METHOD FOR PREPARING DRUG TOXIN PNU-159682 FOR ANTIBODY DRUG CONJUGATE, AND INTERMEDIATES THEREIN

Abstract

The disclosure provides a method for preparing drug toxin PNU-159682 (morpholinyl anthracycline derivative) for antibody-conjugated drugs and intermediates involved in the preparation method. The preparation method of the present disclosure improves the stability, practicability and scalability of the process by introducing protecting groups and changing to reagents that are capable to amplify; the preparation method of the present disclosure minimizes the risk and operational difficulty during scale-up production; production and operation is simple and convenient.

Claims

1. An intermediate compound for synthesis of PNU-159682, a structural formula of which is: ##STR00040## wherein, R is selected from a group consisting of trimethylsilyl TMS, tert-butyldimethylsilyl TBS, tert-butyldiphenylsilyl TBDPS, diethyl isopropylsilyl DEIPS, triisopropylsilyl TIPS, triphenylsilyl TPS, trimethylsilyl Mes, benzyl Bn, p-methoxybenzyl PMB, trityl Tr, 2-tetrahydropyranyl THP, methoxymethyl MOM, 2-ethoxyethyl EE, 2-(trimethylsilyl)ethoxymethyl SEM, Allyl, acetyl Ac, benzoyl Bz, and pivaloyl Pv.

2. An intermediate compound for synthesis of PNU-159682, a structural formula of which is: ##STR00041## wherein, R is selected from a group consisting of trimethylsilyl TMS, tert-butyldimethylsilyl TBS, tert-butyldiphenylsilyl TBDPS, diethyl isopropylsilyl DEIPS, triisopropylsilyl TIPS, Triphenylsilyl TPS, trimethylsilyl Mes, benzyl Bn, p-methoxybenzyl PMB, trityl Tr, 2-tetrahydropyranyl THP, methoxymethyl MOM, 2-ethoxyethyl EE, 2-(trimethylsilyl)ethoxymethyl SEM, Allyl, acetyl Ac, benzoyl Bz, and pivaloyl Pv.

3. A method for synthesizing an intermediate compound as ##STR00042## of PNU-159682, wherein, the method is: in solvent X, in the presence of the reagent Y, a compound of structural formula ##STR00043## is substituted by a protective group R to obtain an intermediate compound of structural formula: ##STR00044## wherein, R is selected from a group consisting of trimethylsilyl TMS, tert-butyldimethylsilyl TBS, tert-butyldiphenylsilyl TBDPS, diethyl isopropylsilyl DEIPS, triisopropylsilyl TIPS, triphenylsilyl TPS, trimethylsilyl Mes, benzyl Bn, p-methoxybenzyl PMB, trityl Tr, 2-tetrahydropyranyl THP, methoxymethyl MOM, 2-ethoxyethyl EE, 2-(trimethylsilyl)ethoxymethyl SEM, Allyl, acetyl Ac, benzoyl Bz, and pivaloyl Pv; Solvent X is selected from a group consisting of dichloromethane, 1,2-dichloroethane, chloroform, carbon tetrachloride, acetonitrile, acetone, ethyl acetate, methyl acetate, water, benzene, ethyl ether, ethylene glycol dimethyl ether, methyl tert-butyl ether, diphenyl ether, 1,4-dioxane, N, N-dimethylformamide, N, N-dimethylacetamide, tetrahydrofuran, 2-methyltetrahydrofuran, and a mixture thereof; Reagent Y is selected from a group consisting of 4-dimethylaminopyridine, pyridine, imidazole, trimethylamine, triethylamine, diisopropyldiamine and a mixture thereof; Reaction temperature is 20° C. subzero to 80° C.

4. The method according to claim 3, wherein the solvent X is N,N-dimethylformamide, the reagent Y is imidazole, and the reaction temperature is 20′C to 25° C.

5. A method for synthesizing an intermediate compound ##STR00045## of PNU-159682, wherein, the method is: a compound of structural formula ##STR00046## is in solvent M, and add oxidizing reagent N to perform an oxidation reaction to obtain a compound of structural formula ##STR00047## wherein, R is selected from a group consisting of trimethylsilyl TMS, tert-butyldimethylsilyl TBS, tert-butyldiphenylsilyl TBDPS, diethyl isopropylsilyl DEIPS, triisopropylsilyl TIPS, triphenylsilyl TPS, trimethylsilyl Mes, benzyl Bn, p-methoxybenzyl PMB, trityl Tr, 2-tetrahydropyranyl THP, methoxymethyl MOM, 2-ethoxyethyl EE, 2-(trimethylsilyl)ethoxymethyl SEM, Allyl, acetyl Ac, benzoyl Bz, and pivaloyl Pv; The solvent M is selected from a group consisting of dichloromethane, 1,2-dichloroethane, chloroform, carbon tetrachloride, acetonitrile, acetone, ethyl acetate, methyl acetate, water, benzene, ether, ethylene glycol dimethyl ether, methyl tert-butyl ether, diphenyl ether, 1,4-dioxane, N,N-dimethylformamide, N,N-dimethylacetamide, tetrahydrofuran, 2-methyltetrahydrofuran and a mixture thereof; The reagent N is selected from a group consisting of Jones reagent, Collins reagent (CrO3.2Py), pyridinium chlorochromate (PCC), pyridine dichromate (PDC), manganese dioxide, DMSO, Dess-Martin oxidant, potassium permanganate, periodic acid, osmium tetroxide, 30% hydrogen peroxide, m-chloroperoxybenzoic acid m-CPBA, tert-butyl hydroperoxide TBHP, acetone peroxy (DMDO) and a mixture thereof; Reaction temperature is 78° C. subzero to 25′C.

6. The method according to claim 5, wherein the reagent N is m-chloroperoxybenzoic acid m-CPBA, the solvent M is methylene chloride, and the reaction temperature is 40° C. subzero to 0° C.

7. A method for synthesizing an intermediate compound ##STR00048## of PNU-159682, wherein, the method is: in solvent O, perform a dehydration cyclization reaction on a compound of structure ##STR00049## in the presence reagent P to generate an intermediate compound ##STR00050## wherein, R is selected from a group consisting of trimethylsilyl TMS, tert-butyldimethylsilyl TBS, tert-butyldiphenylsilyl TBDPS, diethyl isopropylsilyl DEIPS, triisopropylsilyl TIPS, Triphenylsilyl TPS, trimethylsilyl Mes, benzyl Bn, p-methoxybenzyl PMB, trityl Tr, 2-tetrahydropyranyl THP, methoxymethyl MOM, 2-ethoxyethyl EE, 2-(trimethylsilyl)ethoxymethyl SEM, Allyl, acetyl Ac, benzoyl Bz, and pivaloyl Pv; The solvent O is selected from a group consisting of dichloromethane, 1,2-dichloroethane, trichloromethane, carbon tetrachloride, acetonitrile, acetone, ethyl acetate, methyl acetate, benzene, toluene, mesitylene, xylene, chlorobenzene, diethyl ether, ethylene glycol dimethyl ether, methyl tert-butyl ether, diphenyl ether, 1,4-dioxane, N,N-dimethylformamide, N,N-dimethylacetamide, tetrahydrofuran, 2-methyltetrahydrofuran and a mixture thereof; The solvent O is selected from a group consisting of dichloromethane, 1,2-dichloroethane, chloroform, carbon tetrachloride, acetonitrile, acetone, ethyl acetate, methyl acetate, benzene, toluene, mesitylene, xylene, chlorobenzene, diethyl ether, ethylene glycol dimethyl ether, methyl tert-butyl ether, diphenyl ether, 1,4-dioxane, N,N-dimethylformamide, N,N-dimethylacetamide, tetrahydrofuran, 2-methyltetrahydrofuran, and a mixture thereof; The reagent P is selected from a group consisting of dicyclohexylcarbodiimide (DCC), polyphosphoric acid, Burgess reagent, bis[a,a-bis(trifluoromethyl)phenethyl alcohol]-diphenylsulfide, carbon disulfide, iodomethane, sodium hydroxide, potassium hydroxide, lithium hydroxide, sodium hydride, potassium tert-butoxide, sodium tert-butoxide, sodium methoxide, sodium ethoxide, n-butyl lithium, lithium diisopropylamide, cyanuric chloride and a mixture thereof; Reaction temperature is 50° C. subzero to 50′C.

8. The method according to claim 7, wherein the reagent P is Burgess reagent, the solvent O is dichloromethane, and the reaction temperature is 40° C. subzero to 25° C.

9. A method for synthesizing PNU-159682, wherein, the method is: in solvent Q, perform a deprotection reaction on a compound of structure ##STR00051## in the presence reagent S, to generate compound PNU-159682 ##STR00052## wherein, R is selected from a group consisting of trimethylsilyl TMS, tert-butyldimethylsilyl TBS, tert-butyldiphenylsilyl TBDPS, diethyl isopropylsilyl DEIPS, triisopropylsilyl TIPS, triphenylsilyl TPS, trimethylsilyl Mes, benzyl Bn, p-methoxybenzyl PMB, trityl Tr, 2-tetrahydropyranyl THP, methoxymethyl MOM, 2-ethoxyethyl EE, 2-(trimethylsilyl)ethoxymethyl SEM, Allyl, acetyl Ac, benzoyl Bz, and pivaloyl Pv; The solvent Q is selected from a group consisting of dichloromethane, 1,2-dichloroethane, chloroform, carbon tetrachloride, acetonitrile, methanol, ethanol, acetone, ethyl acetate, methyl acetate, benzene, toluene, mesitylene, xylene, chlorobenzene, ethyl ether, ethylene glycol dimethyl ether, methyl tert-butyl ether, diphenyl ether, 1,4-dioxane, N,N-dimethylformamide, N,N dimethylacetamide, tetrahydrofuran, 2-methyltetrahydrofuran and a mixture thereof; The reagent S is selected from a group consisting of hydrochloric acid methanol solution, hydrochloric acid ethanol solution, hydrochloric acid 1,4-dioxane solution, hydrochloric acid ether solution, hydrochloric acid tetrahydrofuran, tetrahydrofuran acetate solution, tetramethylammonium fluoride, tetraethylammonium fluoride, tetra-n-butylammonium fluoride (TBAF), Pd/C catalytic hydrogenation, DDQ, p-toluenesulfonic acid, methanol/sodium hydroxide, methanol/sodium methoxide and a mixture thereof; Reaction temperature is 50° C. subzero to 50° C.

10. The method according to claim 9, wherein the reagent S is tetra-n-butylammonium fluoride (TBAF), the solvent Q is tetrahydrofuran, and the reaction temperature is 30° C. subzero to 25° C.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0039] FIG. 1a is a liquid chromatogram of compound A1 synthesized in the present disclosure.

[0040] FIG. 1b is a graph of liquid chromatography data of compound A1 synthesized in the present disclosure.

[0041] FIG. 2a is a gas chromatogram of compound A1 synthesized in the present disclosure.

[0042] FIG. 2b is a mass spectrum of compound A1 synthesized in the present disclosure.

[0043] FIG. 3 is a NMR spectrum of compound A1 synthesized by the present disclosure.

[0044] FIG. 4a is a liquid chromatogram of compound A2 synthesized in the present disclosure.

[0045] FIG. 4b is a graph of liquid chromatographic data of compound A2 synthesized in the present disclosure.

[0046] FIG. 5a is a gas chromatogram of compound A2 synthesized in the present disclosure.

[0047] FIG. 5b is a mass spectrum of compound A2 synthesized in the present disclosure.

[0048] FIG. 6a is a liquid chromatogram of compound A3 synthesized in the present disclosure.

[0049] FIG. 6b is a graph of liquid chromatography data of compound A3 synthesized in the present disclosure.

[0050] FIG. 7a is a gas chromatogram of compound A3 synthesized in the present disclosure.

[0051] FIG. 7b is a mass spectrum of compound A3 synthesized in the present disclosure.

[0052] FIG. 8 is a NMR spectrum of compound A3 synthesized in the present disclosure.

[0053] FIG. 9a is a liquid chromatogram of the compound PNU-159682 synthesized in the present disclosure.

[0054] FIG. 9b is a graph of liquid chromatographic data of the compound PNU-159682 synthesized in the present disclosure.

[0055] FIG. 10a is a gas chromatogram of the compound PNU-159682 synthesized in the present disclosure.

[0056] FIG. 10b is a mass spectrum of the compound PNU-159682 synthesized in the present disclosure.

[0057] FIG. 11 is a NMR spectrum of the compound PNU-159682 synthesized in the present disclosure.

[0058] FIG. 12a is a liquid chromatogram of compound B1 synthesized in the present disclosure.

[0059] FIG. 12b is a graph of liquid chromatographic data of compound B1 synthesized in the present disclosure.

[0060] FIG. 13a is a gas chromatogram of compound B1 synthesized in the present disclosure.

[0061] FIG. 13b is amass spectrum of compound B1 synthesized in the present disclosure.

[0062] FIG. 14 is a NMR spectrum of compound B1 synthesized in the present disclosure.

[0063] FIG. 15a is a liquid chromatogram of compound C1 synthesized in the present disclosure.

[0064] FIG. 15b is a graph of liquid chromatography data of compound C1 synthesized in the present disclosure.

[0065] FIG. 16a is a gas chromatogram of compound C1 synthesized in the present disclosure.

[0066] FIG. 16b is a mass spectrum of compound C1 synthesized in the present disclosure.

[0067] FIG. 17 is a NMR spectrum of compound C1 synthesized in the present disclosure.

[0068] FIG. 18a is a liquid chromatogram of compound D1 synthesized in the present disclosure.

[0069] FIG. 18b is a graph of liquid chromatography data of compound D1 synthesized in the present disclosure.

[0070] FIG. 19a is a gas chromatogram of compound D1 synthesized in the present disclosure.

[0071] FIG. 19b is a mass spectrum of compound D1 synthesized in the present disclosure.

[0072] FIG. 20 is a NMR spectrum of compound D1 synthesized in the present disclosure.

[0073] FIG. 21a is a gas chromatogram of compound B2 synthesized in the present disclosure.

[0074] FIG. 21b is amass spectrum of compound B2 synthesized in the present disclosure.

[0075] FIG. 22a is a liquid chromatogram of compound B2 synthesized in the present disclosure.

[0076] FIG. 22b is a graph of liquid chromatography data of compound B2 synthesized in the present disclosure.

[0077] FIG. 23a is a gas chromatogram of compound B3 synthesized in the present disclosure.

[0078] FIG. 23b is a mass spectrum of compound B3 synthesized in the present disclosure.

[0079] FIG. 24 is a NMR spectrum of compound B3 synthesized in the present disclosure.

[0080] FIG. 25a is a gas chromatogram of compound C2 synthesized in the present disclosure.

[0081] FIG. 25b is a mass spectrum of compound C2 synthesized in the present disclosure.

[0082] FIG. 26a is a liquid chromatogram of compound C2 synthesized in the present disclosure.

[0083] FIG. 26b is a graph of liquid chromatography data of compound C2 synthesized in the present disclosure.

[0084] FIG. 27a is a gas chromatogram of compound C3 synthesized in the present disclosure.

[0085] FIG. 27b is a mass spectrum of compound C3 synthesized in the present disclosure.

[0086] FIG. 28 is a NMR spectrum of compound C3 synthesized in the present disclosure.

[0087] FIG. 29 is a molecular structure diagram of compound PNU-159682 synthesized in the present disclosure.

DESCRIPTION OF THE EMBODIMENTS

[0088] The technical solution of the present disclosure will be further non-restrictively described in detail below with reference to specific embodiments. It should be pointed out that the following embodiments are merely to illustrate the technical concept and features of the present disclosure, and their purpose is to enable those people familiar with the technology to understand the content of the present disclosure and implement them accordingly, but should not limit the protection scope of the present disclosure. All equivalent variations or modifications made according to the spirit of the present disclosure should be covered by the protection scope of the present disclosure.

[0089] LCMS refers to liquid chromatography mass spectrometry method; HPLC refers to high-performance liquid chromatography detection.

[0090] The raw materials and reagents for the reaction involved in the present disclosure are commercially available or prepared according to the method of the present disclosure.

[0091] The present disclosure provides a method for synthesizing PNU-159682, which includes the following steps:

##STR00023##

[0092] First, dissolve the compound (nemorubicin) of structural formula

##STR00024##

in solvent X, which is selected from a group consisting of dichloromethane, 1,2-dichloroethane, chloroform, carbon tetrachloride, acetonitrile, acetone, ethyl acetate, methyl acetate, water, benzene, ethyl ether, ethylene glycol dimethyl ether, methyl tert-butyl ether, diphenyl ether, 1,4-dioxane, N,N-Dimethylformamnide, N,N-dimethylacetamide, tetrahydrofuran, 2-methyltetrahydrofuran and a mixture thereof, in the presence of one or more of 4-dimethylaminopyridine, pyridine, imidazole, trimethylamine, triethylamine and diisopropyl diamine, it reacts with the substitution reagent with a protective group R, that is, it is substituted with the protective group R to obtain an intermediate compound of the structural formula

##STR00025##

The protective group R is selected from a group consisting of trimethylsilyl TMS, tert-butyldimethylsilyl TBS, tert-butyldiphenylsilyl TBDPS, diethyl isopropylsilyl DEIPS, triisopropylsilyl TIPS, triphenylsilyl TPS, trimethyl Silyl Mes, benzyl Bn, p-methoxybenzyl PMB, trityl Tr, 2-tetrahydropyranyl THP, methoxymethyl MOM, 2-ethoxyethyl EE, 2-(Trimethylsilyl)ethoxymethyl SEM, Allyl, acetyl Ac, benzoyl Bz, pivaloyl Pv; the reaction temperature of the substitution reaction in this step is 20′C subzero to 80° C., Preferably it is 20 to 25° C.

[0093] Dissolve the compound

##STR00026##

obtained in the previous step in an appropriate solvent M, which is selected from a group consisting of dichloromethane, 1,2-dichloroethane, chloroform, carbon tetrachloride, acetonitrile, acetone, ethyl acetate, methyl acetate, water, benzene, ethyl ether, ethylene glycol dimethyl ether, methyl tert-butyl ether, diphenyl ether, 1,4-dioxane, N,N-dimethylformamide, N,N-dimethylacetamide, tetrahydrofuran, 2-methyltetrahydrofuran and a mixture thereof, adding oxidizing reagent N, reagent N is selected from a group consisting of Jones reagent, Collins reagent (CrO3.2Py), pyridinium chlorochromate (PCC), pyridine dichromate (PDC), manganese dioxide, DMSO, Dess-Martin oxidant, potassium permanganate, periodic acid, osmium tetroxide, 30% hydrogen peroxide, m-chloroperoxybenzoic acid m-CPBA, tert-butyl hydroperoxide TBHP, acetone peroxy (DMDO) and mixture thereof,

##STR00027##

is obtained by oxidation reaction, the oxidation reaction temperature is 50′C subzero to 50′C, preferably 40° C. subzero to 25′C. After the reaction is complete, separate the reaction solution, evaporate the solvent under reduced pressure, and then purify or recrystallize by medium pressure chromatography to obtain

##STR00028##

[0094] The obtained compound is dissolved in an appropriate solvent O, which is selected from a group consisting of dichloromethane, 1,2-dichloroethane, chloroform, carbon tetrachloride, acetonitrile, acetone, ethyl acetate, methyl acetate, benzene, toluene, mesitylene, xylene, chlorobenzene, ethyl ether, ethylene glycol dimethyl ether, methyl tert-butyl ether, diphenyl ether, 1,4-dioxane, N,N-dimethylformamide, N,N-dimethylacetamide, tetrahydrofuran, 2-methyltetrahydrofuran and a mixture thereof; Adding dehydrating reagent P, which is selected from a group consisting of dicyclohexylcarbodiimide (DCC), polyphosphoric acid, Burgess reagent, bis[a,a-bis(trifluoromethyl)phenethyl alcohol]-diphenylsulfide, carbon disulfide, methyl iodide, sodium hydroxide, potassium hydroxide, lithium hydroxide, sodium hydride, potassium tert-butoxide, sodium tert-butoxide, sodium methoxide, sodium ethoxide, n-butyl lithium, lithium diisopropylamide, cyanuric chloride and a mixture thereof,

##STR00029##

is obtained by reaction, reaction temperature is 78° C. subzero to 25° C., preferably the reaction temperature is 40° C. subzero to 25° C. After the reaction is complete, perform a separation operation, preferably by evaporating the solvent under reduced pressure, and then purify or recrystallize by medium pressure chromatography to obtain

##STR00030##

[0095] Dissolve the obtained compound

##STR00031##

in an appropriate solvent Q, which is selected from a group consisting of dichloromethane, 1,2-dichloroethane, chloroform, carbon tetrachloride, acetonitrile, methanol, ethanol, acetone, ethyl acetate, methyl acetate, benzene, toluene, mesitylene, xylene, chlorobenzene, diethyl ether, ethylene glycol dimethyl ether, methyl tert-butyl ether, diphenyl ether, 1,4-dioxane, N,N-di methylformamide, N,N-dimethylacetamide, tetrahydrofuran, 2-methyltetrahydrofuran and a mixture thereof, add deprotection reagent S, reagent S is selected from a group consisting of hydrochloric acid methanol solution, hydrochloric acid ethanol solution, 1,4-dioxane hydrochloride solution, hydrochloric acid ether solution, tetrahydrofuran hydrochloride, tetrahydrofuran acetate solution, tetramethylammonium fluoride, tetraethylammonium fluoride, tetra-n-butylammonium fluoride (TBAF), Pd/C catalytic hydrogenation, DDQ, p-toluenesulfonic acid, methanol/sodium hydroxide, methanol/sodium methoxide and a mixture thereof.

##STR00032##

is obtained by deprotection reaction, the reaction temperature is 50° C. subzero to 50° C., preferably 30° C. subzero to 25 r. After the completion of the reaction, a separation operation is carried out, preferably by evaporating the solvent under reduced pressure, and then purifying or recrystallizing by medium pressure chromatography to obtain PNU-159682

##STR00033##

Example 1

[0096] The reaction route is as follows:

##STR00034##

[0097] Step 1:

[0098] When R=TBS, the synthesis of A1:

[0099] 1) Take 20 g of 1.0 eq of Nemorubicin, 8V/160 ml anhydrous DMF, stir at room temperature, and internal temperature of 10-25 degrees.

[0100] 2) Add 2.5 eq/5.28 g imidazole, stir for 2 min, add TBSCl (1.5 eq)/7.02 g in two batches at intervals of 5 min and the temperature is not more than 25 degree, and stir at room temperature.

[0101] 3) Take samples at an interval of 2 hours by HPLC to detect the reaction of the raw materials, and the reaction is complete in about 5-8 hours.

[0102] 4) Post-treatment after the reaction: Take 5V (relative to DMF) water and add dropwise to the reaction system under mechanical stirring. Use an oil pump to remove water and pull dry or freeze-dry with a lyophilizer to obtain a dark red powdery solid product A1.

[0103] Step 2. Synthesis of A2:

[0104] 1) Take 10 g of A1 (1.0 eq), 130 ml of 13V anhydrous DCM, stir and lower the internal temperature to 40 subzero to −35 degree.

[0105] 2) Add m-CPBA (85% 1.02 eq) to the reaction system in three batches at 10 min intervals. After 30 minutes of addition, start sampling and detection. HPLC/LCMS detect separately, and the reaction is complete in about 1 hour.

[0106] 3) HPLC confirms that the reaction is complete, the KI starch test paper detects the remaining oxides in the system, and the test paper does not turn blue, proceed to the next step.

[0107] Step 3. Synthesis of A3

[0108] 4) Dissolve 3.5 eq 1.1 g of Burgess reagent in 20 ml DCM, add dropwise to the reaction system, remove from the low temperature bath after addition, naturally warm to room temperature 20 to 25 degree and react for 4-8 h, check by HPLC/LCMS to confirm that the reaction is complete.

[0109] 5) Post-treatment: Rotary evaporating the system to remove 2/3 of the total volume of solvent and purified on a chromatographic column.

[0110] 6) Purification conditions: DCM-10% EtOH/DCM gradient increase.

[0111] 7) Finally, a dark reddish brown solid product A3 is obtained.

[0112] Step 4:

[0113] 1) Take A3 1.0 eq/16 g, 10V/160 ml anhydrous THF, stir to dissolve, cool to 3 subzero to 2 degree, add TBAF 1.3 eq/27.5 ml (LOM in THF) dropwise to the reaction system, after 5 min, start sampling and LCMS/HPLC-40 detects that the reaction is complete within 30 minutes.

[0114] 2) Post-treatment: add to the system with 10V/160 ml/water, concentrate to remove THF and other solvents. Apply the crude product system directly to the column by wet method, rinse the bottle with a small amount of water, and purify through reversed-phase column under medium pressure.

[0115] 3) Purification conditions: 0˜20 min 10% acetonitrile/ammonium bicarbonate water, 20˜120 min 10%˜90% acetonitrile/ammonium bicarbonate water, after all the products come out, rinse the column

[0116] 4) Mix the product and freeze-dry to obtain 10 g product with a yield of 60%.

Example 2

[0117] The reaction route is as follows:

##STR00035## ##STR00036##

[0118] Step 1:

[0119] When R=TBDPS, the synthesis of B1:

[0120] 1) Nem 1.0 eq 200 mg, 8 V/4 ml anhydrous DMF, stir at room temperature, internal temperature 20 to 25 degree.

[0121] 2) Add 2.5 eq/53 mg of imidazole, stir for 2 min, add TBDPSCl (1.5 eq)/128 mg to the reaction solution, control the temperature not to exceed 25 degree, and stir at room temperature.

[0122] 3) Take samples at an interval of 2 hours by HPLC to detect the reaction of the raw materials, and the reaction is complete in about 5-8 hours.

[0123] 4) Post-treatment after the reaction is complete: Take 10 V (relative to DMF) water and pour the reaction system into the water while stirring at room temperature. Add to the system with ethyl acetate 10 V, (relative to DWF) extraction and liquid separation 3 times, mix the organic phases and wash with water 10 V (relative to DMF) 3 times, concentrate and spin-dry, purify by column to obtain a dark red brown solid product B1 (150 mg, 54.7%1).

[0124] Step 2. Synthesis of B2:

[0125] 1) Take 131 2.0 g (1.0 eq), 13V anhydrous DCM 26 ml, stir and reduce the internal temperature 55 degree subzero to 60 degree subzero.

[0126] 2) Add 0.47 g of m-CPBA (85% 1.01 eq) to the reaction system in three batches at 10 min intervals. After 30 minutes of addition, start sampling and detection, HPLC/LCMS detect separately. The reaction is complete in about 1 to 2 hours, to obtain B2.

[0127] 3) KI starch test paper detects the remaining oxides of the system, the test paper does not turn blue, proceed to the next step.

[0128] Step 3. Synthesis of B3:

[0129] 4) Dissolve 3.5 eq 1.89 g of Burgess reagent in 20 ml DCM, add dropwise to the reaction system, remove from the low temperature bath after adding, naturally warm to room temperature 20 to 25 degree and react for 8-15 h, detect by HPLC/LCMS to confirm that the reaction is complete.

[0130] 5) Post-treatment: Rotary evaporate the system to remove 2/3 of the total volume of solvent and purified on a chromatographic column.

[0131] 6) Purification conditions: DCM-10% EtOH/DCM gradient increase.

[0132] 7) Finally obtain a deep reddish brown solid product B3.

[0133] Step 4, referring to Step 4 of Example 1, PNU-15%82 is obtained with a yield of 50%.

Example 3

[0134] The reaction route is as follows:

##STR00037## ##STR00038##

[0135] Step 1:

[0136] When R-TPS, the synthesis of C1:

[0137] 1) Nem 1.0 eq 150 mg, 8 V/4 ml anhydrous DMF, stir at room temperature, internal temperature 20 to 25 degree.

[0138] 2) Add 3.5 eq/55 mg of imidazole, stir for 2 min, add TPSCl (2.0 eq)/128 mg to the reaction solution, control the temperature not to exceed 25 degrees, and stir at room temperature.

[0139] 3) Take samples at 2 h intervals to detect the reaction of the raw materials by HPLC, and the reaction is basically complete in about 5-8 h.

[0140] 4) Post-treatment after the reaction is complete: Take 10 V (relative to DMF) water and pour the reaction system into the water while stirring at room temperature. Add ethyl acetate 10 V to the system, extract and separate three times (relative to DMF), mix the organic phases and wash 3 times with 10 V (relative to DMF), concentrate and spin dry, purify by column to obtain a dark reddish brown solid product C1 (60 mg, 28.7%).

[0141] Step 2. Synthesis of C2:

[0142] 1) Take C1 2.0 g (1.0 eq), 13V anhydrous DCM 26 ml, stir and reduce the internal temperature 55 degree subzero to 60 degree subzero.

[0143] 2) Add 0.46 g of m-CPBA (85% 1.01 eq) to the reaction system in three batches at 10 min intervals. After 30 minutes of addition, start sampling and detection, HPLC/LCMS detect separately. The reaction is complete in about 1 to 2 hours to obtain C2.

[0144] 3) KI starch test paper detects the remaining oxides of the system, the test paper does not turn blue, proceed to the next step.

[0145] Step 3. Synthesis of C3:

[0146] 4) Dissolve 3.5 eq 1.85 g of Burgess reagent in 20 ml DCM, add dropwise to the reaction system, remove from the low temperature bath after adding, naturally warm to room temperature 20 to 25 degree and react for 8-15 h, detect by HPLC/LCMS to confirm that the reaction is complete.

[0147] 5) Post-treatment: Rotary evaporate the system to remove 2/3 of the total volume of solvent and purify on a chromatographic column.

[0148] 6) Purification conditions: DCM-10% EtOH/DCM gradient increase.

[0149] 7) Finally, obtain a deep reddish-brown solid product C3.

[0150] Step 4, referring to Step 4 of Example 1, obtain PNU-159682 with a yield of 50%.

Example 4

[0151] ##STR00039##

[0152] Step 1:

[0153] When R=TIPS, the synthesis of D1:

[0154] 1) Nem 1.0 eq 150 mg, 8 V/4 ml anhydrous DMF, stir at room temperature, internal temperature 20 to 25 degree.

[0155] 2) Add 2.5 eq/40 mg imidazole, 0.5 eq/14 mg DMAP, stir for 5 min under ice-water bath conditions, TIPSCl (3.0 eq)/135 mg was added to the reaction solution under ice-water bath conditions, and stir at room temperature.

[0156] 3) Take samples at 1 hour intervals to detect the reaction of the raw materials by HPLC, and the reaction is complete in about 3 hours.

[0157] 4) Post-treatment after the reaction is complete: Take a 10 V (relative to DMF) saturated aqueous ammonium chloride solution and pour the reaction system into it under stirring at room temperature. Add ethyl acetate 10 V to the system, (relative to DMF) extraction and separation 3 times, mix the organic phases and wash with water 10 V (relative to DMF) 3 times, concentrate and spin-dry to obtain a dark reddish brown solid product D1 (100 mg, 53.7%).

[0158] Steps 2, 3, 4, refer to Example 1, to obtain PNU-159682 with a yield of 25%.