CN103242348A

CN103242348A - Indoline diketopiperazine spiro-compounds as well as preparation method and use thereof

Info

Publication number: CN103242348A
Application number: CN2013101913845A
Authority: CN
Inventors: 崔承彬; 房士明; 李长伟; 王楠; 吴长景
Original assignee: Institute of Pharmacology and Toxicology of AMMS
Current assignee: Institute of Pharmacology and Toxicology of AMMS
Priority date: 2013-05-22
Filing date: 2013-05-22
Publication date: 2013-08-14
Anticipated expiration: 2033-05-22
Also published as: CN103242348B

Abstract

The invention belongs to the field of medical and chemical industry, relates to indoline diketopiperazine spiro-compounds as well as a preparation method and use thereof, and in particular relates to compounds as shown in formula I or pharmaceutically acceptable salts of the compounds. The compounds have a framework dimerization molecular structure condensed by terracycloindoline diketopiperazine and 1-oxa spiro[4, 5] decane. Two ethylene oxide structures are arranged on the spiro hexatomic ring. The invention further relates to penicillium purpurogenum for preparing the compounds as shown in the formula I. Experiments show that the compounds can be used for preparing cytoskeletal protein inhibitors, apoptosis inducers, tumor cell proliferation inhibitors, tumor cell killing agents or antitumor drugs. The compounds provided by the invention have good antitumor activity.

Description

Indoline diketopiperazine spiro-compound and preparation method and application thereof

Technical Field

The invention belongs to the field of pharmaceutical chemicals, relates to a spiro compound, and particularly relates to an indoline diketopiperazine spiro compound, a composition containing the indoline diketopiperazine spiro compound, and a preparation method and application of the indoline diketopiperazine spiro compound.

Background

Tumor (Tumor) is a new organism formed by the clonal abnormal hyperplasia of a certain cell of local tissue which loses the normal regulation and control of the growth of the certain cell at the gene level under the action of various carcinogenic factors. Tumors are generally classified into two broad categories, benign and malignant. All malignant tumors are collectively referred to as cancer (cancer).

Spiro compounds are very abundant in structural types. Spiro [4,5] alone]The decane compound can form a plurality of different structure types because one or more positions of different positions on the spiro skeleton are substituted by heteroatoms such as oxygen, nitrogen, sulfur and the like. Having 1-oxaspiro [4,5]]Some spiro compounds having a decane skeleton structure have been known so far, but not many have been known which have two ethylene oxide structural fragments on the cyclohexane ring of such spiro skeleton. Wherein, the 6,7 position and the 9,10 position of the cyclohexane are respectively an ethylene oxide segment, the 8 position of the carbon is a ketocarbonyl group or sp³Known 1-oxaspiro [4,5] s having a carbon 2-position on the pentameric spiro ring as a lactone carbonyl or hemiacetal carbon, hybridized oxygen replacing carbon atoms]The decane-based compound is more limited in number. Document 1 (H.W.Fehlhaber et al, Structure of araorosin, a new analytical of a novel latex type, J.Am.chem.Soc.,1988,110(24):8242-128-1, a novel interleukin-1 beta converting enzyme inhibited produced by Penicillium sp.E-2128, J.Antibiot, 2003,56(11): 891-898) describes four spiro compounds with a keto carbonyl group at the 8-carbon position, a lactone carbonyl or hemiacetal carbon at the 2-carbon position, and an unsaturated dodeca aliphatic amide substituent bearing two branched methyl groups attached to the 3-carbon position. Such partial compounds have antibacterial activity (see the above-mentioned documents 1, 2, 3 and 4[ K.Roy, et al, Ananoroson, a novel antibacterial from Pseudomonas bacteria I.Taxolomy, promotion, isolation, chemical and biological Properties, J.Antibiot.,1988, XLI (12): 1780-1784;)]) Some of them also have a selective inhibitory action on interleukin-1 beta converting enzyme activity and an action of inhibiting Lipopolysaccharide (LPS) to induce THP-1 cells to secrete mature interleukin-1 beta converting enzyme (see the above-mentioned document 3). Document 5 (K.Roy, et al, Aranorosol A and Ananorosol B, two new microorganisms from Pseudomonas bacteria: Production, isolation, structural identification and biological properties, J.Antibiol., 1992,45(10): 1592-1598) describes five such carbons 8 as sp.2 with an oxygen substituent attached³The hybrid carbon, carbon 2 is a lactone carbonyl or hemiacetal carbon, the carbon 3 position is connected with a spiro compound with an unsaturated dodecaaliphatic amide substituent carrying two branched methyl groups, and partial compounds have weak antibacterial and antifungal activities.

Many compounds belonging to different structural types are known as indole diketopiperazine compounds (see document 6[ Maryanmin, et al, synthetic research progress of indole diketopiperazine compounds, organic chemistry, 2010, 30(11): 1624-. Indoline diketopiperazines as their dihydro derivatives are also known to belong to various structural classes. However, the tetracyclic indoline diketopiperazine compound formed by connecting a five-membered ring in parallel between the five-membered nitrogen heterocycle of indoline and the six-membered nitrogen heterocycle of diketopiperazine is known to be less. Document 8 (K.Arai, et al, Structure of genes A and B, new alkali synthetic from Petroleum of genes Takeuchi, chem.Pharm.Bull.,1989,37(11):2937-, total synthesis of (+) -asperzine, Tetrahedron,2007,63(35): 8499-8513) describes several such indoline diketopiperazine compounds. Documents 10 and 14 (n.m. gomes, et al, eurocristine, a newdiketopiperazine dimer from the marine span-associated fungus eurotium cristatum, phytochem.lett.,2012,5(4): 717-720) also describe 4 such compounds having a dimeric tetracyclinedione piperazine skeleton. Such partial compounds are known to have weak antitumor activity (see documents 8, 9 and 15[ Chayujing, et al, research on novel antitumor activity products of gentamicin-resistant mutant strain of Penicillium purpurogenum G59, J.Pharmacology, 2011, 38(3):216-222 ]), and are also known to have biological activity of competitively antagonizing the binding of substance P to NK-1 receptor (see document 10). Since substance P plays an important role as an endogenous ligand of NK-1 receptor in pain and inflammation processes, active substances that competitively antagonize substance P binding to NK-1 receptor have potential application values as non-addictive analgesics or anti-inflammatory agents.

However, no document has been found on indoline diketopiperazine spiro compounds having a dimer molecular skeleton formed by condensation-linking the tetracyclic indoline diketopiperazine skeleton structure and the 1-oxaspiro [4,5] decane skeleton structure according to the present invention described below in the molecule.

Disclosure of Invention

The inventors succeeded in obtaining 2 mutant strains of Penicillium purpurogenum BD-1-3 and 3-f-31 by creative labor and diligent efforts, and found indoline diketopiperazine spiro compounds represented by the following formula I from their fermentation products:

wherein,

the Arabic numerals represent corresponding positions of an indoline diketopiperazine skeleton and a spiro skeleton, and the structure is characterized in that molecules contain a dimeric molecular skeleton structure formed by condensation and connection of a tetracyclic indoline diketopiperazine skeleton and a 1-oxaspiro [4,5] decane skeleton, and a spiro six-membered ring of the dimeric molecular skeleton structure has two oxirane structures.

The inventor also surprisingly finds that the compound shown in the formula I can effectively kill tumor cells or inhibit the proliferation of the tumor cells, has good antitumor activity, and thus has good potential as an antitumor and anticancer medicament.

The following invention is thus provided:

one aspect of the present invention pertains to compounds of formula I as described above, or a pharmaceutically acceptable salt thereof,

wherein R is₁-R₃Each independently represents hydrogen, hydroxy, substituted or unsubstituted:

C_1-10(e.g. C)_1-6、C_1-8) Linear or branched, saturated or unsaturated hydrocarbon radical, C_1-10(e.g. C)_1-6、C_1-8) Straight chain orBranched saturated or unsaturated hydrocarbyloxy radical, C_2-18(e.g. C)_2-6、C_2-8) A linear or branched saturated or unsaturated aliphatic acyloxy group, or an aromatic acyloxy group (e.g., benzoyloxy group),

the substituent is selected from hydroxyl, halogen (such as fluorine, chlorine, bromine and iodine), nitro and benzyloxy, and the number of the substituent is 1-3 (such as 1, 2 and 3).

In an embodiment of the present invention, wherein,

R₁represents substituted or unsubstituted C_1-10(e.g. C)_1-6、C_1-8) Straight or branched chain alkyl, preferably 1- (2-methyl) octyl;

R₂and R₃Each independently represents hydrogen, hydroxy, substituted or unsubstituted:

C_1-10(e.g. C)_1-6、C_1-8) Straight or branched alkoxy, C_2-18(e.g. C)_2-6、C_2-8) A linear or branched saturated aliphatic acyloxy group, or an aromatic acyloxy group (e.g., benzoyloxy group),

In a specific embodiment of the present invention, wherein,

R₁represents 1- (2-methyl) octyl;

R₂and R₃Each independently represents hydrogen, hydroxy, methoxy, ethoxy, acetoxy, galloyloxy, tribenzylgalloyloxy, benzoyloxy, p-nitrobenzoyloxy, p-chlorobenzoyloxy, or p-fluorobenzoyloxy.

In a particular embodiment of the invention, the substitution is ortho, para or meta.

In the present invention, said C_1-10The linear or branched saturated or unsaturated hydrocarbon radicals being methyl, ethyl, C₃Straight or branched alkyl, C₄Straight or branched alkyl, C₅Straight or branched alkyl, C₆Straight or branched alkyl, C₇Straight or branched alkyl, C₈Straight or branched alkyl, C₉Straight or branched alkyl, or C₁₀An alkyl group such as a linear or branched alkyl group, or a vinyl group, C₃Straight-chain or branched alkenyl, C₄Straight-chain or branched alkenyl, C₅Straight-chain or branched alkenyl, C₆Straight-chain or branched alkenyl, C₇Straight-chain or branched alkenyl, C₈Straight-chain or branched alkenyl, C₉Straight or branched alkenyl, or C₁₀Alkylene group such as linear or branched alkylene group, or ethynyl group, C₃Alkynyl radical, C₄Straight or branched alkynyl, C₅Straight or branched alkynyl, C₆Straight or branched alkynyl, C₇Straight or branched alkynyl, C₈Straight or branched alkynyl, C₉Straight or branched alkynyl, or C₁₀Straight or branched alkynyl, or phenyl, substituted phenyl, benzyl, substituted benzyl, C₈Straight-chain or branched aromatic hydrocarbon group, C₉Straight or branched aromatic hydrocarbon radicals, or C₁₀An aromatic hydrocarbon group such as a linear or branched aromatic hydrocarbon group.

In the present invention, said C_1-10The linear or branched saturated or unsaturated hydrocarbyloxy groups being methoxy, ethoxy, C₃Straight or branched alkoxy, C₄Straight or branched alkoxy, C₅Straight or branched alkoxy, C₆Straight or branched alkoxy, C₇Straight or branched alkoxy, C₈Straight or branched alkoxy, C₉Linear or branched alkoxy, or C₁₀Linear or branched alkoxy, or is ethyleneoxy, C₃Linear or branched alkenyloxy, C₄Linear or branched alkenyloxy, C₅Linear or branched alkenyloxy, C₆Straight chain orBranched alkyleneoxy radical, C₇Linear or branched alkenyloxy, C₈Linear or branched alkenyloxy, C₉Linear or branched alkenyloxy, or C₁₀An alkenyloxy group such as a linear or branched alkenyloxy group, or an acetylenecarbonyloxy group, C₃Alkenoxy radical, C₄Straight or branched alkynyloxy, C₅Straight or branched alkynyloxy, C₆Straight or branched alkynyloxy, C₇Straight or branched alkynyloxy, C₈Straight or branched alkynyloxy, C₉Straight or branched alkynyloxy, or C₁₀Straight-chain or branched alkynyloxy such as phenoxyl, substituted phenoxyl, benzyloxy, substituted benzyloxy, and C₈Straight or branched chain arene oxy, C₉Linear or branched areneoxy, or C₁₀An aromatic hydrocarbon oxy group such as a straight chain or branched chain aromatic hydrocarbon oxy group.

In the present invention, said C_2-18The linear or branched saturated or unsaturated aliphatic or aromatic acyloxy group is formyloxy, acetoxy, propionyloxy, acryloyloxy, butyryloxy, isobutyryloxy, valeryloxy, isovaleryloxy, succinyloxy, octanoyloxy, decanoyloxy, royal jelly acyloxy, lauroyloxy, 3-hydroxylauranyloxy, myristoyloxy, palmitoyloxy, stearoyloxy, oleoyloxy, or linoleoyloxy, or is benzoyloxy, substituted benzoyloxy, phenylacetyloxy, substituted phenylacetyloxy, or linear or branched C at position 2_2-8Substituted phenylacetyloxy, phenylpropionyloxy, 2-substituted by a straight or branched chain C_2-7Substituted phenylpropoyloxy, substituted in position 3 by a straight or branched chain C_2-7Substituted phenylpropoyloxy, 2-and 3-positions simultaneously substituted by a straight-chain or branched C_2-6Substituted phenylpropoyloxy, phenylbutyloxy, substituted in position 2 by a straight or branched chain C_2-6Substituted phenylbutyloxy, substituted in position 3 by a straight or branched chain C_2-6Substituted phenylbutyloxy, substituted in position 4 by a straight or branched chain C_2-6Substituted phenylbutyryloxy, the 2 and 3 or2 and 4 or 3 and 4 positions being simultaneously substituted by a straight-chain or branched C_2-5Substituted phenylbutyryloxy, or both 2-and 4-positions by a straight chainOr branched C_2-4Substituted phenylbutyloxy and the like.

The second aspect of the invention relates to Penicillium purpurogenum BD-1-3 (Penicillium purpurogenum BD-1-3), the preservation number is CGMCC No.4284, the preservation date is 11.1.2010, the preservation unit is China General Microbiological Culture Center (CGMCC) of the culture Collection management Committee of microorganisms, the address is No. 3 of Xilu No. 1 of the Beijing university of Chaoyang district, the institute of microorganisms of the China academy of sciences, and the postal code is 100101.

The strain is a mutant strain BD-1-3 obtained by performing diethyl sulfate mutagenesis on a wild Penicillium purpurogenum G59 strain of Penicillium fungus of Penicillium purpurogenum which is separated from a sea mud sample of intertidal zone of Bohai Bay of the river Bohai gulf of Tianjin pond donkey colal and identified as Penicillium purpurogenum through taxonomic study. It has the following microbiological characteristics:

after the cells are inoculated on a PDA plate culture medium by streaking and cultured for 3-5 days at 28 ℃, light gray light cyan colonies which grow slightly gray can be seen from the front side of the plate, and orange reddish or pink purple pigment which permeates the bottom of the culture can be seen from the back side of the plate;

culturing the colonies on a Chachi culture medium at 25 deg.C for 12 days with diameter of 17-30mm, and keeping the diameter flat or nearly flat or having several concentric rings; the texture is velvet or flocculent; the conidium surface is dark gray green or dark green, and is similar to olive lemon color, brown olive color or slightly dark olive green; mycelium is orange, yellow or orange-red; dark red, orange or purple red on the back; conidiophores occur in the substrate, fewer aerial hyphae occur, the conidiophores stem (70-) 100-; the broom-shaped branches are double-rotor, even three-rotor or single-rotor, and are close to each other and are nearly parallel; 4-8 stem bases are arranged in each wheel, 9.0-13(-14) multiplied by 2.5-3.0 mu m; each round of phialides has 4-6 phialides, 9.5-13 x (1.8-)2.0-2.4 mu m, and stems are obvious in shape of needles; conidiophores are elliptical, when fully mature, part of conidiophores are approximately spherical, 2.8-3.5(-4.0) multiplied by 2.2-3.0 mu m, and the wall is smooth or slightly rough; the conidiophore chain is loose and is forked or nearly cylindrical.

The third aspect of the invention relates to Penicillium purpurogenum3-f-31 (Penicillium purpurogenum 3-f-31), the collection number is CGMCC No.7286, the collection date is 3.7.2013, the collection unit is China general microbiological culture Collection center (CGMCC), the address is No. 3 of West Lu No. 1 of the Korean area in Beijing, the institute of microbiology of the national academy of sciences, and the postal code is 100101.

The strain is a neomycin resistant mutant strain 3-f-31 of the Penicillium purpurogenum G59, which is obtained by separating a sample of intertidal sea mud from a Penicillium natans donkey colal river Bohai gulf of a Penicillium fungus, identifying the sample as the wild Penicillium purpurogenum G59 of the Penicillium fungus of the Penicillium purpurogenum through taxonomic study and screening 5mg/ml neomycin resistance in 50% (v/v) DMSO. It has the following microbiological characteristics:

after the culture is carried out for 3-5 days at 28 ℃ by streak inoculation on a PDA plate culture medium, a light gray light cyan colony which is slightly cyan and is grown can be seen from the front side of the plate, the color of the bacterial colony is more slightly cyan and has obvious difference compared with the blue mould BD-1-3, and the orange pigment slightly yellow at the bottom of the permeation culture can be seen from the back side of the plate;

A fourth aspect of the present invention relates to a process for the preparation of compound 1 and/or 2, comprising the steps of:

fermenting and culturing the Penicillium purpurogenum BD-1-3 or 3-f-31 to obtain a fermented product containing the compound 1 and/or 2, and separating and purifying the fermented product to obtain the compound 1 and/or 2;

wherein said compound 1 is a compound of formula I according to the first aspect of the invention, wherein R₁=1- (2-methyl) octyl radical, R₂=OH，R₃= H; wherein said compound 2 is a compound of formula I according to the first aspect of the invention, wherein R₁=1- (2-methyl) octyl radical, R₂=R₃=OH。

Specifically, the separation and purification includes conventional methods for separation and purification of natural products, such as liquid-liquid extraction, column chromatography, thin layer chromatography, high performance liquid chromatography, recrystallization, and the like, which are well known to those skilled in the art.

Other production strains of the genus Penicillium capable of producing compounds 1 and/or 2 may also be used for the fermentative culture.

In one embodiment of the present invention, the preparation method comprises the following steps:

1) carrying out fermentation culture on the penicillium purpurogenum to obtain fermentation liquor;

2) filtering the fermentation liquor to obtain filtrate and thalli;

3) extracting the filtrate obtained in the step 2) with ethyl acetate to obtain an ethyl acetate extract of the filtrate;

4) suspending the thallus obtained in the step 2) in 50-95% (v/v) acetone aqueous solution, crushing thallus cells, leaching and filtering, concentrating the filtrate under reduced pressure until the filtrate does not contain acetone, and extracting with ethyl acetate to obtain an ethyl acetate extract;

5) combining the ethyl acetate extracts obtained in the steps 3) and 4), and concentrating under reduced pressure to dryness to obtain an ethyl acetate total extract;

6) dissolving ethyl acetate total extract with mixed solvent of chloroform-methanol at volume ratio of 1:1, or dissolving methanol soluble fraction obtained by dissolving ethyl acetate total extract with a large amount of methanol with mixed solvent of dichloromethane-methanol at volume ratio of 1:2, separating with 100-mesh 200-mesh silica gel column, performing gradient elution under reduced pressure with petroleum ether-chloroform-methanol solvent system or petroleum ether-dichloromethane-acetone-methanol solvent system as eluent to obtain crude component containing the compound, and separating with two times of Sephadex LH-20 column chromatography (first eluting with 95% ethanol by volume fraction and second eluting with mixed solvent of chloroform-methanol at volume ratio of 1: 1) or one time of Sephadex LH-20 column chromatography (eluting with 95% ethanol by volume fraction) and one time of reversed phase silica gel column chromatography ODS (eluting with water-methanol-acetone solvent system) Systematic gradient elution) to obtain column chromatography components containing the main components of the compound;

7) the column chromatography fractions containing the compound were separated and purified by either two reverse phase HPLC (C18 column eluting with methanol-water volume fraction 80:20 for the first time and 86:14 or 82:18 for the second time) or one HPLC (C18 column eluting with 81:19 methanol-water volume fraction) to give the compound.

In one embodiment of the present invention, the preparation method, wherein the aqueous acetone solution in the step 4) is an aqueous acetone solution containing 70% to 90% (v/v) of acetone; specifically, it is an aqueous acetone solution of 75% to 85% (v/v) of acetone, for example, an aqueous acetone solution of 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, or 85% (v/v).

In one embodiment of the present invention, the preparation method, wherein the aqueous acetone solution in the step 4) is an aqueous acetone solution containing 55% to 75% (v/v) of acetone; specifically, it is an aqueous acetone solution containing 60% to 70% (v/v) of acetone, for example, an aqueous acetone solution containing 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70% (v/v) of acetone.

A fifth aspect of the present invention relates to a method for producing a derivative of compound 1 or2, comprising the steps of:

respectively carrying out derivatization reaction on the compound 1 or2 of the invention and appropriate chemical reaction reagents such as halogenated hydrocarbon, ester, acid anhydride, acyl chloride and the like, and separating and purifying reaction products to obtain the derivatives;

Specifically, the separation and purification includes conventional methods for separation and purification, such as column chromatography, thin layer chromatography, high performance liquid chromatography, recrystallization, and the like, which are well known to those skilled in the art.

1) dissolving the compound 1 or2 with acetone or anhydrous pyridine;

2) performing derivatization reaction with methyl iodide, diethyl sulfate, acetic anhydride, gallic acid, tribenzyl galloyl chloride, benzoyl chloride, p-nitrobenzoyl chloride, p-chlorobenzoyl chloride or p-fluorobenzoyl chloride for 1-48 h or 3-10 days under the conditions of keeping out of the sun or heating to keep out of the sun at room temperature and adding hydrochloric acid for catalysis or not adding hydrochloric acid for catalysis;

3) separating and purifying the reaction product by using a silica gel thin layer chromatography (a mixed solvent of chloroform and methanol in a volume ratio of 80: 20-99: 1 is used as a developing agent) to prepare the compound.

In one embodiment of the present invention, the production method, wherein the "warming" in the step 2) means heating to maintain the reaction temperature at 30 ℃ to 80 ℃; specifically, the reaction temperature is maintained at 40 ℃ to 60 ℃, e.g., 40 ℃, 50 ℃, or 60 ℃.

A sixth aspect of the present invention relates to a composition comprising a compound of formula I according to any one of the first aspect of the present invention or a pharmaceutically acceptable salt thereof, optionally together with one or more pharmaceutically acceptable carriers or excipients. In particular, the composition is a pharmaceutical composition. The composition or the pharmaceutical composition can be used for resisting tumors or inhibiting the proliferation of tumor cells or killing the tumor cells.

In a further aspect, the invention relates to the use of a compound of formula I according to any one of the first aspect of the invention or a pharmaceutically acceptable salt thereof as a tool for the preparation of cytoskeletal protein inhibitors, apoptosis inducers, tumor cell proliferation inhibitors, or tumor cell killing agents, as required in life science research such as tumor molecular biology; further, the tumor cell is a leukemia cell or a cancer cell derived from epithelium (for example, a cervical cancer cell, a gastric cancer cell, a breast cancer cell, a lung cancer cell, a liver cancer cell, or a colon cancer cell); still further, the leukemia cells are chronic myelogenous leukemia cells or acute promyelocytic leukemia cells; furthermore, the tumor cells are human chronic myelogenous leukemia K562 cells, human acute promyelocytic leukemia HL-60 cells, human cervical cancer HeLa cells, human gastric cancer BGC-823 cells, or human breast cancer MCF-7 cells.

A further aspect of the present invention relates to the use of a compound of formula I according to any one of the first aspect of the present invention or a pharmaceutically acceptable salt thereof, or a composition according to any one of the sixth aspect of the present invention, in the manufacture of a medicament or agent for killing or inhibiting proliferation of tumor cells; further, the tumor cell is a leukemia cell or a cancer cell derived from epithelium (for example, a cervical cancer cell, a gastric cancer cell, a breast cancer cell, a lung cancer cell, a liver cancer cell, or a colon cancer cell); still further, the leukemia cells are chronic myelogenous leukemia cells or acute promyelocytic leukemia cells.

In an embodiment of the present invention, the tumor cell is a human chronic myelogenous leukemia K562 cell, a human acute promyelocytic leukemia HL-60 cell, a human cervical cancer HeLa cell, a human gastric cancer BGC-823 cell, or a human breast cancer MCF-7 cell.

A further aspect of the present invention relates to a method of killing tumor cells or inhibiting proliferation of tumor cells in vivo or in vitro comprising the step of administering an effective amount of a compound of formula I according to any one of the first aspect of the present invention or a pharmaceutically acceptable salt thereof, or a composition according to any one of the sixth aspect of the present invention; further, the tumor cell is a leukemia cell or a cancer cell derived from epithelium (for example, a cervical cancer cell, a gastric cancer cell, a breast cancer cell, a lung cancer cell, a liver cancer cell, or a colon cancer cell); still further, the leukemia cells are chronic myelogenous leukemia cells or acute promyelocytic leukemia cells.

A further aspect of the invention relates to the use of a compound of formula I according to any one of the first aspect of the invention or a pharmaceutically acceptable salt thereof, or a composition according to any one of the sixth aspect of the invention, in the manufacture of an anti-neoplastic medicament; for example, the tumor is leukemia or a cancer derived from epithelium (e.g., cervical cancer, gastric cancer, breast cancer, lung cancer, liver cancer, or colon cancer); further, the leukemia is chronic myelogenous leukemia, acute promyelocytic leukemia.

A further aspect of the present invention relates to a method of treatment and/or prophylaxis and/or co-treatment of cancer comprising the step of administering to a subject an effective amount of a compound of formula I according to any one of the first aspect of the present invention or a pharmaceutically acceptable salt thereof, or a composition according to any one of the sixth aspect of the present invention; the cancer is leukemia or cancer derived from epithelium (such as cervical cancer, gastric cancer, breast cancer, lung cancer, liver cancer, or colon cancer); further, the leukemia is chronic myelogenous leukemia and acute promyelocytic leukemia.

The compound of the formula I is tested to inhibit human chronic myelogenous leukemia K562 cells, human promyelocytic leukemia HL-60 cells, human cervical carcinoma HeLa cells, human breast cancer MCF-7 cells and human gastric cancer BGC-823 cells by adopting an MTT method. Experiments prove that the compound of the formula I can obviously inhibit the proliferation of various human cancer cells in vitro, thereby having the anti-tumor effect.

A further aspect of the present invention relates to the use of a penicillium purpurogenum as defined above for the preparation of compounds 1 and/or 2 according to the invention; in particular to application of Penicillium purpurogenum BD-1-3 with the preservation number of CGMCC No.4284 and Penicillium purpurogenum3-f-31 with the preservation number of CGMCC No.7286 in preparing the compound 1 and/or 2 of the invention.

The compound of the formula I can be prepared into antitumor drugs by being compatible with various pharmaceutically acceptable carriers, excipients or auxiliary materials, and is used for treating tumors.

The compounds of the present invention may be administered alone or in the form of a pharmaceutical composition. The route of administration may be oral, parenteral or topical. The pharmaceutical composition can be formulated into various suitable dosage forms according to the administration route.

Pharmaceutical compositions of the compounds of the present invention may be administered in any of the following ways: oral, aerosol inhalation, rectal, nasal, buccal, topical, parenteral, e.g. subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intraventricular, intrasternal and intracranial injection or infusion, or via an external reservoir. Among them, oral, intraperitoneal or intravenous administration is preferable.

When administered orally, the compounds of the present invention may be formulated in any orally acceptable dosage form, including but not limited to tablets, capsules, aqueous solutions or suspensions. Among these, carriers for tablets generally include lactose and corn starch, and additionally, lubricating agents such as magnesium stearate may be added. Diluents used in capsule formulations generally include lactose and dried corn starch. Aqueous suspension formulations are generally prepared by mixing the active ingredient with suitable emulsifying and suspending agents. Optionally, some sweetener, aromatic or colorant may be added into the above oral preparation.

When applied topically to the skin, the compounds of the present invention may be formulated in a suitable ointment, lotion, or cream formulation wherein the active ingredient is suspended or dissolved in one or more carriers. Carriers that may be used in ointment formulations include, but are not limited to: mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyethylene oxide, polypropylene oxide, emulsifying wax and water; carriers that can be used in lotions or creams include, but are not limited to: mineral oil, sorbitan monostearate, tween 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.

The compounds of the present invention may also be administered in the form of sterile injectable preparations, including sterile injectable aqueous or oleaginous suspensions or solutions. Among the carriers and solvents that may be employed are water, ringer's solution and isotonic sodium chloride solution. In addition, the sterilized fixed oil may also be employed as a solvent or suspending medium, such as a monoglyceride or diglyceride.

It is further noted that the dosage and method of administration of the compounds of the present invention will depend upon a variety of factors including the age, weight, sex, physical condition, nutritional status, the activity level of the compound, time of administration, metabolic rate, severity of the condition, and the subjective judgment of the treating physician. The preferred dosage is between 0.01-100 mg/kg body weight/day.

In the present invention,

the term "pharmaceutically acceptable salt" refers to pharmaceutically acceptable inorganic or organic salts. The compounds having a basic group in formula I of the present invention may form pharmaceutically acceptable salts with inorganic acids, such as sulfate, hydrochloride, hydrobromide, phosphate; pharmaceutically acceptable salts can also be formed with organic acids such as acetates, oxalates, citrates, gluconates, succinates, tartrates, p-toluenesulfonates, methanesulfonates, benzoates, lactates, maleates, and the like. The compounds having an acidic group in formula I of the present invention may form pharmaceutically acceptable salts with alkali metals or alkaline earth metals, preferably but not limited to sodium, potassium, magnesium or calcium salts.

The term "effective amount" refers to a dose that achieves treatment, prevention, alleviation and/or amelioration of a disease or disorder described herein in a subject.

The term "subject" may refer to a patient or other animal, particularly a mammal, e.g., a human, dog, monkey, cow, horse, etc., that receives a compound of formula I or a pharmaceutical composition of any one of the invention to treat, prevent, alleviate and/or ameliorate a disease or disorder described herein.

The term "disease and/or disorder" refers to a physical condition of the subject that is associated with the disease and/or disorder of the present invention.

Advantageous effects of the invention

The compound of the formula I can effectively kill tumor cells or inhibit the proliferation of the tumor cells, has good antitumor activity and has the potential of being used as an antitumor drug.

Biological material relating to preservation

The Penicillium purpurogenum BD-1-3 has been preserved in China general microbiological culture Collection center (CGMCC) on 11/1 2010; the preservation address is Beijing, Chaoyang district, Beichen Xilu No. 1 institute No. 3, China academy of sciences microbiological research institute, postal code 100101; the preservation number is CGMCC No. 4284; the classification was named Penicillium purpurogenum.

The penicillium purpurogenum3-f-31 has been preserved in China general microbiological culture Collection center (CGMCC) in 2013, 3, 7 and 7 months; the preservation address is Beijing, Chaoyang district, Beichen Xilu No. 1 institute No. 3, China academy of sciences microbiological research institute, postal code 100101; the preservation number is CGMCC No. 7286; the classification was named Penicillium purpurogenum.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by manufacturers, and are all conventional products available on the market.

In the following examples of the present invention,

the compound of the present invention, hereinafter referred to as compound 1, has the following structure as identified by nuclear magnetic resonance or the like: a compound of formula I wherein R₁=1- (2-methyl) octyl radical, R₂=OH，R₃= H; the compound of the present invention, hereinafter referred to as compound 2, has the following structure as identified by nuclear magnetic resonance or the like: a compound of formula I wherein R₁=1- (2-methyl) octyl radical, R₂=R₃=OH；

Wherein the Arabic numerals represent the index; r₁The octyl group of (A) is in the order of 5"→ 12" on the carbon atom bonded to carbon 4", and 14" on the 2-methyl group.

The compounds of the present invention, hereinafter referred to as compound 1a, have the following structure: the compound of the formula I, wherein R₁=1- (2-methyl) octyl radical, R₂=OCH₃，R₃= H; the compounds of the present invention, hereinafter referred to as compound 1b, have the following structure: the compound of the formula I, wherein R₁=1- (2-methyl) octyl radical, R₂=OCH₂CH₃，R₃= H; the compounds of the present invention, hereinafter referred to as compound 1c, have the following structure: the compound of the formula I, wherein R₁=1- (2-methyl) octyl radical, R₂=OCOCH₃，R₃= H; the compounds of the present invention, hereinafter referred to as compound 1d, have the following structure: the compound of the formula I, wherein R₁=1- (2-methyl) octyl radical, R₂= galloyloxy, R₃= H; the compounds of the present invention, hereinafter referred to as compound 1e, have the following structure: the compound of the formula I, wherein R₁=1- (2-methyl) octyl radical, R₂= Tribenzyl Galloyloxy, R₃= H; the compounds of the present invention, hereinafter referred to as compound 1f, have the following structure: the compound of the formula I, wherein R₁=1- (2-methyl) octyl radical, R₂= benzoyloxy, R₃= H; the compound of the invention, hereinafter referred to as compound 1g, has the following structure: the compound of the formula I, wherein R₁=1- (2-methyl) octyl radical, R₂= p-nitrobenzoyloxy, R₃= H; the compounds of the present invention, hereinafter referred to as compound 1h, have the following structure: the compound of the formula I, wherein R₁=1- (2-methyl) octyl radical, R₂= p-chlorobenzoyloxy, R₃= H; the compounds of the present invention, hereinafter referred to as compound 1i, have the following structure: the compound of the formula I, wherein R₁=1- (2-methyl) octyl radical, R₂= p-fluorobenzoyloxy, R₃=H。

The compounds of the present invention, hereinafter referred to as compound 2a, have the following structure: the compound of the formula I, wherein R₁=1- (2-methyl) octyl radical, R₂=OH，R₃=OCH₃(ii) a The compounds of the present invention, hereinafter referred to as compound 2b, have the following structure: the compound of the formula I, wherein R₁=1- (2-methyl) octyl radical, R₂=R₃=OCH₃(ii) a The compounds of the present invention, hereinafter referred to as compound 2c, have the following structure: the compound of the formula I, wherein R₁=1- (2-methyl) octyl radical, R₂=OH，R₃=OCH₂CH₃(ii) a It is called as followsThe compound of the present invention which is compound 2d has the following structure: the compound of the formula I, wherein R₁=1- (2-methyl) octyl radical, R₂=R₃=OCH₂CH₃(ii) a The compounds of the present invention, hereinafter referred to as compound 2e, have the following structure: the compound of the formula I, wherein R₁=1- (2-methyl) octyl radical, R₂=OH，R₃=OCOCH₃(ii) a The compounds of the present invention, hereinafter referred to as compound 2f, have the following structure: the compound of the formula I, wherein R₁=1- (2-methyl) octyl radical, R₂=R₃=OCOCH₃(ii) a The compound of the invention, hereinafter referred to as compound 2g, has the following structure: the compound of the formula I, wherein R₁=1- (2-methyl) octyl radical, R₂=OH，R₃= galloyloxy; the compounds of the present invention, hereinafter referred to as compound 2h, have the following structure: the compound of the formula I, wherein R₁=1- (2-methyl) octyl radical, R₂=R₃= galloyloxy; the compounds of the present invention, hereinafter referred to as compound 2i, have the following structure: the compound of the formula I, wherein R₁=1- (2-methyl) octyl radical, R₂=OH，R₃= tribenzylgalloyloxy; the compounds of the present invention, hereinafter referred to as compound 2j, have the following structure: the compound of the formula I, wherein R₁=1- (2-methyl) octyl radical, R₂=R₃= tribenzylgalloyloxy; the compounds of the present invention, hereinafter referred to as compound 2k, have the following structure: the compound of the formula I, wherein R₁=1- (2-methyl) octyl radical, R₂=OH，R₃= benzoyloxy; the compounds of the present invention, hereinafter referred to as compound 2l, have the following structure: the compound of the formula I, wherein R₁=1- (2-methyl) octyl radical, R₂=R₃= benzoyloxy; the compounds of the present invention, hereinafter referred to as compound 2m, have the following structure: the compound of the formula I, wherein R₁=1- (2-methyl) octyl radical, R₂=OH，R₃= p-nitrobenzoyloxy; the compounds of the present invention, hereinafter referred to as compound 2n, have the following structure: the compound of the formula I, wherein R₁=1- (2-methyl) octyl radical, R₂=R₃= p-nitrobenzoyloxy; the compounds of the present invention, hereinafter referred to as compound 2o, have the following structure: the compound of the formula I, wherein R₁=1- (2-methyl) octyl radical, R₂=OH，R₃= p-chlorobenzoyloxy; the compounds of the present invention, hereinafter referred to as compound 2p, have the following structure: the compound of the formula I, wherein R₁=1- (2-methyl) octyl radical, R₂=R₃= p-chlorobenzoyloxy; the compounds of the present invention, hereinafter referred to as compound 2q, have the following structure: the compound of the formula I, wherein R₁=1- (2-methyl) octyl radical, R₂=OH，R₃= p-fluorobenzoyloxy; the compounds of the present invention, hereinafter referred to as compound 2r, have the following structure: the compound of the formula I, wherein R₁=1- (2-methyl) octyl radical, R₂=R₃= p-fluorobenzoyloxy group.

In the following examples of the present invention,

the melting point was measured with X-4 type precision melting point measuring instrument of Beijing Tian Di Yu science and technology, Ltd, and the temperature was not corrected. The specific polarimetry was measured with PolAAr3005 polarimeter, JASCO P2000 polarimeter, JASCO, Japan, or AutopolII polarimeter, Rudolph Research, USA. Positive and negative ion ESI-MS and HR-ESI-MS were measured with an API3000 liquid chromatography-mass spectrometer (AB, USA) and a 6520Q-TOF mass spectrometer (Agilent, USA), respectively. The Ultraviolet (UV) spectrum was measured with a Cintra20 ultraviolet visible spectrophotometer, GBC Australia, and the Infrared (IR) spectrum was measured with a Tensor27 infrared spectrometer, Bruker, Germany. NMR spectra were measured with a superconducting NMR spectrometer of JNM-ECA-400 model, JEOL, Japan, and INOVA600 model, Varian, USA. The circular double dispersion (CD) spectrum was measured with a JASCO J-815 circular dichrograph, JASCO, Japan, or a MOS-450 circular dichrograph, Biologic Science, France.

All solvents used in all experiments were of analytical grade, with petroleum ether boiling point range of 60-90 ℃.

Example 1: microbial fermentation culture and preparation of compound 1 and compound 2

1. Fermentation culture and extraction treatment of fermented product

1) Production strain

The strain used for the fermentative production of compounds 1 and 2 in this example was Penicillium purpurogenum BD-1-3 (Penicillium purpurogenum BD-1-3) deposited in the general microbiological center of the China Committee for culture Collection of microorganisms with the accession number of 4284 (CGMCC No. 4283).

2) Fermentation culture

A test tube slant is prepared from PDA culture medium (composed of glucose 2%, agar 2%, and NaCl 1.5% and prepared from 20% potato decoction) of Penicillium purpurogenum BD-1-3 stored in a refrigerator at 4 deg.C, an inoculating loop is used to scrape appropriate amount of spores under aseptic condition, the spore is streaked and inoculated onto newly prepared PDA solid culture medium plate, and activated culture is carried out in an incubator at 28 deg.C for 4 days. From the test tube slant after 4 days of activation culture, an appropriate amount of cells were scraped with an inoculating loop, inoculated into 1 500ml Erlenmeyer flask containing 200ml of a liquid fermentation medium (composition: glucose 2%, maltose 1%, mannitol 2%, glutamic acid 1%, peptone 0.5%, yeast extract powder 0.3%), and subjected to primary seed culture in a shaker at 28 ℃ and 200rpm for 48 hours. The culture solution was inoculated at 5% inoculum size into 15 500ml Erlenmeyer flasks each containing 200ml of a liquid fermentation medium (consisting of glucose 2%, maltose 1%, mannitol 2%, glutamic acid 1%, peptone 0.5%, yeast extract 0.3%), and subjected to secondary seed culture at 28 ℃ for 48 hours in a shaker at 200 rpm. The culture solution was inoculated as a seed in an inoculation amount of 5% to 200 500ml Erlenmeyer flasks each containing 200ml of a liquid fermentation medium (composition: glucose 2%, maltose 1%, mannitol 2%, glutamic acid 1%, peptone 0.5%, yeast extract 0.3%), and subjected to shake culture at 28 ℃ and 200rpm for 12 days to obtain about 40L in total of the fermentation broth.

2. Extraction treatment and preparation of ethyl acetate extract

About 40L of the total fermentation broth was filtered through 4 layers of gauze to separate the filtrate and the biomass. About 36L of the filtrate was directly extracted with an equal volume of ethyl acetate 3 times, and the resulting ethyl acetate extracts were combined and concentrated under reduced pressure to give an ethyl acetate extract of the filtrate. The bacteria are fully stirred and suspended by 10L of 80% (v/v) acetone aqueous solution, the bacteria are crushed by ultrasonic for 2h, extracted for 12h at room temperature, filtered by 4 layers of gauze, and the aqueous acetone extracting solution is obtained by filtration. The same extraction of the cells was repeated 3 times in total, and the resulting aqueous acetone extracts were combined and concentrated under reduced pressure until acetone was not contained. About 5L of the residual water layer was extracted with an equal volume of ethyl acetate 3 times, and the resulting ethyl acetate extracts were combined and concentrated under reduced pressure to give an ethyl acetate extract of the cells. Silica gel thin layer chromatography detection results show that the product spots contained in the filtrate and the ethyl acetate extract of the thalli are basically the same, so the two extracts are combined and then are concentrated under reduced pressure to be dry, and 40g of ethyl acetate total extract is obtained. The inhibition rate of the extract on K562 cells under the concentration of 100 mu g/ml is 68.7 percent (the inhibition rate test method is the same as that of the example 4).

3. Separation of ethyl acetate extract and preparation of column chromatography component containing target compound

40g of the total ethyl acetate extract is dissolved by using a proper amount of chloroform-methanol (volume ratio is 1: 1) mixed solvent, 60g of silica gel (100-. Collecting and merging corresponding eluents for concentration according to a thin-layer chromatography detection result to obtain 13 components: fr-1(0.9g, petroleum ether-chloroform volume ratio 2:1 solvent elution fraction), Fr-2(1.3g, petroleum ether-chloroform volume ratio 1:2 solvent elution fraction), Fr-3(9.3g, chloroform elution fraction), Fr-4(2.1g, chloroform elution fraction), Fr-5(1.6g, chloroform-methanol volume ratio 99:1 solvent elution fraction), Fr-6(2.1g, chloroform-methanol volume ratio 99:1 solvent elution fraction), Fr-7(0.6g, chloroform-methanol volume ratio 98:2 solvent elution fraction), Fr-8(6.2g, chloroform-methanol volume ratio 97:3 solvent elution fraction), Fr-9(3.6g, chloroform-methanol volume ratio 95:5 solvent elution fraction), Fr-10(2.7g, chloroform-methanol volume ratio 93:7 solvent elution fraction), and chloroform-methanol elution fraction, Fr-11(2.6g, chloroform-methanol volume ratio 9:1 solvent elution component), Fr-12(3.0g, chloroform-methanol volume ratio 8:2 solvent elution component), Fr-13 (methanol elution component). Wherein 9 components Fr-1, Fr-2, Fr-3, Fr-5, Fr-6, Fr-7, Fr-8, Fr-9 and Fr-10 show strong antitumor activity, and the inhibition rates of the components on K562 cells at a concentration of 100 mu g/ml are respectively 45.7%, 41.9%, 91.0%, 54.9%, 90.7%, 78.9%, 48.6%, 54.5% and 46.1%. The results of analysis by thin layer chromatography showed that one of the components Fr-6 contained the target compounds 1 and 2.

Fr-6(2.1g) is dissolved by using a proper amount of 95% (v/v) ethanol, wet loading is carried out, the obtained solution is added to a Sephadex LH-20 column (column bed: 4.5cm multiplied by 50 cm) pre-loaded with 95% (v/v) ethanol, elution separation is carried out by using 95% (v/v) ethanol as an eluent, eluent is collected according to the detection result of silica gel thin layer chromatography, and is combined and concentrated, and 9 components are sequentially obtained according to the elution order: fr-6-1(30mg), Fr-6-2(45mg), Fr-6-3(85mg), Fr-6-4(300mg), Fr-6-5(460mg), Fr-6-6(236mg), Fr-6-7(280mg), Fr-6-8(210mg), and Fr-6-9(440 mg). Wherein Fr-6-4(300mg) is a target component containing the compounds 1 and 2, and has an inhibition rate of 51.9% on K562 cells at a concentration of 100. mu.g/mL. Dissolving Fr-6-4(300mg) in appropriate amount of mixed solvent of chloroform-methanol at volume ratio of 1:1, wet loading, adding into Sephadex LH-20 column (column bed: 1.5 cm. times.80 cm) pre-packed in mixed solvent of chloroform-methanol at volume ratio of 1:1, eluting with mixed solvent of chloroform-methanol at volume ratio of 1:1 as eluent, collecting eluates containing compounds 1 and 2, mixing, and concentrating to obtain column chromatography component A (190 mg) containing target compounds 1 and 2.

4. Isolation preparation of Compounds 1 and 2

Column chromatography fraction A (190 mg) containing compounds 1 and 2 was dissolved in an appropriate amount of methanol, filtered through a 0.22 μm filter and subjected to preparative reverse phase HPLC separation using a Waters600 high performance liquid chromatograph (Waters 600 controller, Waters600 pump, Waters2998PDA detector, Empower chromatography workstation)(HPLC column: Capcell Pak C18,20mm × 250 mm; column temperature: room temperature; mobile phase: the volume fraction of methanol-water is 80: 20; flow rate: 10 ml/min; detection wavelength: 210 nm), respectively collecting the retention times (t)_R) Corresponding eluates of two elution peaks at 29.9min and 66.2min gave HPLC component B1 containing Compound 1 as the major component (t_R=29.9 min) and HPLC component B2 (t) containing compound 2 as the main component_R=66.2min）。

The HPLC fractions B1 obtained were all dissolved in an appropriate amount of methanol, filtered through a 0.22 μm filter and subjected to semi-preparative reversed-phase HPLC separation using a Waters600 high performance liquid chromatograph (Waters 600 controller, Waters600 pump, Waters2998PDA detector, Empower chromatography workstation) (HPLC column: Capcell Pak C18,

10mm × 250 mm; column temperature: room temperature; mobile phase: methanol-water volume fraction 86: 14; flow rate: 3 ml/min; detection wavelength: 210 nm) and refining to obtain 13mg (retention time t) of the pure compound 1_R=43.7min）。

The HPLC fractions B2 which were obtained were all also dissolved in an appropriate amount of methanol and, after filtration through a 0.22 μm filter, were subjected to a semi-preparative reverse-phase HPLC separation using a Waters600 high performance liquid chromatograph (Waters 600 controller, Waters600 pump, Waters2998PDA detector, Empower chromatography workstation) (HPLC column: Capcell Pak C18,

10mm × 250 mm; column temperature: room temperature; mobile phase: methanol-water volume fraction 82: 18; flow rate: 3 ml/min; detection wavelength: 210 nm) and refining to obtain 5.5mg (retention time t) of pure compound 2_R=37.0min）。

In addition, through the experimental operation of the 50-140L scale of the steps 1 to 4, which are repeatedly carried out for another plurality of times, 350mg of the pure compound 1 and 170mg of the pure compound 2 are obtained in an additional accumulation mode.

5. Physicochemical constants and Popp data for Compounds 1 and 2

Compound 1, white crystalline solid (methanol), mp 131-132 deg.C, is easily soluble in chloroform, soluble in methanol, ethanol, acetone, insoluble in water,

99.7(c0.5, methanol). Positive ion ESI-MSm/z 863[ M + H ]]⁺,885[M+Na]⁺,901[M+K]⁺Negative ion ESI-MS M/z 861[ M-H ]]^-,897[M+Cl]^-,907[M+HCOO]^-. Positive ion HR-ESI-MS M/z, found 863.4969[ M + H [ ]]⁺Calculated value 863.4959 (C)₅₁H₆₇N₄O₈[M+H]⁺) Measured value of 885.4780[ M + Na ]]⁺Calculated value 885.4778 (C)₅₁H₆₆N₄O₈Na[M+Na]⁺) Measured value of 901.4518[ M + K]⁺Calculated value 901.4518 (C)₅₁H₆₆N₄O₈K[M+K]⁺)。UVλ_max nm(logε)in MeOH:298(3.47),244(4.10),209(4.69)。IRν_max cm^-1:3292,3080,2933,2873,1667,1607,1528,1489,1454,1380,1365,1335,1304,1273,1200,1106,1077,1058,1032,983,931,895,862,815,748,703。CDλ_max nm(mdeg)in MeOH at200μg/ml:195.5(-8.37),205.2(0),208.5(+2.04),210.2(0),215.5(-12.86),225.6(0),232.0(+2.54),237.0(0),249.5(-13.62),315.1(0),319.0(+0.31),320.0(+0.32),321.5(+0.30),328.5(+0.40),330.0(+0.42),331.5(+0.38),340.0(+0.47),344.0(+0.50),351.0(+0.54),358.0(+0.22),359.0(+0.31),361.5(+0.29),367.5(+0.37),369.0(+0.37),371.5(+0.32),374.5(+0.37),375.5(+0.37),381.5(+0.18),387.5(+0.44),390.9(0),394.0(+0.09),398.0(+0.13),399.9(0)。600MHz¹H and 150MHz¹³CNMR data: see table 1. NMR data of Compound 1 shown in Table 1 based thereon¹H spectrum,¹³One-dimensional spectra such as C full decoupling spectrum, DEPT spectrum, NOE difference spectrum, etc., and¹H-¹attributing two-dimensional map analysis results such as H COSY, HMQC, HMBC, ROESY and the like.

Compound 2, yellowish crystalline solid (methanol), mp 125-126 deg.C, is easily soluble in chloroform, soluble in methanol, ethanol, acetone, insoluble in water,

65.8(c0.2, methanol). Positive ion ESI-MSm/z 879[ M + H ]]⁺The negative ion ESI-MS M/z is 877[ M-H ]]^-,913[M+Cl]^-. Positive ion HR-ESI-MS M/z, found 879.4902[ M + H [ ]]⁺Calculated value 879.4908 (C)₅₁H₆₇N₄O₉[M+H]⁺) Measured value of 901.4724[ M + Na ]]⁺Calculated value 901.4728 (C)₅₁H₆₆N₄O₉Na[M+Na]⁺) Measured value of 917.4456[ M + K]⁺Calculated value 917.4467 (C)₅₁H₆₆N₄O₉K[M+K]⁺)。UVλ_max nm(logε)in MeOH:297(3.42),209(4.64)。IRν_max cm^-1:3291,2937,2874,1667,1519,1489,1450,1379,1367,1335,1304,1272,1242,1204,1177,1112,1058,1033,984,933,896,862,751。CDλ_max nm(mdeg)inMeOH at200μg/ml:196.5(-12.05),202.3(0),207.0(+4.55),211.3(0),216.0(-8.15),232.6(0),235.5(+0.65),238.1(0),251.5(-8.29),273.7(0),282.5(+0.42),290.0(+0.18),297.5(+0.25),301.6(0),318.5(+0.45),322.0(+0.34),324.5(+0.37),328.5(+0.53),329.5(+0.54),330.5(+0.55),331.5(+0.56),339.5(+0.60),344.0(+0.59),346.5(+0.57),351.0(+0.63),360.5(+0.48),361.5(+0.49),366.5(+0.41),368.5(+0.45),376.5(+0.32),377.5(+0.31),387.5(+0.39),394.0(+0.14),396.5(+0.11),399.1(0)。600MHz¹H and 150MHz¹³C NMR data: see table 1. NMR data of Compound 2 shown in Table 1 based thereon¹H spectrum,¹³One-dimensional spectra such as C full decoupling spectrum, DEPT spectrum, NOE difference spectrum, etc., and¹H-¹attributing two-dimensional map analysis results such as H COSY, HMQC, HMBC, ROESY and the like.

TABLE 1 Compounds 1 and 2 in CDCl₃600MHz of¹H and 150MHz¹³C NMR data

Example 2: fermentation culture of penicillium purpurogenum3-f-31 and separation preparation of compound 1

1. Fermentation culture and extraction treatment of fermented product

1) Production strain

The strain for producing compound 1 by fermentation in this example is Penicillium purpurogenum3-f-31, deposited in the general microbiological center of the China Committee for culture Collection of microorganisms, with the accession number 7286 (CGMCC No. 7286).

2) Fermentation culture

A test tube slant is prepared from PDA culture medium (composed of glucose 2%, agar 2%, and NaCl 1.5% and prepared from 20% potato decoction) of Penicillium purpurogenum3-f-31 stored in a refrigerator at 4 deg.C, an inoculating loop is used to scrape appropriate amount of spores under aseptic condition, and the spore is streaked and inoculated onto newly prepared PDA solid culture medium plate, and cultured in 28 deg.C incubator for 3-5 days. And (3) scraping a proper amount of fresh spores by using an inoculating ring after the spores are formed, putting the fresh spores into a 50ml conical flask filled with a proper amount of sterile water and glass beads, and fully shaking the conical flask to ensure that the glass beads are rotated and beaten to uniformly disperse the spores to obtain a crude spore suspension. Taking 200 mu l of the crude spore suspension, placing the crude spore suspension in a 96-well plate, measuring the OD value under 600nm by using an enzyme-labeling instrument, diluting the crude spore suspension under the detection of the OD value until the OD value reaches 0.5, and recording the dilution times. Accordingly, the whole crude spore suspension was diluted with sterile water by the same factor to prepare a spore suspension of 3-f-31 for inoculation. The spore suspension was inoculated into 120 500ml Erlenmeyer flasks each containing 240ml of a liquid fermentation medium (composition: glucose 2%, maltose 1%, mannitol 2%, glutamic acid 1%, peptone 0.5%, yeast extract powder 0.3%) at an inoculation amount of 140. mu.l per flask, and subjected to shake culture at 28 ℃ and 180rpm for 10 days to obtain about 28.8L of a fermentation broth.

2. Extraction treatment and preparation of ethyl acetate extract

About 28.8L of the total fermentation broth was filtered through 4 layers of gauze to separate the filtrate and the biomass. Extracting the filtrate with equal volume of ethyl acetate for 4 times, mixing the obtained ethyl acetate extractive solutions, and concentrating under reduced pressure to obtain ethyl acetate extract of the filtrate. Adding acetone twice the volume of the thallus to make the volume percentage of the acetone reach about 67 percent, fully stirring and suspending, carrying out ultrasonic treatment for 5 hours to break the thallus, filtering by using 4 layers of gauze, and filtering to obtain an aqueous acetone extracting solution. The same extraction of the cells was repeated for a total of 4 times, and the resulting aqueous acetone extracts were combined and concentrated under reduced pressure until acetone was not contained. About 4L of the residual aqueous layer was extracted 5 times with an equal volume of ethyl acetate, and the resulting ethyl acetate extracts were combined and concentrated under reduced pressure to give an ethyl acetate extract of the cells. Silica gel thin layer chromatography detection results show that the product spots contained in the filtrate and the ethyl acetate extract of the thalli are basically the same, so the two extracts are combined and then are concentrated to be dry under reduced pressure to obtain 29.9g of ethyl acetate total extract. The extract has 78% inhibition rate on K562 cells at 100 μ g/ml concentration.

29.9g of the above ethyl acetate total extract was sufficiently dissolved in a large amount of methanol, and then filtered, concentrated and dried to separate into 6.5g of methanol-insoluble matter and 23.4g of methanol-soluble matter. Wherein, the methanol insoluble substance is mainly a mucoid substance and a part of solid matters remained after cell membrane disruption, has no inhibition effect on K562 cells, while the methanol soluble substance has strong inhibition effect on the K562 cells, and the inhibition rate on the K562 cells under the concentration of 100 mu g/ml is as high as 82.4%.

Dissolving 23.4g of the methanol soluble substance by using a proper amount of mixed solvent with a dichloromethane-methanol volume ratio of 1:2, adding 60g of silica gel (100-. Collecting 75 fractions of eluent per 500ml, mixing corresponding fractions according to the detection result of silica gel thin layer chromatography, and concentrating under reduced pressure to dry to obtain 14 components: fr-1(0.11g, petroleum ether elution component), Fr-2(1.90g, dichloromethane elution component), Fr-3(1.50g, dichloromethane-methanol volume ratio 99:1 solvent elution component), Fr-4(5.45g, dichloromethane-methanol volume ratio 99:1 → 98:2 solvent elution component), Fr-5(7.28g, dichloromethane-methanol volume ratio 98:2 → 97:3 solvent elution component), Fr-6(0.99g, dichloromethane-methanol volume ratio 97:3 → 96:4 solvent elution component), Fr-7(0.90g, dichloromethane-methanol volume ratio 96:4 → 95:5 → 92:8 solvent elution component), Fr-8(0.41g, dichloromethane-methanol volume ratio 92:8 → 9:1 solvent elution component), Fr-9(0.66g, dichloromethane-methanol volume ratio 9:1 → 17:3 solvent elution component), Fr-10(0.69g, dichloromethane-methanol volume ratio 17:3 → 12:3 → 7:3 solvent elution component), Fr-11(0.87g, dichloromethane-methanol volume ratio 7:3 → 1:1 solvent elution component), Fr-12(1.05g, acetone → methanol elution component), Fr-13(0.05g, methanol elution component), Fr-14(1.10g, methanol elution component). Wherein 8 components Fr-3, Fr-4, Fr-5, Fr-6, Fr-7, Fr-8, Fr-9, Fr-10, Fr-11 and Fr-12 show different degrees of antitumor activity, and the inhibition rates of K562 cells at a concentration of 100. mu.g/ml are 30.4%, 67.6%, 73.2%, 58.1%, 58.8%, 26.1%, 54.1%, 54.0%, 43.3% and 31.1%, respectively. The results of analysis by thin layer chromatography showed that one of the components Fr-5 contained the target compound 1.

Dissolving Fr-5(7.28 g) with an appropriate amount of methanol, carrying out wet loading, adding the obtained solution onto a Sephadex LH-20 column (column bed: 5 cm. times.43 cm) pre-loaded in 95% (v/v) ethanol, carrying out elution and chromatographic separation by using 95% (v/v) ethanol as an eluent, collecting eluates according to a thin-layer detection result, combining and concentrating the eluates, and sequentially obtaining 11 components according to the elution order: fr-5-1(600mg), Fr-5-2(2.75g), Fr-5-3(180mg), Fr-5-4(1.7g), Fr-5-5(170mg), Fr-5-6(70mg), Fr-5-7(28mg), Fr-5-8(1.5g), Fr-5-9(130mg), Fr-5-10(15mg), Fr-5-11(10 mg). The inhibition rates of the components on K562 cells at the concentration of 100 mu g/ml are 67.2%, 80.2%, 77.4%, 51.1%, 14.8%, 16.8%, 14.4%, 25.3%, 62.2%, 63.5% and 75.5%, respectively, which shows that 7 components, such as Fr-5-1 to Fr-5-4 and Fr-5-9 to Fr-5-11, are active components with stronger antitumor effect. Wherein Fr-5-1 is a target component containing the compound 1. Fr-5-1(600mg) was dissolved in an appropriate amount of methanol, and then 2g of reversed-phase silicA GEL ODS (YMC. about. GEL ODS-A-HG, 12nm S-50 μm, AAG12S 50) was added thereto, and the mixture was uniformly mixed, dried and ground, and then added to A reduced-pressure glass column packed with 10g of the same reversed-phase silicA GEL ODS (column bed: 2.5 cm. times.20 cm), and then subjected to column chromatography separation using A water-methanol-acetone mixed solvent system as an eluent, by gradually increasing the volume fraction of methanol or acetone in the eluent to decrease the polarity, thereby carrying out column chromatography separation using A reduced-pressure gradient eluent. Collecting and combining corresponding eluent fractions according to a thin-layer chromatography detection result, and respectively concentrating and drying to obtain 7 components: fr-5-1-1(200mg, water-methanol volume ratio 4:1 → 1:4 solvent elution fraction), Fr-5-1-2(14.3mg, water-methanol volume ratio 1:4 solvent elution fraction), Fr-5-1-3(16mg, water-methanol volume ratio 3:17 solvent elution fraction), Fr-5-1-4(50mg, water-methanol volume ratio 1:9 solvent elution fraction), fr-5-1-5(35mg, water-methanol volume ratio 1:9 → 1:19 solvent elution fraction), Fr-5-1-6(130mg, water-methanol volume ratio 1:19 → methanol elution fraction), Fr-5-1-7(80mg, methanol → methanol-acetone volume ratio 1:1 solvent elution fraction). The inhibition rates of the components on K562 cells at the concentration of 100 mu g/ml are respectively 56.7%, 90.4%, 91.4%, 65.6%, 48.4%, 8.6% and 9.1%, which indicates that Fr-5-1-1, Fr-5-1-2, Fr-5-1-3, Fr-5-1-4 and Fr-5-1-5 are active components with stronger antitumor effect. The detection result of the thin layer chromatography shows that Fr-5-1-3 is a column chromatography component containing the compound 1.

4. HPLC separation preparation of Compound 1

Dissolving column chromatography component Fr-5-1-3(16 mg) containing compound 1 in appropriate amount of methanol, and purifying with 022 μm filter, preparative reverse phase HPLC separations were carried out using a Waters600 high performance liquid chromatograph (Waters 600 controller, Waters600 pump, Waters2998PDA detector, Empower chromatography workstation) (HPLC column: Capcell Pak C18,

column, 20mm × 250 mm; column temperature: room temperature; mobile phase: the volume fraction of methanol-water is 81: 19; flow rate: 6 ml/min; detection wavelength: 210 nm) and purification to give Compound 1 (retention time t)_R=106.0 min) pure product 5.1 mg.

In addition, 65mg of the pure compound 1 is obtained by additionally accumulating the experimental operation of the steps 1 to 4 on the scale of 40-120L.

Example 3: derivatization preparation of other inventive compounds 1 a-1I and 2 a-2 r of the formula I

1) Derivatization preparation of Compounds 1 a-1I of the formula I according to the invention

Approximately 10mg of the compound 1 obtained in example 1 or example 2 was weighed, dissolved in 0.5ml of acetone, and 50mg of anhydrous K was added₂CO₂Mixing, and adding 50 mul methyl iodide dropwise under magnetic stirring at 60 ℃ to perform methylation reaction for 6 h. The reaction product was separated and purified by preparative silica gel thin layer chromatography (chloroform-methanol volume ratio 93:7 development) to obtain 1a (1.3 mg, positive ion ESI-MS M/z:877[ M + H ] in addition to recovering 5mg of the starting compound 1]⁺Negative ion ESI-MS M/z 875[ M-H ]]^-）。

Approximately 10mg of the compound 1 obtained in example 1 or example 2 was weighed, dissolved in 0.5ml of acetone, and 50mg of anhydrous K was added₂CO₂Mixing, and adding 50 μ l diethyl sulfate dropwise under magnetic stirring at 60 deg.C for ethylation reaction for 6 hr. The reaction product was separated and purified by preparative silica gel thin layer chromatography (chloroform-methanol volume ratio 93:7 development) to obtain 1b (1.2 mg, positive ion ESI-MS m)/z:891[M+H]⁺，ESI-MS m/z:889[M-H]^-）。

Approximately 10mg of the compound 1 prepared in the above examples 1 and 2 was weighed, dissolved in 0.25ml of anhydrous pyridine, rapidly added with 0.25ml of acetic anhydride, mixed well, and left to stand at room temperature for 24 hours in the dark for acetylation. Separating and purifying the reaction product by preparative silica gel thin layer chromatography (chloroform-methanol volume ratio 92:8 development) to obtain 1c (3 mg, positive ion ESI-MS M/z:905[ M + H ]]⁺Negative ion ESI-MS M/z:903[ M-H ]]^-）。

About 10mg of the compound 1 prepared in example 1 and example 2 was weighed, dissolved in 0.25ml of acetone, mixed with 0.25ml of a 10mg/ml acetone solution of gallic acid, and then mixed with 1 drop of 6N hydrochloric acid, followed by acylation reaction for 7 days while keeping out of the sun at room temperature. The reaction product was separated and purified by preparative silica gel thin layer chromatography (chloroform-methanol volume ratio 80:20 development) to obtain 1d (0.9 mg, positive ion ESI-MS M/z:1015[ M + H ] in addition to 7mg of the starting compound 1]⁺(ii) a Negative ion ESI-MS M/z 1013[ M-H]^-）。

About 10mg of the compound 1 prepared in example 1 and example 2 was weighed, dissolved in 0.2ml of anhydrous pyridine, rapidly added with 16mg of tribenzylgalloyl chloride, mixed and dissolved, and left at 50 ℃ in the dark for 48 hours to carry out acylation reaction. Separating and purifying the reaction product by preparative silica gel thin layer chromatography (chloroform-methanol volume ratio 95:5 development) to obtain 1e (2 mg, positive ion ESI-MS M/z:1285[ M + H ]]⁺(ii) a Negative ion ESI-MS M/z 1283[ M-H ]]^-）。

About 10mg of the compound 1 prepared in example 1 and example 2 was weighed, dissolved in 0.2ml of anhydrous pyridine, rapidly mixed with 10. mu.l of benzoyl chloride, and left to stand at room temperature in the dark for acylation reaction for 24 hours. Separating and purifying the reaction product by preparative silica gel thin layer chromatography (chloroform-methanol volume ratio 93:7 development) to obtain 1f (2.8 mg, positive ion ESI-MS M/z:967[ M + H ]]⁺(ii) a Negative ion ESI-MS M/z:965[ M-H ]]^-）。

Approximately 10mg of the compound 1 prepared in example 1 and example 2 above was weighed and 0.2ml of anhydrous pyridine was addedDissolving pyridine, quickly adding 13mg of p-nitrobenzoyl chloride, uniformly mixing, and standing at room temperature in a dark place for acylation reaction for 10 hours. The reaction product was separated and purified by preparative silica gel thin layer chromatography (chloroform-methanol volume ratio 91:9 development) to give 1g (3.1mg, positive ion ESI-MS M/z:1012[ M + H ]]⁺(ii) a Negative ion ESI-MS M/z 1010[ M-H ]]^-)。

About 10mg of the compound 1 prepared in example 1 and example 2 was weighed, dissolved in 0.2ml of anhydrous pyridine, quickly added with 10. mu.l of p-chlorobenzoyl chloride, mixed well, and left to stand at room temperature in the dark for acylation reaction for 10 hours. Separating and purifying the reaction product by preparative silica gel thin layer chromatography (chloroform-methanol volume ratio 92:8 development) to obtain 1H (3 mg, positive ion ESI-MS M/z:1001[ M + H ]]⁺(ii) a Negative ion ESI-MS M/z 999[ M-H ]]^-）。

About 10mg of the compound 1 prepared in example 1 and example 2 was weighed, dissolved in 0.2ml of anhydrous pyridine, rapidly added with 8. mu.l of p-fluorobenzoyl chloride, mixed well, and left to stand at room temperature in the dark for acylation reaction for 10 hours. Separating and purifying the reaction product by preparative silica gel thin layer chromatography (chloroform-methanol volume ratio 92:8 development) to obtain 1i (2.5 mg, positive ion ESI-MS M/z:985[ M + H ]]⁺(ii) a Negative ion ESI-MS M/z 983[ M-H ]]^-）。

2) Derivatization preparation of the compounds 2 a-2 r of the formula I according to the invention

Approximately 10mg of the compound 2 prepared in example 1 above was weighed, dissolved in 0.5ml of acetone, and 50mg of anhydrous K was added₂CO₂Shaking up, and dripping 50 mul of methyl iodide under magnetic stirring at 60 ℃ to carry out methylation reaction for 6 h. The reaction product was separated and purified by preparative silica gel thin layer chromatography (chloroform-methanol volume ratio 93:7 development) to obtain 2a (2.1 mg, positive ion ESI-MSm/z:893[ M + H ] except that 6mg of the starting compound 2 was recovered]⁺Negative ion ESI-MS M/z 891[ M-H ]]^-) And 2b (1.3 mg, positive ion ESI-MS M/z:907[ M + H ]]⁺，ESI-MS m/z:905[M-H]^-）。

Approximately 10mg of the compound 2 prepared in example 1 above was weighed, dissolved in 0.5ml of acetone, and 50mg of anhydrous K was added₂CO₂Shaking up, and dropwise adding 50 mul diethyl sulfate under magnetic stirring at 60 ℃ to carry out ethylation reaction for 6 h. The reaction product was separated and purified by preparative silica gel thin layer chromatography (chloroform-methanol volume ratio 93:7 development) to obtain 2c (2.3 mg, positive ion ESI-MS M/z:907[ M + H ] M + H) except that 5mg of the starting compound 2 was recovered]⁺Negative ion ESI-MS M/z 905[ M-H ]]^-) And 2d (1.5 mg, positive ion ESI-MS M/z:935[ M + H ]]⁺，ESI-MS m/z:933[M-H]^-）。

About 10mg of the compound 2 prepared in example 1 was weighed, dissolved in 0.25ml of anhydrous pyridine, quickly added with 0.25ml of acetic anhydride, mixed well, and left to stand at room temperature for 24 hours in the dark for acetylation. Separating and purifying the reaction product by preparative silica gel thin layer chromatography (chloroform-methanol volume ratio 92:8 development) to obtain 2e (2.5 mg, positive ion ESI-MS M/z:921[ M + H ]]⁺Negative ion ESI-MS M/z 919[ M-H ]]^-) And 2f (1.4 mg, positive ion ESI-MS M/z:963[ M + H ]]⁺Anion ESI-MS M/z 961[ M-H ]]^-）。

About 10mg of the compound 2 prepared in example 1 was weighed, dissolved in 0.25ml of acetone, dissolved in 4mg of gallic acid, mixed and dissolved, added dropwise with 1 drop of 6N hydrochloric acid and shaken, and left to stand in the dark at room temperature for acylation reaction for 7 days. The reaction product was separated and purified by preparative silica gel thin layer chromatography (chloroform-methanol volume ratio 80:20 development) to obtain 2g (1.1 mg, positive ion ESI-MS M/z:1031[ M + H ] of 4mg of starting compound 2]⁺(ii) a Negative ion ESI-MS M/z 1029[ M-H ]]^-) And 2H (0.9 mg, positive ion ESI-MS M/z:1183[ M + H ]]⁺(ii) a Negative ion ESI-MS M/z 1181[ M-H ]]^-）。

Weighing about 10mg of the compound 2 prepared in the example 1, adding 0.2ml of anhydrous pyridine for dissolving, quickly adding 16mg of tribenzylgalloyl chloride for uniformly mixing and dissolving, and standing for 48 hours at 40 ℃ in the dark for acylation reaction. The reaction product was separated and purified by preparative silica gel thin layer chromatography (chloroform-methanol volume ratio 95:5 development) to give 2i (3 mg, positive ion ESI-MS M/z:1301[ M + H ])]⁺(ii) a Negative ion ESI-MS M/z 1299[ M-H ]]^-) And 2j (1.5 mg, positive ion ESI-MS M/z:1723[ M + H ]]⁺(ii) a Negative ion ESI-MSm/z 1721M-H]^-）。

About 10mg of the compound 2 prepared in example 1 was weighed, dissolved in 0.2ml of anhydrous pyridine, quickly mixed with 5. mu.l of benzoyl chloride, and left to stand at room temperature in the dark for acylation reaction for 24 hours. Separating and purifying the reaction product by preparative silica gel thin layer chromatography (chloroform-methanol volume ratio 93:7 development) to obtain 2k (2 mg, positive ion ESI-MS M/z:983[ M + H ]]⁺(ii) a Negative ion ESI-MS M/z 981[ M-H ]]^-) And 2l (1.7 mg, positive ion ESI-MS M/z:1087[ M + H ]]⁺(ii) a Negative ion ESI-MS M/z 1085[ M-H ]]^-）。

About 10mg of the compound 2 prepared in example 1 was weighed, dissolved in 0.2ml of anhydrous pyridine, rapidly added with 13mg of p-nitrobenzoyl chloride, mixed and dissolved, and left to stand away from light for acylation reaction at room temperature for 10 hours. Separating and purifying the reaction product by preparative silica gel thin layer chromatography (chloroform-methanol volume ratio 91:9 development) to obtain 2M (2 mg, positive ion ESI-MS M/z:1028[ M + H ])]⁺(ii) a Negative ion ESI-MS M/z 1026[ M-H ]]^-) And 2n (1.6 mg, positive ion ESI-MS M/z:1177[ M + H)]⁺(ii) a Negative ion ESI-MSm/z 1175[ M-H ]]^-）。

About 10mg of the compound 2 prepared in example 1 was weighed, dissolved in 0.2ml of anhydrous pyridine, and then mixed with 6. mu.l of p-chlorobenzoyl chloride quickly, and left to stand in the dark at room temperature for acylation reaction for 10 hours. Separating and purifying the reaction product by preparative silica gel thin layer chromatography (chloroform-methanol volume ratio 91:9 development) to obtain 2o (2.1 mg, positive ion ESI-MS M/z:1017[ M + H ]]⁺(ii) a Negative ion ESI-MS M/z 1015[ M-H ]]^-) And 2p (1.9 mg, positive ion ESI-MS M/z:1155[ M + H ]]⁺(ii) a Negative ion ESI-MSm/z 1153[ M-H ]]^-）。

About 10mg of the compound 2 prepared in example 1 was weighed, dissolved in 0.2ml of anhydrous pyridine, and then mixed with 6. mu.l of p-fluorobenzoyl chloride quickly, and left to stand at room temperature in the dark for acylation reaction for 10 hours. Separating and purifying the reaction product by preparative silica gel thin layer chromatography (chloroform-methanol volume ratio 91:9 development) to obtain 2q (2.3 mg, positive ion ESI-MS M/z:1001[ M + H ]]⁺(ii) a Anion ESI-MS m-z:999[M-H]^-) And 2r (1.9 mg, positive ion ESI-MS M/z:1123[ M + H ]]⁺(ii) a Negative ion ESI-MSm/z 1121[ M-H ]]^-）。

Example 4: test of the antitumor Activity of the Compounds of the formula I according to the invention

1. Experimental Material

1) Preparation of solution of sample to be tested

The test samples were compound 1, compound 2 isolated as described in examples 1 and 2 above, and other compounds 1 a-1I and 2 a-2 r of formula I of the present invention derivatized as described in example 3 above. Wherein, the compound 1 and 2 are firstly prepared into mother liquor of 10.0mg/ml by methanol, and then are prepared into methanol solutions of 10.0, 5.0, 2.5, 1.25, 0.625, 0.3125, 0.1562 and 0.0781mg/ml series concentration by dilution by multiple ratio to test the activity, while the compound 1 a-1 i and the compound 2 a-2 r are only prepared into methanol solutions of 2.5mg/ml single concentration to test the activity. A10.0 mg/ml methanol solution of 5-fluorouracil (Aladdin reagent Co., Ltd., lot No. 5402) was used as a positive control, and methanol was used as a blank control.

2) Cell line and subculture of cells

The activity test was carried out using a human chronic myelogenous leukemia K562 cell line, a human acute promyelocytic leukemia HL-60 cell line, a human cervical cancer HeLa cell line, a human gastric cancer BGC-823 cell line, and a human breast cancer MCF-7 cell line (all of which are commercially available, for example, from Yaji Biotech, Inc., Shanghai Smart laboratory facilities, Inc., McMessah, Inc., Shanghai).

K562, HL-60, HeLa, BGC-823 and MCF-7 cells were passaged routinely in RPMI-1640 medium containing 10% fetal bovine serum and 100. mu.g/ml each of penicillin and streptomycin, and maintained in a cell culture chamber with 5% carbon dioxide at 37 ℃.

2. Activity test method

The antitumor activity of the samples was tested using the MTT method in combination with cytomorphological assays. Respectively taking K562, HL-60, HeLa, BGC-823 and MCF-7 cells in logarithmic growth phase, and preparing into cells with density of 2 × 10 with fresh RPMI-1640 culture medium⁴Cell suspension of one/ml, seeded in 96-well plates at 200. mu.l per well. After inoculation, suspension cells K562 and HL-60 were cultured at 37 ℃ for 2h, while adherent cells HeLa, BGC-823 and MCF-7 were cultured at 37 ℃ for 12 h. Thereafter, 2. mu.l of the drug sample solution was added to each well of the sample group, and 2. mu.l of methanol was added to each well of the blank control group, and the incubation was continued at 37 ℃ for 24 hours. And after the culture is finished, observing the morphological change of the tested cells under an optical microscope, observing the morphological characteristics of the presence or absence of typical abnormal cells, apoptotic cells or necrotic cells, and according to the morphological change condition of the tested cells, performing direct comparison and control with a blank control group to preliminarily judge whether the anti-tumor activity of the sample on the tested cells exists or not, and taking a picture if necessary. Thereafter, 20. mu.l of a precooled 5mg/ml MTT solution (prepared in PBS) was added to each well, incubated at 37 ℃ for 4 hours, centrifuged at 4 ℃ and 2000rpm for 10 minutes, the supernatant was aspirated, 150. mu.l of DMSO was added to each well, the resulting mixture was placed on a microplate reader and sufficiently shaken to completely dissolve the MTT purple product, and the OD at 570nm was measured in each well. In the experiment, three parallel holes are respectively arranged on a sample and a blank control group, the average value of three holes OD is taken, and the formula IR% = (OD) is adopted_{Blank space}-OD_{Sample (I)})/OD_{Blank space}X 100%, the inhibition rate (IR%) of the sample on the test cancer cells was calculated. Median Inhibitory Concentration (IC) of Compounds 1 and 2 on cancer cells tested₅₀) The inhibition rate is calculated according to the concentration.

3. Results of the experiment

1) MTT method test results

In the MTT method test, the compounds 1 and 2 showed strong antitumor activity for inhibiting cell proliferation on tested K562, HL-60, HeLa, BGC-823 and MCF-7 cells, and IC of the compounds on the 5 tested cells₅₀The results of the value measurement are shown in table 2 below.

TABLE 2 IC inhibition of human cancer cell proliferation by Compounds 1 and 2₅₀Measurement of value (. mu.M)

The compounds 1 a-1 i and 2 a-2 r also show strong antitumor activity for inhibiting cell proliferation on tested K562, HL-60, HeLa, BGC-823 and MCF-7 cells, and the inhibition rates of the compounds on the cells are all over 40% under the concentration of 25 mu g/ml and are respectively distributed in the interval of 40% -87%.

In the experiment, the inhibition rates of positive control 5-fluorouracil on tested K562, HL-60, HeLa, BGC-823 and MCF-7 cells at the concentration of 100 mu g/ml are 39.3%, 54.2%, 55.5%, 49.6% and 43.2% respectively.

These results indicate that the compounds of the present invention have superior antitumor activity against the above cancer cells in vitro than 5-fluorouracil.

2) Results of cell morphology examination

When the tested cells are treated by the compounds 1 and 2 with the series of concentrations of 100 mu g/ml-0.781 mu g/ml for 24 hours under an optical inverted microscope, part of the cells in the visual field are in the typical shapes of necrotic cells such as cell expansion and cytoplasm agglutination, part of the cells are in the morphological characteristics of apoptotic cells such as snowflake or fragments which are not completely scattered, and part of the cells are in heterotypic shapes such as short thick rods, fusiform or bigement shapes which do not complete cell division: the necrotic morphological cells in the visual field of the high-concentration treatment group of 100 mug/ml are abundant, but as the concentration is reduced, the necrotic cells in the visual field are gradually reduced, and the apoptotic morphological cells and the heterotypic morphological cells are gradually increased and are also gradually increased along with the normal morphological cells; the number of normal morphology cells increased progressively and markedly as the concentration decreased to and further below 3.125. mu.g/ml. The morphological detection results show that the compounds 1 and 2 exert the antitumor effect of inhibiting the proliferation of cancer cells through various ways such as killing cytotoxicity to tested tumor cells, inducing the tested cells to generate apoptosis, acting on skeleton protein to inhibit the division of the tested cells and the like.

In addition, it was observed under an optical inverted microscope that after the cancer cells were treated with 25. mu.g/ml of each of the compounds 1 a-1 i and 2 a-2 r for 24 hours, many of the cells in the field exhibited typical morphology of necrotic cells such as enlarged cell bodies and aggregated cell cytoplasm, and some of the cells exhibited morphological characteristics of apoptotic cells such as snowflake-like or not yet completely scattered flakes, and some of the cells exhibited abnormal morphology such as short thick rods, spindle-like or bigeminal, in which intact cells were not completely divided, while the number of cells exhibiting normal morphology was relatively small in the field. These morphological results show that, like compounds 1 and 2, compounds 1 a-1 i and 2 a-2 r exert their antitumor effects of inhibiting cancer cell proliferation by various pathways such as cytotoxic activity against cancer cells to be tested, induction of apoptosis in test cells, and inhibition of division of test cells by acting on a skeletal protein.

4. Conclusion

The compound of the formula I has strong antitumor activity, and exerts the antitumor effect of inhibiting the proliferation of cancer cells through the ways of killing cytotoxicity to the cancer cells to be tested, inducing the apoptosis of the cancer cells to be tested, acting on a skeleton protein to inhibit the division of the cancer cells to be tested and the like. Therefore, the compound of the formula I can be used as a cytoskeletal protein inhibitor, an apoptosis inducer, a tumor cell proliferation inhibitor or a tumor cell killing agent, and can also be used as an anti-tumor medicament for treating tumors.

Although specific embodiments of the invention have been described in detail, it will be appreciated by those skilled in the art that, based upon the overall teachings of the disclosure, various modifications and alternatives to those details could be developed and still be encompassed by the present invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

Claims

1. A compound of formula I, or a pharmaceutically acceptable salt thereof,

C_1-10(e.g. C)_1-6、C_1-8) Linear or branched saturated or unsaturatedHydrocarbyl radical, C_1-10(e.g. C)_1-6、C_1-8) Linear or branched saturated or unsaturated hydrocarbyloxy radical, C_2-18(e.g. C)_2-6、C_2-8) A linear or branched saturated or unsaturated aliphatic acyloxy group or an aromatic acyloxy group,

2. A compound of formula I according to claim 1, or a pharmaceutically acceptable salt thereof,

wherein,

3. A compound of formula I according to claim 1 or2, or a pharmaceutically acceptable salt thereof,

wherein,

R₁represents 1- (2-methyl) octyl;

4. The Penicillium purpurogenum BD-1-3 (Penicillium purpurogenum BD-1-3) has a preservation number of CGMCC No.4284, a preservation date of 11 months and 1 day 2010 and a preservation place of CGMCC (China general microbiological culture Collection center).

5. The Penicillium purpurogenum3-f-31 (Penicillium purpurogenum 3-f-31) has a preservation number of CGMCC No.7286, a preservation date of 2013, 3 and 7 days, and a preservation place of CGMCC (China general microbiological culture Collection center).

6. A process for the preparation of compound 1 and/or 2 comprising the steps of:

carrying out fermentation culture on the penicillium purpurogenum of claim 4 or claim 5 to obtain a fermentation product containing the compound, and separating and purifying the fermentation product to obtain the compound;

wherein said compound 1 is a compound of formula I according to claim 1, wherein R₁=1- (2-methyl) octyl radical, R₂=OH，R₃= H; wherein said compound 2 is a compound of formula I according to claim 1, wherein R₁=1- (2-methyl) octyl radical, R₂=R₃=OH。

7. The method of claim 6, comprising the steps of:

2) filtering the fermentation liquor to obtain filtrate and thalli;

6) dissolving the total extract with mixed solvent of chloroform-methanol at volume ratio of 1:1, or dissolving the total extract with a large amount of methanol to remove methanol insoluble substances, dissolving the methanol soluble fraction with mixed solvent of dichloromethane-methanol at volume ratio of 1:2, separating with 100-mesh silica gel column, eluting with petroleum ether-chloroform-methanol solvent system or petroleum ether-dichloromethane-acetone-methanol solvent system under reduced pressure to obtain crude fraction containing the compound, separating with two times of Sephadex LH-20 column chromatography (first eluting with 95% ethanol and second eluting with mixed solvent of chloroform-methanol at volume ratio of 1: 1) or one time of Sephadex LH-20 column chromatography (eluting with 95% ethanol) and one time of reversed phase silica gel ODS (eluting with water-methyl ethyl acetate) Gradient elution with an alcohol-acetone solvent system) to obtain column chromatography components containing the main components of the compound;

7) the column chromatography fractions containing the compound are separated and purified by two reverse phase HPLC (C-18 column eluting with methanol-water volume fraction 80:20 for the first time and 86:14 or 82:18 for the second time) or one HPLC (C-18 column eluting with methanol-water volume fraction 81: 19) to yield the compound.

8. A process for the preparation of a derivative of compound 1 or2 comprising the steps of:

respectively carrying out derivatization reaction on the compound 1 or2 and chemical reaction reagents such as halogenated hydrocarbon, ester, acid anhydride or acyl chloride, and separating and purifying reaction products to obtain the derivative;

Preferably, the method comprises the steps of:

1) dissolving the compound 1 or2 with acetone or anhydrous pyridine;

2) performing derivatization reaction with methyl iodide, diethyl sulfate, acetic anhydride, gallic acid, tribenzyl galloyl chloride, benzoyl chloride, p-nitrobenzoyl chloride, p-chlorobenzoyl chloride or p-fluorobenzoyl chloride for 1-48 h or 3-10 days under the conditions of room temperature, light shielding or 30-80 ℃ and hydrochloric acid catalysis or no hydrochloric acid catalysis;

9. A composition comprising a compound of formula I according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof, optionally together with one or more pharmaceutically acceptable carriers or excipients.

10. Use of a compound of formula I according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof or a composition according to claim 9 in the manufacture of an agent such as a cytoskeletal protein inhibitor, an apoptosis-inducing agent, a tumour cell proliferation inhibitor, or a tumour cell killing agent; further, the tumor cell is a leukemia cell or a cancer cell derived from epithelium (for example, a cervical cancer cell, a gastric cancer cell, a breast cancer cell, a lung cancer cell, a liver cancer cell, or a colon cancer cell); still further, the leukemia cells are chronic myelogenous leukemia cells or acute promyelocytic leukemia cells; furthermore, the tumor cells are human chronic myelogenous leukemia K562 cells, human acute promyelocytic leukemia HL-60 cells, human cervical cancer HeLa cells, human gastric cancer BGC-823 cells, or human breast cancer MCF-7 cells.

11. Use of a compound of formula I according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof or a composition according to claim 9 for the preparation of a medicament or agent for killing or inhibiting proliferation of tumor cells; further, the tumor cell is a leukemia cell or a cancer cell derived from epithelium (for example, a cervical cancer cell, a gastric cancer cell, a breast cancer cell, a lung cancer cell, a liver cancer cell, or a colon cancer cell); still further, the leukemia cells are chronic myelogenous leukemia cells or acute promyelocytic leukemia cells; furthermore, the tumor cells are human chronic myelogenous leukemia K562 cells, human acute promyelocytic leukemia HL-60 cells, human cervical cancer HeLa cells, human gastric cancer BGC-823 cells, or human breast cancer MCF-7 cells.

12. A method of killing tumor cells or inhibiting tumor cell proliferation in vivo or in vitro comprising the step of administering an effective amount of a compound of formula I of any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof, or a composition of claim 9; further, the tumor cell is a leukemia cell or a cancer cell derived from epithelium (for example, a cervical cancer cell, a gastric cancer cell, a breast cancer cell, a lung cancer cell, a liver cancer cell, or a colon cancer cell); still further, the leukemia cells are chronic myelogenous leukemia cells or acute promyelocytic leukemia cells; furthermore, the tumor cells are human chronic myelogenous leukemia K562 cells, human acute promyelocytic leukemia HL-60 cells, human cervical cancer HeLa cells, human gastric cancer BGC-823 cells, or human breast cancer MCF-7 cells.

13. Use of a compound of formula I, or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1 to 3, or a composition as claimed in claim 9, in the manufacture of an anti-neoplastic medicament; for example, the tumor is leukemia or a cancer derived from epithelium (e.g., cervical cancer, gastric cancer, breast cancer, lung cancer, liver cancer, or colon cancer); further, the leukemia is chronic myelogenous leukemia and acute promyelocytic leukemia.