CN108164548B - Pyrimidomorpholine derivative and preparation method and application thereof - Google Patents
Pyrimidomorpholine derivative and preparation method and application thereof Download PDFInfo
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- CN108164548B CN108164548B CN201611116574.0A CN201611116574A CN108164548B CN 108164548 B CN108164548 B CN 108164548B CN 201611116574 A CN201611116574 A CN 201611116574A CN 108164548 B CN108164548 B CN 108164548B
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- C07—ORGANIC CHEMISTRY
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- C07D498/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms
- C07D498/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms in which the condensed system contains two hetero rings
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
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Abstract
The invention belongs to the field of chemical medicine, and particularly relates to an anti-medullary thyroid cancer, anti-acute leukemia and anti-inflammatory small molecule compound, and a preparation method and application thereof. The invention aims to solve the technical problems that the selectivity of the medicines on the market for medullary thyroid carcinoma is poor, and the medicines are easy to generate drug resistance and toxic and side effects. The scheme for solving the technical problems is to provide the pyrimido morpholine derivative, wherein the derivative is mainly substituted at the 6-position of pyrimidine, and the structure of the derivative is shown as a formula I. The compound provided by the invention has the advantages of high activity of resisting medullary thyroid carcinoma, low toxic and side effects, and capability of overcoming clinical drug resistance, and has great value in drug development for medullary thyroid carcinoma.
Description
Technical Field
The invention belongs to the technical field of organic synthetic drugs, and particularly relates to a pyrimido morpholine derivative and a preparation method and application thereof.
Background
Protein kinase is a phosphotransferase that functions to transfer the gamma phosphate group of ATP to a specific amino acid residue of a protein to accomplish phosphorylation of the protein. Protein kinases in eukaryotic cells can be divided into five classes according to their phosphorylated amino acid residues: 1) serine/threonine protein kinases; 2) tyrosine protein kinase; 3) a tryptophan protein kinase; 4) a histidine protein kinase; 5) aspartyl/glutamyl protein kinases. Protein kinases are widely present in organisms, regulate cellular DNA replication, energy metabolism, and physiological processes associated with growth and differentiation, occupy an extremely important position in cell signal transduction, and abnormal activities thereof may cause various diseases including tumors, diabetes, and inflammation. Therefore, protein kinase also becomes one of the important targets for treating related diseases.
The RET proto-oncogene, located on the long arm of chromosome 10, contains 21 exons and encodes a single transmembrane tyrosine kinase receptor. Studies have demonstrated the presence of mutations in the RET receptor in thyroid cancer, largely divided into RET gene fusions and single point mutations. RET gene fusion is closely associated with clinically common papillary thyroid carcinoma, while point mutations are associated with multiple endocrine neoplasia syndrome (MEN2) in medullary thyroid carcinoma. Currently, a number of small molecule compounds directed against RET kinase have entered preclinical studies, and the FDA has approved vandetanib and cabozantinib for the treatment of non-surgically resectable malignant locally advanced or metastatic Medullary Thyroid Cancer (MTC). And the secondary mutation of RET kinase, such as RET-V804M/V804L mutation, can cause the medullary thyroid carcinoma patients to generate drug resistance to vandetanib and cabozantinib. Therefore, the development of novel small molecular compounds with inhibitory activity against both RET wild type and drug-resistant mutant types has great application value in clinic.
FLT3(Fms-like tyrosine kinase 3, human Fms-like tyrosine kinase 3) is a receptor tyrosine kinase that plays an important role in the proliferation and differentiation of hematopoietic stem cells. Recent molecular biology studies have found that high expression of FLT3 occurs in approximately one-third of human Acute Myeloid Leukemia (AML) patients. Currently, FLT3 has been an important therapeutic target for treating AML, and its inhibitors are considered to be currently the most promising molecularly targeted drugs for treating AML. FLT3 inhibitors such as SU-5416, PKC-412 and CEP-701 are currently in clinical trials. However, recent studies find that the clinical trial effect of the compounds is not ideal, and one of the main reasons is that the compounds have large toxic and side effects, and the increase of the drug dosage is limited, so that the curative effect is influenced. Therefore, the development of highly potent and low-toxicity FLT3 inhibitors for AML treatment is of particular importance.
Disclosure of Invention
Aiming at the defects that drugs on the market for medullary thyroid carcinoma are easy to generate drug resistance and toxic and side effects clinically at present, the invention aims to solve the technical problem of providing a preparation method and application of a novel micromolecular compound for resisting medullary thyroid carcinoma.
The scheme for solving the technical problems is to provide a pyrimido morpholine derivative, wherein the derivative is mainly substituted at the 6-position of pyrimidine, and the structure of the derivative is shown as a formula I:
wherein X is C or N; n is 1-4;
when R is1Is composed of
When R is2is-H, halogen, -OH, C1-C8 alkyl, C1-C8 alkoxy, -CF3Or C1-C8 alkenyl; when R is2Is composed of
When R is1is-H, halogen, -OH, C1-C8 alkyl, C1-C8 alkoxy, -CF3Or C1-C8 alkenyl;
or R1And R2Form a 5-12 membered heterocycle; the heteroatom of the heterocycle is N, O or S; the number of the heteroatoms is 1-5; the heterocyclic ring is a substituted saturated or unsaturated heterocyclic ring; the substituent of the substituted saturated or unsaturated heterocycle is-H, halogen, -OH, C1-C8 alkyl or C1-C8 alkoxy;
R3～R5independently is-H, halogen, -OH, C1-C8 alkyl, C1-C8 alkoxy, -CF3Or C1-C8 alkenyl;
R6～R10independently C2-C4 acyl, substituted phenyl substituted C1-C8 alkyl, substituted 6-12-membered aryl and substituted 5-10-membered saturated or unsaturated heterocycle, wherein hetero atoms of the heterocycle are N, O or S, and the number of the hetero atoms is 1-4; the substituent of the substituted phenyl, the substituted aryl and the substituted heterocycle is-H and halogenOH, -OH, C1-C8 alkyl, C1-C8 alkoxy, phenyl,
-CF3、-OCF3Or C1-C8 alkenyl.
Preferred embodiments of the present invention are those wherein X is C or N; n is 1-4;
when R is1Is composed of
When R is2is-H, halogen, -OH, C1-C4 alkyl, C1-C4 alkoxy, -CF3Or C1-C4 alkenyl; when R is2Is composed of
When R is1is-H, halogen, -OH, C1-C4 alkyl, C1-C4 alkoxy, -CF3Or C1-C4 alkenyl;
or R1And R2Form a 5-12 membered heterocycle; the heteroatom of the heterocycle is N, O or S; the number of the heteroatoms is 1-5; the heterocyclic ring is a substituted saturated or unsaturated heterocyclic ring; the substituent of the substituted saturated or unsaturated heterocycle is-H, halogen, -OH, C1-C4 alkyl or C1-C4 alkoxy;
R3～R5independently is-H, halogen, -OH, C1-C4 alkyl, C1-C4 alkoxy, -CF3Or C1-C4 alkenyl;
R6～R10independently C2-C4 acyl, substituted phenyl substituted C1-C4 alkyl, substituted 6-12-membered aryl and substituted 5-10-membered saturated or unsaturated heterocycle, wherein hetero atoms of the heterocycle are N, O or S, and the number of the hetero atoms is 1-4; the substituent of the substituted phenyl, the substituted aryl and the substituted heterocycle is-H, halogen, -OH, C1-C4 alkyl, C1-C4 alkoxy, phenyl,
-CF3、-OCF3Or C1-C4 alkenyl.
Preferably, X is C or N; n is 1 or 2;
when R is1Is composed of
When R is2is-H, halogen, -OH, C1-C4 alkyl, C1-C4 alkoxy, -CF3Or C1-C4 alkenyl; when R is2Is composed of
When R is1is-H, halogen, -OH, C1-C4 alkyl, C1-C4 alkoxy, -CF3Or C1-C4 alkenyl;
or R1And R2Form a 5-12 membered heterocycle; the heteroatom of the heterocycle is N, O or S; the number of the heteroatoms is 1-5; the heterocyclic ring is a substituted saturated or unsaturated heterocyclic ring; the substituent of the substituted saturated or unsaturated heterocycle is-H, halogen, -OH, C1-C4 alkyl or C1-C4 alkoxy;
R3～R5independently is-H, halogen, -OH, C1-C4 alkyl, C1-C4 alkoxy, -CF3Or C1-C4 alkenyl;
R6～R10independently C2-C4 acyl, substituted phenyl substituted C1-C4 alkyl, substituted 6-12-membered aryl and substituted 5-10-membered saturated or unsaturated heterocycle, wherein hetero atoms of the heterocycle are N, O or S, and the number of the hetero atoms is 1-4; the substituent of the substituted phenyl, the substituted aryl and the substituted heterocycle is-H, halogen, -OH, C1-C4 alkyl, C1-C4 alkoxy, phenyl,
-CF3、-OCF3Or C1-C4 alkenyl.
Further preferably, X is C or N; n is 1-4;
when R is1Is composed of
When R is2is-H, halogen, -OH, C1-C4 alkyl or C1-C4 alkoxy; when R is2Is composed of
When R is1is-H, halogen, -OH, C1-C4 alkyl or C1-C4 alkoxy;
or R1And R2Form a 5-12 membered heterocycle; the heteroatom of the heterocycle is N, O or S; the number of the heteroatoms is 1-5; the heterocyclic ring is a substituted saturated or unsaturated heterocyclic ring; the substituent of the substituted saturated or unsaturated heterocycle is-H, halogen, -OH, C1-C4 alkyl or C1-C4 alkoxy;
R3～R5independently is-H, halogen, -OH, C1-C4 alkyl, C1-C4 alkoxy, -CF3Or C1-C4 alkenyl;
R6～R10independently C2-C4 acyl, substituted phenyl substituted C1-C4 alkyl, substituted 6-12-membered aryl and substituted 5-10-membered saturated or unsaturated heterocycle, wherein hetero atoms of the heterocycle are N, O or S, and the number of the hetero atoms is 1-4; the substituent of the substituted phenyl, the substituted aryl and the substituted heterocycle is-H, halogen, -OH, C1-C4 alkyl, C1-C4 alkoxy, phenyl,
-CF3、-OCF3Or C1-C4 alkenyl.
Preferably, X is C or N; n is 1-4;
when R is1Is composed of
When R is2is-H, halogen, -OH, C1-C4 alkyl or C1-C4 alkoxy; when R is2Is composed of
When R is1is-H, halogen,-OH, C1-C4 alkyl or C1-C4 alkoxy;
or R1And R2Form a 5-12 membered heterocycle; the hetero atom of the heterocycle is N or O; the number of the heteroatoms is 1-4; the heterocyclic ring is a substituted saturated or unsaturated heterocyclic ring; the substituent of the substituted saturated or unsaturated heterocycle is-H, halogen, -OH, C1-C4 alkyl or C1-C4 alkoxy;
R3～R5independently is-H, halogen, -OH, C1-C4 alkyl, C1-C4 alkoxy, -CF3Or C1-C4 alkenyl;
R6～R10independently C2-C4 acyl, substituted phenyl substituted C1-C4 alkyl, substituted 6-12-membered aryl and substituted 5-10-membered saturated or unsaturated heterocycle, wherein hetero atoms of the heterocycle are N, O or S, and the number of the hetero atoms is 1-4; the substituent of the substituted phenyl, the substituted aryl and the substituted heterocycle is-H, halogen, -OH, C1-C4 alkyl, C1-C4 alkoxy, phenyl,
-CF3、-OCF3Or C1-C4 alkenyl.
Further preferably, X is C or N; n is 1-4;
when R is1Is composed of
When R is2is-H, halogen, -OH, C1-C4 alkyl or C1-C4 alkoxy; when R is2Is composed of
When R is1is-H, halogen, -OH, C1-C4 alkyl or C1-C4 alkoxy;
or R1And R2Form a 5-12 membered heterocycle; the hetero atom of the heterocycle is N or O; the number of the heteroatoms is 1-4; the heterocyclic ring is a substituted saturated or unsaturated heterocyclic ring; the substituent of the substituted saturated or unsaturated heterocycle is-H, C1-C4 alkyl or C1-C4 alkoxy;
R3～R5independently is-H, halogen, -OH, C1-C4 alkyl, C1-C4 alkoxy, -CF3Or C1-C4 alkenyl;
R6～R10independently C2-C4 acyl, substituted phenyl substituted C1-C4 alkyl, substituted 6-12-membered aryl and substituted 5-10-membered saturated or unsaturated heterocycle, wherein hetero atoms of the heterocycle are N, O or S, and the number of the hetero atoms is 1-4; the substituent of the substituted phenyl, the substituted aryl and the substituted heterocycle is-H, halogen, -OH, C1-C4 alkyl, C1-C4 alkoxy, phenyl,
-CF3、-OCF3Or C1-C4 alkenyl.
More preferably, X is C or N; n is 1-4;
when R is1Is composed of
When R is2is-H, halogen, -OH, C1-C4 alkyl or C1-C4 alkoxy; when R is2Is composed of
When R is1is-H, halogen, -OH, C1-C4 alkyl or C1-C4 alkoxy;
or R1And R2Form a 5-12 membered heterocycle; the hetero atom of the heterocycle is N or O; the number of the heteroatoms is 1-4; the heterocyclic ring is a substituted saturated or unsaturated heterocyclic ring; the substituent of the substituted saturated or unsaturated heterocycle is-H or C1-C4 alkyl;
R3～R5independently is-H, halogen, -OH, C1-C4 alkyl, C1-C4 alkoxy, -CF3Or C1-C4 alkenyl;
R6～R10independently C2-C4 acyl, substituted phenyl substituted C1-C4 alkyl, substituted 6-12-membered aryl and substituted 5-10-membered saturated or unsaturated heterocycle, wherein hetero atoms of the heterocycle are N, O or S, and the number of the hetero atoms is 1-4; the substituted phenyl and the substituted arylThe substituent of the substituted heterocycle is-H, halogen, -OH, C1-C4 alkyl, C1-C4 alkoxy, phenyl,
-CF3、-OCF3Or C1-C4 alkenyl.
Preferably, X is C or N; n is 1-4;
when R is1Is composed of
When R is2is-H, halogen, -OH, C1-C4 alkyl or C1-C4 alkoxy; when R is2Is composed of
When R is1is-H, halogen, -OH, C1-C4 alkyl or C1-C4 alkoxy;
or R1And R2Form a 5-12 membered heterocycle; the hetero atom of the heterocycle is N or O; the number of the heteroatoms is 1-4; the heterocyclic ring is a substituted saturated or unsaturated heterocyclic ring; the substituent of the substituted saturated or unsaturated heterocycle is-H or C1-C4 alkyl;
R3～R5independently is-H, halogen, -OH, C1-C4 alkyl or C1-C4 alkoxy;
R6～R10independently C2-C4 acyl, substituted phenyl substituted C1-C4 alkyl, substituted 6-12-membered aryl and substituted 5-10-membered saturated or unsaturated heterocycle, wherein hetero atoms of the heterocycle are N, O or S, and the number of the hetero atoms is 1-4; the substituent of the substituted phenyl, the substituted aryl and the substituted heterocycle is-H, halogen, -OH, C1-C4 alkyl, C1-C4 alkoxy, phenyl,
-CF3、-OCF3Or C1-C4 alkenyl.
Preferably, X is C or N; n is 1-4;
when R is1Is composed of
When R is2is-H, halogen, -OH, C1-C4 alkyl or C1-C4 alkoxy; when R is2Is composed of
When R is1is-H, halogen, -OH, C1-C4 alkyl or C1-C4 alkoxy;
or R1And R2Form a 5-12 membered heterocycle; the hetero atom of the heterocycle is N or O; the number of the heteroatoms is 1-4; the heterocyclic ring is a substituted saturated or unsaturated heterocyclic ring; the substituent of the substituted saturated or unsaturated heterocycle is-H or C1-C4 alkyl;
R3～R5independently is-H, halogen, -OH, C1-C4 alkyl or C1-C4 alkoxy;
R6～R10independently C2-C4 acyl,
m＝1～4；
R11～R19Independently is-H, halogen, -OH, C1-C4 alkyl, C1-C4 alkoxy, phenyl,
-CF3、-OCF3Or C1-C4 alkenyl.
Further preferably, X is C or N; n is 1-4;
when R is1Is composed of
When R is2is-H, halogen, -OH, C1-C4 alkyl or C1-C4 alkoxy; when R is2Is composed of
When R is1is-H, halogen, -OH, C1-C4 alkyl or C1-C4 alkoxy;
or R1And R2Form a 5-12 membered heterocycle; hetero ring of said heterocycleThe atom is N or O; the number of the heteroatoms is 1-4; the heterocyclic ring is a substituted saturated or unsaturated heterocyclic ring; the substituent of the substituted saturated or unsaturated heterocycle is-H or C1-C4 alkyl;
R3～R5independently is-H, halogen, -OH, C1-C4 alkyl or C1-C4 alkoxy;
R6～R10independently C2-C4 acyl,
m is 1 or 2;
R11～R19independently is-H, halogen, -OH, C1-C4 alkyl, C1-C4 alkoxy, phenyl,
-CF3、-OCF3Or C1-C4 alkenyl.
Most preferably, X is C or N; n is 1 or 2;
when R is1Is composed of
When R is2is-H, halogen, -OH, C1-C4 alkyl or C1-C4 alkoxy; when R is2Is composed of
When R is1is-H, halogen, -OH, C1-C4 alkyl or C1-C4 alkoxy;
or R1And R2Form a 5-10 membered heterocycle; the hetero atom of the heterocycle is N or O; the number of the heteroatoms is 1-4; the heterocyclic ring is a substituted saturated or unsaturated heterocyclic ring; the substituent of the substituted saturated or unsaturated heterocycle is-H or C1-C4 alkyl;
R3～R5independently is-H, halogen, -OH, C1-C4 alkyl or C1-C4 alkoxy;
R6～R10independently an acetyl group,
m is 1 or 2;
R11～R19independently is-H, halogen, -OH, C1-C4 alkyl, C1-C4 alkoxy, phenyl,
-CF3、-OCF3Or C1-C4 alkenyl.
The above pyrimido morpholine derivatives when R1Is composed of
When the structure is shown as formula II:
wherein X is C or N; n is 1-4;
R2is-H, halogen, -OH, C1-C8 alkyl, C1-C8 alkoxy, -CF3Or C1-C8 alkenyl;
R3～R5independently is-H, halogen, -OH, C1-C8 alkyl, C1-C8 alkoxy, -CF3Or C1-C8 alkenyl;
R6C2-C4 acyl, substituted phenyl substituted C1-C8 alkyl, substituted 6-12-membered aryl and substituted 5-10-membered saturated or unsaturated heterocycle, wherein hetero atoms of the heterocycle are N, O or S, and the number of the hetero atoms is 1-4; the substituent of the substituted phenyl, the substituted aryl and the substituted heterocycle is-H, halogen, -OH, C1-C8 alkyl, C1-C8 alkoxy, phenyl,
-CF3、-OCF3Or C1-C8 alkenyl.
Preferred embodiments of the present invention are those wherein X is C or N; n is 1-4;
R2is-H, halogen, -OH, C1-C4 alkyl, C1-C4 alkoxy, -CF3Or C1-C4 alkenyl;
R3～R5independently is-H, halogen, -OH, C1-C4 alkyl, C1-C4 alkoxy, -CF3Or C1-C4 alkenyl;
R6C2-C4 acyl, substituted phenyl substituted C1-C4 alkyl, substituted 6-12-membered aryl and substituted 5-10-membered saturated or unsaturated heterocycle, wherein hetero atoms of the heterocycle are N, O or S, and the number of the hetero atoms is 1-4; the substituent of the substituted phenyl, the substituted aryl and the substituted heterocycle is-H, halogen, -OH, C1-C4 alkyl, C1-C4 alkoxy, phenyl,
-CF3、-OCF3Or C1-C4 alkenyl.
Preferably, X is C or N; n is 1 or 2;
R2is-H, halogen, -OH, C1-C4 alkyl, C1-C4 alkoxy, -CF3Or C1-C4 alkenyl;
R3～R5independently is-H, halogen, -OH, C1-C4 alkyl, C1-C4 alkoxy, -CF3Or C1-C4 alkenyl;
R6C2-C4 acyl, substituted phenyl substituted C1-C4 alkyl, substituted 6-12-membered aryl and substituted 5-10-membered saturated or unsaturated heterocycle, wherein hetero atoms of the heterocycle are N, O or S, and the number of the hetero atoms is 1-4; the substituent of the substituted phenyl, the substituted aryl and the substituted heterocycle is-H, halogen, -OH, C1-C4 alkyl, C1-C4 alkoxy, phenyl,
-CF3、-OCF3Or C1-C4 alkenyl.
Further preferably, X is C or N; n is 1-4;
R2is-H, halogen, -OH, C1-C4 alkyl or C1-C4 alkoxy;
R3～R5independently is-H, halogen, -OH, C1-C4 alkyl, C1-C4 alkoxy, -CF3Or C1-C4 alkenyl;
R6is C2-C4 acyl, substituted phenylThe compound is C1-C4 alkyl, substituted 6-12-membered aryl and substituted 5-10-membered saturated or unsaturated heterocycle, wherein hetero atoms of the heterocycle are N, O or S, and the number of the hetero atoms is 1-4; the substituent of the substituted phenyl, the substituted aryl and the substituted heterocycle is-H, halogen, -OH, C1-C4 alkyl, C1-C4 alkoxy, phenyl,
-CF3、-OCF3Or C1-C4 alkenyl.
Preferably, X is C or N; n is 1-4;
R2is-H, halogen, -OH, C1-C4 alkyl or C1-C4 alkoxy;
R3～R5independently is-H, halogen, -OH, C1-C4 alkyl or C1-C4 alkoxy;
R6C2-C4 acyl, substituted phenyl substituted C1-C4 alkyl, substituted 6-12-membered aryl and substituted 5-10-membered saturated or unsaturated heterocycle, wherein hetero atoms of the heterocycle are N, O or S, and the number of the hetero atoms is 1-4; the substituent of the substituted phenyl, the substituted aryl and the substituted heterocycle is-H, halogen, -OH, C1-C4 alkyl, C1-C4 alkoxy, phenyl,
-CF3、-OCF3Or C1-C4 alkenyl.
Preferably, X is C or N; n is 1-4;
R2is-H, halogen, -OH, C1-C4 alkyl or C1-C4 alkoxy;
R3～R5independently is-H, halogen, -OH, C1-C4 alkyl or C1-C4 alkoxy;
R6is C2-C4 acyl,
m＝1～4；
R11～R19Independently is-H, halogen, -OH, C1-C4 alkyl, C1-C4 alkoxy, phenyl,
-CF3、-OCF3Or C1-C4 alkenyl.
Further preferably, X is C or N; n is 1-4;
R2is-H, halogen, -OH, C1-C4 alkyl or C1-C4 alkoxy;
R3～R5independently is-H, halogen, -OH, C1-C4 alkyl or C1-C4 alkoxy;
R6is composed of
R11～R16Independently is-H, halogen, -OH, C1-C4 alkyl, C1-C4 alkoxy, phenyl,
-CF3、-OCF3Or C1-C4 alkenyl.
Preferably, X is C or N; n is 1-4;
R2is-H, halogen, -OH, C1-C4 alkyl or C1-C4 alkoxy;
R3～R5independently is-H, halogen, -OH, C1-C4 alkyl or C1-C4 alkoxy;
R6is composed of
R11～R16Independently is-H, halogen, -OH, C1-C4 alkyl, C1-C4 alkoxy, phenyl, -CF3Or C1-C4 alkenyl.
Most preferably, X is C or N; n is 1 or 2;
R2is-H, halogen, -OH, C1-C4 alkyl or C1-C4 alkoxy;
R3～R5independently is-H, halogen, -OH, C1-C4 alkyl or C1-C4 alkoxy;
R6is composed of
R11～R16Independently is-H, halogen, -OH, C1-C4 alkyl, C1-C4 alkoxy, phenyl, -CF3Or C1-C4 alkenyl.
The above pyrimido morpholine derivatives when R1Is composed of
R6Is composed of
When n is 1, the structure is shown as formula III:
wherein X is C or N;
R2is-H, halogen, -OH, C1-C8 alkyl, C1-C8 alkoxy, -CF3Or C1-C8 alkenyl;
R3～R5independently is-H, halogen, -OH, C1-C8 alkyl, C1-C8 alkoxy, -CF3Or C1-C8 alkenyl;
R11～R15independently is-H, halogen, -OH, C1-C4 alkyl, C1-C4 alkoxy, phenyl,
-CF3、-OCF3Or C1-C4 alkenyl.
Preferred embodiments of the present invention are those wherein X is C or N;
R2is-H, halogen, -OH, C1-C4 alkyl, C1-C4 alkoxy, -CF3Or C1-C4 alkenyl;
R3～R5independently is-H, halogen, -OH, C1-C4 alkyl, C1-C4 alkoxy, -CF3Or C1-C4 alkenyl;
R11～R15independently-H, -halogen, -OH, C1 ℃ -C4 alkyl, C1-C4 alkoxy, phenyl,
-CF3、-OCF3Or C1-C4 alkenyl.
Preferably, X is C or N;
R2is-H, halogen, -OH, C1-C4 alkyl, C1-C4 alkoxy, -CF3Or C1-C4 alkenyl;
R3～R5independently is-H, halogen, -OH, C1-C4 alkyl, C1-C4 alkoxy, -CF3Or C1-C4 alkenyl;
R11～R15independently is-H, halogen, -OH, C1-C4 alkyl, C1-C4 alkoxy, phenyl,
-CF3、-OCF3Or C1-C4 alkenyl.
Further preferably, X is C or N;
R2is-H, halogen, -OH, C1-C4 alkyl or C1-C4 alkoxy;
R3～R5independently is-H, halogen, -OH, C1-C4 alkyl, C1-C4 alkoxy, -CF3Or C1-C4 alkenyl;
R11～R15independently is-H, halogen, -OH, C1-C4 alkyl, C1-C4 alkoxy, phenyl,
-CF3、-OCF3Or C1-C4 alkenyl.
Preferably, X is C or N;
R2is-H, halogen, -OH, C1-C4 alkyl or C1-C4 alkoxy;
R3～R5independently is-H, halogen, -OH, C1-C4 alkyl or C1-C4 alkoxy;
R11～R15independently is-H, halogen, -OH, C1-C4 alkyl, C1-C4 alkoxy, phenyl,
-CF3、-OCF3Or C1-C4 alkenyl.
Most preferably, X is C;
R2is-H, halogen, -OH, C1-C4 alkyl or C1-C4 alkoxy;
R3～R5independently is-H, halogen, -OH, C1-C4 alkyl or C1-C4 alkoxy;
R11～R15independently is-H, halogen, -OH, C1-C4 alkyl, C1-C4 alkoxy, phenyl, -CF3Or C1-C4 alkenyl.
The above pyrimido morpholine derivatives when R1Is composed of
R6Is composed of
When the structure is shown as formula IV:
wherein X is C or N; n is 1-4;
R2is-H, halogen, -OH, C1-C8 alkyl, C1-C8 alkoxy, -CF3Or C1-C8 alkenyl;
R3～R5independently is-H, halogen, -OH, C1-C8 alkyl, C1-C8 alkoxy, -CF3Or C1-C8 alkenyl;
R16is-H, halogen, -OH, C1-C8 alkyl, C1-C8 alkoxy, phenyl,
-CF3、-OCF3Or C1-C8 alkenyl.
Preferred embodiments of the present invention are those wherein X is C, N or O; n is 1-4;
R2is-H, haloElement, -OH, C1-C4 alkyl, C1-C4 alkoxy, -CF3Or C1-C4 alkenyl;
R3～R5independently is-H, halogen, -OH, C1-C4 alkyl, C1-C4 alkoxy, -CF3Or C1-C4 alkenyl;
R16is-H, halogen, -OH, C1-C4 alkyl, C1-C4 alkoxy, phenyl,
-CF3、-OCF3Or C1-C4 alkenyl.
Preferably, X is C or N; n is 1 or 2;
R2is-H, halogen, -OH, C1-C4 alkyl, C1-C4 alkoxy, -CF3Or C1-C4 alkenyl;
R3～R5independently is-H, halogen, -OH, C1-C4 alkyl, C1-C4 alkoxy, -CF3Or C1-C4 alkenyl;
R16is-H, halogen, -OH, C1-C4 alkyl, C1-C4 alkoxy, phenyl,
-CF3、-OCF3Or C1-C4 alkenyl.
Further preferably, X is C or N; n is 1-4;
R2is-H, halogen, -OH, C1-C4 alkyl or C1-C4 alkoxy;
R3～R5independently is-H, halogen, -OH, C1-C4 alkyl, C1-C4 alkoxy, -CF3Or C1-C4 alkenyl;
R16is-H, halogen, -OH, C1-C4 alkyl, C1-C4 alkoxy, phenyl,
-CF3、-OCF3Or C1-C4 alkenyl.
Preferably, X is C or N; n is 1-4;
R2is-H, halogen, -OH, C1-C4 alkyl or C1 ℃ -A C4 alkoxy group;
R3～R5independently is-H, halogen, -OH, C1-C4 alkyl or C1-C4 alkoxy;
R16is-H, halogen, -OH, C1-C4 alkyl, C1-C4 alkoxy, phenyl,
-CF3、-OCF3Or C1-C4 alkenyl.
Preferably, X is C or N; n is 1-4;
R2is-H, halogen, -OH, C1-C4 alkyl or C1-C4 alkoxy;
R3～R5independently is-H, halogen, -OH, C1-C4 alkyl or C1-C4 alkoxy;
R16independently is-H, halogen, -OH, C1-C4 alkyl, C1-C4 alkoxy, phenyl, -CF3Or C1-C4 alkenyl.
Most preferably, X is C or N; n is 1 or 2;
R2is-H, halogen, -OH, C1-C4 alkyl or C1-C4 alkoxy;
R3～R5independently is-H, halogen, -OH, C1-C4 alkyl or C1-C4 alkoxy;
R16independently is-H, halogen, -OH, C1-C4 alkyl, C1-C4 alkoxy, phenyl, -CF3Or C1-C4 alkenyl.
The above pyrimido morpholine derivatives when R1Is composed of
When the structure is shown as formula V:
wherein R is3is-H, halogen, -OH, C1-C8 alkyl, C1-C8 alkoxy, -CF3Or C1-C8 alkenyl;
R8substituted by C2-C4 acyl and substituted phenylC1-C8 alkyl, substituted 6-12-membered aryl and substituted 5-10-membered saturated or unsaturated heterocyclic ring, wherein hetero atoms of the heterocyclic ring are N, O or S, and the number of the hetero atoms is 1-4; the substituent of the substituted phenyl, the substituted aryl and the substituted heterocycle is-H, halogen, -OH, C1-C8 alkyl, C1-C8 alkoxy, phenyl,
-CF3、-OCF3Or C1-C8 alkenyl.
As a preferred embodiment of the present invention, R3is-H, halogen, -OH, C1-C4 alkyl or C1-C4 alkoxy; r8C2-C4 acyl, substituted phenyl substituted C1-C4 alkyl, substituted 6-12-membered aryl and substituted 5-10-membered saturated or unsaturated heterocycle, wherein hetero atoms of the heterocycle are N, O or S, and the number of the hetero atoms is 1-4; the substituent of the substituted phenyl, the substituted aryl and the substituted heterocycle is-H, halogen, -OH, C1-C4 alkyl, C1-C4 alkoxy, phenyl,
-CF3、-OCF3Or C1-C4 alkenyl.
Preferably, R3～R5Independently is C1-C4 alkyl or C1-C4 alkoxy; r8Is C2-C4 acyl,
m＝1～4；R11～R19Independently is-H, halogen, -OH, C1-C4 alkyl, C1-C4 alkoxy, phenyl,
-CF3、-OCF3Or C1-C4 alkenyl.
Further preferably, R3Is C1-C4 alkyl or C1-C4 alkoxy; r8Is composed of
R11～R19Independently is-H, halogen, -OH, C1-C4 alkyl, C1-C4 alkoxy, phenyl,
-CF3、-OCF3Or C1-C4 alkenyl.
Most preferably, R3Is C1-C4 alkyl or C1-C4 alkoxy; r8Is composed of
R11～R17Independently is-H, C1-C4 alkyl or-CF3。
The structural formula of the pyrimido morpholine derivative is as follows:
the invention also provides a preparation method of the pyrimido morpholine derivative.
The preparation route of the compound shown in the formula II is as follows:
wherein X is C or N; n is 1-4;
R2is-H, halogen, -OH, C1-C8 alkyl, C1-C8 alkoxy, -CF3Or C1-C8 alkenyl;
R3～R5independently is-H, halogen, -OH, C1-C8 alkyl, C1-C8 alkoxy, -CF3Or C1-C8 alkenyl;
R6C2-C4 acyl, substituted phenyl substituted C1-C8 alkyl, substituted 6-12-membered aryl and substituted 5-10-membered saturated or unsaturated heterocycle, wherein hetero atoms of the heterocycle are N, O or S, and the number of the hetero atoms is 1-4; the substituent of the substituted phenyl, the substituted aryl and the substituted heterocycle is-H, halogen, -OH, C1-C8 alkyl, C1-C8 alkoxyA phenyl group, a phenyl group,
-CF3、-OCF3Or C1-C8 alkenyl.
The preparation operation steps of the pyrimido morpholine derivative shown in the formula II comprise:
a. reacting the raw material 1 with 30% ammonia water solution in a sealed tube to prepare an intermediate 1; the reaction solvent is any one of n-butanol, isobutanol, isopropanol and the like; the mass volume ratio of the raw material 1 to the 30% ammonia water solution is 5 g: 45 mL; the reaction temperature is 80-90 ℃; the reaction time is 8 h;
b. carrying out demethoxylation reaction on the intermediate 1 and boron tribromide in a dichloromethane solvent to prepare an intermediate 2; the mass-volume ratio of the intermediate 1 to the boron tribromide is 1.5 g: 3 mL; the reaction temperature is 20-35 ℃; the reaction time is 24-72 h;
c. the intermediate 2 reacts with an alkyl bromide reagent in an organic solvent under the catalysis of alkali to prepare an intermediate 3; the alkali is any one of cesium carbonate, potassium carbonate and the like; the molar ratio of the intermediate 2 to the cesium carbonate and the alkyl bromide is 1: 1.5; the organic solvent is any one of acetonitrile, dioxane, ethanol and the like; the reaction temperature is 25-65 ℃; the reaction time is 24-72 h;
d. the intermediate 3 reacts with an alkyl iodide reagent under the catalysis of alkali to prepare an intermediate 4; the alkyl iodide reagent is any one of methyl iodide, ethyl iodide, isopropyl iodide and the like; the alkali is any one of cesium carbonate, potassium carbonate and the like; the organic solvent is N, N-dimethylformamide; the reaction time is 6-12 h; the reaction temperature is 25-35 ℃;
e. under the catalysis of transition metal, alkali and ligand, the intermediate 4 and arylamine or heterocyclic amine are prepared under the protection of nitrogen to obtain an intermediate 5; the ligand is any one of 2-dicyclohexylphosphine-2 ', 6' -dimethoxybiphenyl, 2- (dicyclohexylphosphine) -3, 6-dimethoxy-2 '-4' -6 '-tri-I-propyl-11' -biphenyl and the like; the transition metal is tris (dibenzylideneacetone) dipalladium; the alkali is any one of cesium carbonate, potassium carbonate and the like; the reaction temperature is 90-110 ℃; the molar ratio of the intermediate 4, the transition metal, the base and the ligand is 1:0.1: 2: 0.1; the reaction time is 12-24 h;
f. reducing nitro group of the intermediate 5 into amino group in the presence of palladium carbon and hydrogen, and reacting with isocyanate in the presence of alkali to obtain a compound shown in a formula II; the alkali is any one of triethylamine and N, N-diisopropylethylamine; the reaction temperature is 65-110 ℃, and the reaction time is 6-12 h.
The preparation route of the compound shown in the formula V is as follows:
wherein R is3is-H, halogen, -OH, C1-C8 alkyl, C1-C8 alkoxy, -CF3Or C1-C8 alkenyl;
R8C2-C4 acyl, substituted phenyl substituted C1-C8 alkyl, substituted 6-12-membered aryl and substituted 5-10-membered saturated or unsaturated heterocycle, wherein hetero atoms of the heterocycle are N, O or S, and the number of the hetero atoms is 1-4; the substituent of the substituted phenyl, the substituted aryl and the substituted heterocycle is-H, halogen, -OH, C1-C8 alkyl, C1-C8 alkoxy, phenyl,
-CF3、-OCF3Or C1-C8 alkenyl.
The preparation operation steps of the pyrimido morpholine derivative shown as the formula V comprise:
g. preparing an intermediate 6 from the raw material 2, an amine and a base in the presence of a condensing agent; the alkali is any one of triethylamine, N-diisopropylethylamine and the like; the amine is m-trifluoromethyl aniline and 1-methylpiperidine-4-amine; the condensing agent is any one of HATU (2- (7-azabenzotriazole) -N, N, N ', N' -tetramethyluronium hexafluorophosphate), HBTU (benzotriazol-N, N, N ', N' -tetramethyluronium hexafluorophosphate), HOBT (1-hydroxybenzotriazole) and the like; the reaction temperature is 65 ℃; the reaction time is 8 h;
h. reducing the intermediate 6 with palladium carbon and hydrogen, and then preparing the compound of the formula V with the intermediate 3 under the catalysis of transition metal and ligand; the palladium carbon is 10% of wet palladium carbon; the transition metal is tris (dibenzylideneacetone) dipalladium; the ligand is any one of 2-dicyclohexylphosphine-2 ', 6' -dimethoxybiphenyl, 2- (dicyclohexylphosphine) -3, 6-dimethoxy-2 '-4' -6 '-tri-I-propyl-11' -biphenyl and the like; the reaction temperature is 90-110 ℃, and the reaction time is 8 h;
the invention also provides pharmaceutically acceptable salts or hydrates of the pyrimido morpholine derivatives.
The invention also provides a prodrug of the compound shown in the formula I, the formula II, the formula III, the formula IV or the formula V. Prodrugs, according to the present invention, are derivatives of a compound of formula I, formula II, formula III, formula IV or formula V, as described above, which may themselves be less active or even inactive, but which, upon administration, are converted under physiological conditions (e.g., by metabolism, solvolysis or otherwise) to the corresponding biologically active form.
The invention also provides a composition, which is prepared by adding pharmaceutically acceptable auxiliary components into the pyrimido morpholine derivatives shown in the formulas I, II, III and IV and the salts or hydrates thereof provided by the invention.
The invention also provides the application of the pyrimido morpholine derivative, the salt or hydrate thereof, the prodrug or the composition in preparing tyrosine kinase inhibitors. Preferably, the pyrimido morpholine derivative, its salt or hydrate, prodrug or composition is used for preparing a drug-resistant mutant tyrosine kinase inhibitor.
The invention also provides the application of the pyrimido morpholine derivative, the salt or hydrate thereof, the prodrug or the composition in preparing medicaments for treating medullary thyroid carcinoma.
The invention also provides the application of the pyrimido morpholine derivative, the salt or hydrate thereof, the prodrug or the composition in preparing a medicament for treating acute myeloid leukemia.
The invention also provides the application of the pyrimido morpholine derivative, the salt or hydrate thereof, the prodrug or the composition in preparing anti-inflammatory drugs.
The invention has the beneficial effects that: firstly, the series of compounds are multi-target tyrosine kinase inhibitors of RET, FLT3 and VEGFR, wherein partial compounds can overcome the secondary mutation of RET kinase, namely RET-V804M/V804L, and the mutation can make patients with medullary carcinoma generate drug resistance to cabozantinib and vandetanib. In vitro tests, 1l, 1k, 1w and other compounds show good antitumor effects on TT and MV4-11 cell lines, wherein the compound 1w with the best RET kinase activity also shows good antitumor activity in a xenograft model test of medullary thyroid carcinoma, and meanwhile, 1w also has good anti-inflammatory effects on an ear swelling model of a mouse.
Drawings
FIG. 1 IC of Compound 1w against RET, RET-G691S, RET-M918T, RET-S891A kinase50The curves show that the activity of compound 1w (labeled curve) on the above four kinases is superior to that of sorafenib and cabozantinib.
FIG. 2 IC of Compound 1w against RET-V804L, RET-V804M, RET-Y791F kinase50The curves show that the activity of the compound 1w (labeled curve) on the three kinases is better than that of sorafenib and cabozantinib
FIG. 3 GI of Compound 1w versus Ba/F3 cell line transfected with RET-V804M \ RET-M918T50The activity of compound 1w on the above two cell lines was superior to that of sorafenib and cabozantinib, as can be seen from the curves in the figure
FIG. 4 GI of Compound 1w vs. Ba/F3 cell line transfected with RET \ RET-V804L50The cell activity of compound 1w on RET-transformed Ba/F3 is superior to sorafenib, cabozantinib and vandetanib as can be seen from the curves in the figure; the activity of the compound 1w on Ba/F3 with RET-V804L is better than that of sorafenib and vandetanib; weaker than cabozantinib.
FIG. 5 shows that the compound 1w has slightly stronger antitumor activity than the positive drug cabozantinib when administered for 21 days at a dose of 50mg/kg in a xenograft model experiment on mouse embryonic fibroblasts carrying the NIH3T3-RET-C634Y mutation; meanwhile, the weight of the mice is slightly increased, which indicates that the medicament has small toxic and side effects.
FIG. 6 shows the effect of Compound 1w on the mouse ear swelling model, and as can be seen from the comparative effects of several model groups in the figure, the anti-inflammatory activity of Compound 1w is comparable to that of halomethasone, which is stronger than that of 4a-31 (positive drug).
Detailed Description
Example 1 preparation of 6-chloro-5-methoxypyrimidin-4-amine (intermediate 1).
Adding raw material 1 (5.0 g, 28.1mmol of 4, 6-dichloro-5-methoxypyrimidine), 30% aqueous ammonia solution (45mL) and 15mL of n-butanol into a sealed tube, reacting at 85 ℃ for 8 hours, cooling to normal temperature, concentrating the reaction solution under reduced pressure to obtain white solid, dissolving the white solid into suspension by 150mL of aqueous sodium chloride solution, stirring at normal temperature for 0.5 hour, carrying out suction filtration, and washing the white filter cake with a small amount of ethanol and ether to obtain intermediate 1. The yield thereof was found to be 90%.
1H NMR(400MHz,DMSO-d6)7.97(s,1H),7.29(s,2H),3.71(s, 3H); MS (ESI, positive ion) M/z 159.91[ M + H ]]+。
Example 2 preparation of 4-amino-6-chloropyrimidin-5-ol (intermediate 2).
Dissolving 6-chloro-5-methoxypyrimidine-4-amine (intermediate 1) (1.5g, 9.4mmol) in 150mL of dichloromethane, slowly adding 3mL of boron tribromide by using a constant-pressure dropping funnel at normal temperature, after dropwise addition is finished, stirring reaction liquid at normal temperature for 72 hours, quenching residual boron tribromide in the reaction liquid by using 15mL of methanol, slowly dropwise adding methanol in a quenching process, and concentrating a light yellow reaction liquid under reduced pressure to obtain a orange yellow solid, namely an intermediate 2, wherein the intermediate 2 is good in water solubility and difficult to purify due to large polarity and is directly put into the next step of reaction.
MS (ESI, negative ion) M/z 143.90[ M-H [ ]]-。
Example 3 preparation of 4-chloro-7, 8-dihydro-6H-pyrimidine [5,4-b ] [1,4] oxazine (intermediate 3 a).
Adding the intermediate 2(0.8g, 5.5mmol), cesium carbonate (3.25g,10mmol) and 1, 2-dibromoethane (1mL,11mmol) into 100mL acetonitrile, stirring at normal temperature for 4 hours, heating to 65 ℃ for reaction for 24 hours, filtering the yellowish suspension (with a proper amount of diatomite) after the reaction is finished, obtaining bright yellow filtrate, concentrating, mixing the sample, performing column chromatography, and selecting petroleum ether: ethyl acetate ═ 1:1 eluent purified intermediate 3a (0.3g, 40%).
1H NMR(400MHz,DMSO-d6)8.12(s,1H),7.86(s,1H),4.21(t, J ═ 4.5Hz,2H),3.48(dd, J ═ 7.1Hz, J ═ 4.5Hz,2H), MS (ESI, positive ion) M/z 171.94[ M + H ] M/z]+。
According to a similar preparation method of the intermediate 3a, the intermediate 3b (4-chloro-6, 7,8, 9-tetrahydropyrimidine [5,4-b ] [1,4] oxazepane) is prepared by taking the intermediate 2 and 1, 3-dibromopropane as raw materials and acetonitrile as a solvent. The structure is characterized as follows:
1H NMR(400MHz,DMSO-d6)7.93(s,1H),7.56(s,1H),4.23(t,J＝6.3Hz,2H),3.44-3.36(m,2H),2.07-2.01(m,2H)；MS(ESI,positive ion)m/z:185.93[M+H]+.
example 4 preparation of 4-chloro-8-methyl-7, 8-dihydro-6H-pyrimidine [5,4-b ] [1,4] oxazine (intermediate 4 a).
Dissolving the intermediate 3a (0.8g,4.6mmol) and cesium carbonate (3.25g,10mmol) in 25mL of N, N-dimethylformamide, adding 1mL of iodomethane at normal temperature, continuing to react for 8 hours, concentrating the DMF reaction solution after the reaction is finished, extracting the reaction solution with saturated sodium bicarbonate water solution and dichloromethane, concentrating the dichloromethane extraction solution, mixing the sample, and carrying out column chromatography. Selecting petroleum ether: the ethyl acetate 1:1 eluent was purified to give intermediate 4 a. The yield thereof was found to be 40%.
Preparing an intermediate 4b from the intermediate 3a and 2-iodopropane by the same synthesis method of the intermediate 4 a; preparing an intermediate 4c from the intermediate 3b and methyl iodide; the structure and map representation are shown in table 1:
TABLE 1
EXAMPLE 5 preparation of N- (2-methyl-4-nitrophenyl) -7, 8-dihydro-6H-pyrimidine [5,4-b ] [1,4] oxazin-4-amine (intermediate 5 a).
Adding the intermediate 3a (20mg,0.12mmol), 2-methyl-4-nitroaniline (20mg,0.11mmol), cesium carbonate (80mg,0.23mmol), tris (dibenzylideneacetone) dipalladium (11mg,0.01mmol) and 2- (dicyclohexylphosphine) -3, 6-dimethoxy-2 '-4' -6 '-tri-I-propyl-11' -biphenyl (6mg,0.01mmol) into 18mL of 1, 4-dioxane/water mixed solution (volume ratio is 5: 1), reacting for 24 hours at 100 ℃ under the protection of nitrogen, filtering the reaction solution, concentrating the filtrate, mixing the sample, performing column chromatography, selecting and selecting
Ethyl acetate: methanol-8: 1 eluent purification of intermediate 5a (20mg, 64%).
1H NMR(400MHz,DMSO-d6)8.25(d,J＝9.1Hz,1H),8.11(d,J＝2.0Hz,1H),8.07-8.04(m,1H),7.84(s,1H),7.67(s,1H),7.41(s,1H),4.21(t,J＝4.1Hz,2H),3.48(s,2H),2.36(s,3H)；MS(ESI,positive ion)m/z:288.03[M+H]+.
Preparing an intermediate 5b-5l by adopting the same synthesis method of the intermediate 5 a; the structure and map characterization are shown in table 2:
TABLE 2
Example 6 preparation of 1- (4-chlorobenzene) -3- (4- ((7, 8-dihydro-6H-pyrimidine [5,4-b ] [1,4] oxazin-4-yl) -amino) phenyl) urea (Compound 1 a).
Dissolving the intermediate 5h (20mg,0.07mmol) and 10% palladium carbon (2mg) in 20mL of methanol, reacting for 4 hours at 45 ℃ under the protection of hydrogen, performing suction filtration on a reaction solution after the TLC thin layer monitoring of the reaction of the raw materials is finished, concentrating a filtrate to obtain a red brown solid 15mg, and directly putting the solid into the next reaction without further purification.
Adding red brown solid (15mg, 0.06mmol), 1-chloro-4-phenyl isocyanate (12mg,0.07mmol) and N, N-diisopropylethylamine (20 μ L) into 6mL tetrahydrofuran, reacting at 65 ℃ for 8 hours, directly concentrating the reaction solution, extracting the reaction solution with dichloromethane and saturated aqueous sodium bicarbonate solution, concentrating DCM extract, stirring, and performing column chromatography to purify the product compound 1a (14mg, 60%) with EA: MEOH 80: 1.
1H NMR(400MHz,DMSO-d6)9.35(s,1H),9.06(s,1H),8.11(s,1H),7.74(s,1H),7.61(d,J＝8.4Hz,2H),7.50(d,J＝8.4Hz,2H),7.32(m,4H),6.98(s,1H),4.17(s,2H),3.45(s,2H)；13C NMR(100MHz,DMSO-d6)153.19,149.77,149.71,147.78,139.62,135.55,134.05,129.02,125.33,121.02,119.98,119.82,119.00, 64.41; MS (ESI, positive ion) M/z 397.03[ M + H ]]+。
Preparing a compound 1b-1x by adopting the same synthetic method of the compound 1 a; the structure and map characterization are shown in table 3:
TABLE 3
Example 7 preparation of 3-methoxy-4-nitro-N- (3- (trifluoromethyl) phenyl) benzamide (intermediate 6a)
Adding 3-methoxy-4-nitrobenzoic acid, HATU, DIEA and m-trifluoromethylaniline into tetrahydrofuran, reacting for 4 hours at 65 ℃, directly concentrating the reaction solution, adding saturated sodium bicarbonate water solution for washing, performing suction filtration, washing a filter cake with absolute ethyl alcohol, and drying to obtain an intermediate 6 a.
Intermediates 6b-6c can be prepared by the same synthetic method of the intermediate 6 a; the structures of intermediates 6a-6c are shown below:
the hydrogen spectrum of intermediate 6c is characterized by: 1H NMR (400MHz, DMSO-d)6)10.77(s,1H),8.23(s,1H),8.14(d,J＝8.4Hz,1H),8.06(d,J＝8.5Hz,2H),7.99(dd,J＝8.4,J＝1.7Hz,1H),7.63(t,J＝8.0Hz,1H),7.50(d,J＝7.8Hz,1H),2.60(s,3H).
EXAMPLE 8 preparation of 4- ((7, 8-dihydro-6H-pyrimidine [5,4-b ] [1,4] oxazin-4-yl) amino) -3-methoxy-N- (3- (trifluoromethyl) phenyl) benzamide (Compound 2a)
Dissolving the intermediate 6a (1g) with methanol, adding palladium carbon (0.1g) and nitrogen for replacement, introducing hydrogen, directly filtering the reaction solution with diatomite after the thin-layer monitoring reaction is finished, and concentrating the filtrate to obtain a reddish brown solid; adding the reddish brown solid, the intermediate 3a, the tris (dibenzylideneacetone) dipalladium, the 2-dicyclohexylphosphine-2 ', 6' -dimethoxybiphenyl and the cesium carbonate into dioxane according to the molar ratio of 1:1:0.1:0.1:2, reacting for 24 hours at 95 ℃ under the protection of nitrogen, directly stirring the reaction sample, and performing column chromatography to obtain the compound 2 a.
1H NMR(400MHz,DMSO-d6)10.37(s,1H),8.68(d,J＝8.4Hz,1H),8.23(s,1H),8.06(d,J＝8.4Hz,1H),7.90(s,1H),7.66-7.60(m,4H),7.44(d,J＝7.6Hz,1H),7.32(s,1H),4.24(t,J＝4.2Hz,2H),4.01(s,3H),3.47-3.41(m,2H)；MS(ESI,positive ion)m/z:446.02[M+H]+.
The compounds 2b-2c can be prepared by the same synthetic method of the compound 2 a; the structures and characterizations of compounds 2b-2c are shown in table 4:
TABLE 4
Example 9 preparation of 1- (4- (4- ((7, 8-dihydro-6H-pyrimidine [5,4-b ] [1,4] oxazin-4-yl) amino) -3-methoxyphenyl) piperazin-1-yl) ethan-1-one (Compound 3).
i. Adding 4-fluoro-2-methoxy-1-nitrobenzene, 1-boc-piperazine, triethylamine and HATU into tetrahydrofuran according to the molar ratio of 1:1.2, reacting at 65 ℃ for 4 hours, directly concentrating the reaction solution, adding saturated sodium carbonate aqueous solution, washing, and carrying out suction filtration to obtain a filter cake. Adding 1g of the dried filter cake into dichloromethane for dissolving and clarifying, then adding 3mL of trifluoroacetic acid, reacting overnight at normal temperature for 12 hours, concentrating the reaction solution the next day, extracting the reaction solution with dichloromethane/water, then adding 1mL of triethylamine and 1mL of acetyl chloride into the extracted dichloromethane, reacting at normal temperature for 4 hours, concentrating the dichloromethane reaction solution, mixing the sample, and carrying out column chromatography to obtain an intermediate 7.
j. Reducing nitro in the intermediate 7 by palladium carbon and hydrogen, and then preparing the compound 3 by reacting the reduced nitro with 3a under the catalysis of transition metal and ligand.
1H NMR(400MHz,DMSO-d6)8.16(d,J＝8.8Hz,1H),7.76(s,1H),7.13(s,1H),7.04(s,1H),6.68(d,J＝2.5Hz,1H),6.50(dd,J＝8.8,J＝2.5Hz,1H),4.19(t,J＝4.3Hz,2H),3.85(s,3H),3.58(dd,J＝9.8,J＝4.8Hz,4H),3.44(s,2H),3.10-3.02(m,4H),2.04(s,3H)；MS(ESI,positive ion)m/z:385.19[M+H]+.
Example 10 preparation of 3- ((7, 8-dihydro-6H-pyrimidine [5,4-b ] [1,4] oxazin-4-yl) amino) -4-methyl-N- (1-methyl-1H-pyrazol-5-yl) benzamide (Compound 4a)
i. Adding 4-methyl-3-nitrobenzoic acid, amine, triethylamine and HATU into tetrahydrofuran according to the molar ratio of 1:1.2, reacting for 4 hours at 65 ℃, directly concentrating the reaction solution, adding saturated sodium carbonate aqueous solution, washing, and filtering to obtain a filter cake. Namely the intermediate 8
j. And reducing the nitro group in the intermediate 8 by using palladium carbon and hydrogen, and then preparing the compound 4 by reacting the reduced nitro group with the compound 3a under the catalysis of transition metal and a ligand.
Intermediates 8a, 8b were prepared analogously to intermediates 6a-6c, using a HATU condensing agent, catalyzed by a triethylamine base. The structures and partial hydrogen spectra of intermediates 8a, 8b are as follows:
the hydrogen spectrum of intermediate 8b was 1H NMR (400MHz, DMSO-d)6)10.74(s,1H),8.61(d,J＝1.6Hz,1H),8.24-8.22(m,2H),8.07(d,J＝8.0Hz,1H),7.71(d,J＝8.1Hz,1H),7.63(t,J＝8.0Hz,1H),7.49(d,J＝7.7Hz,1H),2.61(s,3H).
Compounds 4a-4b were prepared in a manner analogous to compounds 2a-2c, and the structural characterization and profiles of compounds 4a-4b are shown in Table 5:
TABLE 5
Example 11 preparation of N- (3- ((7, 8-dihydro-6H-pyrimidine [5,4-b ] [1,4] oxazin-4-yl) amine) -4-methylphenyl) -4-methyl-3- (trifluoromethyl) benzamide (Compound 5 a).
m, adding 4-methyl-3-nitroaniline, acid, triethylamine and HATU into tetrahydrofuran according to the molar ratio of 1:1.2, reacting for 4 hours at 65 ℃, directly concentrating the reaction solution, adding saturated sodium carbonate aqueous solution, washing, and filtering to obtain a filter cake. Namely intermediate 9
And n, reducing the nitro group in the intermediate 9 by using palladium carbon and hydrogen, and then preparing the compound 5a-5b with the 3a under the catalysis of transition metal and ligand.
Compounds 5a-5b were prepared in a manner analogous to compounds 2a-2c, and the structural characterization and profiles of compounds 5a-5b are shown in Table 6:
TABLE 6
EXAMPLE 12 preparation of N- (2-methyl-5- (pyridin-3-yl) phenyl) -7, 8-dihydro-6H-pyrimido [5,4-b ] [1,4] oxazin-4-amine (Compound 6)
o, adding 4-bromo-1-methyl-2-nitrobenzene, 3-pyridine boric acid, tetratriphenylphosphine palladium and cesium carbonate into dioxane according to the proportion of 1:1.2:0.1:2, and reacting for 24 hours at the temperature of 100 ℃ under the protection of nitrogen to obtain an intermediate 10
And n, reducing the nitro group in the intermediate 10 by using palladium carbon and hydrogen, and then catalyzing with 3a in transition metal and ligand to prepare a compound 6.
1H NMR(400MHz,DMSO-d6)8.83(d,J＝1.9Hz,1H),8.54(dd,J＝4.7Hz,J＝1.4Hz,1H),8.01(d,J＝8.0Hz,1H),7.91(s,1H),7.66(s,1H),7.62(s,1H),7.47(dd,J＝7.9,J＝4.9Hz,1H),7.38-7.36(m,1H),7.31(d,J＝7.9Hz,1H),7.02(s,1H),4.20(t,J＝4.3Hz,2H),3.46(m,2H),2.24(s,3H)，MS(ESI,positive ion)m/z:320.05[M+H]+.
Example 13 preparation of N- (4- ((7, 8-dihydro-6H-pyrimidine [5,4-b ] [1,4] oxazin-4-yl) amino) phenyl) -2- (trifluoromethoxy) benzenesulfonamide (compound 7). The reaction scheme is as follows:
and q, reducing the nitro group in the intermediate 5h by using palladium carbon and hydrogen, and then reacting the reduced nitro group with o-O-trifluoromethylbenzenesulfonyl chloride (the molar ratio is 1:1.2) in pyridine at normal temperature to prepare a compound 7.
1H NMR(400MHz,DMSO-d6)10.22(s,1H),8.17(s,1H),7.89(d,J＝7.2Hz,1H),7.72-7.70(m,2H),7.56-7.48(m,4H),7.04(s,1H),6.91(d,J＝8.4Hz,2H),4.14(s,2H),3.42(s,2H)，MS(ESI,positive ion)m/z:468.01[M+H]+.
EXAMPLE 14 preparation of N- (7, 8-dihydro-6H-pyrimidine [5,4-b ] [1,4] oxazin-4-yl) -1, 4-dimethyl-4H-benzo [ b ] [1,2,4] triazolo [4,3-d ] [1,4] oxazin-7-amine (Compound 8)
r, adding acetonitrile into the 2-amino-5-nitrophenol, bromoacetyl bromide and cesium carbonate according to the molar ratio of 1:1.2:1.2, reacting at normal temperature for 2 hours, heating to 78 ℃ for reaction for 12 hours, directly concentrating the reaction solution after the reaction is finished, and carrying out sample mixing column chromatography to obtain an intermediate 11.
And s, adding the intermediate 11 and the Lawson reagent into tetrahydrofuran according to the molar ratio of 1:1.2, reacting for four hours at 65 ℃, directly stirring a reaction solution, and performing column chromatography. Intermediate 12 is obtained.
t, dissolving the intermediate 12(500mg) into bright yellow turbid liquid at normal temperature by using methanol, adding a methanol solution of hydrazine hydrate (1ml) into a dropping funnel at normal temperature, gradually converting the reaction liquid into orange turbid liquid in the process of dripping, continuously stirring for 12 hours at normal temperature, further dripping acetyl chloride (1ml) at normal temperature, gradually converting the reaction liquid into bright yellow turbid liquid in the process of dripping, concentrating the mixed liquid of tetrahydrofuran and methanol after continuously reacting for 8 hours, adding acetic acid, moving to 100 ℃, heating and refluxing for 2 hours, directly stirring the reaction liquid, and carrying out column chromatography to obtain the intermediate 13
u, reducing nitro in the intermediate 13 by palladium carbon and hydrogen, and then catalyzing with 3a in transition metal and ligand to prepare the compound 8.
1H NMR(400MHz,DMSO-d6)8.57(s,1H),7.87(d,J＝2.0Hz,1H),7.82(s,1H),7.61-7.55(m,2H),7.18(s,1H),5.39-5.37(m,1H),4.19(t,J＝4.2Hz,2H),3.45(m,2H),2.68(s,3H),1.72(d,J＝6.5Hz,3H)；MS(ESI,positive ion)m/z:352.14[M+H]+.
Example 15 preparation of N- (4- ((7, 8-dihydro-6H-pyrimidine [5,4-b ] [1,4] oxazin-4-yl) amino) phenyl) -2- (4- (trifluoromethyl) phenyl) benzamide (Compound 9).
The intermediate is reduced by palladium carbon and hydrogen for 5h and then is catalyzed by condensing agent HATU, N, N-diisopropylethylamine and p-trifluoromethylphenylacetic acid to prepare a compound 9.
1HNMR(400MHz，CDCl3)7.94(s，1H)，7.65(d，J＝8.0Hz，2H)，7.56(d，J＝8.9Hz，2H)，7.48(d，J＝7.9Hz，2H)，7.40(d，J＝8.9Hz，2H)，7.09(s，1H)，6.67(s，1H)，4.33～4.21(m，2H)，3.77(s，2H)，3.60(t，J＝5.5Hz，2H)。
Experiment of drug effect
The vandetanib, sorafenib, cabozantinib, halometasone and 4a-31 adopted in the embodiment of the invention have the structural formulas:
example 16 in vitro kinase assays of pyrimido morpholine derivatives
In vitro kinase inhibition assays were performed using the KinaseProfiler service provided by Eurofins and Reaction Biology Corp. The experimental method is briefly described as follows: adding 1w (0.001-10 mu M) of a small molecule to be detected or a blank solvent, a protein kinase to be detected and a corresponding polypeptide substrate into a reaction buffer solution for incubation, wherein the reaction buffer solution consists of 8mM propanesulfonate (MOPS, pH 7.0), 0.2mM Ethylene Diamine Tetraacetic Acid (EDTA), 10mM magnesium acetate and a Km concentration gamma 33P-ATP solution. After the whole reaction was carried out at room temperature for 40min, a 3% phosphate solution was added to the reaction buffer to terminate the reaction. Then, 10. mu.L of the reaction mixture was quantitatively pipetted onto a P30 filter and washed 3 times with 75mM phosphate solution and once with methanol, and the P30 filter was air-dried and scintillation counting was performed by adding scintillation fluid. Half inhibitory concentration IC for inhibitory Activity of Compounds50To represent, IC50Values were fitted from the inhibition ratios corresponding to each concentration gradient. Firstly, the compound 1w with the best cell activity is subjected to a screening test of single-concentration inhibition rate on 358 existing kinases of Eurofins under the condition of 10 mu M concentration, and IC is carried out on the enzyme with higher inhibition activity50The results of the tests are shown in the table below, and the test data is provided by Eurofins.
TABLE 7 inhibitory Activity of Compound 1w on kinases
| Kinase enzymes | Median inhibitory concentration (nM) | Kinase enzymes | Median inhibitory concentration (nM) |
| DDR2 | 55 | PTK5 | 51 |
| FGFR2(N549H) | 21 | |
50 |
| Flt1(h) | 8 | |
10 |
| Flt3(h) | 13 | RET- |
9 |
| |
6 | RET- |
15 |
| |
9 | TrkA | 71 |
| KDR | 892 | TrkB | 68 |
| PDGFRα(D842V) | 38 | |
20 |
| ZAK | 19 | WNK3 | 49 |
As can be seen from Table 7, Compound 1w has a high inhibitory activity against the kinases listed in the above tables, with the most potent being Flt4(6nM), followed by Flt1(8nM), RET (10nM), ZAK (19nM) and the like.
The kinase inhibition experiment aiming at wild type RET and clinically common mutant RET is tested by Reaction Biology Corp company, wherein cabozantinib and sorafenib are positive control drugs, and sorafenib can overcome drug resistance caused by RET-V804M. The data measured by the company are shown in Table 8:
TABLE 8 kinase Activity of Compounds 1w, cabozantinib, Sorafenib on wild-type RET and clinically common mutant RET
As can be seen from table 8, compound 1w has stronger activity on the above seven kinases than cabozantinib and sorafenib.
Example 17 Activity of Compounds on cells transformed with RET and RET mutant kinase Ba/F3
By using gene engineering technology, wild type rat proto-B lymphocyte (Ba/F3) is used as mother board, and TEL/BCR/NMP gene is fused to the catalytic function area of target gene to induce cell to be independent of IL-3 stimulating factor and to be dependent on exogenously transferred fusion or mutation to activate kinase activity, such as RET. If a compound selectively inhibits the activity of a RET recombinant kinase, apoptosis of the corresponding cell will result. The Ba/F3-TEL-RET whole cell library is established, and a high-efficiency and feasible way is provided for researching and finding the action target of the anticancer small molecule drug.
Test compounds were dissolved in DMSO to prepare a 10mM stock solution, which was diluted 3-fold to prepare 5mM, 1.667mM, 0.556mM, 0.185mM, 0.062mM, 0.021mM, 0.007Mm in 24X 16 384 well clear plates, while using the same volume of DMSO solvent as a blank, for a total of 8 concentration gradients, and the plates were stored under vacuum at-20 ℃.
Cells in logarithmic growth phase (1000-2The culture was carried out overnight in an incubator. Cell plates were dosed (0.1. mu.L/well) using a JANUS @ automated workstation with compound final concentrations of 10. mu.M, 3.3. mu.M, 1.1. mu.M, 0.37. mu.M, 0.12. mu.M, 0.04. mu.M, 0.014. mu.M, 0. mu.M, 37 ℃ and 5% CO2 incubator for 72 hours, 10. mu.L of CellTiter-Glo cell proliferation fluorescence detection reagent was added, and left for 10 minutes with an Envision Plate-Reader reading. The results of the cell activity screening test on the pyrimido-morphine derivatives in this series are shown in Table 9, wherein the cell activity of the most active compound 1w is shown in Table 10, and wherein the Ba/F3 cell line was used to determine whether the compound was off-target. All cellular activity data for Ba/F3 are provided by Anhui Primal.
TABLE 9 Activity of partial Compounds on the Ba/F3 cell line transfected with RET plasmid
As can be seen from Table 9, the activity of compound 1w is superior to vandetanib for all cell lines; in addition to the Ba/F3 cell line transformed with RET-V804L being less active than cabozantinib, the other activities were all higher than cabozantinib.
Table 10 shows Ba/F3 cell activities of 1w, sorafenib, cabozantinib, and vandetanib on transfected RET kinase, wherein sorafenib, cabozantinib, and vandetanib were positive controls.
TABLE 10 Activity of Compounds 1w, Sorafenib, Cabotinib and Vandinib on a Ba/F3 cell line transfected with RET kinase
As can be seen from Table 10, compound 1w was more potent than sorafenib, cabozantinib and vandetanib in all cell lines except the Ba/F3 cell line transfected with RET-V804L plasmid.
Example 18 Activity of Compounds on TT, NIH3T3-RET-C634Y cell line
1) Experimental materials:
the main reagents are as follows: RPMI-1640, fetal bovine serum, pancreatin, etc., were purchased from Gibco BRL corporation (Invitrogen corporation, USA), and IMDM medium was purchased from ATCC (American Type Culture Collection). MTT (tetramethylazozolium salt) and DMSO (dimethyl sulfoxide) are products of Sigma company (USA). The pyrimido morpholine derivatives are synthesized by the inventor, and are prepared into 10mM stock solution by 100% DMSO during in vitro experiments, and the stock solution is stored in a refrigerator at the temperature of-20 ℃ in the dark for later use, and is diluted to the required concentration by using complete culture solution when the stock solution is used.
Cell lines and culture: human leukemia cell lines MV4-11 and human thyroid tumor cell lines TT used in the experiment are all purchased from American ATCC (American type culture collection) and are stored in the laboratory. NIH3T3-RET-C634Y cell lineDonation from professor dawn red, Cheng of university of medical science. The thyroid tumor cell line TT is cultured in RPMI-1640 complete medium containing 10% fetal calf serum, 100U/mL penicillin and 100 mu g/mL streptomycin in 5% CO2And cultured at 37 ℃. MV4-11 cell line was cultured in a complete medium of DMEM containing 20% fetal bovine serum, 100U/mL penicillin and 100. mu.g/mL streptomycin at 5% CO2And cultured at 37 ℃.
2) The experimental method comprises the following steps:
adjusting the cell concentration to 1-2 × 10 with the use of whole cell culture fluid4Cell suspension/mL, inoculated into 96-well plate, each well 200 u L cell suspension, cultured overnight. The following day, the supernatants were aspirated (the supernatants were aspirated after centrifugation of the suspension cells) and the cells were then treated with a gradient concentration of test compound, respectively. Setting a negative control group without drug and a solvent control group with the same volume, wherein the concentration of DMSO is 0.1%, each dose group is provided with 3 multiple wells, and the concentration of DMSO is 5% CO at 37 DEG C2Culturing for 72 hours, adding 20 mu L of MTT reagent with the concentration of 5mg/mL into each hole, culturing for 2-4 hours, removing supernatant, adding 150 mu L of DMSO into each hole, shaking and uniformly mixing for 15min, measuring the absorbance (A) value (the A value is in direct proportion to the number of living cells) by using a microplate reader (lambda is 570nm), taking the average value, taking the relative cell proliferation inhibition rate (a control group A570-an experimental group A570)/a control group A570 × 100 percent, repeating the experiment for at least 3 times, expressing the experimental data by the average value, and adopting t test and P test as the statistical data<A difference of 0.05 is statistically significant. IC was used for inhibition of cell proliferation by each of the following compounds50Or inhibition rate.
3) The experimental results are as follows:
by adopting the method, the proliferation inhibition activity test is carried out on the human thyroid tumor cell strain TT, the NIH3T3-RET-C634Y cell strain and the human leukemia cell strain MV4-11, and the NIH3T3 cell strain is used for detecting whether the compound is off-target. The results are shown in tables 11 and 12.
Cell Activity of some Compounds of Table 11 on TT, NIH3T3-RET-C634Y
Table 12 shows the cellular activity of compounds 1l, 1k, 1w against MV 4-11. Among them, compound 1w has high cellular activity against MV4-11, reaching picomolar level.
TABLE 12 inhibitory Activity of Compounds 1l, 1k, 1w on MV4-11
| Compound (I) | Cellular level |
| 1l | 17.8nM |
| 1k | 0.006 |
| 1w | 0.001nM |
Example 19 in vivo model test for medullary thyroid carcinoma
The purpose of this experiment was to examine the in vivo anti-tumor effect of the compounds of the present invention. In this experiment, NOD-SCID mouse subcutaneous medullary thyroid carcinoma model was used to test the in vivo anti-tumor activity of compound 1w of the present invention. The cell line used was NIH3T 3-RET-C634Y. The clinical medicine cabozantinib aiming at medullary carcinoma is taken as a positive control.
1) Experimental materials:
IMDM, fetal bovine serum, pancreatic enzymes, etc., were purchased from Gibco BRL (Invitrogen Corporation, USA), IMDM medium was purchased from ATCC (American Type Culture Collection), NIH3T3-RET-C634Y was a donation from David red, and NOD-SCID mice were purchased from Beijing Huafukang Biotech GmbH, China.
2) The experimental method comprises the following steps:
6-8 week-old NOD-SCID mice were used at about 1 × 1070.1 mL/NIH 3T3-RET-C634Y cell concentration is inoculated on the subcutaneous posterior costal part of the mouse until the tumor grows to 150-200 mm3After (about 10 days), mice were grouped (n-6) and oral gavage was started.
Grouping experiments: drug solvent control group (12.5% castor oil + 12.5% ethanol + 75% water);
positive control cabozantinib: 50mg/kg q.d.;
each group of drugs was dissolved in 12.5% castor oil + 12.5% ethanol + 75% water.
The observation indexes are that the weight of the mouse, the long diameter and the short diameter of the tumor are measured once every 3 days and the tumor volume (length × width) is calculated2× 0.52.52), observing the existence of diarrhea, convulsion, rash, and obvious weight loss.
3) The experimental results are as follows:
the experimentally measured tumor growth curves of the various groups are shown in FIG. 5.
The experimental result shows that the tested compound 1w has obvious in-vivo growth inhibition effect on medullary thyroid carcinoma, can obviously inhibit the growth of tumors 21 days after administration, and is slightly superior to a positive control drug cabozantinib. No adverse reactions such as weight loss, rash, diarrhea and the like of the mice were found in the administration process, indicating that the test compound 1w has very low toxicity in the administration dose range under the test dose.
Example 20 establishment of mouse ear swelling model
1 materials and methods
(1) Material
Animals: SPF grade Balb/C mice, 7-8 weeks old, female. Provided by the animal experiment center of Sichuan university.
The reagent and the drug physiological saline (produced by Sichuan Kelun pharmaceutical industry Co., Ltd.), the compound 1w (synthesized in laboratory), halometasone cream (produced by Australian Mei pharmaceutical preparation), acetone (produced by Fuyu Fine chemical Co., Ltd., Tianjin), TPA (Sigma, 12-O-tetradecanoyl phorbol-13-ethyl ester in USA).
(2) Method of producing a composite material
Grouping experiments: 16 healthy and clean female Balb/C mice are taken and randomly divided into a blank group of mice, a model group, a cream substrate group, a halometasone cream group (the drug content is 0.05 percent), a 1w cream group (the drug content is 0.5 percent) and 4 mice each group.
Experiment dosing: except for the blank group of mice, the other groups were molded 1 day before administration, 10 μ L of TPA (12-O-tetradecanoyl phorbol-13-ethyl ester) acetone solution (10 μ g/mL) was uniformly applied to the inner and outer sides of auricles on both sides of the mice for molding, and molding was performed every 1 day by this method until the end of the experiment (when molding and administration were performed on the same day, administration should be performed 3 hours after molding). On the 1 st day of administration, 4a-31 cream group, 1w cream group and halometasone cream group are evenly smeared with corresponding creams on the inner side and the outer side of an auricle caused by inflammation of the mouse (1mL needle-removing injector is adopted to suck the corresponding creams, and 1 cell of creams are pushed out on each side of the ear caused by inflammation of the mouse to be smeared, and the amount of the creams is equal to that of the creams, and the doses are continuously administered for 7 days and 1 time every day.
Index observation and method: the general physical condition of the mice is observed every day, the change of the ear inflammation of the mice is observed every day, and the appearance change condition of the ears of the mice is photographed and recorded.
As can be seen from the comparative effect of several model groups in FIG. 6, the anti-inflammatory activity of Compound 1w is comparable to that of halomethasone, but stronger than that of 4a-31 (positive drug).
Claims (10)
1. A pyrimido morpholine derivative characterized by: the structure is shown as formula III:
wherein X is C;
R2is-H, halogen, -OH, C1-C4 alkyl or C1-C4 alkoxy；
R3、R4Independently is-H, halogen, -OH, C1-C4 alkyl or C1-C4 alkoxy;
R5is-H or C1-C4 alkyl;
R11～R15independently is-H, halogen, -OH, C1-C4 alkyl, C1-C4 alkoxy, phenyl or-CF3；
The pyrimido morpholine derivatives also include
2. A pyrimido morpholine derivative characterized by: the structure is shown as formula IV:
wherein X is C or N; n is 1 or 2;
R2is-H, halogen, -OH, C1-C4 alkyl or C1-C4 alkoxy;
R3、R4independently is-H, halogen, -OH, C1-C4 alkyl or C1-C4 alkoxy;
R5is-H or C1-C4 alkyl;
R16is-H or C1-C4 alkyl.
3. A pyrimido morpholine derivative having the structural formula:
4. a pyrimido morpholine derivative according to any one of claims 1 to 3 as a pharmaceutically acceptable salt.
5. A composition prepared by adding pharmaceutically acceptable auxiliary components to the pyrimido morpholine derivative according to any one of claims 1 to 3 or the salt according to claim 4.
6. Use of a pyrimido morpholine derivative according to any one of claims 1 to 3, a salt according to claim 4 or a composition according to claim 5 for the preparation of a RET kinase inhibitor.
7. Use of a pyrimido morpholine derivative according to any one of claims 1 to 3, a salt according to claim 4 or a composition according to claim 5 for the preparation of a drug resistant mutant RET kinase inhibitor.
8. Use of a pyrimido morpholine derivative according to any one of claims 1 to 3, a salt according to claim 4 or a composition according to claim 5 for the manufacture of a medicament for the treatment of medullary thyroid carcinoma.
9. Use of a pyrimido morpholine derivative according to any one of claims 1 to 3, a salt according to claim 4 or a composition according to claim 5 for the manufacture of a medicament for the treatment of acute myeloid leukaemia.
10. Use of a pyrimido morpholine derivative according to any one of claims 1 to 3, a salt according to claim 4 or a composition according to claim 5 for the manufacture of an anti-inflammatory medicament.
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