CN1393438A

CN1393438A - Taxane derivative and its preparing process and usage

Info

Publication number: CN1393438A
Application number: CN 01129602
Authority: CN
Inventors: 梁晓天; 郭积玉; 尹大力; 张猛; 朱蔚华; 戴均贵; 韩锐; 刘红岩
Original assignee: Institute of Materia Medica of CAMS
Current assignee: Institute of Materia Medica of CAMS
Priority date: 2001-06-21
Filing date: 2001-06-21
Publication date: 2003-01-29
Anticipated expiration: 2021-06-21
Also published as: CN1393438B

Abstract

A novel taxane derivative and its preparing process, the medical composition containing it and its application to preparing the medicines to treat cancer, and the application of using it to preparing the medicines for treating the drugs-resistant tumor are disclosed.

Description

Taxane derivatives, their preparation and use

The invention relates to a new taxane derivative and a preparation method thereof, a pharmaceutical composition containing the compound and application of the compound in preparing anti-malignant tumor drugs; also relates to the application in the preparation of the medicine for treating the multidrug resistant tumor and the medicine for reversing the multidrug resistant tumor.

Cancer is one of the major diseases that seriously harm human beings. About half of the cancer patients who have been diagnosed early can be treated by surgery or radiation. A significant number of cancers and metastatic cancers require chemotherapy. The chemotherapy of tumor has been advanced in recent years, and can effectively treat various cancers, such as leukemia, malignant lymphoma, malignant sarcoma, etc., and can effectively prolong the survival time of some cancer patients. Among the chemotherapeutic drugs commonly used in the clinic are vinca alkaloids such as Vincristine (VCR), adriamycin such as doxorubicin, cisplatin, podophyllotoxin (etoposide), taxol (taxol), etc. (Ojima, Bounaud PY, Bernacki RJ., J.Modem Drug discovery 1999, 5/6: 45(design taxanes to treat multidrug resistant tumor drugs-manufacture. 1999, May/June: 45)).

At present, although there are nearly 50 anticancer drugs for clinical application, only a few drugs are effective against each tumor because of their respective properties and drug resistance of tumor cells. The biggest problem of tumor chemotherapy is that cancer cells are easy to generate drug resistance, which leads to the failure of chemotherapy. Recent studies have found that: there are various mechanisms by which resistance occurs; after tumor cells develop resistance to a certain chemotherapeutic drug, they also develop cross-resistance to other chemotherapeutic drugs with different chemical structures and mechanisms, which is called "multidrug resistance" (MDR). When cancer cells generate strong drug resistance, if the dosage is increased to continue chemotherapy, the curative effect is not improved, and larger toxic and side effects are generated due to the increase of the dosage. This is a significant cause of the inability of many Cancer patients to treat (Cao LP, Duchrow M, Windholl U, Kujath P, Bruch HP, Broll R. expression of the gene MDR1mRNA encoding P-glycoprotein in formalin-fixed, paraffin-embedded bladder Cancer tissue, European journal of Cancer, 1998, 34 (10): 1612(Expession of MDR1mRNA and encoding P-glycoprotein arrival for male form-fixed partially-embedded bladder Cancer tissues Eur J Cancer, 1998, 34 (10): 1612)).

The current research data indicate that there are several causes of MDR production by tumor cells, and the overexpression of certain membrane proteins by tumor cells is the main cause of MDR production. Membrane proteins associated with MDR, i.e. multidrug resistance proteins, have been found to be 3: P-Glycoprotein (P-Glycoprotein, P-gp, PG-170), multidrug resistance protein (MRP-190) and Lung Resistance Protein (LRP) (Ling, Vancouver. P-Glycoprotein: role in resistance, Journal of American Medicine, 1995, 99 (suppl. 6A): 31s (P-Glycoprotein: Its roll in drug resistance. the Journal of American Journal of Medicine, 1995, 99 (supl 6A): 31 s). The qualitative and quantitative changes of DNA topoisomerase II, a ribozyme, are also the main cause of MDR production by tumor cells against certain drugs (Molecular mechanisms of multidrug resistant tumor chemotherapy, applied pathology research, 1996, 192 (7): 798) (Molecular mechanisms of multidrug resistance and chemotherapy, 1996, 192 (7): 768)), and are the target sites of the co-action of anthracyclines and anti-cancer antibiotics such as epipodophyllotoxins. Glutathione is an important active substance in vivo, and overexpression of glutathione-S-transferase and alteration of the amount of reduced Glutathione (GSH) are also associated with MDR production (O' Brien ML, Tew KD. multidrug resistance)Procedure?Glutathione and its related enzymes of (1), european journal of cancer, 1996, 32A (6): 967 (glutamaterione and related enzymes in Multid)rug resistance. eur J Cancer, 1996, 32A (6): 967)). Changes in Protein Kinases (PKCs) and their isomerases may also contribute to the MDR phenomenon, and many PKC inhibitors have been found to modulate MDR and reverse MDR. In addition, MDR is reported to block or prevent tumor cell apoptosis induced by multiple factors (such as chemotherapeutic drugs, radiation, hormones, etc.) by expression products of some oncogenes (bcl-2, bcr/abl, NF-kB, etc.) associated with apoptosis inhibition, thereby inhibiting tumor cell apoptosis (apoptosis) (Gleave ME, Miayake H, GoldieJ, Nelson C, Tolcher A. Using antisense bcl-2 oligodeoxynucleotide nucleic acid against bcl-2 gene to delay the development of and increase the chemosensitivity of prostate cancer male hormone-dependent cells, J.urology 1999, 54 (supplement, 6A): 36(Targeting bcl-2 gene to delivery gene-expression and enhancement in cancer apoptosis, cell strain No. 54, 18, 3, 7, 3, non-lung cancer cell death promotion, ML., lung cancer cell strain, Z7, ZJ.7, J.6, 3, 4, 7, 3, journal of lung cancer, 1999, 23: 115(Antisense bcl-2 linear generated cell depth in non-small cell volume Cancer cells, 1999, 23: 115)).

Among them, the over-expression of pgp and the above-mentioned resistance-related proteins is the main cause of MDR, so that designing and synthesizing resistance-related protein inhibitors using them as targets is an important strategy for studying drugs for reversing MDR.

MDR reversal agents acting on pgp currently under investigation are: (1) calcium channel blockers (calciumchannelblocker) mainly comprise Verapamide and derivatives thereof, and inhibit pgp synthesis and activity thereof at mdrl translation level; (2) calmodulin antagonists (calmodulin antaronists), including phenothiazine derivatives such as Chlorpromazine, have been clinically tested: (3) cyclosporins, Cyclosporine A and its structural analogues PSC833, SDZ280-466 and the like, block P-glycoprotein, alter the pharmacokinetics of anticancer drugs: (4) quinolines, Quinidine, and the like, have entered clinical trials; (5) anti-estrogen compounds. Other MDR reversal agents are: (1) antisense nucleic acids and ribozymes: disrupting the expression of pgp mRNA from tumor cells by antisense or ribozyme directed against mdrl; (2) the cytokine TNF- α; (3): GSH depleting agent Vitamin K3, BSO (buthionine sulfoximine); (4): protein drug cross-linking agents, and the like.

In addition, taxane compounds, Taxinine and derivatives thereof, have been reported to have excellent activity of reversing multidrug-resistant cancer cells (Hosoyama H, Shigemori H, Tomida a, Tsuruo T, Kobayashi J.taxinineThe derivatives regulate multidrug resistance tumor cell strains, and carry out biological organic and pharmaceutical chemical communication, 1999, 9: 389(Modulation of multidrug resistance in tumor cells bv taxinine derivatives.Bioorganic &Medicinal Chemistry Letters, 1999, 9: 389) paclitaxel derivative ICD5109 has excellent activity against multidrug resistant cancer (Polizzi D, Praresi G, Totoreto M, Suino R, Rivar, Bombardelili E, Zunino F. a taxane compound having stronger tolerance and therapeutic activity against human transplanted tumor,cancer research，1999，59：1036(A novel taxane with improved tolerabilityand therapeutic activity in a panel of human tumor xenografts. Cancer Research,1999, 59: 1036) there are no reports of their clinical trials.

Desirable MDR reversing agents generally require a dose tolerance, can be repeatedly administered to maintain the desired drug concentration, completely reverse MDR, and have fewer side effects. The Verapamul, Cyclosporine A, Quinidine and the like cannot be practically applied to clinical chemotherapy due to the toxic and side effects generated when the Verapamul, Cyclosporine A, Quinidine and the like are used as MDR reversing medicines; other reported drugs are still in the research phase. Therefore, it is very necessary to develop a novel drug for treating malignant tumors, particularly a drug for treating multidrug-resistant tumors and a drug for reversing the multidrug-resistant tumors with good curative effect and small side effect.

The invention aims to provide a novel compound and a derivative thereof, which can be used as a medicament for treating malignant tumors, in particular a medicament for treating multidrug-resistant tumors and a medicament for reversing the multidrug-resistant tumors, which have excellent curative effect and small side effect;

it is another object of the present invention to provide a process for preparing the compounds of the present invention;

it is another object of the present invention to provide a pharmaceutical composition comprising the compound of the present invention.

In order to accomplish the objects of the present invention, the present inventors have now found through studies that novel taxane compounds of the following general formula (I) have activity of killing malignant tumor cells, activity of killing multidrug-resistant tumor cells and activity of reversing the drug resistance of multidrug-resistant tumor cells, and thus can be developed as drugs for treating malignant tumors, particularly for treating multidrug-resistant tumors and reversing the drug resistance of multidrug-resistant tumors.

The present invention relates in a first aspect to a compound of general formula (I) or a stereoisomer thereof or a pharmaceutically acceptable salt thereof.

Wherein R is₁Is hydrogen, hydroxy, R₂Is hydrogen, R₁R₂Is ═ O, R₃Is hydrogen, hydroxy, C_1-8Alkanoyloxy radical, R₄Is hydrogen, R₃R₄Is ═ O, R₅Is C_1-8Alkanoyloxy, benzoyloxy, substituted benzoyloxy, the substituents being hydroxy, methoxy or halogen, there being 1 or 2 identical or different substituents, R₆Is hydrogen, R₇Is hydroxy, alicyclic group C_1-8Alkanoyloxy group, heterocyclic group C_1-8Alkyl acyloxy, aromatic radical C_1-8Alkanoyloxy, including phenyl C_1-8(ii) an alkanoyloxy group which may have 1 or 2 substituents which may be the same or different on the phenyl group, the substituent being an azido group, a methoxy group or a halogen group, at C_1-8The alkyl group may have 1 or 2 identical or different substituents, which may be O, hydroxy, methoxy or halogen, provided that the substituents are not in the same position，R₈Is a hydrogen atom, and is,

R₉is hydrogen, hydroxy, C_1-8An alkyl-acyloxy group(s),

R₁₀is a hydrogen atom, and is,

R₉R₁₀in the formula of ═ O,

R₁₁is hydroxy, C_1-8An alkyl acyloxy group, a benzoyl oxy group, an aryl formyloxy group,

R₁₂is a hydrogen atom, and is,

R₁₁R₁₂is O, but does not include:

the term "phenyl C" in the present invention_1-8C in alkanoyloxy_1-8The alkanoyloxy group "means an alkanoyloxy group having 1 to 8 carbon atoms, such as formyloxy, acetyloxy, propionyloxy, isopropionyloxy, butyryloxy, isobutanoyloxy and the like, valeryloxy containing a branched chain, hexanoyloxy containing a branched chain and the like.

According to the present invention, preferred compounds of formula Ia or stereoisomers thereof include:

wherein,

R₁R₂in the formula of ═ O,

R₃is hydrogen, R₄Is a hydrogen atom, and is,

R₅is C_1-8An alkanoyloxy group which is selected from the group consisting of alkanoyloxy groups,

R₆is a hydrogen atom, and is,

R₇is a cinnamoyloxy, alicyclic group C_1-8Alkanoyloxy group, heterocyclic group C_1-8An alkyl acyloxy group,

Aromatic ring radical C_1-8Alkanoyloxy, including phenyl C_1-8An alkanoyloxy group, on the phenyl radical, may be

With 1 or 2 identical or different substituents which may be azido,

Methoxy or halogen at C_1-8There may be 1 or 2 substituents, which may be the same or different, on the alkyl radical

Substituent can be oxygen ═ O, hydroxyl, methoxy or halogen, provided that the substituent is substituted

The bases are not in the same position,

R₈is a hydrogen atom, and is,

R₉is a hydrogen atom, and is,

R₁₀is a hydrogen atom, and is,

R₁₂is a hydrogen atom, and is,

where n is 0 to 7, preferred compounds of formula Ib or stereoisomers thereof according to the present invention include:wherein,

R₁is a hydrogen atom, and is,

R₂is a hydrogen atom, and is,

R₃is C_1-8An alkanoyloxy group which is selected from the group consisting of alkanoyloxy groups,

R₄is a hydrogen atom, and is,

R₅is C_1-8Alkanoyloxy, benzoyloxy, substituted benzoyloxy, the substituents may be

Is hydroxy, methoxy or halogen, which may have 1 or 2 identical or different substitutions

The base group is a group of a compound,

R₆is a hydrogen atom, and is,

With 1 or 2 identical or different substituents which may be azido,

The bases are not in the same position,

R₈is a hydrogen atom, and is,

R₉is a hydrogen atom, and is,

R₁₀is a hydrogen atom, and is,

R₁₁is C_1-8An alkyl acyloxy group, a benzoyl oxy group, an aryl formyloxy group,

R₁₂is a hydrogen atom, and is,

n-0-7, but excluding:according to the present invention, preferred compounds of formula Ic or stereoisomers thereof include:wherein R is₁Is hydrogen, R₂Is hydrogen, R₃Is C_1-8Alkanoyloxy radical, R₄Is hydrogen, R₅Is C_1-8Alkanoyloxy radical, R₆Is hydrogen, R₇Is a cinnamoyloxy, alicyclic group C_1-8Alkanoyloxy group, heterocyclic group C_1-8Alkyl acyloxy, aromatic radical C_1-8Alkanoyloxy, including phenyl C_1-8An alkyl-acyloxy group(s),there may be 1 or 2 identical or different substituents on the phenyl radical, which may be azido, methoxy or halogen, at C_1-8The alkyl group may have 1 or 2 substituents which may be the same or different, and the substituent may be O, hydroxy, methoxy or halogen, provided that the substituents are not in the same position, R₈Is a hydrogen atom, and is,

R₉is hydroxy, C_1-8An alkyl-acyloxy group(s),

R₁₀is a hydrogen atom, and is,

R₁₂is a hydrogen atom, and is,

where n is 0 to 7, preferred compounds of formula Id or stereoisomers thereof according to the invention include:

wherein,

R₁is a hydrogen atom, and is,

R₂is a hydrogen atom, and is,

R₃R₄is ═ O, R₅Is C_1-8Alkanoyloxy radical, R₆Is hydrogen, R₇Is a cinnamoyloxy, alicyclic group C_1-8Alkanoyloxy group, heterocyclic group C_1-8Alkyl acyloxy, aromatic radical C_1-8Alkanoyloxy, including phenyl C_1-8(ii) an alkanoyloxy group which may have 1 or 2 substituents which may be the same or different on the phenyl group, the substituent being an azido group, a methoxy group or a halogen group, at C_1-8The alkyl group may have 1 or 2 substituents which may be the same or different, and the substituent may be O, hydroxy, methoxy or halogen, provided that the substituents are not in the same position, R₈Is hydrogen, R₉Is a hydrogen atom, and is,

R₁₀is a hydrogen atom, and is,

R₁₂is a hydrogen atom, and is,

n-0-7, preferred compounds of formula Ie or stereoisomers thereof according to the present invention include:wherein the double bond on the A ring is at C₁₂And C₁₃，

R₁Is a hydrogen atom, and is,

R₂in the absence of the presence of the agent,

R₃R₄is ═ O, R₅Is C_1-8Alkanoyloxy radical, R₆Is hydrogen, R₇Is a cinnamoyloxy, alicyclic group C_1-8Alkanoyloxy group, heterocyclic group C_1-8Alkyl acyloxy, aromatic radical C_1-8Alkanoyloxy, including phenyl C_1-8(ii) an alkanoyloxy group which may have 1 or 2 substituents which may be the same or different on the phenyl group, the substituent being an azido group, a methoxy group or a halogen group, at C_1-8The alkyl group may have 1 or 2 substituents which may be the same or different, and the substituent may be O, hydroxy, methoxy or halogen, provided that the substituents are not in the same position, R₈Is hydrogen, R₉Is hydrogen, R₁₀Is hydrogen, R₁₁Is C_1-8An alkyl acyloxy group, a benzoyl oxy group, an aryl formyloxy group,

R₁₂is a hydrogen atom, and is,

n＝0-7，

it will be appreciated that the compounds of formulae Ia to Ie above, or stereoisomers thereof, are particular embodiments or subclasses of the compounds of formula (I) of the present invention and that compounds of formulae Ia to Ie are within the scope of the compounds of formula (I) of the present invention.

According to the present invention, the compounds of general formula (I) according to the present invention include more preferably the following specific compounds or stereoisomers thereof: 5 alpha-deacetyl-14-deacetoxy-13-oxo-5 alpha-phenylpropylAcyloxy-neosequoyins A5 alpha-deacetyl-14-deacetoxy-13-oxo-5 alpha-phenylpropenoyloxy-neosequoyin A5 alpha-deacetyl-14-deacetoxy-13-oxo-5 alpha-phenylacetyloxy-neosequoyin A5 alpha-deacetyl-14-deacetoxy-13-oxo-5 alpha-benzoyloxy-neosequoyin A

5 alpha-deacetyl-14-deacetoxy-13-oxo-5 alpha- (2, 5-dimethoxyphenylacetyloxy) -neosequoyin A

5 alpha-deacetyl-14-deacetoxy-13-oxo-5 alpha-benzoylpropionyloxy-neosequoyin A

5 alpha-deacetyl-14-deacetoxy-13-oxo-5 alpha-phenylbutyloxy-neosequoyin A

5 alpha-deacetyl-5 alpha-phenylpropionyloxy-neosequoyin A5 alpha-deacetyl-5 alpha-phenylpropenoyloxy-neosequoyin A

5 alpha-deacetyl-9 alpha-acetoxy-5 alpha-phenylpropionyl-neosequoyin A5 alpha-deacetyl-14-deacetyloxy-13-oxy-5-3' -indolylpropyloxy-neosequoin A

5 alpha, 14 beta-dideacetyl-14-oxy-5 alpha-phenylpropionyloxy-neosequoyin A

Various methods for preparing the compounds of the general formula (I) of the present invention are involved in the present invention, and hereinafter, for convenience, they will be described in detail. Reaction of preparation Process (A)The corresponding route is as follows: the reaction route of the preparation method (II) is as follows:SINENXAN A a-1

a-2 a-3a-4 a-5

R_b＝Ac or R′CO

I_a SINENXANA R_a＝Ac or Hb-1

R_b＝R′CO or Ac

the reaction route of the b-2 preparation method (III) is as follows:SINENXANA c-1R_a＝Ac or H R_b＝R′CO or Acc-2 I_c

the reaction route of the preparation method (IV) is as follows:

SINENXANA R＝Ac or H

d-1pg is a protecting group R' is a hydrocarbon of various types including aromatic hydrocarbons and aliphatic hydrocarbons

d-2 d-3

d-4 I_dMethod VR' is various hydrocarbons including aromatic hydrocarbon and aliphatic hydrocarbon

d-4 I_e

As can be seen from the above reaction schemes, the present invention actually comprises five preparation methods, wherein the following relates to a preparation method of a compound of the general formula (I), comprising the following steps:

(1) treating neotaxin A with alkaline water solution in water soluble organic solvent for selective hydrolysis to obtain compound of formula a-1, i.e. 14 beta-deacetyl-neotaxin A;

(2) in the presence of an alkaline medium, in an organic solvent, the compound of the formula a-1 reacts with carbon disulfide and methyl iodide to carry out protosulfonyl esterification to generate a compound of the formula-2, namely 14 beta-deacetyl-14 beta-orthosulfonate-neosequoyin A;

(3) deoxidizing the 14 beta-deacetyl-14 beta-ortho sulfonate-neosequoyin A of the compound shown in the formula-2 obtained in the step 2 to generate a compound shown in a formula a-3, namely 14-deoxy-neosequoyin A;

(4) the compound 14-deoxy-sequoyianin A of the formula a-3 obtained in the step 3 is subjected to allylic oxidation to obtain a compound of the formula a-4, namely 14-deoxy-13-oxo-sequoyianin A;

(5) hydrolyzing the 14-deoxy-13-oxo-neotaxin A of the compound of the formula a-4 obtained in the step (4) with alkali, drying, treating with organic alkali in an organic solvent, and reacting with acetic anhydride to selectively acidify to obtain a compound of the formula a-5, namely 5 alpha-deacetyl-14-deoxy-13-oxo-neotaxin A;

(6) reacting the compound of formula a-5 obtained in step 5 with R' COOH in a dry organic solvent in the presence of DCC and an organic base to form an ester, thereby obtaining the compound of formula I_aThe compound, 5 α -deacetyl-14-deoxy-13-oxo-5 α -R' acyl-neosequoyin A.

In other words, the process for the preparation of the compounds of general formula I according to the invention comprises the following steps:

step 1: sequoyins A (Sinenxan A) in organic solvents with KOH or K₂CO₃Treating with water solution to obtain compound (14 beta-deacetyl-neosequoyin A) of formula a-1. The step is carried out in a water-soluble organic solvent, such as: methanol, ethanol, THF, and some mixed solvents, such as: methanol/dichloromethane solution, methanol/acetone solution, etc., wherein mixed solvent is preferred, and methanol/dichloromethane solution is most preferred; the reaction is carried out at 0-40 ℃, preferably 10-15 ℃; KOH or K₂CO₃The aqueous solution is preferably not too concentrated, preferably from 2mol/L to 4 mol/L.

Step 2: the compound of the formula a-1 reacts with carbon disulfide and methyl iodide in an organic solvent in the presence of an alkaline medium to generate a compound of the formula a-2 (14 beta-deacetyl-14 beta-orthosulfonate-neosequoyin A). The reaction is generally carried out in anhydrous organic solvents which do not interfere with the reaction, such as tetrahydrofuran, diethyl ether, dioxane and the like, and tetrahydrofuran is the best; the base used may be anhydrous base such as sodium hydroxide, sodium hydrogen carbonate, potassium tert-butoxide, etc., and sodium hydrogen carbonate is most preferable. The reaction is divided into two stages, wherein in the first stage, alkali is added, and carbon disulfide is generally carried out at room temperature-reflux temperature, preferably the reflux temperature; the second stage, the formation of the orthosulfonate with methyl iodide, is carried out at a relatively low temperature of 20-50 deg.C, preferably 40-50 deg.C.

And step 3: the generated compound of the formula a-2 is reacted with Bu in an organic solvent under the protection of nitrogen₃SnH/AIBN reaction to produce the compound of formula a-3. The reaction adopts organic solvents which do not interfere with the reaction and have higher temperature, such as toluene, benzene, dioxane and the like, and the solvents usually need to be subjected to anhydrous and degassing treatment, and the preferred solvent is toluene; the reaction is generally carried out at 50-100C, preferably 70-80 ℃.

And 4, step 4: the resulting compound of formula a-3 (14-deoxy-sequoyianin A) is treated with PCC, NaOAc, or pyridine chromic anhydride to give a compound of formula a-4. The reaction is carried out under the protection of nitrogen, and the used organic solvent is benzene, dichloromethane and other inert organic solvents which do not interfere with the reaction, and preferably benzene; the reaction is carried out at room temperature-reflux temperature, preferably reflux temperature; the reaction time is not longer, preferably 2-5 h.

And 5: the obtained compound (14-deoxy-13-oxo-neosequoyins A) of the formula a-4 is hydrolyzed by alkali to obtain a product, the product is not required to be purified, and after drying, the product is treated by organic alkali in an organic solvent and reacts with acetic anhydride to carry out selective acylation to obtain the compound (5 alpha-deacetyl-14-deoxy-13-oxo-neosequoyins A) of the formula a-5. The reaction is carried out in two stages, the first stage, usually in methanol or methanol/tetrahydrofuran mixture at 20-40 deg.C with 2-4mol/L KOH or K₂CO₃Treating the aqueous solution to obtain a full hydrolysate; in the second stage, pyridine or dichloromethane/DMAP is used as the organic solvent and alkali, and the reaction is carried out at 20-50 deg.C, preferably 20-30 deg.C.

Step 6: the compound of formula a-5 is reacted with R 'COOH in a dry organic solvent in the presence of DCC and an organic base to form an ester, which gives a compound of formula Ia (5 alpha-deacetyl-14-deoxy-13-oxo-5 alpha-R' acyl-neosequoyine A). The organic solvent used in the reaction is preferably an inert organic solvent which does not interfere with the reaction, such as toluene, dichloromethane and the like, and the best is toluene; the organic base adopts DMAP, 4-PP, triethylamine and the like, and preferably DMAP; the reaction is generally carried out at from 20 to 50 ℃ and preferably from 30 to 35 ℃.

In the present invention, step 5, selective hydrolysis and selective acylation can be carried out by controlling the reaction conditions to obtain 2, 5, 10 different position hydroxyl compounds (e.g., 1mol/L K equivalent of 1 mol/2 times of I-5 compound at-10-0 deg.C)₂CO₃Treating the water solution for 1h to obtain 10 beta-deacetyl-14-deoxy-13-oxo-neosequoyin A); followed by condensation with different acids to give esterification products in different positions.

The invention also includes a process for the preparation of a compound of formula (I) comprising:

(1) the neosequoyins A is treated with alkali in an organic solvent to obtain a full hydrolysate 2 alpha, 5 alpha, 10 beta, 14 beta-tetradeacetyl-neosequoyins A; after drying, the product is treated by organic alkali and reacts with acetic anhydride to carry out selective acylation to generate a compound shown as a formula b-1, namely 5 alpha-deacetyl-neosequoyin A;

(2) reacting the compound b-1 obtained in the step 2 with R' COOH in a dry organic solvent in the presence of DCC and an organic base to form ester, thereby obtaining the compound with the general formula I_bThe compound 5 alpha-deacetyl-5 alpha-R' acyl-neosequoyin A.

In other words, the second process for the preparation of the compounds of general formula (I) according to the invention comprises the following steps:

step 1: the neosequoyins A is hydrolyzed in organic solvent through alkali reaction to obtain total hydrolysate (2 alpha, 5 alpha, 10 beta, 14 beta-tetradeacetyl-neosequoyins A), which is dried, treated with organic alkali and selectively acylated with acetic anhydride to produce compound (5 alpha-deacetyl-neosequoyins A) in the formula b-1. The solvent used for the reaction hydrolysis is methanol, ethanol, or mixed solvent such as methanol/dichloromethane, methanol/acetone, methanol/tetrahydrofuran, etc., wherein methanol, methanol/tetrahydrofuran are preferred; the base used is generally K₂CO₃Or KOH, at 10-60 ℃, preferably 20-40 ℃; the subsequent acylation is generally carried out in pyridine at from 20 to 40 ℃.

Step 2: the resulting hydroxy compound b-1 is reacted with R 'COOH in a dry organic solvent in the presence of DCC and an organic base to form an ester, which gives a compound of formula Ib (5. alpha. -deacetyl-5. alpha. -R' acyl-neosequoyins A). The organic solvent used in the reaction can be an inert organic solvent which does not interfere with the reaction, such as toluene, dichloromethane and the like, the organic base adopts DMAP, 4-DP, triethylamine and the like, and the reaction is generally carried out at the temperature of 20-40 ℃.

In addition, in the method, through controlling the reaction conditions in the step 1, the sequoyianin A can be subjected to selective hydrolysis and selective acylation to obtain monohydroxy or polyhydroxy compounds at different positions of 2, 5 and 10.

The invention also relates to a method for preparing the compound of the general formula (I), which comprises the following steps:

(1) dissolving neosequoyins A in anhydrous ethanol, adding into cultured semen Ginkgo suspension cells at concentration of 10-40mg/L, and reacting for 2-18 days to obtain compound of formula c-1, i.e. 9 alpha-hydroxy-neosequoyins A;

(2) subjecting the compound 9 alpha-hydroxy-neosequoyin A of the formula c-1 obtained in the step 1 to alkali action in an organic solvent to obtain a total hydrolysate (2 alpha, 5 alpha, 10 beta, 14 beta-tetraacetyl-9 alpha-hydroxy-neosequoyin A), drying, treating with organic alkali, and reacting with acetic anhydride to selectively acylate to obtain the compound of the formula c-2, namely 5 alpha-deacetyl-9 alpha-acetyl-neosequoyin A;

(3) reacting the obtained hydroxyl compound with R' COOH in a dry organic solvent in the presence of DCC and organic base to obtain ester_cThe compound, 5 α -deacetyl-5 α -R' acyl-9 α -acetyl-neosequoyin A.

In other words, the present invention also relates to a process for the preparation of a third compound of formula (I), comprising the steps of:

step 1: dissolving neosequoyins A in anhydrous ethanol, adding appropriate amount of the obtained solution into cultured semen Ginkgo suspension cells at appropriate time, and reacting for several days to obtain compound of formula c-1. Culturing suspension cells of Ginkgo biloba according to known method at 18-33 deg.C, preferably 20-30 deg.C, preferably 25 + -2 deg.C; the concentration of neosequoyine A can be 10-40mg/L, preferably 20-30mg/L, preferably 20 mg/L; the adding period of the sequoyins A is 6-18 days, preferably 12-18 days of the cell cycle; the reaction time may be 2 to 18 days, preferably 6 to 10 days.

Step 2: the compound (9 alpha-hydroxy-neosequoyins A) of the formula c-1 is reacted with alkali in an organic solvent to obtain a total hydrolysate (2 alpha, 5 alpha, 10 beta, 14 beta-tetradeacetyl-9 alpha-hydroxy-neosequoyins A), dried, treated with organic alkali, and reacted with acetic anhydride to selectively acylate to obtain the compound (5 alpha-deacetyl-9 alpha-acetyl-neosequoyins A) of the formula c-2. The solvent used for hydrolysis is methanol, ethanol, or mixed solvent such as methanol/dichloromethane, methanol/acetone, methanol/tetrahydrofuran, etc., and the alkali is generally K₂CO₃Or KOH, the reaction temperature is controlled between 20 and 60 ℃, and the reaction temperature is preferably between 20 and 40 ℃. The subsequent acylation is generally carried out in pyridine, at room temperature-40 ℃.

And step 3: the obtained hydroxy compound reacts with R' COOH in a dry organic solvent in the presence of DCC and organic base to form ester, thus obtaining the formula I_c(5 α -deacetyl-5 α -R' acyl-9 α -acetyl-neosequoyin A). The organic solvent used in the reaction can be an inert organic solvent which does not interfere with the reaction, such as toluene, dichloromethane and the like, the organic base adopts DMAP, 4-PP, triethylamine and the like, and the reaction is generally carried out at the temperature of 20-40 ℃.

In addition, in the step 1, the selective hydrolysis and selective acylation can be carried out on the sequoyianin A by controlling the reaction conditions to obtain monohydroxy or polyhydroxy compounds at different positions of 2, 5 and 10

The invention also relates to a third process for the preparation of a compound of formula (I), which comprises the steps of:

(1) the neosequoyins A is treated by organic alkali in organic solvent to obtain full hydrolysate, namely 2 alpha, 5 alpha, 10 beta, 14 beta-tetradeacetyl-neosequoyins A, after drying, the full hydrolysate is selectively acylated with acetic anhydride to generate a compound shown as a formula d-1, namely 5 alpha, 14 beta-dideacetyl-neosequoyins A;

(2) reacting the compound 5 alpha, 14 beta-dideacetyl-neosequoyin A of the formula d-1 obtained in the step 1 with a protecting group in the presence of organic base to obtain a compound of the formula d-2;

(3) the compound of the formula d-2 obtained in the step 2 is put in a dry organic solvent and reacts with R 'COOH in the presence of DCC and organic base to form ester to obtain a compound of the formula d-3, namely 5 alpha, 14 beta-dideacetyl-14 beta-Pg-5 alpha-R' acyl-neosequoyin A, the compound of the formula d-3 can be directly dissolved in the organic solvent after being dried and is deprotected in an acidic medium to obtain a compound of the formula d-4;

(4) deprotecting the compound of formula d-4 obtained in step 3, concentrating and drying the obtained product, dissolving in an organic solvent, and oxidizing with PCC to obtain the compound of formula (I)_dA compound is provided.

It can be seen that, in the present invention, there is also provided a fourth process for the preparation of a compound of formula (I), comprising the steps of:

step 1: the neosequoyins A is treated with alkali in organic solvent to obtain total hydrolysate (2 alpha, 5 alpha, 10 beta, 14 beta-tetradeacetyl-neosequoyins A), dried, treated with organic alkali, and reacted with acetic anhydride to selectively acylate to produce compound (5 alpha, 14 beta-dideacetyl-neosequoyins A) in formula d-1. The conditions for the reaction hydrolysis are the same as in the second preparation method described above. The subsequent acylation is generally carried out in pyridine, at 20-60 ℃ for 2-4h, with a temperature of 40-60 ℃ being preferred.

Step 2: the resulting d-1 compound is reacted with a protecting group such as TMSCl, TESCl, TBDMSCl, etc. in the presence of an organic base to give a compound of formula d-2. The solvent used for this step is generally a dry organic solvent which does not interfere with the reaction, such as: tetrahydrofuran, dichloromethane, DMF and the like, preferably mixed solvent dichloromethane/DMF. As the base, a weak organic base such as imidazole, DMAP or the like is generally used. To increase the selectivity, the reaction is generally carried out at a temperature of from-78 ℃ to-20 ℃ and preferably from-50 ℃ to-30 ℃.

And step 3: the resulting hydroxy compound d-2 is reacted with R 'COOH in a dry organic solvent in the presence of DCC and an organic base to form an ester, yielding the compound of formula d-3 (5 α, 14 β -dideacetyl-14 β -Pg-5 α -R' acyl-neosequoyine A). The organic solvent used in the reaction can be an inert organic solvent which does not interfere with the reaction, such as toluene, dichloromethane and the like, the organic base adopts DMAP, 4-PP, triethylamine and the like, and the reaction is generally carried out at the temperature of 20-40 ℃. The obtained product can be directly dissolved in an organic solvent after being dried, and the deprotection is carried out in an acid medium, thus obtaining the compound shown in the formula d-4. The solvent used is usually methanol, tetrahydrofuran or other relatively polar solvent. The acidic medium is typically diluted with very dilute hydrochloric acid or other organic acid to avoid side reactions.

And 4, step 4: concentrating and drying the deprotected product, dissolving it in an organic solvent, and oxidizing with PCC to obtain formula I_dA compound is provided. The organic solvent used in this step may be benzene, dichloromethane, chloroform, etc., and dried dichloromethane is most preferable.

Finally, the invention also relates to another preparation method of the compound of the general formula (I), which comprises the steps of oxidizing the compound of the formula d-4 by an oxidant, reacting the oxidized compound with an acidic medium, and carrying out double bond transfer on silica gel to obtain the compound of the general formula I_eA compound is provided.

The fifth preparation method of the present invention comprises oxidizing compound d-4 prepared by the method IV with an oxidant, and reacting with an acidic medium to generate double bond transfer on silica gel to obtain the compound of the formula I_eA compound is provided. The organic solvent used in this step can be dried chloroform, dichloromethane, benzene, THF, etc., wherein dichloromethane or benzene is preferred, and dichloromethane is most preferred; the oxidant may be TPAP, PCC, PCC/sodium acetate, etc., preferably PCC or TPAP; the acidic medium may be silica gel, trifluoroacetic acid, p-toluenesulfonic acid, more concentrated hydrochloric acid, HF, etc., with silica gel or HF being preferred, and silica gel being most preferred.

In another aspect, the invention relates to a pharmaceutical composition comprising a therapeutically effective amount of at least one compound of formula (I) and/or a stereoisomer or a pharmaceutically acceptable salt thereof, e.g. a pharmaceutically acceptable acid salt, and a pharmaceutically acceptable carrier.

The invention also relates to the application of the compound of the general formula (I) or the stereoisomer thereof in preparing anti-malignant tumor medicaments, in particular to medicaments for treating multidrug-resistant tumors and medicament-resistant medicaments for reversing the multidrug-resistant tumors.

The compounds of formula (I) or pharmaceutical compositions containing them of the present invention may be administered in unit dosage form by enteral or parenteral routes, such as oral, intramuscular, subcutaneous, nasal, oromucosal, dermal, peritoneal or rectal administration and the like. The administration dosage forms such as tablets, capsules, dripping pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, suppositories, lyophilized injections and the like can be common preparations, sustained-release preparations, controlled-release preparations and various microparticle administration systems. The unit dosage form is formulated into tablets, and various carriers well known in the art can be widely used. Examples of the pharmaceutically acceptable carriers are, for example, diluents and absorbents such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, aluminum silicate and the like; wetting agents and binders such as water, glycerin, polyethylene glycol, ethanol, propanol, starch slurry, dextrin, syrup, honey, glucose solution, acacia slurry, gelatin slurry, sodium carboxymethylcellulose, shellac, methyl cellulose, potassium phosphate, polyvinylpyrrolidone and the like; disintegrating agents such as dried starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene sorbitol fatty acid ester, sodium dodecylsulfate, methyl cellulose, ethyl cellulose, etc.; disintegration inhibitors such as sucrose, glyceryl tristearate, cacao butter, hydrogenated oil and the like; absorption accelerators such as quaternary ammonium salts, sodium lauryl sulfate and the like; lubricants, for example, talc, silica, corn starch, stearate, boric acid, liquid paraffin, polyethylene glycol, and the like. The tablets may be further formulated into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layer and multi-layer tablets. For making the administration units into pills, a wide variety of carriers well known in the art can be used. Examples of the carrier are, for example, diluents and absorbents such as glucose, lactose, starch, cacao butter, hydrogenated vegetable oil, polyvinylpyrrolidone, Gelucire, kaolin, talc and the like; binders such as acacia, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste or batter, etc.; disintegrating agents, such as agar powder, dried starch, alginate, sodium dodecylsulfate, methylcellulose, ethylcellulose, etc. For making the administration unit into suppositories, various carriers well known to those skilled in the art can be widely used. Examples of such carriers are, for example, polyethylene glycol, lecithin, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, semi-synthetic glycerides and the like. In order to encapsulate the administration unit, the compound of the general formula (I) or a stereoisomer thereof as an active ingredient is mixed with the above-mentioned various carriers, and the thus-obtained mixture is placed in a hard gelatin capsule or a soft gelatin capsule. Or making the effective component of the compound of the general formula (I) or the stereoisomer thereof into microcapsules, suspending in an aqueous medium to form a suspension, or filling the microcapsules into hard capsules or making the microcapsules into injections. For preparing the administration unit into preparations for injection, such as solutions, emulsions, lyophilized powders and suspensions, all diluents commonly used in the art can be used, for example, water, ethanol, polyethylene glycol, 1, 3-propanediol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitol fatty acid esters, and the like. In addition, for the preparation of isotonic injection, sodium chloride, glucose or glycerol may be added in an appropriate amount to the preparation for injection, and conventional cosolvents, buffers, pH adjusters and the like may also be added.

In addition, colorants, preservatives, flavors, flavorings, sweeteners or other materials may also be added to the pharmaceutical preparation, if desired.

The dosage of the compound of the general formula (I) or a stereoisomer thereof of the present invention to be administered depends on many factors, such as the nature and severity of the disease to be prevented or treated, the sex, age, body weight and individual response of the patient or animal, the particular compound used, the route of administration and the frequency of administration, etc. The above-mentioned dosage may be administered in a single dosage form or divided into several, e.g. two, three or four dosage forms.

In order to further understand the present invention, the following examples and pharmacological experiments are only used to further illustrate the present invention, but are not meant to be any limitation of the present invention.

Examples example 1: 5 alpha-deacetyl-14-deacetoxy-13-oxo-5 alpha-phenylpropionyloxy-neosequoyin AStep 1: 14 beta-deacetyl-neosequoyin A

503mg (0.9976mmol) of sequoyianin A in a mixture of 30ml of methanol and 4ml of dichloromethane are added 5.0ml (10.0mmol) of 2mol/L KOH at 0 ℃ and stirred for 3.5h, continuing for 5h at room temperature (8 ℃). Adjusting pH to 6-7 with 5% HCl aqueous solution, concentrating under reduced pressure, extracting with ethyl acetate (3 × 50ml), mixing organic phases, and sequentially adding saturated NaHCO₃Solution, brine wash, anhydrous Na₂SO₄And (5) drying. The solvent was evaporated under reduced pressure to give a crude product, which was purified by silica gel column chromatography and eluted with a mixed solvent of petroleum ether/dichloromethane/acetone (10/10/1, 5/20/1) to give the desired product in an amount of 200mg with a yield of 43.4%. Step 2: 14 beta-deacetyl-14 beta-orthosulfonate neosequoyin A

1.6g (3.4632mmol) of 14 β -deacetyl-neosequoyin A in 45mL of tetrahydrofuran (anhydrous treated) were added 831mg (20.78mmol) of NaH (60% in mineral oil), 4.1mL (0.06926mmol) of CS₂Refluxing for 12h, cooling to room temperature, adding 1ml (17.32mmol) of methyl iodide, heating to 40-50 deg.C, stirring for 2.5h, cooling to room temperature, filtering, concentrating, extracting with 200ml of ethyl acetate, sequentially washing with saturated ammonium chloride aqueous solution, brine, and anhydrous Na₂SO₄Drying, vacuum evaporating to remove solvent, purifying with silica gel column chromatography, and petroleum ether/ethyl acetate (6/1, 5/1, 4/1) to obtain desired product 1.82g with yield of 95.2%. And step 3: 14-Deacetoxy-neosequoyin A

Under the protection of nitrogen, 1.53g (2.7717mmol) of 14 beta-deacetyl-14 beta-orthosulfonate-neosequoyin A in 100ml of toluene (treated by anhydrous and degassing treatment) are heated to 75-85 ℃ and after stabilization 7.47ml (27.717mmol) of Bu₃SnH, 46mg (0.27717mmol) of AIBN, stirred for 7 h. Concentrating, diluting with 20ml acetone to 6g of crude silica gel, purifying by silica gel column chromatography, and obtaining 0.986g of the required product with the yield of 79.8% by using petroleum ether/ethyl acetate (8/1, 7/1, 6/1). FABMS: 447.1(M +1)¹HNMR(CDCl₃，δ)：6.06(dd，1H，J＝5.3Hz，12.3Hz，10-H)，5.38(dd，1H，J＝2Hz，6.3Hz，2-H)，5.266(t，1H，J＝3Hz，5-H)，5.250(s，1H，20-H)，4.891(s，1H，20-H)，3.084(d，1H，J＝6Hz，3-H)，2.45(m，1H，，13-H)，2.375(dd，1H，J＝12Hz，14.5Hz，9-H)，2.12(s，3H，10-OAc-CH₃)，2.062(s，3H，2-OAc-CH₃)，2.045(s，3H，5-OAc-CH₃)，2.033(s，3H，18-CH3)，2.094-1.87(m，3H，1-H，13-H，14-H)，1.80(m，2H，6-H，7-H)，1.68(m，1H，6-H)，1.588(s，3H，16-CH₃)，1.588(m，1H，9-H)，1.22(m，7-H)，1.055(s，3H，17-CH₃)，0.849(s，3H，19-CH₃) And 4, step 4: 14-Deacetoxy-13-oxo-neosequoyins A

Under nitrogen protection, 1.26g (20825mmol) of 14-deacetoxy-neosequoyin A was added to 160ml of benzene (anhydrous) in the order of 6.95g (84.75mmol) of anhydrous NaOAc, 18.2g of Celite, 18.2g (84.75mmol) of PCC, heated to 95 ℃ under reflux for 11h, filtered, the residue was washed with 500ml of diethyl ether, the filtrates were combined, concentrated under reduced pressure, purified by column chromatography on silica gel, petroleum ether/acetone (8/1) to give 650mg of the desired product, and 300mg of the starting material was recovered in a yield of 65.7% based on 75.6% conversion of the starting material. FABMS: 461.3(M +1)¹HNMR(CDCl₃，δ)：6.06(dd，1H，J＝12Hz，5.5Hz，10-H)，5.46(dd，1H，J＝2Hz，6.5Hz，2-H)，5.266(s，1H，20-H)，5.226(t，J＝3Hz，1H，5-H)，4.816(s，1H，20-H)，3.187(d，1H，J＝6Hz，3-H)，2.87(dd，1H，J＝6.7Hz，19.7Hz，14-H)，2.48(dd，1H，J＝12Hz，15Hz，9-H)，2.328(d，1H，J＝19.5Hz，14-H)，2.196(s，3H，10-OAc-CH₃)，2.142(dd，1H，J＝2Hz，6.7Hz，1-H)，2.097(s，3H，2-OAc-CH₃)，2.063(s，3H，5-OAc-CH₃)，1.990(s，3H，18-CH₃)，2.14-1.85(m，1H，7-H)，1.79(m，3H，6-H，9-H)，1.691(s，3H，16-CH₃)，1.21(m，7-H)，1.132(s，3H，17-CH₃)，0.903(s，3H，19-CH₃) And 5: 5 alpha-deacetyl-14-deacetoxy-13-oxo-neosequoyin A

32.5mg(0.07065mmol)14-Deacetoxy-13-oxo-neosequoyins A was added to 2.5ml methanol, 0.26ml KOH 4mol/L was added and stirred at room temperature for 38.5h, the pH was adjusted to 6-7 with 4mol/L aqueous HCl, concentrated, extracted with ethyl acetate (3X 15ml), the organic phases were combined, washed with saturated aqueous sodium bicarbonate solution, brine, dried over anhydrous sodium sulfate, the solvent was evaporated under reduced pressure and dried under vacuum. 25.5mg of crude product are obtained, dissolved in 2ml of pyridine, stirred with 0.05ml of acetic anhydride (0.5294mmol), at 0-4 ℃ for 1.5h and at room temperature for 7h, added with 0.1ml of methanol, stirred for 10min, concentrated under reduced pressure, diluted with 50ml of ethyl acetate, 1mol/L hydrochloric acid, saturated CuSO₄Aqueous solution, saturated aqueous sodium bicarbonate solution, brine wash, dried over anhydrous sodium sulfate, concentrated under reduced pressure, purified by TLC, petroleum ether/acetone (2.5/l) to give the desired product 10mg, two-step yield 33.9%, step 6: 5 alpha-deacetyl-14-deacetoxy-13-oxo-5 alpha-phenylpropionyloxy-neosequoyin A

27mg (0.0587mmol) of 5 alpha-deacetyl-14-deacetoxy-13-oxo-neosequoyin A is put into 2ml of toluene, 26mg (0.1262mmol) of DCC and 40mg (0.1393mmol) of phenylpropionic acid are added and stirred for 15min at room temperature, 17mg (0.1393mmol) of DMAP is added for further reaction for 6h, methanol is added for termination of the reaction, the reaction is concentrated and extracted by ethyl acetate, and then 1mol/L of hydrochloric acid, saturated aqueous sodium bicarbonate solution, brine and anhydrous sodium sulfate are sequentially used for washing, the solvent is evaporated under reduced pressure, TLC purification is carried out, petroleum ether/ethyl acetate (1.5/1) is obtained, 30mg of the required product is obtained, and the yield is 84.5%. mp.: 117.2 ℃ FABMS: 551.3(M +1)¹HNMR(CDCl₃，δ)：7.33-7.21(5H，Ph)，6.06(dd，1H，J＝12.3Hz，6Hz，10-H)，5.48(dd，1H，J＝1.5Hz，6.7Hz，2-H)，5.28(s，1H，20-H)，5.24(br.s，1H，5-H)，4.842(s，1H，20-H)，3.167(d，1H，J＝6Hz，3-H)，2.955(t，2H，J＝8Hz，2’-CH₂)，2.81(dd，1H，J＝6.5Hz，20Hz，14-H)，2.63(m，1H，3’-CH₂)，2.53(m，1H，3’-CH₂)，2.50(dd，1H，J＝12Hz，15Hz，9-H)，2.32(d，1H，J＝20Hz，14-H)，2.179(s，3H，10-OAc-CH₃)，2.16(dd，1H，J＝1.5Hz，6.5Hz，1-H)，2.117(s，3H，2-OAc-CH₃)，2.088(s，3H，18-CH₃)，1.91(m，1H，7-H)，1.78(m，3H，6-H，9-H)，1.711(s，3H，16-CH₃)，1.28(m，7-H)，1.154(s，3H，17-CH₃)，0.919(s，3H，19-CH₃) Example 2: 5 alpha-deacetyl-14-deacetoxy-13-oxo-5 alpha-phenylpropenoyloxy-neosequoyin A

The expected product was prepared according to the procedure described in example 1, step 6, substituting cinnamic acid for phenylpropionic acid. White solid, mp: FABMS at 146.8-148 ℃:¹HNMR(CDCl₃，δ)：7.795(d，2H，J＝6.5Hz，Ph-2，6-H)，7.68(d，1H，J＝16Hz，3’H)，7.44(m，3H，Ph-3，4，5-H)，6.48(d，1H，J＝16.5Hz，2’H)，6.10(dd，1H，J＝6Hz，12.3Hz，10-H)，5.517(dd，1H，J＝2Hz，6.5Hz，2-H)，5.317(s，1H，20-H)，5.361(br.s，1H，5-H)，4.836(s，1H，20-H)，3.38(d，1H，J＝7Hz，3-H)，2.86(dd，1H，J＝7Hz，20Hz，14-H)，2.525(dd，1H，J＝12Hz，15Hz，9-H)，2.477(d，1H，J＝20Hz，14-H)，2.26(s，3H，10-OAc-CH₃)，2.10(dd，1H，J＝1.5Hz，6.7Hz，1-H)，2.123(s，3H，2-OAc-CH₃)，2.096(s，3H，18-CH₃)，1.97(m，2H，6-H，7-H)，1.83(m，2H，6-H，9-H)，1.735(s，3H，16-CH₃)，1.344(m，7-H)，1.185(s，3H，17-CH₃)，0.959(s，3H，19-CH₃) Example 3: 5 alpha-deacetyl-14-deacetoxy-13-oxo-5 alpha-phenylacetyloxy-neosequoyin A

The expected product was prepared according to the procedure described in example 1, step 6, substituting phenylacetic acid for phenylpropionic acid.

White solid, mp: FABMS at 51.1-51.9 ℃: 571.5(M + K +1), 537.5(M +1)¹HNMR(CDCl₃，δ)：7.35-7.24(5H，Ph)，6.08(dd，1H，J＝12Hz，5.5Hz，10-H)，5.48(dd，1H，J＝2Hz，6.5Hz，2-H)，5.267(s，1H，20-H)，5.238(t，1H，J＝2Hz，5-H)，4.834(s，1H，20-H)，3.55(q，2H，J＝17Hz，2’CH₂)3.105(d，1H，J＝6.5Hz，3-H)，2.835(dd，1H，J＝7Hz，19.7Hz，14-H)，2.50(dd，1H，J＝11.7Hz，15Hz，9-H)，2.37(d，1H，J＝19.5Hz，14-H)，2.24(s，3H，10-OAc-CH₃)，2.62(dd，1H，J＝2Hz，7Hz，1-H)，2.101(s，3H，2-OAc-CH₃)，2.064(s，3H，18-CH₃)，1.95(m，1H，7-H)，1.80(m，3H，6-H，9-H)，1.703(s，3H，16-CH₃)，1.29(m，1H7-H)，1.154(s，3H，17-CH₃)，0.894(s，3H，19-CH₃) Example 4: 5 alpha-deacetyl-14-deacetoxy-13-oxo-5 alpha-benzoyloxy-neosequoyin A

The expected product was prepared according to the procedure described in example 1, step 6, substituting benzoyl chloride for phenylpropionic acid without DCC. White solid, mp: FABMS at 137-138.5 ℃: 523.4(M +1)¹HNMR(CDCl₃，δ)：7.82(d，2H，J＝7.5Hz，Ph-2，6-H)，7.607(t，1H，J＝7.3Hz，3-H)，7.538(t，2H，J＝7.5Hz，Ph-4，5-H)6.02(dd，1H，J＝12Hz，5.5Hz，10-H)，5.524(d，1H，J＝7Hz，2-H)，5.404(s，1H，5-H)，5.375(s，1H，20-H)，4.815(s，1H，20-H)，3.243(d，1H，J＝7Hz，3-H)，2.836(dd，1H，J＝7Hz，19.5Hz，14-H)，2.41(dd，1H，J＝12.5Hz，14.5Hz，9-H)，2.472(d，1H，J＝20Hz，14-H)，2.21(d，1H，J＝7.5Hz，1-H)，2.077(s，3H，10-OAc-CH₃)，2.002(s，3H，2-OAc-CH₃)，1.951(s，3H，18-CH₃)，2.07-1.915(m，3H，2×6-H，7-H)，1.78(dd，1H，J＝14.7Hz，6.7Hz，9-H)，1.713(s，3H，16-CH₃)，1.35(m，1H，7-H)，1.120(s，3H，17-CH₃)，0.966(s，3H，19-CH₃) Example 5: 5 alpha-deacetyl-14-deacetoxy-13-oxo-5 alpha- (2, 5-dimethoxyphenylacetyloxy) -neosequoyin A

The expected product was prepared according to the procedure described for example 1, substituting 2, 5-dimethoxyphenylacetic acid for phenylpropionic acid in step 6. White solid, mp: FABMS at 69.5-70.5 ℃: 596.3 (M)⁺)¹HNMR(CDCl₃，δ)：6.825-6.75(m，3H，Ph)，6.09(dd，1H，J＝5.5Hz，12Hz，10-H)，5.484(dd，1H，J＝1.5Hz，6.5Hz，2-H)，5.272(s，1H，20-H)，5.252(br.s，1H，5-H)，4.825(s，1H，20-H)，3.769(s，3H，5-OCH₃)，3.759(s，3H，2-0CH₃)，3.596(d，1H，J＝17Hz，2’-H)，3.407(d，1H，J＝17Hz，2’-H)，3.232(d，1H，J＝6.5Hz，3-H)，2.812(dd，1H，J＝7Hz，20Hz，14-H)，2.498(dd，1H，J＝12.3Hz，14.7Hz，9-H)，2.387(d，1H，J＝20Hz，14-H)，2.243(s，3H，10-OAc-CH₃)，2.155(d，1H，J＝5.5Hz，1-H)，2.096(s，3H，2-OAc-CH₃)，2.066(s，3H，18-CH₃)，2.17-1.96(m，1H，7-H)，1.834(m，2H，6-H)，1.768(dd，1H，J＝14.7Hz，5.7Hz，9-H)，1.701(s，3H，16-CH₃)，1.28(m，1H，7-H)，1.140(s，3H，17-CH₃)，0.914(s，3H，19-CH₃) Example 6: 5 alpha-deacetyl-14-deacetoxy-13-oxo-5 alpha-benzoylpropionyloxy-neosequoyin A

The expected product was prepared according to the procedure described in example 1, substituting 4-carbonyl-phenylbutyric acid for phenylpropionic acid in step 6. A white solid. FABMS: 579.3(M +1)¹HNMR(CDCl₃，δ)：7.993(d，2H，J＝7.5Hz，Ph-2，6-H)，7.556-7.444(m，3H，Ph-3，4，5-H)，6.092(dd，1H，J＝12Hz，5.5Hz，10-H)，5.475(dd，1H，J＝1.5Hz，6.3Hz，2-H)，5.265(s，1H，20-H)，5.246(br.s，1H，5-H)，4.827(s，1H，20-H)，3.396(m，1H，3’-CH₂)，3.193(m，2H，3-H，3’-CH₂)，2.79(dd，1H，J＝7Hz，20Hz，14-H)，2.755(m，1H，J＝8Hz，2’-CH₂)，2.56(m，1H，2’-CH₂)2.49(dd，1H，J＝12.3Hz，14.7Hz，9-H)，2.369(d，1H，J＝20Hz，14-H)，2.254(s，3H，10-OAc-CH₃)，2.145(dd，1H，J＝1.7Hz，6.7Hz，1-H)，2.09(s，3H，2-OAc-CH₃)，2.064(s，3H，18-CH₃)，2.01(m，1H，7-H)，1.86(m，2H，6-H)，1.772(dd，1H，J＝5.7Hz，14.7Hz，9-H)，1.696(s，3H，16-CH₃)，1.563(s，3H，17-CH₃)，1.308(m，1H，7-H)，1.133(s，3H，19-CH₃) Example 7: 5 alpha-deacetyl-14-deacetoxy-13-oxo-5 alpha-phenylbutyloxy-neosequoyin A

The expected product was prepared according to the procedure described in example 1, step 6, substituting phenylbutyric acid for phenylpropionic acid. A white solid. FABMS: 565.2(M +1)¹HNMR(CDCl₃，δ)：7.306-7.172(m，5H，Ph)，6.049(dd，1H，J＝11.7Hz，5.7Hz，10-H)，5.461(dd，1H，J＝1.5Hz，6.5Hz，2-H)，5.265(s，1H，20-H)，5.232(t，J＝2.7Hz，1H，5-H)，4.817(s，1H，20-H)，3.396(m，1H，3’-CH₂)，3.162(d，1H，J＝6Hz，3-H)，2.780(dd，1H，J＝6.7Hz，19.7Hz，14-H)，2.696(m，2H，2’-CH₂)，2.481(dd，1H，J＝12Hz，15Hz，9-H)，2.309(d，1H，J＝20Hz，14-H)，2.241(m，2H，3’-CH₂)2.155(s，3H，10-OAc-CH₃)，2.137(dd，1H，J＝1.7Hz，7.3Hz，1-H)，2.094(s，3H，2-OAc-CH₃)，2.060(s，3H，18-CH₃)，1.906(m，3H，7-H，4’-CH₂)，1.76(m，3H，6-H，9-H)，1.726(s，3H，16-CH₃)，1.283(t，1H，J＝3.3Hz，7-H)，1.131(s，3H，17-CH₃)，0.900(s，3H，19-CH₃) Example 8: 5 alpha-deacetyl-5 alpha-phenylpropionyloxy-neosequoyin A

Step A: 2 alpha, 5 alpha, 10 beta, 14 beta-tetradeacetyl-neosequoyin A

50mg (0.099mmol) of sequoyianin A in 2ml of methanol, 0.4ml of 2mol/L KOH (0.8mmol) was added, the mixture was heated to 40 ℃ and stirred for 8 hours, concentrated to dryness under reduced pressure, dissolved in ethyl acetate, insoluble matter was filtered off, the solvent was evaporated under reduced pressure, and dried under vacuum to give 32mg of the desired product with a yield of 96%. And B: 5 alpha-deacetyl-neosequoyin A

60mg (0.1786mmol)2 α, 5 α, 10 β, 14 β -tetraacetoacetyl-neosequoyin A are dissolved in 3ml pyridine, 0.1ml acetic anhydride (0.5294mmol) are added, stirring is carried out at room temperature for 49h, 0.2ml methanol is addedStirring for 10min, concentrating under reduced pressure, diluting with 20ml ethyl acetate, 1mol/L hydrochloric acid, and saturating with CuSO₄Aqueous solution, saturated sodium bicarbonate aqueous solution, brine, anhydrous sodium sulfate drying, vacuum concentration, silica gel column purification, petroleum ether/ethyl acetate (4/1-2/1), desired product 31mg, yield 37.8%. And C: 5 alpha-deacetyl-5 alpha-phenylpropionyloxy-neosequoyin A

78mg (0.52mmol) phenylpropionic acid is added into 2ml toluene, 40mg (0.1948mmol) DCC is added, the mixture is stirred for 15min at room temperature, 30mg (0.06494mmol)2 alpha, 10 beta, 14 beta-triacetoxy-5 alpha-hydroxy-neosequoyin A and 79mg (0.6475mmol) DMAP are added for continuous reaction for 4h, methanol is added for termination reaction, the reaction is concentrated and extracted by ethyl acetate, 1mol/L hydrochloric acid, saturated sodium bicarbonate aqueous solution are used for washing, anhydrous sodium sulfate is used for drying, the solvent is evaporated under reduced pressure, silica gel column purification is carried out, petroleum ether/ethyl acetate (6/1) is carried out, 25mg of the required product is obtained, and the yield is 65%. A light yellow oil. FABMS: 587.2(M + Na), 565.2(M +1)¹HNMR(CDCl₃，δ)：7.308-7.192(m，5H，Ph-H)，6.03(dd，1H，J＝12Hz，5.5Hz，10-H)，5.34(dd，1H，J＝2.5Hz，6.5Hz，2-H)，5.273(s，1H，20-H)，5.299(t，J＝3Hz，1H，5-H)4.985(dd，J＝4.5，9.5Hz，14-H)，4.879(s，1H，20-H)，2.99(m，2H，2’-CH₂)，2.81(d，1H，J＝6.5Hz，3-H)，2.71(m，3H，3’-CH2，13-H)，2.41(dd，1H，J＝4.3，18.7Hz，13-H)，2.367(dd，1H，J＝12.3Hz，14.7Hz，9-H)，2.056(s，3H，10-OAc-CH₃)，2.05(s，3H，2-OAc-CH₃)2.023(s，3H，14-OAc-CH3，)2.009(s，3H，18-CH₃)，1.91(m，1H，7-H)，1.877(d，1H，J＝2.29Hz，1-H)，1.75(m，2H，6-H)，1.65(s，3H，16-CH₃)，1.61(dd，J＝6，15Hz，9-H)，1.106(s，3H，17-CH₃)，0.832(s，3H，19-CH₃) Example 9: 5 alpha-deacetyl-5 alpha-phenylpropenoyloxy-neosequoyin A

The expected product was prepared according to the procedure described in example 8, step C, substituting cinnamic acid for phenylpropionic acid. White solid, mp: 75-78 deg.CFABMS：593(M+1)¹HNMR(CDCl₃，δ)：7.76(d，1H，J＝6.02Hz，3’-H)7.39-7.57(m，5H，Ph)，6.53(d，1H，J＝6.02Hz，2’-H)6.07(dd，1H，J＝12.2Hz，5.7Hz，10-H)，5.36(dd，1H，J＝2.29Hz，6.49Hz，2-H)，5.34(s，1H，20-H)，5.33(br.s，1H，5-H)，5.04(dd，1H，J＝4.58，9.54Hz，14-H)4.93(s，1H，20-H)，2.99(d，J＝6.48Hz，3-H)2.82(dd，1H，J＝9.53Hz，19.45Hz，13-H)，2.47(dd，1H，J＝3.05，19.07Hz，13-H)，2.40(dd，1H，J＝12.21Hz，14.88Hz，9-H)，2.12，(s，3H，10-OAc-CH₃)，2.08(s，3H，2-OAc-CH₃)2.06(s，3H，14-OAc-CH3，)1.99(s，3H，18-CH₃)，1.90(d，1H，J＝2.29Hz，1-H)，1.67(s，3H，16-CH₃)，1.62(dd，J＝5.34，14.88Hz，9-H)，1.11(s，3H，17-CH₃)，0.87(s，3H，19-CH₃) Example 10: 5 alpha-deacetyl-9 alpha-acetoxy-5 alpha-phenylpropionyl-neosequoyin A

Step A: preparation of 9 alpha-hydroxy-neosequoyin A1) culture of Ginkgo biloba suspension cells

For the induction and culture of callus of ginkgo and the establishment of cell suspension culture system, see the literature^[1]. The culture medium is MS^[]+0.5mg/L NAA (alpha-naphthylacetic acid) +0.5 mg/L6-BA (6-benzyladenine) +0.2 mg/L2, 4-D (2, 4-dichlorophenoxyacetic acid), 30g/L sucrose dosage, pH adjusted to 5.8 before disinfection. The shake flasks used were 500mL and 1000mL triangular flasks, 150mL medium was contained in the 500mL triangular flask, 330mL medium was contained in the 1000mL triangular flask, the cell inoculum size was 5g/L culture (dry weight) and shaking culture was performed on a shaker at 110 rpm in the dark, the culture temperature was (25. + -. 2) deg.C. 2) Biotransformation of neotaxin A by a ginkgo biloba cell suspension culture i) substrate is neotaxin A provided by the institute of medicine biosynthesizing department of Chinese medical science, and is obtained by separating from a callus culture of Taxus yunnanensis (Taxus yunnensis), and the purity is more than 95% through HPLC inspection. A certain amount of Sinenxan A is accurately weighed, and is dissolved by absolute ethyl alcohol to prepare a solution with the concentration of 20mg/mL for later use. ii) bioconversion pretest was takenThe said neotaxin A solution 0.5mL was added to a 500mL triangular flask containing suspended cells of ginkgo biloba grown for 15 days, while the other triangular flask was added with 0.5mL absolute ethanol as a blank control. After further 6 days of culture, the culture was filtered under reduced pressure, and the culture was washed with distilled water 3 times, each time 20mL of distilled water, the filtrates were combined, extracted with equal amount of ethyl acetate 3 times, the extracts were concentrated to dryness under reduced pressure, and eluted with a small amount of acetone. The culture is dried in a 50 ℃ oven to constant weight, soaked in ethyl acetate and extracted by ultrasound for 0.5 hour, the solution after filtering the culture is concentrated to dryness under reduced pressure, and is dissolved out by a small amount of acetone. The sample prepared by the above method was subjected to TLC inspection together with the substrate. The developing agent is acetone-petroleum ether (1: 2.5), sprayed with 10% ethanol sulfate solution, and heated until brown spots appear. Repeat 2 times. iii) biotransformation amplification test after 2 preliminary tests confirmed that cultured cells of Ginkgo biloba have the effect of transforming sequoyitin A, amplification test was performed. The volume of the triangular flask is 1000mL, and the total volume is 15 bottles. After 15 days of culture, 1mL of the substrate sequoyianin A solution was added to each flask and harvested for 21 days. Filtering to remove culture, washing with appropriate amount of distilled water for 3 times, mixing filtrates, extracting with equal amount of ethyl acetate for 5 times, concentrating under reduced pressure, and dissolving with acetone. iv) isolation and identification of the biotransformation product 3) the acetone extract was filtered under reduced pressure to remove insoluble material, concentrated and weighed to obtain 450mg residue. Separating by silica gel column chromatography, wherein the silica gel is thin layer chromatography silica gel H (Qingdao ocean chemical plant), the eluent is acetone-petroleum ether (1: 5-1: 1), to obtain a product, and purifying by chromatography¹H-NMR，¹³C-NMR，¹H-¹H COSY，¹³C-¹The product is identified as 2 alpha, 5 alpha, 10 beta, 14 beta-tetraacetoxy-9 alpha-hydroxy-neosequoyin A by spectral techniques such as H COSY, FABMS, infrared and the like. v) the reaction formula, structural formula, yield, molecular weight and specific optical rotation of the 9 alpha-hydroxy-neosequoyin A preparation are as follows:

Sinenxan A 9-hydroxyl-sinenxan A

1(60％)

C₂₈H₄₀O₉

Exact Mass：520.27

Mol.Wt.：520.61

C，64.60；H，7.74；O，27.66

[α]_D ²⁰+44.6°(C＝0.0083，CH₃OH) step B: 2 alpha, 5 alpha, 10 beta, 14 beta-tetradeacetyl-9 alpha-hydroxy-neosequoyin A

135mg (0.260mmol) of 2 α, 5 α, 10 β, 14 β -tetraacetoxy-9 α -hydroxy-neosequoyin A in 10ml of methanol and 1ml of acetone are added 1.2ml of 4mol/L KOH (4.8mmol) and stirred at room temperature for 34h, the pH value is adjusted to 6-7 with 6N aqueous HCl, concentrated, extracted with ethyl acetate (15ml), the organic phase is successively washed with saturated aqueous sodium bicarbonate solution, brine, dried over anhydrous sodium sulfate, the solvent is evaporated off under reduced pressure and dried under vacuum to give 85mg of crude product. Dissolving the crude product in 3ml pyridine, adding 0.23ml acetic anhydride (2.4mmol), stirring at room temperature for 48h, adding 0.2ml methanol, stirring for 10min, concentrating under reduced pressure, diluting with 20ml ethyl acetate, 1mol/L hydrochloric acid, and saturating CuSO₄Aqueous solution, saturated sodium bicarbonate aqueous solution, brine washing, anhydrous sodium sulfate drying, vacuum concentration, silica gel column purification, petroleum ether/ethyl acetate (3/1-1/1), desired product 35mg, yield 28%. And C: 5 alpha-deacetyl-9 alpha-acetoxyYl-5 alpha-phenylpropionyl-neosequoyin A

20mg (0.03846mmol) of 2 alpha, 9 alpha, 10 beta, 14 beta-tetraacetoxy-5 alpha-hydroxy-neosequoyin A is added into 3ml of dichloromethane, 12mg (0.05769mmol) of DCC and 17mg (0.1154mmol) of phenylpropionic acid are added and stirred for 15min at room temperature, 14mg (0.1154mmol) of DMAP is added for continuous reaction for 10h, the temperature is 30-40 ℃ for continuous 4h, methanol is added for stopping the reaction, the mixture is concentrated and extracted by dichloromethane (12ml), and then 1mol/L of hydrochloric acid, saturated sodium bicarbonate aqueous solution, brine, anhydrous sodium sulfate is used for drying, the solvent is evaporated under reduced pressure, silica gel column chromatography purification is carried out, petroleum ether/ethyl acetate (6/1, 5/1) is carried out, 18mg of the required product is obtained, and the yield is 97.8%. FABMS: 675.2(M + Na)¹HNMR(CDCl₃，δ)：7.31-7.199(m，5H，Ph)，5.987(d，1H，J＝10Hz，10-H)，5.793(d，1H，J＝10.5Hz，9-H)，5.404(dd，1H，J＝2.5Hz，7Hz，2-H)，5.34(s，1H，20-H)，5.318(br.s，1H，5-H)，4.962(dd，1H，J＝5，9Hz，14-H)4.898(s，1H，20-H)，3.021(t，1H，J＝7.7Hz，2’-CH₂)，2.92(d，1H，J＝6Hz，3-H)，2.79(m，2H，3’-CH2，13-H)，2.66(m，1H，3’-CH2)，2.42(dd，1H，J＝5，18.5Hz，13-H)，2.15(d，1H，J＝21.5Hz，2’-CH₂)，2.073-2.009(3×s+br.s，12H，9-OAc-CH₃，10-OAc-CH₃，2-OAc-CH3，14-OAc-CH3，18-CH₃)，1.93(d，1H，J＝2Hz，1-H)，1.79-1.64(m，4H，6-H，7-H)，1.672(s，3H，16-CH₃)，1.110(s，3H，17-CH₃)，0.844(s，3H，19-CH₃) Example 11: 5 alpha, 14 beta-dideacetyl-14-oxy-5 alpha-phenylpropionyloxy-neosequoyin AStep A: 2 alpha, 5 alpha, 10 beta, 14 beta-tetradeacetyl-neosequoyin A

50mg (0.099mmol)2 α, 5 α, 10 β, 14 β -tetraacetoxy-sequoyiin A in 2ml methanol, 0.4ml 2mol/L KOH (0.8mmol) was added and the mixture was warmed to 40 ℃ and stirred for 8h, concentrated to dryness under reduced pressure, dissolved in ethyl acetate, filtered off the insoluble matter, evaporated to the solvent under reduced pressure, dried under vacuum to give 32mg of the desired product in 96% yield. And B: 5 alpha, 14 beta-dideacetyl-neosequoyin A

1.6g (4.8mmol)2 alpha, 5 alpha, 10 beta, 14 beta-tetradeacetyl-neosequoyin A in 11ml pyridine is added into 1.8ml acetic anhydride (19.2mmol), stirred for 3h45min at 40-60 deg.C, added with methanol to stop the reaction, diluted with ethyl acetate, 1mol/L hydrochloric acid, saturated CuSO₄Aqueous solution, saturated sodium bicarbonate aqueous solution, brine, anhydrous sodium sulfate drying, decompression concentration, silica gel column purification, petroleum ether/ethyl acetate (1.5/1-1/1), obtain the desired product 0.46 g. The yield thereof was found to be 23%. And C: 5 alpha, 14 beta-dideacetyl-14 beta-TES-neosequoyin A

85mg (0.2021mmol) of 5 alpha, 14 beta-dideacetyl-neosequoyin A and 138mg (2.021mmol) of imidazole in 8ml of a mixed solution of dichloromethane and DMF are reacted at-12 to-14 ℃ for 20 min. The reaction was terminated with 6ml of ice water. Extracting with 3 × 6ml dichloromethane, mixing organic phases, washing with brine, drying with anhydrous sodium sulfate, concentrating under reduced pressure, purifying with silica gel column, and eluting with petroleum ether/ethyl acetate (10/1-8/1) to obtain 36mg of the desired product. The yield thereof was found to be 33.3%. Step D: 5 alpha, 14 beta-dideacetyl-14-oxy-5 alpha-phenylpropionyloxy-neosequoyin A

50mg (0.09349mmol) of 5 alpha, 14 beta-dideacetyl-14 beta-TES-neosequoyin A is added into 2ml of dichloromethane, 56mg (0.374mmol) of benzoic acid and 38mg (0.187mmol) of DCC are added, the mixture is stirred at room temperature for 15min, 46mg (0.374mmol) of DMAP is added for 2h, the mixture is heated to 30-40 ℃ for 6h, the reaction is stopped by 3ml of methanol, the pH value is adjusted to 6-7 by 1mol/L of HCl, the pH value is adjusted to about 3 by 0.5% of HCl solution, the mixture is stirred at room temperature for 1h, the pH value is adjusted to 7 by saturated sodium bicarbonate solution, the mixture is concentrated and extracted by 20ml of ethyl acetate, the organic phase is washed by brine, dried by anhydrous sodium sulfate, and the solvent is removed under reduced pressure, and 43mg of a crude product of 14 beta-hydroxy-2 alpha, 10 beta-diacetyloxy-5 alpha-phenylpropoyloxy-neosequoyi. The crude product was dissolved in 3ml dichloromethane, 40mg (0.1873mmol) PCC was added, stirred at room temperature for 2 hours, passed through a short silica gel column, washed with 50ml diethyl ether, concentrated, and purified by thin layer silica gel plate separation to give 28mg of the desired product. The yield thereof was found to be 71.8%.¹HNMR(CDCl₃，δ)：7.31-7.18(5H，Ar)，5.875(dd，1H，J＝12.5Hz，6Hz，10-H)，5.552(dd，1H，J＝2.5Hz，6.5Hz，2-H)，5.202(s，1H，20-H)，5.183(br.s，1H，5-H)4.854(s，1H，20-H)，2.95(m，4H，13-H，9-H，2’-CH₂)，2.71(d，1H，J＝6Hz，3-H)，2.50(m，3H，13-H，3’-CH₂)，2.099(s，3H，10-OAc-CH₃)，2.053(s，3H，2-OAc-CH₃)，2.027(s，3H，18-CH₃)，1.97(m，2H，1-H，9-H)，1.89-1.73(m，3H，7-H，2×6-H)，1.644(s，3H，16-CH₃)，1.275(s，3H，17-CH₃)，1.126(m，1H，7-H)，0.993(s，3H，19-CH₃) Example 12: 5 alpha-deacetyl-14-deacetyloxy-13-oxy-5-3' -indolylpropyloxy-neosequoin A

The expected product was prepared according to the procedure described for example 1, substituting 3 "-indolpropanic acid for phenylpropionic acid in step 6.¹HNMR(CDCl₃，δ)：8.011(br.s 1H，NH)7.678-7.10(5H，Ar)，6.033(dd，1H，J＝12Hz，5.5Hz，10-H)，5.46(d，1H，J＝6.5Hz，2-H)，5.264(s，1H，20-H)，5.238(br.s，1H，5-H)，4.813(s，1H，20-H)，3.173(d，1H，J＝6.5Hz，3-H)，3.072(m，2H，2’-CH₂)，2.781(dd，1H，J＝7.3Hz，20Hz，14-H)，2.636(m，2H，3’-CH₂)，2.472(dd，1H，J＝11.7Hz，14.7Hz，9-H)，2.325(d，1H，J＝20Hz，14-H)，2.149(s，3H，10-OAc-CH₃)，2.13(1H，1-H)，2.087(s，3H，2-OAc-CH₃)，2.061(s，3H，18-CH₃)，1.89-1.7(m，4H，7-H，2×6-H，9-H)，1.686(s，3H，16-CH₃)，1.24(m，7-H)，1.125(s，3H，17-CH₃)，0.895(s，3H，19-CH₃) Experimental example 1 materials and methods

Drugs and reagents: the novel taxanes of the present invention obtained in example 2, 5 or 10 above were dissolved in DMSO. DMSO is a product of Beijing chemical plant. Vincristine (VCR) is a product of Experimental pharmaceutical factory of Beijing university of medical science. RPMI1640 is a GIBCO product. The calf serum is a product of veterinary prevention and treatment center in Beijing military regions.

Cell lines: KB/V200 (human oral epithelial cancer drug-resistant cell line), established by the institute of medicine of Chinese academy of medicine and cultured for passage.

The instrument comprises the following steps: B10-RAD 550 model enzyme marker.

Determination by MTT method: collecting well-grown tumor drug-resistant cells, and preparing into 1 × 10 with RPMI1640 medium containing 10% calf serum⁴The cell suspension was inoculated in 100. mu.l/well (containing 1000 tumor cells) in 96-well plates at 37 ℃ in 5% CO₂The medicines are added after the incubator is used for 24 hours. The experiment was set up with a blank control and a solvent control, VCR group alone and VCR + test sample group, 4 doses per group, 3 parallel wells per dose. Standing at 37 deg.C for 5% CO₂Culturing in incubator for 4 days. The culture medium was discarded, and 100. mu.l of MTT solution (0.4mg/ml, prepared in RPMI 1640) was added to each well, followed by incubation at 37 ℃ for 4 hours. The supernatant was discarded, 150. mu.l of DMSO was added to each well, the Fomazan particles were dissolved, and after gentle shaking, the OD value was measured at a wavelength of 540nm using an enzyme standard B10-RAD 550.

And (4) calculating a result: plotting the different concentrations of the drug and the inhibition rate on cells to obtain a dose response curve, and calculating the half Inhibitory Concentration (IC)₅₀)。

Results of MTT method human cancer cell killing (KB/V200)

Sample IC₅₀(μmol/L)

EXAMPLE 2 Compound 8.624

EXAMPLE 5 Compound 3.952

EXAMPLE 10 Compound 55.243

Verapamil 61.700

Sample (A)Article IC₅₀(μmol/L)

VCR + Compound of example 2 0.023

VCR + Compound of example 5 < 0.01

VCR + Compound of example 10 0.038

VCR + Verapamil 0.066 notes: increased mdr-1 gene expression and increased P-gp expression are used as main drug resistance mechanism, and Vincristine (VCR) is treated

The drug resistance is over 100 times, and the drug resistance is cross resistance to adriamycin, paclitaxel, colchicine and the like.

Experimental results of control drugs:

the derivatives of the taxane compounds can remarkably reverse drug resistance to multidrug resistant human oral epithelial cancer cells and enhance the activity of anticancer drugs. The activity of the compound of example 2, the compound of example 5 and the compound of example 10 was stronger than that of the known control Verapamul.

Claims

1. A compound of the general formula (1) and stereoisomers thereof,

wherein,

R₁is hydrogen or hydroxyl, and the hydroxyl is hydrogen or hydroxyl,

R₂is a hydrogen atom, and is,

R₁R₂in the formula of ═ O,

R₃is hydrogen, hydroxy, C_1-8An alkanoyloxy group which is selected from the group consisting of alkanoyloxy groups,

R₄is a hydrogen atom, and is,

R₃R₄in the formula of ═ O,

R₅is C_1-8Alkanoyloxy, benzoyloxy, substituted benzoyloxy, the substituents being hydroxy, methoxy or halogen, there being 1 or 2 identical or different substituents, R₆Is hydrogen, R₇Is hydroxy, alicyclic group C_1-8Alkanoyloxy group, heterocyclic group C_1-8Alkyl acyloxy, aromatic rings

Radical C_1-8Alkanoyloxy, including phenyl C_1-8With an alkanoyloxy group, which may have on the phenyl group

1 or 2 identical or different substituents which may be azido, methoxy

Or halogen at C_1-8The alkyl group may have 1 or 2 substituents which may be the same or different,

the substituent may be O, hydroxy, methoxy or halogen, provided that the substituent is absent

In the same position, R₈Is hydrogen, R₉Is hydrogen, hydroxy, C_1-8Alkanoyloxy radical, R₁₀Is hydrogen, R₉R₁₀Is ═ O, R₁₁Is hydroxy, C_1-8An alkyl acyloxy group, a benzoyl oxy group, an aryl formyloxy group,

R₁₂is a hydrogen atom, and is,

R₁₁R₁₂o but excluding the general formula:

2. the compound and its stereoisomers of claim 1, wherein the compound is selected from the group consisting of formula Ia, Ib, Ic, Id and Ie;

wherein the compound of formula Ia or stereoisomers thereof is:

wherein,

R₁R₂in the formula of ═ O,

R₃is hydrogen, R₄Is a hydrogen atom, and is,

R₆is a hydrogen atom, and is,

R₇is an alicyclic group C_1-8Alkanoyloxy group, heterocyclic group C_1-8Alkyl acyloxy, aromatic radical C_1-8

Alkanoyloxy, including phenyl C_1-8Alkanoyloxy, which may have 1 or more in the phenyl group

2 identical or different substituents which may be azido, methoxy or halogen

Element at C_1-8The alkyl group may have 1 or 2 identical or different substituents, substituted

The group may be O, hydroxy, methoxy or halogen, provided that the substituents are not identical

In the position of the position,

R₈is a hydrogen atom, and is,

R₉is a hydrogen atom, and is,

R₁₀is a hydrogen atom, and is,

R₁₂is a hydrogen atom, and is,

n-0-7, and Ib compound or its stereoisomer is:wherein,

R₁is a hydrogen atom, and is,

R₂is a hydrogen atom, and is,

R₄is a hydrogen atom, and is,

Radical, R₆Is hydrogen, R₇Is an alicyclic group C_1-8Alkanoyloxy group, heterocyclic group C_1-8Alkyl acyloxy, aromatic radical C_1-8

Alkanoyloxy radicals, including phenylC_1-8Alkanoyloxy, which may have 1 or more in the phenyl group

2 identical or different substituents which may be azido, methoxy or halogen

In the position of the position,

R₈is a hydrogen atom, and is,

R₉is a hydrogen atom, and is,

R₁₀is a hydrogen atom, and is,

R₁₂is a hydrogen atom, and is,

n-0-7, Ic compounds or stereoisomers thereof are:wherein,

R₁is a hydrogen atom, and is,

R₂is a hydrogen atom, and is,

R₄is a hydrogen atom, and is,

R₆is a hydrogen atom, and is,

2 identical or different substituents which may be azido, methoxy or halogen

In position, R₈Is hydrogen, R₉Is hydroxy, C_1-8Alkanoyloxy radical, R₁₀Is hydrogen, R₁₁Is hydroxy, C_1-8Alkyl acyloxy, benzoyl, aryl formyloxy, R₁₂Is hydrogen, n is 0 to 7,

a compound of formula Id, or a stereoisomer thereof:wherein,

R₁is a hydrogen atom, and is,

R₂is a hydrogen atom, and is,

R₃R₄in the formula of ═ O,

R₆is a hydrogen atom, and is,

R₇alicyclic group C_1-8Alkanoyloxy group, heterocyclic group C_1-8Alkyl acyloxy, aromatic radical C_1-8Alkyl radical

Acyloxy radicals, including phenyl C_1-8Alkanoyloxy, which may have 1 or 2 in the phenyl group

The same or different substituent group can be azido, methoxy or halogen,

at C_1-8The alkyl group may have 1 or 2 substituents which may be the same or different

With the proviso that the substituents are not in the same position, O, hydroxy, methoxy or halogen

In the above-mentioned manner,

R₈is a hydrogen atom, and is,

R₉is a hydrogen atom, and is,

R₁₀is a hydrogen atom, and is,

R₁₂is a hydrogen atom, and is,

n-0-7, the compound of formula Ie or a stereoisomer thereof is:wherein,

on ring AThe double bond being at C₁₂And C₁₃.

R₁Is a hydrogen atom, and is,

R₂in the absence of the presence of the agent,

R₃R₄in the formula of ═ O,

R₆is a hydrogen atom, and is,

2 identical or different substituents which may be azido, methoxy or halogen

In the position of the position,

R₈is a hydrogen atom, and is,

R₉is a hydrogen atom, and is,

R₁₀is a hydrogen atom, and is,

R₁₂is a hydrogen atom, and is,

n＝0-7，

3. the compound and its stereoisomers as claimed in claims 1 or 2, wherein the compound comprises 14-deoxy-13-carbonyl-5-phenylpropanoyl-taxane a, 14-deoxy-13-carbonyl-5-phenylpropenoyl-taxane a, 14-deoxy-13-carbonyl-5-phenylacetyl-taxane a, 14-deoxy-13-carbonyl-5-benzoyl-taxane a, 14-deoxy-13-carbonyl-5-2 ", 5" -dimethoxyphenylacetyl-taxane a, 14-deoxy-13-carbonyl-5-benzoylpropionyl-taxane a, 14-deoxy-13-carbonyl-5-phenylbutyryl-taxane a 5-phenylpropionyl-taxane a, 5-phenylpropenoyl-taxane a or 9-acetyl-5-phenylpropenoyl-taxane a.

4. A process for the preparation of a compound of formula (I) comprising:

(1) treating neotaxin A with alkaline water solution in water soluble organic solvent for selective hydrolysis to obtain compound of formula a-l, i.e. 14 beta-deacetyl-neotaxin A;

(6) the compound of formula a-5 obtained in step 5 is reacted with R 'COOH in a dry organic solvent in the presence of DCC and an organic base to form an ester, and the compound of formula Ia, i.e. 5 alpha-deacetyl-14-deoxy-13-oxo-5 alpha-R' acyl-neosequoyin A, is obtained.

5. The method according to claim 4, wherein the organic solvent used in step (1) is methanol, ethanol, or TlHF and mixed solvent of methanol/dichloromethane solution and methanol/acetone solution; the alkaline aqueous solution is KOH or K₂CO₃An aqueous solution; the reaction is carried out at a temperature of 0 ℃ to 40 ℃, preferably 10 ℃ to 15 ℃.

6. The method according to claim 4, wherein the basic medium used in the step (2) is an anhydrous base comprising sodium hydroxide, sodium hydrogen carbonate, potassium tert-butoxide; the organic solvent comprises tetrahydrofuran, diethyl ether and anhydrous organic solvent which does not interfere the reaction of dioxane.

7. The process as claimed in claim 4, wherein the reaction in step (3) is carried out at a temperature of generally 50 to 100 ℃, preferably 70 to 80 ℃.

8. The method of claim 4, wherein in step (5), the reaction is first carried out in methanol or a mixed solvent of methanol and tetrahydrofuran at 20-40 deg.C with 2-4mol/L KOH or K₂CO₃Treating the aqueous solution to obtain a full hydrolysate; the organic solvent and alkali are pyridine or dichloromethane/DMAP, and the reaction is carried out at 20-50 ℃, preferably 20-30 ℃.

9. The process according to claim 4, wherein in step (6), the organic solvent is an inert organic solvent which does not interfere with the reaction, preferably toluene; the organic base comprises DMAP, 4-PP and triethylamine, preferably DMAP; the reaction is generally carried out at 20 to 50 ℃ and preferably at 30 to 35 ℃.

10. A process for the preparation of a compound of formula (I) comprising the steps of:

(1) the neosequoyins A is hydrolyzed in organic solvent through alkali reaction to obtain full hydrolysate 2 alpha,

5 α, 10 β, 14 β -tetraacetyl-neosequoyin a; after drying, the mixture is treated by organic alkali,

with acetic anhydride to produce the compound of formula b-1, i.e. 5 alpha-deacetyl-

Neosequoyine A;

(2) the compound b-1 obtained in the step 2 is put in a dry organic solvent, DCC and an organic base

Reacting with R' COOH in the presence of oxygen to form an ester to obtain a compound of the formula I_bCompound 5 alpha-deacetyl-5 alpha-R'

Acyl-neosequoyin a.

11. The method according to claim 10, wherein the solvent in step (1) comprises methanol, ethanol, methanol/dichloromethane, methanol/acetone, methanol/tetrahydrofuran mixed solvent, preferably methanol, methanol/tetrahydrofuran; the base used comprises K₂CO₃Or KOH, at 10-60 ℃, preferably 20-40 ℃.

12. The method according to claim 10, wherein the organic solvent in the step (2) is an inert organic solvent which does not interfere with the reaction; the organic base comprises DMAP, 4-PP and triethylamine; the reaction is carried out at 20-40 ℃.

13. A process for the preparation of a compound of formula (I) comprising the steps of:

(3) the resulting hydroxy compound is reacted with R 'COOH in a dry organic solvent in the presence of DCC and an organic base to form an ester, to give the compound of formula Ic, 5 α -deacetyl-5 α -R' acyl-9 α -acetyl-neosequoyin A.

14. The method according to claim 13, wherein in step (1), sequoyianin A is added at a concentration of 10-40mg/L, preferably 20-30 mg/L; the adding period of the sequoyins A is 6-18 days, preferably 12-18 days of the cell cycle; the reaction time may be 2 to 18 days, preferably 6 to 10 days.

15. The method of claim 13, wherein in step (2), the organic solvent is methanol, ethanol, or a mixture thereof, such as methanol/dichloromethane, methanol/acetone, or methanol/tetrahydrofuran, and the base is typically K₂CO₃Or KOH, the reaction temperature is controlled between 20 and 60 ℃, and the reaction temperature is preferably between 20 and 40 ℃. The subsequent acylation is generally carried out in pyridine, at room temperature-40 ℃.

16. The method of claim 13, wherein in step (3), the organic solvent is an inert organic solvent which does not interfere with the reaction, the organic base comprises DMAP, 4-PP, and triethylamine, and the reaction is carried out at 20-40 ℃.

17. A process for the preparation of a compound of formula (I) which comprises the steps of:

(4) and (3) deprotecting the compound of the formula d-4 obtained in the step (3), concentrating and drying the obtained product, dissolving the product in an organic solvent, and oxidizing the product by PCC to obtain the compound of the general formula (I).

18. The method according to claim 17, wherein in step (1), the organic solvent is a dry organic solvent which does not interfere with the reaction, and the organic base is a weak organic base; the reaction is generally carried out at a temperature of from-78 ℃ to-20 ℃ and preferably from-50 ℃ to-30 ℃.

19. The method of claim 17, wherein in step (3), the organic solvent is an inert organic solvent that does not interfere with the reaction, and the organic base comprises DMAP, 4-PP, triethylamine; the reaction is generally carried out at 20 to 40 ℃.

20. A process for preparing the compound of general formula (I) includes oxidizing the compound of formula d-4 with oxidant, reaction with acidic medium, and double bond transfer on silica gel.

21. The process of claim 20, wherein the oxidizing agent comprises TPAP, PCC/sodium acetate, preferably PCC or TPAP; the acidic medium may be silica gel, trifluoroacetic acid, p-toluenesulfonic acid, hydrochloric acid, HF, with silica gel or HF being preferred.

22. A pharmaceutical composition characterized by comprising a therapeutically effective amount of a compound of formula (I) and/or a stereoisomer or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

23. The use of the compound of claim 1 and stereoisomers or pharmaceutically acceptable salts thereof in the preparation of anti-malignant tumor medicaments.

24. The use of the compound of claim 1 and stereoisomers or pharmaceutically acceptable salts thereof in the preparation of medicaments for treating multidrug resistant tumors and medicaments for reversing the resistance of multidrug resistant tumors.