CN107922366A

CN107922366A - Pyruvic dehydrogenase kinase inhibitor and its application

Info

Publication number: CN107922366A
Application number: CN201680047195.2A
Authority: CN
Inventors: 李剑; 黄敏; 耿美玉; 刘毅夫; 唐帅; 兰小晶; 朱进
Original assignee: East China University of Science and Technology; Shanghai Institute of Materia Medica of CAS
Current assignee: East China University of Science and Technology; Shanghai Institute of Materia Medica of CAS
Priority date: 2016-03-23
Filing date: 2016-03-23
Publication date: 2018-04-17
Also published as: WO2017161524A1

Abstract

Pyruvic dehydrogenase kinase inhibitor and its application are provided.Particularly provide Formula X compound R_a‑S‑S‑R_bOr its pharmaceutically acceptable salt, the compound have excellent suppression pyruvic dehydrogenase kinase activity and antitumous effect.Additionally provide the pharmaceutical composition containing the compound and suppress the application of pyruvic dehydrogenase kinase and anti-tumor aspect.

Description

Pyruvic dehydrogenase kinase inhibitor and its application

Technical field

The invention belongs to pharmaceutical chemistry and pharmacotherapeutics field, in particular it relates to Formula X compound and its application in terms of inhibiting pyruvic dehydrogenase kinase.

Background technique

Cancer is one of human diseases of most serious.It is counted according to world health organization (WHO), only just there are 7,600,000 people to die of cancer within 2008, it is contemplated that this number will be increased to 13,200,000 to the year two thousand thirty.Thus huge medical pressure can be brought, and destroys the economic situation of more people.Therefore, the research of anticancer drug is always the most important thing in medicament research and development work.

Pyruvic dehydrogenase kinase (PDHK) is a kind of cyclophorase, the activity of pyruvate dehydrogenase complex (PDC) can be adjusted and the latter has been catalyzed conversion of the pyruvic acid to acetyl coenzyme A, evidence shows that pyruvic dehydrogenase kinase can be such that PDC inactivates by phosphorylation.There are four hypotypes by PDHK: PDHK1-4, wherein PDHK1 and the grade malignancy of cancer are closely related.PDHK1 is activation in kinds cancer such as lung cancer, neck squamous cell carcinoma.PDHK1 is the oncogene transcriptional control such as proto-oncogene (C-MYC) and hypoxia inducible factor (hypoxia inducing factor, HIF), and metabolism and the malignant phenotype of cancer cell are controlled with this.Title has been reported, inhibits PDHK that can effectively improve the effect of tumour cell chemotherapy, and this tumour-specific with height.

JX06It is a kind of irreversible inhibitor of report in 2015, JX06 not yet enters clinical application at present, based on the identification to a hydrophobic pocket is adjoined with ATP binding pocket, JX06 can form disulfide bond with the mercaptan covalent bond on cysteine residues (C240) conservative in enzymatic structure, the conformation of the position R286 is caused to convert by Van der Waals force, to hinder the combination of ATP and enzyme and then generate inhibition to PDHK.Therefore, there is an urgent need in the art to develop new PDHK inhibitor.

Summary of the invention

The purpose of the present invention is to provide a kind of active compound of inhibition pyruvic dehydrogenase kinase and its applications.

The first aspect of the present invention provides a kind of Formula X compound R_a-S-S-R_b(X) purposes, the purposes include:

(a) preparation inhibits pyruvic dehydrogenase kinase (PDHK) active drug or preparation, and/or,

(b) preparation inhibits and/or treats the drug or preparation of tumour cell；

Wherein, R_a、R_bIt is each independently with S hetero atom and the heteroatomic end group group of N, and in the end group group, S hetero atom and N hetero atom constitute " S=C-N " or " S-C=N ", and in the end group group, when using-S-S- as center, the N hetero atom is located at the heteroatomic outside the S, and each S hetero atom and one, the interval-S-S- carbon atom.

In another preferred example, the tumour is the tumour of pyruvic dehydrogenase kinase (PDHK) positive.

In another preferred example, the tumour is selected from the group: lung cancer, nerve metrocyte carcinoma, liver cancer, colon cancer, prostate cancer, breast cancer, kidney.

In another preferred example, the Formula X compound is -1 compound of Formula X,

In formula,

Each R_cIt is separately selected from the group: nothing, substituted or unsubstituted C_1-6Alkyl；

Each R_dIt is separately selected from the group: nothing, substituted or unsubstituted C_1-6Alkyl, substituted or unsubstituted C_6-10Aryl, substituted or unsubstituted C_6-10Heteroaryl, substituted or unsubstituted C_6-10Naphthenic base ,-(CH₂)_m-C_6-10Aryl, m 0-4；

Each R_eIt is separately selected from the group: nothing, substituted or unsubstituted C_1-6Alkyl；

Also, R_c、R_d、R_eMeet following condition:

(1)R_c、R_dRing C is collectively formed with adjacent N, C and S atom, and connect with the S hetero atomIt is connect for singly-bound with N hetero atomFor double bond；Or

(2)R_e、R_dThe 5-6 member ring E that substituted or unsubstituted 7-12 member ring D is collectively formed with adjacent N or replaces, and connect with the S hetero atomIt is connect for double bond with N hetero atomFor singly-bound；

The heterocycle has 1-3 hetero atoms selected from the group below: O, S and N；

The substitution, which refers to, has one or more substituent groups selected from the group below: halogen, C₁-₁₀Alkyl, halogenated C₁-₁₀Alkyl, C₁-₁₀Alkoxy, halogenated C₁-₁₀Alkoxy, C_1-4Alkyl hydroxy, acetyl group, C_1-10Amide groups, C_1-10Carboxyl, C_5-20Aryl, halogenated C_5-20Aryl ,-(L₁)p-Z；

Wherein, each L₁It independently is :-(CH₂) r ,-O- ,-C=O ,-CH₂-(C_1-4Alkyl)-；

P is 0-4, r 0-4；

Z is selected from the group: C_6-20Aryl, C₁-₁₁Alkyl, C_1-10Amide groups, hydroxyl.

In another preferred example, the Formula X compound is compound of formula I:

In formula, n 1-3；

R₁And R₂It is each independently selected from the following group: hydrogen, hydroxyl, substituted or unsubstituted C_1-10Carboxyl, substituted or unsubstituted C₁-₆Ester group, substituted or unsubstituted methylol, substituted or unsubstituted C₁-₄Alkyl, substituted or unsubstituted C₁-₁₀Alkoxy, and R₁And R₂It is not simultaneously hydrogen；

Alternatively, R₁、R₂Saturated or unsaturated 5-8 member aromatic ring or carbocyclic ring is collectively formed.

In another preferred example, the Formula X compound is Formula II compound

In formula,

Ring A is selected from: substituted or unsubstituted C_5-10Carbocyclic ring, substituted or unsubstituted C_5-10Carbon heterocyclic, substituted or unsubstituted C₆-₁₀Aromatic ring, substituted or unsubstituted C₆-₁₀Hetero-aromatic ring.

In another preferred example, ring A is substituted or unsubstituted C₆-₁₀Hetero-aromatic ring, preferably, at 2,3 of pyridine and ring.

In another preferred example, the compound includes whole compounds described in second aspect of the present invention.

The second aspect of the present invention provides a kind of Formula X compound, its optical isomer or its pharmaceutically acceptable salt,

R_a-S-S-R_b (X)

In formula,

Also, R_c、R_d、R_eMeet following condition:

P is 0-4, r 0-4；

In another preferred example, the ring C is two rings, preferably, ring C is selected from the group: 5-10 member carbon heterocyclic and 5-10 member carbocyclic ring, 5-10 member carbon heterocyclic and 5-10 member carbon heterocyclic, 5-10 member carbon heterocyclic and 5-10 member aromatic ring, 5-10 member carbon heterocyclic and 5-10 member heteroaromatic.

In another preferred example, the ring D is two rings, preferably 5-7 member ring and 5-7 member ring, and more preferably ring D is selected from the group: hexatomic ring and hexatomic ring, is more preferably hexa-member heterocycle and phenyl ring, hexa-member heterocycle and six-membered carbon ring, naphthalene.

In another preferred example, the substituent group is-(L₁)p-Z。

In another preferred example, the L₁It is selected from the group :-O-CH₂-、-O-CO-CH₂-。

In another preferred example, the Z is C_6-20Aryl or C_1-10Amide groups.

In another preferred example, R_e、R_dIt is preferably two rings with the adjacent N ring formed.

In another preferred example, described ring C, D and E are saturated or unsaturated, aromatic ring or non-aromatic ring, heterocycle or non-heterocycle.

In another preferred example, the ring E is the 5-6 member ring replaced.

In another preferred example, the ring E is the 5-6 circle heterocyclic ring replaced.

In another preferred example, the ring D is substituted or unsubstituted 7-10 member ring.

In another preferred example, R_a、R_bRespectively identical or differently are as follows:

N is 1-3；

In another preferred example, the Formula X compound is compound of formula I:

In formula, R₁、R₂It is as defined above.

In another preferred example, R₁、R₂Saturated or unsaturated 6 yuan of phenyl ring or carbocyclic ring is collectively formed.

In another preferred example, n=1.

In another preferred example, R₁For substituted or unsubstituted C_1-10Carboxyl, preferable n are 1, R₂For hydrogen.

In another preferred example, R₁For ester group, preferable R₁For methyl esters, n 1, R₂For hydrogen.

In another preferred example, R₁For methyl, preferable n is 1, R₂For hydrogen.

In another preferred example, R₁And R₂It is each independently substituted or unsubstituted methylol or H.

In another preferred example, R₁And R₂It is each independently C_1-10Alkyl-substituted methylol or H, preferably The ground C_1-10Alkyl is selected from the group: methyl, ethyl, normal-butyl, positive decyl.

In another preferred example, R₁And R₂It is each independently N, the methylol or H that N diformamide base replaces.

In another preferred example, R₁And R₂It is each independently the methylol or H of acetyl group substitution, n 2.

In another preferred example, R₁And R₂It is each independently substituted or unsubstituted methylol or H, preferably the substituent group is selected from the group: phenyl, p-methylphenyl, p-methoxyphenyl, naphthalene.

In another preferred example, R₁And R₂It is each independently-CH₂-CH₂The methylol that-OH replaces.

In another preferred example, the Formula X compound is Formula II compound

In formula,

In another preferred example, ring A is the C of saturation_5-7Carbocyclic ring.

In another preferred example, ring A is substituted or unsubstituted 6 yuan of carbon heterocyclics, preferably contains the heterocycle of an oxygen, and contained oxygen atom is in the contraposition of N.

In another preferred example, ring A is substituted or unsubstituted 6 yuan of carbon heterocyclics, preferably, the substituent group is selected from methyl, dimethyl, C_1-10Carboxyl, ethoxycarbonyl, preferably, contraposition of the position of substitution in N atom.

In another preferred example, the Formula X compound is selected from the group:

In another preferred example, the Formula X compound is selected from the group: compound 1,8,9,18,19,21,22,25,26,29 and 33.

In another preferred example, the compound of formula I is selected from the group:

In another preferred example, the Formula II compound is selected from the group:

The third aspect of the present invention provides a kind of pharmaceutical composition, contains in described pharmaceutical composition

(i) the Formula X compound or its pharmaceutically acceptable salt described in second aspect of the present invention；

(ii) pharmaceutically acceptable carrier.

In another preferred example, 0.001-99wt% is contained in described pharmaceutical composition, preferably 0.1-90wt%, the Formula X compound or its pharmaceutically acceptable salt of more preferably 1-80wt% are based on the total weight of the composition.

In another preferred example, the dosage form of the pharmaceutical composition is peroral dosage form or injection.

In another preferred example, the peroral dosage form is selected from: tablet, capsule, film, granule.

In another preferred example, the peroral dosage form is slow-release or non-time-release type dosage form.

In another preferred example, the pharmaceutical composition further includes other active components, and the active constituent is selected from: cis-platinum, taxol or antitumor antibody.

The fourth aspect of the present invention provides a kind of the method for preparing compound of formula I I, comprising steps of

(I-2) in atent solvent, formula B compound is reacted with reagent D, to form compound of formula I, wherein the reagent D is selected from the group: ammonium persulfate, sodium metaperiodate, sodium nitrite, hydrochloric acid, hydrogen peroxide, sulfuric acid, or combinations thereof；

(I-3) isolated or purified optionally is carried out to the compound of formula I formed in step (I-2), thus obtain separation or The compound of formula I of purifying；

In formula, n 1-3；

In another preferred example, in step (I-3), the separation includes recrystallization.

In another preferred example, in step (I-2), it is more preferably 1:1.0-2.0 that the molar percentage of the formula B compound and reagent D, which is 1:0.1-3, preferably 1:0.5-2.5,.

In another preferred example, in step (I-2), (preferably 20-30 DEG C) reacts at -5 to 50 DEG C；It reacts 5-30h (preferably 10-25h), and/or is reacted under agitation.

In another preferred example, the method I is further comprised the steps of:

(I-1) in organic solvent, formula A compound is reacted with carbon disulfide, so that formula B compound be made, wherein n, R₁、R₂It is defined as described above.

In another preferred example, in described (I-1), the organic solvent is selected from the group: tetrahydrofuran, methyltetrahydrofuran, water, or combinations thereof.

In another preferred example, in step (I-1), it is more preferably 1.1-1.8 that the molar percentage of the formula A compound and carbon disulfide, which is 1:0.5-2.5, preferably 1:1.0-2.0,.

In another preferred example, it in step (I-1), is reacted at 20-25 DEG C；It reacts 1-12h (preferably 2-10h)；And/or it is reacted under agitation.

The fifth aspect of the present invention provides a kind of method II of preparation formula II compound, comprising steps of

(II-1) in organic solvent, formula C compound is reacted with reagent D, shape compound of formula II, wherein the reagent D is selected from the group: ammonium persulfate, sodium metaperiodate, sodium nitrite, hydrochloric acid, hydrogen peroxide, sulphur Acid, or combinations thereof, in formula,

In another preferred example, the formula C compound and the molar percentage of reagent D are 1:0.1-3, preferably 1:0.5-2.5, are more preferably 1:1.0-2.0.

In another preferred example, in step (II-1), (preferably 20-30 DEG C) reacts at -5 to 50 DEG C；It reacts 5-30h (preferably 10-25h), and/or is reacted under agitation.

The sixth aspect of the present invention inhibits the active method of pyruvic dehydrogenase kinase with providing a kind of non-therapeutic, comprising steps of

(a) pyruvic dehydrogenase kinase is contacted with Formula X compound, its optical isomer described in second aspect of the present invention, non-raceme, racemic modification or its pharmaceutically acceptable salt, to inhibit the activity of pyruvic dehydrogenase kinase.

In another preferred example, in step (a), Formula X compound, its optical isomer, non-raceme, racemic modification or its pharmaceutically acceptable salt are added in cell culture system, so that it be made to be contacted with pyruvic dehydrogenase kinase.

In another preferred example, the cell is normal cell or tumour cell.

In another preferred example, the cell is mammalian cell.

In another preferred example, the cell is people's cell.

The seventh aspect of the present invention, a kind of method for inhibiting pyruvic dehydrogenase kinase or treating tumour is provided, the method includes: to apply Formula X compound, its optical isomer, non-raceme, racemic modification or its pharmaceutically acceptable salt described in second aspect of the present invention to the object of needs.

In another preferred example, the object includes people and non-human mammal (such as rodent and Primate).

It should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the invention and it can be combined with each other between each technical characteristic specifically described in below (e.g. embodiment), to form a new or preferred technical solution.Due to space limitations, I will not repeat them here.

Detailed description of the invention

Fig. 1 shows molecular level enzyme activity (A549 of the compound respectively in 1 μM of (above) and 0.3 μM (following figure) Cell).

Fig. 2 shows influence of the compound 19,21 under various concentration to A549 cell activity oxygen (ROS).

Fig. 3 shows the influence under compound 19,21 various concentrations to the release of A549 extracellular lactic acid.

Specific embodiment

Present inventor by depth studying extensively, have unexpectedly discovered that Formula X compound can inhibit the activity of pyruvic dehydrogenase kinase (PDHK) significantly for the first time, it activates mitochondria pyruvate dehydrogenase complex (PDC), the glycoxidative conversion that the anaerobic glycolysis for promoting cytoplasm to rely on is relied on to mitochondria, Formula X compound also obtain good result in the proliferation experiment for inhibiting tumour cell.On this basis, the present invention is completed.

Term

As used herein, term " the compounds of this invention " or " compound " refer to Formula X compound represented or its raceme, corresponding isomers or its pharmaceutically acceptable salt.It should be understood that the term further includes the mixture of said components.

Group

Term " C_1-6Alkyl " refers to the linear or branched alkyl group with 1-6 carbon atom, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl group, sec-butyl, tert-butyl or similar group.

Term " C₁-₆Alkoxy " refers to the straight or branched alkoxyl with 1-6 carbon atom, such as methoxyl group, ethyoxyl, propoxyl group, isopropoxy, butoxy, isobutoxy, sec-butoxy, tert-butoxy or similar group.

" naphthenic base " refers to 3 to 8 yuan of full carbon monocycles, 5 yuan/6 yuan of full carbon or 6 yuan/6 yuan fused rings or polycyclic fused ring group, wherein one or more rings can be containing one or more double bonds, but none ring has the pi-electron system of total conjugated.Examples of cycloalkyl has cyclopropyl, cyclobutyl, cyclopenta, cyclohexyl, cyclohexadienyl, adamantyl, cycloheptyl alkyl, cycloheptatriene base etc..

" 5-7 unit monocycle " refers to the monocycle (an only ring structure) with 5-7 member, and the monocycle can be saturation or unsaturated ring, such as naphthenic base, cycloalkenyl, aromatic ring.

" carbocyclic ring " finger ring skeleton is all the saturation or unsaturated ring of carbon atom, wherein one or more rings can contain one or more double bonds.

At least there is a heteroatomic saturation or unsaturated ring selected from the group below: N, S, O or P on " heterocycle " finger ring skeleton, wherein one or more rings can contain one or more double bonds.

" aromatic ring " refers to the aromatic ring of the pi-electron system with conjugation, including isocyclic aryl, heteroaryl.

" heteroaryl " refers to that with 1 hetero atom, as annular atom, remaining annular atom is the aryl of carbon, and hetero atom includes oxygen, sulphur, nitrogen.The ring can be 5- or 6-membered or 7 member rings.The example of heteroaryl groups includes but is not limited to furyl, thienyl, benzofuranyl, benzothienyl, pyridyl group, pyrroles, N- alkyl pyrrole radicals.

" alkoxy " refers to-O- (alkyl).Representative example includes methoxyl group, ethyoxyl, propoxyl group, butoxy etc..

Term " halogen " refers to fluorine, chlorine, bromine, iodine.Term " halogenated " refers to fluoro, chloro, bromo, iodo.

Herein, except place is illustrated, each group of the invention can be unsubstituted or substituted, and " substitution " refers to that one or more hydrogen atoms on group are replaced by substituent group selected from the group below: C₁-C₁₀Alkyl, halogenated C₁-₁₀Alkyl, C₃-C₁₀Naphthenic base, C₁-C₁₀Alkoxy, halogenated C₁-₁₀Alkoxy, halogen, hydroxyl, C_1-10Carboxyl (- COOH), C₁-C₁₀Aldehyde radical, C₂-C₁₀Acyl group, C₂-C₁₀Ester group, amino, phenyl, C_1-4Alkyl hydroxy, C_1-10Amide groups, C_5-20Aryl, halogenated C_5-20Aryl, cyano；The phenyl includes unsubstituted phenyl or the substituted-phenyl with 1-3 substituent group, and the substituent group is selected from: halogen, C₁-C₁₀Alkyl, cyano, OH, nitro, C₃-C₁₀Naphthenic base, C₁-C₁₀Alkoxy, amino.

Formula X compound

The present invention provides a kind of Formula X compound, its optical isomer or its pharmaceutically acceptable salt,

R_a-S-S-R_b (X)

In formula,

Also, R_c、R_d、R_eMeet following condition:

P is 0-4, r 0-4；

Compound of formula I

As used herein, compound of formula I is as follows:

In formula,

N is 1-3；

R₁And R₂It is each independently selected from the following group: hydrogen, hydroxyl, C_1-10Carboxyl, C₁-₆Ester group, substituted or unsubstituted methylol, substituted or unsubstituted C₁-₄Alkyl, substituted or unsubstituted C₁-₁₀Alkoxy, and R₁And R₂It is not simultaneously hydrogen；

Formula II compound

As used herein, Formula II compound is as follows:

In formula,

Pyruvic dehydrogenase kinase

Pyruvic dehydrogenase kinase, that is, Pyruvate Dehydrogenase Kinase (PDHK), it is a kind of cyclophorase, it is able to suppress pyruvate dehydrogenase complex (PDC), it is the key that the glycoxidative control enzyme of grape, can prevent conversion of pyruvate is acetyl coenzyme A, and the latter is the substrate of tricarboxylic acid cycle.The activation energy of PDHK leads to glycolysis and disconnection glycoxidative downstream.The PDHK that four hypotypes have been confirmed the existence of in human cell is respectively that the grade malignancy of PDHK1-4, wherein PDHK1 and cancer is closely related.PDHK1 is activation in kinds cancer such as lung cancer, neck squamous cell carcinoma.PDHK1 is the oncogene transcriptional control such as proto-oncogene (C-MYC) and hypoxia inducible factor (hypoxia inducing factor, HIF), and metabolism and the malignant phenotype of cancer cell are controlled with this.

PDHK1 is generally the tyrosine kinase institute phosphorylation of various tumorigenesis in human cancer cell.Inhibit PDHK that can effectively improve the effect of tumour cell chemotherapy, and the tumour-specific with height.In addition to above-mentioned metabolism anticancer mechanism, also some researches show that the activation of PDC cause active oxygen stress (reactive oxygen species, ROS) increase and induce the apoptosis of tumour cell.Therefore, PDHK is inhibited to can be used as the target spot of killing tumor cell.

Pharmaceutically acceptable salt

The invention also includes the pharmaceutically acceptable salts of Formula X compound.

Term " pharmaceutically acceptable salt " refers to that the compounds of this invention and acid or alkali are formed by the salt for being suitable as drug.Pharmaceutically acceptable salt includes inorganic salts and organic salt.A kind of preferred salt is the salt that the compounds of this invention and acid are formed.The acid for suitably forming salt includes but is not limited to: the inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid, formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulfonic acid, benzene methanesulfonic acid, the organic acids such as benzene sulfonic acid；And the acidic amino acids such as aspartic acid, glutamic acid.

Preparation method

Reaction step is prepared in the present invention and is all made of method well known to those skilled in the art in the prior art, and the response parameter of each step is not particularly limited.Those skilled in the art can suitably make adjustment to reactant, reaction condition of each step reaction involved in reaction equation etc. according to different final expected compounds.

The present invention provides a kind of the method for preparing compound of formula I I, comprising steps of

(I-3) isolated or purified optionally is carried out to the compound of formula I formed in step (I-2), to obtain separation or purifying compound of formula I；

In formula, n 1-3；

In another preferred example, the method I is further comprised the steps of:

In another preferred example, in described (I-1), the organic solvent is selected from the group: tetrahydrofuran, methyl tetrahydro furan Mutter, water, or combinations thereof.

The present invention also provides a kind of method II of preparation formula II compound, comprising steps of

(II-1) in organic solvent, formula C compound is reacted with reagent D, shape compound of formula II, wherein the reagent D is selected from the group: ammonium persulfate, sodium metaperiodate, sodium nitrite, hydrochloric acid, hydrogen peroxide, sulfuric acid, or combinations thereof, in formula,

In another preferred example, compound of formula I preparation method comprising steps of

It is dissolved in substituted morpholine (n=1) or high morpholine (n=2) and suitable carbon disulfide in tetrahydrofuran, is stirred to react at room temperature 10-20 hours.Suitable ammonium persulfate aqueous solution is added into reaction system, is stirred at room temperature 10-20 hours.Organic layer obtains titled reference compound after being spin-dried for re-crystallizing in ethyl acetate or after silica gel column chromatography.The morpholine or high morpholine wherein replaced is to buy or synthesize (see specific embodiment).

In another preferred example, Formula II compound preparation method comprising steps of

The 2- thyroidan of simultaneously ring is dissolved in tetrahydrofuran, suitable ammonium persulfate aqueous solution is then added, room temperature is stirred It mixes 10-20 hours.Organic layer obtains title compound after being spin-dried for re-crystallizing in ethyl acetate or after silica gel column chromatography.The 2- thyroidan wherein replaced is to buy or synthesize (see specific embodiment).

Composition and method of administration

The present invention also provides a kind of for inhibiting the composition of pyruvic dehydrogenase kinase.The composition includes (but being not limited to): pharmaceutical composition, food compositions, dietary supplements, beverage composition for treating dental erosion etc..

In the present invention, the pharmaceutical composition can be directly used for disease treatment, for example, being used for the treatment of anti-tumor aspect.When using pharmaceutical preparation of the present invention, other therapeutic agents, such as anti-tumor drug also can be used simultaneously.

The present invention also provides a kind of pharmaceutical composition, it contains the compounds of this invention and pharmaceutically acceptable carrier or excipient of safe and effective amount.This kind of carrier include (but being not limited to): salt water, buffer, glucose, water, glycerol, ethyl alcohol, pulvis, and combinations thereof.Pharmaceutical preparation should match with administration mode.

By taking pharmaceutical composition as an example, composition of the invention can be made into injection form, such as the aqueous solution with physiological saline or containing glucose and other adjuvants is prepared by conventional method.The pharmaceutical composition of such as tablet and capsule etc can be prepared by conventional method.Pharmaceutical composition such as injection, solution, tablet and capsule preferably aseptically manufacture.Pharmaceutical composition of the invention can also be made into pulvis for Neulized inhalation.The dosage of active constituent is therapeutically effective amount, such as about 5 mg/kg weight of about 1 microgram/kg body weight-daily.In addition, cell autophagy inhibitor of the present invention can be also used together with other therapeutic agents.

For pharmaceutical composition of the invention, required object (such as people and non-human mammal) can be applied to by way of conventional.Representative method of application includes (but being not limited to): oral, injection, Neulized inhalation etc..

When using pharmaceutical composition, it is the medicament administration by safe and effective amount in mammal, the wherein safe and effective amount typically at least about 10 micrograms/kg body weight, and in most cases no more than about 8 mg/kg weight, preferably the dosage is about 1 mg/kg weight of about 10 micrograms/kg body weight-.Certainly, specific dosage is also contemplated that the factors such as administration route, patient health situation, within the scope of these are all skilled practitioners technical ability.

Main advantages of the present invention are:

(1) a kind of new X compound is provided, it can be used for (a) preparation and inhibits pyruvic dehydrogenase kinase (PDHK) active drug or preparation, and/or (b) preparation inhibits and/or treats the drug or preparation of tumour cell；

(2) the compounds of this invention result is simple, and preparation process is succinct, and production cost is low；

(3) the compounds of this invention inside and outside antitumor mechanism is clear, drug effect is significant.

Present invention will be further explained below with reference to specific examples.It should be understood that these examples are only for illustrating the present invention and are not intended to limit the scope of the present invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to normal condition, or according to the normal condition proposed by manufacturer.Unless otherwise stated, otherwise percentage and number are calculated by weight.

Embodiment 1 bis- (4- (2- (methoxy) morpholine) base thiocarbonyls) changes two magisters of sulfur

2.3g compound 1-1 is dissolved in 50mL methanol, under nitrogen protection, 600mg sodium methoxide is added, it is heated overnight at a reflux temperature, after system is cooled to room temperature, with 200mL ethyl acetate and 200mL water stratification, organic phase obtains compound 1-2 1.5g through silica gel column chromatography, compound 1-2 is dissolved in 50mL methanol, it is added 50mg palladium carbon (10%), hydrogen reducing (1atm) is overnight, it is spin-dried for obtaining 0.85g compound 1-3 after filtering, 20mLTHF and 0.5g carbon disulfide is directly added into stir at room temperature, 2g ammonium persulfate is dissolved in 10mL water, it is added after 10 hours, continue to be stirred overnight, organic phase after reaction system 200mL ethyl acetate and 200mL water stratification is spin-dried for, with 1: 3 ethyl acetate and petroleum ether column chromatography for separation obtains 1 yield 47% (calculating according to compound 1-1) of 1g compound.

¹H-NMR(400MHz,CDCl₃)δ5.60-4.40(m,4H),4.11-4.00(m,2H),3.85-3.7(m,4H),3.65-3.42(m,6H),3.42-3.39(s,6H),3.39-3.10(m,2H)；HRMS (EI) m/z calculated value: C₁₄H₂₄N₂O₄S₄[M]⁺, 412.0619, measured value: 412.0618

Embodiment 2 bis- (4- (2- (phenol ylmethyl) morpholine) base thiocarbonyls) changes two magisters of sulfur

1g compound 1-1 is dissolved in 50mLDMF, under nitrogen protection, it is added 0.35g sodium hydride (60%), it is heated overnight at 100 DEG C, after system is cooled to room temperature, with 200mL ethyl acetate and 200mL water stratification, organic phase obtains compound 2-2 0.65g through silica gel column chromatography, compound 2-2 is dissolved in 50mL methanol, it is added 50mg palladium carbon (10%), hydrogen reducing (1atm) is overnight, it is spin-dried for obtaining 0.46g compound 2-3 after filtering, 20mLTHF and 0.4g carbon disulfide is directly added into stir at room temperature, 1g ammonium persulfate is dissolved in 10mL water and is added after 10 hours, continue to be stirred overnight, organic phase after reaction system 200mL ethyl acetate and 200mL water stratification is revolved It is dry, 2 yield 63% (calculating according to compound 2-3) of 0.4g compound is obtained with the ethyl acetate of 1:4 and petroleum ether column chromatography for separation

¹H-NMR(400MHz,CDCl₃)δ7.34-7.27(m,5H),7.02-6.90(m,5H),6.05-4.45(m,4H),4.45-3.03(m,14H)；HRMS (ESI) m/z calculated value: C₂₄H₂₈N₂O₄S₄[M+Na]⁺: 559.0824, measured value: 559.0823

Embodiment 3 bis- (4- (2- (p methoxy phenol ylmethyl) morpholine) base thiocarbonyls) changes two magisters of sulfur

With embodiment 2, the difference is that: it changes p-cresol into p methoxy phenol, obtains 0.09 gram of yellow solid (compound 3), yield is 40%.

¹H-NMR(400MHz,CDCl₃)δ6.91-6.79(m,8H),7.02-6.90(m,5H),5.80-4.21(m,4H),3.91-3.79(m,2H),3.77(s,6H),3.69-3.27(m,4H)；HRMS (ESI) m/z calculated value: C₂₆H₃₂N₂O₆S₄[M+Na]⁺: 619.1035, measured value: 619.1037

Embodiment 4 bis- (4- (2- (alpha-Naphthol ylmethyl) morpholine) base thiocarbonyls) changes two magisters of sulfur

With embodiment 2, the difference is that: it changes p-cresol into alpha-Naphthol, obtains 0.13 gram of yellow solid (compound 4), yield is 53%.

¹H-NMR(400MHz,CDCl₃)δ8.33-8.18(m,2H),7.85-7.75(m,2H),7.57-7.42(m,6H),7.41-7.32(m,2H),6.89-6.68(m,2H),5.88-4.39(m,4H),4.37-4.11(m,8H),3.89-3.39(m,6H)；HRMS (ESI) m/z calculated value: C₂₆H₃₂N₂O₆S₄[M+H]⁺: 659.1137, measured value: 659.1141

Embodiment 5 bis- (4- (2- (methoxy) high morpholine) base thiocarbonyls) changes two magisters of sulfur

With embodiment 1, the difference is that: it changes the N- Benzvlmorpholin (1-1) that 2 chloromethyls replace into the high morpholine of N- benzyl that 2 chloromethyls replace, obtains 0.05 gram of yellow solid (compound 5), yield is 36%.

¹H-NMR(400MHz,CDCl₃)δ5.21-5.10(m,1H),4.92-4.80(m,1H),4.79-4.68(m,1H),4.27-4.08(m,3H),3.99-3.45(m,12H),3.42(s,3H),3.39(s,3H),2.51-1.98(m,4H)；HRMS (EI) m/z calculated value: C₁₆H₂₈N₂O₂S₄[M]⁺, 440.0932, measured value: 440.0930

Embodiment 6 bis- (4- (2- (p-methyl phenol ylmethyl) high morpholine) base thiocarbonyls) changes two magisters of sulfur

With embodiment 2, the difference is that: it changes the N- Benzvlmorpholin that 2 chloromethyls replace into the high morpholine of N- benzyl that 2 chloromethyls replace, obtains 0.04 gram of yellow solid (compound 6), yield is 31%.

¹H-NMR(400MHz,CDCl₃)δ7.15-7.01(m,4H),6.90-6.78(m,4H),5.29-5.20(m,1H),5.05-4.81(m,1H),4.81-4.69(m,1H),4.38-4.11(m,5H),4.11-3.99(m,4H),3.99-3.90 (m,4H),3.90-3.50(m,5H),2.53-2.37(m,1H),2.37-2.32(m,1H),2.28(S,6H),2.24-2.14(m,1H),2.14-2.00(m,1H)；HRMS (ESI) m/z calculated value: C₂₈H₃₆N₂O₄S₄[M+Na]⁺: 615.1450, measured value: 615.1456

Embodiment 7 bis- (4- (2- (phenol ylmethyl) high morpholine) base thiocarbonyls) changes two magisters of sulfur

With embodiment 6, the difference is that: it changes p-cresol into phenol, obtains 0.03 gram of yellow solid (compound 7), yield is 43%.

¹H-NMR(400MHz,CDCl₃)δ7.33-7.21(m,4H),7.04-6.86(m,6H),5.32-5.21(m,1H),5.06-4.83(m,1H),4.83-4.70(m,1H),4.35-4.14(m,5H),4.12-4.04(m,4H),4.02-3.83(m,3H),3.77-3.54(m,5H)；HRMS (ESI) m/z calculated value: C₂₆H₃₂N₂O₄S₄[M+Na]⁺: 587.1134, measured value: 587.1135

Embodiment 8 bis- (4- (2- (ethoxyl methyl) morpholine) base thiocarbonyls) changes two magisters of sulfur

The N- Benzvlmorpholin that 2 chloromethyls of 2g replace is dissolved in 10mL formamide and 2mL water, 200 DEG C are heated to be stirred overnight, excessive ammonium hydroxide is added after cooling, it is spin-dried for after being extracted with 200mL ethyl acetate, 1.3g compound 8-1, it is dissolved in 30mLDMF, 0.8g sodium hydride (60%) and 2g iodoethane is added, it is heated 5 hours at 80 DEG C, with 200mL ethyl acetate and 100mL water stratification, 0.9g compound 8-2 is obtained after organic phase column chromatography for separation, it is directly dissolved in 30mL methanol and hydrogenating reduction 10 hours under 1atm is added after 0.05g palladium carbon, 20mLTHF and 0.5g carbon disulfide is directly added into after being spin-dried for stir at room temperature, 2g ammonium persulfate was dissolved in 10mL water in 10 hours After be added, continue to be stirred overnight, organic phase after reaction system 200mL ethyl acetate and 200mL water stratification revolved Dry, obtaining 8 yield of 0.5g compound with the ethyl acetate of 1:3 and petroleum ether column chromatography for separation is 36% (calculating according to compound 8-1).

¹H-NMR(400MHz,CDCl₃) δ 5.61-4.61 (m, 4H), 4.10-4.00 (m, 2H), 3.86-3.71 (m, 1H), 3.66-3.26 (m, 12H), 1.23 (t, J=7.2Hz, 6H)；HRMS (EI) m/z calculated value: C₁₆H₂₈N₂O₄S₄[M]⁺, 440.0932, measured value: 440.0936

Embodiment 9 bis- (4- (2- (acetoxy-methyl) morpholine) base thiocarbonyls) changes two magisters of sulfur

The N- Benzvlmorpholin that 2 methylols of 0.5g replace is dissolved in 10mL methylene chloride, it is added and 0.5g triethylamine, it is cooled to 0 DEG C, 0.25g chloroacetic chloride is instilled, room temperature is returned to, it is stirred overnight, 100mL methylene chloride is added and 50mL water stratification, organic layer are spin-dried for, column chromatographs to obtain 0.45g compound 10-1,0.22g compound 9, yield 40% are finally obtained with method similarly to Example 9.

¹H-NMR(400MHz,CDCl₃)δ5.36-4.79(m,4H),4.25-4.16(m,4H),4.10-4.03(m,2H),3.91-3.83(m,1H),3.82-3.74(m,2H),3.64-3.48(m,2H),3.45-3.25(m,2H),2.12(s,6H)；HRMS (EI) m/z calculated value: C₁₆H₂₄N₂O₆S₄[M]⁺, 468.0517, measured value: 468.0519

Embodiment 10 bis- (4- (2- hydroxymethyl morpholine) base thiocarbonyls) changes two magisters of sulfur

The N- Benzvlmorpholin for obtaining the substitution of 2 methylols in the method for embodiment 8 uses palladium carbon as raw material in methyl alcohol Add hydrogen to take off benzyl, obtains the morpholine of 2 methylols substitution, obtain 0.1g compound 10, yield 60% with method similarly to Example 1.

¹H-NMR(400MHz,CDCl₃)δ5.67-4.22(m,4H),4.11-4.00(m,2H),3.84-3.74(m,5H),3.72-3.62(m,3H),3.61-3.30(m,4H)；HRMS (ESI) m/z calculated value: C₁₂H₂₀N₂O₄S₄[M+Na]⁺: 407.0198, measured value: 407.0205

Embodiment 11 bis- (4- (2- (dimethylamino formic acid ylmethyl) morpholine) base thiocarbonyls) changes two magisters of sulfur

The N- Benzvlmorpholin replaced using 2 methylols is raw material, through palladium carbon plus hydrogen, and with tert-butoxy protect N after obtain compound 11-1, 0.5g compound 11-1 is dissolved in 10mL THF and is cooled to the n-BuLi of -78 DEG C of 1 equivalents of addition, the dimethylaminoethyl chloride of 1.2 equivalents is added after 1 hour, and slowly return to room temperature, obtain 0.6g compound 12-2, 0.6g compound 11-2 is dissolved in 5mLTFA and is stirred at room temperature 1 hour, 0.3g compound 11-3 is obtained with potassium carbonate is free in methyl alcohol after being spin-dried for, 0.12g compound 11 is finally obtained with method similarly to Example 8 later, yield 20%.

¹H-NMR(400MHz,CDCl₃) δ 5.64-4.61 (m, 4H), 4.26-4.02 (m, 6H), 3.82-3.79 (m, 2H), 3.76 (t, J=11.2Hz 2H), 3.64-3.15 (m, 4H), 2.94 (s, 12H)；HRMS (ESI) m/z calculated value: C₁₈H₃₀N₄O₆S₄[M+Na]⁺: 549.0940, measured value: 549.0943

Embodiment 12 bis- (4- (2- (ethoxyl methyl) high morpholine) base thiocarbonyls) changes two magisters of sulfur

With embodiment 8, the difference is that: it changes the N- Benzvlmorpholin that 2 chloromethyls replace into the high morpholine of N- benzyl that 2 chloromethyls replace, obtains 0.11 gram of yellow oil (compound 12), yield is 36%.

¹H-NMR(400MHz,CDCl₃)δ5.21-5.11(m,1H),4.91-4.82(m,1H),4.77-4.67(m,1H),4.27-4.10(m,3H),3.96-3.45(m,16H),2.51-2.35(m,1H),2.35-2.24(m,1H),2.24-2.14(m,1H),2.14-2.03(m,3.5H),1.26-1.17(m,6H)；HRMS (EI) m/z calculated value: C₁₈H₃₂N₂O₄S₄[M]⁺, 468.1245, measured value: 468.1246

Embodiment 13 bis- (4- (2- (n-butoxy methyl) high morpholine) base thiocarbonyls) changes two magisters of sulfur

With embodiment 12, the difference is that: it changes iodoethane into n-bromide butane, obtains 0.09 gram of yellow oil (compound 13), yield is 30%.

¹H-NMR(400MHz,CDCl₃)δ5.22-5.12(m,1H),4.91-4.80(m,1H),4.78-4.68(m,1H),4.27-4.05(m,3H),4.00-3.74(m,16H),3.66-3.40(m,12H),2.51-2.36(m,1H),2.33-2.23(m,1H),2.23-2.12(m,1H),2.10-1.96(m,1H),1.63-1.56(m,4H),1.42-1.32(m,4H),0.97-0.87(m,6H)；HRMS (ESI) m/z calculated value: C₂₂H₄₀N₂O₄S₄[M+Na]⁺: 547.1763, measured value: 547.1774

Embodiment 14 bis- (4- (2- (n-decyloxy methyl) high morpholine) base thiocarbonyls) changes two magisters of sulfur

With embodiment 12, the difference is that: it changes iodoethane into positive bromo-decane, obtains 0.14 gram of yellow oil (compound 14), yield is 33%.

¹H-NMR(400MHz,CDCl₃) δ 5.21-5.10 (m, 1H), 4.92-4.79 (m, 1H), 4.79-4.67 (m, 1H), 4.29-4.02 (m, 3H), 4.02-3.74 (m, 4H), 3.74-3.37 (m, 12H), 2.50-2.34 (m, 1H), 2.34-2.23 (m, 1H), 2.23-2.11 (m, 1H), 2.11-1.96 (m, 1H), 1.65-1.51 (m, 4H), 1.35-1.22 (m, 28H), 0.88 (t, J=8Hz, 6H)；HRMS (ESI) m/z calculated value: C₃₄H₆₄N₂O₄S₄[M+Na]⁺: 715.3641, measured value: 715.3645

Embodiment 15 bis- (4- (2- (n-decyloxy methyl) high morpholine) base thiocarbonyls) changes two magisters of sulfur

With embodiment 12, the difference is that: it changes iodoethane into benzyl 2- bromoethyl ether, obtains 0.04 gram of yellow oil (compound 15), yield is 19%.

¹H-NMR(400MHz,CDCl₃)δ5.22-5.11(m,1H),4.91-4.80(m,1H),4.78-4.65(m,1H),4.26-4.08(m,3H),4.01-3.40(m,23H),2.51-2.37(m,1H),2.33-2.24(m,1H),2.22-2.10(m,1H),2.10-2.00(m,1H)；HRMS (ESI) m/z calculated value: C₁₈H₃₂N₂O₆S₄[M+Na]⁺: 523.1035, measured value: 523.1041

Embodiment 16 bis- (4- (the high morpholine of 2- methylol) base thiocarbonyls) changes two magisters of sulfur

With embodiment 10, the difference is that: the N- Benzvlmorpholin that 2 chloromethyls replace is changed into the high morpholine of N- benzyl replaced to 2 chloromethyls, obtains 0.05 gram of yellow oil (compound 16), yield is 23%.

¹H-NMR(400MHz,CDCl₃)δ5.08-4.97(m,1H),4.87-4.76(m,1H),4.70-4.60(m,1H),4.25-4.12(m,3H),4.07-3.41(m,12H),2.51-2.37(m,1H),2.35-2.26(m,1H),2.24-2.14(m,1H),2.02-1.94(m,1H)；HRMS (ESI) m/z calculated value: C₁₄H₂₄N₂O₄S₄[M+Na]⁺: 435.0511, measured value: 435.0518

Embodiment 17 bis- (4- (2- (dimethylamino formic acid ylmethyl) high morpholine) base thiocarbonyls) changes two magisters of sulfur

With embodiment 11, the difference is that: change the N- Benzvlmorpholin that 2 methylols replace into 2 methylols The substituted high morpholine of N- benzyl obtains 0.04 gram of yellow solid (compound 17), and yield is 25%.

¹H-NMR(400MHz,CDCl₃)δ5.28-5.18(m,1H),4.92-4.80(m,1H),4.78-4.67(m,1H),4.33-3.97(m,8H),3.96-3.72(m,3H),3.68-3.39(m,4H),2.92(s,12H),2.49-2.35(m,1H),2.35-2.23(m,1H),2.23-2.12(m,1H),2.12-1.98(m,1H)；HRMS (ESI) m/z calculated value: C₂₀H₃₄N₄O₆S₄[M+Na]⁺: 577.1253, measured value: 577.1262

Embodiment 18 bis- (4- (R-3- (ethoxyl methyl) morpholine) base thiocarbonyls) changes two magisters of sulfur

With embodiment 8, the difference is that: the N- Benzvlmorpholin (compound 18-1) that 3 methylols that the N- Benzvlmorpholin (compound 8-1) that 2 methylols replace changes R type into are replaced, 0.07 gram of yellow solid (compound 18) is obtained, yield is 35%

¹H-NMR(400MHz,CDCl₃) δ 5.80-4.41 (m, 4H), 4.15-4.09 (m, 2H), 4.00 (dd, J=12.0,4.0Hz, 2H), 3.93-3.30 (m, 14H), 1.21 (t, J=6.8Hz, 6H)；HRMS (EI) m/z calculated value: C₁₆H₂₈N₂O₄S₄[M]⁺, 440.0932, measured value: 440.0931

Embodiment 19 bis- (4- (R-3- (methoxy) morpholine) base thiocarbonyls) changes two magisters of sulfur

With embodiment 1, the difference is that: the N- Benzvlmorpholin (compound 19-1) that 3 chloromethyls that the N- Benzvlmorpholin (compound 1-1) that 2 chloromethyls replace changes R type into are replaced, 0.15 gram of yellow oil (compound 19) is obtained, yield is 39%

¹H-NMR(400MHz,CDCl₃) δ 5.80-4.48 (m, 4H), 4.17-4.06 (m, 2H), 4.00 (dd, J=12.0,4.0Hz, 2H), 3.90-3.47 (m, 10H), 3.43 (s, 6H)；HRMS (EI) m/z calculated value: C₁₄H₂₄N₂O₄S₄[M]⁺, 412.0619, measured value: 412.0621

Embodiment 20 bis- (4- (R-3- hydroxymethyl morpholine) base thiocarbonyls) changes two magisters of sulfur

With embodiment 10, the difference is that: the morpholine that 3 methylols that the morpholine that 2 methylols replace changes R type into are replaced obtains 0.07 gram of yellow oil (compound 20), and yield is 40%

¹H-NMR(400MHz,CDCl₃)δ5.91-4.50(m,4H),4.22-3.94(m,8H),3.80-3.73(m,2H),3.72-3.46(m,4H)；HRMS (ESI) m/z calculated value: C₁₂H₂₀N₂O₄S₄[M+Na]⁺: 407.0198, measured value: 407.0197

Embodiment 21 bis- (4- high morpholinothio carbonyls) changes two magisters of sulfur

By 1g high morpholine, 1.5g carbon disulfide is dissolved in 50mL tetrahydrofuran, and reaction 10 hours is stirred at room temperature.Then the aqueous solution of 20mL 2.5g ammonium persulfate is added into reaction system, is stirred at room temperature 10 hours.Organic layer be spin-dried for after with obtaining 1.4g compound 21, yield 82% after re-crystallizing in ethyl acetate.

¹H-NMR(400MHz,CDCl₃)δ4.42-4.25(m,7H),4.16-3.97(m,2H),3.93-3.70(m,7H),2.31-2.21(m,2H),2.18-2.06(m,2H)；HRMS (EI) m/z calculated value: C₁₂H₂₀N₂O₂S₄[M]⁺, 352.0408, measured value: 352.0405

Embodiment 22 bis- (4- (2- methyl) morpholinothio carbonyls) changes two magisters of sulfur

With embodiment 21, the difference is that: it changes high morpholine into 2 methyl substituted morpholines, obtains 0.2 g of compound 22, yield is 76%

¹H-NMR(400MHz,CDCl₃)δ5.63-4.65(m,4H),4.10-3.92(m,2H),3.88-3.65(m,4H),3.62-3.36(m,2H),3.32-2.94(m,2H),1.27(s,3H),1.26(s,3H)；HRMS (EI) m/z calculated value: C₁₂H₂₀N₂O₂S₄[M]⁺, 352.0408, measured value: 352.0405

Embodiment 23 bis- (4- (2- methyl) morpholinothio carbonyls) changes two magisters of sulfur

With embodiment 21, the difference is that: it changes high morpholine into benzo morpholine, obtains 0.08 g of compound 23, yield is 42%

¹H-NMR (400MHz, DMSO-d6) δ 8.05 (d, J=8.2Hz, 2H), 7.27 (t, J=8.0Hz, 2H), 7.16-7.04 (m, 4H), 4.28 (t, J=4.8Hz, 2H), 4.03 (t, J=4.8Hz, 2H)；HRMS (EI) m/z calculated value: C₁₈H₁₆N₂O₂S₄[M]⁺, 420.0095, measured value: 420.0093

Embodiment 24 bis- (4- (trans- -1,4 oxazines of octahydro-benzo) base thiocarbonyls) changes two magisters of sulfur

With embodiment 21, the difference is that: it changes high morpholine into trans--octahydro-benzo Isosorbide-5-Nitrae oxazines, obtains 0.03 g of compound 24, yield is 25%

¹H-NMR(400MHz,CDCl₃)δ5.27-5.16(m,2H),4.36-4.24(m,2H),4.23-4.15(m,2H),4.10-4.06(m,2H),4.00-3.89(m,2H),3.86-3.76(m,2H),2.88-2.72(m,2H),2.04-1.98(m,2H),1.88-1.78(m,4H),1.27-1.25(m,8H)；HRMS (EI) m/z calculated value: C₁₈H₂₈N₂O₂S₄[M]⁺, 432.1034, measured value: 432.1036

Embodiment 25 bis- (4- (2-C1-10 carboxyl) morpholinothio carbonyls) changes two magisters of sulfur

With embodiment 21, the difference is that: it changes high morpholine into 2-C1-10 carboxyl morpholine, obtains 0.19 g of compound 25, yield is 85%

¹H-NMR(400MHz,DMSO-d6)δ13.30(brs,2H),5.14-4.90(m,1H),4.79-4.17(m,5H),4.16-3.73(m,6H),3.73-3.63(m,2H)；HRMS (ESI) m/z calculated value: C₁₂H₁₆N₂O₆S₄[M+Na]⁺: 434.9783, measured value: 434.9784

Embodiment 26 bis- (4- (morpholine -2-carboxylate methyl ester) base thiocarbonyls) changes two magisters of sulfur

0.1g compound 25 is dissolved in 15mL methanol, the 0.1mL concentrated sulfuric acid is added in room temperature and is stirred overnight, is layered with 50mL water and 200mL ethyl acetate, organic phase with re-crystallizing in ethyl acetate obtains 0.07g compound 26, yield 65% after being spin-dried for.

¹H-NMR(400MHz,CDCl₃) δ 5.50-5.00 (m, 2H), 4.96-4.72 (m, 2H), 4.34 (dd, J=9.4,2.9Hz, 2H), 4.23-4.11 (m, 2H), 3.93-3.72 (m, 12H)；HRMS (EI) m/z calculated value: C₁₄H₂₀N₂O₆S₄[M]⁺, 440.0204, measured value: 440.0205

Embodiment 27 is bis- (2- ([1,3] thiazole simultaneously [5,4-B] pyridine)) change two magisters of sulfur

By 2- thyroidan [5,4-B] and pyridine 0.5g is dissolved in 50mL THF, and the aqueous solution 20mL of 1g ammonium persulfate is added in room temperature, is stirred overnight, organic layer is spin-dried for, and with re-crystallizing in ethyl acetate, obtains 0.3g compound 27, yield 60%.

¹H-NMR(400MHz,DMSO-d₆) δ 8.61 (d, J=4.6Hz, 2H), 8.39 (d, J=8.3Hz, 2H), 7.65-7.58 (m, 2H)；HRMS (EI) m/z calculated value: C₁₂H₆N₄S₄[M]⁺, 333.9475, measured value: 333.9476

Embodiment 28 bis- (2- cyclopenta thiazoles) changes two magisters of sulfur

1g alpha-chloro pentamethylene and 1.5g ammonium dithiocarbamate are dissolved in 100mL ethyl alcohol heated overnight at reflux, ethyl acetate and water stratification are used after being spin-dried for, organic phase is recrystallized to obtain 1.2g compound 28-2, it takes 1g compound 33-2 to be dissolved in 50mL THF, the aqueous solution 20mL of 1g ammonium persulfate is added in room temperature, is stirred overnight, organic layer is spin-dried for, with re-crystallizing in ethyl acetate, 0.7g compound 28, yield 70% are obtained.

¹H-NMR(400MHz,DMSO-d₆) δ 2.91 (t, J=7.2Hz, 4H), 2.77 (t, J=7.4Hz, 4H), 2.46-2.37 (m, 4H)；HRMS (EI) m/z calculated value: C₁₂H₁₂N₂S₄[M]⁺, 311.9883, measured value: 311.9884

Embodiment 29 bis- (2- cycloheptene and thiazoles) changes two magisters of sulfur

With embodiment 28, the difference is that: it changes alpha-chloro pentamethylene into alpha-brominated cycloheptane, obtains 0.6 g of compound 29, yield is 60%

¹H-NMR(400MHz,CDCl₃)δ2.98-2.89(m,4H),2.83-2.75(m,4H),1.89-1.81(m,4H),1.77-1.64(m,4H)；HRMS (EI) m/z calculated value: C₁₆H₂₀N₂S₄[M]⁺, 368.0509, measured value: 368.0510

Embodiment 30 bis- (2- (6,6- dimethylcyclohexenes and thiazoles)) changes two magisters of sulfur

With embodiment 28, the difference is that: it changes alpha-chloro pentamethylene into 2- bromo- 4,4- dimethylcyclohexanon, obtains 0.8 g of compound 30, yield is 80%

¹H-NMR(400MHz,CDCl₃)δ2.83-2.75(m,4H),2.53(s,4H),1.67-1.58(m,4H),1.03(s,12H)；HRMS (EI) m/z calculated value: C₁₈H₂₄N₂S₄[M]⁺, 396.0822, measured value: 396.0829

Embodiment 31 bis- (2- (6- methylcyclohexene and thiazoles)) changes two magisters of sulfur

With embodiment 28, the difference is that: it changes alpha-chloro pentamethylene into 2- bromo- 4- methyl cyclohexanone, obtains 0.8 g of compound 31, yield is 80%

¹H-NMR(400MHz,CDCl₃)δ2.93-2.79(m,4H),2.79-2.68(m,2H),2.40-2.29(m,2H),1.94-1.79(m,4H),1.56-1.43(m,2H),1.10(s,3H),1.08(s,3H)；HRMS (EI) m/z calculated value: C₁₆H₂₀N₂S₄[M]⁺, 368.0509, measured value: 368.0508

Embodiment 32 is bis- (2- (- 4 hydrogen of 6,7- dihydro-pyrans simultaneously [4,3-D] thiazole)) change two magisters of sulfur

With embodiment 28, the difference is that: it changes alpha-chloro pentamethylene into 3- bromo- tetrahydro pyrone, obtains 0.6 g of compound 32, yield is 60%

¹H-NMR(400MHz,DMSO-d₆)δ4.75(s,4H),3.96-3.88(m,4H),2.83-2.75(m,4H)；HRMS (EI) m/z calculated value: C₁₂H₁₂N₂O₂S₄[M]⁺, 343.9782, measured value: 343.9781

Embodiment 33 bis- (2- (cyclohexene and thiazole -6- Ethyl formates)) changes two magisters of sulfur

With embodiment 28, the difference is that: alpha-chloro pentamethylene is changed into cyclohexanone Ethyl formate, 0.67 g of compound 33 is obtained, yield is 67%

¹H-NMR(400MHz,CDCl₃) δ 4.18 (q, J=7.0Hz, 4H), 3.08-2.99 (m, 4H), 2.99-2.87 (m, 2H), 2.87-2.74 (m, 4H), 2.32-2.21 (m, 2H), 2.07-1.90 (m, 2H), (1.28 t, J=7.1Hz, 6H)；HRMS (ESI) m/z calculated value: C₂₀H₂₄N₂O₄S₄[M+H]⁺: 485.0692, measured value: 485.0700

Embodiment 34 bis- (2- (cyclohexene and thiazole -6- formic acid)) changes two magisters of sulfur

It hydrolyzes the intermediate of embodiment 33 to obtain intermediate 34-1 and is aoxidized to obtain 0.72 g of compound 34 with same method, yield is 72%

¹H-NMR(400MHz,CDCl₃)δ12.48(brs,2H),3.07-2.97(m,2H),2.95-2.83(m,2H),2.83-2.68(m,6H),2.19-2.06(m,2H),1.93-1.79(m,2H)；HRMS (EI) m/z calculated value: C₁₆H₁₆N₂O₄S₄[M]⁺, 427.9993, measured value: 427.9991

Biological assessment

Influence of 35 compound of embodiment (1-34) to pyruvic dehydrogenase kinase (PDHK1) kinase activity

1, experimental method

(1) immobilized substrate: substrate PDHE1 (0.5 μ g/well) dilutes (Na with carbonate buffer solution₂CO₃-NaHCO₃, pH 9.6) and (100 μ L/well) is fixed, set 37 DEG C of shaking tables, 110rpm, fixed 3h.Then, three times without potassium PBST washing, each 5min is patted dry.

(2) enzymatic reaction: 100 μ L HEPES reaction systems: 50mM HEPES (pH 7.5), 10mM MgCl₂, the 1mM μ g enzyme of EGTA, 10 μM of ATP, 0.1；Set 37 DEG C of shaking tables, 110rpm, 1h reaction.Three times, each 5min is patted dry for no potassium PBST washing.

(3) primary antibody is incubated for: phosphorylation PDHE1 (S293) antibody is diluted with PBST, and 100 μ L antibody diluents are added in dilution ratio 1:400, every hole, sets 37 DEG C of shaking tables, and 110rpm is incubated for 1h.Then PBST washing three times, pats dry.

(4) secondary antibody is incubated for: anti-rabbit HRP secondary antibody is diluted with PBST with 1:2000, and 100 μ L antibody diluents are added in every hole, sets 37 DEG C of shaking tables, and 110rpm is incubated for 1h.Then TBST washing three times, pats dry.

(5) it develops the color: the 100 μ l/well of OPD developing solution of 2mg/ml is added (with containing 0.03%H₂O₂0.1M citric acid-sodium citrate buffer solution (pH 5.4) dilution).

(6) 2M H is added₂SO₄50 μ l/well stopped reactions are declined orifice plate microplate reader with wavelengthtunable SPECTRA MAX 190 is read, wavelength 490nm.

(7) interpretation of result

IC50 value uses the random bundled software of microplate reader to acquire with the recurrence of four parametric methods.

2, experimental result

Compound (1-34) is measured the inhibition situation of pyruvic dehydrogenase kinase (PDHK1) by experiment above, the IC measured₅₀Value is shown in Table 1.

The IC of other members of compound 19 and 21 pair pyruvic dehydrogenase kinase family inhibition PDHK2-4 kinase activity₅₀Value is shown in Table 2.

1. compound of table (1-34) inhibits the active IC of PDHK1₅₀Value

2. compound of table (19,21) inhibits the active IC in PDHK2,3,4₅₀Value

The results show that compound (1-34) has certain inhibiting effect to PDHK1 kinases, wherein 1,8,9,18,19,21,22,26,29,31, No. 33 compound activity of compound is preferable, respectively less than 120nM.Compound (19,21) also has inhibiting effect to PDHK2 and PDHK3, but acts on (> 10 μM) to PDHK4 unrestraint.

The influence that 36 compound of embodiment is proliferated kinds of tumor cells

1, experimental method

Tumour cell in logarithmic growth phase is seeded to 96 well culture plates by proper density, and the compound of 10 μ L various concentrations is added after cell is adherent in every 100 μ L system of hole, and each concentration sets three multiple holes.After compound effects 72h, cell training liquid is discarded, 10% (w/v) trichloroacetic acid (100 hole μ L/) is added in 4 DEG C of fixed 1h, then uses distilled water flushing five times.Reverse side is placed into baking oven dry, takes out cooling, and 100 μ LSRB solution (4mg/mL SRB powder is dissolved in 1% glacial acetic acid) is added in every hole, after placing 15min at room temperature, with 1% glacial acetic acid rinse five times with sufficiently remove not with the protein bound SRB of board bottom.Reverse side places into baking oven dry, taking-up cooling, and every hole addition 150 μ L of 10mM Tris solution measures the OD value under 560nm wavelength with microplate reader.The inhibiting rate of compound on tumor cell growth is calculated as follows:

IC₅₀Value uses the random bundled software of microplate reader to acquire with the recurrence of four parametric methods.

2, experimental result

Compound (19,21) the results are shown in Table 3 to the Proliferation Ability of lung cell A549 and human neuroblastoma Kelly.The results show that compound 19,21 has preferable inhibited proliferation.

3. compound of table (19,21) is to A549 cell and Kelly cell inhibitory effect IC₅₀

Influence of 37 compound 1,8,9,18,19,21,22,26 of embodiment in A549 tumour cell to PDHK inhibiting effect

1, experimental method

By cell inoculation in six orifice plates, after processing, degrees of fusion reaches 80% or so, collects cell, is washed twice with cold PBS, 1 × sds gel sample-loading buffer lytic cell 5min is added.Cell pyrolysis liquid heats after ten minutes in boiling water bath, is centrifuged 10 minutes in 4 DEG C of 12000rpm, takes supernatant, abandons precipitating.

Protein sample is placed in various concentration SDS- polyacrylamide gel, after being separated in Tris- glycine-SDS electrophoretic buffer with 80V electrophoresis to albumen Marker, is separated with 120V electrophoresis about 2h.Albumen is transferred to nitrocellulose filter from gel with half-dried blotting or wet robin, shifts 1-2.5h by required molecular weight of albumen size.It is dyed with Ponceaux dyeing liquor and determines albumen transfer situation and protein band position, corresponding purpose band is cut according to albumen Marker molecular weight, then 1h is closed with confining liquid (TBST containing 3%BSA) room temperature, be incubated overnight for 4 DEG C with corresponding antibody (being diluted with the TBST of 3%BSA).With TBST cleaning solution room temperature washing 3 times, each 10min.The secondary antibody (TBST of 1:2000 3%BSA dilutes) with horseradish peroxidase-labeled is added, is incubated at room temperature 1h.Then it is washed three times with TBST, each 10min.After selecting suitable luminescence reagent to be incubated for according to exposure intensity, chromogenic assay is carried out with full automatic gel Image analysis system.

2, experimental result

Cellular level Enzyme activity assay is carried out to the preferable Formula X compound of molecular level enzyme activity.

Fig. 1 shows influence of the compound in 1 μM and 0.3 μM to A549 cell PDHK protein phosphorylation, with JX06 and JX25For positive control.

The results show that in tumour cell (A549), compound effects for 24 hours after, under 1 μM of concentration, other than compound 8, remaining compound has good inhibiting effect to the phosphorylation in the site PDHA1S293 and S232.When concentration is adjusted to 0.3 μM, compound 19,21 still has preferable inhibition, especially best with 21 effect of compound.

The influence of 38 compound of embodiment, 19,21 couples of A549 cell activity oxygen ROS

1, experimental method

Cell in logarithmic growth phase is seeded to 6 well culture plates by proper density, and every hole 2ml, the compound that various concentration is added after adherent or DMSO effect are for 24 hours.Cell culture fluid is sucked, washs attached cell 2 with the PBS of pre-cooling It is secondary, after the PBS that exhausts, 0.25%Trypsin-EDTA vitellophag is added, the training liquid containing serum is added and terminates, cell is transferred in 1.5ml EP pipe, 4 DEG C, 300g is centrifuged 5min, abandons supernatant.DCFH-DA (green skies biotechnology research institute) is diluted according to 1:1000 serum-free medium, makes final concentration of 10 μM.DCFH-DA itself can pass freely through cell membrane without fluorescence, and into after cell, by intracellular esterase hydrolyzed at DCFH, and DCFH cannot pass freely through cell membrane, to stay in intracellular.Intracellular active oxygen can aoxidize non-blooming DCFH and become the DCF for having fluorescence.The level of DCF is detected it is known that intracellular reactive oxygen species.Cell precipitation is suspended again with DCFH-DA dilution, piping and druming uniformly, is incubated for 30 minutes in 37 DEG C of cell incubators.It is mixed by inversion once every 10min, comes into full contact with probe and cell.Cell is washed twice with serum-free cell culture medium, sufficiently to remove the not DCFH-DA by loading cells.It is tested and is analyzed with flow cytometer.

2, experimental result

As a result as shown in Fig. 2, compound 21 can dramatically increase A549 intracellular reactive oxygen level, and have concentration dependent under 1,3,10 μM of concentration.

Compound 19 can increase intracellular reactive oxygen level at 5-10 μM.

The influence of the extracellular lactic acid release of 19,21 pairs of 39 compound of embodiment

1, experimental method

Cell in logarithmic growth phase is seeded to 96 tissue culture plate (degrees of fusion 80%-90%) of Seahorse XF by proper density, every 100 μ L system of hole, after cell is adherent, addition handles (compound) effect certain time (within for 24 hours), and every kind of effect sets three multiple holes or more.200 hole μ L/ XF Calibrant Solution will be added by mentioning the previous day in disposable solid state sensor box, be put into no CO₂, in 37 DEG C of pre-operation station, aquation overnight.

96 host of Seahorse XF and program will open in advance, overnight with systems stabilisation (temperature humidity etc.).(that due to detection is exactly O to configuration metabolism detection liquid₂And H⁺Concentration variation, so detection liquid is no buffer system, detection liquid ingredient is XF Base Medium Minimal DMEM+25mM glucose+2mM pyruvic acid).After to be processed, Kong Zhongpei liquid is carefully sucked, washed once with metabolism detection liquid, the metabolism detection liquid that 200 μ L are added (sufficiently to remove the training liquid that the first step does not exhaust) is discarded.96 tissue culture plate of Seahorse XF for finishing changing metabolism detection liquid is put into no CO₂, 1h in 37 DEG C of pre-operation station, with balance detection system.Editor's detection program, and the disposable solid state sensor box that overnight aquation is completed is put into host, carries out system compensation.After the completion of correction, 96 tissue culture plate of Seahorse XF is put into host and is detected by preset program.

2, experimental result

As a result as shown in figure 3, compound 19 and compound 21 can reduce the extracellular lactic acid emission levels of A549 under 1,3,10 μM of concentration, and has concentration dependent.

All references mentioned in the present invention is incorporated herein by reference, as if each reference was individually incorporated by reference.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can make various modifications or changes to the present invention, these equivalent forms also fall within the scope of the appended claims of the present application.

Claims

A kind of Formula X compound R_a-S-S-R_b(X) purposes, the purposes include:

(a) preparation inhibits pyruvic dehydrogenase kinase (PDHK) active drug or preparation, and/or,

(b) preparation inhibits and/or treats the drug or preparation of tumour cell；

Wherein, R_a、R_bIt is each independently with S hetero atom and the heteroatomic end group group of N, and in the end group group, S hetero atom and N hetero atom constitute " S=C-N " or " S-C=N ", and in the end group group, when using-S-S- as center, the N hetero atom is located at the heteroatomic outside the S, and each S hetero atom and one, the interval-S-S- carbon atom.
Purposes as described in claim 1, which is characterized in that the tumour is the tumour of pyruvic dehydrogenase kinase (PDHK) positive.
Purposes as described in claim 1, which is characterized in that the Formula X compound is -1 compound of Formula X,

In formula,

Each R_cIt is separately selected from the group: nothing, substituted or unsubstituted C_1-6Alkyl；

Each R_dIt is separately selected from the group: nothing, substituted or unsubstituted C_1-6Alkyl, substituted or unsubstituted C_6-10Aryl, substituted or unsubstituted C_6-10Heteroaryl, substituted or unsubstituted C_6-10Naphthenic base ,-(CH₂)_m-C_6-10Aryl, m 0-4；

Each R_eIt is separately selected from the group: nothing, substituted or unsubstituted C_1-6Alkyl；

Also, R_c、R_d、R_eMeet following condition:

(1)R_c、R_dRing C is collectively formed with adjacent N, C and S atom, and connect with the S hetero atomIt is connect for singly-bound with N hetero atomFor double bond；Or

(2)R_e、R_dThe 5-6 member ring E that substituted or unsubstituted 7-12 member ring D is collectively formed with adjacent N or replaces, and connect with the S hetero atomIt is connect for double bond with N hetero atomFor singly-bound；

The heterocycle has 1-3 hetero atoms selected from the group below: O, S and N；

The substitution, which refers to, has one or more substituent groups selected from the group below: halogen, C_1-10Alkyl, halogenated C_1-10Alkyl, C_1-10Alkoxy, halogenated C_1-10Alkoxy, C_1-4Alkyl hydroxy, acetyl group, C_1-10Amide groups, C_1-10Carboxyl, C_5-20Aryl, halogenated C_5-20Aryl ,-(L₁)p-Z；

Wherein, each L₁It independently is :-(CH₂) r ,-O- ,-C=O ,-CH₂-(C_1-4Alkyl)-；

P is 0-4, r 0-4；

Z is selected from the group: C_6-20Aryl, C_1-11Alkyl, C_1-10Amide groups, hydroxyl.
A kind of Formula X compound, its optical isomer or its pharmaceutically acceptable salt,

R_a-S-S-R_b (X)

Wherein, R_a、R_bIt is each independently with S hetero atom and the heteroatomic end group group of N, and in the end group group, S hetero atom and N hetero atom constitute " S=C-N " or " S-C=N ", and in the end group group, when using-S-S- as center, the N hetero atom is located at the heteroatomic outside the S, and each S hetero atom and one, the interval-S-S- carbon atom.
Compound, its optical isomer or its pharmaceutically acceptable salt as claimed in claim 4, which is characterized in that the Formula X compound is -1 compound of Formula X,

In formula,

Each R_cIt is separately selected from the group: nothing, substituted or unsubstituted C_1-6Alkyl；

Each R_dIt is separately selected from the group: nothing, substituted or unsubstituted C_1-6Alkyl, substituted or unsubstituted C_6-10Aryl, substituted or unsubstituted C_6-10Heteroaryl, substituted or unsubstituted C_6-10Naphthenic base ,-(CH₂)_m-C_6-10Aryl, m 0-4；

Each R_eIt is separately selected from the group: nothing, substituted or unsubstituted C_1-6Alkyl；

Also, R_c、R_d、R_eMeet following condition:

(1)R_c、R_dRing C is collectively formed with adjacent N, C and S atom, and connect with the S hetero atomIt is connect for singly-bound with N hetero atomFor double bond；Or

(2)R_e、R_dThe 5-6 member ring E that substituted or unsubstituted 7-12 member ring D is collectively formed with adjacent N or replaces, and connect with the S hetero atomIt is connect for double bond with N hetero atomFor singly-bound；

The heterocycle has 1-3 hetero atoms selected from the group below: O, S and N；

The substitution, which refers to, has one or more substituent groups selected from the group below: halogen, C_1-10Alkyl, halogenated C_1-10Alkyl, C_1-10Alkoxy, halogenated C_1-10Alkoxy, C_1-4Alkyl hydroxy, acetyl group, C_1-10Amide groups, C_1-10Carboxyl, C_5-20Aryl, halogenated C_5-20Aryl ,-(L₁)p-Z；

Wherein, each L₁It independently is :-(CH₂) r ,-O- ,-C=O ,-CH₂-(C_1-4Alkyl)-；

P is 0-4, r 0-4；

Z is selected from the group: C_6-20Aryl, C_1-11Alkyl, C_1-10Amide groups, hydroxyl.
Compound, its optical isomer or its pharmaceutically acceptable salt as claimed in claim 4, which is characterized in that the substituent group is-(L₁) p-Z, wherein L₁, the definition of p, Z it is as claimed in claim 5.
Compound, its optical isomer or its pharmaceutically acceptable salt as claimed in claim 4, which is characterized in that R_a、R_bRespectively identical or differently are as follows:

N is 1-3；

R₁And R₂It is each independently selected from the following group: hydrogen, hydroxyl, substituted or unsubstituted C_1-10Carboxyl, substituted or unsubstituted C_1-6Ester group, substituted or unsubstituted methylol, substituted or unsubstituted C_1-4Alkyl, substituted or unsubstituted C_1-10Alkoxy, and R₁And R₂It is not simultaneously hydrogen；

Alternatively, R₁、R₂Saturated or unsaturated 5-8 member aromatic ring or carbocyclic ring is collectively formed.
Compound, its optical isomer or its pharmaceutically acceptable salt as claimed in claim 4, which is characterized in that the Formula X compound is compound of formula I:

In formula, n, R₁、R₂Definition it is as claimed in claim 7.
Compound, its optical isomer or its pharmaceutically acceptable salt as claimed in claim 4, which is characterized in that the Formula X compound is Formula II compound

In formula,

Ring A is selected from: substituted or unsubstituted C_5-10Carbocyclic ring, substituted or unsubstituted C_5-10Carbon heterocyclic, substituted or unsubstituted C_6-10Aromatic ring, substituted or unsubstituted C_6-10Hetero-aromatic ring.
Compound, its optical isomer or its pharmaceutically acceptable salt as claimed in claim 4, which is characterized in that the Formula X compound is selected from the group:
Compound, its optical isomer or its pharmaceutically acceptable salt as claimed in claim 4, which is characterized in that the Formula X compound is selected from the group: compound 1,8,9,18,19,21,22,25,26,29 and 33.
A kind of pharmaceutical composition contains in described pharmaceutical composition

(i) claim 4-11 any the Formula X compound or its pharmaceutically acceptable salt；

(ii) pharmaceutically acceptable carrier.
A kind of the method for preparing compound of formula I I, which is characterized in that comprising steps of

(I-2) in atent solvent, formula B compound is reacted with reagent D, to form compound of formula I, wherein the reagent D is selected from the group: ammonium persulfate, sodium metaperiodate, sodium nitrite, hydrochloric acid, hydrogen peroxide, sulfuric acid, or combinations thereof；

(I-3) isolated or purified optionally is carried out to the compound of formula I formed in step (I-2), so that separation or purifying compound of formula I is obtained,

In formula, n 1-3；

R₁And R₂It is each independently selected from the following group: hydrogen, hydroxyl, substituted or unsubstituted C_1-10Carboxyl, substituted or unsubstituted C_1-6Ester group, substituted or unsubstituted methylol, substituted or unsubstituted C_1-4Alkyl, substituted or unsubstituted C_1-10Alkoxy, and R₁And R₂It is not simultaneously hydrogen；

Alternatively, R₁、R₂Saturated or unsaturated 5-8 member aromatic ring or carbocyclic ring is collectively formed.
Method as claimed in claim 13, which is characterized in that the method I is further comprised the steps of:

(I-1) in organic solvent, formula A compound is reacted with carbon disulfide, so that formula B compound be made, wherein n, R₁、R₂Definition it is as claimed in claim 13.
A kind of method II of preparation formula II compound, which is characterized in that comprising steps of

(II-1) in organic solvent, formula C compound is reacted with reagent D, shape compound of formula II, wherein the reagent D is selected from the group: ammonium persulfate, sodium metaperiodate, sodium nitrite, hydrochloric acid, hydrogen peroxide, sulfuric acid, or combinations thereof, in formula,

Ring A is selected from: substituted or unsubstituted C_5-10Carbocyclic ring, substituted or unsubstituted C_5-10Carbon heterocyclic, substituted or unsubstituted C_6-10Aromatic ring, substituted or unsubstituted C_6-10Hetero-aromatic ring.
Inhibit the active method of pyruvic dehydrogenase kinase to a kind of non-therapeutic, comprising steps of

(a) pyruvic dehydrogenase kinase is contacted with claim 4-11 any described Formula X compound, its optical isomer, non-raceme, racemic modification or its pharmaceutically acceptable salt, to inhibit the activity of pyruvic dehydrogenase kinase.