THREE-RING PI3K AND/OR MTOR INHIBITOR

Abstract
The present application relates to a compound as represented by general formula (I), a pharmaceutically acceptable salt, ester, solvate or stereoisomer thereof, preparation method of the compounds, a pharmaceutical composition containing the compounds, uses thereof in the preparation of drugs for treating and/or preventing proliferative diseases, and a method using the compounds to treat and/or prevent proliferative diseases. R1, R2, R3, R4, R5, R6, X, A and B in the formula are as defined in the specification.
Description

The present application claims priority to the Chinese applications of No. 201110364054.2, filed on Nov. 17, 2011 and No. 20120072834.4, filed on Mar. 20, 2012. The contents of these prior applications are incorporated herein as a whole by reference. The contents of all the documents cited herein constitute a part of the present application.


TECHNICAL FIELD

The present invention relates to tricyclic PI3K and/or mTOR inhibitors or a pharmaceutically acceptable salt, ester, solvate or stereoisomer thereof, a method for preparing these compounds, a pharmaceutical composition comprising the same, as well as a use of these compounds in the manufacture of a medicament for treating and/or preventing proliferative diseases.


BACKGROUND ART

Tumors are new creatures formed in the body due to actions of a variety of oncogenic factors and the following changes of genetic material of cells resulting in abnormal gene expression and cell proliferation. The tumor cells lose their normal growth regulatory function and have autonomous or relatively autonomous growth ability. The tumor cells can continue to grow even the oncogenic factors disappear and will cause a consumption of large quantities of human's nutrients. If not being found and treated early, the cancer cells will be transferred and grow throughout the body, and release a variety of toxins that cause the body weight loss, anemia, organ failure and even death.


The method of treating tumors mainly includes three aspects: drug therapy, surgery and radiation therapy. Drug therapy is becoming increasingly important in cancer treatment since surgery and radiation therapy are difficult to eradicate the tumor and fail to show obvious effect in patients with the mid-advanced cancer. Conventional anticancer drugs do not distinguish between tumor cells and normal cells, and often cause serious side effects. Targeted drugs specifically target to cancer cells and can accurately act on the tumor, which greatly improves the level of treatment and reduces the adverse response rate, such as making the median survival time of patients with advanced colorectal cancer increased 66.7%, and the efficiency of the treatment of advanced breast cancer increased 71.3%.


As pharmaceutical companies are accelerating the development of targeted antineoplastic drugs, and there is a great market demand for this class of antineoplastic drugs, molecular targeted drugs have become the world's fastest growing unit in the worldwide market.


Phosphatidylinositol 3-kinase (PI3K) pathway is the most common place where human cancer cells mutate, and it can lead to cell proliferation, activation and signal amplification. PI3K and mammalian target of rapamycin (mTOR) are important kinases of PI3K signaling pathway.


PI3K is a member of lipid kinase family, it regulates cell metabolism and growth through phosphorylation at 3-position of phosphatidylinositol to produce phosphatidylinositol-triphosphate (PIP3). This PIP3, which is the second messenger of lipid, can make P13K paired with downstream effectors (especially Akt), resulting in membrane recruitment and phosphorylation, cell proliferation and activation. Therefore, inhibition of phosphatidylinositol 3-kinase can affect the PI3K pathway, thereby inhibiting cancer cell proliferation and activation.


mTOR is a serine/threonine protein kinase present in the cytoplasm, it belongs to the phosphoinositide 3-kinase-related protein kinase family and exists in organism as two complexes forms, namely mTORC1 (rapamycin target) and mTORC2 (not inhibited by rapamycin). mTOR is a cellular signal transduction protein, which regulates the response of tumor cells to nutrients and growth factors, and controls tumor blood supply through the role of vascular endothelial growth factor. mTOR inhibitors can make cancer cells starved, and reduce the tumor volume by inhibiting mTOR.


In Novartis' patent application of WO2006122806 and Pfizer's patent application of WO201003816, a series of compounds inhibiting both PI3K and mTOR have been reported. These compounds have good activity in tumor therapy. However, there is no compound inhibiting both PI3K and mTOR on the current drug market. Therefore, it is needed to develop multi-targeted drugs that have inhibiting activity for both PI3K and mTOR in order to facilitate the treatment of cancer.


SUMMARY OF THE INVENTION

The object of the present invention is to provide a PI3K and/or mTOR inhibitor. Specifically, the present invention relates to:


(1) A compound of general formula (I):




embedded image


or a pharmaceutically acceptable salt, ester, solvate or stereoisomer thereof,


wherein


X is O or S;


A and B are each CR8, R8 is hydrogen, halogen, cyano, hydroxy, carboxyl, —(CH2)nNR10aR10b, —(CH2)nC(O)R11, —(CH2)nS(O)mR11, —(CH2)nS(O)mNR10aR10b, —(CH2)nN(R10a)S(O)mR11, —(CH2)nC(O)NR10aR10b, —(CH2)nOC(O)R11, —(CH2)nC(O)(CH2)nOR11, —(CH2)nN(R10a)C(O)R11, C1-6 alkyl or C1-6 alkoxy, wherein C1-6 alkyl and C1-6 alkoxy may be optionally substituted with 1 to 3 substituents selected from halogen, hydroxy and carboxyl;


R1 is hydrogen, or C1-6 alkyl, C2-8 alkenyl, C2-8 alkynyl, C3-8 cycloalkyl, 6- to 14-membered aryl, 5- to 14-membered heteroaryl, 3- to 14-membered heterocyclic group, 7- to 12-membered spirocyclic group or 7- to 12-membered endocyclic group, all of which except hydrogen may be optionally substituted with 1 to 5 R9a;


R2 is hydrogen, C1-6 alkyl, C2-8 alkenyl, C2-8 alkynyl, C3-8 cycloalkyl, 6- to 14-membered aryl, 5- to 14-membered heteroaryl, 3- to 14-membered heterocyclic group, 7- to 12-membered spirocyclic group or 7- to 12-membered endocyclic group, all of which except hydrogen may be optionally substituted with 1 to 5 R9b;


R3 is hydrogen, carboxyl or C1-6 alkyl, wherein C1-6 alkyl may be optionally substituted with 1 to 3 substituents selected from halogen, hydroxy and carboxyl;


R4 and R5 are each independently hydrogen or C1-6 alkyl, wherein C1-6 alkyl may be optionally substituted with 1 to 3 substituents selected from halogen, hydroxy and carboxyl; or R4 and R5 combine each other to form C3-8 cycloalkyl, 5- to 6-membered monocyclic heteroaryl or 5- to 6-membered monocyclic heterocyclic group;


R6 is hydrogen or C1-6 alkyl, wherein C1-6 alkyl may be optionally substituted with 1 to 3 substituents selected from halogen, hydroxy and carboxyl;


R9a and R9b are each independently


(i) halogen, cyano, hydroxy, —(CH2)nNR10aR10b, —(CH2)nC(O)R11, —(CH2)nS(O)mR11, —(CH2)nS(O)mNR10aR10b, —(CH2)nN(R10a)S(O)mR11, —(CH2)nC(O)NR10aR10b, —(CH2)nOC(O)R11, —(CH2)nC(O)(CH2)nOR11, —(CH2)nN(R10a)C(O)R11;


(ii) C1-6 alkyl, C2-8 alkenyl, C2-8 alkynyl or C1-6 alkoxy, all of which may be optionally substituted with 1 to 3 substituents selected from halogen, hydroxy and cyano;


(iii) C3-8 cycloalkyl, 6- to 14-membered aryl, 5- to 14-membered heteroaryl or 3- to 14-membered heterocyclic group, all of which may be optionally substituted with 1 to 3 substituents selected from halogen, hydroxy, cyano, trifluoromethyl, C1-6 alkyl, C2-8 alkenyl, C2-8 alkynyl, C1-6 alkoxy, —(CH2)nNR10aR10b, —(CH2)nC(O)R11, —(CH2)nC(O)NR10aR10b, —(CH2)nS(O)mR11, —(CH2)nS(O)mNR10aR10b, —(CH2)nN(R10a)S(O)mR11, —(CH2)nOC(O)R11 and —(CH2)nN(R10a)C(O)R11;


R10a and R10b are each independently hydrogen, or C1-6 alkyl, C3-8 cycloalkyl, 6- to 14-membered aryl, 5- to 14-membered heteroaryl or 3- to 14-membered heterocyclic group, all of which except hydrogen may be optionally substituted with 1 to 3 substituents selected from hydroxy, halogen, cyano, carboxyl, —(CH2)nNR10aR10b, sulfamoyl, carbamoyl and sulfamino;


R11 is hydrogen, C1-6 alkyl or C1-6 alkoxy, wherein C1-6 alkyl and C1-6 alkoxy may be optionally substituted with 1 to 3 substituents selected from halogen, cyano, hydroxy, carboxyl, —(CH2)nNR10aR10b, sulfamoyl and carbamoyl;


m is 0, 1 or 2; and


n is 0-4.


(2) The compound of above (1), or a pharmaceutically acceptable salt, ester, solvate or stereoisomer thereof, wherein


R8 is hydrogen, halogen, cyano, hydroxy, carboxyl, —(CH2)nNR10aR10b, —(CH2)nS(O)mNR10aR10b, —(CH2)nN(R10a)S(O)mR11, C1-6 alkyl or C1-6 alkoxy, wherein C1-6 alkyl and C1-6 alkoxy may be optionally substituted with 1 to 3 substituents selected from halogen, hydroxy and carboxyl;


R1 is C3-8 cycloalkyl, 6- to 10-membered aryl, 5- to 10-membered heteroaryl or 5- to 10-membered heterocyclic group, all of which may be optionally substituted with 1 to 3 R9a;


R2 is C3-8 cycloalkyl, 6- to 10-membered aryl, 5- to 10-membered heteroaryl or 5- to 10-membered heterocyclic group, all of which may be optionally substituted with 1 to 3 R9b;


R3, R4, R5 and R6 are each independently hydrogen or C1-6 alkyl, wherein C1-6 alkyl may be optionally substituted with 1 to 3 substituents selected from halogen, hydroxy and carboxyl;


R9a and R9b are each independently


(i) halogen, cyano, hydroxy, —(CH2)n NR10aR10b, —(CH2)nC(O)R11, —(CH2)nS(O)mR11, —(CH2)nS(O)mNR10aR10b, —(CH2)nN(R10a)S(O)mR11, —(CH2)nC(O)NR10aR10b, —(CH2)nOC(O)R11, —(CH2)nC(O)(CH2)OR11, —(CH2)nN(R10a)C(O)R11;


(ii) C1-6 alkyl or C1-6 alkoxy, all of which may be optionally substituted with 1 to 3 substituents selected from halogen, hydroxy and cyano;


(iii) C3-8 cycloalkyl, 6- to 10-membered aryl, 5- to 10-membered heteroaryl or 5- to 10-membered heterocyclic group, all of which may be optionally substituted with 1 to 3 substituents selected from halogen, hydroxy, cyano, trifluoromethyl, C1-6 alkyl, C1-6 alkoxy, —(CH2)nNR10aR10b, —(CH2)nC(O)R11, —(CH2)nC(O)NR10aR10b, —(CH2)nS(O)mR11, —(CH2)nS(O)mNR10aR10b, —(CH2)nN(R10a)S(O)R11, —(CH2)nOC(O)R11 and —(CH2)nN(R10a)C(O)R11;


R10a and R10b are each independently hydrogen or C1-6 alkyl, wherein C1-6 alkyl may be optionally substituted with 1 to 3 substituents selected from hydroxy, halogen, cyano, carboxyl, —(CH2)nNR10aR10b, sulfamoyl, carbamoyl and sulfamino; and


R11 is hydrogen or C1-6 alkyl, wherein C1-6 alkyl may be optionally substituted with 1 to 3 substituents selected from halogen, cyano, hydroxy, carboxyl, —NR10aR10b, sulfamoyl and carbamoyl.


(3) The compound of above (2), or a pharmaceutically acceptable salt, ester, solvate or stereoisomer thereof, wherein


X is O;


A and B are each CH;


R1 is 6- to 10-membered aryl, 5- to 6-membered monocyclic heteroaryl, 9- to 10-membered fused heteroaryl, 5- to 6-membered monocyclic heterocyclic group or 9- to 10-membered fused heterocyclic group, all of which may be optionally substituted with 1 to 3 R9a;


R2 is 6- to 10-membered aryl, 5- to 6-membered monocyclic heteroaryl, 9- to 10-membered fused heteroaryl, 5- to 6-membered monocyclic heterocyclic group or 9- to 10-membered fused heterocyclic group, all of which may be optionally substituted with 1 to 3 R9b;


R3, R4 and R5 are each hydrogen;


R6 is hydrogen or C1-6 alkyl;


R9a and R9b are each independently


(i) halogen, cyano, hydroxy, —(CH2)nNR10aR10b, —(CH2)nC(O)R11, —(CH2)nS(O)mR11, —(CH2)nS(O)mNR10aR10b, —(CH2)nN(R10a)S(O)mR11, —(CH2)nC(O)NR10aR10b, —(CH2)nOC(O)R11, —(CH2)nC(O)(CH2)nOR11, —(CH2)nN(R10a)C(O)R11;


(ii) C1-6 alkyl or C1-6 alkoxy, all of which may be optionally substituted with 1 to 3 substituents selected from halogen, hydroxy and cyano;


(iii) 5- to 10-membered heteroaryl or 5- to 10-membered heterocyclic group, all of which may be optionally substituted with 1 to 3 substituents selected from halogen, hydroxy, cyano, trifluoromethyl, C1-6 alkyl, C1-6 alkoxy, —(CH2)nNR10aR10b, —(CH2)nC(O)R11, —(CH2)nC(O)NR10aR10b, —(CH2)nS(O)mR11, —(CH2)nS(O)mNR10aR10b, —(CH2)nN(R10a)S(O)mR11, —(CH2)nOC(O)R11 and —(CH2)nN(R10a)C(O)R11;


R10a and R10b are each independently hydrogen or C1-6 alkyl;


R11 is hydrogen or C1-6 alkyl, wherein C1-6 alkyl may be optionally substituted with 1 to 3 substituents selected from halogen, cyano, hydroxyl and —(CH2)nNR10aR10b; and


n is 0-3.


(4) The compound of above (3), or a pharmaceutically acceptable salt, ester, solvate or stereoisomer thereof, wherein


R1 is 6- to 10-membered aryl or 5- to 6-membered monocyclic heteroaryl, all of which may be optionally substituted with 1 to 3 R9a;


R2 is 6- to 10-membered aryl, 5- to 6-membered monocyclic heteroaryl or 9- to 10-membered fused heteroaryl, all of which may be optionally substituted with 1 to 3 R9b; and


R9a and R9b are each independently


(i) halogen, cyano, hydroxy, —(CH2)nNR10aR10b, —(CH2)nN(R10a)C(O)R11, —(CH2)nC(O)R11, —(CH2)nS(O)mR11, —(CH2)nC(O)NR10aR10b, —(CH2)nC(O)(CH2)nOR11;


(ii) C1-6 alkyl or C1-6 alkoxy, all of which may be optionally substituted with 1 to 3 substituents selected from halogen, hydroxy and cyano;


(iii) 5- to 6-membered monocyclic heteroaryl or 5- to 6-membered monocyclic heterocyclic group, all of which may be optionally substituted with 1 to 3 substituents selected from halogen, hydroxy, cyano, trifluoromethyl, C1-6 alkyl, C1-6 alkoxy, —(CH2)nNR10aR10b, —(CH2)nC(O)R11, —(CH2)C(O)NR10aR10b, —(CH2)nOC(O)R11 and —(CH2)nN(R10a)C(O)R11.


(5) The compound of above (4), or a pharmaceutically acceptable salt, ester, solvate or stereoisomer thereof, wherein


R1 is phenyl, pyridyl or pyrimidinyl, all of which may be optionally substituted with 1 to 3 R9a;


R2 is phenyl, pyridyl, pyrimidinyl, thienyl, pyrazolyl, indazolyl, indolyl, pyridopyrrolyl, pyrazolopyridyl or quinolyl, all of which may be optionally substituted with 1 to 3 R9b;


R9a and R9b are each independently


(i) cyano, hydroxy, —NR10aR10b, —N(R10a)C(O)R11, —C(O)R11, —S(O)mR11, —C(O)NR10aR10b, —C(O)CH2OR11;


(ii) C1-6 alkyl or C1-6 alkoxy, all of which may be optionally substituted with 1 to 3 substituents selected from halogen, hydroxy and cyano;


(iii) pyrrolyl, pyrazolyl, imidazolyl, piperidinyl, piperazinyl or morpholinyl, all of which may be optionally substituted with 1 to 3 substituents selected from halogen, hydroxy, cyano, trifluoromethyl, C1-6 alkyl, C1-6 alkoxy, —NR10aR10b, —C(O)R11, —C(O)NR10aR10b, —OC(O)R11 and —N(R10a)C(O)R11;


R11 is hydrogen or C1-6 alkyl, wherein C1-6 alkyl may be optionally substituted with 1 to 3 substituents selected from halogen, cyano and hydroxy; and


n is 0.


(6) The compound of above (5), or a pharmaceutically acceptable salt, ester, solvate or stereoisomer thereof, wherein R1 is phenyl optionally substituted with 1 to 3 R9a.


(7) The compound of above (1), or a pharmaceutically acceptable salt, ester, solvate or stereoisomer thereof, wherein general formula (I) is represented by general formula (II) as shown below:




embedded image


wherein R2, R3, R4, R5, R6, X, A and B are as defined in above (1), and R9a is C1-6 alkyl optionally substituted with 1 to 3 halogen atoms.


More preferred compounds of the present invention are as shown in the following Table 1.













No.
Structural Formula







 1


embedded image







 2


embedded image







 3


embedded image







 4


embedded image







 5


embedded image







 6


embedded image







 7


embedded image







 8


embedded image







 9


embedded image







 10


embedded image







 11


embedded image







 12


embedded image







 13


embedded image







 14


embedded image







 15


embedded image







 16


embedded image







 17


embedded image







 18


embedded image







 19


embedded image







 20


embedded image







 21


embedded image







 22


embedded image







 23


embedded image







 24


embedded image







 25


embedded image







 26


embedded image







 27


embedded image







 28


embedded image







 29


embedded image







 30


embedded image







 31


embedded image







 32


embedded image







 33


embedded image







 34


embedded image







 35


embedded image







 36


embedded image







 37


embedded image







 38


embedded image







 39


embedded image







 40


embedded image







 41


embedded image







 42


embedded image







 43


embedded image







 44


embedded image







 45


embedded image







 46


embedded image







 47


embedded image







 48


embedded image







 49


embedded image







 50


embedded image







 51


embedded image







 52


embedded image







 53


embedded image







 54


embedded image







 55


embedded image







 56


embedded image







 57


embedded image







 58


embedded image







 59


embedded image







 60


embedded image







 61


embedded image







 62


embedded image







 63


embedded image







 64


embedded image







 65


embedded image







 66


embedded image







 67


embedded image







 68


embedded image







 69


embedded image







 70


embedded image







 71


embedded image







 72


embedded image







 73


embedded image







 74


embedded image







 75


embedded image







 76


embedded image







 77


embedded image







 78


embedded image







 79


embedded image







 80


embedded image







 81


embedded image







 82


embedded image







 83


embedded image







 84


embedded image







 85


embedded image







 86


embedded image







 87


embedded image







 88


embedded image







 89


embedded image







 90


embedded image







 91


embedded image







 92


embedded image







 93


embedded image







 94


embedded image







 95


embedded image







 96


embedded image







 97


embedded image







 98


embedded image







 99


embedded image







100


embedded image







101


embedded image







102


embedded image







103


embedded image







104


embedded image







105


embedded image







106


embedded image







107


embedded image







108


embedded image







109


embedded image







110


embedded image







111


embedded image







112


embedded image







113


embedded image







114


embedded image







115


embedded image







116


embedded image







117


embedded image







118


embedded image







119


embedded image







120


embedded image







121


embedded image







122


embedded image







123


embedded image







124


embedded image







125


embedded image







126


embedded image







127


embedded image







128


embedded image







129


embedded image







130


embedded image







131


embedded image







132


embedded image







133


embedded image







134


embedded image







135


embedded image







136


embedded image







137


embedded image







138


embedded image







139


embedded image







140


embedded image







141


embedded image







142


embedded image







143


embedded image







144


embedded image







145


embedded image







146


embedded image







147


embedded image







148


embedded image







149


embedded image







150


embedded image







151


embedded image







152


embedded image







153


embedded image







154


embedded image







155


embedded image







156


embedded image







157


embedded image







158


embedded image







159


embedded image







160


embedded image







161


embedded image







162


embedded image







163


embedded image







164


embedded image







165


embedded image







166


embedded image







167


embedded image







168


embedded image







169


embedded image







170


embedded image







171


embedded image







172


embedded image







173


embedded image







174


embedded image







175


embedded image







176


embedded image







177


embedded image







178


embedded image







179


embedded image







180


embedded image







181


embedded image







182


embedded image







183


embedded image







184


embedded image







185


embedded image







186


embedded image







187


embedded image







188


embedded image







189


embedded image







190


embedded image







191


embedded image







192


embedded image







193


embedded image







194


embedded image







195


embedded image







196


embedded image







197


embedded image







198


embedded image







199


embedded image







200


embedded image







201


embedded image







202


embedded image







203


embedded image







204


embedded image







205


embedded image







206


embedded image







207


embedded image







208


embedded image







209


embedded image







210


embedded image







211


embedded image







212


embedded image







213


embedded image







214


embedded image







215


embedded image







216


embedded image







217


embedded image







218


embedded image







219


embedded image







220


embedded image







221


embedded image







222


embedded image







223


embedded image







224


embedded image







225


embedded image







226


embedded image







227


embedded image







228


embedded image







229


embedded image







230


embedded image







231


embedded image







232


embedded image







233


embedded image







234


embedded image







235


embedded image







236


embedded image







237


embedded image







238


embedded image







239


embedded image







240


embedded image







241


embedded image







242


embedded image







243


embedded image







244


embedded image







245


embedded image







246


embedded image







247


embedded image







248


embedded image







249


embedded image







250


embedded image







251


embedded image







252


embedded image







253


embedded image







254


embedded image







255


embedded image







256


embedded image







257


embedded image







258


embedded image







259


embedded image







260


embedded image







261


embedded image







262


embedded image







263


embedded image







264


embedded image







265


embedded image







266


embedded image







267


embedded image







268


embedded image







269


embedded image







270


embedded image







271


embedded image







272


embedded image







273


embedded image







274


embedded image







275


embedded image







276


embedded image







277


embedded image







278


embedded image







279


embedded image







280


embedded image







281


embedded image







282


embedded image







283


embedded image







284


embedded image







285


embedded image







286


embedded image







287


embedded image







288


embedded image







289


embedded image







290


embedded image







291


embedded image







292


embedded image







293


embedded image







294


embedded image







295


embedded image







296


embedded image







297


embedded image







298


embedded image







299


embedded image







300


embedded image







301


embedded image







302


embedded image







303


embedded image







304


embedded image







305


embedded image







306


embedded image







307


embedded image







308


embedded image











The term “halogen” as used herein includes fluorine, chlorine, bromine and iodine.


The term “C1-6 alkyl” may be linear or branched and includes for example “C1-4 alkyl”, “C1-3 alkyl”, “C1-2 alkyl”. Examples include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, 2-methylpropyl, 1-methylpropyl, 1,1-dimethylethyl, n-pentyl, 3-methylbutyl, 2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, n-hexyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl, 2-ethylbutyl, 1,2-dimethylpropyl and the like.


The term “C3-8 cycloalkyl” as used herein includes for example “C3-7 cycloalkyl”, “C3-6 cycloalkyl”, “C4-6 cycloalkyl” and “C5-6 cycloalkyl”. Examples include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl and the like.


The term “C2-8 alkenyl” as used herein may be linear or branched or cyclic, and includes for example “C2-5 alkenyl”, “C2-4 alkenyl”, “C2-3 alkenyl” and “C3-6 cycloalkenyl”. Examples include, but are not limited to, ethenyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 2-methyl-1-propenyl, 1-methyl-2-propenyl, 1-pentenyl, 2-pentenyl, 2-methyl-1-butenyl, 1-methyl-2-butenyl, 3-methyl-2-butenyl, 2-methyl-3-butenyl, 3-methyl-3-butenyl, 1,1-dimethyl-2-propenyl, 1-ethyl-2-propenyl, 3-hex enyl, 2-methyl-1-pentenyl, 3-methyl-1-pentenyl, 1-methyl-2-pentenyl, 3-methyl-2-pentenyl, 1-methyl-3-pentenyl, 2-methyl-3-pentenyl, 4-methyl-3-pentenyl, 1-methyl-4-pentenyl, 2-methyl-4-pentenyl, 1,1-dimethyl-2-butenyl, 1,1-dimethyl-3-butenyl, 1,2-dimethyl-3-butenyl, 1,3-dimethyl-2-butenyl, 2,3-dimethyl-1-butenyl, 2-ethyl-1-butenyl, 2-ethyl-3-butenyl, 2-heptenyl, 3-heptenyl, 4-heptenyl, 1-octenyl, 2-octenyl, 4-octenyl, 1,3-butadienyl, 1,4-pentadienyl, 2,4-pentadienyl, 1,4-hexadienyl, 2,4-hexadienyl, cyclopentenyl, 1,3-cyclopentadienyl, cyclohexenyl, 1,4-cyclohexadienyl, cycloheptenyl, 1,4-cycloheptadienyl, cyclooctenyl and 1,5-cyclooctadienyl and the like. The double bond may be optionally cis- and trans-.


The term “C2-8 alkynyl” as used herein may be linear or branched and includes for example “C2-5 alkynyl”, “C2-4 alkynyl” and “C2-3 alkynyl”. Examples include, but are not limited to, ethynyl, 1-propinyl, 2-propinyl, 2-butynyl, 3-butynyl, 1-methyl-2-propinyl, 2-pentynyl, 3-pentynyl, 2-methyl-3-butynyl, 2-hexynyl, 3-hexynyl, 4-methyl-2-pentynyl, 2-methyl-3-pentynyl, 2-methyl-4-pentynyl, 3-methyl-4-pentynyl, 1,2-dimethyl-3-butynyl, 2,2-dimethyl-3-butynyl, 1-ethyl-2-butynyl, 1-ethyl-3-butynyl, 2-ethyl-3-butynyl, 2-heptynyl, 3-heptynyl, 4-methyl-2-hexynyl, 2-methyl-3-hexynyl, 2-methyl-4-hexynyl, 3-methyl-5-hexynyl, 2-octynyl, 4-octynyl, 4-methyl-2-heptynyl, 2-methyl-3-heptynyl, 5-methyl-3-heptynyl, 1-methyl-4-heptynyl, 2-methyl-5-heptynyl, 2-methyl-6-heptynyl and the like.


The term “C1-6 alkoxy” as used herein refers to “C1-6 alkyl-O-” wherein “C1-6 alkyl” is defined as above.


The term “C1-6 alkyl carbonyl” as used herein refers to “C1-6 alkyl-C(O)—” wherein “C1-6 alkyl” is defined as above.


The term “6- to 14-membered aryl” as used herein includes 6- to 8-membered monocyclic aryl and 8- to 14-membered fused aryl. 6- to 8-membered monocyclic aryl includes for example phenyl and cyclooctatetraenyl. 8- to 14-membered fused aryl includes for example naphthyl, phenanthryl, 2,3-dihydro-1H-indenyl, 1H-indenyl, 1,2,3,4-tetrahydronaphthyl, 1,4-dihydronaphthyl and the like.


The term “5- to 14-membered heteroaryl” as used herein includes 5- to 8-membered monocyclic heteroaryl and 6- to 14-membered fused heteroaryl, wherein heteroatoms are nitrogen, oxygen, sulfur and the like. It includes the case that a carbon atom, a nitrogen atom or a sulfur atom is substituted by oxo.


Examples of 5- to 8-membered monocyclic heteroaryl include, but are not limited to, furyl, thienyl, pyrrolyl, thiazolyl, isothiazolyl, thiadiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, imidazolyl, pyrazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, pyridyl, 2-pyridone, 4-pyridone, pyrimidinyl, 1,4-dioxinyl, 2H-1,2-oxazinyl, 4H-1,2-oxazinyl, 6H-1,2-oxazinyl, 4H-1,3-oxazinyl, 6H-1,3-oxazinyl, 4H-1,4-oxazinyl, pyridazinyl, pyrazinyl, 1,2,3-triazinyl, 1,3,5-triazinyl, 1,2,4,5-tetrazinyl, azepinyl, 1,3-diazepinyl, azacyclooctatetraenyl and the like. It is preferably 5- to 6-membered monocyclic heteroaryl.


Examples of 6- to 14-membered fused heteroaryl include, but are not limited to, benzofuryl, isobenzofuryl, benzothienyl, indolyl, isoindolyl, benzoxazolyl, benzimidazolyl, indazolyl, benzotriazolyl, quinolyl, 2-quinolinone, 4-quinolinone, 1-isoquinolinone, isoquinolyl, acridinyl, phenanthridinyl, benzopyridazinyl, phthalazinyl, quinazolinyl, quinoxalinyl, phenazinyl, pteridinyl, purinyl, naphthyridinyl, phenazinyl, phenothiazinyl and the like. It is preferably 9- to 10-membered fused heteroaryl.


The term “3- to 14-membered heterocyclic group” as used herein includes 3- to 8-membered monocyclic heterocyclic group and 6- to 14-membered fused heterocyclic group, wherein heteroatoms are nitrogen, oxygen, sulfur and the like. It includes the case that a carbon atom, a nitrogen atom or a sulfur atom is substituted by oxo.


Examples of 3- to 8-membered monocyclic heterocyclic group include, but are not limited to, aziridinyl, 2H-aziridinyl, diazirinyl, 3H-diazirinyl, azetidinyl, 1,4-dioxanyl, 1,3-dioxanyl, 1,3-dioxolanyl, 1,4-dioxinyl, tetrahydrofuryl, dihydropyrrolyl, pyrrolidinyl, imidazolidinyl, 4,5-dihydroimidazolyl, pyrazolidinyl, 4,5-dihydropyrazolyl, 2,5-dihydrothienyl, tetrahydrothienyl, 4,5-dihydrothiazolyl, piperidinyl, piperazinyl, morpholinyl, 4,5-dihydrooxazolyl, 4,5-dihydroisoxazolyl, 2,3-dihydroisoxazolyl, 2H-1,2-oxazinyl, 6H-1,3-ox azinyl, 4H-1,3-thiazinyl, 6H-1,3-thiazinyl, 2H-pyranyl, 2H-pyran-2-keto, 3,4-dihydro-2H-pyranyl and the like. It is preferably 5- to 6-membered monocyclic heterocyclic group.


Examples of 6- to 14-membered fused heterocyclic group include, but are not limited to, tetrahydroimidazo[4,5-c]pyridyl, 3,4-dihydroquinazolinyl, 1,2-dihydroquinoxalinyl, benzo[d][1,3]dioxolyl, 1,3-dihydroisobenzofuryl, 2H-chromenyl, 2H-chromene-2-keto, 4H-chromenyl, 4H-chromene-4-keto, chromanyl, 4H-1,3-benzoxazinyl, 4,6-dihydro-1H-furo[3,4-d]imidazolyl, 3a,4,6,6a-tetrahydro-1H-furo[3,4-d]imidazolyl, 4,6-dihydro-1H-thieno[3,4-d]imidazolyl, 4,6-dihydro-1H-pyrrolo[3,4-d]imidazolyl, 4,5,6,7-tetrahydro-1H-benzo[d]imidazolyl and the like. It is preferably 9- to 10-membered fused heterocyclic group.


The term “7- to 12-membered endocyclic group” as used herein refers to 7- to 12-membered endocyclic group in which any two rings share two non-adjacent atoms and all ring atoms are carbon atoms, or 7- to 12-membered endo-heterocyclic group containing at least one hetero atoms such as nitrogen, oxygen and sulfur and the like. The 7- to 12-membered endocyclic group and 7- to 12-membered endo-heterocyclic group may be saturated or partially saturated.


Saturated endocyclic group refers to an endocyclic group in which all rings are saturated, and preferably 7- to 8-membered saturated endocyclic group. Specific examples include, but are not limited to,




embedded image


and the like.


Partially saturated endocyclic group means that at least one ring in the endocyclic group is an unsaturated cyclic group, preferably a 7- to 8-membered partially saturated endocyclic group, specific examples include, but are not limited to,




embedded image


and the like.


The term “7- to 12-membered spirocyclic group” as used herein refers to 7- to 12-membered spirocyclic group wherein at least two rings share one atom and all ring atoms are carbon atoms, or 7- to 12-membered spiro-heterocyclic group containing at least one hetero atom such as nitrogen, oxygen and sulfur and the like. The 7- to 12-membered spirocyclic group and 7- to 12-membered spiro-heterocyclic group may be saturated or partially saturated.


Saturated spirocyclic group ring group means that all rings in the spirocyclic group are saturated cyclic groups. Examples include, but are not limited to,




embedded image


and the like.


Partially saturated spirocyclic group means that at least one ring in the spirocyclic group is an unsaturated cyclic group. Examples include, but are not limited to,




embedded image


and the like.


The compounds of the present invention may be prepared by the following method and/or other synthesis technologies known by a person skilled in the art, but the method is not limited to the following.




embedded image


In the reaction equation, R1, R2, R3, R4, R5, X, A and B are as defined above.


Reaction Steps:


(1) Preparation of Intermediate 1


The raw material 2 was added to a solution of the raw material 1 in alcohols (e.g. methanol, ethanol, isopropanol, tert-butanol, etc.) and heated under reflux. The end of the reaction was determined by thin layer chromatography, the solvent was rotary evaporated to dry, and the residue was subjected to column chromatography to give Intermediate 1.


(2) Preparation of Intermediate 2


Method 1: Intermediate 1 was dissolved in an organic solvent of alcohols (e.g. methanol, ethanol, isopropanol, tert-butanol, etc.) or dichloromethane, and added the raw material 3, then added a small amount of acetic acid to adjust the system to be weakly acidic. After a period of stirring, sodium triacetoxyborohydride was added in batches and stirring was continued. The reaction was quenched with water, and extracted with an organic solvent of halogenated hydrocarbon (such as chlorobenzene, dichlorobenzene, chloromethane, or dichloromethane). The organic phase was concentrated and subjected to column chromatography to give Intermediate 2.


Method 2: Intermediate 1 and the raw material 3 were dissolved in an organic solvent of alcohols (e.g. methanol, ethanol, isopropanol, tert-butanol, etc.), and after a period of stirring at room temperature, sodium borohydride was added in batches and stirring was continued. The reaction was quenched with water, rotary evaporated to remove most of the alcohol solvent, and extracted with an organic solvent of halogenated hydrocarbon (such as chlorobenzene, dichlorobenzene, chloromethane, dichloromethane and the like). The organic phase was concentrated and subjected to column chromatography to provide Intermediate 2.


(3) Preparation of Intermediate 3


In a dry reaction flask, triphosgene and triethylamine were dissolved in tetrahydrofuran or dichloromethane. Intermediate 2 was added under cooling in an ice bath and stirred. The reaction was quenched with saturated aqueous sodium carbonate solution, and extracted with an organic solvent of halogenated hydrocarbon (such as chlorobenzene, dichlorobenzene, chloromethane or dichloromethane and the like), washed with a saturated aqueous solution of sodium carbonate and saturated brine. The organic layer was dried with a drying agent (such as anhydrous calcium sulfate, anhydrous sodium sulfate or anhydrous magnesium sulfate and the like), and purified by column chromatography to give Intermediate Compound 3.


(4) Preparation of Compounds of General Formula I


Intermediate 3, the raw material 4, palladium catalysts (including but not limited to Pd(PPh3)4, Pd(dppf)2Cl2 and Pd(PPh3)2Cl2) and a base (including but not limited to CH3COOK, K3PO4 and K2CO3) were added sequentially to an organic solvent (including but not limited to dioxane, DMSO, DMF and toluene) and a small amount of water (v:v=2˜4:1). The reaction mixture was heated under nitrogen atmosphere. The reaction was quenched with water, and extracted with an organic solvent of halogenated hydrocarbon (such as chlorobenzene, dichlorobenzene, chloromethane or dichloromethane and the like). The organic layer was dried with a drying agent (such as anhydrous calcium sulfate, anhydrous sodium sulfate or anhydrous magnesium sulfate and the like) and subjected to column chromatography to give the compounds of general formula I.


In the reaction, the functional groups in the material compounds, which should not be involved in the reaction, may be protected in advance, and then all or part of the protecting groups can be removed according to conventional methods. For example, if there is a proton of amino acid, it can be protected by a conventional “amino protecting group”.


“A pharmaceutically acceptable salt” of the present compounds of general formula (I) refers to salts formed by the compounds of general formula (I) containing a basic nitrogen atom with an inorganic acid, organic acid or organic protonic acid, including hydrochloride and so on.


“An ester” of the present compounds of general formula (I) refers to those that can be hydrolyzed in the human body to become the parent compounds. It is apparent to those skilled in the art that an ester of the present compounds of general formula (I), which can be easily hydrolysable, may be formed at the free carboxyl or hydroxy group of the compounds, and it can be prepared by a conventional method.


“A solvate” of the present compounds of general formula (I) refers to their association with a solvent. The solvent may be an organic solvent (such as ethanol, methanol, propanol, acetonitrile, etc.) and water. For example, the present compounds can form ethanolate with ethanol, and hydrate with water.


When one or more asymmetric carbon atoms exist in the present compounds of general formula (I), there are enantiomers. When the compounds contain an alkenyl group or a cyclic structure, there are cis/trans isomers. When the compounds contain a ketone or an oxime, there are tautomers. All these enantiomers, diastereomers, racemic isomers, cis/trans isomers, tautomers, geometric isomers, epimerides and mixture thereof are included within the scope of the present invention.


The present compounds of general formula (I), a pharmaceutically acceptable salt, ester, solvate or stereoisomer thereof is useful for treating and/or preventing proliferative diseases including cancer and non-cancer diseases, wherein the cancer disease is selected from a brain tumor, lung cancer, non-small cell lung cancer, squamous cell carcinoma, bladder cancer, stomach cancer, ovarian cancer, peritoneal cancer, pancreatic cancer, breast cancer, head and neck cancer, cervical cancer, endometrial cancer, colorectal cancer, liver cancer, kidney cancer, esophageal adenocarcinoma, esophageal squamous cell carcinoma, solid tumors, non-Hodgkin's lymphoma, glioma, glioblastoma multiforme, gliosarcoma, prostate cancer, thyroid carcinoma, genital tract carcinoma, carcinoma in situ, lymphoma, histiocytic lymphoma, neurofibromatosis, bone cancer, skin cancer, brain cancer, colon cancer, testicular cancer, small cell lung cancer, gastrointestinal stromal tumor, prostate tumor, mast cell tumor, multiple myeloma, melanoma, glioma, glioblastoma, astrocytoma, neuroblastoma, sarcomas; and the non-cancer disease is selected from a skin disease or benign prostate hyperplasia.


The present compounds of general formula (I), a pharmaceutically acceptable salt, ester, solvate or stereoisomer thereof can be made into pharmaceutical preparations with one or more pharmaceutically acceptable carriers. Said pharmaceutical preparations refer to conventional preparations in the clinical use, and can be orally or parenterally applied to patients in need of such treatment. For oral administration, they can be made into conventional solid preparations such as tablets, capsulas, pills, granules, etc., as well as oral liquid preparations, such as oral solutions, oral suspensions, syrups, etc. For parenteral administration, they can be made into injections, including injection solution, a sterile powder for injection, concentrated solution for injection and suspension for injection. For rectal administration, they can be made into suppositories and the like. For transpulmonary administration, they can be made into inhalations or aerosols and the like. For topical or percutaneous administration, they can be made into ointments, pastes, creams, lotions, gels, powders, solutions or transdermal stickers and the like. These preparations can be prepared by a conventional method, adding pharmaceutically acceptable carriers such as excipients, binders, moistening agents, disintegrating agents, thickeners and the like.


The administration amount and frequency of the present compounds can be adjusted according to the judgment of the clinician or pharmacist, for example according to the patient's age, weight, the severity of the symptoms. Generally, the daily dose of the present compounds when administrated in a single dose or divided doses may be 20 mg˜500 mg, preferably 50˜300 mg.


The compounds of Formula (I), a pharmaceutically acceptable salt, ester, solvate, or stereoisomer thereof can be used in combination with one or more anti-cancer agents and immunosuppressants. The anti-tumor agents and immunosuppressants are selected from anti-metabolites, including but not limited to, capecitabine, gemcitabine and pemetrexed disodium; growth factor inhibitors, including but not limited to, pazopanib, imatinib, erlotinib, lapatinib, gefitinib and vandetanib; antibodies, including but not limited to Herceptin and Avastin; mitotic inhibitors, including but not limited to paclitaxel, vinorelbine, docetaxel and doxorubicin; anti-tumor hormones, including but not limited to letrozole, tamoxifen, fulvestrant, flutamide and triptorelin; alkylating agents, including but not limited to cyclophosphamide, nitrogen mustard, melphalan, chlorambucil and carmustine; platinum metals, including but not limited to carboplatin, cisplatin and oxaliplatin; topoisomerase inhibitors, including but not limited to camptothecin, topotecan and irinotecan; immunosuppression categories, including but not limited to everolimus, sirolimus and temsirolimus; purine analogues, including but not limited to, 6-mercaptopurine, 6-thioguanine and azathioprine; antibiotics, including but not limited to streptozotocin D, daunorubicin, doxorubicin, mitoxantrone, bleomycin and plicamycin; adrenal cortex inhibitor, including but not limited to aminoglutethimide.


The present invention also relates to a method for treating and/or preventing proliferative diseases, which comprises administering to a patient a therapeutically effective amount of the present compounds of general formula (I), a pharmaceutically acceptable salt, ester, solvate or stereoisomer thereof.







PREFERRED EMBODIMENTS

The beneficial effects of the present compounds will be further elaborated hereinafter. Other compounds of the present invention have the same beneficial effects with the compounds listed in the experiments, but it should not be understood that the present compounds only have the following beneficial effects.


Experimental Example 1
Enzymatic Inhibitory Activity of the Present Compounds In Vitro

Test Substances


Tested compounds: the present compounds, made by the applicant, see Preparative Examples for the chemical names and structures of each compound;


Control compounds: BGT-226 and BEZ-235 disclosed in WO2006122806 (published on Nov. 23, 2006), which were prepared according the methods disclosed in the application.




embedded image


Experimental Method 1 Activity Test


The abbreviations in the following experiments have the meanings as below:


HEPES: hydroxyethylpiperazine-ethane sulfonic acid;


EDTA: ethylenediaminetetraacetic acid;


CHAPS: 3-((3-Cholamidopropyl)dimethylammonium)-1-propanesulfonate


DTT: dithiothreitol;


PIP2: phosphatidylinositol-4,5-bisphosphate;


ATP: adenosine triphosphate (Sigma);


DMSO: dimethylsulfoxide;


Tween-20: Tween 20;


Determination of the inhibitory activity of PI3K


1. Preparation of test reagents


{circle around (1)} 1× kinase buffer (50 mM HEPES, pH 7.5, 3 mM MgCl2, 1 mM EGTA, 100 mM NaCl, 0.03% CHAPS, 2 mM DTT);


{circle around (2)} 4× kinase solution (added PI3K a kinase to 1× kinase buffer to provide 4× kinase solution with a final concentration of 1.65 nM);


{circle around (3)} 2× substrate solution (added the substrates of PIP2 and ATP into 1× kinase buffer to provide 2× substrate solution with PIP2 final concentration of 50 μM and ATP final concentration of 25 μM);


{circle around (4)} 4× test substance solutions (100× test substance solutions with different concentration gradients were prepared using 100% DMSO, and diluted 25-fold with 1× kinase buffer to give 4× test substance solutions with different concentration gradients);


{circle around (5)} Invitrogen's activity test kit with the test solution was kept stand to room temperature.


2. 2.5 μL of 4× test solution was added to a 384-well plate in parallel;


3. added 2.54 μL of 4× kinase solution and incubated for 10 min;


4. then added 5 μL of 2× solutions of PIP2 and ATP substrates and incubated for 1 h at room temperature;


5. finally, added 10 μL of test solution to quench the reaction, and read after 15 min;


6. IC50 value was obtained via curve fitting.





Inhibition rate (%)=(sample value−minimum)/(maximum−minimum)×100


Maximum refers to the readings of control wells without enzyme, minimum refers to the readings of DMSO control wells.


IC50 value was obtained via curve fitting using a soft of Graphpad 5.0.


Determination of the Inhibitory Activity of mTOR


1. Preparation of test reagents


{circle around (1)} 1× kinase buffer (50 mM HEPES, pH 7.5, 10 mM MgCl2, 1 mM EGTA, 3 mM MnCl, 0.01% Tween-20, 2 mM DTT);


{circle around (2)} 4× kinase solution (added mTOR kinase to 1× kinase buffer to provide 4× kinase solution with a final concentration of 2.5 nM);


{circle around (3)} 2× substrate solution (added the substrates of 4EBP1 and ATP into 1× kinase buffer to provide 2× substrate solution with 4EBP1 final concentration of 50 nM and ATP final concentration of 10.8 μM);


{circle around (4)} 4× test substance solutions (100× test substance solutions with different concentration gradients were prepared using 100% DMSO, and diluted 25-fold with 1× kinase buffer to give 4× test substance solutions with different concentration gradients)


{circle around (5)} preparation of test solution (test solution containing EDTA and 4EBP1 phosphorylated antibody was prepared using activity test kit with assay buffer, the final concentration of EDTA was 8 mM, and the final concentration of 4EBP1 phosphorylated antibody was 2 nM)


2. 2.5 μL of 4× test solution was added to a 384-well plate in parallel;


3. added 2.54 μL of 4× kinase solution and incubated for 10 min; 4. then added 5 μL of 2× solutions of 4EBP1 and ATP substrates and incubated for 1 h at room temperature;


5. finally, added 10 μL of test solution to quench the reaction, and read after 60 min;


6. IC50 value was obtained via curve fitting.





Inhibition rate (%)=(sample value−minimum)/(maximum−minimum)×100


Minimum refers to the readings of control wells without enzyme, maximum refers to the readings of DMSO control wells.


IC50 value was obtained via curve fitting using a soft of Graphpad 5.0.


Experimental Results:









TABLE 2







Enzymatic Inhibitory Activity of Some of the Present


Compounds in vitro (IC50)











Test Substances
PI3Kα (nM)
mTOR (nM)















BGT-226
41.91
1.99



Compound 7
29.3
38.7



Compound 14
3.3
8.5



Compound 22
10.2
17.5

















TABLE 3







Enzymatic Inhibitory Activity of Some of the Present


Compounds in vitro (IC50)











Test Substances
PI3Kα (nM)
mTOR (nM)















BEZ-235
32.5
3.23



Compound 2
88.4
54.3



Compound 3
10.43
8.86



Compound 6
33.8
24



Compound 8
260.3
76.5



Compound 9
872.6
43.7



Compound 11
495.6
31.2



Compound 13
21
30.3



Compound 52
154.3
66.5










Method 2 Binding Assays


Determination of the Inhibitory Activity of PI3K


1. The Final Concentration of the Agents and Preparation of Stock Solutions of the Test Substances


1.1 3×PI3Kα kinase/antibody solution, prepared using kinase buffer containing antibody, with a final concentration of 8.469 nM, 3× substrate 1710 (kinase tracer 1710), with a final concentration of 57.29 nM;


1.2 3×mTOR kinase/antibody solution, prepared using kinase buffer containing antibody, with a final concentration of 24.72 nM, 3× substrate 314 (kinase tracer 314), with a final concentration of 32.50 nM;


1.3 A stock solution of the test substances was prepared with DMSO, the concentration thereof was 10 mM.


2. Experimental Steps


2.1 The stock solution of the test substances was diluted in 3 times of the highest final concentration, and diluted in 4 times with 10 concentration gradients using kinase buffer;


2.2 The gradiently diluted test substance was added to a 384-well plate in an amount of 5 μL well;


2.3 3× the substrate (kinase tracer) solution was added in an amount of 5 μL/well;


2.4 5× kinase/antibody solution was added in an amount of 5 μL well;


2.5 3×PI3Kα kinase solution was incubated for 50 min at room temperature, 3×mTOR kinase solution was incubated for 40 min;


2.6 Envision reading (excitation wavelength: 340 nM, emission wavelength: 615 nM and 665 nM).


3. Data processing





Inhibition rate (%)=(maximum−sample value)/(maximum−minimum)×100


The data was entered into GraphPad Prism 5.0 and plotted to obtain the curve and IC50.


Experimental Results









TABLE 4







Enzymatic Inhibitory Activity of Some of the Present


Compounds in vitro (IC50)











Test Substances
PI3Kα (nM)
mTOR (nM)















Hydrochloride of
8.6
91.94



Compound 58





Compound 86
9.7
257.2



Compound 114
115.0
732.3










In the present invention, when IC50 is 0˜300 nM, the test substances are regarded as having a very good kinase inhibitory activity, when IC50 is 0.3-3 μM, the test substances are regarded as having a good kinase inhibitory activity, when IC50 is higher than 3 μM, the test substances are regarded as not having kinase inhibitory activity.


Experimental Conclusions

It can be seen from Tables 2 to 4 that the present compounds have good enzymatic activity in vitro.


Experimental Example 2
The Cytological Activity Measurement Experiment of the Present Compounds

Tested compounds: the present compounds, made by the applicant, see Preparative Examples for the chemical names and structures of each compound;


Control: paclitaxel, purchased from Sigma Reagent Company, 98% purity.


Experimental Methods


1. Preparation of Reagents and Compounds


1) Preparation of phosphate buffer (PBS):


8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4 and 0.24 g KH2PO4 were weighed, added 800 mL of ultrapure water, adjusted to pH=7.4, further added ultrapure water volume to 1 L, sterilized at high pressure for 20 min.


2) Preparation of XTT test working solution:


100 mg of tetrazolium salt (XTT) powder was weighed and dissolved in dark in 300 mL of phenol red-free serum-free RPMI1640 medium heated to 50° C., filtered and subpackaged, used immediately or within a week. All process should be conducted in dark.


3) Preparation of test compounds


preparation of stock solutions of the test substances: the powder of test substances was dissolved in DMSO in a concentration of 10 mM.


preparation of gradiently diluted stock solutions of test substances: First, 10 mM of the stock solutions of test substances were diluted serially in 4-fold with DMSO to 10 concentrations. 2 μL of DMSO-diluted solution for each compound was taken and added to 998 μL of culture medium containing 10% fetal bovine serum (FBS). The highest concentration of the test substances was 20 μM, the concentration of DMSO was 0.2%, and there were 10 concentration gradients in total.


4) Preparation of positive compound


preparation of stock solution of paclitaxel: the powder of paclitaxel was dissolved in DMSO in a concentration of 10 mM.


preparation of gradiently diluted stock solutions of paclitaxel: First, 10 mM of paclitaxel was diluted serially to 10 concentrations. 2 μL of DMSO-diluted solution of paclitaxel was taken and added to 998 μL of culture medium containing 10% fetal bovine serum. The concentration of DMSO was 0.2%, and there were 10 concentration gradients in total.


2. Cell Culture


1) Cell Recovery:


Freezing tube was taken out from liquid nitrogen and placed in a water bath at 37° C.˜39° C., rapidly melted.


The frozen solution was transferred to a 15 mL sterile centrifuge tube, added 10 times the volume of culture medium and centrifuged at 1000 rpm, 4° C. for 5 min. Discarded the culture medium from the centrifuge tube, and added culture medium containing 10% FBS to resuspend the cells, and then transferred to a culture flask. The medium was changed on the next day.


2) Cell Passage


Cells in logarithmic growth phase were taken, and the culture medium was discarded, added an appropriate volume of PBS to wash once, then add an appropriate volume of digestive fluid containing 0.25% trypsinase and 0.02% EDTA, placed at 37° C. for 2˜5 min, discarded the digestive fluid, washed once with PBS. Added an appropriate volume of culture medium containing 10% FBS to terminate digestion, gently pipetted the cells with a pipette, digested the cells to provide a cell suspension for passage and experiment.


3) Cell Cryopreserving


Cells in logarithmic growth phase were taken and digested with a digestive fluid containing 0.25% trypsinase and 0.02% EDTA to provide a cell suspension, centrifuged at 1000 rpm, 4° C. for 5 min. Discarded the culture medium, added frozen medium containing 10% DMSO and 90% FBS to resuspend the cells, subpackaged into freezing tubes in an amount of 2×106 cells per tube. The freezing tubes were put in a process cooling box and kept at −80° C. for 24 h, then transferred to liquid nitrogen for cryopreservation.


3. Cell Plating


1) Preparation of cell suspension


The culture medium was discarded from the flask, and the cells were washed twice with PBS. Added trypsinase to digest the cell and then collected by centrifugation, resuspended with a medium containing 10% FBS, counted and adjusted to an appropriate concentration (cell viability must be greater than 90%). The cell concentration was 5×104 mL.


2) The cell suspension was added to 96-well plates, each well 100 μL.


3) Incubated in a cell incubator overnight at 37° C., 5% CO2.


4. Drug Treatment


Added compound diluted 2-fold to the cell culture plates, three repeated in total, 100 μL well. The final volume was 200 μL, and the initial concentration was 10 μM, 4-fold dilution, 10 concentration gradients in total, wherein the concentration of BEZ-235 started from 1 μM, 3-fold dilution, 10 concentration gradients in total;


5. Cell Viability Assay by XTT Method


Discarded the culture medium, added XTT test working liquid in an amount of 150 μL well, and kept in a cell incubator at 37° C., 5% CO2 for 2 h, then placed into a microplate reader and read the absorbance at 450 nm.


6. Data Processing


1) The percentage of inhibition was calculated using the following formula:





Inhibition rate (%)=(reading(solvent)−reading(compound))/reading(solvent)−reading(positive control))×100


2) The data was entered into GraphPad Prism 5.0 and plotted to obtain the curve and IC50.


Experimental Results









TABLE 5







Cytologic Activity of Some of the Present Compounds


in vitro (IC50)











Test substances
U87MG (nM)
SKOV-3 (nM)















Compound 3
84.7
155



Compound 7
900.9
895



Compound 14
436.7
890.3



Compound 22
275.1
551.2



Compound 30
499.2
348.9



Compound 52
436.2
466.7



Compound 80
278.9
542.4










In the present invention, when IC50 is 0˜300 nM, the test substances are regarded as having a very good inhibitory activity to cell, when IC50 is 0.3-3 μM, the test substances are regarded as having a good inhibitory activity to cell, and when IC50 is higher than 3 μM, the test substances are regarded as not having inhibitory activity to cell.


Experimental Conclusions

It can be seen from Table 5 that the present compounds have good cytological activity in vitro, and can inhibit the proliferation of tumor cells.


Experimental Example 3
In Vivo Pharmacokinetics Experiment of the Present Compounds in Rats

Test animals: male SD rats, weighing 230-250 g. Three rats were tested for each administration route of each compound.


Test Substances:

    • Control substances: BGT-226 and BEZ-235, dissolved in sterile water for injection;


The present compounds, prepared by the applicant, dissolved in sterile water for injection.


Experimental Methods:


Administration


The test substances were administrated via intravenous bolus injection (IV), dose: 2 mg/kg, administrating volume: 2 mL/kg;


The test substances were administrated via gavage (PO), dose: 4 mg/kg, administrating volume: 4 mL/kg.


Blood Sampling Time Point:


0 h before administration, 0.083 h, 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, and 24 h after administration. About 100 μl of whole blood was taken at each time point, centrifugated at 8,000 rpm in a high-speed centrifuge for 6 minutes to separate plasma, and the resulting plasma was frozen at −80° C. in a refrigerator.


Plasma Samples Analysis:


The samples of BGT-226, hydrochloride of Compound 58 and Compound 86 were treated as follows: 20 μl of plasma was taken and added 80 μl of internal standard solution (KBP-2906), subjected to vortex at 1,500 rev/min for 3 minutes, then to centrifugation at 12,000 rpm/min for 5 minutes. 60 μl of supernatant was taken and added with 1004 of water, then homogenized by vortex and analyzed using LC-MS/MS.


The samples of other compounds were treated as follows: 20 μl of plasma was taken and added 800 μl of internal standard solution (KBP-3957), subjected to vortex at 1,500 rev/min for 10 minutes, then to centrifugation at 12,000 rpm/min for 5 minutes. 400 μl of supernatant was taken and blown to dry under nitrogen, re-dissolved in 200 μL of a mixture of methanol:water (1:1, V/V) and analyzed using LC-MS/MS.









TABLE 6







The evaluation results of pharmacokinetics of the present


compounds in rats (IV)












T ½
AUC
Cl_obs
Vss_obs


Test Substances
(h)
(h*ng/ml)
(L/h/kg)
(L/kg)














BGT-226
4.8
1520
1.3
8.72


BEZ-235
3.19
1439
1.38
5.93


Compound 3
6.48
1702.69
1.14
5.6


Compound 7
0.47
274.63
7.42
4.45


Compound 14
2.08
735.30
2.84
2.97


Compound 22
0.72
356.89
5.57
5.28


Hydrochloride of
3.47
217.7
7.39
36.26


Compound 58






Compound 86
0.2
153.9
13.6
3.58
















TABLE 7







The evaluation results of pharmacokinetics of the present


compounds (PO) in rats












Test
T ½
AUC
Cmax




Substances
(h)
(h*ng/ml)
(ng/ml)
Tmax (h)
F %















BGT-226

852
61.1
6
28


BEZ-235
4.08
2175.69
229.33
2.83
77.44


Compound 3
5.33
1376.23
197.63
0.5
40.4


Compound 7
1.7
63.44
34.94
0.5
11.55


Compound 14
1.39
402.00
147.30
0.5
26.26


Hydrochloride of

258
46.8
4.67
59


Compound 58







Compound 86
1.04
177
250.67
0.33
58









Experimental Conclusions

It can be seen from Tables 6 and 7 that the present compounds have good pharmacokinetic properties and good bioavailability.


Experimental Example 4
The Repeated-Dose Toxicity Test of the Present Compounds in Rats

Tested Substances:

    • the present compound of No. 3, made by the applicant, see Preparative Example for the chemical name and structure thereof;
    • control drug: BEZ-235, prepared according to the method described in WO2006122806.


The test substances were suspended in 0.5% methylcellulose solution to prepare suspensions with a concentration of 0.5 mg/mL, 1.5 mg/mL and 3 mg/mL.


Animals: SD rats, 6-8 weeks old, SPF grade, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., licensed cell No: SCXK (Beijing) 2006-0009.


Experimental Methods

Selected 140 SD rats which meet the test requirements, half male and half female, 20 rats/group, divided into seven dose groups including Compound 3 low, medium and high dose groups, BEZ-235 low, medium and high dose groups and solvent control group. The test substances were administrated as a suspension via gavage, and administrating volume was 10 mL/kg. 0.5% methyl cellulose solution was used in the solvent group, and the administrating volume was 10 mL/kg, administered once daily for consecutive 14 days. Animals were observed daily appearance and general behavior changes.


Experimental Results









TABLE 8







The observation results of vital signs in the repeated-dose toxicity test of


the present compounds in rats













dose
animal
loose

hair


Test Substances
(mg/kg/day)
numbers
stools
lassitude
loss















Compound 3 low-dose
5
20
 0/20
0/20
0/20


group







Compound 3
15
20
 1/20
0/20
0/20


middle-dose group







Compound 3 high-dose
30
20
 1/20
4/20
1/20


group







BEZ-235 low-dose
5
20
 6/20
0/20
0/20


group







BEZ-235 middle-dose
15
20
 7/20
0/20
0/20


group







BEZ-235 high-dose
30
20
15/20
0/20
3/20


group







Solvent control group

20
 0/20
0/20
0/20









It can be seen from Table 8 that, at the low doses, there was no animals with abnormal vital signs in the group of Compound 3, whereas there were six rats with loose stools in BEZ-235 group; at the middle doses, there was one rat with loose stools in the group of Compound 3, whereas there were seven rats with loose stools in BEZ-235 group; at high doses, there were one rat with loose stools, four rats with lassitude and one rat with hair loss in the group of Compound 3, whereas there were fifteen rats with loose stools and three rats with hair loss in BEZ-235 group. Therefore, the present compounds have lower toxicity and are safer than BEZ-235.


Preparative Examples

The above contents of the present invention will be described in further detail by the following Examples, but this should not be construed that the invention is limited to the following Examples.


The abbreviations in the following Examples are defined as follows:


DCM: dichloromethane


Pd(PPh3)4: tetrakis (triphenylphosphine) palladium


DMAP: 4-dimethylaminopyridine


C3Cl6O3: triphosgene


Ac2O: acetic anhydride


TEA: triethylamine


Preparation of ethyl 4,6-dichloro-1,5-naphthyridine-3-carboxylate (material)

The above compound was prepared according to the method disclosed in WO2010038165 A1, and the preparation procedures are as follows:


1. Preparation of 6-bromopyridin-3-amine



embedded image


Iron powder (88 g, 1.571 mmol), concentrated hydrochloric acid (61 mL) and water (287 mL) were sequentially added to a solution of 2-bromo-5-nitropyridine (64 g, 0.317 mol) in ethanol (1 L). The mixture was reacted under reflux for 5 h. The reaction mixture was cooled, filtered and the filtrate was concentrated and then adjusted to approximately pH 7-8 with a saturated aqueous solution of sodium bicarbonate, filtered again, and the filtrate was extracted with 200 mL of dichloromethane. The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure to give 40.5 g of a pale yellow solid.


2. Preparation of diethyl 2-(((6-bromopyridin-3-yl)amino)methylene)malonate



embedded image


6-bromopyridin-3-amine (74 g, 0.43 mol) and diethyl ethoxymethylenemalonate (100 mL) were added to 680 mL of ethanol and heated under reflux for 5 h. The reaction mixture was cooled to precipitate a solid, suction filtrated, and the resulting solid was washed with petroleum ether to give 125.4 g of a pale yellow solid.


3. Preparation of ethyl 6-bromo-4-hydroxy-1,5-naphthyridine-3-carboxylate



embedded image


Diethyl(2-(((6-bromopyridin-3-yl)amino)methylene)malonate (40 g, 0.117 mol) was added to boiling diphenyl ether (214 mL) in batches over 5 min, heated under reflux for 45 min. After confirming a disappearance of the raw materials by a thin layer chromatography plate (ethyl acetate:petroleum ether=1:3), the reaction mixture was cooled and poured into petroleum ether to precipitate a solid, suction filtered to give 24.6 g of a khaki solid.


4. Preparation of ethyl 4,6-dichloro-1,5-naphthyridine-3-carboxylate



embedded image


Ethyl 6-bromo-4-hydroxy-1,5-naphthyridine-3-carboxylate (49.8 g, 0.168 mmol) and N,N-dimethylaniline (8 mL) were added to phosphorus oxychloride (400 mL), and heated under reflux for 3 h. The reaction mixture was cooled, phosphorus oxychloride was distilled off under reduced pressure, and the resulting residue was poured into ice water, and adjusted approximately to pH 8 with a saturated aqueous solution of sodium bicarbonate, then extracted with 200 mL of dichloromethane. The organic phase was dried over anhydrous sodium sulfate, concentrated and subjected to silica gel column chromatography (petroleum ether:ethyl acetate=3:1) to give 20.2 g of a pale yellow solid.


Example 1
Preparation of 9-(6-aminopyridin-3-yl)-3-methyl-1-(3-(trifluoromethyl)phenyl)-3,4-dihydropyrimido[5,4-c][1,5]naphthyridin-1(2H)-one



embedded image


(1) Preparation of ethyl 6-chloro-4-((3-(trifluoromethyl)phenyl)amino-1,5-naphthyridine-3-carboxylate



embedded image


Ethyl 4,6-dichloro-1,5-naphthyridine-3-carboxylate (2.70 g, 10.0 mmol) and 3-(trifluoromethyl)aniline (1.77 g, 11.0 mmol) were dissolved in tert-butanol (50 mL), and to the system added potassium carbonate (4.15 g, 30.0 mmol). The reaction mixture was stirred under reflux for 3 hours. After confirming a disappearance of the raw materials by a thin layer chromatography plate (ethyl acetate:petroleum ether=1:3), the reaction mixture was suction filtered, the solid was washed with 30 mL of dichloromethane, and the resulting wash solution and the filtrate were combined, concentrated under reduced pressure, and the resulting residue was recrystallized from diethyl ether to give 3.7 g of a pale yellow solid.


(2) Preparation of 6-chloro-4-((3-(trifluoromethyl)phenyl)amino)-1,5-naphthyridine-3-carbaldehyde



embedded image


Ethyl 6-chloro-4-((3-trifluoromethyl)phenyl)amino)-1,5-naphthyridine-3-carboxylate (2.5 g, 6.3 mmol) was added to ethanol (80 mL), and to the system added sodium borohydride (0.95 g, 25.1 mmol) in batches, stirred at room temperature for 24 h, ethanol was removed under reduced pressure, added 20 mL of water, and extracted with 150 mL of dichloromethane. The organic layer was dried over anhydrous sodium sulfate, concentrated under reduced pressure. The resulting 1.3 g of the crude product was dissolved in dichloromethane (20 mL), to the solution added manganese dioxide (9.6 g, 110.5 mmol) in batches. The reaction mixture was stirred at room temperature for 8 h. Filtered and the filter cake was washed with 50 mL of dichloromethane, and the resulting wash solution and the filtrate were combined and concentrated. The resulting solid was subjected to silica gel column chromatography (ethyl acetate:petroleum ether=2:1) to give 1.1 g of the product.


(3) Preparation of 6-chloro-3-((methylamino)methyl)-N-(3-(trifluoromethyl)phenyl)-1,5-naphthyridin-4-amine



embedded image


6-chloro-4-((3-(trifluoromethyl)phenyl)amino)-1,5-naphthyridine-3-carbaldehyde (0.75 g, 2.13 mmol) was dissolved in ethanol (20 mL), added 1.2 mL of a solution of methylamine in ethanol (concentration 27%), stirred at room temperature for 4 h, and then added sodium borohydride (0.82 g, 21.7 mmol) to the system, continued stirring at room temperature for 18 h. The reaction was quenched with a saturated aqueous solution of sodium carbonate, concentrated under reduced pressure to remove ethanol, extracted with 80 mL of dichloromethane, the organic layer was dried over anhydrous sodium sulfate, concentrated under reduced pressure to obtain 0.77 g of the titled compound.


(4) Preparation of 9-chloro-3-methyl-1-(3-(trifluoromethyl)phenyl)-3,4-dihydropyrimido[5,4-c][1,5]naphthyridin-1(2H)-one



embedded image


6-chloro-3-((methylamino)methyl)-N-(3-(trifluoromethyl)phenyl)-1,5-naphthyridin-4-amine (0.77 g, 2.10 mmol) was dissolved in dichloromethane (20 mL), to the reaction flask in an ice bath added triphosgene (0.68 g, 2.29 mmol) and triethylamine (0.8 mL), then stirred at room temperature for 2 h. The reaction was quenched with a saturated aqueous solution of sodium carbonate, extracted with 80 mL of dichloromethane, the organic layer was dried over anhydrous sodium sulfate, concentrated under reduced pressure to remove the solvent to give 0.82 g of the titled compound.


(5) Preparation of 9-(6-aminopyridin-3-yl)-3-methyl-1-(3-(trifluoromethyl)phenyl)-3,4-dihydropyrimido[5,4-c][1,5]naphthyridin-1(2H)-one



embedded image


9-chloro-3-methyl-1-(3-(trifluoromethyl)phenyl)-3,4-dihydropyrimido[5,4-c][1,5]naphthyridin-1(2H)-one (0.82 g, 2.09 mmol) obtained in the above step and 6-amino-3-pyridine boronic acid (462 mg, 2.10 mmol) were dissolved in toluene (15 mL) and ethanol (5 mL), to the system added tetrakis(triphenylphosphine) palladium (12 mg) and 2N aqueous sodium carbonate solution (3.2 mL). Refluxed and reacted under nitrogen for 6 h, cooled to room temperature, filtered and the filtrate was concentrated under reduced pressure and dissolved in 30 mL of dichloromethane, washed sequentially with water and saturated brine, dried over anhydrous sodium sulfate, concentrated and subjected to silica gel column chromatography (ethyl acetate:petroleum ether=1:1) to give 373 mg of the product.


Molecular formula: C23H17F3N6O; Molecular weight: 450.14; Mass spectrum (M+H): 450.9



1H-NMR (d6-DMSO, 400 MHz): δ 8.71 (1H, s), 8.30-8.16 (2H, m), 8.10 (1H, t), 7.86 (1H, s), 7.62 (1H, d), 7.55-7.42 (2H, m), 6.92 (1H, dd), 6.45 (2H, s), 6.26 (1H, d), 4.75 (2H, s), 3.00 (3H, s)


Example 2
Preparation of N-(5-(3-methyl-2-oxo-1-(3-(trifluoromethyl)phenyl)-1,2,3,4-tetrahydropyrimido[5,4-c][1,5]naphthyridin-9-yl)pyridin-2-yl)acetamide (Compound 7)



embedded image


(1) Preparation of N-(5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-yl)acetamide



embedded image


4-dimethylaminopyridine (17 mg, 0.139 mmol), triethylamine (0.19 mL, 1.36 mmol) and acetic anhydride (153 mg, 1.50 mmol) were added sequentially to a solution of 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-amine (300 mg, 1.36 mmol) in dichloromethane (15 mL). The reaction mixture was stirred at room temperature for 0.5 h, then diluted with 30 mL of dichloromethane, washed with 50 mL of a saturated aqueous solution of ammonium chloride. The organic phase was dried over anhydrous magnesium sulfate, filtered and concentrated to give 225 mg of the product.


(2) Preparation of N-(5-(3-methyl-2-oxo-1-(3-(trifluoromethyl)phenyl)-1,2,3,4-tetrahydropyrimido[5,4-c][1,5]naphthyridin-9-yl)pyridin-2-yl)acetamide



embedded image


9-chloro-3-methyl-1-(3-(trifluoromethyl)phenyl)-3,4-dihydropyrimido[5,4-c][1,5]naphthyridin-2(1H)-one (314 mg, 0.80 mmol), tetrakis(triphenylphosphine) palladium (15 mg, 0.013 mmol) and 2N aqueous sodium carbonate solution (1.3 mL) were added sequentially to a solution of N-(5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-yl)acetamide (225 mg, 0.858 mmol) in 1,4-dioxane (15 mL), and reacted under nitrogen at 90° C. for 16 h, cooled to room temperature, filtered, the organic layer was concentrated under reduced pressure, dissolved in 50 mL of dichloromethane, washed sequentially with water and saturated brine, dried over anhydrous sodium sulfate, concentrated and subjected to silica gel column chromatography (ethyl acetate) to give 267 mg of the product.


Molecular formula: C25H19F3N6O2; Molecular weight: 492.1; Mass spectrum (M+H): 493.2



1H-NMR (d6-DMSO, 400 MHz): δ 10.64 (1H, s), 8.81 (1H, s), 8.41 (1H, d), 8.37 (1H, d), 8.25 (1H, d), 7.98 (1H, d), 7.82 (1H, s), 7.67-7.62 (1H, m), 7.61-7.52 (2H, m), 7.37 (1H, dd), 4.79 (2H, s), 3.01 (3H, s), 2.10 (3H, s)


Example 3
Preparation of 9-(2-aminopyrimidin-5-yl)-3-methyl-1-(3-(trifluoromethyl)phenyl)-3,4-dihydropyrimido[5,4-c][1,5]naphthyridin-1(2H)-one (Compound 14)



embedded image


9-chloro-3-methyl-1-(3-(trifluoromethyl)phenyl)-3,4-dihydropyrimido[5,4-c][1,5]naphthyridin-1(2H)-one (315 mg, 0.802 mmol) and 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrimidin-2-amine (195 mg, 0.882 mmol) were dissolved in 1,5-dioxane (15 mL), to the system added tetrakis(triphenylphosphine) palladium (12 mg) and 2N aqueous sodium carbonate solution (1.4 mL). Refluxed and reacted under nitrogen for 16 h, cooled to room temperature, filtered, and the organic layer was concentrated under reduced pressure, dissolved in 50 mL of dichloromethane, washed sequentially with water and saturated brine, dried over anhydrous sodium sulfate, concentrated and subjected to silica gel column chromatography (ethyl acetate:petroleum ether=1:1) to give 185 mg of the product.


Molecular formula: C22H16F3N7O; Molecular weight: 451.1; Mass spectrum (M+H): 452.2



1H-NMR (d6-DMSO, 400 MHz): δ 8.74 (1H, s), 8.28 (1H, d), 8.12-8.07 (3H, m), 7.70 (1H, s), 7.67-7.54 (3H, m), 7.06 (2H, s), 4.76 (2H, s), 3.00 (3H, s)


Example 4
3-methyl-9-(1H-pyrrolo[2,3-b]pyridin-5-yl)-1-(3-(trifluoromethyl)phenyl)-3,4-dihydropyrimido[5,4-c][1,5]naphthyridin-1(2H)-one (Compound 22)



embedded image


9-chloro-3-methyl-1-(3-(trifluoromethyl)phenyl)-3,4-dihydropyrimido[5,4-c][1,5]naphthyridin-1(2H)-one (326 mg, 0.83 mmol) and 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrolo[2,3-b]pyridine (244 mg, 1.0 mmol) were dissolved in 1,4-dioxane (12 mL), to the system added tetrakis(triphenylphosphine) palladium (12 mg, 0.0104 mmol) and 2N aqueous sodium carbonate solution (0.6 mL). Refluxed and reacted under nitrogen for 16 h, cooled to room temperature, filtered, and the organic layer was concentrated under reduced pressure, dissolved in 50 mL of dichloromethane, washed sequentially with water and saturated brine, and the organic layer was dried over anhydrous sodium sulfate, concentrated and subjected to silica column chromatography (ethyl acetate) to give 243 mg of the product.


Molecular formula: C25H17F3N6O; Molecular weight: 474.14; Mass spectrum (M+H): 475.2



1H-NMR (d6-DMSO, 400 MHz): δ 11.78 (1H, s), 8.77 (1H, s), 8.34 (1H, d), 8.30 (1H, d), 8.27 (1H, d), 7.83 (1H, s), 7.75-7.69 (1H, m), 7.63-7.58 (3H, m), 7.50 (1H, t), 6.46 (1H, dd), 4.78 (2H, s), 3.01 (3H, s)


Example 5
Preparation of 2-(4-(9-(6-methoxypyridin-3-yl)-3-methyl-2-oxo-3,4-dihydropyrimido[5,4-c][1,5]naphthyridin-1(2H)-yl)phenyl)-2-methylpropionitrile (Compound 30)
(1) Preparation of ethyl 6-chloro-4-((4-(2-cyanopropan-2-yl)phenyl)amino)-1,5-naphthyridine-3-carboxylate



embedded image


Ethyl 4,6-dichloro-1,5-naphthyridine-3-carboxylate (6.4 g, 23.6 mmol) and 2-(4-aminophenyl)-2-methylpropionitrile (5.6 g, 35 mmol) were dissolved in tert-butanol (200 mL), to the system added potassium carbonate (16.4 g, 119 mmol), and the reaction mixture was stirred under reflux for 2 h. After confirming a disappearance of the raw materials by a thin layer chromatography plate (ethyl acetate:petroleum ether=1:3), the reaction mixture was suction filtered, the filtrate was concentrated under reduced pressure, and the resulting residue was washed with diethyl ether to give 8.1 g of a pale yellow solid.


(2) Preparation of 2-(4-((6-chloro-3-(hydroxymethyl)-1,5-naphthyridin-4-yl)amino)phenyl)-2-methylpropionitrile



embedded image


Ethyl 6-chloro-4-((4-(2-cyanopropan-2-yl)phenyl)amino)-1,5-naphthyridine-3-carboxylate (8.1 g, 20.5 mmol) was added to a mixed solvent of ethanol (120 mL) and dichloromethane (12 mL), to the system added sodium borohydride (5.4 g, 143 mmol) in batches, stirred at room temperature for 24 h, ethanol was removed under reduced pressure, adjusted pH of the solution to be neutral with 1 N dilute hydrochloric acid and extracted with 200 mL of ethyl acetate. The organic layer was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the resulting 9.0 g of a crude solid was used directly in the next step.


(3) Preparation of 2-(4-((6-chloro-3-formyl-1,5-naphthyridin-4-yl)amino)phenyl)-2-methylpropionitrile



embedded image


9.0 g of the crude product of 2-(4-((6-chloro-3-(hydroxymethyl)-1,5-naphthyridin-4-yl)amino)phenyl)-2-methylpropionitrile obtained in the above step was dissolved in dichloromethane (250 mL), to the solution added manganese dioxide (67 g, 0.77 mol) in batches, and the reaction mixture was stirred at room temperature for 30 h. Filtered, and the filter cake was washed with 50 mL of dichloromethane, The resulting wash solution and the filtrate were combined and concentrated under reduced pressure to give a solid. The resulting solid was subjected to silica gel column chromatography (ethyl acetate:petroleum ether=2:1) to give 2.5 g of the product.


(4) Preparation of 2-(4-((6-chloro-3-((methylamino)methyl)-1,5-naphthyridin-4-yl)amino)phenyl)-2-methylpropionitrile



embedded image


2-(4-((6-chloro-3-formyl-1,5-naphthyridin-4-yl)amino)phenyl)-2-methylpropionitrile (2.5 g, 7.1 mmol) was dissolved in ethanol (100 mL), added 3 mL of a solution of methylamine in ethanol (concentration 27%), stirred at room temperature for 10 h. Then, to the system added sodium borohydride (1.5 g, 3.97 mol), continued stirring at room temperature for 18 h, and the reaction was quenched with a saturated aqueous solution of sodium carbonate. Concentrated under reduced pressure to remove ethanol, extracted with 150 mL of dichloromethane, the organic layer was dried over anhydrous sodium sulfate, concentrated under reduced pressure to give 2.2 g of a solid.


(5) Preparation of 2-(4-(9-chloro-3-methyl-2-oxo-3,4-dihydropyrimido[5,4-c][1,5]naphthyridin-1(2H)-yl)phenyl)-2-methylpropionitrile



embedded image


2-(4-((6-chloro-3-((methylamino)methyl)-1,5-naphthyridin-4-yl)amino)phenyl)-2-methylpropionitrile (2.0 g, 5.5 mmol) was dissolved in dichloromethane (25 mL), to the reaction flask in an ice bath added triphosgene (1.8 g, 6.1 mmol) and triethylamine (0.7 mL). Then, the mixture was stirred at room temperature for 2 h. The reaction was quenched with a saturated aqueous solution of sodium carbonate, extracted with 100 mL of dichloromethane, the organic layer was dried over anhydrous sodium sulfate, the solvent was removed under reduced pressure to give 1.6 g of a pale yellow solid.


(6) Preparation of 2-(4-(9-(6-methoxypyridin-3-yl)-3-methyl-2-oxo-3,4-dihydropyrimido[5,4-c][1,5]naphthyridin-1(2H)-yl)phenyl)-2-methylpropionitrile



embedded image


2-(4-(9-chloro-3-methyl-2-oxo-3,4-dihydropyrimido[5,4-c][1,5]naphthyridin-1(2H)-yl)phenyl)-2-methylpropionitrile (700 mg, 1.79 mmol) and 6-methoxy-3-pyridine boronic acid (328 mg, 2.15 mmol) were dissolved in toluene (30 mL) and ethanol (10 mL), to the system added tetrakis(triphenylphosphine) palladium (40 mg) and 2N aqueous sodium carbonate solution (2.7 mL). Refluxed and reacted under nitrogen for 3 h, cooled to room temperature, filtered, the organic layer was concentrated under reduced pressure, dissolved in 50 mL of dichloromethane, washed sequentially with water and saturated brine, and the organic layer was dried over anhydrous sodium sulfate, and concentrated. The resulting solid was subjected to silica gel column chromatography (ethyl acetate:petroleum ether=1:1) to give 120 mg of the product.


Molecular formula: C27H24N6O2; Molecular weight: 464.20; MS (M+H): 465.0



1H-NMR (d6-DMSO, 400 MHz): δ 8.75 (1H, s), 8.32 (1H, d), 8.14 (1H, d), 8.00 (1H, d), 7.68 (1H, dd), 7.53-7.46 (2H, m), 7.41-7.35 (2H, m), 6.75 (1H, d), 4.74 (2H, s), 3.87 (3H, s), 2.99 (3H, s), 1.68 (6H, s)


Example 6
Preparation of 2-methyl-2-(4-(3-methyl-2-oxo-9-(quinolin-3-yl)-3,4-dihydropyrimido[5,4-c]1,5]naphthyridin-1(2H)-yl)phenyl)propionitrile (Compound 52)



embedded image


2-(4-(9-chloro-3-methyl-2-oxo-3,4-dihydropyrimido[5,4-c][1,5]naphthyridin-1(2H)-yl)phenyl)-2-methylpropionitrile (700 mg, 1.79 mmol) and quinolin-3-yl boronic acid (372 mg, 2.15 mmol) was dissolved in toluene (30 mL) and ethanol (10 mL), to the system added tetrakis(triphenylphosphine) palladium (70 mg) and 2N aqueous sodium carbonate solution (2.7 mL). Refluxed and reacted under nitrogen for 3 h, cooled to room temperature, filtered, the organic layer was concentrated under reduced pressure, dissolved in 50 mL of dichloromethane, washed sequentially with water and saturated brine, the organic layer was dried over anhydrous sodium sulfate, concentrated and subjected to silica column chromatography (ethyl acetate:petroleum ether=1:1) to give 140 mg of the product.


Molecular formula: C30H24N6O; Molecular weight: 484.20; Mass spectrum (M+H): 485.0



1H-NMR (d6-DMSO, 400 MHz): δ 8.82 (1H, s), 8.69 (1H, d), 8.52 (1H, d), 8.47-8.37 (2H, m), 8.03 (1H, d), 7.98 (1H, d), 7.79 (1H, t), 7.64 (1H, t), 7.53-7.42 (4H, m), 4.78 (2H, s), 3.02 (3H, s), 1.54 (6H, s)


Example 7
Preparation of 9-(6-methoxypyridin-3-yl)-3-methyl-1-(4-(piperazin-1-yl)-3-(trifluoromethyl)phenyl)-3,4-dihydropyrimido[5,4-c][1,5]naphthyridin-2(1H)-one hydrochloride (hydrochloride of Compound 58)



embedded image


embedded image


(1) Methyl 4-((4-(4-(tert-butoxycarbonyl)piperazin-1-yl)-3-(trifluoromethyl)phenyl)amino)-6-chloro-1,5-naphthyridine-3-carboxylate



embedded image


Ethyl 4,6-dichloro-1,5-naphthyridine-3-carboxylate (0.5 g, 1.84 mmol) and tert-butyl 4-(4-amino-2-(trifluoromethyl)phenyl)piperazine-1-carboxylate (0.766 g, 2.22 mmol) were dissolved in a mixture of dichloromethane (5 mL) and tert-butanol (5 mL), to the system added potassium carbonate (0.612 g, 4.43 mmol). The reaction mixture was stirred at room temperature for 24 h, suction filtrated, the solid was washed with 30 mL of dichloromethane, and the resulting wash solution and the filtrate were combined, concentrated under reduced pressure, and the resulting residue was recrystallized from diethyl ether to give 0.73 g of a pale yellow solid.


(2) Preparation of tert-butyl 4-(4-((6-chloro-3-(hydroxymethyl)-1,5-naphthyridin-4-yl)amino)-2-(trifluoromethyl)phenyl)piperazine-1-carboxylate



embedded image


Methyl 4-((4-(4-(tert-butoxycarbonyl)piperazin-1-yl)-3-(trifluoromethyl)phenyl)amino)-6-chloro-1,5-naphthyridine-3-carboxylate (0.565 g, 1.0 mmol) was added to ethanol (10 mL), to the system added sodium borohydride (0.228 g, 6 mmol) in batches, stirred at room temperature for 18 h. Ethanol was removed under reduced pressure, added 10 mL of water, and extracted with 80 mL of dichloromethane. The organic layer was dried over anhydrous sodium sulfate, concentrated under reduced pressure to give 0.568 g of a crude solid product. The solid was directly used in the next step.


(3) Preparation of tert-butyl 4-(4-((6-chloro-3-formyl-1,5-naphthyridin-4-yl)amino)-2-(trifluoromethyl)phenyl)piperazine-1-carboxylate



embedded image


tert-butyl 4-(4-((6-chloro-3-(hydroxymethyl)-1,5-naphthyridin-4-yl)amino)-2-(trifluoromethyl)phenyl) piperazine-1-carboxylate (crude 0.568 g, about 1 mmol) obtained in the above step was dissolved in dichloromethane (10 mL), to the solution added manganese dioxide (2.14 g, 24.6 mmol), and the reaction mixture was stirred at room temperature for 3 h. Filtered, and the solid was washed with 30 mL of dichloromethane. The resulting wash liquid and the filtrate were combined and concentrated, and the resulting solid was subjected to silica gel column chromatography (ethyl acetate:petroleum ether=2:1) to give 0.33 g of a product.


(4) Preparation of tert-butyl 4-(4-((6-chloro-3-((methylamino)methyl)-1,5-naphthyridin-4-yl)amino)-2-(trifluoromethyl)phenyl)piperazine-1-carboxylate



embedded image


tert-butyl 4-(4-((6-chloro-3-formyl-1,5-naphthyridin-4-yl)amino)-2-(trifluoromethyl)phenyl)piperazine-1-carboxylate (0.223 g, 0.416 mmol) was dissolved in 20 mL of ethanol, added 0.3 mL of a solution of methylamine in ethanol (concentration 27%), and stirred at room temperature for 24 h. After confirming a disappearance of the raw materials by LC-MS, to the system added sodium borohydride (0.09 g, 2.38 mmol), continued stirring at room temperature for 18 h. The reaction was quenched with a saturated aqueous solution of sodium carbonate, concentrated under reduced pressure to remove ethanol, extracted with 100 mL dichloromethane. The organic layer was dried over anhydrous sodium sulfate, concentrated under reduced pressure to give 0.193 g of a solid.


(5) Preparation of tert-butyl 4-(4-(9-chloro-3-methyl-2-oxo-3,4-dihydropyrimido[5,4-c][1,5]naphthyridin-1(2H)-yl)-2-(trifluoromethyl)phenyl)piperazine-1-carboxylate



embedded image


tert-butyl 4-(4-((6-chloro-3-((methyl amino)methyl-1,5-naphthyridin-4-yl)amino)-2-(trifluoromethyl)phenyl)piperazine-1-carboxylate (0.452 g, 0.82 mmol) was dissolved in dichloromethane (10 mL), to the reaction flask in an ice bath added triphosgene (0.268 g, 0.90 mmol) and triethylamine (0.38 mL). Then, the mixture was stirred at room temperature for 2 h. The reaction was quenched with a saturated aqueous solution of sodium carbonate, extracted with 80 mL of dichloromethane, the organic layer was dried over anhydrous sodium sulfate, the solvent was removed under reduced pressure, and the resulting 0.473 g of solid was used directly in the next step.


(6) Preparation of tert-butyl 4-(4-(9-(6-methoxy-3-yl)-3-methyl-2-oxo-3,4-dihydropyrimido[5,4-c][1,5]naphthyridin-1(2H)-yl)-2-(trifluoromethyl)phenyl)piperazine-1-carboxylate



embedded image


tert-butyl 4-(4-(9-chloro-3-methyl-2-oxo-3,4-dihydropyrimido[5,4-c][1,5]naphthyridin-1(2H)-yl)-2-(trifluoromethyl)phenyl)piperazine-1-carboxylate (0.139 g, 0.241 mmol) and 6-methoxy-3-pyridine boronic acid (40.7 mg, 0.266 mmol) were dissolved in toluene (6 mL) and ethanol (2 mL), to the system added tetrakis(triphenylphosphine) palladium (5 mg) and 2N sodium carbonate solution (0.36 mL). Refluxed and reacted under nitrogen for 16 h, cooled to room temperature, filtered, and the organic layer was concentrated under reduced pressure and dissolved in 30 mL of dichloromethane, washed sequentially with water and saturated brine, dried over anhydrous sodium sulfate, concentrated and subjected to silica gel column chromatography (ethyl acetate:petroleum ether=2:1) to give 94.4 mg of the product.


(7) Preparation of 9-(6-methoxypyridin-3-yl)-3-methyl-1-(4-(piperazin-1-yl)-3-(trifluoromethyl)phenyl)-3,4-dihydropyrimido[5,4-c][1,5]naphthyridin-2(1H)-one hydrochloride



embedded image


tert-butyl 4-(4-(9-(6-methoxy-3-yl)-3-methyl-2-oxo-3,4-dihydropyrimido[5,4-c][1,5]naphthyridin-1(2H)-yl)-2-(trifluoromethyl)phenyl)piperazine-1-carboxylate (94.4 mg, 0.145 mmol) was dissolved in dichloromethane (10 mL), hydrogen chloride gas was blown to the system for 0.5 h, and a solid was precipitated. The reaction mixture was suction filtered, and the filter cake was washed sequentially with dichloromethane and diethyl ether and dried to give 64 mg of a solid.


Molecular formula: C28H27ClF3N7O2; Molecular weight: 586.01; Mass spectrum (M+H): 586.1



1H-NMR (D2O, 400 MHz): δ 8.77-8.65 (1H, m), 8.17 (1H, d), 7.95 (1H, d), 7.94-7.86 (1H, m), 7.48 (1H, s), 7.32-7.21 (3H, m), 6.69-6.58 (1H, m), 4.77 (2H, s), 3.86 (3H, s), 3.25 (4H, t), 3.04 (3H, s), 2.91-2.91 (4H, br s)


Example 8
Preparation of 3-methyl-1-(4-(piperazin-1-yl)-3-(trifluoromethyl)phenyl)-9-(quinolin-3-yl)-3,4-dihydropyrimido[5,4-c][1,5]naphthyridin-2(1H)-one hydrochloride (hydrochloride of Compound 80)



embedded image


(1) Preparation of tert-butyl 4-(4-(3-methyl-2-oxo-9-(quinolin-3-yl)-3,4-dihydropyrimido[5,4-c][1,5]naphthyridin-1(2H)-yl)-2-(trifluoromethyl)phenyl)piperazine-1-carboxylate



embedded image


Specific operations were conducted according to Example 7 (6), using tert-butyl 4-(4-(9-chloro-3-methyl-2-oxo-3,4-dihydropyrimido[5,4-c][1,5]naphthyridin-1(2H)-yl)-2-(trifluoromethyl)phenyl)piperazine-1-carboxylate (0.449 g, 0.779 mmol), to give 331 mg of the product.


(2) Preparation of 3-methyl-1-(4-(piperazin-1-yl)-3-(trifluoromethyl)phenyl)-9-(quinolin-3-yl)-3,4-dihydropyrimido[5,4-c][1,5]naphthyridin-2(1H)-one hydrochloride



embedded image


Specific operations were conducted according to Example 7 (7), using tert-butyl 4-(4-(3-methyl-2-oxo-9-(quinolin-3-yl)-3,4-dihydropyrimido[5,4-c][1,5]naphthyridin-1(2H)-yl)-2-(trifluoromethyl)phenyl)piperazine-1-carboxylate (0.331 g, 0.494 mmol), to give 261 mg of a product.


Molecular formula: C31H27ClF3N7O; Molecular weight: 606.04; Mass spectrum (M+H): 606.1



1H-NMR (D2O, 400 MHz): δ8.81 (1H, s), 8.75 (1H, s), 8.64 (1H, s), 8.41 (1H, d), 8.20 (1H, d), 8.13-8.00 (3H, m), 7.90-7.85 (1H, m), 7.70 (1H, dd), 7.45-7.40 (2H, m), 4.82 (2H, s), 3.09 (3H, s), 2.93 (4H, brs), 2.48 (4H, brs)


Example 9
Preparation of (R)-1-(1-(2-hydroxypropanoyl)piperidin-4-yl)-9-(6-methoxypyridin-3-yl)-3-methyl-3,4-dihydropyrimido[5,4-c][1,5]naphthyridin-2(1H)-one (Compound 86)



embedded image


9-(6-methoxypyridin-3-yl)-3-methyl-1-(piperidin-4-yl)-3,4-dihydropyrimido[5,4-c][1,5]naphthyridin-2(1H)-one hydrochloride (hydrochloride of Compound 114 (100 mg, 0.227 mmol)) and triethylamine (0.177 mL) were added to dichloromethane (20 mL), stirred at room temperature for 0.5 h, then to the system sequentially added (R)-lactic acid (26.7 mg, 0.296 mmol), 1-hydroxybenzotriazole (37.8 mg, 0.280 mmol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (70.8 mg, 0.37 mmol). The reaction mixture was stirred at room temperature for 2 h. After confirming the reaction was completed by a thin layer chromatography plate (dichloromethane:methanol=10:1), the reaction liquid was washed sequentially with a saturated aqueous solution of sodium carbonate, water and saturated brine, and the organic layer was dried over anhydrous sodium sulfate, concentrated and subjected to silica gel column chromatography (dichloromethane:methanol=10:1) to give 88 mg of a white solid.


Molecular formula: C25H28N6O4; Molecular weight: 476.53; Mass spectrum (M+H): 477.2



1H-NMR (CDCl3, 400 MHz): δ 8.76 (1H, d), 8.59 (1H, s), 8.42 (1H, d), 8.17-8.13 (1H, m), 7.97 (1H, d), 6.91 (1H, d), 5.11-5.01 (1H, m), 4.72 (1H, t), 4.54-4.37 (3H, m), 4.03 (3H, d), 4.00-3.86 (1H, br s), 3.82 (1H, d), 3.06 (3H, s), 2.99-2.68 (3H, m), 2.59-2.30 (2H, m), 2.28-2.10 (1H, m), 1.40 (1.5H, d), 1.27 (1.5H, d)


Example 10
Preparation of 9-(6-methoxypyridin-3-yl)-3-methyl-1-(piperidin-4-yl)-3,4-dihydropyrimido[5,4-c][1,5]naphthyridin-2(1H)-one hydrochloride (hydrochloride of Compound 114)



embedded image


embedded image


(1) Preparation of 6-bromopyridin-3-amine



embedded image


Iron powder (2.8 g, 50 mmol), concentrated hydrochloric acid (1.9 mL) and water (9.1 mL) were sequentially added to a solution of 2-bromo-5-nitropyridine (2.03 g, 10 mmol) in ethanol (48 mL), reacted under reflux for 5 h, cooled, filtered and the filtrate was concentrated, adjusted pH approximately to 7-8 with a saturated aqueous solution of sodium carbonate, and extracted with 100 mL of dichloromethane. The organic layer was dried over anhydrous sodium sulfate, concentrated under reduced pressure to give 1.58 g of a red brown solid.


(2) Preparation of diethyl 2-(((6-bromopyridin-3-yl)amino)methylene)malonate



embedded image


6-bromopyridin-3-amine (40.3 g, 0.233 mol) and diethyl ethoxymethylene malonate (56.5 g, 0.261 mol) were added in 400 mL of ethanol, and reacted under reflux for 5 h, cooled and precipitated a solid. The reaction mixture was suction filtered and the resulting solid was washed with petroleum ether to give 71 g of a pale yellow solid.


(3) Preparation of ethyl 6-bromo-4-hydroxy-1,5-naphthyridine-3-carboxylate



embedded image


Diethyl(2-(((6-bromopyridin-3-yl)amino)methylene)malonate (36 g, 0.105 mol) was added to boiling diphenyl ether (185 mL) in batches over 5 min, and reacted under reflux for 45 min. After confirming a disappearance of the raw materials by a thin layer chromatography plate (ethyl acetate:petroleum ether=1:3), cooled and poured into petroleum ether to precipitate a solid. The reaction mixture was suction filtered to give 17.1 g of a khaki solid.


(4) Preparation of ethyl 4,6-dichloro-1,5-naphthyridine-carboxylate



embedded image


Ethyl 6-bromo-4-hydroxy-1,5-naphthyridine-3-carboxylate (15 g, 50.5 mmol) and N,N-dimethylaniline (5 mL) were added to phosphorus oxychloride (50 mL), reacted under refluxed for 3 h, cooled, phosphorus oxychloride was distilled off under reduced pressure, the residue was poured into ice-water, adjusted pH approximately to 8 with a saturated aqueous solution of sodium carbonate, and then extracted with 100 mL of dichloromethane. The organic layer was dried over anhydrous sodium sulfate, concentrated and subjected to silica gel column chromatography (petroleum ether:ethyl acetate=3:1) to give 5.1 g of a pale yellow solid (yield 37.3%).


(5) Preparation of ethyl 4-(1-((tert-butoxycarbonyl)piperidin-4-yl)amino)-6-chloro-1,5-naphthyridine-3-carboxylate



embedded image


Ethyl 4,6-dichloro-1,5-naphthyridine-3-carboxylate (0.5 g, 1.85 mmol) and tert-butyl 4-aminopiperidine-1-carboxylate (0.45 g, 2.22 mmol) were dissolved in a mixture of dichloromethane (5 mL) and tert-butanol (5 mL). Potassium carbonate was added to the system (0.612 g, 4.43 mmol), and the reaction mixture was stirred at room temperature for 24 h and suction filtrated, the solid was washed with 30 mL of dichloromethane. The washing liquid and the filtrate were combined, concentrated under reduced pressure, and the resulting residue was recrystallized from diethyl ether to give 0.73 g of a pale yellow solid.


(6) Preparation of tert-butyl 4-((6-chloro-3-(hydroxymethyl)-1,5-naphthyridin-4-yl)amino)piperidine-1-carboxylate



embedded image


Ethyl 4-(1-((tert-butoxycarbonyl)piperidin-4-yl)amino)-6-chloro-1,5-naphthyridine-3-carboxylate (0.43 g, 1.0 mmol) was added to ethanol (10 mL), and to the system added sodium borohydride (0.228 g, 6 mmol) in batches, stirred at room temperature for 18 h. Ethanol was removed under reduced pressure, then added 10 mL of water and extracted with 80 mL of dichloromethane. The organic layer was dried over anhydrous sodium sulfate, concentrated under reduced pressure to give 0.39 g of a solid. The solid was used directly in the next step.


(7) Preparation of tert-butyl 4-((6-chloro-3-formyl-1,5-naphthyridin-4-yl)amino)piperidine-1-carboxylate



embedded image


tert-butyl 4-((6-chloro-3-(hydroxymethyl)-1,5-naphthyridin-4-yl)amino)piperidine-1-carboxylate (0.39 g, 1 mmol) was dissolved in dichloromethane (10 mL), and to the solution added manganese dioxide (2.14 g, 32 mmol). The reaction mixture was stirred at room temperature for 3 h, filtered and the solid was washed with 30 mL of dichloromethane. The resulting wash liquid and the filtrate were combined and concentrated, and the resulting solid was recrystallized from methanol to give 0.311 g of a white solid.


(8) Preparation of tert-butyl 4-((6-chloro-3-((methylamino)methyl)-1,5-naphthyridin-4-yl)amino) piperidine-1-carboxylate



embedded image


tert-butyl 4-((6-chloro-3-formyl-1,5-naphthyridin-4-yl)amino)piperidine-1-carboxylate (0.31 g, 0.79 mmol) was dissolved in ethanol, added 0.59 mL of a solution of methylamine in ethanol (concentration 27%), stirred at room temperature for 24 h. After confirming a disappearance of the raw materials by LC-MS, to the system added sodium borohydride (0.181 g, 4.60 mmol), continued to stirring at room temperature for 18 h. The reaction was quenched with a saturated aqueous solution of sodium carbonate, concentrated under reduced pressure to remove ethanol, extracted with 100 mL of dichloromethane. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give 0.27 g of a solid (yield 84.2%).


(9) Preparation of tert-butyl 4-(9-chloro-3-methyl-2-oxo-3,4-dihydropyrimido[5,4-c][1,5]naphthyridin-1(2H)-yl)piperidine-1-carboxylate



embedded image


tert-butyl 4-((6-chloro-3-((methylamino)methyl)-1,5-naphthyridin-4-yl)amino)piperidine-1-carboxylate (0.312 g, 0.77 mmol) was dissolved in dichloromethane (10 mL), to the reaction flask in an ice bath added triphosgene (0.274 g, 0.923 mmol) and triethylamine (0.324 mL). The mixture was stirred at room temperature for 2 h. The reaction was quenched with a saturated aqueous solution of sodium carbonate, and extracted with 80 mL of dichloromethane. The organic layer was dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to give 0.332 g of a solid. The solid was used directly in the next step.


(10) Preparation of tert-butyl 4-(9-(6-methoxypyridin-3-yl)-3-methyl-2-oxo-3,4-dihydropyrimido[5,4-c][1,5]naphthyridin-1(2H)-yl)piperidine-1-carboxylate



embedded image


tert-butyl 4-(9-chloro-3-methyl-2-oxo-3,4-dihydropyrimido[5,4-c][1,5]naphthyridin-1(2H)-yl)piperidine-1-carboxylate (0.104 g, 0.241 mmol) and 6-methoxy-3-pyridine boronic acid (40.7 mg, 0.266 mmol) were dissolved in toluene (6 mL) and ethanol (2 mL), and to the system added tetrakis(triphenylphosphine)palladium (5 mg) and 2N sodium carbonate solution (0.36 mL), reacted under reflux under nitrogen for 16 h, cooled to room temperature, filtered and the organic layer was concentrated under reduced pressure and dissolved in 30 mL of dichloromethane, followed by washing sequentially with water and saturated brine, dried over anhydrous sodium sulfate, concentrated and subjected to silica gel column chromatography (ethyl acetate:petroleum ether=2:1) to give 75 mg of the product.


Preparation of 9-(6-methoxypyridin-3-yl)-3-methyl-1-(piperidin-4-yl)-3,4-dihydropyrimido[5,4-c][1,5]naphthyridin-2(1H)-one hydrochloride



embedded image


tert-butyl 4-(9-(6-methoxypyridin-3-yl)-3-methyl-2-oxo-3,4-dihydropyrimido[5,4-c][1,5]naphthyridin-1(2H)-yl)piperidine-1-carboxylate (75 mg, 0.149 mmol) was dissolved in dichloromethane (10 mL), hydrogen chloride gas was blown to the system for 0.5 h, and a solid was precipitated. The reaction mixture was filtered, the filter cake was washed sequentially with dichloromethane and diethyl ether, and the resulting solid was dried to give 50 mg of the titled compound.


Molecular formula: C22H25ClN6O2; Molecular weight: 440.93; Mass spectrum (M+H): 441.1



1H-NMR (D2O, 400 MHz): δ 8.65 (1H, d), 8.52 (1H, d), 8.34 (1H, d), 8.25-8.20 (1H, m), 8.16 (1H, d), 7.05-7.02 (1H, m), 4.97 (1H, m), 4.56 (2H, s), 3.93 (3H, d), 3.41 (2H, d), 2.98 (3H, s), 2.89-2.67 (4H, m), 2.35 (2H, d)

Claims
  • 1. A compound of general formula (I):
  • 2. The compound of claim 1, or a pharmaceutically acceptable salt, ester, solvate or stereoisomer thereof, wherein R8 is hydrogen, halogen, cyano, hydroxy, carboxyl, —(CH2)nNR10aR10b, —(CH2)nS(O)mNR10aR10b, —(CH2)nN(R10a)S(O)mR11, C1-6 alkyl or C1-6 alkoxy, wherein C1-6 alkyl and C1-6 alkoxy may be optionally substituted with 1 to 3 substituents selected from halogen, hydroxy and carboxyl;R1 is C3-8 cycloalkyl, 6- to 10-membered aryl, 5- to 10-membered heteroaryl or 5- to 10-membered heterocyclic group, all of which may be optionally substituted with 1 to 3 R9a;R2 is C3-8 cycloalkyl, 6- to 10-membered aryl, 5- to 10-membered heteroaryl or 5- to 10-membered heterocyclic group, all of which may be optionally substituted with 1 to 3 R9b;R3, R4, R5 and R6 are each independently hydrogen or C1-6 alkyl, wherein C1-6 alkyl may be optionally substituted with 1 to 3 substituents selected from halogen, hydroxy and carboxyl;R9a and R9b are each independently(1) halogen, cyano, hydroxy, —(CH2)nNR10aR10b, —(CH2)nC(O)R11, —(CH2)nS(O)mR11, —(CH2)nS(O)mNR10aR10b, —(CH2)nN(R10a)S(O)mR11, —(CH2)nC(O)NR10aR10b, —(CH2)nOC(O)R11, —(CH2)nC(O)(CH2)nOR11, —(CH2)nN(R10a)C(O)R11;(2) C1-6 alkyl or C1-6 alkoxy, all of which may be optionally substituted with 1 to 3 substituents selected from halogen, hydroxy and cyano;(3) C3-8 cycloalkyl, 6- to 10-membered aryl, 5- to 10-membered heteroaryl or 5- to 10-membered heterocyclic group, all of which may be optionally substituted with 1 to 3 substituents selected from halogen, hydroxy, cyano, trifluoromethyl, C1-6 alkyl, C1-6 alkoxy, —(CH2)nNR10aR10b, —(CH2)nC(O)R11, —(CH2)nC(O)NR10aR10b, —(CH2)nS(O)mR11, —(CH2)nS(O)mNR10aR10b, —(CH2)nN(R10a)S(O)mR11, —(CH2)nOC(O)R11 and —(CH2)N(R10a)C(O)R11;R10a and R10b are each independently hydrogen or C1-6 alkyl, wherein C1-6 alkyl may be optionally substituted with 1 to 3 substituents selected from hydroxy, halogen, cyano, carboxyl, —(CH2)nNR10aR10b, sulfamoyl, carbamoyl and sulfamino; andR11 is hydrogen or C1-6 alkyl, wherein C1-6 alkyl may be optionally substituted with 1 to 3 substituents selected from halogen, cyano, hydroxy, carboxyl, —NR10aR10b, sulfamoyl and carbamoyl.
  • 3. The compound of claim 2, or a pharmaceutically acceptable salt, ester, solvate or stereoisomer thereof, wherein X is O;A and B are each CH;R1 is 6- to 10-membered aryl, 5- to 6-membered monocyclic heteroaryl, 9- to 10-membered fused heteroaryl, 5- to 6-membered monocyclic heterocyclic group or 9- to 10-membered fused heterocyclic group, all of which may be optionally substituted with 1 to 3 R9a;R2 is 6- to 10-membered aryl, 5- to 6-membered monocyclic heteroaryl, 9- to 10-membered fused heteroaryl, 5- to 6-membered monocyclic heterocyclic group or 9- to 10-membered fused heterocyclic group, all of which may be optionally substituted with 1 to 3 R9b;R3, R4 and R5 are each hydrogen;R6 is hydrogen or C1-6 alkyl;R9a and R9b are each independently(1) halogen, cyano, hydroxy, —(CH2)nNR10aR10b, —(CH2)nC(O)R11, —(CH2)nS(O)mR11, —(CH2)nS(O)mNR10aR10b, —(CH2)nN(R10a)S(O)mR11, —(CH2)nC(O)NR10aR10b, —(CH2)nOC(O)R11, —(CH2)nC(O)(CH2)nOR11, —(CH2)nN(R10a)C(O)R11;(2) C1-6 alkyl or C1-6 alkoxy, all of which may be optionally substituted with 1 to 3 substituents selected from halogen, hydroxy and cyano;(3) 5- to 10-membered heteroaryl or 5- to 10-membered heterocyclic group, all of which may be optionally substituted with 1 to 3 substituents selected from halogen, hydroxy, cyano, trifluoromethyl, C1-6 alkyl, C1-6 alkoxy, —(CH2)nNR10aR10b, —(CH2)nC(O)R11, —(CH2)nC(O)NR10aR10b, —(CH2)nS(O)mR11, —(CH2)nS(O)mNR10aR10b, —(CH2)nN(R10a)S(O)mR11, —(CH2)nOC(O)R11 and —(CH2)nN(R10a)C(O)R11;R10a and R10b are each independently hydrogen or C1-6 alkyl;R11 is hydrogen or C1-6 alkyl, wherein C1-6 alkyl may be optionally substituted with 1 to 3 substituents selected from halogen, cyano, hydroxyl and —(CH2)nNR10aR10b; andn is 0-3.
  • 4. The compound of claim 3, or a pharmaceutically acceptable salt, ester, solvate or stereoisomer thereof, wherein R1 is 6- to 10-membered aryl or 5- to 6-membered monocyclic heteroaryl, all of which may be optionally substituted with 1 to 3 R9a;R2 is 6- to 10-membered aryl, 5- to 6-membered monocyclic heteroaryl or 9- to 10-membered fused heteroaryl, all of which may be optionally substituted with 1 to 3 R9b; andR9a and R9b are each independently(1) halogen, cyano, hydroxy, —(CH2)nNR10aR10b, —(CH2)nN(R10a)C(O)R11, —(CH2)nC(O)R11, —(CH2)nS(O)mR11, —(CH2)nC(O)NR10aR10b, —(CH2)nC(O)(CH2)nOR11;(2) C1-6 alkyl or C1-6 alkoxy, all of which may be optionally substituted with 1 to 3 substituents selected from halogen, hydroxy and cyano;(3) 5- to 6-membered monocyclic heteroaryl or 5- to 6-membered monocyclic heterocyclic group, all of which may be optionally substituted with 1 to 3 substituents selected from halogen, hydroxy, cyano, trifluoromethyl, C1-6 alkyl, C1-6 alkoxy, —(CH2)nNR10aR10b, —(CH2)nC(O)R11, —(CH2)nC(O)NR10aR10b, —(CH2)nOC(O)R11 and —(CH2)nN(R10a)C(O)R11.
  • 5. The compound of claim 4, or a pharmaceutically acceptable salt, ester, solvate or stereoisomer thereof, wherein R1 is phenyl, pyridyl, pyrimidinyl or piperidinyl, all of which may be optionally substituted with 1 to 3 R9a;R2 is phenyl, pyridyl, pyrimidinyl, thienyl, pyrazolyl, indazolyl, indolyl, pyridopyrrolyl, pyrazolopyridyl or quinolyl, all of which may be optionally substituted with 1 to 3 R9b;R9a and R9b are each independently(1) cyano, hydroxy, —NR10aR10b, —N(R10a)C(O)R11, —C(O)R11, —S(O)mR11, —C(O)NR10aR10b, —C(O)CH2OR11;(2) C1-6 alkyl or C1-6 alkoxy, all of which may be optionally substituted with 1 to 3 substituents selected from halogen, hydroxy and cyano;(3) pyrrolyl, pyrazolyl, imidazolyl, piperidinyl, piperazinyl or morpholinyl, all of which may be optionally substituted with 1 to 3 substituents selected from halogen, hydroxy, cyano, trifluoromethyl, C1-6 alkyl, C1-6 alkoxy, —NR10aR10b, —C(O)R11, —C(O)NR10aR10b, —OC(O)R11, —N(R10a)C(O)R11;R11 is hydrogen or C1-6 alkyl, wherein C1-6 alkyl may be optionally substituted with 1 to 3 substituents selected from halogen, cyano and hydroxy; andn is 0.
  • 6. The compound of claim 5, or a pharmaceutically acceptable salt, ester, solvate or stereoisomer thereof, wherein R1 is phenyl optionally substituted with 1 to 3 R9a.
  • 7. The compound of claim 1, or a pharmaceutically acceptable salt, ester, solvate or stereoisomer thereof, wherein general formula (I) is represented by general formula (II) shown below:
  • 8. The compound of claim 1, or a pharmaceutically acceptable salt, ester, solvate or stereoisomer thereof, which is selected from the following compounds:
  • 9. A pharmaceutical composition comprising the compound of claim 1, a pharmaceutically acceptable salt, ester, solvate or stereoisomer thereof and one or more pharmaceutically acceptable carriers, further comprising one or more antitumor agents and immunosuppressants, wherein the antitumor agents and immunosuppressive agents are (1) anti-metabolites selected from the group consisting of capecitabine, gemcitabine and pemetrexed disodium;(2) growth factor inhibitors selected from the group consisting of pazopanib, imatinib, erlotinib, lapatinib, gefitinib and vandetanib;(3) antibodies selected from the group consisting of Herceptin and Avastin;(4) mitotic inhibitors selected from the group consisting of paclitaxel, vinorelbine, docetaxel and doxorubicin;(5) anti-tumor hormones selected from the group consisting of letrozole, tamoxifen, fulvestrant, flutamide and triptorelin;(6) alkylating agents selected from the group consisting of cyclophosphamide, nitrogen mustard, melphalan, chlorambucil and carmustine;(7) platinum metals selected from the group consisting of carboplatin, cisplatin and oxaliplatin;(8) topoisomerase inhibitors selected from the group consisting of camptothecin, topotecan and irinotecan;(9) immunosuppressants selected from the group consisting of everolimus, sirolimus and temsirolimus;(10) purine analogues selected from the group consisting of 6-mercaptopurine, 6-thioguanine and azathioprine;(11) antibiotics selected from the group consisting of streptozotocin D, daunorubicin, doxorubicin, mitoxantrone, bleomycin and plicamycin; or(12) adrenal cortex inhibitor which is aminoglutethimide.
  • 10. (canceled)
  • 11. A method for treating and/or preventing a proliferative disease, comprising administering to a patient a therapeutically effective amount of the compound of claim 1, a pharmaceutically acceptable salt, ester, solvate or stereoisomer thereof.
  • 12. The method of claim 11, wherein the proliferative disease is cancer or a non-cancer disease.
  • 13. The method of claim 12, wherein the cancer is selected from a brain tumor, lung cancer, non-small cell lung cancer, squamous cell carcinoma, bladder cancer, stomach cancer, ovarian cancer, peritoneal cancer, pancreatic cancer, breast cancer, head and neck cancer, cervical cancer, endometrial cancer, colorectal cancer, liver cancer, kidney cancer, esophageal adenocarcinoma, esophageal squamous cell carcinoma, solid tumors, non-Hodgkin's lymphoma, glioma, glioblastoma multiforme, gliosarcoma, prostate cancer, thyroid carcinoma, genital tract carcinoma, carcinoma in situ, lymphoma, histiocytic lymphoma, neurofibromatosis, bone cancer, skin cancer, brain cancer, colon cancer, testicular cancer, small cell lung cancer, gastrointestinal stromal tumor, prostate tumor, mast cell tumor, multiple myeloma, melanoma, glioma glioblastoma, astrocytoma, neuroblastoma, and sarcomas.
  • 14. The method of claim 12, wherein the non-cancer disease is selected from a skin disease or benign prostate hyperplasia.
Priority Claims (2)
Number Date Country Kind
201110364054.2 Nov 2011 CN national
201210072834.4 Mar 2012 CN national
PCT Information
Filing Document Filing Date Country Kind
PCT/CN2012/001551 11/16/2012 WO 00