Patent Application
20220135613
The present disclosure relates to the field of organic synthesis, in particular to a method (method for morpholinyl anthracycline derivatives) for preparing a drug toxin PNU-159682 for antibody drug conjugates and intermediates therein.
Antibody-drug conjugate (ADC for short) is a new type of anti-tumor drug. Its principle is to connect cytotoxin to antibody. Through antibody recognition of specific antigen on the surface of cancer cell, it enters the cancer cells through endocytosis, thereby transporting cytotoxins to the target, achieve the purpose of targeted treatment of malignant tumors. Compared with traditional small-molecule anti-tumor drugs, ADC is more specific and effective because it can rely on the targeted recognition of antibodies and the high activity of toxins.
ADC includes three different components, namely antibody, linker and toxin. The antibody achieves targeting, the linker ensures the stability of the ADC during the blood transport process, and after reaching the target point, the toxin exerts a killing effect on cancer cells. According to the different mechanisms of action, toxins applicable for ADCs comprise microtubule inhibitors, DNA damaging agents, RNA polymerase inhibitors, etc. At present, the toxins used by ADCs commercially available and in clinical trials are mostly microtubule inhibitors, which mainly including dolastatin-based compounds, such as MMAE, MMAF and MMAD, and maytansine-based designed compounds, such as DM1 and DM4. In terms of linkers, the main applications are non-cleavable types, such as valine-citrulline (Valine-Citriline) and cyclohexyl carboxylic acid (MCC). After lysosomal hydrolysis, the drug is still active, and it binds to a certain amino acid residue through the linker
There are many ways to form antibody-drug conjugates. It can be formed either by chemically coupling the amino or sulfhydryl group on the antibody and the drug linker, or by modifying the antibody, then coupling with the drug linker for chemical reaction coupling or enzyme catalyzed reaction coupling after a specific functional group is introduced on the antibody. The molecular structure of the antibody drug toxin involved in the present disclosure is shown below.
For the synthesis method of PNU-159682 currently reported in the literature, refer to WO2012/73217, 2012, A1. The synthesis method uses Nemorubicin to oxidize and cyclize without a protective group. The oxidant is DMDO (acetone peroxy), which is cumbersome and explosive, and the cyclization reagent is trichloroisocyanuric acid. The yield of cyclization is low and it is not easy to purify.
In the prior art, there are many defects such as the occurrence of the secondary reaction of primary alcohol being oxidized, which leads to the low target yield during the scale-up production. In view of the defects of the prior art, the present disclosure provides a novel synthesis method and intermediate compounds involved in each step of the synthesis method.
The above-mentioned objects of the present disclosure are achieved by the following technical solutions.
Dissolve a compound of structural formula
in solvent X, and substitute protective group R in the presence of reagent Y to obtain an intermediate compound of structural formula
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, preferably N, N-dimethylformamide;
The reaction reagent Y is selected from a group consisting of 4-dimethylaminopyridine, pyridine, imidazole, trimethylamine, triethylamine diisopropyldiamine and a mixture thereof, preferably imidazole;
Reaction temperature is 20° C. subzero to 80° C., preferably 20 to 25° C.
Dissolve the intermediate compound
obtained in the previous step in an appropriate solvent M, add oxidizing reagent N, and obtain
by reaction.
Preferably, the appropriate solvent M is selected from a group consisting of dichloromethane, 1,2-dichloroethane, chloroform, carbon tetrachloride, acetonitrile, acetone, ethyl acetate, methyl acetate, water, benzene, 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;
Preferably, 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 Oxidizer, 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.
Preferably, the reagent N is tert-butyl hydroperoxide TBHP, acetone peroxy (DMDO) or m-chloroperoxybenzoic acid m-CPBA, and the solvent M is dichloromethane;
In this step, the reaction temperature is 50° C. subzero to 50° C., preferably 40° C. subzero to 25° C.
Preferably, this step further comprises a step of separating
from the reaction liquid after the reaction is complete.
Preferably, the separation comprises evaporating solvent under reduced pressure, and then purifying or recrystallizing by medium pressure chromatography to obtain
The present disclosure also provides an intermediate compound of structural formula
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, pivaloyl Pv and a mixture thereof; preferably the following compounds:
The compound
is a new compound that has never been reported, and we used a inventive design in the synthesis.
Dissolve the intermediate compound
obtained in the previous step in an appropriate solvent O, add the dehydrating reagent P, and obtain
by reaction.
Preferably, 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.
Preferably, the reagent P is selected from a group consisting of dicyclohexylcarbodiimide (DCC), polyphosphoric acid. Burgess reagent, bis[a,a-bis(trifluoromethyl)phenethyl alcohol]-diphenyl sulfide, 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 chlorid and a mixture thereof.
Preferably, the reagent P is dicyclohexylcarbodiimide (DCC), polyphosphoric acid, Burgess reagent or bis[a,a-bis(trifluoromethyl)phenethyl alcohol]-diphenylsulfide, the solvent O is dichloromethane.
In this step, the reaction temperature is 78° C. subzero to 25° C., preferably the reaction temperature is 40° C. subzero to 25° C.
Preferably, this step further comprises a step of separating
from the reaction liquid after the reaction is complete.
Preferably, the separation comprises evaporating solvent under reduced pressure, and then purifying or recrystallizing by medium-pressure chromatography to obtain
The present disclosure also provides an intermediate compound of structural formula
which is obtained by dehydration and cyclization reaction of compound
wherein R is selected from a group consisting of trimethylsilyl TMS, tert-butyldimethylsilyl TBS, and 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, pivaloyl Pv a mixture thereof; the compound
is a new compound that has never been reported, and we have used inventive designs in the synthesis.
Dissolve the compound
in an appropriate solvent Q, add the deprotection reagent S, and obtain
by the deprotection reaction.
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, pivaloyl Pv 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.
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.
Preferably, in this step, the reaction temperature is 50° C. subzero to 50° C., preferably 30° C. subzero to 25′C.
Preferably, in this step, reagent S is tetrabutylammonium fluoride (TBAF), and reagent Q is tetrahydrofuran.
Preferably, in this step, separate compound
from the reaction liquid after the reaction is complete.
Preferably, the separation comprises evaporating solvent under reduced pressure, and then purifying or recrystallizing by medium-pressure chromatography to obtain
The preparation method of the present disclosure does not directly use the compound Nemorubicin with structural formula
to synthesize PNU-159682, which improves the effectiveness of the N atom reaction, thereby effectively controlling the occurrence of side reactions of primary alcohols being oxidized; minimizing the risk and difficulty in scale-up production; no reverse phase preparation is required, and the preparation and production operation is simple. As mentioned above, this method minimizes the difficulty of operation, makes the quality standard easier to control, and can be applied to the preparation kilogram level. The preparation method of the present disclosure improves the stability, practicability and scalability of the process by introducing protective groups and changing to reagents that are easy to scale up.
As used in this context, the definitions of commonly used organic abbreviations and their corresponding CAS numbers are shown in Table 1:
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.
LCMS refers to liquid chromatography mass spectrometry method; HPLC refers to high-performance liquid chromatography detection.
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.
The present disclosure provides a method for synthesizing PNU-159682, which includes the following steps:
First, dissolve the compound (nemorubicin) of structural formula
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
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.
Dissolve the compound
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,
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
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,
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
Dissolve the obtained compound
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.
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
The reaction route is as follows:
Step 1:
When R=TBS, the synthesis of A1:
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.
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.
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.
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.
Step 2. Synthesis of A2:
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.
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.
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.
Step 3. Synthesis of A3
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.
5) Post-treatment: Rotary evaporating the system to remove 2/3 of the total volume of solvent and purified on a chromatographic column.
6) Purification conditions: DCM-10% EtOH/DCM gradient increase.
7) Finally, a dark reddish brown solid product A3 is obtained.
Step 4:
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.
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.
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
4) Mix the product and freeze-dry to obtain 10 g product with a yield of 60%.
The reaction route is as follows:
Step 1:
When R=TBDPS, the synthesis of B1:
1) Nem 1.0 eq 200 mg, 8 V/4 ml anhydrous DMF, stir at room temperature, internal temperature 20 to 25 degree.
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.
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.
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).
Step 2. Synthesis of B2:
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.
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.
3) KI starch test paper detects the remaining oxides of the system, the test paper does not turn blue, proceed to the next step.
Step 3. Synthesis of B3:
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.
5) Post-treatment: Rotary evaporate the system to remove 2/3 of the total volume of solvent and purified on a chromatographic column.
6) Purification conditions: DCM-10% EtOH/DCM gradient increase.
7) Finally obtain a deep reddish brown solid product B3.
Step 4, referring to Step 4 of Example 1, PNU-15%82 is obtained with a yield of 50%.
The reaction route is as follows:
Step 1:
When R-TPS, the synthesis of C1:
1) Nem 1.0 eq 150 mg, 8 V/4 ml anhydrous DMF, stir at room temperature, internal temperature 20 to 25 degree.
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.
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.
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%).
Step 2. Synthesis of C2:
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.
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.
3) KI starch test paper detects the remaining oxides of the system, the test paper does not turn blue, proceed to the next step.
Step 3. Synthesis of C3:
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.
5) Post-treatment: Rotary evaporate the system to remove 2/3 of the total volume of solvent and purify on a chromatographic column.
6) Purification conditions: DCM-10% EtOH/DCM gradient increase.
7) Finally, obtain a deep reddish-brown solid product C3.
Step 4, referring to Step 4 of Example 1, obtain PNU-159682 with a yield of 50%.
Step 1:
When R=TIPS, the synthesis of D1:
1) Nem 1.0 eq 150 mg, 8 V/4 ml anhydrous DMF, stir at room temperature, internal temperature 20 to 25 degree.
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.
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.
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%).
Steps 2, 3, 4, refer to Example 1, to obtain PNU-159682 with a yield of 25%.
| Number | Date | Country | Kind |
|---|---|---|---|
| 201910779777.5 | Aug 2019 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2020/086631 | 4/24/2020 | WO | 00 |
