WO2023202336A1 - Hpk1激酶抑制剂在预防和/或治疗人的病原体感染中的应用 - Google Patents

Hpk1激酶抑制剂在预防和/或治疗人的病原体感染中的应用 Download PDF

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WO2023202336A1
WO2023202336A1 PCT/CN2023/084751 CN2023084751W WO2023202336A1 WO 2023202336 A1 WO2023202336 A1 WO 2023202336A1 CN 2023084751 W CN2023084751 W CN 2023084751W WO 2023202336 A1 WO2023202336 A1 WO 2023202336A1
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alkyl
cancer
crystal form
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deuterium
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French (fr)
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林星雨
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珠海宇繁生物科技有限责任公司
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    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
    • A61K31/4545—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a six-membered ring with nitrogen as a ring hetero atom, e.g. pipamperone, anabasine
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    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/4439—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. omeprazole
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    • A61K31/551—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having two nitrogen atoms, e.g. dilazep
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    • C07D401/04—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings directly linked by a ring-member-to-ring-member bond
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    • C07B2200/13—Crystalline forms, e.g. polymorphs

Definitions

  • the present invention relates to the field of medical technology, and specifically relates to the application of an HPK1 kinase inhibitor in the preparation of medicines for preventing and/or treating human diseases or conditions caused by or related to pathogen infection.
  • the present invention also relates to the crystal form of the HPK1 kinase inhibitor and its preparation method and application.
  • HPK1 hematopoietic progenitor kinase 1
  • mitogen-activated protein kinase mitogen-activated protein kinase kinase kinase kinase, MAP4K
  • HPK1 kinase participates in many signaling cascades, including growth factor signaling, MAPK signaling, cytokine signaling, apoptosis signaling, growth factor signaling, and antigen receptor signaling.
  • HPK1 is a negative regulator of activation of T cells, B cells and dendritic cells. Inhibiting its function can enhance immune cells and thereby enhance immunological functions. Therefore, the development of HPK1 inhibitors is of great significance.
  • Patent document CN113861188A discloses a pyrazolo[3,4-b]pyridine derivative, its preparation method and its application as an HPK1 inhibitor. Its general formula is as follows: Patent document CN113316576A discloses a HPK1 compound, whose general formula is as follows: Patent document WO2021213317A1 discloses an HPK1 inhibitor and its preparation method and use. The structural formula of the compound is as follows: However, there are few reports on the use of small molecule HPK1 kinase inhibitors in the treatment of viral infections in the current technology. Therefore, the application prospects of HPK1 kinase inhibitors in the treatment of viral infections are very broad.
  • the present invention provides the use of an HPK1 kinase inhibitor in the preparation of medicines for preventing and/or treating human diseases or conditions caused by pathogen infection or related to pathogen infection:
  • the pathogen may be a microorganism, parasite or other vector
  • the parasite is a protozoa, helminth or other vector.
  • the microorganism is selected from: one or more of viruses, chlamydia, rickettsiae, mycoplasma, bacteria, spirochetes, fungi, etc.;
  • the pathogen is a virus, including but not limited to, Adenoviridae, Herpesviridae, HSV2, VZV, EBV, CMV, Poxviridae, Papilloviridae, Parvoviridae, and Hepadnaviridae , Polyomaviridae, Reoviridae, Small Riboviridae, Caliciviridae, Togaviridae, Arenaviridae, Retroviridae, Flaviviridae, Orthomyxoviridae, Paramyxoviridae, Bunyaviridae, Coronaviridae, Astroviridae, Bornaviridae;
  • the above-mentioned pathogens are viruses, such as, but not limited to, Adenoviridae (such as adenovirus), Herpesviridae (such as HSV1 (oral herpes), HSV2 (external genital herpes), VZV (varicella) ), EBV (Eye-Barr virus), CMV (cytomegalovirus)), Poxviridae (such as smallpox virus, vaccinia virus), Papilloviridae (such as human papillomavirus (HPV)), parvovirus Family (such as B19 virus), Hepadnaviridae (such as hepatitis B virus (HBV)), Polyomaviridae (such as polyomavirus), Reoviridae (such as reovirus, rotavirus) , Picornaviridae (such as enterovirus, foot-and-mouth disease virus), Caliciviridae (such as Norwalk virus
  • Adenoviridae
  • the virus is HBV, HIV, HCV, HPV, Ebola virus, Marburg virus, influenza virus, parainfluenza virus, dengue virus and human coronavirus;
  • the human coronavirus includes: SARS-CoV, SARS-CoV-2, MERS-CoV, HCoV-229E, HCoV-NL63, HCoV-OC43 and HCoV-HKU1;
  • the diseases caused by pathogen infection or related to pathogen infection include influenza, SARS, MERS, COVID-19, viral hepatitis, AIDS, dengue fever, Ebola virus disease, and Marburg virus disease.
  • the viral hepatitis includes hepatitis B and hepatitis C.
  • T cell immune response is an important part of the body's immune screening. Activation of CD4+ T cells can help B cells produce neutralizing antibodies and activate CD8+ T cells, and CD8+ T cells can eliminate virus-infected cells (Grifoni A, WeiskopfD, Ramirez S I, Mateus J, Dan J M, Moderbacher C R, Rawlings S A, Sutherland A, Premkumar L, Jadi R S, Marrama D, De Silva A M, Frazier A, Carlin A F, Greenbaum J A, Peters B, Krammer F, Smith D M, Crotty S, Sette A.Targets of T Cell Responses to SARS-CoV-2Coronavirus in Humans with COVID-19 Disease and Unexposed Individuals[J].Cell,2020,181(7):1489-1501.e15.).
  • activated virus-specific effector T cells will produce antiviral cytokines (such as IL-2), thereby helping the body fight the virus (Liao W, Lin J-X, Leonard W J. IL-2Family Cytokines: New Insights into the Complex Roles of IL-2as a Broad Regulator ofT helper Cell Differentiation[J].Current Opinion in Immunology,2011,23(5):598–604, Channappanavar R, Zhao J, Perlman S.T cell-mediated immune response to respiratory coronaviruses [J].Immunologic Research,2014,59(1):118–128).
  • IL-2 antiviral cytokines
  • the HPK1 kinase inhibitor of the present invention can enhance T cell proliferation and continuously increase TCR-induced calcium flow and pro-inflammatory cytokines (such as IL-2) levels. Moreover, it can induce B cell activation and significantly enhance the immune function of T cells and dendritic cells.
  • the HPK1 kinase inhibitor of the present application can be used not only as anti-tumor drugs, but also as anti-viral drugs.
  • the inhibitor is a compound represented by the following general formula I, or its pharmaceutically acceptable salts, stereoisomers, esters, prodrugs, solvates and deuterated compounds:
  • A is selected from CR 10 or N;
  • Q is selected from O or S
  • x and z are independently selected from integers between 0 and 6 (such as 0, 1, 2, 3, 4, 5, 6);
  • y is 0 or 1
  • Ar is selected from an aromatic five-membered heterocyclic group, an aromatic six-membered heterocyclic group or a phenyl group, wherein the aromatic five-membered heterocyclic group is selected from: furyl, thienyl, pyrrolyl, pyrazolyl, Imidazolyl, oxazolyl, thiazolyl or selenothiazolyl, the aromatic six-membered heterocyclic group is selected from: pyridyl, pyridazinyl, pyrimidinyl or pyrazinyl, optionally, the aromatic five-membered heterocyclic group H on the group, aromatic six-membered heterocyclic group or phenyl group can be substituted by the following groups: -D, -SO 2 , -SO 2 N(C 0-10 alkyl)(C 0-10 alkyl ), -N(C 0-10 alkyl)SO 2 (C 0-10 alkyl), -CON(C
  • R 2 is selected from: -H, -D, halogen, -NO 2 , -CN, C 1-10 linear/branched alkyl, C 3-10 cycloalkyl, -N(C 0-10 alkyl) (C 0-10 alkyl), -CF 3 , -OCF 3 , -OCHF 2 , -OCH 2 F or -OC 0-10 alkyl;
  • B 1 , B 2 , B 3 , B 4 and B 5 are independently selected from C or N (when B 1 , B 2 , B 3 , B 4 or B 5 is N, its corresponding R 3 , R 4 , R 5 , R 6 , R 7 do not exist);
  • R 3 , R 4 , R 5 , R 6 and R 7 are independently selected from: -H, -D, halogen, -CN, -OC 0-10 alkyl, -CO(C 0-10 alkyl base), -CON(C 0-10 alkyl)(C 0-10 alkyl), C 1-10 linear/branched alkyl, O or N-containing heteroalkyl, -N(C 0-10 Alkyl) (C 0-10 alkyl), C 3-10 cycloalkyl, -C ⁇ CR 10 , -O heterocycloalkyl, -N heterocycloalkyl, or R 5 and R 4 , R 4 and R 3 , R 3 and R 7 , R 7 and R 6 and the carbon atoms between them form a C 3-8 cycloalkyl group or a C 3-8 heterocycloalkyl group containing -O-, -S-, -N hetero Cycloaromatic group, -O heterocyclic
  • R 8 and R 9 are independently selected from: -H, -D, halogen, C 1-10 linear/branched alkyl;
  • R 10 is selected from: H, -D, C 1-5 linear/branched alkyl, C 3-10 cycloalkyl,
  • one of the C atoms in Ar, R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 and/or R 12 or Multiple H atoms can be replaced by deuterium;
  • one of the heteroatoms among Ar, R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 and/or R 12 connected to or multiple H atoms may be replaced by deuterium;
  • the compound of general formula I contains at least one deuterium atom
  • compounds of general formula I contain at least one deuterium atom.
  • A is CR 10 , in particular CH.
  • Q is O.
  • x is 0.
  • z is 1.
  • y is 1.
  • B 1 , B 2 , B 3 , B 4 and B 5 are all C, that is, in the general formula I, for
  • At least one of B 1 , B 2 , B 3 , B 4 and B 5 is N.
  • B 2 is C, and at least one of B 1 , B 3 , B 4 and B 5 is N.
  • B 2 is C and B 1 is N.
  • B 2 is C and B 3 is N.
  • B 2 is C and B 4 is N.
  • B 2 is C and B 5 is N.
  • B 2 is C
  • B 3 and B 4 are N
  • both B 3 and B 5 are N.
  • Ar is selected from: thiazolyl, selenothiazolyl, imidazolyl, pyrazolyl and pyridinyl.
  • the compound of formula I has the following structure:
  • the E ring is selected from:
  • each R 0 is independently selected from: -H, -D, C 1-10 linear/branched alkyl, -N(C 0-10 alkyl)(C 0-10 alkyl), - OC 0-10 alkyl, -CO(C 0-10 alkyl) or C 3-10 cycloalkyl; wherein the H attached to the above-mentioned C atom or heteroatom can be replaced by deuterium;
  • R 1 is selected from: -H, -D, -O heterocycloalkyl, -N heterocycloalkyl, C 1-10 linear/branched alkyl, C 3-10 cycloalkyl, -OC 0-10 Alkyl, -N(C 0-10 alkyl)(C 0-10 alkyl), -SO 2 (C 0-10 alkyl), -CO(C 0-10 alkyl), -O-phenyl , -S (C 0-10 alkyl), -N heterocyclic aromatic group, -O heterocyclic aromatic group or -S heterocyclic aromatic group;
  • R 2-9 has the corresponding definitions above in the present invention.
  • H connected to the C atoms in R 0 and R 1 may be replaced by deuterium
  • the H connected to the heteroatoms in R 0 and R 1 may be replaced by deuterium;
  • At least one of the R 0-9 contains a deuterium atom
  • the compound of formula II contains at least one deuterium atom.
  • R 1 contains at least one deuterium atom; more specifically, for example, R 1 contains at least one deuterium atom, and R 2 to 9 do not contain deuterium atoms.
  • R 2 contains at least one deuterium atom.
  • R 3 contains at least one deuterium atom.
  • R 4 contains at least one deuterium atom.
  • R 5 contains at least one deuterium atom.
  • R 6 contains at least one deuterium atom.
  • R 8 and/or R 9 contains at least one deuterium atom.
  • each R 0 is independently selected from: C 1-5 linear/branched alkyl or -N(C 0-10 alkyl)(C 0-10 alkyl), wherein the C atom attached to H can be replaced by deuterium.
  • each R 0 is independently selected from: -H, -D, -CH 3 , -CH 2 CH 3 or -NH 2 .
  • R 1 is selected from: -O heterocycloalkyl or -N heterocycloalkyl, -SO 2 (C 0-3 alkyl), -O-phenyl, -S (C 0-4 alkyl) , C 3-6 cycloalkyl or C 3-5 linear/branched alkyl, in which the H attached to the C atom or heteroatom can be replaced by deuterium.
  • R 1 is selected from: -CH 3 ,
  • the H attached to the C atom or N atom can be replaced by deuterium.
  • R 1 is selected from: -CH 2 D, -CHD 2 , -CD 3 ,
  • R 0 and R 1 when R 0 and R 1 are adjacent, R 0 and R 1 form a C 3-8 cycloalkyl group or a C 3-8 heterocycloalkyl group containing -O-, -S- with the carbon atom between them. , -N heterocyclic aryl group, -O heterocyclic aryl group or -S heterocyclic aryl group, phenyl.
  • R 2 is selected from: -H, -D, halogen, -NO 2 , -CN, C 1-5 linear/branched alkyl, C 3-10 cycloalkyl, -N(C 0-10 Alkyl) (C 0-10 alkyl), -CF 3 , -OCF 3 , -OCHF 2 , -OCH 2 F or -OC 0-10 alkyl, where the H attached to the C atom or N atom can be Replaced by deuterium.
  • R 2 is selected from: -NO 2 , -N(C 0-10 alkyl)(C 0-10 alkyl), -OC 0-10 alkyl and -OCF 3 wherein C atom or N atom
  • the H attached to can be replaced by deuterium.
  • R 2 is selected from: -NH 2 , -NHD, -ND 2 or -NO 2 .
  • R 3 is selected from: -H, -D, halogen, -OC 0-10 alkyl, -CO(C 0-10 alkyl), C 1-10 linear/branched alkyl, -N( C 0-10 alkyl) (C 0-10 alkyl) or C 3-10 cycloalkyl, wherein the H attached to the C atom may be replaced by deuterium.
  • R 3 is selected from: -H, -D, halogen, -OC 0-10 alkyl, C 1-10 linear/branched alkyl, wherein the H attached to the C atom can be replaced by deuterium .
  • R 3 is selected from: -H, -D, -F, -OCH 3 , -OCH 2 D, -OCHD 2 , -OCD 3 .
  • R 4 is selected from: -H, -D, halogen, -OC 0-10 alkyl, -CO(C 0-10 alkyl), -CN, C 3-10 cycloalkyl, -C ⁇ CR 10.
  • R 4 is selected from: -H, -D, halogen, -OC 0-10 alkyl, -CN, C 3-10 cycloalkyl or -C ⁇ CR 10 , where the C atom attached H can be replaced by deuterium.
  • the H attached to the C atom in R 10 may be replaced by deuterium.
  • R 4 is selected from: -H, -D, -F, -Cl, -OCH 3 , -OCH 2 D, -OCHD 2 , -OCD 3 , -CN, Or -C ⁇ CR 10 .
  • R 5 , R 6 , and R 7 are independently selected from: -H, -D, halogen, -CN, -OC 0-10 alkyl, -CO(C 0-10 alkyl), C 1-10 Linear/branched alkyl, -N(C 0-10 alkyl)(C 0-10 alkyl), C 3-10 cycloalkyl, -C ⁇ CR 10 , -O heterocycloalkyl or -N Heterocycloalkyl, C 1-5 linear/branched alkyl containing O or N, or the carbon atoms between R 6 , R 7 and R 6 and R 7 form a C 3-8 cycloalkyl or containing - C 3-8 heterocycloalkyl group of O-, -S-, wherein the H attached to the C atom or heteroatom may be replaced by deuterium.
  • R 5 , R 6 , R 7 are independently selected from: -H, -D, halogen, -CN, C 1-3 linear/branched alkyl, -OC 0-3 alkyl, -CO (C 0-3 alkyl), N-containing C 1-3 linear/branched alkyl, or the carbon atoms between R 6 and R 7 and R 6 and R 7 form a C 3-8 cycloalkyl group or -O-containing C 3-8 heterocycloalkyl group, in which the H attached to the C atom or N atom may be replaced by deuterium.
  • R 5 , R 6 , and R 7 are independently selected from: -H, -D, -F, -Cl, -CH 3 , -CH 2 NH 2 , -CH 2 NH (CH 3 ), -CH 2 N(CH 3 ) 2 , -CN, -OCH 3 , -COCH 3 , or the carbon atoms between R 6 , R 7 and R 6 and R 7 form an -O-containing five-membered cycloalkyl group, where, C The H attached to the atom or N atom can be replaced by deuterium.
  • R 5 is selected from: -H, -D, -F, -Cl, -CH 3 , -CH 2 D, -CHD 2 , -CD 3 , -OCH 3 , -COCH 3 , -CH 2 NH 2 , -CH 2 N(CH 3 ) 2 , -CN, -OCH 2 D , -OCHD 2 , -OCD 3 , -COCD 3 , -CH 2 N(CD 3 ) 2 ,, -CH 2 N(CH 3 )(CD 3 ).
  • R 6 is selected from: -H, -D, -F, -Cl, -CH 3 , -CH 2 D, -CHD 2 , -CD 3 , -OCH 3 , -COCH 3 , -CH 2 NH 2 , -CH 2 N(CH 3 ) 2 , -CN, -OCH 2 D , -OCHD 2 , -OCD 3 , -COCD 3 , -CH 2 N(CD 3 ) 2 ,, -CH 2 N(CH 3 )(CD 3 ).
  • R 7 is selected from: -H, -D, -F, -Cl, -CH 3 , -CH 2 D, -CHD 2 , -CD 3 , -OCH 3 , -COCH 3 , -CH 2 NH 2 , -CH 2 N(CH 3 ) 2 , -CN, -OCH 2 D , -OCHD 2 , -OCD 3 , -COCD 3 , -CH 2 N(CD 3 ) 2 , -CH 2 N(CH 3 )(CD 3 ).
  • R 10 is In one embodiment of the present invention, the H attached to the C atom in R 10 may be replaced by deuterium.
  • the alkyl part can be substituted by the following groups: -SO 2 , -SO 2 N(C 0-10 alkyl)(C 0-10 alkyl), -N(C 0-10 alkyl)SO 2 (C 0-10 alkyl), -CON(C 0
  • R 11 and R 12 are independently selected from: -H, -D, -CF 3 , -CHF 2 , -CDF 2 , -CH 2 F , -CD 2 F, -CH 3 , -CH 2 D. -CHD 2 , -CD 3 , -CH 2 CH 3 , -CH 2 CD 3 ,
  • R 8 and R 9 are independently selected from: -H, -D, C 1-10 linear/branched alkyl, wherein the H attached to the C atom may be replaced by deuterium.
  • R 8 and R 9 are independently selected from: -H, -D, C 1-3 linear/branched alkyl, wherein the H attached to the C atom may be replaced by deuterium.
  • R 8 and R 9 are independently selected from: -H, -D, -CH 3 , -CH 2 D, -CHD 2 , -CD 3 .
  • the compound of general formula I has the following structure:
  • any atom not specified as deuterium in the above compounds is present in its natural isotope abundance.
  • the compounds described above have at least, for example, 95% deuterium incorporation at positions designated as "deuterium.”
  • the invention provides a method for preparing a compound of general formula I, which includes the following steps:
  • R 13 is selected from: halogen or R 14 is selected from: -OH or -F;
  • R 15 is selected from: -Br or -SnBu 3 .
  • R 13 in the above step (1) is -Br.
  • R 13 is -Br or When R 13 is -Br, R 15 is -SnBu 3 , when R 13 is When, R 15 is -Br.
  • the present invention also provides pharmaceutically acceptable salts, stereoisomers, esters, prodrugs and solvates of the compounds of the above general formula I.
  • the above-mentioned pharmaceutically acceptable salts include acid addition salts and base addition salts.
  • the above-mentioned acid addition salts include, but are not limited to, salts derived from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid and phosphonic acid, and from organic acids such as aliphatic monocarboxylic acids and dicarboxylic acids. Salts of acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, alkanedioic acids, aromatic acids and aliphatic and aromatic sulfonic acids.
  • inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid and phosphonic acid
  • organic acids such as aliphatic monocarboxylic acids and dicarboxylic acids. Salts of acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, alkanedioic acids, aromatic acids and
  • these salts include, but are not limited to, sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, nitrates, phosphates, monohydrogen phosphates, dihydrogen phosphates, metaphosphates, pyrophosphates Salt, hydrochloride, hydrobromide, iodate, acetate, propionate, octanoate, isobutyrate, oxalate, malonate, succinate, suberate , Sebacate, fumarate, maleate, amygdalate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, phthalic acid salts, benzenesulfonate, tosylate, phenylacetate, citrate, lactate, maleate, tartrate and methanesulfonate, and also include salts of amino acids such as arginine salts, Gluconate, galacturonate,
  • the above-mentioned base addition salts are formed with metals or amines, such as hydroxides of alkali metals and alkaline earth metals, or with organic amines.
  • metals used as cations include, but are not limited to, sodium, potassium, magnesium, and calcium.
  • suitable amines include, but are not limited to, N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine (ethane-1,2-diamine), N- Methylglucosamine and procaine.
  • Base addition salts may be prepared by contacting the free acid form with a sufficient amount of the desired base to form the salt in a conventional manner. The free acid form can be regenerated by contacting the salt form with an acid, and the free acid isolated in a conventional manner.
  • the above-mentioned pharmaceutically acceptable salt is a hydrochloride.
  • the above-mentioned stereoisomers include enantiomers, diastereomers and geometric isomers.
  • Some of the compounds of the present invention have cycloalkyl groups, which may be substituted on more than one carbon atom, in which case all their geometric forms, including cis and trans, and mixtures thereof, are within the scope of the present invention. within the range.
  • solvate refers to the physical combination of a compound of the invention and one or more solvent molecules.
  • This physical bonding includes various degrees of ionic and covalent bonding, including hydrogen bonding.
  • solvates can be isolated, such as when one or more solvent molecules are incorporated into the crystal lattice of a crystalline solid.
  • Solvate includes both solution-phase and isolatable solvates. Representative solvates include ethanolates, methanolates, etc.
  • “Hydrate” is a solvate in which one or more solvent molecules is H2O .
  • the above-mentioned prodrug refers to the form of the compound of formula I that is suitable for administration to patients without excessive toxicity, irritation, allergic reactions, etc. and is effective for its application purpose, including acetal, ester and zwitterionic forms.
  • the prodrug is converted in vivo (eg by hydrolysis in the blood) to give the parent compound of the above formula.
  • the present invention also provides a crystal form of the compound of the above general formula I and its pharmaceutically acceptable salts, stereoisomers, esters, prodrugs and solvates.
  • the present invention provides 4-(3-(((2-amino-5-(1-(1-trideuteromethylpiperidin-4-yl))-1H-pyrazol-4-yl)pyridine- Crystalline form of 3-yl)oxy)methyl)phenyl)-2-methylbut-3-yn-2-ol (which has the following structure).
  • the above-mentioned crystal form is crystal form A, and its XRPD pattern has at least three (or all) positions with 2 ⁇ values of 13.1° ⁇ 0.2°, 16.3° ⁇ 0.2°, 17.5° ⁇ 0.2°, and 23.8° ⁇ 0.2°. ) position has a characteristic peak (main characteristic diffraction peak).
  • the XRPD pattern of the above-mentioned crystal form A also has 2 ⁇ values of 8.1° ⁇ 0.2°, 12.2° ⁇ 0.2°, 15.3° ⁇ 0.2°, 18.0° ⁇ 0.2°, 19.3° ⁇ 0.2°, and 19.5° ⁇ 0.2°. , 21.3° ⁇ 0.2°, 21.6° ⁇ 0.2°, at least three (at least four, at least five, at least six, at least seven, or all) positions have characteristic peaks (secondary characteristic diffraction peaks) ).
  • the above-mentioned crystal form A has an XRPD pattern substantially as shown in FIG. 1 .
  • the DSC spectrum of the above-mentioned crystal form A has an endothermic peak at about 168.8°C.
  • the above-mentioned crystal form A has a DSC pattern substantially as shown in Figure 2.
  • the weight loss of the above-mentioned Form A when heated from room temperature to 170° C. is about 1.1%.
  • the above-mentioned crystal form A has a TGA pattern substantially as shown in FIG. 2 .
  • the above-mentioned crystal form A is an amorphous form.
  • the above-mentioned crystal form is crystal form B, and its XRPD pattern has at least three positions (or There are characteristic peaks (main characteristic diffraction peaks) at all) positions.
  • the XRPD pattern of the above-mentioned crystal form B also has 2 ⁇ values of 7.1° ⁇ 0.2°, 8.8° ⁇ 0.2°, 14.1° ⁇ 0.2°, 17.0° ⁇ 0.2°, 18.0° ⁇ 0.2°, and 18.8° ⁇ 0.2°.
  • the above-mentioned crystal form B has an XRPD pattern substantially as shown in FIG. 6 .
  • the DSC spectrum of the above-mentioned crystal form B has an endothermic peak at at least one of about 59.5°C, 95.6°C, 150.8°C and 160.9°C.
  • the above-mentioned crystal form B has a DSC pattern substantially as shown in FIG. 7 .
  • the weight loss of the above-mentioned crystalline form B when heated from room temperature to 70°C is about 13.2%, and the weight loss when continued to be heated to 170°C is about 8.5%.
  • the above-mentioned crystal form B has a TGA pattern substantially as shown in FIG. 7 .
  • the above-mentioned crystal form B is an EtOAc solvate.
  • the present invention also provides 4-(3-(((2-amino-5-(1-(1-trideuteromethylpiperidin-4-yl))-1H-pyrazol-4-yl)pyridine -Crystalline form of -3-yl)oxy)methyl)phenyl)-2-methylbut-3-yn-2-ol hydrochloride.
  • the above-mentioned crystal form is crystal form A, and its XRPD pattern is in the position where the 2 ⁇ values are 13.0° ⁇ 0.2°, 16.3° ⁇ 0.2°, 17.5° ⁇ 0.2°, 19.4° ⁇ 0.2°, and 23.8° ⁇ 0.2°.
  • the XRPD pattern of the above-mentioned crystal form A also has at least three positions with 2 ⁇ values of 8.1° ⁇ 0.2°, 12.1° ⁇ 0.2°, 15.3° ⁇ 0.2°, 18.0° ⁇ 0.2°, and 21.4° ⁇ 0.2°.
  • the above-mentioned crystal form A has an XRPD pattern substantially as shown in FIG. 11 .
  • the DSC spectrum of the above-mentioned crystal form A has endothermic peaks at about 81.9°C and about 156.0°C.
  • the above-described crystal form A has a DSC pattern substantially as shown in FIG. 12 .
  • the weight loss of the above-mentioned Form A when heated from room temperature to 150° C. was about 8.6%.
  • the above-mentioned crystal form A has a TGA pattern substantially as shown in FIG. 12 .
  • the present invention also provides a method for preparing the above crystal form.
  • the above preparation method is selected from one of: anti-solvent addition, anti-anti-solvent addition, gas-solid diffusion, room temperature suspension stirring, 5°C suspension stirring, slow volatilization, slow cooling, gas-liquid diffusion, and polymer induction. or a combination of multiple.
  • the above antisolvent addition method includes: dissolving the target product in a good solvent, and then adding the antisolvent to the resulting solution (then, for example, volatilizing at room temperature to obtain a solid, or stirring at -20°C to obtain a solid).
  • the above anti-antisolvent addition method includes: dissolving the target product in a good solvent, and then adding the resulting solution to the antisolvent (then, for example, volatilizing at room temperature to obtain a solid, or stirring at -20°C to obtain a solid).
  • the above-mentioned gas-solid diffusion method, room temperature suspension stirring method, 5°C suspension stirring method, slow volatilization method, slow cooling method, and gas-liquid diffusion method all include: dissolving the target product in a solvent and then drying to obtain a solid.
  • the above-mentioned polymer induction method includes: dissolving the target product in a solvent, adding the polymer, and evaporating at room temperature to obtain a solid.
  • the preparation method of the crystal form A of base)oxy)methyl)phenyl)-2-methylbut-3-yn-2-ol is selected from: anti-solvent addition, anti-anti-solvent addition, gas-solid diffusion, room temperature One or a combination of suspension stirring, 5°C suspension stirring, slow volatilization, slow cooling, gas-liquid diffusion, and polymer induction.
  • the good solvent can be selected from: MeOH, acetone, DMSO, EtOAc, EtOH, DCM, CHCl3, THF, IPA, ACN, 1,4-dioxane Ring
  • anti-solvent can be selected from: MTBE, toluene, n-heptane, water.
  • the good solvent in the anti-solvent addition method, can be selected from: MeOH, acetone, DMSO, and the anti-solvent is MTBE.
  • the good solvent in the anti-solvent addition method, can be selected from: EtOH and DCM, and the anti-solvent is toluene.
  • the good solvent in the anti-solvent addition method, can be selected from: CHCl 3 , THF, IPA, anti-solvent
  • the agent is n-heptane.
  • the good solvent in the anti-solvent addition method, can be selected from: acetone, ACN, 1,4-dioxane, and the anti-solvent is water.
  • the good solvent can be selected from: MeOH, MIBK, acetone, anisole, EtOH, THF, EtOAc, DCM, and the antisolvent can be selected from: toluene , n-heptane, water, MTBE.
  • the good solvent may be MIBK and the antisolvent may be toluene.
  • the good solvent in the anti-antisolvent addition method, can be selected from: acetone, anisole, and the antisolvent can be n-heptane.
  • the good solvent can be selected from: EtOH, THF, and the antisolvent can be water.
  • the good solvent can be selected from: EtOAc, DCM, and the antisolvent can be MTBE.
  • the solvent can be selected from: water, DCM, EtOH, MeOH, ACN, THF, CHCl 3 , acetone, DMSO, EtOAc, 1,4-dioxane Ring, IPA.
  • the solvent can be selected from: MTBE, IPAc, n-heptane, toluene, water, EtOH/toluene (for example, in a ratio of 1:3, v/v) , DMSO/MTBE (for example, at a ratio of 1:4, v/v), acetone/water (for example, at a ratio of 1:4, v/v), IPA/n-heptane (for example, at a ratio of 1:4, v) /v), EtOAc/n-heptane (for example, at a ratio of 1:4, v/v), anisole/toluene (for example, at a ratio of 1:4, v/v), DMAc/water (for example, at a ratio of 1:4) 4 ratio, v/v), THF/water (for example, 1:4 ratio, v/v).
  • the solvent can be selected from: MTBE, toluene, water, IPA/n-heptane (for example, in a ratio of 1:2, v/v), MEK/ n-Heptane (for example, at a ratio of 1:2, v/v), EtOAc/toluene (for example, at a ratio of 1:2, v/v), CPME/toluene (for example, at a ratio of 1:2, v/v) , NMP/water (for example, at a ratio of 1:4, v/v), THF/water (for example, at a ratio of 1:4, v/v), ACN/water (for example, at a ratio of 1:2, v/v ), IPAc/DCM (for example, at a ratio of 1:1, v/v), MeOH/toluene (for example, at a ratio of 1:4, v/v), D
  • the solvent can be selected from: EtOH, acetone, IPAc, THF, CPME, anisole, ACN/water (for example, in a ratio of 9:1, v/v ), MeOH/DCM (for example, at a ratio of 1:1, v/v), acetone/EtOAc (for example, at a ratio of 2:1, v/v), THF/water (for example, at a ratio of 4:1, v/ v).
  • the solvent can be selected from: CPME, toluene, ACN/toluene (for example, in a ratio of 1:2, v/v), acetone/n-heptane (for example, in a ratio of 1:2, v/v) 1:1 ratio, v/v), THF/toluene (e.g., 1:2 ratio, v/v), MeOH/water (e.g., 1:1 ratio, v/v), CHCl 3 /MTBE ( For example, in a ratio of 1:1, v/v).
  • the good solvent can be selected from: EtOH, THF, DMSO
  • the anti-solvent can be selected from: n-heptane, MTBE, toluene, cyclohexane, and water.
  • the good solvent is EtOH
  • the antisolvent can be selected from: n-heptane, MTBE, and toluene.
  • the good solvent is THF
  • the antisolvent can be selected from: n-heptane, cyclohexane, and MTBE.
  • the good solvent is DMSO
  • the antisolvent can be selected from: toluene, MTBE, and water.
  • the solvent can be selected from: MEK, ACN/toluene (for example, in a ratio of 4:1, v/v), THF/water (for example, in a ratio of 9:1) 1 in a ratio, v/v), EtOAc, acetone/2-MeTHF (e.g. in a 1:1 ratio, v/v), MeOH/DCM (e.g.
  • polymer in a 1:1 ratio, v/v), can be selected from: polyvinylpyrrolidone, polyvinyl alcohol, polyvinyl chloride, polyvinyl acetate, hypromellose, methylcellulose, polycaprolactone, polyethylene glycol, polymethylmethacrylate, seaweed One or a combination of sodium phosphate and hydroxyethyl cellulose.
  • the polymer in the polymer induction method, is polyvinylpyrrolidone, polyvinyl alcohol, polyvinyl chloride, polyvinyl acetate, hypromellose and methylcellulose (for example (with a mass ratio of 1:1:1:1:1)
  • the solvent can be selected from: MEK, ACN/toluene (for example, in a ratio of 4:1, v/v), THF/water (for example, in a ratio of 9:1 ratio, v/v).
  • the polymer in the polymer induction method, is polycaprolactone, polyethylene glycol, polymethyl methacrylate, sodium alginate and hydroxyethyl cellulose (for example, 1:1:1:1:1 mass ratio), the solvent can be selected from: EtOAc, acetone/2-MeTHF (for example, in a ratio of 1:1, v/v).
  • the good solvent is EtOAc and the antisolvent is toluene.
  • the present invention also provides a pharmaceutical composition, which contains the compound of the above general formula I or its pharmaceutically acceptable salt, stereoisomer, ester, prodrug, solvate, or the above crystal form, and a pharmaceutically acceptable of excipients.
  • auxiliary materials are selected from one or more of carriers, diluents, adhesives, lubricants, wetting agents, etc.
  • the above pharmaceutical composition contains a therapeutically effective amount of a compound of general formula I. In certain embodiments, these pharmaceutical compositions are useful in treating HPK1 kinase-mediated diseases or conditions.
  • the above-mentioned pharmaceutical composition can be tablets (for example, sugar-coated tablets, film-coated tablets, sublingual tablets, orally disintegrating tablets, oral tablets, etc.), pills, powders, granules, capsules (for example, soft capsules, microcapsules), tablets, syrups, emulsions, suspensions, controlled release preparations (for example, instant release preparations, sustained release preparations, sustained release microcapsules), aerosols, films (for example, Orally disintegrating film preparations, oral mucosa-adhesive film preparations), injections (e.g., subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection), intravenous drip preparations, transdermal absorption preparations, ointments, lotions, Adhesive preparations, suppositories (eg, rectal suppositories, vaginal suppositories), pellets, nasal preparations, pulmonary preparations (inhalants), eye drops, and the like.
  • various dosage forms of the above pharmaceutical compositions can be prepared according to conventional production methods in the pharmaceutical field.
  • the active ingredient is mixed with one or more excipients and then formulated into the desired dosage form.
  • the above pharmaceutical composition may contain a weight ratio of 0.1-99.5% (for example, 0.1%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80 %, 90%, 95%, 99%, 99.5%) of active ingredients.
  • 0.1-99.5% for example, 0.1%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80 %, 90%, 95%, 99%, 99.5%
  • the present invention also provides the above-mentioned compound of general formula I and its pharmaceutically acceptable salts, stereoisomers, esters, prodrugs and solvates, or the above-mentioned crystal forms, or the above-mentioned pharmaceutical compositions for the preparation of prevention and/or treatment of tumors. application in medicines.
  • the present invention also provides the above-mentioned compound of general formula I and its pharmaceutically acceptable salts, stereoisomers, esters, prodrugs and solvates, and the above-mentioned crystal forms in combination with PD-1, PD-L1, CTLA-4, TIM- 3.
  • TGF- ⁇ and its receptors LAG3 antagonists or TLR4, TLR7, TLR8, TLR9 and STING agonists in tumor immunotherapy.
  • the present invention also provides the application of the above-mentioned compound of general formula I and its pharmaceutically acceptable salts, stereoisomers, esters, prodrugs and solvates, and the above-mentioned crystal form in combination with CAR-T immunotherapy in tumor immunotherapy. .
  • the above-mentioned CAR-T immunotherapy refers to chimeric antigen receptor T cell immunotherapy. Its basic principle is to use the patient's own immune cells to eliminate cancer cells, which is a type of cell therapy.
  • the above-mentioned tumors are malignant tumors, including but not limited to: lymphoma, blastoma, medulloblastoma, retinoblastoma, sarcoma, liposarcoma, synovial cell sarcoma, neuroendocrine tumors, carcinoid tumors, Gastrinoma, islet cell carcinoma, mesothelioma, schwannoma, acoustic neuroma, meningioma, adenocarcinoma, melanoma, leukemia or lymphoid malignancy, squamous cell carcinoma, epithelial squamous cell carcinoma, lung cancer, Small cell lung cancer, non-small cell lung cancer, adenocarcinoma lung cancer, lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastric cancer, intestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer
  • the present invention also provides a method for preventing and/or treating tumors, which includes administering an effective amount of the compound of the above general formula I of the present invention and its pharmaceutically acceptable salts and stereoisomers to a subject in need. , esters, prodrugs and solvates, or the above-mentioned crystal forms, or the steps of the above-mentioned pharmaceutical compositions of the present invention.
  • tumors have the corresponding definitions set forth above in the present invention.
  • the present invention also provides a method for preventing and/or treating diseases caused by or related to pathogen infection, which includes administering to a subject in need an effective amount of the compound of the above general formula I of the present invention and its pharmaceutical composition.
  • a method for preventing and/or treating diseases caused by or related to pathogen infection which includes administering to a subject in need an effective amount of the compound of the above general formula I of the present invention and its pharmaceutical composition.
  • pathogens and diseases have the corresponding definitions set forth above in the present invention.
  • Figure 1 shows the XRPD pattern of compound A2 free base crystal form A.
  • Figure 2 shows the TGA/DSC spectrum of compound A2 free base crystal form A.
  • Figure 3 shows the 1 H NMR spectrum of compound A2 free base crystal form A.
  • Figure 4 shows the HPLC spectrum of compound A2 free base crystal form A.
  • Figure 5 shows the starting sample of compound A2 free base crystal form A and its XRPD pattern after being left closed for 1 day at 60°C.
  • Figure 6 shows the XRPD pattern of compound A2 free base crystal form B.
  • Figure 7 shows the TGA/DSC spectrum of compound A2 free base crystal form B.
  • Figure 8 shows the 1 H NMR spectrum of compound A2 free base crystal form B.
  • Figure 9 shows the XRPD patterns of compound A2 free base crystal form B before and after being placed at room temperature.
  • Figure 10 shows the XRPD patterns of compound A2 free base crystal form B before and after nitrogen purging.
  • Figure 11 shows the XRPD pattern of compound A2 hydrochloride.
  • Figure 12 shows the TGA/DSC spectrum of compound A2 hydrochloride.
  • Figure 13 shows the XRPD pattern overlay of compound A2 free base crystal form A and compound A2 hydrochloride.
  • Figure 14 shows the concentration of iL-2 in human PBMCs.
  • Figure 15 shows the keratitis lesions in mice after inoculation with HSV virus.
  • Figure 16 shows the clinical scores of mice after inoculation with HSV virus.
  • Figure 17 shows the corneal neovascularization differentiation score of mice after inoculation with HSV virus.
  • Figure 18 shows the changes in the weight of the hamster.
  • Figure 19 shows the changes in lung weight of hamsters.
  • Figure 21 shows the proliferation rate of virus-specific T cells in mice infected with low-dose virus.
  • C 0 alkyl refers to H, therefore, C 0-10 alkyl includes H, C 1 alkyl, C 2 alkyl, C 3 alkyl, C 4 alkyl group, C 5 alkyl, C 6 alkyl, C 7 alkyl, C 8 alkyl, C 9 alkyl, C 10 alkyl.
  • C 1-10 linear/branched alkyl in the present invention includes methyl, ethyl, C 3 linear/branched alkyl, C 4 linear/branched alkyl, C 5 linear/ Branched alkyl, C 6 linear/branched alkyl, C 7 linear/branched alkyl, C 8 linear/branched alkyl, C 9 linear/branched alkyl, C 10 linear/ Branched alkyl.
  • C 3-10 branched alkyl in the present invention includes isopropyl, isobutyl, tert-butyl, and isopentyl.
  • C 3-10 cycloalkyl in the present invention includes C 3 cycloalkyl, C 4 cycloalkyl, C 5 cycloalkyl, C 6 cycloalkyl, C 7 cycloalkyl, C 8 cycloalkyl , C 9 cycloalkyl, C 10 cycloalkyl.
  • C 3-8 cycloalkyl in the present invention includes C 3 cycloalkyl, C 4 cycloalkyl, C 5 cycloalkyl, C 6 cycloalkyl, C 7 cycloalkyl, C 8 cycloalkyl .
  • C 4-8 cycloalkyl in the present invention includes C 4 cycloalkyl, C 5 cycloalkyl, C 6 cycloalkyl, C 7 cycloalkyl, and C 8 cycloalkyl.
  • C 4-6 cycloalkyl in the present invention includes C 4 cycloalkyl, C 5 cycloalkyl, and C 6 cycloalkyl.
  • halogen in the present invention includes fluorine, chlorine, bromine and iodine.
  • heterocycloalkyl in the present invention refers to a non-aromatic saturated monocyclic or polycyclic ring system containing 3 to 10 ring atoms, preferably 5 to 10 ring atoms, in which one or more ring atoms are not carbon atoms. , but for example nitrogen, oxygen or sulfur atoms.
  • Preferred heterocycloalkyl groups contain 5-6 ring atoms.
  • aza, oxa or thia before heterocycloalkyl means that there is at least one nitrogen, oxygen or sulfur atom respectively as a ring atom.
  • heterocyclic aryl in the present invention refers to an aromatic monocyclic or polycyclic ring system containing 5-14 ring atoms, preferably 5-10 ring atoms, in which one or more ring atoms are not carbon atoms, but Such as nitrogen, oxygen or sulfur atoms.
  • Preferred heterocyclic aromatic groups contain 5-6 ring atoms.
  • heterocyclic aromatic groups include pyrazinyl, furyl, thienyl, pyridyl, pyrimidinyl, isoxazolyl, isothiazolyl, oxazolyl, thiazolyl, pyrazolyl, furazyl, and pyrrolyl , pyrazolyl, triazolyl, 1,2,4-thiazodiazolyl, pyrazinyl, pyridazinyl, quinoxalinyl, 2,3-diazanaphthyl, imidazo[1,2 -a]pyridine, imidazo[2,1-b]thiazolyl, benzofuranyl, indolyl, azaindolyl, benzimidazolyl, benzothienyl, quinolyl, imidazolyl, Thienopyridyl, quinazolinyl, thienopyrimidinyl, pyrrolopyridyl, imid
  • D refers to deuterium
  • substituted by deuterium refers to replacing one or more hydrogen atoms with a corresponding number of deuterium atoms.
  • the compounds of the present invention will inherently contain small amounts of deuterated isotopologues. Notwithstanding this variation, the concentrations of such naturally abundant stable hydrogen and carbon isotopes are low and insignificant compared to the extent of stable isotope substitution in the compounds of the present invention. See, for example, Wada, E et al., Seikagaku, 1994, 66:15; Gannes, LZ et al., Comp Biochem Physiol Mol Integr Physiol, 1998, 119:725.
  • any atoms not specified as deuterium are present in their natural isotope abundance.
  • H hydrogen
  • D deuterium
  • that position is understood to have an abundance of deuterium that is at least 3000 times greater than the natural abundance of deuterium, which is 0.015%. (i.e., at least 45% deuterium incorporation).
  • isotopic enrichment coefficient refers to the ratio between the isotopic abundance and the natural abundance of a particular isotope.
  • the compounds of the invention have an isotopic enrichment factor for each specified deuterium atom of at least 3500 (52.5% deuterium incorporation at each specified deuterium atom), at least 4000 (60% deuterium incorporation) , at least 4500 (67.5% deuterium incorporation), at least 5000 (75% deuterium incorporation), at least 5500 (82.5% deuterium incorporation), at least 6000 (90% deuterium incorporation), at least 6333.3 (95% of deuterium incorporation), at least 6466.7 (97% deuterium incorporation), at least 6600 (99% deuterium incorporation), or at least 6633.3 (99.5% deuterium incorporation).
  • isotope refers to a substance whose chemical structure differs only from that of a particular compound of the present invention in its isotopic composition.
  • the overall relative amount of such isotopologues will be less than 49.9% of the compound. In other embodiments, the overall relative amount of such isotopologues will be less than 47.5%, less than 40%, less than 32.5%, less than 25%, less than 17.5%, less than 10%, less than 5% of the compound. %, below 3%, below 1% or below 0.5%.
  • the term "crystalline form" is confirmed by characterization of X-ray powder diffraction patterns.
  • X-ray powder diffraction patterns often change with the conditions of the instrument. It is particularly important to point out that the relative intensity of X-ray powder diffraction patterns may also change with changes in experimental conditions, so the order of peak intensity cannot be used as the only or decisive factor. In fact, the relative intensity of the diffraction peaks in the XRPD pattern is related to the preferred orientation of the crystal.
  • the peak intensities shown in this article are illustrative and not used for absolute comparison.
  • the experimental error of the peak angle is usually 5% or less. The error of these angles should also be taken into account, and an error of ⁇ 0.2° is usually allowed.
  • due to the influence of experimental factors such as sample thickness there will be an overall shift in the peak angle, and a certain shift is usually allowed. Therefore, those skilled in the art can understand that the X-ray powder diffraction pattern of a crystalline form in the present invention does not have to be completely consistent with the X-ray powder diffraction pattern in the embodiments referred to herein.
  • the XRPD pattern is the same as described herein does not mean It means absolutely the same, the same peak position can differ by ⁇ 0.2° and the peak intensity allows certain variability. Any crystalline form having the same or similar pattern as the characteristic peaks in these patterns falls within the scope of the present invention. Those skilled in the art can compare the spectrum listed in the present invention with the spectrum of an unknown crystal form to confirm whether the two sets of patterns reflect the same or different crystal forms.
  • Form A of the present invention is pure and unitary, with substantially no admixture of any other crystalline forms.
  • substantially no when used to refer to a new crystal form means that the crystal form contains less than 20% (weight) of other crystal forms, especially less than 10% (weight) of other crystal forms, and even less Less than 5% (weight) of other crystalline forms refers to less than 1% (weight) of other crystalline forms.
  • room temperature means that the temperature of the item is close to or the same as the temperature of the space (eg, the location of the fume hood in which the item is located). Typically, room temperature is about 20°C to about 30°C, or about 22°C to 27°C, or about 25°C.
  • the antisolvent addition also known as antisolvent crystallization, precipitation crystallization, salting out or forced crystallization
  • the antisolvent addition is usually by adding one or several antisolvents to a solution in which the target product is dissolved in a good solvent, and the product is dissolved in the solution.
  • the solution is in a slightly soluble state, so that the solution reaches a supersaturated state and then crystallizes.
  • Anti-anti-solvent addition is usually done by adding one or several anti-solvents to the solution of the target product after dissolving it with a good solvent.
  • the product is in a slightly soluble state in the solution, so that the solution reaches a supersaturated state.
  • the method of precipitation and crystallization is usually by adding one or several antisolvents to a solution in which the target product is dissolved in a good solvent, and the product is dissolved in the solution.
  • the solution is in a slightly soluble state, so that the solution reaches a supersaturated state
  • the ability of the antisolvent to dissolve the target product is worse than that of the good solvent, for example, the difference is more than 10%, 20%, 30%, 40%, 50%, 60%, 70% or 80%. Therefore, the antisolvent in the system is relative.
  • Good solvents and antisolvents can be polar solvents or non-polar solvents, such as dimethylformamide (DMF), dimethyl sulfoxide (DMSO), water, alcohol solvents, ether solvents, and ketones.
  • DMF dimethylformamide
  • DMSO dimethyl sulfoxide
  • alcohol solvents include but are not limited to methanol, ethanol, propanol, isopropanol or 1,3-propanediol, 1,2-propanediol or chlorobutanol or combinations thereof;
  • ether solvents include but are not limited to tetrahydrofuran, methyl Tert-butyl ether or 1,4-dioxane or combinations thereof;
  • ketone solvents include but are not limited to acetone, methyl ethyl ketone or 4-methyl-2-pentanone or combinations thereof;
  • ester solvents include but are not limited to ethyl acetate Ester, isopropyl acetate, n-butyl acetate or tert-butyl acetate or a combination thereof;
  • alkane solvents include but are not limited to methylene chloride, chloroform, n-hexane, cyclohexane or pentane or n-heptane or
  • Antisolvent addition can be performed by batch, semi-batch, or continuous crystallization operations.
  • the antisolvent is added to the solution (antisolvent crystallization) or the product solution is added to the antisolvent (anti-antisolvent crystallization), either dropwise at a constant rate or slowly at the beginning and then gradually increasing the rate.
  • hPBMs human-Peripheral blood mononuclear cells
  • lymphocytes lymphocytes and monocytes.
  • interleukin-2 refers to interleukin-2, which is a cytokine of the chemokine family and is derived from multicellular sources (mainly produced by activated T cells).
  • Cytokines with pleiotropic effects mainly promoting the growth, proliferation, and differentiation of lymphocytes
  • play an important role in the body's immune response and anti-viral infection and can stimulate T cells that have been activated by specific antigens or mitogenic factors Proliferation; can activate T cells, promote the production of cytokines; stimulate NK cell proliferation, enhance NK killing activity and produce cytokines, induce the production of LAK cells; promote B cell proliferation and secretion of antibodies; activate macrophages.
  • LPS Lipopolysaccharide
  • Glycolipid lipids and polysaccharides.
  • the structure of LPS is composed of antigen-core polysaccharide-lipid A.
  • LPS is an endotoxin (Endotoxin). When it acts on other biological cells such as humans or animals, it will show a variety of biological activities.
  • TLR Toll-like Receptor 4
  • TNF- ⁇ tumor necrosis factor
  • TNF- ⁇ tumor necrosis factor
  • TNF- ⁇ tumor necrosis factor
  • TNF- ⁇ can Kills and inhibits tumor cells, regulates the body's immune function, promotes the killing of tumor cells by T cells and other killer cells, improves the phagocytic ability of neutrophils, increases the production of superoxide anions, enhances ADCC function, and stimulates cells Degranulation and secretion of myeloperoxidase.
  • TNF- ⁇ can also inhibit viral replication, inhibit viral protein synthesis, viral particle production and infectivity, and can kill virus-infected cells.
  • TNF- ⁇ can promote the expression of MHC class I antigens in T cells, enhance the proliferation of IL-2-dependent thymocytes and T cells, promote the differential production of lymphokines such as IL-2, CSF and IFN- ⁇ , and enhance the stimulation of B cells by mitogens or foreign antigens. proliferation and Ig secretion.
  • step 1
  • step 1
  • reaction solution was washed with saturated ammonium chloride (1000Ml*3) and saturated brine (500mL), dried over anhydrous sodium sulfate, and spun to dryness to obtain a yellow oily substance as the target compound (59.4g, crude product), which can be used directly in the next step.
  • reaction LC-MS: 275[M+H]+
  • step 1
  • step 1
  • the XRPD pattern of the above sample is shown in Figure 1, and its XRPD test parameters and result data tables are shown in Tables 1 and 2 respectively, indicating that it is a crystal, named free crystal form A, and can be prepared repeatedly.
  • Tables 1 and 2 The entire list of peaks or corresponding d values in Table 2, or a subset thereof, and an XRPD pattern substantially similar to Figure 1 may be sufficient to characterize the crystalline form.
  • TGA/DSC spectrum of compound A2 free base crystal form A is shown in Figure 2, and the test parameters of TGA and DSC are shown in Table 3.
  • the TGA results show that it loses 1.3% weight when heated from room temperature to 170°C; the DSC results show that it has a sharp endothermic peak at 168.8°C (starting temperature).
  • the XRPD pattern of the above sample is shown in Figure 6, its XRPD test parameters are shown in Table 1 above, and the result data table is shown in Table 6, indicating that it is a crystal and named free crystal form B.
  • Table 6 The entire list of peaks or corresponding d values in Table 6, or a subset thereof, and an XRPD pattern substantially similar to Figure 5 may be sufficient to characterize the crystalline form.
  • TGA/DSC spectrum of compound A2 free base crystal form B is shown in Figure 7, and the test parameters of TGA and DSC are shown in Table 3 above.
  • TGA results show that the sample loses 13.2% weight when heated from room temperature to 70°C, and continues to lose 8.5% when heated to 170°C.
  • the DSC results show that the sample has four endothermic peaks at 59.5°C, 95.6°C, 150.8°C and 160.9°C (peak temperature).
  • the free base crystal form B is an EtOAc solvate.
  • the XRPD pattern of compound A2 hydrochloride is shown in Figure 11.
  • the XRPD test parameters are shown in Table 1 of Example 5.
  • the XRPD result data table is shown in Table 7, indicating that it is a crystal and named hydrochloride crystal form A. .
  • the entire list of peaks or corresponding d values in Table 7, or a subset thereof, and an XRPD pattern substantially similar to Figure 11 may be sufficient to characterize this crystalline form.
  • TGA/DSC spectrum of compound A2 hydrochloride is shown in Figure 12, and the test parameters of TGA and DSC are shown in Table 3 of Example 5.
  • TGA results show that it loses 8.6% weight when heated from room temperature to 150°C;
  • DSC results show that it has two endothermic peaks at 81.9°C and 156.0°C (peak temperature).
  • the test compound dissolve in dimethyl sulfoxide to 20mM, and dilute with 50% acetonitrile aqueous solution (v/v) Dilute the compound stock solution to a concentration of 1.0mM as the working solution; dilute the liver microsomes (20mg/mL) with 50mM dipotassium hydrogen phosphate buffer to 1.27mg/mL as the liver microsome working solution; weigh the reduced coenzyme and add 3307 ⁇ L phosphate buffer solution (50mM) dissolved to 5.0mM as reduced coenzyme working solution.
  • intravenous injection the compound was formulated into a solution of 0.25 mg/ml or 0.5 mg/mL using 10% DMSO/30% PEG400/60% Water and administered in a volume of 2 mL/kg.
  • Oral Administration The compound is formulated into a homogeneous suspension of 0.6 mg/ml or 2.0 mg/mL with 0.5% Methylcellulose and administered in a volume of 5 mL/kg. The specific dosage is shown in the table below.
  • AUC last the in vivo exposures (AUC last ) of A1, A2, C and D were 116hr*ng/mL, 247hr*ng/mL, 74.8hr*ng/mL and 28.7hr*ng/mL respectively, with an average of The total clearance rates (CL) were 129mL/min/kg, 71mL/min/kg, 101mL/min/kg, and 151mL/min/kg respectively.
  • the compound is formulated into a 1.0 mg/mL solution in water or 10% DMSO/30% PEG400/Water for intravenous injection and administered in a volume of 1 mL/kg.
  • For oral administration prepare the compound into a 2.0 mg/mL solution or suspension solution with water or 0.5% methylcellulose/1.7meq 1N hydrochloric acid, and administer it in a volume of 5 mL/kg.
  • AUC last the in vivo exposures (AUC last ) of A1 and A2 were 881hr*ng/mL and 949hr*ng/mL respectively, and the average total clearance rates (CL) were 18.9mL/min/kg and 16.7mL/min respectively. /kg.
  • the in vivo exposure (AUC last ) of A1 and A2 was 388ng*hr/mL and 1758ng*hr/mL respectively.
  • the oral bioavailability in cynomolgus monkeys was 4.61% and 18.8% respectively.
  • hPBMC used in this experiment were purchased from Shanghai Aoneng Biotechnology Co., Ltd. Coat the CD3 antibody onto a 96-well plate in advance and set aside. Cells were seeded in a 96-well plate, 200 ⁇ L per well, 2 ⁇ 106 cells/ml. CD28 antibody was added for activation, and compound A2 with a final concentration of 1 ⁇ M was added, and the supernatant was collected after 24 hours of treatment. IL-2 levels were detected by ELISA method, and the effects of A2 and CD3/CD28 on activating hPBMCs were compared.
  • A2 can effectively enhance human immunity.
  • HSV viral keratitis
  • Viral keratitis modeling was performed on the right eyes of 15 8-week-old C57 mice (weight 22g-25g). During the modeling process, a sterile 25G needle was used to mark the corneas of the right eyes of all animals with the word "#" or the word "rice”. Make parallel scratches, the depth of the scratches must be shallow and cannot penetrate the matrix layer. The scratches between each eyeball should be moderately uniform in depth and evenly distributed in density. Then use a fiber syringe to drop in 4 ⁇ l of mixed HSV virus fluid (the titer is: 2.0*106PFU/ml) to complete the modeling. The day of modeling was designated as D0.
  • the HSV virus of the present invention is cultured and multiplied in the African green monkey kidney cell line and has activity, and the HSV virus suspension titer is good)
  • mice with uniform modeling were selected to be included in the experiment and randomly divided into groups. The results showed that mice in the experimental control group developed severe corneal ulcers, and 2 eyes had corneal perforations on the fifth day of the experiment, which lasted until the end of the experiment. No mice in the group had corneal perforation. See Figure 15 for details.
  • Figure 16 shows the clinical scores of mice within a few days after HSV virus inoculation. From the data in Figure 16, it can be seen that the keratitis lesion scores after 2 weeks of administration of A2 eye drops at concentrations of 0.2% and 1.0% are comparable to those of the model control group. The ratios have statistical differences (P ⁇ 0.01 and P ⁇ 0.05), proving that the medicine of the present invention has a good therapeutic effect on viral keratitis. At the peak of the disease (the 5th day), the clinical score of the control group was more than 6 points.
  • Figure 17 shows the cardiovascular formation scores of the control group, low concentration group (0.2% A2) and high concentration group (1% A2). It can be seen that the score of the control group is above 1 point, and the score of the low concentration group (0.2 The score of %A2) is higher than 0.5 and less than 1, and the score of the high concentration group (1% A2) is lower than 0.5, indicating that the high concentration A2 suspension has a slight advantage over the low concentration A2 clarification liquid.
  • the drug of the present application is effective in mediating.
  • perforation occurred in the model control group of the normal saline eye drops, but not yet in the treatment group.
  • the drug of the present application can effectively prevent corneal perforation.
  • the drug of the present application can effectively prevent corneal perforation.
  • Mediation can effectively reduce corneal edema, corneal ulcer depth, and turbidity during the disease progression stage, and improve the depth of inflammatory infiltration.
  • the high-concentration A2 suspension of the present application has an advantage over the low-concentration A2 clear solution.
  • Ocular surface eye drops of 0.2% and 1.0% A2 can effectively inhibit viral keratitis lesions in mice.
  • the corneal ulcers are graded (scores) based on three criteria: corneal ulcer area, depth, and ulcer shape, and the total score is added up to record.
  • the neovascularization score in the four corneal quadrants ranged from 0 to 2 points, with a maximum score of 8 points.
  • Standards for corneal neovascularization (0 to 2): 0 points, no new blood vessels grow into the cornea; 1 point, new blood vessels grow centripetally, but do not reach the center of the pupil or exceed the radius of the cornea; 2 points, new blood vessels reach the center of the pupil or exceed the corneal radius.
  • Clinical score lesion area score + lesion depth score
  • mice A total of 20 wild-type Syrian golden hamsters were used, 6-8 weeks old, half male and half female.
  • the mice were randomly assigned into two groups, 4 in the control group, and the drug groups were divided into 5 mg/kg group and 20 mg/kg group. There are two groups in total, with 8 animals in each group.
  • the hamsters in the treatment group were orally administered a total daily dose of 5 mg/kg or 20 mg/kg A2, and the control group was orally administered the same amount of blank drug every day.
  • all hamsters were anesthetized.
  • mice after anesthesia, mice were intranasally inoculated with 100uL, 1.375x10 6 PFU/mL SARS-Cov-2 virus. Monitor clinical manifestations and body weight daily.
  • tissues such as lungs
  • viral RNA and infectious virus are quantified.
  • Figure 18 shows the effect of A2 on the body weight of SARS-Cov-2 infected hamsters.
  • the weight of the three groups of hamsters decreased, but the animals in the 20mg/kg group gained weight much faster.
  • lung weight we chose to measure lung weight and normalize it to total body weight.
  • This embodiment uses the increase in lung weight as a reliable and indirect measurement index to characterize the progression of lung inflammation. In other words, changes in lung weight can illustrate the development of lung inflammation.
  • lung weight decreased in all A2 dose groups.
  • the medicine of the invention can prevent Covid 19.
  • mice Female SPFC57BL/6mice mice aged 6-8 weeks (VitalRiver Experimental Animal Technology Co., Ltd., SCXK (Beijing) 2016-0006, Beijing version) were used, infected with H1N1 (PR8 strain), and treated with 1 ⁇ 10 4 TCID50 virus nasal drops Intranasal irritation in mice.
  • the mice were randomly assigned into administration groups and control groups.
  • the administration group was orally administered 3 mg/kg A2, and the control group was orally administered the same amount of blank drug.
  • the weight loss of mice in all groups was monitored daily.
  • the medicine of the present invention can treat infections caused by H1N1.
  • Low-dose virus group 21 4-week-old male SPF grade C57BL/6 mice were divided into three groups, namely: control group, prevention group and treatment group. After the mice were anesthetized, they were treated with 100 ⁇ L containing 4 ⁇ 10 5 PFU. Intrahepatic injection (ih) of DMEM of MHV-A59 virus. The prevention group was administered A2 for the first time 24 hours before challenge, with a dose of 10 mg/kg, and the treatment group was administered A2 for the first time 24 hours after challenge, with a dose of 10 mg/kg, and then continued daily thereafter (prevention group and The dosage of the treatment group was 10 mg/kg per day), and the infected mice were monitored every day.
  • mice in all groups were sacrificed, liver tissue was taken for virus titer determination and histological analysis, and spleen tissue was taken for virus-specific T proliferation detection. The results are shown in Figure 21.
  • High-dose virus group 21 4-week-old male SPF grade C57BL/6 mice were divided into three groups, namely: control group, prevention group and treatment group. After the mice were anesthetized, they were treated with 100 ⁇ L containing 2 ⁇ 10 6 PFU. Intrahepatic injection (ih) of DMEM of MHV-A59 virus. The prevention group was administered A2 for the first time 24 hours before challenge, with a dose of 10 mg/kg. The treatment group was administered A2 for the first time 24 hours after challenge, with a dose of 10 mg/kg, and then was administered continuously every day. The prevention group and The dosage of the treatment group was 10 mg/kg per day, and the weight changes, clinical symptoms of the disease, and mortality of the infected mice were monitored every day.
  • mice in the low-dose virus group are shown in Figure 21.
  • the average proliferation rates of spleen cells in the control group, prevention group and treatment group were 19.19%, 28.18% and 52.95% respectively. It can be seen from the above data , the virus-specific T cells in the treatment group in the low-dose virus group proliferated significantly faster.
  • mice in the high-dose virus group it was found that the weight of the prevention group increased. Specifically, from the 1st to the 8th day of the experiment, the average weight of the mice in the control group decreased from 12.39g to 10.91g. Prevention The average weight of mice in the treatment group increased from 13.39g to 16.34g, while the average weight of mice in the treatment group decreased from 12.48g to 11.39g. In the high-dose virus group, the survival rate of the mice in the prevention group was 42.86%, the survival rate of the mice in the high-dose treatment group was 28.57%, and all the mice in the high-dose control group died.
  • the medicine of the present invention can better prevent and treat diseases caused by MHV.

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Abstract

本发明提供了一种HPK1激酶抑制剂在制备预防和/或治疗人由病原体感染引起或病原体感染相关的疾病或病症的药物中的应用，在制备预防和/或治疗肿瘤的药物中的应用。所述HPK1激酶抑制剂为如下通式Ⅰ所示的化合物，或其药学上可接受的盐、立体异构体、酯、前药、溶剂化物和氘代化合物。

Description

HPK1激酶抑制剂在预防和/或治疗人的病原体感染中的应用 技术领域
本发明涉及医药技术领域，具体涉及一种HPK1激酶抑制剂在制备预防和/或治疗人由病原体感染引起或病原体感染相关的疾病或病症的药物中的应用。本发明还涉及上述HPK1激酶抑制剂的晶型及其制备方法和应用。
背景技术
病毒性疾病的防控和治疗是当今世界非常关注的问题，尽管疫苗在一定程度上起到了预防病毒性传染病的作用，由于病毒本身极易变异，现有的药物远远不能满足需求。全力开发新型有效的抗病毒药物依然任重道远。
HPK1(hematopoietic progenitor kinase 1，)是有丝分裂原激活蛋白激酶(mitogen-activated protein kinase kinase kinase kinase，MAP4K)家族成员之一，在调节细胞存活、细胞迁移、细胞凋亡和自噬中发挥重要作用。HPK1激酶参与很多信号级联反应，包括生长因子信号、MAPK信号、细胞因子信号、凋亡信号、生长因子信号以及抗原受体信号等。HPK1是T细胞、B细胞及树突状细胞激活的负性调节因子，抑制其功能可增强免疫细胞，进而增强免疫学功能，因此，开发HPK1抑制剂具有十分重要的意义。
专利文献CN113861188A公开了一种吡唑并[3,4-b]吡啶类衍生物及其制备方法和作为HPK1抑制剂的应用，其通式如下：专利文献CN113316576A公开了一种HPK1化合物，其通式如下：专利文献WO2021213317A1公开了一种HPK1抑制剂及其制备方法和用途，该化合物的结构式如下：但是，现有技术鲜有将小分子HPK1激酶抑制剂用在抗病毒感染的治疗药物中的报道，因此，将HPK1激酶抑制剂应用在病毒感染的治疗中的应用前景非常广阔。
鉴于此，特提出本发明。
发明内容
本发明提供了一种HPK1激酶抑制剂在制备预防和/或治疗人由病原体感染引起或病原体感染相关的疾病或病症的药物中的应用：
进一步的，所述病原体可以为微生物、寄生虫或其他媒介；
具体地，所述寄生虫为原虫、蠕虫或其他媒介。
优选的，所述微生物选自：病毒、衣原体、立克次体、支原体、细菌、螺旋体、真菌等中的一种或多种；
更优选的，所述病原体为病毒，包括但不限于，腺病毒科、疱疹病毒科、HSV2、VZV、EBV、CMV、痘病毒科、乳多泡病毒科、细小病毒科、嗜肝DNA病毒科、多瘤病毒科、呼肠孤病毒科、小 核糖核酸病毒科、嵌杯样病毒科、披膜病毒科、沙粒病毒科、逆转录病毒科、黄病毒科、正粘病毒科、副粘病毒科、布尼亚病毒科、冠状病毒科、星状病毒科、博尔纳病毒科；
在本发明的具体实施方式中，上述病原体为病毒，例如，但不限于，腺病毒科(如腺病毒)、疱疹病毒科(如HSV1(口腔疱疹)、HSV2(外生殖器疱疹)、VZV(水痘)、EBV(埃-巴二氏病毒)、CMV(巨细胞病毒))、痘病毒科(如天花病毒、牛痘病毒)、乳多泡病毒科(如人乳头瘤病毒(HPV))、细小病毒科(如B19病毒)、嗜肝DNA病毒科(如乙型肝炎病毒(HBV))、多瘤病毒科(如多瘤病毒)、呼肠孤病毒科(如呼肠弧病毒、轮状病毒)、小核糖核酸病毒科(如肠道病毒、口蹄疫病毒)、嵌杯样病毒科(如诺沃克病毒、戊型肝炎病毒)、披膜病毒科(如风疹病毒)、沙粒病毒科(如淋巴细胞性脉络丛脑膜炎病毒)、逆转录病毒科(HIV)、黄病毒科(如登革热病毒、寨卡病毒、乙型脑炎病毒、基孔肯亚病毒、黄热病病毒、丙型肝炎病毒(HCV)、西尼罗病毒等)、正粘病毒科(如流感病毒(如甲型流感病毒、乙型流感病毒、丙型流感病毒等))、副粘病毒科(如1型人副流感病毒(HPV)、2型HPV、3型HPV、4型HPV、仙台病毒、腮腺炎病毒、麻疹病毒、呼吸道合胞病毒、新城疫病毒等)、布尼亚病毒科(如加利福尼亚脑炎病毒、汉坦病毒)、弹状病毒科(如狂犬病毒)、丝状病毒科(如埃博拉病毒、马尔堡病毒)、冠状病毒科(如HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1、SARS-CoV、MERS-CoV、SARS-CoV-2等)、星状病毒科(如星状病毒)、博尔纳病毒科(如博尔纳病毒)。
更优选的，所述病毒为HBV、HIV、HCV、HPV、埃博拉病毒、马尔堡病毒、流感病毒、副流感病毒、登革热病毒和人冠状病毒；
更优选的，所述人冠状病毒包括：SARS-CoV、SARS-CoV-2、MERS-CoV、HCoV-229E、HCoV-NL63、HCoV-OC43和HCoV-HKU1；
更优选的，所述由病原体感染引起或病原体感染相关的疾病包括流感、SARS、MERS、COVID-19、病毒性肝炎、AIDS、登革热、埃博拉病毒病、马尔堡病毒病。
更优选的，所述病毒性肝炎包括乙型肝炎、丙型肝炎。
由现有技术可知，T细胞免疫反应是机体免疫放映的一个重要部分。CD4+T细胞的激活能够帮助B细胞生成中和抗体并激活CD8+T细胞，而CD8+T细胞能够消灭受到病毒感染的细胞(Grifoni A,WeiskopfD,Ramirez S I,Mateus J,Dan J M,Moderbacher C R,Rawlings S A,Sutherland A,Premkumar L,Jadi R S,Marrama D,De Silva A M,Frazier A,Carlin A F,Greenbaum J A,Peters B,Krammer F,Smith D M,Crotty S,Sette A.Targets of T Cell Responses to SARS-CoV-2Coronavirus in Humans with COVID-19 Disease and Unexposed Individuals[J].Cell,2020,181(7):1489-1501.e15.)。在感染部位，活化的病毒特异性效应T细胞就会产生抗病毒细胞因子(如IL-2)，进而帮助机体对抗病毒(Liao W,Lin J-X,Leonard W J.IL-2Family Cytokines:New Insights into the Complex Roles of IL-2as a Broad Regulator ofT helper Cell Differentiation[J].Current Opinion in Immunology,2011,23(5):598–604，Channappanavar R,Zhao J,Perlman S.T cell-mediated immune response to respiratory coronaviruses[J].Immunologic Research,2014,59(1):118–128)。申请人经过创造性劳动发现，本发明的HPK1激酶抑制剂能够增强T细胞增殖能力，使TCR诱导的钙流动和促炎细胞因子(如IL-2)水平的持续升高。并且，能够诱导B细胞活化，同时使T细胞和树突状细胞免疫功能显著增强。本申请的HPK1激酶抑制剂不仅可以用于抗肿瘤药物，也可以应用于抗病毒药物。
进一步的，所述抑制剂为如下通式Ⅰ所示的化合物，或其药学上可接受的盐、立体异构体、酯、前药、溶剂化物和氘代化合物：
其中：
A选自CR10或N；
Q选自O或S；
x和z独立地选自0-6间的整数(如0、1、2、3、4、5、6)；
y为0或1；
Ar选自芳香性五元杂环基团、芳香性六元杂环基团或苯基，其中，芳香性五元杂环基团选自：呋喃基、噻吩基、吡咯基、吡唑基、咪唑基、噁唑基、噻唑基或硒代噻唑基，芳香性六元杂环基团选自：吡啶基、哒嗪基、嘧啶基或吡嗪基，任选地，芳香性五元杂环基团、芳香性六元杂环基团或苯基上的H可被以下基团取代：-D、-SO2、-SO2N(C0-10烷基)(C0-10烷基)、-N(C0-10烷基)SO2(C0-10烷基)、-CON(C0-10烷基)(C0-10烷基)、-N(C0-10烷基)CO(C0-10烷基)、-N(C0-10烷基)COO(C0-10烷基)、-OCON(C0-10烷基)(C0-10烷基)、卤素、-CN、-OCH2F、-OCHF2、-OCF3、C1-10直链/支链烷基、-N(C0-10烷基)(C0-10烷基)、-OC0-10烷基、C3-10环烷基、-O杂环烷基、-N杂环烷基、-N杂环芳香基、-O杂环芳香基或-S杂环芳香基，其中烷基部分可被一个或多个以下基团任意取代：-SO2、-SO2N(C0-10烷基)(C0-10烷基)、-N(C0-10烷基)SO2(C0-10烷基)、-CON(C0-10烷基)(C0-10烷基)、-N(C0-10烷基)CO(C0-10烷基)、-N(C0-10烷基)COO(C0-10烷基)、-OCON(C0-10烷基)(C0-10烷基)、卤素、-CN、-OCH2F、-OCHF2、-OCF3、-N(C0-10烷基)(C0-10烷基)、-OC0-10烷基、-CO(C0-10烷基)、-COO(C0-10烷基)、-N杂环芳香基、-O杂环芳香基或-S杂环芳香基；
R2选自：-H、-D、卤素、-NO2、-CN、C1-10直链/支链烷基、C3-10环烷基、-N(C0-10烷基)(C0-10烷基)、-CF3、-OCF3、-OCHF2、-OCH2F或-OC0-10烷基；
B1、B2、B3、B4和B5独立地选自C或N(当B1、B2、B3、B4或B5为N时，其所对应的R3、R4、R5、R6、R7不存在)；
当存在时，R3、R4、R5、R6和R7独立地选自：-H、-D、卤素、-CN、-OC0-10烷基、-CO(C0-10烷基)、-CON(C0-10烷基)(C0-10烷基)、C1-10直链/支链烷基、含O或N的杂烷基、-N(C0-10烷基)(C0-10烷基)、C3-10环烷基、-C≡C-R10、-O杂环烷基、-N杂环烷基，或R5和R4、R4和R3、R3和R7、R7和R6与其之间的碳原子形成C3-8环烷基或含-O-、-S-的C3-8杂环烷基、-N杂环芳香基、-O杂环芳香基或-S杂环芳香基、苯基，其中烷基部分可被一个或多个以下基团任意取代：-SO2、-SO2N(C0-10烷基)(C0-10烷基)、-N(C0-10烷基)SO2(C0-10烷基)、-CON(C0-10烷基)(C0-10烷基)、-N(C0-10烷基)CO(C0-10烷基)、-N(C0-10烷基)COO(C0-10烷基)、-OCON(C0-10烷基)(C0-10烷基)、卤素、-CN、-OCH2F、-OCHF2、-OCF3、C1-10直链/支链烷基、-N(C0-10烷基)(C0-10烷基)、-OC0-10烷基、C3-10环烷基、-O杂环烷基、-N杂环烷基、-N杂环芳香基、-O杂环芳香基或-S杂环芳香基；
R8和R9独立地选自：-H、-D、卤素、C1-10直链/支链烷基；
R10选自：H、-D、C1-5直链/支链烷基、C3-10环烷基、
R11、R12独立地选自：-H、-D、-CF3、-CHF2H、-CH2F、C1-10直链/支链烷基、-CH＝C(C0-10烷基)(C0-10烷基)、-C≡C(C0-10烷基)、C3-10环烷基、芳香性五元环基团或芳香性六元环基团，或R11、R12与R11和R12之间的碳原子形成C3-8环烷基或含-O-、-S-的C3-8杂环烷基、C4-9稠环烷基、C5-10螺环烷基、C4-9桥环烷基、C3-7环内酰胺、C3-7环内酯、C3-7环酮，其中烷基部分可被一个或多个以下基团任意取代：-SO2、-SO2N(C0-10烷基)(C0-10烷基)、-N(C0-10烷基)SO2(C0-10烷基)、-CON(C0-10烷基)(C0-10烷基)、-N(C0-10烷基)CO(C0-10烷基)、-N(C0-10烷基)COO(C0-10烷基)、-OCON(C0-10烷基)(C0-10烷基)、卤素、-CN、-OCH2F、-OCHF2、-OCF3、-N(C0-10烷基)(C0-10烷基)、-OC0-10烷基、-N杂环芳香基、-O杂环芳香基或-S杂环芳香基；
进一步的，所述Ar、R2、R3、R4、R5、R6、R7、R8、R9、R10、R11和/或R12中的C原子所连接的一个或多个H原子可以被氘取代；
和/或,所述Ar、R2、R3、R4、R5、R6、R7、R8、R9、R10、R11和/或R12中的杂原子所连接的一个或多个H原子可以被氘取代；
和/或，所述通式Ⅰ的化合物含有至少一个氘原子
并且，通式Ⅰ的化合物含有至少一个氘原子。
在本发明的一个实施方式中，A为CR10，特别是CH。
在本发明的一个实施例中，Q为O。
在本发明的一个实施例中，x为0。
在本发明的一个实施例中，z为1。
在本发明的一个实施例中，y为1。
在本发明的一个实施方式中，B1、B2、B3、B4和B5均为C，即通式Ⅰ中，为
在本发明另一实施方式中，B1、B2、B3、B4和B5中至少一个为N。
具体地，B2为C，B1、B3、B4和B5中至少一个为N。
更具体地，B2为C，B1为N。
更具体地，B2为C，B3为N。
更具体地，B2为C，B4为N。
更具体地，B2为C，B5为N。
更具体地，B2为C，B3和B4为N或者B3和B5均为N。
具体地，Ar选自：噻唑基、硒代噻唑基、咪唑基、吡唑基和吡啶基。
在本发明的一个实施方式中，通式Ⅰ的化合物具有如下结构：
其中，E环选自：
E环中，各个R0独立地选自：-H、-D、C1-10直链/支链烷基、-N(C0-10烷基)(C0-10烷基)、-OC0-10烷基、-CO(C0-10烷基)或C3-10环烷基；其中上述C原子或杂原子上所连接的H可以被氘取代；
R1选自：-H、-D、-O杂环烷基、-N杂环烷基、C1-10直链/支链烷基、C3-10环烷基、-OC0-10烷基、-N(C0-10烷基)(C0-10烷基)、-SO2(C0-10烷基)、-CO(C0-10烷基)、-O-苯基、-S(C0-10烷基)、-N杂环芳香基、-O杂环芳香基或-S杂环芳香基；
R2-9具有本发明上述相应定义；
进一步的，所述R0、R1中的C原子上所连接的H可以被氘取代；
和/或，R0、R1中的杂原子上所连接的H可以被氘取代；
和/或，所述R0-9中至少一个含有氘原子；
和/或，所述通式Ⅱ的化合物含有至少一个氘原子。
在本发明的一个实施方式中，通式Ⅱ中，R1中含有至少一个氘原子；更具体地，例如，R1中含有至少一个氘原子，而R2-9不含有氘原子。
在本发明另一个实施方式中，通式Ⅱ中，R2中含有至少一个氘原子。
在本发明另一个实施方式中，通式Ⅱ中，R3中含有至少一个氘原子。
在本发明另一个实施方式中，通式Ⅱ中，R4中含有至少一个氘原子。
在本发明另一个实施方式中，通式Ⅱ中，R5中含有至少一个氘原子。
在本发明另一个实施方式中，通式Ⅱ中，R6中含有至少一个氘原子。
在本发明另一个实施方式中，通式Ⅱ中，R8和/或R9中含有至少一个氘原子。
具体地，各个R0独立地选自：C1-5直链/支链烷基或-N(C0-10烷基)(C0-10烷基)，其中，C原子上所连接的H可以被氘取代。
更具体地，各个R0独立地选自：-H、-D、-CH3、-CH2CH3或-NH2。
具体地，R1选自：-O杂环烷基或-N杂环烷基、-SO2(C0-3烷基)、-O-苯基、-S(C0-4烷基)、C3-6环烷基或C3-5直链/支链烷基，其中C原子或杂原子上所连接的H可以被氘取代。
更具体地，R1选自：-CH3、 其中C原子或N原子上所连接的H可以被氘取代。
更具体地，R1选自： -CH2D、-CHD2、-CD3、
具体地，当R0与R1相邻时，R0和R1与其之间的碳原子形成C3-8环烷基或含-O-、-S-的C3-8杂环烷基、 -N杂环芳香基、-O杂环芳香基或-S杂环芳香基、苯基。
具体地，R2选自：-H、-D、卤素、-NO2、-CN、C1-5直链/支链烷基、C3-10环烷基、-N(C0-10烷基)(C0-10烷基)、-CF3、-OCF3、-OCHF2、-OCH2F或-OC0-10烷基，其中，C原子或N原子上所连接的H可以被氘取代。
更具体地，R2选自：-NO2、-N(C0-10烷基)(C0-10烷基)、-OC0-10烷基和-OCF3其中，C原子或N原子上所连接的H可以被氘取代。
进一步具体地，R2选自：-NH2、-NHD、-ND2或-NO2。
具体地，R3选自：-H、-D、卤素、-OC0-10烷基、-CO(C0-10烷基)、C1-10直链/支链烷基、-N(C0-10烷基)(C0-10烷基)或C3-10环烷基，其中，C原子上所连接的H可以被氘取代。
更具体地，R3选自：-H、-D、卤素、-OC0-10烷基、C1-10直链/支链烷基，其中，C原子上所连接的H可以被氘取代。
进一步具体地，R3选自：-H、-D、-F、-OCH3、-OCH2D、-OCHD2、-OCD3。
具体地，R4选自：-H、-D、卤素、-OC0-10烷基、-CO(C0-10烷基)、-CN、C3-10环烷基、-C≡C-R10、C1-10直链/支链烷基、-N(C0-10烷基)(C0-10烷基)、-O杂环烷基或-N杂环烷基，其中，C原子或N原子上所连接的H可以被氘取代。
更具体地，R4选自：-H、-D、卤素、-OC0-10烷基、-CN、C3-10环烷基或-C≡C-R10，其中，C原子上所连接的H可以被氘取代。在本发明的一个实施例中，R10中的C原子上所连接的H可以被氘取代。
在本发明的一个实施例中，R4选自：-H、-D、-F、-Cl、-OCH3、-OCH2D、-OCHD2、-OCD3、-CN、或-C≡C-R10。
具体地，R5、R6、R7独立地选自：-H、-D、卤素、-CN、-OC0-10烷基、-CO(C0-10烷基)、C1-10直链/支链烷基、-N(C0-10烷基)(C0-10烷基)、C3-10环烷基、-C≡C-R10、-O杂环烷基或-N杂环烷基、含O或N的C1-5直链/支链烷基，或R6、R7与R6和R7之间的碳原子形成C3-8环烷基或含-O-、-S-的C3-8杂环烷基，其中，C原子或杂原子上所连接的H可以被氘取代。
更具体地，R5、R6、R7独立地选自：-H、-D、卤素、-CN、C1-3直链/支链烷基、-OC0-3烷基、-CO(C0-3烷基)、含N的C1-3直链/支链烷基，或R6、R7与R6和R7之间的碳原子形成C3-8环烷基或含-O-的C3-8杂环烷基，其中，C原子或N原子上所连接的H可以被氘取代。
进一步具体地，R5、R6、R7独立地选自：-H、-D、-F、-Cl、-CH3、-CH2NH2、-CH2NH(CH3)、-CH2N(CH3)2、-CN、-OCH3、-COCH3，或R6、R7与R6和R7之间的碳原子形成含-O-五元环烷基，其中，C原子或N原子上所连接的H可以被氘取代。
在本发明的一个实施例中，R5选自：-H、-D、-F、-Cl、-CH3、-CH2D、-CHD2、-CD3、-OCH3、-COCH3、-CH2NH2、-CH2N(CH3)2、-CN、-OCH2D、-OCHD2、-OCD3、-COCD3、-CH2N(CD3)2、、-CH2N(CH3)(CD3)。
在本发明的一个实施例中，R6选自：-H、-D、-F、-Cl、-CH3、-CH2D、-CHD2、-CD3、-OCH3、-COCH3、-CH2NH2、-CH2N(CH3)2、-CN、-OCH2D、-OCHD2、-OCD3、-COCD3、-CH2N(CD3)2、、-CH2N(CH3)(CD3)。
在本发明的一个实施例中，R7选自：-H、-D、-F、-Cl、-CH3、-CH2D、-CHD2、-CD3、-OCH3、-COCH3、-CH2NH2、-CH2N(CH3)2、-CN、-OCH2D、-OCHD2、-OCD3、-COCD3、-CH2N(CD3)2、-CH2N(CH3)(CD3)。
在本发明的一个实施方式中，R10为在本发明的一个实施例中，R10中的C原子上所连接的H可以被氘取代。
具体地，R11和R12独立地选自：-H、-D、-CF3、-CHF2H、-CH2F、C1-10直链/支链烷基、-CH＝C(C0-10烷基)(C0-10烷基)、C3-10环烷基或芳香性六元环基团，或R11、R12与R11和R12之间的碳原子形成C3-8环烷基、C4-7稠环烷基、C5-9螺环烷基、C4-9桥环烷基、C3-7环内酰胺、C3-7环内酯、C3-7环酮，其中C原子上的H可被以下基团取代：-SO2、-SO2N(C0-10烷基)(C0-10烷基)、-N(C0-10烷基)SO2(C0-10烷基)、-CON(C0-10烷基)(C0-10烷基)、-N(C0-10烷基)CO(C0-10烷基)、-N(C0-10烷基)COO(C0-10烷基)、-OCON(C0-10烷基)(C0-10烷基)、卤素、-CN、-OCH2F、-OCHF2、-OCF3、C1-10直链/支链烷基、-N(C0-10烷基)(C0-10烷基)、-OC0-10烷基、-CO(C0-10烷基)、C3-10环烷基、-O杂环烷基、-N杂环烷基、-N杂环芳香基、-O 杂环芳香基或-S杂环芳香基，其中所述烷基部分可被一个或多个以下基团任意取代：-SO2、-SO2N(C0-10烷基)(C0-10烷基)、-N(C0-10烷基)SO2(C0-10烷基)、-CON(C0-10烷基)(C0-10烷基)、-N(C0-10烷基)CO(C0-10烷基)、-N(C0-10烷基)COO(C0-10烷基)、-OCON(C0-10烷基)(C0-10烷基)、卤素、-CN、-OCH2F、-OCHF2、-OCF3、-N(C0-10烷基)(C0-10烷基)、-OC0-10烷基、-CO(C0-10烷基)、-N杂环芳香基、-O杂环芳香基或-S杂环芳香基；其中，C原子或杂原子上所连接的H可以被氘取代。
更具体地，R11和R12独立地选自：-H、-D、-CF3、-CHF2、-CH2F、C1-5直链/支链烷基、-CH＝CH(C0-10烷基)、C3-10环烷基或芳香性六元环基团，或R11、R12与R11和R12之间的碳原子形成C3-6环烷基、C4-6稠环烷基、C5-8螺环烷基、C4-8桥环烷基、C3-7环内酰胺、C3-7环内酯、C3-7环酮，其中烷基部分可被以下基团取代：-SO2、-SO2N(C0-10烷基)(C0-10烷基)、-N(C0-10烷基)SO2(C0-10烷基)、-CON(C0-10烷基)(C0-10烷基)、-N(C0-10烷基)CO(C0-10烷基)、-N(C0-10烷基)COO(C0-10烷基)、-OCON(C0-10烷基)(C0-10烷基)、卤素、-CN、-OCH2F、-OCHF2、-OCF3、C1-10直链/支链烷基、-N(C0-10烷基)(C0-10烷基)、-OC0-10烷基、-CO(C0-10烷基)、C3-10环烷基、-O杂环烷基、-N杂环烷基、-N杂环芳香基、-O杂环芳香基或-S杂环芳香基，其中所述烷基部分可被一个或多个以下基团任意取代：-SO2、-SO2N(C0-10烷基)(C0-10烷基)、-N(C0-10烷基)SO2(C0-10烷基)、-CON(C0-10烷基)(C0-10烷基)、-N(C0-10烷基)CO(C0-10烷基)、-N(C0-10烷基)COO(C0-10烷基)、-OCON(C0-10烷基)(C0-10烷基)、卤素、-CN、-OCH2F、-OCHF2、-OCF3、-N(C0-10烷基)(C0-10烷基)、-OC0-10烷基、-CO(C0-10烷基)、-N杂环芳香基、-O杂环芳香基或-S杂环芳香基；其中，C原子或杂原子上所连接的H可以被氘取代。
进一步具体地，R11和R12独立地选自：-H、-D、-CF3、-CHF2、-CH2F、-CH3、-CH2CH3、-CH＝CH2、或R11、R12与R11和R12之间的碳原子形成 其中，C原子或N原子上所连接的H可以被氘取代。
更进一步具体地，R11和R12独立地选自：-H、-D、-CF3、-CHF2、-CDF2、-CH2F、-CD2F、-CH3、-CH2D、-CHD2、-CD3、-CH2CH3、-CH2CD3、
具体地，R8和R9独立地选自：-H、-D、C1-10直链/支链烷基，其中，C原子上所连接的H可以被氘取代。
更具体地，R8和R9独立地选自：-H、-D、C1-3直链/支链烷基，其中，C原子上所连接的H可以被氘取代。
进一步具体地，R8和R9独立地选自：-H、-D、-CH3、-CH2D、-CHD2、-CD3。
具体地，通式Ⅰ的化合物具有如下结构：





















具体地，上述化合物中，任何不被指明为氘的原子以其天然同位素丰度存在。
具体地，上述化合物中，被指明为“氘”的位置具有至少(例如)95％的氘掺入。
本发明提供一种通式Ⅰ的化合物的制备方法，其包括如下步骤：
(1)与发生缩合反应生成R13选自：卤素或R14选自：-OH或-F；
(2)与Ar-R15发生缩合反应生成 R15选自：-Br或-SnBu3。
具体地，上述步骤(1)中R13为-Br。
具体地，上述步骤(2)中R13为-Br或当R13是-Br时，R15为-SnBu3，当R13是时，R15为-Br。
本发明还提供上述通式Ⅰ的化合物的药学上可接受的盐、立体异构体、酯、前药、溶剂化物。
具体地，上述药学上可接受的盐中包括酸加成盐和碱加成盐。
具体地，上述酸加成盐包括但不限于来自无机酸诸如盐酸、硝酸、磷酸、硫酸、氢溴酸、氢碘酸和膦酸的盐，以及来自有机酸如脂肪族单羧酸和二羧酸、苯基取代的链烷酸、羟基链烷酸、链烷二酸、芳香酸和脂肪族和芳香族磺酸的盐。因此，这些盐包括但是不限于硫酸盐、焦硫酸盐、硫酸氢盐、亚硫酸盐、亚硫酸氢盐、硝酸盐、磷酸盐、磷酸一氢盐、磷酸二氢盐、偏磷酸盐、焦磷酸盐、盐酸盐、氢溴酸盐、碘酸盐、乙酸盐、丙酸盐、辛酸盐、异丁酸盐、乙二酸盐、丙二酸盐、琥珀酸盐、辛二酸盐、癸二酸盐、富马酸盐、马来酸盐、苦杏仁酸盐、苯甲酸盐、氯代苯甲酸盐、甲基苯甲酸盐、二硝基苯甲酸盐、酞酸盐、苯磺酸盐、甲苯磺酸盐、苯基乙酸盐、柠檬酸盐、乳酸盐、马来酸盐、酒石酸盐和甲磺酸盐，还包含氨基酸的盐如精氨酸盐、葡糖酸盐、半乳糖醛酸盐等。酸加成盐可以通过以常规方式使游离碱形式与足够量的所需酸接触形成盐的方式制备。可通过使盐形式与碱接触重新生成游离碱形式，并且以常规方式分离该游离碱。
具体地，上述碱加成盐与金属或者胺形成，诸如碱金属和碱土金属的氢氧化物，或者与有机胺形成。用作阳离子的金属的例子包括但是不限于钠、钾、镁、和钙。适当的胺的例子包括但不限于N,N′-二苄基乙二胺、氯普鲁卡因、胆碱、二乙醇胺、乙二胺(乙烷-1,2-二胺)、N-甲基葡糖胺和普鲁卡因。碱加成盐可通过以常规方式使游离酸形式与足够量的所需碱接触形成盐的方式制备。可通过使盐形式与酸接触重新生成游离酸形式，并且以常规方式分离游离酸。
在本发明的一个实施例中，上述药学上可接受的盐为盐酸盐。
具体地，上述立体异构体包括对映体、非对映体和几何异构体的形式存在。本发明的一些化合物具有环烷基，其可在超过一个碳原子上被取代，在这种情况下，其所有的几何形式，包括顺式和反式，及其混合物，都处在本发明的范围内。
具体地，上述溶剂化物是指本发明的化合物与一种或多种溶剂分子的物理结合。该物理结合包括各种程度的离子和共价键合，包括氢键合。在某些情况下，溶剂化物可被分离出来，例如当一个或多个溶剂分子掺入到结晶固体的晶格中。“溶剂化物”包括溶液相的和可分离的溶剂化物。代表性的溶剂化物包括乙醇化物、甲醇化物等。“水合物”是其中一个或多个溶剂分子为H2O的溶剂化物。
具体地，上述前药指适于对患者给药的无过分毒性、刺激性和变态反应等的并且对其应用目的有效的式Ⅰ化合物形式，包括缩醛、酯和两性离子形式。前药在体内转化(例如通过在血液中水解)得到上式的母体化合物。
本发明还提供一种上述通式Ⅰ的化合物及其药学上可接受的盐、立体异构体、酯、前药和溶剂化物的晶型。
具体地，本发明提供4-(3-(((2-氨基-5-(1-(1-三氘代甲基哌啶-4-基)-1H-吡唑-4-基)吡啶-3-基)氧基)甲基)苯基)-2-甲基丁-3-炔-2-醇(其具有如下结构)的晶型。
具体地，上述晶型为晶型A，其XRPD图谱在2θ值为13.1°±0.2°、16.3°±0.2°、17.5°±0.2°、23.8°±0.2°的位置中至少三个(或全部)位置处具有特征峰(主要特征衍射峰)。
具体地，上述晶型A的XRPD图谱还在2θ值为8.1°±0.2°、12.2°±0.2°、15.3°±0.2°、18.0°±0.2°、19.3°±0.2°、19.5°±0.2°、21.3°±0.2°、21.6°±0.2°的位置中的至少三个(至少四个，至少五个，至少六个，至少七个，或全部)位置处具有特征峰(次要特征衍射峰)。
具体地，上述晶型A具有基本上如图1所示的XRPD图谱。
具体地，上述晶型A的DSC图谱，在约168.8℃具有吸热峰。
具体地，上述晶型A具有基本上如图2所示的DSC图谱。
具体地，上述晶型A从室温加热至170℃重量损失为约1.1％。
具体地，上述晶型A具有基本上如图2所示的TGA图谱。
具体地，上述晶型A为无水晶型。
具体地，上述晶型为晶型B，其XRPD图谱在2θ值为5.7°±0.2°、11.3°±0.2°、22.7°±0.2°、23.5°±0.2°的位置中的至少三个(或全部)位置处具有特征峰(主要特征衍射峰)。
具体地，上述晶型B的XRPD图谱还在2θ值为7.1°±0.2°、8.8°±0.2°、14.1°±0.2°、17.0°±0.2°、18.0°±0.2°、18.8°±0.2°的位置中的至少三个(至少四个，至少五个，或全部)位置处具有特征峰(次要特征衍射峰)。
具体地，上述晶型B具有基本上如图6所示的XRPD图谱。
具体地，上述晶型B的DSC图谱，在约59.5℃、95.6℃、150.8℃及160.9℃中的至少一处具有吸热峰。
具体地，上述晶型B具有基本上如图7所示的DSC图谱。
具体地，上述晶型B从室温加热至70℃重量损失为约13.2％，且继续加热至170℃重量损失为约8.5％。
具体地，上述晶型B具有基本上如图7所示的TGA图谱。
具体地，上述晶型B为EtOAc溶剂合物。
具体地，本发明还提供4-(3-(((2-氨基-5-(1-(1-三氘代甲基哌啶-4-基)-1H-吡唑-4-基)吡啶-3-基)氧基)甲基)苯基)-2-甲基丁-3-炔-2-醇盐酸盐的晶型。
具体地，上述晶型为晶型A，其XRPD图谱在2θ值为13.0°±0.2°、16.3°±0.2°、17.5°±0.2°、19.4°±0.2°、23.8°±0.2°的位置中的至少三个(至少四个，或全部)位置处具有特征峰(主要特征衍射峰)。
具体地，上述晶型A的XRPD图谱还在2θ值为8.1°±0.2°、12.1°±0.2°、15.3°±0.2°、18.0°±0.2°、21.4°±0.2°的位置中的至少三个(至少四个，或全部)位置处具有特征峰(次要特征衍射峰)。
具体地，上述晶型A具有基本上如图11所示的XRPD图谱。
具体地，上述晶型A的DSC图谱，在约81.9℃、约156.0℃处具有吸热峰。
具体地，上述晶型A具有基本上如图12所示的DSC图谱。
具体地，上述晶型A从室温加热至150℃重量损失为约8.6％。
具体地，上述晶型A具有基本上如图12所示的TGA图谱。
本发明还提供上述晶型的制备方法。
具体地，上述制备方法选自：反溶剂添加、反-反溶剂添加、气固扩散、室温悬浮搅拌、5℃悬浮搅拌、缓慢挥发、缓慢降温、气液扩散、高聚物诱导中的一种或多种的组合。
具体地，上述反溶剂添加法包括：将目标产物用良溶剂溶解，然后向所得溶液中加入反溶剂(然后，例如，在室温下挥发得到固体，或在-20℃搅拌得到固体)。
具体地，上述反-反溶剂添加法包括：将目标产物用良溶剂溶解，然后将所得溶液加入反溶剂中(然后，例如，在室温下挥发得到固体，或在-20℃搅拌得到固体)。
具体地，上述气固扩散法、室温悬浮搅拌法、5℃悬浮搅拌法、缓慢挥发法、缓慢降温法、气液扩散法均包括：将目标产物用溶剂溶解，然后干燥得到固体。
具体地，上述高聚物诱导法包括：将目标产物用溶剂溶解，加入高聚物，室温下挥发得到固体。
具体地，上述4-(3-(((2-氨基-5-(1-(1-三氘代甲基哌啶-4-基)-1H-吡唑-4-基)吡啶-3-基)氧基)甲基)苯基)-2-甲基丁-3-炔-2-醇的晶型A的制备方法选自：反溶剂添加、反-反溶剂添加、气固扩散、室温悬浮搅拌、5℃悬浮搅拌、缓慢挥发、缓慢降温、气液扩散、高聚物诱导中的一种或多种的组合。
具体地，对于上述晶型A的制备，对于反溶剂添加法，良溶剂可以选自：MeOH、丙酮、DMSO、EtOAc、EtOH、DCM、CHCl3、THF、IPA、ACN、1,4-二氧六环，反溶剂可以选自：MTBE、甲苯、正庚烷、水。
在本发明的一个实施方式中，在反溶剂添加法中，良溶剂可以选自：MeOH、丙酮、DMSO，反溶剂为MTBE。
在本发明的另一个实施方式中，在反溶剂添加法中，良溶剂可以选自：EtOH、DCM，反溶剂为甲苯。
在本发明的另一个实施方式中，在反溶剂添加法中，良溶剂可以选自：CHCl3、THF、IPA，反溶 剂为正庚烷。
在本发明的另一个实施方式中，在反溶剂添加法中，良溶剂可以选自：丙酮、ACN、1,4-二氧六环，反溶剂为水。
具体地，对于上述晶型A的制备，对于反-反溶剂添加法，良溶剂可以选自：MeOH、MIBK、丙酮、苯甲醚、EtOH、THF、EtOAc、DCM，反溶剂可以选自：甲苯、正庚烷、水、MTBE。
在本发明的一个实施方式中，在反-反溶剂添加法中，良溶剂可以为MIBK，反溶剂可以为甲苯。
在本发明另一个实施方式中，在反-反溶剂添加法中，良溶剂可以选自：丙酮、苯甲醚，反溶剂可以为正庚烷。
在本发明另一个实施方式中，在反-反溶剂添加法中，良溶剂可以选自：EtOH、THF，反溶剂可以为水。
在本发明另一个实施方式中，在反-反溶剂添加法中，良溶剂可以选自：EtOAc、DCM，反溶剂可以为MTBE。
具体地，对于上述晶型A的制备，对于气固扩散法，溶剂可以选自：水、DCM、EtOH、MeOH、ACN、THF、CHCl3、丙酮、DMSO、EtOAc、1,4-二氧六环、IPA。
具体地，对于上述晶型A的制备，对于室温悬浮搅拌法，溶剂可以选自：MTBE、IPAc、正庚烷、甲苯、水、EtOH/甲苯(例如以1:3的比例，v/v)、DMSO/MTBE(例如以1:4的比例，v/v)、丙酮/水(例如以1:4的比例，v/v)、IPA/正庚烷(例如以1:4的比例，v/v)、EtOAc/正庚烷(例如以1:4的比例，v/v)、苯甲醚/甲苯(例如以1:4的比例，v/v)、DMAc/水(例如以1:4的比例，v/v)、THF/水(例如以1:4的比例，v/v)。
具体地，对于上述晶型A的制备，对于5℃悬浮搅拌法，溶剂可以选自：MTBE、甲苯、水、IPA/正庚烷(例如以1:2的比例，v/v)、MEK/正庚烷(例如以1:2的比例，v/v)、EtOAc/甲苯(例如以1:2的比例，v/v)、CPME/甲苯(例如以1:2的比例，v/v)、NMP/水(例如以1:4的比例，v/v)、THF/水(例如以1:4的比例，v/v)、ACN/水(例如以1:2的比例，v/v)、IPAc/DCM(例如以1:1的比例，v/v)、MeOH/甲苯(例如以1:4的比例，v/v)、DCM/MTBE(例如以1:4的比例，v/v)、THF/正庚烷(例如以1:4的比例，v/v)。
具体地，对于上述晶型A的制备，对于缓慢挥发法，溶剂可以选自：EtOH、丙酮、IPAc、THF、CPME、苯甲醚、ACN/水(例如以9:1的比例，v/v)、MeOH/DCM(例如以1:1的比例，v/v)、丙酮/EtOAc(例如以2:1的比例，v/v)、THF/水(例如以4:1的比例，v/v)。
具体地，对于上述晶型A的制备，对于缓慢降温法，溶剂可以选自：CPME、甲苯、ACN/甲苯(例如以1:2的比例，v/v)、丙酮/正庚烷(例如以1:1的比例，v/v)、THF/甲苯(例如以1:2的比例，v/v)、MeOH/水(例如以1:1的比例，v/v)、CHCl3/MTBE(例如以1:1的比例，v/v)。
具体地，对于上述晶型A的制备，对于气液扩散法，良溶剂可以选自：EtOH、THF、DMSO，反溶剂可以选自：正庚烷、MTBE、甲苯、环己烷、水。
在本发明的一个实施方式中，在气液扩散法中，良溶剂为EtOH，反溶剂可以选自：正庚烷、MTBE、甲苯。
在本发明另一个实施方式中，在气液扩散法中，良溶剂为THF，反溶剂可以选自：正庚烷、环己烷、MTBE。
在本发明另一个实施方式中，在气液扩散法中，良溶剂为DMSO，反溶剂可以选自：甲苯、MTBE、水。
具体地，对于上述晶型A的制备，对于高聚物诱导法，溶剂可以选自：MEK、ACN/甲苯(例如以4:1的比例，v/v)、THF/水(例如以9:1的比例，v/v)、EtOAc、丙酮/2-MeTHF(例如以1:1的比例，v/v)、MeOH/DCM(例如以1:1的比例，v/v)，高聚物可以选自：聚乙烯吡咯烷酮、聚乙烯醇、聚氯乙烯、聚乙酸乙烯酯、羟丙甲纤维素、甲基纤维素、聚己内酯、聚乙二醇、聚甲基丙烯酸甲酯、海藻酸钠、羟乙基纤维素中的一种或多种的组合。
在本发明的一个实施方式中，在高聚物诱导法中，高聚物为聚乙烯吡咯烷酮、聚乙烯醇、聚氯乙烯、聚乙酸乙烯酯、羟丙甲纤维素和甲基纤维素(例如以1:1:1:1:1的质量比)的混合物，溶剂可以选自：MEK、ACN/甲苯(例如以4:1的比例，v/v)、THF/水(例如以9:1的比例，v/v)。
在本发明另一个实施方式中，在高聚物诱导法中，高聚物为聚己内酯、聚乙二醇、聚甲基丙烯酸甲酯、海藻酸钠和羟乙基纤维素(例如以1:1:1:1:1的质量比)的混合物，溶剂可以选自：EtOAc、丙酮/2-MeTHF(例如以1:1的比例，v/v)。
具体地，上述4-(3-(((2-氨基-5-(1-(1-三氘代甲基哌啶-4-基)-1H-吡唑-4-基)吡啶-3-基)氧基)甲基)苯 基)-2-甲基丁-3-炔-2-醇的晶型B的制备方法为反溶剂添加法。
在本发明的一个实施例中，对于上述晶型B的制备，良溶剂为EtOAc，反溶剂为甲苯。
本发明还提供一种药物组合物，其包含上述通式Ⅰ的化合物或其药学上可接受的盐、立体异构体、酯、前药、溶剂化物，或上述晶型，以及药学上可接受的辅料。
具体地，上述辅料选自：载体、稀释剂、粘合剂、润滑剂、润湿剂等中的一种或多种。具体地，上述药物组合物包含治疗有效量的通式Ⅰ的化合物。在某些实施方案中，这些药物组合物可用于治疗HPK1激酶介导的疾病或病症。
具体地，上述药物组合物可以为片剂(例如，糖衣片剂、膜包衣片剂、舌下片剂、口腔崩解片、口腔片剂等)、丸剂、粉剂、颗粒剂、胶囊剂(例如，软胶囊、微胶囊)、锭剂、糖浆剂、乳剂、混悬剂、控制释放制剂(例如，瞬时释放制剂、缓释制剂、缓释微囊)、气雾剂、膜剂(例如，口服崩解膜剂、口腔粘膜-粘附膜剂)、注射剂(例如，皮下注射、静脉注射、肌内注射、腹膜内注射)、静脉滴注剂、透皮吸收制剂、软膏剂、洗剂、粘附制剂、栓剂(例如，直肠栓剂、阴道栓剂)、小药丸、鼻制剂、肺制剂(吸入剂)、眼睛滴剂等等。
具体地，上述药物组合物的各种剂型可以按照药学领域的常规生产方法制备。例如使活性成分与一种或多种辅料混合，然后将其制成所需的剂型。
具体地，上述药物组合物可以含有重量比为0.1-99.5％(例如，0.1％、1％、5％、10％、20％、30％、40％、50％、60％、70％、80％、90％、95％、99％、99.5％)的活性成分。
本发明还提供上述通式Ⅰ的化合物及其药学上可接受的盐、立体异构体、酯、前药和溶剂化物，或上述晶型，或上述药物组合物在制备预防和/或治疗肿瘤的药物中的应用。
本发明还提供上述通式Ⅰ的化合物及其药学上可接受的盐、立体异构体、酯、前药和溶剂化物、上述晶型联合PD-1、PD-L1、CTLA-4、TIM-3、TGF-β及其受体、LAG3拮抗剂或TLR4、TLR7、TLR8、TLR9、STING激动剂在肿瘤免疫疗法中的应用。
本发明还提供上述通式Ⅰ的化合物及其药学上可接受的盐、立体异构体、酯、前药和溶剂化物、上述晶型与CAR-T免疫疗法相结合在肿瘤免疫疗法中的应用。
具体地，上述CAR-T免疫疗法是指：嵌合抗原受体T细胞免疫疗法，其基本原理为利用病人自身的免疫细胞来清除癌细胞，属于一种细胞疗法。
具体地，上述肿瘤为恶性肿瘤，其包括但不限于：淋巴瘤、母细胞瘤、髓母细胞瘤、视网膜母细胞瘤、肉瘤、脂肪肉瘤、滑膜细胞肉瘤、神经内分泌肿瘤、类癌肿瘤、胃泌素瘤、胰岛细胞癌、间皮瘤、神经鞘瘤、听神经瘤、脑膜瘤、腺癌、黑素瘤、白血病或淋巴样恶性肿瘤、鳞状细胞癌、上皮鳞状细胞癌、肺癌、小细胞肺癌、非小细胞肺癌、腺癌肺癌、肺鳞癌、腹膜癌、肝细胞癌、胃癌、肠癌、胰腺癌、成胶质细胞瘤、子宫颈癌、卵巢癌、肝癌、膀胱癌、肝癌、乳腺癌、转移性乳腺癌、结肠癌、直肠癌、结肠直肠癌、子宫癌、唾液腺癌、肾癌、前列腺癌、外阴癌、甲状腺癌、肝癌、肛门癌、阴茎癌、梅克尔细胞癌、食管癌、胆道肿瘤、头颈部癌和血液恶性肿瘤。
本发明还提供一种预防和/或治疗肿瘤的方法，其包括向有此需要的受试者施用有效量的本发明上述通式Ⅰ的化合物及其药学上可接受的盐、立体异构体、酯、前药和溶剂化物，或上述晶型，或本发明上述药物组合物的步骤。
具体地，肿瘤具有本发明上述相应定义。
本发明还提供一种预防和/或治疗治疗由病原体感染引起或病原体感染相关的疾病的方法，其包括向有此需要的受试者施用有效量的本发明上述通式Ⅰ的化合物及其药学上可接受的盐、立体异构体、酯、前药和溶剂化物，或上述晶型，或本发明上述药物组合物的步骤。
具体地，病原体和疾病具有本发明上述相应定义。
附图说明
图1所示为化合物A2游离碱晶型A的XRPD图谱。
图2所示为化合物A2游离碱晶型A的TGA/DSC图谱。
图3所示为化合物A2游离碱晶型A的1H NMR图谱。
图4所示为化合物A2游离碱晶型A的HPLC图谱。
图5所示为化合物A2游离碱晶型A的起始样品及在60℃条件下闭口放置1天后的XRPD图谱。
图6所示为化合物A2游离碱晶型B的XRPD图谱。
图7所示为化合物A2游离碱晶型B的TGA/DSC图谱。
图8所示为化合物A2游离碱晶型B的1H NMR图谱。
图9所示为化合物A2游离碱晶型B在室温下放置前后的XRPD图谱。
图10所示为化合物A2游离碱晶型B经氮气吹扫前后的XRPD图谱。
图11所示为化合物A2盐酸盐的XRPD图谱。
图12所示为化合物A2盐酸盐的TGA/DSC图谱。
图13所示为化合物A2游离碱晶型A和化合物A2盐酸盐的XRPD图谱叠图。
图14所示为人PBMCs的iL-2的浓度。
图15所示为小鼠接种HSV病毒后的角膜炎病变情况。
图16所示为小鼠接种HSV病毒后的临床评分情况。
图17所示为小鼠接种HSV病毒后的角膜新生血管分化评分情况。
图18所示为仓鼠的体重变化情况。
图19所示为仓鼠的肺部重量变化情况。
图20所示为H1N1感染小鼠的Kaplan-Meier生存图(n＝6)，35％的虚线是人道主义的终点，任何体重下降低于这个标准(-35％)的老鼠都将被安乐死。
图21所示为给予低剂量病毒感染的小鼠的病毒特异性T细胞增殖率。
具体实施方式
除非另有定义，本发明中所使用的所有科学和技术术语具有与本发明涉及技术领域的技术人员通常理解的相同的含义。
本发明中术语“C0-10烷基”，C0烷基是指H，因此，C0-10烷基包括H、C1烷基、C2烷基、C3烷基、C4烷基、C5烷基、C6烷基、C7烷基、C8烷基、C9烷基、C10烷基。
本发明中术语“C1-10直链/支链烷基”，包括甲基、乙基、C3直链/支链烷基、C4直链/支链烷基、C5直链/支链烷基、C6直链/支链烷基、C7直链/支链烷基、C8直链/支链烷基、C9直链/支链烷基、C10直链/支链烷基。
本发明中术语“C3-10支链烷基”，包括异丙基、异丁基、叔丁基、异戊基。
本发明中术语“C3-10环烷基”，包括C3环烷基、C4环烷基、C5环烷基、C6环烷基、C7环烷基、C8环烷基、C9环烷基、C10环烷基。
本发明中术语“C3-8环烷基”，包括C3环烷基、C4环烷基、C5环烷基、C6环烷基、C7环烷基、C8环烷基。
本发明中术语“C4-8环烷基”，包括C4环烷基、C5环烷基、C6环烷基、C7环烷基、C8环烷基。
本发明中术语“C4-6环烷基”，包括C4环烷基、C5环烷基、C6环烷基。
本发明术语“卤素”，包括氟、氯、溴、碘。
本发明术语“杂环烷基”是指含3-10个环原子，优选5-10个环原子的非芳香的饱和单环或多环环系，其中的一个或多个环原子不是碳原子，而是例如氮、氧或硫原子。优选的杂环烷基含有5-6个环原子。杂环烷基前的前缀氮杂、氧杂或硫杂分别是指至少有一个氮、氧或硫原子作为环原子。
本发明术语“杂环芳香基”是指含5-14个环原子，优选5-10个环原子的芳香单环或多环环系，其中的一个或多个环原子不是碳原子，而是例如氮、氧或硫原子。优选的杂环芳香基含有5-6个环原子。代表性的杂环芳香基包括吡嗪基、呋喃基、噻吩基、吡啶基、嘧啶基、异噁唑基、异噻唑基、噁唑基、噻唑基、吡唑基、呋咱基、吡咯基、吡唑基、三唑基、1,2,4-硫杂二唑基、吡嗪基、哒嗪基、喹喔啉基、2,3-二氮杂萘基、咪唑并[1,2-a]吡啶、咪唑并[2,1-b]噻唑基、苯并呋咱基、吲哚基、氮杂吲哚基、苯并咪唑基、苯并噻吩基、喹啉基、咪唑基、噻吩并吡啶基、喹唑啉基、噻吩并嘧啶基、吡咯并吡啶基、咪唑并吡啶基、异喹啉基、苯并氮杂吲哚基、1,2,4-三嗪基、苯并噻唑基等。
在本发明中，“D”指氘；“被氘取代”指的是用相应数量的氘原子取代一个或多个氢原子。
应认识到，取决于合成中使用的化学材料的来源，在合成的化合物中存在天然同位素丰度的一些变化。因此，本发明的化合物将固有地包含少量的氘化的同位素体。尽管存在这种变异，但与本发明化合物的稳定同位素取代的程度相比较，这种天然丰度的稳定氢和碳同位素的浓度还是很低的和无关紧要的。参见例如Wada,E等,Seikagaku,1994,66:15；Gannes,LZ等.,Comp Biochem Physiol Mol Integr Physiol,1998,119:725。
在本发明的化合物中，其中任何不被指明为氘的原子以其天然同位素丰度存在。除非另有说明，当一个位置被特别地指明为“H”或“氢”时，该位置应理解为具有按照其天然丰度同位素组成的氢。同样，除非另有说明，当一个位置被特别地指明为“D”或“氘”时，该位置应理解为具有大于氘的天然丰度(其为0.015％)至少3000倍的丰度的氘(即，至少45％的氘掺入)。
本文使用的术语“同位素富集系数”是指特定同位素的同位素丰度与天然丰度之间的比率。
在其它实施方式中，本发明的化合物对于各个指定的氘原子的同位素富集系数为至少3500(在各个指定的氘原子处52.5％的氘掺入)、至少4000(60％的氘掺入)、至少4500(67.5％的氘掺入)、至少5000(75％的氘掺入)、至少5500(82.5％的氘掺入)、至少6000(90％的氘掺入)、至少6333.3(95％的氘掺入)、至少6466.7(97％的氘掺入)、至少6600(99％的氘掺入)或至少6633.3(99.5％的氘掺入)。
术语“同位素体”是指其中化学结构与本发明的特定化合物只在其同位素组成方面不同的物质。
术语“化合物”，当涉及本发明的化合物时，是指除了在分子的组成原子当中可能有同位素变化之外具有相同化学结构的分子的集合。因此本技术领域的技术人员很清楚，由含有指明的氘原子的具体化学结构所表示的化合物也包含较少量的在该结构的一个或多个指定氘位置处具有氢原子的同位素体。在本发明化合物中这样的同位素体的相对量将取决于多种因素，包括用来制造所述化合物的氘化试剂的同位素纯度和在用来制备所述化合物的各个合成步骤中氘的掺入效率。然而，如上文所述，这样的同位素体的总体相对量将低于化合物的49.9％。在其他实施方式中，这样的同位素体的总体相对量将低于化合物的47.5％，低于40％，低于32.5％，低于25％，低于17.5％，低于10％，低于5％，低于3％，低于1％或低于0.5％。
本发明所用到的部分缩写的解释如下：
XRPD：X射线粉末衍射
DSC：差式扫描量热
TGA：热重分析
1H HMR：液态核磁氢谱
在本发明中，术语“晶型”是通过X射线粉末衍射图表征证实的。本领域技术人员能够理解，这里所讨论的理化性质可以被表征，其中的实验误差取决于仪器的条件、样品的准备和样品的纯度等。特别是，本领域技术人员公知，X射线衍射图通常会随着仪器的条件而有所改变。特别需要指出的是，X射线粉末衍射图的相对强度也可能随着实验条件的变化而变化，所以峰强度的顺序不能作为唯一或决定性因素。事实上，XRPD图谱中衍射峰的相对强度与晶体的择优取向有关，本文所示的峰强度为说明性而非用于绝对比较。另外，峰角度的实验误差通常在5％或更少，这些角度的误差也应该被考虑进去，通常允许有±0.2°的误差。另外，由于样品厚度等实验因素的影响，会造成峰角度的整体偏移，通常允许一定的偏移。因而，本领域技术人员可以理解的是，本发明中一个晶型的X射线粉末衍射图不必和本文所指的实施例中的X射线粉末衍射图完全一致，本文所述“XRPD图相同”并非指绝对相同，相同峰位置可相差±0.2°且峰强度允许一定可变性。任何具有和这些图谱中的特征峰相同或相似的图的晶型均属于本发明的范围之内。本领域技术人员能够将本发明所列的图谱和一个未知晶型的图谱相比较，以证实这两组图谱反映的是相同还是不同的晶型。
在一些实施方案中，本发明的晶型A是纯的、单一的，基本没有混合任何其他晶型。本发明中，“基本没有”当用来指新晶型时指这个晶型含有少于20％(重量)的其他晶型，尤其指少于10％(重量)的其他晶型，更指少于5％(重量)的其他晶型，更指少于1％(重量)的其他晶型。
需要说明的是，本发明中提及的数值及数值范围不应被狭隘地理解为数值或数值范围本身，本领域技术人员应当理解其可以根据具体技术环境的不同，在不背离本发明精神和原则的基础上围绕具体数值有所浮动，本发明中，这种本领域技术人员可预见的浮动范围多以术语“约”来表示。当在本发明数值前使用术语“约”并且指代所述数值时，其意指该值的±10％的范围内，优选±5％的范围内，更优选±2％的范围内，优选±1％的范围内的任意值。例如，“约10”应解释为意指9-11，优选为9.5-10.5，更优选为9.8-10.2，更优选为9.9-10.1。
在本发明中，术语“室温”是指物品的温度与空间(例如所述物品位于其中的通风橱的场所)的温度接近或相同。通常，室温为约20℃至约30℃，或约22℃至27℃，或约25℃。
反溶剂添加(也称为反溶剂结晶、沉淀析晶、盐析或逼晶)法通常是通过在用良溶剂溶解了目标产物的溶液中加入一种或几种反溶剂，产品在所述溶液中处于微溶状态，从而使溶液达到过饱和状态后析出结晶的方法。反-反溶剂添加通常是通过在用良溶剂溶解了目标产物的溶液后，将其加入一种或几种反溶剂，产品在所述溶液中处于微溶状态，从而使溶液达到过饱和状态后析出结晶的方法。
反溶剂溶解目标产物的能力比良溶剂差，比如差超过10％、20％、30％、40％、50％、60％、70％或者80％，所以，体系中的反溶剂是相对而言。良溶剂和反溶剂可以为极性溶剂或非极性溶剂，如可以选自：二甲基甲酰胺(DMF)、二甲基亚砜(DMSO)、水、醇类溶剂、醚溶剂、酮类溶剂、酯类溶剂、烷烃类溶剂、芳烃类溶剂、腈类溶剂的一种或多种。其中，醇类溶剂包括但不限于甲醇、乙醇、丙醇、异丙醇或1,3-丙二醇、1,2-丙二醇或三氯叔丁醇或其组合；醚溶剂包括但不限于诸如四氢呋喃、甲基 叔丁基醚或1,4-二氧六环或其组合；酮类溶剂包括但不限于丙酮、甲乙酮或4-甲基-2-戊酮或其组合；酯类溶剂包括但不限于乙酸乙酯、乙酸异丙酯、乙酸正丁酯或乙酸叔丁酯或其组合；烷烃类溶剂包括但不限于二氯甲烷、氯仿、正己烷、环己烷或戊烷或正庚烷或其组合；芳烃类溶剂包括但不限于苯、甲苯或其组合；腈类溶剂包括但不限于乙腈、丙二腈。
反溶剂添加、反-反溶剂添加可以通过间歇、半间歇或连续结晶操作。反溶剂加入溶液(反溶剂结晶)中或产品溶液加入反溶剂(反-反溶剂结晶)中，可以以恒定速率滴加，也可以是起始时缓慢滴加，然后逐渐递加速率。
在本发明中，术语“hPBMs”(human-Peripheral blood mononuclearcell)是指人外周血中具有单个核的细胞，包括淋巴细胞和单核细胞。
本发明中，术语“iL-2”(Interleukin-2)是指白细胞介素-2，是趋化因子家族的一种细胞因子，它是由多细胞来源(主要由活化T细胞产生)，又具有多向性作用的细胞因子(主要促进淋巴细胞生长、增殖、分化)；对机体的免疫应答和抗病毒感染等有重要作用，能刺激已被特异性抗原或致丝裂因数启动的T细胞增殖；能活化T细胞，促进细胞因子产生；刺激NK细胞增殖，增强NK杀伤活性及产生细胞因子，诱导LAK细胞产生；促进B细胞增殖和分泌抗体；激活巨噬细胞。
本发明中，术语“LPS”(Lipopolysaccharide)是革兰氏阴性细菌细胞壁外壁的组成成分，是由脂质和多糖构成的物质(糖脂质)。LPS的结构为抗原-核心多糖-类脂A组成，LPS是一种内毒素(Endotoxin)，当其作用于人类或动物等其他生物细胞时，就会表现出多种的生物活性。LPS的生理作用是通过存在于宿主细胞的细胞膜表面的Toll样受体(Toll-like Receptor、TLR)4(TLR4)而体现的。
本发明中TNF-α(tumor necrosis factor)是指一种肿瘤坏死因子，由活化的巨噬细胞，NK细胞及T淋巴细胞产生，巨噬细胞产生的TNF命名为TNF-α，TNF-α能够杀伤、抑制肿瘤细胞，对机体免疫功能的调节作用，能促进T细胞及其它杀伤细胞对肿瘤细胞的杀伤，提高中性粒细胞的吞噬能力，增加过氧化物阴离子产生，增强ADCC功能，刺激细胞脱颗粒和分泌髓过氧化物酶。TNF-α还能抑制病毒复制，抑制病毒蛋白合成、病毒颗粒的产生和感染性，并可杀伤病毒感染细胞。以及促进髓样白血病细胞向巨噬细胞分化，如促进髓样白血病细胞ML-1、单核细胞白血病细胞U937、早幼粒白血病细胞HL60的分化。TNF-α能够促进T细胞MHCⅠ类抗原表达，增强IL-2依赖的胸腺细胞、T细胞增殖能力，促进IL-2、CSF和IFN-γ等淋巴因子差生，增强有丝分裂原或外来抗原刺激B细胞的增殖和Ig分泌。
本文所引用的各种出版物、专利和公开的专利说明书，其公开内容通过引用整体并入本文。
下面将结合本发明实施例，对本发明的技术方案进行清楚、完整地描述，显然，所描述的实施例仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1：化合物A2的合成
实验步骤如下：
步骤1：
在500mL的单口瓶中加入1(11.3g,23.7mmol)、联硼酸频哪醇酯(9.06g,35.6mmol)、双(二苯基膦)二茂铁二氯化钯(1.74g,2.37mmol)、醋酸钾(6.99g,71.3mmol)和二甲基亚砜(150mL)，氮气保护，95℃下反应16小时。加水(300mL)淬灭，用乙酸乙酯(150mL×3)萃取，饱和食盐水(150mL×2)洗涤，旋干得黑色固体为目标产物(13.0g,粗品)。LC-MS:463[M+Na]+
步骤2:
在500mL的三口瓶中加入3(15g,74.6mmol)、三乙胺(22.7g,223.8mmol)和二氯甲烷(150mL),冰浴下加入甲基磺酰氯(12.1g,111.9mmol)并在冰浴下反应1个小时。加水(300mL)淬灭，用二氯甲烷萃取(100mL×3),饱和食盐水洗涤，无水硫酸钠干燥，旋干并通过柱层析纯化(石油醚：乙酸乙酯＝40：1)得黄色固体为目标产物(20.7g,粗品)。LC-MS：280[M+H]+
步骤3:
在500mL单口瓶中加入5(7.27g,49.4mmol)和DMF(200mL)，在0℃下分批加入60％含量的NaH(2.96g,74.1mmol),室温下反应1小时，加入4(20.7g,74.1mmol)，氮气保护，70℃下反应16h。加水(500mL)淬灭，用乙酸乙酯(200mL×3)萃取，有机相用饱和食盐水洗涤(200mL×2)，无水硫酸钠干燥，旋干，柱层析分离(石油醚：乙酸乙酯＝30：1)，得到白色固体为目标产物(9.8g,收率：61.2％)。LC-MS：330[M+H]+
步骤4:
在500mL单口瓶中，将6(9.8g,35.8mmol)溶于DCM(80mL)，0℃下滴加入TFA(16mL)，室温反应16h。低温浓缩，加入DCM(200mL)稀释，加入冰水淬灭，0℃下用氨水调节PH＝10，用DCM(200mL×3)萃取，有机相用饱和食盐水洗涤(200mL×2)，无水硫酸钠干燥，旋干得白色固体为目标产物6.59g(收率：96.6％)。LC-MS：230[M+H]+
步骤5:
在250mL三口瓶中，将7(5.53g,24.0mmol)和TEA(10mL,72.1mmol)溶于THF(80mL)，室温反应1小时。冰浴降温至0℃，滴加入CD3I(1.65mL,26.4mmol)，氮气保护，室温反应2小时。加水淬灭，DCM(100mL×3)萃取，有机相用饱和食盐水洗涤(100mL×2)，无水硫酸钠干燥，旋干得黄色油状物为目标产物3.1g(收率：52.2％)。LC-MS：248[M+H]+
步骤6:
在500mL的单口瓶中加入2(7.13g,16.2mmol)、8(2.67g,10.8mmol)、xphosPdGⅡ(850mg,1.08mmol)、xphos(515mg,1.08mmol)、磷酸钾(4.58g,21.6mmol)和DMF/H2O(150mL/30mL)，氮气保护，95℃下反应2.5小时。加水(300mL)淬灭，用乙酸乙酯(150mL×3)萃取，饱和食盐水(150mL×2)洗涤，无水硫酸钠干燥，旋干并通过Flash纯化得黑色油状物为目标产物(3.3g,粗品)。LC-MS:563[M+H]+
步骤7:
在250mL三口瓶中，加入9(3.3g,7.14mmol)、Fe(2.0g,35.7mmol)、氯化铵(1.93g,35.7mmol)、乙醇(40mL)和水(8mL)，85℃下反应2.5小时。抽滤，滤液浓缩得黑色固体粗品为目标产物3.5g。LC-MS：533[M+H]+
步骤8:
在250mL三口瓶中，将10(3.5g,6.55mmol)溶于四氢呋喃(40mL)，0℃下滴加入HCl/Dioxane(8mL)，室温反应1小时。低温浓缩，加入DCM(100mL)稀释，加入冰水淬灭，0℃下用氨水调节pH＝10，用DCM(100mL×3)萃取，有机相用饱和食盐水洗涤(100mL×2)，无水硫酸钠干燥，旋干，通过Prep-HPLC纯化得白色固体为目标产物450mg(收率：15.3％)。LC-MS：449[M+H]+,1H NMR(400MHz,MeOD)δ8.38(s,1H),8.01(s,1H),7.81(s,1H),7.73(d,J＝1.5Hz,1H),7.54(s,1H),7.48(td,J＝4.7,1.7Hz,1H),7.38(s,2H),5.21(s,2H),4.51(t,J＝6.9Hz,1H),3.59(d,J＝12.6Hz,2H),3.19(s,2H),2.37–2.32(m,4H),1.56(s,6H).
实施例2：化合物B2的合成
实验步骤如下：
步骤1：
在2000mL的单口瓶中加入1(25.0g,102mmol)、氯乙醛的水溶液(12.0g,154mmol)和丙酮(500mL)，50℃下反应16小时。旋干，柱层析分离(MeOH in DCM,from 0％to 10％,v/v)得到黄色油状物为目标产物(9.20g,收率：53.4％)。LC-MS：169[M+H]+
步骤2：
在1000mL的单口瓶中加入3(4.00g,23.8mmol)、三乙胺(6.01g,59.5mmol)、四氢呋喃(200mL)。室温下加入氘代碘甲烷(3.62g,25.0mmol)，室温下搅拌2小时。加入水(250mL)淬灭旋干，用乙酸乙酯萃取(150mL x 3)无水硫酸钠干燥，旋干。柱层析分离(MeOH in DCM,from 0％to 10％,v/v)得到黄色固体为目标产物(2.70g,收率：61.3％)。LC-MS：186[M+H]+
步骤3：
在500mL的三口瓶中加入4(2.70g,14.6mmol)和四氢呋喃(100mL)，氮气保护，-78℃滴加正丁基锂(2.4M in tetrahydrofuran,7.30mL,17.5mmol)，保持温度不变继续搅拌1h，然后滴加三正丁基氯化亚锡(7.14g,21.9mmol)，-78℃继续反应1h。反应完成后用饱和氯化铵水溶液(100mL)淬灭反应，乙酸乙酯(120mL x 3)萃取，有机层用饱和食盐水洗涤，无水硫酸钠干燥，抽滤，减压浓缩的黄色油状物无目标产物。可直接用于下一步。(7.05g，粗品)。LC-MS：476[M+H]+
步骤4：
在3L的单口烧瓶中加入6(59.4g,707mmol)，四氢呋喃(1.5L)，分别加入DHP(68.4g,813mmol)以及PPTS(3.55g,14.1mmol)，室温下反应16个小时。饱和碳酸氢钠(1000mL*3)淬洗涤，饱和食盐水洗涤(500mL)，无水硫酸钠干燥，旋干得无色油状物为目标产物(120g,粗品)，可直接用于下步反应。LC-MS：169[M+H]+
步骤5:
在3L的单口瓶中加入7(35.8g,213.15mmol)、8(47.5g,203.00mmol)、双三苯基膦二氯化钯(1.43g,2.03mmol)、碘化亚铜(1.93g，10.16mmol)、三乙胺(40.01g,406.00mmol)和无水二氯甲烷(1L)，在氮气保护下，室温反应16小时。反应液用饱和氯化铵(1000Ml*3)以及饱和食盐水(500mL)洗涤，无水硫酸钠干燥，旋干，得黄色油状物为目标化合物(59.4g，粗品)，可直接用于下一步反应。LC-MS：275[M+H]+
步骤6:
在2000mL三口瓶中加入9(50.3g,184mmol)、四氢呋喃(500mL)、10(40.1g,184mmol)和三苯基膦(72.3g，276mmol)氮气保护，室温搅拌下加入偶氮二甲酸二乙酯(55.8g,276mmol)，室温反应16小时，旋干，通过柱层析(石油醚：乙酸乙酯＝10：1)得到黄色固体为目标产物(70.2g,收率：80.6％)。LC-MS：476[M+H]+
步骤7:
在1000mL单口瓶中加入11(5.05g,10.7mmol)、5(9.15g,19.3mmol)、双(三苯基膦)二氯化钯(II)(376mg,0.535mmol)、碘化亚铜(305mg，1.61mmol)和1,4-二氧六环(200mL)，氮气保护，在90℃条件下搅拌5小时。加入乙酸乙酯(400mL)稀释，用饱和氯化铵水溶液(300mL x 3)洗涤，旋干，柱层析分离(MeOH in DCM,from 0％to 5％,v/v)，得到黄色固体为目标产物(2.50g,收率：40.5％)LC-MS：580[M+H]+
步骤8:
在500ml单口瓶中加入12(2.50g,4.32mmol)、还原铁粉(1.21g，21.6mmol)、氯化铵(1.14g，21.6mmol)、乙醇(100mL)和水(20mL)80℃反应2小时。加入二氯甲烷(100mL)稀释，抽滤，旋干，柱层析分离(MeOH in DCM,from 0％to 12％,v/v),得到黄色固体为目标产物(1.40mg,59.1％)。LC-MS：550[M+H]+
步骤9:
在250ml单口瓶中加入13(1.02g,1.86mmol)、四氢呋喃(25mL)，冰浴下搅拌，然后滴加4M/L的盐酸气的1,4-二氧六环溶液(5mL)，室温反应20分钟，旋干，溶于二氯甲烷，氨水调pH到9，用二氯甲烷(30mL x 3)旋干，通过高压液相制备得淡黄色固体为目标产物(350mg,40.5％)。LC-MS：466[M+H]+1H NMR(400MHz,DMSO)δ7.88(s,1H),7.77(d,J＝1.8Hz,1H),7.54(d,J＝5.4Hz,2H),7.42–7.37(m,1H),7.34(dd,J＝5.4,1.6Hz,2H),6.10(s,2H),5.50(s,1H),5.22(s,2H),2.96–2.78(m,3H),2.02(dd,J＝16.9,6.7Hz,4H),1.72(qd,J＝12.5,3.6Hz,2H),1.47(s,6H).
实施例3：化合物C的合成
实验步骤如下：
步骤1:
在2000mL的单口瓶中加入1(7.86g,30.0mmol)、四氢呋喃(500mL)，0℃下分批加入氘代氢化锂铝(3.15g,75.0mmol)，0℃下反应1小时。加入醋酸(50mL)淬灭，用乙酸乙酯(500mL×3)萃取,饱和食 盐水(500mL×3)洗涤，旋干，得黄色固体为目标产物(3.50g,粗品)。LC-MS:259[M+Na]+
步骤2:
在250mL三颈圆底烧瓶中加入3(3.50g,14.8mmol)、双三苯基膦二氯化钯(519mg,0.740mmol)、碘化亚铜(281mg,1.48mmol)、三乙胺(4.48g,44.4mmol)和二氯甲烷(60mL)，氮气保护室温下加入3(2.50g,14.8mmol)，室温下反应16小时。加入二氯甲烷(400mL)稀释，用饱和氯化铵水溶液洗涤(300mLx 3),旋干，通过柱层析(石油醚：乙酸乙酯＝4：1)，得黄色固体为目标产物(1.03g,收率：25.2％)。LC-MS:299[M+Na]+
步骤3:
在250mL的三口烧瓶中加入4(1.03g,3.73mmol)、四氢呋喃(30mL)、5(854mg,3.92mmol)和三苯基膦(1.47g,5.60mmol)。氮气保护0℃下滴入偶氮二甲酸二乙丙酯(1.13g,5.60mmol)。氮气保护室温下反应16小时。旋干，通过柱层析(石油醚：乙酸乙酯＝10：1)得黄色固体为目标产物(1.30g,收率：73.2％)。LC-MS：499[M+Na]+
步骤4:
在100mL单口瓶中加入6(300mg,0.630mmol)、双联频哪醇硼酸酯(240mg,0.945mmol)、Pd(dppf)Cl2(23.5mg,0.0315mmol)、乙酸钾(154mg,1.58mmol)和二甲基亚砜(10mL)氮气氛围下90℃反应16小时。加水淬灭(80mL)，用乙酸乙酯萃取(50mL x 3)，饱和食盐水洗涤(50mL x 2)，无水硫酸钠干燥，旋干得棕色油状物为目标产物(400mg,粗品)。LC-MS：443[M+H]+
步骤5:
在100mL单口瓶中加入7(400mg,0.905mmol)、8(200mg,0.905mmol)、XPhosPdG2(35.6mg,0.0453mmol)、XPhos(43.2mg,0.0905mmol)、磷酸钾(384mg,1.81mmol)、DMF(10mL)和水(2mL)氮气保护90℃下反应2小时。加水淬灭(80mL)，用乙酸乙酯萃取(50mL x 3)，饱和食盐水洗涤(50mLx 2)，无水硫酸钠干燥，旋干，通过柱层析(二氯甲烷：甲醇＝18：1)得黄色固体为目标产物(250mg，收率：49.2％)。LC-MS：562[M+H]+
步骤6:
在100ml单口瓶中加入9(250mg,0.446mmol)、还原铁粉(125mg，2.23mmol)、氯化铵(118mg，2.23mmol)、乙醇(10mL)和水(2mL)80℃反应2小时。加入二氯甲烷(50mL)稀释，抽滤，旋干，柱层析分离(二氯甲烷：甲醇＝10：1)，得到黄色固体为目标产物(220mg,93.0％)。LC-MS：532[M+H]+
步骤7:
在250ml单口瓶中加入10(220mg,0.414mmol)、四氢呋喃(10mL)，冰浴下搅拌，然后滴加4M/L的盐酸的1,4-二氧六环溶液(3mL)，室温反应20分钟，旋干，溶于二氯甲烷，氨水调pH到9，用二氯甲烷(30mL x 3)旋干，通过高压液相制备得淡黄色固体为目标产物(85.0mg,45.9％)。LC-MS：448[M+H]+1H NMR(400MHz,DMSO)δ8.10(s,1H),7.79(d,J＝1.8Hz,1H),7.75(s,1H),7.58–7.49(m,2H),7.44–7.30(m,3H),5.66(s,2H),5.49(s,1H),4.14–4.00(m,1H),2.86(d,J＝11.5Hz,2H),2.21(s,3H),1.99(d,J＝3.1Hz,6H),1.46(s,6H).
实施例4：化合物D的合成
实验步骤如下：
步骤1：
在100mL的三口瓶中加入1(3.0g,30.61mmol和四氢呋喃(30mL),氮气保护，-78℃滴加正丁基锂(2.4M in tetrahydrofuran,12.75mL 30.61mmol),保持温度不变继续搅拌1h,然后滴加2(2.0g,30.61mmol),-78℃继续反应1h,升温至室温在反应2h。反应完成后用饱和氯化铵水溶液(30mL)淬灭反应，乙酸乙酯(100mLx3)萃取，有机层用饱和食盐水洗涤，无水硫酸钠干燥，抽滤，减压浓缩的黄色油状物无目标产物，可直接用于下一步。(6.1g,粗品)。LC-MS：163[M+H]+
步骤2：
在250mL的单口烧瓶中加入3(6.1g,37.65mmol)，四氢呋喃(50mL)，分别加入DHP(4.76g,56.63mmol)以及PPTS(158.8mg,0.63mmol)，室温下反应16个小时。饱和碳酸氢钠(100mLx 3)淬洗涤，饱和食盐水洗涤(100mL)，无水硫酸钠干燥，旋干得无色油状物为目标产物(8.5g,粗品)，可直接用于下步反应。LC-MS：247[M+H]+
步骤3:
在250mL的单口烧瓶中加入4(8.5g,34.55mmol),甲醇和二氯甲烷(10mL/10mL)和碳酸钾(7.15g,51.83mmol),室温下反应3个小时。抽滤，浓缩滤液，旋干得无色油状物为目标产物(6.3g,粗品)，可直接用于下步反应。LC-MS：175[M+H]+
步骤4:
在250mL的单口瓶中加入5(6.3g,36.21mmol)，6(8.05g,34.40mmol)，双三苯基膦二氯化钯(238mg,0.34mmol)，碘化亚铜(322mg，1.69mmol)，三乙胺(6.95g,68.8mmol)和无水二氯甲烷(80mL)，在氮气保护下，室温反应16小时。反应液用饱和氯化铵(200mL x 3)以及饱和食盐水(100mL)洗涤，无水硫酸钠干燥，旋干，通过柱层析(石油醚：乙酸乙酯＝10：1)得黄色油状物为目标化合物(3.2g)LC-MS：281[M+H]+
步骤5:
在100mL三口瓶中加入7(1.5g,5.36mmol)，8(1.752g，8.04mmol)，三苯基膦(2.106g，8.04mmol)和无水四氢呋喃(20mL)，氮气保护，室温搅拌下加入偶氮二甲酸二异丙酯(1.624g,8.04mmol)，室温反应16小时，旋干，通过柱层析(石油醚：乙酸乙酯＝4：1)得到黄色固体为目标产物(450mg,收率：17.50％)。LC-MS：481[M+H]+
步骤6:
在100mL的单口瓶中加入9(450mg,0.94mmol),连硼酸频哪醇酯(359mg,1.41mmol),双(二苯基膦基)二茂铁二氯化钯(35mg,0.047mmol)，醋酸钾(276mg,2.82mmol)和二甲基亚砜(10mL)，氮气保护，90℃下反应16小时。加水(30mL)淬灭，用乙酸乙酯(50mL×3)萃取,饱和食盐水(100mL×2)洗涤，旋干得黑色固体为目标产物(500mg,粗品)。LC-MS:447[M+H]+
步骤7:
在100mL单口瓶中加入10(363mg,0.81mmol),11(178g,0.73mmol),氯(2-二环己基膦基-2',4',6'-三异丙基-1,1'-联苯基)[2-(2'-氨基-1,1'-联苯)]钯(II)(59mg,0.081mmol)，2-二环己基磷-2,4,6-三异丙基联苯(39mg,0.082mmol)和磷酸钾(343mg,1.62mmol)，DMF(10mL)和H2O(2mL)，氮气保护，在95℃条件下搅拌1.5h)。加水(30mL)淬灭，乙酸乙酯(100mL×3)萃取，合并有机相并用饱和食盐水(100mL×3)洗涤，无水硫酸钠干燥，旋干，通过TCL分离(二氯甲烷：甲醇＝15：1)，得到棕色液体为目标产物(266mg,收率：57.9％)LC-MS：566[M+H]+
步骤8:
在100ml单口瓶中加入12(266mg,0.47mmol)，铁粉(201mg，3.77mmol)，氯化铵(203mg，3.77mmol)，乙醇(10mL)和水(2mL),80℃反应1h。冷却至室温，抽滤，滤液浓缩。通过TLC分离(甲醇:二氯甲烷＝1：10，v/v),得到黄色固体为目标产物(95mg,37.6％)LC-MS：536[M+H]+
步骤9:
在50ml单口瓶中加入13(95mg,0.17mmol)，四氢呋喃(5mL)，冰浴下搅拌，然后滴加4M/L的盐酸1,4-二氧六环溶液(0.01mL，0.34mmol)，室温反应1h，0℃旋干，通过高压液相制备得黑色固体为目标产物(15mg,18.7％)。LC-MS：452[M+H]+1H NMR(400MHz,DMSO)δ8.20(s,1H),8.10(s,1H),7.80(d,J＝1.7Hz,1H),7.76(s,1H),7.52(d,J＝1.4Hz,2H),7.40(s,1H),7.35–7.32(m,2H),5.67(s,2H),5.18(s,2H),4.09(dd,J＝10.4,5.1Hz,1H),2.89(d,J＝11.1Hz,2H),2.24(s,3H),2.10(d,J＝11.3Hz,2H),2.00(d,J＝3.3Hz,4H).
实施例5：化合物A2游离碱晶型A
将实施例1制备的化合物A2过硅胶柱得到1.6克粗品(纯度80％左右)，再由制备高效液相分离，浓缩至100毫升，加碳酸氢钠溶液调至pH＝9,二氯甲烷(100毫升)萃取三次。有机相用饱和氯化钠溶液(80毫升)洗涤一次，硫酸钠干燥，旋干得到淡黄色固体570毫克(纯度97％)。该固体用石油醚/乙酸乙酯(3：1)50毫升打浆，过滤，得到类白色固体460毫克(纯度>99％)。
重复制备：
(1)称取230.9mg上述游离碱样品于20mL玻璃瓶中，加入1mL的IPAc，得到澄清溶液；
(2)在室温下搅拌(1000rpm)约5分钟后，析出大量固体，继续加入1mL的IPAc；
(3)在室温下悬浮搅拌约1天后，离心(10000rpm，2min)得到固体；
(4)在室温下真空干燥3小时，共收集到195.4mg样品(回收率：84.6％)。
上述样品的XRPD图谱如图1所示，其XRPD测试参数和结果数据表分别如表1和2所示，表明其为晶体，命名为游离态晶型A，且可重复制备得到。表2中峰或相应的d值的整个列表或其子集、以及基本上类似于图1的XRPD图谱可足以表征该晶型。
化合物A2游离碱晶型A的TGA/DSC图谱如图2所示，TGA和DSC的测试参数如表3所示。图2中，TGA结果表明，其从室温加热至170℃失重1.3％；DSC结果表明，其在168.8℃(起始温度)处有一个尖锐的吸热峰。
化合物A2游离碱晶型A的1H NMR图谱如图3所示。1H NMR结果表明，残留溶剂IPAc与游离碱晶型A的摩尔比为0.01:1(对应TGA失重为0.2％)。
化合物A2游离碱晶型A的HPLC图谱如图4所示，结果如表4所示。推测化合物A2游离碱晶型A为无水晶型。
表1 XRPD测试参数
表2化合物A2游离碱晶型A的XRPD数据表
表3 TGA和DSC的测试参数
表4化合物A2游离碱晶型A的HPLC结果
游离碱晶型A的固态稳定性
上述游离碱晶型A的起始样品及样品在60℃条件下闭口放置1天后的XRPD图谱如图5所示，HPLC结果如表5所示。
表5化合物A2游离碱晶型A的HPLC结果
实施例6：化合物A2游离碱晶型B
(1)称取9.9游离碱样品(如实施例5中所述)于20mL玻璃瓶中，加入2mL的EtOAc溶解样品，用0.45μm的PTFE滤膜过滤，得到澄清API溶液；
(2)向API溶液中逐滴滴加反溶剂Toluene，边滴加边磁力搅拌(～1000rpm)，共加入10mL的Toluene；
(3)得到澄清溶液在室温下搅拌～2小时候，转移至5℃搅拌约15小时，仍为澄清溶液；
(4)转移至-20℃下搅拌约6小时候，仍为澄清溶液；
(5)将该澄清溶液转移至室温下挥发8天后得到固体。
上述样品的XRPD图谱如图6所示，其XRPD测试参数如上表1所示，结果数据表如表6所示，表明其为晶体，命名为游离态晶型B。表6中峰或相应的d值的整个列表或其子集、以及基本上类似于图5的XRPD图谱可足以表征该晶型。
表6化合物A2游离碱晶型B的XRPD数据表
化合物A2游离碱晶型B的TGA/DSC图谱如图7所示，TGA和DSC的测试参数如上表3所示。TGA结果表明，样品从室温加热至70℃失重13.2％，继续加热至170℃失重8.5％。DSC结果表明，样品在59.5℃、95.6℃、150.8℃及160.9℃(峰值温度)处有四个吸热峰。
化合物A2游离碱晶型B的1H NMR图谱如图8所示。1H NMR结果表明，残留溶剂EtOAc与游离碱的摩尔比为0.8:1(对应TGA失重为18.1％)。
化合物A2游离碱晶型B在室温下放置前后的XRPD图谱如图9所示。结果表明，游离碱晶型B在室温下闭口放置5天后，出现游离碱晶型A的衍射峰，表明游离碱晶型B在室温下放置后，有向游离碱晶型A转变的趋势。
化合物A2游离碱晶型B经氮气吹扫前后的XRPD图谱如图10所示。结果表明，游离碱晶型B(含晶型A的一个衍射峰)在30℃下氮气吹扫20分钟后，晶型发生转变。推测游离碱晶型B在氮气吹扫后，脱去EtOAc导致晶型转变。
综合表征结果，推测游离碱晶型B为EtOAc溶剂合物。
实施例7：化合物A2盐酸盐制备：
称取100毫克(0.223mmol)化合物A2溶于5毫升无水二氯甲烷，室温搅拌5分钟，逐滴加入1.11毫升盐酸乙醚溶液(1N),滴加完毕继续在室温搅拌30分钟。TLC显示原料全部消失。通氮气10分钟，10℃减压浓缩得类白色固体(纯度>99％)。该固体置于冻干机12小时，得到108毫克的A2的盐酸盐(类白色，纯度>99％)。
化合物A2盐酸盐的XRPD图谱如图11所示，XRPD测试参数如实施例5表1所示，其XRPD结果数据表如表7所示，表明其为晶体，命名为盐酸盐晶型A。表7中峰或相应的d值的整个列表或其子集、以及基本上类似于图11的XRPD图谱可足以表征该晶型。
化合物A2盐酸盐的TGA/DSC图谱如图12所示，TGA和DSC的测试参数如实施例5表3所示。TGA结果表明，其从室温加热至150℃失重8.6％；DSC结果表明，其在81.9℃及156.0℃(峰值温度)处有两个吸热峰。
表7化合物A2盐酸盐的XRPD数据表
化合物A2游离碱晶型A和化合物A2盐酸盐的XRPD图谱叠图如图13所示，二者的XRPD结果基本一致，但盐酸盐的结晶度较低。
实施例8
微粒体代谢产物鉴定操作步骤：
称取化合物(测试化合物及编号如下所示，其中化合物A1和B1参考CN110396087A中公开的相关方法制备)溶于二甲基亚砜中配成20mM溶解，用50％乙腈水溶液(v/v)稀释化合物储备液至1.0mM浓度作为工作液；用50mM磷酸氢二钾缓冲液稀释肝微粒体(20mg/mL)至1.27mg/mL作为肝微粒体工作液；称取还原型辅酶，加入3307μL磷酸缓冲液(50mM)溶解至5.0mM作为还原型辅酶工作液。对于T＝60分钟样本，加入4μL供试品工作液(1.0mM)，再加入316μL肝微粒体工作液(1.27mg/mL)，最后加入80μL还原型辅酶工作液启动反应；对于T＝0分钟样本，加入316μL肝微粒体工作液(1.27mg/mL)，再加入80μL还原型辅酶工作液启动反应，无供试品孵育；在37℃孵育60分钟后，加入1200μL终止液终止酶反应；对于T＝0分钟样本，加入4μL供试品工作液(1.0mM)。将样本板放在涡旋仪上，600转/分钟涡旋5分钟。然后在4000转/分钟条件下离心10分钟，移取上清，混合后室温用氮气吹干。用300μL of10％乙腈(0.1％FA)溶剂复溶氮气吹干剩余物，然后在4000转/分钟转速下离心15分钟，移取上清液至检测板待质谱分析。质谱分析采用LC/Q-Exactive plus来检测。
选择7-乙氧基香豆素(10μM)作为阳性对照化合物，采用与化合物平行的操作步骤。
60min后母核剩余百分比如下表所示：
表8化合物稳定性考察结果
实施例9药代动力学实验
大鼠药代动力学实验
6只雄性Sprague Dawley大鼠上先后单次单剂量分别通过静脉注射(iv,n＝3)和口服(po,n＝3)给药。静脉注射用10％DMSO/30％PEG400/60％Water将化合物配制成0.25mg/ml或0.5mg/mL的溶液，以2mL/kg的体积给药。口服给药用0.5％Methylcellulose将化合物配制成0.6mg/ml或2.0mg/mL的均匀混悬液，以5mL/kg的体积给药。具体给药剂量如下表所示。
表9给药剂量
给药后，于0.0833、0.25、0.5、1、2、4、8和24小时收集静脉注射和口服的血液样本，并运用LC-MS/MS检测样本中化合物的浓度，定量下限为1ng/mL，采用WinNolin非房室模型计算化合物的药代参数。
本实施例中所涉及的化合物结构及编号如下所示：
结果
静脉注射给药后，A1、A2、C和D的体内暴露量(AUClast)分别为116hr*ng/mL、247hr*ng/mL、74.8hr*ng/mL、28.7hr*ng/mL，平均总清除率(CL)分别为129mL/min/kg、71mL/min/kg、101mL/min/kg、151mL/min/kg。
口服给药后，A1、A2、C和D的体内暴露量(AUClast)分别为79.8ng*hr/mL、572ng*hr/mL、42.8ng*hr/mL、9.94ng*hr/mL。与静脉注射的数据对比，大鼠的口服生物利用度分别为6.84％、23.2％、9.52％、5.2％。
食蟹猴药代动力学实验
3只雄性食蟹猴单次单剂量通过静脉注射(iv,1mg/kg,n＝3)，洗脱一周后，口服(po,10mg/kg,n＝3)给药。静脉注射用水或者10％DMSO/30％PEG400/Water将化合物配制成1.0mg/mL的溶液，以1mL/kg的体积给药。口服给药用水或者0.5％甲基纤维素/1.7meq 1N盐酸将化合物配制成2.0mg/mL的溶液或者悬浮溶液，以5mL/kg的体积给药。
给药后，于0.0833、0.25、0.5、1、2、4、8、12和24小时收集静脉注射和口服的血液样本，并运用LC-MS/MS检测样本中化合物的浓度，定量下限为1ng/mL，采用WinNolin非房室模型计算化合物的药代参数。
结果
静脉注射给药后，A1和A2体内暴露量(AUClast)分别为881hr*ng/mL和949hr*ng/mL，平均总清除率(CL)分别为18.9mL/min/kg和16.7mL/min/kg。
口服给药后，A1和A2体内暴露量(AUClast)分别为388ng*hr/mL和1758ng*hr/mL。与静脉注射的数据对比，食蟹猴的口服生物利用度分别为4.61％和18.8％。
实施例10药效学评价
本实施例中所涉及的化合物结构及编号如下所示：
用于hPBMCs的药效学评价
本实验用hPBMC购自上海澳能生物科技有限公司。预先将CD3抗体coating到96孔板，备用。细胞接种于96孔板，每孔200μL，2×106个/ml。加入CD28抗体活化，并加入终浓度为1μM的化合物A2，处理24小时后收集上清液。通过ELISA方法检测IL-2水平，比较A2与CD3/CD28激活hPBMCs的作用。
结果
结果如图14所示，当使用1uM的A2激活PBMCs细胞后，浓度比(A2刺激-未刺激的)/(受刺激的-未受刺激的)＝5.89，说明A2刺激hPBMS表达的IL-2为CD3/CD28(ELISA)刺激hPBMCs表达的iL-2的5.89倍。
结论
A2能够有效增强人体免疫能力。
病毒性角膜炎(HSV)的药效学评价
将15只8周龄C57小鼠(体重22g～25g)的右眼进行病毒性角膜炎造模，造模过程中使用无菌25G针头在所有动物右眼的角膜上划#字/米字/平行划痕，划痕深度须在浅层不可穿透基质层，各眼球之间划痕深浅适度均一且密度均匀分布，再用纤维注射器滴入混匀好的4μl HSV病毒液(滴度为:2.0*106PFU/ml)完成造模。造模当天定为D0，于造模1天后，对动物右眼进行病毒性角膜炎评分并挑选出造模均一的9只小鼠纳入试验，将9只小鼠随机分为对照组、1％A2组和0.2％A2组共3组，每组3只。根据分组在各动物右眼眼表给予相应药液溶媒或不同浓度3024滴眼进行治疗，隔天给药，给药当天每只眼睛滴药3次，每次给药10μl，连续用药2周。将造模后1天、3天、5天、7天、9天、11天和14天的角膜炎病变程度做评分并进行分析比较。
(本发明的HSV病毒在非洲绿猴肾细胞系中的培养和增殖，具有活性，且HSV病毒悬液滴度良好)
结果
造模1天后各小鼠右眼出现病毒性角膜炎典型的角膜溃疡体征，选择其中9只造模均一的小鼠纳入试验并随机分组。结果发现，实验对照组的小鼠发生严重角膜溃疡病变，并在实验第五天有2眼出现角膜穿孔并持续到实验结束，而低浓度组(0.2％A2)和高浓度(1％A2)组的小鼠均未出现角膜穿孔现象。具体情况见图15。
图16显示了小鼠HSV病毒接种后几天内的临床评分情况，从图16的数据可以看出，给予浓度为0.2％和1.0％A2滴眼2周的角膜炎病变评分与模型对照组相比均有统计学差异(P<0.01和P<0.05)，证明本发明的药物病毒角膜炎均有较好的治疗作用，在疾病最高峰(第5天)，对照组临床得分6分以上，而低浓度组(0.2％A2)和高浓度(1％A2)组的临床得分均在6分以下，进一步说明了两个浓度的A2对于病毒角膜炎均有较好的治疗作用，表现为在疾病进展期能够有效降低角膜水肿、角膜溃疡深度和浑浊度，并改善角膜炎症浸润深度，在疾病高峰期和整个用药期间未出现角膜穿孔病变。图14和15说明了0.2％和1.0％A2眼表滴眼能有效抑制小鼠的病毒性角膜炎病变。
图17显示了对照组、低浓度组(0.2％A2)和高浓度组(1％A2)的心血管形成得分情况，可以看出，对照组的得分情况在1分以上，低浓度组(0.2％A2)的得分情况高于0.5，小于1，高浓度组(1％A2)的得分情况低于0.5，说明高浓度A2悬浊液相对于低浓度A2澄清液，效果有些微优势。
根据图15～17的结果显示，本申请的药物介导有效，在疾病最高峰，生理盐水点眼的模型对照组出现穿孔，治疗组尚未出现，本申请的药物能够有效阻止角膜穿孔，本申请药物介导在疾病进展期有效降低角膜水肿，角膜溃疡深度，浑浊度，改善炎症浸润深浅，另外，本申请的A2的高浓度悬浊液相对于低浓度A2澄清液，效果有优势。
结论
0.2％和1.0％A2眼表滴眼能有效抑制小鼠的病毒性角膜炎病变。
疾病模型评分标准：
根据角膜溃疡面积、深度及溃疡形态三个标准进行分级(即为分值)，相加记录总分。
(1)角膜溃疡面积:1分＝1～25％，2分＝26～50％，3分＝51～75％，4分＝76％～100％。
(2)病变深度:1分＝角膜轻度混浊，虹膜纹理尚清，2分＝角膜浅层灰白色混浊，窥见虹膜纹理，3分＝角膜深层灰白色混浊，窥不清虹膜纹理，4分＝角膜全层致密灰白色混浊，窥不清前房。
(3)角膜溃疡形态:1分＝表面轻度不规则，2分＝角膜表面粗糙，轻度肿胀，3分＝重度水肿，后弹力层膨出，4分＝角膜穿孔或重度后弹力层膨出。
(4)如有前房积脓和/或前房出血时，加1分。
角膜新生血管分化评估标准:
4个角膜象限的新生血管评分为0～2分，最大评分为8分。角膜新生血管化的标准(0～2):0分，无新生血管长入角膜，1分，新生血管向心性生长，但未达到瞳孔中央或未超过角膜半径；2分，新生血管达到瞳孔中央或超过角膜半径。
临床评分＝病变面积评分+病变深度评分
Covid 19的药效学评价
使用野生型的叙利亚金仓鼠共一共20只，6-8周龄，雌雄各半，将小鼠随机分配成两组，对照组4只，给药组分为5mg/kg组和20mg/kg组共两组，每组8只。在第0天(D0)开始，给药组仓鼠每日口服总量为5mg/kg或20mg/kg的A2，对照组每天口服等量的空白药物，同时，在第0天，将所有仓鼠麻醉，麻醉后用100uL，1.375x106PFU/mL的SARS-Cov-2病毒鼻内接种小鼠。每日监测其临床表现和体重。在研究结束时，收集称重组织(如肺部)，然后将病毒RNA和感染性病毒定量。
结果
图18表明了A2对SARS-Cov-2感染的仓鼠体重的影响。从图中可以看出，三组仓鼠的体重均有减少，但是，20mg/kg组的动物体重增加的速度要快得多。为了量化肺部炎症，本发明选择测量肺重量，并归一化为总体重。本实施例以肺重量增加作为肺重量的增加为一个可靠的、间接的衡量指标表征肺部炎症进展情况，也就是说，肺重量变化，可以说明肺部炎症的发展。如图19所示，所有A2剂量组的肺重量均下降。这些结果表明，A2加速了仓鼠体内SARS-CoV-2病毒的清除，说明了A2能够预防疾病。
结论
本发明的药物可以预防Covid 19。
H1N1的药效学评价
使用6-8周龄雌性SPFC57BL/6mice小鼠(VitalRiver实验动物技术有限公司，SCXK(京)2016-0006，北京版)，感染H1N1(PR8毒株)，用1×104TCID50病毒滴鼻液滴鼻刺激小鼠。将小鼠随机分配为给药组和对照物，给药组口服3mg/kgA2，对照组口服等量的空白药物，每日监测所有组的小鼠的体重减轻情况。
结果
结果如图20所示，A2(3mg/kg)组的生存率明显高于空白对照组。
结论
本发明的药物可以治疗H1N1所引起的感染。
MHV的药效学评价
考察A2对MHV的预防和治疗作用：
低剂量病毒组：将21只4周龄雄性SPF级C57BL/6小鼠分为三组，分别为：对照组、预防组和治疗组，将小鼠麻醉后，用100μL含4×105PFU MHV-A59病毒的DMEM肝内注射(i.h.)。预防组攻毒前24小时第一次给药A2，剂量为10mg/kg，治疗组攻毒后24小时第一次给药A2，剂量为10mg/kg，之后每日连续给药(预防组和治疗组的给药剂量均为每日10mg/kg)，每天监测感染小鼠。感染后第0天、第4天眼眶后静脉丛采血，取血清检测丙氨酸转氨酶(ALT)/谷丙转氨酶(GPT)水平。感染后第8天，处死所有组小鼠，取肝脏组织进行病毒滴度测定和组织学分析，并取脾脏组织进行病毒特异性T增殖检测，结果如图21所示。
高剂量病毒组：将21只4周龄雄性SPF级C57BL/6小鼠分为三组，分别为：对照组、预防组和治疗组，将小鼠麻醉后，用100μL含2×106PFU MHV-A59病毒的DMEM肝内注射(i.h.)。预防组攻毒前24小时第一次给药A2，剂量为10mg/kg，治疗组攻毒后24小时第一次给药A2，剂量为10mg/kg，之后每日连续给药，预防组和治疗组的给药剂量均为每日10mg/kg，每天监测感染小鼠的体重变化、疾病的临床症状和死亡率。
结果
低剂量病毒组的小鼠的特异性T细胞增殖率如图21所示，对照组、预防组和治疗组脾细胞平均增殖率分别为19.19％、28.18％和52.95％，从上述数据可以看出，低剂量病毒组中的治疗组病毒特异性T细胞明显增殖更快。
观察高剂量病毒组的小鼠，观察过程中可发现，预防组的体重有所增加，具体的，从实验第1到第8天，对照组小鼠平均体重从12.39g降低到10.91g，预防组小鼠平均体重从13.39g增加到16.34g，治疗组小鼠平均体重从12.48g降低到11.39g。高剂量病毒组中的预防组的小鼠的存活率为42.86％，高剂量治疗组的小鼠存活率为28.57％，高剂量对照组小鼠全部死亡。从上述高剂量病毒组的实验结果来看，治疗组和对照组的体重略微降低，预防组存活率更高，当延长观察期发现，预防组的生存曲线存在优于治疗组的趋势。
结论
本发明的药物能够更好的预防和治疗MHV引起的疾病。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换等，均应包含在本发明的保护范围之内。
本发明中描述的前述实施例和方法可以基于本领域技术人员的能力、经验和偏好而有所不同。
本发明中仅按一定顺序列出方法的步骤并不构成对方法步骤顺序的任何限制。

Claims (29)

  1. 一种HPK1激酶抑制剂在制备预防和/或治疗人由病原体感染引起或病原体感染相关的疾病或病症的药物中的应用。
  2. 根据权利要求1所述的应用，其特征在于，所述病原体可以为微生物、寄生虫或其他媒介；
    优选的，所述微生物选自：病毒、衣原体、立克次体、支原体、细菌、螺旋体、真菌等中的一种或多种；
    更优选的，所述病原体为病毒，包括但不限于，腺病毒科、疱疹病毒科、HSV2、VZV、EBV、CMV、痘病毒科、乳多泡病毒科、细小病毒科、嗜肝DNA病毒科、多瘤病毒科、呼肠孤病毒科、小核糖核酸病毒科、嵌杯样病毒科、披膜病毒科、沙粒病毒科、逆转录病毒科、黄病毒科、正粘病毒科、副粘病毒科、布尼亚病毒科、冠状病毒科、星状病毒科、博尔纳病毒科。
  3. 根据权利要求1所述的应用，其特征在于，所述抑制剂为如下通式Ⅰ所示的化合物，或其药学上可接受的盐、立体异构体、酯、前药、溶剂化物和氘代化合物：
    其中：
    A选自CR10或N；
    Q选自O或S；
    x和z独立地选自0-6间的整数；
    y为0或1；
    Ar选自芳香性五元杂环基团、芳香性六元杂环基团或苯基，其中，芳香性五元杂环基团选自：呋喃基、噻吩基、吡咯基、吡唑基、咪唑基、噁唑基、噻唑基或硒代噻唑基，芳香性六元杂环基团选自：吡啶基、哒嗪基、嘧啶基或吡嗪基；任选地，芳香性五元杂环基团、芳香性六元杂环基团或苯基上的H可被以下基团取代：-D、-SO2、-SO2N(C0-10烷基)(C0-10烷基)、-N(C0-10烷基)SO2(C0-10烷基)、-CON(C0-10烷基)(C0-10烷基)、-N(C0-10烷基)CO(C0-10烷基)、-N(C0-10烷基)COO(C0-10烷基)、-OCON(C0-10烷基)(C0-10烷基)、卤素、-CN、-OCH2F、-OCHF2、-OCF3、C1-10直链/支链烷基、-N(C0-10烷基)(C0-10烷基)、-OC0-10烷基、C3-10环烷基、-O杂环烷基、-N杂环烷基、-N杂环芳香基、-O杂环芳香基或-S杂环芳香基，其中烷基部分可被一个或多个以下基团任意取代：-SO2、-SO2N(C0-10烷基)(C0-10烷基)、-N(C0-10烷基)SO2(C0-10烷基)、-CON(C0-10烷基)(C0-10烷基)、-N(C0-10烷基)CO(C0-10烷基)、-N(C0-10烷基)COO(C0-10烷基)、-OCON(C0-10烷基)(C0-10烷基)、卤素、-CN、-OCH2F、-OCHF2、-OCF3、-N(C0-10烷基)(C0-10烷基)、-OC0-10烷基、-CO(C0-10烷基)、-COO(C0-10烷基)、-N杂环芳香基、-O杂环芳香基或-S杂环芳香基；
    R2选自：-H、-D、卤素、-NO2、-CN、C1-10直链/支链烷基、C3-10环烷基、-N(C0-10烷基)(C0-10烷基)、-CF3、-OCF3、-OCHF2、-OCH2F或-OC0-10烷基；
    B1、B2、B3、B4和B5独立地选自C或N；
    R3、R4、R5、R6和R7独立地选自：-H、-D、卤素、-CN、-OC0-10烷基、-CO(C0-10烷基)、-CON(C0-10烷基)(C0-10烷基)、C1-10直链/支链烷基、含O或N的杂烷基、-N(C0-10烷基)(C0-10烷基)、C3-10环烷基、-C≡C-R10、-O杂环烷基、-N杂环烷基，或R5和R4、R4和R3、R3和R7、R7和R6与其之间的碳原子形成C3-8环烷基或含-O-、-S-的C3-8杂环烷基、-N杂环芳香基、-O杂环芳香基或-S杂环芳香基、苯基；
    R8和R9独立地选自：-H、-D、卤素、C1-10直链/支链烷基；
    R10选自：H、-D、C1-5直链/支链烷基、C3-10环烷基、
    R11、R12独立地选自：-H、-D、-CF3、-CHF2H、-CH2F、C1-10直链/支链烷基、-CH＝C(C0-10烷 基)(C0-10烷基)、-C≡C(C0-10烷基)、C3-10环烷基、芳香性五元环基团或芳香性六元环基团，或R11、R12与R11和R12之间的碳原子形成C3-8环烷基或含-O-、-S-的C3-8杂环烷基、C4-9稠环烷基、C5-10螺环烷基、C4-9桥环烷基、C3-7环内酰胺、C3-7环内酯、C3-7环酮；
  4. 根据权利要求3所述的应用，其特征在于，所述Ar、R2、R3、R4、R5、R6、R7、R8、R9、R10、R11和/或R12中的C原子所连接的一个或多个H原子可以被氘取代；
    和/或,所述Ar、R2、R3、R4、R5、R6、R7、R8、R9、R10、R11和/或R12中的杂原子所连接的一个或多个H原子可以被氘取代；
    和/或，所述通式Ⅰ的化合物含有至少一个氘原子。
  5. 根据权利要求3所述的应用，其特征在于，所述化合物具有如下结构：
    其中，E环选自：
    E环中，各个R0独立地选自：-H、-D、C1-10直链/支链烷基、-N(C0-10烷基)(C0-10烷基)、-OC0-10烷基、-CO(C0-10烷基)或C3-10环烷基；其中上述C原子或杂原子上所连接的H可以被氘取代；
    R1选自：-H、-D、-O杂环烷基、-N杂环烷基、C1-10直链/支链烷基、C3-10环烷基、-OC0-10烷基、-N(C0-10烷基)(C0-10烷基)、-SO2(C0-10烷基)、-CO(C0-10烷基)、-O-苯基、-S(C0-10烷基)、-N杂环芳香基、-O杂环芳香基或-S杂环芳香基。
  6. 根据权利要求5所述的应用，其特征在于，所述R0、R1中的C原子上所连接的H可以被氘取代；
    和/或，R0、R1中的杂原子上所连接的H可以被氘取代；
    和/或，所述R0-9中至少一个含有氘原子；
    和/或，所述通式Ⅱ的化合物含有至少一个氘原子。
  7. 根据权利要求5所述的应用，其特征在于，所述R1、R4、R8、R9中至少一个含有氘原子。
  8. 根据权利要求5所述的应用，其特征在于，R1选自： -CH3、 -CH2D、-CHD2、-CD3、
  9. 根据权利要求5所述的应用，其特征在于，R4选自：-H、-D、-F、-Cl、-OCH3、-OCH2D、-OCHD2、-OCD3、-CN、或-C≡C-R10。
  10. 根据权利要求9所述的应用，其特征在于，R11和R12独立地选自：-H、-D、-CF3、-CHF2、-CH2F、-CH3、-CH2CH3、-CH＝CH2、 或R11、R12与R11和R12之间的碳原子形成 其中，C原子或N原子上所连接的H可以被氘取代；
    优选地，R11和R12独立地选自：-H、-D、-CF3、-CHF2、-CDF2、-CH2F、-CD2F、-CH3、-CH2D、-CHD2、-CD3、-CH2CH3、-CH2CD3、
  11. 根据权利要求5所述的应用，其特征在于，各个R0独立地选自：-H、-D、-CH3、-CH2CH3或-NH2。
  12. 根据权利要求5所述的应用，其特征在于，R2选自：-NO2、-N(C0-10烷基)(C0-10烷基)、-OC0-10烷基和-OCF3；其中，C原子或N原子上所连接的H可以被氘取代；
    优选地，R2选自：-NH2、-NHD、-ND2或-NO2。
  13. 根据权利要求5所述的应用，其特征在于，R3选自：-H、-D、卤素、-OC0-10烷基、-CO(C0-10烷基)、C1-10直链/支链烷基，其中，C原子上所连接的H原子可以被氘取代；
    优选地，R3选自：-H、-D、-F、-OCH3、-OCH2D、-OCHD2、-OCD3。
  14. 根据权利要求5所述的应用，其特征在于，R5、R6、R7独立地选自：-H、-D、卤素、-CN、C1-3直链/支链烷基、-OC0-3烷基、-CO(C0-3烷基)、含N的C1-3直链/支链烷基，或R6、R7与R6和R7之间的碳原子形成C3-8环烷基或含-O-的C3-8杂环烷基，其中，C原子或N原子上所连接的H原子可以被氘取代；
    优选地，R5、R6、R7独立地选自：-H、-D、-F、-Cl、-CH3、-CH2D、-CHD2、-CD3、-OCH3、-COCH3、-CH2NH2、-CH2N(CH3)2、-CN、-OCH2D、-OCHD2、-OCD3、-COCD3、-CH2N(CD3)2、-CH2N(CH3)(CD3)。
  15. 根据权利要求5所述的应用，其特征在于，R8和R9独立地选自：-H、-D、C1-3直链/支链烷基，其中，C原子上所连接的H原子可以被氘取代；
    优选地，R8和R9独立地选自：-H、-D、-CH3、-CH2D、-CHD2、-CD3。
  16. 根据权利要求1所述的应用，其特征在于，所述化合物选自如下结构：




















  17. 一种权利要求1-16任一项所述的化合物或其药学上可接受的盐、立体异构体、酯、前药、溶剂化物的晶型。
  18. 如权利要求17所述的晶型，其特征在于，所述晶型为4-(3-(((2-氨基-5-(1-(1-三氘代甲基哌啶-4-基)-1H-吡唑-4-基)吡啶-3-基)氧基)甲基)苯基)-2-甲基丁-3-炔-2-醇的晶型，其XRPD图谱在2θ值为13.1°±0.2°、16.3°±0.2°、17.5°±0.2°、23.8°±0.2°的位置中至少三个位置处具有特征峰。
  19. 如权利要求18所述的晶型，其特征在于，所述晶型的XRPD图谱还在2θ值为8.1°±0.2°、12.2°±0.2°、15.3°±0.2°、18.0°±0.2°、19.3°±0.2°、19.5°±0.2°、21.3°±0.2°、21.6°±0.2°的位置中的至少三个位置处具有特征峰。
  20. 如权利要求18所述的晶型，其特征在于，所述晶型具有基本上如图1所示的XRPD图谱。
  21. 如权利要求17所述的晶型，其特征在于，所述晶型为4-(3-(((2-氨基-5-(1-(1-三氘代甲基 哌啶-4-基)-1H-吡唑-4-基)吡啶-3-基)氧基)甲基)苯基)-2-甲基丁-3-炔-2-醇的晶型，其XRPD图谱在2θ值为5.7°±0.2°、11.3°±0.2°、22.7°±0.2°、23.5°±0.2°的位置中的至少三个位置处具有特征峰。
  22. 如权利要求21所述的晶型，其特征在于，所述晶型的XRPD图谱还在2θ值为7.1°±0.2°、8.8°±0.2°、14.1°±0.2°、17.0°±0.2°、18.0°±0.2°、18.8°±0.2°的位置中的至少三个位置处具有特征峰。
  23. 如权利要求21所述的晶型，其特征在于，所述晶型具有基本上如图6所示的XRPD图谱。
  24. 如权利要求17所述的晶型，其特征在于，所述晶型为4-(3-(((2-氨基-5-(1-(1-三氘代甲基哌啶-4-基)-1H-吡唑-4-基)吡啶-3-基)氧基)甲基)苯基)-2-甲基丁-3-炔-2-醇盐酸盐的晶型，其XRPD图谱在2θ值为13.0°±0.2°、16.3°±0.2°、17.5°±0.2°、19.4°±0.2°、23.8°±0.2°的位置中的至少三个位置处具有特征峰。
  25. 如权利要求24所述的晶型，其特征在于，所述晶型的XRPD图谱还在2θ值为8.1°±0.2°、12.1°±0.2°、15.3°±0.2°、18.0°±0.2°、21.4°±0.2°的位置中的至少三个位置处具有特征峰。
  26. 如权利要求24所述的晶型，其特征在于，所述晶型具有基本上如图11所示的XRPD图谱。
  27. 一种药物组合物，其包含权利要求1-16任一项所述的化合物或其药学上可接受的盐、立体异构体、酯、前药、溶剂化物，或权利要求17-26任一项所述的晶型，以及药学上可接受的辅料。
  28. 如权利要求1-16任一项所述的化合物或其药学上可接受的盐、立体异构体、酯、前药、溶剂化物，或权利要求17-26任一项所述的晶型，或权利要求23所述的药物组合物在制备预防和/或治疗肿瘤的药物中的应用。
  29. 如权利要求28所述的应用，其特征在于，所述肿瘤选自：淋巴瘤、母细胞瘤、髓母细胞瘤、视网膜母细胞瘤、肉瘤、脂肪肉瘤、滑膜细胞肉瘤、神经内分泌肿瘤、类癌肿瘤、胃泌素瘤、胰岛细胞癌、间皮瘤、神经鞘瘤、听神经瘤、脑膜瘤、腺癌、黑素瘤、白血病或淋巴样恶性肿瘤、鳞状细胞癌、上皮鳞状细胞癌、肺癌、小细胞肺癌、非小细胞肺癌、腺癌肺癌、肺鳞癌、腹膜癌、肝细胞癌、胃癌、肠癌、胰腺癌、成胶质细胞瘤、子宫颈癌、卵巢癌、肝癌、膀胱癌、肝癌、乳腺癌、转移性乳腺癌、结肠癌、直肠癌、结肠直肠癌、子宫癌、唾液腺癌、肾癌、前列腺癌、外阴癌、甲状腺癌、肝癌、肛门癌、阴茎癌、梅克尔细胞癌、食管癌、胆道肿瘤、头颈部癌和血液恶性肿瘤。
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