CN109134512B - Laragzole analogue with C-18 fluoro, preparation method thereof and application of Laragzole analogue in preparation of antitumor agent - Google Patents

Abstract

The invention belongs to the field of pharmacy, and relates to a fluoro analog of a marine natural product cyclic lipopeptide (namely a marine natural product Largazole) shown in a formula (I), a preparation method and a pharmaceutical application thereof.

Description

Laragzole analogue with C-18 fluoro, preparation method thereof and application of Laragzole analogue in preparation of antitumor agent

Technical Field

The invention belongs to the field of pharmacy, and relates to a novel fluoro analog of a marine natural product cyclic lipopeptide (namely a marine natural product Largazole), a preparation method thereof, and application of a medicament containing the compound or a composition thereof as an anti-tumor therapeutic agent.

Background

Data show that cancer becomes a serious disease which is subsequent to cardiovascular and cerebrovascular diseases and harms human health, the number of cancer attacks and deaths in China has been on the rise from the 70 th generation of the 20 th century, the number of cancer attacks is estimated to be more 300 million times and the number of deaths in year is estimated to be 250 million times in 2020, and cancer accounts for the first cause of death in urban residents in China, so that research and discovery of low-toxicity and high-efficiency tumor treatment medicines have important clinical and commercial values.

At present, a plurality of anti-tumor drugs are developed internationally, and more than 80 anti-tumor drugs are commonly used clinically. With the continuous and deep research on tumors, people recognize the traditional chemotherapy drugs with cytotoxicity, and while killing the tumor cells, the traditional chemotherapy drugs with cytotoxicity can bring more harm to certain normal tissues, organs and cells of human bodies, such as bone marrow, digestive tract, liver, kidney and the like, which greatly restricts the clinical application of the traditional chemotherapy drugs. The development of new antineoplastic drugs is currently moving from traditional cytotoxic drugs to specific antineoplastic drugs, i.e. molecular targeted therapeutics, directed at aberrant signaling system targets within cancer cells. With the continuous understanding of tumor signal networks, some molecular targeted drugs have been developed and enter clinical application, and remarkable results are achieved. Wherein, Histone Deacetylase (HDACs) is a protein which plays an important role in regulating and controlling the growth of tumor cells. Histone Acetyltransferases (HATs) and Histone Deacetylases (HDACs) are responsible for regulating and controlling the dynamic balance of core histone acetylation and deacetylation, thereby ensuring the normal function of human cells and preventing canceration. However, studies have shown that HDACs are overexpressed in most tumor cells, which results in histone being in a low acetylation state, and imbalance of histone acetylation state is closely related to the occurrence and development of tumors, and HDACs inhibitors are found to achieve the purpose of treating cancer mainly through the action mechanisms of cell cycle arrest, apoptosis induction, angiogenesis inhibition, autophagy induction, synergistic action, and the like.

The HDACs inhibitors found so far are mainly of the following types by structure 1. short chain fatty acids including butyric acid, phenylbutyric acid and isovaleric acid and their salts; 2. hydroximic acids including trichostatin A (TSA) and vorinostat (SAHA) and its derivatives CBHA and MM232, etc.; 3. cyclic tetrapeptide structures without epoxy ketone groups, including FR90I228, apicidin and cyclic tetrapeptide structures containing epoxy ketone groups, including trapoxin B, and the like; 4. amides, including MS-275, CI-994 and cso55, and the like (as shown below).

HDACs share a subset of 18 HDACs in mammalian cells and are classified into the following 4 major classes based on homology to yeast HDAC sequences: the class I HDAC family includes HDAC1, HDAC2, HDAC3 and HDAC8, similar to the yeast Rpd3 protein; the class II HDAC family includes HDAC4, HDAC5, HDAC6, HDAC7, HDAC9 and HDAC10, similar to the yeast Hda1 protein; the HDAC family III has similar sequences with a yeast transcription inhibitor Sir 2; class IV is only HDAC 11. Of these, the HDAC family of I, II and IV are Zn2+ dependent targets, whereas HDAC class III are conserved nicotinamide adenine dinucleotide (NAD +) dependent targets.

Practice shows that most of the existing HDACs inhibitors have poor selectivity for HDACs subtypes, and have exposed more potential adverse reactions, such as basically equivalent activity of Vorinostat (SAHA) on HDACs 1-9, which causes erythropenia, thrombocytopenia, abnormal electrocardiogram and the like, and greatly restricts the clinical efficacy of the inhibitors. With the continuous and intensive research on HDAC and tumorigenesis and development research, especially the continuous disclosure of the structure and function of each subtype of HDACs, a single subtype or a plurality of subtypes belonging to the same class of selective histone deacetylase inhibitors are more advantageous in exerting therapeutic effects and reducing side effects.

The HDACs inhibitor drugs that have been clinically used at present are mainly: vorinostat (SAHA), which has high inhibitory activity on HDAC1, HDAC2, HDAC3, HDAC4, HDAC6, HDAC7, HDAC9 and HDAC10, is approved by the U.S. FDA for the treatment of cutaneous T-lymphomas in 2006, while the hydroxamic acid inhibitor Belinostat, which is an inhibitor of hydroxamic acids, is also approved by the U.S. FDA for clinical use in 2014; romidepsin (FK-228) belongs to a selective HDAC inhibitor of type I, has a better selective inhibition effect on HDAC type I, has stronger inhibition activity on HDAC2 and HDAC1 than on HDAC4 and HDAC6, has a disulfide bond in its structure that is reduced to a thiol group in vivo and then exerts a binding effect on a metal ion, and is approved by the FDA in the united states for clinical treatment of CTTL patients in 2010; sidalaniline, an amide HDACs inhibitor approved for marketing in china 1 month of 2015 for the treatment of Peripheral T Cell Lymphoma (PTCL).

The marine natural product Largazole is a natural product with a sixteen-membered ring peptide lactone structure which is obtained by Hendrink Luesch et al, the natural substance research institute of Florida State university, for the first time, and is proved to be a powerful histone deacetylase inhibitor, particularly has excellent selective inhibition effect on type I histone deacetylase and can effectively inhibit the proliferation of tumor cells, and preclinical studies show that the Largazole with a proper dosage can selectively kill the tumor cells without influencing normal cells (J.Am.chem.Soc.2008,130, 13506). It is similar to romidepsin (FK-228) which has a 16-membered macrocyclic structure, and hydrolysis of its thioester side chain can generate an activated thiol structure similar to that of FK228 which exerts its pharmaceutically active structure in vivo, which can coordinate to histone deacetylase which catalyzes Zn2+ (org. lett.2010,12,1368).

Largazole, because of its unique structure, good pharmacological activity and specific targeting property, has been found to raise the hot tide of structural modification of Largazole, so far, there are a lot of reports on its synthetic modification and metabolic activity (nat. prod. rep.2012,29,449), and meanwhile, the X-diffraction crystal structure of Largazole free thiol and HDAC8 complex is also publicly reported (j.am. chem. soc.2011, 133,12474). However, the Largazole fluoro-analogue is not deeply researched, and a large amount of new drug development researches show that the introduction of F element into an active molecule can increase the activity and the in vivo metabolic stability of the active molecule, which are caused by the following reasons: 1. the sizes of fluorine atoms and hydrogen atoms are very close, and the sizes and the shapes of molecules are hardly changed after the fluorine atoms and the hydrogen atoms are introduced; 2. the introduction of fluorine atoms generates polarity of nonpolar carbon-carbon double bonds (C ═ C); 3. f atoms of strong electronegativity may participate in the formation of hydrogen bonds; 4. the introduction of fluorine atoms can generate strong lipophilicity, and is particularly beneficial to the permeation of cell membranes; 5. the introduction of an F atom into the double bond is more stable and more tolerant to enzymes than a conventional C ═ C double bond. Thus, the introduction of fluorine atoms, especially at the olefinic double bond, in the development of reactive molecules tends to produce unexpected results.

Although Largazole has been shown to be an anti-tumor therapeutic, further structural modifications are necessary to improve its HDACs-inhibiting effect, reduce its toxicity and physicochemical properties. Based on the current state of the art, the inventors of the present application intend to provide a Largazole fluoro analog having an antitumor effect, a preparation method thereof, and a use of a drug containing the compound or a composition thereof as an antitumor therapeutic agent.

Disclosure of Invention

The invention aims to provide a Largazole fluoro analogue with an anti-tumor effect based on the current situation of the prior art, and particularly relates to a novel marine natural product cyclic lipopeptide (namely, a marine natural product Largazole) fluoro analogue, a preparation method thereof, and application of a medicament containing the compound or a composition thereof as an anti-tumor therapeutic agent.

The present invention provides a compound represented by the general formula (I) or a salt thereof:

wherein:

R¹selected from H, R³，R³S，R³CO，R³NHCO；

R²Selected from H, Me, Et, R³S，R³SS，SH，CH₂SR³，CH₂SSR³，CH₂SH，R³O, Bn, or substituted Bn;

R³selected from hydrogen, C₁-C₁₀Alkyl, aryl, wherein C₁-C₁₀May have one or more oxygen or nitrogen atoms embedded in the alkyl chain; or C₁-C₁₀An aromatic group can be inserted into the alkyl chain; or R³Can also be selected from C₁-C₁₀A combination of cyclic alkyl and aryl groups of (a);

R¹and R²A ring may also be formed;

in addition, the invention provides a preparation method of the compound shown in the general formula (I), which is carried out according to the following synthetic route:

wherein the content of the first and second substances,

and a sixth step: preparing corresponding thiazole heterocycle-containing fluoroalkene ester 7 by carrying out a condensation reaction on an S-configuration alcohol 6, wherein the condensation reaction refers to reacting the S-configuration alcohol 6 with an amino acid with an amino protecting group under the action of a base and an acid activator under the conditions of a proper solvent and a reaction temperature to prepare the corresponding thiazole heterocycle-containing fluoroalkene ester 7, wherein the base can be Diisopropylethylamine (DIPEA) or 4-dimethylamino piperidine (DMAP); the activating agent is acyl chloride, HOBT, HOAT, EDCI or DCC; wherein the solvent is non-protonic organic solvent such as tetrahydrofuran, diethyl ether, dichloromethane, DMF, etc., and the reaction temperature is-10-100 ℃;

the seventh step: preparing a macrocyclic compound 8 with a corresponding configuration from the ester 7 through hydrolysis reaction, deprotection reaction and intramolecular condensation ring-closing reaction; the hydrolysis reaction is as follows: under alkaline conditions, in a polar solvent, ester 7 is subjected to selective methyl ester hydrolysis, and the corresponding intermediate acid is obtained through neutralization; the alkaline condition is alkali such as KOH, NaOH, LiOH, Ba (OH)₂Or Bu₃SnOH and the like; the polar solvent is a mixed solvent of 1, 2-dichloroethane, THF, 1, 4-dioxane, DMF, DMSO, methanol, ethanol, isopropanol, water and the like or a combination of the solvents; the reaction conditions include reaction temperature, preferably the reaction temperature is-10-100 ℃;

the deprotection reaction refers to: the corresponding intermediate acid obtained by hydrolysis reaction of the ester 7 is subjected to deprotection reaction to prepare a corresponding carboxyl-containing organic amine compound; the deprotection reaction refers to that the corresponding intermediate acid uses a secondary amine compound as an organic base, such as: diethylamine, morpholine, piperidine and the like in an organic solvent such as dichloromethane, 1, 2-dichloroethane, THF, 1, 4-dioxane, DMF, DMSO, methanol, ethanol or isopropanol and the like, and the amino protecting group in the intermediate can be selectively removed by controlling the reaction temperature to be 10 ℃ below zero to 100 ℃ so as to prepare the corresponding amine compound containing carboxyl;

the intramolecular condensation ring-closing reaction is as follows: ester 7 is hydrolyzed, deprotected to prepare corresponding carboxyl-containing organic amine compound, and then intramolecular condensation cyclization reaction is carried out to prepare corresponding configuration fluoroolefin substituted macrocyclic compound 8, wherein the intramolecular condensation cyclization reaction is as follows: organic amine compound containing carboxyl group prepared by hydrolysis reaction and deprotection reaction is prepared by reacting in the presence of condensing agent such as HATU, HOAT, HOBt, DIPEA or any combination of the three in proper organic solvent such as: the macrocyclic compound 8 of the fluoroolefin with the corresponding configuration can be prepared in solvents such as dichloromethane, 1, 2-dichloroethane, THF, 1, 4-dioxane, DMF, DMSO or acetonitrile and the like, and the reaction temperature is controlled to be-10-100 ℃;

eighth step: macrocyclic compound 8 of fluoroolefin with corresponding configuration is subjected to sulfhydryl protecting group removal reaction and acylation reaction to prepare Largazole fluoroolefin analogue 9, namely the compound shown in the general formula (I):

the removing reaction of the sulfhydryl protecting group refers to that the macrocyclic compound 8 is subjected to the removal of the sulfhydryl protecting group in an organic solvent such as dichloromethane, 1, 2-dichloroethane, THF, 1, 4-dioxane, DMF, DMSO or acetonitrile and the like at a preferred reaction temperature range such as-10-100 ℃ under the single or synergistic action of triisopropylsilane and trifluoroacetic acid to obtain a free thiol intermediate;

the acylation reaction refers to that macrocyclic compound 8 is subjected to a mercapto protecting group removal reaction to prepare a free thiol intermediate, and the free thiol intermediate is subjected to an acylation reaction with an acylating agent in an organic solvent such as dichloromethane, 1, 2-dichloroethane, THF, 1, 4-dioxane, DMF, DMSO or acetonitrile and the like at a preferable reaction temperature range such as-10-100 ℃ under the action of a base to synthesize the Largazole fluoroolefin analogue, wherein the base comprises an inorganic base or an organic base such as NaHCO₃、KHCO₃、K₂CO₃、Na₂CO3、Cs₂CO₃Or triethylamine, diisopropylethylamine, pyridine, DMAP and the like, wherein the acylating agent is C1-10 alkyl acid chloride, aryl acid chloride, C1-10 alkoxy carbonyl chloride, C1-10 alkyl amino carbonyl chloride, aryl acid chloride, aryl oxygen carbonyl chloride, aryl amino carbonyl chloride;

wherein in the above description reference is made to functional groups, chemical reagents or solvent designations, with reference to the international general nomenclature or the common usage, the definitions of functional groups, chemical reagents or solvent designations are as follows:

Ac：Acetyl；

Bn：Benzyl；

Boc：tert-Butoxycarbonyl；

Cbz：Benzyloxycarbonyl；

DIBALH：Diisobutylaluminium hydride；

DCE：Dichloromethane；

DCM：Dichloromethane；

DIPEA：Diisopropylethyamine；

DME：1,2-Ethanedioldimethylether；

DMAP：4-Dimethylamino pyridine；

DMF：N,N-Dimethylformamide；

DMP：Dess-Martin periodinane；

DMSO：Dimethylsulfoxide；

DPPA：Diphenylphosphonic azide；

DMPU：1,3-Dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone；

EA：Ethyl Acetate；

EDCI：Dimethylaminopropyl-N’-enthylcarbodiimide hydrochloride；

Fmoc-Cl：9-Fluorenylmethylchloroformate；

Fmoc：9-Fluorenylmethylformyl；

HATU：2-(7-Aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium

Hexafluorophosphate；

HOAT：1-Hydroxy-7-azabenzotriazole；

HOBT：1-Hydroxybenzotriazole；

LDA：Lithium diisopropylamide；

MeCN：Acetonitrile；

NaHMDS：Sodiumbis(trimethylsilyl)amide；

Py：Pridine；

THF：Tetrahydrofuran；

TIPS：Triisopropylsilane；

TFA：Trifluoroacetic acid；

TMSOTf：Trimethylsilyltrifluoromethanesulfonate；

Tol：Toluene。

the present inventors have performed in vitro assays for the inhibitory activity of HDACs on a subset of the compounds, and examined the inhibitory activity of 6 commercially available HDACs (e.g., HDAC1,2,3,6,8, and 10) separately and compared them to Largazole; the results show that the compound of the invention has strong and selective effect of inhibiting HDACs; IC of representative Compounds for HDACs₅₀The values are shown in Table 1;

TABLE 1

The invention also provides a pharmaceutical composition consisting of the compound and more than one adjuvant, wherein the pharmaceutical composition contains the compound shown in the general formula, and further the pharmaceutical composition is used for inhibiting the cell proliferation of mammals, namely the pharmaceutical composition is used for taking the medicine shown in the general formula with effective treatment dose to the mammals with tumors, wherein the tumors of the mammals comprise solid tumors, cancers, lymphomas, Hodgkin's disease, tumor diseases, new tumor diseases and the like.

The invention provides a pharmaceutical composition, which contains a therapeutically effective dose of a compound with a general formula or a salt thereof and a pharmaceutical carrier, and an application of the pharmaceutical composition in preparing an anti-tumor medicament. In other words, the invention also provides compositions containing an effective amount of the above-mentioned compounds, the salts of the compounds of formula (I) of the invention being in the free form and in the form of acid addition salts or carboxylates. Examples of acid addition salts include inorganic acid salts such as: sulfate, nitrate, hydrobromide, hydroiodide, phosphate, etc., or organic acid salts such as tartrate, acetate, methanesulfonate, benzenesulfonate, toluenesulfonate, citrate, maleate, fumarate, lactate, etc.

Detailed Description

The present invention will be further described with reference to the following examples, which are not intended to limit the scope of the present invention.

Example 1 Synthesis of benzyl substituted Largazole fluoro analogs

First step, preparation of compound 2:

the compound trithiol (1.28g,4.62mmol) and 20ml of anhydrous dichloromethane were added to a 100ml dry reaction flask, dissolved with stirring at room temperature, triethylamine (0.9ml,6.50mmol) was added, followed by addition of acrolein (0.43ml,6.50mmol) dropwise and reaction with stirring at room temperature for 1h (follow-up by TLC). Stirring was stopped and the solvent was spin dried to give a crude white product which was used directly in the preparation of compound 2 without further purification. R_f＝0.13(PE:EA ＝40:1).¹H-NMR(400MHz,CDCl3):δ9.56(brs,1H),7.23–7.43(m,15H),2.47(t, J＝7.0Hz,2H),2.37(t,J＝6.7Hz,2H).

Second step preparation of compound 3:

PPh was added to a 100ml dry reaction flask₃(3.15g,12.0mmol), ethyl dibromofluoroacetate (0.83ml,6.0mmol) and 30ml of anhydrous THF were dissolved with stirring at room temperature, a 1.0M hexane solution of diethyl zinc (12.0ml,12.0mmol) was rapidly dropped thereto, the mixture was stirred for 10min, and then compound 2(1.0g,3.0mmol) was rapidly added thereto, and the reaction was allowed to proceed overnight. Adding 10Quenching with ml anhydrous ethanol, separating out solid, stirring for 10min, concentrating under reduced pressure, adding 100ml anhydrous ether, stirring at room temperature for 30min, filtering with diatomaceous earth, washing with ether, concentrating the filtrate, and performing silica gel column chromatography on the residue (elution condition: PE/EA is 40: 1) to obtain white solid, wherein the E formula structure is 0.325g, and the yield is 26%; the yield of the Z-type structure was 58% and was 0.730 g. The Z-type structure:

R_f＝0.35(PE/EA＝40:1).¹H-NMR(400MHz,CDCl₃):δ7.40(m,6H),7.25(m,6H), 7.18(m,3H),5.75(dt,J＝20.9,7.9Hz,1H),4.23(q,J＝7.1Hz,2H),2.56(m,2H), 2.26(t,J＝7.2Hz,2H),1.29(t,J＝7.1Hz,3H).¹³C-NMR(150MHz,CDCl₃):δ 160.58(d,²J_C-F＝34.5Hz),147.91(d,¹J_C-F＝252Hz),144.65,129.48,127.81,126.60, 121.24(d,²J_C-F＝19.6Hz),66.71,61.28,31.30,24.55(d,³J_C-F＝5.1Hz),14.00. ¹⁹F-NMR(376MHz,CDCl₃):δ-121.29(d,22.6Hz).ESI-MS(M/Z):443.6[M+Na]⁺. HRMS-ESI(M/Z):[M+Na]⁺Calcd.for C₂₆H₂₅FO₂443.1452, found 443.1452. E formula structure:

R_f＝0.27(PE/EA＝40:1).¹H-NMR(400MHz,CDCl₃):δ7.41(m,6H),7.28(m,6H), 7.22(m,3H),5.97(dt,J＝32.8,7.2Hz,1H),4.25(q,J＝7.1Hz,2H),2.25(m,4H), 1.31(t,J＝7.1Hz,3H).¹³C-NMR(150MHz,CDCl₃):δ155.83(d,²J_C-F＝34.5Hz), 143.62(d,¹J_C-F＝256.5Hz),139.93,124.82,123.19,121.99,113.57(d,²J_C-F F＝11.4 Hz),62.15,56.85,25.79,18.90,9.39.¹⁹F-NMR(376MHz,CDCl₃):δ-128.95(d,J＝ 32.8Hz).ESI-MS(M/Z):443.6[M+Na]⁺.HRMS-ESI(M/Z):[M+Na]⁺Calcd.for C₂₆H₂₅FO₂SNa:443.1452,found:443.1454.

third step preparation of compound 4:

a500 ml dry reaction flask was charged with Compound Z-3(4.028g,9.60mmol), and 100ml of dry toluene was added under argon, and dissolved with stirring. Cooled to-78 deg.C, 1.5M diisobutylaluminum hydride (22.0ml,33.5mmol) was added dropwise, after the addition was completed, the temperature was kept for reaction for 1h, quenched by careful addition of 50ml methanol, returned to room temperature, added with 100ml saturated potassium sodium tartrate and stirred at room temperature overnight. Standing for liquid separation, extracting the aqueous phase with EA (100 ml. times.2), combining the organic phases, washing with saturated sodium chloride, drying over anhydrous sodium sulfate, filtering, concentrating, and subjecting the residue to silica gel column chromatography (elution conditions: PE/DCM/EA ═ 35: 5: 1) to give compound 4, wherein 4 of compound Z-configuration is 2.74g of white solid, yield 76%. R_f＝0.35 (PE/DCM/EA＝35:5:1).¹H-NMR(400MHz,CDCl₃):δ9.13(d,J＝18.2Hz,1H),7.43 (m,6H),7.27(m,9H),5.75(dt,J＝32.1,7.2Hz,1H),2.35(m,4H).¹³C-NMR(100 MHz,DMSO-d6):δ183.49(d,J＝24.9Hz),156.48(d,J＝261Hz),146.88,144.53, 129.54,128.71(d,J＝10.2Hz),127.99(d,J＝10.1Hz),127.29,126.86,67.09,30.22, 24.01.¹⁹F-NMR(376MHz,CDCl₃):δ-132.15(dd,J＝32.1,18.2Hz).ESI-MS (M/Z):399.6[M+Na]⁺.HRMS-ESI(M/Z):[M+Na]⁺Calcd.for C₂₄H₂₁FOSNa: 399.1189,found:399.1192.

Fourth step preparation of compound 5:

a250 ml dry reaction flask was charged with compound Z-configured 4(1.12g,4.46mmol), and 50ml of anhydrous dichloromethane was added under argon protection and dissolved with stirring. Titanium tetrachloride (0.82ml,7.43 mmol) was added dropwise under an ice salt bath, and the mixture was stirred to react for 0.5 hour to give an orange suspension. Cooled to-40 ℃, DIPEA (1.23ml, 7.43mmol) was added dropwise and reacted at this temperature for 2 h; the temperature is reduced to-90 ℃, about 20ml of a solution of compound 67(1.44g, 3.71mmol) in dry dichloromethane is slowly added dropwise and the temperature is maintained for reaction for 3h, and 20ml of saturated ammonium chloride solution is added for quenching and the temperature is returned to room temperature. 20ml of water was added, the mixture was allowed to stand for liquid separation, the aqueous phase was extracted with methylene chloride (20 ml. times.3), the organic phases were combined, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was subjected to silica gel column chromatography (elution conditions: PE/EA ═ 8: 1) to give two yellow isomers, compound 5, 5S (1.027g, yield 44%) and compound 5R (0.956g, yield 40%).

Wherein compound 5S:

R_f＝0.19(PE/EA＝4:1).¹H-NMR(400MHz,CDCl₃):δ7.29(m,20H),5.33(m,1H), 4.86(dd,J＝36.8,7.1Hz,1H),4.63(brs,1H),3.67(m,1H),3.47(dd,J＝17.9,8.8 Hz,1H),3.36(dd,J＝11.2,7.2Hz,1H),3.21(m,1H),3.03(m,1H),2.95(d,J＝4.1 Hz,1H),2.87(d,J＝11.6Hz,1H),2.19(m,4H).¹³C-NMR(150MHz,CDCl₃):δ 201.27,172.02,158.60(d,¹J_C-F＝257.2Hz),144.87,136.32,129.60,129.45,128.97, 127.88,127.34,126.63,104.96(d,³J_C-F＝12.9Hz),68.32,66.63,42.81,36.79,32.18, 31.52,22.72.¹⁹F-NMR(376MHz,CDCl₃):δ-129.15(dd,J＝32.1,18.2Hz).ESI-MS (M/Z):650.4[M+Na]⁺.HRMS-ESI(M/Z):[M+Na]⁺Calcd.for C₃₆H₃₄FNO₂S₃Na: 650.1628,found:650.1621.

fifth step preparation of compound 6:

a50 ml dry reaction flask was charged with the compound-substituted thiazolamine described in the above reaction formula (0.603g, 1.636mmol), DMAP (0.520g,4.253mmol) and 20ml of anhydrous dichloromethane, and stirred well at room temperature for 5 min. About 10ml of an anhydrous dichloromethane solution containing compound 5S (1.027g,1.636mmol) was added dropwise thereto, and the reaction was stirred at room temperature for 1 hour (TLC follow-up). Stirring was stopped, the mixture was concentrated, and the residue was subjected to silica gel column chromatography (elution conditions: PE/EA: 1:2) to obtain 0.759g of a white solid in 65% yield. R_f＝0.26(PE/ EA＝1：1).

(c＝0.4,CHCl₃).¹H-NMR(400MHz,CDCl₃):δ7.89(s, 1H),4.79(m,2H),4.68(m,2H),4.49(brs,1H),3.87(d,J＝11.4Hz,1H),3.79(s, 3H),3.27(d,J＝11.4Hz,1H),2.56(m,2H),2.15(m,5H),1.63(s,3H).¹³C-NMR (100MHz,DMSO-d6):δ173.62,171.49,167.63,162.78,158.92(d,J＝257.4Hz), 148.20,144.83,129.58,127.91,126.67,122.56,104.67(d,J＝12.3Hz),84.49,67.09, 66.87,66.62,53.01,41.54,40.80,39.72,31.49,24.02,22.70(d,J＝3.5Hz). ¹⁹F-NMR(376MHz,CDCl₃):δ-124.95(dd,J＝36.2,20.3Hz).ESI-MS(M/Z): 712.4[M+Na]⁺.HRMS-ESI(M/Z):[M+Na]⁺Calcd.for C₃₆H₃₆FN₃O₄S₃Na: 712.1744,found:712.1748.

Sixth step preparation of compound 7:

the compound Fmoc-L-Phe-OH (0.079g,0.20mmol) and DMAP (0.014g,0.11mmol) were put into a 50mL dry and clean single-neck reaction flask, argon gas was introduced into the reaction flask, 15mL of anhydrous dichloromethane was added into a disposable syringe, the mixture was dissolved by stirring in an ice bath, the temperature was reduced to 0 ℃ and 2,4, 6-trichlorobenzoyl chloride (0.04mL,0.25mmol) and DIPEA (0.05mL,0.3mmol) were added dropwise into the reaction flask via a disposable syringe, and the reaction was carried out for 1 hour while maintaining the temperature. After dissolving compound 6(0.280g,0.406mmol) in 5mL of anhydrous THF solution, dropwise adding to the reaction system, returning to room temperature for 6h, stopping stirring, concentrating, and subjecting the crude product to silica gel column chromatography (elution conditions: PE/EA ═ 1:1) to obtain 0.063g of white solid with a yield of 60%.

R_f＝0.26(PE/EA＝1：1).

(c＝0.097,CHCl₃).1HNMR(600MHz, CDCl3)δ7.89(s,1H),7.76(d,J＝7.5Hz,2H),7.53(t,J＝7.2Hz,2H),7.39(d,J＝ 7.4Hz,8H),7.33–7.25(m,9H),7.19(dd,J＝16.7,7.7Hz,6H),7.04(d,J＝6.9Hz, 2H),6.60(s,1H),5.77–5.57(m,1H),5.26(d,J＝7.5Hz,1H),4.81(dt,J＝36.0,7.0 Hz,1H),4.70–4.61(m,2H),4.52(dd,J＝12.9,6.3Hz,1H),4.45–4.27(m,2H), 4.18(t,J＝6.8Hz,1H),3.86(d,J＝11.3Hz,1H),3.78(s,3H),3.25(d,J＝11.3Hz, 1H),3.03(m,J＝48.0,13.9,6.1Hz,2H),2.66(m,J＝19.6,14.9,6.5Hz,2H),2.18(t, J＝6.5Hz,3H),2.15–2.07(m,2H),1.63(s,4H).¹³C NMR(151MHz,CDCl3)δ 172.56(s),169.70(s),167.54(s),167.03(s),162.15(s),154.99(d,J＝11.5Hz), 154.46(d,J＝14.7Hz),152.56(d,J＝74.3Hz),147.53(d,J＝54.5Hz),144.14(s), 143.09(d,J＝14.9Hz),140.69(s),134.75(s),128.94(s),128.73(s),127.94(s), 127.29(s),127.11(s),126.50(d,J＝7.5Hz),126.07(s),124.44(d,J＝7.6Hz), 121.71(s),119.37(s),109.04(d,J＝12.3Hz),83.92(s),69.32(d,J＝29.3Hz),66.25 (d,J＝46.7Hz),54.26(s),52.28(s),46.50(s),40.89(s),40.41(s),37.41(s),37.18 (s),30.43(s),23.36(s),22.31(s)..¹⁹F-NMR(376MHz,CDCl₃):δ-126.27(dd,J＝ 35.4,21.1Hz).ESI-MS(M/Z):1059.2[M+H]⁺.HRMS-ESI(M/Z):[M+Na]⁺Calcd. for C₆₀H₅₅FN₄O₇S₃Na:1081.3109,found:1081.3126.

Seventh step preparation of compound 8:

compound 7(0.37g,0.35mmol) was added to a 100mL clean dry reaction flask, and 45mL THF/H was added₂O is 4:1, dissolved by stirring, cooled to 0 ℃ and 5.25mL of 0.1M LiOH in THF/H was added dropwise₂The reaction was carried out by TLC detection while maintaining the temperature for 1 hour for a mixed solution of O4: 1, and after the completion of the reaction, 5mL of a 0.1M diluted hydrochloric acid solution was added to acidify the reaction solution, EA was extracted (100 mL. times.3), washed with 50mL of saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and the crude product was subjected to silica gel column chromatography (elution conditions: EA: MEOH: AcOH 20:1:1) to obtain 0.256g of a white solid. Dissolving the obtained product in a clean and dry 50mL single-mouth reaction bottle, introducing argon for protection, adding 15mL of anhydrous dichloromethane into a disposable syringe, stirring at room temperature for 5min, dropwise adding diisoamine (1.50mL,14.561mmol) into the disposable syringe, reacting at room temperature overnight, detecting by TLC, and after the reaction is finished, concentrating under reduced pressureAfter condensation, 10mL of anhydrous toluene was added, concentrated under reduced pressure, and repeated twice to remove excess diisoamine. The crude product was made up under argon, HATU (0.157 g,0.412mmol) and HOAT (0.057g,0.416mmol) were added, 400mL of dry dichloromethane were added via a one-shot syringe, DIPEA (0.13mL,0.786mmol) was added dropwise and the reaction was allowed to proceed at room temperature for 30 h. And (3) detecting the reaction by TLC, concentrating after the reaction is finished, and passing the crude product through a silica gel short column (elution conditions: firstly PE/EA is 2: 1, and then washing with EA) to obtain 30mg of white solid, wherein the total yield of the three steps is 11%.

R_f＝0.15(PE/EA＝2:3).

(c＝0.021,CHCl₃)¹H NMR(600MHz, CDCl3)δ7.61(s,1H),7.38(d,J＝7.6Hz,6H),7.27(dd,J＝11.0,4.3Hz,6H),7.20 (t,J＝7.3Hz,3H),7.13(d,J＝7.7Hz,1H),6.94–6.70(m,5H),6.15(d,J＝8.3Hz, 1H),5.71(m,J＝18.9,10.1,1.9Hz,1H),5.05(d，12Hz，1H),5.00(dt,36Hz， 7.5Hz，1H),4.88(m,1H),4.17(dd,J＝17.4,2.8Hz,1H),4.08(d,J＝11.3Hz,1H), 3.25(d,j＝11.3Hz,1H),3.20(dd,J＝14.0,23Hz,1H),3.06(dd,J＝14.0,5.9Hz,1H), 2.99(dd,J＝16.4,10.2Hz,1H),2.62(dd,J＝16.4,2.1Hz,1H),2.17(dt,J＝11.0,4.0 Hz,2H),2.10(dt,J＝14.4,7.2Hz,2H),1.79(s,3H).¹³C NMR(150MHz,CDCl3)δ 173.67(s),168.89(s),168.16(s),166.97(s),163.73(s),155.33(s),153.63(s), 147.26(s),144.78(s),135.11(s),129.60(d,J＝8.8Hz),127.88(d,J＝7.6Hz), 126.65(s),125.93(s),123.68(s),109.17(d,J＝12.5Hz),84.19(s),70.18(d,J＝30.9 Hz),66.61(s),54.19(s),42.46(s),40.98(s),37.70(s),37.46(s),31.12(s),24.98(s), 22.88(d,J＝3.8Hz).¹⁹F-NMR(376MHz,CDCl₃):δ-124.43(dd,J＝36.2,20.3Hz). ESI-MS(M/Z):827.2[M+Na]⁺.HRMS-ESI(M/Z):[M+Na]⁺Calcd.for C₄₄H₄₁FN₄O₄S₃Na:827.2166,found:827.2166.

Step 8 preparation of Compound 9:

adding compound 8(49mg,0.061mmol) into a 50mL clean dry single-mouth reaction bottle, introducing argon for protection, adding 10mL of anhydrous dichloromethane into a disposable syringe, and stirring and dissolving for 30min in an ice-water bath. Triisopropylsilane (25. mu.l, 0.12mmol) was added dropwise at 0 ℃ using a disposable syringe, followed by trifluoroacetic acid (0.27mL,3.7 mmol). Naturally heating to room temperature for reaction for 1h, detecting by TLC, concentrating under reduced pressure, and performing rapid silica gel column chromatography (EA) on a crude product to obtain a white solid 20mg with a yield of 59%.

R_f＝0.42(PE/EA＝2：3).¹HNMR(600MHz,CDCl3)δ7.66(s,1H),7.19(m, 2H),6.84(m,5H),6.15(s,1H),5.78(dd,J＝17.9,9.4Hz,1H),5.16(dt,J＝36.3,7.5 Hz,1H),5.04(dd,J＝17.4,8.2Hz,1H),4.93(m,1H),4.27(d,J＝17.1Hz,1H),4.11 (d,J＝11.2Hz,1H),3.26(d,J＝11.3Hz,1H),3.22(dd,J＝14.0,3.0Hz,1H),3.12– 3.08(m,1H),3.07(d,J＝5.9Hz,1H),2.67(d,J＝14.8Hz,1H),2.54(dd,J＝14.4, 7.1Hz,1H),2.40(m,2H),1.83(s,3H).ESI-MS(M/Z):563.0[M+H]⁺.HRMS-ESI (M/Z):[M+H]⁺Calcd.for C₂₅H₂₇FN₄O₄S₃H:563.1251,found:563.1254.

Adding the mercaptan (0.033g,0.06mmol) into a 50mL clean and dry single-mouth reaction flask, adding 10mL anhydrous dichloromethane into a disposable syringe, stirring for 30min in an ice-water bath, and dropping Et with a microsyringe at 0 deg.C₃N (16uL,0.070mmol) and octanoyl chloride (51uL,0.3mmol) were allowed to return to room temperature. Stirring reaction at room temperature for 4h, TLC tracing reaction, adding 2mL methanol to quench reaction after reaction, concentrating, adding 50mL ethyl acetate to dissolve, washing with saturated sodium bicarbonate (10mL × 1), water (10mL × 1), saturated sodium chloride, drying with anhydrous sodium sulfate, filtering, concentrating, and performing silica gel column chromatography to the residue (elution condition: MeOH: CH)₂Cl₂1:200) to yield 13mg of a white amorphous solid in 61% yield.

R_f＝0.42(DCM/EA＝3:1).

(c＝0.025,CHCl₃).¹HNMR(150MHz, CDCl3)δ7.64(s,1H),7.14(d,J＝7.7Hz,2H),7.00–6.74(m,5H),6.15(d,J＝8.0 Hz,1H),5.76(dd,J＝19.2,9.9Hz,1H),5.12(dt,J＝36.3Hz，7.5Hz,1H),5.06(d,J＝ 9.0Hz,1H),4.92(t,J＝8.1Hz,1H),4.92(t,J＝8.1Hz,1H),4.24(dd,J＝17.4,2.1 Hz,1H),4.10(d,J＝11.2Hz,1H),3.23(d,J＝11.2Hz,1H),3.21(d,J＝2.8Hz, 1H)，3.06(m,2H),2.88(t,J＝7.1Hz,2H),2.66(d,J＝16.0Hz,1H),2.53(t,J＝7.5 Hz,2H),2.36(dd,J＝14.4,7.2Hz,2H),1.83(s,3H),1.64(dd,J＝14.0,7.0Hz,3H), 1.27(m,8H),0.89(dd,J＝9.0,4.6Hz,3H).¹³C NMR(150MHz,CDCl3)δ 198.61(s),173.12(s),168.27(s),167.47(s),166.35(s),163.04(s),146.63(d,J＝ 28.5Hz),134.52(s),129.03(s),127.22(s),125.29(s),123.03(s),107.82(d,J＝23.4 Hz),83.63(s),69.52(d,J＝30.9Hz),53.57(s),43.52(s),41.84(s),40.37(s),37.00 (d,J＝18.3Hz),30.98(s),29.07(s),28.27(s),27.27(s),24.99(s),24.35(s),23.20 (s),21.95(s),13.45(d,J＝8.6Hz).¹⁹F-NMR(376MHz,CDCl₃):δ-124.76(dd,J＝ 36.1,19.3Hz).ESI-MS(M/Z):689.2[M+H]⁺.HRMS-ESI(M/Z):[M+H]⁺Calcd.for C₃₃H₄₁FN₄O₅S₃:689.2296,found:689.2302.。

EXAMPLE 2 Synthesis of methylthiomethyl-substituted Largazole fluoro analog thiol

Synthesis of methylthiomethyl-substituted Largazol fluoro analogs was prepared by the method of example 1 and experimental procedure in which Fmoc-L-Phe-OH was replaced with the corresponding Fmoc-L-MeS-OH in the sixth experimental step. EXAMPLE 3 Synthesis of methylthiomethyl-substituted Largazole fluoro analogs

Synthesis of methylthiomethyl-substituted Largazol fluoro analogs by the procedure and experimental procedures of example 1 in the eighth experimental procedure of example 1, the methylthiomethyl-substituted Largazol fluoro analog thiol obtained in example 2 was used as the starting material.

EXAMPLE 4 Synthesis of de-isopropyl Largazole fluoro analogs

Synthesis of methylthiomethyl substituted Largazol fluoro analogs was prepared according to the procedure and experimental procedure of example 1, substituting Fmoc-L-Phe-OH with the corresponding Fmoc-L-Gly-OH in the sixth experimental step.

Example 5 test experiment

The present inventors have performed in vitro assays for the inhibitory activity of HDACs on a subset of the compounds, and examined the inhibitory activity of 6 commercially available HDACs (e.g., HDAC1,2,3,6,8, and 10) separately and compared them to Largazole;

principle of testing for inhibitory activity: the fluorophore 4-amino-7-coumarin is coupled to the acetylated peptide fragment (Lys-Ac-AMC) when the fluorophore does not produce emission under excitation. The structure after HDAC deacetylation is the reaction site specifically recognized by trypsin by using Lys-Ac-AMC as a substrate, so that AMC is released after enzyme-linked reaction, and emitted light is generated under the excitation light;

the in vitro inhibition HDAC activity test of the compound comprises the following specific steps: HDAC proteins were purchased from BPS Bioscience, modified Tris-HCl (pH 7.0) as the reaction buffer. All small molecule compounds were formulated in 100% DMSO. For HDAC1,2,3,6, HDAC was formulated in buffer at a concentration as an enzyme solution; trypsin and an acetylated peptide fragment substrate coupled with a fluorescent group are prepared in a buffer solution according to a certain concentration to be used as a substrate solution. The compound was added to reaction wells in a 384-well plate at the designed concentration, then 15uL of HDAC enzyme solution was added to the reaction wells, and the reaction was incubated at room temperature for 15 minutes, followed by addition of 10uL of substrate solution to start the reaction, and after incubation at room temperature for 1 hour, the fluorescence intensity (emission wavelength 355nM, absorption wavelength 460nM) was measured with a microplate reader; results data were analyzed by GraphPadPrism software;

for HDAC8,10, HDACs were formulated in buffer at certain concentrations as enzyme solutions; the acetylated peptide fragment substrate coupled with the fluorescent group is prepared in a buffer solution according to a certain concentration to be used as a substrate solution. Adding the compound into reaction wells in a 384-well plate according to the designed concentration, adding 15uL of HDAC enzyme solution into the reaction wells, incubating for 15 minutes at room temperature, adding 10uL of substrate solution to start reaction, incubating for 4 hours at room temperature, adding 15uL of trypsin solution, incubating for 90 minutes at 37 ℃, and measuring the fluorescence intensity (emission wavelength of 355nM, absorption wavelength of 460nM) by a microplate reader; results data were analyzed by GraphPadPrism software;

IC of representative Compounds for HDACs₅₀The results, shown in table 1, show that the compounds of the present invention have a strong and selective effect of inhibiting HDACs.

TABLE 1

Claims (7)

1. A compound or salt thereof:

2. a process for the preparation of the compound according to claim 1, characterized by the following synthetic route:

wherein R is²Is CH₂SMe，R¹Is H;

and a sixth step: preparing corresponding thiazole heterocycle-containing fluoroalkene ester 7 by carrying out condensation reaction on the S-configuration alcohol 6, wherein the condensation reaction refers to the reaction of the S-configuration alcohol 6 and amino acid with an amino protecting group under the action of an aprotic organic solvent and an acid activator at the reaction temperature of-10-100 ℃ to prepare the corresponding thiazole heterocycle-containing fluoroalkene ester 7;

the seventh step: preparing a macrocyclic compound 8 with a corresponding configuration from the ester 7 through hydrolysis reaction, deprotection reaction and intramolecular condensation ring-closing reaction; the hydrolysis reaction is as follows: under alkaline conditions, in a polar solvent, ester 7 is subjected to selective methyl ester hydrolysis, and the corresponding intermediate acid is obtained through neutralization; the reaction temperature is-10-100 ℃;

the deprotection reaction refers to: the corresponding intermediate acid obtained by hydrolysis reaction of the ester 7 is subjected to deprotection reaction to prepare a corresponding carboxyl-containing organic amine compound;

the intramolecular condensation ring-closing reaction is as follows: ester 7 is subjected to hydrolysis reaction and deprotection reaction to prepare a corresponding carboxyl-containing organic amine compound, and then subjected to intramolecular condensation ring-closing reaction to prepare a fluoroolefin-substituted macrocyclic compound 8 with a corresponding configuration;

eighth step: macrocyclic compounds 8 of fluoroolefins of the corresponding configuration are subjected to a thiol protecting group removal reaction to prepare Largazole fluoroolefin analogues 9, i.e. said compounds.

3. A process for the preparation of a compound according to claim 2, wherein,

and a sixth step: wherein the base is Diisopropylethylamine (DIPEA), 4-Dimethylaminopiperidine (DMAP); the acid activating agent is acyl chloride, HOBT, HOAT, EDCI or DCC; the aprotic organic solvent is selected from tetrahydrofuran, diethyl ether, dichloromethane or DMF;

the seventh step: the alkaline condition refers to that the alkali is KOH, NaOH, LiOH, Ba (OH)₂Or Bu₃SnOH; the polar solvent is 1, 2-dichloroethane, THF, 1, 4-dioxane, DMF, DMSO, methanol, ethanol, isopropanol or water or a mixed solvent of the solvent combination;

the deprotection reaction refers to that corresponding intermediate acid uses organic base diethylamine, morpholine or piperidine, in organic solvent dichloromethane, 1, 2-dichloroethane, THF, 1, 4-dioxane, DMF, DMSO, methanol, ethanol or isopropanol, reaction temperature is controlled to be-10-100 ℃, amino protecting groups in the intermediate are selectively removed, and corresponding amine compounds containing carboxyl are prepared;

the intramolecular condensation ring-closing reaction is as follows: in the presence of a condensing agent, wherein the condensing agent is HATU, HOAT, HOBt, DIPEA or any combination of the three, in an organic solvent of dichloromethane, 1, 2-dichloroethane, THF, 1, 4-dioxane, DMF, DMSO or acetonitrile, controlling the reaction temperature to be-10-100 ℃ to prepare a macrocyclic compound 8 of fluoroolefins with corresponding configuration;

eighth step: the removing reaction of the sulfhydryl protecting group means that the macrocyclic compound 8 is subjected to the removing of the sulfhydryl protecting group in organic solvents of dichloromethane, 1, 2-dichloroethane, THF, 1, 4-dioxane, DMF, DMSO or acetonitrile at the reaction temperature of-10-100 ℃ under the single or synergistic action of triisopropylsilane and trifluoroacetic acid.

4. The compound or salt thereof according to claim 1, wherein the acid addition salt comprises an inorganic acid salt and an organic acid salt.

5. The compound or salt according to claim 4, wherein the inorganic acid salt is selected from the group consisting of: sulfates, nitrates, hydrobromides, hydroiodides, phosphates; the organic acid salt is selected from: tartrate, acetate, mesylate, besylate, tosylate, citrate, maleate, fumarate, lactate.

6. A pharmaceutical composition comprising a therapeutically effective amount of a compound of claim 1 or a salt thereof and a pharmaceutically acceptable carrier.

7. Use of the compound of claim 1 or a salt thereof for the preparation of an antitumor agent.