阅读说明：本技术 用作分子伴侣介导的自噬调节剂的化合物 (Compounds useful as chaperone mediated modulators of autophagy ) 是由 安娜·玛利亚·奎尔沃 埃夫里皮迪思·加瓦肖蒂斯 于 2018-08-30 设计创作，主要内容包括：公开了式I化合物或其药学上可接受的盐。某些式I化合物和盐作为CMA调节剂具有活性。本公开提供了含有式I化合物的药物组合物。(Compounds of formula I or pharmaceutically acceptable salts thereof are disclosed. Certain compounds and salts of formula I are active as CMA modulators. The present disclosure provides pharmaceutical compositions containing compounds of formula I.)

1. A compound of formula I

Or a pharmaceutically acceptable salt thereof, wherein

X is O andis a single bond, or XIs N andis an aromatic bond;

R¹、R³and R⁴Independently selected from hydrogen, halogen, hydroxy, C₁-C₆Alkyl and C₁-C₆An alkoxy group;

R²is halogen;

R⁵、R⁶、R⁸and R⁹Independently selected from hydrogen, halogen, hydroxy, C₁-C₆Alkyl and C₁-C₆An alkoxy group;

R⁷is-NR¹⁰COR¹¹or-NR¹⁰SO₂R¹¹Or is or

R⁷Is phenyl, naphthyl, and a monocyclic or bicyclic heteroaryl, each of which is optionally substituted with:

halogen, hydroxy, -cyano, -CHO, -COOH, amino and C₁-C₆Alkyl, wherein any carbon-carbon single bond is optionally replaced by a carbon-carbon double or triple bond, and any methylene group is optionally replaced by O, S or NR¹²Substituted, and optionally substituted with one or more substituents independently selected from halo, hydroxy, cyano, amino, and oxo; and

is selected from-NR¹⁰COR¹¹And NR¹⁰SO₂R¹¹One of the substituents of (a) or (b),

R¹⁰independently at each occurrence, selected from hydrogen and C₁-C₆An alkyl group;

R¹¹independently at each occurrence, selected from hydrogen, C₁-C₆Alkyl radical, C₁-C₂Haloalkyl, monocyclic aryl and heteroaryl, each of which is optionally independently selected from halogen, hydroxy, cyano, C₁-C₆Alkyl radical, C₁-C₆Alkoxy radical, C₁-C₂Haloalkyl and C₁-C₂One or more substituents of haloalkoxy; and is

R¹²Is hydrogen, C₁-C₆Alkyl or (C)₃-C₇Cycloalkyl) C₀-C₂An alkyl group.

2. The compound or salt of claim 1 wherein X is O andis a single bond.

3. The compound or salt of claim 1 wherein X is N andis an aromatic bond.

4. A compound or salt according to any one of claims 1-3, wherein R¹、R³And R⁴Are all hydrogen.

5. A compound or salt according to any one of claims 1-4, wherein R²Is chlorine.

6. A compound or salt according to any one of claims 1-5, wherein R⁵、R⁶、R⁸And R⁹Are all hydrogen.

7. The compound or salt of any one of claims 1 to 6, wherein

R⁷is-NR¹⁰COR¹¹or-NR¹⁰SO₂R¹¹Or is or

R⁷Is phenyl, naphthyl, pyrrolyl, pyrazolyl, thienyl, furyl, imidazolyl, thiazolyl, triazolyl, pyridyl, pyrimidinyl, benzimidazolyl, imidazopyridinyl, indolyl, indazolyl, quinolinyl or isoquinolinyl, each of which is optionally substituted by:

independently selectFrom halogen, hydroxy, cyano, -CHO, -COOH, amino and C₁-C₆One or more substituents of alkyl, wherein any carbon-carbon single bond is optionally replaced by a carbon-carbon double or triple bond, and any methylene group is optionally replaced by O, S or NR¹²Substituted, and optionally substituted with one or more substituents independently selected from halo, hydroxy, cyano, amino, and oxo; and

is selected from-NR¹⁰COR¹¹And NR¹⁰SO₂R¹¹One of the substituents of (a) or (b),

R¹⁰independently at each occurrence, selected from hydrogen and C₁-C₆An alkyl group; and is

R¹¹Independently at each occurrence, selected from hydrogen, C₁-C₆Alkyl radical, C₁-C₂Haloalkyl, C₃-C₇Cycloalkyl, monocyclic aryl and heteroaryl, each of which is optionally independently selected from halogen, hydroxy, cyano, C₁-C₆Alkyl radical, C₁-C₆Alkoxy radical, C₁-C₂Haloalkyl and C₁-C₂One or more substituents of haloalkoxy; and is

R¹²Is hydrogen, C₁-C₆Alkyl or C₃-C₇A cycloalkyl group.

8. The compound or salt of any one of claims 1 to 7, wherein

R¹¹Independently at each occurrence, selected from hydrogen, C₁-C₆Alkyl radical, C₁-C₂Haloalkyl, C₃-C₇Cycloalkyl, phenyl and pyridyl, each of which is optionally independently selected from halogen, hydroxy, cyano, C₁-C₆Alkyl radical, C₁-C₆Alkoxy radical, C₁-C₂Haloalkyl and C₁-C₂One or more substituents of haloalkoxy.

9. The compound or salt of any one of claims 1 to 6, wherein

R⁷is-NR¹⁰COR¹¹or-NR¹⁰SO₂R¹¹；

R¹⁰Is hydrogen or methyl; and is

R¹¹Is C₁-C₆Alkyl or CF₃。

10. The compound or salt of any one of claims 1 to 6, wherein

R⁷Is phenyl, optionally substituted with:

independently selected from halogen, hydroxy, C₁-C₄Alkyl radical, C₁-C₄One or more substituents of alkoxy, trifluoromethyl and trifluoromethoxy; and

is selected from-NR¹⁰COR¹¹And NR¹⁰SO₂R¹¹A substituent of (1).

11. A compound or salt according to claim 1, wherein

X is O andis a single bond;

R¹、R³、R⁴、R⁵、R⁶、R⁸and R⁹Is hydrogen;

R²is chlorine; and is

R⁷Is phenyl, optionally independently selected from hydroxy and C₁-C₂One or more substituents of the alkoxy group.

12. A compound or salt according to claim 1, wherein

X is N andis a fragranceA group bond;

R¹、R³、R⁴、R⁵、R⁶、R⁸and R⁹Is hydrogen;

R²is chlorine;

R⁷is phenyl optionally substituted by halogen, -NR¹⁰COR¹¹Or NR¹⁰SO₂R¹¹；

R¹⁰Is hydrogen; and is

R¹¹Is selected from C₁-C₆Alkyl radical, C₁-C₂Haloalkyl and phenyl, each of said phenyl optionally substituted with one or more halogens.

13. A compound or salt according to claim 1, wherein

X is N andis an aromatic bond;

R¹、R³、R⁴、R⁵、R⁶、R⁸and R⁹Is hydrogen;

R²is chlorine;

R⁷is-NR¹⁰COR¹¹or-NR¹⁰SO₂R¹¹；

R¹⁰Is hydrogen; and is

R¹¹Is selected from C₁-C₆Alkyl radical, C₁-C₂Haloalkyl and phenyl, each of said phenyl optionally substituted with one or more halogens.

14. The compound or salt thereof according to claim 1, wherein the compound is

15. The compound or salt thereof according to claim 1, wherein the compound is

16. The compound of claim 1, or a salt thereof, wherein the compound is a compound of table 1.

17. A pharmaceutical composition comprising a compound or salt according to any one of claims 1-16 and a pharmaceutically acceptable carrier.

18. A method of selectively activating chaperone-mediated autophagy in a subject in need thereof, the method comprising administering to the subject an effective amount of a compound according to any one of claims 1 to 16.

19. The method of claim 18, wherein the subject has parkinson's disease, huntington's disease, alzheimer's disease, frontotemporal dementia, prion disease, amyotrophic lateral sclerosis, retinal degeneration, leber's congenital amaurosis, diabetes, acute liver failure, NASH, liver cirrhosis, alcoholic fatty liver, renal failure and chronic kidney disease, emphysema, sporadic inclusion body myositis, spinal cord injury, traumatic brain injury, lysosomal storage disease, cardiovascular disease, or immunosenescence.

20. Use of a compound of any one of claims 1 to 16 for selectively activating chaperone-mediated autophagy in a subject in need thereof.

21. Use of a compound of any one of claims 1 to 16 for the treatment of parkinson's disease, huntington's disease, alzheimer's disease, frontotemporal dementia, prion diseases, amyotrophic lateral sclerosis, retinal degeneration, leber's congenital amaurosis, diabetes, acute liver failure, NASH, liver cirrhosis, alcoholic fatty liver, renal failure and chronic kidney disease, emphysema, sporadic inclusion body myositis, spinal cord injury, traumatic brain injury, lysosomal storage disease, cardiovascular disease, or immunosenescence.

Technical Field

The present disclosure relates to 3-phenyl-benzoxazines and 3-phenyl-quinazolines as CMA modulators and pharmaceutical compositions containing these compounds.

Background

Autophagy is a process by which unwanted or dysfunctional proteins present in the cytosol are degraded in the lysosomal compartment. In chaperone-mediated autophagy (CMA), proteins are individually selected and targeted from the cytosol to the lysosomal lumen by crossing the membrane directly. CMA plays a role in cellular quality control by facilitating the removal of damaged or abnormal proteins as well as the redundant subunits of the multi-protein complex. The functions after CMA activation are: decomposing protein to provide amino acids as fuel during periods of starvation for long periods of time; removing oxidized protein during oxidative stress; and removal of damaged proteins after toxic chemical exposure. CMA also has a regulatory function in cells, as CMA can regulate the activity of other cellular processes (i.e., glycolysis, lipogenesis, lipolysis, cell cycle, DNA repair, etc.) by degrading key proteins involved in each of these processes.

CMA is a multi-step process. The chaperone, heat shock homologous protein 70(Hsc70), recognizes and binds the pentapeptide motif (e.g., KFERQ) of the protein substrate to be degraded. Once bound to Hsc70, the protein substrate is targeted to the surface of the lysosome where it interacts with the cytosolic tail in the form of a monomer of the membrane associated type 2A membrane protein (LAMP-2A) receptor of the membrane-bound lysosome. When the Hsc70 protein substrate complex binds to the LAMP-2 receptor, this triggers the formation of multimeric complexes ("translocation complexes") of LAMP-2A with the relevant lysosomal proteins. Only after the translocation complex is formed can the protein substrate cross the membrane from the cytosol to the lysosome. Once the substrate translocates into the lysosomal cavity, LAMP-2A detaches from the translocation complex and the protein substrate undergoes degradation.

Both decreased and enhanced CMA activity is associated with human disease. In particular, problems with the function of the translocation complex contribute to the development of disease pathology. For example, a decrease in CMA activity correlates with: neurodegenerative diseases such as tauopathies (frontotemporal dementia, Alzheimer's Disease), Parkinson's Disease, Huntington's Disease, prion diseases, amyotrophic lateral sclerosis, retinal and macular degeneration, leber congenital amaurosis, diabetes, acute liver failure, NASH, cirrhosis, alcoholic steatohepatitis, renal failure and chronic kidney Disease, emphysema, sporadic inclusion body myositis, spinal cord injury, traumatic brain injury, lysosomal storage disorders including, but not limited to, cystinosis, galactosialidosis, mucomucomucomucomucositiosis), cardiovascular diseases or immunosenescence. Alternatively, up-regulation of CMA activity is associated with the survival and proliferation of cancer cells, and also occurs, for example, in lupus. However, known small molecules that modulate CMA are non-specific and affect the activity of other cellular quality control mechanisms. Accordingly, there is a need for compounds that modulate CMA activity to treat diseases and conditions associated with increased or decreased CMA activity.

Disclosure of Invention

The inventors of the present invention have discovered a class of compounds and salts of formula I that modulate CMA. Some compounds activate RAR receptors. Some compounds inactivate RAR receptors.

Retinoic Acid Receptors (RARs) are nuclear hormone receptors that act as transcription factors, regulating cell division, cell growth, and cell death. There are three types of RAR (RAR α, RAR β, and RAR γ) identified in mammals that are encoded by different genes. The expression of RAR β and RAR γ is tissue-dependent, while RAR α is ubiquitously expressed. The natural ligands for RAR are all-trans retinoic acid (ATRA) and 9-cis retinoic acid (9-cis RA).

RAR α signaling inhibits LAMP-2A transcription and expression of other CMA genes. When RAR α is activated upon binding of an RAR α agonist (e.g., ATRA, 9-cis RA, or derivatives thereof), transcription of LAMP-2A is reduced and there are fewer LAMP-2A receptors involved in CMA. Alternatively, an antagonist that binds to RAR α would potentially block inhibition of transcription of LAMP-2A, resulting in the presence of more LAMP-2A receptors involved in CMA.

The disclosure encompasses compounds and salts of formula I

Or a pharmaceutically acceptable salt thereof, wherein

X is O andis a single bond, or X is N andis an aromatic bond;

R¹、R³and R⁴Independently selected from hydrogen, halogen, hydroxy, C₁-C₆Alkyl and C₁-C₆An alkoxy group;

R²is halogen;

R⁵、R⁶、R⁸and R⁹Independently selected from hydrogen, halogen, hydroxy, C₁-C₆Alkyl and C₁-C₆An alkoxy group;

R⁷is-NR¹⁰COR¹¹or-NR¹⁰SO₂R¹¹Or is or

R⁷Is phenyl, naphthyl, and a monocyclic or bicyclic heteroaryl, each of which is optionally substituted with: halogen, hydroxy, -cyano, -CHO, -COOH, amino and C₁-C₆Alkyl, wherein any carbon-carbon single bond is optionally replaced by a carbon-carbon double or triple bond, and any methylene group is optionally replaced by O, S or NR¹²Substituted, and optionally substituted with one or more substituents independently selected from halo, hydroxy, cyano, amino, and oxo; and is selected from the group consisting of-NR¹⁰COR¹¹And NR¹⁰SO₂R¹¹A substituent of (a);

R¹⁰independently at each occurrence, selected from hydrogen and C₁-C₆An alkyl group;

R¹¹independently at each occurrence, selected from hydrogen, C₁-C₆Alkyl radical, C₁-C₂Haloalkyl, monocyclic aryl and heteroaryl, each of which is optionally independently selected from halo, hydroxy, cyano, heteroaryl, optionally substituted with one or more substituents selected from halogen, hydroxy, cyano, halogen,C₁-C₆Alkyl radical, C₁-C₆Alkoxy radical, C₁-C₂Haloalkyl and C₁-C₂One or more substituents of haloalkoxy; and is

R¹²Is hydrogen, C₁-C₆Alkyl or (C)₃-C₇Cycloalkyl) C₀-C₂An alkyl group.

Pharmaceutical compositions comprising a compound or salt of formula I and a pharmaceutically acceptable carrier are disclosed.

The present disclosure provides pharmaceutical compositions comprising a compound of formula I or a pharmaceutically acceptable salt of formula I and a pharmaceutically acceptable carrier.

The present disclosure further provides a method of selectively activating chaperone-mediated autophagy in a subject in need thereof by administering to the subject a therapeutically effective amount of a compound of formula I or a salt thereof. The present disclosure provides the use of a compound of formula I, or a salt thereof, for activating chaperone-mediated autophagy in a subject in need thereof.

Drawings

FIG. 1A-FIG. 1C: FIG. 1A shows the effect of CA39 and CA39.1(CA3901) on CMA; fig. 1B shows the effect of CA77 and CA77.1(CA7701) on CMA.

Fig. 2A-2B: FIG. 2A shows the concentration of CA77.1 in the plasma of ICR (CD-1) mice after intravenous and oral administration of a 10mg/kg dose; FIG. 2B shows the concentration of CA77.1 in the brain of ICR (CD-1) mice after intravenous and oral administration of a 10mg/kg dose.

Fig. 3A-3B: figure 3A shows CMA activation dose-dependence in cells 12 hours after administration of CA39, CA77, or CA 77.1; figure 3B shows CMA activation dose-dependence in cells 24 hours after administration of CA39, CA77, or CA 77.1.

Detailed Description

Chemical description and terminology

Before setting forth the invention in detail, it may be helpful to provide a definition of certain terms used in this disclosure. Compounds are described using standard nomenclature. 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 to which this invention belongs. Unless the context clearly prohibits, each compound name encompasses the free acid or free base form of the compound as well as all pharmaceutically acceptable salts of the compound.

The term "compound of formula I" encompasses all compounds satisfying formula I, including any enantiomers, racemates and stereoisomers as well as all pharmaceutically acceptable salts of such compounds. A dash ("-") that is not between two letters or symbols is used to indicate a point of attachment for a substituent.

The terms "a" and "an" do not denote a limitation of quantity, but rather denote the presence of at least one of the referenced item. The term "or" means "and/or". The open transition phrase "comprising" encompasses the intermediate transition phrase "consisting essentially of … …" and the closed phrase "consisting of … …". A claim reciting one of these three transitional phrases, or having an alternative transitional phrase (such as "comprising" or "including"), can be written with any other transitional phrase, unless context or art clearly excludes it. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. The endpoints of all ranges are inclusive of the stated range and independently combinable. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention as used herein. Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

A dash ("-") that is not between two letters or symbols is used to indicate a point of attachment for a substituent. For example, - (C ═ O) OH is linked through the carbon of the keto group (C ═ O).

Bonds represented by a combination of solid and dashed lines, i.e.May be a single bond or a double bond.

An "alkyl" group is a branched or straight chain saturated aliphatic hydrocarbon group having the indicated number of carbon atoms, typically from 1 to about 8 carbon atoms. As used herein, the term C₁-C₆Alkyl indicates alkyl having 1, 2, 3, 4, 5, or 6 carbon atoms. Other embodiments include alkyl groups having 1 to 8 carbon atoms, 1 to 4 carbon atoms, or 1 or 2 carbon atoms, e.g., C₁-C₄Alkyl and C₁-C₂An alkyl group. When referred to herein as C₀-C_nAlkyl with another group (e.g., -C)₀-C₂When alkyl (phenyl)) groups are used in combination, the indicated group (in this case phenyl) is directly bound via a single covalent bond (C)₀Alkyl) or linked by an alkyl chain having the indicated number of carbon atoms (in this case 1, 2, 3 or 4 carbon atoms). The alkyl groups may also be interrupted by other groups such as-O-C₀-C₄Alkyl radical (C)₃-C₇Cycloalkyl) is attached. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, 3-methylbutyl, tert-butyl, n-pentyl, and sec-pentyl.

An "alkoxy" group is an alkyl group as defined above having the indicated number of carbon atoms covalently bonded to the group it is substituted with through an oxygen bridge (-O-). Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, 2-butoxy, tert-butoxy, n-pentoxy, 2-pentoxy, 3-pentoxy, isopentoxy, neopentoxy, n-hexoxy, 2-hexoxy, 3-hexoxy, and 3-methylpentoxy.

"aryl" refers to an aromatic group containing only carbon in one or more aromatic rings. Typical aryl groups contain 1 to 3 separate, fused, or pendant rings and 6 to about 18 ring atoms, with no heteroatoms as ring members. When indicated, such aryl groups may be further substituted with carbon or non-carbon atoms or groups. Aryl groups include, for example, phenyl, naphthyl (including 1-naphthyl, 2-naphthyl), and biphenyl.

"cycloalkyl" is a saturated hydrocarbon ring group having the indicated number of carbon atoms. Monocyclic cycloalkyl groups typically have from 3 to about 7(3, 4, 5, 6, or 7) carbon ring atoms. Cycloalkyl substituents may be pendant to a substituted nitrogen, sulfur, oxygen, or carbon atom, or a substituted carbon atom that may have two substituents may have a cycloalkyl group attached as a spiro group. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl as well as bridged or caged saturated cyclic groups such as norbornane or adamantane.

"haloalkyl" includes both branched and straight-chain alkyl groups having the indicated number of carbon atoms substituted with 1 or more halogen atoms (up to the maximum allowable number of halogen atoms). Examples of haloalkyl groups include, but are not limited to, trifluoromethyl, difluoromethyl, 2-fluoroethyl, and pentafluoroethyl.

"haloalkoxy" is a haloalkyl group as defined herein attached through an oxygen bridge (the oxygen of an alcohol group).

"halo" or "halogen" indicates any of fluorine, chlorine, bromine and iodine.

"heteroaryl" is a stable, monocyclic aromatic ring having the indicated number of ring atoms, containing 1 to 4, or in some embodiments 1 to 2, heteroatoms selected from N, O and S, the remaining ring atoms being carbon or a stable bicyclic ring system, containing at least 5 to 7-membered aromatic rings, containing 1 to 4, or in some embodiments 1 to 2, heteroatoms selected from N, O and S, the remaining ring atoms being carbon. Monocyclic heteroaryl groups typically have 5 to 7 ring atoms. In certain embodiments, heteroaryl is a 5 or 6 membered heteroaryl having 1, 2, 3, or 4 heteroatoms selected from N, O and S and no more than 2O atoms and 1S atom.

As used herein, the term "substituted" means that any one or more hydrogens on the designated atom or group is replaced with a selection from the indicated group, provided that the designated atom's normal valence is not exceeded. When the substituent is oxo (i.e., ═ O), then 2 hydrogens on the atom are replaced. When an oxo group replaces an aromatic moiety, the corresponding partially unsaturated ring replaces the aromatic ring. For example, pyridyl substituted with oxo is pyridone. Combinations of substituents and/or variables are permissible only if such combinations result in stable compounds or useful synthetic intermediates. A stable compound or stable structure is intended to indicate a compound that is sufficiently robust to survive isolation from the reaction mixture and formulation into an effective therapeutic agent. Unless otherwise indicated, substituents are designated as core structures. For example, it is understood that when aminoalkyl is listed as a possible substituent, the point of attachment of this substituent to the core structure is in the alkyl moiety.

In certain embodiments, groups that may be "substituted" or "optionally substituted" include, but are not limited to: monocyclic aryl groups such as phenyl; monocyclic heteroaryl groups such as pyrrolyl, pyrazolyl, thienyl, furyl, imidazolyl, thiazolyl, triazolyl, pyridyl, pyrimidyl; bicyclic heteroaryls such as benzimidazolyl, imidazopyridinizinyl, imidazopyridazinyl, indolyl, indazolyl, quinolinyl, isoquinolinyl; and C₁-C₆Alkyl, wherein any carbon-carbon single bond is optionally replaced by a carbon-carbon double or triple bond, and any methylene group is optionally replaced by O, S or NR¹²And (6) replacing.

Suitable groups that may be present in a "substituted" or "optionally substituted" position include, but are not limited to: halogen; a cyano group; CHO; COOH; a hydroxyl group; an oxo group; an amino group; an alkyl group having from 1 to about 6 carbon atoms; alkoxy groups having one or more oxygen linkages and from 1 to about 8 or from 1 to about 6 carbon atoms; haloalkyl having one or more halogens and from 1 to about 8,1 to about 6, or 1 to about 2 carbon atoms; and haloalkoxy groups having one or more oxygen linkages and one or more halogens and from 1 to about 8,1 to about 6, or 1 to about 2 carbon atoms.

A "pharmaceutical composition" is a composition that includes at least one active agent (e.g., a compound or salt of formula I) and at least one other substance (e.g., a carrier). The pharmaceutical composition optionally contains one or more additional active agents. Pharmaceutical compositions, when specified, meet GMP (good manufacturing practice) standards for human or non-human drugs by the FDA in the united states.

"pharmaceutically acceptable salts" encompass derivatives of the disclosed compounds in which the parent compound is modified by making inorganic and organic, non-toxic, acid or base addition salts thereof. Salts of the compounds of the present invention may be synthesized from the parent compound, which contains a basic or acidic moiety, by conventional chemical methods. In general, such salts can be prepared by reacting the free acid forms of these compounds with a stoichiometric amount of the appropriate base (e.g., Na, Ca, Mg, or K hydroxide, carbonate, bicarbonate, etc.) or by reacting the free base forms of these compounds with a stoichiometric amount of the appropriate acid. Such reactions are usually carried out in water or an organic solvent or a mixture of both. Generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol or acetonitrile are preferred where feasible. Salts of the compounds of the present invention further comprise solvates of the compounds and salts of the compounds.

Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; an alkali metal or organic salt of an acidic residue such as a carboxylic acid; and the like. Pharmaceutically acceptable salts include the conventional non-toxic salts and the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. For example, conventional non-toxic acid salts include salts derived from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, nitric acid, and the like; and salts prepared from organic acids such as acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, pamoic acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, sulfanilic acid, 2-acetoxybenzoic acid, fumaric acid, p-toluenesulfonic acid, methanesulfonic acid, ethanesulfonic acid, oxalic acidIsethionic acid or HOOC- (CH)₂)_n-COOH (wherein n is 0-4), and the like.

The term "carrier" as applied to the pharmaceutical compositions/combinations of the present disclosure refers to a diluent, excipient, or vehicle used to provide the active compound. To be pharmaceutically acceptable, the carrier must be safe, non-toxic, and not biologically or otherwise undesirable.

Description of the chemistry

The present disclosure provides compounds and salts of formula I. The term "formula I" encompasses pharmaceutically acceptable salts of formula I unless the context clearly indicates otherwise. In certain instances, the compounds of formula I may contain one or more asymmetric elements, such as stereogenic centers, stereogenic axes, and the like, for example asymmetric carbon atoms, such that the compounds may exist in different stereoisomeric forms. These compounds may be in racemic or optically active form, for example. For compounds having two or more asymmetric elements, these compounds may additionally be mixtures of diastereomers. For compounds having asymmetric centers, it is understood that all optical isomers and mixtures thereof are contemplated. In addition, compounds having carbon-carbon double bonds may exist in the Z and E forms, all isomeric forms of which are encompassed by the present disclosure. In these cases, a single enantiomer (i.e., the optically active form) may be obtained by asymmetric synthesis, synthesis from optically pure precursors, or resolution of racemates. Resolution of the racemate may also be accomplished, for example, by conventional methods such as crystallization in the presence of a resolving agent or chromatography using, for example, a chiral HPLC column.

When a compound exists in various tautomeric forms, the invention is not limited to any one of the specific tautomers, but encompasses all tautomeric forms.

The present disclosure includes all isotopes of atoms occurring in the compounds of the present invention. Isotopes include those atoms having the same atomic number but different mass numbers. By way of general example and not limitation, isotopes of hydrogen include tritium and deuterium, and isotopes of carbon include¹¹C、¹³C and¹⁴C。

the disclosure encompasses compounds and salts of formula I wherein the variables (e.g., X, R)¹、R²、R³、R⁴、R⁵、R⁶、R⁷、R⁸、R⁹、R¹⁰、R¹¹、R¹²) With any of the definitions set forth below. Any of the variable definitions set forth below may be combined with any of the other variable definitions set forth below as long as a stable compound is produced.

CA77 and CA39 are provided as comparative examples and are outside the scope of formula I.

X variable and

x is O andis a single bond.

X is N andis an aromatic bond.

^{1 12}R-R variable

(1)R¹、R³And R⁴Are all hydrogen.

(2)R⁵、R⁶、R⁸And R⁹Are all hydrogen.

(3)R¹、R³、R⁴、R⁵、R⁶、R⁸And R⁹Is hydrogen.

(4)R²Is chlorine.

(5)R⁷Is phenyl, naphthyl, pyrrolyl, pyrazolyl, thienyl, furyl, imidazolyl, thiazolyl, triazolyl, pyridyl, pyrimidinyl, benzimidazolyl, imidazopyridinyl, indolyl, indazolyl, quinolinyl or isoquinolinyl, each of which is optionally substituted by: independently selected from halogen, hydroxy, cyano, -CHO, -COOH, amino and C₁-C₆One or more substituents of alkyl, wherein any carbon-carbon single bond is optionally replaced by a carbon-carbon double or triple bond, and any methylene group is optionally replaced by O, S or NR¹²Substituted, and optionally substituted with one or more substituents independently selected from halo, hydroxy, cyano, amino, and oxo; and is selected from the group consisting of-NR¹⁰COR¹¹And NR¹⁰SO₂R¹¹A substituent of (1).

(6)R⁷Is phenyl, optionally independently selected from halogen, hydroxy, C₁-C₄Alkyl radical, C₁-C₄Alkoxy, trifluoromethyl and trifluoromethoxy; and is selected from the group consisting of-NR¹⁰COR¹¹And NR¹⁰SO₂R¹¹A substituent of (1).

(7)R⁷Is phenyl, optionally selected from hydroxy and C₁-C₂One or more substituents of the alkoxy group.

(8)R⁷Is phenyl optionally substituted by halogen, -NR¹⁰COR¹¹Or NR¹⁰SO₂R¹¹。

(9)R⁷Is 4-fluorophenyl.

(10)R⁷is-NR¹⁰COR¹¹or-NR¹⁰SO₂R¹¹。

(11)R⁷is-NR¹⁰COR¹¹。

(12)R⁷is-NR¹⁰SO₂R¹¹。

(13)R¹⁰Independently at each occurrence, selected from hydrogen and C₁-C₆An alkyl group.

(14)R¹⁰Is hydrogen or methyl.

(15)R¹⁰Is hydrogen.

(16)R¹¹Independently at each occurrence, selected from hydrogen, C₁-C₆Alkyl radical, C₁-C₂Haloalkyl, C₃-C₇Cycloalkyl, monocyclic aryl and heteroaryl, each of which is optionally independently selected from halogen, hydroxy, cyano, C₁-C₆Alkyl radical, C₁-C₆Alkoxy radical, C₁-C₂Haloalkyl and C₁-C₂One or more substituents of haloalkoxy.

(17)R¹¹Independently at each occurrence, selected from hydrogen, C₁-C₆Alkyl radical, C₁-C₂Haloalkyl, C₃-C₇Cycloalkyl, phenyl and pyridyl, each of which is optionally independently selected from halogen, hydroxy, cyano, C₁-C₆Alkyl radical, C₁-C₆Alkoxy radical, C₁-C₂Haloalkyl and C₁-C₂One or more substituents of haloalkoxy.

(18)R¹¹Is selected from C₁-C₆Alkyl radical, C₁-C₂Haloalkyl and phenyl, each of said phenyl optionally substituted with one or more halogens.

(19)R¹¹Is C₁-C₆Alkyl or CF₃。

(20)R¹¹is-CH₃、CF₃、-CH(CH₃)₂、-(CH₂)₂CH₃Or 4-fluorophenyl.

(21)R¹²Is hydrogen, C₁-C₆Alkyl or C₃-C₇A cycloalkyl group.

In certain embodiments, R⁷Selected from one of the following groups:

the present disclosure encompasses the following compounds and pharmaceutically acceptable salts thereof:

the present disclosure encompasses the following compounds and pharmaceutically acceptable salts thereof:

certain compounds of the present disclosure have advantages over comparative compounds CA77 and CA39, including improved pharmaceutical properties, such as bioavailability.

Another aspect of the above example (formula I) is a compound or salt of table 1.

Pharmaceutical preparation

The compounds disclosed herein may be administered as pure chemicals, but are preferably administered as pharmaceutical compositions. Accordingly, the present disclosure provides pharmaceutical compositions comprising a compound or pharmaceutically acceptable salt of a CMA modulator (e.g., a compound of formula I) and at least one pharmaceutically acceptable carrier. In certain implementations, the pharmaceutical composition is in a dosage form comprising: from about 0.1mg to about 2000mg, from about 10mg to about 1000mg, from about 100mg to about 800mg or from about 200mg to about 600mg of a compound of formula I and optionally from about 0.1mg to about 2000mg, from about 10mg to about 1000mg, from about 100mg to about 800mg or from about 200mg to about 600mg of an additional active agent in unit dosage form.

The compounds disclosed herein can be administered orally, topically, parenterally, by inhalation or spray, sublingually, transdermally, buccally, rectally, as an ophthalmic solution, or by other means in dosage unit formulations containing conventional pharmaceutically acceptable carriers. The pharmaceutical composition may be formulated in any pharmaceutically useful form, for example as an aerosol, cream, gel, pill, capsule, tablet, syrup, transdermal patch or ophthalmic solution. Some dosage forms, such as tablets and capsules, are subdivided into suitably sized unit doses containing appropriate quantities of the active ingredient, e.g., an effective amount to achieve the desired purpose.

The carrier comprises excipients and diluents and must be of sufficiently high purity and low toxicity to render it suitable for administration to the patient being treated. The carrier may be inert or the carrier may have its own pharmaceutical benefit. The amount of carrier used in conjunction with the compound is sufficient to provide the actual amount of material administered per unit dose of the compound.

Types of carriers include, but are not limited to, binders, buffers, colorants, diluents, disintegrants, emulsifiers, flavoring agents, glidants, lubricants, preservatives, stabilizers, surfactants, tableting agents, and wetting agents. Some carriers may be listed as more than one type, for example vegetable oils may be used as lubricants in some formulations and as diluents in other formulations. Exemplary pharmaceutically acceptable carriers include sugars, starch, cellulose, powdered tragacanth, malt, gelatin; talc powder and vegetable oil. The optional active agent may be included in a pharmaceutical composition that does not substantially interfere with the activity of the compounds of the present disclosure.

The pharmaceutical composition/combination may be formulated for oral administration. These compositions contain between 0.1 and 99 weight percent (wt.%) of the compound of formula I and typically at least about 5 wt.% of the compound of formula I. Some embodiments contain from about 25 wt.% to about 50 wt.% or from about 5 wt.% to about 75 wt.% of the compound of formula (ila).

Method of treatment

The present disclosure also provides a method of selectively activating chaperone-mediated autophagy (CMA) in a subject in need thereof, the method comprising administering to the subject a compound of formula I in an amount effective to activate CMA in the subject.

The subject may be suffering from, for example, neurodegenerative diseases such as tauopathies (frontotemporal dementia, alzheimer's disease), parkinson's disease, huntington's disease, prion diseases, amyotrophic lateral sclerosis, retinal degeneration, leber's congenital amaurosis, diabetes, acute liver failure, nonalcoholic steatohepatitis (NASH), cirrhosis, alcoholic fatty liver, renal failure and chronic kidney disease, emphysema, sporadic inclusion body myositis, spinal cord injury, traumatic brain injury, lysosomal storage disorders, cardiovascular disease, and immunosenescence. Lysosomal storage disorders include, but are not limited to, cystinosis, galactosialiosis, and mucolipidosis. The subject may also have a disease or condition in which CMA is upregulated, such as cancer or lupus. The subject may have a reduced CMA compared to a normal subject prior to administration of the compound. Preferably, the compound does not affect macroautophagy or other autophagy pathways. In macrophages, proteins and organelles are sequestered in double-membrane vesicles and delivered to lysosomes for degradation. In CMA, protein substrates are selectively identified and targeted to lysosomes through interaction with cytosolic chaperones.

The present disclosure also provides a method of protecting cells from oxidative stress, protein toxicity, and/or lipotoxicity in a subject in need thereof, the method comprising administering to the subject any of the compounds disclosed herein or the combination of compounds of formula I in an amount effective to protect cells from oxidative stress, protein toxicity, and/or lipotoxicity. The subject may suffer from one or more chronic conditions, for example associated with increased oxidative stress and oxidation and a background prone to protein toxicity. Protected cells may include, for example, heart cells, kidney and liver cells, neurons and glia, muscle cells, fibroblasts, and/or immune cells. The compounds can, for example, selectively activate chaperone-mediated autophagy (CMA). In one embodiment, the compound does not affect macroautophagy.

In one embodiment, the subject is a mammal. In certain embodiments, the subject is a human, e.g., a human patient undergoing medical treatment. The subject may also be a companion to a non-human mammal, such as a companion animal (e.g., cats and dogs) or livestock animal.

For diagnostic or research applications, a variety of mammals will be suitable subjects, including rodents (e.g., mice, rats, hamsters), rabbits, primates, and pigs (e.g., inbred pigs), among others. Additionally, for in vitro applications, such as in vitro diagnostic and research applications, bodily fluids (e.g., blood, plasma, serum, interstitial fluid, saliva, stool, and urine) as well as cell and tissue samples of the subject described above would be suitable for use.

An effective amount of a pharmaceutical composition can be an amount sufficient to inhibit the progression of a disease or disorder, cause regression of a disease or disorder, alleviate symptoms of a disease or disorder, or significantly alter the level of a marker of a disease or disorder. For example, levels of dopamine transporter (DAT) and vesicular monoamine transporter 2(VMAT2) in the brain of parkinson's patients are reduced both in the prodromal phase and at diagnosis, and can also be used to monitor disease progression by brain imaging. Thus, as observed by brain imaging, a therapeutically effective amount of a compound of formula I comprises an amount effect that slows the decrease in brain DAT or VMAT2 levels. Accumulation of tau protein in the brain of patients with frontotemporal dementia has been observed by PET imaging and therefore a therapeutically effective amount of a compound of formula I comprises an amount sufficient to reduce tau brain deposits or slow the rate of tau brain deposits. Markers for effective treatment of NASH, cirrhosis, and alcoholic fatty liver include reduced lipid content and fibrosis in liver biopsies. Markers for effective treatment of cancer include reduction in tumor size (e.g., as observed by MRI), reduction in the number or size of metastases. Markers for effective treatment of emphysema include improvements in volume and velocity parameters in spirometry. Markers for effective treatment of immunosenescence include restoration of T cell activation in vitro. Markers for effective treatment of renal dysfunction include plasma creatine levels and normalization of plasma to urine creatine ratio.

An effective amount of a compound or pharmaceutical composition described herein will also provide a sufficient concentration of a compound of formula I when administered to a subject. A sufficient concentration is the concentration of the compound of formula I required to prevent or counter CMA-mediated or other diseases or conditions in a patient for which the compound of formula I is effective. This amount can be determined experimentally (e.g., by measuring the blood concentration of the compound) or theoretically by calculating bioavailability.

The method of treatment comprises providing a dose of a compound of formula I to a subject or patient. Dosage levels of about 0.1mg to about 140mg of each compound per kilogram of body weight per day can be used to treat the conditions indicated above (about 0.5mg to about 7g per patient per day). The amount of compound that can be combined with the carrier material to produce a single dosage form will vary depending upon the subject being treated and the particular mode of administration. Dosage unit forms will typically contain between about 1mg to about 500mg of each active compound. In certain embodiments, 25mg to 500mg or 25mg to 200mg of a compound of formula I is provided to the patient per day. The frequency of administration may also vary depending on the compound used and the particular disease being treated. However, for the treatment of most diseases and conditions, a dosage regimen of 4 times per day or less may be used, and in certain embodiments, a dosage regimen of 1 or 2 times per day is used.

It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, health, sex, diet, time of administration, route and rate of excretion, drug combination and the severity of the particular disease undergoing therapy.

In one embodiment, the present invention provides a method of treating a lysosomal storage disease in a patient identified as in need of such treatment, comprising providing to the patient an effective amount of a compound of formula I. The compounds of formula I provided herein may be administered alone as the sole active agent or in combination with one or more other active agents.

Examples

Examples 1-3 provide detailed synthetic methods for representative compounds. The remaining compounds of the present disclosure can be prepared by these methods using variations in starting materials and reaction conditions that will be apparent to those of ordinary skill in the art of organic chemical synthesis.

Example 1.Synthesis of N- (4- (6-chloroquinoxalin-2-yl) phenyl) acetamide (CA77.1)

In a dry and argon purged 50mL round bottom flask, 2, 6-dichloroquinoxaline (250mg, 1.25mmol), (4-acetamidophenyl) boronic acid (293mg, 1.64mmol, 1.30 equivalents) and potassium carbonate (1.0M; 1.25mL, 1.25mmol, 1.00 equivalents) were dissolved in dioxane (8.40 mL). The mixture was degassed (argon sparge for 20 minutes). Palladium four (73mg, 63. mu. mol, 5.0 mol%) was then added. The flask was purged with more argon and a reflux condenser was placed on top. The mixture was heated at 100 ℃ overnight under an argon atmosphere. TLC analysis indicated complete conversion. Filtering the mixture; the resulting solid was purified using combiFlash (MeOH in DCM gradient 0-10%). The product was recrystallized using a mixture of dichloromethane and methanol.

TLC:R_f0.16 (CH with 2% MeOH)₂Cl₂)。¹H-NMR(600MHz,dmso-d₆):10.20(s,1H),9.58(s,1H),8.32–8.31(m,2H),8.18(d,J＝2.4Hz,1H),8.13(d,J＝8.9Hz,1H),7.89(dd,J＝8.9,2.4Hz,1H),7.82(d,J＝8.8Hz,2H),2.11(s,3H)。¹³C-NMR(151MHz,CDCl₃) Delta 168.6,150.9,144.5,141.6,141.0,140.1,133.5,131.0,130.8,129.9,128.1,127.5,119.0, 24.1. HRMS (for C)₁₆H₁₂ClN₃O, M + H) calculated: 298.0742, found: 298.0743.

example 2.3- ([1,1' -Biphenyl)]-4-yl) -7-chloro-2H-benzo [ b][1,4]Synthesis of oxazines (CA39)

To acetonitrile (40mL) containing 2-amino-5-chlorophenol (1g, 6.96mmol) was added K₂CO₃(1.92g, 13.92 mmol). 1- ([1,1' -biphenyl) is reacted at room temperature]-4-yl) -2-bromoethan-1-one (2.15g, 8.36mmol) in acetonitrile (20mL) was added dropwise to this mixture. The reaction mixture was then stirred at room temperature overnight. The solvent was then evaporated and the residue was dissolved in dichloromethane (20 mL). The organic layer was washed with water, brine, and over Na₂SO₄And (5) drying. The desired compound was isolated by silica gel chromatography (hexane/ethyl acetate: 15/1). Recrystallization from isopropanol gave a pale yellow powder (a-1, CA39, 1.2g, 53.9%).

¹H-NMR(300M,CDCl₃)δppm:8.03(d,J＝0.03Hz,2H),7.75(d,J＝0.02Hz,2H),7.68(d,J＝0.02Hz,2H),7.48-7.53(m,2H),7.37-7.45(m,2H),7.01-7.05(dd,J＝0.01Hz,0.03Hz,1H),6.96(d,J＝0.01Hz,1H),5.13(s,2H)。¹³C-NMR(151MHz,DMSO-d6)δppm:158.11,146.84,144.08,140.00,133.90,133.30,132.48,128.95x 2,128.51,128.05,127.44x 2,127.15x2,126.94x2,62.83。1HRMS：C₂₀H₁₄ClNO (M + H) calculated 320.0837, found: 320.0839.

example 3.2- ([1,1' -Biphenyl)]Synthesis of (E) -4-yl) -6-chloroquinoxaline (CA39.1)

With 2, 6-dichloroquinoxaline (200mg, 1mmol), (4-acetyl)Aminophenyl) boronic acid (1257.4mg, 1.3mmol) and K₂CO₃(276mg, 2mmol, 2N in water) was filled into a nitrogen purged vessel. The vessel was flushed a second time with nitrogen and tetrakis (triphenylphosphine) palladium (0) (115.6mg, 0.1mmol) was added. Solvent dioxane (10mL) was then added and the reaction was degassed and blanketed with nitrogen and stirred at 110 ℃ overnight. The solvent was then evaporated and the residue was loaded onto silica gel for purification (hexane/acetone: 5/1-2/1). The resulting crude product was then recrystallized from hot ethanol to yield a light yellow powder (CA39.1, 181.3mg, 57%).

¹H-NMR(300M,CDCl₃)δppm:9.41(s,1H),8.33(d,J＝0.03Hz,2H),8.15(d,J＝0.03Hz,2H),7.85(d,J＝0.03Hz,2H),7.75-7.78(dd,J₁＝0.03Hz,J₂＝0.01Hz,2H),7.72(d,J＝0.02Hz,2H),7.50-7.55(m,2H),7.46(d,J＝0.03Hz,1H)。¹³C-NMR(75MHz,CDCl₃)δppm:151.60,144.14,143.31,141.91,141.00,140.21,135.30,135.25,131.44,130.91,129.03x2,128.18,127.99x2,127.98x2,127.24x2。HRMS：C₂₀H₁₃ClN₂Calcd for (M + H) 317.0840, found: 317.0839.

example 4Measurement of CMA Activity in vitro

The light-activatable CMA reporter gene assay was constructed by inserting a 21 amino acid sequence of ribonuclease A bearing a CMA targeting motif into the N-terminal multiple cloning site of the light-activatable protein mCherry1 or the light-switchable protein Dendra 2.

NIH 3T3 fibroblasts were stably transduced with a photoconversion CMA reporter gene, photoswitched by exposing KFERQ-Dendra to 3.5MA (constant current) LED (Norlux, 405nm) for 10 min and fixed in 3% formaldehyde at the desired time. Test cells were exposed to the indicated concentrations of compound for 12 hours (fig. 3A) or 24 hours (fig. 3B). The cells were imaged using a high content microscope (Operetta, Perkin Elmer) or by capturing images with an Axiovert 200 fluorescence microscope (Zeiss) with an apotome and equipped with a 63 × 1.4NA oil objective and a set of filters of red (excitation 570/30nm, emission 615/30nm), cyan (excitation 365/50nm and emission 530/45nm) and green (excitation 475/40nm and emission 535/45nm) (Chroma corp)). After optical sectioning by apotome, images were acquired with a high resolution CCD camera. CMA activity was measured as the average number of fluorescent spots (CMA-active lysosomes) per cell. Values are expressed relative to values in untreated cells assigned an arbitrary value of 1 and are the average of >2,500 cells counted under each condition. In all cases, the s.d. < 0.01% mean. Table 2 provides a comparison of compound CA39.1 and its comparative example CA39 and CA77.01 and its comparative example CA77.

Example 5Pharmacokinetic comparison of CA77 and CA77.1

All Animal work was approved and performed according to the guidelines set by the Committee for Animal protection and Use of the Albert Einstein College of Medicine Institutional Animal Care and Use Committee.

ICR (CD-1) male mice were fasted for at least three hours prior to the study and were given ad libitum access to water. Animals were housed in a controlled environment and under target conditions: the temperature is 18 to 29 ℃ and the relative humidity is 30 to 70%. Temperature and relative humidity were monitored daily. An electronic time-controlled lighting system is used to provide a 12-hour light/12-hour dark cycle. 3 mice per indicated time point. ICR (CD-1) mice were administered CA77 intravenously at 1mg/kg and orally at 30mg/kg or CA77.1 intravenously at 1mg/kg (FIG. 2A) and orally at 10mg/kg (FIG. 2B). Three (3) mice were included in each dose and time group. Mice were sacrificed and plasma and brain were obtained at 0.083, 0.25, 0.50, 1.0, 2.0, 4.0, 8.0 and 24.0 hours post-administration and drug concentrations were determined using LC-MS/MS. Brains were removed and homogenized in cold Phosphate Buffered Saline (PBS) containing 5% w/v BSA using a tissue homogenizer. Aliquots of 100 microliters of brain sample were dispensed into glass culture tubes and mixed with ethyl acetate (800 μ l), vortexed, and centrifuged. The organic layer was transferred to a fresh culture tube, dried under nitrogen, and reconstituted in the mobile phase for quantification. Pharmacokinetic parameters were determined by standard methods using Phoenix WinNonlin 6.3 software.

Tables 3A and 3B provide the concentration of CA77 in ICR (CD-1) mouse plasma (3A) and mouse brain (3B) after intravenous administration of 1mg/kg CA77. Table 3C provides the brain/plasma ratio of CA77 after intravenous administration of 1mg/kg CA77. Plasma and brain pharmacokinetic parameters for CA77 are provided in table 4. The shaded cells indicate data that is not included in the statistical information due to the abnormal value. BLQ is below the quantitation limit.

Tables 5A and 5B provide the concentration of CA77.1 in ICR (CD-1) mouse plasma (5A) and mouse brain (5B) after intravenous administration of 1mg/kg CA 77.1. Table 5C provides the brain/plasma ratio of CA77.1 after intravenous administration of 1mg/kg CA 77.1. Plasma and brain pharmacokinetic parameters for CA77.1 are provided in table 6. A cell contains data not present in the statistical information.

Example 6.Metabolic stability of human, rat and mouse microsomes.

Microsomal stability was determined in human, rat and mouse liver microsomes. The compound at a final concentration of 3. mu.M was incubated with 0.5mg/mL microsomal protein and 1mM NADPH for 0, 5, 15, 30 and 60 minutes. As a negative control, the test compound was incubated with microsomes in the absence of NADPH. The sample was quenched with methanol and centrifuged at 2500rpm for 25 minutes to precipitate the protein. The supernatant was analyzed by LC-MS/MS (N ═ 3). The ln peak area ratio (compound peak area/internal standard peak area) was plotted versus time, and the gradient of the line determined the elimination rate constant [ k ═ 1 (slope)]. Root of herbaceous plantHalf-life (t) is calculated according to the following equation_1/2In minutes), incubation volume (V, in. mu.L/mg protein) and in vitro intrinsic Clearance (CL)_intIn microliters/min/mg protein):

half life (t)_1/2) (minute) ═ 0.693/k (1)

V (μ L/mg) ═ incubation volume (μ L)/protein under incubation (mg) (2)

Intrinsic Clearance (CL)_int) (microliter/min/mg protein) ═ V0.693/t_1/2 (3)

Table 7 provides a comparison of the stability of CA77 and CA77.1 in human microsomes. Table 8 provides a comparison of the stability of CA39 and CA39.1 in human microsomes. Testosterone, diclofenac (diclofenac) and propafenone (propafenone) were provided as controls. R²Is a correlation coefficient of a linear regression used to determine kinetic constants. T is_1/2Is half-life and CL_int(mic)Is the intrinsic clearance. CL_{int (liver)}＝CL_int(mic)Mg microsomal protein/g liver weight/kg body weight. For humans, the liver weight/kg body weight is 20 g/kg.