WO2017161524A1

WO2017161524A1 - Pyruvate dehydrogenase kinase inhibitor and application therefor

Info

Publication number: WO2017161524A1
Application number: PCT/CN2016/077137
Authority: WO
Inventors: 李剑; 黄敏; 耿美玉; 刘毅夫; 唐帅; 兰小晶; 朱进
Original assignee: 华东理工大学; 中国科学院上海药物研究所
Priority date: 2016-03-23
Filing date: 2016-03-23
Publication date: 2017-09-28
Also published as: CN107922366A

Abstract

Provided is a pyruvate dehydrogenase kinase inhibitor and an application therefor. Specifically provided is a compound R_a-S-S-R_b of formula X or a pharmaceutically acceptable salt thereof, the compound having excellent inhibition of pyruvate dehydrogenase kinase activity and anti-tumour effects. Also provided is a pharmaceutical composition containing said compound and applications for inhibiting pyruvate dehydrogenase kinase activity and resisting tumours.

Description

丙酮酸脱氢酶激酶抑制剂及其应用Pyruvate dehydrogenase kinase inhibitor and its application

技术领域Technical field

本发明属于药物化学和药物治疗学领域，具体地，本发明涉及式X化合物和其在抑制丙酮酸脱氢酶激酶方面的应用。The present invention is in the field of medicinal chemistry and pharmacotherapeutics, and in particular, the present invention relates to compounds of formula X and their use in inhibiting pyruvate dehydrogenase kinase.

背景技术Background technique

癌症是最严重的人类疾病之一。据世界健康组织(WHO)统计，仅2008年就有760万人死于癌症，预计到2030年这一数字将增加至1320万。由此会带来巨大的医疗压力，并摧毁更多人的经济状况。因此，抗癌药物的研究一直是药物研发工作中的重中之重。Cancer is one of the most serious human diseases. According to the World Health Organization (WHO), 7.6 million people died of cancer in 2008 alone, and this number is expected to increase to 13.2 million by 2030. This will bring huge medical pressure and destroy the economic situation of more people. Therefore, research on anticancer drugs has always been a top priority in drug research and development.

丙酮酸脱氢酶激酶(PDHK)是一种线粒体酶，能够调节丙酮酸脱氢酶复合物(PDC)的活性而后者催化了丙酮酸向乙酰辅酶A的转化，证据表明丙酮酸脱氢酶激酶可以通过磷酸化使PDC失活。PDHK有四个亚型：PDHK1-4，其中PDHK1与癌症的恶性程度紧密相关。PDHK1在多种癌症如肺癌，头颈鳞状上皮癌中都是激活的。PDHK1为原癌基因(C-MYC)和低氧诱导因子(hypoxia inducing factor，HIF)等致癌基因转录调控，以此控制癌细胞的代谢和恶性表型。已有报道称,抑制PDHK可以有效地提高肿瘤细胞化疗的效果,并且这具有高度的肿瘤特异性。Pyruvate dehydrogenase kinase (PDHK) is a mitochondrial enzyme that regulates the activity of pyruvate dehydrogenase complex (PDC), which catalyzes the conversion of pyruvate to acetyl-CoA. Evidence suggests pyruvate dehydrogenase kinase PDC can be inactivated by phosphorylation. PDHK has four subtypes: PDHK1-4, of which PDHK1 is closely related to the degree of malignancy of cancer. PDHK1 is activated in a variety of cancers such as lung cancer, head and neck squamous cell carcinoma. PDHK1 is a transcriptional regulation of oncogenes such as protooncogene (C-MYC) and hypoxia inducing factor (HIF), thereby controlling the metabolism and malignant phenotype of cancer cells. It has been reported that inhibition of PDHK can effectively improve the effect of tumor cell chemotherapy, and this has a high degree of tumor specificity.

JX06

是2015年报道的一种不可逆抑制剂，目前JX06尚未进入临床应用，基于对与ATP结合口袋毗邻一个疏水口袋的识别，JX06能够与酶结构中保守的半胱氨酸残基(C240)上的硫醇共价结合形成二硫键，通过范德华力引起R286位置的构象变换，从而阻碍ATP与酶的结合进而对PDHK产生抑制。因此，本领域迫切需要研究出新的PDHK抑制剂。JX06

Is an irreversible inhibitor reported in 2015, JX06 has not yet entered clinical application, based on the recognition of a hydrophobic pocket adjacent to the ATP binding pocket, JX06 is able to interact with a cysteine residue (C240) conserved in the enzyme structure. The thiol covalently binds to form a disulfide bond, which causes a conformational shift at the position of R286 by van der Waals force, thereby hindering the binding of ATP to the enzyme and inhibiting PDHK. Therefore, there is an urgent need in the art to develop new PDHK inhibitors.

发明内容Summary of the invention

本发明的目的在于提供一种抑制丙酮酸脱氢酶激酶活性的化合物及其应用。It is an object of the present invention to provide a compound which inhibits pyruvate dehydrogenase kinase activity and uses thereof.

本发明的第一方面，提供了一种式X化合物R_a-S-S-R_b(X)的用途，所述用途包括：In a first aspect of the invention there is provided a use of a compound of formula X R _a -SSR _b (X), said use comprising:

(a)制备抑制丙酮酸脱氢酶激酶(PDHK)活性的药物或制剂，和/或， (a) preparing a medicament or preparation that inhibits pyruvate dehydrogenase kinase (PDHK) activity, and/or,

(b)制备抑制和/或***细胞的药物或制剂；(b) preparing a medicament or preparation for inhibiting and/or treating tumor cells;

其中，R_a、R_b各自独立地为具有S杂原子和N杂原子的端基团，并且所述端基团中，S杂原子和N杂原子构成“S＝C-N”或“S-C＝N”，并且所述的端基团中，当以-S-S-为中心时，所述N杂原子位于所述S杂原子的外侧，并且所述的各S杂原子与所述-S-S-间隔一个碳原子。Wherein R _a and R _b are each independently an end group having an S hetero atom and an N hetero atom, and in the terminal group, the S hetero atom and the N hetero atom constitute "S=CN" or "SC=N" And said end group, when centered on -SS-, said N hetero atom is located outside said S hetero atom, and said each S hetero atom is separated from said -SS- carbon atom.

在另一优选例中，所述的肿瘤为丙酮酸脱氢酶激酶(PDHK)阳性的肿瘤。In another preferred embodiment, the tumor is a pyruvate dehydrogenase kinase (PDHK) positive tumor.

在另一优选例中，所述的肿瘤选自下组：肺癌、神经母细胞癌、肝癌、结肠癌、***癌、乳腺癌、肾癌。In another preferred embodiment, the tumor is selected from the group consisting of lung cancer, neuroblastoma, liver cancer, colon cancer, prostate cancer, breast cancer, and kidney cancer.

在另一优选例中，所述的式X化合物为式X-1化合物，In another preferred embodiment, the compound of formula X is a compound of formula X-1,

式中，In the formula,

各R_c分别独立地选自下组：无、取代或未取代的C_1-6烷基；Each R _{c is} independently selected from the group consisting of: unsubstituted, substituted or unsubstituted C _1-6 alkyl;

各R_d分别独立地选自下组：无、取代或未取代的C_1-6烷基、取代或未取代的C_6-10芳基、取代或未取代的C_6-10杂芳基、取代或未取代的C_6-10环烷基、-(CH₂)_m-C_6-10芳基，m为0-4；Each R _{d is} independently selected from the group consisting of unsubstituted, substituted or unsubstituted C _1-6 alkyl, substituted or unsubstituted C _6-10 aryl, substituted or unsubstituted C _6-10 heteroaryl, a substituted or unsubstituted C _6-10 cycloalkyl, -(CH ₂ ) _m -C _6-10 aryl, m is 0-4;

各R_e分别独立地选自下组：无、取代或未取代的C_1-6烷基；Each R _{e is} independently selected from the group consisting of: unsubstituted, substituted or unsubstituted C _1-6 alkyl;

并且，R_c、R_d、R_e满足下列条件：Moreover, R _c , R _d , and R _e satisfy the following conditions:

(1)R_c、R_d与相邻的的N、C和S原子共同形成环C，并且与所述S杂原子连接的

为单键而与N杂原子连接的

为双键；或者(1) R _c , R _d together with adjacent N, C and S atoms form a ring C and are bonded to the S hetero atom

Connected to an N hetero atom for a single bond

For double keys; or

(2)R_e、R_d与相邻的N共同形成取代或未取代的7-12元环D、或取代的5-6元环E，并且与所述S杂原子连接的

为双键而与N杂原子连接的

为单键；(2) R _e , R _d together with an adjacent N form a substituted or unsubstituted 7-12 membered ring D, or a substituted 5-6 membered ring E, and is bonded to the S hetero atom

Connected to an N hetero atom for a double bond

For a single button;

所述杂环具有1-3个选自下组的杂原子：O、S、和N；The heterocyclic ring has 1-3 heteroatoms selected from the group consisting of O, S, and N;

所述取代是指具有一个或多个选自下组的取代基：卤素、C₁-₁₀烷基、卤代C₁-₁₀烷基、C₁-₁₀烷氧基、卤代C₁-₁₀烷氧基、C_1-4烷基羟基、乙酰基、C_1-10酰胺基、C_1-10羧基、C_5-20芳基、卤代C_5-20芳基、-(L₁)p-Z；The substitution means having one or more substituents selected from the group consisting of halogen, C ₁ _-10 alkyl, halogenated C ₁ _-10 alkyl, C ₁ _-10 alkoxy, halogenated C ₁ - ₁₀ Alkoxy, C _1-4 alkylhydroxy, acetyl, C _1-10 amide, C _1-10 carboxyl, C _5-20 aryl, halogenated C _5-20 aryl, -(L ₁ )pZ ;

其中，所述各L₁独立地为：-(CH₂)r、-O-、-C＝O、-CH₂-(C_1-4烷基)-；Wherein each of L _{1 is} independently: -(CH ₂ )r, -O-, -C=O, -CH ₂ -(C _1-4 alkyl)-;

p为0-4，r为0-4；p is 0-4, r is 0-4;

Z选自下组：C_6-20芳基、C₁-₁₁烷基、C_1-10的酰胺基、羟基。 Z is selected from the group consisting of C _6-20 aryl, C ₁ _-11 alkyl, C _1-10 amide, and hydroxy.

在另一优选例中，所述式X化合物为式I化合物：In another preferred embodiment, the compound of formula X is a compound of formula I:

式中，n为1-3；Where n is 1-3;

R₁和R₂各自独立地选自下组：氢、羟基、取代或未取代的C_1-10羧基、取代或未取代的C₁-₆酯基、取代或未取代的羟甲基、取代或未取代的C₁-₄烷基、取代或未取代的C₁-₁₀烷氧基，且R₁和R₂不同时为氢；R ₁ and R ₂ are each independently selected from the group consisting of: hydrogen, hydroxyl, a substituted or unsubstituted C _1-10 carboxyl group, a substituted or unsubstituted C ₁ - ₆ ester group, a substituted or unsubstituted hydroxymethyl, substituted unsubstituted C ₁ - ₄ alkyl group, a substituted or unsubstituted C ₁ - ₁₀ alkoxy, and R ₁ and R ₂ are not simultaneously hydrogen;

或者，R₁、R₂共同形成饱和或不饱和的5-8元芳环或碳环。Alternatively, R ₁ and R ₂ together form a saturated or unsaturated 5-8 membered aromatic or carbocyclic ring.

在另一优选例中，所述式X化合物为式II化合物In another preferred embodiment, the compound of formula X is a compound of formula II

式中，In the formula,

环A选自：取代或未取代的C_5-10碳环、取代或未取代的C_5-10碳杂环、取代或未取代的C₆-₁₀芳环、取代或未取代的C₆-₁₀杂芳环。Ring A is selected from: a substituted or unsubstituted C _5-10 carbon ring, a substituted or unsubstituted C _5-10 heterocycle carbons, a substituted or unsubstituted C ₆ - ₁₀ aromatic ring, a substituted or unsubstituted C ₆ - ₁₀ heteroaryl rings.

在另一优选例中，环A为取代或未取代的C₆-₁₀杂芳环，较佳地，在所成吡啶的2,3位并环。In another preferred embodiment, Ring A is a substituted or unsubstituted C ₆ _-10 heteroaryl ring, preferably a ring at the 2,3 position of the pyridine formed.

在另一优选例中，所述化合物包括本发明第二方面所述的全部化合物。In another preferred embodiment, the compound includes all of the compounds described in the second aspect of the invention.

本发明的第二方面，提供一种式X化合物、其光学异构体或其药学上可接受的盐，In a second aspect of the invention, there is provided a compound of formula X, an optical isomer thereof, or a pharmaceutically acceptable salt thereof,

R_a-S-S-R_b (X)R _a -SSR _b (X)

在另一优选例中，所述的式X化合物为式X-1化合物， In another preferred embodiment, the compound of formula X is a compound of formula X-1,

式中，In the formula,

为单键而与N杂原子连接的

Connected to an N hetero atom for a single bond

For double keys; or

为双键而与N杂原子连接的

Connected to an N hetero atom for a double bond

For a single button;

p为0-4，r为0-4；p is 0-4, r is 0-4;

Z选自下组：C_6-20芳基、C₁-₁₁烷基、C_1-10的酰胺基、羟基。Z is selected from the group consisting of C _6-20 aryl, C ₁ _-11 alkyl, C _1-10 amide, and hydroxy.

在另一优选例中，所述环C为二环，较佳地，环C选自下组：5-10元碳杂环并5-10元碳环、5-10元碳杂环并5-10元碳杂环、5-10元碳杂环并5-10元芳环、5-10元碳杂环并5-10元芳杂环。In another preferred embodiment, the ring C is a bicyclic ring, preferably, the ring C is selected from the group consisting of a 5-10 membered heterocyclic ring and a 5-10 membered carbocyclic ring, and a 5-10 membered carbocyclic ring. a 10-membered carbon heterocycle, a 5-10 membered heterocyclic ring and a 5-10 membered aromatic ring, a 5-10 membered heterocyclic ring and a 5-10 membered aromatic heterocyclic ring.

在另一优选例中，所述环D为二环，较佳地为5-7元环并5-7元环，更佳地环D选自下组：六元环并六元环，更佳地为六元杂环并苯环、六元杂环并六元碳环、萘基。In another preferred embodiment, the ring D is a bicyclic ring, preferably a 5-7 membered ring and a 5-7 membered ring, and more preferably the ring D is selected from the group consisting of a six-membered ring and a six-membered ring. Preferably, it is a six-membered heterocyclic acene ring, a six-membered heterocyclic ring and a six-membered carbocyclic ring, and a naphthyl group.

在另一优选例中，所述取代基为-(L₁)p-Z。In another preferred embodiment, the substituent is -(L ₁ )pZ.

在另一优选例中，所述L₁选自下组：-O-CH₂-、-O-CO-CH₂-。In another preferred embodiment, the L _{1 is} selected from the group consisting of -O-CH ₂ -, -O-CO-CH ₂ -.

在另一优选例中，所述Z为C_6-20芳基或C_1-10的酰胺基。In another preferred embodiment, the Z is a C _6-20 aryl group or a C _1-10 amide group.

在另一优选例中，R_e、R_d与相邻的N形成的环优选为二环。 In another preferred embodiment, the ring formed by R _e , R _d and the adjacent N is preferably a bicyclic ring.

在另一优选例中，所述环C、D和E为饱和或不饱和的、芳环或非芳环的、杂环或非杂环的。In another preferred embodiment, the rings C, D and E are saturated or unsaturated, aromatic or non-aromatic, heterocyclic or non-heterocyclic.

在另一优选例中，所述环E为取代的5-6元环。In another preferred embodiment, the ring E is a substituted 5-6 membered ring.

在另一优选例中，所述环E为取代的5-6元杂环。In another preferred embodiment, the ring E is a substituted 5-6 membered heterocyclic ring.

在另一优选例中，所述环D为取代或未取代的7-10元环。In another preferred embodiment, the ring D is a substituted or unsubstituted 7-10 membered ring.

在另一优选例中，R_a、R_b各自相同或不同地为：

In another preferred embodiment, R _a and R _b are each the same or different:

n为1-3；n is 1-3;

式中，R₁、R₂的定义如上所述。In the formula, R ₁ and R ₂ are as defined above.

在另一优选例中，R₁、R₂共同形成饱和或不饱和的6元苯环或碳环。In another preferred embodiment, R ₁ and R ₂ together form a saturated or unsaturated 6-membered benzene or carbocyclic ring.

在另一优选例中，n＝1。In another preferred embodiment, n=1.

在另一优选例中，R₁为取代或未取代的C_1-10羧基，较佳的n为1，R₂为氢。In another preferred embodiment, R ₁ is a substituted or unsubstituted C _1-10 carboxyl group, preferably n is 1, and R ₂ is hydrogen.

在另一优选例中，R₁为酯基，较佳的R₁为甲酯，n为1，R₂为氢。In another preferred embodiment, R ₁ is an ester group, preferably R ₁ is a methyl ester, n is 1, and R ₂ is hydrogen.

在另一优选例中，R₁为甲基，较佳的n为1，R₂为氢。In another preferred embodiment, R ₁ is a methyl group, preferably n is 1, and R ₂ is hydrogen.

在另一优选例中，R₁和R₂各自独立地为取代或未取代的羟甲基或H。In another preferred embodiment, R ₁ and R ₂ are each independently a substituted or unsubstituted hydroxymethyl group or H.

在另一优选例中，R₁和R₂各自独立地为C_1-10烷基取代的羟甲基或H，较佳地所述C_1-10烷基选自下组：甲基、乙基、正丁基、正癸基。In another preferred embodiment, R ₁ and R ₂ are each independently a C _1-10 alkyl-substituted hydroxymethyl group or H, preferably the C _1-10 alkyl group is selected from the group consisting of methyl, B. Base, n-butyl, n-decyl.

在另一优选例中，R₁和R₂各自独立地为N，N二甲酰胺基取代的羟甲基或H。In another preferred embodiment, R ₁ and R ₂ are each independently N,N diformamide-substituted hydroxymethyl or H.

在另一优选例中，R₁和R₂各自独立地为乙酰基取代的羟甲基或H，n为2。In another preferred embodiment, R ₁ and R ₂ are each independently an acetyl-substituted hydroxymethyl group or H, and n is 2.

在另一优选例中，R₁和R₂各自独立地为取代或未取代的羟甲基或H，较佳地所述取代基选自下组：苯基、对甲苯基、对甲氧基苯基、萘基。In another preferred embodiment, R ₁ and R ₂ are each independently a substituted or unsubstituted hydroxymethyl group or H, preferably the substituent is selected from the group consisting of phenyl, p-tolyl, p-methoxy. Phenyl, naphthyl.

在另一优选例中，R₁和R₂各自独立地为-CH₂-CH₂-OH取代的羟甲基。In another preferred embodiment, R ₁ and R ₂ are each independently -CH ₂ -CH ₂ -OH substituted hydroxymethyl.

式中，In the formula,

在另一优选例中，环A为饱和的C_5-7碳环。In another preferred embodiment, Ring A is a saturated C _5-7 carbocyclic ring.

在另一优选例中，环A为取代或未取代的6元碳杂环，较佳地为含一个氧的杂环，且所含氧原子在N的对位。In another preferred embodiment, Ring A is a substituted or unsubstituted 6-membered carbon heterocycle, preferably an oxygen-containing heterocycle, and the oxygen atom is contained in the para position of N.

在另一优选例中，环A为取代或未取代的6元碳杂环，较佳地，所述取代基选自甲基、二甲基、C_1-10羧基、乙酯基，较佳地，取代位置在N原子的对位。In another preferred embodiment, Ring A is a substituted or unsubstituted 6-membered carbon heterocyclic ring. Preferably, the substituent is selected from the group consisting of methyl, dimethyl, C _1-10 carboxyl, ethyl ester groups, preferably. Ground, the substitution position is in the para position of the N atom.

在另一优选例中，所述式X化合物选自下组：In another preferred embodiment, the compound of formula X is selected from the group consisting of

在另一优选例中，所述式X化合物选自下组：化合物1、8、9、18、19、21、22、25、26、29和33。In another preferred embodiment, the compound of formula X is selected from the group consisting of

compounds

1, 8, 9, 18, 19, 21, 22, 25, 26, 29 and 33.

在另一优选例中，所述式I化合物选自下组：In another preferred embodiment, the compound of formula I is selected from the group consisting of

在另一优选例中，所述式II化合物选自下组：In another preferred embodiment, the compound of formula II is selected from the group consisting of

本发明的第三方面，提供了一种药物组合物，所述药物组合物中含有According to a third aspect of the present invention, a pharmaceutical composition comprising the pharmaceutical composition

(i)本发明第二方面所述的式X化合物或其药学上可接受的盐；(i) a compound of formula X according to the second aspect of the invention, or a pharmaceutically acceptable salt thereof;

(ii)药学上可接受的载体。(ii) a pharmaceutically acceptable carrier.

在另一优选例中，所述药物组合物中含有0.001-99wt％，较佳地0.1-90wt％，更佳地1-80wt％的式X化合物或其药学上可接受的盐，按组合物的总重量计。In another preferred embodiment, the pharmaceutical composition contains 0.001 to 99% by weight, preferably 0.1 to 90% by weight, more preferably 1 to 80% by weight, of the compound of the formula X or a pharmaceutically acceptable salt thereof, by composition Total weight.

在另一优选例中，所述的药物组合物的剂型为口服剂型、或注射剂。In another preferred embodiment, the pharmaceutical composition is in the form of an oral dosage form, or an injection.

在另一优选例中，所述口服剂型选自：片剂、胶囊剂、膜剂、颗粒剂。In another preferred embodiment, the oral dosage form is selected from the group consisting of a tablet, a capsule, a film, and a granule.

在另一优选例中，所述口服剂型为缓释型或非缓释型剂型。In another preferred embodiment, the oral dosage form is a sustained release or non-release type.

在另一优选例中，所述的药物组合物还包括其他活性成分，所述活性成分选自：顺铂、紫杉醇、或抗肿瘤的抗体。In another preferred embodiment, the pharmaceutical composition further comprises other active ingredients selected from the group consisting of: cisplatin, paclitaxel, or an anti-tumor antibody.

本发明的第四方面，提供了一种制备式I化合物的方法I，包括步骤：In a fourth aspect of the invention, there is provided a process I for the preparation of a compound of formula I, comprising the steps of:

(I-2)在惰性溶剂中，将式B化合物与试剂D进行反应，从而形成式I化合物，其中，所述试剂D选自下组：过硫酸铵、高碘酸钠、亚硝酸钠、盐酸、双氧水、硫酸、或其组合；(I-2) reacting a compound of the formula B with a reagent D in an inert solvent to form a compound of the formula I, wherein the reagent D is selected from the group consisting of ammonium persulfate, sodium periodate, sodium nitrite, Hydrochloric acid, hydrogen peroxide, sulfuric acid, or a combination thereof;

(I-3)任选地对步骤(I-2)中形成的式I化合物进行分离或纯化，从而获得分离的或纯化的式I化合物；(I-3) optionally separating or purifying the compound of the formula I formed in the step (I-2) to obtain a separated or Purified compound of formula I;

式中，n为1-3；Where n is 1-3;

在另一优选例中，在步骤(I-3)，所述的分离包括重结晶。In another preferred embodiment, in the step (I-3), the separation comprises recrystallization.

在另一优选例中，在步骤(I-2)中，所述式B化合物与试剂D的摩尔百分比为1：0.1-3，较佳地为1：0.5-2.5，更佳地为1：1.0-2.0。In another preferred embodiment, in the step (I-2), the molar percentage of the compound of the formula B to the reagent D is 1:0.1-3, preferably 1:0.5-2.5, more preferably 1: 1.0-2.0.

在另一优选例中，在步骤(I-2)中，在-5至50℃下(优选20-30℃)反应；反应5-30h(较佳地为10-25h)，和/或在搅拌条件下进行反应。In another preferred embodiment, in the step (I-2), the reaction is carried out at -5 to 50 ° C (preferably 20 to 30 ° C); the reaction is carried out for 5 to 30 hours (preferably 10 to 25 hours), and/or The reaction was carried out under stirring.

在另一优选例中，所述方法I还包括步骤：In another preferred embodiment, the method 1 further includes the steps of:

(I-1)在有机溶剂中，将式A化合物与二硫化碳进行反应，从而制得式B化合物，其中，n、R₁、R₂的定义如前所述。(I-1) A compound of the formula B is obtained by reacting a compound of the formula A with carbon disulfide in an organic solvent, wherein n, R ₁ and R ₂ are as defined above.

在另一优选例中，所述(I-1)中，所述有机溶剂选自下组：四氢呋喃、甲基四氢呋喃、水、或其组合。In another preferred embodiment, in the (I-1), the organic solvent is selected from the group consisting of tetrahydrofuran, methyltetrahydrofuran, water, or a combination thereof.

在另一优选例中，在步骤(I-1)中，所述式A化合物与二硫化碳的摩尔百分比为1：0.5-2.5，较佳地为1：1.0-2.0，更佳地为1.1-1.8。In another preferred embodiment, in the step (I-1), the molar percentage of the compound of the formula A to carbon disulfide is 1:0.5 to 2.5, preferably 1:1.0 to 2.0, more preferably 1.1 to 1.8. .

在另一优选例中，在步骤(I-1)中，在20-25℃下进行反应；反应1-12h(较佳地为2-10h)；和/或在搅拌条件下进行反应。In another preferred embodiment, in the step (I-1), the reaction is carried out at 20 to 25 ° C; the reaction is carried out for 1 to 12 hours (preferably 2 to 10 hours); and/or the reaction is carried out under stirring.

本发明的第五方面，提供了一种制备式II化合物的方法II，包括步骤：According to a fifth aspect of the invention, there is provided a process II for the preparation of a compound of formula II, comprising the steps of:

(II-1)在有机溶剂中，将式C化合物与试剂D进行反应，形成式II化合物，其中所述试剂D选自下组：过硫酸铵、高碘酸钠、亚硝酸钠、盐酸、双氧水、硫酸、或其组合，式中，(II-1) reacting a compound of the formula C with a reagent D in an organic solvent to form a compound of the formula II, wherein the reagent D is selected from the group consisting of ammonium persulfate, sodium periodate, sodium nitrite, hydrochloric acid, Hydrogen peroxide, sulfur Acid, or a combination thereof, in the formula,

在另一优选例中，所述式C化合物与试剂D的摩尔百分比为1：0.1-3，较佳地为1：0.5-2.5，更佳地为1：1.0-2.0。In another preferred embodiment, the molar percentage of the compound of the formula C to the reagent D is from 1:0.1 to 3, preferably from 1:0.5 to 2.5, more preferably from 1:1.0 to 2.0.

在另一优选例中，在步骤(II-1)中，在-5至50℃下(优选20-30℃)反应；反应5-30h(较佳地为10-25h)，和/或在搅拌条件下进行反应。In another preferred embodiment, in the step (II-1), the reaction is carried out at -5 to 50 ° C (preferably 20 to 30 ° C); the reaction is carried out for 5 to 30 hours (preferably 10 to 25 hours), and/or The reaction was carried out under stirring.

本发明的第六方面，提供了一种非治疗性地抑制丙酮酸脱氢酶激酶活性的方法，包括步骤：In a sixth aspect of the invention, a method for non-therapeutic inhibition of pyruvate dehydrogenase kinase activity comprising the steps of:

(a)将丙酮酸脱氢酶激酶与本发明第二方面所述的式X化合物、其光学异构体、非消旋体、外消旋体、或其药学上可接受的盐进行接触，从而抑制丙酮酸脱氢酶激酶的活性。(a) contacting pyruvate dehydrogenase kinase with a compound of formula X according to the second aspect of the invention, an optical isomer thereof, a non-racemate, a racemate, or a pharmaceutically acceptable salt thereof, Thereby inhibiting the activity of pyruvate dehydrogenase kinase.

在另一优选例中，在步骤(a)中，将式X化合物、其光学异构体、非消旋体、外消旋体、或其药学上可接受的盐添加到细胞培养体系中，从而使其与丙酮酸脱氢酶激酶进行接触。In another preferred embodiment, in the step (a), the compound of the formula X, its optical isomer, the non-racemic form, the racemate, or a pharmaceutically acceptable salt thereof is added to the cell culture system, Thereby it is brought into contact with pyruvate dehydrogenase kinase.

在另一优选例中，所述的细胞为正常细胞或肿瘤细胞。In another preferred embodiment, the cell is a normal cell or a tumor cell.

在另一优选例中，所述的细胞为哺乳动物细胞。In another preferred embodiment, the cell is a mammalian cell.

在另一优选例中，所述的细胞为人细胞。In another preferred embodiment, the cell is a human cell.

本发明的第七方面，提供了一种抑制丙酮酸脱氢酶激酶或***的方法，所述的方法包括：给需要的对象施用本发明第二方面所述的式X化合物、其光学异构体、非消旋体、外消旋体、或其药学上可接受的盐。According to a seventh aspect of the invention, there is provided a method of inhibiting pyruvate dehydrogenase kinase or treating a tumor, the method comprising: administering to a subject in need thereof a compound of the formula X according to the second aspect of the invention, which is optically different A conformation, a racemate, a racemate, or a pharmaceutically acceptable salt thereof.

在另一优选例中，所述的对象包括人和非人哺乳动物(如啮齿动物和灵长动物)。In another preferred embodiment, the subject includes human and non-human mammals (e.g., rodents and primates).

应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。It is to be understood that within the scope of the present invention, the various technical features of the present invention and the various technical features specifically described hereinafter (as in the embodiments) may be combined with each other to constitute a new or preferred technical solution. Due to space limitations, we will not repeat them here.

附图说明DRAWINGS

图1显示了化合物分别在1μM(上图)和0.3μM(下图)时的分子水平酶活(A549 细胞)。Figure 1 shows the molecular level enzyme activity of the compound at 1 μM (top panel) and 0.3 μM (bottom panel) (A549) cell).

图2显示了化合物19、21在不同浓度下对A549细胞活性氧(ROS)的影响。Figure 2 shows the effect of compounds 19, 21 on reactive oxygen species (ROS) in A549 cells at various concentrations.

图3显示了化合物19、21不同浓度下对A549细胞外乳酸释放的影响。Figure 3 shows the effect of different concentrations of Compounds 19 and 21 on the release of lactic acid from A549 cells.

具体实施方式detailed description

本申请发明人经过广泛而深入地研究，首次意外地发现了式X化合物可以显著地抑制丙酮酸脱氢酶激酶(PDHK)的活性，活化线粒体丙酮酸脱氢酶复合体(PDC)，促进细胞质依赖的无氧糖酵解向线粒体依赖的糖氧化转化，式X化合物在抑制肿瘤细胞的增殖实验中也取得良好结果。在此基础上，完成了本发明。The inventors of the present application have extensively and intensively studied, and for the first time, unexpectedly discovered that the compound of the formula X can significantly inhibit the activity of pyruvate dehydrogenase kinase (PDHK), activate the mitochondrial pyruvate dehydrogenase complex (PDC), and promote cytoplasm. The dependent anaerobic glycolysis is oxidatively transformed into mitochondria-dependent sugars, and the compound of formula X also achieves good results in the inhibition of tumor cell proliferation experiments. On the basis of this, the present invention has been completed.

术语the term

如本文所用，术语“本发明化合物”、或“化合物”指式X所示的化合物、或其消旋体、对应异构体、或其药学上可接受的盐。应理解，该术语还包括上述组分的混合物。The term "compound of the invention", or "compound", as used herein, refers to a compound of formula X, or a racemate thereof, a corresponding isomer, or a pharmaceutically acceptable salt thereof. It should be understood that the term also includes mixtures of the above components.

基团Group

术语“C_1-6烷基”指具有1-6个碳原子的直链或支链烷基，例如甲基、乙基、丙基、异丙基、丁基、异丁基、仲丁基、叔丁基、或类似基团。The term "C _1-6 alkyl" refers to a straight or branched alkyl group having from 1 to 6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl. , tert-butyl, or the like.

术语“C₁-₆烷氧基”指具有1-6个碳原子的直链或支链烷氧基，例如甲氧基、乙氧基、丙氧基、异丙氧基、丁氧基、异丁氧基、仲丁氧基、叔丁氧基、或类似基团。The term "C ₁ - ₆ alkoxy" refers to straight or branched chain alkoxy group having 1 to 6 carbon atoms, such as methoxy, ethoxy, propoxy, isopropoxy, butoxy, Isobutoxy, sec-butoxy, tert-butoxy, or the like.

“环烷基”指3至8元全碳单环、全碳5元/6元或6元/6元稠合环或多环稠合环基团，其中一个或多个环可以含有一个或多个双键，但没有一个环具有完全共轭的π电子***。环烷基实例有环丙基、环丁基、环戊基、环己基、环己二烯基、金刚烷基、环庚烷基、环庚三烯基等。"Cycloalkyl" means a 3 to 8 membered all carbon monocyclic, all carbon 5 membered/6 membered or 6 membered/6 membered fused or polycyclic fused ring group wherein one or more of the rings may contain one or Multiple double bonds, but none of the rings have a fully conjugated π-electron system. Examples of cycloalkyl groups are cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexadienyl, adamantyl, cycloheptyl, cycloheptatriene and the like.

“5-7元单环”指具有5-7元的单环(仅有一个环结构)，所述的单环可以是饱和或不饱和环，如环烷基、环烯基、芳环。"5-7 membered monocyclic" refers to a monocyclic ring having 5-7 members (only one ring structure), and the monocyclic ring may be a saturated or unsaturated ring such as a cycloalkyl group, a cycloalkenyl group, or an aromatic ring.

“碳环”指环骨架皆为碳原子的饱和或不饱和环，其中一个或多个环可以含有一个或多个双键。"Carbocycle" refers to a saturated or unsaturated ring in which the ring skeleton is a carbon atom, wherein one or more of the rings may contain one or more double bonds.

“杂环”指环骨架上至少存在一个选自下组的杂原子的饱和或不饱和环：N、S、O或P，其中一个或多个环可以含有一个或多个双键。"Heterocycle" means a saturated or unsaturated ring having at least one heteroatom selected from the group consisting of N, S, O or P, wherein one or more of the rings may contain one or more double bonds.

“芳环”指具有共轭的π电子***的芳环，包括碳环芳基、杂芳基。 "Aromatic ring" refers to an aromatic ring having a conjugated π-electron system, including carbocyclic aryl, heteroaryl.

“杂芳基”指具有1个杂原子作为环原子，其余的环原子为碳的芳基，杂原子包括氧、硫、氮。所述环可以是5元或6元或7元环。杂芳基基团的实例包括但不限于呋喃基、噻吩基、苯并呋喃基、苯并噻吩基、吡啶基、吡咯、N-烷基吡咯基。"Heteroaryl" means an aryl group having one hetero atom as a ring atom and the remaining ring atoms being carbon, the hetero atom including oxygen, sulfur, nitrogen. The ring may be a 5- or 6- or 7-membered ring. Examples of heteroaryl groups include, but are not limited to, furyl, thienyl, benzofuranyl, benzothienyl, pyridyl, pyrrole, N-alkylpyrrolyl.

“烷氧基”指-O-(烷基)。代表性实例包括甲氧基、乙氧基、丙氧基、丁氧基等。"Alkoxy" means -O-(alkyl). Representative examples include methoxy, ethoxy, propoxy, butoxy, and the like.

术语“卤素”是指氟、氯、溴、碘。术语“卤代的”是指氟代的、氯代的、溴代的、碘代的。The term "halogen" means fluoro, chloro, bromo, iodo. The term "halogenated" means fluoro, chloro, bromo, iodo.

在本文中，除特别说明之处，本发明的各个基团可以未取代的或取代的，所述“取代”指基团上的一个或多个氢原子被选自下组的取代基取代：C₁-C₁₀烷基、卤代C₁-₁₀烷基、C₃-C₁₀环烷基、C₁-C₁₀烷氧基、卤代C₁-₁₀烷氧基、卤素、羟基、C_1-10羧基(-COOH)、C₁-C₁₀醛基、C₂-C₁₀酰基、C₂-C₁₀酯基、氨基、苯基、C_1-4烷基羟基、C_1-10酰胺基、C_5-20芳基、卤代C_5-20芳基、氰基；所述的苯基包括未取代的苯基或具有1-3个取代基的取代苯基，所述取代基选自：卤素、C₁-C₁₀烷基、氰基、OH、硝基、C₃-C₁₀环烷基、C₁-C₁₀烷氧基、氨基。Herein, unless otherwise stated, each group of the present invention may be unsubstituted or substituted, and the "substituted" means that one or more hydrogen atoms on the group are substituted with a substituent selected from the group consisting of: C ₁ -C ₁₀ alkyl, halogenated C ₁ _-10 alkyl, C ₃ -C ₁₀ cycloalkyl, C ₁ -C ₁₀ alkoxy, halogenated C ₁ _-10 alkoxy, halogen, hydroxy, C _1-10 carboxyl (-COOH), C ₁ -C ₁₀ aldehyde group, C ₂ -C ₁₀ acyl group, C ₂ -C ₁₀ ester group, amino group, phenyl group, C _1-4 alkyl hydroxy group, C _1-10 amide a C _5-20 aryl group, a halogenated C _5-20 aryl group, a cyano group; the phenyl group includes an unsubstituted phenyl group or a substituted phenyl group having 1 to 3 substituents selected from the group consisting of From: halogen, C ₁ -C ₁₀ alkyl, cyano, OH, nitro, C ₃ -C ₁₀ cycloalkyl, C ₁ -C ₁₀ alkoxy, amino.

式X化合物Compound of formula X

本发明提供了一种式X化合物、其光学异构体或其药学上可接受的盐，The present invention provides a compound of the formula X, an optical isomer thereof or a pharmaceutically acceptable salt thereof,

R_a-S-S-R_b (X)R _a -SSR _b (X)

式中，In the formula,

各R_d分别独立地选自下组：无、取代或未取代的C_1-6烷基、取代或未取代的C_6-10芳基、取代或未取代的C_6-10杂芳基、取代或未取代的C_6-10环烷基、-(CH₂)_m-C_6-10芳基，m为0-4； Each R _{d is} independently selected from the group consisting of unsubstituted, substituted or unsubstituted C _1-6 alkyl, substituted or unsubstituted C _6-10 aryl, substituted or unsubstituted C _6-10 heteroaryl, a substituted or unsubstituted C _6-10 cycloalkyl, -(CH ₂ ) _m -C _6-10 aryl, m is 0-4;

为单键而与N杂原子连接的

Connected to an N hetero atom for a single bond

For double keys; or

为双键而与N杂原子连接的

Connected to an N hetero atom for a double bond

For a single button;

p为0-4，r为0-4；p is 0-4, r is 0-4;

式I化合物Compound of formula I

如本文所用，式I化合物如下所示：As used herein, the compound of formula I is as follows:

式中，In the formula,

n为1-3；n is 1-3;

R₁和R₂各自独立地选自下组：氢、羟基、C_1-10羧基、C₁-₆酯基、取代或未取代的羟甲基、取代或未取代的C₁-₄烷基、取代或未取代的C₁-₁₀烷氧基，且R₁和R₂不同时为氢；R ₁ and R ₂ are each independently selected from the group consisting of: hydrogen, hydroxy, C _1-10 carboxy, C ₁ - ₆ ester group, a substituted or unsubstituted hydroxymethyl, substituted or unsubstituted C ₁ - ₄ alkyl a substituted or unsubstituted C ₁ _-10 alkoxy group, and R ₁ and R _{2 are} not simultaneously hydrogen;

式II化合物 Compound of formula II

如本文所用，式II化合物如下所示：As used herein, the compound of formula II is as follows:

式中，In the formula,

丙酮酸脱氢酶激酶Pyruvate dehydrogenase kinase

丙酮酸脱氢酶激酶即Pyruvate Dehydrogenase Kinase(PDHK)，是一种线粒体酶，能够抑制丙酮酸脱氢酶复合物(PDC)，是葡萄糖氧化的关键控制酶，可以阻止丙酮酸转化为乙酰辅酶A，而后者是三羧酸循环的底物。PDHK的活化能导致糖酵解和其下游糖氧化的脱节。在人类细胞中现已证实存在四个亚型的PDHK分别为PDHK1-4，其中PDHK1与癌症的恶性程度紧密相关。PDHK1在多种癌症如肺癌，头颈鳞状上皮癌中都是激活的。PDHK1为原癌基因(C-MYC)和低氧诱导因子(hypoxia inducing factor，HIF)等致癌基因转录调控，以此控制癌细胞的代谢和恶性表型。Pyruvate Dehydrogenase Kinase (PDHK), a pyruvate dehydrogenase kinase, is a mitochondrial enzyme that inhibits pyruvate dehydrogenase complex (PDC) and is a key control enzyme for glucose oxidation, preventing the conversion of pyruvate to acetyl-CoA. The latter is a substrate for the tricarboxylic acid cycle. Activation of PDHK can lead to a dissociation of glycolysis and its downstream sugar oxidation. It has been confirmed in human cells that there are four subtypes of PDHK, PDHK1-4, respectively, of which PDHK1 is closely related to the degree of malignancy of cancer. PDHK1 is activated in a variety of cancers such as lung cancer, head and neck squamous cell carcinoma. PDHK1 is a transcriptional regulation of oncogenes such as protooncogene (C-MYC) and hypoxia inducing factor (HIF), thereby controlling the metabolism and malignant phenotype of cancer cells.

PDHK1在人体癌细胞内一般为各种致瘤的酪氨酸激酶所磷酸化。抑制PDHK可以有效地提高肿瘤细胞化疗的效果,并且具有高度的肿瘤特异性。除了上述代谢抗癌机制，还有研究表明PDC的活化导致活性氧应激(reactive oxygen species，ROS)增加并诱导肿瘤细胞的凋亡。因此，抑制PDHK可以作为杀伤肿瘤细胞的靶点。PDHK1 is generally phosphorylated by various tumorigenic tyrosine kinases in human cancer cells. Inhibition of PDHK can effectively improve the effect of tumor cell chemotherapy and has a high degree of tumor specificity. In addition to the metabolic anticancer mechanisms described above, studies have shown that activation of PDC leads to an increase in reactive oxygen species (ROS) and induces apoptosis in tumor cells. Therefore, inhibition of PDHK can be used as a target for killing tumor cells.

药学上可接受的盐Pharmaceutically acceptable salt

本发明还包括式X化合物的药学上可接受的盐。The invention also includes pharmaceutically acceptable salts of the compounds of formula X.

术语“药学上可接受的盐”指本发明化合物与酸或碱所形成的适合用作药物的盐。药学上可接受的盐包括无机盐和有机盐。一类优选的盐是本发明化合物与酸形成的盐。适合形成盐的酸包括但并不限于：盐酸、氢溴酸、氢氟酸、硫酸、硝酸、磷酸等无机酸，甲酸、乙酸、丙酸、草酸、丙二酸、琥珀酸、富马酸、马来酸、乳酸、苹果酸、酒石酸、柠檬酸、苦味酸、甲磺酸、苯甲磺酸，苯磺酸等有机酸；以及天冬氨酸、谷氨酸等酸性氨基酸。 The term "pharmaceutically acceptable salt" refers to a salt of the compound of the present invention which is formed with an acid or a base and which is suitable for use as a medicament. Pharmaceutically acceptable salts include inorganic and organic salts. A preferred class of salts are the salts of the compounds of the invention with acids. Suitable acids for forming salts include, but are not limited to, mineral 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, Organic acids such as maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulfonic acid, benzoic acid, and benzenesulfonic acid; and acidic amino acids such as aspartic acid and glutamic acid.

制备方法Preparation

本发明中制备反应步骤均采用现有技术中本领域技术人员熟知的方法，对各个步骤的反应参数没有特别限制。本领域技术人员可以根据不同的最终预期的化合物，对反应式中涉及的各步骤反应的反应物、反应条件等适当作出调整。The preparation steps in the present invention are all those known to those skilled in the art in the prior art, and the reaction parameters of the respective steps are not particularly limited. Those skilled in the art can appropriately adjust the reactants, reaction conditions and the like of the respective steps involved in the reaction formula according to different final expected compounds.

本发明提供了一种制备式I化合物的方法I，包括步骤：The present invention provides a process I for the preparation of a compound of formula I, comprising the steps of:

(I-3)任选地对步骤(I-2)中形成的式I化合物进行分离或纯化，从而获得分离的或纯化的式I化合物；(I-3) optionally isolating or purifying the compound of formula I formed in step (I-2) to obtain an isolated or purified compound of formula I;

式中，n为1-3；Where n is 1-3;

在另一优选例中，所述(I-1)中，所述有机溶剂选自下组：四氢呋喃、甲基四氢呋喃、水、或其组合。In another preferred embodiment, in the above (I-1), the organic solvent is selected from the group consisting of tetrahydrofuran and methyltetrahydrofuran. Oral, water, or a combination thereof.

本发明还提供了一种制备式II化合物的方法II，包括步骤：The invention also provides a method II for preparing a compound of formula II, comprising the steps of:

(II-1)在有机溶剂中，将式C化合物与试剂D进行反应，形成式II化合物，其中所述试剂D选自下组：过硫酸铵、高碘酸钠、亚硝酸钠、盐酸、双氧水、硫酸、或其组合，式中，(II-1) reacting a compound of the formula C with a reagent D in an organic solvent to form a compound of the formula II, wherein the reagent D is selected from the group consisting of ammonium persulfate, sodium periodate, sodium nitrite, hydrochloric acid, Hydrogen peroxide, sulfuric acid, or a combination thereof, wherein

在另一优选例中，式I化合物的制备方法包括步骤：In another preferred embodiment, the method of preparing a compound of formula I comprises the steps of:

将取代的吗啉(n＝1)或高吗啉(n＝2)和适量的二硫化碳，溶于四氢呋喃中，室温下搅拌反应10-20小时。向反应体系中加入适量的过硫酸铵水溶液，室温搅拌10-20小时。有机层旋干后用乙酸乙酯重结晶或经硅胶柱层析后得到题述化合物。其中取代的吗啉或高吗啉为购买或合成(见具体实施例)。The substituted morpholine (n = 1) or high morpholine (n = 2) and an appropriate amount of carbon disulfide are dissolved in tetrahydrofuran, and the reaction is stirred at room temperature for 10-20 hours. An appropriate amount of an aqueous solution of ammonium persulfate was added to the reaction system, and the mixture was stirred at room temperature for 10-20 hours. The organic layer was dried to dryness and then crystallised from ethyl acetate. Wherein the substituted morpholine or homomorpholine is purchased or synthesized (see specific examples).

在另一优选例中，式II化合物的制备方法包括步骤：In another preferred embodiment, the method of preparing a compound of formula II comprises the steps of:

将并环的2-巯基噻唑溶于四氢呋喃中，然后加入适量的过硫酸铵水溶液，室温搅拌10-20小时。有机层旋干后用乙酸乙酯重结晶或经硅胶柱层析后得到标题化合物。其中取代的2-巯基噻唑为购买或合成(见具体实施例)。The cyclo-2-mercaptothiazole is dissolved in tetrahydrofuran, then an appropriate amount of aqueous ammonium persulfate solution is added, and the mixture is stirred at room temperature. Mix for 10-20 hours. The organic layer was dried with EtOAc (EtOAc)EtOAc. The substituted 2-mercaptothiazole is either purchased or synthesized (see specific examples).

组合物和施用方法Composition and method of administration

本发明还提供了一种用于抑制丙酮酸脱氢酶激酶的组合物。所述的组合物包括(但并不限于)：药物组合物、食品组合物、膳食补充剂、饮料组合物等。The invention also provides a composition for inhibiting pyruvate dehydrogenase kinase. Such compositions include, but are not limited to, pharmaceutical compositions, food compositions, dietary supplements, beverage compositions, and the like.

在本发明中，所述的药物组合物可直接用于疾病治疗，例如，用于抗肿瘤方面的治疗。在使用本发明药物制剂时，还可同时使用其他治疗剂，如抗肿瘤的药物等。In the present invention, the pharmaceutical composition can be directly used for the treatment of diseases, for example, for the treatment of anti-tumor. When the pharmaceutical preparation of the present invention is used, other therapeutic agents such as antitumor drugs and the like can also be used at the same time.

本发明还提供了一种药物组合物，它含有安全有效量的本发明化合物以及药学上可接受的载体或赋形剂。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、粉剂、及其组合。药物制剂应与给药方式相匹配。The invention also provides a pharmaceutical composition comprising a safe and effective amount of a compound of the invention together with a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to, saline, buffer, dextrose, water, glycerol, ethanol, powders, and combinations thereof. The pharmaceutical preparation should be matched to the mode of administration.

以药物组合物为例，本发明的组合物可以被制成针剂形式，例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。诸如片剂和胶囊之类的药物组合物，可通过常规方法进行制备。药物组合物如针剂、溶液、片剂和胶囊宜在无菌条件下制造。本发明的药物组合也可以被制成粉剂用于雾化吸入。活性成分的给药量是治疗有效量，例如每天约1微克/千克体重-约5毫克/千克体重。此外，本发明细胞自噬抑制剂还可与其他治疗剂一起使用。Taking the pharmaceutical composition as an example, the composition of the present invention can be prepared into an injection form, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as tablets and capsules can be prepared by conventional methods. Pharmaceutical compositions such as injections, solutions, tablets and capsules are preferably manufactured under sterile conditions. The pharmaceutical combination of the invention may also be formulated as a powder for nebulization. The amount of active ingredient administered is a therapeutically effective amount, for example from about 1 microgram per kilogram body weight to about 5 milligrams per kilogram body weight per day. Furthermore, the autophagy inhibitors of the invention may also be used with other therapeutic agents.

对于本发明的药物组合物，可通过常规的方式施用于所需的对象(如人和非人哺乳动物)。代表性的施用方式包括(但并不限于)：口服、注射、雾化吸入等。For the pharmaceutical composition of the present invention, it can be administered to a subject (e.g., human and non-human mammal) by a conventional means. Representative modes of administration include, but are not limited to, oral, injection, nebulization, and the like.

使用药物组合物时，是将安全有效量的药物施用于哺乳动物，其中该安全有效量通常至少约10微克/千克体重，而且在大多数情况下不超过约8毫克/千克体重，较佳地该剂量是约10微克/千克体重-约1毫克/千克体重。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是熟练医师技能范围之内的。When a pharmaceutical composition is used, a safe and effective amount of the medicament is administered to the mammal, wherein the safe and effective amount is usually at least about 10 micrograms per kilogram of body weight, and in most cases no more than about 8 milligrams per kilogram of body weight, preferably The dose is from about 10 micrograms per kilogram of body weight to about 1 milligram per kilogram of body weight. Of course, specific doses should also consider factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled physician.

本发明的主要优点在于：The main advantages of the invention are:

(1)提供了一类新型式X化合物，其可用于(a)制备抑制丙酮酸脱氢酶激酶(PDHK)活性的药物或制剂，和/或，(b)制备抑制和/或***细胞的药物或制剂；(1) Providing a novel class of compounds of formula X which are useful for (a) preparing a medicament or formulation that inhibits pyruvate dehydrogenase kinase (PDHK) activity, and/or, (b) preparing for inhibiting and/or treating tumor cells Drug or preparation;

(2)本发明化合物结果简单，制备工艺简洁，生产成本低；(2) The compound of the invention has simple results, simple preparation process and low production cost;

(3)本发明化合物体内外抗肿瘤机制明确、药效显著。 (3) The anti-tumor mechanism of the compound of the present invention is clear in vivo and in vitro, and the drug effect is remarkable.

下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。The invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are not intended to limit the scope of the invention. The experimental methods in the following examples which do not specify the specific conditions are usually in accordance with conventional conditions or according to the conditions recommended by the manufacturer. Percentages and parts are by weight unless otherwise stated.

实施例1双(4-(2-(甲氧基甲基)吗啉)基硫代羰基)化二硫的制备Example 1 Preparation of bis(4-(2-(methoxymethyl)morpholine)thiocarbonyl) disulfide

将2.3g化合物1-1溶于50mL甲醇，在氮气保护下，加入600mg甲醇钠，在回流温度下加热过夜，待体系冷至室温后，用200mL乙酸乙酯和200mL水分层，有机相经硅胶柱层析得到化合物1-2 1.5g，将化合物1-2溶于50mL甲醇，加入50mg钯碳(10％)，氢气还原(1atm)过夜，过滤后旋干得到0.85g化合物1-3，直接加入20mLTHF和0.5g二硫化碳于室温下搅拌，将2g过硫酸铵溶于10mL水中，于10小时后加入，继续搅拌过夜，将反应体系用200mL乙酸乙酯和200mL水分层后有机相旋干，用1:3的乙酸乙酯和石油醚柱层析分离得到1g化合物1产率47％(依照化合物1-1计算)。2.3 g of compound 1-1 was dissolved in 50 mL of methanol, and 600 mg of sodium methoxide was added under nitrogen atmosphere, and heated at reflux temperature overnight. After the system was cooled to room temperature, 200 ml of ethyl acetate and 200 mL of water layer were used, and the organic phase was passed through. Silica gel column chromatography gave compound 1-2 1.5 g, compound 1-2 was dissolved in 50 mL of methanol, 50 mg of palladium carbon (10%) was added, hydrogen was reduced (1 atm) overnight, filtered and then dried to give 0.85 g of compound 1-3. Directly add 20 mL of THF and 0.5 g of carbon disulfide, stir at room temperature, dissolve 2 g of ammonium persulfate in 10 mL of water, add after 10 hours, continue stirring overnight, and then spin the organic phase with 200 mL of ethyl acetate and 200 mL of water. Separation of 1:3 ethyl acetate and petroleum ether column chromatography gave 1 g of Compound 1 yield 47% (calculated according to compound 1-1).

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计算值:C₁₄H₂₄N₂O₄S₄[M]⁺,412.0619，测量值：412.0618 ¹ H-NMR (400MHz, CDCl ₃ ) δ 5.60-4.40 (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 calcd: C ₁₄ H ₂₄ N ₂ O ₄ S ₄ [M] ⁺ , 412.0619, Measured: 412.0618

实施例2双(4-(2-(苯酚基甲基)吗啉)基硫代羰基)化二硫的制备 Example 2 Preparation of bis(4-(2-(phenolmethyl))morpholine)thiocarbonyl)disulfide

将1g化合物1-1溶于50mLDMF中，在氮气保护下，加入0.35g氢化钠(60％)，在100℃下加热过夜，待体系冷至室温后，用200mL乙酸乙酯和200mL水分层，有机相经硅胶柱层析得到化合物2-2 0.65g，将化合物2-2溶于50mL甲醇，加入50mg钯碳(10％)，氢气还原(1atm)过夜，过滤后旋干得到0.46g化合物2-3，直接加入20mLTHF和0.4g二硫化碳于室温下搅拌，将1g过硫酸铵溶于10mL水中于10小时后加入，继续搅拌过夜，将反应体系用200mL乙酸乙酯和200mL水分层后有机相旋干，用1:4的乙酸乙酯和石油醚柱层析分离得到0.4g化合物2产率63％(依照化合物2-3计算)1 g of compound 1-1 was dissolved in 50 mL of DMF, and 0.35 g of sodium hydride (60%) was added under nitrogen atmosphere, and heated at 100 ° C overnight. After the system was cooled to room temperature, 200 mL of ethyl acetate and 200 mL of water layer were used. The organic phase was subjected to silica gel column chromatography to give compound 2-2 0.65 g, Compound 2-2 was dissolved in 50 mL of methanol, 50 mg of palladium carbon (10%) was added, hydrogen was reduced (1 atm) overnight, filtered and then dried to give 0.46 g of compound. 2-3, directly add 20mL of THF and 0.4g of carbon disulfide at room temperature, 1g of ammonium persulfate dissolved in 10mL of water after 10 hours, continue to stir overnight, the reaction system with 200mL of ethyl acetate and 200mL of water layer organic The phase was dried and separated by a 1:4 ethyl acetate and petroleum ether column chromatography to yield 0.4 g of compound 2 yield 63% (calculated according to compound 2-3)

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计算值:C₂₄H₂₈N₂O₄S₄[M+Na]⁺:559.0824,测量值：559.0823 ¹ H-NMR (400 MHz, CDCl ₃ ) δ 7.34 - 7.27 (m, 5H), 7.02 - 6.90 (m, 5H), 6.05 - 4.45 (m, 4H), 4.45 - 3.03 (m, 14H); ESI) m/z calcd. for _C24 H ₂₈ N ₂ O ₄ S ₄ [M+Na] ⁺ : 559.0824, Measured: 559.0823

实施例3双(4-(2-(对甲氧基苯酚基甲基)吗啉)基硫代羰基)化二硫的制备Example 3 Preparation of bis(4-(2-(p-methoxyphenolylmethyl)morpholine)thiocarbonyl) disulfide

同实施例2，不同之处在于：将对甲苯酚换成对甲氧基苯酚，得到0.09克黄色固体(化合物3)，收率是40％。The same as Example 2 except that p-cresol was replaced with p-methoxyphenol to obtain 0.09 g of a yellow solid (Compound 3) in a yield of 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计算值:C₂₆H₃₂N₂O₆S₄[M+Na]⁺:619.1035,测量值：619.1037 ^{1 H-NMR (400MHz, CDCl} 3) 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 calcd.: C ₂₆ H ₃₂ N ₂ O ₆ S ₄ [M+Na] ⁺ : 619.1035, Measured: 619.1037

实施例4双(4-(2-(α-萘酚基甲基)吗啉)基硫代羰基)化二硫的制备Example 4 Preparation of bis(4-(2-(α-naphthylmethyl)morpholine)thiocarbonyl) disulfide

同实施例2，不同之处在于：将对甲苯酚换成α-萘酚，得到0.13克黄色固体(化合物4)，收率是53％。The same as Example 2 except that p-cresol was replaced with α-naphthol to obtain 0.13 g of a yellow solid (Compound 4) in a yield of 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计算值:C₂₆H₃₂N₂O₆S₄[M+H]⁺:659.1137,测量值：659.1141 ^{1 H-NMR (400MHz, CDCl} 3) 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: C ₂₆ H ₃₂ N ₂ O ₆ S ₄ [M+H] ⁺ : 659.1137, measured value: 659.1141

实施例5双(4-(2-(甲氧基甲基)高吗啉)基硫代羰基)化二硫的制备Example 5 Preparation of bis(4-(2-(methoxymethyl)-high morpholine) thiocarbonyl) disulfide

同实施例1，不同之处在于：将2位氯甲基取代的N-苄基吗啉(1-1)换成2位氯甲基取代的N-苄基高吗啉，得到0.05克黄色固体(化合物5)，收率是36％。The same as in Example 1, except that the 2-position chloromethyl-substituted N-benzylmorpholine (1-1) was replaced with the 2-position chloromethyl-substituted N-benzylhighmorpholine to obtain 0.05 g of yellow. Solid (Compound 5) in a yield of 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计算值:C₁₆H₂₈N₂O₂S₄[M]⁺,440.0932,测量值：440.0930 ^{1 H-NMR (400MHz, CDCl} 3) 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: C ₁₆ H ₂₈ N ₂ O ₂ S ₄ [M] ⁺ , 440.0932, measured value: 440.0930

实施例6双(4-(2-(对甲基苯酚基甲基)高吗啉)基硫代羰基)化二硫的制备Example 6 Preparation of bis(4-(2-(p-methylphenolylmethyl)-high morpholine) thiocarbonyl) disulfide

同实施例2，不同之处在于：将2位氯甲基取代的N-苄基吗啉换成2位氯甲基取代的N-苄基高吗啉，得到0.04克黄色固体(化合物6)，收率是31％。The same as in Example 2 except that the 2-position chloromethyl-substituted N-benzyl morpholine was replaced with the 2-position chloromethyl-substituted N-benzyl homomorpholine to obtain 0.04 g of a yellow solid (Compound 6). The yield was 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计算值:C₂₈H₃₆N₂O₄S₄[M+Na]⁺:615.1450,测量值：615.1456 ¹ H-NMR (400MHz, CDCl ₃ ) δ 7.15-7.01 (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 calc.: C ₂₈ H ₃₆ N ₂ O ₄ S ₄ [M+Na] ⁺ : 615.1450, Measured: 615.1456

实施例7双(4-(2-(苯酚基甲基)高吗啉)基硫代羰基)化二硫的制备Example 7 Preparation of bis(4-(2-(phenolmethyl)) morpholine) thiocarbonyl) disulfide

同实施例6，不同之处在于：将对甲苯酚换成苯酚，得到0.03克黄色固体(化合物7)，收率是43％。The same as Example 6, except that p-cresol was changed to phenol to obtain 0.03 g of a yellow solid (compound 7) in a yield of 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计算值:C₂₆H₃₂N₂O₄S₄[M+Na]⁺:587.1134,测量值：587.1135 ¹ H-NMR (400MHz, CDCl ₃ ) 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 For C ₂₆ H ₃₂ N ₂ O ₄ S ₄ [M+Na] ⁺ : 587.1134, Measured: 587.1135

实施例8双(4-(2-(乙氧基甲基)吗啉)基硫代羰基)化二硫的制备Example 8 Preparation of bis(4-(2-(ethoxymethyl)morpholine) thiocarbonyl) disulfide

将2g 2位氯甲基取代的N-苄基吗啉溶于10mL甲酰胺和2mL水中，加热至200℃搅拌过夜，冷却后加入过量的氨水，用200mL乙酸乙酯萃取后旋干得,1.3g化合物8-1，将其溶于30mLDMF，加入0.8g氢化钠(60％)并2g碘乙烷，在80℃下加热5小时，用200mL乙酸乙酯和100mL水分层，有机相柱层析分离后得到0.9g化合物8-2，直接溶于30mL甲醇加入0.05g钯碳后于1atm下加氢还原10小时，旋干后直接加入20mLTHF和0.5g二硫化碳于室温下搅拌，将2g过硫酸铵溶于10mL水中于10小时后加入，继续搅拌过夜，将反应体系用200mL乙酸乙酯和200mL水分层后有机相旋干，用1:3的乙酸乙酯和石油醚柱层析分离得到0.5g化合物8收率为36％(依照化合物8-1计算)。2 g of 2-chloromethyl-substituted N-benzylmorpholine was dissolved in 10 mL of formamide and 2 mL of water, heated to 200 ° C and stirred overnight. After cooling, excess ammonia water was added, extracted with 200 mL of ethyl acetate and then dried. g compound 8-1, dissolved in 30 mL of DMF, added 0.8 g of sodium hydride (60%) and 2 g of ethyl iodide, heated at 80 ° C for 5 hours, with 200 mL of ethyl acetate and 100 mL of water layer, organic phase column After separation, 0.9 g of compound 8-2 was obtained, directly dissolved in 30 mL of methanol, added with 0.05 g of palladium carbon, and then hydrogenated at 1 atm for 10 hours. After spinning, directly add 20 mL of THF and 0.5 g of carbon disulfide at room temperature to stir, 2 g of persulfuric acid. Ammonium was dissolved in 10 mL of water and added after 10 hours. Stirring was continued overnight. The reaction system was organically phased with 200 mL of ethyl acetate and 200 mL of water. Dry, using a 1:3 ethyl acetate and petroleum ether column chromatography to afford 0.5 g of compound 8 yield 36% (calculated according to compound 8-1).

¹H-NMR(400MHz,CDCl₃)δ5.61-4.61(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计算值:C₁₆H₂₈N₂O₄S₄[M]⁺,440.0932,测量值：440.0936 ^{1 H-NMR (400MHz, CDCl} 3) δ5.61-4.61 (m, 4H), 4.10-4.00 (m, 2H), 3.86-3.71 (m, 1H), 3.66-3.26 (m, 12H), 1.23 ( t, J = 7.2 Hz, 6H); HRMS (EI) m/z calc.: C ₁₆ H ₂₈ N ₂ O ₄ S ₄ [M] ⁺ , 440.0932, Measured: 440.0936

实施例9双(4-(2-(乙酰氧基甲基)吗啉)基硫代羰基)化二硫的制备Example 9 Preparation of bis(4-(2-(acetoxymethyl)morpholine)thiocarbonyl) disulfide

将0.5g 2位羟甲基取代的N-苄基吗啉溶于10mL二氯甲烷中，加入和0.5g三乙胺，降温至0℃，将0.25g乙酰氯滴入，回到室温，搅拌过夜，加入100mL二氯甲烷和50mL水分层，有机层旋干，柱层析得到0.45g化合物10-1，用与实施例9同样的方法最后得到0.22g化合物9，收率40％。0.5 g of 2-hydroxymethyl-substituted N-benzylmorpholine was dissolved in 10 mL of dichloromethane, and 0.5 g of triethylamine was added thereto, and the temperature was lowered to 0 ° C, and 0.25 g of acetyl chloride was added dropwise, and returned to room temperature, and stirred. After overnight, 100 mL of dichloromethane and 50 mL of a water layer were added, and the organic layer was evaporated to dryness to give 0.45 g of Compound 10-1, which was obtained in the same manner as in Example 9 to obtain 0.22 g of Compound 9 in a yield of 40%.

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计算值:C₁₆H₂₄N₂O₆S₄[M]⁺,468.0517,测量值：468.0519 ^{1 H-NMR (400MHz, CDCl} 3) 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: C ₁₆ H ₂₄ N ₂ O ₆ S ₄ [M] ⁺ , 468.0517, measured value: 468.0519

实施例10双(4-(2-羟甲基吗啉)基硫代羰基)化二硫的制备Example 10 Preparation of bis(4-(2-hydroxymethylmorpholine)thiocarbonyl) disulfide

以实施例8的方法得到2位羟甲基取代的N-苄基吗啉为原料，在甲醇中用钯碳加氢脱苄，得到2位羟甲基取代的吗啉，用与实施例1同样的方法得到0.1g化合物10，收率60％。The 2-hydroxymethyl-substituted N-benzylmorpholine was obtained as a raw material by the method of Example 8, and palladium carbon was used in methanol. Hydrodebenzylation was carried out to obtain a hydroxymethyl group-substituted morpholine. In the same manner as in Example 1, 0.1 g of Compound 10 was obtained in a yield of 60%.

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计算值:C₁₂H₂₀N₂O₄S₄[M+Na]⁺:407.0198,测量值：407.0205 ¹ H-NMR (400MHz, CDCl ₃ ) δ 5.67-4.22 (m, 4H), 4.11-4.00 (m, 2H), 3.84-3.74 (m, 5H), 3.72-3.62 (m, 3H), 3. 3.30 (m, 4H); HRMS (ESI) m / z _{_{_{Calcd: C 12 H 20 N 2 O}}} 4 S 4 [m + Na] +: 407.0198, found: 407.0205

实施例11双(4-(2-(二甲氨基甲酸基甲基)吗啉)基硫代羰基)化二硫的制备Example 11 Preparation of bis(4-(2-(dimethylaminomethyl)methyl)morpholine)thiocarbonyl) disulfide

以2位羟甲基取代的N-苄基吗啉为原料，经钯碳加氢，并用叔丁氧基保护N后得到化合物11-1，将0.5g化合物11-1溶于10mL THF并降温至-78℃加入1当量的正丁基锂，1小时后再加入1.2当量的二甲氨基甲酰氯，并缓慢回到室温，得到0.6g化合物12-2，将0.6g化合物11-2溶于5mLTFA中室温搅拌1小时，旋干后用碳酸钾在甲醇中游离得0.3g化合物11-3，之后用与实施例8同样的方法最后得到0.12g化合物11，收率20％。The N-benzylmorpholine substituted with 2-hydroxymethyl group was used as a raw material, hydrogenated by palladium carbon, and N was protected with tert-butoxy group to obtain compound 11-1. 0.5 g of compound 11-1 was dissolved in 10 mL of THF and cooled. 1 equivalent of n-butyllithium was added to -78 ° C, and 1.2 equivalents of dimethylcarbamoyl chloride was added after 1 hour, and slowly returned to room temperature to obtain 0.6 g of compound 12-2, and 0.6 g of compound 11-2 was dissolved. After stirring at room temperature for 1 hour in 5 mL of TFA, the mixture was evaporated to dryness, and then, then,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,

¹H-NMR(400MHz,CDCl₃)δ5.64-4.61(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计算值:C₁₈H₃₀N₄O₆S₄[M+Na]⁺:549.0940,测量值：549.0943 ^{1 H-NMR (400MHz, CDCl} 3) δ5.64-4.61 (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 calc.: C ₁₈ H ₃₀ N ₄ O ₆ S ₄ [M+Na] ⁺ : 549.0940, Measured: 549.0943

实施例12双(4-(2-(乙氧基甲基)高吗啉)基硫代羰基)化二硫的制备Example 12 Preparation of bis(4-(2-(ethoxymethyl))highmorpholinyl)thiocarbonyl)disulfide

同实施例8，不同之处在于：将2位氯甲基取代的N-苄基吗啉换成2位氯甲基取代的N-苄基高吗啉，得到0.11克黄色油状物(化合物12)，收率是36％。The same as Example 8, except that the 2-position chloromethyl-substituted N-benzyl morpholine was replaced with the 2-position chloromethyl-substituted N-benzyl homomorpholine to obtain 0.11 g of a yellow oil (Compound 12). ), the 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计算值:C₁₈H₃₂N₂O₄S₄[M]⁺,468.1245,测量值：468.1246 ¹ H-NMR (400 MHz, CDCl ₃ ) 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 calcd.: C ₁₈ H ₃₂ N ₂ O ₄ S ₄ [M] ⁺ , 468.1245, Measured: 468.1246

实施例13双(4-(2-(正丁氧基甲基)高吗啉)基硫代羰基)化二硫的制备Example 13 Preparation of bis(4-(2-(n-butoxymethyl)-high morpholine) thiocarbonyl) disulfide

同实施例12，不同之处在于：将碘乙烷换成正溴丁烷，得到0.09克黄色油状物(化合物13)，收率是30％。The same as Example 12 except that ethyl iodide was changed to n-bromobutane to obtain 0.09 g of a yellow oil (compound 13) in a yield of 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计算值:C₂₂H₄₀N₂O₄S₄[M+Na]⁺:547.1763,测量值：547.1774 ^{1 H-NMR (400MHz, CDCl} 3) 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: C ₂₂ H ₄₀ N ₂ O ₄ S ₄ [ M+Na] ⁺ : 547.1763, measured value: 547.1774

实施例14双(4-(2-(正癸氧基甲基)高吗啉)基硫代羰基)化二硫的制备Example 14 Preparation of bis(4-(2-(n-decyloxymethyl)-high morpholine) thiocarbonyl) disulfide

同实施例12，不同之处在于：将碘乙烷换成正溴癸烷，得到0.14克黄色油状物(化合物14)，收率是33％。The same procedure as in Example 12 except that ethyl iodide was changed to n-bromo-decane to give 0.14 g of a yellow oil (comp. 14) in a yield of 33%.

¹H-NMR(400MHz,CDCl₃)δ5.21-5.10(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计算值:C₃₄H₆₄N₂O₄S₄[M+Na]⁺:715.3641,测量值：715.3645 ^{1 H-NMR (400MHz, CDCl} 3) δ5.21-5.10 (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 = 8 Hz, 6H); HRMS (ESI) m/z calc.: C ₃₄ H ₆₄ N ₂ O ₄ S ₄ [M+Na] ⁺ : 715.3641, measured value: 715.3645

实施例15双(4-(2-(正癸氧基甲基)高吗啉)基硫代羰基)化二硫的制备Example 15 Preparation of bis(4-(2-(n-decyloxymethyl)-high morpholine) thiocarbonyl) disulfide

同实施例12，不同之处在于：将碘乙烷换成苄基2-溴乙基醚，得到0.04克黄色油状物(化合物15)，收率是19％。The same procedure as in Example 12 was carried out except that ethyl iodide was changed to benzyl 2-bromoethyl ether to give 0.04 g of a yellow oil (compound 15) in a yield of 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计算值:C₁₈H₃₂N₂O₆S₄[M+Na]⁺:523.1035,测量值：523.1041 ¹ H-NMR (400 MHz, CDCl ₃ ) δ 5.22-5.11 (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 For C ₁₈ H ₃₂ N ₂ O ₆ S ₄ [M+Na] ⁺ : 523.1035, Measured: 523.1041

实施例16双(4-(2-羟甲基高吗啉)基硫代羰基)化二硫的制备Example 16 Preparation of bis(4-(2-hydroxymethylhomomorpholine)thiocarbonyl) disulfide

同实施例10，不同之处在于：将2位氯甲基取代的N-苄基吗啉换成对2位氯甲基取代的N-苄基高吗啉，得到0.05克黄色油状物(化合物16)，收率是23％。The same as Example 10 except that the 2-position chloromethyl-substituted N-benzyl morpholine was replaced with the 2-position chloromethyl-substituted N-benzyl homomorpholine to obtain 0.05 g of a yellow oil (compound). 16), the yield was 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计算值:C₁₄H₂₄N₂O₄S₄[M+Na]⁺:435.0511,测量值：435.0518 ¹ H-NMR (400 MHz, CDCl ₃ ) δ 5.08 - 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 For C ₁₄ H ₂₄ N ₂ O ₄ S ₄ [M+Na] ⁺ : 435.0511, Measured: 435.0518

实施例17双(4-(2-(二甲氨基甲酸基甲基)高吗啉)基硫代羰基)化二硫的制备Example 17 Preparation of bis(4-(2-(dimethylaminomethyl)methyl) morpholine)thiocarbonyl) disulfide

同实施例11，不同之处在于：将2位羟甲基取代的N-苄基吗啉换成2位羟甲基取代的N-苄基高吗啉，得到0.04克黄色固体(化合物17)，收率是25％。Same as Example 11, except that the 2-hydroxymethyl substituted N-benzyl morpholine was replaced with the 2-hydroxymethyl group. Substituted N-benzyl high morpholine gave 0.04 g of a yellow solid (compound 17) in a yield of 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计算值:C₂₀H₃₄N₄O₆S₄[M+Na]⁺:577.1253,测量值：577.1262 ¹ H-NMR (400 MHz, CDCl ₃ ) δ 5.28-5.18 (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 _{_{_{Calcd: C 20 H 34 N 4 O}}} 6 S 4 [m + Na] +: 577.1253, found: 577.1262

实施例18双(4-(R-3-(乙氧基甲基)吗啉)基硫代羰基)化二硫的制备Example 18 Preparation of bis(4-(R-3-(ethoxymethyl)morpholine) thiocarbonyl) disulfide

同实施例8，不同之处在于：将2位羟甲基取代的N-苄基吗啉(化合物8-1)换成R型的3位羟甲基取代的N-苄基吗啉(化合物18-1)，得到0.07克黄色固体(化合物18)，收率是35％Same as Example 8, except that the N-benzyl morpholine (compound 8-1) substituted with 2-hydroxymethyl group was replaced with the N-benzyl morpholine substituted at the 3-position of the R type. 18-1), 0.07 g of a yellow solid (compound 18) was obtained in a yield of 35%

¹H-NMR(400MHz,CDCl₃)δ5.80-4.41(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计算值:C₁₆H₂₈N₂O₄S₄[M]⁺,440.0932,测量值：440.0931 ¹ H-NMR (400 MHz, CDCl ₃ ) δ 5.80-4.41 (m, 4H), 4.15 - 4.09 (m, 2H), 4.40 (dd, J = 12.0, 4.0 Hz, 2H), 3.93 - 3.30 (m, 14H), 1.21 (t, J = 6.8 Hz, 6H); HRMS (EI) m/z: C ₁₆ H ₂₈ N ₂ O ₄ S ₄ [M] ⁺ , 440.0932, Measured: 440.0931

实施例19双(4-(R-3-(甲氧基甲基)吗啉)基硫代羰基)化二硫的制备Example 19 Preparation of Bis(4-(R-3-(methoxymethyl)morpholine)ylthiocarbonyl)disulfide

同实施例1，不同之处在于：将2位氯甲基取代的N-苄基吗啉(化合物1-1)换成R型的3位氯甲基取代的N-苄基吗啉(化合物19-1)，得到0.15克黄色油状物(化合物19)，收率是39％The same as in Example 1, except that the 2-position chloromethyl-substituted N-benzylmorpholine (Compound 1-1) was replaced with the R-type 3-position chloromethyl-substituted N-benzylmorpholine (compound). 19-1), 0.15 g of a yellow oil (compound 19) was obtained in a yield of 39%.

¹H-NMR(400MHz,CDCl₃)δ5.80-4.48(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计算值:C₁₄H₂₄N₂O₄S₄[M]⁺,412.0619,测量值：412.0621 ^{1 H-NMR (400MHz, CDCl} 3) δ5.80-4.48 (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 calc.: C ₁₄ H ₂₄ N ₂ O ₄ S ₄ [M] ⁺ , 412.0619, Measured: 412.0621

实施例20双(4-(R-3-羟甲基吗啉)基硫代羰基)化二硫的制备Example 20 Preparation of bis(4-(R-3-hydroxymethylmorpholine)thiocarbonyl) disulfide

同实施例10，不同之处在于：将2位羟甲基取代的吗啉换成R型的3位羟甲基取代的吗啉，得到0.07克黄色油状物(化合物20)，收率是40％The same as Example 10 except that the 2-hydroxymethyl-substituted morpholine was replaced with the R-form 3-hydroxymethyl-substituted morpholine to obtain 0.07 g of a yellow oil (Compound 20) in a yield of 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计算值:C₁₂H₂₀N₂O₄S₄[M+Na]⁺:407.0198,测量值：407.0197 ¹ H-NMR (400 MHz, CDCl ₃ ) δ 5.91-4.50 (m, 4H), 4.22-3.94 (m, 8H), 3.80-3.73 (m, 2H), 3.72-3.46 (m, 4H); ESI) m / z _{_{_{Calcd: C 12 H 20 N 2 O}}} 4 S 4 [m + Na] +: 407.0198, found: 407.0197

实施例21双(4-高吗啉基硫代羰基)化二硫的制备Example 21 Preparation of bis(4-highmorpholinylthiocarbonyl) disulfide

将1g高吗啉，1.5g二硫化碳，溶于50mL四氢呋喃中，在室温搅拌反应10小时。然后向反应体系中加入20mL 2.5g过硫酸铵的水溶液，室温搅拌10小时。有机层旋干后用乙酸乙酯重结晶后得到1.4g化合物21，产率82％。1 g of high morpholine, 1.5 g of carbon disulfide, was dissolved in 50 mL of tetrahydrofuran, and the reaction was stirred at room temperature for 10 hours. Then, 20 mL of an aqueous solution of 2.5 g of ammonium persulfate was added to the reaction system, and the mixture was stirred at room temperature for 10 hours. The organic layer was dried and then crystallised from 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计算值:C₁₂H₂₀N₂O₂S₄[M]⁺,352.0408,测量值：352.0405 ^{1 H-NMR (400MHz, CDCl} 3) 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 _{_{_{Calcd: C 12 H 20 N 2 O}}} 2 S 4 [m] +, 352.0408, found: 352.0405

实施例22双(4-(2-甲基)吗啉基硫代羰基)化二硫的制备 Example 22 Preparation of bis(4-(2-methyl)morpholinylthiocarbonyl) disulfide

同实施例21，不同之处在于：将高吗啉换成2位甲基取代的吗啉，得到0.2克化合物22，收率是76％The same as in Example 21 except that the high morpholine was changed to the methyl substituted morpholine at the 2-position to obtain 0.2 g of the compound 22 in a yield of 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计算值:C₁₂H₂₀N₂O₂S₄[M]⁺,352.0408,测量值：352.0405 ^{1 H-NMR (400MHz, CDCl} 3) 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 calcd: C ₁₂ H ₂₀ N ₂ O ₂ S ₄ [M] ⁺ , 352.0408, measured value :352.0405

实施例23双(4-(2-甲基)吗啉基硫代羰基)化二硫的制备Example 23 Preparation of bis(4-(2-methyl)morpholinylthiocarbonyl) disulfide

同实施例21，不同之处在于：将高吗啉换成苯并吗啉，得到0.08克化合物23，收率是42％Same as Example 21 except that the high morpholine was changed to benzomorpholine to obtain 0.08 g of the compound 23 in a yield of 42%.

¹H-NMR(400MHz,DMSO-d6)δ8.05(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计算值:C₁₈H₁₆N₂O₂S₄[M]⁺,420.0095,测量值：420.0093 ¹ H-NMR (400 MHz, DMSO-d6) δ 8.05 (d, J = 8.2 Hz, 2H), 7.27 (t, J = 8.0 Hz, 2H), 7.16-7.04 (m, 4H), 4.28 (t, J = 4.8 Hz, 2H), 4.03 (t, J = 4.8 Hz, 2H); HRMS (EI) m/z calc.: C ₁₈ H ₁₆ N ₂ O ₂ S ₄ [M] ⁺ , 420.0095, Measured: 420.0093

实施例24双(4-(反式-八氢-苯并1,4噁嗪)基硫代羰基)化二硫的制备Example 24 Preparation of bis(4-(trans-octahydro-benzo-1,4oxazinyl)thiocarbonyl) disulfide

同实施例21，不同之处在于：将高吗啉换成反式-八氢-苯并1,4噁嗪，得到0.03克化合物24，收率是25％Same as Example 21 except that the high morpholine was changed to trans-octahydro-benzo-1,4 oxazine to obtain 0.03 g of the compound 24 in a yield of 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计算值:C₁₈H₂₈N₂O₂S₄[M]⁺,432.1034,测量值：432.1036 ^{1 H-NMR (400MHz, CDCl} 3) 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 calcd. for C ₁₈ H ₂₈ N ₂ O ₂ S ₄ [M] ⁺ , 432.1034, Measured: 432.1036

实施例25双(4-(2-C1-10羧基)吗啉基硫代羰基)化二硫的制备Example 25 Preparation of bis(4-(2-C1-10carboxy)morpholinylthiocarbonyl) disulfide

同实施例21，不同之处在于：将高吗啉换成2-C1-10羧基吗啉，得到0.19克化合物25，收率是85％Same as Example 21 except that the high morpholine was changed to 2-C1-10 carboxymorpholine to obtain 0.19 g of the compound 25 in a yield of 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计算值:C₁₂H₁₆N₂O₆S₄[M+Na]⁺:434.9783,测量值：434.9784 ¹ H-NMR (400MHz, DMSO-d6) δ 13.30 (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 _{_{_{Calcd: 2 O 6 S 4 [m}}} + Na] + C 12 H 16 N: 434.9783, found: 434.9784

实施例26双(4-(吗啉-2-羧酸甲酯)基硫代羰基)化二硫的制备Example 26 Preparation of bis(4-(morpholine-2-carboxylic acid methyl ester) thiocarbonyl) disulfide

将0.1g化合物25溶于15mL甲醇，于室温加入0.1mL浓硫酸搅拌过夜，用50mL水和200mL乙酸乙酯分层，有机相旋干后用乙酸乙酯重结晶得到0.07g化合物26，收率65％。0.1 g of the compound 25 was dissolved in 15 mL of methanol, and 0.1 mL of concentrated sulfuric acid was added thereto at room temperature, and the mixture was stirred overnight with 50 mL of water and 200 mL of ethyl acetate. 65%.

¹H-NMR(400MHz,CDCl₃)δ5.50-5.00(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计算值:C₁₄H₂₀N₂O₆S₄[M]⁺,440.0204,测量值：440.0205 ¹ H-NMR (400 MHz, CDCl ₃ ) δ 5.50-5.00 (m, 2H), 4.96 - 4.72 (m, 2H), 4.34 (dd, J = 9.4, 2.9 Hz, 2H), 4.23-4.11 (m, 2H), 3.93-3.72 (m, 12H); HRMS (EI) m/z calcd: C ₁₄ H ₂₀ N ₂ O ₆ S ₄ [M] ⁺ , 440.0204, Measured: 440.0205

实施例27双(2-([1,3]噻唑并[5,4-B]吡啶))化二硫的制备Example 27 Preparation of bis(2-([1,3]thiazolo[5,4-B]pyridine))disulfide

将2-巯基噻唑[5,4-B]并吡啶0.5g溶于50mL THF，于室温加入1g过硫酸铵的水溶液20mL，搅拌过夜，有机层旋干，用乙酸乙酯重结晶，得到0.3g化合物27，收率60％。0.5 g of 2-mercaptothiazole [5,4-B]pyridine was dissolved in 50 mL of THF, and 20 mL of an aqueous solution of 1 g of ammonium persulfate was added thereto at room temperature, and the mixture was stirred overnight. Compound 27, yield 60%.

¹H-NMR(400MHz,DMSO-d₆)δ8.61(d,J＝4.6Hz,2H),8.39(d,J＝8.3Hz,2H),7.65-7.58(m,2H)；HRMS(EI)m/z计算值:C₁₂H₆N₄S₄[M]⁺,333.9475,测量值：333.9476 ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ 8.61 (d, J = 4.6 Hz, 2H), 8.39 (d, J = 8.3 Hz, 2H), 7.65-7.58 (m, 2H); HRMS (EI) m/z calculated: C ₁₂ H ₆ N ₄ S ₄ [M] ⁺ , 333.9475, Measured: 333.9476

实施例28双(2-环戊烯并噻唑)化二硫的制备Example 28 Preparation of bis(2-cyclopentenylthiazole) disulfide

将1gα-氯代环戊烷和1.5g二硫代氨基甲酸铵溶于100mL乙醇加热回流过夜，旋干后用乙酸乙酯和水分层，有机相经重结晶得1.2g化合物28-2，取1g化合物33-2溶于50mL THF，于室温加入1g过硫酸铵的水溶液20mL，搅拌过夜，有机层旋干，用乙酸乙酯重结晶，得到0.7g化合物28，收率70％。1 g of α-chlorocyclopentane and 1.5 g of ammonium dithiocarbamate were dissolved in 100 mL of ethanol and heated to reflux overnight. After spin-drying, ethyl acetate and a water layer were obtained, and the organic phase was recrystallized to obtain 1.2 g of compound 28-2. 1 g of the compound 33-2 was dissolved in 50 mL of THF, and 20 ml of an aqueous solution of 1 g of ammonium persulfate was added thereto at room temperature, and the mixture was stirred overnight. The organic layer was evaporated and evaporated to ethylamine.

¹H-NMR(400MHz,DMSO-d₆)δ2.91(t,J＝7.2Hz,4H),2.77(t,J＝7.4Hz,4H),2.46-2.37(m,4H)；HRMS(EI)m/z计算值:C₁₂H₁₂N₂S₄[M]⁺,311.9883,测量值：311.9884 ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ 2.91 (t, J = 7.2 Hz, 4H), 2.77 (t, J = 7.4 Hz, 4H), 2.46-2.37 (m, 4H); HRMS (EI) ) m / z calculated: C ₁₂ H ₁₂ N ₂ S ₄ [M] ⁺ , 311.9883, measured value: 311.9884

实施例29双(2-环庚烯并噻唑)化二硫的制备Example 29 Preparation of bis(2-cycloheptenylthiazole) disulfide

同实施例28，不同之处在于：将α-氯代环戊烷换成α-溴代环庚烷，得到0.6克化合物29，收率是60％Same as Example 28, except that α-chlorocyclopentane was replaced with α-bromocycloheptane to obtain 0.6 g of compound 29 in a yield of 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计算值:C₁₆H₂₀N₂S₄[M]⁺,368.0509,测量值：368.0510 ¹ H-NMR (400 MHz, CDCl ₃ ) δ 2.98-2.89 (m, 4H), 2.83-2.75 (m, 4H), 1.89-1.81 (m, 4H), 1.77-1.64 (m, 4H); EI) m/z calcd for C ₁₆ H ₂₀ N ₂ S ₄ [M] ⁺ , 368.0509, Measured: 368.0510

实施例30双(2-(6,6-二甲基环己烯并噻唑))化二硫的制备Example 30 Preparation of Bis(2-(6,6-Dimethylcyclohexenylthiazole)) Disulfide

同实施例28，不同之处在于：将α-氯代环戊烷换成2-溴-4,4-二甲基环己酮，得到0.8克化合物30，收率是80％Same as Example 28, except that α-chlorocyclopentane was changed to 2-bromo-4,4-dimethylcyclohexanone to obtain 0.8 g of compound 30 in a yield of 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计算值:C₁₈H₂₄N₂S₄[M]⁺,396.0822,测量值：396.0829 ¹ H-NMR (400 MHz, CDCl ₃ ) δ 2.83 - 2.75 (m, 4H), 2.53 (s, 4H), 1.67-1.58 (m, 4H), 1.03 (s, 12H); HRMS (EI) m/ z Calculated for C ₁₈ H ₂₄ N ₂ S ₄ [M] ⁺ , 396.0822, Measured: 396.0829

实施例31双(2-(6-甲基环己烯并噻唑))化二硫的制备Example 31 Preparation of bis(2-(6-methylcyclohexenylthiazole)) disulfide

同实施例28，不同之处在于：将α-氯代环戊烷换成2-溴-4-甲基环己酮，得到0.8克化合物31，收率是80％Same as Example 28, except that α-chlorocyclopentane was changed to 2-bromo-4-methylcyclohexanone to obtain 0.8 g of compound 31 in a yield of 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计算值:C₁₆H₂₀N₂S₄[M]⁺,368.0509,测量值：368.0508 ¹ H-NMR (400 MHz, CDCl ₃ ) δ 2.93-2.79 (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 calc.: C ₁₆ H ₂₀ N ₂ S ₄ [M] ⁺ , 368.0509, Measured: 368.0508

实施例32双(2-(6,7-二氢-4氢-吡喃并[4,3-D]噻唑))化二硫的制备Example 32 Preparation of bis(2-(6,7-dihydro-4hydro-pyrano[4,3-D]thiazole)) disulfide

同实施例28，不同之处在于：将α-氯代环戊烷换成3-溴-四氢吡喃酮，得到0.6克化合物32，收率是60％Same as Example 28, except that α-chlorocyclopentane was changed to 3-bromo-tetrahydropyranone to obtain 0.6 g of compound 32 in a yield of 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计算值:C₁₂H₁₂N₂O₂S₄[M]⁺,343.9782,测量值：343.9781 ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ 4.75 (s, 4H), 3.96-3.88 (m, 4H), 2.83-2.75 (m, 4H); HRMS (EI) m/z calc. ₁₂ H ₁₂ N ₂ O ₂ S ₄ [M] ⁺ , 343.9782, Measured: 343.9781

实施例33双(2-(环己烯并噻唑-6-甲酸乙酯))化二硫的制备Example 33 Preparation of bis(2-(cyclohexenethiazole-6-carboxylate)) disulfide

同实施例28，不同之处在于：将α-氯代环戊烷换成对环己酮甲酸乙酯，得到0.67克化合物33，收率是67％The same as Example 28 except that α-chlorocyclopentane was changed to ethyl p-cyclohexanonecarboxylate to obtain 0.67 g of compound 33 in a yield of 67%.

¹H-NMR(400MHz,CDCl₃)δ4.18(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计算值:C₂₀H₂₄N₂O₄S₄[M+H]⁺:485.0692,测量值：485.0700 ¹ H-NMR (400MHz, CDCl ₃ ) δ 4.18 (q, J = 7.0 Hz, 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 = 7.1 Hz, 6H); HRMS (ESI) m/z calc.: C ₂₀ H ₂₄ N ₂ O ₄ S ₄ [M+H] ⁺ :485.0692, measured value: 485.0700

实施例34双(2-(环己烯并噻唑-6-甲酸))化二硫的制备Example 34 Preparation of bis(2-(cyclohexenylthiazole-6-carboxylic acid)) disulfide

将实施例33的中间体水解得到中间体34-1用同样方法氧化得到0.72克化合物34，收率是72％The intermediate of Example 33 was hydrolyzed to give Intermediate 34-1 which was oxidized in the same manner to give 0.72 g of Compound 34 in a yield of 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计算值:C₁₆H₁₆N₂O₄S₄[M]⁺,427.9993,测量值：427.9991 ¹ H-NMR (400 MHz, CDCl ₃ ) δ 12.48 (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 calc.: C ₁₆ H ₁₆ N ₂ O ₄ S ₄ [M] ⁺ , 427.9993, Measured: 427.9991

生物学评价Biological evaluation

实施例35化合物(1-34)对丙酮酸脱氢酶激酶(PDHK1)激酶活性的影响Effect of Compound (1-34) of Example 35 on Pyruvate Dehydrogenase Kinase (PDHK1) Kinase Activity

1、实验方法1. Experimental method

(1)固定底物：底物PDHE1(0.5μg/well)用碳酸盐缓冲液稀释(Na₂CO₃-NaHCO₃，pH 9.6)进行固定(100μL/well)，置37℃摇床，110rpm，固定3h。然后，不含钾PBST洗涤三次，每次5min，拍干。(1) Fixed substrate: substrate PDHE1 (0.5 μg/well) was fixed with carbonate buffer (Na ₂ CO ₃ -NaHCO ₃ , pH 9.6) (100 μL/well), shaken at 37 ° C, 110 rpm , fixed for 3h. Then, the potassium PBST was washed three times, each time for 5 min, and patted dry.

(2)酶促反应：100μL HEPES反应体系：50mM HEPES(pH 7.5)，10mM MgCl₂，1mM EGTA，10μM ATP，0.1μg酶；置37℃摇床，110rpm，1h反应。无钾PBST洗涤三次，每次5min，拍干。(2) Enzymatic reaction: 100 μL HEPES reaction system: 50 mM HEPES (pH 7.5), 10 mM MgCl ₂ , 1 mM EGTA, 10 μM ATP, 0.1 μg of enzyme; 37 ° C shaker, 110 rpm, 1 h reaction. Wash the potassium-free PBST three times for 5 min each time and pat dry.

(3)一抗孵育：磷酸化PDHE1(S293)抗体用PBST稀释，稀释比例为1:400，每孔加入100μL抗体稀释液，置37℃摇床，110rpm，孵育1h。然后PBST洗涤三次，拍干。(3) Primary antibody incubation: Phosphorylated PDHE1 (S293) antibody was diluted with PBST at a dilution ratio of 1:400, 100 μL of antibody dilution was added to each well, and shaken at 37 ° C, 110 rpm, and incubated for 1 h. The PBST was then washed three times and patted dry.

(4)二抗孵育：抗兔HRP二抗，用PBST以1：2000稀释，每孔加入100μL抗体稀释液，置37℃摇床，110rpm，孵育1h。然后TBST洗涤三次，拍干。(4) Incubation of secondary antibody: anti-rabbit HRP secondary antibody, diluted 1:2000 with PBST, 100 μL of antibody dilution was added to each well, and shaken at 37 ° C, 110 rpm, and incubated for 1 h. The TBST was then washed three times and patted dry.

(5)显色：加入2mg/ml的OPD显色液100μl/well(用含有0.03％H₂O₂的0.1M柠檬酸-柠檬酸钠缓冲液(pH 5.4)稀释)。(5) Color development: 2 mg/ml of OPD chromogenic solution 100 μl/well (diluted with 0.1 M citric acid-sodium citrate buffer (pH 5.4) containing 0.03% H ₂ O ₂ ) was added.

(6)加入2M H₂SO₄ 50μl/well中止反应，用可调波长式微孔板酶标仪 SPECTRA MAX 190读数，波长为490nm。(6) Add 2M H ₂ SO ₄ 50 μl/well to stop the reaction, and read with a tunable wavelength microplate microplate reader SPECTRA MAX 190 at a wavelength of 490 nm.

(7)结果分析(7) Analysis of results

IC50值采用酶标仪随机附带软件以四参数法回归求得。The IC50 value was obtained by four-parameter regression using the software attached to the microplate reader.

2、实验结果2, the experimental results

化合物(1-34)对丙酮酸脱氢酶激酶(PDHK1)的抑制情况通过以上实验进行测定，测得的IC₅₀值见表1。The inhibition of pyruvate dehydrogenase kinase (PDHK1) by the compound (1-34) was measured by the above experiment, and the IC ₅₀ values measured are shown in Table 1.

化合物19和21对丙酮酸脱氢酶激酶家族其他成员抑制PDHK2-4激酶活性的IC₅₀值见表2。 Compound 19 and 21 other members of the pyruvate dehydrogenase kinase family kinase activity inhibition IC PDHK2-4 ₅₀ values in Table 2.

表1.化合物(1-34)抑制PDHK1活性的IC₅₀值Table 1. IC ₅₀ values of Compound (1-34) for inhibiting PDHK1 activity

表2.化合物(19、21)抑制PDHK2、3、4活性的IC₅₀值 Table 2. Compound (19, 21) inhibiting activity IC ₅₀ value PDHK2,3,4

结果显示，化合物(1-34)对PDHK1激酶均有一定的抑制作用，其中化合物1、8、9、18、19、21、22、26、29、31、33号化合物活性较好，均小于120nM。化合物(19、21)对PDHK2和PDHK3也有抑制作用，但是对PDHK4无抑制作用(>10μM)。The results showed that the compound (1-34) had a certain inhibitory effect on PDHK1 kinase, and the

compounds

1, 8, 9, 18, 19, 21, 22, 26, 29, 31, 33 had better activity and were less than 120nM. Compounds (19, 21) also inhibited PDHK2 and PDHK3, but did not inhibit PDHK4 (>10 μM).

实施例36化合物对多种肿瘤细胞增殖的影响Effect of the compound of Example 36 on proliferation of various tumor cells

1、实验方法1. Experimental method

处于对数生长期的肿瘤细胞按合适密度接种至96孔培养板，每孔100μL体系，待细胞贴壁后，加入10μL不同浓度的化合物，每个浓度设三个复孔。化合物作用72h后，弃去细胞培液，加入10％(w/v)三氯乙酸(100μL/孔)于4℃固定1h，随后用蒸馏水冲洗五次。反面放置至烘箱中干燥，取出冷却，每孔加入100μLSRB溶液(4mg/mL SRB粉末溶于1％冰醋酸)，室温下放置15min后，用1％冰醋酸冲洗五次以充分去除未与板底蛋白结合的SRB。反面放置至烘箱中干燥，取出冷却，每孔加入10mM Tris溶液150μL,用酶标仪测定560nm波长下的OD值。按以下公式计算化合物对肿瘤细胞生长的抑制率：Tumor cells in logarithmic growth phase were inoculated into 96-well culture plates at a suitable density, 100 μL per well. After the cells were attached, 10 μL of different concentrations of compounds were added, and three replicate wells were set for each concentration. After the compound was allowed to act for 72 hours, the cell culture solution was discarded, and 10% (w/v) trichloroacetic acid (100 μL/well) was added thereto, and fixed at 4 ° C for 1 hour, followed by rinsing with distilled water five times. The reverse side was placed in an oven to be dried, taken out and cooled, and 100 μl of LSRB solution (4 mg/mL SRB powder dissolved in 1% glacial acetic acid) was added to each well. After standing at room temperature for 15 minutes, it was washed five times with 1% glacial acetic acid to completely remove the bottom and the bottom. Protein-bound SRB. The reverse side was placed in an oven to be dried, taken out and cooled, and 150 μL of a 10 mM Tris solution was added to each well, and the OD value at a wavelength of 560 nm was measured with a microplate reader. The inhibition rate of the compound on tumor cell growth was calculated according to the following formula:

IC₅₀值采用酶标仪随机附带软件以四参数法回归求得。IC ₅₀ values were obtained by four-parameter regression using software attached to the microplate reader.

2、实验结果2, the experimental results

化合物(19、21)对肺癌细胞A549和人神经母细胞瘤Kelly的增殖抑制结果见表3。结果显示，化合物19、21有较好的增殖抑制作用。The results of inhibition of proliferation of lung cancer cell A549 and human neuroblastoma Kelly by compound (19, 21) are shown in Table 3. The results showed that the compounds 19 and 21 had a good proliferation inhibiting effect.

表3.化合物(19、21)对A549细胞和Kelly细胞增殖抑制IC₅₀ Table 3. Compound (19, 21) inhibits proliferation of A549 cells and Kelly cells IC ₅₀

实施例37化合物1、8、9、18、19、21、22、26在A549肿瘤细胞中对PDHK抑制作用的影响Effect of

Compounds

1, 8, 9, 18, 19, 21, 22, 26 of Example 37 on PDHK Inhibition in A549 Tumor Cells

1、实验方法1. Experimental method

将细胞接种于六孔板，经处理后，融合度达到80％左右，收集细胞，用冷的PBS洗两次，加入1×SDS凝胶上样缓冲液裂解细胞5min。细胞裂解液在沸水浴中加热10分钟后，于4℃12000rpm离心10分钟，取上清，弃沉淀。The cells were seeded in a six-well plate. After treatment, the degree of fusion reached about 80%. The cells were collected, washed twice with cold PBS, and lysed with 1×SDS gel loading buffer for 5 min. After the cell lysate was heated in a boiling water bath for 10 minutes, it was centrifuged at 12,000 rpm for 10 minutes at 4 ° C, and the supernatant was taken, and the precipitate was discarded.

蛋白样品置于不同浓度SDS-聚丙烯酰胺凝胶中，在Tris-甘氨酸-SDS电泳缓冲液中以80V电泳至蛋白Marker分开后，用120V电泳约2h进行分离。用半干印迹法或湿转法将蛋白从凝胶转移至硝酸纤维素膜，按所需蛋白分子量大小转移1-2.5h。用丽春红染色液染色确定蛋白转印情况和蛋白条带位置，依据蛋白Marker分子量裁剪相应目的条带，然后用封闭液(含3％BSA的TBST)室温封闭1h，与相应的抗体(用3％BSA的TBST稀释)4℃孵育过夜。用TBST洗涤液室温洗涤3次，每次10min。加入用辣根过氧化物酶标记的二抗(1：2000用3％BSA的TBST稀释)，室温孵育1h。然后用TBST洗三次，每次10min。根据曝光强度选择合适的发光试剂孵育后，用全自动凝胶成像分析***进行显色分析。The protein samples were placed in different concentrations of SDS-polyacrylamide gel, separated into protein Marker by electrophoresis at 80 V in Tris-glycine-SDS running buffer, and separated by electrophoresis at 120 V for about 2 h. The protein is transferred from the gel to the nitrocellulose membrane by semi-dry blotting or wet transfection and transferred for 1-2.5 h according to the desired protein molecular weight. The protein transfer and protein band position were determined by staining with Ponceau red staining solution. The corresponding target band was cut according to the molecular weight of the protein Marker, and then blocked with a blocking solution (TBST containing 3% BSA) for 1 h at room temperature, and the corresponding antibody (using The TBST dilution of 3% BSA was incubated overnight at 4 °C. The TBST washing solution was washed 3 times at room temperature for 10 min each time. A secondary antibody labeled with horseradish peroxidase (1:2000 diluted with 3% BSA in TBST) was added and incubated for 1 h at room temperature. Then wash three times with TBST for 10 min each time. After incubation with an appropriate luminescent reagent according to the exposure intensity, a color analysis was performed using a fully automated gel imaging analysis system.

2、实验结果2, the experimental results

对分子水平酶活较好的式X化合物进行细胞水平酶活检测。Cell-level enzyme activity assays are performed on compounds of formula X with better enzyme activity at the molecular level.

图1显示了化合物在1μM和0.3μM时对A549细胞PDHK蛋白磷酸化的影响，以JX06和JX25

为阳性对照。Figure 1 shows the effect of compounds on phosphorylation of PDHK protein in A549 cells at 1 μM and 0.3 μM, with JX06 and JX25

As a positive control.

结果显示，在肿瘤细胞(A549)中，化合物作用24h后，在1μM的浓度下，除了化合物8外，其余化合物对PDHA1S293和S232位点的磷酸化都有较好的抑制作用。当浓度调整为0.3μM时，化合物19、21仍然有较好抑制，尤以化合物21效果最好。The results showed that in the tumor cells (A549), after compound treatment for 24 h, at the concentration of 1 μM, except for compound 8, the other compounds had a good inhibitory effect on the phosphorylation of PDHA1S293 and S232 sites. When the concentration was adjusted to 0.3 μM, the compounds 19 and 21 were still well inhibited, and in particular, the compound 21 was the best.

实施例38化合物19、21对A549细胞活性氧ROS的影响Effect of Compounds 19 and 21 of Example 38 on Reactive Oxygen ROS in A549 Cells

1、实验方法1. Experimental method

处于对数生长期的细胞按合适密度接种至6孔培养板，每孔2ml，贴壁后，加入不同浓度的化合物或DMSO作用24h。吸去细胞培养液，用预冷的PBS洗涤贴壁细胞2 次，吸净PBS后，加入0.25％Trypsin-EDTA消化细胞，加入含血清培液终止，将细胞转移到1.5ml EP管中，4℃，300g离心5min，弃上清。按照1:1000用无血清培液稀释DCFH-DA(碧云天生物技术研究所)，使终浓度为10μM。DCFH-DA本身没有荧光，可以自由穿过细胞膜，进入细胞后，被细胞内的酯酶水解成DCFH，而DCFH不能自由通过细胞膜，从而留在了细胞内。细胞内的活性氧可以氧化无荧光的DCFH变成有荧光的DCF。检测DCF的水平就可以知道细胞内的活性氧水平。用DCFH-DA稀释液将细胞沉淀重新混悬，吹打均匀，37℃细胞培养箱内孵育30分钟。每隔10min颠倒混匀一次，使探针和细胞充分接触。用无血清细胞培养液洗涤细胞两次，以充分去除未被细胞装载的DCFH-DA。用流式细胞仪进行实验并分析。Cells in logarithmic growth phase were inoculated to a 6-well culture plate at a suitable density of 2 ml per well. After adherence, different concentrations of compound or DMSO were added for 24 h. Aspirate the cell culture medium and wash the adherent cells with pre-cooled PBS 2 After PBS was added, 0.25% Trypsin-EDTA was added to digest the cells, and the suspension was added to the serum-containing medium. The cells were transferred to a 1.5 ml EP tube, centrifuged at 300 g for 5 min at 4 ° C, and the supernatant was discarded. DCFH-DA (Biyuntian Biotechnology Research Institute) was diluted with 1:1000 in serum-free medium to a final concentration of 10 μM. DCFH-DA itself has no fluorescence and can pass through the cell membrane freely. After entering the cell, it is hydrolyzed into DCFH by the esterase in the cell, and DCFH cannot pass through the cell membrane freely, thus remaining in the cell. The reactive oxygen species in the cells can oxidize the non-fluorescent DCFH into a fluorescent DCF. The level of active oxygen in the cells can be known by detecting the level of DCF. The cell pellet was resuspended in DCFH-DA dilution, blown evenly, and incubated in a 37 °C cell incubator for 30 minutes. Mix once every 10 min to bring the probe and cells into full contact. The cells were washed twice with serum-free cell culture medium to sufficiently remove DCFH-DA that was not loaded with cells. Experiments and analysis were performed using a flow cytometer.

2、实验结果2, the experimental results

结果如图2所示，化合物21在1、3、10μM浓度下，均可显著增加A549细胞内活性氧水平，并具浓度依赖性。As a result, as shown in Fig. 2, Compound 21 significantly increased the level of reactive oxygen species in A549 cells at a concentration of 1, 3, and 10 μM in a concentration-dependent manner.

化合物19在5-10μM时能增加细胞内活性氧水平。 Compound 19 increased intracellular reactive oxygen species levels at 5-10 [mu]M.

实施例39化合物19、21对细胞外乳酸释放的影响Effect of Compounds 19 and 21 of Example 39 on Extracellular Lactic Acid Release

1、实验方法1. Experimental method

处于对数生长期的细胞按合适密度接种至Seahorse XF 96细胞培养板(融合度为80％-90％)，每孔100μL体系，待细胞贴壁后，加入处理(化合物)作用一定时间(24h以内)，每种作用设三个复孔以上。提前一天将一次性固态传感器盒中加入XF Calibrant Solution 200μL/孔，放入无CO₂，37℃的预工作站中，过夜水化。Cells in logarithmic growth phase were inoculated to Seahorse XF 96 cell culture plate (combination degree 80%-90%) at a suitable density, 100 μL per well. After the cells were attached, the treatment (compound) was added for a certain period of time (24h). Within), each effect is set to three or more holes. XF Calibrant Solution 200 μL/well was added to the disposable solid-state sensor box one day in advance, placed in a pre-workstation without CO ₂ , 37 ° C, and hydrated overnight.

Seahorse XF 96主机和程序都要提前开启，过夜以稳定***(温度湿度等)。配置代谢检测液(由于检测的就是O₂和H⁺浓度变化，所以检测液是无缓冲体系，检测液成分为XF Base Medium Minimal DMEM+25mM葡萄糖+2mM丙酮酸)。待处理结束后，小心地吸去孔中培液，用代谢检测液洗涤一次，弃去(为充分去除第一步未吸尽的培液)，加入200μL的代谢检测液。将换好代谢检测液的Seahorse XF 96细胞培养板放入无CO₂，37℃的预工作站中1h，以平衡检测体系。编辑检测程序，并将过夜水化完成的一次性固态传感器盒放入主机，进行***校正。校正完成后，将Seahorse XF 96细胞培养板放入主机按预设的程序进行检测。Both Seahorse XF 96 mainframes and programs must be turned on beforehand to stabilize the system (temperature, humidity, etc.). The metabolic test solution was configured (since the change in the concentration of O ₂ and H ^{+ was} detected, the test solution was an unbuffered system, and the test solution was XF Base Medium Minimal DMEM + 25 mM glucose + 2 mM pyruvate). After the treatment is completed, carefully remove the medium in the well, wash it once with the metabolic test solution, discard it (to fully remove the unwashed liquid in the first step), and add 200 μL of the metabolic test solution. The Seahorse XF 96 cell culture plate with the metabolic test solution was placed in a pre-workstation without CO ₂ at 37 ° C for 1 h to balance the detection system. Edit the test procedure and place the disposable solid-state sensor cartridge that has been hydrated overnight into the main unit for system calibration. After the calibration is complete, place the Seahorse XF 96 cell culture plate in the host and test it according to the preset procedure.

2、实验结果2, the experimental results

结果如图3所示，化合物19和化合物21在1、3、10μM浓度下均可降低A549细胞外乳酸释放水平，并具浓度依赖性。 As a result, as shown in Fig. 3, both Compound 19 and Compound 21 reduced the release level of lactic acid in A549 cells at a concentration of 1, 3, and 10 μM in a concentration-dependent manner.

在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。 All documents mentioned in the present application are hereby incorporated by reference in their entirety in their entireties in the the the the the the the the In addition, it should be understood that various modifications and changes may be made by those skilled in the art in the form of the appended claims.

Claims

一种式X化合物R_a-S-S-R_b (X)的用途，所述用途包括：Use of a compound of formula X R _a -SSR _b (X), said use comprising:

(a)制备抑制丙酮酸脱氢酶激酶(PDHK)活性的药物或制剂，和/或，(a) preparing a medicament or preparation that inhibits pyruvate dehydrogenase kinase (PDHK) activity, and/or,

(b)制备抑制和/或***细胞的药物或制剂；(b) preparing a medicament or preparation for inhibiting and/or treating tumor cells;

其中，R_a、R_b各自独立地为具有S杂原子和N杂原子的端基团，并且所述端基团中，S杂原子和N杂原子构成“S＝C-N”或“S-C＝N”，并且所述的端基团中，当以-S-S-为中心时，所述N杂原子位于所述S杂原子的外侧，并且所述的各S杂原子与所述-S-S-间隔一个碳原子。Wherein R _a and R _b are each independently an end group having an S hetero atom and an N hetero atom, and in the terminal group, the S hetero atom and the N hetero atom constitute "S=CN" or "SC=N" And said end group, when centered on -SS-, said N hetero atom is located outside said S hetero atom, and said each S hetero atom is separated from said -SS- carbon atom.
如权利要求1所述的用途，其特征在于，所述的肿瘤为丙酮酸脱氢酶激酶(PDHK)阳性的肿瘤。The use according to claim 1, wherein the tumor is a pyruvate dehydrogenase kinase (PDHK)-positive tumor.
如权利要求1所述的用途，其特征在于，所述的式X化合物为式X-1化合物，The use according to claim 1 wherein said compound of formula X is a compound of formula X-1.

式中，In the formula,

各R_c分别独立地选自下组：无、取代或未取代的C_1-6烷基；Each R _{c is} independently selected from the group consisting of: unsubstituted, substituted or unsubstituted C _1-6 alkyl;

各R_d分别独立地选自下组：无、取代或未取代的C_1-6烷基、取代或未取代的C_6-10芳基、取代或未取代的C_6-10杂芳基、取代或未取代的C_6-10环烷基、-(CH₂)_m-C_6-10芳基，m为0-4；Each R _{d is} independently selected from the group consisting of unsubstituted, substituted or unsubstituted C _1-6 alkyl, substituted or unsubstituted C _6-10 aryl, substituted or unsubstituted C _6-10 heteroaryl, a substituted or unsubstituted C _6-10 cycloalkyl, -(CH ₂ ) _m -C _6-10 aryl, m is 0-4;

各R_e分别独立地选自下组：无、取代或未取代的C_1-6烷基；Each R _{e is} independently selected from the group consisting of: unsubstituted, substituted or unsubstituted C _1-6 alkyl;

并且，R_c、R_d、R_e满足下列条件：Moreover, R _c , R _d , and R _e satisfy the following conditions:

(1)R_c、R_d与相邻的的N、C和S原子共同形成环C，并且与所述S杂原子连接的
为单键而与N杂原子连接的
为双键；或者(1) R _c , R _d together with adjacent N, C and S atoms form a ring C and are bonded to the S hetero atom
Connected to an N hetero atom for a single bond
For double keys; or

(2)R_e、R_d与相邻的N共同形成取代或未取代的7-12元环D、或取代的5-6元环E，并且与所述S杂原子连接的
为双键而与N杂原子连接的
为单键；(2) R _e , R _d together with an adjacent N form a substituted or unsubstituted 7-12 membered ring D, or a substituted 5-6 membered ring E, and is bonded to the S hetero atom
Connected to an N hetero atom for a double bond
For a single button;

所述杂环具有1-3个选自下组的杂原子：O、S、和N；The heterocyclic ring has 1-3 heteroatoms selected from the group consisting of O, S, and N;

所述取代是指具有一个或多个选自下组的取代基：卤素、C_1-10烷基、卤代C_1-10烷基、C_1-10烷氧基、卤代C_1-10烷氧基、C_1-4烷基羟基、乙酰基、C_1-10酰胺基、C_1-10羧基、C_5-20芳基、卤代C_5-20芳基、-(L₁)p-Z； The substitution means having one or more substituents selected from the group consisting of halogen, C _1-10 alkyl, halogenated C _1-10 alkyl, C _1-10 alkoxy, halogenated C _1-10 Alkoxy, C _1-4 alkylhydroxy, acetyl, C _1-10 amide, C _1-10 carboxyl, C _5-20 aryl, halogenated C _5-20 aryl, -(L ₁ )pZ ;

其中，所述各L₁独立地为：-(CH₂)r、-O-、-C＝O、-CH₂-(C_1-4烷基)-；Wherein each of L _{1 is} independently: -(CH ₂ )r, -O-, -C=O, -CH ₂ -(C _1-4 alkyl)-;

p为0-4，r为0-4；p is 0-4, r is 0-4;

Z选自下组：C_6-20芳基、C_1-11烷基、C_1-10的酰胺基、羟基。Z is selected from the group consisting of C _6-20 aryl, C _1-11 alkyl, C _1-10 amide, and hydroxy.
一种式X化合物、其光学异构体或其药学上可接受的盐，A compound of formula X, an optical isomer thereof, or a pharmaceutically acceptable salt thereof,

R_a-S-S-R_b (X)R _a -SSR _b (X)

其中，R_a、R_b各自独立地为具有S杂原子和N杂原子的端基团，并且所述端基团中，S杂原子和N杂原子构成“S＝C-N”或“S-C＝N”，并且所述的端基团中，当以-S-S-为中心时，所述N杂原子位于所述S杂原子的外侧，并且所述的各S杂原子与所述-S-S-间隔一个碳原子。Wherein R _a and R _b are each independently an end group having an S hetero atom and an N hetero atom, and in the terminal group, the S hetero atom and the N hetero atom constitute "S=CN" or "SC=N" And said end group, when centered on -SS-, said N hetero atom is located outside said S hetero atom, and said each S hetero atom is separated from said -SS- carbon atom.
如权利要求4所述的化合物、其光学异构体或其药学上可接受的盐，其特征在于，所述的式X化合物为式X-1化合物，The compound according to claim 4, an optical isomer thereof or a pharmaceutically acceptable salt thereof, wherein the compound of the formula X is a compound of the formula X-1,

式中，In the formula,

各R_c分别独立地选自下组：无、取代或未取代的C_1-6烷基；Each R _{c is} independently selected from the group consisting of: unsubstituted, substituted or unsubstituted C _1-6 alkyl;

各R_d分别独立地选自下组：无、取代或未取代的C_1-6烷基、取代或未取代的C_6-10芳基、取代或未取代的C_6-10杂芳基、取代或未取代的C_6-10环烷基、-(CH₂)_m-C_6-10芳基，m为0-4；Each R _{d is} independently selected from the group consisting of unsubstituted, substituted or unsubstituted C _1-6 alkyl, substituted or unsubstituted C _6-10 aryl, substituted or unsubstituted C _6-10 heteroaryl, a substituted or unsubstituted C _6-10 cycloalkyl, -(CH ₂ ) _m -C _6-10 aryl, m is 0-4;

各R_e分别独立地选自下组：无、取代或未取代的C_1-6烷基；Each R _{e is} independently selected from the group consisting of: unsubstituted, substituted or unsubstituted C _1-6 alkyl;

并且，R_c、R_d、R_e满足下列条件：Moreover, R _c , R _d , and R _e satisfy the following conditions:

(1)R_c、R_d与相邻的的N、C和S原子共同形成环C，并且与所述S杂原子连接的
为单键而与N杂原子连接的
为双键；或者(1) R _c , R _d together with adjacent N, C and S atoms form a ring C and are bonded to the S hetero atom
Connected to an N hetero atom for a single bond
For double keys; or

(2)R_e、R_d与相邻的N共同形成取代或未取代的7-12元环D、或取代的5-6元环E，并且与所述S杂原子连接的
为双键而与N杂原子连接的
为单键；(2) R _e , R _d together with an adjacent N form a substituted or unsubstituted 7-12 membered ring D, or a substituted 5-6 membered ring E, and is bonded to the S hetero atom
Connected to an N hetero atom for a double bond
For a single button;

所述杂环具有1-3个选自下组的杂原子：O、S、和N；The heterocyclic ring has 1-3 heteroatoms selected from the group consisting of O, S, and N;

所述取代是指具有一个或多个选自下组的取代基：卤素、C_1-10烷基、卤代C_1-10烷基、C_1-10烷氧基、卤代C_1-10烷氧基、C_1-4烷基羟基、乙酰基、C_1-10酰胺基、C_1-10羧基、C_5-20芳基、卤代C_5-20芳基、-(L₁)p-Z；The substitution means having one or more substituents selected from the group consisting of halogen, C _1-10 alkyl, halogenated C _1-10 alkyl, C _1-10 alkoxy, halogenated C _1-10 Alkoxy, C _1-4 alkylhydroxy, acetyl, C _1-10 amide, C _1-10 carboxyl, C _5-20 aryl, halogenated C _5-20 aryl, -(L ₁ )pZ ;

其中，所述各L₁独立地为：-(CH₂)r、-O-、-C＝O、-CH₂-(C_1-4烷基)-；Wherein each of L _{1 is} independently: -(CH ₂ )r, -O-, -C=O, -CH ₂ -(C _1-4 alkyl)-;

p为0-4，r为0-4； p is 0-4, r is 0-4;

Z选自下组：C_6-20芳基、C_1-11烷基、C_1-10的酰胺基、羟基。Z is selected from the group consisting of C _6-20 aryl, C _1-11 alkyl, C _1-10 amide, and hydroxy.
如权利要求4所述的化合物、其光学异构体或其药学上可接受的盐，其特征在于，所述取代基为-(L₁)p-Z，其中，L₁、p、Z的定义如权利要求5所述。The compound according to claim 4, an optical isomer thereof or a pharmaceutically acceptable salt thereof, wherein the substituent is -(L ₁ )pZ, wherein L ₁ , p, Z are as defined Claim 5
如权利要求4所述的化合物、其光学异构体或其药学上可接受的盐，其特征在于，R_a、R_b各自相同或不同地为：
The compound according to claim 4, an optical isomer thereof or a pharmaceutically acceptable salt thereof, wherein R _a and R _b are each the same or different:

n为1-3；n is 1-3;

R₁和R₂各自独立地选自下组：氢、羟基、取代或未取代的C_1-10羧基、取代或未取代的C_1-6酯基、取代或未取代的羟甲基、取代或未取代的C_1-4烷基、取代或未取代的C_1-10烷氧基，且R₁和R₂不同时为氢；R ₁ and R ₂ are each independently selected from the group consisting of hydrogen, hydroxy, substituted or unsubstituted C _1-10 carboxy, substituted or unsubstituted C _1-6 ester, substituted or unsubstituted hydroxymethyl, substituted Or unsubstituted C _1-4 alkyl, substituted or unsubstituted C _1-10 alkoxy, and R ₁ and R _{2 are} not simultaneously hydrogen;

或者，R₁、R₂共同形成饱和或不饱和的5-8元芳环或碳环。Alternatively, R ₁ and R ₂ together form a saturated or unsaturated 5-8 membered aromatic or carbocyclic ring.
如权利要求4所述的化合物、其光学异构体或其药学上可接受的盐，其特征在于，所述式X化合物为式I化合物：The compound according to claim 4, an optical isomer thereof or a pharmaceutically acceptable salt thereof, wherein the compound of the formula X is a compound of the formula I:

式中，n、R₁、R₂的定义如权利要求7所述。Wherein n, R ₁ and R ₂ are as defined in claim 7.
如权利要求4所述的化合物、其光学异构体或其药学上可接受的盐，其特征在于，所述式X化合物为式II化合物The compound according to claim 4, an optical isomer thereof, or a pharmaceutically acceptable salt thereof, wherein the compound of formula X is a compound of formula II

式中，In the formula,

环A选自：取代或未取代的C_5-10碳环、取代或未取代的C_5-10碳杂环、取代或未取代的C_6-10芳环、取代或未取代的C_6-10杂芳环。Ring A is selected from a substituted or unsubstituted C _5-10 carbocyclic ring, a substituted or unsubstituted C _5-10 carbocyclic ring, a substituted or unsubstituted C _6-10 aromatic ring, a substituted or unsubstituted C _{6- 10} heteroaryl rings.
如权利要求4所述的化合物、其光学异构体或其药学上可接受的盐，其特征在于，所述式X化合物选自下组： The compound according to claim 4, an optical isomer thereof or a pharmaceutically acceptable salt thereof, wherein the compound of the formula X is selected from the group consisting of:
如权利要求4所述的化合物、其光学异构体或其药学上可接受的盐，其特征在于，所述式X化合物选自下组：化合物1、8、9、18、19、21、22、25、26、29和33。The compound according to claim 4, an optical isomer thereof or a pharmaceutically acceptable salt thereof, wherein the compound of the formula X is selected from the group consisting of compounds 1, 8, 9, 18, 19, 21, 22, 25, 26, 29 and 33.
一种药物组合物，所述药物组合物中含有a pharmaceutical composition comprising the pharmaceutical composition

(i)权利要求4-11任一所述的式X化合物或其药学上可接受的盐；(i) a compound of formula X according to any one of claims 4-11, or a pharmaceutically acceptable salt thereof;

(ii)药学上可接受的载体。(ii) a pharmaceutically acceptable carrier.
一种制备式I化合物的方法I，其特征在于，包括步骤：A method I for preparing a compound of formula I, comprising the steps of:

(I-2)在惰性溶剂中，将式B化合物与试剂D进行反应，从而形成式I化合物，其中，所述试剂D选自下组：过硫酸铵、高碘酸钠、亚硝酸钠、盐酸、双氧水、硫酸、或其组合；(I-2) reacting a compound of the formula B with a reagent D in an inert solvent to form a compound of the formula I, wherein the reagent D is selected from the group consisting of ammonium persulfate, sodium periodate, sodium nitrite, Hydrochloric acid, hydrogen peroxide, sulfuric acid, or a combination thereof;

(I-3)任选地对步骤(I-2)中形成的式I化合物进行分离或纯化，从而获得分离的或纯化的式I化合物，(I-3) optionally isolating or purifying the compound of formula I formed in step (I-2) to obtain an isolated or purified compound of formula I,

式中，n为1-3；Where n is 1-3;

R₁和R₂各自独立地选自下组：氢、羟基、取代或未取代的C_1-10羧基、取代或未取代的C_1-6酯基、取代或未取代的羟甲基、取代或未取代的C_1-4烷基、取代或未取代的C_1-10烷氧基，且R₁和R₂不同时为氢；R ₁ and R ₂ are each independently selected from the group consisting of hydrogen, hydroxy, substituted or unsubstituted C _1-10 carboxy, substituted or unsubstituted C _1-6 ester, substituted or unsubstituted hydroxymethyl, substituted Or unsubstituted C _1-4 alkyl, substituted or unsubstituted C _1-10 alkoxy, and R ₁ and R _{2 are} not simultaneously hydrogen;

或者，R₁、R₂共同形成饱和或不饱和的5-8元芳环或碳环。Alternatively, R ₁ and R ₂ together form a saturated or unsaturated 5-8 membered aromatic or carbocyclic ring.
如权利要求13所述的方法，其特征在于，所述方法I还包括步骤：The method of claim 13 wherein said method 1 further comprises the step of:

(I-1)在有机溶剂中，将式A化合物与二硫化碳进行反应，从而制得式B化合物，其中，n、R₁、R₂的定义如权利要求13所述。 (I-1) A compound of the formula B is obtained by reacting a compound of the formula A with carbon disulfide in an organic solvent, wherein n, R ₁ and R ₂ are as defined in claim 13.
一种制备式II化合物的方法II，其特征在于，包括步骤：A method II for preparing a compound of formula II, comprising the steps of:

(II-1)在有机溶剂中，将式C化合物与试剂D进行反应，形成式II化合物，其中所述试剂D选自下组：过硫酸铵、高碘酸钠、亚硝酸钠、盐酸、双氧水、硫酸、或其组合，式中，(II-1) reacting a compound of the formula C with a reagent D in an organic solvent to form a compound of the formula II, wherein the reagent D is selected from the group consisting of ammonium persulfate, sodium periodate, sodium nitrite, hydrochloric acid, Hydrogen peroxide, sulfuric acid, or a combination thereof, wherein

环A选自：取代或未取代的C_5-10碳环、取代或未取代的C_5-10碳杂环、取代或未取代的C_6-10芳环、取代或未取代的C_6-10杂芳环。Ring A is selected from a substituted or unsubstituted C _5-10 carbocyclic ring, a substituted or unsubstituted C _5-10 carbocyclic ring, a substituted or unsubstituted C _6-10 aromatic ring, a substituted or unsubstituted C _{6- 10} heteroaryl rings.
一种非治疗性地抑制丙酮酸脱氢酶激酶活性的方法，包括步骤：A method for non-therapeutic inhibition of pyruvate dehydrogenase kinase activity, comprising the steps of:

(a)将丙酮酸脱氢酶激酶与权利要求4-11任一所述的式X化合物、其光学异构体、非消旋体、外消旋体、或其药学上可接受的盐进行接触，从而抑制丙酮酸脱氢酶激酶的活性。 (a) subjecting a pyruvate dehydrogenase kinase to a compound of the formula X according to any one of claims 4 to 11, an optical isomer thereof, a non-racemic form, a racemate, or a pharmaceutically acceptable salt thereof Contact, thereby inhibiting the activity of pyruvate dehydrogenase kinase.