CN115073467B - Pyrimidine imidazole compound, and pharmaceutical composition and application thereof - Google Patents

Pyrimidine imidazole compound, and pharmaceutical composition and application thereof Download PDF

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CN115073467B
CN115073467B CN202110276327.1A CN202110276327A CN115073467B CN 115073467 B CN115073467 B CN 115073467B CN 202110276327 A CN202110276327 A CN 202110276327A CN 115073467 B CN115073467 B CN 115073467B
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徐石林
唐炜
饶丹妮
李恒
温翠云
黄河
任雪莲
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Shanghai Institute of Materia Medica of CAS
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Abstract

The invention provides a pyrimidoimidazole compound, a medicinal composition and application thereof, and in particular provides a pyrimidoimidazole compound shown as a formula (I), or pharmaceutically acceptable salt or stereoisomer thereof, or prodrug molecule thereof, wherein the definition of each group is as described in the specification. The compounds of the present invention may be used as ZAP70 selective inhibitors.

Description

Pyrimidine imidazole compound, and pharmaceutical composition and application thereof
Technical Field
The invention belongs to the field of chemical medicines, and particularly relates to a pyrimidine imidazole compound, a medicinal composition thereof and application thereof.
Background
ZAP70 (Zeta-chain-associated protein,70 kDa) kinase, a cytoplasmic tyrosine protein kinase of 70kDa in molecular weight, belongs to a member of the Syk kinase (Spleen tyrosine kianse) family, including Syk and ZAP 70. ZAP70 proteins are expressed mainly in T cells and Natural Killer (NK) cells, playing an important role in T cell proliferation and immune response. Upon exposure of the T cell receptor (T cell antigen receptor, TCR) to the activation signal of antigen presenting cells, the ITAMs zeta chain phosphorylates and binds to the SH2 domain of ZAP70 protein, phosphorylating ZAP70 protein and activating downstream cascades, promoting T cell proliferation, cellular calcium ion flux and IL-2 (interleukin-2) production. T cells play an important role in combating pathogen and tumor immunity, ZAP70 deficiency is detected in patients with severe combined immunodeficiency syndrome, and furthermore, T cell activation and loss of downstream signaling function are observed in ZAP 70-deficient T cells, suggesting that ZAP70 plays a critical role in T cell development and activation. Since excessive TCR activation by gene mutations (e.g., R360P) can lead to numerous immune diseases, inhibition of T cell activation is an important research direction for immunotherapy. Because ZAP70 plays a key role in TCR signaling pathways, ZAP70 is an important potential target for developing therapies for autoimmune diseases and organ transplant rejection.
Since ZAP70 protein is mainly expressed in T cells, NK cells and basophils and is highly expressed in mature T cells, the same family protein Syk is low expressed in mature T cells. Thus, when ZAP70 activity is inhibited, NK cells and Syk expressed in basophils are able to compensate for the immune function of both types of cells, and the function of T cells is more dependent on ZAP70 than Syk, suggesting that inhibiting ZAP70 function only affects T cell immune activity without unduly impairing innate immune function.
At present, ZAP70 inhibitors in the prior art are reversible inhibitors, and the inhibition effect of the inhibitors as drugs in vivo is often not ideal as that of in vitro experiments, so that the present field has urgent need to provide a ZAP70 irreversible inhibitor, which has important significance for treating immune diseases.
Disclosure of Invention
The purpose of the present invention is to provide a ZAP70 covalent bond inhibitor, wherein the inhibition effect on ZAP70 can be improved because the compound of the present invention forms a bond with ZAP70 through covalent bond.
In a first aspect of the present invention there is provided a pyrimidoimidazole compound of the formula (i) or a pharmaceutically acceptable salt or stereoisomer thereof, or a prodrug molecule thereof:
wherein,
w is selected from the group consisting of: - (X) m -Ra-(X) m -; wherein m is selected from the group consisting of: 0.1, 2,3,4, 5 or 6;
each X is independently selected from the group consisting of: o, NR, cr=cr, C (R) 2 S, S (O) or S (O) 2 ；
Ra is selected from the group consisting of: a chemical bond, or a group selected from the group consisting of: a substituted or unsubstituted benzene ring, a substituted or unsubstituted 4-7 membered saturated nitrogen-containing heterocyclic ring, or a substituted or unsubstituted 5-7 membered heteroaromatic ring;
R 1 selected from the group consisting of: a substituted or unsubstituted 6-10 membered aryl, or a substituted or unsubstituted 5-12 membered heteroaryl;
R 2 selected from the group consisting of: -C (O) Rb, wherein said Rb is selected from the group consisting of: H. OH, NH 2 Substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C2-C6 alkenyloxy, substituted or unsubstituted C2-C6 alkynyloxy;
r is selected from the group consisting of: H. a substituted or unsubstituted C1-C6 alkyl group, or two R groups located on the same C atom together form an oxygen atom (=o);
the substituents refer to the substitution of one or more hydrogen atoms on the group with a substituent selected from the group consisting of: halogen, oxygen atom (=o), carboxyl, hydroxyl, amino, nitro, cyano, C1-C6 alkoxy, C1-C6 alkylamino, C1-C6 alkoxycarbonyl, C1-C6 amido, C2-C12 ester group; or a group selected from the group consisting of: C1-C10 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, C6-C10 aryl, 5-6 membered heteroaryl, 3-12 membered heterocyclyl, 3-12 membered cycloalkyl;
wherein the hetero atoms in each heterocycle are selected from N, O, S.
In another preferred embodiment, W is selected from the group consisting of: -NH (CH) 2 ) n –Ra-(X) m -, wherein n is optionally selected from 1,2,3; and Ra is selected from the group consisting of: a substituted or unsubstituted 4-7 membered saturated ring containing an N atom, a substituted or unsubstituted benzene ring, or a substituted or unsubstituted 5-7 membered aromatic heterocycle.
In another preferred embodiment, R is 1 Has a structure shown in the following formula:
wherein A is 1 、A 2 、A 3 、A 4 And A 5 Each independently selected from the group consisting of: H. f, cl, C 1 ～C 2 Alkyl, fluoromethyl, C 1 ～C 4 Alkoxy, N-dimethylamino, 5-to 6-membered saturated heterocyclic ring, or substituted by A 6 Substituted 4-7 membered saturated heterocyclic ring, wherein each A 6 Each independently selected from the group consisting of: methyl, dimethylamino, 4-methylpiperazino, 2-fluoroethyl, C 1 ～C 2 Alkyl-4-methylpiperazinyl, C 1 ～C 4 -N(A 7 )(A 8 ) Wherein A is 7 、A 8 Optionally from methyl, ethyl.
In another preferred embodiment, R is 2 Selected from the group consisting of:
in another preferred embodiment, the compound has the structure of formula (ii):
wherein,
A 1 ,A 2 ,A 3 ,A 4 ,A 5 each independently selected from the group consisting of: H. f, cl methyl, trifluoromethyl, methoxy, ethoxy, isopropoxy, pyrrolyl, piperidinyl, morpholinyl, thiarphinyl, N, N-dimethylamino, 4-methylpiperazinyl, 4-methylpiperidinyl, 2- (4-methylpiperazinyl) ethyl, 4- (4-methylpiperazinyl) piperidinyl, 4- (N, N-dimethyl) piperidinyl, 4-methylpiperazinyl, 1- (2-fluoroethyl) azetidin-3-amino, 4- (N, N-diethyl) butyl;
the W is 1 Selected from the group consisting of:
n is optionally from 0,1,2,3; x is selected from the group consisting of: halogen atom, trifluoromethyl, methyl.
In another preferred embodiment, A 1 ,A 2 ,A 4 And A 5 Each independently selected from the group consisting of: H. methyl, methoxy, ethoxy; and A is 3 Selected from the group consisting of:
w is selected from the group consisting of:
x is selected from the group consisting of: cl, F, methyl, trifluoromethyl; said R is 2 Selected from the group consisting of:
in another preferred embodiment, the compound is selected from the group consisting of:
/>
in a second aspect of the present invention there is provided a pharmaceutical composition comprising as an active ingredient a compound according to the first aspect of the present invention, or a pharmaceutically acceptable salt or stereoisomer thereof, or a prodrug molecule thereof.
In another preferred embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or diluent.
In a third aspect, the invention provides an application of the pyrimidoimidazole compound and pharmaceutically acceptable salt or stereoisomer or prodrug molecules thereof in preparing medicines for treating tumors or immune diseases.
In another preferred embodiment, the autoimmune disease is selected from the group consisting of: rheumatoid arthritis, polyneuritis, dermatomyositis, pemphigus, scleroderma, autoimmune hemolytic anemia, ankylosing spondylitis, ulcerative colitis, crohn's disease, systemic lupus erythematosus, dermatomyositis, multiple sclerosis, type I diabetes, sjogren's syndrome and vasculitis, or immune-related inflammatory skin diseases; preferably, the immune-related inflammatory skin disease is selected from the group consisting of: atopic dermatitis, eczema, alopecia areata, psoriasis, vitiligo, lichen planus, lichen sclerosus, panniculitis, acne, and hidradenitis suppurativa.
It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.
Drawings
FIG. 1 is the inhibition of T cell division by compound RDN 009;
FIG. 2 is the inhibition of T cell differentiation by compound RDN 009;
FIG. 3 is the inhibition of T cell activation CD25 by compound RDN 009;
FIG. 4 is the inhibition of T cell activation CD69 by compound RDN 009;
FIG. 5 is the inhibition of T cell secretion cytokines IFN-gamma and IL-4 by compound RDN 009;
FIG. 6 shows the results of elution of the compounds of the present invention after binding to peptide fragments.
Detailed Description
We propose to enhance the binding activity to ZAP70 by designing a ZAP70 small molecule covalent inhibitor to form a covalent bond with cys amino acids of the catalytic domain in its protein. Based on the above design, the inventors completed the present invention by multi-step synthesis using 2,4, 6-trichloropyrimidine as a starting material.
Terminology
In the chemical compounds of the present invention, when any variable (e.g., R 1 R, etc.) occur more than once in any component, then the definition of each occurrence is independent of the definition of each other occurrence. Also, combinations of substituents and variables are permissible provided that such combinations stabilize the compounds. The lines drawn from the substituents into the ring system indicate that the bond referred to may be attached to any substitutable ring atom. If the ring system is polycyclic, it means that such bonds are only attached to any suitable carbon atom adjacent to the ring. It is to be understood that substituents and substitution patterns of the compounds of this invention may be selected by one of ordinary skill in the art to provide compounds that are chemically stable and that may be readily synthesized from readily available starting materials by techniques in the art and methods set forth below. If the substituent itself is substituted with more than one group, it is understood that these groups may be on the same carbon atom or on different carbon atoms, as long as the structure is stabilized. The phrase "optionally substituted with one or more substituents" is considered to be equivalent to the phrase "optionally substituted with at least one substituent" and in this case preferred embodiments will have from 0 to 3 substituents.
The term "alkyl" as used herein is meant to include both branched and straight chain saturated aliphatic hydrocarbon groups having a specified number of carbon atoms. For example, "C 1 -C 5 Alkyl "medium" C 1 -C 5 The definition of "includes groups having 1,2,3,4, or 5 carbon atoms arranged in a straight or branched chain. For example, "C 1 -C 5 The alkyl group includes, in particular, methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, pentyl. The term "cycloalkyl" refers to a monocyclic saturated aliphatic hydrocarbon group having a specified number of carbon atoms. For example, "cycloalkyl" includes cyclopropyl, methyl-cyclopropyl, 2-dimethyl-cyclobutyl, 2-ethyl-cyclopentyl, cyclohexyl, and the like.
The term "heterocycle" or "heterocyclyl" as used herein refers to a 5-to 6-membered aromatic or non-aromatic heterocycle containing 1 to 4 heteroatoms selected from O, N and S, and includes bicyclic groups. "heterocyclyl" thus includes the heteroaryl groups mentioned above, as well as the dihydro and tetrahydro analogs thereof. Further examples of "heterocyclyl" include, but are not limited to: imidazolyl, indolyl, isothiazolyl, isoxazolyl, oxadiazolyl, oxazolyl, oxetanyl, pyranyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolyl, quinoxalinyl, tetrazolyl, thiadiazolyl, thiazolyl, thienyl, triazolyl, l, 4-dioxanyl, pyrrolidinyl, dihydroimidazolyl, dihydroisoxazolyl, dihydroisothiazolyl, dihydrooxadiazolyl, dihydrooxazolyl, dihydropyrazinyl, dihydropyrazolyl, dihydropyridinyl, dihydropyrimidinyl, dihydropyrrolyl, dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl, dihydrothienyl, dihydrotriazolyl, dihydroazetidinyl, tetrahydrofuranyl and tetrahydrothienyl, and N-oxides thereof. The attachment of the heterocyclic substituent may be through a carbon atom or through a heteroatom.
As understood by those of skill in the art, "halo" or "halogen" as used herein is meant to include fluorine, chlorine, bromine and iodine.
Unless otherwise defined, alkyl, cycloalkyl, aryl, and heterocyclyl substituents may be unsubstituted or substituted. For example, (C) 1 -C 6 ) The alkyl group may be substituted with one, two or three substituents selected from OH, halogen, alkoxy, dialkylamino or heterocyclyl groups such as morpholino, piperidinyl and the like.
The present invention includes the free forms of the compounds of formulas I-II, as well as pharmaceutically acceptable salts and stereoisomers thereof. Some specific exemplary compounds herein are protonated salts of amine compounds. The term "free form" refers to an amine compound in a non-salt form. Included are pharmaceutically acceptable salts including not only the exemplary salts of the specific compounds described herein, but also all typical pharmaceutically acceptable salts of the compounds of formulas i-II in free form. The free form of the particular salt of the compound may 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 solution, such as dilute aqueous NaOH, dilute aqueous potassium carbonate, dilute aqueous ammonia, and dilute aqueous sodium bicarbonate. The free forms differ somewhat from their respective salt forms in certain physical properties, such as solubility in polar solvents, but for the purposes of this invention such acid and base salts are otherwise pharmaceutically comparable to their respective free forms.
Pharmaceutically acceptable salts of the present invention can be synthesized from the compounds of the present invention containing a basic moiety or an acidic moiety by conventional chemical methods. Typically, salts of basic compounds are prepared by ion exchange chromatography or by reacting the free base with a stoichiometric or excess of an inorganic or organic acid in the form of the desired salt in a suitable solvent or combination of solvents. Similarly, salts of acidic compounds are formed by reaction with suitable inorganic or organic bases.
Thus, pharmaceutically acceptable salts of the compounds of the invention include the conventional non-toxic salts of the compounds of the invention formed by the reaction of a basic compound of the invention with an inorganic or organic acid. For example, conventional nontoxic salts include salts derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like, and also salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxy-benzoic, fumaric, toluenesulfonic, methanesulfonic, ethanedisulfonic, oxalic, isethionic, trifluoroacetic and the like.
If the compounds of the present invention are acidic, suitable "pharmaceutically acceptable salts" refer to salts prepared from pharmaceutically acceptable non-toxic bases including inorganic and organic bases, salts derived from inorganic bases include aluminum, ammonium, calcium, copper, iron, ferrous, lithium, magnesium, manganese, manganous, potassium, sodium, zinc, and the like. Ammonium, calcium, magnesium, potassium and sodium salts are particularly preferred. Salts derived from pharmaceutically acceptable organic non-toxic bases including salts of primary, secondary and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins such as arginine, betaine, caffeine, choline, N' -dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, aminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydroxycobalamin, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine and the like.
Berg et al, "Pharmaceutical Salts" j.pharm.sci.1977:66:1-19 describe in more detail the preparation of pharmaceutically acceptable salts as described above and other typical pharmaceutically acceptable salts.
Since under physiological conditions the deprotonated acidic moiety, e.g. carboxyl, in the compound may be anionic, and this charge may then be balanced out by the protonated or alkylated basic moiety, e.g. tetravalent nitrogen atom, which is internally cationic, it should be noted that the compounds of the present invention are potentially internal salts or zwitterions.
Pyrimidoimidazoles as ZAP70 inhibitors
The invention provides a pyrimidoimidazole compound with a structure shown in a formula (I), or pharmaceutically acceptable salt or stereoisomer or prodrug molecules thereof:
wherein R is 1 Any one selected from:
wherein A is 1 ,A 2 ,A 3 ,A 4 ,A 5 Any one selected from:
H；
F,Cl；
C1-C2 alkyl;
a fluorine-containing methyl group;
C 1 ～C 4 alkoxy group；
N, N-dimethylamino;
5-6 membered saturated heterocycle;
quilt A 6 Substituted 4-7 membered saturated heterocyclic ring, wherein A 6 Any one selected from: methyl, dimethylamino, 4-methylpiperazinyl, 2-fluoroethyl;
C 1 ～C 2 -4-methylpiperazinyl;
C 1 ～C 4 -N A 7 A 8 wherein A is 7 ,A 8 Optionally from methyl, ethyl;
the hetero atom in the heterocycle is selected from N, O and S;
R 2 any one selected from:
w is optionally selected from:
–NH(CH 2 ) n NH-, wherein n is optionally selected from 1,2,3;
a 4-7 membered saturated ring containing an N atom;
containing N or O substituted benzene rings or aromatic heterocyclic rings.
In another preferred embodiment, the compound has the structure of formula (ii):
wherein,
A 1 ,A 2 ,A 3 ,A 4 ,A 5 any one selected from:
H；
F,Cl；
methyl, trifluoromethyl;
methoxy, ethoxy, isopropoxy;
pyrrolyl, piperidinyl, morpholinyl, thiarphinyl, N-dimethylamino;
4-methylpiperazinyl, 4-methylpiperidinyl, 2- (4-methylpiperazinyl) ethyl, 4- (4-methylpiperazinyl) piperidinyl, 4- (N, N-dimethylpiperidinyl, 4-methylpiperazinyl, 1- (2-fluoroethyl) azetidin-3-amino, 4- (N, N-diethyl) butyl;
the W is selected from:
n is optionally from 0,1,2,3; x is optionally selected from halogen atom, trifluoromethyl and methyl.
The R is 2 Selected from:
in another preferred embodiment, the compound has the structure of formula (ii):
wherein,
A 1 ,A 2 ,A 4 ,A 5 any one selected from:
H；
a methyl group;
methoxy, ethoxy;
A 3 any one selected from:
w is optionally selected from:
x is optionally selected from Cl, F, methyl, trifluoromethyl.
The R is 2 Selected from:
preparation of pyrimidoimidazoles
In addition to standard methods known in the literature or exemplified in experimental procedures, the compounds of the present invention can be prepared using the reactions shown in the schemes below. Accordingly, the following illustrative schemes are for purposes of illustration and are not limited to the listed compounds or any particular substituents. The number of substituents shown in the schemes does not necessarily correspond to the number used in the claims and for clarity the single substituents are shown attached to compounds which allow multiple substituents under the definition of Wen Zhongshi I-II above.
The compounds of the formula I-II according to the invention can be synthesized from 2,4, 6-trichloropyrimidine as starting material by 7-step reactions.
In one embodiment, the compounds contemplated herein and pharmaceutically acceptable salts thereof may be used to treat or control rheumatoid arthritis, polyneuritis, dermatomyositis, pemphigus, scleroderma, autoimmune hemolytic anemia, ankylosing spondylitis, ulcerative colitis, crohn's disease, systemic lupus erythematosus, dermatomyositis, multiple sclerosis, type I diabetes, sjogren's syndrome, vasculitis, atopic dermatitis, eczema, alopecia areata, psoriasis, vitiligo, lichen planus, scleroatrophic lichen, panniculitis, acne, and suppurative sweat gland.
Drug metabolites and prodrugs
Metabolites of the compounds and pharmaceutically acceptable salts thereof, as well as prodrugs that can be converted in vivo to the structures of the compounds and pharmaceutically acceptable salts thereof, are also encompassed by the claims of the present application.
In the present invention, "pharmaceutically acceptable excipient, carrier or diluent" includes, but is not limited to, any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye/colorant, flavoring agent, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent or emulsifying agent, and the like, which is approved by the relevant government regulatory agency as acceptable for human or livestock use.
According to the present invention, the drug prepared in the above-mentioned use may contain, as another active ingredient, other agents useful for preventing or treating autoimmune diseases and immune-related inflammatory skin diseases, in addition to the small molecule compound of the present invention. Examples of such agents include, but are not limited to, vitamin D derivatives, vitamin a derivatives, glucocorticoids, calcineurin inhibitors, or non-steroidal anti-inflammatory drugs, and the like. When the medicament comprises a plurality of active ingredients, each active ingredient may be administered simultaneously, sequentially or separately at the discretion of the physician.
In addition, the small molecule compounds of the invention may be administered to a patient by a variety of routes, such as orally, transdermally, subcutaneously, intranasally, intravenously, intramuscularly, intrathecally, regionally or topically (e.g., mucosal). The most suitable route of administration in any given case will depend on the nature and severity of the subject and the disease, as well as the physical condition of the subject, etc. In one embodiment, the small molecule compounds of the invention may be administered intravenously. In another embodiment, the small molecule compounds of the invention may be administered orally. Accordingly, the medicament of the invention can be prepared into different dosage forms according to different administration modes. For example, in one embodiment, the medicament may be prepared as a tablet, capsule, pill, granule, aerosol, spray or injection.
The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental methods, in which specific conditions are not noted in the following examples, are generally conducted under conventional conditions or under conditions recommended by the manufacturer. Percentages and parts are by weight unless otherwise indicated.
Example 1
5- ((3- (2-chloroacetamido) benzyl) amino) -7- ((3, 5-dimethoxyphenyl) amino) imidazo [1,2-c ] pyrimidine-8-amide (RDN 009)
Step 1.4-amino-2, 6-dichloropyrimidine-5-carboxamide (2)
2,4, 6-trichloropyrimidine-5-carboxylic acid (5.0 g,22 mmol) was dissolved in dry tetrahydrofuran (150 mL) at 0deg.C, catalyzed by the addition of two drops of DMF, followed by dropwise addition of oxalyl chloride (4.5 mL,44 mmol) and stirring at room temperature was continued for 1 hour. The solution was then concentrated to dryness to give the crude acid chloride. The crude acid chloride was dissolved in dry tetrahydrofuran (50 mL) and added dropwise to a solution of aqueous ammonia (23 mL) in tetrahydrofuran at 0 ℃. After stirring at room temperature for 2 hours, water was added to quench the mixture, and the organic phase was concentrated by spin-drying and purified by column chromatography to give 3.97g (87%) of the compound as a white solid.
MS(ESI)m/z 205.0[M-H] - .
Step 2.4-amino-2-chloro-6- (((3, 5-dimethoxyphenyl) amino) pyrimidine-5-carboxamide (3)
2 (1.0 g,4.83 mmol) and 3, 5-dimethoxyaniline (0.89 g,5.80 mmol) were dissolved in 1, 4-dioxane (60 mL), DIPEA (1.50 mL,9.66 mmol) was added to the mixture, and the mixture was stirred at 80℃overnight. After the reaction was completed, the resulting precipitate was collected by suction filtration, washed with water and dried under reduced pressure to obtain 0.6g (38%) as a white solid.
1 H NMR(500MHz,DMSO-d6)δ9.67(s,1H),7.70(s,1H),7.20(s,1H),6.78(d,J＝2.15Hz,2H),6.19(t,J＝2.10Hz,1H),3.72(s,6H).
Step 3.5-chloro-7- ((3, 5-dimethoxyphenyl) amino) imidazo [1,2-c ] pyrimidine-8-amide (4)
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3 (250 mg,0.77 mmol) and NaHCO 3 (65 mg,0.77 mmol) was dissolved in t-BuOH (50 mL), naI (5 mg) was added to the mixture, and then 40% chloroacetaldehyde (0.6 mL,2.31 mmol) was added, and the reaction stirred at 80℃overnight. After the reaction was completed, the mixture was cooled to room temperature, concentrated under reduced pressure, and purified by column chromatography to obtain 100mg (37%) of a yellow solid.
1 H NMR(500MHz,chloroform-d)δ11.60(s,1H),7.51(d,J＝1.65Hz,1H),7.44(d,J＝1.65Hz,1H),6.80(d,J＝2.15Hz,2H),6.28(t,J＝2.20Hz,1H),3.82(s,6H).
Step 4.5- ((3-aminobenzyl) amino) -7- ((3, 5-dimethoxyphenyl) amino) imidazo [1,2-c ] pyrimidine-8-amide (5)
To a solution of 4 (1.6 g,4.66 mmol) and 3- (aminomethyl) aniline (0.8 mg,7.00 mmol) in DMF (50 mL) was added K 2 CO 3 (1.9 g,0.87 mmol) and the mixture was stirred at 80℃for 1 hour. The mixture was then cooled to room temperature, poured into water (300 mL) and extracted with EtOAc (3×150 mL), concentrated under reduced pressure and chromatographed on silica gel to give 1.4g (70%) of an orange solid.
1 H NMR(500MHz,DMSO-d 6 ):δ12.52(s,1H),9.55(d,J＝3.55Hz,1H),8.88(t,J＝5.80Hz,1H),7.94(d,J＝1.75Hz,1H),7.42(d,J＝3.55Hz,1H),7.37(d,J＝1.65Hz,1H),6.96(t,J＝7.70Hz,1H),6.82(d,J＝2.25Hz,2H),6.54(brs,1H),6.51(d,J＝7.45Hz,1H),6.45(dd,J＝8.00,1.30Hz,1H),6.12(t,J＝2.20Hz,1H),5.04(s,2H),4.71(d,J＝5.65Hz,2H),3.61(s,6H).
MS(ESI,[M+H] + )m/z 434.3.
Step 5.5- ((3- (2-chloroacetamido) benzyl) amino) -7- ((3, 5-dimethoxyphenyl) amino) imidazo [1,2-c ] pyrimidine-8-amide RDN009
5 (100 mg,0.23 mmol) was dissolved in DCM, then cooled to 0deg.C, DIPEA (96 μL,0.69 mmol) was added followed by dropwise addition of 2-chloroacetyl chloride (22 μL,0.28 mmol) and the reaction stirred at room temperature for 30 min. After the reaction was completed, the reaction solution was concentrated under reduced pressure, and the crude product was purified by reverse phase HPLC to give 53mg (45%) of a white solid.
1 H NMR(500MHz,DMSO-d 6 )δ12.35(s,1H),10.26(s,1H),9.05(brs,1H),7.95(brs,1H),7.57-7.50(m,2H),7.42(brs,1H),7.29(t,J＝7.95Hz,1H),7.12(d,J＝7.60Hz,1H),6.77(d,J＝1.80Hz,2H),6.13(t,J＝2.20Hz,1H),4.83(d,J＝5.60Hz,2H),4.20(s,2H).3.60(s,6H).
13 C NMR(151MHz,DMSO-d 6 )δ169.10,165.09,161.00,154.99,147.55,145.98,141.73,139.27,139.24,129.51,123.14,118.79,118.05,108.55,98.89,95.57,82.78,55.42,44.65,44.01.
HRMS(ESI,[M+H] + )calcd for C 24 H 25 ClN 7 O 4 ,510.1578；found,509.9892.
Example 2
Step 1. Tert-butyl (4- (((8-carbamoyl-7- ((3, 5-dimethoxyphenyl) amino) imidazo [1,2-c ] pyrimidin-5-yl) amino) methyl) benzyl) carbamate (RDN 1147)
To a solution of RDN2106 (100 mg,0.29 mmol) and tert-butyl (4- (aminomethyl) benzyl) carbamate (136 mg,0.58 mmol) in DMF (5 mL) was added K 2 CO 3 (120 mg,0.87 mmol). After stirring at 80℃for 1 hour, the mixture was stirredCooled to room temperature. Then, 2mL of water was added to quench the reaction. The mixture was poured into water (30 mL) and then extracted with EtOAc (3×15 mL). The organic phase was then concentrated under high vacuum and purified by silica gel column chromatography to give 140mg (92%) of an orange solid.
1H NMR(500MHz,DMSO-d 6 ):δ12.29(s,1H),9.00(t,J＝5.35Hz,1H),7.92(d,J＝1.75Hz,1H),7.40(d,J＝1.75Hz,1H),7.33(d,J＝7.95Hz,2H),7.18(d,J＝7.95Hz,2H),6.80(d,J＝2.20Hz,1H),6.15(t,J＝2.20Hz,1H),4.77(d,J＝5.40Hz,2H),4.07(d,J＝6.05Hz,2H),3.62(s,6H),1.36(s,9H).
MS(ESI,[M+H] + )m/z 548.3.
Step 2 5- ((4- (aminomethyl) benzyl) amino) -7- ((3, 5-dimethoxyphenyl) amino) imidazo [1,2-c ] pyrimidine-8-amide (RDN 1152)
Compound RDN1147 (140 mg,0.25 mmol) was dissolved in DCM (2 mL), trifluoroacetic acid (2 mL) was added to the mixture, followed by stirring at room temperature for 1h and then concentration under reduced pressure. Subsequently, the crude product was dissolved in DCM, cooled to 0 ℃ and DIPEA (133 μl,0.75 mmol) was added followed by dropwise addition of acryloyl chloride (25 μl,0.31 mmol), the mixture was stirred at room temperature for 30min, after completion of the reaction it was concentrated under reduced pressure and then purified by reverse phase HPLC to give 80mg (62%) of a white solid.
1 H NMR(500MHz,DMSO-d 6 ):δ12.33(s,1H),8.99(t,J＝5.55Hz,1H),8.56(t,J＝5.90Hz,1H),7.92(d,J＝1.75Hz,1H),7.39(d,J＝1.65Hz,2H),7.34(d,J＝8.10Hz,2H),7.21(d,J＝8.10Hz,1H),6.80(d,J＝2.20Hz,2H),6.24(dd,J＝17.10,10.15Hz,1H),6.16(t,J＝2.20Hz,1H),6.10(dd,J＝17.05,2.10Hz,1H),5.60(dd,J＝10.15,2.15Hz,1H),4.78(d,J＝5.65Hz,2H),4.30(d,J＝5.95Hz,2H),3.63(s,6H).
MS(ESI,[M+H] + )m/z 502.2.
HRMS(ESI,[M+H] + )calcd for C 26 H 28 N 7 O 4 ,502.2125；found,502.0402.
Example 3
(E) -7- (((3, 5-Dimethoxyphenyl) amino) -5- ((3- (4- (dimethylamino) but-2-enamino) benzyl) amino) imidazo [1,2-c ] pyrimidine-8-amide (WCY 010)
Compound RDN1137 (100 mg,0.23 mmol), (E) -4- (dimethylamino) but-2-enoic acid (36 mg,0.28 mmol) and HATU (106 mg,0.28 mmol) were dissolved in DCM (5 mL) to which DIPEA (96 μl,0.69 mmol) was added. The mixture was stirred at room temperature overnight, after completion of the reaction, it was concentrated under reduced pressure, and the crude product was purified by reverse phase HPLC to give 20mg (62%) of a white solid.
1 H NMR(500MHz,DMSO-d 6 ):δ12.51(s,1H),10.03(s,1H),9.56(d,J＝3.60Hz,1H),8.99(t,J＝5.80Hz,1H),7.94(d,J＝1.70Hz,1H),7.62(d,J＝8.25Hz,1H),7.60(s,1H),7.44(d,J＝3.60Hz,1H),7.38(d,J＝1.60Hz,1H),7.27(t,J＝7.80Hz,1H),7.08(d,J＝7.70Hz,1H),6.78(d,J＝2.20Hz,2H),6.70(dt,J＝15.40,6.95Hz,1H),6.23(d,J＝15.40Hz,1H),6.11(t,J＝2.20Hz,1H),4.83(d,J＝5.60Hz,2H),3.60(s,6H),3.07(d,J＝5.60Hz,1H),2.18(s,6H).
MS(ESI,[M+H] + )m/z 545.4.
HRMS(ESI,[M+H] + )calcd for C 28 H 33 N 8 O 4 ,545.2547；found,545.1116.
Example 4
Step 1.5- ((4-aminobenzyl) amino) -7- ((3, 5-dimethoxyphenyl) amino) imidazo [1,2-c ] pyrimidine-8-amide (RDN 2015)
RDN2106 (289 mg,0.83 mmol) and 4- (aminomethyl) aniline (204 mg,1.67 mmol) were dissolved in DMF (8 mL) and then K was added thereto 2 CO 3 (344 mg,2.49 mmol) and reactingThe solution was stirred at 80℃for 1 hour. After the reaction was completed, the mixture was cooled to room temperature, quenched with water (50 mL), and then extracted with EtOAc (3×20 mL). The organic phase was concentrated under reduced pressure and purified by silica gel column chromatography to give 1.4g (70%) of an orange solid.
1 H NMR(500MHz,DMSO-d 6 ):δ＝12.38(s,1H),9.03(t,J＝5.80Hz,1H),7.92(d,J＝1.75Hz,1H),7.40-7.37(m,3H),7.11(d,J＝8.35Hz,2H),6.78(d,J＝2.2Hz,2H),6.15(t,J＝2.2Hz,1H),4.78(d,J＝5.65Hz,1H),3.64(s,6H).
MS(ESI,[M+H] + )m/z 434.2.
Step 2.5- ((4-acrylamidobenzyl) amino) -7- ((3, 5-dimethoxyphenyl) amino) imidazo [1,2-c ] pyrimidine-8-amide (WCY 007)
Compound RDN2015 (140 mg,0.25 mmol) was dissolved in DCM (2 mL), cooled to 0 ℃ and DIPEA (133 μl,0.75 mmol) was added followed by dropwise addition of acryloyl chloride (25 μl,0.31 mmol), and the mixture was stirred at room temperature for 30min, after completion of the reaction concentrated under reduced pressure, followed by purification by reverse phase HPLC to give 80mg (62%) of a white solid.
1 H NMR(500MHz,DMSO-d 6 ):δ12.46(s,1H),10.14(s,1H),8.94(t,J＝5.45Hz,1H),7.91(d,J＝1.35Hz,1H),7.61(d,J＝8.40Hz,2H),7.37(d,J＝1.35Hz,1H),7.34(d,J＝8.45Hz,2H),6.81(d,J＝2.05Hz,2H),6.41(d,J＝16.95,10.15Hz,1H),6.41(d,J＝16.95,10.15Hz,1H),6.23(d,J＝16.95,1.80Hz,1H),6.15(s,3H),6.23(d,J＝10.15,1.80Hz,1H),4.77(d,J＝5.55Hz,2H),3.64(s,6H).
MS(ESI,[M+H] + )m/z 488.3.
HRMS(ESI,[M+H] + )calcd for C25H26N7O4,488.1968；found,488.0143.
Example 5
Step 7- (((3, 5-Dimethoxyphenyl) amino) -5- ((3-propanamido benzyl) amino) imidazo [1,2-c ] pyrimidine-8-carboxamide RDN1135
Compound RDN1137 (50 mg,0.12 mmol), propiolic acid (9 mg,0.13 mmol), DCC (27 mg,0.13 mmol) and 4-DMAP (1 mg,0.01 mmol) were dissolved in DCM (5 mL) and the mixture stirred at room temperature overnight after the reaction was complete, concentrated under reduced pressure and the crude product was purified by reverse phase HPLC to give 10mg (18%) of a white solid.
1 H NMR(500MHz,DMSO-d 6 ):δ12.41(s,1H),10.77(s,1H),9.01(brs,1H),7.92(brs,1H),7.58(brs,1H),7.52(d,J＝7.60Hz,1H),7.40(brs,1H),7.27(t,J＝7.40Hz,1H),7.12(d,J＝6.95Hz,1H),6.76(brs,2H),6.12(s,1H),4.80(d,J＝5.10Hz,1H),4.36(s,1H),3.60(s,6H).
MS(ESI,[M+H] + )m/z 486.2.
HRMS(ESI,[M+H] + )calcd for C25H23N7O4；found,486.0091.
Example 6
5- ((3-acrylamidobenzyl) amino) -7- ((3, 5-dimethoxyphenyl) amino) imidazo [1,2-c ] pyrimidine-8-amide WCY006
The synthesis was as in example 1.
1 H NMR(500MHz,DMSO-d 6 ):δ12.44(s,1H),10.11(s,1H),9.02(brs,1H),7.94(s,1H),7.64-7.61(m,2H),7.39(brs,1H),7.28(t,J＝7.55Hz,1H),7.09(d,J＝7.80Hz,1H),6.78(d,J＝2.20Hz,2H),6.39(dd,J＝16.85,10.10Hz,1H),6.22(dd,J＝16.90,1.90Hz,1H),6.12(t,J＝2.10Hz,1H),5.72(dd,J＝10.10,1.90Hz,1H),4.83(d,J＝5.65Hz,2H),3.60(s,6H).
MS(ESI,[M+H] + )m/z 488.3.
HRMS(ESI,[M+H] + )calcd for C25H26N7O4,488.1968；found,488.0339.
Example 7
5- ((4- (2-chloroacetamido) benzyl) amino) -7- ((3, 5-dimethoxyphenyl) amino) imidazo [1,2-c ] pyrimidine-8-amide WCY008
The synthesis was as in example 1.
1 H NMR(500MHz,DMSO-d 6 ):δ12.43(s,1H),10.29(s,1H),8.96(brs,1H),7.91(s,1H),7.53(d,J＝8.35Hz,2H),7.36(t,J＝4.15Hz,2H),7.36(d,J＝8.20Hz,2H),6.80(brs,2H),6.15(brs,1H),4.77(d,J＝5.05Hz,2H),4.23(s,2H),3.63(s,6H).
HRMS(ESI,[M+H] + )calcd for C 24 H 25 ClN 7 O 4 ,510.1578；found,509.9869.
Example 8
5- ((3- (acrylamidomethyl) benzyl) amino) -7- ((3, 5-dimethoxyphenyl) amino) imidazo [1,2-c ] pyrimidine-8-amide WCY011
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The synthesis was as in example 1.
1 H NMR(500MHz,DMSO-d 6 ):δ12.40(s,1H),8.98(s,1H),8.58-8.54(m,1H),7.91(s,1H),7.37(s,1H),7.28(d,J＝2.50Hz,1H),7.27(s,1H),7.16-7.14(m,1H),6.80(d,J＝2.20Hz,2H),6.22(dd,J＝17.10,10.15Hz,1H),6.14(t,J＝2.10Hz,1H),6.07(dd,J＝17.05,2.10Hz,1H),5.58(dd,J＝17.05,2.20Hz,1H),4.80(d,J＝5.60Hz,2H),4.30(d,J＝6.00Hz,2H),3.62(s,6H).
MS(ESI,[M+H] + )m/z 502.2.
HRMS(ESI,[M+H] + )calcd for C 26 H 28 N 7 O 4 ,502.2125；found,502.0431.
Example 9
5- ((((1-propenylpiperidin-3-yl) methyl) amino) -7- (((3, 5-dimethoxyphenyl) amino) imidazo [1,2-c ] pyrimidine-8-amide WCY 012)
The synthesis was as in example 1.
1 H NMR(500MHz,DMSO-d 6 ):δ12.20(s,1H),8.55(d,J＝22.1Hz,1H),7.91(s,1H),7.41(d,J＝7.15Hz,1H),6.84(d,J＝10.85Hz,2H),6.79-6.57(m,1H),6.22(d,J＝8.75Hz,1H),6.02(d,J＝16.76Hz,1H),5.63-5.53(m,1H),4.37(d,J＝12.51Hz,1H),3.75(s,6H),3.57-3.52(m,1H),3.51-3.43(m,1H),3.42-3.36(m,1H),3.07-2.92(m,1H),2.80-2.53(m,1H),1.89(brs,1H),1.73-1.70(m,1H),1.37-1.23(m,1H).
MS(ESI,[M+H] + )m/z 480.3.
HRMS(ESI,[M+H] + )calcd for C 24 H 30 N 7 O 4 ,480.2281；found,480.0645.
Example 10
5- ((3-acrylamidophenyl) amino) -7- ((3, 5-dimethoxyphenyl) amino) imidazo [1,2-c ] pyrimidine-8-amide WCY013
The synthesis was as in example 1.
1 H NMR(500MHz,DMSO-d 6 ):δ12.44(s,1H),10.08(s,1H),8.56-8.51(m,1H),7.85(d,J＝1.75Hz,1H),7.57-7.51(m,2H),7.35(d,J＝1.60Hz,1H),7.24(t,J＝7.70Hz,1H),6.96(d,J＝7.55Hz,1H),6.85(d,J＝2.20Hz,2H),6.42(dd,J＝16.95,10.15Hz,1H),6.24(dd,J＝16.95,1.90Hz,1H),6.16(t,J＝2.20Hz,1H),5.74(dd,J＝10.15Hz,1.80,1H),3.79-3.73(m,2H),3.65(s,6H),2.98-2.94(m,1H).
MS(ESI,[M+H] + )m/z 502.3.
HRMS(ESI,[M+H] + )calcd for C26H28N7O4,502.2125；found,502.0466.
Example 11
5- ((3-acrylamidopropyl) amino) -7- ((3, 5-dimethoxyphenyl) amino) imidazo [1,2-c ] pyrimidine-8-amide WCY014
The synthesis was as in example 1.
1 H NMR(500MHz,DMSO-d 6 ):δ12.29(s,1H),8.50(brs,1H),8.13(t,J＝5.20Hz,1H),7.87(brs,1H),7.40(brs,1H),6.87(d,J＝2.20Hz,2H),6.20-6.13(m,2H),6.06(dd,J＝17.10,2.25Hz,1H),5.56(dd,J＝10.00,2.25Hz,1H),3.74(s,6H),3.24(q,J＝6.70Hz,2H),1.86(m,2H).
MS(ESI,[M+H] + )m/z 440.3.
HRMS(ESI,[M+H] + )calcd for C21H26N7O4,440.1968；found,440.0438.
Example 12
5- ((3- (but-2-ylamido) benzyl) amino) -7- ((3, 5-dimethoxyphenyl) amino) imidazo [1,2-c ] pyrimidine-8-amide RDN1124
The synthesis was as in example 2.
1 H NMR(500MHz,DMSO-d 6 ):δ12.43(s,1H),10.55(s,1H),8.99(t,J＝5.65Hz,1H),7.93(d,J＝1.60Hz,1H),7.57(s,1H),7.53(d,J＝8.00Hz,1H),7.39(d,J＝1.60Hz,1H),7.25(t,J＝7.85Hz,1H),7.09(d,J＝7.55Hz,1H),6.77(d,J＝2.15Hz,2H),6.12(t,J＝2.15Hz,1H),4.80(d,J＝5.65Hz,2H),3.60(s,6H),2.01(s,3H).
MS(ESI,[M+H] + )m/z 500.2.
HRMS(ESI,[M+H] + )calcd for C26H26N7O4,500.1968；found,500.0258.
Example 13
5- ((1- (3-acrylamidophenyl) ethyl) amino) -7- ((3, 5-dimethoxyphenyl) amino) imidazo [1,2-c ] pyrimidine-8-amide RDN1132
The synthesis was as in example 1.
MS(ESI,[M+H] + )m/z 502.3.
Example 14
7- (((3, 5-Dimethoxyphenyl) amino) -5- ((3-methacrylamidobenzyl) amino) imidazo [1,2-c ] pyrimidine-8-amide RDN1141
The synthesis was as in example 1.
MS(ESI,[M+H] + )m/z 502.3.
Example 15
5- ((((1-propenylpiperidin-4-yl) methyl) amino) -7- (((3, 5-dimethoxyphenyl) amino) imidazo [1,2-c ] pyrimidine-8-amide RDN 1146)
The synthesis was as in example 1.
1 H NMR(500MHz,DMSO-d 6 ):δ12.23(s,1H),8.57(s,1H),7.90(s,1H),7.39(s,1H),6.85-6.77(m,3H),6.21(brs,1H),6.07(dd,J＝16.70,2.40Hz,1H),5,64(dd,J＝10.40,2.35Hz,1H),4.43(d,J＝12.50Hz,1H),4.07(d,J＝13.20Hz,1H),3.74(s,6H),3.43(q,J＝5.65Hz,2H),3.00(t,J＝12.50Hz,1H),2.59(t,J＝12.10Hz,1H),2.10-1.98(m,1H),1.84(d,J＝12.70Hz,2H),1.18-1.03(m,2H).
MS(ESI,[M+H] + )m/z 480.4.
HRMS(ESI,[M+H] + )calcd for C 24 H 30 N 7 O 4 ,480.2281；found,480.0630.
Example 16
5- ((((1-propenoylpyrrolidin-3-yl) methyl) amino) -7- (((3, 5-dimethoxyphenyl) amino) imidazo [1,2-c ] pyrimidine-8-amide RDN 1153)
The synthesis was as in example 1.
1 H NMR(500MHz,DMSO-d 6 ):δ12.25-12.17(m,1H),8.69-8.59(m,1H),7.89(d,J＝11.09Hz,1H),7.42-7.40(m,1H),6.84(d,J＝1.95Hz,2H),6.56-6.48(m,1H),6.22-6.20(m,1H),6.14-6.09(m,1H),5.65(td,J＝10.19,2.37Hz,1H),3.74(s,6H),3.69-3.48(m,4H),3.38-3.29(m,1H),3.19(dd,J＝12.30,6.92Hz,1H),2.79-2.59(m,1H),2.14-1.98(m,1H),1.80-1.61(m,1H).
MS(ESI,[M+H] + )m/z 466.2.
HRMS(ESI,[M+H] + )calcd for C 23 H 28 N 7 O 4 ,466.2125；found,466.0507.
Example 17
5- ((3-acrylamido-4-chlorobenzyl) amino) -7- ((3, 5-dimethoxyphenyl) amino) imidazo [1,2-c ] pyrimidine-8-amide RDN1156
The synthesis was as in example 1.
MS(ESI,[M+H] + )m/z 522.2.
Example 18
Methyl (E) -4- ((3- ((((8-carbamoyl-7- ((3, 5-dimethoxyphenyl) amino) imidazo [1,2-c ] pyrimidin-5-yl) amino) methyl) phenyl) amino) -2-enoate RDN2022
RDN1137 (50 mg,0.12 mmol) and methyl (E) -4-bromobut-2-enoate (25 mg,0.14 mmol) were dissolved in DMF (3 mL) followed by DIPEA (0.36, 60. Mu.L) and the reaction was heated to 60℃and after one hour the reaction was purified directly by reverse phase HPLC to give 10mg (16.4%) of a white solid.
MS(ESI,[M+H] + )m/z 532.2.
Example 19
7- (((3, 5-Dimethoxyphenyl) amino) -5- ((3- (vinylsulfonamide) benzyl) amino) imidazo [1,2-c ] pyrimidine-8-amide RDN 2024)
The synthesis was as in example 1.
MS(ESI,[M+H] + )m/z 524.1.
Example 20
(E) -7- (((3, 5-dimethoxyphenyl) amino) -5- ((3- ((4- (methylamino) -4-oxetan-2-2-1-yl) amino) benzyl) amino) imidazo [1,2-c ] pyrimidine-8-amide RDN 2032)
The synthesis was as in example 17.
HRMS(ESI,[M+H] + )calcd for C 27 H 31 N 8 O 4 ,531.2390；found,531.0736.
Example 21
7- (((3, 5-Dimethoxyphenyl) amino) -5- ((3-propanamido benzyl) amino) imidazo [1,2-c ] pyrimidine-8-amide RDN2033
The synthesis was as in example 1.
HRMS(ESI,[M+H] + )calcd for C 25 H 27 N 7 O 4 ,490.2125；found,490.0588
Example 22
5- ((3-acrylamidobenzyl) amino) -7- ((4-morpholinophenyl) amino) imidazo [1,2-c ] pyrimidine-8-amide RDN2050
The synthesis was as in example 1.
MS(ESI,[M+H] + )m/z 513.4.
Example 23
5- ((3- (2-chloroacetamido) benzyl) amino) -7- ((4-morpholinophenyl) amino) imidazo [1,2-c ] pyrimidine-8-amide RDN2051
The synthesis was as in example 1.
1 H NMR(600MHz,CD 3 OD)δ7.84-7.80(m,2H),7.51(d,J＝2.05Hz,1H),7.39-7.40(m,4H),7.10((d,J＝7.05Hz,1H),7.04(brs,2H),4.76(s,2H),4.19(s,2H),3.90(t,J＝4.55Hz,4H),3.26(brs,4H).
MS(ESI,[M+H] + )m/z 535.1.
HRMS(ESI,[M+H] + )calcd for C 26 H 28 ClN 8 O 3 ,535.1895；found,534.9447.
Example 24
5- ((3-acrylamidobenzyl) amino) -7- ((4- (dimethylamino) phenyl) amino) imidazo [1,2-c ] pyrimidine-8-amide RDN2062
The synthesis was as in example 1.
MS(ESI,[M+H] + )m/z 471.1.
Example 25
5- ((3- (2-chloroacetamido) benzyl) amino) -7- ((4- (dimethylamino) phenyl) amino) imidazo [1,2-c ] pyrimidine-8-amide RDN2065
The synthesis was as in example 1.
1 H NMR(500MHz,chloroform-d)δ11.78(s,1H),7.56(s,1H),7.44(d,J＝7.45Hz,1H),7.36-7.29(m,4H),7.21(s,1H),7.11(d,J＝7.55Hz,1H),6.77-6.75(m,2H),4.70(d,J＝5.45Hz,2H),4.20(s,2H),3.49(s,2H),2.94(s,6H).
MS(ESI,[M+H] + )m/z 493.1.
HRMS(ESI,[M+H] + )calcd for C 24 H 26 ClN 8 O 2 ,493.1789；found,493.1897.
Example 26
5- ((3-acrylamidobenzyl) amino) -7- ((4- (4-methylpiperazin-1-yl) phenyl) amino) imidazo [1,2-c ] pyrimidine-8-amide RDN2078
The synthesis was as in example 1.
MS(ESI,[M+H] + )m/z 526.3.
Example 27
5- ((3- (2-chloroacetamido) benzyl) amino) -7- ((4- (4-methylpiperazin-1-yl) phenyl) amino) imidazo [1,2-c ] pyrimidine-8-amide RDN2081
The synthesis was as in example 1.
1 H NMR(500MHz,CD 3 OD)δ7.77-7.38(m,2H),7.42-7.38(m,2H),7.37-7.30(m,3H),7.12(d,J＝7.40Hz,1H),6.91(d,J＝8.90Hz,2H),4.77(s,2H),4.19(s,2H),3.80-3.77(m,2H),3.62-3.59(m,2H),3.28-3.25(m,2H),3.01-2.95(m,5H).
MS(ESI,[M+H] + )m/z 548.2.
HRMS(ESI,[M+H] + )calcd for C 27 H 31 ClN 9 O 2 ,548.2211；found,548.0922.
Example 28
5- ((3-acrylamidobenzyl) amino) -7- ((4- (4-methyl-1, 4-diaza-1-yl) phenyl) amino) imidazo [1,2-c ] pyrimidine-8-amide RDN2104
The synthesis was as in example 1.
MS(ESI,[M+H] + )m/z 540.3.
Example 29
5- ((3- (2-chloroacetamido) benzyl) amino) -7- ((4- (4-methyl-1, 4-diaza-1-yl) phenyl) amino) imidazo [1,2-c ] pyrimidine-8-amide RDN2115
The synthesis was as in example 1.
1 H NMR(600MHz,CD 3 OD)δ7.76-7.74(m,2H),7.42-7.37(m,2H),7.34-7.29(m,3H),7.11(d,J＝7.85Hz,1H),6.72-6.70(m,2H),4.75(s,2H),4.19(s,2H),3.83-3.80(m,1H),3.70-3.64(m,2H),3.53-3.49(m,3H),3.34-3.28(m,2H),2.95(s,3H),2.30-2.19(m,2H).
MS(ESI,[M+H] + )m/z 562.1.
HRMS(ESI,[M+H] + )calcd for C 28 H 33 ClN 9 O 2 ,562.2367；found,562.0583.
Example 30
5- ((3-acetamidobenzyl) amino) -7- ((3, 5-dimethoxyphenyl) amino) imidazo [1,2-c ] pyrimidine-8-amide WCY2026
The synthesis was as in example 1.
MS(ESI,[M+H] + )m/z 476.3.
Example 31
5- ((3-acrylamido-4-methylbenzyl) amino) -7- ((3, 5-dimethoxyphenyl) amino) imidazo [1,2-c ] pyrimidine-8-amide WCY2035
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The synthesis was as in example 1.
MS(ESI,[M+H] + )m/z 502.3
Example 32
5- ((3-acrylamido-4-fluorobenzyl) amino) -7- ((3, 5-dimethoxyphenyl) amino) imidazo [1,2-c ] pyrimidine-8-amide WCY2039
The synthesis was as in example 1.
MS(ESI,[M+H] + )m/z 506.2
Example 33
5- ((3-acrylamidobenzyl) amino) -7- ((3-methoxy-4-morpholinophenyl) amino) imidazo [1,2-c ] pyrimidine-8-amide RDN2149
The synthesis was as in example 1.
HRMS(ESI,[M+H] + )calcd for C 28 H 30 N 8 O 4 ,543.2390；found,543.1168.
Example 34
5- ((3-acrylamido-4-chlorobenzyl) amino) -7- ((3-methoxy-4-morpholinophenyl) amino) imidazo [1,2-c ] pyrimidine-8-amide RDN2150
The synthesis was as in example 1.
HRMS(ESI,[M+H] + )calcd for C 28 H 29 ClN 8 O 4 ,577.2000；found,577.0571.
Example 35
5- ((4-acrylamidobenzyl) amino) -7- ((4- (4- (dimethylamino) piperidin-1-yl) phenyl) amino) imidazo [1,2-c ] pyrimidine-8-amide RDN2087
The synthesis was as in example 1.
MS(ESI,[M+H] + )m/z 554.3.
Example 36
5- ((4- (2-chloroacetamido) benzyl) amino) -7- ((4- (4- (dimethylamino) piperidin-1-yl) phenyl) amino) imidazo [1,2-c ] pyrimidine-8-amide RDN2088
The synthesis was as in example 1.
HRMS(ESI,[M+H] + )calcd for C 29 H 34 ClN 9 O 2 ,576.2524；found,576.1032.
Example 37
5- ((3-acrylamido-2-chlorobenzyl) amino) -7- ((3, 5-dimethoxyphenyl) amino) imidazo [1,2-c ] pyrimidine-8-amide RDN3051
The synthesis was as in example 1.
1 H NMR(500MHz,DMSO-d 6 ):δ12.42(s,1H),9.76(s,1H),7.28(t,J＝6.35Hz,1H),7.95(d,J＝1.65Hz,1H),7.75(d,J＝7.40Hz,1H),7.40(d,J＝1.60Hz,1H),7.33-7.27(m,2H),6.71(d,J＝2.20Hz,2H),6.66(dd,J＝17.00,10.10Hz,1H),6.29(dd,J＝17.00,1.90Hz,1H),6.11(t,J＝2.20Hz,1H),5.80(dd,J＝10.10,1.80Hz,1H),4.90(d,J＝5.45Hz,2H),3.59(s,6H).
MS(ESI,[M+H] + )m/z 522.1.
Example 38
5- (7-acrylamido-3, 4-dihydroisoquinolin-2 (1-hydro) -yl) -7- ((3, 5-dimethoxyphenyl) amino) imidazo [1,2-c ] pyrimidine-8-amide RDN3053
The synthesis was as in example 1.
1 H NMR(500MHz,MeOD):δ7.84(d,J＝2.35Hz,1H),7.68(brs,1H),7.56(d,J＝2.35Hz,1H),7.37-7.34(m,1H),7.22(d,J＝8.30Hz,1H),6.88(d,J＝6.90Hz,2H),6.46-6.40(m,1H),6.39-6.35(m,1H),6.34(t,J＝2.05Hz,1H),5.78(dd,J＝9.45,2.10Hz,1H),4.96(s,2H),4.02(t,J＝5.80Hz,2H),3.84(s,6H),3.11(t,J＝5.15Hz,2H).
MS(ESI,[M+H] + )m/z 514.2.
Example 39
5- ((3-acrylamidobenzyl) (methyl) amino) -7- ((3, 5-dimethoxyphenyl) amino) imidazo [1,2-c ] pyrimidine-8-amide RDN3054
The synthesis was as in example 1.
1 H NMR(500MHz,DMSO-d 6 ):δ12.44(s,1H),10.19(s,1H),9.69(s,1H),7.67-7.65(m,2H),7.60-7.57(m,2H),7.37-7.33(m,2H),7.09(d,J＝7.65Hz,1H),6.79(d,J＝2.15Hz,2H),6.39(dd,J＝16.90,10.15Hz,1H),6.24(dd,J＝10.15,1.90Hz,1H),6.15(t,J＝2.15Hz,1H),5.74(dd,J＝10.15,1.90Hz,1H),4.90(s,2H),3.67(s,6H),3.23(s,3H).
MS(ESI,[M+H] + )m/z 502.1.
Example 40
5- ((3-acrylamido-2-fluorobenzyl) amino) -7- ((3, 5-dimethoxyphenyl) amino) imidazo [1,2-c ] pyrimidine-8-amide RDN3058
The synthesis was as in example 1.
1 H NMR(500MHz,MeOD):δ7.86(t,J＝7.45Hz,1H),7.83(d,J＝2.25Hz,1H),7.49(d,J＝2.25Hz,1H),7.16(t,J＝6.65Hz,1H),7.10-7.06(m,1H),6.76(d,t,J＝2.20Hz,2H),6.54(dd,J＝17.00,10.20Hz,1H),6.39(dd,J＝17.00,1.60Hz,1H),6.25(t,t,J＝2.20Hz,1H),5.81(dd,J＝10.20,1.60Hz,1H),3.70(s,6H).
MS(ESI,[M+H] + )m/z 506.2.
Example 41
5- ((5-acrylamido-2-chlorobenzyl) amino) -7- ((3, 5-dimethoxyphenyl) amino) imidazo [1,2-c ] pyrimidine-8-amide RDN3063
The synthesis was as in example 1.
1 H NMR(500MHz,DMSO-d 6 ):δ12.41(s,1H),10.22(s,1H),9.00(t,J＝5.75Hz,1H),7.96(d,J＝1.70Hz,1H),7.84(dd,J＝8.75,2.35Hz,1H),7.52(d,J＝2.30Hz,1H),7.46(d,J＝8.75Hz,1H),7.43(d,J＝1.60Hz,1H),6.66(d,J＝2.20Hz,2H),6.34(dd,J＝16.95,10.10Hz,1H),6.20(dd,J＝16.95,2.00Hz,1H),6.10(t,J＝2.15Hz,1H),5.72(dd,J＝10.10,2.00Hz,1H),4.85(d,J＝5.50Hz,2H),3.59(s,6H).
MS(ESI,[M+H] + )m/z 522.2.
Example 42
5- (((5-acrylamidopyridin-3-yl) methyl) amino) -7- ((3, 5-dimethoxyphenyl) amino) imidazo [1,2-c ] pyrimidine-8-amide RDN3070
The synthesis was as in example 1.
1 H NMR(500MHz,MeOD):δ8.98(brs,1H),8.43(s,1H),8.36(s,1H),7.76(d,J＝1.95Hz,1H),7.48(d,J＝1.95Hz,2H),6.42(d,J＝3.00Hz,1H),6.20(d,J＝2.05Hz,2H),5.86(dd,J＝8.90,2.75Hz,2H),4.94(s,2H),3.71(s,6H).
MS(ESI,[M+H] + )m/z 489.1.
Example 43
5- ((((1-propenoyl-1, 2,3, 4-tetrahydroquinolin-5-yl) methyl) amino) -7- (((3, 5-dimethoxyphenyl) amino) imidazo [1,2-c ] pyrimidine-8-amide RDN 3071)
The synthesis was as in example 1.
1 H NMR(500MHz,DMSO-d 6 ):δ12.32(brs,1H),8.89(brs,1H),7.99(brs,1H),7.41(brs,1H),7.16-7.14(m,2H),7.04(brs,1H),6.71(d,J＝2.20Hz,2H),6.53-6.46(m,1H),6.22(dd,J＝16.80,2.15Hz,1H),6.14(t,J＝2.00Hz,1H),5.70(dd,J＝10.25,2.15Hz,1H),4.79(d,J＝5.35Hz,2H),3.76(t,J＝6.20Hz,2H),3.63(s,6H),2.76(t,J＝6.65Hz,2H),1.94-1.87(m,2H).
MS(ESI,[M+H] + )m/z 528.1.
Example 44
Test of ZAP70 kinase inhibition rate of pyrimidine imidazole compounds
Inhibition rate assays of all final compounds against ZAP70 and Syk were performed by chemartner company (china) at the upper sea, ATP concentration of K m . Proteins ZAP70 and Syk were purchased from Carna. FAM-22 peptide fragments and cross-package bases were purchased mainly from GL Biochem and Seleckchem.
1. Preparation of 1x kinase base buffer and stop buffer to test kinase
1) 1x kinase base buffer 50mM HEPES,pH 7.5 0.0015%Brij-35
2) Stop buffer 100mM HEPES,pH 7.5 0.015%Brij-35.2% coating reagent #3 50mM EDTA
2. Preparation of the Compounds
1) The compound was diluted with 100% dmso to 50X of the highest inhibitor concentration ultimately required in the reaction. 100 μl of this compound dilution was transferred to one well of a 96-well plate.
2) In the same 96-well plate, 100 μl of 100% dmso was added to both wells for no compound control and no enzyme control. The plate is labeled as the source plate.
3) Intermediate plates were prepared and 10 μl of compound was transferred from the source plate to a new 96-well plate as an intermediate plate. Mu.l of 1 Xkinase buffer was added to each well of the intermediate plate. The compounds were mixed on the shaker for 10 minutes on the intermediate plate.
3. Preparation of assay plate
1) Mu.l of each well was transferred from the 96-well intermediate plate into 384-well plates in duplicate. For example, A1 of a 96-well plate is transferred to A1 and A2 of a 384-well plate. A2 of the 96-well plate was transferred to A3 and A4 of the 384-well plate, and so on.
4. Kinase reaction
1) A 2.5x enzyme solution was prepared and kinase was added to the 1x kinase base buffer.
2) Preparation of 2.5x peptide solution FAM-labeled peptide and ATP were added to the 1x kinase base buffer.
3) Assay plates already contained 5 μl of compound in 10% dmso.
4) Transfer of 2.5 Xenzyme solution into assay plate 10. Mu.l of 2.5 Xenzyme solution was added to each well of 384 well assay plate.
5) Incubate for 10 minutes at room temperature.
6) Transfer of 2.5x peptide solution into assay plate 10 μl of 2.5x peptide solution was added to each well of 384 well assay plates.
7) The kinase reaction was then incubated at 28℃for 30 minutes. Add 25. Mu.l stop buffer to stop the reaction.
5. The caliper reading collects data on the caliper.
6. Curve fitting
1) The conversion data is copied from the Caliper program.
2) The converted value is converted into a suppression value. Percent inhibition = (maximum conversion)/(max-min) ×100."max" represents DMSO control; "min" represents low control force.
3) Data were fit in XLfit excel add-on version 4.3.1 to obtain IC50 values.
The formula used is: y=bottom+ (top-bottom)/(1+ (IC) 50 /X)^HillSlope)
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The results show that the compound provided by the invention has a better inhibition effect on ZAP 70.
Example 45
Homologous female BALB/c mice (6-8 weeks old, 18-20g weight) were derived from Shanghai laboratory animal center, proc, shanghai laboratory animal center SPF-grade house rearing. All experiments strictly followed the Shanghai pharmaceutical institute biological ethics committee requirements. Single-core spleen cell suspensions were prepared using the BALB/c mice and prepared using EasySep TM Separation and purification of mouse T cell sorting kit (Stemcell) to obtain CD4 + T cell (purity)>98%). Culturing CD4 with RPMI-1640 culture solution + T cells, and RDN009 and CD3 antibody (5. Mu.g/ml, thermo Fisher Scientific) and CD28 (2. Mu.g/ml, thermo Fisher Scientific) antibodies were added at the indicated concentrations. After incubation for 24 hours or 48 hours, 0.5. Mu. Ci/well [ 3 H]Thymidine treatment of cell culture broth and detection of radioactivity by Beta counter (Microbeta Trilux, perkinElmer Life Sciences) was used to calculate CD4 + The proliferation rate of T cells and the results are shown in fig. 2. The results show that the compounds of the invention have CD4 inhibition + T cell differentiation.
After this example was completed, cell supernatants were collected for subsequent analysis of inflammatory factors and cell activation levels.
Example 46
CD4 + T cells were labeled with CFSE for 5 min at 37 ℃ and then washed 3 times with RPMI 1640 medium containing 20% fbs. The labeled cells were then incubated with CD3 antibody (5. Mu.g/ml) and CD28 antibody (2. Mu.g/ml) in RPMI 1640 medium containing 10% FBS for 72 hours, and finally the fluorescence intensity of CFSE was detected by flow cytometry, and the cell cycle was analyzed. The results are shown in FIG. 1, which shows that the compounds of the present invention have CD4 inhibition + Effect of T cell division rate.
Example 47
For CD4 in the foregoing cell proliferation assay + T cells were blocked using anti-CD16/CD32 antibodies (eBioscience) and stained with the following reagents, respectively: fluorescein isothiocyanate labeled antibody CD3 (FITC-conjugated CD 3), phycoerythrin labeled antibody CD25 (PE-conjugated CD 25), allophycocyanin labeled antibody CD4 (APC-conjugated CD 4) and Brilliant violet labeled antibody CD6 (BV 421-conjugated CD 69) (BD Biosciences). Finally, the fluorescence intensity of the cells was measured by flow cytometry, and the data was analyzed by FlowJo software. The results are shown in FIGS. 3 and 4, which show that CD4 after treatment of the above cells with the compounds of the present invention + CD25 + Cell and CD4 + CD69 + There is a significant decrease in the proportion of cells that activate.
Example 48
The cell supernatants isolated in example 38 were analyzed for secretion of IL-4 and IFN-gamma according to ELISA kit (BD Pharmingen) instructions. OD values were measured at 450nm using a microplate reader. The results are shown in FIG. 5, which shows that the compounds of the present invention effectively reduce inflammatory responses, as the amount of inflammatory factors secreted is reduced after the cells are treated with the compounds of the present invention.
Example 49
Small molecule covalent inhibitors and ZAP-70 protein (Carna, cat. No. 08-377) in buffer (0.01% triton. Times. 100,50mM HEPES,5mM MgCl) 2 ,1mM MnCl 2 ) At a molar concentration ratio of 1:1.4 at a temperature of 28 DEG CIncubate for 5 minutes at this temperature. Then dithiothreitol (DTT, final concentration 2 mM) was added and incubated for an additional 10 minutes. 0.1. Mu.g trypsin was added and the digestion was stopped by digestion at 37℃for 18 hours and 2% by volume trifluoroacetic acid. The peptide fragment obtained was dissolved in HPLC buffer (0.1%formic acid,v/v) and isolated using the EASY-nLC 1200UHPLC system (ThermoFisher Scientific). The elution conditions were: 30min,5% -90% HPLC buffer (0.1%formic acid in 80%acetonitrile,v/v). The eluted peptide fragments were analyzed by Q exact HF-X mass spectrometry, and finally the ZAP-70 sequence was retrieved by Mascot 2.3 analysis, identifying the site of covalent modification of the small molecule. The results are shown in FIG. 6, which shows that the compounds of the present invention are irreversible inhibitors after acting on ZAP70, whereas similar compounds in the prior art are reversible inhibitors, suggesting that the compounds of the present invention are a better ZAP70 inhibitor.
All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the claims appended hereto.

Claims (12)

1. A pyrimidoimidazole compound of formula (i) or a pharmaceutically acceptable salt or stereoisomer thereof:
wherein,
the W is-NH-W 1 -: the W is 1 Selected from the group consisting of:n is 1, n1 is 0;
x is selected from the group consisting of: a halogen atom;
said R is 2 Selected from the group consisting of:
said R is 1 Has a structure shown in the following formula:
wherein A is 1 And A 5 Is H, A 2 And A 4 Each independently selected from the group consisting of: H. f, cl, C 1 ～C 2 Alkyl, fluoromethyl, C 1 ～C 4 An alkoxy group; wherein, C is as follows 1 ～C 2 Alkyl is unsubstituted or substituted with 1 OH; a is that 3 Selected from the group consisting of: H. saturated heterocyclic ring with 5-6 membered, or A 6 Substituted 4-7 membered saturated heterocyclic ring, wherein each A 6 Each independently selected from the group consisting of: methyl, dimethylamino, 4-methylpiperazino, 2-fluoroethyl, C 1 ～C 2 Alkyl-4-methylpiperazinyl, C 1 ～C 4 -N(A 7 )(A 8 ) Wherein A is 7 、A 8 Optionally from methyl, ethyl;
wherein the heteroatoms in each heterocycle are selected from N, O; and at least one is an N atom.
2. A compound according to claim 1, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein R 1 Has a structure shown in the following formula:
wherein A is 1 And A 5 Is H, A 2 、A 4 Each independently selected from the group consisting of: H. f, cl, C 1 ～C 2 Alkyl, fluoromethyl, C 1 ～C 4 An alkoxy group; wherein, C is as follows 1 ～C 2 Alkyl is unsubstituted or substituted with 1 OH;
A 3 selected from the group consisting of: H. 5-6 membered saturated heterocycle; the hetero atoms in each heterocycle are selected from N, O; and at least one is an N atom.
3. A compound according to claim 1, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein R 1 Has a structure shown in the following formula:
wherein A is 1 And A 5 Is H, A 2 、A 4 Each independently selected from the group consisting of: H. f, cl, C 1 ～C 2 Alkyl, fluoromethyl, C 1 ～C 4 An alkoxy group;
A 3 selected from the group consisting of: H. quilt A 6 Substituted 4-7 membered saturated heterocyclic ring, wherein each A 6 Each independently selected from the group consisting of: methyl, dimethylamino, 4-methylpiperazino, 2-fluoroethyl, C 1 ～C 2 Alkyl-4-methylpiperazinyl; the hetero atoms in each heterocycle are selected from N, O; and at least one is an N atom.
4. A compound according to claim 1, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein R 2 Selected from the group consisting of:
5. the compound of claim 1, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein the compound has the structure of formula (ii):
wherein,
A 2 、A 4 each independently selected from the group consisting of: H. f, cl methyl, trifluoromethyl, methoxy, ethoxy, isopropoxy;
A 3 selected from the group consisting of: pyrrolyl, piperidinyl, morpholinyl, thiarphinyl, 4-methylpiperazinyl, 4- (4-methylpiperazinyl) piperidinyl, 4- (N, N-dimethyl) piperidinyl, 4-methylpiperazinyl.
6. The compound of claim 5, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein a 1 And A 5 Is H, A 2 And A 4 Each independently selected from the group consisting of: H. methyl, methoxy, ethoxy; and A is 3 Selected from the group consisting of:
7. a compound of the formula, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein the compound has the structure of formula (ii):
wherein,
A 1 、A 5 each independently is H;
A 2 、A 4 each independently is methoxy;
A 3 is H;
the W is 1 Is thatn=1, and n1=0;
x is selected from the group consisting of: a halogen atom;
said R is 2 Selected from the group consisting of:
8. a compound selected from the group consisting of:
9. a pharmaceutical composition comprising as an active ingredient a compound according to any one of claims 1 to 8, or a pharmaceutically acceptable salt or stereoisomer thereof.
10. Use of a pyrimidoimidazole compound as defined in any one of claims 1 to 8, and a pharmaceutically acceptable salt or stereoisomer thereof for the preparation of ZAP-70 inhibitors; the ZAP-70 inhibitor is used for treating immune diseases, wherein the immune diseases are autoimmune diseases or immune-related inflammatory skin diseases.
11. The use according to claim 10, wherein the autoimmune disease is selected from the group consisting of: rheumatoid arthritis, polyneuritis, pemphigus, scleroderma, autoimmune hemolytic anemia, ankylosing spondylitis, ulcerative colitis, crohn's disease, systemic lupus erythematosus, dermatomyositis, multiple sclerosis, type I diabetes, sjogren's syndrome and vasculitis, or inflammatory skin diseases associated with immunity.
12. The use according to claim 10, wherein the immune-related inflammatory skin disease is selected from the group consisting of: atopic dermatitis, eczema, alopecia areata, psoriasis, vitiligo, lichen planus, lichen sclerosus, panniculitis, acne, and hidradenitis suppurativa.
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