CN103450062B - A dual-target pharmaceutical compound for treating tumors and its preparation method and application - Google Patents

Abstract

The invention discloses a dual-target drug compound for treating tumors and its preparation method and use. Drugs for tumors of cancer and gastric cancer.

Description

A dual-target pharmaceutical compound for treating tumors and its preparation method and application

This application is a divisional application with application number: 201310028119.5, application date: 2013.1.25, and name "Ras and HDAC dual inhibitors and their preparation methods and uses".

technical field

The pharmaceutical field of the present invention, specifically relates to hydroxamic acid farnesyl thiosalicylic acid derivatives and pharmaceutically acceptable salts thereof that can be used as dual inhibitors of Ras and HDAC, their preparation methods, and pharmaceuticals containing these derivatives The compositions and their medical applications especially relate to the prevention, delay or treatment of diseases mediated by Ras or HDAC alone or both, especially in tumor drugs.

Background technique

All-trans farnesylthiosalicylic acid (abbreviation: FTA, trade name: Salirasib), as a new farnesyl transferase-based Ras protein inhibitor, is similar to farnesyl cysteine on the cell membrane in vivo, and can Competitive substitution of F-Ras and F-Ras muteins for galectin binding, inhibition of Ras-initiated downstream signaling pathway Z (including Raf and PI3K signaling pathways) and mTOR (stimulator of tumorigenesis, which can rely on or independently Open the PI3K signaling pathway), thereby inducing tumor cell apoptosis and inhibiting the growth of tumor cells. Studies have shown that FTA can inhibit the proliferation and migration of a variety of tumors (glioma, liver cancer, lung cancer, pancreatic cancer, breast cancer, colon cancer, etc.) (Cancer Chemother Pharmacol, 2008, 61 (1): 89-96; Cancer Chemother Pharmacol, 2009, 65(2): 235-241; J Thorac Oncol, 2011, 6(8): 1435-1437). Although FTA is already in Phase II clinical research, because it cannot effectively prevent and reverse the development of malignant tumors, and the clinical treatment effect is not high, and the dosage is large, it usually needs to be combined with other anti-tumor drugs with cytotoxicity in clinical practice. treat. The reason may be that the inhibition of the Ras protein target alone is difficult to achieve the ideal blocking of the malignant proliferation of tumor cells, and may cause tumor drug resistance.

Farnesylthiosalicylic acid (FTA, Salirasib)

The orderly regulation of gene transcription is the premise for the body cells to maintain normal functions. If the gene transcription regulation function is disordered, the cells may become cancerous. Histone deacetylases (histone deacetylases, HDACs) and histone acetyltransferases (histone acetyltransferases, HATs) are two families in eukaryotic cells that control the acetylation level of histone tails in chromatin, the core histone amino-terminal tail The included lysine residues are HAT and HDAC acetylation and deacetylation substrates, and the acetylation and deacetylation of the ε-amino group of lysine residues represents the main molecular epigenetic mechanism controlling gene expression. In tumor cells, overexpression of HDACs leads to enhanced binding of histones to DNA, which causes chromosome heterogeneity and affects gene transcription. At the same time, overexpressed HDACs can inhibit the expression of cell cycle inhibitor p21 ^CIP1 or p27 ^KIP1 , reduce the stability of tumor suppressor p53, and promote the hypoxia inducible factor-l (HIF-l ) and vascular endothelial growth factor (vascular endothelial growth factor, VEGF) expression. Studies have found that HDACs inhibitors (HDACi) inhibit the enzymatic activity of HDACs, hinder the deacetylation of histones, relax the chromosome structure, and promote the binding of transcription factors and DNA, which can effectively inhibit the proliferation of tumor cells, lead to cell cycle arrest, induce Tumor cell differentiation and apoptosis, improve the sensitivity of radiotherapy and chemotherapy. Therefore, HDACs have become new targets for anticancer drug design, and the development of HDACs inhibitors (HDACi) is regarded as an effective strategy for tumor therapy.

According to clinical studies, the Ras inhibitor FTA can significantly block the growth cycle of tumor cells under the action of HDAC inhibitor valproic acid, inhibit the expression of Survivin (apoptosis inhibitor protein) and the transcription of Aurora A oncogene, especially for the presence of K-ras The curative effect of gene mutation lung and intestinal cancer is more clear (Int.J.Cancer2011, 128, 691–701); Ras inhibitor Sorafenib can not only significantly inhibit Ras/Raf/MEK/ERK and PI3K/ The Akt pathway can also inhibit the expression of anti-apoptotic proteins, synergize with the cell killing effect through Bax configuration changes and translocation to mitochondria, stimulate mitochondria to release cytochrome C, thereby promoting cell apoptosis (Clin. Cancer Res. 2008, 14 (17 ):5385–5399). Based on these studies, we consider the introduction of HDACs inhibitor structural fragments on the carboxyl group of the Ras inhibitor FTA, so that it not only has Ras inhibitory activity, but also targets HDACs therapy, effectively inhibits tumor cell proliferation, induces its differentiation and apoptosis, and leads to cell Cycle arrest, inhibit Ras mutant protein and downstream Ras/Raf/MEK/ERK, PI3K/Akt signaling pathways, so as to obtain Ras and HDACs multi-target anti-tumor drugs with high efficiency, low toxicity and synergistic effect.

In order to obtain compounds with better antitumor activity than FTA, we carried out research on the structural modification of FTA. The invention discloses a class of hydroxamic acid FTA derivatives with dual inhibitory activities of Ras and HDAC and pharmaceutically acceptable salts thereof, which have medicinal value, and there is no report on such compounds so far.

Contents of the invention

The object of the present invention is to provide a dual inhibitor of Ras and HDAC with dual inhibitory activity of Ras and HDAC, its preparation method and application.

Technical solution of the present invention is:

A dual inhibitor of Ras and HDAC is characterized in that it has the structure of the following general formula I:

In general formula Ⅰ: -NH-A-CO- is selected from the configuration of glycine residues, L- or D-type α-alanine residues, β-alanine residues, L- or D-type valine residues amino acid residues, L- or D-type leucine residues, L- or D-type isoleucine residues, L- or D-type methionine residues, L- or D-type half Cystine residues, L-or D-type phenylalanine residues, L-or D-type tyrosine residues, L-or D-type tryptophan residues, L-or D-type sperm Amino acid residues, L- or D-type proline residues, L- or D-type histidine residues; or selected from -NH(CH ₂ ) _n CO-, n=3～7; or selected since m=0～5, R=H, methyl, ethyl, propyl, butyl, pentyl; or selected from or from o=0～5; or selected from

-NH-A-CO- in the structure of the general formula I is selected from the following:

-NH-A-CO-=-NHCH ₂ CO-;

Or -NH-A-CO-=-NH(CH ₂ ) ₂ CO-;

Or -NH-A-CO-=-NH(CH ₂ ) ₃ CO-;

Or -NH-A-CO-=-NH(CH ₂ ) ₄ CO-;

Or -NH-A-CO-=-NH(CH ₂ ) ₅ CO-;

or -NH-A-CO-=

or -NH-A-CO-=

or -NH-A-CO-=

or -NH-A-CO-=

or -NH-A-CO-=

or -NH-A-CO-=

or -NH-A-CO-=

or -NH-A-CO-=

A dual inhibitor of Ras and HDAC is characterized in that it has the structure of the following general formula II:

In the general formula II: -NH-A-CO- is selected from the configuration of glycine residues, L- or D-type α-alanine residues, β-alanine residues, L- or D-type valine residues amino acid residues, L- or D-type leucine residues, L- or D-type isoleucine residues, L- or D-type methionine residues, L- or D-type half Cystine residues, L-or D-type phenylalanine residues, L-or D-type tyrosine residues, L-or D-type tryptophan residues, L-or D-type sperm Amino acid residues, L- or D-type proline residues, L- or D-type histidine residues; or selected from -NH(CH ₂ ) _n CO-, n=3～7; or selected since or from o=0～5; or selected from X is selected from -O-, or from -NH-, or from -NCH ₃ -; m=1-8.

-NH-A-CO-, X and m in the structure of the general formula II are selected from the following combinations:

-NH-A-CO-=-NHCH ₂ CO-, X=NH, m=3;

or -NH-A-CO-= X=NH, m=2;

or -NH-A-CO-= X=NH, m=1;

Or -NH-A-CO-=-NHCH ₂ CO-, X=NH, m=1;

Or -NH-A-CO-=-NHCH ₂ CO-, X=NH, m=2;

A method for preparing a dual inhibitor of Ras and HDAC, characterized in that:

The preparation method of the general formula I comprises the following steps:

A. First prepare FTA acid chloride (1) by FTA under the action of thionyl chloride;

B. Reaction with H ₂ NA-COOMe in triethylamine in dichloromethane solution to obtain compound (2);

C. compound (2) reacts with hydroxylamine hydrochloride under the methanol solution of potassium hydroxide to obtain general formula Ⅰ;

The reaction scheme of general formula I is as follows:

Among them, in the general formula I: -NH-A-CO- is selected from the configuration of -NH-A-CO- selected from the configuration of glycine residues, L- or D-type α-alanine residues, β - Alanine residues, L- or D-type valine residues, L- or D-type leucine residues, L- or D-type isoleucine residues, L- or D-type Methionine residues, L- or D-cysteine residues, L- or D-phenylalanine residues, L- or D-tyrosine residues, L- or D- Type tryptophan residues, L- or D-type arginine residues, L- or D-type proline residues, L- or D-type histidine residues; or selected from -NH(CH ₂ ) _n CO-, n=3～7; or selected from m=0～5, R=H, methyl, ethyl, propyl, butyl, pentyl; or selected from or from o=0～5; or selected from

A method for preparing a dual inhibitor of Ras and HDAC, characterized in that:

The preparation method of the general formula II comprises the following steps:

A. Compound (2) is hydrolyzed in methanol solution containing NaOH to obtain compound (3);

B. Compound (3) reacts with HX(CH2) under the action of condensing agent 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and 4-dimethylaminopyridine (DMAP) mCOOMe reacts to generate compound (4);

C. The last compound (4) reacts with hydroxylamine hydrochloride under the methanol solution of potassium hydroxide to obtain general formula II;

The general formula II reaction scheme is as follows:

Among them, in the general formula II: -NH-A-CO- is selected from the configuration of glycine residues, L- or D-type α-alanine residues, β-alanine residues, L- or D- Valine residues, L-or D-leucine residues, L-or D-type isoleucine residues, L-or D-methionine residues, L-or D- Cysteine residues, L- or D-phenylalanine residues, L- or D-tyrosine residues, L- or D-tryptophan residues, L- or D- Arginine residues, L- or D-proline residues, L- or D-histidine residues; or selected from -NH(CH ₂ ) _n CO-, n=3～7; or from or from o=0～5; or selected from X is selected from -O-, or from -NH-, or from -NCH ₃ -; m=1-8.

A pharmaceutical composition is characterized in that it is a pharmaceutical composition containing the compound of general formula I or II.

The application of a dual inhibitor of Ras and HDAC in the preparation of tumor drugs for treating chronic inflammation, liver cancer, pancreatic cancer, lung cancer, breast cancer, brain cancer, colon cancer and stomach cancer.

The application of a dual inhibitor of Ras and HDAC in the preparation of tumor drugs for treating chronic inflammation, liver cancer, pancreatic cancer, lung cancer, breast cancer, brain cancer, colon cancer and stomach cancer.

Specifically, the hydroxamic acid FTA derivatives shown in general formula I are preferably selected from the following compounds:

N-(2-(hydroxyamino)-2-oxaethyl)-farnesylthiosalicylic acid amide (compound number: Ⅰ ₁ , the same below)

N-(3-(hydroxyamino)-3-oxapropyl)-farnesylthiosalicylic acid amide (Ⅰ ₂ )

N-(4-(hydroxyamino)-4-oxabutyl)-farnesylthiosalicylic acid amide (Ⅰ ₃ )

N-(5-(hydroxyamino)-5-oxapentyl)-farnesylthiosalicylic acid amide (Ⅰ ₄ )

N-(6-(hydroxyamino)-6-oxahexyl)-farnesylthiosalicylic acid amide (Ⅰ ₅ )

(S)-N-(1-(hydroxyamino)-1-oxapropyl-2-yl)-farnesylthiosalicylic acid amide (Ⅰ ₆ )

(S)-N-(1-(hydroxyamino)-3-methyl-1-oxabutyl-2-yl)-farnesylthiosalicylic acid amide (Ⅰ ₇ )

(S)-N-(1-(hydroxyamino)-4-methylthio-1-oxabutyl-2-yl)-farnesylthiosalicylic acid amide (Ⅰ ₈ )

(S)-N-(1-(hydroxyamino)-4-methyl-1-oxapentyl-2-yl)-farnesylthiosalicylic acid amide (Ⅰ ₉ )

(S)-N-(1-(hydroxyamino)-3-methyl-1-oxapentyl-2-yl)-farnesylthiosalicylic acid amide (Ⅰ ₁₀ )

(E)-N-(4-(3-(hydroxyamino)-3-oxa-1-propenyl)phenyl)-farnesylthiosalicylic acid amide (Ⅰ ₁₁ )

N-(4-(3-(hydroxyamino)-3-oxapropyl)phenyl)-farnesylthiosalicylic acid amide (Ⅰ ₁₂ )

N-(4-(hydroxycarbamoyl)phenyl)-farnesylthiosalicylic acid amide (Ⅰ ₁₃ )

N-(4-(hydroxycarbamoyl)benzyl)-farnesylthiosalicylic acid amide (Ⅰ ₁₄ )

The hydroxamic acid FTA derivatives shown in the general formula II are preferably selected from the following compounds:

N-(2-(4-(hydroxylamine)-4-oxabutylamino)-2-oxaethyl)-farnesylthiosalicylic acid amide (Ⅱ ₁ )

(S)-N-(1-(3-(hydroxylamine)-3-oxapropylamino)-1-oxapropyl-2-yl)-farnesylthiosalicylic acid amide (Ⅱ ₂ ) ( S)-N-(1-(2-(Hydroxylamine)-2-oxaethylamino)-3-methyl-1-oxabutyl-2-yl)-farnesylthiosalicylic acid amide (II ₃ )

N-(2-(2-(hydroxylamine)-2-oxaethylamino)-2-oxaethyl)-farnesylthiosalicylic acid amide (Ⅱ ₄ )

N-(2-(3-(hydroxylamine)-3-oxapropylamino)-2-oxaethyl)-farnesylthiosalicylic acid amide (Ⅱ ₅ )

The preferred compound codes of the general formula I above and their corresponding structures are shown in Table 1

Table 1 Preferred compound codes of general formula I and their corresponding structures

The preferred compound codes of the general formula II above and their corresponding structures are shown in Table 2

Table 2 Preferred compound codes of general formula II and their corresponding structures

Said compound includes all conformational isomers, optical isomers and racemates, diastereoisomers, tautomers and stereoisomers of compounds of general formula I and II, as well as the above forms hybrid.

The compounds of the present invention can be formulated alone or in combination with one or more pharmaceutically acceptable carriers for administration. For example, solvents, diluents, etc., can be administered in oral dosage forms, such as tablets, capsules, dispersible powders, granules, and the like. Various dosage forms of the pharmaceutical composition of the present invention can be prepared according to well-known methods in the field of pharmacy. These pharmaceutical formulations may contain, for example, 0.05% to 90% by weight, more usually between about 15% and 60% by weight of active ingredient in combination with a carrier. The dosage of the compound of the present invention can be 0.005-5000 mg/kg/day, and the dosage can also be used beyond this dosage range according to the severity of the disease or different dosage forms.

The compounds of the present invention can be combined with other antineoplastic drugs such as alkylating agents (such as cyclophosphamide or cisplatin), antimetabolites (such as 5-fluorouracil or hydroxyurea), topoisomerase inhibitors (such as camptothecin), mitotic Inhibitors (such as paclitaxel or vinblastine), DNA intercalating agents (such as doxorubicin) are used in combination, and radiation therapy can also be used in combination. These other antineoplastic drugs or radiation therapy may be administered simultaneously or at different times with the compounds of the present invention. These combination treatments can produce synergistic effects that can help improve therapeutic outcomes.

The part pharmacological test result of compound of the present invention is as follows:

(1) Tetramethylazolium blue colorimetric method (MTT) in vitro anti-tumor test

The results of pharmacological experiments show that the compounds of the present invention have different degrees of inhibitory effects on the proliferation of human tumor cells, most of the compounds have significantly stronger anti-tumor activities than the lead compound FTA, and most of the compounds have slightly stronger or equivalent anti-tumor activities than the positive control drug SAHA. After a series of tumor cell tests, it was found that these compounds had strong effects on pancreatic cancer cells PANC-1, liver cancer cells SMMC-7721 and human glioma cells U251, especially Ⅰ _1-3 , Ⅰ _11-12 and Ⅱ in Table 4 The inhibitory rate of compounds _1-2 at the concentration of 25μmol/L was much higher than that of the lead FTA.

Table 3 The inhibition rate % (25 μ mol/L) of some compounds of the present invention to tumor cell proliferation

ND: Not detected.

(2) Ras downstream p-Raf, p-Akt, p-ERK inhibitory activity test

The experimental results showed that compounds Ⅰ ₁ - Ⅰ ₁₄ or compounds Ⅱ 1 - Ⅱ 5 all inhibited Ras downstream p-Raf, p-Akt, and p-ERK to varying degrees, and retained the original inhibitory activity of the mother nuclear FTA on Ras downstream signaling pathways , in which compounds I ₁ -I ₃ , I ₁₁ -I ₁₄ , and II ₂ can significantly inhibit the phosphorylation of Akt, ERK, and Raf molecules at concentrations of 6.125 μM and 12.5 μM, suggesting that the new hydroxamic acid FTA derivatives still retain Inhibitory activity on Ras downstream signaling pathway.

(3) HDACs inhibitory activity test

The experimental results show that compounds Ⅰ ₁ - Ⅰ ₁₄ or compounds Ⅱ ₁ - Ⅱ ₅ have different degrees of inhibitory activity on HDACs, among which compounds Ⅰ ₁ - _{Ⅰ 3} , Ⅰ ₆ , Ⅰ ₁₁ , Ⅰ ₁₄ , and Ⅱ ₂ have HDACs inhibitory activity data As shown in Table 4, compounds I ₁ -I ₃ , I ₁₁ , I ₁₄ , and II ₂ all showed slightly stronger or equivalent inhibitory activity than the positive control drug SAHA, suggesting that the new hydroxamic acid FTA derivatives not only have the ability to inhibit Ras downstream signals Pathway inhibitory activity, and HDACs inhibitory activity, so as to obtain multi-target anti-tumor effect of Ras and HDACs with synergistic effect.

Table 4 Some compounds of the present invention in vitro enzyme inhibition test results

Detailed ways

In order to further clarify the present invention, a series of examples are given below, these examples are completely illustrative, they are only used to specifically describe the present invention, and should not be construed as limiting the present invention. The FTA used in the present invention is prepared in a laboratory, and the content is >98%.

Example 1 Preparation of N-(2-(hydroxylamino)-2-oxaethyl)-farnesylthiosalicylic acid amide (I ₁ )

Preparation of farnesylthiosalicylic acid chloride (1)

Dissolve 0.36g (1.00mmol) of FTA in 10mL of anhydrous CH ₂ Cl ₂ , add 0.40mL (5.51mmol) of thionyl chloride to it, stir at 55°C for 1 hour, and concentrate to obtain a yellow oily farnesylthio Salicylic Acid Chloride (1).

Preparation of N-(2-(methoxy)-2-oxaethyl)-farnesylthiosalicylic acid amide (2a)

Dissolve 0.09g (1.00mmol) of glycine methyl ester and 0.2mL (1.50mmol) of triethylamine in 5mL of anhydrous CH ₂ Cl ₂ , add dropwise the 10 mL of anhydrous CH ₂ Cl ₂ solution prepared above under ice-cooling, Afterwards, the reaction was stirred at room temperature for 1.5 h, and the reaction solution was washed with 10 mL of water and saturated NaCl solution, and CH ₂ Cl ₂ was dried with anhydrous sodium sulfate, filtered, and spin-dried to obtain 0.37 g of a yellow oil with a yield of 86%.

Preparation of N-(2-(hydroxyamino)-2-oxaethyl)-farnesylthiosalicylic acid amide (Ⅰ ₁ )

Dissolve 1.74g (25mmol) of hydroxylamine hydrochloride in 10mL of methanol, slowly add 1.40g (25mmol) of potassium hydroxide in 10mL of methanol solution under ice-cooling, stir at room temperature for 1h and then filter, add 0.21g (0.5 mmol) 5 mL of methanol solution of compound (2a), stirred at room temperature for 0.5 h, then added 0.06 g (1.0 mmol) of potassium hydroxide, continued to react at room temperature for 24 h, concentrated, and column chromatography gave 0.16 g of oily substance, yield 69%.

¹ H NMR(CDCl ₃ ,300MHz):δ7.80(d,1H,J=7.8Hz,Ar-H),7.24(m,2H,Ar-H),7.12(m,1H,Ar-H), 5.22(m,1H,SCH ₂ CH ),5.01(m,2H,2×CH ₂ CH =CCH ₃ ),4.56(m,2H,NHC H ₂ ),3.82(d,2H,J=7.2Hz , SC H ₂ ), 2.10-1.76 (m, 8H, 2× CHCH ₂ CH ₂ CH), 1.69-1.56 (m, 12H, 4×CH ₃ ); ESI-MS (m/z): 431[ M+H] ⁺ .

Example 2 Preparation of N-(3-(hydroxylamino)-3-oxapropyl)-farnesylthiosalicylic acid amide (I ₂ )

Preparation of N-(3-(methoxy)-3-oxapropyl)-farnesylthiosalicylic acid amide (2b)

With reference to the preparation method of N-(2-(methoxy)-2-oxaethyl)-farnesylthiosalicylic acid amide (2a) in Example 1, the method is replaced by methyl 3-alanine Glycine methyl ester in (1) was reacted with (1) to obtain yellow oily substance (2b) with a yield of 85%.

Preparation of N-(3-(hydroxyamino)-3-oxapropyl)-farnesylthiosalicylic acid amide (Ⅰ ₂ )

Referring to the preparation method of I ₁ in Example 1, the oily substance (I ₂ ) was obtained by replacing 2a in the method 2b with hydroxylamine hydrochloride, and the yield was 73%.

¹ H NMR (CDCl ₃ , 300MHz): δ8.24(m,1H,NH),8.09(m,1H,NH),7.65(m,2H,ArH),7.37(m,2H,ArH),5.41( m,1H,SCH ₂ CH ) ,5.18(m,2H,2×CH ₂ CH =CCH ₃ ),3.76(d,2H,J=7.2Hz,SC H ₂ ),3.18(m,4H,2 ×CH ₂ ),2.05-1.83(m,8H,2×CHC H ₂ CH ₂ CH),1.69-1.55(m,12H,4×CH ₃ ); ESI-MS(m/z):445[M +H] ⁺ .

Example 3 Preparation of N-(4-(hydroxylamino)-4-oxabutyl)-farnesylthiosalicylic acid amide (I ₃ )

Preparation of N-(4-(methoxy)-4-oxabutyl)-farnesylthiosalicylic acid amide (2c)

With reference to the preparation method of N-(2-(methoxy)-2-oxaethyl)-farnesylthiosalicylic acid amide (2a) in Example 1, the method is replaced by methyl 4-aminobutyrate Glycine methyl ester in (1) was reacted with (1) to obtain yellow oil (2c), the yield was 83%.

Preparation of N-(4-(hydroxyamino)-4-oxabutyl)-farnesylthiosalicylic acid amide (Ⅰ ₃ )

Referring to the preparation method of I ₁ in Example 1, the oily substance (I ₃ ) was obtained by replacing 2a in the method 2c with hydroxylamine hydrochloride, and the yield was 70%.

¹ H NMR(CDCl ₃ ,300MHz):δ7.98(d,1H,J=7.8Hz,Ar-H),7.60-7.57(m,2H,Ar-H),7.39(m,1H,Ar-H ),5.46(m,1H,SCH ₂ CH ),5.20(m,2H,2×CH ₂ CH =CCH ₃ ),3.78(d,2H,J=7.2Hz, SCH ₂ ),3.52(m ,2H, _NHC H ₂ ),2.34(m,2H, CH ₂ CONH),2.21(m,2H,NHCH ₂ CH ₂ ),2.00-1.87(m,8H,2×CHCH 2 CH ₂ CH ),1.68-1.57(m,12H,4×CH ₃ ); ESI-MS(m/z):459[M+H] ⁺ .

Example 4 Preparation of N-(5-(hydroxyamino)-5-oxapentyl)-farnesylthiosalicylic acid amide (I ₄ )

Preparation of N-(5-(methoxy)-5-oxapentyl)-farnesylthiosalicylic acid amide (2d)

Referring to the preparation method of N-(2-(methoxy)-2-oxaethyl)-farnesylthiosalicylic acid amide (2a) in Example 1, the method is replaced by methyl 5-aminovalerate Glycine methyl ester in (1) was reacted with (1) to obtain yellow oil (2d), with a yield of 77%.

Preparation of N-(5-(hydroxyamino)-5-oxapentyl)-farnesylthiosalicylic acid amide (Ⅰ ₄ )

Referring to the preparation method of I ₁ in Example 1, the oily substance (I ₄ ) was obtained by reacting 2a in 2d instead of 2a with hydroxylamine hydrochloride, and the yield was 71%.

¹ H NMR(CDCl ₃ ,300MHz):δ7.88(d,1H,J=7.8Hz,Ar-H),7.67-7.58(m,2H,Ar-H),7.32(m,1H,Ar-H ),5.34(m,1H,SCH ₂ CH ),5.28(m,2H _, 2×CH ₂ CH =CCH ₃ ),3.81(d,2H,J=7.2Hz,SCH 2 ),3.40(m ,2H,NHC H ₂ ),2.32(m,2H, CH ₂ CONH),2.01-1.89(m,8H,2×CHC H ₂ CH ₂ CH),1.59-1.70(m,12H,4×CH ₃ ),1.56(m,2H,NHCH ₂ CH ₂ ),1.53(m,2H, CH ₂ CH ₂ CONH);ESI-MS(m/z):473[M+H] ⁺ .

The preparation of embodiment 5N-(6-(hydroxylamino)-6-oxahexyl)-farnesyl thiosalicylic acid amide (I5)

Preparation of N-(6-(methoxy)-6-oxahexyl)-farnesylthiosalicylic acid amide (2e)

Referring to the preparation method of N-(2-(methoxy)-2-oxaethyl)-farnesylthiosalicylic acid amide (2a) in Example 1, the method is replaced by methyl 6-aminocaproate Glycine methyl ester in (1) was reacted with (1) to obtain yellow oil (2e), with a yield of 72%.

Preparation of N-(6-(hydroxyamino)-6-oxahexyl)-farnesylthiosalicylic acid amide (Ⅰ ₅ )

Referring to the preparation method of I ₁ in Example 1, the oil (I ₅ ) was obtained by replacing 2a in the method 2e with hydroxylamine hydrochloride, and the yield was 73%.

¹ H NMR(CDCl ₃ ,300MHz):δ8.11(d,1H,J=7.8Hz,Ar-H),7.60-7.58(m,2H,Ar-H),7.31(m,1H,Ar-H ),5.90(m,1H,SCH ₂ CH ),5.15(m,2H,2×CH ₂ CH =CCH ₃ ),3.74(d,2H,J=7.2Hz, SCH ₂ ),3.54(m ,2H,NHC H ₂ ),2.43(m,2H, CH ₂ CONH),2.13-1.82(m,8H,2×CHC H ₂ CH ₂ CH),1.70-1.59(m,12H,4×CH ₃ ),1.56(m,2H,NHCH ₂ CH ₂ ),1.32(m,2H, CH ₂ CH ₂ CONH);ESI-MS(m/z):487[M+H] ⁺ .

Example 6 Preparation of (S)-N-(1-(hydroxylamino)-1-oxapropyl-2-yl)-farnesylthiosalicylic acid amide (I6)

Preparation of (S)-N-(1-(methoxy)-1-oxapropyl-2-yl)-farnesylthiosalicylic acid amide (2f)

Referring to the preparation method of N-(2-(methoxy)-2-oxaethyl)-farnesyl thiosalicylic acid amide (2a) in Example 1, it is replaced by L-alpha alanine methyl ester The glycine methyl ester in the method is reacted with (1) to obtain a yellow oily substance (2f), and the yield is 70%.

Preparation of (S)-N-(1-(hydroxyamino)-1-oxapropyl-2-yl)-farnesylthiosalicylic acid amide (Ⅰ ₆ )

Referring to the preparation method of I ₁ in Example 1, the oily substance (I ₆ ) was obtained by replacing 2a in the method 2f with hydroxylamine hydrochloride, and the yield was 65%.

¹ H NMR(CDCl ₃ ,300MHz):δ7.84(d,1H,J=7.8Hz,Ar-H),7.66-7.55(m,2H,Ar-H),7.35(m,1H,Ar-H ),5.42(m,1H,SCH ₂ CH ),5.25(m,2H,2×CH ₂ CH =CCH ₃ ),4.71(m,1H,NHC H ),3.81(m,2H, SCH ₂ ),2.01-1.87(m,8H,2×CHCH H ₂ CH ₂ CH) _, 1.78-1.46(m,12H,4×CH=CC H ₃ ),1.48(m,3H,NHHCCH 3 );ESI -MS(m/z):445[M+H] ⁺ .

Example 7 Preparation of (S)-N-(1-(hydroxyamino)-3-methyl-1-oxabutyl-2-yl)-farnesylthiosalicylic acid amide (I ₇ )

Preparation of (S)-N-(1-(methoxy)-3-methyl-1-oxabutyl-2-yl)-farnesylthiosalicylic acid amide (2g)

With reference to the preparation method of N-(2-(methoxy)-2-oxaethyl)-farnesylthiosalicylic acid amide (2a) in Example 1, the method is replaced by L-valine methyl ester Glycine methyl ester in (1) was reacted to obtain a yellow oil (2g), the yield was 65%.

Preparation of (S)-N-(1-(hydroxyamino)-3-methyl-1-oxabutyl-2-yl)-farnesylthiosalicylic acid amide (Ⅰ ₇ )

Referring to the preparation method of I ₁ in Example 1, the oil (I ₇ ) was prepared by reacting 2 g of 2a in the alternative method with hydroxylamine hydrochloride, with a yield of 68%.

¹ H NMR(CDCl ₃ ,300MHz):δ8.02(d,1H,J=7.8Hz,Ar-H),7.78-7.40(m,2H,Ar-H),7.23(m,1H,Ar-H ),5.44(m,1H,SCH ₂ CH ),5.04(m,2H,2×CH ₂ CH =CCH ₃ ),4.53(m,1H,NHC H ),3.80(d,2H,J=7.2 Hz, SC H ₂ ), 2.03-1.78(m, 9H, 2× CHCH ₂ CH ₂ CH,NHCHC H ), 1.71-1.60(m, 12H, 4×CH=CC H ₃ ), 0.94(m, 6H, CH( CH ₃ ) ₂ ); ESI-MS(m/z): 473[M+H] ⁺ .

Example 8 Preparation of (S)-N-(1-(hydroxyamino)-4-methylthio-1-oxabutyl-2-yl)-farnesylthiosalicylic acid amide (I ₈ )

Preparation of (S)-N-(1-(methoxy)-4-methylthio-1-oxabutyl-2-yl)-farnesylthiosalicylic acid amide (2h)

Referring to the preparation method of N-(2-(methoxy)-2-oxaethyl)-farnesylthiosalicylic acid amide (2a) in Example 1, it is replaced by L-methionine methyl ester The glycine methyl ester in the method is reacted with (1) to obtain a yellow oil (2h), and the yield is 69%.

Preparation of (S)-N-(1-(hydroxyamino)-4-methylthio-1-oxabutyl-2-yl)-farnesylthiosalicylic acid amide (Ⅰ ₈ )

Referring to the preparation method of I1 in Example 1, the oily substance (I ₈ ) was obtained by reacting 2a in the 2h substitution method with hydroxylamine hydrochloride, and the yield was 72%.

¹ H NMR(CDCl ₃ ,300MHz):δ7.95(d,1H,J=7.6Hz,Ar-H),7.70-7.54(m,2H,Ar-H),7.14(m,1H,Ar-H ),5.60(m,1H,SCH ₂ CH ),5.32(m,2H,2×CH ₂ CH =CCH ₃ ),4.81(m,1H,NHC H ),3.91(m,2H, SCH ₂ CH=CCH ₃ ),2.46(t,2H,J=7.6Hz, CH ₂ SCH ₃ ),2.13(s,3H,SCH ₃ ),2.08-1.80(m,10H,CHCH 2 _, 2×CHCH 3 ₂ C H ₂ CH),1.70-1.40(m,12H,4×CH=CC H ₃ ); ESI-MS(m/z):505[M+H] ⁺ .

Example 9 Preparation of (S)-N-(1-(hydroxyamino)-4-methyl-1-oxapentyl-2-yl)-farnesylthiosalicylic acid amide (I ₉ )

Preparation of (S)-N-(1-(methoxy)-4-methyl-1-oxapentyl-2-yl)-farnesylthiosalicylic acid amide (2i)

Referring to the preparation method of N-(2-(methoxy)-2-oxaethyl)-farnesyl thiosalicylic acid amide (2a) in Example 1, the method is replaced by L-leucine methyl ester Glycine methyl ester in (1) was reacted with (1) to obtain yellow oil (2i), with a yield of 66%.

Preparation of (S)-N-(1-(hydroxyamino)-4-methyl-1-oxapentyl-2-yl)-farnesylthiosalicylic acid amide (Ⅰ ₉ )

Referring to the preparation method of I ₁ in Example 1, the oil (I ₉ ) was obtained by replacing 2a in the method 2i with hydroxylamine hydrochloride, and the yield was 71%.

¹ H NMR(CDCl ₃ ,300MHz):δ8.05(d,1H,J=7.8Hz,Ar-H),7.82-7.52(m,2H,Ar-H),7.48(m,1H,Ar-H ),5.39(m,1H,SCH ₂ CH ),5.13(m,2H,2×CH ₂ CH =CCH ₃ ),4.26(m,1H,NHC H ),3.95(d,2H,J=7.2 Hz,SC H ₂ ),,2.21-1.80(m,10H,2× CHCH ₂ CH ₂ CH,NHCHC H ₂ ),1.68-1.44(m,12H,4×CH=CC H ₃ ),1.31( m,2H, CH ₂ CH ₃ ),0.84(t,3H,J=7.4Hz,CH ₂ CH ₃ );ESI-MS(m/z):487[M+H] ⁺ .

Example 10 Preparation of (S)-N-(1-(hydroxyamino)-3-methyl-1-oxapentyl-2-yl)-farnesylthiosalicylic acid amide (I ₁₀ )

Preparation of (S)-N-(1-(methoxy)-3-methyl-1-oxapentyl-2-yl)-farnesylthiosalicylic acid amide (2j)

Referring to the preparation method of N-(2-(methoxy)-2-oxaethyl)-farnesylthiosalicylic acid amide (2a) in Example 1, it is replaced by L-isoleucine methyl ester The glycine methyl ester in the method is reacted with (1) to obtain a yellow oil (2j) with a yield of 69%.

Preparation of (S)-N-(1-(hydroxyamino)-3-methyl-1-oxapentyl-2-yl)-farnesylthiosalicylic acid amide (Ⅰ ₁₀ )

Referring to the preparation method of I ₁ in Example 1, the oily substance (I ₁₀ ) was obtained by replacing 2a in the method 2j with hydroxylamine hydrochloride, and the yield was 70%.

¹ H NMR(CDCl ₃ ,300MHz):δ7.87(d,1H,J=7.8Hz,Ar-H),7.53-7.43(m,2H,Ar-H),7.31(m,1H,Ar-H ),5.33(m,1H,SCH ₂ CH ),5.10(m,2H,2×CH ₂ CH =CCH ₃ ),4.78(m,1H,NHC H ),3.65(d,2H,J=7.2 Hz,SC H ₂ ),2.66(m,1H,NHCHC H ),2.05-1.72(m,8H,2×CHCH H ₂ CH ₂ CH ),1.63-1.41(m,12H,4×CH=CC H ₃ ),1.03(m,6H, CH ₃ CHC H ₃ );ESI-MS(m/z):487[M+H] ⁺ .

Example 11 (E)-N-(4-(3-(hydroxylamino)-3-oxapropyl-1-enyl)phenyl)-farnesylthiosalicylic acid amide (I ₁₁ ) preparation

Preparation of (E)-N-(4-(3-(methoxy)-3-oxapropyl-1-enyl)phenyl)-farnesylthiosalicylic acid amide (2k)

Referring to the preparation method of N-(2-(methoxy)-2-oxaethyl)-farnesyl thiosalicylic acid amide (2a) in Example 1, in the alternative method of methyl p-aminophenylacrylate Glycine methyl ester, and then reacted with (1) to obtain a yellow oily substance (2k), with a yield of 72%.

Preparation of (E)-N-(4-(3-(hydroxyamino)-3-oxapropyl-1-enyl)phenyl)-farnesylthiosalicylic acid amide (Ⅰ ₁₁ )

Referring to the preparation method of I ₁ in Example 1, the oily substance (I ₁₁ ) was prepared by reacting 2a in the 2k alternative method with hydroxylamine hydrochloride, and the yield was 72%.

¹ H NMR (CDCl ₃ , 300MHz): 7.90(m,1H,ArH),7.72(m,2H,ArH),7.55(m,2H,ArH,ArCH),7.40(m,2H,ArH),7.30( m,1H,ArH),5.23(m,1H,SCH ₂ CH ),5.04(m,2H,2×CH ₂ CH =CCH ₃ ),3.50(d,2H,J=7.2Hz,SCH ₂ ) ,1.97-2.04(m,8H,CH ₂ ),1.55-1.69(m,12H,CH ₃ );ESI-MS(m/z):519[M+H] ⁺ .

Example 12 Preparation of N-(4-(3-(hydroxyamino)-3-oxapropyl)phenyl)-farnesylthiosalicylic acid amide (I ₁₂ )

Preparation of N-(4-(3-(methoxy)-3-oxapropyl)phenyl)-farnesylthiosalicylic acid amide (2l)

With reference to the preparation method of N-(2-(methoxy)-2-oxaethyl)-farnesylthiosalicylic acid amide (2a) in Example 1, the method is replaced by methyl p-aminophenylpropionate Glycine methyl ester in (1) was reacted with (1) to obtain yellow oil (2l), the yield was 66%.

Preparation of N-(4-(3-(hydroxyamino)-3-oxapropyl)phenyl)-farnesylthiosalicylic acid amide (Ⅰ ₁₂ )

Referring to the preparation method of I ₁ in Example 1, the oily substance (I ₁₂ ) was prepared by reacting 2a in the alternative method of 2l with hydroxylamine hydrochloride, and the yield was 68%.

¹ H NMR(CDCl ₃ ,300MHz):7.92(m,1H,ArH),7.75(m,2H,ArH),7.51(m,2H,ArH),7.44(m,2H,ArH),7.36(m, 1H,ArH),5.26(m,1H,SCH ₂ CH ),5.06(m,2H,2×CH ₂ CH =CCH ₃ ),3.50(d,2H,J=7.2Hz,SCH ₂ ),2.93 (m,2H,ArCH ₂ ),2.74(m,2H,CH ₂ CO),2.00(m,8H,CH ₂ ),1.54(m,12H,CH ₃ ); ESI-MS(m/z):521 [M+H] ⁺ .

Example 13 Preparation of N-(4-(hydroxycarbamoyl)phenyl)-farnesylthiosalicylic acid amide (I ₁₃ )

Preparation of N-(4-(methoxyformyl)phenyl)-farnesylthiosalicylic acid amide (2m)

With reference to the preparation method of N-(2-(methoxy)-2-oxaethyl)-farnesyl thiosalicylic acid amide (2a) in Example 1, in the alternative method of methyl p-aminobenzoate Glycine methyl ester was reacted with (1) to obtain a yellow oil (2m), with a yield of 69%.

Preparation of N-(4-(hydroxycarbamoyl)phenyl)-farnesylthiosalicylic acid amide (Ⅰ ₁₃ )

Referring to the preparation method of I ₁ in Example 1, the oily substance (I ₁₃ ) was obtained by reacting 2a in the alternative method of 2m with hydroxylamine hydrochloride, and the yield was 73%.

¹ H NMR(CDCl ₃ ,300MHz):8.13(m,1H,Ar-H),7.97(d,1H,J=7.8Hz,Ar-H),7.78(m,2H,Ar-H),7.27- 7.40(m,4H,Ar-H),5.24(m,1H,SCH ₂ CH ),5.09(m,2H,2×CH ₂ CH =CCH ₃ ),3.58(d,2H,J=7.2Hz ,SCH ₂ ),1.99-2.05(m,8H,4×CH ₂ ),1.45-1.68(m,12H,4×CH ₃ );ESI-MS(m/z):493[M+H] ⁺ .

Example 14 Preparation of N-(4-(hydroxycarbamoyl)benzyl)-farnesylthiosalicylic acid amide (I ₁₄ )

Preparation of N-(4-(methoxyformyl)benzyl)-farnesylthiosalicylic acid amide (2n)

With reference to the preparation method of N-(2-(methoxy)-2-oxaethyl)-farnesylthiosalicylic acid amide (2a) in Example 1, it is replaced by methyl p-aminomethylbenzoate The glycine methyl ester in the method is reacted with (1) to obtain a yellow oil (2n), and the yield is 65%.

Preparation of N-(4-(hydroxycarbamoyl)benzyl)-farnesylthiosalicylic acid amide (Ⅰ ₁₄ )

Referring to the preparation method of I ₁ in Example 1, the oily substance (I ₁₄ ) was obtained by reacting 2a in the 2n substitution method with hydroxylamine hydrochloride, and the yield was 67%.

¹ H NMR (CDCl ₃ , 300MHz): 7.95 (m, 1H, ArH), 7.78 (m, 2H, ArH), 7.53 (m, 2H, ArH, ArCH), 7.41 (m, 2H, ArH), 7.28 ( m,1H,ArH),5.26(m,1H,SCH ₂ CH ),5.05(m,2H,2×CH ₂ CH =CCH ₃ ),4.39(s,2H,ArCH ₂ ),3.53(d, 2H,J=7.2Hz,SCH ₂ ),1.97-2.02(m,8H,CH ₂ ),1.51-1.67(m,12H,CH ₃ );ESI-MS(m/z):507[M+H] ⁺ .

Example 15 Preparation of N-(2-(4-(hydroxylamine)-4-oxabutylamino)-2-oxaethyl)-farnesylthiosalicylic acid amide (Ⅱ1)

Preparation of N-(2-(hydroxyl)-2-oxaethyl)-farnesylthiosalicylic acid amide (3a)

Dissolve 0.43g (1.00mmol) of 2a in 10mL of MeOH, add 2mL of 1M NaOH aqueous solution to it, stir at 60°C for 1.5h, evaporate methanol in the reaction solution, and then add 2M hydrochloric acid solution dropwise to adjust the pH to 3-4 , and then extracted with ethyl acetate (3×50mL), combined the organic layers, washed the organic layer with 50mL of saturated brine, dried over anhydrous sodium sulfate, filtered, and spin-dried to obtain 0.36g of oil, with a yield of 87%.

Preparation of N-(2-(4-methoxy-4-oxabutylamino)-2-oxaethyl)-farnesylthiosalicylic acid amide (4a)

Dissolve 0.42g (1.01mmol) of 3a and 0.12g (1.00mmol) of methyl 4-aminobutyrate in 10mL of anhydrous CH ₂ Cl ₂ , add 0.02g (0.16mmol) of DMAP to it, and slowly Add dropwise 0.20g (1.04mmol) of EDC in 5mL of anhydrous CH ₂ Cl ₂ solution, react at room temperature for 24h, wash the reaction solution with 20mL of 1M hydrochloric acid solution, 20mL of saturated brine, dry the organic layer with anhydrous sodium sulfate, filter, and dry to obtain Oil, yield 65%.

Preparation of N-(2-(4-(hydroxylamine)-4-oxabutylamino)-2-oxaethyl)-farnesylthiosalicylic acid amide (Ⅱ ₁ )

Referring to the preparation method of I ₁ in Example 1, the oily substance (II ₁ ) was obtained by replacing 2a in the method 4a with hydroxylamine hydrochloride, and the yield was 68%.

H NMR(CDCl ₃ ,300MHz):δ7.98(d,1H,J=7.8Hz,Ar-H),7.75-7.46(m,2H,Ar-H),7.31(m,1H,Ar-H) ,5.40(m,1H,SCH ₂ CH ),5.09(m,2H,2×CH ₂ CH =CCH ₃ ),4.48(m,2H,NHC H ₂ CONH),3.77(d,2H,J= 7.2Hz, SCH ₂ ), 3.20(m, 2H, NHC H ₂ ), 2.33(t, 2H, J=7.6Hz, CH ₂ CO), 1.92-1.74(m, 10H, 2×CHCH H ₂ CH ₂ CH,NHCH ₂ CH ₂ ),1.57-1.46(m,12H,4×CH ₃ ); ESI-MS(m/z):516[M+H] ⁺ .

Example 16 (S)-N-(1-(3-(hydroxylamine)-3-oxapropylamino)-1-oxapropyl-2-yl)-farnesylthiosalicylic acid amide (Ⅱ ₂ ) Preparation of

Preparation of (S)-N-(1-(hydroxyl)-1-oxapropyl-2-yl)-farnesylthiosalicylic acid amide (3b)

Referring to the preparation method of N-(2-(hydroxyl)-2-oxaethyl)-farnesylthiosalicylic acid amide (3a) in Example 11, hydrolyze 2f in place of 2a in the method to obtain an oil (3b), yield 80%.

(S)-N-(1-(3-(Methoxy)-3-oxapropylamino)-1-oxapropyl-2-yl)-farnesylthiosalicylic acid amide (4b) preparation of

Referring to the preparation method of N-(2-(4-methoxy-4-oxabutylamino)-2-oxaethyl)-farnesylthiosalicylic acid amide (4a) in Example 11, Substitute 3a and 4-aminobutyric acid methyl ester in the method with 3b and 3-aminopropionic acid methyl ester, and condense to obtain oil (4b) with a yield of 66%.

(S)-N-(1-(3-(hydroxylamine)-3-oxapropylamino)-1-oxapropyl-2-yl)-farnesylthiosalicylic acid amide (Ⅱ ₂ ) preparation

Referring to the preparation method of I ₁ in Example 1, the oily substance (II ₂ ) was obtained by replacing 2a in the method 4b with hydroxylamine hydrochloride, and the yield was 68%.

H NMR(CDCl ₃ ,300MHz):δ7.83(d,1H,J=7.8Hz,Ar-H),7.60-7.51(m,2H,Ar-H),7.40(m,1H,Ar-H) ,5.63(m,1H,SCH ₂ CH ),5.16(m,2H,2×CH ₂ CH =CCH ₃ ),4.71(m,1H,NHC H CH ₃ ),3.62(d,2H,J= 7.2Hz, SCH ₂ ), 2.28(m, 2H, NHC H ₂ ), 2.23(t, 2H, J=7.6Hz, CH ₂ CO), 1.97-1.74(m, 8H, 2×CHCH H ₂ CH ₂ CH),1.64-1.41(m,15H,4×CH=CC H ₃ ,NHCHC H ₃ ); ESI-MS(m/z):516[M+H] ⁺ .

Example 17 (S)-N-(1-(2-(hydroxylamine)-2-oxaethylamino)-3-methyl-1-oxabutyl-2-yl)-farnesylthio Preparation of salicylic acid amide (Ⅱ ₃ )

Preparation of (S)-N-(1-(hydroxyl)-3-methyl-1-oxabutyl-2-yl)-farnesylthiosalicylic acid amide (3c)

Referring to the preparation method of N-(2-(hydroxyl)-2-oxaethyl)-farnesylthiosalicylic acid amide (3a) in Example 11, hydrolyze 2g of 2a in the alternative method to obtain an oil (3c), yield 77%.

(S)-N-(1-(2-(Methoxy)-2-oxaethylamino)-3-methyl-1-oxabutyl-2-yl)-farnesylthiowater Preparation of citric acid amide (4c)

Referring to the preparation method of N-(2-(4-methoxy-4-oxabutylamino)-2-oxaethyl)-farnesylthiosalicylic acid amide (4a) in Example 11, 3c and glycine methyl ester were substituted for 3a and 4-aminobutyric acid methyl ester in the condensation reaction to obtain oil (4c) with a yield of 61%.

(S)-N-(1-(2-(Hydroxylamine)-2-oxaethylamino)-3-methyl-1-oxabutyl-2-yl)-farnesylthiosalicylic acid Preparation of Amide (II ₃ )

Referring to the preparation method of I ₁ in Example 1, the oily substance (II ₃ ) was obtained by replacing 2a in the method 4c with hydroxylamine hydrochloride, and the yield was 72%.

H NMR(CDCl ₃ ,300MHz):δ8.14(d,1H,J=7.8Hz,Ar-H),7.60-7.56(m,2H,Ar-H),7.36(m,1H,Ar-H) ,5.47(m,1H,SCH ₂ CH ),5.17(m,2H,2×CH ₂ CH =CCH ₃ ),4.68(m,1H,NHC H ),4.42(m,2H,NHC H ₂ CONH ),3.72(d,2H,J=7.2Hz,SC H ₂ ),2.13-1.84(m,9H,2×CHCH H ₂ CH ₂ CH,NHCHC H ),1.66-1.49(m,12H,4× CH=CC H ₃ ),1.02(m,6H,CH ₂ ( CH ₃ ) ₂ ); ESI-MS(m/z):530[M+H] ⁺ .

Example 18 Preparation of N-(2-(2-(hydroxylamino)-2-oxaethylamino)-2-oxaethyl)-farnesylthiosalicylic acid amide (II ₄ )

Preparation of N-(2-(2-(methoxy)-2-oxaethylamino)-2-oxaethyl)-farnesylthiosalicylic acid amide (4d)

Referring to the preparation method of N-(2-(4-methoxy-4-oxabutylamino)-2-oxaethyl)-farnesylthiosalicylic acid amide (4a) in Example 11, Substitution of 3a and 4-aminobutyric acid methyl ester by 3a and glycine methyl ester in the condensation reaction gave oil (4d) with a yield of 56%.

Preparation of N-(2-(2-(hydroxylamino)-2-oxaethylamino)-2-oxaethyl)-farnesylthiosalicylic acid amide (Ⅱ ₄ )

Referring to the preparation method of I ₁ in Example 1, the oily substance (II ₄ ) was obtained by replacing 2a in the method 4d with hydroxylamine hydrochloride, and the yield was 73%.

H NMR(CDCl ₃ ,300MHz):δ7.90(d,1H,J=7.8Hz,Ar-H),7.60-7.53(m,2H,Ar-H),7.36(m,1H,Ar-H) ,5.43(m,1H,SCH ₂ CH ),5.22(m,2H,2×CH ₂ CH =CCH ₃ ),4.66(m,1H,NHC H ₂ ),3.77(d,2H,J=7.2 Hz, SC H ₂ ), 2.03-1.81 (m, 8H, 2× CHCH ₂ CH ₂ CH), 1.66-1.43 (m, 12H, 4×CH=CC H ₃ ); ESI-MS (m/z ):488[M+H] ⁺ .

Example 19 Preparation of N-(2-(3-(hydroxylamino)-3-oxapropylamino)-2-oxaethyl)-farnesylthiosalicylic acid amide (Ⅱ ₅ )

Preparation of N-(2-(3-(methoxy)-3-oxapropylamino)-2-oxaethyl)-farnesylthiosalicylic acid amide (4e)

Referring to the preparation method of N-(2-(4-methoxy-4-oxabutylamino)-2-oxaethyl)-farnesylthiosalicylic acid amide (4a) in Example 11, Substitution of 3a and 4-aminobutyric acid methyl ester in 3a and 3-aminopropionic acid methyl ester, condensation reaction gave oil (4e), yield 59%.

Preparation of N-(2-(3-(hydroxylamino)-3-oxapropylamino)-2-oxaethyl)-farnesylthiosalicylic acid amide (Ⅱ ₅ )

Referring to the preparation method of I ₁ in Example 1, the oily substance (II ₅ ) was obtained by replacing 2a in the method 4e with hydroxylamine hydrochloride, and the yield was 71%.

H NMR(CDCl ₃ ,300MHz):8.01(d,1H,J=7.6Hz,Ar-H),7.70-7.39(m,4H,Ar-H),7.30-7.25(m,3H,Ar-H) ,5.68(m,1H,SCH ₂ CH ),5.27(m,2H,2×CH ₂ CH =CCH ₃ ),4.73(m,1H,NHC H ₂ ),3.79(d,2H,J=7.4 Hz,SC H ₂ ), _3.54 (m,2H,NHC H ₂ ),2.59(t,2H,J=7.6Hz, CH ₂ CONH),2.04-1.78(m,8H,2×CHCH 2 CH ₂ CH),1.68-1.50(m,12H,4×CH=CC H ₃ ); ESI-MS(m/z):502[M+H] ⁺ .

Example 20

Tetramethylazolium blue colorimetric method (MTT) anti-tumor test in vitro

The anti-proliferation activity of the compound of the present invention on 8 kinds of human cancer cell lines was evaluated by tetramethylazolium blue colorimetric method (MTT). MTT method has been widely used in large-scale antitumor drug screening, cytotoxicity test and tumor radiosensitivity determination. FTA and SAHA were selected as positive control drugs. SAHA is an anti-tumor drug widely used clinically at present, and its target is HDAC, so it is selected as a positive control drug.

Human cancer cell lines: liver cancer cell SMMC-7721, pancreatic cancer cell PANC-1, lung cancer cell H460, breast cancer cell MCF-7, brain cancer cell U251, ovarian cancer cell SKOV-3, bladder cancer cell EJ, gastric cancer cell SGC- 7901.

The experimental method is as follows: Take a bottle of cells in good exponential growth phase, add 0.25% trypsin to digest, make the adherent cells fall off, and make a suspension containing 2×10 ⁴ to 4×10 ⁴ cells per ml. The cell suspension was inoculated on a 96-well plate, 180 μL per well, and cultured in a constant temperature CO ₂ incubator for 24 hours. Change the medium, add test compound Ⅰ ₁ - Ⅰ ₁₄ or compound Ⅱ ₁ - Ⅱ ₅ (the compound is dissolved in DMSO and diluted with PBS, the concentration of the test compound is 6.25×10 ^-6 , 1.25×10 ^-5 , 2.5×10 ^-5 , 5×10 ^-5 mol/L), 20 μL per well, cultured for 48 hours. Add MTT into the 96-well plate, 20 μL per well, and react in the incubator for 4 hours. Aspirate the supernatant, add DMSO, 150 μL per well, and shake on a plate shaker for 5 min. The absorbance of each well was measured at a wavelength of 570nm by an enzyme-linked immunosorbent detector, and the cell inhibition rate was calculated. The experimental results are shown in Table 3.

Cell inhibition rate = (OD value of negative control group – OD value of test substance group) / OD value of negative control group × 100%.

Example 21

Ras downstream p-Raf, p-Akt, p-ERK inhibitory activity test

Western blotting was used to detect the p-Raf, p-Akt, p-ERK inhibitory activity of test compounds Ⅰ ₁ - Ⅰ ₁₄ or compound Ⅱ ₁ - Ⅱ ₅ on the Ras downstream of tumor cell PANC-1. Cells in good exponential growth phase were taken to make a suspension containing 1.5×10 ⁵ cells per milliliter, seeded on a 96-well plate, and cultured in a constant temperature CO ₂ incubator for 24 hours. Change the medium, add 6.25 μM, 12.5 μM test compound, add the same amount of PBS as the negative control, and continue to incubate for 8 hours. Digest with trypsin and wash twice with PBS. Resuspend the sample in PBS, discard the supernatant, put the cells in a 2mL EP tube, add protein lysate, 200μL/tube, react in an ice bath for 30min after repeated pipetting, centrifuge and take the supernatant into a 2mL EP tube, and use SDS-PAGE (12% gel concentration) and transferred to nitrocellulose membrane. Put the membrane in the prepared 5% non-fat milk powder blocking solution, rinse the residual milk powder with a small amount of Blot wash after blocking, dilute the primary antibody to the working concentration with TBST, 500 μL/strip, and react for 1 hour at room temperature on a shaker. Place at 4°C overnight. After the reaction, the ziplock bag was cut open, the antibody was discarded, and each membrane was placed in a dish and washed 4 times with TBST. Dilute the peroxidase-labeled secondary antibody with TBST to the concentration of the working solution, 500 μL/strip. Seal the bag and react on a shaker according to the previous method. After the reaction, discard the secondary antibody and wash it 4 times with TBST. PIERCE luminescence solution A + B were mixed in equal volumes and poured into the prepared ziplock bag. After reacting for 5 minutes, the membrane was transferred to the cassette of BIO-RAD gel imager for exposure and imaging.

Example 22

HDACs inhibitory activity test

The inhibitory activity of the compounds on HDAC in vitro was tested by ELISA enzyme-linked immunosorbent assay. EpiQuik ^TM HADC Activity/Inhibition Assay Kit was purchased from Epigentek, and the test compounds were prepared as solutions with three concentrations of 1nM, 10nM and 100nM, and 10μL of HDACs buffer and 5μL of Hela cell nucleus extract were incubated at 37°C for 5min Finally, 25 μL of HDAC fluorescent substrate was added and incubated at 37°C for 45 min, then 25 μL of HDAC Assay developer was added to the reaction well to terminate the reaction, and incubated at 37°C for 20 min, and the absorbance was measured at 405 nm using a microplate reader. Compounds were tested in triplicate at each concentration for each compound.

Hela cell nuclear extract operation method: take the culture medium containing 10% calf serum to cultivate the Hela cell line, blow down the cells with a pipette, collect the supernatant by centrifugation, and save the cell pellet for later use. Add 200 μL of cell protein extraction reagent added with phenylmethylsulfonyl fluoride (PMSF) to every 20 μL of cell pellet (about 2× ¹⁰⁶ cells), vortex at high speed to completely suspend and disperse the cells, bathe in ice for 5-10 minutes, and add the cell slurry 10 μL of protein extraction reagent, vortexed at high speed and then centrifuged at 4°C for 5 min at high speed. Completely suck off the residual supernatant, then add 50 μL of PMSF-added nuclear protein extraction reagent, repeat high-speed vortexing and centrifugation to remove the supernatant, and then extract the obtained Hela nuclear protein.

Data analysis method: a. Calculate the average signal value of each sample; b. Subtract the average background signal value from the signal value of each sample concentration; c. Calculate the inhibition rate of each sample. After the 100% active well value is subtracted from the well value corresponding to different concentrations of each test compound, divide by the 100% active well value, and then multiplied by 100 to obtain the inhibition rate of each test compound at different concentrations. Inhibition rate = (100% active well value - test compound corresponding well value) / 100% active well value × 100. The IC ₅₀ of the test compound was obtained by fitting the concentration and the corresponding inhibition rate in Excel through nonlinear regression.

Claims (1)

1. A preparation method for a dual-target drug compound for treating tumors, characterized in that: the drug compound has the structure of the following general formula II: In the general formula II: -NH-A-CO- is selected from the configuration of glycine residues, L- or D-type α-alanine residues, β-alanine residues, L- or D-type valine residues amino acid residues, L- or D-type leucine residues, L- or D-type isoleucine residues, L- or D-type methionine residues, L- or D-type half Cystine residues, L-or D-type phenylalanine residues, L-or D-type tyrosine residues, L-or D-type tryptophan residues, L-or D-type sperm Amino acid residues, L- or D-type proline residues, L- or D-type histidine residues; or selected from -NH(CH ₂ ) _n CO-, n=3～7; or selected since or from o=0～5; or selected from X is selected from -O-, or from -NH-, or from -NCH ₃ -; m=1～8; The preparation method of the general formula II comprises the following steps: A. Compound (2) is hydrolyzed in a methanol solution containing NaOH to obtain compound (3); B. Compound (3) reacts with HX (CH ₂ ) _m COOMe reacts to generate compound (4); C. last compound (4) reacts with hydroxylamine hydrochloride under the methanol solution of potassium hydroxide to obtain general formula II; The general formula II reaction scheme is as follows: Among them, in the general formula II: -NH-A-CO- is selected from the configuration of glycine residues, L- or D-type α-alanine residues, β-alanine residues, L- or D- Valine residues, L-or D-leucine residues, L-or D-type isoleucine residues, L-or D-methionine residues, L-or D- Cysteine residues, L- or D-phenylalanine residues, L- or D-tyrosine residues, L- or D-tryptophan residues, L- or D- Arginine residues, L- or D-proline residues, L- or D-histidine residues; or selected from -NH(CH ₂ ) _n CO-, n=3～7; or from or from o=0～5; or selected from X is selected from -O-, or from -NH-, or from -NCH ₃ -; m=1-8.