CN109646442B

CN109646442B - Application of acetate compound in preparation of cycloguanosine synthetase acetylation drugs

Info

Publication number: CN109646442B
Application number: CN201910086264.6A
Authority: CN
Inventors: 张学敏; 周涛; 何新华; 李爱玲; 李涛
Original assignee: Biomedical Analysis Center of AMMS
Current assignee: Biomedical Analysis Center of AMMS
Priority date: 2019-01-29
Filing date: 2019-01-29
Publication date: 2023-01-24
Anticipated expiration: 2039-01-29
Also published as: CN109646442A

Abstract

The invention discloses an acetate compound shown in the following formula I, and application of pharmaceutically acceptable salts or solvates thereof in preparation of cGAS acetylation medicines.

。

Description

Application of acetate compound in preparation of cycloguanosine synthetase acetylation drugs

Technical Field

The invention relates to the field of medicines, and in particular relates to application of acetate compounds shown in a formula I in preparation of a cycloguanosine synthetase acetylation medicine.

Background

The presence of DNA in the cytoplasm is generally recognized by the body as a signal for microbial infection or tissue damage, and thus cytoplasmic DNA as a danger signal can elicit a strong innate immune response (Annual review of immunology,2011, 29, 185-214). Recognition of cytoplasmic DNA is an important mechanism by which the body defends against microbial infections. cGAS (cyclic GMP-AMP synthsase) is a major cytoplasmic DNA receptor that rapidly recognizes DNA stimuli and activates immune responses. After binding to DNA, activated cGAS can catalyze ATP and GTP to produce cyclic small molecule cGAMP (cyclic GMP-AMP) (Science, 2013, 339, 786-791. cGAMP can bind and activate endoplasmic reticulum protein STING (also known as MITA, MPYS, ERIS) as a second messenger (Nature, 2008, 455, 674-678, molecular and cellular biology,2008, 28, 5014-5026 proc Natl Acad Sci U S a,2009, 106,8653-8658, immunity,2008, 29, 538-550), STING can mediate activation of downstream TBK1 and IRF3 and produce type I interferon (Science calling, 2012,5, pe9). Type I interferons play an important role in antiviral responses, and induce the expression of a range of interferon-induced genes, ISGs (Proc Natl Acad Sci USA,2015, 112, E5699-5705).

In addition to microbial infections, cellular damage or cellular endogenous retroviruses can also produce cytoplasmic self DNA (Current opinion in immunology,2014, 31, 121-126, nature reviews immunology,2016, 16, 207-219. Metazoans have evolved dnases which can clear cellular DNA from themselves, thereby preventing immune responses caused by inappropriate activation of cGAS. For example, TREX1 can degrade DNA in the cytoplasm, and loss of TREX1 function is found in autoimmune disease patients such as AGS syndrome (Aicardi-Gouti res syndrome) and systemic lupus erythematosus (Current opinion in immunology,2014, 31, 121-126, annals of neurology,1984, 15, 49-54, nature reviews immunology,2015, 15, 429-440 lancet,2014, 384, 1878-1888. AGS syndrome patients accumulate their own DNA in the cytoplasm due to mutations containing the TREX1 gene, and can chronically stimulate cGAS to produce type I interferon (Nature genetics,2006, 38, 917-920, proc Natl Acad Sci usa,2015, 112, E5699-5705 journal of immunology,2015, 195, 1939-1943 cell,2008, 134, 587-598. The excessive interferon produced can cause systemic inflammation and other autoimmune reactions in the body (2012, 24, 499-505). Knockout of Trex1 in mice produces a severe autoimmune response that is dependent on the cGAS-STING pathway (Proc Natl Acad Sci USA,2015, 112, journal of immunology,2015, 195, 1939-1943). These studies indicate that cGAS inhibition can be used to treat autoimmune diseases caused by self-DNA. However, lack of understanding of the mechanisms of cGAS regulation has hampered the discovery of effective therapeutic approaches.

Disclosure of Invention

In one aspect of the invention, the use of the acetate compound shown in formula I, and pharmaceutically acceptable salts or solvates thereof in the preparation of cGAS acetylation drugs is provided:

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wherein Ar is phenyl or 6,7-dihydro-4H-thieno [3,2-C ] pyridin-2-yl, X is substituted or unsubstituted carbonyl, substituted or unsubstituted aminoacyl, substituted or unsubstituted C1-C8 alkyl, substituted or unsubstituted acylamino, carboxyl, wherein the substituents contained therein are selected from C1-C6 oxyalkyl, a saturated or unsaturated 5-or 6-membered heterocyclic group containing 1 to 3 heteroatoms selected from N, O and S, a halogen atom.

Preferably, when Ar is phenyl, the structure of the compound of formula I may be represented by formula II below, wherein Y is oxygen or nitrogen atom, R' is hydrogen atom, substituted or unsubstituted C1-C8 alkyl group, wherein the substituents in the substituted C1-C8 alkyl group are selected from saturated or unsaturated 5-or 6-membered heterocyclic group containing 1 to 3 heteroatoms selected from N, O and S, C1-C6 oxyalkyl;

or Y and R' together with the carbon atom to which they are attached form a saturated or unsaturated 5-or 6-membered heterocyclic group containing 1 to 3 heteroatoms selected from N, O and S.

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Further preferably, when Ar is phenyl, the structure of the compound of formula I may be represented by formula II, wherein Y is oxygen or nitrogen atom, R' is hydrogen atom, substituted or unsubstituted C1-C3 alkyl, wherein the substituents in the substituted C1-C3 alkyl are selected from saturated or unsaturated 5-or 6-membered heterocyclic group containing 1 to 3 heteroatoms selected from N, O and S, C1-C3 oxyalkyl.

Preferably, when Ar is 6,7-dihydro-4H-thieno [3,2-C ] pyridin-2-yl, the structure of the compound of formula I may be represented by formula III below, wherein R "is a substituted or unsubstituted C1-C6 alkyl, wherein the substituent in the substituted C1-C6 alkyl is selected from the group consisting of a saturated or unsaturated 5-or 6-membered heterocyclic group containing 1 to 3 heteroatoms selected from N, O and S, a phenyl group substituted with a halogen atom, a C3-C6 cycloalkyl substituted carbonyl group.

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Further preferably, when Ar is 6,7-dihydro-4H-thieno [3,2-C ] pyridin-2-yl, where R "is a substituted or unsubstituted methylene or ethylene group, wherein the substituents are selected from the group consisting of saturated or unsaturated 5 or 6 membered heterocyclic group containing 1 to 3 heteroatoms selected from N, O and S, phenyl substituted with halogen atoms, C3-C6 cycloalkyl substituted carbonyl.

Preferably, the halogen atom is selected from fluorine, chlorine, bromine or iodine, preferably fluorine, chlorine or bromine, more preferably fluorine or chlorine.

Preferably, the compound of formula I according to the invention is selected from the following compounds:

in still another aspect of the present invention, there is provided a cGAS acetylated pharmaceutical composition comprising a compound of formula I, a pharmaceutically acceptable salt or solvate thereof, as an active ingredient, and a pharmaceutically acceptable carrier.

The cGAS acetylation medicine composition can be used for treating multiple sclerosis, lupus erythematosus, tumors, liver cirrhosis, pulmonary fibrosis, and genetic diseases with cGAS over-activation, such as Aicardi-Gouti syndrome.

The cGAS acetylated pharmaceutical composition according to the present invention may be formulated in various preparation forms including, but not limited to, capsules, tablets, injections, suppositories, infusion solutions, liniments, emulsions and the like.

Advantageous effects

The invention discovers for the first time that the cGAS acetylation can inhibit cGAS from synthesizing cGAMP, and further can inhibit the activation of a downstream signal path, thereby inhibiting the generation of I-type interferon, having the functions of treating autoimmune diseases such as lupus erythematosus, multiple sclerosis and psoriasis, tumors, hepatic fibrosis, pulmonary fibrosis and other diseases, and providing a treatment strategy with a brand new mechanism for the treatment of the diseases; the compound shown in the formula I has the function of acetylating cGAS, and has the application of treating autoimmune diseases such as lupus erythematosus, multiple sclerosis and psoriasis, and diseases such as tumor, hepatic fibrosis and pulmonary fibrosis.

Drawings

FIG. 1 is a graph showing an acetylsalicylic acid (aspirin) acetylated cGAS protein immunoblot assay (western blot) in example 5;

FIG. 2 is a graph comparing the inhibition of cGAS enzyme activity in cells by acetylsalicylic acid (aspirin) in example 6;

FIG. 3 is a graph of an activated immunoblot assay (western blot) of the cGAS pathway in the acetylsalicylic acid (aspirin) -inhibited cells of example 7;

FIG. 4 is a graph of the compound of example 4 and prasugrel acetylated cGAS protein immunoblot assay (western blot) of example 8;

FIGS. 5a and 5b are graphs comparing the compound of example 4 of example 9 and prasugrel, respectively, for cGAS enzyme activity inhibition assays.

Detailed Description

Hereinafter, the present invention will be described in detail. Before the description is made, it should be understood that the terms used in the present specification and the appended claims should not be construed as limited to general and dictionary meanings, but interpreted based on the meanings and concepts corresponding to technical aspects of the present invention on the basis of the principle that the inventor is allowed to define terms appropriately for the best explanation. Accordingly, the description proposed herein is just a preferable example for the purpose of illustrations only, not intended to limit the scope of the invention, so it should be understood that other equivalents and modifications could be made thereto without departing from the spirit and scope of the invention.

The inventor finds that the cGAS acetylation can inhibit cGAS from synthesizing cGAMP, further inhibit the activation of a downstream signal path, thereby inhibiting the generation of type I interferon, and has the effects of treating autoimmune diseases such as lupus erythematosus, multiple sclerosis and psoriasis, and diseases such as tumor, hepatic fibrosis and pulmonary fibrosis. Based on this finding, the present inventors screened compounds represented by formula I, which can be effectively used for cGAS acetylation, and further achieved the purpose of treating diseases such as lupus erythematosus.

In addition, according to the application of the compound shown in the formula I in preparing cGAS acetylation medicines, the invention develops a novel medicine composition, wherein the compound shown in the formula I, the pharmaceutically acceptable salt or solvate thereof and a pharmaceutically acceptable carrier are contained as active ingredients.

The pharmaceutically acceptable salt is a conventional non-toxic salt formed by reacting the compound of formula (I) with an inorganic acid or an organic acid. For example, the conventional non-toxic salts can be prepared by reacting the compound of formula (I) with inorganic acids including hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, sulfamic acid, phosphoric acid and the like, or organic acids including citric acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, naphthalenesulfonic acid, ethanesulfonic acid, naphthalenedisulfonic acid, maleic acid, malic acid, malonic acid, fumaric acid, succinic acid, propionic acid, oxalic acid, trifluoroacetic acid, stearic acid, pamoic acid, hydroxymaleic acid, phenylacetic acid, benzoic acid, salicylic acid, glutamic acid, ascorbic acid, p-aminobenzenesulfonic acid, 2-acetoxybenzoic acid, isethionic acid and the like; or sodium salt, potassium salt, calcium salt, aluminum salt or ammonium salt formed by the compound of the general formula (I) and propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, aspartic acid or glutamic acid after forming ester and then forming inorganic base; or the methylamine salt, ethylamine salt or ethanolamine salt formed by the compound of the general formula (I) and organic base; or the compound of the general formula (I) forms ester with lysine, arginine and ornithine and then forms corresponding inorganic acid salt with hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid and phosphoric acid or forms corresponding organic acid salt with formic acid, acetic acid, picric acid, methanesulfonic acid and ethanesulfonic acid.

The term "pharmaceutically acceptable carrier" refers to any formulation or carrier medium capable of delivering an effective amount of an active agent of the present invention, without interfering with the biological activity of the active agent, and without toxic side effects to the host or patient, and representative carriers include water, oils, vegetables and minerals, cream bases, lotion bases, ointment bases, and the like. These include suspending agents, viscosity enhancers, skin penetration enhancers, and the like. Their preparation is known to those skilled in the cosmetic or topical pharmaceutical field. For additional information on the carrier, reference may be made to Remington: the Science and Practice of Pharmacy,21st Ed., lippincott, williams & Wilkins (2005), the contents of which are incorporated herein by reference.

The term "effective amount" or "therapeutically effective amount" with respect to a drug or pharmacologically active agent refers to a sufficient amount of the drug or agent that is non-toxic but achieves the desired effect. For oral dosage forms of the invention, an "effective amount" of one active agent in a composition is the amount required to achieve the desired effect when combined with another active agent in the composition. The determination of an effective amount varies from person to person, depending on the age and general condition of the recipient and also on the particular active substance, and an appropriate effective amount in an individual case can be determined by a person skilled in the art according to routine tests.

Various dosage forms of the pharmaceutical composition of the present invention can be prepared according to conventional preparation methods in the pharmaceutical field. The unit dose of the preparation formula comprises 0.05-200mg of the compound shown in the general formula (I), and preferably, the unit dose of the preparation formula comprises 0.1-100 mg of the compound shown in the general formula (I).

The compounds and pharmaceutical compositions of the present invention may be administered to mammals, including humans and animals, clinically, by oral, nasal, dermal, pulmonary, or gastrointestinal routes of administration. Most preferably oral. The optimal daily dosage is 0.01-200mg/kg body weight, and can be administered in one time or 0.01-100mg/kg body weight in several times. Regardless of the method of administration, the optimal dosage for an individual will depend on the particular treatment. Usually, the dosage is increased gradually starting from a small dosage until the most suitable dosage is found.

The following examples are given by way of illustration of embodiments of the invention and are not to be construed as limiting the invention, and it will be understood by those skilled in the art that modifications may be made without departing from the spirit and scope of the invention. Unless otherwise specified, materials, reagents, instruments and the like used in the following examples are commercially available products unless otherwise specified.

Example 1.2 Synthesis of- ((2-morpholinoethyl) aminoacyl) phenol acetate

Acetylsalicylic acid (3.6g, 20mmol) was added to anhydrous dimethylformamide (50 ml), HOBt (8.3g, 22mmol), EDCI (22mmol, 4.2g) were added, and then stirred at room temperature for 2 hours, 2-morpholinoethylamine (2.9g, 20mmol) was added, and the reaction was allowed to stand at room temperature overnight. The next day, the dimethylformyl group was distilled off under reduced pressureThe amine, the residue was extracted with dichloromethane 100ml × 3, the organic phases were combined, 50ml × 3 was washed with saturated aqueous ammonium chloride solution, and dried over anhydrous sodium sulfate overnight. Column chromatography (eluent petroleum ether: ethyl acetate = 3:1-1:1) gave off-white solid 5.5g. The yield thereof was found to be 94%. ¹ H NMR(400MHz, DMSO-d6):8.80(br s,1H),8.06(d,1H),7.88-7.90(m,2H),7.34(m,1H), 3.52-3.54(d,2H),3.60-3.64(m,4H),2.42-2.46(m,6H),2.39(s,3H).

Example 2 Synthesis of 2- (morpholine-4-acyl) phenol acetate

The synthesis was the same as in example 1, except that the starting materials for the reaction were different. The yield thereof was found to be 90%. ¹ H NMR(400 MHz,DMSO-d6):8.06(d,1H),7.88-7.90(m,2H),7.34(m,1H),3.50-3.54(m, 4H),3.61-3.64(m,4H),2.39(s,3H).

Example 3.2- ((2-methoxyethyl) aminoacyl) phenol acetate

The synthesis was the same as in example 1, except that the starting materials for the reaction were different. The yield thereof was found to be 96%. ¹ H NMR(400 MHz,DMSO-d6):8.06(d,1H),7.88-7.90(m,2H),7.34(m,1H),3.71(d,2H), 3.30(s,3H),3.28(d,2H),2.39(s,3H).

Example 4.5- (thien-2-ylmethyl) -4,5,6,7-tetrahydrothieno [3,2-c ] pyridin-2-ylacetate

Tetrahydrothienopyridine hydrochloride (3.5g, 20mmol) was dissolved in methylene chloride (30 ml), triethylamine (4.8g, 48mmol) was added, the mixture was stirred for 2 hours, 2-bromomethylthiophene (3.5g, 20mmol) was added, and the mixture was heated to 45 ℃ to react for 6 hours. Cooling to room temperature, adding saturated ammonium chloride solution while stirring, collecting organic layer, and collecting aqueous phase with dichloromethaneAlkane extraction 100ml × 3. The organic phases were combined and dried over anhydrous sodium sulfate overnight. Separating by column chromatography to obtain light yellow oily substance, dissolving in ethyl acetate, cooling to 0 deg.C, and slowly adding ethyl acetate hydrochloride solution dropwise to obtain white solid. Suction filtration gave 3.9g, yield 66%. ¹ H NMR(400 MHz,DMSO-d6):11.8(br s,1H),7.72(d,1H),7.49(d,1H),7.46(d,1H),7.17(m, 1H),4.69(s,2H),4.19(s,2H),3.72-3.37(m,2H),3.29-3.09(m,2H)。

Example 5 acetylsalicylic acid (Aspirin Aspirin) acetylation cGAS assay

The in vitro purified cGAS recombinant protein was mixed with a gradient concentration of aspirin (available from Beijing coupling technology) in HEPES (20 mM), pH 7.5, mgCl ₂ (5 mM) was incubated at 37 ℃ for 2 hours, the loading buffer was added and the sample was boiled in boiling water for 10 minutes. cGAS acetylation was detected by immunoblotting. Detection by broad-spectrum acetylation antibody showed a distinct gradient of increased cGAS acetylation. It was demonstrated that aspirin can directly acetylate cGAS. Figure 1 is acetylsalicylic acid (aspirin) acetylated cGAS protein.

Example 6 cGAMP Synthesis inhibition assay by acetylsalicylic acid (aspirin)

After differentiating THP1 cells with PMA for 72 hours, the cells were treated with DMSO or aspirin (4 mM) for 24 hours. HT-DNA (1. Mu.g/ml) was added and the stimulation was performed for about 2 hours. After stimulation was complete, cells were washed 2 times with PBS or saline. 800 μ l of cold extraction solution (volume ratio methanol: acetonitrile: water = 40. The whole extract was placed in a refrigerator at-20 ℃ and allowed to stand for 30 minutes. After 30 minutes of extraction, the mixture was centrifuged at 12000rpm at 4 ℃ for 10 minutes. The supernatant was taken, dried and assayed for cGAMP by LC-MS/MRM. The results show that aspirin treatment can significantly inhibit cGAMP synthesis in cells. Figure 2 is a graph of acetylsalicylic acid (aspirin) inhibiting cGAS enzyme activity in cells.

Example 7 acetylsalicylic acid (aspirin) inhibition of Interferon Signaling pathway activation assay

After differentiating THP1 cells with PMA for 72 hours, the cells were treated with DMSO or 4mM aspirin for 24 hours. HSV-1 (MOI =10 1) was added, stimulating at different time points. After stimulation was complete, cells were washed 2 times with PBS or saline. After lysing the cells with M2 cell lysate for 30 minutes on ice, they were centrifuged at 12000rpm at 4 ℃ for 15 minutes. And taking the supernatant, adding a loading buffer solution, and boiling the sample in boiling water for 10 minutes. Phosphorylation of IRF3 was detected by immunoblotting. The results show that aspirin treatment can significantly inhibit IRF3 activation in cells caused by HSV-1 stimulation. Figure 3 is a graph showing that acetylsalicylic acid (aspirin) inhibits the activation of the cGAS pathway in cells.

Example 8.5- (Thien-2-ylmethyl) -4,5,6,7-tetrahydrothieno [3,2-c ] pyridin-2-ylacetate (the compound of example 4) and prasugrel acetylated cGAS assay

In vitro purified cGAS recombinant protein and 5- (thiophene-2-ylmethyl) -4,5,6,7-tetrahydrothieno [3,2-c]Pyridin-2-ylacetate (compound of example 4, 0.1 mM) or prasugrel (0.1 mM, available from Beijing coupling technology) was added to HEPES (20 mM) pH 7.5, mgCl ₂ (5 mM) solution was incubated at 37 ℃ for 2 hours, loading buffer was added and the sample was boiled in boiling water for 10 minutes. cGAS acetylation was detected by immunoblotting. A significant increase in cGAS acetylation was shown by broad spectrum acetylation antibody detection. It was demonstrated that the compound of example 4 and prasugrel can directly acetylate cGAS. Figure 4 is the compound of example 4 and prasugrel acetylated cGAS protein.

Example 9 Compounds from example 4 and assay for inhibition of cGAS Synthesis cGAMP by Pralagrel

The in vitro purified cGAS recombinant protein was mixed with gradient concentrations of either Zhou-0312 or prasugrel HEPES (20 mM) pH 7.5, mgCl ₂ (5 mM) solution at 37 degrees C were incubated for 2 hours. Then HT-DNA (0.2. Mu.g/. Mu.l), ATP (2 mM), GTP (2 mM) were added and incubated at 37 ℃ for 2 hours. 4 volumes of cold extraction solution were added (volume ratio methanol: acetonitrile = 40. The whole extract was placed in a refrigerator at-2 ℃ and allowed to stand for 30 minutes. After 30 minutes of extraction, centrifuge at 12000rpm at 4 ℃ for 10 minutes. The supernatant was removed, dried and assayed for cGAMP by LC-MS/MRM. The results show that the compound of example 4 and prasugrel can significantly inhibit cGAMP synthesis in cells. Figure 5a shows cGAS enzyme activity inhibition by the compound of example 4 figure 5b shows cGAS enzyme activity inhibition by prasugrel.

Example 10 cGAS acetylation experiments of the compounds of example 1, example 2 and example 3

Experimental procedure As in example 5, the percent acetylation was calculated by comparing the western blot acetylation bands and the results are given in Table 1 below.

TABLE 1

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Claims

1. Use of an acetate compound, a pharmaceutically acceptable salt thereof, for the manufacture of a cGAS acetylation medicament for the treatment or prevention of diseases of the immune response caused by inappropriate activation of cGAS, said acetate compound being selected from the group consisting of:

the diseases of the immune response caused by inappropriate activation of cGAS are lupus erythematosus and Aicardi-Gouties syndrome.

2. Use of an acetate compound, a pharmaceutically acceptable salt thereof, as the only active ingredient in the manufacture of a cGAS acetylation drug for the treatment or prevention of diseases of the immune response caused by inappropriate activation of cGAS