CN111233774B - Amino pyrimidine compound - Google Patents

Amino pyrimidine compound Download PDF

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CN111233774B
CN111233774B CN201811432384.9A CN201811432384A CN111233774B CN 111233774 B CN111233774 B CN 111233774B CN 201811432384 A CN201811432384 A CN 201811432384A CN 111233774 B CN111233774 B CN 111233774B
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CN111233774A (en
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张贵民
唐贞波
白文钦
杨伟河
马永杰
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Lunan Pharmaceutical Group Corp
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
    • C07D239/02—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings
    • C07D239/24—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
    • C07D239/28—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms
    • C07D239/46—Two or more oxygen, sulphur or nitrogen atoms
    • C07D239/50—Three nitrogen atoms

Abstract

The invention provides an aminopyrimidine compound with a novel structure shown in a formula (I) and a preparation method and application thereof. The aminopyrimidine compound is a compound shown in a formula (I), or pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer or tautomer thereof and the like. The aminopyrimidine compound provided by the invention has a good proliferation inhibition effect on various cancer cells, has a low tumor cell inhibition concentration, remarkably improves the activity of the compound, has good selectivity on the tumor cells, has good solubility, and is expected to become a specific medicine for treating malignant tumor cell abnormal proliferation diseases caused by EGFR mutation.

Description

Amino pyrimidine compound
Technical Field
The invention belongs to the field of pharmaceutical chemistry, and particularly relates to a 2,4,6-triaminopyrimidine EGFR inhibitor, and a preparation method and application thereof.
Background
Protein tyrosine kinases are a class of enzymes that catalytically transfer phosphate groups from ATP to tyrosine residues located on protein substrates, which play a role in normal cell growth. Many growth factor receptor proteins act through tyrosine kinases and affect signals through this process, thereby regulating cell growth. However, under certain conditions, these receptors become aberrant, either mutated or overexpressed, causing uncontrolled cell proliferation, resulting in tumor growth, ultimately leading to the well-known disease, cancer. Growth factor receptor protein tyrosine kinase inhibitors act to treat cancer by inhibiting the phosphorylation process described above.
The epidermal growth factor receptor EGFR, also known as c-erbB1/Her1, and the products of the neu oncogene (also known as c-erbB2/Her 2) are members of the EGF receptor superfamily. The EGF receptor superfamily belongs to the large family of receptor tyrosine kinases. They interact with specific growth factors or natural ligands into EGF or TGF α on the cell surface, thereby activating receptor tyrosine kinases. Typically this will activate a cascade of downstream signaling proteins, resulting in altered gene expression and increased growth rates.
The EGF receptor is a transmembrane glycoprotein with a molecular weight of 170,000 and is found on many types of epithelial cells and is activated by at least 3 of the rations EGF \ TGF-alpha (transforming growth factor alpha) and amphiregulin (ampheriregulin). Both Epidermal Growth Factor (EGF) and transforming growth factor alpha (TGF-. Alpha.) have been shown to bind to EGF receptors and cause cell proliferation and tumor growth. These growth factors do not bind to Her2 (Ulrich and Schlesinger,1990, CELL61, 203). Unlike several families of growth factors that induce receptor dimerization by virtue of their self-dimerization properties (e.g., PDGF), monomeric growth factors such as EGF have 2 receptor binding sites, and thus, can cross-link two adjacent EGF receptors (Lemmon et al, 1997, EMBO J.16, 281).
When bound to a growth factor ligand, such as Epidermal Growth Factor (EGF), the receptor may homodimerize with additional EGFR molecules or heterodimerize with another family member, such as erbB2 (HER 2), erbB3 (HER 3), or erbB4 (HER 4). Homodimerization and/or heterodimerization of erbB receptors results in phosphorylation of key tyrosine residues in the intracellular domain and in stimulation of many intracellular signaling pathways involved in cell proliferation and survival. Dysregulation of erbB family signaling promotes proliferation, invasion, metastasis, angiogenesis, and tumor cell survival, and has been described in many human cancers (including lung, head and neck, and breast cancers, among others).
EGFR is a member of the erbB receptor family of transmembrane protease, the serine kinase. When bound to a growth factor ligand, such as Epidermal Growth Factor (EGF), the receptor may homodimerize with additional EGFR molecules or heterodimerize with another family member, such as erbB2 (HER 2), erbB3 (HER 3), or erbB4 (HER 4). Dysregulation of erbB family signaling promotes proliferation, invasion, metastasis, angiogenesis, and tumor cell survival, and has been described in many human cancers, including those of lung, head and neck, and breast cancers. Thus, the erbB family represents a logical target for the development of anticancer drugs, and many agents targeting EGFR or erbB2 are now clinically available, including gefitinib, erlotinib, lapatinib, and the like.
EGFR belongs to a tyrosine kinase inhibitor receptor, and plays a crucial role in the processes of cell proliferation, angiogenesis, tumor invasion, metastasis and apoptosis. Studies have shown that overexpression or abnormal mutations of EGFR are present in many solid tumors and that tumor growth can be inhibited by inhibiting the kinase activity of EGFR tyrosine. Through the analysis of EGFR structure, specific parts of EGFR are selected as targets to interfere the signal conduction of EGFR, so that the aim of inhibiting the growth of tumor cells is fulfilled, and the EGFR structure is a new idea for developing anti-tumor drugs.
The tyrosine kinase inhibitor selectively targets the intracellular tyrosine kinase catalytic region of EGFR, competitively binds with ATP to a pocket of the kinase, inhibits the phosphorylation of tyrosine, and interrupts downstream signal conduction caused by the catalysis of the kinase, thereby achieving the purpose of blocking the overexpression of the EGFR. Inhibitors can be classified into reversible inhibitors and irreversible inhibitors according to the binding mode of the kinase inhibitor to the target protein.
Currently, tumor-targeted small molecule drugs directed against EGFR targets have been developed up to 3 rd generation. The 1 st generation EGFR inhibitor is an irreversible inhibitor, such as gefitinib and erlotinib, and is mainly used for treating NSCLC. Although the NSCLC treatment effect of the 1 st generation drug is significant, drug resistance is generated due to the EGFR-T790M mutation in more than 50% of patients. In order to solve the problem of drug resistance, reversible 2 nd generation EGFR inhibitors afatinib, crizotinib and the like are successively disclosed. However, these drugs have poor selectivity for EGFR-T790M and wild-type EGFR, and have severe side effects such as diarrhea, rash, etc. Therefore, in order to solve the problems of the first two generations of inhibitors, researchers further develop a third generation EGFR inhibitor, wherein WZ4002 is the reported first third generation EGFR inhibitor and is an inhibitor based on a pyrimidine structure as a mother nucleus, and in vitro cell experiments show that the inhibitor has good selectivity and inhibitory activity; the 5-trifluoromethyl-2,4-diaminopyrimidine screened by the Sjin task group is taken as an inhibitor of a mother nucleus, and a better in vivo and in vitro experimental result is shown; the baric drug of astrazepam, oseltamiib (AZD 9291), also employs an aminopyrimidine ring as the parent nucleus and is the first third-generation EGFR inhibitor approved by the FDA for marketing.
Figure GDA0004064996830000031
In addition, in 2009, w.j.zhou found novel mutation selective small molecule inhibitors of WZ4002, WZ8040, etc.:
Figure GDA0004064996830000032
emily j. Hanan et al obtained novel kinase inhibitors through a ring closure strategy:
Figure GDA0004064996830000033
kwangho lee et al also developed a series of EGFR kinase inhibitors based on this (US 2012157426), and found that I-1 has excellent inhibitory activity:
Figure GDA0004064996830000041
they further designed compound I-4 (KR 20130133202, CN 103269704):
Figure GDA0004064996830000042
although irreversible pyrimidine-type EGFR inhibitors have achieved good clinical results to date, it is anticipated that the problems of achieving selective resistance and drug toxicity in patients have not yet been completely solved, and there is a continuing need in the art for the development of new drugs having EGFR inhibitory activity.
Disclosure of Invention
The invention aims to provide a 2,4,6-triaminopyrimidine EGFR inhibitor with a novel structure, the compound provided by the invention has a strong in-vitro inhibition effect on various cancer cells, and has a patent drug potential, and a key intermediate and a target compound are efficiently synthesized by a simple method.
Research shows that the 2-site arylamine of the pyrimidine 2,4,6-triaminopyrimidine compound shown as the formula (I) in the invention can form a strong hydrogen bond with amino acid of a target spot in an EGFR tyrosine kinase binding region in the structure, and the binding region is in horseshoe conformation, so that the affinity of the compound and the EGFR target spot is greatly enhanced. And 2-site arylamine introduced substituent can improve the hydrogen bond effect of the compound and the hinge region of the EGFR, so as to enhance the affinity of the compound and the hinge region of the EGFR. In addition, in-vitro experiments of Ostinib show that the Ostinib has poor solubility, and the 6-site arylamine substituent of the compound provided by the invention can extend into a solvent exposure area, so that the affinity of the compound and a solvent is improved, the solubility of the compound is greatly improved, and the compound is helpful for rapidly permeating membrane tissues of cancerous cells to better exert the cytotoxicity of the compound.
Interpretation of terms
The following sets forth definitions of various terms used to describe the present application. These definitions apply to the terms used throughout the specification and claims, unless otherwise limited in specific instances either individually or as part of a larger group.
The term "alkyl" in the present invention refers to a saturated straight or branched chain hydrocarbon group, in certain embodiments, containing 1 to 6 or 1 to 3 carbon atoms, respectively, C 1-6 Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, t-butyl, neopentyl, n-hexyl, or the like, and C 1-3 Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, or the like.
The term "halo" in the present invention refers to a group formed by replacing a hydrogen atom on a carbon atom with a halogen atom, wherein the halogen atom includes but is not limited to F, cl, br, I.
The term "alkoxy" in the present invention refers to an-O-alkyl group, wherein said alkyl group includes but is not limited to C 1-3 Alkyl radical, C 1-6 Alkyl and C 3-6 Specific examples of cycloalkyl groups include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, cyclopropoxy, cyclobutoxy, cyclopentyloxy, cyclohexyloxy, and halo forms thereof.
The term "aryl" in the present invention refers to a monocyclic or polycyclic carbocyclic ring system having one or more fused or non-fused aromatic rings, including but not limited to phenyl, naphthyl, tetrahydronaphthyl, and the like, and having hydrogen atoms on ring carbonsMay also be substituted with one to more substituents including, but not limited to, C 1-3 Alkyl, alkoxy, amino, amido, urea, oxo, halo, pyrazolyl, imidazolyl, triazolyl, CN, -NHC (O) (C) 1-3 Alkyl), acyl, sulfonyl, sulfonamide, - (CH) 2 ) m (C 3-7 Heterocycloalkyl); each m is independently 0, 1,2 or 3.
The term "heterocycloalkyl" in the context of the present invention refers to a monocyclic or polycyclic non-aromatic ring system containing 2 to 6 ring carbon atoms and 1 to 3 ring heteroatoms selected from N, O, S.
The term "cycloalkyl" in the context of the present invention is intended to mean a monovalent radical of a monocyclic or polycyclic, saturated or partially unsaturated, carbocyclic compound, C 3-6 Cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like, and the hydrogen atoms on the ring carbons thereof may also be substituted with one or more substituents including, but not limited to, C 1-3 Alkyl, alkoxy, amino, hydroxyamino, amido, urea, oxo, halo, pyrazolyl, imidazolyl, triazolyl, CN, -NHC (O) (C) 1-3 Alkyl), acyl, sulfonyl, sulfonamide, - (CH) 2 ) m (C 3-7 Heterocycloalkyl); each m is independently 0, 1,2 or 3.
The term "heteroaryl" in the present invention refers to an aromatic ring system containing 1 to 6 carbons and at least one heteroatom selected from the group consisting of N, S, O; and the hydrogen atoms on the ring carbons may also be substituted with one to more substituents including, but not limited to, C 1-3 Alkyl, alkoxy, amino, hydroxyamino, amido, urea, oxo, halo, pyrazolyl, imidazolyl, triazolyl, CN, -NHC (O) (C) 1-3 Alkyl), acyl, sulfonyl, sulfonamide, - (CH) 2 ) m (C 3-7 Heterocycloalkyl); each m is independently 0, 1,2 or 3. Heteroaryl groups of specific embodiments include, but are not limited to, pyrazolyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyridyl, imidazolyl, thiazolyl, thienyl, furanyl, quinolinyl, isoquinolinyl, benzimidazolyl and the like.
Hair brushThe term "alkylamine" as used herein refers to the group-alkyl-NR a R b Wherein alkyl includes but is not limited to C 1-3 Alkyl radical, C 1-3 Haloalkyl, C 1-3 Alkoxy and C 3-6 Cycloalkyl radical, R a And R b Each independently selected from H, C 1-3 Alkyl or C 3-6 A cycloalkyl group.
The term "acyl" in the present invention refers to a-C (O) -substituent, wherein the substituent includes, but is not limited to, C 1-3 Alkyl radical, C 1-3 Haloalkyl, hydroxy, C 1-3 Alkoxy, -NH-C 3-6 Cycloalkyl, -NH-aryl, -NH-substituted aryl, -NH-heteroaryl, -NH-C 1-3 Alkyl, -NH-C 1-3 Alkylamines, and the like.
The term "amido" in the context of the present invention refers to the group-C (O) NR a R b Wherein R is a And R b Each independently selected from H, C 1-3 Alkyl or C 3-6 A cycloalkyl group.
The term "urea" according to the invention refers to the group-NR a C(O)NR b Wherein R is a And R b Each independently selected from H, C 1-3 Alkyl or C 3-6 A cycloalkyl group.
The term "pharmaceutically acceptable salt" as used herein refers to a salt of a compound of the present invention, prepared from a compound of the present invention having a specified substituent group, and a relatively nontoxic acid or base. The present invention contains relatively basic functional groups and acid addition salts can be obtained by contacting the neutral forms of such compounds with a sufficient amount of an acid in neat solution or in a suitable inert solvent, and pharmaceutically acceptable acid addition salts include inorganic and organic acid salts. The inorganic acid salts include, but are not limited to, hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate nitrate, phosphate, acid phosphate; the organic acid salts include, but are not limited to, formates, acetates, trifluoroacetates, propionates, pyruvates, glycolates, oxalates, malonates, fumarates, maleates, lactates, malates, citrates, tartrates, methanesulfonates, ethanesulfonates, benzenesulfonates, salicylates, picrates, glutamates, salicylates, ascorbates, camphorates, camphorsulfonates, and the like.
The term "solvate" as used herein refers to a form containing stoichiometric or non-stoichiometric amounts of an additive solvent selected from the group consisting of water, ethanol, isopropanol, ether, acetone, and the like.
The term "prodrug" as used herein refers to a compound that is metabolically convertible in the body to provide any of the compounds described by the formulae herein, and various forms of the drug are known in the art.
An "effective amount" as used herein refers to a dosage which is capable of achieving the desired therapeutic effect in the desired subject, but which does not unduly adversely affect the effect, and the specific dosage can generally be determined as desired by one of ordinary skill in the art.
"treatment" as used herein refers to a method of alleviating or alleviating the symptoms of a disease or its complications; by preventing is meant reducing or eliminating the onset of symptoms or complications of a disease, condition, or disorder.
It is to be understood that other terms not explained above, but appearing in the present invention, are to be defined as commonly understood by one of ordinary skill in the art.
The invention provides a 2,4,6-triaminopyrimidine compound shown as a formula (I), or a pharmaceutically acceptable salt, a hydrate, a solvate, a prodrug, a stereoisomer or a tautomer thereof.
Figure GDA0004064996830000071
Wherein:
R 1 selected from the group consisting of: c 3-6 Cycloalkylene radical, C 3-6 Heterocycloalkylene, C 1-6 Alkylene radical, C 1-6 Halogenoalkylene group, C 1-6 Alkyleneoxy, halogeno C 1-6 Alkyleneoxy, thio C 1-6 Alkyleneoxy, arylene, heteroarylene;
R 2 selected from the group consisting of: H. halogen, hydroxy, C 1-3 Alkyl, aryl, heteroaryl, and heteroaryl,-C(O)-R 4 、-S(O) 2 -R 4 、C 1-3 Alkoxy, -NR 5 R 6 ；
Wherein R is 4 Is selected from C 1-3 Alkyl radical, C 1-3 Haloalkyl, hydroxy, -NHR 7 、C 1-3 An alkoxy group; wherein R is 7 Is selected from C 3-6 Cycloalkyl, aryl, heteroaryl, C 1-3 Alkyl radical, C 1-3 An alkylamine;
wherein R is 5 Independently selected from H or C 1-3 An alkyl group; r is 6 Independently selected from H, C 1-3 Alkyl radical, C 3-6 Cycloalkyl, C 1-3 Haloalkyl, aryl, heteroaryl;
wherein said C 3-6 The cycloalkyl, aryl, heteroaryl may also be substituted by one or more R 3 The group is further substituted;
wherein R is 3 Independently selected from C 1-3 Alkyl, alkoxy, amino, hydroxyamino, amido, urea, oxo, halo, pyrazolyl, imidazolyl, triazolyl, CN, -NHC (O) (C) 1-3 Alkyl), acyl, sulfonyl, sulfonamide, - (CH) 2 ) m (C 3-7 Heterocycloalkyl); each m is independently 0, 1,2 or 3.
In a preferable embodiment of the invention, the aminopyrimidine compound shown in formula (I) and pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer or tautomer thereof are shown in the specification, wherein R is 1 Is selected from C 3-6 Heterocycloalkylene, C 1-6 Alkylene radical, C 1-6 Alkyleneoxy, arylene, heteroarylene; r is 2 、R 3 、R 4 、R 5 、R 6 、R 7 As defined for the compounds of formula (I).
In a preferred embodiment of the present invention, the aminopyrimidine compound shown in formula (I), pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer or tautomer thereof, R is 1 Is selected from C 1-6 Alkylene or arylene; r is 2 、R 3 、R 4 、R 5 、R 6 、R 7 As defined for the compounds of formula (I).
In a preferable embodiment of the invention, the aminopyrimidine compound shown in formula (I) and pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer or tautomer thereof are shown in the specification, wherein R is 2 Is selected from-S (O) 2 -R 4 Wherein R is 4 Is selected from C 1-3 Alkyl radical, C 1-3 Haloalkyl, hydroxy, -NHR 7 、C 1-3 An alkoxy group; other groups are as defined for compounds of formula (I).
In a preferred embodiment of the present invention, the aminopyrimidine compound shown in formula (I), pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer or tautomer thereof, R is 2 Selected from-C (O) -R 4 Wherein R is 4 Is selected from C 1-3 Alkyl radical, C 1-3 Haloalkyl, hydroxy, -NHR 7 、C 1-3 An alkoxy group; other groups are as defined for compounds of formula (I).
In a preferable embodiment of the invention, the aminopyrimidine compound shown in formula (I) and pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer or tautomer thereof are shown in the specification, wherein R is 2 is-C (O) -R 4 or-S (O) 2-R 4 Wherein R is 4 is-NHR 7 ，-NHR 7 Preferably selected from N, N-dimethylethylenediamine, cyclohexylamine, aniline, para-fluoroaniline, 2-aminopyridine, 4-methoxy-3-methylaniline, 2-aminobenzimidazole or 4- (4- (4-methylpiperazin-1 yl) -piperidin-1 yl) aniline; other groups are as defined for compounds of formula (I).
In a preferred embodiment of the present invention, the aminopyrimidine compound shown in formula (I), pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer or tautomer thereof, R is 1 Is C 3-6 Heterocycloalkylene, C 1-6 Alkylene radical, C 1-6 Alkyleneoxy, arylene, heteroarylene; the R is 2 is-C (O) -R 4 or-S (O) 2-R 4 (ii) a Wherein R is 4 is-NHR 7 ，-NHR 7 Preferably selected from 2-aminopyridine or 4-methoxy-3-methylaniline.
In a preferred embodiment of the invention, the aminopyrimidine compound shown in the formula (I) is selected from the following group:
Figure GDA0004064996830000081
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Figure GDA0004064996830000091
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Figure GDA0004064996830000101
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Figure GDA0004064996830000111
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Figure GDA0004064996830000121
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Figure GDA0004064996830000131
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Figure GDA0004064996830000141
in a preferable scheme of the invention, the aminopyrimidine compound shown in the formula (I) is selected from the following group:
Figure GDA0004064996830000142
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Figure GDA0004064996830000151
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Figure GDA0004064996830000161
in a preferable scheme of the invention, the aminopyrimidine compound shown in the formula (I) is selected from the following group:
Figure GDA0004064996830000171
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Figure GDA0004064996830000181
in a preferred embodiment of the invention, the aminopyrimidine compound shown in the formula (I) is selected from the following group:
Figure GDA0004064996830000182
/>
Figure GDA0004064996830000191
in a preferable scheme of the invention, the pharmaceutically acceptable salt is an inorganic acid salt or an organic acid salt, and the inorganic acid salt is selected from hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, nitrate, phosphate and acid phosphate; the organic acid salt is selected from formate, acetate, trifluoroacetate, propionate, pyruvate, glycollate, oxalate, malonate, fumarate, maleate, lactate, malate, citrate, tartrate, methanesulfonate, ethanesulfonate, benzenesulfonate, salicylate, picrate, glutamate, salicylate, ascorbate, camphorate, and camphorsulfonate.
In a second aspect of the invention, a preparation method of the aminopyrimidine compound shown in the formula (I) is provided. The compounds of the present application can be prepared using a variety of means known to those skilled in the art. The present application can be synthesized using the methods described herein and organic chemical synthesis methods or variations thereof as understood by those skilled in the art. Preferred methods include, but are not limited to, the following.
Preferably, the general synthesis method of the aminopyrimidine compounds shown in the formula (I) comprises the following steps:
Figure GDA0004064996830000201
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wherein R is 1 And R 2 As defined for the first aspect of the invention; x is selected from F, cl, br, I, preferably Cl, br, I.
The preferable scheme is that the preparation method of the aminopyrimidine compound shown in the formula (I) comprises the following specific steps:
step 1: the compound 1, namely 2,4,6-trichloropyrimidine and the compound 2, namely 3-chloro-4-fluoroaniline have substitution reaction at the 4-position of a pyrimidine ring to generate an intermediate III, namely 2,6-dichloro-4- (3-chloro-4-fluoroaniline) -pyrimidine;
step 2: carrying out substitution reaction on the intermediate III and the compound 3 at the 2-position of a pyrimidine nucleus ring under the action of a catalyst to generate an intermediate II; wherein R in the compound 3 1 And R 2 As defined for the first aspect of the invention;
and step 3: the intermediate II, namely 2,4-disubstituted pyrimidine compound reacts with the compound 4 to generate the compound shown in the formula (I).
The preferable scheme is that the preparation method of the aminopyrimidine compound shown in the formula (I) specifically comprises the following steps:
step 1: adding a compound 1, namely 2,4,6-trichloropyrimidine into a solvent, stirring until the mixture is dissolved, cooling in an ice bath, slowly dropwise adding a compound 2, namely 3-chloro-4-fluoroaniline, removing the ice bath after the dropwise adding is finished, controlling the temperature for reaction, reducing the pressure and concentrating after the TLC tracking detection reaction is finished, and performing column chromatography separation on residues to obtain an intermediate III, namely 2,6-dichloro-N- (3-chloro-4-fluorophenyl) pyrimidine-4-amine;
step 2: adding the intermediate III and the compound 3 into a solvent, adding a strong acid catalyst, carrying out temperature-controlled reflux reaction, carrying out TLC detection and tracking until the reaction is complete, carrying out reduced pressure concentration, and carrying out column chromatography on residuesCarrying out chromatographic separation to obtain an intermediate II; wherein R in the compound 3 1 And R 2 As defined for the first aspect of the invention;
and step 3: adding the intermediate II, concentrated hydrochloric acid and the compound 4 acrylamide into a solvent, placing the mixture into a closed microwave reactor, reacting at a certain temperature until the reaction is finished, cooling to room temperature, adding ethyl acetate and water, extracting, separating liquid, washing an organic phase with water, drying with a proper amount of anhydrous sodium sulfate, concentrating under reduced pressure, and separating residues by column chromatography to obtain the compound TM shown in the formula (I).
In a preferred embodiment, the reaction solvent in step 1 is dichloromethane, acetone, methanol, ethanol, or ethyl acetate, and dichloromethane is particularly preferred.
In a preferred embodiment, the reaction temperature in step 1 is 15 ℃ to 50 ℃, and 25 ℃ is particularly preferred.
In a preferred embodiment, the molar ratio of compound 1 to compound 2 in step 1 is 1.05 to 1.5, wherein 1.
Preferably, the reaction solvent in step 2 is ethanol, isopropanol, tert-butanol, N-dimethylformamide, and particularly preferably isopropanol.
Preferably, the catalyst in step 2 is hydrochloric acid, sulfuric acid, perchloric acid, hydrobromic acid, and hydrochloric acid is particularly preferred.
In a preferred embodiment, the molar ratio of the intermediate iii to the compound 3 in the step 2 is 1.01 to 1.5, wherein 1.
In a preferred embodiment, the reaction solvent in step 3 is dimethyl sulfoxide, N-dimethylformamide, or N-octanol, and N-octanol is particularly preferred.
In a preferred embodiment, the reaction temperature in step 3 is 120 to 180 ℃, and 150 ℃ is particularly preferred.
In a preferred embodiment, the molar ratio of the intermediate II 2,4-disubstituted pyrimidine compound to the compound 4 acrylamide in step 3 is 1.
Preferably, the compound 3 described in step 2 can be obtained by purchase or experimental synthesis.
In the preferred embodiment, when said R is 1 Is selected from C 3-6 Heterocycloalkylene, C 1-6 Alkylene radical, C 1-6 Alkyleneoxy, arylene, heteroarylene, R 2 Selected from-C (O) -R 4 When compound 3 is prepared by the following method:
Figure GDA0004064996830000211
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wherein, R is 4 ，R 7 As defined for the first aspect of the invention;
in the preferred embodiment, when said R is 1 Is selected from C 3-6 Heterocycloalkylene, C 1-6 Alkylene radical, C 1-6 Alkyleneoxy, arylene, heteroarylene, R 2 Is selected from-S (O) 2 -R 4 When compound 3 is prepared by the following method:
Figure GDA0004064996830000212
wherein, R is 4 ，R 7 As defined for the first aspect of the invention;
in a third aspect of the present invention, there is provided a pharmaceutical composition comprising: a therapeutically effective amount of one or more of aminopyrimidine compounds of formula (I), or pharmaceutically acceptable salts, tautomers, optical isomers, and pharmaceutically acceptable solvates thereof, and optionally pharmaceutically acceptable carriers, excipients, adjuvants, or diluents.
Preferably, the pharmaceutical composition can be used for treating diseases related to abnormal expression or high activity of tyrosine kinase.
Preferably, the diseases associated with abnormal expression or high tyrosine kinase activity of tyrosine kinase include, but are not limited to, abnormal cell proliferation, morphological changes, hyperkinesia, angiogenesis diseases, tumor growth, and tumor metastasis diseases.
In a preferred embodiment, the EGFR inhibitor comprises an inhibitory effective amount of one or more aminopyrimidines of formula (I), or a pharmaceutically acceptable salt, tautomer, optical isomer, or pharmaceutically acceptable solvate thereof, according to the present invention, and optionally a pharmaceutically acceptable carrier, excipient, adjuvant, or diluent.
The compounds of the invention can be used directly for prophylaxis and therapy or, preferably, in the form of pharmaceutical compositions. Although the active ingredients can be administered separately, they are preferably in the form of pharmaceutical preparations or compositions. Accordingly, the present invention provides a pharmaceutical formulation comprising a compound of the present invention in association with a pharmaceutically acceptable diluent, excipient or carrier (collectively referred to herein as "carrier" material). The pharmaceutical compositions of the present invention may take the form of pharmaceutical formulations as described hereinafter. The present invention therefore relates to a pharmaceutical composition comprising at least one compound of formula (I) and conventional excipients.
Exemplary compositions for oral administration include: suspending agents which may include, for example, microcrystalline cellulose for imparting bulk, alginic acid or sodium alginate as a suspending agent, methylcellulose as a viscosity enhancing agent, and sweetening or flavoring agents such as those known in the art; such as immediate release tablets which may contain, for example, microcrystalline cellulose, dicalcium phosphate, starch, magnesium stearate, calcium sulfate, sorbitol, glucose and/or lactose and/or other excipients, binders, extenders, disintegrants, diluents and lubricants such as those known in the art. Suitable binders include: starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes and the like. Disintegrants include, but are not limited to: starch, methyl cellulose, agar, bentonite, xanthan gum and the like. The compounds of formula (I) may also be delivered orally by sublingual and/or buccal means. Molded, compressed or lyophilized tablets are exemplary forms that may be used. Exemplary compositions include those formulated with the compounds of the present invention with fast dissolving diluents such as mannitol, lactose, sucrose and/or cyclodextrins. These formulations may also include high molecular weight excipients such as cellulose (microcrystalline powdered cellulose) or polyethylene glycol (PEG). These formulations may also include excipients to aid mucosal adhesion, such as hydroxypropyl cellulose (HPC), hydroxypropyl methylcellulose (HPMC), sodium carboxymethyl cellulose (SCMC), maleic anhydride copolymers (e.g., gantrez) and agents to control release such as polyacrylic copolymers (e.g., carbopo 1934). Lubricants, glidants, flavoring agents, coloring agents and stabilizers may also be added to facilitate preparation and use. Lubricants used in these dosage forms include: sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like. For oral administration in liquid form, the oral pharmaceutical composition can be combined with any oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like.
The pharmaceutical formulations of the invention include those suitable for oral, parenteral [ including subcutaneous, intradermal, intramuscular, intravenous (bolus or infusion) and intraarticular ], inhalation (including fine particle powders or sprays which may be produced by means of various types of metered dose pressurised aerosols), nebuliser or inhaler, rectal, intraperitoneal and topical (including dermal, buccal, sublingual and intraocular) administration, although the most suitable route may depend, for example, on the condition and state of the recipient.
Formulations of the invention suitable for oral administration may be presented as discrete units containing a predetermined amount of the active ingredient, for example, as capsules, cachets, pills or tablets; a powder or granules; solutions or suspensions in aqueous or non-aqueous liquids, such as elixirs, tinctures, suspensions or syrups; or an oil-in-water emulsion or a water-in-oil emulsion. The active ingredient can also be made into bolus, electuary or paste.
Tablets may be made by compression or molding with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, lubricant, surfactant or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide sustained or controlled release of the active ingredient therein. The compounds of the invention may be administered in a form suitable for immediate release or sustained release. Immediate or sustained release can be achieved by using suitable pharmaceutical compositions comprising the compounds of the invention or, especially in the case of sustained release, by using devices such as subcutaneous implants or osmotic pumps. The compounds of the invention may also be administered in liposomes. Preferred unit dose formulations are those containing an effective dose (as described below) or an appropriate fraction thereof of the active ingredient.
It will be appreciated that in addition to the ingredients particularly mentioned above, the formulations of the invention may include other agents conventional in the art having regard to the type of formulation in question, for example, those suitable for oral administration may include flavouring agents.
The formulations may be conveniently presented in unit dosage form and may be manufactured by any of the methods well known in the art of pharmacy. All methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general, the active ingredient is combined uniformly and intimately with liquid carriers and/or finely divided solid carriers to prepare formulations, which are then, if necessary, shaped to give the desired formulation.
The compounds of the invention may also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids, 1,2-dipalmitoylphosphatidylcholine, phosphatidylethanolamine (cephalin), phosphatidylserine, phosphatidylinositol, diphosphatidylglycerol (cardiolipin), or phosphatidylcholine (lecithin).
Formulations for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain antioxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may contain suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and the compositions may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example saline or water-for-injection, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described. Exemplary compositions for parenteral administration include: injectable solutions or suspensions which may contain, for example, suitable non-toxic parenterally acceptable diluents or solvents such as polyethylene glycol, ethanol, 1,3-butanediol, water, ringer's solution, sodium chloride, and the like, or other suitable dispersing or wetting agents and suspending agents, including synthetic mono-or diglycerides and fatty acids, including oleic acid and Cremaphor.
Exemplary compositions for nasal, aerosol or inhalation administration include solutions in saline, which may contain, for example, benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, and/or other solubilizing or dispersing agents such as those known in the art.
Formulations for rectal administration may be presented as suppositories with conventional carriers such as cocoa butter/synthetic glycerides or polyethylene glycols. These carriers are typically solid at ambient conditions, but liquefy and/or dissolve in the rectal lumen to release the drug.
Formulations for topical administration, e.g. buccal or sublingual administration, in the buccal cavity include lozenges comprising the active ingredient in a flavoured base, such as sucrose and acacia or tragacanth, and pastilles comprising the active ingredient in a base of gelatin and glycerol or sucrose and acacia. Exemplary compositions for topical administration include topical carriers such as Plastibase (mineral oil gelled with polyethylene).
In a fourth aspect, the present invention provides the use of an aminopyrimidine compound of formula (I), in particular for the preparation of a tyrosine kinase inhibitor, for the non-therapeutic inhibition of tyrosine kinase activity in vitro, for the non-therapeutic inhibition of tumor cell growth in vitro or a combination thereof, for the treatment or prevention of a disease in which EGFR plays a role.
In a preferred embodiment, the aminopyrimidine compounds shown in formula (I) are used for preparing medicines for treating or preventing diseases in which EGFR plays a role.
Preferably, the EGFR-affecting disease comprises cancer or a metabolic system-related disease, particularly, the cancer includes, but is not limited to, non-small cell lung cancer, ovarian cancer, cervical cancer, colorectal cancer, breast cancer, pancreatic cancer, glioma, glioblastoma, melanoma, prostate cancer, leukemia, lymphoma, non-hodgkin lymphoma, gastric cancer, lung cancer, hepatocellular carcinoma, gastric cancer, gastrointestinal stromal tumor (GIST), thyroid cancer, cholangiocarcinoma, endometrial cancer, kidney cancer, anaplastic large cell lymphoma, acute Myeloid Leukemia (AML), multiple myeloma, melanoma, or mesothelioma; the diseases related to the metabolic system include but are not limited to diabetes, atherosclerosis, obesity and the like, wherein non-small cell lung cancer is particularly preferred.
In a preferred embodiment, the treatment refers to a method of alleviating or alleviating the symptoms of the disease and its complications; by preventing is meant reducing or eliminating the onset of symptoms or complications of a disease, condition, or disorder.
Compared with the prior art, the invention has the main advantages that:
(1) Compared with the existing similar compounds, the compound provided by the invention has lower tumor cell inhibition concentration, and the activity of the compound is remarkably improved.
(2) Compared with the existing aminopyrimidine compounds, the aminopyrimidine compound shown in the formula (I) provided by the invention has greatly improved selectivity on some tumor cells.
(3) Compared with the known AZD9291 compound, the aminopyrimidine compound shown in the formula (I) provided by the invention has obviously improved solubility.
(4) Compared with the known similar compounds, the aminopyrimidine compound shown in the formula (I) provided by the invention has the advantages of shorter synthetic route and simpler operation, and is suitable for industrial amplification production.
It is to be understood that within the scope of the present invention, the above-described technical features of the present invention and the technical features specifically described below (including the embodiments) may be combined with each other to constitute a new or preferred technical solution.
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
The present invention is further illustrated by the following examples, it being understood that these examples are intended to be illustrative only and are not intended to be limiting, and that the invention is not limited thereto.
Abbreviations used herein:
EDCI:1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride
HOBt: 1-carbonyl benzotriazole
DCM: methylene dichloride
TFA: trifluoroacetic acid
TLC: thin layer chromatography
1 H NMR: hydrogen spectrum of nuclear magnetic resonance
13 C-NMR: nuclear magnetic resonance carbon spectrum
Example 1 preparation of TM-1
Figure GDA0004064996830000251
Compound 3: adding tert-butoxycarbonyl protected glycine (21.03g, 120mmol) into dichloromethane (30 mL), sequentially adding EDCI (25.31g, 132mmol), HOBt (17.82g, 132mmol) and N, N-dimethylethylenediamine (11.63g, 132mmol), stirring at room temperature for 36h, detecting by TLC, removing the solvent under reduced pressure, re-dissolving the obtained solid in 120mL of mixed solution (DCM: TFA = 7:3), stirring at room temperature for 0.5h, detecting by TLC, removing the solvent by rotary evaporation under reduced pressure, and separating by column chromatography to obtain the product compound 3 with yield of 76.42% and HPLC:99.56%.
An intermediate III: 2,4,6-trichloropyrimidine (compound 1) (55.03g, 300mmol) is added into dichloromethane (300 ml) and stirred until dissolved, the temperature is reduced to 0-5 ℃ in an ice bath, 3-chloro-4-fluoroaniline (45.71g, 315mmol, compound 2) is slowly dripped in, after the dripping is finished, the ice bath is removed, the temperature is naturally raised to room temperature, after the TLC tracking detection reaction is finished, the decompression and the concentration are carried out, and the residue is separated by column chromatography to obtain an intermediate III, the yield is 85.61%, HPLC (high performance liquid chromatography) 99.71% and white solid.
An intermediate II: adding the intermediate III (100 mmol) and the compound 3 (14.43g, 110mmol) into isopropanol (150 ml), adding concentrated hydrochloric acid (1 ml), refluxing, TLC detecting until the reaction is complete, decompressing and concentrating, and separating the residue by column chromatography to obtain the intermediate II with the yield of 72.16%, HPLC:99.62% and white solid.
TM-1: adding the intermediate II (85.11g, 12mmol), concentrated hydrochloric acid (0.5 ml) and acrylamide (1.02g, 14.4 mmol) into n-octanol (20 ml), placing in a closed microwave reactor, setting the temperature at 140 ℃, finishing the reaction after 0.5h, cooling to room temperature, adding 200ml of each of ethyl acetate and water, extracting, separating liquid, washing an organic phase once with water, drying with a proper amount of anhydrous sodium sulfate, concentrating under reduced pressure, and carrying out column chromatography separation on the residue to obtain the compound TM-1 of the formula I, wherein the yield is 62.39%, and the HPLC (high performance liquid chromatography) is 99.76%.
1 H NMR(400MHz,DMSO-d 6 )δ：11.27(s,1H),10.45(s,1H),8.84(s,1H),8.41(s,1H),7.40～7.34(m,3H),6.53(t,J＝10.6Hz,1H),6.03(d,J＝6.4Hz,2H),4.69(s,1H),4.15(s,2H),3.58(s,2H),2.15(s,6H)； 13 C-NMR(400MHz,DMSO-d 6 ):171.4,70.6,168.5,162.2,159.1,148.4,137.5,130.9,126.7,122.2,118.7,118.1,114.8,72.5,70.0,56.2,45.3(2C)，MS-ESI m/z:421.14(M+H) + 。
Example 2 preparation of TM-2
Figure GDA0004064996830000261
Compound 3: adding tert-butoxycarbonyl protected glycine (21.03g, 120mmol) into dichloromethane (40 mL), sequentially adding EDCI (25.31g, 132mmol), HOBt (17.82g, 132mmol) and cyclohexylamine (13.09g, 132mmol), stirring at room temperature for 24h, detecting by TLC, removing solvent under reduced pressure, re-dissolving the obtained solid in 150mL of mixed solution (DCM: TFA = 7:3), stirring at room temperature for 0.5h, detecting by TLC, removing solvent by rotary evaporation under reduced pressure, and separating by column chromatography to obtain the product compound 3 with the yield of 73.62% and HPLC:99.62%.
Intermediate III: 2,4,6-trichloropyrimidine (compound 1) (55.03g, 300mmol) is added into ethyl acetate (300 ml) and stirred until dissolved, the temperature is reduced to 0-5 ℃ in an ice bath, 3-chloro-4-fluoroaniline (45.72g, 315mmol, compound 2) is slowly dripped in, the ice bath is removed after the dripping is finished, the temperature is naturally raised to room temperature, after the TLC tracking detection reaction is finished, the pressure is reduced and the concentration is carried out, and the residue is separated by column chromatography to obtain an intermediate III, the yield is 85.91%, HPLC is 99.73%, and white solid is obtained.
An intermediate II: adding the intermediate III (29.25g, 100mmol) and the compound 3 (17.16g, 110mmol) into isopropanol (150 ml), adding concentrated hydrochloric acid (1 ml), refluxing, detecting by TLC until the reaction is complete, concentrating under reduced pressure, and separating the residue by column chromatography to obtain the intermediate II with the yield of 72.16%, HPLC (high performance liquid chromatography) 99.69% and white solid.
TM-2: adding the intermediate II (4.95g, 12mmol), concentrated hydrochloric acid (0.5 ml) and acrylamide (1.02g, 14.4 mmol) into n-octanol (20 ml), placing the n-octanol in a closed microwave reactor, setting the temperature to 140 ℃, finishing the reaction after 1.5h, cooling to room temperature, adding 200ml of each of ethyl acetate and water, extracting and separating the liquid, washing an organic phase once by using water, drying by using a proper amount of anhydrous sodium sulfate, concentrating under reduced pressure, and separating the residue by using column chromatography to obtain the compound TM-2 of the formula I, wherein the yield is 62.33 percent and the HPLC (high performance liquid chromatography) is 99.66 percent.
1 H NMR(400MHz,DMSO-d 6 )δ：11.07(s,1H),10.69(s,1H),8.73(s,1H),7.44～7.38(m,3H),6.45(s,1H),6.50(t,J＝10.6Hz,1H),6.08(d,J＝6.4Hz,2H),4.79(s,1H),4.10(s,2H),3.55～3.47(m,1H),1.74～1.69(m,2H),1.50～1.43(m,4H),1.23～1.11(m,4H)； 13 C-NMR(400MHz,DMSO-d 6 ):δ171.4,170.3,168.5,162.1,159.6,148.7,137.2,130.3,126.8,122.5,118.7,118.4,114.4,72.3,53.2,50.1,35.6(2C),25.6,26.7(2C)，MS-ESI m/z:446.92(M+H) + 。
Example 3 preparation of TM-3
Figure GDA0004064996830000281
Compound 3: adding tert-butoxycarbonyl protected glycine (21.03g, 120mmol) into dichloromethane (30 mL), sequentially adding EDCI (25.31g, 132mmol), HOBt (17.82g, 132mmol) and aniline (12.29g, 132mmol), stirring at room temperature for 24h, detecting by TLC to finish the reaction, removing the solvent under reduced pressure, re-dissolving the obtained solid in 120mL of mixed solution (DCM: TFA = 7:3), stirring at room temperature for 0.5h, detecting by TLC to finish the reaction, removing the solvent by rotary evaporation under reduced pressure, and separating by column chromatography to obtain the product compound 3 with the yield of 75.61% and HPLC of 99.71%.
An intermediate III: 2,4,6-trichloropyrimidine (compound 1) (55.03g, 300mmol) is added into acetone (300 ml) and stirred until dissolution, the temperature is reduced to 0-5 ℃ in an ice bath, 3-chloro-4-fluoroaniline (45.72g, 315mmol, compound 2) is slowly dripped, the ice bath is removed after the dripping is finished, the temperature is naturally raised to room temperature, after the TLC tracking detection reaction is finished, the pressure is reduced and the concentration is carried out, and the residue is separated by column chromatography to obtain an intermediate III, the yield is 84.67%, HPLC is 99.69%, and white solid is obtained.
An intermediate II: adding intermediate III (29.25g, 100mmol) and compound 3 (16.52g, 110mmol) into isopropanol (150 ml), adding concentrated hydrochloric acid (1 ml), refluxing, detecting by TLC until the reaction is complete, concentrating under reduced pressure, and separating the residue by column chromatography to obtain intermediate II with yield of 73.39%, HPLC (high performance liquid chromatography) of 99.76% and white solid.
TM-3: adding the intermediate II (4.86g, 12mmol), concentrated hydrochloric acid (0.5 ml) and acrylamide (1.02g, 14.4mmol) into n-octanol (20 ml), placing in a closed microwave reactor, setting the temperature at 140 ℃, finishing the reaction after 1.0h, cooling to room temperature, adding 200ml of each of ethyl acetate and water, extracting, separating liquid, washing an organic phase once with water, drying with a proper amount of anhydrous sodium sulfate, concentrating under reduced pressure, and separating residues by column chromatography to obtain the compound TM-3 of the formula I, wherein the yield is 67.51 percent and the HPLC is 99.73 percent.
1 H NMR(400MHz,DMSO-d 6 )δ：11.07(s,1H),10.33(s,1H),8.73(s,1H),7.58(d,J＝6.4Hz 2H),7.33～7.20(m,5H),7.01～6.95(m,1H),6.69(s,1H),6.50(t,J＝10.6Hz,1H),6.03(d,J＝6.4Hz,2H),4.73(s,1H),4.05(s,2H)； 13 C-NMR(400MHz,DMSO-d 6 ):δ171.5,170.2,168.6,162.2,159.5,148.8,137.1,134.3,130.4,128.8(2C),127.5,126.6,122.3,120.5(2C),118.5,118.2,114.5,72.4,53.5，MS-ESI m/z:440.87(M+H) + 。
Example 4 preparation of TM-4
Figure GDA0004064996830000291
Compound 3: adding tert-butoxycarbonyl protected glycine (21.03g, 120mmol) into dichloromethane (50 mL), sequentially adding EDCI (25.31g, 132mmol), HOBt (17.82g, 132mmol) and para-fluoroaniline (14.67g, 132mmol), stirring at room temperature for reaction 36h, detecting by TLC for reaction, removing the solvent under reduced pressure, re-dissolving the obtained solid in 150mL of mixed solution (DCM: TFA = 7:3), stirring at room temperature for reaction for 0.5h, detecting by TLC for reaction, removing the solvent by rotary evaporation under reduced pressure, and separating by column chromatography to obtain the product compound 3 with the yield of 71.66% and HPLC:99.59%.
An intermediate III: 2,4,6-trichloropyrimidine (compound 1) (55.03g, 300mmol) is added into ethyl acetate (300 ml) and stirred until dissolved, the temperature is reduced to 0-5 ℃ in an ice bath, 3-chloro-4-fluoroaniline (45.72g, 315mmol, compound 2) is slowly dripped in, the ice bath is removed after the dripping is finished, the temperature is naturally raised to room temperature, after the TLC tracking detection reaction is finished, the decompression concentration is carried out, the residue is separated by column chromatography to obtain an intermediate III, the yield is 83.57%, the HPLC (high performance liquid chromatography) ratio is 99.67%, and white solid is obtained.
An intermediate II: adding intermediate III (29.25g, 100mmol) and compound 3 (18.50g, 110mmol) into N, N-dimethylformamide (150 ml), adding concentrated hydrochloric acid (1 ml), refluxing, detecting by TLC until the reaction is complete, concentrating under reduced pressure, and separating the residue by column chromatography to obtain intermediate II with yield of 73.27%, HPLC:99.76%, and white solid.
TM-4: adding the intermediate II (5.09g, 12mmol), concentrated hydrochloric acid (0.5 ml) and acrylamide (1.02g, 14.4 mmol) into n-octanol (20 ml), placing in a closed microwave reactor, setting the temperature to 140 ℃, finishing the reaction after 0.5h, cooling to room temperature, adding 200ml of each of ethyl acetate and water, extracting, separating liquid, washing the organic phase once with water, drying with a proper amount of anhydrous sodium sulfate, concentrating under reduced pressure, and separating the residue by column chromatography to obtain the compound TM-4 of the formula I, namely a white solid with the yield of 71.36% and the HPLC of 99.74%.
1 H NMR(400MHz,DMSO-d 6 )δ：10.99(s,1H),10.45(s,1H),8.82(s,1H),7.58(d,J＝6.4Hz 2H),7.29～7.10(m,5H),6.71(s,1H),6.48(t,J＝10.6Hz,1H),6.06(d,J＝6.4Hz,2H),4.69(s,1H),4.15(s,2H)； 13 C-NMR(400MHz,DMSO-d 6 ):δ171.3,170.5,168.7,159.3,148.6,137.2,134.5,130.2,129.5,127.3,126.8,122.5,120.7(2C),118.6,118.5,117.4(2C),114.2,72.2,53.6，MS-ESI m/z:458.83(M+H) + 。
Example 5 preparation of TM-5
Figure GDA0004064996830000301
Compound 3: adding tert-butoxycarbonyl protected glycine (21.03g, 120mmol) into dichloromethane (40 mL), sequentially adding EDCI (25.31g, 132mmol), HOBt (17.82g, 132mmol) and 2-aminopyridine (12.42g, 132mmol), stirring at room temperature for 24h, detecting by TLC, removing the solvent under reduced pressure, re-dissolving the obtained solid in 120mL of mixed solution (DCM: TFA = 7:3), stirring at room temperature for 0.5h, detecting by TLC, removing the solvent by rotary evaporation under reduced pressure, and separating by column chromatography to obtain the product compound 3 with yield of 73.41% and HPLC:99.64%.
Intermediate III: 2,4,6-trichloropyrimidine (compound 1) (55.03g, 300mmol) is added into dichloromethane (300 ml) and stirred until dissolved, the temperature is reduced to 0-5 ℃ in an ice bath, 3-chloro-4-fluoroaniline (45.72g, 315mmol, compound 2) is slowly dripped in, after the dripping is finished, the ice bath is removed, the temperature is naturally raised to room temperature, after the TLC tracking detection reaction is finished, the pressure is reduced and the concentration is carried out, and the residue is separated by column chromatography to obtain an intermediate III, the yield is 86.52%, HPLC is 99.71%, and white solid is obtained.
An intermediate II: adding the intermediate III (29.25g, 100mmol) and the compound 3 (16.62g, 110mmol) into N, N-dimethylformamide (150 ml), adding concentrated hydrochloric acid (1 ml), refluxing, TLC detecting and tracking till the reaction is complete, decompressing and concentrating, and separating the residue by column chromatography to obtain the intermediate II with the yield of 73.28%, HPLC (high performance liquid chromatography) 99.76% and white solid.
TM-5: adding the intermediate II (4.89g, 12mmol), concentrated hydrochloric acid (0.5 ml) and acrylamide (1.02g, 14.4 mmol) into n-octanol (20 ml), placing the n-octanol in a closed microwave reactor, setting the temperature to 140 ℃, finishing the reaction after 2.5h, cooling to room temperature, adding 200ml of each of ethyl acetate and water, extracting, separating liquid, washing an organic phase once with water, drying with a proper amount of anhydrous sodium sulfate, concentrating under reduced pressure, and carrying out column chromatography separation on residues to obtain a compound TM-5 of the formula I, namely a white solid with the yield of 71.36% and the HPLC (99.74%).
1 H NMR(400MHz,DMSO-d 6 )δ：11.21(s,1H),10.40(s,1H),8.92(s,1H),8.39(d,J＝20.6Hz 1H),8.20(d,J＝14.4Hz 1H),8.08(dd,J＝7.8Hz 1H),7.35～7.18(m,4H),6.69(s,1H),6.47(t,J＝10.6Hz,1H),6.03(d,J＝6.4Hz,2H),4.73(s,1H),3.98(s,2H)； 13 C-NMR(400MHz,DMSO-d 6 ):δ171.5,170.8,168.9,162.4,159.8,152.8,148.9,145.7,139.5,134.7,130.7,126.5,124.4,122.3,118.8,118.9(2C),114.4,72.6,53.7，MS-ESI m/z:441.11(M+H) + 。
Example 6 preparation of TM-6
Figure GDA0004064996830000311
Compound 3: adding tert-butoxycarbonyl protected glycine (21.03g, 120mmol) into dichloromethane (50 mL), sequentially adding EDCI (25.31g, 132mmol), HOBt (17.82g, 132mmol) and 4-methoxy-3-methylaniline (18.09g, 132mmol), stirring at room temperature for 36h, detecting by TLC, removing the solvent under reduced pressure, re-dissolving the obtained solid in 150mL of mixed solution (DCM: TFA = 7:3), stirring at room temperature for 1h, detecting by TLC, removing the solvent by rotary evaporation under reduced pressure, and separating by column chromatography to obtain the product compound 3 with the yield of 71.36% and HPLC:99.62%.
Intermediate III: 2,4,6-trichloropyrimidine (compound 1) (55.03g, 300mmol) is added into dichloromethane (300 ml) and stirred until dissolved, the temperature is reduced to 0-5 ℃ in an ice bath, 3-chloro-4-fluoroaniline (45.72g, 315mmol, compound 2) is slowly dripped, the ice bath is removed after the dripping is finished, the temperature is naturally raised to room temperature, after the TLC tracking detection reaction is finished, the pressure is reduced and the concentration is carried out, and the remainder is separated by column chromatography to obtain an intermediate III, the yield is 87.52%, HPLC (high performance liquid chromatography) is 99.81%, and white solid is obtained.
An intermediate II: adding the intermediate III (29.25g, 100mmol) and the compound 3 (21.37g, 110mmol) into N, N-dimethylformamide (145.5 ml), adding concentrated hydrochloric acid (1 ml), refluxing, detecting by TLC until the reaction is complete, concentrating under reduced pressure, and separating the residue by column chromatography to obtain the intermediate II with the yield of 69.57%, HPLC:99.70% and white solid.
TM-6: adding the intermediate II (5.40g, 12mmol), concentrated hydrochloric acid (0.5 ml) and acrylamide (1.02g, 14.4 mmol) into n-octanol (20 ml), placing in a closed microwave reactor, heating to 140 ℃, finishing reaction for 2.0h, cooling to room temperature, adding 200ml of each of ethyl acetate and water, extracting, separating liquid, washing an organic phase once with water, drying with a proper amount of anhydrous sodium sulfate, concentrating under reduced pressure, and separating residues by column chromatography to obtain the compound TM-6 of the formula I, wherein the yield is 73.29 percent and the HPLC (high performance liquid chromatography) is 99.77 percent.
1 H NMR(400MHz,DMSO-d 6 )δ：11.12(s,1H),10.28(s,1H),8.65(s,1H),7.59(s,1H),7.29～7.18(m,5H),6.79(s,1H),6.47(dd,J＝10.6,8.8Hz,1H),5.99(d,J＝6.4Hz,2H),4.68(s,1H),4.10(s,2H)；3.32(s,3H),2.15(s,3H)； 13 C-NMR(400MHz,DMSO-d 6 ):δ172.9,169.3,167.4,162.2,158.4,149.6,137.5,135.1,131.0,128.1,125.4,123.3,122.8,121.7,119.6,118.7,118.4,115.1,114.8,72.0,56.4,54.4,19.7，MS-ESI m/z:484.14(M+H) + 。
Example 7 preparation of TM-7
Figure GDA0004064996830000321
Compound 3: t-butoxycarbonyl protected glycine (21.03g, 120mmol) was added to dichloromethane (60 mL), EDCI (25.31g, 132mmol), HOBt (17.82g, 132mmol) and 4- (4- (4-methylpiperazin-1 yl) -piperidin-1 yl) aniline (30.20g, 132mmol) were sequentially added, the reaction was stirred at room temperature for 36h, TLC detection was completed, the solvent was removed under reduced pressure, the resulting solid was redissolved in 150mL of a mixed solution (DCM: TFA 7:3), TLC detection was completed after stirring at room temperature for 1h, the solvent was removed by rotary evaporation under reduced pressure, and the product compound 3 was isolated by column chromatography in 67.49% yield and 99.69%.
Intermediate III: 2,4,6-trichloropyrimidine (compound 1) (55.03g, 300mmol) is added into dichloromethane (300 ml) and stirred until dissolved, the temperature is reduced to 0-5 ℃ in an ice bath, 3-chloro-4-fluoroaniline (45.72g, 315mmol, compound 2) is slowly dripped in, after the dripping is finished, the ice bath is removed, the temperature is naturally raised to room temperature, after the TLC tracking detection reaction is finished, the decompression concentration is carried out, and the residue is separated by column chromatography to obtain an intermediate III, the yield is 86.37%, the HPLC is 99.83%, and white solid is obtained.
An intermediate II: adding intermediate III (29.25g, 100mmol) and compound 3 (36.46g, 110mmol) into N, N-dimethylformamide (150 ml), adding concentrated hydrochloric acid (1 ml), refluxing, detecting by TLC until the reaction is complete, concentrating under reduced pressure, and separating the residue by column chromatography to obtain intermediate II with yield of 68.53%, HPLC:99.77% and white solid.
TM-7: adding the intermediate II (7.05g, 12mmol), concentrated hydrochloric acid (0.5 ml) and acrylamide (1.02g, 14.4mmol) into N, N-dimethylformamide (20 ml), placing in a closed microwave reactor, setting the temperature at 140 ℃, finishing the reaction after 0.5h, cooling to room temperature, adding 200ml of ethyl acetate and 200ml of water respectively, extracting, separating, washing an organic phase once with water, drying with a proper amount of anhydrous sodium sulfate, concentrating under reduced pressure, and separating residues by column chromatography to obtain the compound TM-7 of the formula I, wherein the yield is 77.62%, and the HPLC (high performance liquid chromatography) is 99.82%.
1 H NMR(400MHz,DMSO-d 6 )δ：10.89(s,1H),10.40(s,1H),8.81(s,1H),7.57(d,J＝6.4Hz 2H),7.28～7.11(m,5H),6.74(s,1H),6.50(t,J＝10.6Hz,1H),6.04(d,J＝6.4Hz,2H),4.70(s,1H),4.18(s,2H)；3.11(m,4H)；2.74(m,1H)；2.45(m,8H)；2.36(s,3H)；1.82(m,4H)； 13 C-NMR(400MHz,DMSO-d 6 ):δ171.5,170.7,168.6,159.4,148.8,137.3,134.1,130.4,129.3,127.1,126.4,122.7,120.6(2C),118.4,118.3,117.7(2C),114.1,72.4,71.1,63.4(2C),56.7(2C),54.3(2C),53.3,45.3,29.4(2C)，MS-ESI m/z:622.14(M+H) + 。
Example 8 preparation of TM-8
Figure GDA0004064996830000331
Compound 3: adding tert-butoxycarbonyl protected glycine (21.03g, 120mmol) into dichloromethane (40 mL), sequentially adding EDCI (25.31g, 132mmol), HOBt (17.82g, 132mmol) and 2-aminobenzimidazole (17.57g, 132mmol), stirring at room temperature for 24h, detecting by TLC, removing the solvent under reduced pressure, re-dissolving the obtained solid in 120mL of mixed solution (DCM: TFA = 7:3), stirring at room temperature for 0.5h, detecting by TLC, removing the solvent by rotary evaporation under reduced pressure, and separating by column chromatography to obtain the product compound 3 with the yield of 68.51% and HPLC:99.62%.
An intermediate III: 2,4,6-trichloropyrimidine (compound 1) (55.03g, 300mmol) is added into dichloromethane (300 ml) and stirred until dissolved, the temperature is reduced to 0-5 ℃ in an ice bath, 3-chloro-4-fluoroaniline (45.72g, 315mmol, compound 2) is slowly dripped in, after the dripping is finished, the ice bath is removed, the temperature is naturally raised to room temperature, after the TLC tracking detection reaction is finished, the decompression concentration is carried out, the residue is separated by column chromatography to obtain an intermediate III, the yield is 85.32%, HPLC is 99.82%, and white solid is obtained.
An intermediate II: adding the intermediate III (29.25g, 100mmol) and the compound 3 (20.92g, 110mmol) into N, N-dimethylformamide (145.5 ml), adding concentrated hydrochloric acid (1 ml), refluxing, detecting by TLC until the reaction is complete, concentrating under reduced pressure, and separating the residue by column chromatography to obtain the intermediate II with the yield of 66.25%, HPLC:99.79% and white solid.
TM-8: adding the intermediate II (5.36g, 12mmol), concentrated hydrochloric acid (0.5 ml) and acrylamide (1.02g, 14.4 mmol) into n-octanol (20 ml), placing the n-octanol in a closed microwave reactor, setting the temperature to 140 ℃, finishing the reaction after 1.5h, cooling to room temperature, adding 200ml of each of ethyl acetate and water, extracting, separating liquid, washing an organic phase once with water, drying with a proper amount of anhydrous sodium sulfate, concentrating under reduced pressure, and carrying out column chromatography separation on residues to obtain a compound TM-8 of the formula I, namely a white solid, wherein the yield is 79.13% and the HPLC (high performance liquid chromatography) is 99.86%.
1 H NMR(400MHz,DMSO-d 6 )δ：11.10(s,1H),10.70(s,1H),8.75(s,1H),7.30～7.19(m,7H),6.47(s,1H),6.52(t,J＝10.6Hz,1H),6.06(d,J＝6.4Hz,2H),5.40(s,1H),4.75(s,1H),4.01(s,2H)； 13 C-NMR(400MHz,DMSO-d 6 ):δ171.4,170.3,168.5,162.1,159.6,148.7,146.4,137.2,136.8(2C),130.3,126.8,123.2(2C),122.5,118.7,118.4,115.5(2C),114.4,72.3,53.2，MS-ESI m/z:480.16(M+H) + 。
Example 9 preparation of TM-9
Figure GDA0004064996830000341
Compound 3: adding tert-butoxycarbonyl protected aminopropionic acid (22.68g, 120mmol) into dichloromethane (40 mL), sequentially adding EDCI (25.31g, 132mmol), HOBt (17.82g, 132mmol) and 4-methoxy-3-methylaniline (18.09g, 132mmol), stirring at room temperature for 24h, detecting by TLC, removing the solvent under reduced pressure, redissolving the obtained solid in 150mL of mixed solution (DCM: TFA = 7:3), stirring at room temperature for 1h, detecting by TLC, removing the solvent by rotary evaporation under reduced pressure, and separating by column chromatography to obtain the product compound 3 with yield of 72.56% and HPLC:99.64%.
An intermediate III: 2,4,6-trichloropyrimidine (compound 1) (55.03g, 300mmol) is added into dichloromethane (300 ml) and stirred until dissolved, the temperature is reduced to 0-5 ℃ in an ice bath, 3-chloro-4-fluoroaniline (45.72g, 315mmol, compound 2) is slowly dripped in, after the dripping is finished, the ice bath is removed, the temperature is naturally raised to room temperature, after the TLC tracking detection reaction is finished, the decompression concentration is carried out, the residue is separated by column chromatography to obtain an intermediate III, the yield is 86.35%, the HPLC is 99.85%, and white solid is obtained.
An intermediate II: adding the intermediate III (29.25g, 100mmol) and the compound 3 (22.91g, 110mmol) into isopropanol (150 ml), adding concentrated hydrochloric acid (1 ml), refluxing, tracking by TLC detection until the reaction is complete, concentrating under reduced pressure, and separating the residue by column chromatography to obtain the intermediate II with the yield of 66.47%, HPLC (high performance liquid chromatography) of 99.79% and white solid.
TM-9: adding the intermediate II (5.57g, 12mmol), concentrated hydrochloric acid (0.5 ml) and acrylamide (1.02g, 14.4 mmol) into n-octanol (20 ml), placing the n-octanol in a closed microwave reactor, setting the temperature to 140 ℃, finishing the reaction after 1.5h, cooling to room temperature, adding 200ml of each of ethyl acetate and water, extracting and separating the liquid, washing an organic phase once by using water, drying by using a proper amount of anhydrous sodium sulfate, concentrating under reduced pressure, and carrying out column chromatography separation on the residue to obtain a compound TM-9 of the formula I, namely a white solid, wherein the yield is 79.21 percent and the HPLC (high performance liquid chromatography) is 99.86 percent.
1 H NMR(400MHz,DMSO-d 6 )δ：11.08(s,1H),10.24(s,1H),8.70(s,1H),7.55(s,1H),7.28～7.16(m,5H),6.69(s,1H),6.50(dd,J＝10.6,8.8Hz,1H),5.87(d,J＝6.4Hz,2H),4.64(s,1H),3.98～3.80(m,2H)；3.34(s,3H),2.68～2.54(m,2H)；2.18(s,3H)； 13 C-NMR(400MHz,DMSO-d 6 ):δ173.0,168.8,167.9,162.4,158.9,149.8,137.7,135.5,130.8,128.3,125.7,123.0,122.5,121.5,119.4,118.9,118.6,115.3,114.6,72.2,56.6,39.7,35.5,19.9，MS-ESI m/z:498.16(M+H) + 。
Example 10 preparation of TM-14
Figure GDA0004064996830000351
Compound 3: adding tert-butoxycarbonyl protected aminopropionic acid (22.68g, 120mmol) into dichloromethane (30 mL), sequentially adding EDCI (25.3g, 132mmol), HOBt (17.82g, 132mmol) and 2-aminopyridine (12.42g, 132mmol), stirring at room temperature for 24h, detecting by TLC, removing the solvent under reduced pressure, re-dissolving the obtained solid in 120mL of mixed solution (DCM: TFA = 7:3), stirring at room temperature for 0.5h, detecting by TLC, removing the solvent by rotary evaporation under reduced pressure, and separating by column chromatography to obtain the product compound 3 with yield of 71.06% and HPLC of 99.67%.
Intermediate III: 2,4,6-trichloropyrimidine (compound 1) (55.03g, 300mmol) is added into dichloromethane (300 ml) and stirred until dissolved, the temperature is reduced to 0-5 ℃ in an ice bath, 3-chloro-4-fluoroaniline (45.72g, 315mmol, compound 2) is slowly dripped in, after the dripping is finished, the ice bath is removed, the temperature is naturally raised to room temperature, after the TLC tracking detection reaction is finished, the pressure is reduced and the concentration is carried out, and the residue is separated by column chromatography to obtain an intermediate III, the yield is 85.34%, HPLC is 99.83%, and white solid is obtained.
An intermediate II: adding the intermediate III (29.25g, 100mmol) and the compound 3 (18.17g, 110mmol) into ethanol (150 ml), adding concentrated hydrochloric acid (1 ml), refluxing, detecting by TLC until the reaction is complete, concentrating under reduced pressure, and separating the residue by column chromatography to obtain the intermediate II with the yield of 66.47%, HPLC (high performance liquid chromatography) of 99.77% and white solid.
TM-14: adding the intermediate II (5.06g, 12mmol), concentrated hydrochloric acid (0.5 ml) and acrylamide (1.02g, 14.4 mmol) into n-octanol (20 ml), placing the n-octanol into a closed microwave reactor, setting the temperature to 140 ℃, finishing the reaction after 1.5h, cooling to room temperature, adding 200ml of each of ethyl acetate and water, extracting and separating the liquid, washing an organic phase once by using water, drying by using a proper amount of anhydrous sodium sulfate, concentrating under reduced pressure, and carrying out column chromatography separation on the residue to obtain a compound TM-14 of the formula I, wherein the yield is 73.65 percent and the HPLC (high performance liquid chromatography) is 99.82 percent.
1 H NMR(400MHz,DMSO-d 6 )δ：11.17(s,1H),10.41(s,1H),8.92(s,1H),8.39(d,J＝20.6Hz 1H),8.17(d,J＝14.4Hz 1H),8.05(dd,J＝7.8Hz 1H),7.35～7.18(m,4H),6.68(s,1H),6.47(t,J＝10.6Hz,1H),6.02(d,J＝6.4Hz,2H),4.74(s,1H),3.98(m,2H),2.74(m,2H)； 13 C-NMR(400MHz,DMSO-d 6 ):δ171.2,170.8,168.4,162.9,159.5,152.8,148.7,145.7,139.8,134.7,130.2,126.6,124.4,122.8,118.5,118.7(2C),114.4,72.6,53.7,37.8，MS-ESI m/z:455.13(M+H) + 。
Example 11 preparation of TM-17
Figure GDA0004064996830000361
Compound 3: adding tert-butoxycarbonyl protected p-aminobenzoic acid (28.56g, 120mmol) into dichloromethane (30 mL), sequentially adding EDCI (25.31g, 132mmol), HOBt (17.82g, 132mmol) and N, N-dimethylethylenediamine (11.63g, 132mmol), stirring at room temperature for 24h, detecting by TLC, removing the solvent under reduced pressure, redissolving the obtained solid in 120mL of mixed solution (DCM: TFA = 7:3), stirring at room temperature for 0.5h, detecting by TLC, removing the solvent by rotary evaporation under reduced pressure, and separating by column chromatography to obtain the product compound 3 with the yield of 72.34% and HPLC of 99.76%.
An intermediate III: 2,4,6-trichloropyrimidine (compound 1) (55.03g, 300mmol) is added into dichloromethane (300 ml) and stirred until dissolved, the temperature is reduced to 0-5 ℃ in an ice bath, 3-chloro-4-fluoroaniline (45.72g, 315mmol, compound 2) is slowly dripped in, after the dripping is finished, the ice bath is removed, the temperature is naturally raised to room temperature, after the TLC tracking detection reaction is finished, the decompression and the concentration are carried out, and the residue is separated by column chromatography to obtain an intermediate III, the yield is 84.69%, HPLC (high performance liquid chromatography) 99.76% and white solid.
An intermediate II: adding the intermediate III (29.25g, 100mmol) and the compound 3 (21.26g, 110mmol) into N, N-dimethylformamide (120 ml), adding concentrated hydrochloric acid (1 ml), refluxing, detecting by TLC until the reaction is complete, concentrating under reduced pressure, and separating the residue by column chromatography to obtain the intermediate II with yield of 65.79%, HPLC:99.79% and white solid.
TM-17: adding the intermediate II (5.39g, 12mmol), concentrated hydrochloric acid (0.5 ml) and acrylamide (1.02g, 14.4 mmol) into dimethyl sulfoxide (20 ml), placing the mixture into a closed microwave reactor, setting the temperature to 140 ℃, finishing the reaction after 2.5h, cooling to room temperature, adding 200ml of each of ethyl acetate and water, extracting and separating the solution, washing an organic phase once by using water, drying by using a proper amount of anhydrous sodium sulfate, concentrating under reduced pressure, and carrying out column chromatography separation on residues to obtain a compound TM-17 of the formula I, wherein the yield is 74.36 percent and the HPLC (high performance liquid chromatography) is 99.80 percent.
1 H NMR(400MHz,DMSO-d 6 )δ：11.06(s,1H),10.12(s,1H),8.72(s,1H),7.35～7.18(m,7H),6.67(s,1H),6.42(t,J＝10.6Hz,1H),6.03(d,J＝6.4Hz,2H),4.75(s,1H),4.27(s,2H),2.27(m,4H)； 13 C-NMR(400MHz,DMSO-d 6 ):δ172.1,167.2(2C),166.1,159.6,152.3,139.7,130.9,129.2,128.3(2C),124.6,124.1,121.2,117.1(2C),116.2(2C),118.6,72.1,69.3,42.1(2C),MS-ESI m/z:483.56(M+H) + 。
Example 12 preparation of TM-18
Figure GDA0004064996830000371
Compound 3: adding tert-butoxycarbonyl protected p-aminobenzoic acid (28.56g, 120mmol) into dichloromethane (40 mL), sequentially adding EDCI (25.31g, 132mmol), HOBt (17.82g, 132mmol) and cyclohexylamine (13.09g, 132mmol), stirring at room temperature for 24h, detecting by TLC, removing solvent under reduced pressure, re-dissolving the obtained solid in 120mL of mixed solution (DCM: TFA = 7:3), stirring at room temperature for 0.5h, detecting by TLC, removing solvent by rotary evaporation under reduced pressure, and separating by column chromatography to obtain the product compound 3 with yield of 71.62% and HPLC of 99.79%.
An intermediate III: adding 2,4,6-trichloropyrimidine (compound 1) (55.03g, 300mmol) into ethyl acetate (300 ml), stirring until dissolving, cooling to 0-5 ℃ in an ice bath, slowly dropwise adding 3-chloro-4-fluoroaniline (45.72g, 315mmol, compound 2), removing the ice bath after dropwise adding, naturally heating to room temperature, after TLC tracking detection reaction is finished, concentrating under reduced pressure, and carrying out column chromatography separation on residues to obtain an intermediate III with yield of 83.57%, HPLC (high performance liquid chromatography) of 99.81% and a white solid.
An intermediate II: adding the intermediate III (29.25g, 100mmol) and the compound 3 (24.01g, 110mmol) into N, N-dimethylformamide (120 ml), adding concentrated hydrochloric acid (1 ml), refluxing, detecting by TLC until the reaction is complete, concentrating under reduced pressure, and separating the residue by column chromatography to obtain the intermediate II with yield of 61.37%, HPLC (high performance liquid chromatography) of 99.73% and white solid.
TM-18: adding the intermediate II (5.54g, 12mmol), concentrated hydrochloric acid (0.5 ml) and acrylamide (1.02g, 14.4 mmol) into n-octanol (20 ml), placing the n-octanol in a closed microwave reactor, setting the temperature to 140 ℃, finishing the reaction after 2.5h, cooling to room temperature, adding 200ml of each of ethyl acetate and water, extracting and separating the liquid, washing the organic phase once with water, drying with a proper amount of anhydrous sodium sulfate, concentrating under reduced pressure, and carrying out column chromatography separation on the residue to obtain a compound TM-18 of the formula I, wherein the yield is 76.25 percent and the HPLC is 99.87 percent.
1 H NMR(400MHz,DMSO-d 6 )δ：11.09(s,1H),10.36(s,1H),8.83(s,1H),7.35～7.18(m,7H),6.65(s,1H),6.43(t,J＝10.6Hz,1H),6.06(d,J＝6.4Hz,2H),4.72(s,1H),1.83～1.49(m,10H)； 13 C-NMR(400MHz,DMSO-d 6 ):δ172.1,167.4(3C),166.2,159.6,152.5,146.3,139,8,131.3,129.4,128.4,121.3,118.6,116.1(3C),117.2,72.3，47.7,33.6(2C),28.2,22.7(2C),MS-ESI m/z:505.32(M+H) + 。
Example 13 preparation of TM-20
Figure GDA0004064996830000381
Compound 3: adding tert-butoxycarbonyl protected p-aminobenzoic acid (28.56g, 120mmol) into dichloromethane (30 mL), sequentially adding EDCI (25.31g, 132mmol), HOBt (17.82g, 132mmol) and 2-aminopyridine (12.42g, 132mmol), stirring at room temperature for 24h, detecting by TLC, removing the solvent under reduced pressure, re-dissolving the obtained solid in 120mL of mixed solution (DCM: TFA = 7:3), stirring at room temperature for 0.5h, detecting by TLC, removing the solvent by rotary evaporation under reduced pressure, and separating by column chromatography to obtain the product compound 3 with the yield of 75.49% and HPLC 99.78%.
Intermediate III: 2,4,6-trichloropyrimidine (compound 1) (55.03g, 300mmol) is added into ethyl acetate (300 ml) and stirred until dissolved, the temperature is reduced to 0-5 ℃ in an ice bath, 3-chloro-4-fluoroaniline (45.72g, 315mmol, compound 2) is slowly dripped in, the ice bath is removed after the dripping is finished, the temperature is naturally raised to room temperature, after the TLC tracking detection reaction is finished, the pressure is reduced and the concentration is carried out, and the residue is separated by column chromatography to obtain an intermediate III, the yield is 82.59%, HPLC is 99.83%, and white solid is obtained.
An intermediate II: adding intermediate III (29.25g, 100mmol) and compound 3 (23.46g, 110mmol) into isopropanol (150 ml), adding concentrated hydrochloric acid (1 ml), refluxing, TLC detecting until the reaction is complete, concentrating under reduced pressure, and separating the residue by column chromatography to obtain intermediate II with yield of 65.33%, HPLC:99.81% and white solid.
TM-20: adding the intermediate II (5.63g, 12mmol), concentrated hydrochloric acid (0.5 ml) and acrylamide (1.02g, 14.4 mmol) into n-octanol (20 ml), placing the n-octanol into a closed microwave reactor, setting the temperature to 140 ℃, finishing the reaction after 1.5h, cooling to room temperature, adding 200ml of each of ethyl acetate and water, extracting and separating the liquid, washing the organic phase once by using water, drying the organic phase by using a proper amount of anhydrous sodium sulfate, concentrating the organic phase under reduced pressure, and separating the residue by column chromatography to obtain the compound TM-20 of the formula I, wherein the yield is 78.65 percent and the HPLC (high performance liquid chromatography) is 99.83 percent.
1 H NMR(400MHz,DMSO-d 6 )δ：11.03(s,1H),10.20(s,1H),8.74(s,1H),8.12(s,1H),7.71～7.54(m,3H),7.45～7.38(m,2H),7.25～7.17(m,5H),6.71(s,1H),6.55(dd,J＝10.6,8.8Hz,1H),5.82(d,J＝6.4Hz,2H),4.59(s,1H),. 13 C-NMR(400MHz,DMSO-d 6 ):172.8,168.8,167.7,162.8,158.6,149.7,145.7,142.4,137.5,135.7,131.2(2C),130.4,128.5,125.8,124.3,123.4,122.3,121.2,118.4,115.1,114.8,113.5(2C),72.4,MS-ESI m/z:503.13(M+H) + 。
Example 14 preparation of TM-21
Figure GDA0004064996830000391
Compound 3: adding tert-butoxycarbonyl protected p-aminobenzoic acid (28.56g, 120mmol) into dichloromethane (50 mL), sequentially adding EDCI (25.31g, 132mmol), HOBt (17.82g, 132mmol) and 4-methoxy-3-methylaniline (18.09g, 132mmol), stirring at room temperature for 24h, detecting by TLC, removing the solvent under reduced pressure, redissolving the obtained solid in 150mL of mixed solution (DCM: TFA = 7:3), stirring at room temperature for 1h, detecting by TLC, removing the solvent by rotary evaporation under reduced pressure, and separating by column chromatography to obtain the product compound 3 with yield of 76.35% and HPLC:99.81%.
Intermediate III: 2,4,6-trichloropyrimidine (compound 1) (55.03g, 300mmol) is added into dichloromethane (300 ml) and stirred until dissolved, the temperature is reduced to 0-5 ℃ in an ice bath, 3-chloro-4-fluoroaniline (45.72g, 315mmol, compound 2) is slowly dripped in, after the dripping is finished, the ice bath is removed, the temperature is naturally raised to room temperature, after the TLC tracking detection reaction is finished, the decompression concentration is carried out, the residue is separated by column chromatography to obtain an intermediate III, the yield is 83.79%, the HPLC is 99.80%, and white solid is obtained.
An intermediate II: adding intermediate III (29.25g, 100mmol) and compound 3 (28.17g, 110mmol) into isopropanol (150 ml), adding concentrated hydrochloric acid (1 ml), refluxing, detecting by TLC until the reaction is complete, concentrating under reduced pressure, and separating the residue by column chromatography to obtain intermediate II with yield of 61.37%, HPLC of 99.82% and white solid.
TM-21: adding the intermediate II (6.15g, 12mmol), concentrated hydrochloric acid (0.5 ml) and acrylamide (1.02g, 14.4 mmol) into n-octanol (20 ml), placing the n-octanol in a closed microwave reactor, setting the temperature to 140 ℃, finishing the reaction after 1.5h, cooling to room temperature, adding 200ml of each of ethyl acetate and water, extracting and separating the solution, washing the organic phase once with water, drying with a proper amount of anhydrous sodium sulfate, concentrating under reduced pressure, and carrying out column chromatography separation on the residue to obtain the compound TM-21 of the formula I, namely a white solid with the yield of 76.52% and the HPLC (99.85%).
1 H NMR(400MHz,DMSO-d 6 )δ：11.03(s,1H),10.20(s,1H),8.74(s,1H),7.71～7.54(m,4H),7.45(s,1H),7.25～7.17(m,5H),6.71(s,1H),6.55(dd,J＝10.6,8.8Hz,1H),5.82(d,J＝6.4Hz,2H),4.59(s,1H),3.37(s,3H),2.15(s,3H).13C-NMR(400MHz,DMSO-d6):172.8,168.8,167.7,162.8,158.6,149.7,142.4,137.5,135.7,131.2(2C),130.4,128.5,125.8,124.3,123.4,122.3,121.2,119.5,118.7,118.4,115.1,114.8,113.5(2C),72.4,56.8,19.7，MS-ESI m/z:546.16(M+H) + 。
Example 15 preparation of TM-23
Figure GDA0004064996830000401
Compound 3: adding tert-butoxycarbonyl protected p-aminobenzoic acid (28.56g, 120mmol) into dichloromethane (40 mL), sequentially adding EDCI (25.31g, 132mmol), HOBt (17.82g, 132mmol) and 2-aminobenzimidazole (17.57g, 132mmol), stirring at room temperature for 24h, detecting by TLC, removing the solvent under reduced pressure, re-dissolving the obtained solid in 150mL of mixed solution (DCM: TFA = 7:3), stirring at room temperature for 0.5h, detecting by TLC, removing the solvent by rotary evaporation under reduced pressure, and separating by column chromatography to obtain the product compound 3 with yield of 74.32% and HPLC of 99.79%.
Intermediate III: 2,4,6-trichloropyrimidine (compound 1) (55.03g, 300mmol) is added into dichloromethane (300 ml) and stirred until dissolved, the temperature is reduced to 0-5 ℃ in an ice bath, 3-chloro-4-fluoroaniline (45.72g, 315mmol, compound 2) is slowly dripped in, after the dripping is finished, the ice bath is removed, the temperature is naturally raised to room temperature, after the TLC tracking detection reaction is finished, the pressure is reduced and the concentration is carried out, and the residue is separated by column chromatography to obtain an intermediate III, the yield is 85.49%, HPLC is 99.83%, and white solid is obtained.
An intermediate II: adding the intermediate III (29.25g, 100mmol) and the compound 3 (27.75g, 110mmol) into N, N-dimethylformamide (150 ml), adding concentrated hydrochloric acid (1 ml), refluxing, detecting by TLC until the reaction is complete, concentrating under reduced pressure, and separating the residue by column chromatography to obtain the intermediate II with the yield of 65.37%, HPLC:99.79% and white solid.
TM-23: adding the intermediate II (6.52g, 12mmol), concentrated hydrochloric acid (0.5 ml) and acrylamide (1.02g, 14.4mmol) into N, N-dimethylformamide (20 ml), placing in a closed microwave reactor, setting the temperature to 150 ℃, finishing the reaction after 1.5h, cooling to room temperature, adding 200ml of ethyl acetate and 200ml of water respectively, extracting, separating, washing an organic phase once with water, drying with a proper amount of anhydrous sodium sulfate, concentrating under reduced pressure, and separating residues by column chromatography to obtain a compound TM-23 of the formula I, which is a white solid, with the yield of 74.39% and the HPLC rate of 99.86%.
1 H NMR(400MHz,DMSO-d 6 )δ：11.13(s,1H),10.22(s,1H),8.76(s,1H),7.71～7.52(m,4H),7.45(s,1H),7.25～7.17(m,7H),6.73(s,1H),6.56(dd,J＝10.6,8.8Hz,1H),5.87(d,J＝6.4Hz,2H),4.49(s,1H).13C-NMR(400MHz,DMSO-d6):171.5,167.2,166.5,164.8,159.3,152.8,147.7,146.5,140.6,138.9(2C),131.3,129.5,128.6(2C),124.6,123.6(2C),121.5,118.5,117.3,116.1(3C),115.1(3C),72.6，MS-ESI m/z:542.32(M+H) + 。
Example 16 preparation of TM-28
Figure GDA0004064996830000421
Compound 3: adding tert-butoxycarbonyl protected p-4-aminopiperazine-1-carboxylic acid (29.89g, 120mmol) to dichloromethane (30 mL), sequentially adding EDCI (25.31g, 132mmol), HOBt (17.82g, 132mmol) and aniline (12.29g, 132mmol), stirring at room temperature for 24h, detecting by TLC, removing the solvent under reduced pressure, redissolving the obtained solid in 120mL of mixed solution (DCM: TFA = 7:3), stirring at room temperature for 1h, detecting by TLC, removing the solvent by rotary evaporation under reduced pressure, and separating by column chromatography to obtain the product compound 3 with the yield of 76.85% and HPLC:99.81%.
Intermediate III: 2,4,6-trichloropyrimidine (compound 1) (55.03g, 300mmol) is added into ethyl acetate (300 ml) and stirred until dissolved, the temperature is reduced to 0-5 ℃ in an ice bath, 3-chloro-4-fluoroaniline (45.72g, 315mmol, compound 2) is slowly dripped in, the ice bath is removed after the dripping is finished, the temperature is naturally raised to room temperature, after the TLC tracking detection reaction is finished, the pressure is reduced and the concentration is carried out, and the residue is separated by column chromatography to obtain an intermediate III, the yield is 83.47%, HPLC is 99.81%, and white solid is obtained.
An intermediate II: adding the intermediate III (29.25g, 100mmol) and the compound 3 (24.23g, 110mmol) into isopropanol (150 ml), adding concentrated hydrochloric acid (1 ml), refluxing, tracking by TLC detection until the reaction is complete, concentrating under reduced pressure, and separating the residue by column chromatography to obtain the intermediate II with the yield of 66.52%, HPLC (high performance liquid chromatography) of 99.82% and white solid.
TM-28: adding the intermediate II (5.72g, 12mmol), concentrated hydrochloric acid (0.5 ml) and acrylamide (1.02g, 14.4 mmol) into n-octanol (20 ml), placing the n-octanol in a closed microwave reactor, setting the temperature to 140 ℃, finishing the reaction after 2.5h, cooling to room temperature, adding 200ml of each of ethyl acetate and water, extracting and separating the liquid, washing the organic phase once with water, drying with a proper amount of anhydrous sodium sulfate, concentrating under reduced pressure, and carrying out column chromatography separation on the residue to obtain the compound TM-28 of the formula I, wherein the yield is 76.21 percent and the HPLC is 99.83 percent.
1 H NMR(400MHz,DMSO-d 6 )δ：11.18(s,1H),10.09(s,1H),8.56(s,1H),7.64～7.47(m,5H),7.45(s,1H),7.23～7.15(m,4H),6.69(s,1H),6.52(dd,J＝10.6,8.8Hz,1H),5.76(d,J＝6.4Hz,2H),4.49(s,1H),3.26(s,4H),2.75(s,4H).13C-NMR(400MHz,DMSO-d6):172.5,166.9,159.4,157.2,155.6,152.7,140.6,135.7,131.7,129.5(2C),121.3(3C),118.5,117.8,116.5,77.6,56.1(2C),47.5(2C)，MS-ESI m/z:510.43(M+H) + 。
Example 17 preparation of TM-29
Figure GDA0004064996830000431
Compound 3: boc-protected p-4-aminopiperazine-1-carboxylic acid (29.89g, 120mmol) was added to dichloromethane (50 mL), EDCI (25.31g, 132mmol), HOBt (17.8g, 132mmol) and 4-methoxy-3-methylaniline (18.09g, 132mmol) were sequentially added, the reaction was stirred at room temperature for 24h, TLC detection was completed, the solvent was removed under reduced pressure, the resulting solid was redissolved in 150mL of a mixed solution (DCM: TFA = 8978 xft 8978), after stirring at room temperature for 1h, TLC detection was completed, the solvent was removed by rotary evaporation under reduced pressure, and the product compound 3 was isolated by column chromatography in a yield of 75.32%, HPLC:99.78%.
An intermediate III: 2,4,6-trichloropyrimidine (compound 1) (55.03g, 300mmol) is added into dichloromethane (300 ml) and stirred until dissolved, the temperature is reduced to 0-5 ℃ in an ice bath, 3-chloro-4-fluoroaniline (45.72g, 315mmol, compound 2) is slowly dripped in, after the dripping is finished, the ice bath is removed, the temperature is naturally raised to room temperature, after the TLC tracking detection reaction is finished, the decompression concentration is carried out, the residue is separated by column chromatography to obtain an intermediate III, the yield is 85.34%, HPLC is 99.86%, and white solid is obtained.
An intermediate II: adding the intermediate III (29.25g, 100mmol) and the compound 3 (29.08g, 110mmol) into isopropanol (145.5 ml), adding concentrated hydrochloric acid (1 ml), refluxing, detecting and tracking by TLC until the reaction is complete, concentrating under reduced pressure, and separating the residue by column chromatography to obtain the intermediate II with yield of 67.35%, HPLC (high performance liquid chromatography) of 99.85% and white solid.
TM-29: adding the intermediate II (6.24g, 12mmol), concentrated hydrochloric acid (0.5 ml) and acrylamide (1.02g, 14.4 mmol) into n-octanol (20 ml), placing the n-octanol in a closed microwave reactor, setting the temperature to 140 ℃, finishing the reaction after 1h, cooling to room temperature, adding 200ml of each of ethyl acetate and water, extracting, separating liquid, washing an organic phase once by using water, drying by using a proper amount of anhydrous sodium sulfate, concentrating under reduced pressure, and carrying out column chromatography separation on residues to obtain a compound TM-29 of the formula I, wherein the yield is 78.65 percent and the HPLC (high performance liquid chromatography) is 99.86 percent.
1 H NMR(400MHz,DMSO-d 6 )δ：11.03(s,1H),10.20(s,1H),8.74(s,1H),7.71～7.54(m,2H),7.45(s,1H),7.25～7.17(m,3H),6.71(s,1H),6.55(dd,J＝10.6,8.8Hz,1H),5.82(d,J＝6.4Hz,2H),4.59(s,1H),3.64～3.57(m,4H),3.37(s,3H),2.83～3.79(m,4H),2.15(s,3H).13C-NMR(400MHz,DMSO-d6):172.8,168.8,167.7,162.8,158.6,149.7,142.4,137.5,135.7,130.4,128.5,125.8,124.3,123.4,122.3,121.2,119.5,118.7,118.4,115.1,114.8,72.4,58.9(2C),56.8,44.8(2C),19.7，MS-ESI m/z:554.20(M+H) + 。
Example 18 preparation of TM-30
Figure GDA0004064996830000441
Compound 3: adding tert-butoxycarbonyl protected p-4-aminopiperazine-1-carboxylic acid (29.89g, 120mmol) into dichloromethane (30 mL), sequentially adding EDCI (25.31g, 132mmol) and HOBt (17.82g, 132mmol) 2-aminopyridine (12.42g, 132mmol), stirring at room temperature for 24h, detecting by TLC, removing the solvent under reduced pressure, redissolving the obtained solid in 120mL of mixed solution (DCM: TFA = 7:3), stirring at room temperature for 0.5h, detecting by TLC, removing the solvent by reduced pressure rotary evaporation, and separating by column chromatography to obtain the product compound 3 with the yield of 77.61% and HPLC:99.82%.
Intermediate III: 2,4,6-trichloropyrimidine (compound 1) (55.03g, 300mmol) is added into dichloromethane (300 ml) and stirred until dissolved, the temperature is reduced to 0-5 ℃ in an ice bath, 3-chloro-4-fluoroaniline (45.72g, 315mmol, compound 2) is slowly dripped in, after the dripping is finished, the ice bath is removed, the temperature is naturally raised to room temperature, after the TLC tracking detection reaction is finished, the decompression concentration is carried out, the residue is separated by column chromatography to obtain an intermediate III, the yield is 86.53%, the HPLC is 99.82%, and white solid is obtained.
An intermediate II: adding intermediate III (29.25g, 100mmol) and compound 3 (24.33g, 110mmol) into isopropanol (145.5 ml), adding concentrated hydrochloric acid (1 ml), refluxing, detecting by TLC until the reaction is complete, concentrating under reduced pressure, and separating the residue by column chromatography to obtain intermediate II with yield of 69.51%, HPLC:99.82% and white solid.
TM-30: adding the intermediate II (5.73g, 12mmol), concentrated hydrochloric acid (0.5 ml) and acrylamide (1.02g, 14.4 mmol) into n-octanol (20 ml), placing in a closed microwave reactor, setting the temperature at 140 ℃, finishing the reaction after 1.5h, cooling to room temperature, adding 200ml of each of ethyl acetate and water, extracting, separating liquid, washing the organic phase once with water, drying with a proper amount of anhydrous sodium sulfate, concentrating under reduced pressure, and separating the residue by column chromatography to obtain the compound TM-30 of the formula I, namely a white solid with the yield of 76.33% and the HPLC (99.83%).
1 H NMR(400MHz,DMSO-d 6 )δ：10.96(s,1H),1.54(s,1H),8.56(s,1H),8.13(s,1H),7.61～7.51(m,3H),7.23～7.15(m,3H),6.70(s,1H),6.57(dd,J＝10.6,8.8Hz,1H),5.84(d,J＝6.4Hz,2H),4.52(s,1H),3.62～3.58(m,4H),2.84～2.77(m,4H).13C-NMR(400MHz,DMSO-d6):171.8,167.8,166.7,163.8,159.6,148.7,145.6,142.4,137.5,130.4,128.5,125.7,124.2,123.8,122.5,121.7,118.6,115.1,114.8,59.5(2C),48.4(2C)，MS-ESI m/z:511.16(M+H) + 。
Example 19 preparation of TM-32
Figure GDA0004064996830000451
Compound 3: adding tert-butoxycarbonyl protected p-3-aminopyrrolidine-1-carboxylic acid (26.53g, 120mmol) into dichloromethane (30 mL), sequentially adding EDCI (25.31g, 132mmol), HOBt (17.82g, 132mmol) and N, N-dimethylethylenediamine (11.64g, 132mmol), stirring at room temperature for 24h, detecting by TLC, removing the solvent under reduced pressure, re-dissolving the obtained solid in 120mL of mixed solution (DCM: TFA = 3238 zft 3238), stirring at room temperature for 0.5h, detecting by TLC, removing the solvent under reduced pressure, and separating by column chromatography to obtain the product compound 3 with a yield of 73.52% and HPLC:99.86%.
Intermediate III: 2,4,6-trichloropyrimidine (compound 1) (55.03g, 300mmol) is added into dichloromethane (300 ml) and stirred until dissolved, the temperature is reduced to 0-5 ℃ in an ice bath, 3-chloro-4-fluoroaniline (45.72g, 315mmol, compound 2) is slowly dripped, the ice bath is removed after the dripping is finished, the temperature is naturally raised to room temperature, after the TLC tracking detection reaction is finished, the pressure is reduced and the concentration is carried out, and the remainder is separated by column chromatography to obtain an intermediate III, the yield is 85.34%, HPLC (high performance liquid chromatography) is 99.80%, and white solid is obtained.
An intermediate II: adding the intermediate III (29.25g, 100mmol) and the compound 3 (20.49g, 110mmol) into N, N-dimethylformamide (150 ml), adding concentrated hydrochloric acid (1 ml), refluxing, TLC detecting and tracking till the reaction is complete, decompressing and concentrating, and separating the residue by column chromatography to obtain the intermediate II with the yield of 71.66%, HPLC (high performance liquid chromatography) 99.85% and white solid.
TM-32: adding the intermediate II (5.31g, 12mmol), concentrated hydrochloric acid (0.5 ml) and acrylamide (1.02g, 14.4 mmol) into N, N-dimethylformamide (20 ml), placing in a closed microwave reactor, setting the temperature to 140 ℃, finishing the reaction after 1.0h, cooling to room temperature, adding 200ml of ethyl acetate and water respectively, extracting, separating liquid, washing an organic phase once with water, drying with a proper amount of anhydrous sodium sulfate, concentrating under reduced pressure, and separating residues by column chromatography to obtain a compound TM-32 of the formula I, namely a white solid with the yield of 74.62% and the HPLC of 99.85%.
1 H NMR(400MHz,DMSO-d 6 )δ：10.79(s,1H),10.51(s,1H),8.63(s,1H),7.26～7.17(m,3H),6.72(s,1H),6.56(dd,J＝10.6,8.8Hz,1H),5.73(d,J＝6.4Hz,2H),4.51(s,1H),4.31(d,J＝5.7Hz,2H),3.62～3.40(m,4H),2..26(m,4H).1.62(d,J＝5.4Hz,2H).13C-NMR(400MHz,DMSO-d6):172.6,166.6,160.3,159.2,155.1,152.6,140.6,131.2,129.3,121.4,118.5,117.5,116.2,72.3，71.1,56.3,54.4,46.5,42.6(2C),34.8,MS-ESI m/z:476.25(M+H) + 。
Example 20 preparation of TM-35
Figure GDA0004064996830000461
Compound 3: adding tert-butoxycarbonyl protected p-3-aminopyrrolidine-1-carboxylic acid (26.53g, 120mmol) into dichloromethane (30 mL), adding EDCI (25.31g, 132mmol), HOBt (17.82g, 132mmol) and p-fluoroaniline (14.68g, 132mmol) in sequence, stirring at room temperature for 24h, detecting by TLC, removing the solvent under reduced pressure, re-dissolving the obtained solid in 120mL of mixed solution (DCM: TFA = 7:3), stirring at room temperature for 0.5h, detecting by TLC, removing the solvent by reduced pressure rotary evaporation, and separating by column chromatography to obtain the product compound 3 with the yield of 77.64% and HPLC:99.83%.
Intermediate III: 2,4,6-trichloropyrimidine (compound 1) (55.03g, 300mmol) is added into dichloromethane (300 ml) and stirred until dissolved, the temperature is reduced to 0-5 ℃ in an ice bath, 3-chloro-4-fluoroaniline (45.72g, 315mmol, compound 2) is slowly dripped in, after the dripping is finished, the ice bath is removed, the temperature is naturally raised to room temperature, after the TLC tracking detection reaction is finished, the decompression concentration is carried out, the residue is separated by column chromatography to obtain an intermediate III, the yield is 84.67%, HPLC is 99.86%, and white solid is obtained.
An intermediate II: adding the intermediate III (29.25g, 100mmol) and the compound 3 (24.56g, 110mmol) into isopropanol (150 ml), adding concentrated hydrochloric acid (1 ml), refluxing, detecting by TLC until the reaction is complete, concentrating under reduced pressure, and separating the residue by column chromatography to obtain the intermediate II with the yield of 72.91%, HPLC (high performance liquid chromatography) of 99.82% and white solid.
TM-35: adding the intermediate II (5.75g, 12mmol), concentrated hydrochloric acid (0.5 ml) and acrylamide (1.02g, 14.4 mmol) into n-octanol (20 ml), placing in a closed microwave reactor, setting the temperature at 140 ℃, finishing the reaction after 1.5h, cooling to room temperature, adding 200ml of each of ethyl acetate and water, extracting, separating liquid, washing the organic phase once with water, drying with a proper amount of anhydrous sodium sulfate, concentrating under reduced pressure, and separating the residue by column chromatography to obtain the compound TM-35 of the formula I, namely a white solid with the yield of 71.46% and the HPLC (99.87%).
1 H NMR(400MHz,DMSO-d 6 )δ：11.26(s,1H),10.63(s,1H),8.77(s,1H),7.53～7.15(m,7H),6.73(s,1H),6.56(dd,J＝10.6,8.8Hz,1H),5.79(d,J＝6.4Hz,2H),4.51(s,1H),4.32(d,J＝5.7Hz,2H),3.64～3.47(m,4H).13C-NMR(400MHz,DMSO-d6):172.5,166.8,160.2,158.6(2C),153.7,152.6,159.6,140.2,131.5(2C),129.2,123.5(2C),121.7,118.2,117.7,115.9(3C),116.2,72.3，56.7,54.3,46.3,34.5,MS-ESI m/z:513.52(M+H) + 。
Example 21 preparation of TM-36
Figure GDA0004064996830000471
Compound 3: tert-butoxycarbonyl protected p-3-aminopyrrolidine-1-carboxylic acid (26.53g, 120mmol) was added to dichloromethane (30 mL), EDCI (25.31g, 132mmol), HOBt (17.82g, 132mmol), 2-aminopyridine (12.42g, 132mmol) were added sequentially, the reaction stirred at room temperature for 36h, TLC detection was complete, the solvent was removed under reduced pressure, the resulting solid was redissolved in 120mL of mixed solution (DCM: TFA = 7:3), after stirring at room temperature for 0.5h, TLC detection was complete, the solvent was removed by rotary evaporation under reduced pressure, and the product compound 3 was isolated by column chromatography in 71.43% yield and HPLC:99.81%.
Intermediate III: 2,4,6-trichloropyrimidine (compound 1) (55.03g, 300mmol) is added into dichloromethane (300 ml) and stirred until dissolved, the temperature is reduced to 0-5 ℃ in an ice bath, 3-chloro-4-fluoroaniline (45.72g, 315mmol, compound 2) is slowly dripped in, after the dripping is finished, the ice bath is removed, the temperature is naturally raised to room temperature, after the TLC tracking detection reaction is finished, the pressure is reduced and the concentration is carried out, and the residue is separated by column chromatography to obtain an intermediate III, the yield is 85.62%, HPLC is 99.83%, and white solid is obtained.
An intermediate II: adding intermediate III (29.25g, 100mmol) and compound 3 (22.69g, 110mmol) into isopropanol (145.5 ml), adding concentrated hydrochloric acid (1 ml), refluxing, detecting by TLC until the reaction is complete, concentrating under reduced pressure, and separating the residue by column chromatography to obtain intermediate II with yield of 76.24%, HPLC:99.85% and white solid.
TM-36: adding the intermediate II (5.55g, 12mmol), concentrated hydrochloric acid (0.5 ml) and acrylamide (1.02g, 14.4 mmol) into n-octanol (20 ml), placing the n-octanol in a closed microwave reactor, setting the temperature to 140 ℃, finishing the reaction after 0.5h, cooling to room temperature, adding 200ml of each of ethyl acetate and water, extracting, separating liquid, washing an organic phase once with water, drying with a proper amount of anhydrous sodium sulfate, concentrating under reduced pressure, and carrying out column chromatography separation on the residue to obtain a compound TM-36 of the formula I, namely a white solid, wherein the yield is 75.31% and the HPLC (high performance liquid chromatography) is 99.87%.
1 H NMR(400MHz,DMSO-d 6 )δ：11.31(s,1H),10.43(s,1H),8.71(s,1H),8.10(s,1H),7.61～7.53(m,3H),7.15～7.10(m,3H),6.76(s,1H),6.52(dd,J＝10.6,8.8Hz,1H),5.53(d,J＝6.4Hz,2H),4.54(s,1H),3.75～3.59(m,4H),2.84～2.75(m,1H),2.12～1.88(m,2H).13C-NMR(400MHz,DMSO-d6):170.8,168.8,166.7,161.8,157.6,149.7,146.7,142.4,137.5,132.9,128.6,125.5,124.8,123.9,123.3,121.2,117.4,115.1,114.5,65.4,57.5,42.3,38.2，MS-ESI m/z:496.15(M+H) + 。
Example 22 preparation of TM-37
Figure GDA0004064996830000481
Compound 3: adding tert-butoxycarbonyl protected p-3-aminopyrrolidine-1-carboxylic acid (26.53g, 120mmol) into dichloromethane (50 mL), sequentially adding EDCI (25.31g, 132mmol), HOBt (17.82g, 132mmol) and 4-methoxy-3-methylaniline (18.09g, 132mmol), stirring at room temperature for reaction for 36h, detecting by TLC, removing the solvent under reduced pressure, re-dissolving the obtained solid in 150mL of mixed solution (DCM: TFA = 3238 zft 3238), stirring at room temperature for 0.5h, detecting by TLC under reduced pressure, removing the solvent by rotary evaporation, and separating by column chromatography to obtain the product compound 3 with a yield of 76.53%, HPLC:99.83%.
Intermediate III: 2,4,6-trichloropyrimidine (compound 1) (55.03g, 300mmol) is added into dichloromethane (300 ml) and stirred until dissolved, the temperature is reduced to 0-5 ℃ in an ice bath, 3-chloro-4-fluoroaniline (45.72g, 315mmol, compound 2) is slowly dripped in, after the dripping is finished, the ice bath is removed, the temperature is naturally raised to room temperature, after the TLC tracking detection reaction is finished, the decompression concentration is carried out, the residue is separated by column chromatography to obtain an intermediate III, the yield is 82.57%, HPLC is 99.88%, and white solid is obtained.
An intermediate II: adding the intermediate III (29.25g, 100mmol) and the compound 3 (27.42g, 110mmol) into N, N-dimethylformamide (145.5 ml), adding concentrated hydrochloric acid (1 ml), refluxing, detecting by TLC until the reaction is complete, concentrating under reduced pressure, and separating the residue by column chromatography to obtain the intermediate II with the yield of 77.36%, HPLC:99.87% and white solid.
TM-37: adding the intermediate II (6.06g, 12mmol), concentrated hydrochloric acid (0.5 ml) and acrylamide (1.02g, 14.4mmol) into n-octanol (20 ml), placing the n-octanol into a closed microwave reactor, setting the temperature to 140 ℃, finishing the reaction after 1h, cooling to room temperature, adding 200ml of each of ethyl acetate and water, extracting, separating, washing an organic phase once by using water, drying by using a proper amount of anhydrous sodium sulfate, concentrating under reduced pressure, and separating residues by column chromatography to obtain a compound TM-37 of the formula I, namely a white solid, wherein the yield is 79.56%, and the HPLC is 99.85%.
1 H NMR(400MHz,DMSO-d 6 )δ：11.08(s,1H),10.33(s,1H),8.71(s,1H),7.60～7.55(m,2H),7.42(s,1H),7.27～7.19(m,3H),6.71(s,1H),6.52(dd,J＝10.6,8.8Hz,1H),5.87(d,J＝6.4Hz,2H),4.53(s,1H),3.72～3.58(m,4H),3.367(s,3H),2.84～2.75(m,1H),2.16～1.75(m,2H),2.18(s,3H).13C-NMR(400MHz,DMSO-d6):171.8,168.3,166.7,162.2,158.4,149.6,142.2,136.5,135.2,130.1,128.7,125.2,124.5,123.7,122.3,121.8,119.8,118.5,116.1,115.8,74.1,66.4,57.5,41.3,37.2,19.3，MS-ESI m/z:539.18(M+H) + 。
Example 23 preparation of TM-40
Figure GDA0004064996830000491
Compound 3: adding tert-butoxycarbonyl protected p-4-aminocyclobutane-2-carboxylic acid (23.17g, 120mmol) into dichloromethane (50 mL), sequentially adding EDCI (25.31g, 132mmol), HOBt (17.82g, 132mmol) and 4-methoxy-3-methylaniline (18.09g, 132mmol), stirring at room temperature for 24h, detecting by TLC, removing the solvent under reduced pressure, re-dissolving the obtained solid in 150mL of mixed solution (DCM: TFA = 3238 zft 3238), stirring at room temperature for 0.5h, detecting by TLC, removing the solvent under reduced pressure, performing rotary evaporation, and performing column chromatography to obtain the product compound 3 with the yield of 3262 zft 3262% and HPLC:99.85%.
An intermediate III: 2,4,6-trichloropyrimidine (compound 1) (55.03g, 300mmol) is added into acetone (300 ml) and stirred until dissolved, the temperature is reduced to 0-5 ℃ in an ice bath, 3-chloro-4-fluoroaniline (45.72g, 315mmol, compound 2) is slowly dripped in, after the dripping is finished, the ice bath is removed, the temperature is naturally raised to room temperature, after the TLC tracking detection reaction is finished, the decompression concentration is carried out, and the residue is separated by column chromatography to obtain an intermediate III, the yield is 84.37%, the HPLC is 99.83%, and white solid is obtained.
An intermediate II: adding intermediate III (29.25g, 100mmol) and compound 3 (24.22g, 110mmol) into isopropanol (150 ml), adding concentrated hydrochloric acid (1 ml), refluxing, detecting by TLC until the reaction is complete, concentrating under reduced pressure, and separating the residue by column chromatography to obtain intermediate II with yield of 75.62%, HPLC:99.85% and white solid.
TM-40: adding the intermediate II (5.72g, 12mmol), concentrated hydrochloric acid (0.5 ml) and acrylamide (1.02g, 14.4 mmol) into N, N-dimethylformamide (20 ml), placing the mixture into a closed microwave reactor, setting the temperature to 140 ℃, finishing the reaction after 1.5h, cooling to room temperature, adding 200ml of ethyl acetate and water respectively, extracting and separating the solution, washing an organic phase once with water, drying the organic phase with a proper amount of anhydrous sodium sulfate, concentrating the organic phase under reduced pressure, and separating the residue by column chromatography to obtain a compound TM-40 of the formula I, namely a white solid with the yield of 77.26% and the HPLC (99.87%).
1 H NMR(400MHz,DMSO-d 6 )δ：11.21(s,1H),10.12(s,1H),8.71(s,1H),7.66～7.51(m,2H),7.49(s,1H),7.26～7.14(m,3H),6.78(s,1H),6.56(dd,J＝10.6,8.8Hz,1H),5.80(d,J＝6.4Hz,2H),4.55(s,1H),3.74～3.55(m,4H),3.36(s,3H),2.81～2.75(m,1H),2.09～1.75(m,2H),2.16(s,3H).13C-NMR(400MHz,DMSO-d6):172.4,167.8,166.7,160.8,157.5,148.6,143.4,138.5,136.7,134.4,128.2,125.7,124.6,123.5,122.4,121.8,119.3,118.3,115.7,114.2,72.5,65.7,58.6,42.5,38.3,19.5，MS-ESI m/z:510.15(M+H) + 。
Example 24 preparation of TM-43
Figure GDA0004064996830000501
Compound 3: adding tert-butoxycarbonyl protected p-4-aminocyclobutane-2-carboxylic acid (23.17g, 120mmol) into dichloromethane (30 mL), sequentially adding EDCI (25.31g, 132mmol), HOBt (17.82g, 132mmol) and 2-aminopyridine (12.42g, 132mmol), stirring at room temperature for 24h, detecting by TLC, removing the solvent under reduced pressure, redissolving the obtained solid in 120mL of mixed solution (DCM: TFA = 7:3), stirring at room temperature for 1h, detecting by TLC, removing the solvent by rotary evaporation under reduced pressure, and separating by column chromatography to obtain the product compound 3 with a yield of 73.62% and an HPLC of 99.83%.
Intermediate III: 2,4,6-trichloropyrimidine (compound 1) (55.03g, 300mmol) is added into dichloromethane (300 ml) and stirred until dissolved, the temperature is reduced to 0-5 ℃ in an ice bath, 3-chloro-4-fluoroaniline (45.72g, 315mmol, compound 2) is slowly dripped in, after the dripping is finished, the ice bath is removed, the temperature is naturally raised to room temperature, after the TLC tracking detection reaction is finished, the decompression and the concentration are carried out, and the residue is separated by column chromatography to obtain an intermediate III, the yield is 85.67%, HPLC (high performance liquid chromatography) 99.87% and white solid.
An intermediate II: adding the intermediate III (29.25g, 100mmol) and the compound 3 (19.49g, 110mmol) into isopropanol (145.5 ml), adding concentrated hydrochloric acid (1 ml), refluxing, detecting by TLC until the reaction is complete, concentrating under reduced pressure, and separating the residue by column chromatography to obtain the intermediate II with the yield of 76.29%, HPLC (high performance liquid chromatography) 99.82% and white solid.
TM-43: adding the intermediate II (5.20g, 12mmol), concentrated hydrochloric acid (0.5 ml) and acrylamide (1.02g, 14.4mmol) into N, N-dimethylformamide (20 ml), placing in a closed microwave reactor, setting the temperature to 140 ℃, finishing the reaction for 1.5h, cooling to room temperature, adding 200ml of ethyl acetate and water respectively, extracting, separating, washing an organic phase once by using water, drying by using a proper amount of anhydrous sodium sulfate, concentrating under reduced pressure, and carrying out column chromatography on residues to obtain a compound TM-43 of the formula I, namely a white solid, wherein the yield is 75.35%, and HPLC (high performance liquid chromatography) is 99.89%.
1 H NMR(400MHz,DMSO-d 6 )δ：11.13(s,1H),10.21(s,1H),8.71(s,1H),8.17(s,1H),7.74～7.56(m,3H),7.20～7.12(m,3H),6.70(s,1H),6.54(dd,J＝10.6,8.8Hz,1H),5.86(d,J＝6.4Hz,2H),5.75～5.64(m,2H),4.55(s,1H),2.92～2.83(m,2H).13C-NMR(400MHz,DMSO-d6):171.8,168.4,167.9,162.5,158.2,149.7,145.7,142.4,137.5,130.4,128.5,125.5,124.6,123.4,122.4,121.5,118.4,115.6,114.5,95.4,88.4,33.6，MS-ESI m/z:467.12(M+H) + 。
Example 25 preparation of TM-44
Figure GDA0004064996830000521
Compound 3: adding tert-butoxycarbonyl protected p-4-aminocyclobutane-2-carboxylic acid (23.17g, 120mmol) into dichloromethane (50 mL), sequentially adding EDCI (25.31g, 132mmol), HOBt (17.82g, 132mmol), 4- (4- (4-methylpiperazin-1 yl) -piperidin-1 yl) aniline (30.20g, 132mmol), stirring at room temperature for 24h, detecting by TLC, removing the solvent under reduced pressure, re-dissolving the obtained solid in 150mL of mixed solution (DCM: TFA = 7:3), stirring at room temperature for 0.5h, detecting by TLC, removing the solvent by reduced pressure rotary evaporation, and separating by column chromatography to obtain the product compound 3 with 76.33% yield and HPLC:99.837%.
Intermediate III: 2,4,6-trichloropyrimidine (compound 1) (55.03g, 300mmol) is added into dichloromethane (300 ml) and stirred until dissolved, the temperature is reduced to 0-5 ℃ in an ice bath, 3-chloro-4-fluoroaniline (45.72g, 315mmol, compound 2) is slowly dripped in, after the dripping is finished, the ice bath is removed, the temperature is naturally raised to room temperature, after the TLC tracking detection reaction is finished, the decompression concentration is carried out, the residue is separated by column chromatography to obtain an intermediate III, the yield is 86.52%, HPLC is 99.86%, and white solid is obtained.
An intermediate II: adding the intermediate III (29.25g, 100mmol) and the compound 3 (39.32g, 110mmol) into isopropanol (145.5 ml), adding concentrated hydrochloric acid (1 ml), refluxing, detecting by TLC until the reaction is complete, concentrating under reduced pressure, and separating the residue by column chromatography to obtain the intermediate II with the yield of 73.51%, HPLC (high performance liquid chromatography) 99.86% and white solid.
TM-44: adding the intermediate II (7.33g, 12mmol), concentrated hydrochloric acid (0.5 ml) and acrylamide (1.02g, 14.4 mmol) into n-octanol (20 ml), placing the n-octanol in a closed microwave reactor, setting the temperature to 140 ℃, finishing the reaction after 1.5h, cooling to room temperature, adding 200ml of each of ethyl acetate and water, extracting, separating liquid, washing an organic phase once by using water, drying by using a proper amount of anhydrous sodium sulfate, concentrating under reduced pressure, and carrying out column chromatography separation on residues to obtain a compound TM-44 of the formula I, namely a white solid with the yield of 77.52% and the HPLC (99.90%).
1 H NMR(400MHz,DMSO-d 6 )δ：11.26(s,1H),10.17(s,1H),8.83(s,1H),7.20～7.12(m,7H),6.73(s,1H),6.56(dd,J＝10.6,8.8Hz,1H),5.87(d,J＝6.4Hz,2H),5.75～5.64(m,2H),4.51(s,1H),2.83(m,4H),2.45(m,8H),2.32(s,3H),1.72(m,4H).13C-NMR(400MHz,DMSO-d6):172.4,171.4,166.7,160.3,159.1,152.6,145.7,140.6,131.3,129.5,128.2,122.4(2C),121.4,118.5,117.9,116.7,114.6(2C),91.2,83.6,73.1,57.6,55.7(2C),50.5(2C),47.9(2C),43.3,32.6,28.5(2C),MS-ESI m/z:646.23(M+H) + 。
Example 26 preparation of TM-47
Figure GDA0004064996830000531
Compound 3: adding tert-butoxycarbonyl protected p-4-aminocyclobutane-2-carboxylic acid (23.17g, 120mmol) into dichloromethane (40 mL), sequentially adding EDCI (25.31g, 132mmol), HOBt (17.82g, 132mmol) and 2-aminobenzimidazole (17.57g, 132mmol), stirring at room temperature for 24h, detecting by TLC, removing the solvent under reduced pressure, redissolving the obtained solid in 120mL of mixed solution (DCM: TFA = 7:3), stirring at room temperature for 0.5h, detecting by TLC, removing the solvent by reduced pressure rotary evaporation, and separating by column chromatography to obtain the product compound 3 with the yield of 71.53% and HPLC:99.87%.
An intermediate III: 2,4,6-trichloropyrimidine (compound 1) (55.03g, 300mmol) is added into dichloromethane (300 ml) and stirred until dissolved, the temperature is reduced to 0-5 ℃ in an ice bath, 3-chloro-4-fluoroaniline (45.72g, 315mmol, compound 2) is slowly dripped in, after the dripping is finished, the ice bath is removed, the temperature is naturally raised to room temperature, after the TLC tracking detection reaction is finished, the decompression concentration is carried out, the residue is separated by column chromatography to obtain an intermediate III, the yield is 83.42%, HPLC is 99.86%, and white solid is obtained.
An intermediate II: adding the intermediate III (29.25g, 100mmol) and the compound 3 (23.79g, 110mmol) into N, N dimethylformamide (150 ml), adding concentrated hydrochloric acid (1 ml), refluxing, detecting by TLC until the reaction is completed, concentrating under reduced pressure, and separating the residue by column chromatography to obtain the intermediate II with yield of 76.35%, HPLC (high performance liquid chromatography) of 99.82% and white solid.
TM-47: adding the intermediate II (5.67g, 12mmol), concentrated hydrochloric acid (0.5 ml) and acrylamide (1.02g, 14.4 mmol) into n-octanol (20 ml), placing in a closed microwave reactor, setting the temperature to 140 ℃, finishing the reaction after 1.5h, cooling to room temperature, adding 200ml of each of ethyl acetate and water, extracting, separating liquid, washing the organic phase once with water, drying with a proper amount of anhydrous sodium sulfate, concentrating under reduced pressure, and separating the residue by column chromatography to obtain the compound TM-47 of the formula I, which is a white solid, with the yield of 72.16% and HPLC of 99.91%.
1 H NMR(400MHz,DMSO-d 6 )δ：11.06(s,1H),10.43(s,1H),8.69(s,1H),7.74～7.37(m,4H),7.26～7.16(m,3H),6.76(s,1H),6.46(dd,J＝10.6,8.8Hz,1H),5.89(d,J＝6.4Hz,2H),5.79～5.62(m,4H),4.52(s,1H),3.29～3.01(m,2H).13C-NMR(400MHz,DMSO-d6):172.5,171.5,166.5,160.3,159.7,152.6,147.6,140.2,138.7,131.2,129.4,123.5(2C),121.5,118.5,117.8,116.2,115.2(2C),91.2,83.6,72.6,32.6,MS-ESI m/z:506.36(M+H) + 。
Example 27 preparation of TM-52
Figure GDA0004064996830000541
Compound 3: adding tert-butoxycarbonyl protected 2-aminoethanesulfonic acid (27.01g, 120mmol) into dichloromethane (50 mL), sequentially adding EDCI (25.31g, 132mmol), HOBt (17.82g, 132mmol) and 4-methoxy-3-methylaniline (18.09g, 132mmol), stirring at room temperature for 24h, detecting by TLC, removing the solvent under reduced pressure, redissolving the obtained solid in 150mL of mixed solution (DCM: TFA = 7:3), stirring at room temperature for 1h, detecting by TLC, removing the solvent by rotary evaporation, and separating by column chromatography to obtain the product compound 3 with a yield of 77.62% and an HPLC (high performance liquid chromatography) of 99.83%.
An intermediate III: 2,4,6-trichloropyrimidine (compound 1) (55.03g, 300mmol) is added into dichloromethane (300 ml) and stirred until dissolved, the temperature is reduced to 0-5 ℃ in an ice bath, 3-chloro-4-fluoroaniline (45.72g, 315mmol, compound 2) is slowly dripped in, after the dripping is finished, the ice bath is removed, the temperature is naturally raised to room temperature, after the TLC tracking detection reaction is finished, the decompression and the concentration are carried out, and the residue is separated by column chromatography to obtain an intermediate III, the yield is 86.37%, HPLC (high performance liquid chromatography) 99.89% and white solid.
An intermediate II: adding intermediate III (29.25g, 100mmol) and compound 3 (26.87g, 110mmol) into N, N-dimethylformamide (145.5 ml), adding concentrated hydrochloric acid (1 ml), refluxing, detecting by TLC until the reaction is complete, concentrating under reduced pressure, and separating the residue by column chromatography to obtain intermediate II with yield of 73.46%, HPLC:99.87%, and white solid.
TM-52: adding the intermediate II (6.01g, 12mmol), concentrated hydrochloric acid (0.5 ml) and acrylamide (1.02g, 14.4mmol) into N, N-dimethylformamide (20 ml), placing in a closed microwave reactor, setting the temperature at 140 ℃, finishing the reaction after 1h, cooling to room temperature, adding 200ml of each of ethyl acetate and water, extracting, separating, washing an organic phase once with water, drying with a proper amount of anhydrous sodium sulfate, concentrating under reduced pressure, and carrying out column chromatography separation on residues to obtain a compound TM-52 of the formula I, namely a white solid, wherein the yield is 75.33%, and HPLC (high performance liquid chromatography) is 99.90%.
1 H NMR(400MHz,DMSO-d 6 )δ：11.03(s,1H),10.20(s,1H),8.73(s,1H),7.75～7.61(m,2H),7.41(s,1H),7.27～7.18(m,3H),6.74(s,1H),6.56(dd,J＝10.6,8.8Hz,1H),5.85(d,J＝6.4Hz,2H),4.53(s,1H),3.67～3.58(m,4H),3.47(s,3H),2.17(s,3H).13C-NMR(400MHz,DMSO-d6):172.5,168.4,167.7,162.6,158.1,149.5,142.3,137.5,135.7,130.4,128.3,125.2,124.8,123.1,121.3,119.5,118.2,115.8,114.8,65.4,58.5,42.8,19.4，MS-ESI m/z:534.13(M+H) + 。
Example 28 preparation of TM-53
Figure GDA0004064996830000551
Compound 3: adding tert-butoxycarbonyl protected 2-aminoethanesulfonic acid (27.01g, 120mmol) into dichloromethane (30 mL), sequentially adding EDCI (25.31g, 132mmol), HOBt (17.82g, 132mmol) and 2-aminopyridine (12.42g, 132mmol), stirring at room temperature for 24h, detecting by TLC, removing the solvent under reduced pressure, re-dissolving the obtained solid in 120mL of mixed solution (DCM: TFA = 7:3), stirring at room temperature for 0.5h, detecting by TLC, removing the solvent by rotary evaporation under reduced pressure, and separating by column chromatography to obtain the product compound 3 with the yield of 78.46% and HPLC:99.85%.
An intermediate III: 2,4,6-trichloropyrimidine (compound 1) (55.03g, 300mmol) is added into dichloromethane (300 ml) and stirred until dissolved, the temperature is reduced to 0-5 ℃ in an ice bath, 3-chloro-4-fluoroaniline (45.72g, 315mmol, compound 2) is slowly dripped, the ice bath is removed after the dripping is finished, the temperature is naturally raised to room temperature, after the TLC tracking detection reaction is finished, the pressure is reduced and the concentration is carried out, and the remainder is separated by column chromatography to obtain an intermediate III, the yield is 83.21%, HPLC (high performance liquid chromatography) is 99.87%, and white solid is obtained.
An intermediate II: adding intermediate III (29.25g, 100mmol) and compound 3 (22.14g, 110mmol) into isopropanol (145.5 ml), adding concentrated hydrochloric acid (1 ml), refluxing, TLC detecting to complete the reaction, decompression concentrating, and separating the residue by column chromatography to obtain intermediate II in 75.42% yield, HPLC in 99.89% and white solid.
TM-53: adding the intermediate II (5.49g, 12mmol), concentrated hydrochloric acid (0.5 ml) and acrylamide (1.02g, 14.4 mmol) into n-octanol (20 ml), placing in a closed microwave reactor, setting the temperature to 140 ℃, finishing the reaction after 0.5h, cooling to room temperature, adding 200ml of each of ethyl acetate and water, extracting, separating liquid, washing an organic phase once with water, drying with a proper amount of anhydrous sodium sulfate, concentrating under reduced pressure, and separating residues through column chromatography to obtain a compound TM-53 of the formula I, wherein the yield is 72.35 percent and the HPLC is 99.88 percent.
1 H NMR(400MHz,DMSO-d 6 )δ：11.22(s,1H),11.21(s,1H),8.64(s,1H),8.18(s,1H),7.70～7.58(m,3H),7.21～7.19(m,3H),6.70(s,1H),6.57(dd,J＝10.6,8.8Hz,1H),5.85(d,J＝6.4Hz,2H),4.66(s,1H),3.62～3.58(m,4H).13C-NMR(400MHz,DMSO-d6):170.8,169.8,168.7,163.8,155.6,149.4,145.5,142.3,136.8,130.8,128.5,126.3,124.1,122.2,117.6,115.1,114.8,95.4,65.3,42.8，MS-ESI m/z:491.09(M+H) + 。
Example 29 preparation of TM-55
Figure GDA0004064996830000561
Compound 3: adding tert-butoxycarbonyl protected 2-aminoethanesulfonic acid (27.01g, 120mmol) into dichloromethane (40 mL), sequentially adding EDCI (25.31g, 132mmol), HOBt (17.82g, 132mmol) and 2-aminobenzimidazole (17.57g, 132mmol), stirring at room temperature for 24h, detecting by TLC, removing the solvent under reduced pressure, re-dissolving the obtained solid in 150mL of mixed solution (DCM: TFA = 7:3), stirring at room temperature for 0.5h, detecting by TLC, removing the solvent by rotary evaporation under reduced pressure, and separating by column chromatography to obtain the product compound 3 with yield of 74.36% and HPLC of 99.81%.
Intermediate III: 2,4,6-trichloropyrimidine (compound 1) (55.03g, 300mmol) is added into dichloromethane (300 ml) and stirred until dissolved, the temperature is reduced to 0-5 ℃ in an ice bath, 3-chloro-4-fluoroaniline (45.72g, 315mmol, compound 2) is slowly dripped in, after the dripping is finished, the ice bath is removed, the temperature is naturally raised to room temperature, after the TLC tracking detection reaction is finished, the decompression concentration is carried out, the residue is separated by column chromatography to obtain an intermediate III, the yield is 79.53%, HPLC is 99.86%, and white solid is obtained.
An intermediate II: adding the intermediate III (29.25g, 100mmol) and the compound 3 (26.42g, 110mmol) into N, N-dimethylformamide (145.5 ml), adding perchloric acid (2 ml), refluxing, detecting by TLC until the reaction is complete, concentrating under reduced pressure, and separating the residue by column chromatography to obtain the intermediate II with the yield of 72.16%, HPLC:99.83% and white solid.
TM-55: adding the intermediate II (5.96g, 12mmol), concentrated hydrochloric acid (0.5 ml) and acrylamide (1.02g, 14.4 mmol) into n-octanol (20 ml), placing the n-octanol in a closed microwave reactor, setting the temperature to 140 ℃, finishing the reaction after 1h, cooling to room temperature, adding 200ml of each of ethyl acetate and water, extracting, separating liquid, washing an organic phase once by using water, drying by using a proper amount of anhydrous sodium sulfate, concentrating under reduced pressure, and carrying out column chromatography separation on residues to obtain a compound TM-55 of the formula I, namely a white solid, wherein the yield is 76.31% and the HPLC (high performance liquid chromatography) is 99.89%.
1 H NMR(400MHz,DMSO-d 6 )δ：11.37(s,1H),11.16(s,1H),8.76(s,1H),7.73～7.46(m,4H),7.21～7.19(m,4H),6.73(s,1H),6.57(dd,J＝10.6,8.8Hz,1H),5.85(d,J＝6.4Hz,2H),4.69(s,1H),3.62(S,4H).13C-NMR(400MHz,DMSO-d6):172.5,166.9,160.3,159.2,152.6,149.6,140.3,131.3,138.8(2C),129.1,123.1(2C),121.5,118.4,117.6,116.5,115.6(2C),72.5,55.7,40.5,MS-ESI m/z:530.56(M+H) + 。
Example 30 preparation of TM-58
Figure GDA0004064996830000571
Compound 3: adding tert-butoxycarbonyl protected 5-aminopyridyl-2-sulfonic acid (33.18g, 120mmol) to dichloromethane (50 mL), sequentially adding EDCI (25.31g, 132mmol), HOBt (17.82g, 132mmol), and 4-methoxy-3-methylaniline (18.09g, 132mmol), stirring at room temperature for 24h, detecting by TLC, removing the solvent under reduced pressure, redissolving the obtained solid in 150mL of a mixed solution (DCM: TFA = 7:3), stirring at room temperature for 0.5h, detecting by TLC, removing the solvent under reduced pressure, and separating by column chromatography to obtain the product compound 3 with a yield of 75.42% and an HPLC:99.83%.
Intermediate III: 2,4,6-trichloropyrimidine (compound 1) (55.03g, 300mmol) is added into dichloromethane (300 ml) and stirred until dissolved, the temperature is reduced to 0-5 ℃ in an ice bath, 3-chloro-4-fluoroaniline (45.72g, 315mmol, compound 2) is slowly dripped in, after the dripping is finished, the ice bath is removed, the temperature is naturally raised to room temperature, after the TLC tracking detection reaction is finished, the pressure is reduced and the concentration is carried out, and the residue is separated by column chromatography to obtain an intermediate III, the yield is 82.43%, HPLC is 99.85%, and white solid is obtained.
An intermediate II: adding the intermediate III (29.25g, 100mmol) and the compound 3 (32.27g, 110mmol) into isopropanol (145.5 ml), adding concentrated hydrochloric acid (1 ml), refluxing, detecting by TLC until the reaction is complete, concentrating under reduced pressure, and separating the residue by column chromatography to obtain the intermediate II with the yield of 73.26%, HPLC (high performance liquid chromatography) 99.85% and white solid.
TM-58: adding the intermediate II (6.59g, 12mmol), concentrated hydrochloric acid (0.5 ml) and acrylamide (1.02g, 14.4 mmol) into n-octanol (20 ml), placing the n-octanol into a closed microwave reactor, setting the temperature to 140 ℃, finishing the reaction after 1.5h, cooling to room temperature, adding 200ml of each of ethyl acetate and water, extracting and separating the liquid, washing the organic phase once by using water, drying the organic phase by using a proper amount of anhydrous sodium sulfate, concentrating the organic phase under reduced pressure, and separating the residue by column chromatography to obtain the compound TM-58 of the formula I, wherein the yield is 77.34 percent and the HPLC (high performance liquid chromatography) is 99.91 percent.
1 H NMR(400MHz,DMSO-d 6 )δ：10.89(s,1H),10.37(s,1H),8.64(s,1H),7.70～7.58(m,3H),7.47(s,1H),7.28～7.20(m,5H),6.74(s,1H),6.51(dd,J＝10.6,8.8Hz,1H),5.83(d,J＝6.4Hz,2H),4.64(s,1H),3.40(s,3H),2.17(s,3H).13C-NMR(400MHz,DMSO-d6):171.6,167.3,167.1,162.5,158.3,149.5,137.6,135.9,132.2(2C),131.4,128.9,125.5,124.7,123.4,120.2,119.5,119.1,118.5,115.7,114.2,113.1(2C),72.8,56.5,19.3，MS-ESI m/z:583.12(M+H) + 。
Example 31 preparation of TM-59
Figure GDA0004064996830000581
Compound 3: tert-butoxycarbonyl protected 5-aminopyridyl-2-sulfonic acid (33.18g, 120mmol) was added to dichloromethane (30 mL), EDCI (25.31g, 132mmol), HOBt (17.82g, 132mmol), 2-aminopyridine (12.42g, 132mmol) were sequentially added, the reaction was stirred at room temperature for 24h, TLC detection was completed, the solvent was removed under reduced pressure, the resulting solid was redissolved in 120mL of a mixed solution (DCM: TFA = 7:3), and after stirring at room temperature for 0.5h, TLC detection was completed, the solvent was removed by rotary evaporation under reduced pressure, and product compound 3 was isolated by column chromatography in 76.12% yield and HPLC 99.85%.
Intermediate III: 2,4,6-trichloropyrimidine (compound 1) (55.03g, 300mmol) is added into dichloromethane (300 ml) and stirred until dissolved, the temperature is reduced to 0-5 ℃ in an ice bath, 3-chloro-4-fluoroaniline (45.72g, 315mmol, compound 2) is slowly dripped in, after the dripping is finished, the ice bath is removed, the temperature is naturally raised to room temperature, after the TLC tracking detection reaction is finished, the pressure is reduced and the concentration is carried out, and the residue is separated by column chromatography to obtain an intermediate III, the yield is 81.33%, HPLC is 99.87%, and white solid is obtained.
An intermediate II: adding intermediate III (29.25g, 100mmol) and compound 3 (27.53g, 110mmol) into isopropanol (145.5 ml), adding concentrated hydrochloric acid (1 ml), refluxing, detecting by TLC until the reaction is complete, concentrating under reduced pressure, and separating the residue by column chromatography to obtain intermediate II with yield of 75.62%, HPLC:99.86% and white solid.
TM-59: adding the intermediate II (6.08g, 12mmol), concentrated hydrochloric acid (0.5 ml) and acrylamide (1.02g, 14.4 mmol) into dimethyl sulfoxide (20 ml), placing in a closed microwave reactor, heating to 140 ℃, reacting for 1.5h, cooling to room temperature, adding 200ml of each of ethyl acetate and water, extracting, separating, washing the organic phase once with water, drying with an appropriate amount of anhydrous sodium sulfate, concentrating under reduced pressure, and separating the residue by column chromatography to obtain the compound TM-59 of formula I, wherein the yield is 79.51%, and the HPLC is 99.89%.
1 H NMR(400MHz,DMSO-d 6 )δ：11.04(s,1H),10.17(s,1H),8.65(s,1H),8.02(s,1H),7.73～7.58(m,3H),7.44～7.37(m,2H),7.25～7.17(m,4H),6.71(s,1H),6.54(dd,J＝10.6,8.8Hz,1H),5.72(d,J＝6.4Hz,2H),4.64(s,1H).13C-NMR(400MHz,DMSO-d6):172.8,167.5,166.7,160.8,157.6,148.7,145.8,142.4,137.6,135.7,131.2(2C),130.4,128.5,124.3,124.8,123.3,120.2,118.4,116.1,113.8,112.5(2C)，MS-ESI m/z:540.09(M+H) + 。
Example 32 preparation of TM-63
Figure GDA0004064996830000591
Compound 3: adding tert-butoxycarbonyl protected 5-aminopyridyl-2-sulfonic acid (33.18g, 120mmol) to dichloromethane (50 mL), successively adding EDCI (25.31g, 132mmol), HOBt (17.82g, 132mmol), and 4- (4- (4-methylpiperazin-1-yl) -piperidin-1-yl) aniline (30.20g, 132mmol), stirring at room temperature for 24h, detecting by TLC, removing the solvent under reduced pressure, re-dissolving the obtained solid in 150mL of a mixed solution (DCM: TFA = 7:3), stirring at room temperature for 0.5h, detecting by TLC, removing the solvent by reduced pressure distillation, and isolating to obtain the product compound 3 with a yield of 77.29% and an HPLC:99.87%.
Intermediate III: 2,4,6-trichloropyrimidine (compound 1) (55.03g, 300mmol) is added into dichloromethane (300 ml) and stirred until dissolved, the temperature is reduced to 0-5 ℃ in an ice bath, 3-chloro-4-fluoroaniline (45.72g, 315mmol, compound 2) is slowly dripped in, after the dripping is finished, the ice bath is removed, the temperature is naturally raised to room temperature, after the TLC tracking detection reaction is finished, the pressure is reduced and the concentration is carried out, and the residue is separated by column chromatography to obtain an intermediate III, the yield is 79.36%, HPLC is 99.85%, and white solid is obtained.
An intermediate II: adding the intermediate III (29.25g, 100mmol) and the compound 3 (47.36g, 110mmol) into N, N-dimethylformamide (150 ml), adding concentrated hydrochloric acid (1 ml), refluxing, detecting by TLC until the reaction is complete, concentrating under reduced pressure, and separating the residue by column chromatography to obtain the intermediate II with yield of 76.59%, HPLC (high performance liquid chromatography) of 99.89% and white solid.
TM-63: adding the intermediate II (8.24g, 12mmol), concentrated hydrochloric acid (0.5 ml) and acrylamide (1.02g, 14.4 mmol) into n-octanol (20 ml), placing the n-octanol in a closed microwave reactor, setting the temperature to 140 ℃, finishing the reaction after 0.5h, cooling to room temperature, adding 200ml of each of ethyl acetate and water, extracting, separating liquid, washing an organic phase once with water, drying with a proper amount of anhydrous sodium sulfate, concentrating under reduced pressure, and carrying out column chromatography separation on residues to obtain a compound TM-63 of the formula I, namely a white solid with the yield of 76.33% and the HPLC (99.85%).
1 H NMR(400MHz,DMSO-d 6 )δ：11.02(s,1H),10.13(s,1H),8.87(s,1H),8.59(s,1H),8.04(s,1H),7.32～7.26(m,4H),6.69(s,1H),6.52(dd,J＝10.6,8.8Hz,1H),6.36～6.29(m,4H),5.71(d,J＝6.4Hz,2H),4.67(s,1H),2.81(m,4H),2.46(m,8H),2.31(m,3H),1.68～1.43(m,4H).13C-NMR(400MHz,DMSO-d6):172.4,167.6,166.5,159.5,152.6,149.6,148.6,140.3,139.6(2C),136.5,131.6,129.1,127.6,121.5(2C),120.6,118.2,117.6(3C),116.2,115.2(3C),72.4,57.7,55.4(2C),50.6(2C),47.6(2C),43.6,28.3(2C),MS-ESI m/z:721.16(M+H) + 。
Example 33 preparation of TM-64
Figure GDA0004064996830000611
Compound 3: the tert-butoxycarbonyl protected 5-aminopyridyl-2-carboxylic acid (33.18g, 120mmol) was added to dichloromethane (30 mL), EDCI (25.31g, 132mmol), HOBt (17.82g, 132mmol), and N, N-dimethylethylenediamine (11.63g, 132mmol) were added in this order, and the mixture was stirred at room temperature for 24h, after completion of TLC detection, the solvent was removed under reduced pressure, the resulting solid was redissolved in 120mL of a mixed solution (DCM: TFA = 7:3), and after stirring at room temperature for 0.5h, the reaction was completed by TLC detection, and the solvent was removed by rotary evaporation under reduced pressure, and the product, compound 3, was isolated by column chromatography, at 75.16% yield, and HPLC:99.83%.
Intermediate III: 2,4,6-trichloropyrimidine (compound 1) (55.03g, 300mmol) is added into dichloromethane (300 ml) and stirred until dissolved, the temperature is reduced to 0-5 ℃ in an ice bath, 3-chloro-4-fluoroaniline (45.72g, 315mmol, compound 2) is slowly dripped in, after the dripping is finished, the ice bath is removed, the temperature is naturally raised to room temperature, after the TLC tracking detection reaction is finished, the decompression and the concentration are carried out, and the residue is separated by column chromatography to obtain an intermediate III, the yield is 81.31%, HPLC (high performance liquid chromatography) 99.86% and white solid.
An intermediate II: adding intermediate III (29.25g, 100mmol) and compound 3 (21.37g, 110mmol) into isopropanol (145.5 ml), adding concentrated hydrochloric acid (1 ml), refluxing, detecting by TLC until the reaction is complete, concentrating under reduced pressure, and separating the residue by column chromatography to obtain intermediate II with yield of 77.56%, HPLC:99.88% and white solid.
TM-64: adding the intermediate II (5.40g, 12mmol), concentrated hydrochloric acid (0.5 ml) and acrylamide (1.02g, 14.4 mmol) into N, N-dimethylformamide (20 ml), placing in a closed microwave reactor, setting the temperature to 140 ℃, finishing the reaction after 1.5h, cooling to room temperature, adding 200ml of ethyl acetate and water respectively, extracting, separating liquid, washing an organic phase once with water, drying with a proper amount of anhydrous sodium sulfate, concentrating under reduced pressure, and separating residues by column chromatography to obtain a compound TM-64 of the formula I, namely a white solid with the yield of 77.69% and the HPLC of 99.89%.
1 H NMR(400MHz,DMSO-d 6 )δ：11.23(s,1H),10.46(s,1H),8.87(s,1H),8.57(s,1H),8.19(s,1H),7.73～7.58(m,4H),6.73(s,1H),6.59(dd,J＝10.6,8.8Hz,1H),5.76(d,J＝6.4Hz,2H),4.67(s,1H),4.16(s,2H),2.31(m,6H),.13C-NMR(400MHz,DMSO-d6):172.9,167.6,166.4,161.3,159.7,152.9,147.6,140.2(2C),134.5,131.3,129.6,123.6,122.2,121.8,118.5,117.8,116.3,72.3,68.6,43.2(2C),MS-ESI m/z:483.76(M+H) + 。
Example 34 preparation of TM-68
Figure GDA0004064996830000621
Compound 3: adding tert-butoxycarbonyl protected 5-aminopyridyl-2-carboxylic acid (33.18g, 120mmol) to dichloromethane (40 mL), sequentially adding EDCI (25.31g, 132mmol), HOBt (17.82g, 132mmol), and 4-methoxy-3-methylaniline (18.09g, 132mmol), stirring at room temperature for 24h, detecting by TLC, removing the solvent under reduced pressure, redissolving the obtained solid in 150mL of mixed solution (DCM: TFA = 7:3), stirring at room temperature for 0.5h, detecting by TLC, removing the solvent by rotary evaporation, and separating by column chromatography to obtain the product compound 3 with a yield of 76.53% and an HPLC:99.85%.
An intermediate III: 2,4,6-trichloropyrimidine (compound 1) (55.03g, 300mmol) is added into dichloromethane (300 ml) and stirred until dissolved, the temperature is reduced to 0-5 ℃ in an ice bath, 3-chloro-4-fluoroaniline (45.72g, 315mmol, compound 2) is slowly dripped in, after the dripping is finished, the ice bath is removed, the temperature is naturally raised to room temperature, after the TLC tracking detection reaction is finished, the decompression concentration is carried out, and the residue is separated by column chromatography to obtain an intermediate III, the yield is 80.16%, the HPLC is 99.85%, and white solid is obtained.
An intermediate II: adding the intermediate III (29.25g, 100mmol) and the compound 3 (28.29g, 110mmol) into isopropanol (145.5 ml), adding concentrated hydrochloric acid (1 ml), refluxing, detecting by TLC until the reaction is complete, concentrating under reduced pressure, and separating the residue by column chromatography to obtain the intermediate II with yield of 72.51%, HPLC (high performance liquid chromatography) of 99.89% and white solid.
TM-68: adding the intermediate II (6.16g, 12mmol), concentrated hydrochloric acid (0.5 ml) and acrylamide (1.02g, 14.4 mmol) into n-octanol (20 ml), placing the n-octanol in a closed microwave reactor, setting the temperature to 140 ℃, finishing the reaction after 2.5h, cooling to room temperature, adding 200ml of each of ethyl acetate and water, extracting and separating the solution, washing the organic phase once with water, drying with a proper amount of anhydrous sodium sulfate, concentrating under reduced pressure, and carrying out column chromatography separation on the residue to obtain the compound TM-68 of the formula I, namely a white solid with the yield of 76.31% and the HPLC (99.90%).
1 H NMR(400MHz,DMSO-d 6 )δ：10.88(s,1H),10.20(s,1H),8.71(s,1H),7.71～7.50(m,4H),7.47(s,1H),7.25～7.17(m,4H),6.71(s,1H),6.55(dd,J＝10.6,8.8Hz,1H),5.83(d,J＝6.4Hz,2H),4.55(s,1H),3.37(s,3H),2.15(s,3H).13C-NMR(400MHz,DMSO-d6):171.8,168.5,167.7,162.2,158.6,149.8,142.4,137.5,135.7,132.2(2C),130.4,127.5,125.8,123.3,123.6,122.3,118.5,118.7,118.4,116.2,114.8,113.5(2C),72.4,56.8,19.7，MS-ESI m/z:547.15(M+H) + 。
Example 35 preparation of TM-69
Figure GDA0004064996830000631
Compound 3: tert-butoxycarbonyl protected 5-aminopyridyl-2-carboxylic acid (33.18g, 120mmol) was added to methylene chloride (30 mL), EDCI (25.31g, 132mmol), HOBt (17.82g, 132mmol), and cyclohexylamine (13.09g, 132mmol) were sequentially added, the reaction was stirred at room temperature for 24h, the reaction was terminated by TLC detection, the solvent was removed under reduced pressure, the resulting solid was redissolved in 120mL of a mixed solution (DCM: TFA = 7:3), the reaction was stirred at room temperature for 1h, the reaction was terminated by TLC detection, the solvent was removed by rotary evaporation under reduced pressure, and the product, compound 3, was isolated by column chromatography, at a yield of 75.69%, and HPLC:99.86%.
Intermediate III: 2,4,6-trichloropyrimidine (compound 1) (55.03g, 300mmol) is added into dichloromethane (300 ml) and stirred until dissolved, the temperature is reduced to 0-5 ℃ in an ice bath, 3-chloro-4-fluoroaniline (45.72g, 315mmol, compound 2) is slowly dripped in, after the dripping is finished, the ice bath is removed, the temperature is naturally raised to room temperature, after the TLC tracking detection reaction is finished, the decompression and the concentration are carried out, and the residue is separated by column chromatography to obtain an intermediate III, the yield is 81.47%, HPLC (high performance liquid chromatography) 99.87% and white solid.
An intermediate II: adding the intermediate III (29.25g, 100mmol) and the compound 3 (24.12g, 110mmol) into N, N-dimethylacetamide (150 ml), adding concentrated hydrochloric acid (1 ml), refluxing, detecting by TLC until the reaction is complete, concentrating under reduced pressure, and separating the residue by column chromatography to obtain the intermediate II with the yield of 75.36%, HPLC (high performance liquid chromatography) 99.86% and white solid.
TM-69: adding the intermediate II (5.70g, 12mmol), concentrated hydrochloric acid (0.5 ml) and acrylamide (1.02g, 14.4mmol) into n-octanol (20 ml), placing in a closed microwave reactor, setting the temperature to 140 ℃, finishing the reaction after 0.5h, cooling to room temperature, adding 200ml of each of ethyl acetate and water, extracting, separating liquid, washing an organic phase once with water, drying with a proper amount of anhydrous sodium sulfate, concentrating under reduced pressure, and separating residues by column chromatography to obtain a compound TM-69 of the formula I, namely a white solid with the yield of 78.22% and the HPLC (high performance liquid chromatography) of 99.91%.
1 H NMR(400MHz,DMSO-d 6 )δ：11.26(s,1H),10.51(s,1H),8.85(s,1H),8.67(s,1H),8.42(s,1H),7.76～7.52(m,4H),6.73(s,1H),6.57(dd,J＝10.6,8.8Hz,1H),5.91(d,J＝6.4Hz,2H),4.71(s,1H),3.37(s,3H),1.78～1.39(m,10H).13C-NMR(400MHz,DMSO-d6):172.1,167.6,166.5,160.3,159.7,152.5,147.6,140.3(2C),134.6,131.2,129.4,123.4,122.5,121.7,118.6,117.6,116.2,72.4,47.6,33.6(2C),28.1,22.6(2C),MS-ESI m/z:509.23(M+H) + 。
Example 36 preparation of TM-70
Figure GDA0004064996830000641
Compound 3: adding tert-butoxycarbonyl protected 2- (aminomethoxy) acetic acid (22.92g, 120mmol) into dichloromethane (30 mL), sequentially adding EDCI (25.31g, 132mmol), HOBt (17.82g, 132mmol) and N, N-dimethylethylenediamine (11.63g, 132mmol), stirring at room temperature for 24h, detecting by TLC, removing the solvent under reduced pressure, redissolving the obtained solid in 120mL of mixed solution (DCM: TFA = 7:3), stirring at room temperature for 0.5h, detecting by TLC, removing the solvent by reduced pressure rotary evaporation, and separating by column chromatography to obtain the product compound 3 with the yield of 76.36% and HPLC:99.85%.
Intermediate III: 2,4,6-trichloropyrimidine (compound 1) (55.03g, 300mmol) is added into dichloromethane (300 ml) and stirred until dissolved, the temperature is reduced to 0-5 ℃ in an ice bath, 3-chloro-4-fluoroaniline (45.72g, 315mmol, compound 2) is slowly dripped in, after the dripping is finished, the ice bath is removed, the temperature is naturally raised to room temperature, after the TLC tracking detection reaction is finished, the decompression and the concentration are carried out, and the residue is separated by column chromatography to obtain an intermediate III, the yield is 83.41%, HPLC (high performance liquid chromatography) 99.86% and white solid.
An intermediate II: adding the intermediate III (29.25g, 100mmol) and the compound 3 (17.74g, 110mmol) into isopropanol (150 ml), adding concentrated hydrochloric acid (1 ml), refluxing, detecting by TLC until the reaction is complete, concentrating under reduced pressure, and separating the residue by column chromatography to obtain the intermediate II with yield of 76.43%, HPLC (high performance liquid chromatography) of 99.89% and white solid.
TM-70: adding the intermediate II (5.01g, 12mmol), concentrated hydrochloric acid (0.5 ml) and acrylamide (1.02g, 14.4 mmol) into n-octanol (20 ml), placing the n-octanol in a closed microwave reactor, setting the temperature to 140 ℃, finishing the reaction after 1.5h, cooling to room temperature, adding 200ml of each of ethyl acetate and water, extracting and separating the liquid, washing an organic phase once by using water, drying by using a proper amount of anhydrous sodium sulfate, concentrating under reduced pressure, and carrying out column chromatography separation on the residue to obtain a compound TM-70 of the formula I, namely a white solid, wherein the yield is 79.16 percent and the HPLC (high performance liquid chromatography) is 99.90 percent.
1 H NMR(400MHz,DMSO-d 6 )δ：10.89(s,1H),10.17(s,1H),8.62(s,1H),7.36～7.27(m,3H),6.73(s,1H),6.56(dd,J＝10.6,8.8Hz,1H),5.89(d,J＝6.4Hz,2H),5.27(d,J＝6.4Hz,2H),4.76(s,1H),4.19(m,4H),2.35(m,6H).13C-NMR(400MHz,DMSO-d6):172.6,169.3,166.1,160.2,159.7,152.3,140.3,131.2,129.5,121.3,118.6,117.6,116.3,80.6,72.6,68.5,67.2,43.2(2C),MS-ESI m/z:451.17(M+H) + 。
Example 37 preparation of TM-71
Figure GDA0004064996830000651
Compound 3: adding tert-butoxycarbonyl protected 2- (aminomethoxy) acetic acid (22.92g, 120mmol) into dichloromethane (40 mL), sequentially adding EDCI (25.31g, 132mmol), HOBt (17.82g, 132mmol) and 2-aminobenzimidazole (17.57g, 132mmol), stirring at room temperature for 24h, detecting by TLC, removing the solvent under reduced pressure, re-dissolving the obtained solid in 150mL of mixed solution (DCM: TFA = 7:3), stirring at room temperature for 1h, detecting by TLC, removing the solvent by rotary evaporation under reduced pressure, and separating by column chromatography to obtain the product compound 3 with the yield of 73.52% and HPLC:99.82%.
Intermediate III: 2,4,6-trichloropyrimidine (compound 1) (55.03g, 300mmol) is added into dichloromethane (300 ml) and stirred until dissolved, the temperature is reduced to 0-5 ℃ in an ice bath, 3-chloro-4-fluoroaniline (45.72g, 315mmol, compound 2) is slowly dripped in, after the dripping is finished, the ice bath is removed, the temperature is naturally raised to room temperature, after the TLC tracking detection reaction is finished, the pressure is reduced and the concentration is carried out, and the residue is separated by column chromatography to obtain an intermediate III, the yield is 81.36%, HPLC is 99.87%, and white solid is obtained.
An intermediate II: intermediate III (29.25g, 100mmol) and compound 3 (24.22g, 110mmol) were added to N, N dimethylformamide (145.5 ml), concentrated hydrochloric acid (1 ml) was added, reflux reaction was carried out, TLC detection followed reaction completed, concentration under reduced pressure was carried out, and the residue was separated by column chromatography to give intermediate II in 77.23% yield, HPLC:99.85%, and white solid.
TM-71: adding the intermediate II (5.72g, 12mmol), concentrated hydrochloric acid (0.5 ml) and acrylamide (1.02g, 14.4 mmol) into dimethyl sulfoxide (20 ml), placing the mixture into a closed microwave reactor, setting the temperature at 180 ℃, finishing the reaction after 0.5h, cooling to room temperature, adding 200ml of each of ethyl acetate and water, extracting and separating the solution, washing an organic phase once with water, drying with a proper amount of anhydrous sodium sulfate, concentrating under reduced pressure, and carrying out column chromatography separation on the residue to obtain a compound TM-71 of the formula I, namely a white solid with the yield of 76.53% and the HPLC (99.89%).
1 H NMR(400MHz,DMSO-d 6 )δ：11.26(s,1H),10.57(s,1H),8.73(s,1H),7.73～7.43(m,4H),7.36～7.23(m,4H),6.74(s,1H),6.52(dd,J＝10.6,8.8Hz,1H),5.81(d,J＝7.2Hz,2H),5.32(d,J＝6.4Hz,2H),4.65(s,1H),4.32(d,J＝5.3Hz,2H).13C-NMR(400MHz,DMSO-d6):172.3,169.3,166.7,160.2,159.3,152.9,147.6,140.3,138.9(2C),131.2,129.2,123.5(2C),121.7,118.6,117.7,116.5,115.2(2C),80.2,72.4,67.6,MS-ESI m/z:510.86(M+H) + 。
Example 38 preparation of TM-74
Figure GDA0004064996830000661
Compound 3: the tert-butoxycarbonyl protected 2- (aminomethoxy) acetic acid (22.92g, 120mmol) was added to dichloromethane (30 mL), EDCI (25.31g, 132mmol), HOBt (17.82g, 132mmol) and 2-aminopyridine (12.42g, 132mmol) were sequentially added, the reaction was stirred at room temperature for 24h, the TLC detection reaction was completed, the solvent was removed under reduced pressure, the resulting solid was redissolved in 120mL of a mixed solution (DCM: TFA = 7:3), the reaction was stirred at room temperature for 1.5h, the TLC detection reaction was completed, the solvent was removed by rotary evaporation under reduced pressure, and the product compound 3 was isolated by column chromatography in a yield of 76.47%, HPLC:99.85%.
Intermediate III: 2,4,6-trichloropyrimidine (compound 1) (55.03g, 300mmol) is added into dichloromethane (300 ml) and stirred until dissolved, the temperature is reduced to 0-5 ℃ in an ice bath, 3-chloro-4-fluoroaniline (45.72g, 315mmol, compound 2) is slowly dripped in, after the dripping is finished, the ice bath is removed, the temperature is naturally raised to room temperature, after the TLC tracking detection reaction is finished, the decompression concentration is carried out, the residue is separated by column chromatography to obtain an intermediate III, the yield is 80.35%, HPLC is 99.86%, and white solid is obtained.
An intermediate II: adding the intermediate III (29.25g, 100mmol) and the compound 3 (19.93g, 110mmol) into isopropanol (150 ml), adding concentrated hydrochloric acid (1 ml), refluxing, tracking by TLC detection until the reaction is complete, concentrating under reduced pressure, and separating the residue by column chromatography to obtain the intermediate II with the yield of 78.57%, HPLC (high performance liquid chromatography) of 99.86% and white solid.
TM-74: adding the intermediate II (5.25g, 12mmol), concentrated hydrochloric acid (0.5 ml) and acrylamide (1.02g, 14.4 mmol) into dimethyl sulfoxide (20 ml), placing the mixture into a closed microwave reactor, setting the temperature to 160 ℃, finishing the reaction after 1.0h, cooling to room temperature, adding 200ml of each of ethyl acetate and water, extracting and separating the solution, washing an organic phase once with water, drying with a proper amount of anhydrous sodium sulfate, concentrating under reduced pressure, and carrying out column chromatography separation on the residue to obtain a compound TM-74 of the formula I, wherein the yield is 78.26 percent and the HPLC (high performance liquid chromatography) is 99.90 percent.
1 H NMR(400MHz,DMSO-d 6 )δ：10.97(s,1H),10.31(s,1H),8.67(s,1H),8.49(s,1H),8.21(s,1H),7.91(s,1H),7.47(s,1H),7.25～7.17(m,4H),6.73(s,1H),6.52(dd,J＝10.6,8.8Hz,1H),5.86(d,J＝6.4Hz,2H),5.32(d,J＝6.4Hz,2H),4.67(s,1H),4.32(d,J＝5.3Hz,2H).13C-NMR(400MHz,DMSO-d6):172.6,169.2,166.6,160.1,159.3,152.8,150.2,146.3,140.2,138.6,131.4,129.6,121.3,119.5,118.6,117.6,116.2,115.7,80.6,72.2,67.2,MS-ESI m/z:471.36(M+H) + 。
Example 39 preparation of TM-76
Figure GDA0004064996830000671
Compound 3: adding tert-butoxycarbonyl protected 2- (aminomethoxy) acetic acid (22.92g, 120mmol) into dichloromethane (30 mL), sequentially adding EDCI (25.31g, 132mmol), HOBt (17.82g, 132mmol) and cyclohexylamine (13.09g, 132mmol), stirring at room temperature for 24h, detecting by TLC, removing the solvent under reduced pressure, re-dissolving the obtained solid in 120mL of mixed solution (DCM: TFA = 7:3), stirring at room temperature for 0.5h, detecting by TLC, removing the solvent by rotary evaporation under reduced pressure, and separating by column chromatography to obtain the product compound 3 with the yield of 79.37% and HPLC:99.87%.
An intermediate III: 2,4,6-trichloropyrimidine (compound 1) (55.03g, 300mmol) is added into dichloromethane (300 ml) and stirred until dissolved, the temperature is reduced to 0-5 ℃ in an ice bath, 3-chloro-4-fluoroaniline (45.72g, 315mmol, compound 2) is slowly dripped, the ice bath is removed after the dripping is finished, the temperature is naturally raised to room temperature, after the TLC tracking detection reaction is finished, the pressure is reduced and the concentration is carried out, and the remainder is separated by column chromatography to obtain an intermediate III, the yield is 81.46%, HPLC (high performance liquid chromatography) is 99.87%, and white solid is obtained.
An intermediate II: adding the intermediate III (29.25g, 100mmol) and the compound 3 (20.49g, 110mmol) into isopropanol (145.5 ml), adding concentrated hydrochloric acid (1 ml), refluxing, detecting by TLC until the reaction is complete, concentrating under reduced pressure, and separating the residue by column chromatography to obtain the intermediate II with the yield of 79.33%, HPLC (high performance liquid chromatography) 99.84% and white solid.
TM-76: adding the intermediate II (5.31g, 12mmol), concentrated hydrochloric acid (0.5 ml) and acrylamide (1.02g, 14.4mmol) into dimethyl sulfoxide (20 ml), placing in a closed microwave reactor, setting the temperature to 140 ℃, finishing the reaction after 1.5h, cooling to room temperature, adding 200ml of each of ethyl acetate and water, extracting, separating, washing the organic phase once with water, drying with a proper amount of anhydrous sodium sulfate, concentrating under reduced pressure, and separating the residue by column chromatography to obtain the compound TM-76 of the formula I, namely a white solid with the yield of 79.15% and the HPLC (high performance liquid chromatography) of 99.91%.
1 H NMR(400MHz,DMSO-d 6 )δ：11.13(s,1H),10.27(s,1H),8.69(s,1H),7.37～7.21(m,3H),6.76(s,1H),6.49(dd,J＝10.6,8.8Hz,1H),5.81(d,J＝6.4Hz,2H),5.36(d,J＝6.4Hz,2H),4.67(s,1H),4.37(d,J＝5.3Hz,2H),1.78～1.71(m,4H),1.46～1.39(m,6H).13C-NMR(400MHz,DMSO-d6):172.5,168.6,166.4,160.2,159.4,152.6,140.3,131.2,129.3,121.6,118.5,117.7,116.2,80.6,72.3,67.4,47.5,33.6(2C),28.1,22.7(2C),MS-ESI m/z:476.83(M+H) + 。
Example 40 preparation of TM-81
Figure GDA0004064996830000681
Compound 3: boc-protected 2- (aminomethoxy) sulfonic acid (27.24g, 120mmol) was added to dichloromethane (50 mL), EDCI (25.31g, 132mmol), HOBt (17.82g, 132mmol), and 4- (4- (4-methylpiperazin-1 yl) -piperidin-1 yl) aniline (30.20g, 132mmol) were sequentially added thereto, and after stirring at room temperature for reaction for 24h, the TLC detection reaction was terminated, the solvent was removed under reduced pressure, the resulting solid was redissolved in 150mL of a mixed solution (DCM: TFA = 7:3), and after stirring at room temperature for reaction for 1h, the TLC detection reaction was terminated, and the solvent was removed by rotary evaporation under reduced pressure, and the product compound 3 was isolated by column chromatography in 76.45% yield and 99.85% HPLC.
Intermediate III: 2,4,6-trichloropyrimidine (compound 1) (55.03g, 300mmol) is added into dichloromethane (300 ml) and stirred until dissolved, the temperature is reduced to 0-5 ℃ in an ice bath, 3-chloro-4-fluoroaniline (45.72g, 315mmol, compound 2) is slowly dripped in, after the dripping is finished, the ice bath is removed, the temperature is naturally raised to room temperature, after the TLC tracking detection reaction is finished, the decompression concentration is carried out, the residue is separated by column chromatography to obtain an intermediate III, the yield is 80.33%, HPLC is 99.86%, and white solid is obtained.
An intermediate II: adding the intermediate III (29.25g, 100mmol) and the compound 3 (43.73g, 110mmol) into isopropanol (150 ml), adding concentrated hydrochloric acid (1 ml), refluxing, detecting by TLC until the reaction is complete, decompressing and concentrating, and separating the residue by column chromatography to obtain the intermediate II with the yield of 76.57%, HPLC (high performance liquid chromatography) of 99.87% and white solid.
TM-81: adding the intermediate II (7.84g, 12mmol), concentrated hydrochloric acid (0.5 ml) and acrylamide (1.02g, 14.4 mmol) into n-octanol (20 ml), placing in a closed microwave reactor, setting the temperature to 140 ℃, finishing the reaction after 0.5h, cooling to room temperature, adding 200ml of each of ethyl acetate and water, extracting, separating liquid, washing an organic phase once with water, drying with a proper amount of anhydrous sodium sulfate, concentrating under reduced pressure, and separating residues through column chromatography to obtain a compound TM-81 of the formula I, wherein the yield is 77.43 percent, and the HPLC is 99.89 percent.
1 H NMR(400MHz,DMSO-d 6 )δ：11.03(s,1H),10.15(s,1H),8.61(s,1H),8.04(s,1H),7.32～7.26(m,4H),6.69(s,1H),6.52(dd,J＝10.6,8.8Hz,1H),6.34～6.28(m,4H),5.71(d,J＝6.4Hz,2H),5.49(d,J＝6.4Hz,2H),5.16(d,J＝6.4Hz,2H),4.67(s,1H),2.81(m,4H),2.46(m,8H),2.31(m,3H),1.68～1.43(m,4H).13C-NMR(400MHz,DMSO-d6):172.4,166.7,160.2,159.5,152.6,140.2,139.6(2C),131.5,129.2,127.4,121.3,118.5,117.8,117.3(2C),116.1,115.3(3C),91.5,79.7,72.3,57.9,55.6(2C),50.5(2C),47.6(2C),43.4,28.3,,MS-ESI m/z:688.29(M+H) + 。
Example 41 preparation of TM-83
Figure GDA0004064996830000691
Compound 3: adding tert-butoxycarbonyl protected 2- (aminomethoxy) sulfonic acid (27.24g, 120mmol) to dichloromethane (30 mL), adding EDCI (25.31g, 132mmol), HOBt (17.82g, 132mmol) and 2-aminopyridine (12.42g, 132mmol) in this order, stirring at room temperature for 24h, detecting by TLC, removing the solvent under reduced pressure, redissolving the obtained solid in 120mL of a mixed solution (DCM: TFA = 7:3), stirring at room temperature for 0.5h, detecting by TLC, removing the solvent by rotary evaporation under reduced pressure, and separating by column chromatography to obtain the product compound 3 with a yield of 78.53% and HPLC of 99.87%.
Intermediate III: 2,4,6-trichloropyrimidine (compound 1) (55.03g, 300mmol) is added into dichloromethane (300 ml) and stirred until dissolved, the temperature is reduced to 0-5 ℃ in an ice bath, 3-chloro-4-fluoroaniline (45.72g, 315mmol, compound 2) is slowly dripped in, after the dripping is finished, the ice bath is removed, the temperature is naturally raised to room temperature, after the TLC tracking detection reaction is finished, the decompression concentration is carried out, the residue is separated by column chromatography to obtain an intermediate III, the yield is 81.43%, HPLC is 99.89%, and white solid is obtained.
An intermediate II: adding intermediate III (29.25g, 100mmol) and compound 3 (23.90g, 110mmol) into isopropanol (145.5 ml), adding concentrated hydrochloric acid (1 ml), refluxing, detecting by TLC until the reaction is complete, concentrating under reduced pressure, and separating the residue by column chromatography to obtain intermediate II with yield of 78.59%, HPLC:99.86% and white solid.
TM-83: adding the intermediate II (5.68g, 12mmol), concentrated hydrochloric acid (0.5 ml) and acrylamide (1.02g, 14.4 mmol) into N, N-dimethylformamide (20 ml), placing in a closed microwave reactor, setting the temperature to 150 ℃, finishing the reaction after 0.5h, cooling to room temperature, adding 200ml of ethyl acetate and water respectively, extracting, separating liquid, washing an organic phase once with water, drying with a proper amount of anhydrous sodium sulfate, concentrating under reduced pressure, and separating residues by column chromatography to obtain a compound TM-83 of the formula I, namely a white solid with the yield of 79.37% and the HPLC of 99.90%.
1 H NMR(400MHz,DMSO-d 6 )δ：11.23(s,1H),10.56(s,1H),8.69(s,1H),8.13(s,1H),7.46(s,1H),7.23～7.15(m,5H),6.73(s,1H),6.52(dd,J＝10.6,8.8Hz,1H),5.79(d,J＝6.4Hz,2H),4.71(s,1H),5.47(d,J＝6.4Hz,2H),5.21(d,J＝5.7Hz,2H),3.37(s,3H),2.15(s,3H).13C-NMR(400MHz,DMSO-d6):171.8,168.5,167.7,162.2,158.6,149.8,142.4,137.5,135.7,132.2(2C),130.4,127.5,125.8,123.3,123.6,122.3,118.5,118.7,118.4,116.2,114.8,113.5(2C),72.4,56.8,19.7，MS-ESI m/z:507.26(M+H) + 。
Example 42 preparation of TM-86
Figure GDA0004064996830000711
Compound 3: tert-butoxycarbonyl protected 2- (aminomethoxy) sulfonic acid (27.24g, 120mmol) was added to dichloromethane (30 mL), EDCI (25.31g, 132mmol), HOBt (17.82g, 132mmol), 4-methoxy-3-methylaniline (18.09g, 132mmol) were sequentially added, the mixture was stirred at room temperature for 24h, TLC detection was completed, the solvent was removed under reduced pressure, the resulting solid was redissolved in 120mL of a mixed solution (DCM: TFA = 7:3), and after stirring at room temperature for 0.5h, TLC detection was completed, the solvent was removed by reduced pressure rotary evaporation, and the product compound 3 was isolated by column chromatography in 75.46% yield and HPLC 99.89%.
Intermediate III: 2,4,6-trichloropyrimidine (compound 1) (55.03g, 300mmol) is added into dichloromethane (300 ml) and stirred until dissolved, the temperature is reduced to 0-5 ℃ in an ice bath, 3-chloro-4-fluoroaniline (45.72g, 315mmol, compound 2) is slowly dripped in, after the dripping is finished, the ice bath is removed, the temperature is naturally raised to room temperature, after the TLC tracking detection reaction is finished, the decompression concentration is carried out, the residue is separated by column chromatography to obtain an intermediate III, the yield is 82.57%, HPLC is 99.86%, and white solid is obtained.
An intermediate II: adding the intermediate III (29.25g, 100mmol) and the compound 3 (28.63g, 110mmol) into N, N-dimethylformamide (150 ml), adding concentrated hydrochloric acid (1 ml), refluxing, detecting by TLC until the reaction is complete, concentrating under reduced pressure, and separating the residue by column chromatography to obtain the intermediate II with the yield of 77.32%, HPLC (high performance liquid chromatography) 99.83% and white solid.
TM-86: adding the intermediate II (6.20g, 12mmol), concentrated hydrochloric acid (0.5 ml) and acrylamide (1.02g, 14.4mmol) into N, N-dimethylformamide (20 ml), placing in a closed microwave reactor, setting the temperature to 150 ℃, finishing the reaction for 1.0h, cooling to room temperature, adding 200ml of ethyl acetate and 200ml of water respectively, extracting, separating, washing an organic phase once by using water, drying by using a proper amount of anhydrous sodium sulfate, concentrating under reduced pressure, and carrying out column chromatography on residues to obtain a compound TM-86 of the formula I, wherein the yield is 80.51%, and the HPLC (high performance liquid chromatography) is 99.91%.
1 H NMR(400MHz,DMSO-d 6 )δ：10.93(s,1H),10.20(s,1H),8.70(s,1H),7.75～7.54(m,2H),7.45(s,1H),7.24～7.17(m,3H),6.71(s,1H),6.55(dd,J＝10.6,8.8Hz,1H),5.82(d,J＝6.4Hz,2H),5.57～5.44(m,2H),4.77～4.69(m,2H),4.59(s,1H),3.47(s,3H),2.14(s,3H).13C-NMR(400MHz,DMSO-d6):172.6,168.8,167.7,162.1,158.6,149.7,142.4,137.4,135.7,130.2,128.5,125.9,124.3,123.4,121.2,119.8,118.7,115.1,114.8,65.4,58.5,42.3,19.7，MS-ESI m/z:550.12(M+H) + 。
Example 43 preparation of TM-89
Figure GDA0004064996830000721
Intermediate III: 2,4,6-trichloropyrimidine (compound 1) (55.03g, 300mmol) is added into dichloromethane (300 ml) and stirred until dissolved, the temperature is reduced to 0-5 ℃ in an ice bath, 3-chloro-4-fluoroaniline (45.72g, 315mmol, compound 2) is slowly dripped in, after the dripping is finished, the ice bath is removed, the temperature is naturally raised to room temperature, after the TLC tracking detection reaction is finished, the decompression concentration is carried out, the residue is separated by column chromatography to obtain an intermediate III, the yield is 82.46%, HPLC (high performance liquid chromatography) is 99.85%, and white solid is obtained.
An intermediate II: adding intermediate III (29.25g, 100mmol) and compound 3 (18.50g, 110mmol) into N, N-dimethylformamide (150 ml), adding concentrated hydrochloric acid (1 ml), refluxing, detecting by TLC until the reaction is complete, concentrating under reduced pressure, and separating the residue by column chromatography to obtain intermediate II with yield of 77.52%, HPLC:99.89% and white solid.
TM-89: adding the intermediate II (5.09g, 12mmol), concentrated hydrochloric acid (0.5 ml) and acrylamide (1.02g, 14.4 mmol) into N, N-dimethylformamide (20 ml), placing in a closed microwave reactor, setting the temperature to 150 ℃, finishing the reaction after 1.0h, cooling to room temperature, adding 200ml of ethyl acetate and water respectively, extracting, separating liquid, washing an organic phase once with water, drying with a proper amount of anhydrous sodium sulfate, concentrating under reduced pressure, and separating residues by column chromatography to obtain a compound TM-89 of the formula I, wherein the yield is 80.43 percent, and the HPLC (high performance liquid chromatography) is 99.91 percent.
1 H NMR(400MHz,DMSO-d 6 )δ：10.93(s,1H),10.20(s,1H),8.70(s,1H),7.96～7.73(m,4H),7.35～7.23(m,3H),6.76(s,1H),6.57(dd,J＝10.6,8.8Hz,1H),6.15(m,2H),5.91(d,J＝6.4Hz,2H),4.63(s,1H),3.39(m,2H),1.14(s,3H).13C-NMR(400MHz,DMSO-d6):172.4,167.4,166.5,159.5,152.6,140.2,132.3,131.2,129.5,121.5,118.4,117.6,117.2,116.1,109.7,72.3,40.3,12.6，MS-ESI m/z:458.56(M+H) + 。
Example 44 preparation of TM-90,
Figure GDA0004064996830000731
intermediate III: 2,4,6-tribromopyrimidine (compound 1) (31.68g, 100mmol) is added into dichloromethane (200 ml) and stirred until dissolved, the temperature is reduced to 0-5 ℃ by ice bath, 3-chloro-4-fluoroaniline (15.23g, 105mmol, compound 2) is slowly dripped, the ice bath is removed after the dripping is finished, the temperature is naturally raised to room temperature, after the TLC tracking detection reaction is finished, the decompression concentration is carried out, the residue is separated by column chromatography to obtain an intermediate III, the yield is 81.76%, the HPLC is 99.86%, and white solid is obtained.
An intermediate II: adding the intermediate III (38.14g, 100mmol) and the compound 3 (15.20g, 110mmol) into N, N-dimethylformamide (150 ml), adding concentrated hydrochloric acid (1 ml), refluxing, detecting by TLC until the reaction is complete, concentrating under reduced pressure, and separating the residue by column chromatography to obtain an intermediate II with the yield of 75.57 percent, HPLC (high performance liquid chromatography) of 99.83 percent and a white solid.
TM-90: adding the intermediate II (5.26g, 12mmol), concentrated hydrochloric acid (0.5 ml) and acrylamide (1.02g, 14.4mmol) into N, N-dimethylformamide (20 ml), placing in a closed microwave reactor, setting the temperature to 150 ℃, finishing the reaction for 1.0h, cooling to room temperature, adding 200ml of ethyl acetate and 200ml of water respectively, extracting, separating, washing an organic phase once with water, drying with a proper amount of anhydrous sodium sulfate, concentrating under reduced pressure, and separating residues by column chromatography to obtain the compound TM-90 of the formula I, wherein the yield is 79.86%, and the HPLC (high performance liquid chromatography) is 99.89%.
1 H NMR(400MHz,DMSO-d 6 )δ：10.86(s,1H),10.16(s,1H),8.69(s,1H),7.63(s,1H),7.43～7.36(m,3H),7.23(s,1H),6.62(dd,J＝10.6,8.8Hz,1H),6.41(m,2H),5.89(d,J＝6.4Hz,2H),4.69(s,1H),3.71(m,3H),2.64(s,3H).13C-NMR(400MHz,DMSO-d6):172.4,167.4,166.5,159.3,152.7,149.2,14.5,131.7,131.2,131.1,129.2,126.1,121.5,118.7,118.4,117.6,116.1,72.4,56.3，14.5,MS-ESI m/z:428.33(M+H) + 。
Example 45 preparation of TM-92
Figure GDA0004064996830000741
Intermediate III: 2,4,6-tribromopyrimidine (compound 1) (31.68g, 100mmol) is added into dichloromethane (200 ml) and stirred until dissolved, the temperature is reduced to 0-5 ℃ in ice bath, 3-chloro-4-fluoroaniline (15.23g, 105mmol, compound 2) is slowly dripped in, after the dripping is finished, the ice bath is removed, the temperature is naturally raised to room temperature, after the TLC tracing detection reaction is finished, the decompression and concentration are carried out, and the residue is separated by column chromatography to obtain an intermediate III, the yield is 82.56%, HPLC (high performance liquid chromatography) 99.87%, and white solid is obtained.
An intermediate II: intermediate III (38.14g, 100mmol) and compound 3 (22.14g, 110mmol) were added to N, N-dimethylformamide (150 ml), concentrated hydrochloric acid (1 ml) was added, the reaction was refluxed, TLC detection was followed until completion, concentration was carried out under reduced pressure, and the residue was separated by column chromatography to give intermediate II in 76.25% yield, HPLC:99.85%, white solid.
TM-92: adding the intermediate II (6.02g, 12mmol), concentrated hydrochloric acid (0.5 ml) and acrylamide (1.02g, 14.4 mmol) into N, N-dimethylformamide (20 ml), placing the mixture into a closed microwave reactor, setting the temperature to 150 ℃, finishing the reaction after 1.0h, cooling to room temperature, adding 200ml of ethyl acetate and water respectively, extracting and separating the liquid, washing an organic phase once with water, drying with a proper amount of anhydrous sodium sulfate, concentrating under reduced pressure, and separating residues by column chromatography to obtain a compound TM-92 of the formula I, wherein the yield is 79.87% and the HPLC (high performance liquid chromatography) is 99.88%.
1 H NMR(400MHz,DMSO-d 6 )δ：10.81(s,1H),10.32(s,1H),8.75(s,1H),7.69(m,2H),7.24～7.17(m,3H),6.76(m,2H),6.72(s,1H),6.52(dd,J＝10.6,8.8Hz,1H),5.84(d,J＝6.4Hz,2H),5.49(m,3H),4.71(s,1H),3.27(s,3H).13C-NMR(400MHz,DMSO-d6):172.2,167.6,166.7,159.5,152.7,148.1,140.2,131.2,129.2(3C),128.9,121.5,118.4,117.6,117.2(2C),116.1,87.6,72.3,57.7，MS-ESI m/z:491.73(M+H) + 。
Example 46 preparation of TM-94
Figure GDA0004064996830000751
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Compound 3: adding tert-butoxycarbonyl protected 2-chloroethylamine (28.65g, 120mmol) into N, N dimethylformamide (100 mL), sequentially adding potassium carbonate (41.46 g) and 4-methoxy-3-methylaniline (18.09g, 132mmol), stirring at 100 ℃ for 24h, detecting by TLC to finish the reaction, removing the solvent under reduced pressure, re-dissolving the obtained solid in 120mL of mixed solution (DCM: TFA = 7:3), stirring at room temperature for 0.5h, detecting by TLC to finish the reaction, removing the solvent by rotary evaporation under reduced pressure, and separating by column chromatography to obtain the product compound 3 with the yield of 56.32% and HPLC of 99.83%.
An intermediate III: 2,4,6-tribromopyrimidine (compound 1) (31.68g, 100mmol) is added into dichloromethane (200 ml) and stirred to dissolve, the temperature is reduced to 0-5 ℃ in ice bath, 3-chloro-4-fluoroaniline (15.23g, 105mmol, compound 2) is slowly dripped, after the dripping is finished, the ice bath is removed, the temperature is naturally raised to room temperature, after the TLC tracing detection reaction is finished, the decompression and concentration are carried out, and the residue is separated by column chromatography to obtain an intermediate III, the yield is 81.47%, HPLC (high performance liquid chromatography) 99.89% and white solid.
An intermediate II: intermediate III (38.14g, 100mmol) and compound 3 (19.83g, 110mmol) were added to N, N-dimethylformamide (150 ml), concentrated hydrochloric acid (1 ml) was added, the reaction was refluxed, TLC detection was followed until completion of the reaction, and the residue was concentrated under reduced pressure, and column chromatography gave intermediate II in 76.43% yield, HPLC:99.87%, as a white solid.
TM-94: adding the intermediate II (5.77g, 12mmol), concentrated hydrochloric acid (0.5 ml) and acrylamide (1.02g, 14.4 mmol) into N, N-dimethylformamide (20 ml), placing in a closed microwave reactor, setting the temperature to 150 ℃, finishing the reaction after 1.0h, cooling to room temperature, adding 200ml of ethyl acetate and water respectively, extracting, separating liquid, washing an organic phase once with water, drying with a proper amount of anhydrous sodium sulfate, concentrating under reduced pressure, and separating residues by column chromatography to obtain a compound TM-94 of the formula I, namely a white solid with the yield of 78.96% and the HPLC of 99.87%.
1 H NMR(400MHz,DMSO-d 6 )δ：10.87(s,1H),10.26(s,1H),8.76(s,1H),7.36～7.21(m,3H),6.71(s,1H),6.55(dd,J＝10.6,8.8Hz,1H),6.43(s,1H),6.13(m,2H),5.82(d,J＝6.4Hz,2H),4.64(s,1H),3.77(s,3H),2.34(s,3H).13C-NMR(400MHz,DMSO-d6):172.4,166.5,160.2,159.7,152.7,147.2,140.2,139.7,131.2,129.2,125.4,121.5,118.4,117.6,116.1,115.2,114.3,111.6,72.6,56.3,49.2(2C),14.6,MS-ESI m/z:470.64(M+H) + 。
Example 47 preparation of TM-98
Figure GDA0004064996830000761
Compound 3: adding tert-butoxycarbonyl protected chloromethoxymethylamine (30.57g, 120mmol) into dichloromethane (30 mL), sequentially adding potassium carbonate (41.46 g) and 4-fluoroaniline (14.67g, 132mmol), stirring at room temperature for 24h, detecting by TLC, removing solvent under reduced pressure, dissolving the obtained solid in 120mL of mixed solution (DCM: TFA = 7:3), stirring at room temperature for 0.5h, detecting by TLC, removing solvent by reduced pressure rotary evaporation, and separating by column chromatography to obtain product 3 with yield of 65.37% and HPLC:99.79%.
An intermediate III: 2,4,6-tribromopyrimidine (compound 1) (31.68g, 100mmol) is added into dichloromethane (200 ml) and stirred until dissolved, the temperature is reduced to 0-5 ℃ by ice bath, 3-chloro-4-fluoroaniline (15.23g, 105mmol, compound 2) is slowly dripped, the ice bath is removed after the dripping is finished, the temperature is naturally raised to room temperature, after the TLC tracking detection reaction is finished, the pressure is reduced and the concentration is carried out, and the residue is separated by column chromatography to obtain an intermediate III, the yield is 81.49%, HPLC is 99.87%, and white solid is obtained.
An intermediate II: adding the intermediate III (38.14g, 100mmol) and the compound 3 (15.63g, 110mmol) into N, N-dimethylformamide (150 ml), adding concentrated hydrochloric acid (1 ml), refluxing, detecting by TLC until the reaction is complete, concentrating under reduced pressure, and separating the residue by column chromatography to obtain the intermediate II with the yield of 76.47%, HPLC:99.86% and white solid.
TM-98: adding the intermediate II (5.31g, 12mmol), concentrated hydrochloric acid (0.5 ml) and acrylamide (1.02g, 14.4mmol) into N, N-dimethylformamide (20 ml), placing in a closed microwave reactor, setting the temperature to 150 ℃, finishing the reaction for 1.0h, cooling to room temperature, adding 200ml of ethyl acetate and 200ml of water respectively, extracting, separating, washing an organic phase once with water, drying with a proper amount of anhydrous sodium sulfate, concentrating under reduced pressure, and separating residues by column chromatography to obtain the compound TM-98 of the formula I, wherein the yield is 78.75%, and the HPLC is 99.89%.
1 H NMR(400MHz,DMSO-d 6 )δ：10.74(s,1H),10.16(s,1H),8.92(s,1H),7.34～7.29(m,3H),6.76(m,2H),6.73(s,1H),6.52(dd,J＝10.6,8.8Hz,1H),6.43(m,2H),5.81(d,J＝6.4Hz,2H),5.26(m,4H),4.68(s,1H).13C-NMR(400MHz,DMSO-d6):172.4,166.8,160.3,159.5,152.7,143.2,140.2,131.2,129.2,121.5,118.4,117.6,116.1(2C),115.2(2C),78.8,78.6,72.3，MS-ESI m/z:432.6(M+H) + 。
Example 48 preparation of TM-100
Figure GDA0004064996830000771
Intermediate III: 2,4,6-tribromopyrimidine (compound 1) (31.68g, 100mmol) is added into dichloromethane (200 ml) and stirred until dissolved, the temperature is reduced to 0-5 ℃ by ice bath, 3-chloro-4-fluoroaniline (15.23g, 105mmol, compound 2) is slowly dripped, the ice bath is removed after the dripping is finished, the temperature is naturally raised to room temperature, after the TLC tracking detection reaction is finished, the pressure is reduced and the concentration is carried out, and the residue is separated by column chromatography to obtain an intermediate III, the yield is 80.87%, HPLC is 99.85%, and white solid is obtained.
An intermediate II: adding the intermediate III (38.14g, 100mmol) and the compound 3 (19.61g, 110mmol) into N, N-dimethylformamide (150 ml), adding concentrated hydrochloric acid (1 ml), refluxing, detecting by TLC until the reaction is complete, concentrating under reduced pressure, and separating the residue by column chromatography to obtain the intermediate II with yield of 76.59 percent and HPLC (high performance liquid chromatography) of 99.89 percent as a white solid.
TM-100: adding the intermediate II (5.75g, 12mmol), concentrated hydrochloric acid (0.5 ml) and acrylamide (1.02g, 14.4 mmol) into N, N-dimethylformamide (20 ml), placing in a closed microwave reactor, setting the temperature to 150 ℃, finishing the reaction after 1.0h, cooling to room temperature, adding 200ml of ethyl acetate and water respectively, extracting, separating liquid, washing an organic phase once with water, drying with a proper amount of anhydrous sodium sulfate, concentrating under reduced pressure, and separating residues by column chromatography to obtain the compound TM-100 of the formula I, wherein the yield is 79.68%, and the HPLC (high performance liquid chromatography) is 99.88%.
1 H NMR(400MHz,DMSO-d 6 )δ：10.87(s,1H),10.32(s,1H),8.76(s,1H),8.49～8.31(m,2H),7.40～7.29(m,2H),7.21～7.15(m,3H),6.73(s,1H),6.57(dd,J＝10.6,8.8Hz,1H),5.86(d,J＝6.4Hz,2H),4.67(s,1H),3.31(s,1H),2.81～2.72(m,4H),1.91～1.65(m,2H).13C-NMR(400MHz,DMSO-d6):172.4,166.7,160.2,159.5,152.7,145.2,140.2,138.8,137.5,131.1,129.1,124.6,122.7,121.6,118.4,117.6,116.2,72.4,59.6,52.5,50.8,33.2,MS-ESI m/z:468.57(M+H) + 。
Example 49 preparation of TM-104
Figure GDA0004064996830000781
Compound 3: adding tert-butoxycarbonyl protected 3-chloro-cyclobutylamine (31.77g, 120mmol) into dichloromethane (30 mL), sequentially adding potassium carbonate (41.46 g) and 2-aminobenzimidazole (17.57g, 132mmol), stirring at room temperature for 24h, detecting by TLC, removing solvent under reduced pressure, re-dissolving the obtained solid in 120mL of mixed solution (DCM: TFA = 7:3), stirring at room temperature for 0.5h, detecting by TLC, removing solvent by reduced pressure rotary evaporation, and separating by column chromatography to obtain the product compound 3 with the yield of 75.56% and HPLC:99.89%.
Intermediate III: 2,4,6-tribromopyrimidine (compound 1) (31.68g, 100mmol) is added into dichloromethane (200 ml) and stirred to dissolve, the temperature is reduced to 0-5 ℃ in ice bath, 3-chloro-4-fluoroaniline (15.23g, 105mmol, compound 2) is slowly dripped, after the dripping is finished, the ice bath is removed, the temperature is naturally raised to room temperature, after the TLC tracing detection reaction is finished, the pressure is reduced and the concentration is carried out, and the residue is separated by column chromatography to obtain an intermediate III, the yield is 81.65%, HPLC is 99.89%, and white solid is obtained.
An intermediate II: intermediate III (38.14g, 100mmol) and compound 3 (22.25g, 110mmol) were added to N, N-dimethylformamide (150 ml), concentrated hydrochloric acid (1 ml) was added, the reaction was refluxed, TLC detection was followed until completion of the reaction, and the residue was concentrated under reduced pressure, and column chromatography gave intermediate II in 77.62% yield, HPLC:99.87%, as a white solid.
TM-104: adding the intermediate II (6.03g, 12mmol), concentrated hydrochloric acid (0.5 ml) and acrylamide (1.02g, 14.4 mmol) into N, N-dimethylformamide (20 ml), placing in a closed microwave reactor, setting the temperature to 150 ℃, finishing the reaction after 1.0h, cooling to room temperature, adding 200ml of ethyl acetate and water respectively, extracting, separating liquid, washing an organic phase once with water, drying with a proper amount of anhydrous sodium sulfate, concentrating under reduced pressure, and separating residues by column chromatography to obtain a compound TM-104 of the formula I, wherein the yield is 79.23 percent and the HPLC (high performance liquid chromatography) is 99.87 percent.
1 H NMR(400MHz,DMSO-d 6 )δ：10.85(s,1H),10.32(s,1H),8.87(s,1H),7.75(m,2H),7.25(m,2H),7.23～7.15(m,3H),6.73(s,1H),6.53(dd,J＝10.6,8.8Hz,2H),5.83(d,J＝6.4Hz,2H),4.67(s,1H),3.17(m,2H),2.24(m,4H).13C-NMR(400MHz,DMSO-d6):172.2,166.7,160.2,159.5,152.7,141.6,140.2,138.8,131.2,129.1,123.2(2C),121.5,118.2,117.6,116.2,115.2(2C),72.5,49.6,38.4(2C),MS-ESI m/z:492.46(M+H) + 。
In vitro cytotoxic Activity assay for cancer cells
To investigate the ability of the target compound synthesized in this experiment to inhibit tumor cell proliferation, we determined the in vitro cytotoxicity of the compound of the present invention against four tumor cells, human colon cancer cell (HT 29), lung cancer cell (a 549), breast cancer cell (MCF 7), head and neck squamous cell carcinoma cell (HN 5), and austinib (AZD 9291) was used as a positive control. The assay used was a standard MTT assay.
The experimental method specifically comprises the following steps:
human colon cancer cells, lung cancer cells, breast cancer cells and head and neck squamous carcinoma cells (Shanghai and Nippon Biotechnology Co., ltd.) are trypsinized for 10min, then liquid is discarded, 5% serum culture solution is used for blowing and beating, the cell concentration is adjusted to 300-400/mu L, the tested compounds are sequentially added, a blank group only containing the culture solution is reserved, and the mixture is cultured in an incubator for 24h. Discarding supernatant, adding diluted target compounds with different concentrations into a 96-well plate (Shanghai horizontal macro biotechnology, inc.), adding cell sap only into a control group, setting three auxiliary wells for each group of concentration, mixing uniformly, continuing to culture for 36h, observing cell morphology change in different time periods, adding a prepared MTT solution into each well for color development after the cells and the tested compounds fully act, and continuing to culture for 6h. Discarding the supernatant, dissolving in 100 μ L DMSO to form purple crystals, selecting 490nm, and adding enzymeThe absorbance was measured with a standard meter (HBS-1096A enzyme labeling Analyzer, nanjing DeFei laboratory instruments Co., ltd.). By the formula: cell inhibition (%) = (1-A experiment/A control) × 100%, and the IC was determined 50 Values (cell viability vs log dose mapping).
The results of in vitro toxicity tests on the inhibition of proliferation of four cancer cells, namely human colon cancer cell (HT 29), lung cancer cell (A549), breast cancer cell (MCF 7) and head and neck squamous carcinoma cell (HN 5), are shown in Table 1.
TABLE 1 test for tumor cell toxicity of compounds
Figure GDA0004064996830000791
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Figure GDA0004064996830000801
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Figure GDA0004064996830000811
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Figure GDA0004064996830000821
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Figure GDA0004064996830000831
Note that the values in table a are the average values of 3 experiments; b the action time of the medicine is 36h
The proliferation inhibiting effect of other compounds not listed in the table on cancer cells is similar to that of the compounds in the table, and all show good cytotoxicity.
Compared with a control compound AZD9291, the novel anti-tumor active compound provided by the invention has four cancer thin cells of human colon cancer cells (HT 29), lung cancer cells (A549), breast cancer cells (MCF 7) and head and neck squamous carcinoma cells (HN 5)The cell growth inhibition activity is equivalent to that of AZD9291, and some compounds show outstanding inhibition effect on lung cancer cells, for example, the inhibition effect of compounds TM-6, TM-9, TM-14, TM-21, TM-58 and TM-68 on lung cancer cells is greatly improved compared with that of AZD 9291. Wherein, the proliferation inhibition effect of the compound TM-21 on four tumor cells in the experiment is better than that of a control drug AZD9291, the inhibition effect on three tumor cells of HT29, MCF7 and A549 is nearly ten times that of the AZD9291, wherein the inhibition effect on IC of lung cancer (A549) is nearly ten times that of the AZD9291 50 Reaches 2.66nM, and shows excellent inhibiting activity and selectivity.
Solubility test of Compounds
To investigate the solubility of the compounds according to the invention, the solubility of the compounds according to the invention and of the control compound ADZ9291 in different media was tested. 10ml of medium were measured: adding excessive tested compound into 6 groups of penicillin bottles by using water, 0.1mmol/L HCl solution, phosphate buffer solution with pH value of 6.8, simulated gastric juice (SGF), simulated fasted state intestinal juice (FaSSIF) and simulated feeding state intestinal juice (FeSSIF) to prepare 6 corresponding groups of supersaturated solution, placing the prepared solution into a constant-temperature water bath with the temperature of 25 ℃ for continuously oscillating and stirring for 2 hours until the solution is balanced, filtering by using a 0.45 mu m filter membrane, taking 1ml of supernatant, additionally taking 9ml of purified water, adding the purified water into the supernatant, oscillating and uniformly mixing, and detecting by using HPLC (high performance liquid chromatography), wherein the specific result is shown in Table 2:
TABLE 2 solubility of the compounds of the formula (I) according to the invention in various media
Figure GDA0004064996830000841
/>
Figure GDA0004064996830000851
/>
Figure GDA0004064996830000861
/>
Figure GDA0004064996830000871
The experimental result shows that compared with a reference compound AZD9291, the compound of the invention has better solubility in water, 0.1mmol/L HCl solution, phosphate buffer solution with pH of 6.8 and three biological solvents, and particularly, the solubility in water is greatly improved compared with the prior AZD9291 on the market, thereby being beneficial to the development of a preparation process; the solubility in biological solvent is improved, which is beneficial for the absorption of the medicine by patients and improves the bioavailability.
Bioavailability test of compounds
Bioavailability of each test compound was investigated using the following method: a test compound was dissolved in a 0.2M phosphate buffer solution to prepare a 2mg/ml solution as an administration formulation, and 6 male SD rats (200-240 g in body weight, purchased from Shanghai Spire-Bikeka laboratory animals Co., ltd.) were divided into two groups of A, B, and after fasting but 12 hours of free drinking water, blank plasma was collected at time 0; respectively intragastrically administering a compound TM-21 to rats in group A at a dose of 10mg/kg; taking rats in group B, and administrating compound TM-21 via tail vein at administration dose of 2mg/kg; after administration, continuously taking 300 mu l of blood from fundus venous plexus for 5min,30min,1h,2h,4h,8h,12h and 24h, placing the blood into a test tube distributed with heparin anticoagulation, centrifuging at 8000rpm/min for 10min, and taking upper plasma; precisely measuring 100 mu l of plasma sample in a blank centrifuge tube, adding 10 mu l of ginkgolide B internal standard solution (5.85 mu g/ml), mixing uniformly, adding 1ml of ethyl acetate extract, performing vortex oscillation for 3min, and centrifuging for 10min at 14000r/min, extracting an upper organic phase, and performing LC-MS/MS analysis; according to the blood concentration-time data obtained by the test, a non-atrioventricular model of WinNonlin is adopted to process pharmacokinetic data to calculate pharmacokinetic parameters, and a control compound AZD9291 is used for carrying out pharmacokinetic and bioavailability investigation by the same experimental method, and the specific experimental results are shown in a table 3:
TABLE 3 pharmacokinetic parameters of the Compound of formula (I) of the invention in rats
Figure GDA0004064996830000881
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Figure GDA0004064996830000891
According to the experimental results of the pharmacokinetic studies, it can be seen that the compound of formula (I) has relatively high bioavailability, and compared with the control AZD9291, the bioavailability of the compound of the invention is better than that of AZD9291, wherein the bioavailability of TM-6, TM-9, TM-14, TM-21, TM-58 and TM-68 is about twice that of AZD9291, and the compound shows excellent bioavailability characteristics.

Claims (9)

1.2,4,6-triaminopyrimidine compounds shown as formula (I) or pharmaceutically acceptable salts thereof;
Figure FDA0004100328480000011
wherein:
R 1 selected from: c 3-6 Cycloalkylene radical, C 3-6 Heterocycloalkylene, C 1-6 Alkylene radical, C 1-6 Alkyleneoxy, arylene and heteroarylene, wherein arylene is phenylene, heteroarylene is pyridylene, and C 3-6 The heteroatom in the heterocycloalkylene group is selected from N;
R 2 selected from the group consisting of: -C (O) -R 4 、-S(O) 2 -R 4 、C 1-3 Alkoxy, -NR 5 R 6 ；
Wherein R is 4 Is selected from C 1-3 Alkyl radical, C 1-3 Haloalkyl, -NHR 7 、C 1-3 An alkoxy group; wherein-NHR 7 Selected from the group consisting of N, N-dimethylethylenediamino, cyclohexylamino, anilino, p-fluoroanilino, pyridin-2-amino, 4-methoxy-3-methylanilino, benzimidazol-2-amino or 4- (4- (4-methylpiperazin-1-yl) -piperidin-1-yl) anilino;
wherein R is 5 Independently selected from H or C 1-3 An alkyl group; r is 6 Independently selected from H, C 1-3 Alkyl radical, C 3-6 Cycloalkyl, C 1-3 Halogenated alkyl, aryl and heteroaryl, wherein the aryl is phenyl, and the heteroaryl is pyridyl or benzimidazolyl;
wherein C is as defined above 3-6 The cycloalkyl, aryl, heteroaryl may also be substituted by one or more R 3 The group is further substituted;
wherein R is 3 Independently selected from methyl, methoxy and fluoro.
2. The aminopyrimidine compound according to claim 1 or a pharmaceutically acceptable salt thereof wherein the compound of formula (I) is selected from the group consisting of:
Figure FDA0004100328480000021
3. a process for the preparation of aminopyrimidines according to any of claims 1-2 including the steps of:
Figure FDA0004100328480000031
wherein R is 1 And R 2 As defined for formula (I); x is selected from F, cl, br and I.
4. The method for preparing aminopyrimidine compounds according to claim 3 wherein X is Cl, comprising the steps of:
step 1: the compound 1, namely 2,4,6-trichloropyrimidine and the compound 2, namely 3-chloro-4-fluoroaniline have substitution reaction at the 4-position of a pyrimidine ring to generate an intermediate III, namely 2,6-dichloro-4- (3-chloro-4-fluoroaniline) -pyrimidine;
step 2: carrying out substitution reaction on the intermediate III and the compound 3 at the 2-position of a pyrimidine ring under the action of a catalyst to generate an intermediate II; wherein R in the compound 3 1 And R 2 As defined for formula (I);
and 3, step 3: the intermediate II, namely 2,4-disubstituted pyrimidine compound reacts with the compound 4 to generate the compound shown in the formula (I).
5. The method for preparing aminopyrimidine compounds according to claim 3, wherein X is Cl, comprising the steps of:
step 1: adding a compound 1, namely 2,4,6-trichloropyrimidine into a solvent, stirring until the compound 1 is dissolved, cooling in an ice bath, slowly dropwise adding a compound 2, namely 3-chloro-4-fluoroaniline, removing the ice bath after the dropwise adding is finished, controlling the temperature for reaction, performing TLC (thin layer chromatography) tracking detection reaction, performing reduced pressure concentration, and performing column chromatography separation on a residue to obtain an intermediate III, namely 2,6-dichloro-N- (3-chloro-4-fluorophenyl) pyrimidine-4-amine;
and 2, step: adding the intermediate III and the compound 3 into a solvent, adding a strong acid catalyst, performing temperature-controlled reflux reaction, detecting and tracking by TLC (thin layer chromatography) until the reaction is complete, performing reduced pressure concentration, and performing column chromatography separation on the residue to obtain an intermediate II; wherein R in the compound 3 1 And R 2 As defined for formula (I);
and step 3: adding the intermediate II, concentrated hydrochloric acid and the compound 4 acrylamide into a solvent, placing the mixture into a closed microwave reactor, reacting at a certain temperature until the reaction is finished, cooling to room temperature, adding ethyl acetate and water, extracting, separating liquid, washing an organic phase with water, drying with a proper amount of anhydrous sodium sulfate, concentrating under reduced pressure, and separating residues by column chromatography to obtain the compound TM shown in the formula (I).
6. A process for preparing aminopyrimidines according to claim 3 wherein when R is the same as R 1 Is selected from C 3-6 Heterocycloalkylene, C 1-6 Alkylene radical, C 1-6 Alkyleneoxy, arylene, heteroarylene, R 2 Selected from-C (O) -R 4 ，R 4 Is selected from-NHR 7 When compound 3 is prepared by the following method:
Figure FDA0004100328480000041
wherein, the-NHR 7 As defined in claim 1.
7. A process for preparing aminopyrimidines according to claim 3 wherein when R is 1 Is selected from C 3-6 Heterocycloalkylene, C 1-6 Alkylene radical, C 1-6 Alkyleneoxy, arylene, heteroarylene, R 2 Is selected from-S (O) 2 -R 4 ，R 4 Is selected from-NHR 7 Compound 3 was prepared by the following method:
Figure FDA0004100328480000042
wherein, the-NHR 7 As defined in claim 1.
8. A pharmaceutical composition, comprising: a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1-2, optionally together with a pharmaceutically acceptable carrier, excipient, adjuvant, or diluent.
9. Use of a compound of formula (I), or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1-2, for the non-therapeutic inhibition of tumor cell proliferation in vitro.
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