CRYSTAL FORM, PREPARATION METHOD, AND APPLICATION OF 4′-SUBSTITUTED NUCLEOSIDE

Abstract

The present invention discloses a crystal form, a preparation method and an application of a 4′-substituted nucleoside compound I having the following structure, including salts, prodrugs, and compositions thereof. Animal pharmacokinetic studies demonstrated that the effective drug concentrations of Compound Ia and Compound Ig in HIV target cells, peripheral blood mononuclear cells (PBMC), were effective in inhibiting HIV replication after 7 and 5 days, respectively. Therefore, Compound I can be used as a long-acting drug for preventing and treating AIDS. R is selected from ethynyl, azide, and cyano groups, X is selected from hydrogen and fluorine, and B is selected from B1 and B2.

Claims

1-9. (canceled)

10. A method for prevention or treatment of AIDS, the method comprising administering to a patient in need thereof a compound of formula I, or a pharmaceutically acceptable salt, a solvate, a crystalline form, a prodrug, or a composition thereof; ##STR00018## ##STR00019## ##STR00020## ; wherein: R is ethynyl, azide or cyano; X is hydrogen or fluorine; and B is B1 or B2.

11. The method of claim 10, wherein the compound has one of the following formulas: ##STR00021## ##STR00022## ##STR00023## ##STR00024## ##STR00025## ##STR00026## ##STR00027## .

12. The method of claim 11, wherein the compound of formula Ia is used in combination with a HIV inhibitor, and the HIV inhibitor is selected form the group consisting of the compound of formula Ig, tenofovir, tenofovir alafenamide, rilpivirine, dolutegravir, bictegravir, and albuvirtide.

13. The method of claim 11, wherein the compound of formula Ig is used in combination with a HIV inhibitor, and the HIV inhibitor is selected form the group consisting of the compound Ia, Ib, Ic, Id, Ie, If, tenofovir, tenofovir alafenamide, rilpivirine, dolutegravir, bictegravir, and albuvirtide.

14. The method of claim 10, wherein the compound is administered to the patient in need thereof with an interval of 2 days or more.

15. A method for preparing a compound of formula Ia: ##STR00028## the method comprising: a) fluorinating a compound of formula 1 with a fluorinating agent to form a compound of formula 2; ##STR00029## ##STR00030## wherein: R.sup.1 is a protecting group for a hydroxyl group and comprises benzyl or substituted benzyl; R.sup.2 is a protecting group for a terminal alkyne and comprises ##STR00031## ; the fluorinating agent comprises diethylaminosulfur trifluoride (DAST); or the hydroxyl group is activated by a leaving group and substituted by a fluoride ion to achieve fluorination; and a solvent for the fluorination reaction is an aprotic organic solvent; and b) removing the protecting group from the compound of formula 2 to form the compound of formula Ia; wherein a reagent for removing the protecting group from the compound of formula 2 is selected from metallic sodium-liquid ammonia, acids, and sulfonic acids.

16. A crystal form A of a compound of formula Ia: ##STR00032## the crystal form A having diffraction peaks in a CuKα X-ray powder diffraction (XRPD) spectrum when a diffraction angle 2θ is 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; and an error range of 2θ is ±0.2.

17. The crystal form A of claim 16, wherein the CuKα X-ray powder diffraction (XRPD) spectrum is shown in FIG. 7.

18. A method for prevention or treatment of AIDS, the method comprising administering to a patient in need thereof a pharmaceutical composition comprising the crystal form A of the compound of formula Ia of claim 16.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0035] FIG. 1 is a histogram of anti-HIV activity of the compound Ia;

[0036] FIG. 2 is a histogram of the toxicity of the compound Ia to inhibit proliferation of HIV-infected cells;

[0037] FIG. 3 is a histogram of anti-HIV activity of the compound Ib;

[0038] FIG. 4 is a histogram of the toxicity of the compound Ib to inhibit proliferation of HIV-infected cells;

[0039] FIG. 5 is a histogram of anti-HIV activity of the compound Ig-TP;

[0040] FIG. 6 is a histogram of the toxicity of the compound Ig-TP to inhibit proliferation of HIV-infected cells; and

[0041] FIG. 7 is an XRPD spectrum of the compound Ia.

DETAILED DESCRIPTION

[0042] To further illustrate, embodiments detailing 4′-substituted nucleoside derivatives and use thereof are described below. It should be noted that the following embodiments are intended to describe and not to limit the disclosure.

Example 1

Preparation of Compound 1

[0043] The compound 1 is prepared based on a method according to a literature (K. Fukuyama et al Org. Lett.2015,17, 828-831).

Example 2

[0044] Preparation of compound Ia

##STR00017##

[0045] Raw material 1 (9.64 g, 15.97 mmol, 1.0eq) and pyridine (7.7 mL, 95.8 mmol, 6.0eq) were added to 100 mL of dry toluene; diethylaminosulfur trifluorid (DAST) (15.4 g, 95.8 mmol, 6.0eq) was dissolved in 40 mL of dry toluene; the resulting solution was added dropwise to the reaction solution at 0° C. in the presence of nitrogen; after the addition was complete, the reaction solution was heated to 50° C., refluxed for 6 hours, detected by liquid chromatography- mass spectrometry (LC-MS) until the reaction was completed, and cooled; the saturated sodium bicarbonate solution was added dropwise to the cooled reaction solution at the low temperature for quenching; the resulting solution was adjusted to pH 7-8, followed by addition of ethyl acetate (EA); the resulting organic phase was washed with water, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, condensed, beated with polyethylene (PE), and filtered to yield 2.44 g of crude compound 2 which was directly used in the next step. 1H NMR (400 MHz, CDCl3) δ 8.01 (d, J = 2.0 Hz, 1H), 7.44 - 7.28 (m, 10H), 6.53 (dd, J = 11.4, 5.1 Hz, 1H), 5.86 (s, 2H), 5.37 (dt, J = 53.3, 4.9 Hz, 1H), 4.73 (s, 2H), 4.69 - 4.48 (m, 3H), 3.77 (dt, J = 26.1, 6.2 Hz, 2H), 0.99 (t, J = 7.9 Hz, 9H), 0.63 (q, J = 8.0 Hz, 6H); 19F NMR (376 MHz, CDCl3) δ -50.83, -196.65.

[0046] The compound 2 (7.37 g crude product, 12.17 mmol, 1.0eq) and ammonium fluoride (2.25 g, 60.83 mmol, 1.5eq) were added to 50 mL of methanol, allowed to react at 50° C. for 6 hours in the presence of nitrogen, detected by liquid chromatography- mass spectrometry (LC-MS) until the reaction was completed; the resulting solvent was removed with a rotary evaporator, followed by addition of EA; the resulting organic phase was washed with water, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, condensed, poured into a column using a dry packing method to yield 3.24 g of compound 3 (the total yield of the compounds 12 to 15 is about 30-40 %). 1H NMR (400 MHz, CDCl3) δ 7.94 (d, J = 2.2 Hz, 1H), 7.42 - 7.27 (m, 10H), 6.54 (dd, J = 12.1, 5.1 Hz, 1H), 6.03 (s, 2H), 5.56 - 5.21 (m, 1H), 4.82 - 4.49 (m, 5H), 3.76 (dt, J = 26.5, 6.2 Hz, 2H), 2.73 (s, 1H).; 19F NMR (376 MHz, CDCl3) δ -50.78, -195.84; m/z(ESI) (M+H).sup.+=492.2, m/z(ESI) (M+Na).sup.+=514.3.

[0047] The compound 3 (1.71 g, 3.48 mmol) was dissolved in 26 mL of chloroform, followed by dropwise addition of 6.84 mL of methanesulfonic acid; the mixture was allowed to react at room temperature for 5 hours in the presence of nitrogen, detected by liquid chromatography- mass spectrometry (LC-MS) until the reaction was completed; the saturated sodium bicarbonate solution was added dropwise to the resulting solution at a low temperature for quenching, adjusted to pH 7-8, followed by addition of EA. The organic phase is separated from the resulting solution, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, condensed, and poured into a column (DCM: MeOH=20:1-10:1) using a dry packing method to yield a compound Ia (748 mg, 69%). 1H NMR (400 MHz, MeOD) δ 8.25 (d, J = 1.7 Hz, 1H), 6.46 (dd, J = 8.9, 5.7 Hz, 1H), 5.35 (dt, J = 53.9, 5.8 Hz, 1H), 4.75 (dd, J = 22.3, 5.9 Hz, 1H), 3.88 (dd, J = 12.3, 1.8 Hz, 1H), 3.80 (d, J = 12.3 Hz, 1H), 3.22 (s, 1H).; 19F NMR (376 MHz, MeOD) δ -53.22, -201.69; m/z(ESI) (M+H).sup.+=312.2, m/z(ESI)(M+Na).sup.+=334.1.

[0048] Referring to FIG. 7, the crystal A of the compound Ia had diffraction peaks at 2θ angles (±0.2): 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 in a diffraction spectrum using CuKα radiation with a wavelength of λ=1.5418 Å.

TABLE-US-00001 Diffraction peaks at 2θ angles and corresponding relative intensities (%) of the crystal A of compound Ia 20 (±0.2°) Relative intensities 1% 6.13 4.5 7.77 7.7 9.43 100.0 10.61 28.0 11.88 26.7 12.44 23.7 12.80 14.6 13.88 58.2 14.28 22.8 14.66 23.2 15.51 30.5 17.09 27.6 17.55 12.6 17.88 5.9 18.95 65.2 21.18 6.4 21.86 7.4 22.40 11.2 22.87 9.7 23.52 79.3 24.93 29.6 25.69 24.5 26.63 23.5 28.60 52.1 30.46 13.4

Example 3

Measurement of Anti-HIV Activity of the Compounds Ia and Ib

Materials and Methods

3.1 Compound

[0049] The compound Ia or compound Ib was dissolved in DMSO to achieve a final concentration of 10 mg/ml and stored at -20° C.

3.2 HIV-1

[0050] The plasmids used for packaging pseudotyped single-cycle infectious HIV-1 (HIV-luc/JRFL) are as follows: the plasma pLAI-Δenv-Luc contains HIV-1 isolate in which Env and Vpr genes are deleted and luciferase gene is inserted into the Nef locus; and the plasma pJRFL contains CCR5-tropic HIV-1 env gene; the two plasmas are co-transfected into HEK293T cells; the supernatant was collected, filtered, and packaged; and the virion capture was quantified by p24.sup.gag ELISA.

3.3 Detection of HIV-1 Infection

[0051] CD4.sup.+ T cells (Hut/CCR5) were cultured in RPMI 1640 medium (Gibco) containing 10% fetal bovine serum (Gibco), 100 U/mL penicillin (Invitrogen), and 100 U/mL streptomycin (Invitrogen). 1×10.sup.5 Hut/CCR5 cells were added to the compound Ia or Ib, infected with HIV-luc/JRFL (1 ng p24.sup.gag), incubated at 37° C. for 3 hours, washed to remove free virus, and resuspended in cell culture medium, followed by addition of the corresponding compound to be tested, incubated at 37° C. for 3 days, and collected for measurement of the luciferase activity and calculation of the percentage of viral inhibition. AZT was used as a control group (as shown in FIGS. 1 and 3).

3.4 Measurement of Cell Cytotoxicity (MTT Assay)

[0052] CD4.sup.+ T cells (Hut/CCR5) were plated into the wells of a 96-well plate (1x10.sup.4 uL/well), followed by addition of different compounds to be tested; the 96-well plate was incubated at 37° C. in an incubator for 3 days; 20 uL of MTT solution was added into the wells (5 mg/well) of the 96-well plate; the 96-well plate was incubated at 37° C. in the incubator for 4 hours; 100 uL of Formazan solution was added into the wells of the 96-well plate; the 96-well plate was incubated at 37° C. in the incubator until the complete dissolution of Formazan was observed by a common optical microscope; and the absorbance of the solution was measured at 570 nm (as shown in FIGS. 2 and 4).

Example 4

Measurement of Anti-HIV Activity of the Compound Ig-TP

Materials and Methods

4.1 Measurement of the Anti-HIV Activity and Cell Cytotoxicity of the Compound Ig-TP (CL-002-TP)

4.2 Measurement Method

[0053] CD4.sup.+ T cells (Hut/CCR5) were cultured in 1640 medium (Gibco) containing 10% fetal bovine serum (Gibco), 100 U/mL penicillin (Invitrogen), and 100 U/mL streptomycin (Invitrogen). 1×10.sup.5 Hut/CCR5 cells were added to the compound Ig-TP (CL-002-TP), infected with HIV-luc/JRFL (1 ng p24.sup.gag), incubated at 37° C. for 3 hours, washed to remove free virus, and resuspended in cell culture medium, followed by addition of the corresponding compound to be tested, incubated at 37° C. for 3 days, and collected for measurement of the luciferase activity and calculation of the percentage of virus inhibition. AZT was used as a control group (as shown in FIGS. 1 and 3).

4.3 Experimental Results

[0054] The cytotoxicity of the compound Ig-TP on CD4.sup.+ T cells (Hut/CCR5) was measured using MTT assay. The results show that the compound Ig-TP has a low toxicity to CD4.sup.+ T cells, with a cytotoxic concentration (CC50) value of 2,328 ng/mL (8,134 nM in FIG. 5). The results also show that the compound Ig-Tp is potent in inhibiting HIV viral proliferation (as shown in FIG. 6, IC50 = 0.373 ng/ml, 1.3 nM).

Example 5

[0055] Active metabolites of compounds Ia, 3TC, and Ig in PBMC of rhesus monkey

5.1 Experimental Materials

5.2 Drugs and Reagents

[0056] Compounds Ia, 3TC, and Ig (CL-002); [0057] Ia-TP, 3TC-TP, Ig-TP; [0058] Anticoagulant EDTAK2, which is purchased from Shanghai Institute of Chemical Reagents; [0059] Normal saline, which is purchased from Shanghai Chemical Reagent Station Sub-assembly Factory; and [0060] Lymphocyte separation medium (Ficoll-Paque PLUS, 1.077 g/mL, 6 × 100 mL), GE Healthcare.

5.3 Experimental Equipment

[0061] 3 mL pasteur pipette; [0062] 50 ml horizontal centrifuge, no braking; [0063] Electronic analytical balance, OHAUS, USA; [0064] KQ5200 Ultrasonic Cleaner, Kunshan Ultrasonic Instrument Co., Ltd.; and [0065] 10 uL, 200 uL, and 1 mL pipettes, Eppendorf, Germany.

5.4 Laboratory Animals

[0066] Two male rhesus macaques weighing 8 and 9 kg, were provided by Henan Normal University.

5.5 Preparation of Blank Plasma From Rhesus Monkeys

[0067] The two male rhesus macaques were fasted without access to water for 12 hours; 15-20 mL of venous blood was collected from the hind limbs of each male rhesus macaque; 3 mL of venous blood was centrifuged at 3000 rpm for 10 minutes to separate the plasma from the venous blood, and stored below -20° C. The PBMC was separated from the remaining venous blood with Ficoll-Paque separation medium, condensed to 0.3 mL of cell suspension, counted, and stored below -20° C.

[0068] 5.6 Pharmacokinetics of the compounds Ia, Ig (CL-002) and 3TC after oral administration into rhesus monkeys: 130 mg of the compound CL-002 was added to 21.7 mL of normal saline, and sonicated at 40° C. for 15 minutes to yield a liquid medicine with a concentration of 6 mg/mL; the liquid medicine was diluted to 1 mg/mL in a ratio of 1:5; 6 mg/mL of the liquid medicine is used as a high-dose group and 1 mg/mL of the liquid medicine is used as a low-dose group; the dosage volume was 1 mL/kg; and the compound CL-002 was administered to the rhesus monkeys at 6 mg/kg and 1 mg/kg, respectively (the compound 3TC was administered to the rhesus monkeys at 20 mg/kg; and the compound Ia was to the rhesus monkeys at 6 mg/kg).

[0069] The two male rhesus macaques were randomly divided into a high-dose group and a low-dose group, and one rhesus macaque in each group; the two healthy rhesus macaques are fasted without access to water 12 hours; 1 mg/kg and 6 mg/kg of the compound CL-002 (or 20 mg/kg of 3TC) were orally administered to the rhesus macaques; 15-20 ml of venous blood was collected from the hind limbs of rhesus macaque at 1 h, 6 h, 24 h, 3 days, 5 days, and 7 days after administration; 1 mL of venous blood was centrifuged at 3000 rpm for 10 min to separate the plasma from the venous blood; and stored below -20° C. The PBMC was separated from the remaining venous blood with Ficoll-Paque separation medium, condensed to 0.3 mL of cell suspension, counted, and stored frozen below -20° C.

[0070] 5.7 Measurement of the compounds Ia, 3TC and Ig: 50 .Math.L of the plasma was aspirated; 50 .Math.L of internal standard solution (3TC, 50 ng/mL) and 200 .Math.L of methanol were added to the 50 .Math.L of the plasma in order, vortexed on a vortex mixer for 30 seconds, and centrifuged at 13000 rpm for 5 minutes; 250 .Math.L of the supernatant was transferred to a 10 mL round-bottom glass tube, dried in a stream of nitrogen at 50° C., redissolved in 250 mL of methanol solution (methanol: water = 1:1), vortexed for 60 minutes, transferred into a 1.5 mL EP tube, centrifuged at 13000 rpm for 5 minutes, filtered, and centrifuged to obtain 10 .Math.L of supernatant for LM-MS/MS analysis.

5.8 Measurement of the Compounds Ia, 3TC and Ig in PBMC

[0071] The PBMC sample was crushed with an ultrasonic cell crusher, followed by addition of 3 times volume of acetonitrile, vortexed for 60 seconds, followed by 1 volume of the water, vortexed for 60 seconds, and centrifuged at 3000 rpm for 5 minutes; the supernatant was aspirated, transferred into a 10 mL round-bottom glass tube, dried in a stream of nitrogen at 40° C., and redissolved in 500 .Math.L of methanol solution (methanol: water = 2:8 (v/v)); the resulting solution was vortexed for 60 seconds, transferred into a 1.5 mL EP tube, centrifuged at 13000 rpm for 5 minutes, filtered, and centrifuged to obtain 10 L of supernatant for LM-MS/MS analysis.

[0072] 5.9 Experimental results

TABLE-US-00002 Drug concentration in PBMC after oral administration of compound Ia or 3TC in rhesus monkeys Drug Compound Ia-TP Compound 3TC-TP Concentration (pmol/10.sup.6 cells) (pmol/10.sup.6 cells) dosage 6 mg/kg 20 mg/kg 1 h 0.34 0.51 6h 0.51 2.45 24 h 0.92 ND 72 h 0.57 ND 120 h 0.11 ND 168 h >0.10 ND ND: Not Detected

TABLE-US-00003 Drug concentration in plasma after oral administration of compound Ig (CL-002) in rhesus monkeys Drug Compound Ig Concentration (Pmol/10.sup.6 cells) dosage 1 mg/kg 6 mg/kg 1 h 196.50 2068.29 6 h 3.15 204.48 24 h ND 6.54 72 h ND ND 120 h ND ND 168 h ND ND ND: Not Detected

TABLE-US-00004 Drug concentration in PBMC after oral administration of compound Ig (CL-002) in rhesus monkeys Drug Compound Ig Compound Ig-TP Concentration (pmol/10.sup.6 cells) (pmol/10.sup.6 cells) dosage 1 mg/kg 6 mg/kg 1 mg/kg 6 mg/kg 1 h 0.025 0.44 0.39 0.23 6 h ND 0.09 2.10 4.07 24 h ND ND 0.29 0.44 72 h ND ND 0.66 1.85 120 h ND ND 0.54 1.35 168 h ND ND ND ND ND: Not Detected