CN111909107A - IDO/HDAC dual inhibitor and pharmaceutical composition and application thereof - Google Patents

Abstract

The invention discloses a heterocyclic hydroximes compound with a specific structure, a pharmaceutically acceptable salt, an isomer, a prodrug or a solvate thereof, and a pharmaceutical composition containing the heterocyclic hydroximes compound as an active ingredient, and further experiments prove that the compound and the pharmaceutical composition have double inhibition effects on indoleamine-2,3-dioxygenase and histone deacetylase, can be applied to preparation of drugs for treating diseases related to abnormal regulation of 2, 3-indoleamine dioxygenase and histone deacetylase, wherein the diseases comprise cancer, neurodegenerative diseases, AIDS, senile dementia, malaria, diabetes and the like, and have wide application prospects.

Description

IDO/HDAC dual inhibitor and pharmaceutical composition and application thereof

technical field

The invention belongs to the field of chemical medicine, and particularly relates to heterocyclic hydroxime compounds of specific structures and pharmaceutically acceptable salts, isomers, prodrugs or solvates thereof, and pharmaceutical compositions containing such compounds, and Their use in the preparation of medicines.

Background technique

Indoleamine-2,3-dioxygenase (IDO) is an extrahepatic monomeric enzyme containing heme, which catalyzes the tryptophan kynurenine metabolic pathway. The rate-limiting step in the process of oxidative reaction of tryptophan converts tryptophan into N-formylkynurenine, which is hydrolyzed to generate kynuric acid, which then enters the kynuric acid metabolic pathway to generate metabolites such as quinolinic acid and kynurenic acid. Excessive expression of IDO results in local tryptophan depletion, increased metabolites such as kynuric acid, inhibits the proliferation of T cells, activates regulatory T cells, and then creates an immune-tolerant tumor microenvironment, making the body unable to recognize and treat tumor cells. Clear. In addition, a large number of studies have shown that IDO is highly expressed in various tumors and antigen-presenting cells, which is closely related to the poor prognosis and poor survival rate of patients. Therefore, inhibiting the activity of IDO can effectively prevent the degradation of tryptophan around tumor cells and promote the proliferation of T cells, thereby enhancing the body's ability to attack tumor cells. However, IDO inhibitors have only moderate activity when used alone, and have good efficacy when used in combination with chemotherapy drugs or other immune checkpoint inhibitors. However, drug combination therapy may have problems such as complex pharmacokinetics, inability to reach the target at the same time, and drug-drug interactions. Dual-target or multi-target drugs can reduce the occurrence of drug resistance, enhance drug activity and improve the therapeutic index.

Histone deacetylases (HDACs) catalyze the deacetylation of histone amino-terminal lysines, causing chromatin condensation and transcriptional repression. Currently, 18 different isoforms of histone deacetylases are known, which are divided into 4 major categories according to germline: class I includes HDAC1, HDAC2, HDAC3 and HDAC8, which only exist in the nucleus; class II includes HDAC4, HDAC5 , HDAC6, HDAC7, HDAC9, HDAC10 and HDAC11, shuttle between the nucleus and cytoplasm during signal transduction; class IV is HDAC11; class III is SIRT1-SIRT7 is very different from class I, II and IV, and its Activity is independent of Zn ²⁺ , but rather dependent on coenzyme I (NAD), which cannot be inhibited by class I and II HDAC inhibitors. HDAC inhibitors can induce tumor cell apoptosis, differentiation and autophagy, inhibit tumor cell proliferation, inhibit tumor cell adhesion and metastasis, and inhibit angiogenesis. Currently, there are four HDAC inhibitors on the market for the treatment of various hematological malignancies. It is worth mentioning that recent studies have shown that HDAC inhibitors can improve the ability of the immune system to recognize tumors and reverse tumor immunosuppression. Therefore, IDO/HDAC dual inhibitors can effectively combine the advantages of tumor immune checkpoint inhibitors and epigenetic drugs to improve antitumor efficacy.

SUMMARY OF THE INVENTION

Purpose of the invention: Aiming at the problems of inaccurate curative effect and large toxic and side effects of existing drugs, the present invention provides indoleamine-2,3-dioxygenase and histone deacetylase of heterocyclic hydroxime compounds The invention also provides a preparation method of the indoleamine-2,3-dioxygenase/histone deacetylase dual inhibitor, and a pharmaceutical composition containing the inhibitor, and their pharmaceutical uses.

Technical scheme: The heterocyclic hydroxime compound of the present invention is selected from the compounds shown in the following formulas I to VII:

and pharmaceutically acceptable salts, isomers, prodrugs or solvates thereof.

The preparation method of the heterocyclic hydroxime compound comprises the following steps:

(1) the compound shown in formula (A) and isocyanate are reacted in an organic solvent to obtain the compound shown in formula (B), and the reaction process is as follows:

(2) deprotection reaction of the compound shown in formula (B) under the effect of a base obtains the compound shown in formula (C), and the reaction process is as follows:

Further, in step (1), the organic solvent is selected from dichloromethane, tetrahydrofuran (THF), N,N-dimethylformamide (DMF), ethylene glycol dimethyl ether, 1,2-dichloromethane One or more of ethane, dimethyl phthalate (DMP), methanol, ethanol, petroleum ether, n-hexane and diethyl ether.

In step (2), the alkali is selected from potassium carbonate, sodium carbonate, sodium bicarbonate, magnesium carbonate, calcium carbonate, cesium carbonate, sodium hydroxide, potassium hydroxide, lithium hydroxide, cesium hydroxide, magnesium hydroxide, One or more of imidazole hydroxide, triethylamine, diisopropylethylamine, piperidine, lutidine, N-methylmorpholine, DABCO and pyridine.

The present invention also provides a pharmaceutical composition, which contains an effective amount of the above-mentioned heterocyclic hydroxime compound or a pharmaceutically acceptable salt, prodrug or solvate thereof, and a pharmaceutically acceptable carrier.

The present invention also provides the application of the above heterocyclic hydroxime compounds and their pharmaceutically acceptable salts or their prodrug molecules in preparing medicines for treating or preventing tumors.

The tumors include lymphoma, non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, gastric cancer, pancreatic cancer, breast cancer, prostate cancer, liver cancer, skin cancer, epithelial cell cancer, leukemia and cervical cancer. Proliferative disease.

The heterocyclic hydroxime compounds and pharmaceutically acceptable salts thereof involved in the present invention can effectively inhibit the growth of various tumor cells, and can effectively inhibit the production of 2,3-indoleamine dioxygenase and histone deacetylase. It has dual inhibitory effects and can be used to prepare corresponding antitumor drugs. As understood by those in the art, the compounds involved in the present application and their pharmaceutically acceptable salts can be used to prepare and treat hyperproliferative diseases such as tumors in humans and other mammals.

The medicaments prepared by the heterocyclic hydroxime compounds and the pharmaceutically acceptable salts thereof involved in the present invention can be used for treating mammals related to abnormal regulation of 2,3-indoleamine dioxygenase and histone deacetylase Diseases include cancer, neurodegenerative diseases, AIDS, Alzheimer's, malaria and diabetes, among others.

One of ordinary skill in the art would know the meaning of the following terms or abbreviations.

The term "pharmaceutically acceptable salts" refers to salts which are suitable, within the scope of sound medical judgment, for use in contact with mammalian, especially human tissue, without undue toxicity, irritation, allergic reaction, etc. and commensurate with a reasonable benefit/risk ratio, Medically acceptable salts such as amines, carboxylic acids and other types of compounds are well known in the art.

The term "solvate" refers to a mixture of a compound and a solvent, eg, a crystal is a solvate.

The term "prodrug" refers to a compound that is rapidly transformed in vivo by hydrolysis in blood to yield the parent compound of the above formula.

Beneficial effects: the present invention provides heterocyclic hydroxime compounds with specific structures and pharmaceutically acceptable salts, isomers, prodrugs or solvates thereof, and the heterocyclic hydroxime compounds containing the above-mentioned heterocyclic hydroxime compounds as active ingredients. The pharmaceutical composition is further verified by experiments that the above-mentioned compounds and pharmaceutical compositions have dual inhibitory effects on indoleamine-2,3-dioxygenase and histone deacetylase, and can be used in the preparation of therapeutic 2,3- The medicines for diseases related to abnormal regulation of indoleamine dioxygenase and histone deacetylase include cancer, neurodegenerative diseases, AIDS, senile dementia, malaria and diabetes, etc., and have broad application prospects.

Detailed ways

The present application will be described in detail below with reference to specific embodiments.

The present invention includes the free forms of formulae (I) to (VII), as well as pharmaceutically acceptable salts and stereoisomers. Some of the specific exemplary compounds herein are protonated salts of amine compounds. The term "free form" refers to the amine compound in non-salt form. Included are pharmaceutically acceptable salts not only exemplary salts of the particular compounds described herein, but also typical pharmaceutically acceptable salts of all compounds represented by formulae (I)-(VII) in free form. The free forms of specific salts of the compounds can be isolated using techniques known in the art. For example, the free form can be regenerated by treating the salt with a suitable dilute aqueous base such as sodium hydroxide, sodium carbonate, ammonia and potassium bicarbonate. The free forms differ somewhat from their respective salt forms in certain physical properties such as solubility in polar solvents, but the acid and base salts are otherwise pharmaceutically equivalent to their respective free forms for the purposes of the invention.

The pharmaceutically acceptable salts of the present invention can be synthesized from compounds of the present invention containing a basic or acidic moiety by conventional chemical methods. Generally, salts of basic compounds are prepared by ion exchange chromatography or by reacting the free base and a stoichiometric amount or excess of the desired inorganic or organic acid in the form of the desired salt in a suitable solvent or combination of solvents. Similarly, salts of compounds are formed by reaction with an appropriate inorganic or organic base.

Accordingly, pharmaceutically acceptable salts of the compounds of the present invention include conventional non-toxic salts of the compounds of the present invention formed by reacting a basic compound of the present invention with an inorganic or organic acid. For example, conventional non-toxic salts include those derived from inorganic acids such as hydrochloric, sulfuric, hydrobromic, sulfamic, phosphoric, nitric, etc., as well as those derived from organic acids such as acetic, propionic, succinic, glycolic, acetic, hard Fatty acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, pamoic acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, p-aminobenzenesulfonic acid, fumaric acid , 2-acetoxy-benzoic acid, fumaric acid, p-toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, isethionic acid, trifluoroacetic acid, etc. prepared salts.

Example 1

The reaction formula is as follows;

Step 1. 1-(4-(4-(3-Bromo-4-fluorophenyl)-5-oxo-4,5-dihydro-1,2,4-oxadiazole-3-substituted)- 1,2,5-Oxadiazole-3-substituted)-3-(3-methoxyphenyl)urea

A solution of m-methoxybenzene isocyanate (14.9 mg, 0.10 mmol) in tetrahydrofuran (1 mL) was slowly added dropwise to a solution of starting material (30 mg, 0.0877 mmol) in tetrahydrofuran (1 mL) at 0 °C under argon, The temperature was raised to room temperature for 16 h, concentrated, water (1 mL) was added, then ethyl acetate (1 mL) was added, the layers were separated, the aqueous phase was extracted with ethyl acetate (2*2 mL), the organic phases were combined, and saturated brine (2 mL) was used. Washed, dried over anhydrous sodium sulfate, concentrated, and subjected to column chromatography to obtain the target compound (322 mg, 66%). MS (EI, m/z): 491 (M ⁺ +1).

Step 2. (Z)-(N-(3-Bromo-4-fluorophenyl)-N'-hydroxy-4-(3-(3-methoxyphenyl)ureido)-1,2,5 -Oxadiazole-3-carbimide

The above raw material (20 mg) was dissolved in tetrahydrofuran (1 mL), NaOH aqueous solution (0.5 mL, 2 M) was added to react for 2 h, water (1 mL), ethyl acetate (5 mL) were added, the layers were separated, and the aqueous phase was washed with ethyl acetate (2* 5 mL), the organic phases were combined, washed with saturated brine (2 mL), dried over anhydrous sodium sulfate, concentrated, and subjected to column chromatography to obtain the target compound I (18 mg, 95%). ¹ H NMR (400MHz, DMSO): δ11.53(s,1H), 9.85(s,1H), 9.55(s,1H), 9.01(s,1H), 7.33(s,1H), 7.28-7.26( m,2H),7.16-7.05(m,1H),6.94(t,J=8.2Hz,1H),6.77(d,J=6.2Hz,1H),6.65-6.60(m,1H),3.65(s , 3H) ppm. ¹³ C NMR (125MHz, DMSO): δ163.9, 162.3, 158.3, 154.1, 148.3, 146.1, 136.6, 133.8, 128.7, 119.6, 118.2, 117.8, 115.8, 113.2, 110.8, 109.3, 58.3 ppm MS. (EI,m/z): 465(M ⁺ +1).

Example 2

(Z)-(N-(3-Bromo-4-fluorophenyl)-N'-hydroxy-4-(3-(2-methoxyphenyl)ureido)-1,2,5-oxadi oxazol-3-carbimide

The synthetic method refers to Example 1.

¹ H NMR (400MHz, DMSO): δ 11.56(s, 1H), 9.70(s, 1H), 9.50(s, 1H), 8.95(s, 1H), 7.89(t, J=8.5Hz, 1H) ,7.12-7.06(m,1H),6.87(t,J=8.3Hz,1H),6.87(d,J=6.2Hz,1H),6.69-6.63(m,1H),3.78(s,3H)ppm . ¹³ C NMR (125MHz, DMSO): δ ¹³ C NMR (125MHz, DMSO): δ 163.1, 156.3, 152.1, 149.3, 147.1, 141.3, 135.8, 128.7, 124.1, 121.2, 120.3, 113.2, 110.8, 109.3, 58.4ppm .MS(EI,m/z): 465(M ⁺ +1).

Example 3

(Z)-(N-(3-Bromo-4-fluorophenyl)-N'-hydroxy-4-(3-(4-hydroxyphenyl)ureido)-1,2,5-oxadiazole- 3-methylimidrine

The synthetic method refers to Example 1.

¹ H NMR (400MHz, DMSO): δ11.23(s,1H), 9.88(s,1H), 9.55(s,1H), 9.43(s,1H), 9.21(s,1H), 7.38(d, J=5.3Hz, 2H), 6.93 (t, J=8.2Hz, 1H), 6.87 (d, J=6.2Hz, 1H), 6.75 (d, J=5.3Hz, 2H) ppm. ¹³ C NMR (125MHz ,DMSO):δ162.9,,155.3,153.1,150.2,147.5,142.1,134.6,132.2,123.5,119.5,117.5,117.8,116.8,115.2,109.9ppm.MS(EI,m/z):451(M ⁺ +1).

Example 4

(Z)-(N-(3-Bromo-4-fluorophenyl)-4-(3-(3,4-dimethoxyphenyl)ureido)-N'-hydroxy-1,2,5 -Oxadiazole-3-carbimide

The synthetic method refers to Example 1.

¹ H NMR (400MHz, DMSO): δ11.38(s,1H), 9.88(s,1H), 9.65(s,1H), 9.05(s,1H), 7.23(s,1H), 6.94-6.89( m, 2H), 6.83 (d, J=6.2Hz, 1H), 6.69-6.62 (m, 1H), 3.88 (s, 3H), 3.62 (s, 3H) ppm. ¹³ C NMR (125MHz, DMSO): δ ¹³ C NMR (125MHz, DMSO): δ 163.1, 155.3, 152.1, 150.1, 147.3, 146.1, 142.0, 134.3, 129.8, 119.7, 118.2, 116.5, 114.2, 111.9, 110.8, 107.2, 58.4, 56.9 ppm MS (EI ,m/z):495(M ⁺ +1).

Example 5

(Z)-(N-(3-Bromo-4-fluorophenyl)-N'-hydroxy-4-(3-(4-trifluoromethoxy)phenyl)ureido)-1,2,5 -Oxadiazole-3-carbimide

The synthetic method refers to Example 1.

¹ H NMR (400MHz, DMSO): δ11.23(s,1H), 9.89(s,1H), 9.54(s,1H), 9.02(s,1H), 7.22(m,2H), 6.96-6.93( m, 3H), 6.83 (t, J=8.5Hz, 1H), 6.72 (m, 1H) ppm. ¹³ C NMR (125 MHz, CDCl ₃ ): δ 165.4, 155.8, 151.2, 148.0, 146.4, 142.3, 134.6, 132.6 ,130.2,119.3,118.0,117.2,114.5,110.8ppm.MS(EI,m/z):518(M ⁺ +1).

Example 6

(Z)-(N-(3-Bromo-4-fluorophenyl)-N'-hydroxy-4-(3-(3-trifluoromethoxy)phenyl)ureido)-1,2,5 -Oxadiazole-3-carbimide

The synthetic method refers to Example 1.

¹ H NMR (400MHz, DMSO): δ11.28(s,1H), 9.89(s,1H), 9.43(s,1H), 9.12(s,1H), 7.34(s,1H), 7.23-7.20( m, 2H), 6.93 (t, J=8.5Hz, 1H), 6.83-6.79 (m, 2H), 6.68-6.65 (m, 1H) ppm. ¹³ C NMR (125MHz, DMSO): δ163.2, 160.8, 155.3 ,151.9,147.4,142.3,136.6,134.8,129.2,119.8,118.0,117.2,113.5,110.8,110.2,109.3ppm.MS(EI,m/z):518(M ⁺⁺ 1).

Example 7

(Z)-(N-(3-Bromo-4-fluorophenyl)-N'-hydroxy-4-(3-(piperidin-4-yl)ureido)-1,2,5-oxadi oxazol-3-carbimide

The synthetic method refers to Example 1.

¹ H NMR (400MHz, DMSO): δ11.27(s,1H), 9.88(s,1H), 9.33(s,1H), 9.09(s,1H), 7.32(s,1H), 7.23-7.20( ¹³ C NMR (125MHz, DMSO): δ 163.1, 156.3, 154.9, 147.3, 142.3, 134.6, 120.8, 118.8, 117.2, 110.7, 49.5, 48.5, 32.9, 31.2 ppm. MS (EI, m/z): 442 (M ⁺ +1).

Example 8

Determination of HDAC enzymatic activity

Assay principle: The biochemical activity of a compound is determined according to the degree of its inhibition of deacetylation of HDAC enzymes. Following the action of a fluorescently labeled substrate containing an acetylated lysine side chain with the HDAC enzyme, the fluorescent substrate is deacetylated. After the deacetylated fluorescently labeled substrate is cleaved by the enzyme, a fluorescent substance is released, and the fluorescent substance generates an emission light of 460 nm under the excitation of 360 nm light.

Specific steps: the substrate of HDAC is diluted to 200M with reaction buffer (the reaction concentration is 20M), the HDAC enzyme is diluted to an appropriate concentration, the heterocyclic urea compounds prepared by the present invention of different concentrations are added, and the reaction is carried out at 37° C. for 30 minutes, and then Add the same volume of 2 times the concentration of substrate developer (developer), incubate at room temperature for 15 minutes, and finally measure the reading with a microplate reader, the excitation light is 360 nm, the emission light is 460 nm, and the data is processed with Prime 4 software. The test results are shown in Table 1.

Example 9

Determination of IDO1 enzymatic activity

First, add 15 μl of sodium hydrogen phosphate buffer (pH: 7-8) into the wells of the microplate, add 5 μl of reaction buffer containing an appropriate amount of IDO1 enzyme and the heterocyclic urea compound prepared by the present invention, mix well, and react at room temperature for 3 After 1 hour, use the Envision MultilabelReader of PE Company to detect the light absorption value at 320nm wavelength, calculate the inhibition rate of the heterocyclic urea compound on the enzyme reaction according to the absorption ratio, and use the GraphPad software to analyze and calculate the heterocyclic urea compound. the _IC50 value. The test results are shown in Table 1.

Table 1

The _IC50 in the above table refers to the concentration of inhibitor at half inhibition (50% inhibitory concentration).

It can be seen from the results in the above table that the above compounds have a certain HDAC enzyme inhibitory activity compared with SAHA, and have a stronger IDO1 inhibitory activity compared with INCB24360.

Example 10

Determination of IDO1 enzymatic activity at the cellular level

In a carbon dioxide incubator at 37°C, culture and subculture HeLa cells with DMEM medium supplemented with antibiotics, inoculate into 96-well plates for overnight culture, and then add 1 μl of heterocyclic urea compounds of appropriate concentration to each well, and then add 100 μl of interferon γ (final concentration 100ng/ml). After culturing cells for 72 h, 70 μl of cell culture supernatant was added to the 96-well bottom plate, 5 μl of trichloroacetic acid (6.1N) was added to each well, incubated at 50°C for 30 min, taken out, and centrifuged at 25,000 rpm for 10 min. Transfer 20 μl/well of the above centrifuged supernatant into a 384 microwell plate, then add 20 μl of 2% dimethylaminobenzaldehyde acetic acid solution to each well, shake and mix well, measure the absorbance at 480 nm, and measure the value. The test results are shown in Table 2.

Table 2

sample IC₅₀(μM) INCB24360 0.055 I 0.043 II 0.046 III 0.033 IV 0.039 V 0.021 VI 0.032 VII 0.018

Example 11

Experiments to detect the activity of compounds on cancer cells

Experimental principle: The inhibition of cancer cell growth by compounds was detected by MTT method. The principle of the MTT method is that yellow thiazolan can penetrate into the cell through the cell membrane, and succinyl dehydrogenase in the mitochondria of living cells can reduce exogenous MTT to insoluble blue-purple needle-like Formazan crystals and deposit in the cells. In , the crystals can be dissolved by dimethyl sulfoxide (DMSO), and the light absorption value is detected by enzyme-linked immunosorbent assay at 490nm/570nm wavelength, which can indirectly reflect the number of cells.

Experimental materials: The cancer cell lines used are Hela (human cervical cancer cells), MCF-7 (human breast cancer cells), BGC-823 (human gastric cancer cells), A549 (human lung cancer cells), HT1080 (human fibrosarcoma cells) ), A431 (human epidermal squamous cell carcinoma cells), DU145 (human prostate cancer cells), U937 (human leukemia cells), Pac-1 (human pancreatic cancer cells), MOLT-4 (human acute lymphoblastic leukemia cells) ; cultured with DMEM+10%FBS medium or cultured with 1640+10%FBS respectively.

Experimental method and result analysis:

Experimental group: 190μl of cell suspension + 10μl of different concentrations of drugs (final concentration of 10 ^-5 to 10 ^-10 M)

Blank control group: 200 μl PBS

Negative control group: 190 μl cell suspension + 10 μl 2% DMSO (the final concentration of DMSO is 0.1%)

Positive control group: 190 μl cell suspension + 10 μl compounds of different concentrations

a) The cells were seeded in a 96-well plate with a seeding amount of 1500 cells/well, 190 μl/well, and cultured overnight in a 37°C 5% CO ₂ incubator;

b) The next day, 10 μl of different drugs were added to each well, and the final drug concentration was 10 ^-5 to 10 ^-10 M, and three parallel wells were set up; incubated in a 37°C, 5% CO ₂ incubator for 72 hours;

c) Add 20 μl of 5 mg/ml MTT to each well and incubate for 4 hours at 37°C in a 5% CO ₂ incubator;

d) Discard the supernatant, add 100 μl of DMSO to each well, and shake;

e) Reading at 570 nm, calculating cell viability, calculating GI ₅₀ according to the results, and obtaining Table 3 below.

table 3

The GI ₅₀ in the above table represents the drug concentration required for 50% growth inhibition of cells (50% growth inhibition).

It can be seen from the results in the above table that the above-mentioned drugs have certain cytotoxicity compared with the positive control (SAHA).

Claims (5)

1. A heterocyclic hydroxime compound, selected from the compounds shown in the following formulae I～VII:

and pharmaceutically acceptable salts, isomers, prodrugs or solvates thereof. 2. a pharmaceutical composition is characterized in that, containing the heterocyclic hydroxime compound or its pharmaceutically acceptable salt, prodrug or solvate of claim 1 in an effective amount, and pharmaceutically acceptable Carrier. 3. The application of the heterocyclic hydroxime compound according to claim 1 in the preparation of a medicine for treating diseases related to abnormal regulation of 2,3-indoleamine dioxygenase and histone deacetylase. 4. The application according to claim 3, wherein the diseases include hyperproliferative diseases, neurodegenerative diseases, AIDS, Alzheimer's disease, malaria, diabetes and the like. 5. application according to claim 3 is characterized in that, described transition proliferative disease comprises lymphoma, non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, gastric cancer, pancreatic cancer, breast cancer, prostate cancer, liver cancer, skin cancer, epithelial cell cancer, leukemia and cervical cancer.