2020007070A1 PCT/CN2019/078992 CN2019078992W WO2020007070A1 WO 2020007070 A1 WO2020007070 A1 WO 2020007070A1 CN 2019078992 W CN2019078992 W CN 2019078992W WO 2020007070 A1 WO2020007070 A1 WO 2020007070A1
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compound
hiv
preparation
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medicament
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常俊标
杜锦发
朱凯凯
王凯
李建永
张春霞
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河南真实生物科技有限公司
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Definitions

  • the invention relates to the crystal form, preparation and application of 4'-substituted nucleosides, and belongs to the field of medicinal chemistry.
  • HIV infection causes AIDS. From the first AIDS patient found in the United States in 1981, there are currently approximately 39 million AIDS patients in the world. AIDS has become a major threat to human health. HIV replication is accomplished through processes such as adsorption, invasion and husking, reverse transcription, integration of viral RNA and protein synthesis, assembly, release, and maturation. Each of these steps can be used as targets for HIV drugs. After more than 30 years of research, including the combined drug, 40 AIDS treatment drugs have been approved by the FDA for clinical use. According to the mechanism of action of these drugs, they can be mainly divided into: nucleoside and non-nucleoside reverse transcriptase inhibitors, protease inhibitors, invasion inhibitors and integrase inhibitors.
  • nucleosides with 3'-OH such as the compounds Id (CAStoddart et Antimicrob. Agents Chemother. 2015, 59, 4190) and Ig (Q. Wang et al. Eur. J. Med. Chem.
  • R is selected from ethynyl, azide, cyano;
  • X is selected from hydrogen, fluorine;
  • B is selected from B1 or B2.
  • the compounds Ia-Ig are preferably selected from the following:
  • the present invention takes Compound Ia as a representative, and studies the pharmacokinetics of Compound Ia after gavage in rhesus monkeys. It was found that compound Ia could not be detected in plasma 24 hours after gavage of 6 mg / kg of compound Ia in rhesus monkeys. However, the active ingredient of compound Ia and the triphosphate Ia-TP of Ia were administered for 168 hours (7 days The concentration of Ia-TP in HIV target cells and peripheral blood mononuclear cells of rhesus monkeys was still greater than 0.11 nM, and the half-life of effective drugs in target cells was more than 72 hours (Table 1).
  • Compound Ia can be used as a long-acting drug for the prevention or treatment of AIDS.
  • Compound Ia has long-lasting properties, which is likely related to its introduction of 2-fluoro and a large substituent at the 4'-position to improve its metabolic stability.
  • Compounds Ia-If are all 4'-substituted 2-fluoroadenine analogs, and therefore have potential as long-acting drugs for the prevention or treatment of AIDS.
  • the present invention measured the anti-HIV activity of compound Ig's triphosphate (Ig-TP) in CD4 + T cells (Hut / CCR5).
  • the concentration that inhibits 50% of HIV is 1.3 nM (see Figure 5.
  • EC 50 0.373 ng / mL).
  • the concentration that inhibited 50% of cell proliferation was 8134 nM (see Figure 6.
  • CC 50 2328 ng / mL).
  • the present invention further studies the pharmacokinetics of compound Ig administered to rhesus monkeys, and the results show that after administering 1mg / kg and 6mg / kg of compound Ig to rhesus monkeys, the concentration of compound Ig in plasma increases with increasing dose.
  • the concentration of Compound Ig dropped to 6.54 nM level, which was below the lower limit of detection after the third day (Table 2).
  • the concentration of compound Ig in HIV target cells and PBMC decreased to 0.09 pmol / 10 6 cells after 6 hours of administration, and was lower than the lower limit of detection after 24 hours.
  • the concentration of the active ingredient (Ig-TP, Table 3) in their PBMCs is still 1.35 nM, which is still higher than the drugs required to inhibit 50% of HIV Concentration (0.373ng / mL, 1.3nM), it can be seen that the active drug concentration in HIV target cells can still effectively inhibit HIV replication 5 days after compound Ig is administered to rhesus monkeys. Therefore, the compound Ig can be used as a long-acting drug for preventing or treating AIDS. Or its pharmaceutically acceptable salt, prodrug or composition thereof
  • the compound I according to the present invention including Ia-Ig and their salts, solvates, crystal forms, or prodrugs or compositions, as active ingredients, can be applied to the preparation of long-acting drugs for the prevention and treatment of AIDS. Patients can take medicine every 2 days or more.
  • Compound I can also be used in combination with other HIV inhibitors to form a composition as a long acting drug for the prevention or treatment of HIV infection.
  • the other HIV inhibitors include, but are not limited to: nucleoside inhibitors, non-nucleoside reverse transcriptase inhibitors, protease inhibitors, and integrase inhibitors; for example, optionally, the nucleoside inhibitors include: Zidovudine (AZT), didanosine (ddI), zalcitabine (ddC), stavudine (d4T), lamivudine (3TC), abacavir (Abacavir), enqu Tabin (FTC), tenofovir (TDF), EFdA (MK-8591); optionally, the non-nucleoside reverse transcriptase inhibitors include: nevirapine, delavirdine, efavirenz, Etraviron; optionally, the protease inhibitors include: saquinavir, indin
  • the HIV inhibitor is preferably selected from the group consisting of compound Ig, tenofovir , Lipivirin, Duluthway, Bictegravir, Abbottweiler.
  • the HIV inhibitor is preferably selected from the group consisting of compounds Ia-If, tenofovir or tenofovir alafenol Amine, Ripivirin, Dulutevir, Bictegravir, Abbottavier.
  • the term "protected” refers to a group that is added to an oxygen, nitrogen, or phosphorus atom to prevent it from reacting further or for other purposes.
  • Those skilled in the art of organic synthesis are aware of various oxygen and nitrogen protecting groups.
  • any hydroxyl or amino group can be protected or unprotected.
  • a hydroxyl or amino group is referred to as “protected”
  • “Pharmaceutically acceptable salt” includes any salt of a compound provided herein, such as sodium, potassium, ammonium, and the like. It retains the biological properties of the compound and is non-toxic or otherwise undesirable for medical use.
  • prodrug in the present invention refers to any compound that produces a biologically active compound due to a spontaneous chemical reaction, an enzyme-catalyzed reaction, and / or a metabolic process or a combination of any one when administered to a biological system.
  • Standard prodrugs are formed using groups that are attached to functional groups such as -OH, -NH 2 and are associated with the drug and cleave in vivo. Examples of prodrugs include esters and the like.
  • Compounds capable of forming an "ester” with the nucleoside I of the present invention include various organic acids, such as acetic acid and 3'-OH to form acetate, and propionic acid and 3'-OH to form propionate.
  • the prodrugs described in the present invention are exemplary, but not limiting, and those skilled in the art can prepare other known kinds of prodrugs.
  • the "solvate” according to the present invention includes the above-mentioned nucleoside formula I or a salt thereof in a case where the solvent is water by combining a non-covalent intermolecular force with a stoichiometric amount or a non-stoichiometric amount of a solvent. Hydrate.
  • the monophosphate group, diphosphate group, and triphosphate group structures of the present invention are shown below.
  • R 1 is a hydroxy protecting group, including but not limited to benzyl or substituted benzyl;
  • R 2 is H, or an alkyne terminal protecting group, including but not limited to the following groups:
  • the fluorinating reagent of Compound 1 can be selected from, but not limited to, DAST-type fluorinating agents or other fluorinating reagents; or the fluorination of the hydroxyl group is achieved by activating the hydroxyl group with a leaving group and then replacing it with a fluoride ion; Can be selected from aprotic organic solvents;
  • the reagent for removing the benzyl protecting group of Compound 2 is selected from the group consisting of metal sodium-liquid ammonia or similar methods, or acids and sulfonic acids, such as methanesulfonic acid.
  • FIG. 1 is a histogram of the activity of compound Ia for suppressing HIV
  • FIG. 3 is a histogram of the activity of Compound Ib in inhibiting HIV
  • Compound 1 was prepared according to literature methods (K. Fukuyama et al. Org. Lett. 2015, 17, 828-831).
  • reaction was detected by LC-MS, the reaction was stopped, cooled, and saturated sodium bicarbonate solution was added dropwise at low temperature to quench Off, adjust the pH to 7-8, add EA extraction, wash the organic phase with water, wash with saturated sodium chloride solution, dry with anhydrous sodium sulfate, filter, concentrate, beaten with PE, and filter to obtain 7.44g of crude compound 2, which is directly used in the next step.
  • the stable crystal A of compound Ia has a characteristic peak and its relative intensity (%) at the following characteristic peak diffraction angle 2 ⁇ :
  • HIV-1 virus
  • the plasmid used to package pseudotyped single-cycle infectious HIV-1 (HIV-luc / JRFL) is as follows: pLAI- ⁇ env-Luc contains the backbone gene sequence of HIV-1, but the env and vpr genes are deleted and inserted at the nef gene site The Luciferase reporter gene; pJRFL contains the CCR5 tropic HIV-1 env gene; the plasmid was co-transfected into HEK293T cells. Collect cell supernatant and filter aliquots. Virus particles were quantified using p24 gag capture ELISA.
  • CD4 + T cells (Hut / CCR5) were cultured in complete 1640 medium (Gibco), which contained 10% fetal bovine serum (Gibco), 100U / ml penicillin (Invitrogen), and 100U / ml streptomycin (Gibco). Invitrogen). 1 ⁇ 10 5 Hut / CCR5 cells were added with the test compound Ia or Ib, and then infected with HIV-luc / JRFL (1ng p24 gag ), and incubated at 37 ° C for 3 hours. The free virus was washed away, then resuspended in culture medium, and added Corresponding concentrations of test compounds were cultured at 37 ° C for 3 days. Finally, cells were collected to detect luciferase activity, and the virus replication and inhibition rates were calculated. AZT served as a compound control (see Figures 1 and 3 for results).
  • CD4 + T cells (Hut / CCR5) were cultured in complete 1640 medium (Gibco), which contained 10% fetal bovine serum (Gibco), 100U / ml penicillin (Invitrogen), and 100U / ml streptomycin (Gibco). Invitrogen). Add 1 ⁇ 10 5 Hut / CCR5 cells to compound Ig-TP (CL-002-TP), and then infect HIV-luc / JRFL (1ng p24 gag ), incubate for 3 hours at 37 ° C, wash away the free virus, and then use the medium Resuspend, add the test compound at the corresponding concentration, and incubate at 37 ° C for 3 days. Finally, collect cells to measure luciferase activity, and calculate the virus replication and inhibition rate. AZT served as a compound control.
  • the MTT method was used to investigate the cytotoxicity of compound Ig-TP on CD4 + T cells (Hut / CCR5).
  • the results showed that the compound Ig-TP was very toxic to CD4 + T cells, with a CC 50 of 2328 ng / ml and 8134 nM (Figure 5).
  • Ia-TP 3TC-TP, Ig-TP;
  • EDTAK 2 anticoagulant tube purchased from Shanghai Chemical Reagent Institute
  • the experimental animals were two Taihangshan macaques, males, weighing 8 and 9 kg, respectively, provided by Henan Normal University.
  • PBMC Peripheral blood mononuclear cells
  • Compound CL-002 (or 3TC) gavage solution was prepared: 130 mg of compound CL-002 was weighed, added to 21.7 ml of physiological saline, and sonicated at 40 ° C for 15 minutes, and a drug solution with a concentration of 6 mg / ml was prepared as a large dose.
  • the administration volume is 1ml / kg
  • the CL-002 administration doses are: 6mg / kg and 1mg / kg (3TC dose is 20mg / kg; Ia dose is 6mg / kg).
  • Two healthy macaque monkeys were randomly divided into two groups (low and high dose groups), one in each group, fasting for 12 hours before administration and water, and 6 g / kg and 1 mg / kg of compound CL-002 (g Or 20 mg / kg of 3TC), and 1 to 6 hours, 24 hours, 3 days, 5 days, and 7 days after administration, 15-20 ml of blood was collected from hind limb veins, 1 ml of plasma was centrifuged, and frozen and stored below -20 ° C.
  • Peripheral blood mononuclear cells (PBMC) were separated by Ficoll-Paque separation solution, concentrated to about 0.3 ml of cell fluid, counted, and frozen and stored below -20 ° C.

Abstract

本发明公开了结构如下的4'-取代核苷化合物I的晶型、制备和应用,包括它们的盐，前药和组合物。动物药代动力学研究结果证明，化合物Ia和Ig在HIV靶细胞，外周血单核细胞(PBMC)内的有效药物浓度分别在7天和5天后仍能有效地抑制HIV的复制。因此，化合物I可以作为预防和治疗艾滋病的长效药物。 R选自乙炔基，叠氮和氰基；X选自氢和氟；B选自B1和B2。

Description

4’-取代核苷的晶型、制备和应用 技术领域
本发明涉及4’-取代核苷的晶型、制备和应用,属于药物化学领域。
背景技术
HIV感染引起艾滋病发作,从1981年美国发现第一例艾滋病患者，到目前世界上大约有三千九百万艾滋病病人。艾滋病已成为人类健康的一个重大威胁。艾滋病病毒(HIV)的复制经过吸附、侵入和脱壳、逆转录、整合病毒RNA和蛋白质的合成、装配、释放和成熟等过程完成。其中每个环节均可作为抑制HIV药物的靶点。经过三十多年的研究，包括合并用药在内，已有40个艾滋病治疗药被美国FDA批准用于临床。根据这些药物的作用机理，它们主要可以分为：核苷和非核苷类逆转录酶抑制剂、蛋白酶抑制剂、侵入抑制剂和整合酶抑制剂等几大类。这些药物能有效地抑制HIV的复制，但都不能治愈艾滋病，而且，使用这些药物治疗一段时间后，病毒的耐药性就会不断产生，使现有药物治疗失去作用。所以用不同作用机理的HIV抑制剂组合治疗艾滋病已成为一种标准的治疗策略。但是，即使联合用药治疗艾滋病，新耐药性仍会不断出现。因此，仍然需要继续寻找具有新作用机理的艾滋病治疗药物。另外，到目前为止，所有艾滋病治疗药物都必须每天至少服药一次，长期服药成为病人的负担，因此，开发长效艾滋病治疗药物十分必要。
2’-脱氧核苷是用于临床的主要一类HIV抑制剂，它们通过抑制逆转录酶发挥药物作用。目前临床用核苷类HIV抑制剂都是3'-脱氧核苷。由于没有3'-OH，药物嵌入HIV的DNA后，使HIV的DNA延长终止，从而成为HIV的DNA合成终止剂，并因此达到抑制HIV的复制效果。近年来，具有3’-OH的核苷，如化合物Id(C.A.Stoddart et al Antimicrob.Agents Chemother.2015,59,4190)和Ig(Q.Wang et al Eur.J.Med.Chem.2011,46,4178)相继被报道具有显著抑制HIV的活性。其结构特点是在4’-位都有一个大的取代基,如化合物Id中的乙炔基和Ig中的叠氮基。由于这些大取代基的引入，使这些核苷磷酸酯被 结合进入病毒DNA后，因其位阻效应，使病毒的DNA的延长变得缓慢或终止，达到抑制HIV复制的目的。
腺嘌呤核苷中，2-氟的引进使4’-乙炔基-2’-脱氧腺苷的抑制HIV活性(EdA的EC 50＝11nM)提高(Id，FEdA的EC50＝0.05nM)了2200倍(E.Michailidis et al J.Biol.Chem.2009,284,35681)。而2-氟引入泰诺福韦对其抗HIV活性的影响正好相反。文献报道，替诺福韦(Tenofovir)的膦酸二酰胺和膦酸胺酯前药的抗HIV活性相当(F.Pertusati,et al Eur.J.Med.Chem.2014,78,259),其EC 50在0.019和0.03之间。而2-氟取代的泰诺福韦膦酸二酰胺的抗HIV活性则降低了183-1377倍(EC 50＝5.51uM)。所以，2-氟取代对产物的抑制HIV活性的影响是不可预测的。美国专利(J.Chang US8835615,2014)披露了化合物Ia的化学结构，但是，没有其合成方法和生物活性的相关报道。因此，对化合物Ia进行化学合成和抗病毒活性的研究十分必要。
发明内容
为了寻找安全有效的长效抗艾滋病药物，发明人首次合成了化合物Ia和Ib,并测试了它们的抗HIV的活性和安全性。发现，化合物Ia和Ib具有显著的抗HIV活性(Ia:EC 50＝0.9nM，见图1；Ib:EC 50＝0.62nM，见图3)，并且在测试的剂量范围内没有明显的细胞毒性(Ia:CC 50>8000nM，见图2；Ib:CC 50>149000nM，见图4)。
化合物Ia和Ib是通式I所述的一类4’-取代的核苷的代表，
Figure PCTCN2019078992-appb-000001
R选自乙炔基，叠氮，氰基；X选自氢，氟；B选自B1或B2。
通式化合物I的类似物中，优先选自如下化合物Ia-Ig：
Figure PCTCN2019078992-appb-000002
因它们都在4’-位有一个大的取代基，除了都有显著的抗病毒活性潜力外，它们还具有优越的代谢稳定性。
为了研究化合物I作为长效艾滋病治疗药物的可行性，本发明以化合物Ia为代表，研究了化合物Ia在猕猴灌胃后的药代动力学。发现，猕猴灌胃6mg/kg化合物Ia 24小时后，血浆中已检测不到化合物Ia，但是，化合物Ia的活性成份、Ia的三磷酸酯(Ia-TP),在灌胃168小时(7天)后猕猴的HIV靶细胞、外周血单核细胞里Ia-TP的浓度仍然大于0.11nM,有效药物在靶细胞内的半衰期超过72小时(表1)。而临床用药拉米夫定(3TC)在灌胃20mg/kg 24小时后，猕猴的血浆和靶细胞PBMC中都不再检测到3TC或其有效成份，3TC的三磷酸酯(3TC-TP)。因此，与临床应用的艾滋病治疗药物不同，化合物Ia可作为预防或治疗艾滋病的长效药物。化合物Ia具有长效的特性，很可能与其2-氟的引进和4’-位有一个大取代基使其代谢稳定性提高有关。化合物Ia-If都是4’- 取代的2-氟腺嘌呤类似物，因此，都有作为艾滋病预防或治疗的长效药物的潜力。
为了研究化合物Ig作为预防或治疗艾滋病的长效药物的可行性，本发明测定了化合物Ig的三磷酸酯(Ig-TP)在CD4 +T细胞(Hut/CCR5)中的抗HIV的活性，其抑制50％艾滋病病毒的浓度是1.3nM(见图5.EC 50＝0.373ng/mL)。抑制50％细胞增殖的浓度是8134nM(见图6.CC 50＝2328ng/mL)。
本发明进一步研究了猕猴灌胃化合物Ig的药代动力学，结果表明，猕猴灌胃1mg/kg和6mg/kg化合物Ig后，血浆中化合物Ig的浓度随剂量增加而增加。大剂量组24小时后，化合物Ig的浓度降至6.54nM水平，第三天以后低于检测下限(表2)。大剂量组在用药6小时后，HIV靶细胞，PBMC内化合物Ig的浓度降至0.09pmol/10 6cells水平，24小时后低于检测下限。猕猴灌胃6mg/Kg化合物Ig后120小时(5天)，在其PBMC中的有效成份(Ig-TP，表3)的浓度仍为1.35nM，仍然高于抑制50％艾滋病病毒所需要的药物浓度(0.373ng/mL,1.3nM)，可见，猕猴灌胃化合物Ig后5天，HIV靶细胞内的活性药物浓度仍能有效地抑制HIV的复制。因此，化合物Ig可以作为预防或治疗艾滋病的长效药物。将其或其医药上可接受的盐、前药或其组合物
本发明所述化合物I，包括Ia-Ig以及它们的盐，溶剂化物，晶体形式或前药或组合物，作为活性成分，可应用于预防和治疗艾滋病的长效药物的制备。病人服药间隔可为2天或2天以上。
化合物I还可以与其它HIV抑制剂联用形成组合物作为长效药物用于预防或治疗HIV感染。所述其它HIV抑制剂包括，但不限于：核苷抑制剂、非核苷类反转录酶抑制剂、蛋白酶抑制剂、整合酶抑制剂；比如：可选地，所述核苷抑制剂包括：齐多夫定(AZT)、去羟肌苷(ddI)、扎西他滨(ddC)、司他夫定(d4T)、拉米夫定(3TC)、阿巴卡韦(Abacavir)、恩曲他滨(FTC)、替诺福韦(TDF)，EFdA(MK-8591)；可选地，所述非核苷类反转录酶抑制剂包括：奈韦拉平、地拉韦定、依非韦仑、依曲韦仑；可选地，所述蛋白酶抑制剂包括：沙奎那韦、茚地那韦、利托那韦、奈非那韦、氨普那韦、洛匹那韦、阿扎那韦、福沙普利那韦、替拉那韦、大诺那韦；可选地，所述整合酶抑制剂包括：雷特格韦、埃替拉韦、度鲁特韦。
化合物Ia与其它HIV抑制剂形成的组合物在制备预防或治疗艾滋病的长效药物中的应用，所述HIV抑制剂优先选自：化合物Ig，替诺福韦或者替诺福韦艾拉酚胺，利匹韦林，度鲁特韦，Bictegravir，艾博卫泰。
化合物Ig与其它HIV抑制剂形成的组合物在制备预防或治疗艾滋病长效药物中的应用，所述HIV抑制剂优先选自：化合物Ia-If，替诺福韦或者替诺福韦艾拉酚胺，利匹韦林，度鲁特韦，Bictegravir，艾博卫泰。
除非另外定义，否则本文所用的所有化学术语都采用所属领域的技术人员所了解的它们的一般含义。
除非另外定义，否则术语“经保护”是指加到氧、氮或磷原子中以防止它进一步反应或达成其它目的的基团。有机合成领域的技术人员知道多种氧和氮保护基。在本文披露或描述的任何结构，尤其是核苷或核苷酸中，任何羟基或氨基都可以经保护或未经保护。当羟基或氨基称为“经保护”时，意味着此基团如所属领域的技术人员所了解经例如酰基、膦酰基、磷酸酯基等可去除的基团保护。
“医药学上可接受的盐”包括本文提供的化合物的任何盐，例如钠盐、钾盐、铵盐等。它保留所述化合物的生物学性质并且无毒或不在其它方面是医药用途所不希望的。
本发明所述“前药”是指当投予生物***时由于自发的化学反应、酶催化反应和/或代谢过程或任一者的组合而产生生物学活性化合物的任何化合物。标准的前药是使用连接到例如-OH、-NH 2等官能团、与药物缔合并在体内裂解的基团形成。前药的例子包括酯等。能够与本发明核苷I形成“酯”的化合物包括各种有机酸，如乙酸与3’-OH可形成乙酸酯，丙酸与3’-OH可形成丙酸酯。在本发明中描述的前药是例示性的，但不是限制性的，并且所属领域的技术人员可以制备其它已知种类的前药。
本发明所述“溶剂化物”包括通过非共价的分子间作用力结合化学计算量或非化学计算量的溶剂的上述的核苷式I或其盐在溶剂是水的情况下溶剂化物，是水合物。
本发明所述单磷酸酯基团、二磷酸酯基团、三磷酸酯基团结构分别如下所示
Figure PCTCN2019078992-appb-000003
制备化合物Ia的方法如下:
(a)、化合物1与氟化试剂氟化得到化合物2，
Figure PCTCN2019078992-appb-000004
R 1是羟基保护基，包括但不限于苄基或取代苄基；
R 2是H,或者炔端保护基，包括但不限于如下基团：
Figure PCTCN2019078992-appb-000005
化合物1的氟化试剂可选自但不限于DAST类氟化剂或其他氟化试剂；或者羟基的氟化通过离去基团对羟基的活化，然后由氟离子取代实现；氟化反应的溶剂可选自非质子性有机溶剂；
(b)、化合物2通过脱去苄基保护基生成化合物Ia
脱去化合物2的苄基类保护基的试剂选自金属钠-液氨或类似方法，或者酸类、磺酸类，如甲磺酸。
动物药代动力学研究结果证明，化合物Ia和Ig在HIV靶细胞，外周血单核细胞(PBMC)内的有效药物浓度分别在7天和5天后仍能有效地抑制HIV的复制。因此，化合物I及其药学上可接受的盐，或前药或组合物可以作为预防或治疗艾滋病的长效药物。其用药间隔为2天或者2天以上。
附图说明
图1为化合物Ia抑制HIV的活性柱状图；
图2为化合物Ia抑制细胞增殖的毒性柱状图；
图3为化合物Ib抑制HIV的活性柱状图；
图4为化合物Ib抑制细胞增殖的毒性柱状图；
图5为化合物Ig-TP抑制HIV-1的活性柱状图；
图6为化合物Ig-TP对CD4 +T细胞的毒性柱状图；
图7化合物Ia的CuKα-XRPD图谱。
具体实施方式
以下对本发明的具体实施方式进行详细说明。应当理解的是，此处所描述的具体实施方式仅用于示例性地对本发明进行说明，并不用于限制本发明。
实施例1、化合物1的制备：
化合物1根据文献方法制备(K.Fukuyama et al Org.Lett.2015,17,828-831)。
实施例2、化合物Ia的制备：
Figure PCTCN2019078992-appb-000006
将原料1(9.64g,15.97mmol,1.0eq)，吡啶(7.7mL,95.8mmol,6.0eq)加入到100mL干燥的甲苯中，将DAST(15.4g,95.8mmol,6.0eq)溶解于40mL干燥的甲苯中，氮气保护，0℃下滴加至反应液中，滴加完毕，升至50℃回流反应6h，LC-MS检测反应完毕，停止反应，冷却，低温下滴加饱和碳酸氢钠溶液淬 灭，调pH至7～8，加EA萃取，有机相用水洗，饱和氯化钠溶液洗，无水硫酸钠干燥，过滤，浓缩,PE打浆，过滤得化合物2 7.44g粗品，直接用于下一步。 1H NMR(400MHz,CDCl 3)δ8.01(d,J＝2.0Hz,1H),7.44–7.28(m,10H),6.53(dd,J＝11.4,5.1Hz,1H),5.86(s,2H),5.37(dt,J＝53.3,4.9Hz,1H),4.73(s,2H),4.69–4.48(m,3H),3.77(dt,J＝26.1,6.2Hz,2H),0.99(t,J＝7.9Hz,9H),0.63(q,J＝8.0Hz,6H)； 19F NMR(376MHz,CDCl 3)δ-50.83,-196.65。
将化合物2(7.37g粗品,12.17mmol,1.0eq)，氟化铵(2.25g,60.83mmol,1.5eq)加入50mL甲醇中，氮气保护，50℃反应6h，LC-MS检测反应完毕，旋蒸除去溶剂，加入EA溶解，有机相分别用水洗，饱和氯化钠溶液洗，无水硫酸钠干燥，过滤，浓缩，干法过柱，得化合物3(3.24g，从化合物12到化合物15，总产率约为30～40％)。 1H NMR(400MHz,CDCl 3)δ7.94(d,J＝2.2Hz,1H),7.42–7.27(m,10H),6.54(dd,J＝12.1,5.1Hz,1H),6.03(s,2H),5.56–5.21(m,1H),4.82–4.49(m,5H),3.76(dt,J＝26.5,6.2Hz,2H),2.73(s,1H).； 19F NMR(376MHz,CDCl 3)δ-50.78,-195.84；m/z(ESI)[M+H] +＝492.2,m/z(ESI)[M+Na] +＝514.3。
将化合物3(1.71g，3.48mmol)纯品溶于26mL氯仿中，室温下滴加甲基磺酸6.84mL，氮气保护，室温反应5h，LC-MS检测反应完毕，低温下滴加饱和碳酸氢钠溶液淬灭，调pH至7～8，加EA萃取，分液，有机相用饱和氯化钠溶液洗，无水硫酸钠干燥，过滤，浓缩，干法过柱(DCM:MeOH＝20:1-10:1)得化合物Ia(748mg,69％)。 1H NMR(400MHz,MeOD)δ8.25(d,J＝1.7Hz,1H),6.46(dd,J＝8.9,5.7Hz,1H),5.35(dt,J＝53.9,5.8Hz,1H),4.75(dd,J＝22.3,5.9Hz,1H),3.88(dd,J＝12.3,1.8Hz,1H),3.80(d,J＝12.3Hz,1H),3.22(s,1H).； 19F NMR(376MHz,MeOD)δ-53.22,-201.69；m/z(ESI)[M+H] +＝312.2，m/z(ESI)[M+Na] +＝334.1。
使用Cu-Kα辐射，在
Figure PCTCN2019078992-appb-000007
化合物Ia晶体A的X射线粉末衍射(XRPD)在衍射角2θ为6.13，7.77，9.43，10.61，11.88，12.44，12.80，13.88，14.28，14.66，15.51，17.09，17.55，17.88，18.95，21.18，21.86，22.40，22.87， 23.52，24.93，25.69，26.63，28.60，30.46处有特征峰，其中2θ值误差范围为±0.2(见图7)。
化合物Ia的稳定晶体A在下列特征峰衍射角2θ处具有特征峰及其相对强度(％)：
Figure PCTCN2019078992-appb-000008
Figure PCTCN2019078992-appb-000009
实施例3、化合物Ia和Ib抗病毒活性的测定
材料和方法
3.1.化合物：
将化合物Ia或Ib溶解于DMSO,－20℃保存,stock 10mg/ml；
3.2.HIV-1病毒：
包装pseudotyped single-cycle infectious HIV-1(HIV-luc/JRFL)所用到的质粒如下：pLAI-Δenv-Luc包含HIV-1的骨架基因序列，但删除了env和vpr基因，在nef基因位点***了Luciferase报告基因；pJRFL包含了CCR5嗜性的HIV-1的env基因；质粒共转染进入HEK293T细胞。收集细胞上清并过滤分装。病毒颗粒均用p24 gag capture ELISA进行定量。
3.3.HIV-1感染检测：
CD4 +T细胞(Hut/CCR5)培养在完全1640培养基(Gibco)中，其含质量百分含量10％胎牛血清(Gibco)，100U/ml盘尼西林(Invitrogen)和100U/ml链霉素(Invitrogen)。1×10 5Hut/CCR5细胞加入待测化合物Ia或Ib，然后感染HIV-luc/JRFL(1ng p24 gag)，37℃孵育3小时，洗去游离的病毒，然后利用培养基重悬，并加入相应浓度的待测化合物，37℃培养3天，最后收集细胞检测luciferase活性，计算病毒的复制和抑制率。AZT作为化合物对照(结果见图1和3)。
3.4细胞毒性检测(MTT Assay)：
将CD4 +T细胞(Hut/CCR5)铺至96孔板(1x10 4/ul/well)中，加入不同浓度的待测化合物，在37℃细胞培养箱内孵育3天后，加入20ul的MTT溶液(5mg/ml)，在37℃细胞培养箱内孵育4小时，然后每孔加入100ul的Formazan溶解液在培养箱中继续孵育，直至普通光学显微镜下观察发现Formazan全部溶解，570nm处测定吸光度(结果见图2和4)。
实施例4、化合物Ig-TP抗病毒活性的测定
材料和方法
4.1.化合物Ig-TP(CL-002-TP))抗HIV-1活性和细胞毒性的测定
4.2测定方法
CD4 +T细胞(Hut/CCR5)培养在完全1640培养基(Gibco)中，其含质量百分含量10％胎牛血清(Gibco)，100U/ml盘尼西林(Invitrogen)和100U/ml链霉素(Invitrogen)。1×10 5Hut/CCR5细胞加入化合物Ig-TP(CL-002-TP)，然后感染HIV-luc/JRFL(1ng p24 gag)，37℃孵育3小时，洗去游离的病毒，然后利用培养基重悬，并加入相应浓度的待测化合物，37℃培养3天，最后收集细胞检测luciferase活性，计算病毒的复制和抑制率。AZT作为化合物对照。
4.3实验结果
采用MTT方法考察化合物Ig-TP对CD4 +T细胞(Hut/CCR5)的细胞毒性。结果表明化合物Ig-TP对CD4 +T细胞毒性很小，CC 50为2328ng/ml，8134nM(图5)。活性数据表明化合物Ig-TP(CL-002-TP)对HIV-1具有很好的增殖抑制作用(图6,IC 50＝0.373ng/ml，1.3nM)。
实施例5、化合物Ia,3TC和化合物Ig在猕猴PBMC内活性代谢物研究
5.1实验材料
5.2药品与试剂
化合物Ia,3TC,化合物Ig(CL-002)；
Ia-TP,3TC-TP，Ig-TP；
EDTAK 2抗凝管，购自上海化学试剂研究所；
生理盐水，购自上海化学试剂站分装厂；
Ficoll-Paque plus(1.077g/ml)淋巴细胞分离液(6x100ml)，GE Healthcare公司；
5.3仪器
3ml巴士吸管；
50ml水平离心机，no braking；
电子分析天平，美国OHAUS公司；
KQ5200超声波清洗仪，昆山市超声仪器有限公司；
10ul、200ul、1ml移液枪，德国Eppendorf公司。
5.4实验动物
实验动物为太行山猕猴2只，雄性，体重分别为8和9kg，由河南师范大 学提供。
5.5猕猴空白血浆制备
取健康猕猴2只，禁食12h，不禁水，每只动物后肢静脉取血15-20ml，取3ml，3000rpm离心10min分离血浆，-20℃以下冷冻保存。剩余用Ficoll-Paque分离液分离外周血单核细胞(PBMC)，浓缩至0.3ml左右细胞液，计数，-20℃以下冷冻保存。
5.6猕猴灌胃Ia,Ig(CL-002)和3TC后药代动力学
化合物CL-002(或者3TC)灌胃溶液配制：称取130mg化合物CL-002，加至21.7ml生理盐水，40℃超声15min，配制成浓度为6mg/ml的药液作为大剂量。按1:5稀释至1mg/ml的药液作为小剂量。给药容积为1ml/kg，CL-002给药剂量分别为:6mg/kg和1mg/kg(3TC剂量为20mg/kg；Ia剂量为6mg/kg)。
取健康猕猴2只，随机分为2组(低、高剂量组)，每组1只，给药前禁食12h，不禁水，分别灌胃6mg/kg和1mg/kg的化合物CL-002(或者20mg/kg的3TC)，并分别于给药后1h、6h、24h、3天、5天、7天后肢静脉取血15-20ml，取1ml离心分离血浆，-20℃以下冷冻保存。剩余用Ficoll-Paque分离液分离外周血单核细胞(PBMC)，浓缩至0.3ml左右细胞液，计数，-20℃以下冷冻保存。
5.7血浆中化合物Ia,3TC和化合物Ig的测定
精密吸取50μL血浆样品，依次加入50μL内标(3TC，50ng/ml)溶液、200μL甲醇，于涡旋混合器上涡旋30s混合均匀，13000r·min -1离心5min，小心吸取250μL上清液转移至另一10ml圆底玻璃管中，置50℃氮气流吹干，250μL复溶(甲醇：水＝1:1)，涡旋60s后，转移至1.5ml EP管中，13000r·min -1离心5min，过滤，再离心，取上清进样10μL，进行LC-MS/MS分析。
5.8 PBMC中Ia-TP,3TC-TP和Ig-TP的测定
PBMC样品用超声细胞粉碎仪破碎，加入3倍体积乙腈，涡旋60s后，加入1倍体积水，涡旋60s，13000r·min -1离心5min，小心吸取全部上清液转移至10ml圆底玻璃管中，置40℃氮气流吹干，加500μL复溶(甲醇：水＝2:8)，涡旋60s后，转移至1.5ml EP管中，13000r·min -1离心5min，过滤，再离心，取上清进样10μL，进行LC-MS/MS分析。
5.9实验结果
表1.猕猴灌胃化合物Ia或3TC后PBMC中药物浓度
药物 化合物Ia-TP 3TC-TP
浓度 (pmol/10 6cells) (pmol/10 6cells)
剂量 6mg/kg 20mg/kg
1h 0.34 0.51
6h 0.51 2.45
24h 0.92 ND
72h 0.57 ND
120h 0.11 ND
168h >0.10 ND
ND：Not Detected.
表2.猕猴灌胃化合物Ig(CL-002)后血浆中药物浓度
Figure PCTCN2019078992-appb-000010
ND：Not Detected.
表3.猕猴灌胃化合物Ig(CL-002)后PBMC中药物浓度
Figure PCTCN2019078992-appb-000011
Figure PCTCN2019078992-appb-000012
ND：Not Detected.

Claims (9)

  1. 结构如通式I所述的化合物在药物制备中的应用，其特征在于，将其或其医药上可接受的盐、溶剂化物、晶体形式或前药或其组合物作为活性成分，用于制备预防或治疗艾滋病的长效药物：
    Figure PCTCN2019078992-appb-100001
    R选自乙炔基，叠氮或氰基；X选自氢或氟；B选自B1或B2。
  2. 如权利要求1所述化合物I在药物制备中的应用，其特征在于，选自如下化合物：
    Figure PCTCN2019078992-appb-100002
  3. 如权利要求2所述化合物I在药物制备中的应用，其特征在于，化合物Ia与其它HIV抑制剂联用形成组合物，所述HIV抑制剂选自：化合物Ig，替诺福韦或者替诺福韦艾拉酚胺，利匹韦林，度鲁特韦，Bictegravir，艾博卫泰。
  4. 如权利要求2所述化合物I在药物制备中的应用，其特征在于，化合物Ig与其它HIV抑制剂联用形成组合物，所述HIV抑制剂选自：化合物Ia-If其中之一，替诺福韦或者替诺福韦艾拉酚胺，利匹韦林，度鲁特韦，Bictegravir，艾博卫泰。
  5. 如权利要求1-4其中之一所述化合物I在药物制备中的应用，其特征在于，其用药间隔为2天或2天以上。
  6. 一种制备化合物Ia的方法，其特征在于，包括如下步骤：
    (a)、化合物1与氟化试剂氟化得到化合物2，
    Figure PCTCN2019078992-appb-100003
    R 1是羟基保护基，包括但不限于苄基或取代苄基；
    R 2是H,或者炔端保护基，包括但不限于如下基团：
    Figure PCTCN2019078992-appb-100004
    化合物1的氟化试剂选自但不限于DAST类氟化剂或其他氟化试剂；或者羟基的氟化通过离去基团对羟基的活化，然后由氟离子取代实现；氟化反应的溶剂可选自非质子性有机溶剂；
    (b)、化合物2通过脱去苄基保护基生成化合物Ia
    脱去化合物2的苄基类保护基的试剂选自金属钠-液氨，或者选自酸类、磺酸类。
  7. 化合物Ia的晶型A，其特征在于，其CuKα-XRPD图谱在2θ为6.13，7.77，9.43，10.61，11.88，12.44，12.80，13.88，14.28，14.66，15.51，17.09，17.55，17.88，18.95，21.18，21.86，22.40，22.87，23.52，24.93，25.69，26.63，28.60，30.46处有特征峰，其中2θ值误差范围为±0.2。
  8. 如权利要求7所述化合物Ia的晶型A，其特征在于，其CuKα-XRPD图谱如附图7所示。
  9. 如权利要求7或8所述化合物Ia的晶型A在药物制备中的应用，其特征在于，作为活性成分，用于制备预防或治疗艾滋病的药物。
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