HK1166984B

HK1166984B - Fused pyrimidines as akt inhibitors

Info

Publication number: HK1166984B
Application number: HK12107786.9A
Authority: HK
Inventors: M．芬内曼; T.贝尔; T.迈尔; S.赫尔德; G．贝内克; F．德默尔; A．齐尔希; A．施特鲁布; T．贝克尔斯; S．英斯; H.雷温克尔; 刘宁姝; U.伯默
Original assignee: 拜耳医药股份有限公司
Priority date: 2009-02-13
Filing date: 2010-02-05
Publication date: 2015-09-25

Description

Fused pyrimidines as AKT inhibitors

Technical Field

The present invention relates to fused pyrimidine compounds, which are useful in the pharmaceutical industry for the preparation of pharmaceutical compositions.

Background

Cancer is the second most common cause of death in the united states, resulting in 450000 deaths annually. Despite substantial progress in identifying some possible environmental and genetic causes of cancer, there remains a need for additional therapeutic modalities for cancer and related diseases. In particular, there is a need for therapeutic methods for treating diseases associated with growth disorders/proliferation.

Cancer is a complex disease resulting from the process of selection of cells with acquired functional capacity (e.g., increased viability/resistance to apoptosis and unlimited proliferation potential). Therefore, it is preferable to develop a drug for cancer therapy against a significant feature of an existing tumor.

One pathway that has been shown to mediate important survival signals in mammalian cells includes receptor tyrosine kinases such as platelet derived growth factor receptor (PDGF-R), human epidermal growth factor 2/3 receptor (HER2/3), or insulin-like growth factor 1 receptor (IGF-1R). These receptors activate the phosphatidylinositol 3-kinase (Pi3K)/Akt pathway after activation by the ligand, respectively. The phosphatidylinositol 3-kinase (Pi3K)/Akt protein kinase pathway is critical to control cell growth, proliferation and survival, which contributes to tumor development. Thus, within the class of serine-threonine specific signal transduction kinases, Akt (protein kinase B; PKB) with the isoenzymes Akt1(PKB α), Akt2(PKB β) and Akt3(PKB γ) has a high therapeutic implication. Akt is mainly activated in a Pi 3-kinase dependent manner, and this activation is regulated by the tumor suppressor PTEN (phosphatase and tensin homolog), which essentially acts as a functional antagonist of Pi 3K.

This Pi3K/Akt pathway regulates essential cellular functions (e.g., transcription, translation, growth, and survival) and is implicated in human diseases including diabetes and cancer. In a wide range of tumor entities, such as breast and prostate cancer, this pathway is often over-activated. Upregulation may be due to overexpression or constitutive activation of receptor tyrosine kinases (e.g. EGFR, HER2/3) which are upstream and involved in their direct activation or gain-of-function or loss-of-function variants of some components, such as PTEN deletion. This pathway is more frequently targeted for genomic alterations, including mutations, amplifications, and rearrangements, than any other pathway in human cancer, possibly except the p53 and retinoblastoma pathways. Alterations in the Pi3K/Akt pathway trigger a cascade of biological events that promote tumor development, survival, angiogenesis, and metastasis.

Activation of Akt kinases promotes increased nutrient uptake, turning the cell toward glucose-dependent metabolism (which redirects lipid precursors and amino acids to anabolic processes that maintain cell growth and proliferation). These metabolic phenotypes with overactivated Akt lead to malignancies that exhibit a metabolic shift to aerobic glycolysis (Warburg effect). In this regard, the Pi3K/Akt pathway is considered to be critical for survival despite unfavorable growth conditions such as glucose deficiency or hypoxia.

Another aspect of the activated PI3K/Akt pathway is to protect cells from programmed cell death (apoptosis) and is therefore thought to transduce survival signals. The Pi3K/Akt pathway, particularly Akt itself, is a target for cancer therapy since it acts as a regulator of anti-apoptotic signal transduction in tumor cells. Activated Akt phosphorylates and regulates several targets, such as BAD, GSK3 or FKHRL1 (which affect different signal transduction pathways, such as cell survival, protein synthesis or cell movement). This Pi3K/Akt pathway also plays an important role in the resistance of tumor cells to conventional anticancer therapies. Thus, blocking the Pi3K/Akt pathway can simultaneously inhibit proliferation of tumor cells (e.g., by inhibiting metabolic effects) and sensitize them to pro-apoptotic agents.

Akt inhibition selectively sensitizes tumor cells to apoptotic stimuli such as Trail, camptothecin, and doxorubicin. Depending on the genetic background/molecular mechanisms (molecular applications) of the tumor, Akt inhibitors can also induce apoptotic cell death in monotherapy.

Akt inhibitors are described in international patent applications WO2004096131, WO2005100344, WO2006036395, WO2006065601, WO2006091395 and WO 2006135627. In a recent publication, y.li et al (bioorg.med.chem.lett.2009, 19, 834-836 and references cited therein) detail the difficulty of finding the optimal Akt inhibitors. Due to the potential use of Akt inhibitors in a variety of disease conditions, such as cancer, it would be particularly desirable to provide new Akt inhibitors to replace those existing Akt inhibitors.

Disclosure of Invention

A solution to the above problem is to provide alternative inhibitors of Akt. It has now been found that novel fused pyrimidine compounds, detailed below, have AKT inhibitor activity.

According to a first aspect, the present invention relates to a compound of formula (I), or an N-oxide, a salt, a tautomer or a stereoisomer of said compound, or a salt of said N-oxide, tautomer or stereoisomer:

wherein ring B fused to the pyrimidine group is selected from:

indicates the point of connection, and indicates,

r1 is hydrogen, 1-4C-alkyl (optionally substituted by halogen, hydroxy, amino, mono-1-4C-alkylamino OR di-1-4C-alkylamino), halogen, trifluoromethyl, cyano, 3-7C-cycloalkyl, 2-4C-alkenyl, 2-4C-alkynyl, C (O) NR11R12, -C (O) OR2, OR is a monocyclic 5-OR 6-membered heteroarylene (hetereoarylene) comprising 1 nitrogen atom and optionally 1,2 OR 3 additional heteroatoms independently selected from oxygen, nitrogen and sulfur,

r2 is hydrogen, 1-4C-alkyl (optionally substituted by halogen, hydroxy, amino, mono-1-4C-alkylamino or di-1-4C-alkylamino) or 3-7C-cycloalkyl,

r4 is phenyl, thienyl, pyridyl, oxazolyl or thiazolyl, and wherein R4 is optionally substituted with R5,

r5 is 1-4C-alkyl, halogen, 1-4C-alkoxy or NR11R12,

r6 is hydrogen or 1-4C-alkyl,

n is a number of 1 or2,

m is a number of 1 or2,

r7 is a group of the formula-W-Y,

w is a monocyclic 5-or 6-membered heteroarylene comprising 1 nitrogen atom and optionally 1,2 or 3 additional heteroatoms independently selected from oxygen, nitrogen and sulfur, or is a bicyclic 9-membered heteroarylene comprising 1 nitrogen atom and optionally 1,2 or 3 additional heteroatoms independently selected from oxygen, nitrogen and sulfur, and wherein the bicyclic heteroarylene is optionally substituted with R8,

r8 is hydrogen, 1-4C-alkyl, 1-4C-haloalkyl, 3-7C-cycloalkyl, 1-4C-alkoxy, halogen, cyano or NR11R12,

y is hydrogen, aryl, or monocyclic 5-or 6-membered heteroaryl comprising 1 nitrogen atom and optionally 1,2 or 3 additional heteroatoms independently selected from oxygen, nitrogen, sulfur, and wherein said heteroaryl is optionally substituted with R9 and optionally further substituted with R9A,

r9 is 1-4C-alkyl, 1-4C-alkoxy, halogen, hydroxy, 1-4C-haloalkyl, NR11R12, cyano or C (O) NH₂，

R9A is 1-4C-alkyl or halogen,

r10 is hydrogen or 1-4C-alkyl (optionally substituted by halogen, hydroxy, amino, mono-1-4C-alkylamino or di-1-4C-alkylamino),

r11, R12, which are identical or different, are hydrogen, 1-4C-alkyl (optionally substituted by halogen, hydroxy, amino, mono-1-4C-alkylamino or di-1-4C-alkylamino) or 3-7C-cycloalkyl.

Another aspect of the invention relates to a compound of claim 1, or an N-oxide, salt, tautomer or stereoisomer of said compound, or a salt of said N-oxide, tautomer or stereoisomer, wherein

R1 is hydrogen, 1-4C-alkyl (optionally substituted by halogen, hydroxy, amino, mono-1-4C-alkylamino OR di-1-4C-alkylamino), halogen, trifluoromethyl, cyano, 3-7C-cycloalkyl, C (O) NR11R12, -C (O) OR2,

r2 is hydrogen or 1-4C-alkyl,

r5 is 1-4C-alkyl, halogen, 1-4C-alkoxy or NR11R12,

r6 is hydrogen or 1-4C-alkyl,

n is a number of 1 or2,

m is a number of 1 or2,

r7 is a group of the formula-W-Y,

y is hydrogen, aryl, or a monocyclic 5-or 6-membered heteroaryl group comprising 1 nitrogen atom and optionally 1,2 or 3 additional heteroatoms independently selected from oxygen, nitrogen, sulfur, and wherein the heteroaryl group is optionally substituted with R9 and optionally further substituted with R9A,

R9A is 1-4C alkyl or halogen,

r11, R12, which are identical or different, are hydrogen or 1-4C-alkyl.

Yet another aspect of the present invention relates to a compound of claim 1, or an N-oxide, salt, tautomer or stereoisomer of said compound, or a salt of said N-oxide, tautomer or stereoisomer, wherein

r2 is hydrogen or 1-4C-alkyl,

r4 is phenyl, thienyl, pyridyl, oxazolyl or thiazolyl,

r6 is hydrogen or 1-4C-alkyl,

n is a number of 1 or2,

m is a number of 1 or2,

r7 is a group of the formula-W-Y,

w is a 1,2, 4-triazolylene group,

y is a monocyclic 5-or 6-membered heteroaryl group comprising 1 nitrogen atom and optionally 1,2 or 3 additional heteroatoms independently selected from oxygen, nitrogen, sulfur, and wherein said heteroaryl group is optionally substituted with R9 and optionally further substituted with R9A,

R9A is 1-4C-alkyl,

r10 is hydrogen or 1-4C-alkyl,

Another aspect of the present invention relates to a compound of general formula (I) according to claim 1, or an N-oxide, salt, tautomer or stereoisomer of said compound, or a salt of said N-oxide, tautomer or stereoisomer, wherein

R1 is hydrogen, 1-4C-alkyl (optionally substituted by halogen, hydroxy, amino, mono-1-4C-alkylamino OR di-1-4C-alkylamino), halogen, trifluoromethyl, cyano, 3-7C-cycloalkyl, 2-4C-alkenyl, 2-4C-alkynyl, C (O) NR11R12, -C (O) OR2, OR is a monocyclic 5-OR 6-membered heteroarylene comprising 1 nitrogen atom and optionally 1,2 OR 3 additional heteroatoms independently selected from oxygen, nitrogen and sulfur,

r5 is 1-4C-alkyl, halogen, 1-4C-alkoxy or NR11R12,

r6 is hydrogen or 1-4C-alkyl,

n is a number of 1 or2,

m is a number of 1 or2,

r7 is a group of the formula-W-Y,

y is hydrogen, aryl, or monocyclic 5-or 6-membered heteroaryl comprising 1 nitrogen atom and optionally 1,2 or 3 additional heteroatoms independently selected from oxygen, nitrogen, sulfur, and wherein said heteroaryl is optionally substituted with R9,

Yet another aspect of the present invention relates to a compound of general formula (I) according to claim 1, or an N-oxide, a salt, a tautomer or a stereoisomer of said compound, or a salt of said N-oxide, tautomer or stereoisomer, wherein

r2 is hydrogen or 1-4C-alkyl,

r5 is 1-4C-alkyl, halogen, 1-4C-alkoxy or NR11R12,

r6 is hydrogen or 1-4C-alkyl,

n is a number of 1 or2,

m is a number of 1 or2,

r7 is a group of the formula-W-Y,

r11, R12, which are identical or different, are hydrogen or 1-4C-alkyl.

r2 is hydrogen or 1-4C-alkyl,

r4 is phenyl, thienyl, pyridyl, oxazolyl or thiazolyl,

r6 is hydrogen or 1-4C-alkyl,

n is a number of 1 or2,

m is a number of 1 or2,

r7 is a group of the formula-W-Y,

w is a 1,2, 4-triazolylene group,

y is a monocyclic 5-or 6-membered heteroaryl group comprising 1 nitrogen atom and optionally 1,2 or 3 additional heteroatoms independently selected from oxygen, nitrogen, sulfur, and wherein said heteroaryl group is optionally substituted with R9,

R10 is hydrogen or 1-4C-alkyl,

R1 is hydrogen or 1-4C-alkyl,

r4 is a phenyl group, and R4 is a phenyl group,

r6 is a hydrogen atom or a salt thereof,

n is a number of 1 or2,

m is a number of 1 or2,

r7 is a group of the formula-W-Y,

w is a 1,2, 4-triazolylene group,

y is pyridin-2-yl, 2-pyrazinyl or 2-pyrimidinyl optionally substituted with R9 and optionally further substituted with R9A,

r9 is hydrogen, 1-4C-alkyl, halogen, 1-4C-haloalkyl,

R9A is 1-4C-alkyl,

r10 is hydrogen or 1-4C-alkyl.

R1 is hydrogen, 1-4C-alkyl,

r4 is a phenyl group, and R4 is a phenyl group,

r6 is a hydrogen atom or a salt thereof,

n is a number of 1 or2,

m is a number of 1 or2,

r7 is a group of the formula-W-Y,

w is a 1,2, 4-triazolylene group,

y is a pyridin-2-yl group,

r10 is hydrogen or 1-4C-alkyl.

R1 is hydrogen or a methyl group,

r4 is a phenyl group, and R4 is a phenyl group,

r6 is a hydrogen atom or a salt thereof,

n is a number of 1 or2,

m is a number of 1 or2,

r7 is a group of the formula-W-Y,

w is a 1,2, 4-triazolylene group,

r9 is hydrogen, methyl, F, Cl, Br, CF₃，

R9A is a methyl group, or a salt thereof,

r10 is hydrogen or methyl.

R1 is hydrogen or a methyl group,

r4 is a phenyl group, and R4 is a phenyl group,

r6 is a hydrogen atom or a salt thereof,

n is a number of the radicals 2,

m is a number of 2, and m is,

r7 is a group of the formula-W-Y,

w is a 1,2, 4-triazolylene group,

y is a pyridin-2-yl group,

r10 is hydrogen or methyl.

A preferred aspect of the invention is a compound according to claim 1, or an N-oxide, salt, tautomer or stereoisomer of said compound, or a salt of said N-oxide, tautomer or stereoisomer, said compound being selected from:

5-methyl-8-phenyl-7- {4- [4- (5-pyridin-2-yl-1H- [1, 2, 4] triazol-3-yl) -piperidin-1-ylmethyl ] -phenyl } -imidazo [1, 2-c ] pyrimidine,

8-phenyl-7- {4- [4- (5-pyridin-2-yl-1H- [1, 2, 4] triazol-3-yl) -piperidin-1-ylmethyl ] -phenyl } -imidazo [1, 2-c ] pyrimidine,

8-phenyl-7- {4- [4- (5-pyridin-2-yl-1H- [1, 2, 4] triazol-3-yl) -piperidin-1-ylmethyl ] -phenyl } - [1, 2, 4] triazolo [4, 3-c ] pyrimidine, pharmaceutically acceptable salts thereof, pharmaceutical compositions thereof, and methods of use,

3-methyl-8-phenyl-7- {4- [4- (5-pyridin-2-yl-1H- [1, 2, 4] triazol-3-yl) -piperidin-1-ylmethyl ] -phenyl } - [1, 2, 4] triazolo [4, 3-c ] pyrimidine, optionally substituted phenyl,

7- (4- {4- [5- (5, 6-dimethyl-pyridin-2-yl) -1H- [1, 2, 4] triazol-3-yl ] -piperidin-1-ylmethyl } -phenyl) -5-methyl-8-phenyl-imidazo [1, 2-c ] pyrimidine,

5-methyl-8-phenyl-7- {4- [3- (5-pyridin-2-yl-1H- [1, 2, 4] triazol-3-yl) -azetidin-1-ylmethyl ] -phenyl } -imidazo [1, 2-c ] pyrimidine,

5-methyl-8-phenyl-7- {4- [4- (5-pyrazin-2-yl-1H- [1, 2, 4] triazol-3-yl) -piperidin-1-ylmethyl ] -phenyl } -imidazo [1, 2-c ] pyrimidine,

5-methyl-8-phenyl-7- {4- [4- (5-pyrimidin-2-yl-1H- [1, 2, 4] triazol-3-yl) -piperidin-1-ylmethyl ] -phenyl } -imidazo [1, 2-c ] pyrimidine,

7- (4- {4- [5- (5-bromo-pyridin-2-yl) -1H- [1, 2, 4] triazol-3-yl ] -piperidin-1-ylmethyl } -phenyl) -8-phenyl-imidazo [1, 2-c ] pyrimidine,

7- (4- {4- [5- (5, 6-dimethyl-pyridin-2-yl) -1H- [1, 2, 4] triazol-3-yl ] -piperidin-1-ylmethyl } -phenyl) -8-phenyl-imidazo [1, 2-c ] pyrimidine,

8-phenyl-7- {4- [5- (pyrazin-2-yl) -1H- [1, 2, 4] triazol-3-yl) -piperidin-1-ylmethyl ] -phenyl } -imidazo [1, 2-c ] pyrimidine,

7- (4- {4- [5- (5-fluoro-pyridin-2-yl) -1H- [1, 2, 4] triazol-3-yl ] -piperidin-1-ylmethyl } -phenyl) -8-phenyl-imidazo [1, 2-c ] pyrimidine,

7- (4- {4- [5- (5-methyl-pyridin-2-yl) -1H- [1, 2, 4] triazol-3-yl ] -piperidin-1-ylmethyl } -phenyl) -8-phenyl-imidazo [1, 2-c ] pyrimidine,

7- (4- {4- [5- (6-chloro-pyridin-2-yl) -1H- [1, 2, 4] triazol-3-yl ] -piperidin-1-ylmethyl } -phenyl) -8-phenyl-imidazo [1, 2-c ] pyrimidine,

7- (4- {4- [5- (4, 6-dimethyl-pyridin-2-yl) -1H- [1, 2, 4] triazol-3-yl ] -piperidin-1-ylmethyl } -phenyl) -8-phenyl-imidazo [1, 2-c ] pyrimidine,

8-phenyl-7- (4- {4- [5- (4-trifluoromethyl-pyridin-2-yl) -1H- [1, 2, 4] triazol-3-yl ] -piperidin-1-ylmethyl } -phenyl) -imidazo [1, 2-c ] pyrimidine,

8-phenyl-7- {4- [3- (5-pyridin-2-yl-1H- [1, 2, 4] triazol-3-yl) -azetidin-1-ylmethyl ] -phenyl } -imidazo [1, 2-c ] pyrimidine.

An aspect of the invention is a compound of claim 1, or an N-oxide, salt, tautomer or stereoisomer of said compound, or a salt of said N-oxide, tautomer or stereoisomer, selected from the group consisting of:

3-methyl-8-phenyl-7- {4- [4- (5-pyridin-2-yl-1H- [1, 2, 4] triazol-3-yl) -piperidin-1-ylmethyl ] -phenyl } - [1, 2, 4] triazolo [4, 3-c ] pyrimidine.

One embodiment of the above aspect of the invention relates to a compound of formula (I), or an N-oxide, salt, tautomer or stereoisomer of said compound, or a salt of said N-oxide, tautomer or stereoisomer, wherein ring B fused to the pyrimidine ring is the following ring system:

r1, R4 and R10 are as defined above.

Yet another embodiment of the above aspects of the invention relates to a compound of formula (I), or an N-oxide, salt, tautomer or stereoisomer of said compound, or a salt of said N-oxide, tautomer or stereoisomer, wherein ring B fused to the pyrimidine ring is the following ring system:

r1, R4 and R10 are as defined above.

In a further embodiment of the above aspects, the invention relates to a compound of formula (I) wherein m is 1 and n is 1.

In a further embodiment of the above aspects, the invention relates to a compound of formula (I) wherein m is 1 and n is 2.

In a further embodiment of the above aspects, the invention relates to a compound of formula (I) wherein m is 2 and n is 2.

In a further embodiment of the above aspects, the invention relates to a compound of formula (I) wherein m is 1 and n is 1, or, m is 2 and n is 2.

In another embodiment, the invention relates to compounds of formula (I) wherein R1 is hydrogen, 1-4C-alkyl (optionally substituted by halogen, hydroxy, amino, mono-1-4C-alkylamino OR di-1-4C-alkylamino), halogen, trifluoromethyl, cyano, 3-7C-cycloalkyl, 2-4C-alkenyl, 2-4C-alkynyl, C (O) NR11R12, -C (O) OR 2.

In a further embodiment of the above aspects, the invention relates to compounds of formula (I) wherein R1 is hydrogen, 1-4C-alkyl (optionally substituted by halogen, hydroxy, amino, mono-1-4C-alkylamino or di-1-4C-alkylamino), halogen, trifluoromethyl, cyano or-C (O) NR11R 12.

In a further embodiment of the above aspects the invention relates to compounds of formula (I) wherein R1 is hydrogen, 1-4C-alkyl, halogen, trifluoromethyl, cyano or-C (O) NR11R 12.

In a further embodiment of the above aspects, the invention relates to compounds of the formula (I) in which R1 is hydrogen or 1-4C-alkyl, in particular hydrogen or methyl.

In a further embodiment of the above aspects, the invention relates to a compound of formula (I) wherein R4 is phenyl.

In a further embodiment of the above aspects, the invention relates to a compound of formula (I) wherein R6 is hydrogen.

In a further embodiment of the above aspects the invention relates to compounds of formula (I) wherein R8 is hydrogen, 1-4C-alkyl, 1-4C-haloalkyl, 3-7C-cycloalkyl, 1-4C-alkoxy, halogen, cyano or NR11R 12.

In a further embodiment of the above aspects, the invention relates to compounds of formula (I) wherein R8 is hydrogen, 1-4C-haloalkyl, halogen, cyano or amino, in particular R8 is hydrogen.

In a further embodiment of the above aspects, the invention relates to a compound of formula (I) wherein W is monocyclic 5-or 6-membered-heteroarylene, in particular 5-membered-heteroarylene.

In a further embodiment of the above aspects, the invention relates to a compound of formula (I) wherein W is 1,2, 4-triazolylene.

In yet another embodiment of the above aspects, the invention relates to a compound of formula (I) wherein W is a bicyclic heteroarylene comprising 1 nitrogen atom and optionally 1,2 or 3 additional heteroatoms independently selected from oxygen, nitrogen and sulfur, and wherein the bicyclic heteroarylene is optionally substituted with R8 and Y is hydrogen.

In a further embodiment of the above aspects, the invention relates to a compound of formula (I) wherein Y is monocyclic 5-or 6-membered heteroaryl comprising 1 nitrogen atom and optionally 1,2 or 3 additional heteroatoms independently selected from oxygen, nitrogen, sulfur, and wherein said heteroaryl is optionally substituted with R9, and R9 is as defined in any claim.

In another embodiment, Y is a 6-membered heteroaryl group comprising 1 nitrogen atom and optionally 1 or2 additional nitrogen atoms, in particular a pyridyl, pyrimidinyl or pyrazinyl group, more in particular a 2-pyridyl, 2-pyrimidinyl or 2-pyrazinyl group.

In a further embodiment of the above aspects, the invention relates to a compound of formula (I), wherein Y is pyridin-2-yl, pyrimidin-2-yl or pyrazin-2-yl optionally substituted with R9 and optionally further substituted with R9A, and R9 is 1-4C-alkyl, 1-4C-alkoxy, halogen, hydroxy, 1-4C-haloalkyl, NR11R12, cyano or-C (O) NH₂And R9A is 1-4C-alkyl or halogen.

In a further embodiment of the above aspects, the invention relates to compounds of formula (I) wherein Y is pyridin-2-yl optionally substituted with R9, and R9 is 1-4C-alkyl, 1-4C-alkoxy, halogen, hydroxy, 1-4C-haloalkyl, NR11R12, cyano or-C (O) NH₂。

In a further embodiment of the above aspects, the invention relates to compounds of the formula (I), wherein R9 is 1-4C-alkyl, 1-4C-alkoxy, halogen, hydroxy, 1-4C-haloalkyl, NR11R12, cyano or-C (O) NH₂Especially 1-4C-alkyl, 1-4C-haloalkyl or halogen, more especially CH₃、F、Cl、Br、CF₃。

In a further embodiment of the above aspects the invention relates to compounds of formula (I) wherein R9A is 1-4C-alkyl or halogen, especially 1-4C-alkyl, more especially CH₃。

In a further embodiment of the above aspects the invention relates to compounds of formula (I) wherein R10 is hydrogen or 1-4C-alkyl (optionally substituted by halogen, hydroxy, amino, mono-1-4C-alkylamino or di-1-4C-alkylamino), especially hydrogen or 1-4C-alkyl, more especially hydrogen or CH₃。

In a further embodiment of the above aspects the invention relates to compounds of formula (I), wherein R11, R12, which may be the same or different, are hydrogen, 1-4C-alkyl (optionally substituted by halogen, hydroxy, amino, mono-1-4C-alkylamino or di-1-4C-alkylamino) or 3-7C-cycloalkyl.

In a further embodiment of the above aspects, the invention relates to compounds of the formula (I), wherein R11, R12, which may be identical or different, are hydrogen or 1-4C-alkyl.

Definition of

1-4C-alkyl is a straight-chain or branched alkyl group having 1 to 4 carbon atoms. Examples are methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl.

The mono-1-4C-alkylamino or di-1-4C-alkylamino group contains, in addition to the nitrogen atom, 1 or2 of the abovementioned 1-4C-alkyl groups. Examples are methylamino, ethylamino, isopropylamino, dimethylamino, diethylamino and diisopropylamino groups.

The mono-1-4C-alkylaminocarbonyl or di-1-4C-alkylaminocarbonyl radical comprises, in addition to a carbonyl radical, one of the above-mentioned mono-1-4C-alkylamino or di-1-4C-alkylamino radicals. Examples are N-methylaminocarbonyl, N-dimethylaminocarbonyl, N-ethylaminocarbonyl, N-propylaminocarbonyl, N-diethylaminocarbonyl and N-isopropylaminocarbonyl.

Within the meaning of the present invention, halogen is iodine, bromine, chlorine or fluorine, preferably bromine, chlorine or fluorine, in the case of which it serves as a leaving group, preferably bromine or iodine.

1-4C-haloalkyl is a straight-chain or branched alkyl group having 1 to 4 carbon atoms in which at least 1 hydrogen is substituted by a halogen atom. Examples are chloromethyl or 2-bromoethyl. For partially or fully fluorinated C's included in the term "haloalkyl₁-C₄Alkyl groups, the following partially or fully fluorinated groups are considered, for example: fluoromethyl, difluoromethyl, trifluoromethyl, fluoroethyl, 1-difluoroethyl, 1, 2-difluoroethyl, trifluoromethyl, and the like,1, 1, 1-trifluoroethyl, tetrafluoroethyl and pentafluoroethyl, preferably trifluoromethyl.

1-4C-alkoxy denotes a group which, in addition to an oxygen atom, also comprises a linear or branched alkyl group having 1 to 4 carbon atoms. Examples which may be mentioned are the butoxy, isobutoxy, sec-butoxy, tert-butoxy, propoxy, isopropoxy, ethoxy and methoxy groups, preferably methoxy.

3-7C-cycloalkyl represents cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or cycloheptyl, preferably cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl.

3-7C-Cycloalkoxy represents cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy or cycloheptyloxy.

2-4C-alkenyl is a straight-chain or branched alkenyl group having 2 to 4 carbon atoms. Examples are but-2-enyl, but-3-enyl (homoallyl), prop-1-enyl, prop-2-enyl (allyl) and vinyl groups.

2-4C-alkynyl is a straight-chain or branched alkynyl group having 2 to 4 carbon atoms. Examples are but-2-ynyl, but-3-ynyl (homopropargyl), prop-1-ynyl, 1-methylprop-2-ynyl (1-methylpropargyl), prop-2-ynyl (propargyl) and ethynyl groups.

The term "monocyclic 5-or 6-membered heteroaryl" includes, but is not limited to: a 5-membered heteroaryl group, a furyl group, a thienyl group, a pyrrolyl group, an oxazolyl group, an isoxazolyl group, a thiazolyl group, an isothiazolyl group, an imidazolyl group, a pyrazolyl group, a triazolyl group (1, 2, 4-triazolyl group, 1, 3, 4-triazolyl group or 1,2, 3-triazolyl group), a thiadiazolyl group (1, 3, 4-thiadiazolyl group, 1,2, 5-thiadiazolyl group, 1,2, 3-thiadiazolyl group or 1,2, 4-thiadiazolyl group), and an oxadiazolyl group (1, 3, 4-oxadiazolyl group, 1,2, 5-oxadiazolyl group, 1,2, 3-oxadiazolyl group or 1,2, 4-oxadiazolyl group), as well as a 6-membered heteroaryl group, a pyridyl group, a pyrimidinyl group, a pyrazinyl group and a pyridazinyl group. Preferred 5-or 6-membered heteroaryl groups are furyl, thienyl, pyrrolyl, thiazolyl, oxazolyl, thiadiazolyl, oxadiazolyl, pyridyl, pyrimidinyl, pyrazinyl or pyridazinyl. More preferred 5-or 6-membered heteroaryl groups are furan-2-yl, thiophen-2-yl, pyrrol-2-yl, thiazolyl, oxazolyl, 1, 3, 4-thiadiazolyl, 1, 3, 4-oxadiazolyl, pyridin-2-yl, pyridin-4-yl, pyrimidin-2-yl, pyrimidin-4-yl, pyrazin-2-yl or pyridazin-3-yl.

The term "monocyclic 5-membered heteroarylene" is a divalent group in which any one hydrogen atom is removed from the above "heteroaryl" and may include, but is not limited to: a 5-membered heteroaryl group, a furanylene group, a thiophenylene group, a pyrrolylene group, an oxazolylene group, an isoxazolylene group, a thiazolyl ene group, an isothiazolylene group, an imidazolyl group, a pyrazolyl ene group, a triazolylene group (1, 2, 4-triazolylene group, 1, 3, 4-triazolylene group or 1,2, 3-triazolylene group), a thiadiazolylene group (1, 3, 4-thiadiazolylene group, 1,2, 5-thiadiazolylene group, 1,2, 3-thiadiazolylene group or 1,2, 4-thiadiazolylene group), and an oxadiazoylene group (1, 3, 4-oxadiazoylene group, 1,2, 5-oxadiazolene group, 1,2, 3-oxadiazolene group or 1,2, 4-oxadiazolene group). Preferred 5-membered heteroaryl groups are triazolylene, pyrazolyl or imidazolyl. More preferred 5-membered heteroaryl groups are 1,2, 4-triazolylene, pyrazolyl or imidazolyl, most preferably 1,2, 4-triazolylene.

The NR10R11 group includes an amino group (NH)₂) Monoalkylamino, dialkylamino, especially amino (NH)₂) mono-1-4C-alkylamino, di-1-4C-alkylamino, e.g. NH, N (H) CH₃、N(CH₃)₂、N(H)CH₂CH₃And N (CH)₃)CH₂CH₃。

Unless otherwise indicated, generally, the heteroaryl or heteroarylene group includes all possible isomeric forms thereof, e.g., positional isomers thereof. Thus, for some illustrative, non-limiting examples, the term pyridyl or pyridinylene includes pyridin-2-yl, pyridinylene-2-yl, pyridin-3-yl, pyridinylene-3-yl, pyridin-4-yl and pyridinylene-4-yl; alternatively, the term thienyl or thienylene includes thien-2-yl, thien-3-yl, and thien-3-yl.

Unless otherwise specified, components optionally substituted as described herein may be substituted one or more times independently of each other at any possible position. Similarly, it will be understood that for any heteroaryl group, if chemically suitable, the heteroaryl group may be attached to the rest of the molecule via any suitable atom. Unless otherwise indicated, the following substituents are preferred: methyl, ethyl, trifluoromethyl, fluorine, chlorine, bromine, methoxy, amino, methylamino, dimethylamino.

Unless otherwise indicated, heteroaryl or heteroarylene groups mentioned herein may be substituted at any possible position (e.g. at any substitutable ring carbon atom or ring nitrogen atom) by their particular substituent or parent molecular group.

Unless otherwise indicated, preferably, rings containing quaternizable amino-or imino-type ring nitrogen atoms (-N ═) may not be quaternized on these amino-or imino-type ring nitrogen atoms with the substituents or parent molecular groups mentioned.

Unless otherwise stated, it is assumed that any heteroatom of a heteroaryl or heteroarylene ring having an unsaturated valence mentioned herein has a hydrogen atom to saturate its valence.

When any variable occurs more than 1 time in any constituent, the respective definitions are independent.

Unless otherwise indicated, the term "suitable leaving group" means, for example, a sulfonic acid group, such as, i.e., a methanesulfonic acid group, a trifluoromethanesulfonic acid group or a p-toluenesulfonic acid group, or a halogen, such as iodine, bromine or chlorine.

The salts of the compounds according to the invention include all inorganic and organic acid addition salts and salts with bases, in particular all pharmaceutically acceptable inorganic and organic acid addition salts and salts with bases, especially all pharmaceutically acceptable inorganic and organic acid addition salts and salts with bases which are customary for pharmacy.

Examples of acid addition salts include, but are not limited to: hydrochloride, hydrobromide, phosphate, nitrate, sulphate, salt of sulphamic acid, formate, acetate, propionate, citrate, D-gluconate, benzoate, 2- (4-hydroxybenzoyl) benzoate, butyrate, salicylate, sulphosalicylate, lactate, maleate, laurate, malate, fumarate, succinate, oxalate, malonate, pyruvate, acetoacetate, tartrate, stearate, benzenesulfonate, tosylate, mesylate, triflate, 3-hydroxy-2-naphthoate, benzenesulfonate, napadisylate and trifluoroacetate.

Examples of salts with bases include, but are not limited to: lithium, sodium, potassium, calcium, aluminum, magnesium, titanium, meglumine, optionally derived from NH₃Or ammonium salts of organic amines having 1 to 16C atoms, such as salts of ethylamine, diethylamine, triethylamine, ethyldiisopropylamine, monoethanolamine, diethanolamine, triethanolamine, dicyclohexylamine, dimethylaminoethanol, procaine, dibenzylamine, N-methylmorpholine, arginine, lysine, ethylenediamine, N-methylpiperidine and guanidine.

The salts include water-insoluble salts, and in particular water-soluble salts.

The compounds of formula (I) and salts thereof of the present invention may comprise (e.g. when isolated in crystalline form) various amounts of solvent according to the skilled person. Accordingly, the scope of the present invention also includes all solvates, in particular all hydrates, of the compounds of formula (I) according to the invention, and all solvates, in particular all hydrates, of the salts of the compounds of formula (I) according to the invention.

The term "combination" in the present invention is used as known to the person skilled in the art and may exist in fixed combination, non-fixed combination or kit-of-parts form.

The "fixed combination" in the present invention is used as known to the person skilled in the art and is defined as a combination wherein the first active ingredient and the second active ingredient are present together in one unit dosage form or in a single entity. An example of a "fixed combination" is a pharmaceutical composition wherein the first active ingredient and the second active ingredient are present in the form of a mixture, e.g. a formulation, for simultaneous administration. Another example of a "fixed combination" is a pharmaceutical combination wherein the first active ingredient and the second active ingredient are present as non-mixed units.

The non-fixed combination or "kit" according to the invention is used as known to the person skilled in the art and is defined as a combination in which the first active ingredient and the second active ingredient are present in more than one unit. An example of a non-fixed combination or kit is a combination wherein the first active ingredient and the second active ingredient are present separately. The components of the ambulatory combination or kit are administered separately, sequentially, simultaneously, concurrently or chronologically staggered.

The term "(chemotherapeutic agent) anticancer agent" includes, but is not limited to, (i) alkylating/carbamylating agents, such as cyclophosphamide (Endoxan)) Ifosfamide (Holoxan)) Thiotepa Lederle) Melphalan (Alkeran)) Or chloroethylnitrosourea (BCNU); (ii) platinum derivatives, e.g. cisplatin (platinum)BMS), oxaliplatin (Eloxatin)) Satraplatin or carboplatin (Cabroplat)BMS); (iii) antimitotic/tubulin inhibitors, e.g. vinca alkaloids (vincristine, vinblastine, vinorelbine), taxanes, e.g. paclitaxel (Taxol)) Docetaxel (Taxotere)) And analogs and novel formulations and conjugates thereof (e.g., nanoparticle formulation Abraxane @)Wherein paclitaxel is bound to albumin), epothilones, such as epothilone B (Patupilone)) Azabepilone (Ixabepilone), Azaepothilone) Or ZK-EPO (fully synthetic epothilone B analogs); (iv) topoisomerase inhibitors, e.g. anthracyclines (e.g. doxorubicin/Adribilastin)) Epipodophyllotoxins (e.g. etoposide/Etopophos)) And camptothecin analogs (e.g., irinotecan/Camptosar)Or topotecan/Hycamtin) (ii) a (v) Pyrimidine antagonists, e.g. 5-fluorouracil (5-FU), capecitabine (Xeloda)) Cytosine/Cytosine Alexan) Or gemcitabine (Gemzar)) (ii) a (vi) Purine antagonists, e.g. 6-mercaptopurine (Puri-Nethol)) 6-thioguanine or fludarabine (Fludara)) And (vii) folic acid antagonists, e.g., methotrexate (Farmitrexat)) Or pemetrexed (Alimta))。

The term "target-specific anti-cancer agent" includes, but is not limited to, (i) kinase inhibitors, such as imatinib (Glivec)) ZD-1839/gefitinib (Iressa)) Bay43-9006 (sorafenib, Nexavar)) SU 11248/sunitinib (Sutent)) OSI-774/erlotinib (Tarceva)) Dasatinib (spraycel)) Lapatinib (Lapatinib) (Tykerb)) Or, see also below, Vatalanib, Vandanib (Vandanib), Vandanib (Zactima)) Or Pazopanib (Pazopanib); (ii) proteasome inhibitors, e.g. PS-341/bortezomib (Velcade)) (ii) a (iii) Histone deacetylase inhibitors, such as SAHA (Zolinza)) PXD101, MS275, MGCD0103, depsipeptide/FK 228, NVP-LBH589, valproic acid (VPA), CRA/PCI24781, ITF2357, SB939 and butyrate, (iv) heat shock protein 90 inhibitors such as 17-allylaminogeldanamycin (17-AAG) or 17-dimethylamino geldanamycin (17-DMAG); (v) vascular Targeting Agents (VTA) such as combretastatin A4 phosphate or AVE8062/AC7700 and anti-angiogenic agents, e.g., VEGF antibodies such as bevacizumab (Avastin)) Or KDR tyrosine kinase inhibitors such as PTK787/ZK222584 (Vatalanib)) Or vandetanib (Zactima)) Or pazopanib; (vi) monoclonal antibodies, e.g. trastuzumab (Herceptin)) Rituximab (MabThera/Rituxan)) Arbizumab (Campath)) Tositumomab (Bexxar)) C225/cetuximab (Erbitux)) Avastin (see above) or Panitumumab (Venitumumab) (Vectibix)) And variants and conjugates of monoclonal antibodies such as gemtuzumab ozogamicin (Mylotarg)) Or ibritumomab tiuxetan (Zevalin)) And antibody fragments; (vii) oligonucleotide-based therapeutic agents such as G-3139/Obblimers (Genasense)) Or the DNMT1 inhibitor MG 98; (viii) toll-like receptor/TLR 9 agonists such as PromuneTLR 7 agonists such as imiquimod (Aldara)) Or isatoribine and analogs thereof, or TLR 7/8 agonists such as resiquimod, and immunostimulatory RNAs such as TLR 7/8 agonists; (ix) proteinAn enzyme inhibitor; (x) Hormonal therapeutic agents, for example antiestrogens (such as tamoxifen or raloxifene), antiandrogens (such as flutamide or Casodex), LHRH analogues (such as leuprolide, goserelin or triptorelin) and aromatase inhibitors (such as Femara, Arimedex or Aromasin).

Other "target-specific anti-cancer agents" include: bleomycin, retinoids such as all-trans retinoic acid (ATRA), DNA methyltransferase inhibitors such as 5-aza-2' -deoxycytidine (decitabine, Dacogen)) And 5-azacytidine (Vidaza)) Atranol, cytokines such as interleukin-2, interferons such as interferon alpha 2 or interferon gamma, bcl2 antagonists (e.g., ABT-737 or the like), death receptor agonists such as TRAIL, DR4/5 agonistic antibodies, FasL, and TNF-R agonists (e.g., TRAIL receptor agonists such as mapatumamab (mapatumamab) or lexalimumab (lexatuumamab)).

Specific examples include, but are not limited to, 5FU, actinomycin D, abarelix, abciximab, aclarubicin, adapalene, alemtuzumab, altretamine, aminoglutethimide, apremide, amrubicin, anastrozole, ancitabine, artemisinin, azathioprine, basiliximab, bendamustine, bevacizumab, BEXXAR, bicalutamide, bleomycin, bortezomib, uridine, busulfan, CAMPATH, capecitabine, carboplatin, carboquinone, carmustine, cetrorelix, chlorambucil, nitrogen mustard, cisplatin, cladribine, clomiphene, cyclophosphamide, dacarbazine, daclizumab, dactinomycin, dasatinib, daunorubicin, decitabine, delorelin, dexrazoxane, docetaxel, floxuridine, doxorubicin, droxifene, tasapride, etasone, efonin, efletirimate, efluorefluorin, efluoromethionine, efluoride, eflomicrin, etil, efloxapigenin, etil, doxycycline, and pharmaceutically acceptable salts, Epirubicin, epithisterol, eptaplatin, ERBITUX, erlotinib, estramustine, etoposide, exemestane, fadrozole, finasteride, floxuridine, flucytosine, fludarabine, fluorouracil, flutamide, formestane, foscarnet, fosfestrol, fotemustine, fulvestrant, gefitinib, GENASENSE, gemcitabine, GLIVEC, goserelin, guanolimus, HERCEPTIN, idarubicin, idoxuridine, ifosfamide, imatinib, improsulfasin, infliximab, irinotecan, IXABEPILONE, lanreotide, lapatinib, letrozole, LEUPROLIDE (LEUPROLIDE), lobaplatin, lomustine, roripreline (LEUPROLIDE), melphalan, mercaptopurine, methotrexate, metripiprepidoplatin, platinum, timifenesione, timothipristone, tebufenozide, tebuformone, tefuramisole, mizoriginol, mitomycin, and other, MYLOTARG, narcotinib, NEBAZUMAB, nedaplatin, nilutamide, nimustine, octreotide, oxymetafilne, oxaliplatin, paclitaxel, palivizumab, panitumumab, PATUPILONE, pazopanib, pemetrexed, pefilgratin, pemetrexed, pentostatin, phosphoramide, piposulfan, pirarubicin, plicamycin, prednimustine, procarbazine, propafenib, propiospirammonium, raloxifene, ranitidine, ranimustine, ranpirnase, lanreose, ranizosin, rituximab, rifampin, ritroscovitine, romurtopeptide, RUBOXIURIN, sargramostim, satraplatin, sirolimus, sorafenib, spiromustine, streptozocin, sunitinib, tamoxifen, temozine, tipepinasil, tegafur, tezomepinasin, teflunomide, tebucindomethacin, texate, tebucindomethacin, tioxate, tioxaposide, temoposide, temab, temoposide, temab, temopo, Toremifene, TRAIL, trastuzumab, troosulfan, triimidyl, tritrexate, triptorelin, tramadol, uredepa, valrubicin, vatalanib, vandetanib, verteporfin, vinblastine, vincristine, vindesine, vinorelbine, vorozole, ZEVALIN, and ZOLINZA.

The compounds of the present invention and salts thereof may exist in tautomeric forms, tautomers being included in embodiments of the present invention. In particular, for example, those compounds of the invention comprising a pyrazole group may exist in the form of a 1H tautomer or a 2H tautomer, or even in the form of a mixture of any amount of these two tautomers, or, for example, those compounds comprising a triazole group may exist in the form of a 1H tautomer, a 2H tautomer or a 4H tautomer, or even in the form of a mixture of any amount of said 1H, 2H and 4H tautomers:

the compounds of the present invention and their salts include stereoisomers. Each stereocenter (stereogenic center) present in the stereoisomer may have either the absolute configuration R or the absolute configuration S (according to the Cahn-Ingold-Prelog rule). Thus, in formula (Ia)^*) In the case of the compounds of (1), the stereoisomers (1S) and (1R) and their salts are part of the present invention.

The invention also includes all mixtures of the above stereoisomers in any ratio, including racemates.

Some of the compounds and salts of the present invention may exist in different crystalline forms (polymorphs) which are within the scope of the present invention.

In addition, the present invention includes derivatives of the compounds of formula (I) and salts (bioprecursors or prodrugs) thereof that are converted in a biological system to the compounds of formula (I) or salts thereof. The biological system is, for example, a mammalian organism, particularly a human individual. The biological precursor is converted, for example by metabolic processes, into a compound of formula (I) or a salt thereof.

Intermediates for the synthesis of compounds according to claims 1 to 4 as described below and their use for the synthesis of compounds according to claims 1 to 4 are further aspects of the present invention.

The compounds of the present invention can be prepared as follows.

Reaction scheme 1

As shown in scheme 1, compounds of formula (I), wherein B, R4, R7, m, n and R10 have the above-mentioned meanings and R6 is hydrogen or 1-4C-alkyl, can be obtained by reductive amination of the corresponding compounds of formula (III), wherein R has the meaning of-C (O) R6, with amine derivatives of formula (II), wherein R7 has the above-mentioned meaning. Can be carried out according to standard methods, for example by using NaBH (OAc)₃Or NaBH₃CN is subjected to said reductive amination in a suitable solvent such as 1, 2-Dichloroethane (DCE), Tetrahydrofuran (THF), N-methylpyrrolidone (NMP), Dimethylformamide (DMF), methanol or suitable mixtures of the above solvents.

Amine derivatives of formula (II) (wherein R7, m and n have the above-mentioned meanings) are known or can be prepared according to known methods (in some cases they may contain one or more protecting groups to protect other functional groups, such as, but not limited to, NH functional groups). The amine derivative of formula (II) may be prepared as a suitable salt, for example as the hydrochloride salt, which may be the monohydrochloride or the dihydrochloride. The reaction using the salt of the amine derivative of formula (II) requires the addition of a suitable base, such as triethylamine. Unless otherwise stated, in order to calculate the amount of base required for the reaction using the salt of the amine derivative of formula (II), it is assumed that the salt of the amine derivative of formula (II) is a divalent salt, such as a dihydrochloride.

The use of a compound of formula (II) or a salt thereof for the synthesis of a compound according to claims 1-4 is an aspect of the present invention.

The compounds of the formula (III), in which R has the meaning of-C (O) H, can be obtained in a 1-step process or in a 2-step process from the corresponding compounds of the formula (III), in which R has the meaning of-C (O) O (1-4C-alkyl). The ester groups are selectively reduced to aldehyde groups in a 1-step process according to methods known to those skilled in the art, for example by using DIBALH at low temperatures, for example-80 ℃ to-60 ℃. Alternatively, in a 2-step process, according to known methods, for example by using LiAlH₄Or NaBH₄Reducing the ester group to an alcohol group (-CH)₂OH) and then following methods known to those skilled in the art, for example with SO₃The resulting alcohol is selectively oxidized to the-C (O) H group by pyridine complex or Dess-Martin reagent (Dess-Martin Periodinane).

Alternatively, as an alternative to the above reaction sequence, the compounds of formula (I), wherein B, R4, R7, m, n and R10 have the above meaning and R6 is hydrogen or 1-4C-alkyl, can be obtained by reaction of the corresponding compounds of formula (IIIa), wherein X is a suitable leaving group, such as a halogen atom or a sulfonate, with amine derivatives of formula (II), wherein R7, m and n have the above meaning. This reaction is preferably carried out in an inert solvent such as DMF at a temperature of 60-100 ℃ in the presence of a base such as triethylamine.

The compounds of formula (IIIa), wherein X is a suitable leaving group, for example a halogen atom, can be obtained from the corresponding compounds of formula (III), wherein R is-CH (R6) OH and R6 is hydrogen or 1-4C-alkyl, by halogenation reactions. Such halogenation reactions can be achieved, for example, by using PBr₃In dichloromethane.

Alternatively, a compound of formula (IIIa), wherein X is a suitable leaving group, e.g. a halogen atom, may be prepared from the corresponding compound of formula (III), wherein R is-CH, by benzylic halogenation₂R6, and R6 is hydrogen or 1-4C-alkyl). Benzylic halogenation can be carried out, for example, by using N-bromosuccinimide (NBS).

Alternatively, compounds of formula (IIIa), wherein X is a suitable leaving group, for example a sulfonate such as mesylate, triflate or p-toluenesulfonate, may be obtained from the corresponding compounds of formula (III), wherein R is-CH (R6) OH and R6 is hydrogen or 1-4C-alkyl, by sulfonylation. Such sulfonylation may be carried out, for example, by using a suitable sulfonic anhydride or sulfonyl halide such as methanesulfonyl chloride in the presence of a suitable base such as triethylamine in a suitable solvent such as dichloromethane or dimethylformamide or a mixture thereof.

Compounds of the formula (III), in which R is-CH (R6) OH and R6 is hydrogen or 1-4C-alkyl, can be prepared, for example, according to methods known to the person skilled in the art, for example by reacting NaBH₄Or LiAlH₄Reduction, obtained from the corresponding compound of formula (III) wherein R is-C (O) R6.

Alternatively, compounds of formula (III) wherein R is-CH (R6) OH and R6 is hydrogen or 1-4C-alkyl can be prepared from the corresponding compounds of formula (III) wherein R is-CH (R6) OH by benzylic oxidation (benzylation)₂R6), which benzylic oxidation can be achieved, for example, by using catalytic or equimolar amounts of SeO₂The process is carried out.

In yet another alternative embodiment, a compound of formula (III), wherein R is-CH (1-4C-alkyl) OH, may be obtained from the corresponding compound of formula (III), wherein R is-C (O) H, by the addition of a suitable organometallic reagent, such as, but not limited to, a Grignard reagent or a lithium reagent.

For the reactions in scheme 1, to synthesize compounds of formula (III) wherein B, R4 and R10 have the above-mentioned meanings and R is-c (o) R6 or-CH (R6) OH, some or all of the precursors may be optionally protected with suitable protecting groups known to those skilled in the art. Deprotection of a compound of formula (III) wherein B, R4 and R10 have the above meanings and R is a protected ketone, aldehyde or alcohol group, can be carried out according to art-known methods of deprotecting groups to give the corresponding deprotected compound.

Compounds of the formula (IIIb) (in which R1, R4 and R10 have the abovementioned meanings and R has-C (O) O (1-4C-alkyl), -C (O) R6, -CH (R6) OH or-CH) can be obtained as shown in scheme 2₂R6 and R6 is hydrogen or 1-4C-alkyl).

Reaction scheme 2

Compounds of the formula (III), in which R1, R4 and R10 have the abovementioned meanings and R has the meaning-C (O) O (1-4C-alkyl), -C (O) R6, -CH (R6) OH or-CH 2R6 and R6 is hydrogen or 1-4C-alkyl, are corresponding compounds of the formula (IV), in which X1 is Cl, Br, I or-OS (O)₂CF₃And Ra and Rb independently have the meanings as defined above for R1) with a corresponding compound of formula (V) in which M is for example-b (oh)₂-Sn (1-4C-alkyl)₃-ZnCl, -ZnBr, -ZnI, or

If M has the formula-B (OH)₂The transition metal-catalyzed C-C bond-forming reaction can be carried out, for example, in dioxane and Cs₂CO₃In a mixture of aqueous solutions at a temperature of from 60 ℃ to the boiling point of the solvent, preferably at 115 ℃, and by using a Pd catalyst such as, but not limited to, 1' -bis (diphenylphosphino) ferrocene]Palladium dichloride or Pd (PPh)₃)₄The process is carried out.

The compounds of the formula (IV), in which R4, R10, Ra, Rb and X1 have the abovementioned meanings, are obtainable by condensation of compounds of the general formula (VI), in which R4, R10 and X1 have the abovementioned meanings, with aldehydes or ketones of the formula (VII), in which Ra and Rb have the abovementioned meanings and X2 is Cl, Br or I. The condensation reaction may be carried out in a suitable solvent, such as ethanol, at elevated temperature, such as 100 ℃. The elevated temperature may be obtained by conventional heating or by using microwave radiation. The aldehydes or ketones of the general formula (VII) are commercially available or can be prepared according to methods known to the person skilled in the art.

The compounds of the general formula (VI) in which R4, R10 and X1 have the abovementioned meanings can be obtained from the compounds of the general formula (VIII) by amination. The amination reaction can be carried out by reacting the compound of the formula (VIII) with a suitable ammonia source, for example a solution of ammonia in ethanol or aqueous ammonia, which can be used as a solvent for the reaction, in a suitable solvent, for example ethanol, at elevated temperature, for example 100-120 ℃.

The compounds of the formula (VIII) in which R4, R10 and X1 have the abovementioned meanings can be prepared by halogenation, for example by reaction with POCl₃(in the case where X1 has the meaning of Cl), POBr₃(in the case where X1 has the meaning of Br) from a compound of formula (IX).

The compounds of formula (IX), wherein R4 and R10 have the above-mentioned meanings, can be obtained by condensation reaction of a compound of formula (X) with an ester of formula R10-c (o) ary in the presence of a suitable base, such as NaOEt, in a suitable solvent, such as ethanol, at elevated temperature, such as 50 ℃. Compounds of formula (IX) or R10-C (O) ORy are commercially available or can be prepared according to methods known to those skilled in the art.

Compounds of the formula (IIIc) (in which R1, R4 and R10 have the abovementioned meanings and R has-C (O) O (1-4C-alkyl), -C (O) R6, -CH (R6) OH or-CH) can be obtained as shown in scheme 3₂R6 and R6 is hydrogen or 1-4C-alkyl).

Reaction scheme 3

Compounds of the formula (III), in which R1, R4 and R10 have the abovementioned meanings and R has the meaning-C (O) O (1-4C-alkyl), -C (O) R6, -CH (R6) OH or-CH₂R6 and R6 is hydrogen or 1-4C-alkyl) by transition metal catalysis of the corresponding compounds of the formula (XI) in which X1 is Cl, Br, I or-OS (O)₂CF₃) With a corresponding compound of formula (V) in which M is, for example-B(OH)₂-Sn (1-4C-alkyl)₃-ZnCl, -ZnBr, -ZnI, or

Compounds of formula (XI) can be prepared by reacting a compound of formula (XII) with a compound of formula R1(ORy)₃Wherein R1 has the meaning given above and Ry is 1-4C-alkyl. The condensation reaction may be carried out in a suitable solvent at elevated temperature (e.g. the boiling point of the solvent) where the orthoester may be used as the solvent for the reaction.

Compounds of the general formula (XII), wherein R4, R10 and X1 have the above-mentioned meanings, can be obtained from compounds of the general formula (VIII) by reaction with a suitable hydrazine source, such as hydrazine hydrate. The reaction may be carried out by reacting a compound of formula (VIII) with the hydrazine source in a suitable solvent, such as ethanol, at elevated temperature, such as 50 ℃.

Alternatively, the compounds of formula (I) (wherein R1 ═ halogen) may be further functionalized to provide further compounds of formula (I) (wherein R1 is 1-4C-alkyl, cyano, 3-7C-cycloalkyl, 2-4C-alkenyl, 2-4C-alkynyl, -C (o) OR2, OR monocyclic 5-OR 6-membered heteroarylene). This conversion can be achieved by metal-mediated reactions well known to those skilled in the art, such as those described above.

A preferred aspect of the present invention is a process for the preparation of the compounds of claims 1-4 according to the examples.

Optionally, the compound of formula (I) may be converted into a salt thereof, or, optionally, a salt of the compound of formula (I) may be converted into the free compound. The corresponding methods are conventional to the person skilled in the art.

It is known to the person skilled in the art that if several reaction centers are present on the starting materials or intermediate compounds, it may be necessary to temporarily shield one or more reaction centers by protecting groups in order to allow the reaction to proceed specifically at the desired reaction center. Detailed descriptions of the use of a number of proven Protecting Groups are found, for example, in T.W. Greene, Protective Groups in Organic Synthesis, John Wiley & Sons, 1999, 3 rd edition, or P.Kocienski, Protective Groups, Thieme Medical Publishers, 2000.

The compounds of the invention are isolated and purified according to methods known in the art, for example by evaporation of the solvent in vacuo and recrystallization of the resulting residue from a suitable solvent, or by subjecting it to a conventional purification method, for example column chromatography on a suitable support material.

The salts of the compounds of formula (I) of the present invention can be obtained by dissolving the free compound in a suitable solvent (e.g., a ketone such as acetone, methyl ethyl ketone or methyl isobutyl ketone, an ether such as diethyl ether, tetrahydrofuran or dioxane, a chlorinated hydrocarbon such as dichloromethane or chloroform, or a low molecular weight aliphatic alcohol such as methanol, ethanol or isopropanol) containing the desired acid or base, or thereafter adding the desired acid or base thereto. The acids or bases can be used in equimolar quantitative ratios or ratios different therefrom, depending on whether mono-or poly-acids or bases are considered and on which salt is desired. The salt is obtained by filtration, reprecipitation, precipitation with a non-solvent for the salt, or by evaporation of the solvent. The resulting salt may be converted to the free compound, which in turn may be converted to a salt. Thus, for example, pharmaceutically unacceptable salts which can be obtained as process products in industrial scale manufacture can be converted into pharmaceutically acceptable salts according to methods known to those skilled in the art.

Optionally, the compound of formula (I) may be converted to its N-oxide. The N-oxide may also be introduced via an intermediate. The N-oxide may be prepared by treating the appropriate precursor with an oxidizing agent (e.g., m-chloroperbenzoic acid) in an appropriate solvent (e.g., dichloromethane) at an appropriate temperature (e.g., 0 ℃ to 40 ℃, with room temperature generally preferred). Other corresponding methods of forming the N-oxide are conventional to those skilled in the art.

Pure diastereomers and pure enantiomers of the compounds of the invention and salts thereof may be obtained, for example, by asymmetric synthesis, by use of chiral starting compounds in the synthesis, and by resolution of enantiomeric and diastereomeric mixtures obtained in the synthesis.

Enantiomeric and diastereomeric mixtures can be resolved into the pure enantiomers and pure diastereomers according to methods known to those skilled in the art. Preferably, the diastereomer mixture is separated by crystallization, in particular fractional crystallization, or by chromatography. For example, enantiomeric mixtures may be separated by forming diastereomers with a chiral auxiliary, resolving the resulting diastereomers, and removing the chiral auxiliary. As chiral auxiliary, the enantiomeric bases can be separated by the formation of diastereomeric salts, for example, chiral acids such as mandelic acid, and chiral bases can be used to separate the enantiomeric acids. Alternatively, diastereomeric derivatives such as diastereomeric esters can be formed from mixtures of enantiomers of alcohols or mixtures of enantiomers of acids, respectively, by using chiral acids or chiral alcohols, respectively, as chiral auxiliary agents. Furthermore, diastereomeric complexes or diastereomeric clathrates can be used to separate enantiomeric mixtures. Alternatively, mixtures of enantiomers can be resolved in chromatography using chiral separation columns. Another suitable method of separating the enantiomers is enzymatic separation.

It will be understood by those skilled in the art that the present invention is not limited to the particular embodiments described herein, but covers all modifications of the described embodiments which come within the spirit and scope of the invention as defined by the appended claims.

Commercial use

The compounds of formula (I) and stereoisomers of the compounds of formula (I) of the present invention are hereinafter referred to as the compounds of the present invention. In particular, the compounds of the present invention are pharmaceutically acceptable. The compounds of the present invention possess valuable pharmaceutical properties which render them commercially useful. In particular, they inhibit the Pi3K/Akt pathway and exhibit cellular activity. They are expected to be commercially useful in the treatment of diseases (e.g., diseases that rely on Pi3K/Akt being overactivated).

Abnormal activation of the PI3K/AKT pathway is an essential step in initiating and maintaining human tumors, and therefore, its inhibition (e.g., with AKT inhibitors) is considered an effective method for treating human tumors. For a recent review see Garcia-Echeverria et al (Oncogene, 2008, 27, 551-.

Cell activity and similar terms are used in the present invention as known to those skilled in the art, e.g., induction of apoptosis or chemosensitization.

Chemosensitization and similar terms are used in the present invention as known to those skilled in the art. These stimuli include, for example, effectors of death receptors and survival pathways, as well as cytotoxic/chemotherapeutic and targeting agents, and finally radiotherapy. According to the present invention, induction of apoptosis and like terms are used to identify compounds that perform programmed cell death in cells contacted therewith or in combination with other compounds conventionally used in therapy.

Apoptosis is used in the present invention as known to those skilled in the art. Induction of apoptosis in cells contacted with a compound of the invention may not necessarily be associated with inhibition of cell proliferation. Preferably, the inhibition of proliferation and/or induction of apoptosis is specific for cells with abnormal cell growth.

In addition, the compounds of the invention inhibit protein kinase activity in cells and tissues, leading to the transfer to dephosphorylated substrate proteins and, as a functional consequence thereof, for example, to the induction of apoptosis, cell cycle arrest and/or sensitization to chemotherapeutic and target-specific cancer drugs. In a preferred embodiment, inhibition of the Pi3K/Akt pathway induces the cellular effects mentioned herein, either alone or in combination with standard cytotoxic drugs or targeted cancer drugs.

The compounds of the invention exhibit anti-proliferative and/or pro-apoptotic and/or chemosensitizing properties. Thus, the compounds of the present invention are useful for the treatment of hyperproliferative disorders, particularly cancer. Thus, the compounds of the present invention are useful for producing anti-proliferative and/or pro-apoptotic and/or chemosensitizing effects in a mammal, such as a human, suffering from a hyperproliferative disorder, such as cancer.

The compounds of the invention exhibit anti-proliferative and/or pro-apoptotic properties in mammals, such as humans, by inhibiting the metabolic activity of cancer cells that are capable of surviving even under adverse growth conditions (e.g., glucose deprivation, hypoxia, or other chemical stress).

Thus, the compounds of the present invention can be used for the treatment, amelioration or prevention of diseases with benign or malignant manifestations as described herein, e.g. for the inhibition of cell tumors.

The tumor is used in the present invention as known to those skilled in the art. Benign tumors are defined as hyperproliferation of cells that fail to form aggressive, metastatic tumors in vivo. Conversely, a malignant tumor is defined as a cell with various cellular and biochemical abnormalities capable of forming a systemic disease (e.g., forming tumor metastases in distant organs).

Preferably, the compounds of the present invention are useful for the treatment of malignant tumors. Examples of malignancies that can be treated with the compounds of the present invention include solid tumors and hematological tumors. Solid tumors may be, for example, tumors of the breast, bladder, bone, brain, central and peripheral nervous system, colon, endocrine glands (such as thyroid and adrenal cortex), esophagus, endometrium, germ cells, head and neck, kidney, liver, lung, larynx and hypopharynx, mesothelioma, ovary, pancreas, prostate, rectum, kidney, small intestine, soft tissue, testis, stomach, skin, ureter, vagina and vulva. Malignant tumors include hereditary cancers such as retinoblastoma and nephroblastoma (Wilms tumor). Furthermore, malignant tumors include primary tumors in the organs and corresponding secondary tumors in distant organs (tumor metastases). Hematological tumors can be, for example, aggressive and indolent forms of leukemia and lymphoma, i.e., non-hodgkin's disease, chronic and acute myeloid leukemia (CML/AML), Acute Lymphocytic Leukemia (ALL), hodgkin's disease, multiple myeloma, and T-cell type lymphoma. Also included are myelodysplastic syndromes, plasmacytomas, paraneoplastic syndromes and cancers of unknown primary site and AIDS-related malignancies.

It should be noted that malignant tumors do not necessarily require the formation of metastases in the distal organs. Some tumors exert devastating effects on the primary organ itself through their aggressive growth properties. These can lead to structural destruction of tissues and organs, ultimately resulting in failure of function and death of the organ in question.

Drug resistance is particularly important for frequent failure of standard cancer therapies. This resistance is caused by various cellular and molecular mechanisms. One aspect of drug resistance is caused by constitutive activation of anti-apoptotic survival signals by PKB/Akt, a key signal transduction kinase. Inhibition of the Pi3K/Akt pathway results in re-sensitization to standard chemotherapeutic drugs or target-specific cancer therapeutics. Thus, the commercial use of the compounds of the present invention is not limited to first-line treatment of cancer patients. In preferred embodiments, cancer patients resistant to cancer chemotherapeutic agents or target-specific anticancer agents are also amenable to treatment with these compounds, e.g., for 2-line or 3-line treatment cycles. In particular, the compounds of the present invention can be used in combination with standard chemotherapeutic or targeting drugs to re-sensitize tumors to these agents.

The compounds of the present invention are characterized by unexpectedly valuable and desirable effects associated therewith, e.g., excellent therapeutic window, excellent bioavailability (e.g., good oral absorption), low toxicity, and/or other beneficial effects associated with their therapeutic and pharmaceutical properties, in terms of their properties, functions, and uses mentioned herein.

The compounds of the invention are useful for the treatment, prevention or amelioration of diseases having the above-mentioned benign and malignant manifestations, such as benign or malignant neoplasms, in particular cancer, especially cancer that is sensitive to inhibition of the Pi3K/Akt pathway.

The invention also includes the use of the compounds for treating, preventing or ameliorating a mammal, including a human, suffering from one of the conditions, disorders or diseases described above. Said use is characterized by administering to a subject in need of such treatment a pharmacologically active and therapeutically effective and tolerable amount of one or more compounds of the invention.

The invention also includes the use of such compounds for the treatment, prevention or amelioration of diseases responsive to inhibition of the Pi3K/Akt pathway in a mammal, including a human, which comprises administering to such mammal a pharmacologically active and therapeutically effective and tolerable amount of one or more of the compounds of the invention.

The invention also includes the use of the compounds for the treatment of hyperproliferative diseases with benign or malignant manifestations and/or disorders responsive to induction of apoptosis (e.g., cancer, particularly any of those cancer diseases described above) in a mammal comprising administering to said mammal a pharmacologically active and therapeutically effective and tolerable amount of one or more of the compounds of the invention.

The invention also includes the use of the compounds for inhibiting cellular hyperproliferation or arresting abnormal cell growth in a mammal comprising administering to said mammal a pharmacologically active and therapeutically effective and tolerable amount of one or more compounds of the invention.

The invention also comprises the use of said compounds for inducing apoptosis in the treatment of benign or malignant neoplasia, in particular cancer, comprising administering to a subject in need of such treatment a pharmacologically active and therapeutically effective and tolerable amount of one or more compounds of the invention.

The invention also includes the use of the compounds for inhibiting protein kinase activity in a cell, comprising administering to a patient in need of such treatment a pharmacologically active and therapeutically effective and tolerable amount of one or more compounds of the invention.

The invention also includes the use of the compounds for sensitizing to a chemotherapeutic agent or a target-specific anti-cancer agent in a mammal comprising administering to the mammal a pharmacologically active and therapeutically effective and tolerable amount of one or more of the compounds of the invention.

The invention also includes the use of such compounds for the treatment of benign and/or malignant neoplasms, particularly cancer, in a mammal, including a human, comprising administering to such mammal a pharmacologically active and therapeutically effective and tolerable amount of one or more of the compounds of the present invention.

The invention also relates to the use of said compounds for producing pharmaceutical compositions for the treatment, prevention and/or amelioration of one or more of the diseases mentioned.

The invention also relates to the use of said compounds for the preparation of a pharmaceutical composition for the treatment, prevention or amelioration of hyperproliferative diseases and/or disorders responsive to the induction of apoptosis, such as benign or malignant tumors, in particular cancer.

The invention also relates to the use of a compound of the invention for the preparation of a pharmaceutical composition for the treatment, prevention or amelioration of benign or malignant neoplasia, in particular cancer, such as any of those cancer diseases described above.

The invention also relates to a compound of the invention or a pharmaceutically acceptable salt thereof for use in the treatment and/or prevention of (hyper) proliferative diseases and/or disorders responsive to induction of apoptosis, including benign neoplasia and malignant neoplasia, including cancer.

The invention also relates to the use of a compound of the invention or a pharmaceutically acceptable salt thereof for the preparation of a pharmaceutical composition for the treatment, prevention or amelioration of a disease mediated by a dysfunction of a protein kinase or protein kinases and/or a condition responsive to induction of apoptosis.

The invention also relates to pharmaceutical compositions comprising a compound of the invention or a pharmaceutically acceptable salt thereof for the treatment and/or prevention of (hyper) proliferative diseases and/or disorders responsive to induction of apoptosis, including benign neoplasia and malignant neoplasia, including cancer.

The invention also relates to the use of the compounds and pharmaceutically acceptable salts of the invention for the preparation of pharmaceutical compositions useful for sensitizing against chemotherapeutic anti-cancer agents and/or target-specific anti-cancer agents.

The invention also relates to the use of a compound of the invention for the preparation of a pharmaceutical composition useful for sensitization against radiotherapy of those diseases described herein, in particular cancer.

The invention also relates to the use of a compound of the invention for the preparation of a pharmaceutical composition useful for the treatment of a disease that is sensitive to treatment with a protein kinase inhibitor and that is different from a cell tumour. These non-malignant diseases include, but are not limited to, benign prostatic hyperplasia, neurofibromatosis, skin disorders, and myelodysplastic syndromes.

The invention also relates to pharmaceutical compositions comprising one or more compounds of the invention and a pharmaceutically acceptable carrier or diluent.

The invention also relates to pharmaceutical compositions comprising one or more compounds of the invention and pharmaceutically acceptable adjuvants and/or excipients.

The pharmaceutical compositions of the present invention are prepared according to methods known in the art and well known to those skilled in the art. As pharmaceutical compositions, the compounds of the invention (═ active compounds) are used either alone or, preferably, in combination with suitable pharmaceutical auxiliaries and/or excipients, for example in the form of tablets, coated tablets, dragees, pills, cachets, granules, capsules, caplets (caplets), suppositories, patches (e.g. as TTS), emulsions (e.g. microemulsions or lipid emulsions), suspensions (e.g. nanosuspensions), gels, solubilizers (solublisates) or solutions (e.g. sterile solutions), or encapsulated in liposomes or in the form of β -cyclodextrins or β -cyclodextrin derivative inclusion complexes or the like, the active compounds advantageously being present in an amount of from 0.1% to 95%, and where, by appropriate selection of the auxiliaries and/or excipients, a pharmaceutical administration form (e.g. a sustained release form or an enteric form) can be obtained which is well suited to the active compound and/or to the desired onset of action In solution form).

The person skilled in the art is familiar, on account of his/her expert knowledge, with adjuvants, vehicles, excipients, diluents, carriers or auxiliaries which are suitable for the desired pharmaceutical preparations, formulations or compositions. In addition to solvents, gel-forming agents, ointment bases and other active formulating excipients, such as antioxidants, dispersants, emulsifiers, preservatives, solubilizers (e.g. glycerol polyoxyethylene triricinoleate 35, PEG 400, Tween 80, Captisol, Solutol HS15, etc.), colorants, complexing agents, penetration enhancers, stabilizers, fillers, binders, thickeners, disintegrants, buffers, pH adjusting agents (e.g. to obtain a neutral, basic or acidic formulation), polymers, lubricants, coating agents, propellants, tonicity adjusting agents, surfactants, flavoring agents, sweeteners or dyes, may also be used.

In particular, adjuvants and/or excipients of the type suitable for the desired formulation and the desired mode of administration are used.

The compounds, pharmaceutical compositions or combinations of the present invention may be administered in any of the recognized modes of administration available in the art. Illustrative examples of suitable modes of administration include intravenous, oral, nasal, parenteral, topical, transdermal and rectal. Oral administration and intravenous administration are preferred.

In general, the pharmaceutical compositions of the present invention can be administered such that the dosage of the active compound is within the conventional range for inhibitors of the Pi3K/Akt pathway. In particular, the dose of the active compound is preferably 0.01 to 4000 mg/day for an average adult patient weighing 70 kg. In this connection, it should be noted that the dosage depends, for example, on the particular compound used, the species treated, the age, body weight, general health, sex and diet of the individual treated, the mode and time of administration, the rate of excretion, the severity of the disease to be treated and the drug combination.

The pharmaceutical composition may be administered in a single dose per day or in multiple sub-doses per day, for example 2-4 doses per day. Individual dosage units of the pharmaceutical composition may contain, for example, from 0.01mg to 4000mg, preferably from 0.1mg to 2000mg, more preferably from 0.5 to 1000mg, most preferably from 1 to 500mg of the active compound. In addition, the pharmaceutical compositions may be adapted for weekly, monthly, or even less frequent administration, for example by using implants (e.g. subcutaneous or intramuscular implants), by using the active compound in the form of a sparingly soluble salt, or by using an active compound coupled to a polymer.

The choice of the optimal dosage regimen and the duration of the drug treatment, in particular the optimal dosage of the active compound and the mode of administration required in each case, can be determined by the person skilled in the art.

The present invention also relates to a combination comprising one or more first active ingredients selected from the compounds of the present invention, and one or more second active ingredients selected from chemotherapeutic anti-cancer agents and target-specific anti-cancer agents, e.g. for use in the treatment, prevention or amelioration of diseases that are responsive or sensitive to inhibition of the Pi3K/Akt pathway, e.g. hyperproliferative diseases with benign or malignant manifestations and/or disorders responsive to induction of apoptosis, in particular cancer, e.g. any of those cancer diseases mentioned above.

The invention also relates to the use of a pharmaceutical composition comprising one or more compounds of the invention as the only active ingredient or ingredients and a pharmaceutically acceptable carrier or diluent in the manufacture of a pharmaceutical product for the treatment and/or prevention of the above mentioned diseases.

Depending on the particular disease to be treated or prevented, other therapeutically active agents that are conventionally administered for the treatment or prevention of such diseases may optionally be administered in combination with the compounds of the present invention. As used herein, other therapeutic agents that are commonly administered for the treatment or prevention of a particular disease are known to be appropriate for the disease being treated.

The above-mentioned anticancer agents as a combination partner (partner) of the compounds of the invention are intended to include pharmaceutically acceptable derivatives thereof, such as their pharmaceutically acceptable salts.

The total daily dosage and administration form of the one or more other therapeutic agents administered in combination is known to those skilled in the art. The total daily dose may vary within wide limits.

In practicing the present invention, the compounds of the invention can be administered separately, sequentially, simultaneously, concurrently or staggered in time (e.g., in the form of a combined unit dosage form, in the form of separate unit dosage forms, in the form of adjacent separate unit dosage forms, in the form of a fixed or non-fixed combination, in the form of a kit or in the form of an admixture) in combination therapy with one or more standard therapeutic agents (chemotherapeutic anti-cancer agents and/or target-specific anti-cancer agents), particularly anti-cancer agents known in the art such as any of those described above.

The invention herein also relates to a combination comprising a first active ingredient which is at least one compound of the invention and a second active ingredient which is at least one art-known anti-cancer agent, such as one or more of those described above, for use in therapy, separately, sequentially, simultaneously, concurrently or chronologically staggered, such as the treatment of any of those diseases described herein.

The invention also relates to a pharmaceutical composition comprising a first active ingredient which is at least one compound of the invention and a second active ingredient which is at least one anti-cancer agent known in the art, such as one or more of those described above, and optionally a pharmaceutically acceptable carrier or diluent, for separate, sequential, simultaneous, concurrent or temporally staggered use in therapy.

The invention also relates to a combination product comprising:

a.) at least one compound of the invention formulated with a pharmaceutically acceptable carrier or diluent, and

b.) at least one art-known anti-cancer agent, such as one or more of those described above, formulated with a pharmaceutically acceptable carrier or diluent.

The invention also relates to a kit comprising: a formulation of a first active ingredient which is a compound of the invention and a pharmaceutically acceptable carrier or diluent; a formulation of a second active ingredient which is an anti-cancer agent known in the art, for example one of those described above, and a pharmaceutically acceptable carrier or diluent; for simultaneous, concurrent, sequential, separate, or temporally staggered use in therapy. Optionally, the kit comprises instructions for its use in therapy, e.g., for treating hyperproliferative diseases and diseases responsive or sensitive to inhibition of the Pi3K/Akt pathway, such as benign or malignant tumors, particularly cancer, more specifically any of those cancer diseases described above.

The present invention also relates to a combined preparation for simultaneous, concurrent, sequential or separate administration comprising at least one compound of the invention and at least one anti-cancer agent known in the art.

The invention also relates to a combination, composition, formulation or kit of the invention having Pi3K/Akt pathway inhibitory activity.

Furthermore, the present invention also relates to a method of treating a hyperproliferative disease and/or a disorder responsive to induction of apoptosis, such as cancer, in a patient in a combination therapy comprising administering to said patient in need thereof a combination, composition, formulation or kit as described herein.

In addition, the present invention also relates to a method of treating a hyperproliferative disease with benign or malignant manifestations and/or a disorder responsive to induction of apoptosis, such as cancer, in a patient in need thereof, comprising administering separately, simultaneously, concurrently, sequentially or time-staggered in a combination therapy to said patient a pharmacologically active and therapeutically effective and tolerable amount of a pharmaceutical composition comprising a compound of the present invention and a pharmaceutically acceptable carrier or diluent, and a pharmacologically active and therapeutically effective and tolerable amount of one or more art-known anti-cancer agents, such as one or more of those described herein.

Still further, the present invention relates to a method of treating, preventing or ameliorating a hyperproliferative disease and/or a disorder responsive to induction of apoptosis, such as a benign or malignant neoplasm (e.g., cancer, particularly any of those cancer diseases described herein), in a patient, comprising separately, simultaneously, concurrently, sequentially or chronologically staggered administration of an amount of a first active compound and an amount of at least one second active compound to said patient in need thereof, the first active compound is a compound of the invention, the at least one second active compound is a standard therapeutic agent, in particular at least one art-known anti-cancer agent, such as one or more of those chemotherapeutic anti-cancer agents and target-specific anti-cancer agents described herein, wherein the amounts of the first active compound and the second active compound produce a therapeutic effect.

Still further, the present invention relates to a method of treating, preventing or ameliorating a hyperproliferative disease and/or a disorder responsive to induction of apoptosis, such as a benign or malignant neoplasm (e.g., cancer, particularly any of those cancer diseases described herein), in a patient comprising administering a combination of the present invention.

Furthermore, the present invention relates to the use of a composition, combination, formulation or kit of the invention in the manufacture of a pharmaceutical product, e.g. a commercial package or medicament, for the treatment, prevention or amelioration of a hyperproliferative disease (e.g. cancer) and/or a disorder responsive to induction of apoptosis, in particular those diseases described herein, e.g. malignant or benign neoplasias.

The present invention also relates to a commercial package comprising one or more compounds of the invention and instructions for simultaneous, concurrent, sequential or separate use with one or more chemotherapeutic anti-cancer agents and/or target-specific anti-cancer agents, such as any of those described herein.

The present invention also relates to a commercial package consisting essentially of, as the only active ingredient, one or more compounds of the invention and instructions for simultaneous, concurrent, sequential or separate use with one or more chemotherapeutic anti-cancer agents and/or target-specific anti-cancer agents, such as any of those described herein.

The present invention also relates to a commercial package comprising one or more chemotherapeutic anti-cancer agents and/or target-specific anti-cancer agents (such as any of those described herein) and instructions for use simultaneously, concurrently, sequentially or separately with one or more compounds of the invention.

The compositions, combinations, formulations, preparations, kits or packages mentioned in the context of the combination therapy of the invention may also comprise more than one compound of the invention and/or more than one mentioned art-known anticancer agent.

The first and second active ingredients of the combination or kit of the invention may be provided as separate formulations (i.e. independently of each other) which are thereafter used together in a combination therapy simultaneously, concurrently, sequentially, separately or chronologically staggered; or as separate components packaged in combination and provided for simultaneous, concurrent, sequential, separate, or staggered in time for use in combination therapy.

The type of pharmaceutical formulation of the first and second active ingredients of the combination or kit of the invention may be the same, i.e. both ingredients are formulated as separate tablets or capsules, or may be different, i.e. suitable for different forms of administration, e.g. one active ingredient is formulated as a tablet or capsule and the other is formulated for e.g. intravenous administration.

The amounts of the first and second active ingredients of the combination, composition or kit of the invention may together comprise an amount that is therapeutically effective for the treatment, prevention or amelioration of a hyperproliferative disease and/or a disorder responsive to induction of apoptosis, particularly one of those diseases described herein, e.g. a malignant or benign neoplasm, particularly a cancer, such as any of those cancer diseases described herein.

In addition, the compounds of the present invention may be used for pre-or post-operative treatment of cancer.

Furthermore, the compounds of the present invention may be used in combination with radiation therapy.

The combination of the invention may refer to a composition comprising one or more compounds of the invention and one or more other active anti-cancer agents in a fixed combination (fixed unit dosage form), or alternatively, the combination may refer to a pharmaceutical package comprising two or more active ingredients in separate dosage forms (non-fixed combination). In the case of pharmaceutical packs containing two or more active ingredients, the active ingredients are preferably packaged in blister cards (blister cards), which is suitable for improving compliance.

Each blister card preferably contains a 1 day treatment medication to be taken. If the medicaments are to be taken at different times of day, the medicaments may be placed in different portions of the blister card according to the different time periods during the day during which the medicaments are to be taken (e.g., morning and evening or morning, midday and evening). The blister cavities of the doses taken together at a particular time of day are arranged to be within a corresponding time period of the day. Of course, the times of day are also indicated on the blister in a clearly visible manner. Of course, it is also possible to e.g. indicate the period during which the medicament is taken, e.g. indicate the time.

The daily portion may be taken as a line of blister cards and the time of day is then indicated in this column in chronological order.

The doses that must be taken together at a particular time of day are placed together at the appropriate time on the blister sheet, preferably with a reduced separation interval so that they can be easily pushed out of the blister and with the effect that the dosage form cannot be forgotten to be removed from the blister.

The following examples illustrate the invention in more detail, but without limitation. Other compounds of the invention whose preparation is not explicitly described may be prepared in a similar manner.

The compounds described in the examples and their salts represent preferred embodiments of the invention and claims covering all sub-combinations of the groups of the compounds of formula (I) disclosed in the specific examples.

The term "according to" is used in the experimental part to mean "analogously" to the method indicated. Experimental part

The following table lists abbreviations not explained in the text and used in this paragraph and in the examples section. NMR peak patterns are described in terms of their behavior in the spectra, without taking into account possible higher order effects. Chemical names were generated using AutoNom2000 applied in MDL ISIS Draw. In some cases, accepted nomenclature for commercially available reagents is used in place of the nomenclature generated by AutoNom 2000. The compounds and intermediates prepared according to the process of the invention may require purification. Purification of organic compounds is well known to those skilled in the art, and several purification methods may exist for the same compound. In some cases, purification may not be necessary. In some cases, the compound may be purified by crystallization. In some cases, the impurities may be stirred out using a suitable solvent. In some cases, the compound may be purified by chromatography. In some cases, the compound may be purified by preparative HPLC. In some cases, the purification methods described above can provide those compounds of the invention having a sufficiently basic or acidic functionality in the form of a salt, for example, in the case of a sufficiently basic compound of the invention, for example, in the form of a trifluoroacetate or formate salt, or, in the case of a sufficiently acidic compound of the invention, for example, in the form of an ammonium salt. Such salts can be converted to their free base or free acid forms, respectively, by various methods known to those skilled in the art, or can be used in the form of a salt for subsequent biological assays. It will be understood that the particular form of the compounds of the invention isolated as described herein (e.g., salts, free bases, etc.) is not necessarily the only form in which the compounds may be used in biological assays to quantify a particular biological activity.

Abbreviations	Means of
		Ac	Acetyl group
ACN	Acetonitrile
		br	Broad peak
d	Double peak
		DBU	Diaza (1, 3) bicyclo [5.4.0]Undecane
dd	Double doublet
		DCM	Methylene dichloride
DIBAL	Diisobutylaluminum hydride
		DMAP	4-dimethylaminopyridine

DMF	N, N-dimethylformamide
		DMSO	Dimethyl sulfoxide
Eq.	Equivalent weight
		ESI	Electrospray ionization
EtOAc	Ethyl acetate
		HBTU	2- (1H-benzotriazol-1-yl) -1, 1, 3, 3-tetramethyluronium hexafluorophosphate
HPLC	High performance liquid chromatography
		LC-MS	Liquid chromatography-mass spectrometry
m	Multiple peaks
		MS	Mass spectrometry
NBS	N-bromosuccinimide
		NIS	N-iodosuccinimide
NMP	N-methyl pyrrolidone
		NMR	Nuclear magnetic resonance spectroscopy: chemical shifts (. delta.) are provided in ppm
q	Quartet peak
		qn	Quintuple peak
rf	Refluxing
		r.t. or rt	At room temperature
RT	Retention time (minutes), unless stated, was determined by UPLC according to standard procedures
		s	Single peak
t	Triplet peak
		TLC	Thin layer chromatography
THF	Tetrahydrofuran (THF)

The meaning of the other abbreviations is conventional to those skilled in the art.

The aspects of the invention described in the present application are illustrated by the following examples, but are in no way meant to limit the invention in any way.

Examples

UPLC-MS Standard operation

Unless otherwise stated, the analysis UPLC-MS was performed under the following conditions.

The instrument comprises the following steps: waters Acquity UPLC-MS ZQ 4000; column: acquity UPLC BEH C181.750 × 2.1 mm; eluent A: water + 0.05% formic acid, eluent B: acetonitrile + 0.05% formic acid; gradient: 0-1.6min 1-99% B, 1.6-2.0min 99% B; flow rate: 0.8 ml/min; temperature: 60 ℃; sample introduction amount: 2 mu l of the solution; DAD scan: 210-400 nm.

Unless negative ion mode (ES-) is indicated, the reported mass numbers (m/z) are from positive ion mode electrospray ionization.

Examples of intermediates

Intermediate example 1.0: 4- (5-methyl-8-phenyl-imidazo [1, 2-c ] pyrimidin-7-yl) -benzaldehyde

Step 1: 2-methyl-5-phenyl-pyrimidine-4, 6-diol

To a mixture of NaOEt (19.7g, 0.275mol) in EtOH (140mL) was added 2-phenyl-malonamide (10g, 55 mmol). To the resulting thick suspension, EtOAc (90.4mL, 110mmol) was added dropwise. The mixture was diluted with EtOH (60mL) and heated at 50 ℃ for 2h with thorough stirring. The reaction was cooled to 0 ℃ and quenched with ice-cold water (175 mL). The resulting solution was treated with concentrated aqueous hydrochloric acid (90mL) and the resulting precipitate was filtered off, washed with 0.5M aqueous hydrochloric acid (40mL) and dried to give the crude title compound which was used in the next step without further purification.

Step 2: 4, 6-dibromo-2-methyl-5-phenyl-pyrimidine

2-methyl-5-phenyl-pyrimidine-4, 6-diol (3.69g) and POBr were heated at 180 deg.C₃(10.4g, 2Eq.) for 30 minutes. After cooling to 0 ℃, the reaction was quenched with ice-cold water and stirred well, and the resulting suspension was filtered. The residue was co-evaporated with toluene and dried to give the crude title compound, which was used in the next step without further purification.

And step 3: 6-bromo-2-methyl-5-phenyl-pyrimidin-4-ylamine

A mixture of 4.6-dibromo-2-methyl-5-phenyl-pyrimidine (3.2g) and a solution of ammonia in EtOH (2M, 50mL) was heated at 100 ℃ overnight. Reaction control (LC-MS) showed incomplete conversion, so aqueous ammonia (25%, 10Eq.) was added and the mixture was heated at 120 ℃. After cooling, the volatiles were removed in vacuo and the residue was co-evaporated with toluene and dried to give the crude title compound which was used in the next step without further purification.

And 4, step 4: 4- (6-amino-2-methyl-5-phenyl-pyrimidin-4-yl) -benzaldehyde

6-bromo-2-methyl-5-phenyl-pyrimidin-4-ylamine (2.59g) was suspended in dioxane (65mL) and 4-formylphenylboronic acid (1.90g, 9.8mmol), Cs, were added successively under a stream of nitrogen₂CO₃Aqueous solution (2M, 39.2mmol) and Pd (dppf) Cl₂(0.417g, 0.5 mmol). The reaction was heated at 115 ℃ for 6 days under nitrogen. After cooling, the reaction was stirred at rt for a further 2 days, then filtered through a pad of silica gel and washed with NH₄The filtrate was partitioned between aqueous Cl and EtOAc. By CH₂Cl₂The aqueous phase was extracted and the organic phase was washed with brine and dried (MgSO₄) And concentrated in vacuo. Purification by silica gel chromatography gave the title compound.

And 5: 4- (5-methyl-8-phenyl-imidazo [1, 2-c ] pyrimidin-7-yl) -benzaldehyde

4- (6-amino-2-methyl-5-phenyl-pyrimidin-4-yl) -benzaldehyde (500mg, 1.7mmol) was suspended in EtOH (4mL), treated with chloral (50% in water, 10Eq.) and the mixture was heated at 100 ℃ for 45min under microwave irradiation. After cooling, the mixture was concentrated in vacuo and co-evaporated with toluene. Purification by chromatography on silica gel (eluent: toluene: dioxane 7: 3) gave the title compound.

Intermediate example 2.0: 4- (8-phenyl-imidazo [1, 2-c ] pyrimidin-7-yl) -benzaldehyde

Step 1: 5-phenyl-pyrimidine-4, 6-diols

To a mixture of NaOEt (79g, 1.1mol) in EtOH (650mL) was added 2-phenyl-malonamide (40g, 0.22 mol). To the resulting suspension was added dropwise ethyl formate (36mL, 0.44 mol). The mixture was heated at 50 ℃ for 2h, then the reaction was cooled to 0 ℃ and quenched with water (700mL) and aqueous hydrochloric acid (6N, 360 mL). The resulting precipitate was filtered off, washed with 0.5N aqueous hydrochloric acid and dried to give the crude title compound, which was used in the next step without further purification.

¹H NMR(300MHz，d6-DMSO)：δ11.95(br s，2H)，8.07(s，1H)，7.2-7.5(m，5H)ppm。

Step 2: 4, 6-dibromo-5-phenyl-pyrimidines

5-phenyl-pyrimidine-4, 6-diol (34.8g) and POBr were heated at 180 deg.C₃(106g, 2Eq.) for 40 minutes. After cooling to rt, the reaction was carefully quenched with water (500 mL). The precipitate was filtered and dried to give the crude title compound which was used in the next step without further purification.

And step 3: 6-bromo-5-phenyl-pyrimidin-4-ylamine

A mixture of 4, 6-dibromo-5-phenyl-pyrimidine (52.5g) and a solution of ammonia in EtOH (2M, 835mL) was heated at reflux for 30 hours. After 2 hours, 6 hours and 20 hours, an additional part of 15Eq. ammonia in MeOH (7M) was added. After cooling, the volatiles were removed in vacuo and the residue was suspended in water (300 mL). The solid was filtered off and dried to give the crude title compound which was used in the next step without further purification.

And 4, step 4: 4- (6-amino-5-phenyl-pyrimidin-4-yl) -benzaldehyde

To a mixture of 6-bromo-5-phenyl-pyrimidin-4-ylamine (15g, 60mmol) and 4-formylphenylboronic acid (11.7g, 78mmol) in dioxane (400mL) was added Cs₂CO₃Solution (2M, 120mL) and Pd (dppf) Cl₂(2.4g, 3 mmol). In thatThe reaction was heated at reflux for 18 hours under an inert gas atmosphere. After cooling, the reaction was stopped by partitioning between water and EtOAc. The aqueous phase was extracted with EtOAc and dried (MgSO)₄) The combined organic phases were concentrated in vacuo. Purification by silica gel chromatography gave the title compound.

¹H NMR(300MHz，d6-DMSO)：δ9.92(s，1H)，8.5(s，1H)，7.71(d，2H)，7.32-7.42(m，5H)，7.17(d，2H)ppm。

And 5: 4- (8-phenyl-imidazo [1, 2-c ] pyrimidin-7-yl) -benzaldehyde

4- (6-amino-2-methyl-5-phenyl-pyrimidin-4-yl) -benzaldehyde (7.5g, 27.2mmol) was suspended in EtOH (55mL), treated with chloral (50% in water, 272mmol) and the mixture heated at 100 ℃ for 20 min under microwave irradiation. After cooling, the mixture was concentrated in vacuo and purified by silica gel chromatography to give the title compound.

¹H NMR(300MHz，d6-DMSO)：δ9.98(s，1H)，9.57(s，1H)，8.2(s，1H)，7.8(d，2H)，7.73(s，1H)，7.55(d，2H)，7.4(m，5H)ppm。

Intermediate example 3.0: 4- (8-phenyl- [1, 2, 4] triazolo [4.3-c ] pyrimidin-7-yl) -benzaldehyde

Step 1: (6-bromo-5-phenyl-pyrimidin-4-yl) -hydrazine

A mixture of 4, 6-dibromo-5-phenyl-pyrimidine (20g) and EtOH (150mL) was treated with hydrazine hydrate (9.56g, 191mmol) and the reaction was heated at 50 ℃ for 45 minutes. The reaction was concentrated in vacuo and the residue was co-evaporated with toluene (2 ×) and dried to give the crude title compound which was used in the next step without further purification.

MS (M + 1): 265.0, 267.0(Br isotope)

Step 2: 7-bromo-8-phenyl- [1, 2, 4] triazolo [4, 3-c ] pyrimidine

(6-bromo-5-phenyl-pyrimidin-4-yl) -hydrazine (11g, 41.5mmol) was suspended in triethyl orthoformate (300mL) and the reaction was heated under reflux overnight. Almost all volatiles were removed in vacuo, the resulting suspension was cooled, and the solids were removed by filtration. The filtrate was concentrated in vacuo and the resulting residue was recrystallized from EtOH to give the title compound.

¹H NMR(300MHz，d6-DMSO)：δ9.78(s，1H)，8.64(s，1H)，7.51-7.6(m，5H)。

MS (M + 1): 275.1, 277.1(Br isotope)

And step 3: 4- (8-phenyl- [1, 2, 4] triazolo [4.3-c ] pyrimidin-7-yl) -benzaldehyde

Purging 7-bromo-8-phenyl- [1, 2, 4] with nitrogen]Triazolo [4, 3-c]Pyrimidine (0.55g, 2mmol), 4-formylphenylboronic acid (0.39g, 2.6mmol) and Cs₂CO₃(2.6g, 8mmol) in dioxane (20mL) and water (2mL) with Pd (dppf) Cl₂(82mg, 0.1 mmol). The reaction was heated at reflux overnight. After cooling, the reaction was filtered through Celite (Celite), washed with EtOH (200mL), and washed with NH₄The mixture was diluted with aqueous Cl and extracted twice with EtOAc. The combined organic phases were washed with brine, dried and concentrated in vacuo. Purification by chromatography gave the title compound.

¹H NMR(300MHz，CDCl₃)：δ10.0(s，1H)，9.5(s，1H)，7.8(d，2H)，7.63(d，2H)，7.38-7.45(m，5H)ppm。

MS(M+1)：301.2。

Intermediate example 4.0: 4- (3-methyl-8-phenyl- [1, 2, 4] triazolo [4.3-c ] pyrimidin-7-yl) -benzaldehyde

Step 1: 7-bromo-3-methyl-8-phenyl- [1, 2, 4] triazolo [4, 3-c ] pyrimidine

(6-bromo-5-phenyl-pyrimidin-4-yl) -hydrazine (10g, 37.7mmol) was suspended in triethyl orthoformate (300mL) and the reaction was heated under reflux overnight. Almost all volatiles were removed in vacuo, the resulting suspension was cooled, and the solids were removed by filtration. The filtrate was concentrated in vacuo and the resulting residue was recrystallized from EtOH to give the title compound.

¹H NMR (300MHz, d 6-DMSO): δ 9.62(s, 1H), 7.55(m, 5H), 2.49 (signal partially masked by solvent).

MS (M + 1): 289.1, 291.1(Br isotope)

Step 2: 4- (3-methyl-8-phenyl [1, 2, 4] triazolo [4.3-c ] pyrimidin-7-yl) -benzaldehyde

Purging 7-bromo-3-methyl-8-phenyl- [1, 2, 4] with nitrogen]Triazolo [4, 3-c]Pyrimidine (0.825g, 3mmol), 4-formylphenylboronic acid (0.584g, 3.9mmol), and Cs₂CO₃(3.9g, 12mmol) in dioxane (30mL) and water (3mL) with Pd (dppf) Cl₂(123mg, 0.15 mmol). The reaction was heated at reflux overnight. After cooling, the reaction was filtered through celite, washed with EtOH (200mL), and NH₄The mixture was diluted with aqueous Cl and extracted twice with EtOAc. The combined organic phases were washed with brine, dried and concentrated in vacuo. Purification by chromatography gave the title compound.

¹H NMR (300MHz, d 6-DMSO): δ 10.0(s, 1H), 9.8(s, 1H), 7.82(d, 2H), 7.6(d, 2H), 7.4(m, 5H), 2.52 (signal partially masked by solvent) ppm.

MS(M+1)：315.1。

Intermediate example 5.0: 2- (5-piperidin-4-yl-2H- [1, 2, 4] triazol-3-yl) -pyridine dihydrochloride (alternative methods described in US4011218 or WO 2005100344)

Step 1: pyridine-2-carbohydrazolamide

A solution of pyridine-2-carbonitrile 20g (192mmol), hydrazine hydrate (3eq.) in ethanol (50ml) was stirred at room temperature for 18 hr. The reaction was then diluted with water, extracted with ethyl acetate, dried over anhydrous sodium sulfate, and concentrated in vacuo to give the desired compound.

MS(M+1)：137.28

¹H NMR(300MHZ，CDCl₃)：δ8.53(d，1H，J＝8&2.3Hz)，8.02(d，1H，J＝7.8&2.1Hz)，7.72(t，1H，J＝8.2&2Hz)，7.29(t，1H，J＝8.4&2.1Hz)，5.42(bs，2H)，4.60(bs，2H)ppm。

Step 2: 4- ({ (2Z) -2- [ amino (pyridin-2-yl) methylidene ] hydrazino } carbonyl) piperidine-1-carboxylic acid tert-butyl ester

To a solution of 37g (167mM) of 1- (tert-butoxycarbonyl) piperidine-4-carboxylic acid in dichloromethane (300ml) was added carbonyldiimidazole (1eq.) in small portions over 30 min. Pyridine-2-carbohydrazolamide was then added to the reaction mixture and stirred at room temperature for 3 hr. The dichloromethane was evaporated and then the reaction was stirred in water for 30 min. The precipitated solid was filtered and dried to give the desired compound.

MS(M+1)：348.07

¹H NMR(300MHZ，CDCl3)：δ10.75(s，1H)，8.56(d，1H，J＝4.5Hz)，8.10(d，1H，J＝8.3Hz)，7.75(dt，1H，J＝8.2&1.3Hz)，7.34(dt，1H，J＝7.9&1.5Hz)，4.18(bs，2H)，3.46(s，1H)，2.88(t，2H)，1.91(m，2H)，1.72(m，4H)，1.47(s，9H)ppm。

And step 3: 4- [5- (pyridin-2-yl) -1H-1, 2, 4-triazol-3-yl ] piperidine-1-carboxylic acid tert-butyl ester

45g (129mmol) of tert-butyl 4- ({ (2Z) -2- [ amino (pyridin-2-yl) methylidene ] hydrazino } carbonyl) piperidine-1-carboxylate from step 2 was melted at 220 ℃ for 1hr under a nitrogen atmosphere. The reaction was then cooled to 150 ℃ and ethanol was added until the solid dissolved. The ethanol was then evaporated to give the desired crude compound, which contained 2- (5-piperidin-4-yl-2H- [1, 2, 4] triazol-3-yl) -pyridine as an impurity.

MS(M+1)：330.5

¹H NMR(300MHZ，DMSO)：δ9.11(s，1H)，8.74(dd，1H，J＝4.8&1.3Hz)，8.17(dt，2H，J＝8.2&2.1Hz)，7.66(dt，1H，J＝8.0&1.3Hz)，3.34(m，2H)，3.18(m，1H)，3.06(m，2H)，2.20(m，2H)，1.99(m，2H)，1.28(s，9H)ppm。

And 4, step 4: 2- (5-piperidin-4-yl-2H- [1, 2, 4] triazol-3-yl) -pyridine dihydrochloride

To a solution of crude tert-butyl 4- [5- (pyridin-2-yl) -1H-1, 2, 4-triazol-3-yl ] piperidine-1-carboxylate 39g (111mmol) in 50ml methanol was added a solution of HCl in dioxane (100ml) and stirred at room temperature for 3 hr. The precipitated solid was then filtered and washed with cold acetonitrile to give 2- (5-piperidin-4-yl-2H- [1, 2, 4] triazol-3-yl) -pyridine dihydrochloride as a white solid.

¹H NMR(300MHZ，DMSO)：δ9.11(s，1H)，8.97(s，1H)，8.74(dd，1H，J＝4.8&1.3Hz)，8.17(dt，2H，J＝8.2&2.1Hz)，7.66(dt，1H，J＝8.0&1.3Hz)，3.34(m，2H)，3.18(m，1H)，3.06(m，2H)，2.20(m，2H)，1.99(m，2H)ppm。

Intermediate example 6.0: 2- (5-piperidin-4-yl-2H- [1, 2, 4] triazol-3-yl) -pyridin-1-oxide hydrochloride

The intermediate is prepared according to the preparation method of 2- (5-piperidine-4-yl-2H- [1, 2, 4] triazole-3-yl) -pyridine dihydrochloride.

1H-NMR(300MHz，d6-DMSO).：9.15(br s，1H)，8.93(br s，1H)，8.43-8.46(m，1H)，8.22-8.25(m，1H)，7.5-7.53(m，1H)，3.23-3.28(m，2H)，2.95-3.15(m，3H)，2.09-2.15(m，2H)，1.86-1.99(m，2H)。

Intermediate example 7.0: 4- (5-piperidin-4-yl-2H- [1, 2, 4] triazol-3-yl) -pyridine hydrochloride

This intermediate was prepared according to the procedure for the preparation of 2- (5-piperidin-4-yl-2H- [1, 2, 4] triazol-3-yl) -pyridine dihydrochloride (intermediate example 5.0).

Intermediate example 8.0: 2-methyl-6- (5-piperidin-4-yl-2H- [1, 2, 4] triazol-3-yl) -pyridine hydrochloride

1H-NMR(300MHz，d6-DMSO).：9.16-9.24(m，2H)，8.04-8.15(m，2H)，7.59(d，1H)，3.15-3.30(m，3H)，2.96-3.06(m，2H)，2.64(s，3H)，2.14-2.18(m，2H)，1.93-2.04(m，2H)ppm。

Intermediate example 9.0: 5-methyl-2- (5-piperidin-4-yl-2H- [1, 2, 4] triazol-3-yl) -pyridine hydrochloride

MS(M+1)：244

1H-NMR(300MHz，d6-DMSO).：8.70-8.66(m, 2H), 8.54(d, 1H), 7.99(s, 1H), 7.46(d, 1H), 3.27-3.32(m, 2H), 2.97-3.16(m, 3H), [3H masked by solvent]，2.12-2.16(m，2H)，1.86-1.99(m，2H)ppm。

Intermediate example 10.0: 2, 4-dimethyl-6- (5-piperidin-4-yl-2H- [1, 2, 4] triazol-3-yl) -pyridine hydrochloride

MS(M+1)：258.33；

UPLC-MS：RT＝0.47min；m/z＝258.33。

Intermediate example 11.0: 2.3-dimethyl-6- (5-piperidin-4-yl-2H- [1, 2, 4] triazol-3-yl) -pyridine hydrochloride

UPLC-MS：RT＝0.50min；m/z＝258.27。

Intermediate example 12.0: 5-fluoro-2- (5-piperidin-4-yl-2H- [1, 2, 4] triazol-3-yl) -pyridine hydrochloride

UPLC-MS：RT＝0.49min；m/z＝248.22。

Intermediate example 13.0: 5-chloro-2- (5-piperidin-4-yl-2H- [1, 2, 4] triazol-3-yl) -pyridine hydrochloride

Intermediate example 14.0: 4-chloro-2- (5-piperidin-4-yl-2H- [1, 2, 4] triazol-3-yl) -pyridine hydrochloride

MS(M+1)：264.23；

UPLC-MS：RT＝0.58min；m/z＝264.23。

Intermediate example 15.0: 2-chloro-6- (5-piperidin-4-yl-2H- [1, 2, 4] triazol-3-yl) -pyridine hydrochloride

UPLC-MS：RT＝0.50min；m/z＝264.21。

Intermediate example 16.0: 4-methoxy-2- (5-piperidin-4-yl-2H- [1, 2, 4] triazol-3-yl) -pyridine hydrochloride

Intermediate example 17.0: 2-methoxy-5- (5-piperidin-4-yl-2H- [1, 2, 4] triazol-3-yl) -pyridine hydrochloride

MS(M+1)：260.26；

UPLC-MS：RT＝0.55min；m/z＝260.26。

Intermediate example 18.0: 4-Ethyl-2- (5-piperidin-4-yl-2H- [1, 2, 4] triazol-3-yl) -pyridine hydrochloride

MS(M+1)：258.29；

UPLC-MS：RT＝0.60min；m/z＝258.29。

Intermediate example 19.0: 2- (5-piperidin-4-yl-2H- [1, 2, 4] triazol-3-yl) -6-trifluoromethyl-pyridine hydrochloride

MS(M+1)：298；

1H-NMR (300MHz, d6-DMSO, characteristic Signal).:9.16(br s，1H)，8.93(br s，1H)，8.28(d，1H)，8.19(t，1H)，7.93(dd，1H)ppm。

intermediate example 20.0: 2- (5-piperidin-4-yl-2H- [1, 2, 4] triazol-3-yl) -4-trifluoromethyl-pyridine hydrochloride

¹H NMR(300MHz，400MHz).：9.22(m，1H)，9.0(m，1H)，8.93(d，1H)，8.21(s，1H)，7.86(d，1H)，3.29(m，2H)，3.15(m，1H)，3.02(m，2H)，2.13-2.17(m，2H)，1.91-2.01(m，2H)ppm。

Intermediate example 21.0: 2- (5-piperidin-4-yl-2H- [1, 2, 4] triazol-3-yl) -pyrimidine hydrochloride

Prepared according to the procedure for the preparation of 2- (5-piperidin-4-yl-2H- [1, 2, 4] triazol-3-yl) -pyridine dihydrochloride (intermediate example 5.0).

¹H-NMR(300MHz，d6-DMSO).：9.17-8.98(m，2H)，8.93(d，2H)，7.57(t，1H)，3.24-3.29(m，2H)，2.95-3.18(m，3H)，2.11-2.16(m，2H)，1.88-2.00(m，2H)ppm。

Intermediate example 22.0: 2- (5-piperidin-4-yl-2H- [1, 2, 4] triazol-3-yl) -pyrazine hydrochloride

MS(M+1)：231.21；

UPLC-MS：RT＝0.51min；m/z＝231.21。

Intermediate example 23.0: 4- (5-Furan-2-yl-1H- [1, 2, 4] triazol-3-yl) -piperidine hydrochloride

Intermediate example 24.0: 4- (5-Thien-2-yl-2H- [1, 2, 4] triazol-3-yl) -piperidine hydrochloride

Intermediate example 25.0: 4- [5- (1H-pyrrol-2-yl) -1H- [1, 2, 4] triazol-3-yl ] -piperidine hydrochloride

Step 1: 1H-pyrrole-2-carbohydrazolamide

A solution of 10g of 1H-pyrrole-2-carbonitrile and 1eq of sodium methoxide in 20ml of ethanol was stirred for 10 min. Then adding waterHydrazine (3eq.) and the resulting reaction mixture was stirred at room temperature for 18 h. The reaction mixture was then diluted with water, extracted with ethyl acetate, and extracted with Na₂SO₄Drying and concentration in vacuo afforded the desired compound.

Step 2: 4- [5- (1H-pyrrol-2-yl) -1H- [1, 2, 4] triazol-3-yl ] piperidine (3 steps)

Further synthesis was carried out analogously to the synthesis of 2- (5-piperidin-4-yl-2H- [1, 2, 4] triazol-3-yl) -pyridine dihydrochloride (intermediate example 5.0, steps 2-4) except that pyridine-2-carbohydrazolamide was replaced with 1H-pyrrole-2-carbohydrazolamide in step 2.

Intermediate example 26.0: 4- (5-phenyl-1H- [1, 2, 4] triazol-3-yl) -piperidine hydrochloride

Prepared according to the procedure for the preparation of 4- [5- (1H-pyrrol-2-yl) -1H- [1, 2, 4] triazol-3-yl ] -piperidine hydrochloride (intermediate example 25.0).

Intermediate example 27.0: 4- (5-Thiazol-2-yl-1H- [1, 2, 4] triazol-3-yl) -piperidine hydrochloride

Step 1: 2-trimethylsilyl-thiazoles

To a mixture of 40.6ml of n-butyllithium (1.6M in hexane) and 18ml of diethyl ether, a solution of 5.03g of thiazole dissolved in 59ml of diethyl ether was added dropwise at-70 ℃. After 30min, 6.41g of trimethylsilyl chloride dissolved in 59ml of diethyl ether were added at-70 ℃. The reaction mixture was stirred at-70 ℃ for 1h and allowed to warm to room temperature. With saturated NaHCO₃The mixture was washed with Na₂SO₄Dried and the solvent evaporated. The residue was distilled to give the desired product.

Step 2: thiazol-2-yl-iminocarbonylhydrazines

10.0g of 2-trimethylsilyl-thiazole and 11.5g of tolylsulfonyl cyanide were stirred at 70 ℃ for 5 h. The mixture was diluted with THF and 9.83g of hydrazine hydrate was added at 10 ℃. The reaction mixture was stirred at room temperature overnight. The solvent was removed by evaporation and the residue was purified by silica gel chromatography (dichloromethane/methanol) to give the desired product.

And step 3: 4- [ N' - (imino-thiazol-2-yl-methyl) -hydrazinocarbonyl ] -piperidine-1-carboxylic acid tert-butyl ester

8.65g of mono-tert-butyl piperidine-1, 4-dicarboxylate are dissolved in methylene chloride and 6.12g of 1, 1-carbonyldiimidazole are added in portions. 5.45g thiazol-2-yl-iminocarbonylhydrazine are added slowly and the mixture is stirred at room temperature for 18 hours. The solvent was removed by evaporation and the residue was washed with water. The crude product was dried and used without further purification.

And 4, step 4: 4- (5-Thiazol-2-yl-1H- [1, 2, 4] triazol-3-yl) -piperidine-1-carboxylic acid tert-butyl ester

8.00g of 4- [ N' - (imino-thiazol-2-yl-methyl) -hydrazinocarbonyl ] -piperidine-1-carboxylic acid tert-butyl ester were heated to 220 ℃. The clear melt was stirred at this temperature for 15 min. The melt was cooled to 80 ℃ and 42ml of ethanol were carefully added. Removal of the solvent gave a crude product which was a mixture of the desired product and 4- (5-thiazol-2-yl-1H- [1, 2, 4] triazol-3-yl) -piperidine. This mixture was used in the next reaction without further purification.

And 5: 4- (5-Thiazol-2-yl-1H- [1, 2, 4] triazol-3-yl) -piperidine hydrochloride

7.59g of the crude product mixture obtained in step 4 was dissolved in dioxane and a 4M solution of hydrogen chloride in dioxane (68ml) was slowly added. The product appeared as an oil. After addition of 542ml of methanol, the oil was dissolved. The solution was stirred overnight until the crystalline product precipitated.

Intermediate example 28.0: 2- (3-piperidin-4-yl- [1, 2, 4] oxadiazol-5-yl) -pyridine hydrochloride

Can be prepared according to the method provided in WO 2006065601.

Intermediate example 29.0: 2- (5-piperidin-4-yl-2H-pyrazol-3-yl) -pyridine hydrochloride

Can be prepared according to the method provided in WO 2004096131.

Intermediate example 30.0: 2- (5-azetidin-3-yl-2H- [1, 2, 4] triazol-3-yl) -pyridine hydrochloride

Method A

Step 1: pyridine-2-carbohydrazolamide

A solution of pyridine-2-carbonitrile 20g (192mmol) and hydrazine hydrate (3Eq.) in ethanol (50mL) was stirred at room temperature for 18 hr. The reaction was then diluted with water, extracted with ethyl acetate and dried (Na)₂SO₄) The organic portion was concentrated in vacuo to afford the desired compound.

MS(M+1)：137.07

¹H NMR(300MHz，CDCl₃)：δ8.53(d，1H)，8.02(d，1H)，7.72(t，1H)，7.29(t，1H)，5.42(bs，2H)，4.60(bs，2H)ppm。

Step 2: 3- [ 1-amino-1-pyridin-2-yl-meth- (Z) -ylidene-hydrazinocarbonyl ] -azetidine-1-carboxylic acid tert-butyl ester

To a solution of 1- (tert-butoxycarbonyl) azetidine-3-carboxylic acid in dichloromethane (0.56mL/mmol 1- (tert-butoxycarbonyl) azetidine-3-carboxylic acid) was added carbonyldiimidazole (1Eq.) in small portions over 30 min. Pyridine-2-carbohydrazolamide was then added to the reaction mixture and stirred at room temperature for 3 hr. The mixture was concentrated in vacuo, then the reaction was stirred in water for 30 min. The precipitated solid was filtered and dried to give the desired compound.

MS(M+1)：319.93

¹H NMR(300MHz，CDCl₃)：δ10.90(s，1H)，8.53(d，1H)，8.04(d，1H)，7.75-7.70(m，1H)，7.24(d，1H)，6.44(s，2H)，4.24-4.17(m，4H)，4.09-4.03(m，1H)，1.45(s，9H)ppm。

And step 3: 3- (5-pyridin-2-yl-1H- [1, 2, 4] triazol-3-yl) -azetidine-1-carboxylic acid tert-butyl ester

Melting the 3- [ 1-amino-1-pyridin-2-yl-meth- (Z) -ylidene-hydrazinocarbonyl ] -azetidine-1-carboxylic acid tert-butyl ester obtained in step 2 at 220 ℃ for 1hr under a nitrogen atmosphere. The reaction was then cooled until ethanol could be safely added to the still warm melt. Sufficient ethanol was added until the solid dissolved. Evaporation of the ethanol afforded the crude desired compound, which was used in the next step without further purification.

MS(M+1)：302.35

¹H NMR(300MHz，CDCl₃)：δ12.97(bs，1H)，8.76(d，1H)，8.24(d，1H)，7.89(t，1H)，7.45(d，1H)，4.3-4.27(m，4H)，4.03-4.0(m，1H)，1.46(s，9H)ppm。

And 4, step 4: 2- (5-azetidin-3-yl-2H- [1, 2, 4] triazol-3-yl) -pyridine hydrochloride

Tert-butyl 3- (5-pyridin-2-yl-1H- [1, 2, 4] triazol-3-yl) -azetidine-1-carboxylate (3.13g, 10.39mmol) was suspended in a solution of HCl in dioxane (4M, 80mL) and stirred at rt overnight. The reaction mixture was diluted with ether, filtered, and the residue was suspended in acetonitrile and stirred at rt for 45 min. The solid (hygroscopic) was isolated by filtration, partially dissolved in warm methanol and diethyl ether added to give a yellow sticky solid precipitate which could not be filtered. The mixture was concentrated in vacuo and dried in a vacuum oven (40 ℃) to afford the desired compound as a pale yellow solid.

MS(M+1)：202.13

¹H NMR(300MHz，d6-DMSO)：δ9.61(bs，1H)，9.25(bs，1H)，8.76(d，1H)，8.16(m，2H)，7.75(d，1H)，4.10-4.27(m，5H)ppm。

Method B

Step 1: 3-Hydrazinocarbonyl-azetidine-1-carboxylic acid tert-butyl ester

1- (tert-Butoxycarbonyl) azetidine-3-carboxylic acid (5g, 24.8mmol) was suspended in dichloromethane (15mL) and 1, 1' -carbonyldiimidazole (4.56g, 28.1mmol) was added portionwise. The resulting mixture was stirred at rt for 30min, then a solution of hydrazine hydrate (1.94mL, 39.9mmol) in dichloromethane (5mL) was added dropwise. After the addition was complete, the mixture was stirred at rt for 30 min. With saturated Na₂CO₃The reaction mixture was washed with aqueous (2 ×), brine and dried (Na)₂SO₄) And concentrated in vacuo to give a white crystalline solid, triturated with ether overnight, filtered, and air dried for 5h to give a white solid.

Step 2: 3- (5-pyridin-2-yl-1H- [1, 2, 4] triazol-3-yl) -azetidine-1-carboxylic acid tert-butyl ester

3-Hydrazinocarbonyl-azetidine-1-carboxylic acid tert-butyl ester (2.74g, 12.73mmol) and 2-cyano-pyridine (1.45g, 13.95mmol) were dissolved in 2-ethoxyethanol (30mL) and a 30 wt% solution of NaOMe in MeOH (1.19mL, 6.38mmol) was added. The resulting mixture was heated to 130 ℃ and stirred overnight. After cooling, by addingThe mixture was neutralized with acetic acid and washed with EtOAc and saturated NaHCO₃The aqueous solution was partitioned. Drying (Na)₂SO₄) The organic phase was concentrated in vacuo to afford a yellow solid. Further purification was by trituration with ether followed by recrystallization from MeOH.

And step 3: 2- (5-azetidin-3-yl-2H- [1, 2, 4] triazol-3-yl) -pyridine hydrochloride

Prepared as described above in method a, step 4.

Intermediate example 31: 2- [5- (azetidin-3-yl) -1H-1, 2, 4-triazol-3-yl ] -6-methylpyridine dihydrochloride

This intermediate was prepared in analogy to example 44.0, method a.

Step 1: 6-methylpyridine-2-carbohydrazolamide

11.97g (101.4mmol) of 6-methylpyridine-2-carbonitrile were dissolved in 25mL of ethanol. After addition of 36.3mL (304.05mmol) of hydrazine hydrate (w ═ 30%), the reaction mixture was stirred at room temperature for 24 hours. The precipitated product (K1 ═ 1.45g) was filtered off and the filtrate was evaporated to 1/3 of its volume. After dilution with water, the reaction mixture was extracted 3 times with ethyl acetate. The combined organic extracts were washed with brine and dried (Na)₂SO₄). Removal of the solvent gave the desired product K2(11.11 g). The overall yield was 78.9%.

MS(ES+，M+1)：151

¹H-NMR (300MHz, d 6-DMSO): 7.65(d, 1H), 7.90(dd, 1H), 7.12(d, 1H), 5.65(br., 2H), 5.19(br., 2H), 2.51(s, 3H, under signal from solvent) ppm.

Step 2: 3- ({2- [ amino (6-methylpyridin-2-yl) methylene ] hydrazino } carbonyl) azetidine-1-carboxylic acid tert-butyl ester

To a solution of 8.21g (54.7mmol) of 1- (tert-butoxycarbonyl) azetidine-3-carboxylic acid in 80mL of dichloromethane was added 8.86g (54.7mmol) of carbonyldiimidazole over 30 min. After stirring for 5 minutes, 11g (54.7mmol) of 6-methylpyridine-2-carbohydrazolamide were added and the reaction mixture was stirred at room temperature for 3 hours. The solvent was evaporated and the residue was treated with water. The precipitate formed is filtered off and dried to yield 16.47g (81.3%) of the desired compound as a mixture of tautomers.

MS(CI，M+1)：334

¹H-NMR (300MHz, d 6-DMSO): 10.09, 9.87(s, combined 1H), 7.64-7.89(m, 2H), 7.22-7.31(m, 1H), 6.59(br., 2H), 3.80-4.10(m, 4H), 3.25-3.45(m, 1H, under the signal of water in the solvent, 2.52(s, 3H), 1.35 ("s", 9H) ppm.

And step 3: 3- [3- (6-methylpyridin-2-yl) -1H-1, 2, 4-triazol-5-yl ] azetidine-1-carboxylic acid tert-butyl ester

16.4g (49.3mmol) of tert-butyl 3- ({2- [ amino (6-methylpyridin-2-yl) methylidene ] hydrazino } carbonyl) azetidine-1-carboxylate were heated under nitrogen to the melting point (220 ℃ C.) and held there for 90 minutes. During the cooling phase (at about 135 ℃), ethanol was carefully added to the reaction mixture. The reaction mixture was stirred at room temperature overnight and the ethanol was evaporated. The residue was heated again to 220 ℃ for 1 hour due to incomplete reaction and work-up was repeated to give 12.91g (74.58%) of crude expected product (by-product was cyclized compound with Boc group removed).

¹H-NMR (300MHz, d 6-DMSO): 14.30(br., 1H), 7.75-7.89(m, 2H), 7.31(d, 1H), 3.82-4.49(m, 4H), 3.32-3.48(m, 1H, part under the water signal of the solvent), 2.52(s, 3H), 1.39(s, 9H) ppm.

And 4, step 4: 2- [5- (azetidin-3-yl) -1H-1, 2, 4-triazol-3-yl ] -6-methylpyridine dihydrochloride

11.6g (36.8mmol) of tert-butyl 3- [3- (6-methylpyridin-2-yl) -1H-1, 2, 4-triazol-5-yl ] azetidine-1-carboxylate were dissolved in 150mL of dioxane. 27.6mL of HCl in dioxane (4M) was added dropwise and the reaction mixture was stirred at room temperature overnight. The reaction mixture was evaporated to dryness to give 13.1g (76.6%) of the desired salt, 60% pure, which was used without further purification.

Intermediate example 32.0: 2- (5-pyrrolidin-3-yl-2H- [1, 2, 4] triazol-3-yl) -pyridine hydrochloride

This intermediate was prepared according to the procedure for the preparation of 2- (5-azetidin-3-yl-2H- [1, 2, 4] triazol-3-yl) -pyridine hydrochloride (intermediate example 30.0).

MS(M+1)：216

Intermediate example 33.0: 5-fluoro-2-piperidin-4-yl-1H-benzimidazole hydrochloride

To a mixture of piperidine-4-carboxylic acid (18.33g, 0.14mol) and 4-fluorobenzene-1, 2-diamine (18.01g, 0.14mol) was added polyphosphoric acid (138.39g), and the mixture was heated at 180 ℃ (internal temperature) for 2 hours 45 minutes. The reaction mixture was cooled, heated to 80 ℃ and quenched by the careful addition of water (300 mL). The mixture was made basic (pH 8) by adding concentrated aqueous NaOH. Sequentially using 3: 7 isopropanol: CH₂Cl₂(2X 200mL) and CH₂Cl₂The aqueous phase was extracted (150mL) and dried (Na)₂SO₄) The combined organic phases were concentrated. The aqueous phase was re-extracted with n-butanol (2X 200mL) and dried (Na)₂SO₄) Organic layer, and concentrate. The crude product was stirred in ether, filtered, and dried to give crude productpino-5-fluoro-2-piperidin-4-yl-1H-benzimidazole. Further purification was performed by making hydrochloride salt thereof.

Thus, 10g of crude 5-fluoro-2-piperidin-4-yl-1H-benzimidazole was dissolved in MeOH (85mL), and a solution of HCl in dioxane (20mL) was added dropwise and the title compound was obtained by filtration.

MS(M+1)：220.1

¹H NMR(d6-DMSO+D₂O)：7.78(m，1H)，7.6(m，1H)，7.38(m，1H)，3.55(m，1H)，3.4(m，2H)，3.08(m，1H)，2.3(m，2H)，2.08(m，2H)ppm。

In analogy to the above 5-fluoro-2-piperidin-4-yl-1H-benzimidazole dihydrochloride, the following intermediates were prepared by substituting the appropriate diamine for 4-fluoro-benzene-1, 2-diamine.

Intermediate example 34.0: 2-piperidin-4-yl-1H-benzimidazole-5-carbonitrile hydrochloride

Step 1: 4- (2-amino-4-cyano-phenylcarbamoyl) -piperidine-1-carboxylic acid tert-butyl ester

To a solution of mono-tert-butyl piperidine-1, 4-dicarboxylate (14.1g, 0.061mol) in DMF (282mL) were added HBTU (27.76g, 0.073mol), DMAP (10.2g, 0.084mol) and diisopropylethylamine (24.2 mL). The reaction mixture was stirred at rt for 30min, then 3, 4-diaminobenzonitrile (8g, 0.059mol) was added. The mixture was stirred at rt overnight, then quenched by pouring into water (2L). By CH₂Cl₂The mixture was extracted successively with 1M aqueous HCl and 10% Na₂CO₃The organic phase is washed with an aqueous solution and dried (Na)₂SO₄) And concentrated in vacuo. Purification by silica gel chromatography gave the title compound.

Step 2: 4- (5-cyano-1H-benzimidazol-2-yl) -piperidine-1-carboxylic acid tert-butyl ester

A solution of 4- (2-amino-4-cyano-phenylcarbamoyl) -piperidine-1-carboxylic acid tert-butyl ester (6g) in EtOH (61mL) and 2M aqueous NaOH solution (61mL) was heated at 75 deg.C (bath temperature) overnight. The heating was stopped, the reaction was cooled (ice-water bath), and quenched by pouring into saturated aqueous citric acid (250 mL). By CH₂Cl₂(5X) extract the mixture and dry (Na)₂SO₄) The combined organic extracts were filtered and concentrated in vacuo. Purification by silica gel chromatography gave the title compound.

And step 3: 2-piperidin-4-yl-1H-benzimidazole-5-carbonitrile hydrochloride

To a solution of 4- (5-cyano-1H-benzoimidazol-2-yl) -piperidine-1-carboxylic acid tert-butyl ester (3.2g, 10mmol) in dioxane (13mL) was added a solution of HCl in dioxane (25%, 14.3 mL). The resulting precipitate was filtered to give the title compound.

MS(M+1)：227.1

¹H NMR(400MHz，d6-DMSO+D₂O).：8.22(s，1H)，7.85(d，1H)，7.77(d，1H)，3.42-3.5(m，3H)，3.12(m，2H)，2.34(m，2H)，2.09(m，2H)ppm。

Intermediate example 35.0: 9-piperidin-4-yl-9H-purin-6-ylamine hydrochloride

Prepared according to the method provided in WO 2006065601.

MS(M+1)：219.2

Intermediate example 36.0: 2-piperidin-4-yl-quinoxaline hydrochloride

To a stirred solution of 4-quinoxalin-2-yl-piperidine-1-carboxylic acid tert-butyl ester (200mg, 0.64mmol, commercially available) in 0.5mL dioxane/MeOH (2: 3) was added a solution of HCl in dioxane (1.6mL, 10Eq.) at rt. The mixture was stirred for 2h, then the solid was filtered, washed, and dried to give the title compound.

MS(M+1)：214.2

¹H NMR(300MHz，d6-DMSO+D₂O).：δ9.45(br s，1H)，9.15(br s，1H)，8.95(s，1H)，8.08(m，2H)，7.85(m，2H)，3.35-3.45(m，3H)，3.06(m，2H)，2.1-2.2(m，4H)ppm。

Intermediate example 37.0: 4-methyl-2- (5-piperidin-4-yl-2H- [1, 2, 4] triazol-3-yl) -pyridine hydrochloride

UPLC-MS：RT＝0.47min；m/z＝244.27；

¹H NMR(300MHz，d6-DMSO).：9.01-9.23(2×br s，2H)，8.49(d，1H)，7.86(s，1H)，7.28(d，1H)，3.24-3.28(m，2H)，2.95-3.13(m，3H)，2.37(s，3H)，2.09-2.15(m，2H)，1.87-2.01(m，2H)ppm。

The compounds of formula (I) can typically be prepared according to the following general methods or their preparation is illustrated by the following specific examples. The preparation of other examples not listed here can be effected analogously, or with modifications or alterations, by these methods or by known methods.

General procedure 1: reductive amination (using amine salts)

To a solution of 0.75mmol of the aldehyde intermediate in THF (6mL) was added triethylamine (2 Eq.). The reaction mixture was stirred for 5 minutes, then the amine salt (1.5Eq.) and acetic acid (2.5Eq.) were added. The reaction mixture was stirred for 10 minutes, then NaBH (OAc) was added portionwise over 40 minutes₃(6 Eq.). The reaction mixture was stirred at room temperature overnight, then quenched with methanol and concentrated in vacuo. The residue was dissolved in chloroform and washed with water, dried, and concentrated in vacuo. Purification according to standard techniques gives the desired compound.

In the case of the use of the amine free base, the above general procedure can be modified by omitting triethylamine.

General procedure 2: amination by mesylate intermediate (using amine salt)

To a stirred solution of the benzyl alcohol intermediate (0.52mmol) in 15mL of dichloromethane was added methanesulfonyl chloride (1.1eq) followed by triethylamine (1.5eq) at 0 ℃. The reaction mixture was stirred at room temperature for 3 h. The reaction was quenched with water and extracted with DCM. The organic layer was dried and concentrated. Then used in the next reaction without further purification. The crude product was dissolved in 5mL of DMF. To this solution was added amine hydrochloride (1eq) and triethylamine (4 eq). The reaction mixture was heated at 80 ℃ for 3 h. The reaction mixture was quenched with water and extracted with ethyl acetate. The organic layer was dried and concentrated. Purification by standard techniques gives the desired compound.

In the case of the use of amine free base, the above general procedure can be modified by reducing the number of equivalents of triethylamine from 4 to 2.

Example 1.0: 5-methyl-8-phenyl-7- {4- [4- (5-pyridin-2-yl-1H- [1, 2, 4] triazol-3-yl) -piperidin-1-ylmethyl ] -phenyl } -imidazo [1, 2-c ] pyrimidine

0.350g (1.12mmol)4- (5-methyl-8-phenyl-imidazo [1, 2-c) is stirred at 40 deg.C]Pyrimidin-7-yl) -benzaldehyde, 406mg (1.33mmol)2- (5-piperidin-4-yl-2H- [1, 2,4]Triazol-3-yl) -pyridine dihydrochloride, 392. mu.L triethylamine and 92. mu.L AcOH in 35mL DCE. After cooling to rt, 283mg NaBH (OAc) is added₃. The reaction mixture was stirred for 2h and then heated to 45 ℃. After 1 hour, an additional 283mg NaBH (OAc) was added₃And heating was continued at 45 ℃. After cooling to rt, the reaction was concentrated in vacuo and purified by chromatography followed by preparative HPLC to give the title compound.

MS(M+1)：527.1

¹H NMR(400MHz，d6-DMSO).：13.8&14.3(br s，br s，1H)，8.65(br s，1H)，7.9-8.05(m，3H)，7.67(s，1H)，7.3-7.55(m，8H)，7.2(d，2H)，3.46(s，2H)，2.88(s，3H)，2.75-2.88(m，3H)，2.08(m，2H)，1.95(m，2H)，1.78(m，2H)ppm。

Example 2.0: 8-phenyl-7- {4- [4- (5-pyridin-2-yl-1H- [1, 2, 4] triazol-3-yl) -piperidin-1-ylmethyl ] -phenyl } -imidazo [1, 2-c ] pyrimidine

2.93mL (20.9mmol) of triethylamine was added to 3.02g of 2- (5-piperidin-4-yl-2H- [1, 2, 4%]Triazol-3-yl) -pyridine dihydrochloride in 80mL MeOH. To this solution was added 2.50g (8.35mmol) of 4- (8-phenyl-imidazo [1, 2-c)]Solution of pyrimidin-7-yl) -benzaldehyde in 80mL of DMF, then 1.2mL of glacial acetic acid and 7.45g of NaBH (OAc) are added₃. The resulting mixture was stirred at rt. After 1 hour, 2 hours and 3 hours, respectively, another 2 equivalent parts of NaBH (OAc) are added₃. The reaction was stirred for 3 days, then the volatiles were removed in vacuo and the residue was taken up in CH₂Cl₂And water. Separating the organic phase with CH₂Cl₂The aqueous phase is extracted. The combined organic portions were dried and concentrated, and the residue was purified by chromatography to give the title compound (762 mg).

MS(M+1)：513.1；

¹H NMR(300MHz，d6-DMSO).：13.9，14.3(br s，br s，1H)，9.5(s，1H)，8.67(m，1H)，8.10(s，1H)，8.03(d，1H)，7.95(m，1H)，7.65(m，1H)，7.46(m，1H)，7.3-7.4(m，10H)，7.2(d，2H)，3.47(s，2H)，2.7-2.85(m，3H)，2.09(m，2H)，1.95(m，2H)，1.8(m，2H)ppm。

Example 3.0: 8-phenyl-7- {4- [4- (5-pyridin-2-yl-1H- [1, 2, 4] triazol-3-yl) -piperidin-1-ylmethyl ] -phenyl } - [1, 2, 4] triazolo [4.3-c ] pyrimidine

In analogy to example 1, this example was prepared by reacting 4- (8-phenyl- [1, 2, 4] triazolo [4, 3-c ] pyrimidin-7-yl) -benzaldehyde with 2- (5-piperidin-4-yl-2H- [1, 2, 4] triazol-3-yl) -pyridine dihydrochloride.

MS(M+1)：514.2

¹H NMR(300MHz，CDCl₃).：9.45(s，1H)，8.67(m，1H)，8.42(s，1H)，8.15(d，1H)，7.83(t，1H)，7.35-7.45(m，8H)，7.25-7.3(m，2H)，3.58(s，2H)，3.0(m，2H)，2.9(m，1H)，2.0-2.2(m，6H)ppm。

Example 4.0: 3-methyl-8-phenyl-7- {4- [4- (5-pyridin-2-yl-1H- [1, 2, 4] triazol-3-yl) -piperidin-1-ylmethyl ] -phenyl } - [1, 2, 4] triazolo [4.3-c ] pyrimidine

In analogy to example 1, this example was prepared by reacting 4- (3-methyl-8-phenyl- [1, 2, 4] triazolo [4, 3-c ] pyrimidin-7-yl) -benzaldehyde with 2- (5-piperidin-4-yl-2H- [1, 2, 4] triazol-3-yl) -pyridine dihydrochloride.

MS(M+1)：528.2；

¹H NMR(300MHz，CDCl₃).：9.30(s，1H)，8.62(m，1H)，8.15(d，1H)，7.82(t，1H)，7.32-7.42(m，8H)，7.25(m，2H)，3.52(s，2H)，2.95(m，2H)，2.85(m，1H)，2.52(s，3H)，1.9-2.18(m，6H)ppm。

Example 5.0: 7- (4- {4- [5- (5, 6-dimethyl-pyridin-2-yl) -1H- [1, 2, 4] triazol-3-yl ] -piperidin-1-ylmethyl } -phenyl) -5-methyl-8-phenyl-imidazo [1, 2-c ] pyrimidine

Reacting 4- (5-methyl-8-phenyl-imidazo [1, 2-c ]]Pyrimidin-7-yl) -benzaldehyde (150mg, 0.479mmol) and 2, 3-dimethyl-6- [3- (piperidin-4-yl) -1H-1, 2, 4-triazol-5-yl]A mixture of pyridine dihydrochloride (174mg) in NMP (2mL) was treated with triethylamine (0.147mL) and AcOH (0.066mL) and stirred at rt overnight. Addition of NaBH (OAc)₃(0.264g), and the mixture was stirred for another 5 hours. The reaction was partitioned between DCM and saturated aqueous sodium bicarbonate solution, the organic layer was dried and concentrated in vacuo. The residue was purified by preparative reverse phase HPLC to give the title compound containing formic acid as an impurity.

UPLC-MS：RT＝0.69min；m/z＝553.5(ES-)；

¹H NMR(300MHz，d6-DMSO).：14.05&13.71(br s), 8.03(s, 1H), 7.71(d, 1H), 7.50-7.63(m, 2H), 7.26-7.32(m, 7H), 7.16(d, 2H), 3.49(s, 2H), 2.80-2.84(m, 6H), [3H masked by solvent]，2.26(s，3H)，2.00-2.18(m，2H)，1.90-1.94(m，2H)，1.68-1.79(m，2H)ppm。

The following examples were prepared analogously by using the appropriate aldehyde and amine intermediates:

biological research

The following assays may be used to illustrate the commercial use of the compounds of the present invention.

Biological assay 1.0: akt1 kinase assay

The inhibitory activity of Akt1 of compounds of the invention can be quantified using the Akt1 TR-FRET assay described in the following paragraphs.

Full-length His-tagged human recombinant kinase expressed in insect cells Akt1 was purchased from Invitrogen (cat # PV 3599). The biotinylated peptide biotin-Ahx-KKLNRTLSFAEPG (C-terminus in amide form) was used as a substrate for kinase reactions, and is available, for example, from Biosynthan GmbH (Berlin-Buch, Germany).

For the assay, 50nl of a 100-fold concentrated solution of the test compound in DMSO was pipetted into a black, small-volume 384-well microtiter plate (Greiner Bio-One, Frickenhausen, Germany), Akt1 was added in assay buffer [50mM TRIS/HCl pH 7.5, 5mM MgCl₂1mM dithiothreitol, 0.02% (v/v) Triton X-100(Sigma)]And the mixture was incubated at 22 ℃ for 15min to pre-bind the test compound to the enzyme before the kinase reaction started. Then, the kinase reaction was started by adding adenosine triphosphate (ATP, 16.7 μ M ═ 10 μ M final concentration in 5 μ l assay volume) and a solution of substrate (1.67 μ M ═ 1 μ M final concentration in 5 μ l assay volume) in assay buffer (3 μ l), and incubating the resulting mixture at 22 ℃ for a reaction time of 60 min. In the assay, the concentration of Akt1 was adjusted according to the activity of the enzyme batch, and the concentration of Akt1 was chosen appropriately so that the assay was in the linear range, with a typical enzyme concentration being about 0.05 ng/. mu.l (final in 5. mu.l assay volume)Concentration).

The reaction was stopped by adding 5. mu.l of a solution of HTRF detection reagents (200nM streptavidin-XL 665[ Cisbio ] and 1.5nM anti-phosphoserine antibody [ Millipore, cat. #35-001] and 0.75 nMLLANCE Eu-W1024 labeled anti-mouse IgG antibody [ Perkin Elmer ]) in aqueous EDTA (100mM EDTA, 0.1% (W/v) bovine serum albumin in 50mM HEPES/NaOH pH 7.5).

The resulting mixture was incubated at 22 ℃ for 1h to allow the biotinylated phosphorylated peptide to bind to streptavidin-XL 665 and the antibody. The amount of phosphorylated substrate was then assessed by measuring the resonance energy transfer from the anti-mouse IgG-Eu-chelator to streptavidin-XL 665. Thus, the fluorescence emission at 620nm and 665nm after excitation at 350nm is determined in an HTRF reader, for example Rubystar (BMG Labtechnologies, Offenburg, Germany) or Viewlux (Perkin-Elmer). The ratio of the emission at 665nm to that at 622nm was taken as a measure of the amount of phosphorylated substrate. Data were normalized (enzyme reaction without inhibitor 0% inhibition, all other assay components except enzyme 100% inhibition). Typically, the values of the test compounds at each concentration are determined in duplicate on the same microtiter plate at 10 different concentrations in the range of 20 μ M to 1nM (20 μ M, 6.7 μ M, 2.2 μ M, 0.74 μ M, 0.25 μ M, 82nM, 27nM, 9.2nM, 3.1nM and 1nM, the dilution series being prepared by 1: 3 serial dilution at the level of stock solution concentrated 100-fold prior to the determination), and the IC is calculated using the software itself according to a 4-parameter fit₅₀The value is obtained.

Biological assay 2.0: akt2 kinase assay

The inhibitory activity of Akt2 of compounds of the invention was quantified using the Akt2 TR-FRET assay described in the following paragraphs.

His-tagged human recombinant kinase expressed in insect cells and activated by PDK1 full-length Akt2 was purchased from Invitrogen (cat # PV 3975). The biotinylated peptide biotin-Ahx-KKLNRTLSFAEPG (C-terminus in amide form) was used as a substrate for kinase reactions, and is available, for example, from Biosynthan GmbH (Berlin-Buch, Germany).

For the assay, 50nl of a 100-fold concentrated solution of the test compound in DMSO was pipetted into a black, small-volume 384-well microtiter plate (Greiner Bio-One, Frickenhausen, Germany), Akt2 was added in assay buffer [50mM TRIS/HCl pH 7.5, 5mM MgCl₂1mM dithiothreitol, 0.02% (v/v) Triton X-100(Sigma)]And the mixture was incubated at 22 ℃ for 15min to pre-bind the test compound to the enzyme before the kinase reaction started. Then, the kinase reaction was started by adding adenosine triphosphate (ATP, 16.7 μ M ═ 10 μ M final concentration in 5 μ l assay volume) and a solution of substrate (1.67 μ M ═ 1 μ M final concentration in 5 μ l assay volume) in assay buffer (3 μ l), and incubating the resulting mixture at 22 ℃ for a reaction time of 60 min. In the assay, the concentration of Akt2 was adjusted according to the activity of the enzyme batch, and the concentration of Akt2 was chosen appropriately so that the assay was in the linear range, with a typical enzyme concentration being about 0.2 ng/. mu.l (final concentration in 5. mu.l assay volume).

The resulting mixture was incubated at 22 ℃ for 1h to allow the biotinylated phosphorylated peptide to bind to streptavidin-XL 665 and the antibody. The amount of phosphorylated substrate was then assessed by measuring the resonance energy transfer from the anti-mouse IgG-Eu-chelator to streptavidin-XL 665. Thus, the fluorescence emission at 620nm and 665nm after excitation at 350nm is determined in a TR-FRET reader, for example Rubystar (BMG Labtechnologies, Offenburg, Germany) or Viewlux (Perkin-Elmer). The ratio of the emission at 665nm to that at 622nm was taken as a measure of the amount of phosphorylated substrate. Data were normalized (enzyme reaction without inhibitor 0% inhibition, all other assay components except enzyme 100% inhibition). Usually, in the same microdropletOn a fixed plate, the value of the test compound at each concentration was determined in duplicate at 10 different concentrations in the range of 20. mu.M to 1nM (20. mu.M, 6.7. mu.M, 2.2. mu.M, 0.74. mu.M, 0.25. mu.M, 82nM, 27nM, 9.2nM, 3.1nM and 1nM, the dilution series being prepared by 1: 3 serial dilution at the level of stock solution concentrated 100-fold before the determination), and the IC was calculated using the own software according to 4-parameter fitting₅₀The value is obtained.

Preferred compounds of the invention show in either Akt1 or Akt2 kinase assays: IC (integrated circuit)₅₀< 5. mu.M, more preferably, IC₅₀< 0.5. mu.M, even more preferably, IC₅₀＜0.05μM。

Cell assay: p-PRAS40 and p-AKT assay

Evaluation of cellular AKT Activity was carried out with the HEK293-AKT and HEK293-PRAS40 cell lines. These cell lines express AKT or PRAS40 as fusions with green fluorescent protein (GFP, a suitable TR-FRET acceptor for an excited state Tb fluorophore), respectively. Using a LanthaScreen^TMTb-anti-AKT (S473) and Tb-anti-pPRAS 40[ pThr246 [ ]]Antibodies measure the efficacy of AKT inhibitors on the phosphorylation state of GFP-PRAS40 or GFP-AKT fusion proteins in cell lysates.

Biological assay 3.1: p-PRAS40 assay

HEK293-PRAS40 cells (PerkinElmer #6007688) were seeded in 384-well MTPs at 20000 cells/well. After overnight incubation at 37 ℃, test compounds diluted in growth medium were added to the cells. After 1 hour of treatment, cells were stimulated with Insulin (Insulin #12585-014 Invitrogen) at a final concentration of 500pM for 40 min. Thereafter, the cells were lysed with a buffer containing 20mM Tris, pH 7.4, 5mM EDTA, 150mM NaCl, 1% NP-40, phosphatase/protease inhibitor and 5nM Tb-anti-AKT. After 2 hours incubation at room temperature, TR-FRET values were measured using a PHERAStar plate reader (BMG LABTECH) and IC was calculated using an emission ratio of 520/490nm₅₀。

Biological assay 3.2: p-AKT assay

The phospho-AKT assay was performed similarly to the p-PRAS40 protocol, except that the cell line was HEK293-AKT and stimulation was 5ng/mL IGF-1.

Preferred compounds of the invention show: IC (integrated circuit)₅₀< 10. mu.M, more preferably, IC₅₀＜1μM。

Biological assay 4.0: tumor cell proliferation assay

The compounds were tested in a cell-based assay that measures the ability of the compounds to inhibit tumor cell proliferation 72h after exposure to the drug. Cell viability was determined using the Cell Titer-Glo luminescent Cell viability kit from Promega (Cat. # G7573). Cells were seeded at 1000-. For each cell line assayed, cells were seeded on separate plates for luminescence assay at time points t 0 hours and t 72 hours. After overnight incubation at 37 ℃, the plate was moved to an orbital shaker by adding 100 μ L of CellTiter-Glo solution/well, shaken at room temperature for 10 minutes, and then read on a Wallac Victor 21420 Multi-label HTS Counter using a luminescence assay window (maximum light detection determined at 428 nM) to determine the luminescence value for the t-0 sample. Dose plates for the t-72 hour time point were treated with a final volume of 50 μ Ι _ of compound diluted in growth medium. The cells were then incubated at 37 ℃ for 72 hours. The luminescence value of the sample was determined by adding 150 μ L of Promega CellTiter-Glo solution, placing the cells on a shaker, shaking at room temperature for 10 minutes, and then reading the luminescence using a Victor luminometer, to give t-72 hours luminescence. Data were processed using a template specific for luciferase assays. Briefly, for both treated and untreated samples, t-0 values were subtracted from those determined at the 72 hour time point. The percent difference between the drug-treated and control luminescence was used to determine the percent inhibition of growth.

The following further cellular assays may be used to further illustrate the commercial use of the compounds of the present invention.

Biological assay 5.0: cellular PI3K/Akt pathway assay

To investigate the cellular activity of the compounds of the invention, an enzyme-linked immunosorbent assay (ELISA) -based assay can be used to investigate the inhibitory potency on Akt phosphorylation. The assay is based on a sandwich ELISA kit (PathScan)^TM Phospho-Akt1(Ser473)；Cell Signaling，USA；#7160)。

This ELISA kit detects endogenous levels of phosphorylated Akt protein. phospho-Akt (Ser473) antibody (Cell Signaling, USA; #9271) has been coated onto microwells. After incubation with the cell lysate, the coated antibody captures phosphorylated Akt protein. After extensive washing, Akt1 monoclonal antibody (Cell Signaling, USA; #2967) was added to detect the captured phospho-Akt 1 protein. Bound detection antibody was then recognized using HRP-linked anti-mouse antibody (HRP: horseradish peroxidase; CellSignaling, USA; # 7076). An HRP substrate (═ 3, 3 ', 5, 5' -Tetramethylbenzidine (TMB); Cell Signaling, USA; #7160) was added for color development. The magnitude of this developed optical density is proportional to the amount of phosphorylated Akt protein.

MCF7 cells (ATCC HTB-22) were seeded at a density of 10000 cells/well in 96-well flat-bottom plates. 24 hours after inoculation, the cells were serum-starved using low serum medium (IMEM medium, FCS (FCS: fetal calf serum) containing 0.1% charcoal treatment). After 24 hours, 1. mu.l each of the compound dilution solutions (test compound dissolved in Dimethylsulfoxide (DMSO) as a 10mM solution, then diluted) was added to each well of a 96-well plate and incubated at 37 ℃ with 5% CO₂Was incubated for 48h in a humid atmosphere. To stimulate Akt phosphorylation, beta-heregulin (20 ng/mL. beta. -HRG) was added in parallel with the compound. Wells containing unstimulated control cells (no β -heregulin stimulation) were incubated with or without the diluted compound. Wells containing untreated control cells (no compound) were filled with medium containing 0.5% v: v DMSO and stimulated with or without β -heregulin.

Cells were harvested and lysed in 1 Xcell lysis buffer (20mM Tris (pH 7.5), 150mM NaCl, 1mM ethylenediaminetetraacetic acid (EDTA), 1mM ethylene glycolAlcohol bis (2-aminoethyl) -N, N, N ', N' -tetraacetic acid (EGTA), 1 vol% Triton X-100, 2.5mM sodium pyrophosphate, 1mM beta-glycerophosphate, 1mM Na₃VO₄1. mu.g/mL leupeptin) was dissolved with brief sonication. The lysate was centrifuged for 10min at 4 ℃ and the supernatant was transferred to a new tube. Mu.l of sample diluent (0.1 vol% Tween-20, 0.1 vol% sodium azide in Phosphate Buffered Saline (PBS)) was added to the microcentrifuge tube and 100. mu.l of the cell lysate was transferred to this tube and vortexed. 100 μ l of each diluted cell lysate was added to the appropriate ELISA well and incubated overnight at 4 ℃. The plate was washed 4 times with 1 XWash buffer (1 vol% tween-20, 0.33 vol% thymol in PBS). Next, 100. mu.l of detection antibody (Akt1(2H10) monoclonal detection antibody; Cell Signaling, USA; #2967) was added to each well and incubation continued at 37 ℃ for 1H. This washing step is repeated between steps. Mu.l of a secondary antibody (anti-mouse IgGHRP-linked antibody; Cell Signaling, USA; #7076) was added to each well and incubated at 37 ℃ for 30 min. Then, 100. mu.l of TMB substrate (0.05% 3, 3 ', 5, 5' -tetramethylbenzidine, 0.1% hydrogen peroxide, complexed polypeptide, in buffer solution; Cell Signaling, USA; #7160) was added to each well and incubated at 25 ℃ for 30 min. Finally, 100. mu.l of STOP solution (0.05 vol% of alpha and beta unsaturated carbonyl) was added to each well and the plate was gently shaken. The absorbance was measured at λ 450nm (Wallac Victor 2; Perkin Elmer, USA) within 30min after addition of the STOP solution. Data analysis was performed using a statistical program (Excel; Microsoft, USA).

Biological assay 6.0: cell pGSK3 assay:

to investigate the cellular activity of the compounds of the invention, an ELISA-based assay was used for phosphorylated protein glycogen synthase kinase 3(GSK 3). This assay is based on a solid phase sandwich ELISA that measures endogenous levels of phosphorylated GSK3 using a phospho-GSK 3(Ser9) specific antibody (BioSource International, inc.; Catalog # KHO 0461). Following incubation with cell lysates, the coated antibody captured phosphorylated GSK3 protein. After extensive washing, the GSK3 polyclonal antibody was added to detect the captured phospho-GSK 3 protein. The bound detection antibody was then recognized using a secondary antibody (anti-rabbit IgG-HRP). A second incubation was performed and washed to remove all excess anti-rabbit IgG-HRP, then a solution of substrate was added and the bound enzyme acted on the substrate to produce color. The intensity of this colored product is directly proportional to the concentration of GSK-3 β [ pS9] present in the original sample.

MCF7 cells (ATCC HTB-22) were seeded at a density of 10000 cells/well in 96-well flat-bottom plates. After 24 hours, 1. mu.l each of the compound dilution solutions (test compound dissolved in Dimethylsulfoxide (DMSO) as a 10mM solution, then diluted) was added to each well of a 96-well plate and incubated at 37 ℃ with 5% CO₂Was incubated for 48h in a humid atmosphere.

Cells were harvested and extracted in cell extraction buffer (10mM Tris, pH 7.4, 100mM NaCl, 1mM EDTA, 1mM EGTA, 1mM NaF, 20mM Na₄P₂O₇、2mMNa₃VO₄1% Triton X-100, 10 vol% glycerol, 0.1 vol% SDS, 0.5 vol% deoxycholate, and 1mM phenylmethylsulfonyl fluoride (PMSF). The lysate was centrifuged for 10min at 4 ℃ and the supernatant was transferred to a new tube. Add 50. mu.l of sample diluent (standard dilution buffer, Biosource) and transfer 100. mu.l of cell lysate to this tube and vortex. 100 μ l of each diluted cell lysate was added to the appropriate ELISA well plate and incubated for 3h at room temperature. The plate was washed 4 times with 1 × wash buffer (Biosource). Mu.l of detection antibody (GSK3(Ser9) detection antibody; BioSource) was added to each well and incubated at room temperature for 30 min. This washing step is repeated between steps. Mu.l of HRP-linked secondary antibody (anti-mouse IgG HRP-linked antibody) was added to each well and incubated at room temperature for 30 min. Mu.l of TMB substrate (0.05 vol% 3, 3 ', 5, 5' -tetramethylbenzidine, 0.1 vol% hydrogen peroxide, complexed polypeptide in buffer; Biosource) was added to each well and incubated at room temperature for 30 min. Finally, 100. mu.l of STOP solution (0.05 vol% of alpha and beta unsaturated carbonyl) was added to each well and the plate was gently shaken for several seconds. After addition of STOP solutionThe absorbance was measured at λ 450nm (Wallac Victor 2; Perkin Elmer, USA) within 30 min.

Data analysis was performed using a statistical program (Excel; Microsoft, USA) and IC inhibition by pGSK3 was calculated₅₀。

Biological assay 7.0: cell proliferation/cytotoxicity assay:

the antiproliferative activity of the compounds described herein can be assessed using the OvCAR3, HCT116 and A549 cell lines and Alamar Blue (Resazurin) cell viability assay (O' Brien et al, Eur J Biochem 267, 5421-5426, 2000). Resazurin is reduced by cellular dehydrogenase activity (associated with surviving proliferating cells) to fluorescent resorufin. Test compounds were dissolved in DMSO as 10mM solutions and then diluted. Cells such as HCT116 or a549 cells were seeded at a density of 10000 cells/well (OvCAR3 cells), 1000 cells/well (HCT116 cells) or 2000 cells/well (a549 cells) in a volume of 200 μ Ι/well in a 96-well flat-bottom plate. 24 hours after inoculation, 1. mu.l each of the compound dilutions was added to each well of a 96-well plate. Dilutions of each compound were tested in at least two replicates. Wells containing untreated control cells were filled with 200 μ l DMEM (Dulbecco modified Eagle medium) containing 0.5 vol% v: v DMSO. Then, at 37 ℃ in a solution containing 5 vol% CO₂The cells were incubated with the substance for 72h in a humid atmosphere. To determine the viability of the cells, 20. mu.l of resazurin solution (90mg/l) was added. After incubation at 37 ℃ for 4h, fluorescence was measured by extinction at a lambda of 544nm and emission at a lambda of 590nm (Wallac Victor 2; Perkin Elmer, USA). To calculate cell viability, the emission value of untreated cells was set to 100% viability, and the fluorescence intensity of treated cells was correlated to the value of untreated cells. Viability is expressed in% values. Determination of the corresponding IC of the cytotoxic Activity of a Compound from concentration-Effect curves Using non-Linear regression₅₀The value is obtained. Data analysis was performed using a biometric program (GraphPad Prism, USA).

Biological assay 8.0: chemosensitization assay

The ability of the compounds disclosed herein to sensitize cancer cells to apoptotic stimuli can be evaluated. Akt inhibitors were tested to determine efficacy on apoptosis induction, both alone and in combination with chemotherapeutic agents and targeted cancer therapeutics.

Cancer cells were treated at 2X 10³To 1X 10⁴Individual cells/well were plated at concentrations in their respective growth media in 96-well plates. After 48-72 hours, apoptosis assays were performed as follows:

for assays in combination with chemotherapeutic agents, particularly preferably topoisomerase inhibitors (e.g., doxorubicin, etoposide, camptothecin or mitoxantrone) or antimitotic/tubulin inhibitors (e.g., vincristine), the compounds are added at the respective indicated concentrations and at CO₂The plates were incubated in an incubator at 37 ℃ for 18 hours. For standard combination assays using chemotherapeutic treatment, the concentrations indicated for each were added simultaneously.

For the combined assay involving the addition of a targeted pro-apoptotic agent such as the death receptor ligand TRAIL/Apo2L (research diagnostics), compound was added, TRAIL was added 1.5 hours later, and the plates were incubated for 3-4 hours after TRAIL addition. For the time course, plates were incubated with TRAIL ligand for 2, 3, 4 and 6 hours, and the assay was terminated.

For both protocols, the total final volume did not exceed 250. mu.l. At the end of the incubation time, the cells were pelleted by centrifugation (200 Xg; 10min at rt) and the supernatant was discarded. Resuspend cells and use lysis buffer (Cell Death Detection ELISA)^PLUSRoche, Cat.No.11774425001) was incubated at rt for 30 min. After repeated centrifugation (200 Xg; 10min, at rt), an aliquot of the supernatant was transferred to streptavidin-coated wells of a microplate. Then, nucleosomes in the supernatant were incubated (2h, rt) with anti-histone antibodies (biotin-labeled) and anti-DNA antibodies (peroxidase-conjugated; Cell Death Detection ELISA)^PLUSRoche, directory number 11774425001). The antibody-nucleosome complex is bound to a microplate. Washing the immobilized antibody-histone at rtComplexes were performed 3 times to remove non-immunoreactive cellular components. Adding substrate solution (2, 2' -azino-bis [ 3-ethylbenzothiazoline-6-sulfonic Acid (ABTS); Cell Death detection ELISA)^PLUSRoche, catalog No.11774425001) and incubate the sample at rt for 15 min. The amount of colored product was determined spectrophotometrically (absorbance at. lambda. 405 nm). Data are expressed as percent activity of the control, cisplatin was used as a positive control. The apoptosis induction of 50 μ M cisplatin was subjectively defined as 100 cisplatin units (100 CPU).

The following table sets forth selected data for selected embodiments of the present invention.

TABLE 1

Examples	IC₅₀p-Akt (biological assay 5.0), μ M
		1.0	＜0.05
2.0	0.008
		3.0	＜0.5
4.0	＜0.5

TABLE 2

Examples	IC₅₀Akt1 (biological assay 1.0), μ M	IC₅₀Akt2 (biological assay 2.0), μ M
			1.0	0.004	0.053
2.0	0.008	0.042
			5.0	0.003	0.027
6.0	0.003	0.081
			7.0	0.012	0.334
8.0	0.014	0.414
			9.0	0.018	0.185
10.0	0.007	0.038
			11.0	0.029	0.445
11.1	0.019	0.098
			12.0	0.008	0.050
13.0	0.016	0.132
			14.0	0.007	0.016
15.0	0.022	0.222
			16.0	0.012	0.052

Claims

1. A compound of formula (I), or a salt of said compound,

wherein ring B fused to the pyrimidine group is selected from:

indicates the point of connection, and indicates,

r1 is hydrogen or 1-4C-alkyl,

r4 is a phenyl group, and R4 is a phenyl group,

r6 is a hydrogen atom or a salt thereof,

n is a number of 1 or2,

m is a number of 1 or2,

r7 is a group of the formula-W-Y,

w is a 1,2, 4-triazolylene group,

r9 is hydrogen, 1-4C-alkyl, halogen, 1-4C-haloalkyl,

R9A is 1-4C-alkyl,

r10 is hydrogen or 1-4C-alkyl.

2. Process for the preparation of compounds of the general formula (I), characterized in that an aldehyde or ketone of the formula (III) is reacted with an amine (II) or a salt thereof to give a compound of the formula (I)

Wherein B, R4, R6, R7, m, n and R10 have the meanings stated in claim 1, and R has the meaning of-C (O) R6.

3. A process for the preparation of a compound of the general formula (I), characterized in that a compound of the formula (IIIa) is reacted with an amine (II) or a salt thereof to give a compound of the formula (I)

Wherein B, R4, R6, R7, m, n and R10 have the meanings stated in claim 1, and X is a suitable leaving group.

4. Compounds of the general formulae (III) and (IIIa),

wherein B, R4, R6 and R10 have the meanings indicated in claim 1, R has the meaning of-C (O) O (1-4C-alkyl), -C (O) R6, -CH (R6) OH or-CH₂R6 and X is a suitable leaving group.

5. The use of compounds of the general formulae (III) and (IIIa) according to claim 4 for preparing compounds of the general formula (I) according to claim 1,

6. Use of a compound of the general formula (II) according to one of claims 2 or 3 or a salt thereof for the preparation of a compound of the general formula (I) according to claim 1,

wherein R7, m and n have the meanings indicated in claim 1.

7. Use of a compound of general formula (I) according to claim 1 or a pharmaceutical composition comprising a compound of general formula (I) according to claim 1 for the preparation of a medicament for the treatment or prevention of a disease.

8. The use of claim 7, wherein the disease is a hyperproliferative disease and/or a disorder responsive to induction of apoptosis.

9. The use of claim 8, wherein the hyperproliferative disease and/or disorder responsive to induction of apoptosis is cancer.

10. Pharmaceutical compositions comprising at least one compound of general formula (I) according to claim 1, and at least one pharmaceutically acceptable adjuvant.

11. A combination comprising one or more first active ingredients selected from compounds of general formula (I) according to claim 1, and one or more second active ingredients selected from chemotherapeutic anti-cancer agents and target-specific anti-cancer agents.

12. Use of a compound of general formula (I) according to claim 1 for the preparation of a pharmaceutical composition for the treatment of benign and/or malignant neoplasia.