HK1165425B

HK1165425B - 7-cycloalkylaminoquinolones as gsk-3 inhibitors

Info

Publication number: HK1165425B
Application number: HK12106295.5A
Authority: HK
Inventors: 冈田京子; 佐藤太朗; 河野靖志; 野村昌弘
Original assignee: 杏林制药株式会社
Priority date: 2009-03-11
Filing date: 2010-03-10
Publication date: 2016-07-15

Abstract

Provided herein are aminoquinolones of formula I and compositions containing the compounds. The compounds and compositions provided herein are useful in the prevention, amelioration or treatment of GSK-3 inhibitors mediated diseases.

Description

7-cycloalkylaminoquinolones as GSK-3 inhibitors

Technical Field

Compounds, compositions and methods for treating GSK-3 mediated diseases are provided. The compounds provided herein are aminoquinolones that are GSK-3 inhibitors.

Background

Glycogen synthase kinase-3 (GSK-3) is a serine/threonine protein kinase having α and β isoforms each encoded by a different gene [ Coghlan et al, Chemistry & Biology, 7, 793-; and Kim and Kimmel, curr. opinion Genetics dev., 10, 508-. GSK-3 is implicated in a variety of diseases, including diabetes, alzheimer's disease, CNS disorders such as bipolar disorder and neurodegenerative disease, and cardiomyocyte hypertrophy [ see, e.g., WO 99/65897; WO 00/38675; and Haq et al, j.cell Biol. (2000)151, 117 ]. These diseases may be caused by or may result in abnormal operation of some of the cell signaling pathways in which GSK-3 plays a role.

GSK-3 has been found to phosphorylate and modulate the activity of a number of regulatory proteins. These include glycogen synthase (which is the rate-limiting enzyme required for glycogen synthesis), microtubule-associated protein Tau, the gene transcription factor beta-catenin, the translation initiation factor e1F-2B, and ATP citrate lyase, axin, heat shock factor-1, c-Jun, c-myc, c-myb, CREB and CEPB alpha. These different targets involve GSK-3 in many aspects of cellular metabolism, proliferation, differentiation and development.

Small molecule inhibitors of GSK-3 have been reported recently [ WO99/65897 (Chiron) and WO00/38675(SmithKline Beecham) ], however, there is a continuing need to find more effective therapeutic agents for treating GSK-3 mediated diseases.

SUMMARY

1. Provided herein are compounds represented by formula (I):

or a pharmaceutically acceptable salt thereof, wherein

R¹Is a lower alkyl group;

R²is hydrogen or lower alkyl;

m is 1, 2 or 3;

n is 1 or 2;

ar is aryl or heteroaryl optionally substituted with one to three substituents each independently selected from the group Q;

wherein Q is halo, hydroxy, cyano, nitro, oxo, thio, hydroxycarbonyl, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, aralkyl, heteroaralkyl, alkoxy, haloalkoxy, cycloalkoxy, heterocyclyloxy, aryloxy, heteroaryloxy, aralkoxy, heteroaralkoxy, alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, unsubstituted or substituted aminocarbonyl, alkylcarbonyloxy, arylcarbonyloxy, aralkylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, aralkoxycarbonyloxy, unsubstituted or substituted aminocarbonyloxy, unsubstituted or substituted amino, alkylthio, cycloalkylthio, arylthio, heteroarylthio, aralkylthio, heteroarylalkylthio, alkylsulfinyl, cycloalkylsulfinyl, alkyl-sulfinyl, alkyl-haloalkoxy, aryl-haloalkoxy, heteroaryl, aryl-haloalkoxy, heteroarylthio, heteroaryl, alkoxy-haloalkoxy, heterocyclic-alkoxy-carbonyl, heterocyclic-alkoxy-carbonyl, Arylsulfinyl, heteroarylsulfinyl, aralkylsulfinyl, heteroaralkylsulfinyl, alkylsulfonyl, cycloalkylsulfonyl, arylsulfonyl, heteroarylsulfonyl, aralkylsulfonyl, heteroaralkylsulfonyl, alkoxysulfonyl, aryloxysulfonyl, unsubstituted or substituted aminosulfonyl or hydroxysulfonyl.

Also provided herein are pharmaceutical compositions containing a compound of formula (I) and a pharmaceutically acceptable carrier.

Also provided herein are methods of treating, preventing, or alleviating one or more symptoms of a GSK-3 mediated disease by administering the compounds and compositions provided herein.

Detailed description of the embodiments

A. Definition of

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. All patents, applications, published applications and other publications are incorporated by reference in their entirety. Where there are multiple definitions for a term herein, that section controls unless otherwise stated.

As used herein, the terms alkyl, alkoxy, carbonyl, and the like are used as is commonly understood by those skilled in the art.

As used herein, an alkyl carbon chain (if not specified) contains from 1 to 20 carbons, 1 to 16 carbons, or 1 to 6 carbons and is straight or branched. In some embodiments, the alkyl carbon chain contains from 1 to 6 carbons. Alkyl groups exemplified herein include, but are not limited to, methyl, ethyl, propyl, isopropyl, isobutyl, n-butyl, sec-butyl, tert-butyl, isopentyl, neopentyl, tert-pentyl, isohexyl. As used herein, lower alkyl refers to carbon chains having from about 1 carbon up to about 6 carbons.

As used herein, an alkenyl carbon chain (if not specified) contains from 2 to 20 carbons, 2 to 16 carbons, or 2 to 6 carbons and is straight or branched. In some embodiments, the alkenyl carbon chain contains from 2 to 6 carbons. In some embodiments, alkenyl carbon chains of from 2 to 20 carbons contain 1 to 8 double bonds, and in some embodiments, alkenyl carbon chains of from 2 to 16 carbons contain 1 to 5 double bonds. In some embodiments, an alkenyl carbon chain of 2 to 6 carbons contains 1 to 2 double bonds. Alkenyl groups exemplified herein include, but are not limited to, ethenyl, 1-propenyl, 2-propenyl, isopropenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1, 3-butadienyl. As used herein, lower alkenyl refers to carbon chains having from about 2 carbons up to about 6 carbons.

As used herein, alkynyl carbon chains (if not specified) contain from 2 to 20 carbons, 2 to 16 carbons, or 2 to 6 carbons and are straight or branched. In some embodiments, the alkynyl carbon chain contains from 2 to 6 carbons. In some embodiments, alkynyl carbon chains of from 2 to 20 carbons contain 1 to 8 triple bonds, and in some embodiments, alkynyl carbon chains of from 2 to 16 carbons contain 1 to 5 triple bonds. In some embodiments, alkynyl carbon chains of from 2 to 6 carbons contain 1 to 2 triple bonds. Alkynyl groups exemplified herein include, but are not limited to, ethynyl, 1-propynyl, and 2-propynyl. As used herein, lower alkynyl refers to carbon chains having from about 2 carbons up to about 6 carbons.

As used herein, "alkoxy" contains from 1 to 20 carbons, 1 to 16 carbons, or 1 to 6 carbons and is straight or branched. In some embodiments, the alkoxy carbon chain contains from 1 to 6 carbons. Alkoxy groups exemplified herein include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, isobutoxy, n-butoxy, sec-butoxy, tert-butoxy, isopentoxy, neopentyloxy, tert-pentyloxy, and isohexyloxy.

As used herein, "aralkyl" refers to an alkyl group in which one of the hydrogen atoms of the alkyl group is substituted with an aryl group.

As used herein, "halo," "halogen," or "halide" refers to F, Cl, Br, or I.

As used herein, "aryl" refers to an aromatic monocyclic or polycyclic group containing from 6 to 19 carbon atoms. Aryl groups include, but are not limited to, groups such as fluorenyl, substituted fluorenyl, phenyl, substituted phenyl, naphthyl, and substituted naphthyl.

As used herein, "cycloalkyl" refers to a saturated monocyclic or polycyclic ring system, in some embodiments, 3 to 10 carbon atoms, in other embodiments, 3 to 6 carbon atoms; cycloalkenyl and cycloalkynyl refer to monocyclic or polycyclic ring systems comprising at least one double bond and at least one triple bond, respectively. In some embodiments, cycloalkenyl and cycloalkynyl groups can include 3 to 10 carbon atoms, in other embodiments cycloalkenyl groups contain 4 to 7 carbon atoms, and in other embodiments cycloalkynyl groups contain 8 to 10 carbon atoms. The ring systems of cycloalkyl, cycloalkenyl and cycloalkynyl can include one ring or two or more rings that can be joined together in a fused, bridged or spiro-linked fashion.

As used herein, "heterocyclyl" refers to a monocyclic or polycyclic non-aromatic ring system, in one embodiment referring to a 3 to 10 member monocyclic or polycyclic non-aromatic ring system, in another embodiment referring to a 4 to 7 member monocyclic or polycyclic non-aromatic ring system, in a further embodiment referring to a 5 to 6 member monocyclic or polycyclic non-aromatic ring system, wherein one or more, in some embodiments 1 to 3, of the atoms of the ring system are heteroatoms, i.e., elements other than carbon, including, but not limited to, nitrogen, oxygen, or sulfur. In embodiments where the heteroatom is nitrogen, the nitrogen is optionally substituted with alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, heteroaralkyl, cycloalkyl, heterocyclyl, cycloalkylalkyl, heterocycloalkyl, acyl, aminocarbonyl, alkoxycarbonyl, guanidino, or the nitrogen may be quaternized to form an ammonium group, where the substituents are selected as above.

As used herein, "heteroaryl" refers to a monocyclic or polycyclic aromatic ring system, in some embodiments 5 to about 15 members, wherein one or more, in one embodiment 1 to 3, of the atoms in the ring system are heteroatoms, i.e., elements other than carbon, including but not limited to nitrogen, oxygen, or sulfur. The heteroaryl group may be optionally fused to a benzene ring. Heteroaryl groups include, but are not limited to, furyl, imidazolyl, pyrimidinyl, tetrazolyl, thienyl, pyridyl, pyrrolyl, N-methylpyrrolyl, quinolinyl, and isoquinolinyl.

As used herein, "fused heterocyclic aryl" refers to an aryl group fused to a heterocyclic group. In one embodiment, a fused heterocyclic aryl group is one in which the heterocyclic group contains from about 5 to about 6 ring atoms and the aryl group is phenyl. The fused heterocyclic aryl group may be bonded through any atom of the ring system. Representative fused heterocyclic aromatic groups include 1, 3-benzodioxol-4-yl, 1, 3-benzodioxol-5-yl, 1, 3-benzodioxol-6-yl, 1, 3-benzodioxol-7-yl, 4-indolinyl, 5-indolinyl, 6-indolinyl, 7-indolinyl.

As used herein, "fused arylheterocyclyl" refers to a fused heterocyclyl fused to an aryl. In one embodiment, a fused arylheterocyclyl is one in which the aryl group is phenyl and the heterocyclyl contains about 5 to about 6 ring atoms. The fused aromatic heterocyclic group may be bonded through any atom of the ring system. Representative fused aromatic heterocyclic groups include 1-indolinyl, 2-indolinyl, 3-indolinyl, 1, 2,3, 4-tetrahydroquinolin-1-yl, 1, 2,3, 4-tetrahydroquinolin-2-yl, 1, 2,3, 4-tetrahydroquinolin-3-yl, and 1, 2,3, 4-tetrahydroquinolin-4-yl.

As used herein, "haloalkyl" refers to an alkyl group in which one or more of the hydrogen atoms are substituted with a halogen. "lower haloalkyl" refers to a lower alkyl group wherein one or more of the hydrogen atoms are replaced with halogen. Such groups include, but are not limited to, chloromethyl, trifluoromethyl, and 1-chloro-2-fluoroethyl.

As used herein, "heteroaralkyl" refers to an alkyl group in which one of the hydrogen atoms is substituted with a heteroaryl group.

As used herein, "haloalkoxy" refers to RO-, wherein R is haloalkyl.

As used herein, "cycloalkoxy" refers to RO-, wherein R is cycloalkyl.

As used herein, "aryloxy" refers to RO-, wherein R is aryl.

As used herein, "heteroaryloxy" refers to RO-, wherein R is heteroaryl.

As used herein, "heterocyclyloxy" refers to RO-, wherein R is heterocyclyl.

As used herein, "aralkoxy" refers to RO-, where R is aralkyl.

As used herein, "heteroarylalkoxy" refers to RO-, wherein R is heteroarylalkyl.

As used herein, "alkylcarbonyl" refers to RCO-, where R is alkyl.

As used herein, "arylcarbonyl" refers to RCO-, wherein R is aryl.

As used herein, "heteroarylcarbonyl" refers to RCO-, wherein R is heteroaryl.

As used herein, "alkoxycarbonyl" refers to RCO-, where R is alkoxy.

As used herein, "aryloxycarbonyl" refers to RCO-where R is aryloxy.

As used herein, "aralkoxycarbonyl" refers to RCO-, where R is aralkoxy.

As used herein, "unsubstituted or substituted aminocarbonyl" refers to-c (o) NR 'R, where R' and R are independently hydrogen, alkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl.

As used herein, "alkylcarbonyloxy" refers to-OC (O) R, where R is alkyl.

As used herein, "arylcarbonyloxy" refers to-OC (O) R, where R is aryl.

As used herein, "aralkylcarbonyloxy" refers to-OC (O) R, where R is aralkyl.

As used herein, "alkoxycarbonyloxy" refers to-OC (O) OR, where R is alkyl.

As used herein, "aryloxycarbonyloxy" refers to-OC (O) OR, where R is aryl.

As used herein, "aralkyloxycarbonyloxy" refers to-OC (O) OR, where R is aralkyl.

As used herein, "unsubstituted or substituted aminocarbonyloxy" refers to — oc (o) NR 'R, where R' and R are independently hydrogen, alkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl.

As used herein, "unsubstituted or substituted amino" refers to — NR 'R, where R' and R are independently hydrogen, alkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, unsubstituted or substituted aminocarbonyl, alkylsulfonyl, cycloalkylsulfonyl, arylsulfonyl, heteroarylsulfonyl, aralkylsulfonyl, or heteroaralkylsulfonyl.

As used herein, "alkylthio" refers to-SR, where R is alkyl.

As used herein, "cycloalkylthio" refers to — SR, where R is cycloalkyl.

As used herein, "arylthio" refers to-SR, where R is aryl.

As used herein, "heteroarylthio" refers to-SR, where R is heteroaryl.

As used herein, "aralkylthio" refers to-SR, wherein R is aralkyl.

As used herein, "heteroarylalkylthio" refers to-SR, where R is heteroarylalkyl.

As used herein, "alkylsulfinyl" refers to-S (O) R, where R is alkyl.

As used herein, "cycloalkylsulfinyl" refers to-S (O) R, where R is cycloalkyl.

As used herein, "arylsulfinyl" refers to-S (O) R, where R is aryl.

As used herein, "heteroarylsulfinyl" refers to-S (O) R, wherein R is heteroaryl.

As used herein, "aralkylsulfinyl" refers to-S (O) R, where R is aralkyl.

As used herein, "heteroaralkylsulfinyl" refers to-S (O) R, where R is heteroaralkyl.

As used herein, "alkylsulfonyl" refers to-S (O)₂R, wherein R is alkyl.

As used herein, "cycloalkylsulfonyl" refers to-S (O)₂R, wherein R is cycloalkyl.

As used herein, "arylsulfonyl" refers to-S (O)₂R, wherein R is aryl.

As used herein, "heteroarylsulfonyl" refers to-S (O)₂R, wherein R is heteroaryl.

As used herein, "alkoxysulfonyl" refers to-S (O)₂R, wherein R is alkoxy.

As used herein, "aryloxysulfonyl" refers to-S (O)₂R, wherein R is aryloxy.

As used herein, "aralkylsulfonyl" refers to-S (O)₂R, wherein R is aralkyl.

As used herein, "heteroaralkylsulfonyl" refers to-S (O)₂R, wherein R is heteroaralkyl.

As used herein, "unsubstituted or substituted aminosulfonyl" refers to-S (O)₂NR 'R, wherein R' and R are independently hydrogen, alkyl, aryl, heteroaryl, aralkyl or heteroaralkyl.

As used herein, pharmaceutically acceptable salts include, but are not limited to, amine salts such as, but not limited to, N '-dibenzylethylenediamine, chloroprocaine, choline, ammonia, diethanolamine and other hydroxyalkylamines, ethylenediamine, N-methylglucamine, procaine, N-benzylphenethylamine, 1-p-chlorobenzyl-2-pyrrolidin-1' -ylmethylbenzimidazole, diethylamine and other alkylamines, piperazine and tris (hydroxymethyl) methylamine; alkali metal salts such as, but not limited to, lithium, potassium, and sodium; alkaline earth metal salts such as, but not limited to, barium, calcium, and magnesium; transition metal salts such as, but not limited to, zinc; and inorganic salts such as, but not limited to, disodium hydrogen phosphate and disodium phosphate; and also include, but are not limited to, salts of inorganic acids such as, but not limited to, hydrochlorides and sulfates; and salts of organic acids such as, but not limited to, acetate, lactate, malate, tartrate, citrate, ascorbate, succinate, butyrate, valerate, mesylate, and fumarate.

It is to be understood that the compounds provided herein may contain chiral centers. Such chiral centers may have either the (R) or (S) configuration, or may be mixtures thereof. Thus, the compounds provided herein can be enantiomerically pure, or stereoisomeric or diastereomeric mixtures.

As used herein, substantially pure means sufficiently homogeneous to exhibit no readily detectable impurities as detected by standard analytical methods such as Thin Layer Chromatography (TLC), gel electrophoresis, High Performance Liquid Chromatography (HPLC), Nuclear Magnetic Resonance (NMR), and Mass Spectrometry (MS), which are used by those skilled in the art to assess the purity, or sufficiently pure that further purification does not appreciably alter the physical and chemical properties of the substance, such as enzymatic and biological activity. Methods of purifying compounds to produce substantially chemically pure compounds are known to those skilled in the art. However, a substantially chemically pure compound may be a mixture of stereoisomers. In such instances, further purification may increase the specific activity of the compound. The present disclosure is intended to include all such possible isomers, as well as racemic and optically pure forms thereof. Optically active (+) and (-), (R) -and (S) -, or (D) -and (L) -isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques, such as reverse phase HPLC. When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, and unless otherwise specified, compounds that include both E and Z geometric isomers are intended. Likewise, all tautomeric forms are also intended to be included.

As used herein, IC₅₀Refers to the amount, concentration or dose of a particular test compound that achieves 50% inhibition of the maximal response in an assay that measures the response.

As used herein, EC₅₀Refers to the dose, concentration or amount of a particular test compound that elicits a dose-dependent response that is 50% of the maximum expression of the particular response induced, driven or enhanced by that particular test compound.

As used herein, treatment refers to any method of ameliorating or otherwise beneficially altering one or more of the symptoms of a disease or disorder. Treatment also includes any pharmaceutical use of the compositions herein, such as for the treatment of diabetes.

As used herein, ameliorating the symptoms of a particular disorder by administration of a particular compound or pharmaceutical composition refers to any alleviation, whether permanent or temporary, persistent or transient, attributed to or associated with the administration of the composition.

As used herein, the term "preventing" refers to preventing the recurrence of a particular disease or disorder in a patient already suffering from the disease or disorder, and/or extending the period of time in which a patient suffering from the disease or disorder remains in remission.

As used herein, the term "GSK-3 mediated disease" or "GSK-3 mediated condition" refers to any disease or other deleterious condition or state in which GSK-3 is known to play a role. Such diseases or conditions include, but are not limited to, diabetes, conditions associated with diabetes, chronic neurodegenerative conditions including dementias such as alzheimer's disease, parkinson's disease, progressive supranuclear palsy, subacute sclerosis global brain inflammatory parkinsonism, postencephalitic parkinsonism, boxer encephalitis (pugilistic cephalitis), guam parkinsonism-dementia complex, pick's disease, corticobasal degeneration, frontotemporal dementia, huntington's chorea, AIDS-related dementia, amyotrophic lateral sclerosis, multiple sclerosis, neurotrauma tic disorders such as acute stroke, epilepsy, mood disorders such as depression, schizophrenia and bipolar disorder, rheumatoid arthritis, inflammatory bowel disease, ulcerative colitis, crohn's disease, sepsis, pancreatic cancer, ovarian cancer, and osteoporosis.

B. Compound (I)

Provided herein are compounds of formula (I):

or a pharmaceutically acceptable salt thereof, wherein

R¹Is a lower alkyl group;

R²is hydrogen or lower alkyl;

m is 1, 2 or 3;

n is 1 or 2;

wherein Q is halo, hydroxy, cyano, nitro, oxo, thio, hydroxycarbonyl, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, aralkyl, heteroaralkyl, alkoxy, haloalkoxy, cycloalkoxy, heterocyclyloxy, aryloxy, heteroaryloxy, aralkoxy, heteroaralkoxy, alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, unsubstituted or substituted aminocarbonyl, alkylcarbonyloxy, arylcarbonyloxy, aralkylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, aralkoxycarbonyloxy, unsubstituted or substituted aminocarbonyloxy, unsubstituted or substituted amino, alkylthio, cycloalkylthio, arylthio, heteroarylthio, aralkylthio, heteroarylalkylthio, alkylsulfinyl, cycloalkylsulfinyl, alkyl-sulfinyl, alkyl-haloalkoxy, aryl-haloalkoxy, heteroaryl, aryl-haloalkoxy, heteroarylthio, heteroaryl, alkoxy-haloalkoxy, heterocyclic-alkoxy-carbonyl, heterocyclic-alkoxy-carbonyl, Arylsulfinyl, heteroarylsulfinyl, aralkylsulfinyl, heteroaralkylsulfinyl, alkylsulfonyl, cycloalkylsulfonyl, arylsulfonyl, heteroarylsulfonyl, aralkylsulfonyl, heteroaralkylsulfonyl, alkoxysulfonyl, aryloxysulfonyl, unsubstituted or substituted aminosulfonyl, alkoxysulfonyl, aryloxysulfonyl, or hydroxysulfonyl.

In one embodiment, R²Is hydrogen.

In another embodiment, Ar is heteroaryl optionally substituted with one to three substituents each independently selected from Q groups.

In another embodiment, m + n is 3 or 4.

In some embodiments, the compound is formula (Ia);

or a pharmaceutically acceptable salt thereof, wherein

n is 1 or 2;

In one embodiment, the compound is formula (Ia), wherein Ar is heteroaryl optionally substituted with one to three substituents each independently selected from Q groups.

In one embodiment, the compound is formula (Ia), wherein Ar is pyridinyl, pyrimidinyl, pyrazolyl, or imidazolyl optionally substituted with one to three substituents each independently selected from Q groups.

In further embodiments, the compound is selected from:

C. preparation of the Compounds

The compounds provided herein can be prepared by methods known to those skilled in the art as shown below and according to procedures analogous to those described in the examples section herein below and conventional modifications thereof.

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The compound represented by the general formula (1) according to the present invention can be prepared by synthesis 1 or a combination of conventional methods.

Wherein Ar, R¹、R²M and n are as described above.

The conversion from formula (2) and formula (3) to formula (1) (i.e., process 1-a) is carried out using a base (such as triethylamine, pyridine, isopropylamine, 1, 8-diazabicycloundecyne) at room temperature to 180 ℃ for 1-48 hours in a suitable solvent (such as N, N-dimethylformamide, dimethylsulfoxide, N-methylpyrrolidinone, toluene, acetonitrile, tetrahydrofuran, methanol, ethanol, or mixtures thereof) or without a solvent.

The compound represented by the general formula (1) according to the present invention can also be prepared by synthesis 2.

Wherein Ar, R¹、R²M and n are as described above.

The conversion from the general formula (4) to the general formula (5) (i.e., process 2-A) can be carried out by a method similar to that of process 1-A.

The conversion from formula (5) to formula (6) (i.e., process 2-B) can be performed as follows: the general formula (5) is first reacted with thionyl chloride, thionyl bromide, acetic anhydride, ethyl chlorocarbonate, methyl chlorocarbonate or the like in a suitable solvent such as N, N-dimethylformamide, dichloromethane, chloroform, tetrahydrofuran or a mixture thereof at-15 ℃ to room temperature for 5min to 3 hours. This converts the carboxyl groups into reactive derivative groups. The reaction can be carried out in the absence or presence of a base such as pyridine and triethylamine. Subsequently, the reaction product is reacted with an aqueous ammonia solution in a suitable solvent (such as N, N-dimethylformamide, dichloromethane, chloroform, tetrahydrofuran or a mixture thereof) at a temperature of from 0 ℃ to 100 ℃ for 5min to 24 hours.

The conversion from formula (6) to formula (1) (i.e., process 2-C) can be performed as follows: the general formula (6) is reacted with a dehydroxylating agent such as trifluoroacetic anhydride and phosphorus oxychloride in a suitable solvent such as dichloromethane, chloroform, tetrahydrofuran or a mixture thereof at-78 to 50 c for 1 to 24 hours in the presence of a base such as pyridine and triethylamine.

The compound represented by the general formula (3) according to the present invention can also be prepared by synthesizing 3.

Wherein Ar, m and n are as described above, and X^aIs a leaving group (such as a halogen atom, p-toluenesulfonyloxy group, methanesulfonyloxy group, trifluoromethanesulfonyloxy group).

The conversion from formula (7) to formula (8) (i.e., process 3-a) can be performed as follows: using a reducing agent such as sodium borohydride, lithium borohydride, diisobutylaluminum hydride, lithium aluminum hydride in a suitable solvent such as tetrahydrofuran, methanol, ethanol or a mixture thereof at 0 ℃ to 50 ℃ for 30min to 24 hours.

The conversion from formula (8) and compound (9a) to formula (10) (i.e., process 3-B) can be performed as follows: the reaction is carried out in a suitable solvent such as tetrahydrofuran, 1, 4-dioxane, toluene, benzene or a mixture thereof using a Mitsunobu reagent such as diethyl azodicarboxylate, diisopropyl azodicarboxylate, cyanomethylene tributylphosphine, N' -tetramethylazodicarboxamide and a phosphine agent such as triphenylphosphine, tributylphosphine, if necessary, at 0 ℃ to 50 ℃ for 1 to 24 hours.

Alternatively, the transformation may be performed as follows: the conversion from formula (8) to formula (11) (i.e., process 3-C) can be performed as follows: in a suitable solvent such as N, N-dimethylformamide, dimethylsulfoxide, toluene, acetonitrile, tetrahydrofuran, dichloromethane or a mixture thereof, in the presence of a base such as triethylamine, pyridine, isopropylamine and 1, 8-diazabicycloundecene, using a halogenating agent such as thionyl chloride, phosphorus oxychloride or thionyl bromide or a sulfonating agent such as methanesulfonyl chloride, p-toluenesulfonyl chloride or trifluoromethanesulfonic anhydride at 0 ℃ to room temperature for 1-24 hours.

The conversion from formula (11) and compound (9b) to formula (10) (i.e., process 3-D) can be performed as follows: in a suitable solvent (such as N, N-dimethylformamide, dimethyl sulfoxide, N-methylpyrrolidone, toluene, acetonitrile, tetrahydrofuran, methanol, ethanol or a mixture thereof) at 0 ℃ to 100 ℃ for 1 to 12 hours.

The conversion from formula (10) to formula (3) (i.e., process 3-E) can be performed as follows: using a hydrazine reagent (such as NH) in a suitable solvent (such as methanol, ethanol, toluene, acetonitrile, tetrahydrofuran, water, or mixtures thereof)₂NH₂-H₂O、MeNHNH₂、PhNHNH₂) Acids (such as hydrochloric acid, formic acid) or bases (such as KOH, NaOH, K)₂CO₃) At 0 to 100 ℃ for 30min to 12 hours.

Alternatively, the transformation may be performed as follows: the conversion from formula (11) to formula (12) (i.e., process 3-F) can be performed as follows: : in a suitable solvent such as dimethylsulfoxide, toluene, tetrahydrofuran, N-dimethylformamide, acetonitrile, dichloromethane or a mixture thereof, with an azide such as sodium azide, trimethylsilyl azide, diphenylphosphine azide (diphenylphosphinyl azide) at room temperature to 80 ℃ for 1 to 24 hours. The conversion from formula (12) to formula (3) (i.e., process 3-G) can be performed as follows: hydrogenation is carried out in a suitable solvent such as methanol, ethanol, ethyl acetate, tetrahydrofuran, N-dimethylformamide, acetic acid or a mixture thereof, using a metal catalyst such as palladium on activated carbon, platinum oxide and raney nickel at room temperature to 80 ℃ for 1 to 24 hours. The reaction may be carried out under a hydrogen atmosphere at a pressure of from normal pressure to 0.5 MPa.

In synthesis 3, among the compounds represented by the general formula (7), the compound represented by the general formula (7a) can be prepared by synthesis 4.

Wherein Ar and n are as described above, and PG^aIs a protecting group (such as methyl, ethyl, benzyl, trimethylsilyl, acetyl) and X^bIs a halogen atom.

The conversion from general formula (13) and general formula (14) to general formula (15) (i.e., process 4-a) can be performed as follows: general formula (14) is reacted with an alkyllithium such as n-butyllithium, isobutyllithium, t-butyllithium, lithium diisopropylamide at-78 deg.c to-40 deg.c for 10min to 1 hour in a suitable solvent such as diethyl ether, tetrahydrofuran or a mixture thereof, and then the reaction product is reacted with general formula (13) at-78 deg.c to room temperature for 1 to 12 hours in a suitable solvent such as diethyl ether, tetrahydrofuran or a mixture thereof.

The conversion from formula (15) to formula (16) (i.e., process 4-B) can be performed as follows: in a suitable solvent such as water, acetonitrile, tetrahydrofuran, dichloromethane, water or a mixture thereof, in the presence of an acid such as hydrochloric acid, trifluoromethylacetic acid (trifloromethyl acetic acid), formic acid at-10 ℃ to 50 ℃ for 30min to 12 hours.

The conversion from formula (16) to formula (7a) (i.e., process 4-C) can be performed as follows: hydrogenation is carried out in a suitable solvent such as methanol, ethanol, ethyl acetate, tetrahydrofuran, N-dimethylformamide, acetic acid or a mixture thereof at room temperature to 80 ℃ for 1 to 24 hours using a metal catalyst such as palladium on activated carbon, platinum oxide and raney nickel. The reaction may be carried out under a hydrogen atmosphere at a pressure of from normal pressure to 0.5 MPa.

In synthesis 3, among the compounds represented by the general formula (7), the compound represented by the general formula (7b) can be prepared by synthesis 5.

Wherein Ar and X^bM and n are as described above, and PG^bRepresents an alkyl or aralkyl group, or forms a cyclic ketal (such as 1, 3-dioxane or 1, 3-dioxolane).

The conversion from general formula (17) and general formula (14) to general formula (18) (i.e., process 5-A) can be performed by a method similar to process 4-A.

The conversion from formula (18) to formula (19) (i.e., process 5-B) can be performed as follows: formula (18) is first reacted with a halogenating agent (thionyl chloride, phosphorus chloride) or a sulfonating agent (methanesulfonyl chloride, p-toluenesulfonyl chloride) in a suitable solvent (such as toluene, acetonitrile, tetrahydrofuran, dichloromethane or a mixture thereof) in the presence of a base (such as triethylamine, pyridine, isopropylamine and 1, 8-diazabicycloundecane) at-10 ℃ to room temperature for 30min to 5 hours.

Alternatively, the transformation may be performed as follows: in a suitable solvent such as water, acetonitrile, tetrahydrofuran, dichloromethane or mixtures thereof, in the presence of an acid such as hydrochloric acid, trifluoromethylacetic acid, formic acid at room temperature to 100 ℃ for 30min to 12 hours.

The conversion from formula (19) to formula (20) (i.e., Process 5-C) can be carried out by a method similar to that of Process 4-C.

The conversion from formula (20) to formula (7b) (i.e., process 5-D) can be performed as follows: in a suitable solvent such as water, acetonitrile, tetrahydrofuran, dichloromethane, water or mixtures thereof in the presence of an acid such as hydrochloric acid, trifluoromethyl acetic acid, formic acid at-10 ℃ to 50 ℃ for 30min to 12 hours.

In synthesis 3, among the compounds represented by the general formula (8), the compound represented by the general formula (8a) can be prepared by synthesis 6.

Wherein n is as described above, andrepresents pyrroloyl (pyrroyl), imidazoloyl (imidazoyl), triazoloyl (triazolyl) or tetrazoloyl (tetrazolyl) optionally substituted with one to three substituents.

The conversion from formula (21) and formula (22) to formula (23) (i.e., process 6-a) can be performed as follows: in a suitable solvent (such as tetrahydrofuran, dichloromethane, toluene, diethyl ether, ethanol, acetonitrile) using a catalyst (such as NaOEt, AcOH, copper reagent, lewis acid) at 0 ℃ to 50 ℃ for 30min to 24 hours.

The conversion from formula (23) to formula (8a) (i.e., Process 6-B) can be carried out by a method similar to that of Process 3-A.

Among the compounds represented by the general formula (3) according to the present invention, the compounds represented by the general formulae (3b) and (3c) can also be prepared by synthesis 7.

Wherein Ar and n are as described above, and X^cIs a halogen atomOr magnesium halide (MgCl, MgBr, MgI), and R^aIs acyl (such as formyl, acetyl or benzoyl)

The conversion from formula (24) and formula (25) to formula (26) (i.e., process 7-a) can be performed as follows: when X is present^cRepresenting a halogen atom, with an alkyllithium (such as n-butyllithium, iso-butyllithium, tert-butyllithium) and a magnesium salt (such as MgCl) in a suitable solvent (such as diethyl ether, tetrahydrofuran or mixtures thereof) in the presence of a copper reagent (such as CuCN, CuI) at-78 ℃ to room temperature₂) Or a grignard reagent such as i-PrMgCl, i-PrMgBr for 1 to 12 hours, and then reacting the reaction product with the general formula (24) in a suitable solvent such as diethyl ether, tetrahydrofuran or a mixture thereof at-78 ℃ to room temperature for 1 to 12 hours.

Alternatively, when X^cWhen representing a magnesium halide (MgCl, MgBr, MgI), the reaction product of formula (25) is reacted with a copper reagent (such as CuCN, CuI) in a suitable solvent (such as diethyl ether, tetrahydrofuran or mixtures thereof) at-78 ℃ to room temperature for 1 to 12 hours, and then the reaction product is reacted with formula (24) in a suitable solvent (such as diethyl ether, tetrahydrofuran or mixtures thereof) at-78 ℃ to room temperature for 1 to 12 hours.

The conversion from formula (26) to formula (27) (i.e., Process 7-B) can be carried out by a method similar to that of Process 4-C.

The conversion from general formula (27) to general formula (28) (i.e., process 7-C) can be performed with an acid reagent (such as acetic acid, trifluoroacetic acid, benzoic acid, p-nitrobenzoic acid) by a method similar to process 3-B.

The conversion from formula (28) to formula (29) (i.e., process 7-D) can be performed as follows: in a suitable solvent (such as methanol, ethanol, tetrahydrofuran, water or mixtures thereof) in a base (such as K)₂CO₃NaOH, KOH, pyridine) at 0 ℃ to 50 ℃ for 1-24 hours.

The conversion from formula (29) to formula (30a) or formula (27) to formula (30B) (i.e., process 7-E) can be carried out by a method similar to process 3-B.

The conversion from formula (30a) to formula (3a) or formula (30b) to formula (3b) (i.e., process 7-F) can be carried out by a method similar to process 3-E.

In synthesis 1 and synthesis 2, the compounds represented by general formulae (2) and (4) can be prepared by synthesis 8.

Wherein R is¹And R²As described above, and R^bIs an alkyl group (such as methyl, ethyl).

The conversion from formula (31) and formula (32) to formula (33) (i.e., process 8-a) can be performed as follows: the reaction of formula (31) with a mixture of acetic anhydride and orthoester such as ethyl orthoformate and ethyl orthoacetate at 100 to 150 ℃ for 1 to 8 hours, and then the reaction product with formula (32) in a suitable solvent such as toluene, tetrahydrofuran, methanol, ethanol, t-butanol or a mixture thereof at room temperature for 1 to 24 hours. The reaction can be carried out in the absence or presence of a base (potassium carbonate, sodium carbonate, t-BuOK, t-BuONa, triethylamine and pyridine).

The conversion from formula (33) to formula (34) (i.e., process 8-B) can be performed as follows: in a suitable solvent such as N, N-dimethylformamide, dimethylsulfoxide, tetrahydrofuran or a mixture thereof, using a base such as potassium hydride, sodium hydride, potassium carbonate, sodium carbonate, t-BuOK and t-BuONa, or a fluorine salt such as potassium fluoride and sodium fluoride at 0 ℃ to 150 ℃ for 1 to 24 hours.

The conversion from general formula (34) to general formula (35) (i.e., process 8-C) can be performed by general nitration, for example, a reaction using a nitrosating agent such as potassium nitrate, sodium nitrate, and nitric acid in concentrated sulfuric acid. The reaction can be carried out at 0 ℃ to 50 ℃ for 1 to 8 hours.

The conversion from formula (35) to formula (36) (i.e., process 8-D) can be performed as follows: in the presence of an acid such as hydrochloric acid and acetic acid, in a suitable solvent such as tetrahydrofuran, methanol, ethanol, water or a mixture thereof or without a solvent, using a metal such as reduced iron, tin and zinc at room temperature to 120 ℃ for 1 to 48 hours.

Alternatively, the transformation may be performed as follows: hydrogenation is carried out in a suitable solvent such as methanol, ethanol, ethyl acetate, tetrahydrofuran, N-dimethylformamide, acetic acid or a mixture thereof, using a metal catalyst such as palladium on activated carbon, platinum oxide and raney nickel at room temperature to 80 ℃ for 1 to 24 hours. The reaction may be carried out under a hydrogen atmosphere at a pressure of from normal pressure to 0.5 MPa.

In addition, the transformation can be carried out as follows: reduction with sodium dithionite in a suitable solvent such as water, methanol, ethanol, tetrahydrofuran or mixtures thereof at room temperature to 100 ℃ for 1-24 hours.

The conversion from formula (36) to formula (4) (i.e., process 8-E) can be carried out by general hydrolysis, for example, reaction with a base (such as KOH, NaOH, LiOH) or an acid (such as hydrochloric acid, sulfuric acid, trifluoroacetic acid) in a suitable solvent (such as water, methanol, ethanol, tetrahydrofuran, or a mixture thereof). The reaction can be carried out at 0 ℃ to 50 ℃ for 1 to 8 hours.

The conversion from formula (4) to formula (37) (i.e., process 8-F) can be carried out by a method similar to process 2-B.

The conversion from formula (37) to formula (2) (i.e., process 8-G) can be carried out by a method similar to process 2-C.

Optical isomers of the compound represented by the general formula (1) can be synthesized using the optically active material compounds according to the above syntheses 1 to 3 and 7.

The racemic compound represented by the general formula (1) can be synthesized by separation and recrystallization using an optically active acid or base.

The above compounds can be prepared by chromatographic techniques using chiral supports (choral supports).

D. Pharmaceutical composition

The pharmaceutical compositions provided herein contain a therapeutically effective amount of one or more of the compounds provided herein, which compounds are useful in preventing, treating, or ameliorating one or more of the symptoms of a GSK-3 mediated disease.

Pharmaceutical compositions of the compounds provided herein can be administered systemically or topically, or orally or parenterally (such as rectally, subcutaneously, intramuscularly, intravenously, or transdermally).

The compositions contain one or more compounds provided herein. The compounds may be formulated into suitable pharmaceutical preparations for oral administration or for parenteral administration in sterile solutions or suspensions, such as solutions, suspensions, tablets, dispersible tablets, pills, capsules, granules, powders, fine powders, injections, sustained release formulations or elixirs, as well as transdermal patch preparations and dry powder inhalers. In some embodiments, the compositions provided herein can be prepared according to conventional pharmaceutical preparation techniques by the addition of suitable excipients, fillers, binders, disintegrants, coatings, sugar coatings, pH adjusting agents, solubilizing agents, or aqueous or non-aqueous solvents. Typically, the above compounds are formulated into pharmaceutical compositions using techniques and procedures well known in the art (see, e.g., Ansel Introduction to pharmaceutical Dosage Forms, seventh edition 1999).

In the compositions, an effective concentration of one or more compounds or pharmaceutically acceptable salts is mixed with a suitable pharmaceutical carrier or vehicle. As described above, the compounds may be derivatized to the corresponding salts, esters, enols, ethers or esters, acids, bases, solvates, hydrates or prodrugs prior to formulation. When administered, the concentration of the compound in the composition is effective to deliver an amount that treats, prevents, or ameliorates one or more symptoms of a GSK-3 mediated disease.

Typically, the compositions are formulated for single dose administration. To formulate the composition, a weight fraction of the compound is dissolved, suspended, dispersed, or otherwise mixed in a vehicle of choice at an effective concentration such that the condition being treated is reduced or ameliorated. Pharmaceutical carriers or vehicles provided herein that are suitable for administration of a compound include any such carrier known to those of skill in the art to be suitable for the particular mode of administration.

In addition, the compounds may be formulated as separate pharmaceutically active ingredients in a composition or may be combined with other active ingredients. Liposomal suspensions (including tissue-targeting liposomes, such as tumor-targeting liposomes) may also be suitable as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art. For example, liposomal formulations can be prepared as known in the art. Briefly, liposomes such as Multilamellar Liposomes (MLV) can be formed by drying egg phosphatidylcholine and brain phosphatidylserine (7: 3 molar ratio) inside the flask. A solution of the compounds provided herein in Phosphate Buffered Saline (PBS) lacking divalent cations was added and the flask was shaken until the lipid film was dispersed. The resulting vesicles were washed to remove unencapsulated compounds, pelleted by centrifugation, and then resuspended in PBS.

The active compound is included in a pharmaceutically acceptable carrier in an amount sufficient to exert a therapeutically useful effect in the treated patient without undesirable side effects. Therapeutically effective concentrations can be determined empirically by testing compounds in vitro and in vivo systems as described herein, and then inferring dosages for humans therefrom.

The concentration of the active compound in the pharmaceutical composition will depend on absorption, inactivation, and excretion rates of the active compound, the physicochemical properties of the compound, the dosage regimen and amount administered, and other factors known to those skilled in the art. For example, the amount delivered is sufficient to ameliorate one or more of the symptoms of a GSK-3 mediated disease.

In some embodiments, a therapeutically effective dose should result in a serum concentration of the active ingredient of from about 0.1ng/ml to about 50-100 μ g/ml. In one embodiment, the pharmaceutical composition provides a dose of from about 0.001mg to about 2000mg of the compound per kilogram of body weight per day. Pharmaceutical dosage unit forms are prepared to provide from about 1mg to about 1000mg, and in some embodiments, from about 10mg to about 500mg of the primary active ingredient or combination of primary ingredients per dosage unit form.

The active ingredient may be administered at one time, or may be divided into a number of smaller doses to be administered at intervals. It will be understood that the precise dose and duration of treatment will vary with the condition to be treated and may be measured empirically using known testing protocols or by extrapolation from in vivo or in vitro test data. Notably, concentrations and dose values may also vary with the severity of the condition to be alleviated. It is also to be understood that for any particular subject, the particular dosage regimen should be adjusted over time according to the individual needs and professional judgment or guidance of the individual administering the composition, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition.

Thus, an effective concentration or amount of one or more of the compounds described herein, or a pharmaceutically acceptable salt thereof, is mixed with a pharmaceutical carrier or vehicle suitable for systemic, local or local administration to form a pharmaceutical composition. Included are compounds in an amount effective to ameliorate one or more symptoms of, or treat or prevent, a kinase-mediated disease, including, but not limited to, a GSK-3 mediated disease. The concentration of the active compound in the composition will depend on absorption, inactivation, excretion rate of the active compound, dosage regimen, amount administered, particular dosage form, and other factors known to those skilled in the art.

The compositions are intended to be administered by a suitable route, including orally, parenterally, rectally, topically (topically), and topically (locally). For oral administration, capsules and tablets may be formulated. The compositions are in liquid, semi-liquid or solid form and are formulated in a manner suitable for each route of administration.

Solutions or suspensions for parenteral, intradermal, subcutaneous or topical application may include any of the following components: sterile diluents such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol, dimethylacetamide or other synthetic solvents; antimicrobial agents such as benzyl alcohol and methyl paraben; antioxidants such as ascorbic acid and sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates and phosphates; and agents for tonicity such as sodium chloride or dextrose. Parenteral preparations may be packaged in ampoules, disposable syringes or single or multiple dose vials made of glass, plastic or other suitable material.

In cases where the compound exhibits insufficient solubility, the method used to solubilize the compound may be used. Such methods are known to those skilled in the art and include, but are not limited to, the use of co-solvents, such as dimethyl sulfoxide (DMSO), with surfactants, such as TWEENOr dissolved in an aqueous sodium bicarbonate solution.

When the compounds are mixed or added, the resulting mixture may be a solution, suspension, emulsion, or the like. The form of the resulting mixture depends on a number of factors, including the intended mode of administration and the solubility of the compound in the selected carrier or vehicle. The effective concentration is sufficient to ameliorate the symptoms of the disease, disorder or condition being treated and can be determined empirically.

Pharmaceutical compositions for administration to humans and animals are provided in unit dosage forms such as tablets, capsules, pills, powders, granules, sterile injectable solutions or suspensions, and oral solutions or suspensions, and aqueous and oily emulsions, containing a suitable amount of a compound or a pharmaceutically acceptable salt thereof. The pharmaceutically active compounds and salts thereof are formulated and administered in unit dose form or in multi-dose form. Unit dosage forms, as used herein, refer to physically discrete units suitable for human and animal subjects and packaged separately as is known in the art. Each unit dose contains a predetermined amount of the therapeutically active compound in combination with a desired pharmaceutical carrier, vehicle or diluent sufficient to produce the desired therapeutic effect. Examples of unit dosage forms include ampoules and syringes and individually packaged tablets or capsules. The unit dosage form may be administered in portions or multiples thereof. A multi-dose form is a number of identical unit dose forms packaged in separate containers for administration as separate unit dose forms. Examples of multi-dose forms include vials, bottles of tablets or capsules, or pints or gallon bottles. Thus, a multi-dose form is a number of unit doses packaged without separation.

Sustained release formulations may also be prepared. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing the compound provided herein, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly (2-hydroxyethyl methacrylate), or poly (vinyl alcohol)), polylactides, copolymers of L-glutamic acid and ethyl L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT^TM(injectable microspheres comprising lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D- (-) -3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid are capable of releasing molecules for more than 100 days, some hydrogels release proteins for shorter periods of time. When the encapsulated compound remains in the body for a long time, the compound may denature or aggregate due to exposure to moisture at 37 ℃, resulting in a loss of biological activity and possible changes in its structure. For stabilization, rational strategies can be devised depending on the mechanism of action involved. For example, if the aggregation mechanism is found to be intermolecular S — S bond formation through thio-disulfide interchange, stabilization can be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions.

Dosage forms or compositions may be prepared containing from 0.005% to 100% of the active ingredient with the remainder being comprised of non-toxic carriers. For oral administration, pharmaceutically acceptable non-toxic compositions are formed by any commonly used excipient, such as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, talc or talcum, cellulose derivatives, gelatin, agar, pectin, acacia, olive oil, sesame oil, cocoa butter, ethylene glycol, croscarmellose sodium, glucose, sucrose, magnesium carbonate or sodium saccharin. Such compositions include solutions, suspensions, tablets, capsules, powders and sustained release formulations such as, but not limited to, implants and microencapsulated delivery systems, and biodegradable, biocompatible polymers such as collagen, ethylene vinyl acetate, polyanhydrides, polyglycolic acid, polyorthoesters, polylactic acid, and others. Methods for preparing the compositions are known to those skilled in the art. Desirable compositions may contain from about 0.001% to about 10%, in some embodiments from about 0.1% to about 85%, or from about 75% to about 95%, of the active ingredient.

The active compound or pharmaceutically acceptable salt can be formulated with a carrier that protects the compound from rapid expulsion from the body, such as a time release formulation or coating.

The composition may include other active compounds to achieve a desired combination of properties. The compounds provided herein, or pharmaceutically acceptable salts thereof as described herein, may also be advantageously administered for therapeutic or prophylactic purposes together with another pharmacological agent known in the art to have value in the treatment of one or more of the diseases or medical conditions referred to above, such as GSK-3 mediated diseases. It is to be understood that such combination therapy constitutes additional aspects of the compositions and methods of treatment provided herein.

Lactose-free compositions provided herein can contain excipients well known in the art and listed, for example, in the United States Pharmacopeia (USP) sp (xxi)/nf (xvi). Generally, lactose-free compositions contain the active ingredient, binder/filler and lubricant in pharmaceutically compatible and pharmaceutically acceptable amounts. An exemplary lactose-free dosage form contains the active ingredient, microcrystalline cellulose, pregelatinized starch, and magnesium stearate.

Also included are anhydrous pharmaceutical compositions and dosage forms containing the compounds provided herein. For example, the addition of water (e.g., 5%) is widely accepted in the pharmaceutical arts as a means of simulating long-term storage to determine properties such as shelf life or formulation stability over time. See, e.g., Jens t. carstensen, Drug Stability: principles & Practice, second edition, Marcel Dekker, NY, NY, 1995, pages 379-80. In fact, water and heat accelerate the decomposition of some compounds. Thus, the effect of water on the formulation can be very important, as moisture and/or humidity are commonly encountered during manufacture, handling, packaging, storage, transport and use of the formulation.

The anhydrous pharmaceutical compositions and dosage forms described herein can be prepared using anhydrous or low moisture containing ingredients and low moisture or low humidity conditions. In some embodiments, pharmaceutical compositions and dosage forms comprising lactose and at least one active ingredient comprising a primary or secondary amine may be anhydrous if the active ingredient is expected to be in substantial contact with moisture and/or humidity during manufacture, packaging, and/or storage.

Anhydrous pharmaceutical compositions should be prepared and stored to maintain their anhydrous nature. Accordingly, in some embodiments, the anhydrous composition is packaged using known materials to prevent exposure to water so that it can be contained in a suitable kit. Examples of suitable packaging include, but are not limited to, sealed foil, plastic, unit dose containers (e.g., vials), blister packs, and strip packs.

Oral dosage form

Oral pharmaceutical dosage forms are solid, gel or liquid. The solid dosage forms are tablets, capsules, granules and bulk powders. Types of oral tablets include compressed chewable lozenges and tablets which may be enteric-coated, sugar-coated or film-coated. The capsules may be hard or soft gelatin capsules, while granules and powders may be provided in non-effervescent or effervescent form in combination with other ingredients known to those skilled in the art.

In some embodiments, the formulation is a solid dosage form, such as a capsule or tablet. Tablets, pills, capsules, lozenges, and the like may contain any of the following ingredients or compounds of similar properties: binders, diluents, disintegrants, lubricants, glidants, sweeteners, and flavoring agents.

Examples of the binder include microcrystalline cellulose, tragacanth gum, glucose solution, acacia syrup, gelatin solution, sucrose and starch paste. Lubricants include talc, starch, magnesium or calcium stearate, Pinaceae and stearic acid. Diluents include, for example, lactose, sucrose, starch, kaolin, salt, mannitol, and dicalcium phosphate. Glidants include, but are not limited to, colloidal silicon dioxide. Disintegrants include croscarmellose sodium, sodium starch glycolate, alginic acid, corn starch, potato starch, bentonite, methylcellulose, agar and carboxymethylcellulose. Colorants include, for example, any of the approved certified water-soluble FD and C dyes, mixtures thereof; and water insoluble FD and C dyes suspended in aluminum hydroxide. Sweeteners include sucrose, lactose, mannitol, and artificial sweeteners such as saccharin, and many spray dried flavors. Flavoring agents include natural flavors extracted from plants such as fruits and synthetic mixtures of compounds that produce pleasant sensations, such as, but not limited to, mint and methyl salicylate. Wetting agents include propylene glycol monostearate, sorbitan monooleate, diethylene glycol monododecanoate, and polyoxyethylene lauryl ether. Enteric coatings include fatty acids, fats, waxes, shellac, ammoniated shellac, and cellulose acetate phthalate. The film coat comprises hydroxyethyl cellulose, sodium carboxymethyl cellulose, polyethylene glycol 4000 and cellulose acetate phthalate.

Compositions that can protect the compound from the acidic environment of the stomach provide the compound if oral administration is desired. For example, the composition may be formulated in an enteric coating that maintains the integrity of the composition in the stomach and releases the active compound in the intestine. The compositions may also be formulated in combination with antacids or other such ingredients.

When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier such as a fatty oil. In addition, the dosage unit forms can contain a variety of other materials that modify the physical form of the dosage unit, such as sugar coatings and other enterically absorbed medicaments. The compounds may also be administered as elixirs, suspensions, syrups, wafers, sprays, chewing gums or other components. In addition to the active compounds, syrups may contain sucrose as a sweetening agent and also some preservatives, dyes and pigments and flavouring agents.

The active material may also be mixed with other active materials that do not impair the desired action, or with materials that supplement the desired action, such as antacids, H2 blockers, and diuretics. The active ingredient is a compound described herein or a pharmaceutically acceptable salt thereof. Higher concentrations of up to about 98% by weight of the active ingredient may be included.

Pharmaceutically acceptable carriers included in tablets are binders, lubricants, diluents, disintegrating agents, coloring agents, flavoring agents, and wetting agents. Enteric coated tablets (due to the enteric coating) resist the action of gastric acid and dissolve or disintegrate in the neutral or alkaline intestine. Sugar-coated tablets are compressed tablets of a pharmaceutically acceptable material employing different layers. Film coated tablets are compressed tablets that have been coated with a polymer or other suitable coating. Multiple compressed tablets are compressed tablets prepared by more than one compression cycle using the previously mentioned pharmaceutically acceptable substances. Coloring agents may also be used in the above dosage forms. Flavoring agents and sweeteners are used in compressed tablets, sugar-coated tablets, multiple compressed tablets, and chewable tablets. Flavoring and sweetening agents are particularly useful in the formulation of chewable tablets and lozenges.

Liquid oral dosage forms include aqueous solutions, emulsions, suspensions, solutions and/or suspensions reconstituted from non-effervescent granules and effervescent preparations reconstituted from effervescent granules. Aqueous solutions include, for example, elixirs and syrups. The emulsion is oil-in-water or water-in-oil.

Elixirs are clear, sweetened, aqueous alcoholic preparations. Pharmaceutically acceptable carriers used in elixirs include solvents. Syrups are concentrated aqueous solutions of sugars (e.g., sucrose) and may contain preservatives. Emulsions are two-phase systems in which one liquid is dispersed as small droplets throughout the other liquid. Pharmaceutically acceptable carriers used in emulsions are non-aqueous liquids, emulsifiers and preservatives. Suspensions employ pharmaceutically acceptable suspending agents and preservatives. Pharmaceutically acceptable materials used in non-effervescent granules to reconstitute liquid oral dosage forms include diluents, sweeteners and wetting agents. Pharmaceutically acceptable materials used in effervescent granules reconstituted into liquid oral dosage forms include organic acids and sources of carbon dioxide. Coloring agents and flavoring agents are used in all of the above dosage forms.

Solvents include glycerin, sorbitol, ethanol, and syrup. Examples of preservatives include glycerol, methyl and propyl parabens, benzoic acid, sodium benzoate and alcohol. Examples of non-aqueous liquids used in the emulsion include mineral oil and cottonseed oil. Examples of emulsifying agents include gelatin, gum acacia, gum tragacanth, bentonite, and surfactants such as polyoxyethylene sorbitan monooleate. Suspending agents include sodium carboxymethylcellulose, pectin, tragacanth, magnesium aluminium silicate and acacia. Diluents include lactose and sucrose. Sweeteners include sucrose, syrup, glycerin, and artificial sweeteners such as saccharin. Wetting agents include propylene glycol monostearate, sorbitan monooleate, diethylene glycol monostearate and polyoxyethylene lauryl ether. Organic acids include citric acid and tartaric acid. Sources of carbon dioxide include sodium bicarbonate and sodium carbonate. Colorants include any approved water-soluble FD and C dyes and mixtures thereof. Flavoring agents include natural flavors extracted from plants such as fruits and synthetic mixtures of compounds that produce a pleasant taste sensation.

For solid dosage forms, solutions or suspensions in, for example, propylene carbonate, vegetable oils or triglycerides are encapsulated in capsules. Such solutions and their formulation and encapsulation are disclosed in U.S. patent nos. 4,328,245, 4,409,239 and 4,410,545. For liquid dosage forms, for example, a solution in polyethylene glycol may be diluted with a sufficient amount of a pharmaceutically acceptable liquid carrier, such as water, to facilitate measurement for administration.

Alternatively, a liquid or semi-solid oral formulation may be prepared by: the active compound or salt is dissolved or dispersed in vegetable oils, glycols, triglycerides, propylene glycol esters (e.g., propylene carbonate) and other such carriers, and these solutions or suspensions are encapsulated in hard or soft gelatin capsule shells. Other useful formulations include, but are not limited to, those containing the compounds provided herein, dialkylated mono-or polyalkylene glycols including, but not limited to, 1, 2-dimethoxymethane, diglyme, triglyme, tetraglyme, polyethylene glycol-350-dimethyl ether, polyethylene glycol-550-dimethyl ether, polyethylene glycol-750-dimethyl ether, where 350, 550, and 750 refer to the approximate average molecular weight of the polyethylene glycol, and one or more antioxidants such as Butylated Hydroxytoluene (BHT), Butylated Hydroxyanisole (BHA), propyl gallate, vitamin E, hydroquinone, hydroxycoumarins, ethanolamine, lecithin, cephalin, ascorbic acid, malic acid, sorbitol, phosphoric acid, thiodipropionic acid and its esters, and dithiocarbamates.

Other formulations include, but are not limited to, hydroalcoholic solutions comprising pharmaceutically acceptable acetals. The alcohol used in these formulations is any pharmaceutically acceptable water miscible solvent having one or more hydroxyl groups, including, but not limited to, propylene glycol and ethanol. Acetals include, but are not limited to, di (lower alkyl) acetals of lower alkyl aldehydes such as acetaldehyde diethyl acetal.

In all embodiments, tablet and capsule formulations may be coated as known to those skilled in the art to modify or maintain dissolution of the active ingredient. Thus, for example, the formulations may be coated with conventional enteric digestible coatings such as phenyl salicylate, waxes and cellulose acetate phthalate.

Injections, solutions and emulsions

Parenteral administration, generally characterized as injection, either subcutaneous, intramuscular, or intravenous, is also contemplated herein. Injectables can be prepared in conventional forms, such as liquid solutions or suspensions, solid forms suitable for solution in liquid or suspension prior to injection, or as emulsions. Suitable excipients are, for example, water, saline, dextrose, glycerol or ethanol. In addition, if desired, the pharmaceutical compositions to be administered may also contain minor amounts of non-toxic substances such as wetting or emulsifying agents, pH buffering agents, stabilizing agents, dissolution enhancing agents and other such agents, such as, for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate and cyclodextrins. It is also contemplated herein to implant a sustained release or sustained release system to maintain a constant level of dosage. Briefly, the compounds provided herein are dispersed in a solid inner matrix, e.g., polymethyl methacrylate, polybutyl methacrylate, plasticized or unplasticized polyvinyl chloride, plasticized nylon, plasticized polyethylene terephthalate, natural rubber, polyisoprene, polyisobutylene, polybutadiene, polyethylene, ethylene-vinyl acetate copolymers, silicone rubber, polydimethylsiloxane, silicone carbonate copolymers, hydrophilic polymers such as hydrogels of esters of acrylic and methacrylic acid, collagen, crosslinked polyvinyl alcohol, and crosslinked partially hydrolyzed polyvinyl acetate, the solid inner matrix being surrounded by an outer polymeric film, e.g., polyethylene, polypropylene, ethylene/propylene copolymers, ethylene/ethyl acrylate copolymers, ethylene/vinyl acetate copolymers, silicone rubber, polyethylene-vinyl acetate copolymers, Polydimethylsiloxane, neoprene, chlorinated polyethylene, polyvinyl chloride, copolymers of vinyl chloride and vinyl acetate, vinylidene chloride, ethylene and propylene, ionomers polyethylene terephthalate, butyl rubber epichlorohydrin rubber, ethylene/vinyl alcohol copolymers, ethylene/vinyl acetate/vinyl alcohol terpolymers and ethylene/ethyleneoxyethanol copolymers, which are insoluble in body fluids. The compound diffuses through the outer polymer membrane during the release rate controlling step. The percentage of active compound contained in such parenteral compositions is highly dependent on its particular properties, as well as the activity of the compound and the needs of the subject.

Parenteral administration of the composition includes intravenous, subcutaneous and intramuscular administration. Formulations for parenteral administration include sterile solutions to be injected, sterile dry soluble preparations such as lyophilized powders for combination with a solvent just prior to use, including subcutaneous tablets for injection, sterile suspensions to be injected, sterile dry insoluble preparations for combination with a vehicle just prior to use, and sterile emulsions. The solution may be aqueous or non-aqueous.

If administered intravenously, suitable carriers include physiological saline or Phosphate Buffered Saline (PBS), and the solution contains thickening and solubilizing agents such as glucose, polyethylene glycol, polypropylene glycol, and mixtures thereof.

Pharmaceutically acceptable carriers for use in parenteral formulations include aqueous vehicles, non-aqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifying agents, sequestering or chelating agents and other pharmaceutically acceptable materials.

Examples of aqueous vehicles include sodium chloride injection, ringer's injection, isotonic glucose injection, sterile water injection, ringer's injection of glucose and lactate. Non-aqueous parenteral vehicles include fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil and peanut oil. Antimicrobial agents at bacteriostatic or fungistatic concentrations must be added to parenteral formulations packaged in multi-dose containers, including phenols or cresols, mercurial preparations, benzyl alcohol, chlorobutanol, methyl and propyl parabens, thimerosal, benzalkonium chloride and benzethonium chloride. Isotonic agents include sodium chloride and dextrose. Buffers include phosphates and citrates. The antioxidant comprises sodium bisulfate. Local anesthetics include procaine hydrochloride. Suspending and dispersing agents include sodium carboxymethylcellulose, hydroxypropylmethylcellulose and polyethyleneA pyrrolidone. The emulsifier comprises polysorbate 80 (TWEEN)80). Sequestering or chelating agents for metal ions include EDTA. The drug carrier also comprises ethanol, polyethylene glycol and propylene glycol used for water-miscible solvent medium and sodium hydroxide, hydrochloric acid, citric acid or lactic acid used for pH adjustment.

The concentration of the pharmaceutically active compound is adjusted so that the injection provides an effective amount to produce the desired pharmacological effect. The precise dosage will depend on the age, weight and condition of the patient or animal, as is known in the art.

The unit dose parenteral formulations are packaged in ampoules, vials or syringes with needles. As known and practiced in the art, all formulations for parenteral administration must be sterile.

Illustratively, intravenous or intra-arterial infusion of a sterile aqueous solution containing the active compound is an effective mode of administration. Another embodiment is a sterile aqueous or sterile oily solution or sterile suspension containing the active material, injected as required to produce the desired pharmacological effect.

Injections are designed for local and systemic administration. A therapeutically effective dose is typically formulated to contain the active compound at a concentration of at least about 0.1% w/w up to about 90% w/w or more, such as greater than 1% w/w to the treated tissue. The active ingredient may be administered at one time, or may be divided into a number of smaller doses to be administered at intervals. It will be understood that the precise dose and duration of treatment will vary with the condition to be treated and may be measured empirically from in vivo or in vitro test data using known test protocols or by extrapolation. It is noted that concentrations and dosage values may also vary with the age of the individual being treated. It will also be understood that for any particular subject, the particular dosage regimen should be adjusted over time according to the individual needs and the professional judgment of the individual administering or the professional judgment directing the administration of the formulation, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed formulation.

The compounds may be suspended in micronized form or other suitable forms or may be derivatized to produce a more soluble active product or to produce a prodrug. The form of the resulting mixture depends on many factors, including the desired mode of administration and the solubility of the compound in the selected carrier or vehicle. The effective concentration is sufficient to ameliorate symptoms of the condition and can be determined empirically.

Freeze-dried powder

Also contemplated herein are lyophilized powders, which can be reconstituted for administration as solutions, emulsions, and other mixtures. Lyophilized powders may also be reconstituted and formulated as a solid or gel.

Sterile lyophilized powders are prepared by dissolving a compound provided herein, or a pharmaceutically acceptable salt thereof, in a suitable solvent. The solvent may contain excipients to improve stability, or other pharmacological components of the powder or reconstituted solution prepared from the powder. Excipients that may be used include, but are not limited to, dextrose, sorbitol, fructose, corn syrup, xylitol, glycerol, glucose, sucrose, or other suitable agents. The solvent may also contain a buffering agent, such as citrate, sodium or potassium phosphate or other such buffering agents known to those skilled in the art, in one embodiment, the buffering agent is at about neutral pH. Subsequent filter sterilization of the solution followed by lyophilization under standard conditions known to those skilled in the art provides the desired dosage form. Typically, the resulting solution will be dispensed into vials for freeze drying. Each vial will contain a single dose (i.e., 10-1000mg or 100-500mg) or multiple doses of the compound. The lyophilized powder can be stored under suitable conditions, such as at about 4 ℃ to room temperature.

Reconstitution of the lyophilized powder with water for injection provides a formulation for parenteral administration. For reconstitution, about 1-50mg, about 5-35mg, or about 9-30mg of the lyophilized powder is added per mL of sterile water or other suitable carrier. The exact amount depends on the compound selected. The amount may be determined empirically.

Topical application

Topical mixtures were prepared as described for topical and systemic administration. The resulting mixture may be a solution, suspension, emulsion, or the like, and is formulated as a cream, gel, ointment, emulsion, solution, elixir, lotion, suspension, tincture, paste, foam, aerosol, douche, spray, suppository, bandage, skin patch, or any other formulation suitable for topical administration.

The compounds or pharmaceutically acceptable salts thereof can be formulated as aerosols for topical application, such as by inhalation (see, e.g., U.S. patent nos. 4,044,126, 4,414,209, and 4,364,923, which describe aerosols for delivery of steroids for the treatment of inflammatory diseases, particularly asthma). These formulations for administration to the respiratory tract may be in the form of a solution for an aerosol or nebulizer, or as a microfine powder for insufflation, alone or in combination with an inert carrier such as lactose. In such cases, the particles of the formulation will have a diameter of less than 50 microns or less than 10 microns.

The compounds may be formulated for topical or local application, such as to the skin and mucous membranes, such as in the form of gels, creams and lotions in the eye, and for ophthalmic application or for intracisternal or intraspinal application. Are contemplated for transdermal delivery, and also for ocular or mucosal administration, or for topical administration for inhalation therapy. Nasal solutions of the active compound alone or in combination with other pharmaceutically acceptable excipients may also be administered.

These solutions (especially those intended for ocular use) can be formulated as 0.001% -10% isotonic solutions, pH about 5-7, and suitable salts.

Compositions for other routes of administration

Other routes of administration are also contemplated herein, such as topical application, transdermal patches, and rectal administration.

For example, pharmaceutical dosage forms for rectal administration are rectal suppositories for systemic action, rectal capsules and tablets. Rectal suppositories are used herein mean solids for insertion into the rectum which melt or soften at body temperature to release one or more pharmacologically or therapeutically active ingredients. Pharmaceutically acceptable substances used in rectal suppositories are melting point enhancing bases or vehicles and agents. Examples of bases include cocoa butter (cocoa butter), glycerins, carbowaxes (polyethylene glycols) and suitable mixtures of mono-, di-and triglycerides of fatty acids. Combinations of substrates may be used. Agents that increase the melting point of suppositories include spermaceti and wax. Rectal suppositories may be prepared by compression methods or by moulding. An exemplary weight of the rectal suppository is about 2 to 3 gm.

Tablets and capsules for rectal administration are manufactured using the same pharmaceutically acceptable substances and by the same methods as the orally administered formulations.

Sustained release composition

The active ingredients provided herein can be administered by controlled release methods or delivery devices well known to those of ordinary skill in the art. Examples include, but are not limited to, those described in U.S. Pat. nos. 3,845,770, 3,916,899, 3,536,809, 3,598,123, and 4,008,719, 5,674,533, 5,059,595, 5,591,767, 5,120,548, 5,073,543, 5,639,476, 5,354,556, 5,639,480, 5,733,566, 5,739,108, 5,891,474, 5,922,356, 5,972,891, 5,980,945, 5,993,855, 6,045,830, 6,087,324, 6,113,943, 6,197,350, 6,248,363, 6,264,970, 6,267,981, 6,376,461, 6,419,961, 6,589,548, 6,613,358, 6,699,500, and 6,740,634, each of which is incorporated herein by reference. Such dosage forms may be used to provide sustained or controlled release of one or more active ingredients using, for example, hydroxypropylmethylcellulose (hypromellose), other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes, microspheres, or combinations thereof, to provide desired release characteristics in varying proportions. Suitable controlled release formulations known to those of ordinary skill in the art, including those described herein, can be readily selected for use with the active ingredients provided herein.

All controlled release drug products have the common goal of improving drug therapy over that achieved by their non-controlled counterparts. In one embodiment, the use of optimally designed controlled release formulations in drug therapy is characterized by the use of a minimum amount of drug product in a minimum amount of time to cure or control the condition. In some embodiments, advantages of controlled release formulations include prolonged drug activity, reduced dosing frequency, and increased patient compliance. In addition, controlled release formulations can be used to affect the onset time or other properties of an effect, such as the blood concentration of the drug, and thus can affect the occurrence of side (e.g., adverse) effects.

Most controlled release formulations are designed to initially release an amount of drug (active ingredient) that rapidly produces the desired therapeutic effect, and gradually and continuously release other amounts of drug to maintain this level of therapeutic or prophylactic effect over an extended period of time. In order to maintain this constant level of drug in the body, the drug must be released from the dosage form at a rate that replaces the amount of drug that is metabolized and excreted from the body. Controlled release of the active ingredient can be stimulated by a variety of conditions, including, but not limited to, pH, temperature, enzymes, water, or other physiological conditions or compounds.

In some embodiments, the agent may be administered using intravenous infusion, implanted osmotic pumps, transdermal patches, liposomes, or other forms of administration. In one embodiment, a pump may be used (see Sefton, CRC crit. Ref. biomed. Eng.14: 201 (1987); Buchwald et al, Surgery 88: 507 (1980); Saudek et al, N.Engl. J. Med.321: 574 (1989)). In another embodiment, polymeric materials may be used. In yet another embodiment, a Controlled Release system may be placed in the vicinity of the therapeutic target, i.e., so that only a fraction of the systemic dose is required (see, e.g., Goodson, medical applications of Controlled Release Release in medicine, Vol.2, p.115-138 (1984)).

In some embodiments, the controlled release device is introduced to the subject at an inappropriate immune activation site or near a tumor. Other controlled release systems are discussed in the review by Langer (Science 249: 1527-. The active ingredient may be dispersed in a solid inert matrix such as, for example, polymethyl methacrylate, polybutyl methacrylate, plasticized or unplasticized polyvinyl chloride, plasticized nylon, plasticized polyethylene terephthalate, natural rubber, polyisoprene, polyisobutylene, polybutadiene, polyethylene, ethylene-vinyl acetate copolymers, silicone rubber, polydimethylsiloxane, silicone carbonate copolymers, hydrogels of hydrophilic polymers such as esters of acrylic and methacrylic acid, collagen, crosslinked polyvinyl alcohol and crosslinked partially hydrolyzed polyvinyl acetate, surrounded by an outer polymer film such as, for example, polyethylene, polypropylene, ethylene/propylene copolymers, ethylene/ethyl acrylate copolymers, ethylene/vinyl acetate copolymers, silicone rubber, polydimethylsiloxane, polyvinyl chloride, plasticized nylon, plasticized polyethylene terephthalate, natural rubber, polyisoprene, polyethylene terephthalate, Neoprene, chlorinated polyethylene, polyvinyl chloride, copolymers of vinyl chloride and vinyl acetate, vinylidene chloride, ethylene and propylene, ionomer polyethylene terephthalate, butyl rubber epichlorohydrin rubber, ethylene/vinyl alcohol copolymers, ethylene/vinyl acetate/vinyl alcohol terpolymers, and ethylene/ethyleneoxyethanol copolymers, the outer polymer film being insoluble in body fluids. The active ingredient then diffuses through the outer polymer film during the release rate controlling step. The percentage of active ingredient contained in such parenteral compositions is highly dependent on its particular properties, as well as the needs of the subject.

Targeted formulations

The compounds provided herein, or pharmaceutically acceptable salts thereof, may also be formulated to target a particular tissue, recipient, or other area of the body of the subject being treated. Many such targeting methods are well known to those skilled in the art. All such targeting methods for the present compositions are contemplated herein. Non-limiting examples of targeting methods are found, for example, in U.S. Pat. nos. 6,316,652, 6,274,552, 6,271,359, 6,253,872, 6,139,865, 6,131,570, 6,120,751, 6,071,495, 6,060,082, 6,048,736, 6,039,975, 6,004,534, 5,985,307, 5,972,366, 5,900,252, 5,840,674, 5,759,542 and 5,709,874.

In one embodiment, liposomal suspensions (including tissue-targeting liposomes, such as tumor-targeting liposomes) may also be suitable as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art. For example, liposome formulations can be prepared as described in U.S. Pat. No. 4,522,811. Briefly, liposomes such as Multilamellar Liposomes (MLV) can be formed by drying egg phosphatidylcholine and brain phosphatidylserine (7: 3 molar ratio) inside the flask. A solution of the compounds provided herein in Phosphate Buffered Saline (PBS) without divalent cations was added and the flask was shaken until the lipid film was dispersed. The resulting vesicles were washed to remove unencapsulated compounds, pelleted by centrifugation, and then resuspended in PBS.

Article of manufacture

The compounds or pharmaceutically acceptable salts can be packaged as an article of manufacture containing packaging material, a compound provided herein, or a pharmaceutically acceptable salt thereof, for use in treating, preventing, or ameliorating one or more symptoms associated with kinase activity, including but not limited to GSK-3 activity, and a label indicating that the compound or pharmaceutically acceptable salt thereof is for use in treating, preventing, or ameliorating one or more symptoms mediated by a kinase, including but not limited to a GSK-3 mediated disease.

Articles provided herein include packaging materials. Packaging materials for packaging pharmaceutical products are well known to those skilled in the art. See, for example, U.S. patent nos. 5,323,907, 5,052,558, and 5,033,252. Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, tubes, inhalers, pumps, pouches, vials, containers, syringes, bottles, and any packaging material suitable for the selected formulation and the desired mode of administration and treatment. Various formulations of the compounds and compositions provided herein are contemplated.

E. Method of treatment

Methods of use of the compounds and compositions are also provided. The methods involve the use of compounds and compositions in vitro and in vivo.

In some embodiments, provided herein are methods for inhibiting the effects of GSK-3 by administering the compounds and compositions provided herein. In one embodiment, a method comprises contacting GSK-3 with a compound provided herein.

F. Evaluation of Compound Activity

A) GSK inhibition

The GSK3 inhibitory activity of the compounds provided herein can be readily detected using the assays described herein as well as assays generally known to those of ordinary skill in the art.

Exemplary methods for identifying specific inhibitors of GSK3 include cell-free GSK3 kinase assays and cell-based GSK3 kinase assays. Cell-free GSK3 kinase assays detect inhibitors that act by interacting directly with the polypeptide GSK3, whereas cell-based GSK3 kinase assays identify inhibitors that act by interacting directly with GSK3 itself or by interfering with GSK3 expression or post-translational processing required to produce mature, active GSK 3. U.S. application No. 20050054663 describes an exemplary cell-free GSK3 kinase assay and a cell-based GSK3 kinase assay. Exemplary assays for use herein are briefly discussed below:

10-25ng of recombinant full length human GSK3 β (Upstate) were incubated at 30 degrees Celsius for 1 hour in 20mM MOPS, pH 7.0, 10mM magnesium acetate, 0.2mM EDTA, 2mM EGTA, 30mM magnesium chloride, 62.5 μ M glycogen phosphate synthase peptide-2, 5 μ M ATP, 10mM β -glycerophosphate, 1mM sodium orthovanadate and 1mM dithiothreitol, in the presence or absence of varying concentrations of the compound. The KinaseGlo luciferase reaction was performed.

After completion of the kinase reaction, an equal volume of KinasGlo luciferase reagent (Promega) was added and luminescence read in 5-10 minutes using a luminescence plate reader. Compound activity was expressed as% inhibition relative to the maximum inhibition observed at the maximum dose, and IC50 values were then calculated using curve fitting software (graphpadpizm).

Examples

Example 1:

(3S) -8-amino-9-fluoro-2, 3-dihydro-3-methyl-7-oxo-10- [ (1S, 3S) -3- (2-pyridinyl) cyclopentyl Amino group]-7H-pyrido [1, 2, 3-de][1，4]Benzoxazine-6-carbonitriles

Step 1: 3- (2-pyridinyl) -2-cyclopenten-1-ones

To a solution of 2-bromopyridine (5.00g, 31.6mmol) in THF (30ml) at-78 deg.C was added n-BuLi (2.55M in hexane, 12.5mL, 31.9mmol) dropwise and stirred for 10 min. To the reaction mixture was added 3-ethoxy-2-cyclopentenone (2.00g, 15.9mmol) in THF (10mL) dropwise at-78 deg.C and stirred at 0 deg.C for 2 h. The reaction mixture was acidified with 2M HCl (10mL) and the aqueous layer was washed with EtOAc. The aqueous layer was basified with 1M aqueous NaOH solution and then with CH₂Cl₂And (4) extracting. The organic layer was washed with brine and over anhydrous Na₂SO₄Drying, followed by removal of the solvent. The crude product was purified by column chromatography (hexane/EtOAc ═ 1/3) to afford 3- (2-Pyridyl) -2-cyclopenten-1-one (1.57g, 58%) as a brown solid.

¹H-NMR(CDCl₃-d，400MHz)2.60-2.65(2H，m)，3.16(2H，dt，J＝7.9，1.8Hz)，6.82(1H，t，J＝1.8Hz)，7.32-7.37(1H，m)，7.70(1H，d，J＝7.9Hz)，7.79(1H，td，J＝7.9，1.8Hz)，8.73(1H，d，J＝4.9Hz)。

EIMS(+)159[M]⁺。

Step 2: 3- (2-pyridinyl) cyclopent-1-ones

A mixture of 3- (2-pyridyl) -2-cyclopenten-1-one (1.43g, 8.98mmol) and 10% Pd/C (142mg) in EtOH (30mL) was stirred under a hydrogen atmosphere (0.3MPa) for 6.5 hours. The catalyst was removed by filtration through celite and the filtrate was concentrated in vacuo. The crude product was purified by column chromatography (hexanes/EtOAc ═ 1/3) to give 3- (2-pyridyl) cyclopent-1-one as a colorless oil (3.26g, 94%).

¹H-NMR(CDCl₃-d，400MHz)2.13-2.24(1H，m)，2.26-2.35(1H，m)，2.36-2.55(2H，m)，2.56-2.75(2H，m)，3.56(1H，qui，J＝6.7Hz)，7.10-7.23(2H，m)，7.64(1H，td，J＝7.3，1.2Hz)，8.57(1H，d，J＝4.2Hz)。

EIMS(+)161[M]⁺。

And step 3: cis-3- (2-pyridyl) cyclopentanol

To a solution of 3- (2-pyridyl) cyclopent-1-one (3.17g, 19.7mmol) in MeOH (80ml) at 0 deg.C was added NaBH in portions₄(745mg, 19.7mmol) and stirred at room temperature for 2 h. The reaction mixture was poured into ice water and then extracted with EtOAcAnd (6) taking. The organic layer was passed over anhydrous Na₂SO₄Drying, followed by removal of the solvent. The crude product was purified by column chromatography (hexane/EtOAc ═ 1/1 → 1/3) to give cis-3- (2-pyridyl) cyclopentanol (1.90g, 59%) as a colorless oil.

¹H-NMR(CDCl₃-d，400MHz)1.70-2.00(4H，m)，2.12-2.30(2H，m)，3.42(1H，dd，J＝15.2，9.7Hz)，4.35(1H，t，J＝4.3Hz)，6.47(1H，brs)，7.10(1H，dd，J＝7.3，5.5Hz)，7.17(1H，d，J＝7.3Hz)，7.59(1H，t，J＝7.3Hz)，8.51(1H，d，J＝4.3Hz)。

EIMS(+)163[M]⁺。

And 4, step 4: trans-N- [3- (2-pyridyl) cyclopentyl ] phthalimide

A solution of cis-3- (2-pyridyl) cyclopentanol (1.86g, 11.4mmol) in THF (100mL) was cooled to 0 deg.C and treated with phthalimide (1.85g, 12.5mmol), diethyl azodicarboxylate (2.2M in toluene, 5.7mL, 11.4mmol), and triphenylphosphine (3.29g, 12.5 mmol). The reaction mixture was stirred at room temperature overnight. The solvent was removed and the crude product was purified by column chromatography (hexane/EtOAc ═ 3/1 → 2/1 → 1/1) to give trans-N- [3- (2-pyridyl) cyclopentyl ] phthalimide (1.52g, 41%) as a colorless powder.

¹H-NMR(CDCl₃-d，400MHz)1.87-2.02(1H，m)，2.15-2.41(4H，m)，2.47(1H，ddd，J＝13.4，8.6，6.1Hz)，3.81(1H，qui，J＝8.6Hz)，4.92-5.03(1H，m)，7.11(1H，dd，J＝7.3，5.2Hz)，7.22(1H，d，J＝7.3Hz)，7.60(1H，td，J＝7.3，1.2Hz)，7.69-7.73(2H，m)，7.81-7.85(2H，m)，8.57(1H，d，J＝4.3Hz)。

EIMS(+)292[M]⁺。

And 5:

(1S, 3S) -N- [3- (2-pyridyl) cyclopentyl ] phthalimide and (1R, 3R) -N- [3- (2-pyridyl) cyclopentyl ] phthalimide

By preparative HPLC utilizing a Chiralpak IA column (. phi.20X 250mm) and MeCN-CH₂Cl₂(9/1) separating trans-N- [3- (2-pyridyl) cyclopentyl ] group as eluent at a flow rate of 5mL/min]Phthalimide (10.0g) for 1 h. The UV detector is set at 300nm, the volume of the sample injection quantitative ring is 5mL, and the sample injection amount is 68-70mg in MeCN-CH₂Cl₂(9/1) in solution.

(1S, 3S) -N- [3- (2-pyridyl) cyclopentyl]Phthalimide; 4.78g, > 99% purity > 99% enantiomeric excess (Chiralpak IA column (. phi.4.6X 250mm), MeCN-CH₂Cl₂(9/1)，0.5mL/min，Rt＝10.3min)

(1R, 3R) -N- [3- (2-pyridyl) cyclopentyl group]Phthalimide; 4.85g, > 99% purity > 99% enantiomeric excess (Chiralpak IA column (. phi.4.6X 250mm), MeCN-CH₂Cl₂(9/1)，0.5mL/min，Rt＝13.4min)

Step 6: (1S, 3S) -3- (2-pyridinyl) cyclopentylamine

A mixture of (1S, 3S) -N- [3- (2-pyridyl) cyclopentyl ] phthalimide (4.12g, 14.1mmol) and hydrazine hydrate (2.0mL, 41.2mmol) in EtOH (100mL) was stirred at reflux for 3 h. After cooling, the reaction mixture was filtered and the solvent was subsequently removed. The residue was suspended in EtOAc and stirred at reflux for 1 h. After cooling, the precipitate was filtered, followed by removal of the solvent to give (1S, 3S) -3- (2-pyridyl) cyclopentylamine (2.10g, 92%) as a yellow oil.

¹H-NMR(CDCl₃-d，400MHz)1.40-1.63(3H，m)，1.77-1.92(2H，m)，2.10-2.28(3H，m)，3.49(1H，qui，J＝8.6Hz)，3.66(1H，qui，J＝6.1Hz)，7.08(1H，dd，J＝7.9，5.5Hz)，7.16(1H，d，J＝7.9Hz)，7.57(1H，td，J＝7.9，1.2Hz)，8.54(1H，d，J＝4.3Hz)。

CIMS(+)163[M+H]⁺。

And 7:

(3S) -8-amino-9-fluoro-2, 3-dihydro-3-methyl-7-oxo-10- [ (1S, 3S) -3- (2-pyridyl) cyclopentylamino ] -7H-pyrido [1, 2, 3-de ] [1, 4] benzoxazine-6-carbonitrile

(3S) -8-amino-9, 10-difluoro-2, 3-dihydro-3-methyl-7-oxo-7H-pyrido [1, 2, 3-de ] in anhydrous DMSO (8mL) with stirring at 100 deg.C][1，4]A mixture of benzoxazine-6-carbonitrile (1.30g, 4.69mmol), (1S, 3S) -3- (2-pyridyl) cyclopentylamine (913mg, 5.63mmol), and triethylamine (1.30mL, 9.33mmol) was 7 h. After cooling, the reaction mixture was poured into ice water and then diluted with CH₂Cl₂-MeOH extraction. The organic layer was washed with water and brine and passed over anhydrous Na₂SO₄Drying, followed by removal of the solvent. The crude product was purified by column chromatography (EtOAc) to afford (3S) -8-amino-9-fluoro-2, 3-dihydro-3-methyl-7-oxo-10- [ (1S, 3S) -3- (2-pyridyl) cyclopentylamino]-7H-pyrido [1, 2, 3-de][1，4]Benzoxazine-6-carbonitrile (364mg, 19%) as a yellow powder.

¹H-NMR(DMSO-d₆，400MHz)1.35(3H，d，J＝6.7Hz)，1.66-1.82(2H，m)，1.96-2.06(1H，m)，2.06-2.20(3H，m)，3.43(1H，t，J＝8.3Hz)，4.05(1H，dd，J＝11.0，1.8Hz)，4.32(1H，dd，J＝11.0，1.8Hz)，4.39-4.52(2H，m)，5.17(1H，dd，J＝7.9，1.8Hz)，6.94(2H，brs)，7.17(1H，dd，J＝7.9，4.9Hz)，7.26(1H，d，J＝7.9Hz)，7.67(1H，td，J＝7.9，1.8Hz)，8.46(1H，s)，8.48(1H，d，J＝4.3Hz)。

ESIMS(+)420[M+H]⁺。

HRESIMS(+)420.18345(C₂₃H₂₃FN₅O₂Calculated value of 420.18358).

Analysis C65.57% H5.24% N16.31%, C₂₃H₂₂FN₅O₂，0.2H₂Calculated for O, C65.30% H5.34% N16.55%.

Example 2

(3S) -8-amino-9-fluoro-2, 3-dihydro-3-methyl-7-oxo-10- [ (1R, 3R) -3- (2-pyridinyl) cyclopentyl Amino group]-7H-pyrido [1, 2, 3-de][1，4]Benzoxazine-6-carbonitriles

Step 1: (1R, 3R) -3- (2-pyridinyl) cyclopentylamine

A mixture of (1R, 3R) -N- [3- (2-pyridyl) cyclopentyl ] phthalimide (4.17g, 14.3mmol) and hydrazine hydrate (2.0mL, 41.2mmol) in EtOH (100mL) was stirred at reflux for 4 h. After cooling, the reaction mixture was filtered and the solvent was subsequently removed. The residue was suspended in EtOAc and stirred at reflux for 1 h. After cooling, the precipitate was filtered, followed by removal of the solvent to give (1R, 3R) -3- (2-pyridyl) cyclopentylamine (1.62g, 70%) as a yellow oil.

CIMS(+)163[M+H]⁺。

Step 2:

(3S) -8-amino-9-fluoro-2, 3-dihydro-3-methyl-7-oxo-10- [ (1R, 3R) -3- (2-pyridyl) cyclopentylamino ] -7H-pyrido [1, 2, 3-de ] [1, 4] benzoxazine-6-carbonitrile

(3S) -8-amino-9, 10-difluoro-2, 3-dihydro-3-methyl-7-oxo-7H-pyrido [1, 2, 3-de ] in anhydrous DMSO (8mL) was stirred at 100 deg.C][1，4]A mixture of benzoxazine-6-carbonitrile (1.40g, 5.05mmol), (1R, 3R) -3- (2-pyridyl) cyclopentylamine (985mg, 6.07mmol), and triethylamine (1.40mL, 10.0mmol) was 7 h. After cooling, the reaction mixture was poured into ice water and then diluted with CH₂Cl₂-MeOH extraction. The organic layer was washed with water and brine and passed over anhydrous Na₂SO₄Drying, followed by removal of the solvent. The crude product was purified by column chromatography (EtOAc) to afford (3S) -8-amino-9-fluoro-2, 3-dihydro-3-methyl-7-oxo-10- [ (1R, 3R) -3- (2-pyridyl) cyclopentylamino]-7H-pyrido [1, 2, 3-de][1，4]Benzoxazine-6-carbonitrile (325mg, 15%) as a yellow powder.

¹H-NMR(DMSO-d₆，400MHz)1.35(3H，d，J＝6.7Hz)，1.66-1.82(2H，m)，1.95-2.04(1H，m)，2.05-2.23(3H，m)，3.42(1H，qui，J＝7.9Hz)，4.06(1H，dd，J＝11.0，1.8Hz)，4.33(1H，dd，J＝11.0，1.8Hz)，4.40-4.52(2H，m)，5.17(1H，dd，J＝7.9，1.8Hz)，6.94(2H，brs)，7.17(1H，dd，J＝7.3，4.9Hz)，7.26(1H，d，J＝7.9Hz)，7.67(1H，td，J＝7.3，1.8Hz)，8.46(1H，s)，8.48(1H，d，J＝4.3Hz)。

ESIMS(+)420[M+H]⁺。

HRESIMS(+)420.18368(C₂₃H₂₃FN₅O₂Calculated value of 420.18358).

Analysis C65.61% H 5.28％ N 16.19％，C₂₃H₂₂FN₅O₂Calcd for C65.86% H5.29% N16.70%.

Example 3:

(3S) -8-amino-9-fluoro-2, 3-dihydro-3-methyl-7-oxo-10- [ cis-3- (2-pyridinyl) cyclopentylamino Base of]-7H-pyrido [1, 2, 3-de][1，4]Benzoxazine-6-carbonitriles

Step 1: trans-3- (2-pyridyl) cyclopentanol

To a solution of 3- (2-pyridyl) cyclopent-1-one (3.17g, 19.7mmol) in MeOH (80ml) at 0 deg.C was added NaBH in portions₄(745mg, 19.7mmol) and stirred at room temperature for 2 h. The reaction mixture was poured into ice water and then extracted with EtOAc. The organic layer was passed over anhydrous Na₂SO₄Drying, followed by removal of the solvent. The crude product was purified by column chromatography (hexane/EtOAc ═ 1/1 → 1/3) to give trans-3- (2-pyridyl) cyclopentanol (965mg, 30%) as a colorless oil.

¹H-NMR(CDCl₃-d，400MHz)1.50-1.78(2H，m)，1.80-1.93(1H，m)，2.04-2.13(2H，m)，2.16-2.33(2H，m)，3.55(1H，qui，J＝7.9Hz)，4.52-4.62(1H，m)，7.09(1H，dd，J＝7.3，5.5Hz)，7.17(1H，d，J＝7.3Hz)，7.58(1H，td，J＝7.3，1.8Hz)，8.54(1H，d，J＝4.3Hz)。

EIMS(+)163[M]⁺。

Step 2: cis-N- [3- (2-pyridyl) cyclopentyl ] phthalimide

A solution of trans-3- (2-pyridyl) cyclopentanol (957mg, 5.86mmol) in THF (25mL) was cooled to 0 deg.C and treated with phthalimide (1.29g, 8.77mmol), cyanomethylenetri-n-butylphosphine (2.12g, 8.78 mmol). The reaction mixture was stirred at 70 ℃ for 8 h. The solvent was removed and the crude product was purified by column chromatography (hexane/EtOAc ═ 3/1 → 1/1) and triturated with hexane-EtOAc to give cis-N- [3- (2-pyridyl) cyclopentyl ] phthalimide (901mg, 53%) as a light yellow powder.

¹H-NMR(CDCl₃-d，400MHz)2.10-2.40(5H，m)，2.56(1H，dd，J＝22.6，12.2Hz)，3.28-3.40(1H，m)，4.80-4.90(1H，m)，7.13(1H，dd，J＝7.9，5.9Hz)，7.35(1H，d，J＝7.9Hz)，7.63(1H，t，J＝7.9Hz)，7.71(2H，dd，J＝5.5，3.0Hz)，7.83(2H，dd，J＝5.5，3.0Hz)，8.55(1H，d，J＝4.3Hz)。

EIMS(+)292[M]⁺。

And step 3: cis-3- (2-pyridyl) cyclopentylamine

The title compound (498mg, 99%) was prepared from cis-N- [3- (2-pyridyl) cyclopentyl ] phthalimide (905mg, 3.10mmol) in a manner similar to that described for the preparation of trans-3- (2-pyridyl) cyclopentylamine.

¹H-NMR(CDCl₃-d，400MHz)1.52-1.74(3H，m)，1.95-2.13(3H，m)，2.31-2.40(1H，m)，3.26(1H，qui，J＝7.9Hz)，3.48(1H，qui，J＝6.1Hz)，7.09(1H，dd，J＝7.9，4.9Hz)，7.18(1H，d，J＝7.9Hz)，7.58(1H，td，J＝7.9，1.2Hz)，8.54(1H，d，J＝4.3Hz)。

CIMS(+)163[M+H]⁺。

And 4, step 4:

(3S) -8-amino-9-fluoro-2, 3-dihydro-3-methyl-7-oxo-10- [ cis-3- (2-pyridyl) cyclopentylamino ] -7H-pyrido [1, 2, 3-de ] [1, 4] benzoxazine-6-carbonitrile

(3S) -8-amino-9, 10-difluoro-2, 3-dihydro-3-methyl-7-oxo-7H-pyrido [1, 2, 3-de ] in anhydrous DMSO (3mL) was stirred at 80 deg.C][1，4]Benzoxazine-6-carbonitrile (180mg, 0.649mmol), cis-3- (2-pyridyl) cyclopentylamine (158mg, 0.973mmol), and triethylamine (0.200mL, 0.976mmol) were combined for 8 h. After cooling, the reaction mixture was poured into ice water and then diluted with CH₂Cl₂-MeOH extraction. The organic layer was washed with water and brine and passed over anhydrous Na₂SO₄Drying, followed by removal of the solvent. The crude product was purified by column chromatography (hexane/EtOAc ═ 1/3) to give (3S) -8-amino-9-fluoro-2, 3-dihydro-3-methyl-7-oxo-10- [ cis-3- (2-pyridyl) cyclopentylamino]-7H-pyrido [1, 2, 3-de][1，4]Benzoxazine-6-carbonitrile (mixture of diastereomers, 55.6mg, 20%) as a yellow powder.

¹H-NMR(DMSO-d₆，400MHz)1.34(3H×1/2，d，J＝6.7Hz)，1.37(3H×1/2，d，J＝6.7Hz)，1.65-1.93(5H，m)，2.03-2.12(1H，m)，2.27-2.37(2H，m)，3.34-3.40(1H，m)，3.99-4.10(1H，m)，4.36(1H，dd J＝11.6.3.7Hz)，4.40-4.49(2H，m)，6.56(1H×1/2，d，J＝7.3Hz)，6.70(1H×1/2，d，J＝7.3Hz)，6.93(2H，brs)，7.22(1H，t，J＝6.1Hz)，7.30(1H，d，J＝7.3Hz)，7.70(1H，t，J＝7.3Hz)，8.45(1H，s)，8.55(1H，t，J＝5.5Hz)。

ESIMS(+)420[M+H]⁺。

HRESIMS(+)420.18374(C₂₃H₂₃FN₅O₂Calculated value of 420.18358).

Example 4:

(3S) -8-amino-9-fluoro-2, 3-dihydro-3-methyl-7-oxo-10- [ trans-3- (1H-pyrazol-1-yl) cyclopentyl Amino group]-7H-pyrido [1, 2, 3-de][1，4]Benzoxazine-6-carbonitrile (F)

Step 1:

3- (1H-pyrazol-1-yl) cyclopent-1-one

Pyrazole (6.85g, 100mmol), 2-cyclopent-1-one (19.3g, 235mmol) and ScCl were stirred at room temperature₃(1.42g, 9.38mmol) in anhydrous CH₂Cl₂(3mL) for 30 min. The reaction mixture was poured into water and then diluted with CH₂Cl₂And (4) extracting. The organic layer was washed with brine and over anhydrous Na₂SO₄Drying, followed by removal of the solvent. The crude product was purified by column chromatography (hexanes/EtOAc ═ 4/1) to give 3- (1H-pyrazol-1-yl) cyclopent-1-one (14.1g, 94%) as a yellow oil.

¹H-NMR(CDCl₃-d，400MHz)2.27-2.38(1H，m)，2.39-2.67(3H，m)，2.75(1H，dd，J＝18.3，7.3Hz)，2.84(1H，dd，J＝18.3，7.3Hz)，4.97(1H，qui，J＝6.1Hz)，6.28(1H，t，J＝7.9，1.8Hz)，7.45(1H，d，J＝1.8Hz)，7.54(1H，s)。

Step 2:

cis-3- (1H-pyrazol-1-yl) cyclopentanol

To a solution of 3- (1H-pyrazol-1-yl) cyclopent-1-one (14.09g, 93.8mmol) in MeOH (150ml) at 0 deg.C was added NaBH in portions₄(1.77g, 46.8mmol) and stirred at room temperature for 30 min. The reaction mixture was poured into 0.5M HCl and saturated NaHCO₃Basified with aqueous solution and then treated with CH₂Cl₂And (4) extracting. The organic layer was passed over anhydrous Na₂SO₄Drying, followed by removal of the solvent. The crude product was purified by column chromatography (hexane/EtOAc ═ 2/1 → 1/1) to give cis-3- (1H-pyrazol-1-yl) cyclopentanol (10.41g, 73%) as a colorless oil.

¹H-NMR(CDCl₃-d，400MHz)1.80-1.91(1H，m)，1.96-2.19(3H，m)，2.19-2.33(2H，m)，4.35-4.42(1H，m)，4.80(1H，td，J＝7.9，4.3Hz)，5.75(1H，d，J＝9.8Hz)，6.20(1H，t，J＝1.8Hz)，7.43(1H，d，J＝1.8Hz)，7.51(1H，s)。

EIMS(+)219[M]⁺。

And step 3:

trans-N- [3- (1H-pyrazol-1-yl) cyclopentyl ] phthalimide

A solution of cis-3- (1H-pyrazol-1-yl) cyclopentanol (5.01g, 32.9mmol) in THF (150mL) was cooled to 0 deg.C and treated with phthalimide (5.33g, 36.2mmol), diisopropyl azodicarboxylate (7.1mL, 36.1mmol), and triphenylphosphine (9.48g, 36.1 mmol). The reaction mixture was stirred at 0 ℃ for 4 h. The reaction mixture was poured into water and then extracted with EtOAc. The organic layer was passed over anhydrous Na₂SO₄Drying, followed by removal of the solvent. The crude product was purified by column chromatography (Chromatrex, hexanes/EtOAc ═ 5/1) to give trans-N- [3- (1H-pyrazol-1-yl) cyclopentyl]Phthalimide (4.85g, 52%) was a colorless powder.

¹H-NMR(CDCl₃-d，400MHz)2.16-2.33(3H，m)，2.45-2.56(2H，m)，2.56-2.65(1H，m)，5.00-5.10(1H，m)，5.16(1H，qui，J＝7.3Hz)，6.25(1H，t，J＝1.8Hz)，7.47(1H，d，J＝1.8Hz)，7.55(1H，d，J＝1.8Hz)，7.72(2H，dd，J＝5.5，3.1Hz)，7.84(2H，dd，J＝5.5，3.1Hz)。

And 4, step 4:

trans-N- [3- (1H-pyrazol-1-yl) cyclopentyl ] phthalimide (F) and trans-N- [3- (1H-pyrazol-1-yl) cyclopentyl ] phthalimide (R)

trans-N- [3- (1H-pyrazol-1-yl) cyclopentyl ] phthalimide (3.12g) was separated by preparative HPLC using a Chiralpak IA column (20 x 250mm) and MeCN as eluent at a flow rate of 5mL/min for 1H. The UV detector was set at 300nm, the sample loop volume was 5mL, and the sample size was 80mg in MeCN solution.

trans-N- [3- (1H-pyrazol-1-yl) cyclopentyl ] phthalimide (F); 1.56g, > 99% purity, and > 99% enantiomeric excess (Chiralpak IA column (. phi.4.6X 250mm), MeCN, 0.5mL/min, Rt 10.3min)

trans-N- [3- (1H-pyrazol-1-yl) cyclopentyl ] phthalimide (R); 1.5g, > 99% purity, and > 99% enantiomeric excess (Chiralpak IA column (. phi.4.6X 250mm), MeCN, 0.5mL/min, Rt ═ 15.2min)

And 5: trans-3- (1H-pyrazol-1-yl) cyclopentylamine (F)

Trans-N- [3- (1H-pyrazol-1-yl) cyclopentyl in EtOH (30mL) was stirred at reflux]A mixture of phthalimide (F) (1.53g, 5.44mmol) and hydrazine hydrate (0.53mL, 10.9mmol) was used for 4 h. After cooling, the reaction mixture was filtered and the solvent was subsequently removed. The crude product was purified by column chromatography (Chromatrex, CH)₂Cl₂EtOH ═ 30/1) to give trans-3- (1H-pyrazol-1-yl) cyclopentylamine (427mg, 52%) as a yellow oil.

¹H-NMR(CDCl₃-d，400MHz)1.30-1.50(3H，m)，1.88-1.98(1H，m)，2.01-2.10(1H，m)，2.15-2.25(1H，m)，2.27-2.42(2H，m)，3.74(1H，qui，J＝6.1Hz)，4.88(1H，qui，J＝7.3Hz)，6.22(1H，d，J＝1.8Hz)，7.40(1H，d，J＝1.8Hz)，7.51(1H，s)。

Step 6:

(3S) -8-amino-9-fluoro-2, 3-dihydro-3-methyl-7-oxo-10- [ trans-3- (1H-pyrazol-1-yl) cyclopentylamino ] -7H-pyrido [1, 2, 3-de ] [1, 4] benzoxazine-6-carbonitrile (F)

(3S) -8-amino-9, 10-difluoro-2, 3-dihydro-3-methyl-7-oxo-7H-pyrido [1, 2, 3-de ] in anhydrous DMSO (12mL) was stirred at 100 deg.C][1，4]A mixture of benzoxazine-6-carbonitrile (580mg, 2.09mmol), trans-3- (1H-pyrazol-1-yl) cyclopentylamine (380mg, 2.51mmol) and diisopropylethylamine (0.80mL, 4.59mmol) for 7H. After cooling, the reaction mixture was poured into ice water and then diluted with CH₂Cl₂-MeOH extraction. The organic layer was washed with water and brine and passed over anhydrous Na₂SO₄Drying, followed by removal of the solvent. The crude product was purified by column chromatography (EtOAc) to give (3S) -8-amino-9-fluoro-2, 3-dihydro-3-methyl-7-oxo-10- [ trans-3- (1H-pyrazol-1-yl) cyclopentylamino]-7H-pyrido [1, 2, 3-de][1，4]Benzoxazine-6-carbonitrile (125mg, 15%) as a yellow powder.

¹H-NMR(DMSO-d₆，400MHz)1.35(3H，d，J＝6.7Hz)，1.60-1.73(1H，m)，1.86-1.98(1H，m)，2.11-2.29(4H，m)，4.04(1H，d，J＝10.3Hz)，4.31(1H，d，J＝10.3Hz)，4.44(1H，q，J＝6.7Hz)，4.55(1H，q，J＝6.7Hz)，4.89(1H，qui，J＝6.7Hz)，5.31(1H，d，J＝8.5Hz)，6.20(1H，t，J＝1.8Hz)，6.95(2H，brs)，7.43(1H，s)，7.74(1H，d，J＝1.8Hz)，8.46(1H，s)。

ESIMS(+)409[M+H]⁺。

HRESIMS(+)409.17920(C₂₁H₂₂FN₆O₂Calculated value of 409.17883).

Analysis C61.70%, H5.17%, N20.25%, C₂₁H₂₁FN₆O₂，0.1H₂Calculated for O, C61.48%, H5.21%, N20.49%.

Example 5:

(3S) -8-amino-9-fluoro-2, 3-dihydro-3-methyl-7-oxo-10- [ trans-3- (1H-pyrazol-1-yl) cyclopentyl Amino group]-7H-pyrido [1, 2, 3-de][1，4]Benzoxazine-6-carbonitrile (R)

Step 1: trans-3- (1H-pyrazol-1-yl) cyclopentylamine (R)

Trans-N- [3- (1H-pyrazol-1-yl) cyclopentyl in EtOH (30mL) was stirred at reflux]A mixture of phthalimide (R) (1.49g, 5.30mmol) and hydrazine hydrate (0.51mL, 10.5mmol) was used for 3 h. After cooling, the reaction mixture was filtered and the solvent was subsequently removed. The crude product was purified by column chromatography (Chromatrex, CH)₂Cl₂EtOH ═ 30/1) to give trans-3- (1H-pyrazol-1-yl) cyclopentylamine (517mg, 65%) as a yellow oil.

CIMS(+)152[M+H]⁺。

Step 2:

(3S) -8-amino-9-fluoro-2, 3-dihydro-3-methyl-7-oxo-10- [ trans-3- (1H-pyrazol-1-yl) cyclopentylamino ] -7H-pyrido [1, 2, 3-de ] [1, 4] benzoxazine-6-carbonitrile (R)

(3S) -8-amino-9, 10-difluoro-2, 3-dihydro-3-methyl-7-oxo-7H-pyrido [1, 2, 3-de ] in anhydrous DMSO (14mL) with stirring at 100 deg.C][1，4]A mixture of benzoxazine-6-carbonitrile (702mg, 2.53mmol), trans-3- (1H-pyrazol-1-yl) cyclopentylamine (460mg, 3.04mmol) and diisopropylethylamine (0.970mL, 5.57mmol) was added for 7H. After cooling, the reaction mixture was poured into ice water and then diluted with CH₂Cl₂-MeOH extraction. The organic layer was washed with water and brine and passed over anhydrous Na₂SO₄Drying, followed by removal of the solvent. The crude product was purified by column chromatography (hexane/EtOAc ═ 1/10 → EtOAc) to give (3S) -8-amino-9-fluoro-2, 3-dihydro-3-methyl-7-oxo-10- [ trans-3- (1H-pyrazol-1-yl) cyclopentylamino]-7H-pyrido [1, 2, 3-de][1，4]Benzoxazine-6-carbonitrile (108mg, 10%) as a yellow powder.

¹H-NMR(DMSO-d₆，400MHz)1.35(3H，d，J＝6.7Hz)，1.63-1.75(1H，m)，1.86-1.98(1H，m)，2.07-2.30(4H，m)，4.05(1H，d，J＝11.5Hz)，4.31(1H，d，J＝10.9Hz)，4.44(1H，q，J＝6.7Hz)，4.54(1H，q，J＝6.7Hz)，4.89(1H，qui，J＝6.7Hz)，5.30(1H，d，J＝8.5Hz)，6.20(1H，t，J＝1.8Hz)，6.94(2H，brs)，7.42(1H，s)，7.74(1H，d，J＝1.8Hz)，8.46(1H，s)。

ESIMS(+)409[M+H]⁺。

HRESIMS(+)409.17826(C₂₁H₂₂FN₆O₂Calculated value of 409.17883).

Analysis of C61.16% H5.08% N20.06%, C₂₁H₂₁FN₆O₂，0.3H₂Count of O, C60.95% H5.26% N20.31%.

Example 6:

(3S) -8-amino-9-fluoro-2, 3-dihydro-3-methyl-7-oxo-10- [ cis-4- (3-pyridinyl) cyclohexylamino Base of]-7H-pyrido [1, 2, 3-de][1，4]Benzoxazine-6-carbonitriles

Step 1: 4-hydroxy-4- (3-pyridyl) cyclohexanone vinyl acetal

To a solution of n-BuLi (1.59M in hexane, 45mL, 71.6mmol) in dry ether (150mL) at 78 deg.C was added dropwise a solution of 3-bromopyridine (11.24g, 71.1mmol) in dry ether (70 mL). After stirring for 10min, a solution of cyclohexane-1, 4-dione monovinylacetal (9.25g, 59.2mmol) in THF (70mL) was added at 78 ℃ over a period of 20min and stirred for 3 h. Water was added to the reaction mixture. CH for aqueous layer₂Cl₂Extract and wash the organic layer with brine. The extract was combined with the organic layer. After drying and concentration, the crude product was purified by column chromatography (hexane/EtOAc ═ 2/1) to give 4-hydroxy-4- (3-pyridyl) cyclohexanone vinyl acetal (6.55g, 47%) as a pale yellow powder.

¹H-NMR(CDCl₃-d，400MHz)1.65-1.78(3H，m)，1.84(2H，d，J＝11.6Hz)，2.05-2.23(4H，m)，3.96-4.05(4H，m)，7.27(1H，dd，J＝7.9，4.8Hz)，7.84(1H，dt，J＝7.9，1.2Hz)，8.49(1H，dd，J＝4.9.1.2Hz)，8.78(1H，d，J＝1.2Hz)。

EIMS(+)235[M]⁺。

Step 2: 4- (3-pyridinyl) cyclohex-3-en-1-one vinyl acetal

Thionyl chloride (10mL) was added to a solution of 4-hydroxy-4- (3-pyridyl) cyclohexanone vinyl acetal (6.53g.27.8mmol) in pyridine (60mL) at-10 ℃. Stirring at 0 ℃ for 30miAfter n, the reaction mixture was poured into ice. The aqueous layer was adjusted to pH 8 with 2M aqueous NaOH and CH₂Cl₂And (4) extracting. After drying and concentration, the crude product was purified by column chromatography (hexane/EtOAc ═ 2/1) to give 4- (3-pyridyl) cyclohex-3-en-1-one vinyl acetal (4.85g, 62%) as a colorless oil.

¹H-NMR(CDCl₃-d，400MHz)1.94(2H，d，J＝6.7Hz)，2.47-2.51(2H，m)，2.64-2.70(2H，m)，4.03(4H，s)，6.03-6.07(1H，m)，7.23(1H，dd，J＝7.9，5.5Hz)，7.67(1H，dt，J＝7.9，1.8Hz)，8.46(1H，dd，J＝4.9.1.8Hz)，8.66(1H，d，J＝1.8Hz)。

And step 3: 4- (3-pyridyl) cyclohexanone vinyl acetal

A mixture of 4- (3-pyridyl) cyclohex-3-en-1-one vinyl acetal (4.80g, 22.1mmol) in EtOAc (50mL) containing 10% Pd/C (458mg) was hydrogenated at atmospheric pressure for 3 h. After filtration of the catalyst, the solution was concentrated in vacuo to give 4- (3-pyridyl) cyclohexanone vinyl acetal (4.81g, 99%) as a yellow solid.

¹H-NMR(CDCl₃-d，400MHz)1.66-1.76(6H，m)，1.80(2H，d，J＝11.6Hz)，1.88(4H，d，J＝11.0Hz)，2.60(1H，t，J＝11.0Hz)，3.99(4H，s)，7.22(1H，dd，J＝7.9，4.9Hz)，7.56(1H，d，J＝7.9Hz)，8.44(1H，d，J＝4.9Hz)，8.50(1H，d，J＝1.2Hz)。

EIMS(+)219[M]⁺。

And 4, step 4: 4- (3-pyridyl) cyclohexanone

To 4- (3-pyridyl) cyclohexanone vinyl acetal (4.79g,21.9mmol) of CF₃CO₂Water (1mL) was added to the cold solution of H (20 mL). After stirring at room temperature for 6h, the reaction mixture was added dropwise to saturated NaHCO₃An aqueous solution. The resulting mixture was adjusted to pH 8 with 2M aqueous NaOH and CH₂Cl₂And (4) extracting. After drying and concentration, the crude product was purified by column chromatography (hexane/EtOAc ═ 1/1 → 0/1) to give 4- (3-pyridyl) purified cyclohexanone (3.23g, 84%) as a colorless powder.

¹H-NMR(CDCl₃-d，400MHz)1.90-2.04(2H，m)，2.22-2.30(2H，m)，2.47-2.61(4H，m)，3.07(1H，tt，J＝12.2，3.1Hz)，7.23-7.30(1H，m)，7.55(1H，d，J＝7.9Hz)，8.50(1H，dd，J＝4.9，1.2Hz)，8.55(1H，d，J＝1.2Hz)。

EIMS(+)175[M]⁺。

And 5: trans-4- (3-pyridinyl) cyclohexanols

To a solution of 4- (3-pyridyl) cyclohexanone (3.22g, 18.4mmol) in MeOH (25ml) at 0 deg.C was added NaBH in portions₄(348mg, 9.30mmol) and stirred at room temperature for 30 min. The reaction mixture was poured into 0.5M HCl and saturated NaHCO₃Basified with aqueous solution and then treated with CH₂Cl₂And (4) extracting. The organic layer was passed over anhydrous Na₂SO₄Drying, followed by removal of the solvent. The crude product was purified by column chromatography (hexane/EtOAc ═ 5/1) to give trans-4- (3-pyridinyl) cyclohexanol (2.48g, 76%) as a colorless powder.

¹H-NMR(CDCl₃-d，400MHz)1.38-1.61(4H，m)，1.90-1.98(2H，m)，2.09-2.16(2H，m)，2.53(1H，tt，J＝11.6，3.7Hz)，3.65-3.76(1H，m)，7.22(1H，dd，J＝7.9，4.9Hz)，7.51(1H，dt，J＝7.9，1.8Hz)，8.44(1H，dd，J＝4.9，1.8Hz)，8.48(1H，d，J＝1.8Hz)。

CIMS(+)178[M+H]⁺。

Step 6: cis-N- [4- (3-pyridyl) cyclohexyl ] phthalimide

A solution of trans-4- (3-pyridyl) cyclohexanol (1.38g, 7.79mmol) in THF (70mL) was cooled to 0 deg.C and treated with phthalimide (1.26g, 8.58mmol), diisopropyl azodicarboxylate (1.7mL, 8.63mmol) and triphenylphosphine (1.26g, 8.56 mmol). The reaction mixture was stirred at room temperature for 6 h. The reaction mixture was poured into water and then extracted with EtOAc. The organic layer was passed over anhydrous Na₂SO₄Drying, followed by removal of the solvent. The crude product was purified by column chromatography (hexane/EtOAc ═ 3/1 → 2/1) to give cis-N- [4- (3-pyridyl) cyclohexyl]Phthalimide (1.54g, 64%) was a colorless powder.

¹H-NMR(CDCl₃-d，400MHz)1.65-1.75(2H，m)，1.90-2.03(2H，m)，2.26-2.48(4H，m)，3.10-3.18(1H，m)，4.25-4.35(1H，m)，7.30(1H，dd，J＝7.9，4.9Hz)，7.69(2H，dd，J＝5.5，3.0Hz)，7.80(2H，dd，J＝5.5，3.0Hz)，7.84(1H，d，J＝7.9Hz)，8.47(1H，d，J＝4.9Hz)，8.66(1H，s)。

EIMS(+)306[M]⁺。

And 7: cis-4- (3-pyridyl) cyclohexylamine

cis-N- [4- (3-pyridyl) cyclohexyl in EtOH (30mL) was stirred at reflux]A mixture of phthalimide (1.37g, 4.47mmol) and hydrazine hydrate (0.45mL, 9.00mmol) was used for 3 h. After cooling, the reaction mixture was filtered and the solvent was subsequently removed. The crude product was purified by column chromatography (Chromatrex, CH)₂Cl₂EtOH ═ 20/1) to give cisFormula-4- (3-pyridyl) cyclohexylamine (705mg, 89%) as a yellow oil.

¹H-NMR(CDCl₃-d，400MHz)1.48(2H，brs)，1.64-1.80(6H，m)，1.83-1.95(2H，m)，2.55-2.65(1H，m)，3.23-3.30(1H，m)，7.22(1H，dd，J＝7.9，4.9Hz)，7.57(1H，d，J＝7.9Hz)，8.43(1H，dd，J＝4.8，1.8Hz)，8.51(1H，d，J＝1.8Hz)。

EIMS(+)176[M]⁺。

And 8:

(3S) -8-amino-9-fluoro-2, 3-dihydro-3-methyl-7-oxo-10- [ cis-4- (3-pyridinyl) cyclohexylamino ] -7H-pyrido [1, 2, 3-de ] [1, 4] benzoxazine-6-carbonitrile

(3S) -8-amino-9, 10-difluoro-2, 3-dihydro-3-methyl-7-oxo-7H-pyrido [1, 2, 3-de ] in anhydrous DMSO (14mL) was stirred at 100 deg.C][1，4]A mixture of benzoxazine-6-carbonitrile (792mg, 2.86mmol), cis-4- (3-pyridyl) cyclohexylamine (605mg, 3.43mmol), and diisopropylethylamine (1.0mL, 5.74mmol) was allowed to stand for 8 h. After cooling, the reaction mixture was poured into ice water and then diluted with CH₂Cl₂-MeOH extraction. The organic layer was washed with water and brine and passed over anhydrous Na₂SO₄Drying, followed by removal of the solvent. The crude product was purified by column chromatography (EtOAc) to give (3S) -8-amino-9-fluoro-2, 3-dihydro-3-methyl-7-oxo-10- [ cis-4- (3-pyridyl) cyclohexylamino]-7H-pyrido [1, 2, 3-de][1，4]Benzoxazine-6-carbonitrile (152mg, 12%) as a yellow powder.

¹H-NMR(DMSO-d₆，400MHz)1.36(3H，d，J＝6.7Hz)，1.59-1.89(8H，m)，2.62-2.71(1H，m)，4.01-4.13(2H，m)，4.40(1H，d，J＝11.5Hz)，4.44-4.51(1H，m)，4.92(1H，d，J＝8.5Hz)，6.97(2H，brs)，7.33(1H，dd，J＝7.9，4.8Hz)，7.66(1H，d，J＝7.9Hz)，8.40(1H，dd，J＝4.2，1.8Hz)，8.47-8.50(2H，m)。

ESIMS(+)434[M+H]⁺。

HRESIMS(+)434.20011(C₂₄H₂₅FN₅O₂Calculated value of 434.19923).

Analysis C66.38% H5.58% N15.88%, C₂₄H₂₄FN₅O₂Calculated for (C) 66.50% H5.58% N16.16%.

Example 7:

(3S) -8-amino-9-fluoro-2, 3-dihydro-3-methyl-7-oxo-10- [ trans-4- (3-pyridinyl) cyclohexylamino Base of]-7H-pyrido [1, 2, 3-de][1，4]Benzoxazine-6-carbonitriles

Step 1: cis-4- (3-pyridyl) cyclohexanol

To a solution of 4- (3-pyridyl) cyclohexanone (3.22g, 18.4mmol) in MeOH (25ml) at 0 deg.C was added NaBH in portions₄(348mg, 9.30mmol) and stirred at room temperature for 30 min. The reaction mixture was poured into 0.5M HCl and saturated NaHCO₃Basified with aqueous solution and then treated with CH₂Cl₂And (4) extracting. The organic layer was passed over anhydrous Na₂SO₄Drying, followed by removal of the solvent. The crude product was purified by column chromatography (hexane/EtOAc ═ 5/1) to give cis-4- (3-pyridinyl) cyclohexanol (510mg, 16%) as a colourless powder.

¹H-NMR(CDCl₃-d，400MHz)1.63-1.76(5H，m)，1.86-1.98(4H，m)，2.52-2.62(1H，m)，4.13-4.18(1H，m)，7.23(1H，dd，J＝7.9，4.3Hz)，7.56(1H，dt，J＝7.9，1.8Hz)，8.44(1H，dd，J＝4.9，1.8Hz)，8.50(1H，d，J＝2.4Hz)。

CIMS(+)178[M+H]⁺。

Step 2: cis-4- (3-pyridyl) cyclohexane methanesulfonate

Stirring anhydrous CH at room temperature₂Cl₂A mixture of cis-4- (3-pyridyl) cyclohexanol (302mg, 1.70mmol), methanesulfonyl chloride (0.290mL, 3.76mmol) and triethylamine (1.0mL, 3.66mmol) in (15mL) was 2.5 h. The reaction mixture was poured into ice water and saturated NaHCO₃Basified with aqueous solution and then treated with CH₂Cl₂And (4) extracting. The organic layer was passed over anhydrous Na₂SO₄Drying, followed by removal of the solvent. The crude product was purified by column chromatography (hexane/EtOAc ═ 1/2) to give cis-4- (3-pyridyl) cyclohexane methanesulfonate (423mg, 97%) as a colorless powder.

¹H-NMR(CDCl₃-d，400MHz)1.73-1.95(6H，m)，2.25(2H，d，J＝14.5Hz)，2.58-2.68(1H，m)，3.06(3H，s)，5.05-5.12(1H，m)，7.20-7.28(1H，m)，7.55(1H，d，J＝7.9Hz)，8.46(1H，dd，J＝4.8，1.2Hz)，8.49(1H，d，J＝1.2Hz)。

EIMS(+)255[M]⁺。

And step 3: trans-4- (3-pyridinyl) cyclohexyl azide

A mixture of cis-4- (3-pyridyl) cyclohexane methanesulfonate (407mg, 1.59mmol), sodium azide (507mg, 7.80mmol) in anhydrous DMF (8mL) was stirred at 60 ℃ for 4.5 h. The reaction mixture was poured into ice water and then diluted with CH₂Cl₂And (4) extracting. The organic layer was passed over anhydrous Na₂SO₄Drying, followed by removal of the solvent. The crude product was purified by column chromatography (hexane)EtOAc (═ 2/1) to give trans-4- (3-pyridyl) cyclohexyl azide (245mg, 76%) as a colorless oil.

¹H-NMR(CDCl₃-d，400MHz)1.45-1.63(4H，m)，1.95-2.06(2H，m)，2.10-2.21(2H，m)，2.50-2.61(1H，m)，3.32-3.42(1H，m)，7.22(1H，dd J＝7.9，4.8Hz)，7.49(1H，d，J＝7.9Hz)，8.46(1H，dd，J＝4.8，1.8Hz)，8.47(1H，d，J＝1.8Hz)。

CIMS(+)203[M+H]

And 4, step 4: trans-4- (3-pyridyl) cyclohexylamine

A solution of trans-4- (3-pyridyl) cyclohexyl azide (238mg, 1.18mmol) in EtOH (20mL) was treated with hydrogen at atmospheric pressure over 10% Pd/C (21.2mg) for 2 h. The catalyst was removed by filtration through celite and the filtrate was concentrated and dried under vacuum to give trans-4- (3-pyridyl) cyclohexylamine (202mg, 97%) as a yellow oil.

¹H-NMR(CDCl₃-d，400MHz)1.20-1.33(2H，m)，1.87-2.04(4H，m)，2.51(1H，tt，J＝12.1，3.0Hz)，2.75(1H，tt，J＝11.5，3.0Hz)，7.21(1H，dd J＝7.9，4.8Hz)，7.51(1H，dd，J＝7.9，1.8Hz)，8.44(1H，dd，J＝4.8，1.2Hz)，8.48(1H，d，J＝1.2Hz)。

EIMS(+)176[M]⁺。

And 5:

(3S) -8-amino-9-fluoro-2, 3-dihydro-3-methyl-7-oxo-10- [ trans-4- (3-pyridinyl) cyclohexylamino ] -7H-pyrido [1, 2, 3-de ] [1, 4] benzoxazine-6-carbonitrile

(3S) -8-amino-9, 10-difluoro-2, 3-dihydro-3-methyl-7-oxo-7H-pyrido [1, 2, 3-de ] in anhydrous DMSO (4mL) was stirred at 100 deg.C][1，4]A mixture of benzoxazine-6-carbonitrile (290mg, 1.05mmol), trans-4- (3-pyridyl) cyclohexylamine (220mg, 1.25mmol) and diisopropylethylamine (0.4mL, 2.30mmol) was allowed to stand for 8 h. After cooling, the reaction mixture was poured into ice water and then diluted with CH₂Cl₂-MeOH extraction. The organic layer was washed with water and brine and passed over anhydrous Na₂SO₄Drying, followed by removal of the solvent. The crude product was purified by column chromatography (EtOAc) to give (3S) -8-amino-9-fluoro-2, 3-dihydro-3-methyl-7-oxo-10- [ trans-4- (3-pyridyl) cyclohexylamino]-7H-pyrido [1, 2, 3-de][1，4]Benzoxazine-6-carbonitrile (33.2mg, 7%) as a yellow powder.

¹H-NMR(DMSO-d₆，400MHz)1.35(3H，d，J＝6.7Hz)，1.42-1.48(2H，m)，1.50-1.63(2H，m)，1.80-1.88(2H，m)，2.01-2.08(2H，m)，3.70-3.82(1H，m)，4.06(1H，d，J＝11.9Hz)，4.33(1H，d，J＝11.9Hz)，4.42-4.50(1H，m)，4.96(1H，J＝7.9Hz)，6.95(2H，brs)，7.29(1H，dd，J＝7.3，4.8Hz)，7.68(1H，d，J＝7.9Hz)，8.39(1H，d，J＝4.8Hz)，8.45-8.50(2H，m)。

ESIMS(+)434[M+H]⁺。

HRESIMS(+)434.20016(C₂₄H₂₅FN₅O₂Calculated value of 434.19923).

Analysis C65.33% H5.65% N15.23%, C₂₄H₂₄FN₅O₂Calculated for (C) 66.50% H5.58% N16.16%.

Example 8:

(3S) -8-amino-9-fluoro-2, 3-dihydro-3-methyl-7-oxo-10- [ (1S, 3S) -3- (3-pyridinyl) cyclopentyl Amino group]-7H-pyrido [1, 2, 3-de][1，4]Benzoxazine-6-carbonitriles

Step 1: (1S, 4S) -4- (3-pyridinyl) -2-cyclopenten-1-ol

To a solution of 3-iodopyridine (13.0g, 63.3mmol) in THF (30mL) at 3-9 deg.C was added i-PrMgCl (38.0mL, 76.0 mmol). After stirring at room temperature for a further 30min, CuCN (567mg, 6.33mmol) was added to the solution at 2 ℃. After stirring at 3 ℃ for a further 30min, (1R, 4S) -4-hydroxycyclopent-2-enyl ester of acetic acid (3.00g, 21.1mmol) in THF (30mL) was added to the resulting solution. The entire mixture was stirred at room temperature for 2 hours and with saturated NH under vigorous stirring₄And (5) diluting with Cl. The product was extracted three times with EtOAc and the combined layers were passed over anhydrous Na₂SO₄And (5) drying. After concentration under reduced pressure, the residue was purified by column chromatography (NH, hexane/EtOAc ═ 4/1) to give (1S, 4S) -4- (3-pyridyl) -2-cyclopenten-1-ol as a yellow oil.

¹H-NMR(CDCl₃-d，400MHz)1.81(1H，brs)，2.04-2.14(1H，m)，2.34(1H，ddd，J＝14.2，8.1，2.6Hz)，4.14-4.20(1H，m)，5.08(1H，brs)，6.03(1H，dd，J＝5.5，1.2Hz)，6.07-6.12(1H，m)，7.22(1H，dd，J＝8.3，5.2Hz)，7.42(1H，dt，J＝7.9，1.8Hz)，7.43(1H，d，J＝2.4Hz)，8.46(1H，dd，J＝4.3，1.2Hz)。

EIMS(+)：161.1[M]⁺。

Step 2: (1R, 3R) -3- (3-pyridyl) cyclopentanol

A mixture of (1S, 4S) -4- (3-pyridyl) -2-cyclopenten-1-ol (1.86g, 11.5mmol) and 10% Pd/C (186mg) in EtOH (35mL) was stirred under a hydrogen atmosphere for 3 hours. The catalyst was removed by filtration through celite and the filtrate was concentrated in vacuo. This gave (1R, 3R) -3- (3-pyridyl) cyclopentanol (1.82g, 97%) as a yellow oil.

¹H-NMR(CDCl₃-d，400MHz)1.56-1.88(4H，m)，2.08-2.36(4H，m)，3.37-3.49(1H，m)，4.53-4.59(1H，m)，7.24-7.30(1H，m)，7.58(1H，dt，J＝7.9，1.8Hz)，8.46(1H，d，J＝4.3Hz)，8.53(1H，s)。

CIMS(+)：164.1[M+H]⁺。

And step 3: acetic acid (1R, 3R) -3- (3-pyridyl) cyclopentyl ester

A solution of (1R, 3R) -3- (3-pyridyl) cyclopentanol (3.47g, 21.3mmol) in THF (100mL) was cooled to 4 deg.C and treated with acetic acid (1.34mL, 23.4mmol), diisopropyl azodicarboxylate (4.61mL, 23.4mmol), and triphenylphosphine (6.14g, 23.4 mmol). The reaction mixture was stirred at room temperature for 12 h. The reaction mixture was diluted with EtOAc and washed with H₂O and brine wash. The organic layer is passed over Na₂SO₄And (5) drying. After concentration under reduced pressure, the residue was purified by column chromatography (NH, hexane/EtOAc ═ 6/1) to give (1R, 3R) -3- (3-pyridyl) cyclopentyl acetate (4.39g, 100%) as a colorless oil.

¹H-NMR(CDCl₃-d，400MHz)1.70-2.18(8H，m)，2.55-2.65(1H，m)，3.03-3.15(1H，m)，5.23-5.31(1H，m)，7.24(1H，dd，J＝7.9，4.9Hz)，7.58(1H，dt，J＝7.9，1.8Hz)，8.46(1H，dd，J＝4.9，1.8Hz)，8.53(1H，d，J＝2.4Hz)。

CIMS(+)：206.1[M+H]⁺。

And 4, step 4: (1S, 3R) -3- (3-pyridyl) cyclopentanol

To a solution of acetic acid (1R, 3R) -3- (3-pyridyl) cyclopentyl ester (5.43g, 26.5mmol) in MeOH (80mL) at 4 deg.C was added 1N aqueous NaOH (26mL) and stirred at the same temperature for 2 h. Adding H to the reaction mixture₂O, then with CH₂Cl₂Extracted four times and the combined layers were passed over Na₂SO₄And (5) drying. After concentration under reduced pressure, the residue was purified by column chromatography (EtOAc) to give (1S, 3R) -3- (3-pyridyl) cyclopentanol (3.99g, 92%) as a colorless oil.

¹H-NMR(CDCl₃-d，400MHz)1.60-2.18(6H，m)，2.44-2.54(1H，m)，3.02-3.13(1H，m)，4.45-4.54(1H，m)，7.23(1H，dd，J＝7.3，4.9Hz)，7.65(1H，d，J＝7.9Hz)，8.43(1H，dd，J＝4.9，1.2Hz)，8.52(1H，d，J＝1.8Hz)。

CIMS(+)：164.1[M+H]⁺。

And 5: (1R, 3R) -N- [3- (3-pyridyl) cyclopentyl ] phthalimide

A solution of (1S, 3R) -3- (3-pyridyl) cyclopentanol (3.95g, 24.2mmol) in THF (80mL) was cooled to 4 deg.C and treated with phthalimide (3.91g, 26.6mmol), diisopropyl azodicarboxylate (5.24mL, 26.6mmol) and triphenylphosphine (6.98g, 26.6 mmol). The reaction mixture was stirred at room temperature for 2 h. The reaction mixture was diluted with EtOAc and washed with H₂O and brine wash. The organic layer is passed over Na₂SO₄And (5) drying. After concentration under reduced pressure, the residue was purified by column chromatography (hexane/EtOAc ═ 4/1) to give (1R, 3R) -N- [3- (3-pyridyl) cyclopentyl]Phthalimide (6.27g, 89%) was a colorless powder.

¹H-NMR(CDCl₃-d，400MHz)1.66-1.82(1H，m)，2.09(1H，dt，J＝17.9，6.7Hz)，2.16-2.43(3H，m)，2.45-2.56(1H，m)，3.67-3.79(1H，m)，4.87-4.99(1H，m)，7.22-7.28(1H，m)，7.59(1H，d，J＝7.3Hz)，7.69-7.76(2H，m)，7.81-7.89(2H，m)，8.46(1H，dd，J＝4.9，1.2Hz)，8.56(1H，d，J＝1.8Hz)。

CIMS(+)：293.1[M+H]⁺。

Step 6: (1R, 3R) -3- (3-pyridinyl) cyclopentylamine

(1R, 3R) -N- [3- (3-pyridyl) cyclopentyl ] in EtOH (40mL) was stirred at room temperature]A mixture of phthalimide (3.00g, 10.3mmol) and hydrazine hydrate (1.00mL, 20.5mmol) was used for 1 h. The reaction mixture was filtered, followed by removal of the solvent. The crude product was purified by column chromatography (NH, CHCl)₃MeOH 50/1) to give (1R, 3R) -3- (3-pyridyl) cyclopentylamine (1.63g, 97%) as a yellow oil.

¹H-NMR(CDCl₃-d，400MHz)1.33-1.75(4H，m)，1.81-1.95(2H，m)，2.05-2.30(2H，m)，3.29-3.41(1H，m)，3.62-3.71(1H，m)，7.21(1H，dd，J＝7.3，4.9Hz)，7.52(1H，d，J＝7.9Hz)，8.43(1H，dd，J＝4.9，1.2Hz)，8.49(1H，d，J＝1.8Hz)。

CIMS(+)：163.1[M+H]⁺。

And 7:

(3S) -8-amino-9-fluoro-2, 3-dihydro-3-methyl-7-oxo-10- [ (1S, 3S) -3- (3-pyridinyl) cyclopentylamino ] -7H-pyrido [1, 2, 3-de ] [1, 4] benzoxazine-6-carbonitrile

(3S) -8-amino-9, 10-difluoro-2, 3-dihydro-3-methyl-7-oxo-7H-pyrido [1, 2, 3-de ] in anhydrous DMSO (18mL) was stirred at 91 deg.C][1，4]A mixture of benzoxazine-6-carbonitrile (1g, 3.61mmol), (1R, 3R) -3- (3-pyridyl) cyclopentylamine (702mg, 4.33mmol), and diisopropylethylamine (1.38mL, 7.94mmol) for 7 h. After cooling, the reaction mixture was poured into ice water and then diluted with CH₂Cl₂MeOH extraction. The organic layer was washed with water and brine and passed over anhydrous Na₂SO₄Drying, followed by removal of the solvent. The crude product was purified by column chromatography (EtOAc) to afford (3S) -8-amino-9-fluoro-2, 3-dihydro-3-methyl-7-oxo-10- [ (1S, 3S) -3- (3-pyridyl) cyclopentylamino]-7H-pyrido [1, 2, 3-de][1，4]Benzoxazine-6-carbonitrile (247mg, 16%) as a yellow powder.

¹H-NMR(DMSO-d₆，400MHz)1.35(3H，d，J＝6.7Hz)，1.54-1.80(2H，m)，1.86-1.98(1H，m)，2.02-2.29(3H，m)，4.06(1H，d，J＝9.7Hz)，4.33(1H，d，J＝10.3Hz)，4.40-4.54(2H，m)，5.23(1H，d，J＝6.1Hz)，6.94(2H，brs)，7.30(1H，dd，J＝7.9，4.2Hz)，7.67(1H，d，J＝7.9Hz)，8.38(1H，d，J＝3.0Hz)，8.43-8.50(2H，m)。

ESIMS(+)：420.2[M+H]⁺。

HRESIMS(+)：420.18332(C₂₃H₂₃FN₅O₂Calculated value of 420.18358).

Example 9:

(3S) -8-amino-9-fluoro-2, 3-dihydro-3-methyl-7-oxo-10- [ trans-3- (3-pyridinyl) cyclohexylamino Base of]-7H-pyrido [1, 2, 3-de][1，4]Benzoxazine-6-carbonitriles

Step 1: 3- (3-pyridinyl) -2-cyclohexen-1-one

To n-BuLi (1.54M in hexane, 145mL.224mmol) in Et at-75-68 deg.C₂To a solution of O (600mL) was added 3-bromopyridine (22.0mL, 224mmol) dropwise, and stirred for 20 min. 3-ethoxy-2-cyclohexenone (25.0mL, 187mmol) was added dropwise to the reaction mixture at-75-69 ℃ and stirred at room temperature for 1 h. The reaction mixture was acidified with 2M HCl (300mL) and the aqueous layer was washed with brineAcOEt washing. The aqueous layer was basified with 1M aqueous NaOH solution and then with CH₂Cl₂And (4) extracting. The organic layer was washed with brine and over anhydrous Na₂SO₄Drying, followed by removal of the solvent. The crude product was purified by column chromatography (hexane/EtOAc ═ 1/2) to give 3- (3-pyridyl) -2-cyclohexen-1-one as a yellow powder.

¹H-NMR(CDCl₃-d，400MHz)2.15-2.26(2H，m)，2.52(2H，t，J＝6.7Hz)，2.75-2.84(2H，m)，6.43(1H，s)，7.36(1H，dd，J＝7.9，4.9Hz)，7.82(1H，dt，J＝5.0，2.6Hz)，8.64(1H，dd，J＝4.9，1.2Hz)，8.80(1H，d，J＝1.8Hz)。

EIMS(+)：173.1[M]⁺。

Step 2: cis-3- (3-pyridyl) cyclohexanol

A mixture of 3- (3-pyridyl) -2-cyclohexen-1-one (19.1g, 110mmol) and 10% Pd/C (1.91g) in EtOH (300mL) was stirred under a hydrogen atmosphere for 16 h. The catalyst was removed by filtration through celite and the filtrate was concentrated in vacuo. To the crude product in MeOH (360ml) at 3 deg.C was added NaBH in portions₄(2.08g, 55.0mmol) and stirred at the same temperature for 30 min. The reaction mixture was poured into ice water and then extracted with AcOEt. The organic layer was passed over anhydrous Na₂SO₄Drying, followed by removal of the solvent. The crude product was purified by column chromatography (NH, hexane/EtOAc ═ 2/1) to give cis-3- (3-pyridyl) cyclohexanol (14.6g, 75%) as a colorless oil.

¹H-NMR(CDCl₃-d，400MHz)1.22-1.55(4H，m)，1.79-1.99(2H，m)，2.03-2.23(2H，m)，2.62(1H，tt，J＝12.4，3.2Hz)，3.76(1H，t，J＝10.3Hz)，7.23(1H，dd，J＝7.9，4.8Hz)，7.52(1H，d，J＝7.9Hz)，8.42-8.52(2H，m)。

CIMS(+)：178.1[M+H]⁺。

And step 3: trans-N- [3- (3-pyridyl) cyclohexyl ] phthalimide

A solution of cis-3- (3-pyridyl) cyclohexanol (5.00g, 28.2mmol) in THF (100mL) was cooled to 4 deg.C and treated with phthalimide (4.56g, 31.0mmol), diisopropyl azodicarboxylate (6.10mL, 31.0mmol) and triphenylphosphine (8.13g, 31.0 mmol). The reaction mixture was stirred at room temperature for 2 h. The reaction mixture was diluted with EtOAc and washed with H₂O and brine wash. The organic layer is passed over Na₂SO₄And (5) drying. After concentration under reduced pressure, the residue was purified by column chromatography (hexane/EtOAc ═ 4/1) to give trans-N- [3- (3-pyridyl) cyclohexyl]Phthalimide (5.18g, 60%) was a colorless powder.

¹H-NMR(CDCl₃-d，400MHz)1.45-1.63(1H，m)，1.74-1.86(2H，m)，1.94(1H，tt，J＝13.3，4.2Hz)，2.16-2.28(2H，m)，2.36(1H，ddd，J＝24.5，12.4，3.9Hz)，2.72(1H，td，J＝12.7，5.0Hz)，3.43(1H，brs)，4.39(1H，tt，J＝11.5，3.8Hz)，7.29(1H，dd，J＝7.9，4.8Hz)，7.67-7.88(5H，m)，8.48(1H，d，J＝4.2Hz)，8.67(1H，d，J＝1.8Hz)。

CIMS(+)：307.1[M+H]⁺。

And 4, step 4: trans-3- (3-pyridyl) cyclohexylamine

A mixture of trans-N- [3- (3-pyridyl) cyclohexyl ] phthalimide (3.00g, 9.79mmol) and hydrazine hydrate (0.95mL, 19.6mmol) in EtOH (40mL) was stirred at room temperature for 1.5 h. The reaction mixture was filtered, followed by removal of the solvent. The crude product was purified by column chromatography (NH, EtOAc) to give trans-3- (3-pyridyl) cyclohexylamine (1.08g, 60%) as a colorless oil.

¹H-NMR(CDCl₃-d，400MHz)1.35-1.95(10H，m)，2.95-3.07(1H，m)，3.38(1H，t，J＝3.6Hz)，7.21(1H，dd，J＝7.9，4.8Hz)，7.53(1H，d，J＝7.9Hz)，8.43(1H，dd，J＝4.8，1.2Hz)，8.50(1H，d，J＝1.8Hz)。

CIMS(+)：177.1[M+H]⁺。

And 5:

(3S) -8-amino-9-fluoro-2, 3-dihydro-3-methyl-7-oxo-10- [ trans-3- (3-pyridinyl) cyclohexylamino ] -7H-pyrido [1, 2, 3-de ] [1, 4] benzoxazine-6-carbonitrile

(3S) -8-amino-9, 10-difluoro-2, 3-dihydro-3-methyl-7-oxo-7H-pyrido [1, 2, 3-de ] in anhydrous DMSO (12mL) was stirred at 84 deg.C][1，4]A mixture of benzoxazine-6-carbonitrile (800mg, 2.89mmol), trans-3- (3-pyridyl) cyclohexylamine (612mg, 3.47mmol) and diisopropylethylamine (1.11mL, 6.36mmol) was used for 9 h. After cooling, the reaction mixture was poured into ice water and then diluted with CH₂Cl₂-MeOH extraction. The organic layer was washed with water and brine and passed over anhydrous Na₂SO₄Drying, followed by removal of the solvent. The crude product was purified by column chromatography (EtOAc) to give (3S) -8-amino-9-fluoro-2, 3-dihydro-3-methyl-7-oxo-10- [ trans-3- (3-pyridyl) cyclohexylamino]-7H-pyrido [1, 2, 3-de][1，4]Benzoxazine-6-carbonitrile (309mg, 24%) as a yellow powder.

¹H-NMR(DMSO-d₆，400MHz)1.37(3H，dd，J＝6.7，4.3Hz)，1.47-1.93(8H，m)，2.80-2.95(1H，m)，4.06-4.22(2H，m)，4.35-4.52(2H，m)，4.98(1H，d，J＝7.9Hz)，6.95(2H，brs)，7.29(1H，dd，J＝7.9，4.8Hz)，7.64(1H，d，J＝6.7Hz)，8.38(1H，dd，J＝4.8，1.2Hz)，8.44(1H，s)，8.49(1H，s)。

ESIMS(+)：434.2[M+H]⁺。

HRESIMS(+)：434.19998(C₂₄H₂₅FN₅O₂Calculated value of 434.19923).

Example 10:

(3S) -8-amino-9-fluoro-2, 3-dihydro-3-methyl-7-oxo-10- [ cis-3- (3-pyridinyl) cyclohexylamino ] -7H-pyrido [1, 2, 3-de ] [1, 4] benzoxazine-6-carbonitrile

Step 1: trans-3- (3-pyridinyl) cyclohexanols

A mixture of 3- (3-pyridyl) -2-cyclohexen-1-one (19.1g, 110mmol) and 10% Pd/C (1.91g) in EtOH (300mL) was stirred under a hydrogen atmosphere for 16 h. The catalyst was removed by filtration through celite and the filtrate was concentrated in vacuo. To the crude product in MeOH (360ml) at 3 deg.C was added NaBH in portions₄(2.08g, 55.0mmol) and stirred at the same temperature for 30 min. The reaction mixture was poured into ice water and then extracted with AcOEt. The organic layer was passed over anhydrous Na₂SO₄Drying, followed by removal of the solvent. The crude product was purified by column chromatography (NH, hexane/EtOAc ═ 2/1) to give trans-3- (3-pyridinyl) cyclohexanol (4.19g, 21%) as a colorless powder.

¹H-NMR(CDCl₃-d，400MHz)1.38-2.10(8H，m)，3.00-3.15(1H，m)，4.26(1H，brs)，7.22(1H，dd，J＝7.9，4.8Hz)，7.52(1H，d，J＝7.9Hz)，8.40-8.53(2H，m)。

CIMS(+)：178.1[M+H]⁺。

Step 2: cis-N- [3- (3-pyridyl) cyclohexyl ] phthalimide

A solution of trans-3- (3-pyridyl) cyclohexanol (3.99g, 22.5mmol) in THF (90mL) was cooled to 4 deg.C and treated with phthalimide (3.65g, 24.8mmol), diisopropyl azodicarboxylate (4.88mL, 24.8mmol) and triphenylphosphine (6.50g, 24.8 mmol). The reaction mixture was stirred at room temperature for 30 min. The reaction mixture was diluted with EtOAc and washed with H₂O and brine wash. The organic layer is passed over Na₂SO₄And (5) drying. After concentration under reduced pressure, the residue was purified by column chromatography (hexane/EtOAc ═ 4/1) to give cis-N- [3- (3-pyridyl) cyclohexyl]Phthalimide (1.66g, 24%) was a colorless powder.

¹H-NMR(CDCl₃-d，400MHz)1.45-1.64(2H，m)，1.79-1.98(3H，m)，2.01-2.11(1H，m)，2.24-2.38(1H，m)，2.46(1H，q，J＝12.2Hz)，2.68-2.79(1H，m)，4.33(1H，tt，J＝12.2，3.7Hz)，7.23(1H，dd，J＝7.3，4.9Hz)，7.57(1H，d，J＝7.9Hz)，7.67-7.75(2H，m)，7.78-7.86(2H，m)，8.45(1H，dd，J＝4.9，1.2Hz)，8.51(1H，d，J＝1.8Hz)。

EIMS(+)：306.1[M]⁺。

And step 3: cis-3- (3-pyridyl) cyclohexylamine

A mixture of cis-N- [3- (3-pyridyl) cyclohexyl ] phthalimide (1.64g, 5.35mmol) and hydrazine hydrate (0.52mL, 10.7mmol) in EtOH (25mL) was stirred at room temperature for 2 h. The reaction mixture was filtered, followed by removal of the solvent. The crude product was purified by column chromatography (NH, EtOAc) to give cis-3- (3-pyridyl) cyclohexylamine (348g, 37%) as a colorless oil.

¹H-NMR(CDCl₃-d，400MHz)1.11(1H，ddd，J＝24.5，12.4，3.3Hz)，1.20-1.70(4H，m)，1.71-2.19(5H，m)，2.62(1H，tt，J＝12.1，3.3Hz)，2.83(1H，tt，J＝11.2，3.8Hz)，7.22(1H，dd，J＝7.9，4.8Hz)，7.51(1H，d，J＝7.9Hz)，8.40-8.91(2H，m)。

EIMS(+)：176.1[M]⁺。

And 4, step 4:

(3S) -8-amino-9, 10-difluoro-2, 3-dihydro-3-methyl-7-oxo-7H-pyrido [1, 2, 3-de) in anhydrous DMSO (8mL) was stirred at 93 deg.C][1，4]A mixture of benzoxazine-6-carbonitrile (457mg, 1.65mmol), cis-3- (3-pyridyl) cyclohexylamine (348mg, 1.97mmol) and diisopropylethylamine (0.63mL, 3.63mmol) was used for 9 h. After cooling, the reaction mixture was poured into ice water and then diluted with CH₂Cl₂-MeOH extraction. The organic layer was washed with water and brine and passed over anhydrous Na₂SO₄Drying, followed by removal of the solvent. The crude product was purified by column chromatography (EtOAc) to give (3S) -8-amino-9-fluoro-2, 3-dihydro-3-methyl-7-oxo-10- [ cis-3- (3-pyridyl) cyclohexylamino]-7H-pyrido [1, 2, 3-de][1，4]Benzoxazine-6-carbonitrile (96.0mg, 13%) as a yellow powder.

¹H-NMR(DMSO-d₆，400MHz)1.44-1.68(8H，m)，1.81(2H，dd，J＝37.2，11.2Hz)，1.95-2.08(2H，m)，2.69(1H，t，J＝11.2Hz)，3.75-3.88(1H，m)，4.04(1H，d，J＝10.9Hz)，4.32(1H，d，J＝11.5Hz)，4.38-4.50(1H，m)，4.95(1H，d，J＝9.7Hz)，6.94(2H，brs)，7.30(1H，dd，J＝7.3，4.8Hz)，7.60-7.68(1H，m)，8.39(1H，d，J＝4.2Hz)，8.45(2H，s)。

ESIMS(+)：434.2[M+H]⁺。

HRESIMS(+)：434.19890(C₂₄H₂₅FN₅O₂Calculated value of 434.19923).

Example 11: GS activation

HepG2 cells were obtained from the JCRB cell bank (Japanese Collection of research Bioresources) and in standard medium (Low glucose Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum supplemented with 100U/mL penicillin and 100. mu.g/mL streptomycin) at 37 ℃ in a humid atmosphere and 5% CO₂And growing under atmospheric pressure. HepG2 cells were harvested with 0.25% trypsin solution containing 1mM EDTA at 1X 10 per well⁵Cells were seeded in 12-well plates. Following 3 days of culture, cells were washed once with Phosphate Buffered Saline (PBS) and incubated with serum-free low glucose DMEM supplemented with 100U/mL penicillin and 100. mu.g/mL streptomycin. After 3h of culture, different concentrations of GSK-3 inhibitor and 2.5. mu. Ci/mL D2-substituted glucose in serum-free low-glucose DMEM³H]Glucose (new england nucleus, boston, MA, USA). Vehicle control of DMSO (0.3%, final concentration) was also used. The total volume of reaction medium per well was 1.0mL of serum-free low glucose DMEM. After incubation at 37 ℃ for 3h, the medium was aspirated and the cells were washed twice with PBS, and 0.25mL of 1N KOH containing 0.4mg/mL of carrier glycogen was added. After incubation at 37 ℃ for 30min, 0.25mL of 48.8% (w/v) KOH was added to the wells for cell lysis. After incubation at 95 ℃ for 30min, 1.5mL of 95% (v/v) ethanol was added to the cell lysate. Total glycogen was precipitated overnight at-20 ℃. The glycogen precipitate was recovered by centrifugation at 19,000x g for 30min at 4 ℃. The precipitate was washed once with 1mL of 70% (v/v) ethanol and resuspended in 0.5mL of water. Glycogen pair [2 ] was evaluated using a liquid scintillation counter (Packard Instrument Co., Meriden, CT, USA)³H]Binding of glucose.

EC for glycogen synthesis of Compounds of examples 7,9 and 10₅₀Is 0.3. mu.M or less.

Example 12: animal studies: oral glucose tolerance test

Male Cr1j CD1(ICR) mice were obtained from the Japanese Charles river laboratory (Negroside, Japan). All mice were given a standard diet (clean Japan, tokyo, Japan) and tap water ad libitum. All institutional guidelines for animal care and use were applied in this study. Test compounds were suspended in 0.3% sodium carboxymethylcellulose (CMC-Na; Sigma, St. Louis, Mo.). After fasting for 15-17 hours, 7-week-old ICR mice were orally administered test compound (3, 10, 30, 100, or 300mg/kg) or vehicle (0.3% CMC-Na). Glucose solution (5g/kg) was administered orally 30 minutes after test compound treatment. Blood samples were collected from the tail vein using EDTA · K-containing capillaries before test compound treatment and at 0, 0.5, 1 and 2 hours after glucose uptake. Blood samples were centrifuged at 2,500Xg for 5 minutes and the separated plasma was stored on ice and analyzed on the same day. Plasma glucose levels were determined using the glucose CII-assay (Wako Pure Chemical Industries, Osaka, Japan). The sum of plasma glucose levels at 0.5 and 1h after glucose intake was compared to the sum of vehicle-treated plasma glucose levels and the results were expressed as a percentage decrease. The title compound of example 2 (10mg/kg) gave a result of 22%.

Claims

1. A compound of formula (I):

or a pharmaceutically acceptable salt thereof, wherein

R¹Is C1-6 alkyl;

R²is hydrogen or C1-6 alkyl;

m is 1, 2 or 3;

n is 1 or 2;

wherein m + n is 3 or 4;

ar is pyridyl, pyrimidinyl, pyrazolyl, or imidazolyl optionally substituted with one to three substituents each independently selected from Q groups;

wherein Q is halo, hydroxy, cyano, hydroxycarbonyl, C1-6 alkyl, C1-6 haloalkyl, C1-20 alkoxy, C1-6 alkoxycarbonyl, or amino.

2. The compound of claim 1, wherein R²Is hydrogen.

3. A compound according to claim 1, having formula (Ia):

or a pharmaceutically acceptable salt thereof,

wherein n is 1 or 2;

wherein Q is halo, hydroxy, cyano, hydroxycarbonyl, C1-6 alkyl, C1-6 haloalkyl, C1-6 alkoxy, C1-6 alkoxycarbonyl, or amino.

4. The compound of claim 1, selected from:

5. a pharmaceutical composition comprising a compound of any one of claims 1-4 and a pharmaceutically acceptable carrier.

6. Use of a compound according to any one of claims 1 to 4 in the manufacture of a medicament for the treatment, prevention or amelioration of a glycogen synthase kinase-3 mediated disease.

7. The use of claim 6, wherein the glycogen synthase kinase-3 mediated disease is selected from the group consisting of: diabetes mellitus; disorders associated with diabetes; chronic neurodegenerative disorders; a neurotrauma disease; mood disorders; rheumatoid arthritis; inflammatory bowel disease; crohn's disease; sepsis; pancreatic cancer; ovarian cancer and osteoporosis.

8. The use according to claim 7, wherein the chronic neurodegenerative disorder is dementia.

9. The use of claim 7, wherein the inflammatory bowel disease is ulcerative colitis.

10. The use according to claim 8, wherein the dementia is selected from the group consisting of Alzheimer's disease, Parkinson's disease, progressive supranuclear palsy, boxer encephalitis, pick's disease, corticobasal degeneration, frontotemporal dementia, Huntington's disease, dementia associated with acquired immune deficiency syndrome, amyotrophic lateral sclerosis, and multiple sclerosis.

11. The use of claim 10, wherein the parkinson's disease is selected from subacute sclerosis holoencephalic inflammatory parkinsonism, postencephalitic parkinsonism, or guam parkinsonism-dementia syndrome.

12. The use according to claim 7, wherein the neurotrauma disease is selected from stroke and epilepsy.

13. Use according to claim 7, wherein the mood disorder is selected from depression, schizophrenia and bipolar disorder.