Neuroactive steroids and methods of use thereof
11104701 · 2021-08-31
Assignee
Inventors
- Gabriel Martinez Botella (Wayland, MA)
- Boyd L. Harrison (Princeton Junction, NJ)
- Albert Jean Robichaud (Boston, MA, US)
- Francesco G. Salituro (Marlborough, MA)
Cpc classification
A61P25/14
HUMAN NECESSITIES
A61P29/00
HUMAN NECESSITIES
A61P25/18
HUMAN NECESSITIES
A61P1/14
HUMAN NECESSITIES
A61P9/10
HUMAN NECESSITIES
C07J41/0061
CHEMISTRY; METALLURGY
A61P43/00
HUMAN NECESSITIES
C07J9/005
CHEMISTRY; METALLURGY
A61P25/28
HUMAN NECESSITIES
C07J9/00
CHEMISTRY; METALLURGY
International classification
Abstract
Compounds are provided according to Formula (I): ##STR00001##
and pharmaceutically acceptable salts thereof, and pharmaceutical compositions thereof; wherein R.sup.1, R.sup.2, R.sup.3a, R.sup.3b, R.sup.4, R.sup.5a, R.sup.5b, R.sup.6a, and R.sup.6b are as defined herein. Compounds of the present invention are contemplated useful for the prevention and treatment of a variety of CNS-related conditions.
Claims
1. A method for treating a CNS-related condition in a subject in need thereof, the method comprising administering to the subject an effective amount of a compound or pharmaceutically acceptable salt thereof, wherein the compound is selected from the group consisting of ##STR00139## where the absolute stereochemistry of the compound is as shown; ##STR00140## where the absolute stereochemistry of the compound is as shown; ##STR00141## where the absolute stereochemistry of the compound is as shown; ##STR00142## where the absolute stereochemistry of the compound is as shown; ##STR00143## where the absolute stereochemistry of the compound is as shown; ##STR00144## where the absolute stereochemistry of the compound is as shown; ##STR00145## where the absolute stereochemistry of the compound is as shown; ##STR00146## where the absolute stereochemistry of the compound is as shown; ##STR00147## where the absolute stereochemistry of the compound is as shown; and ##STR00148## where the absolute stereochemistry of the compound is as shown; and wherein the CNS-related condition is selected from the group consisting of: a) a cognitive disorder selected from the group consisting of dementia and Alzheimer's disease; and b) a movement disorder selected from the group consisting of Parkinson's disease and Huntington's disease.
2. The method of claim 1, wherein the compound is ##STR00149## where the absolute stereochemistry of the compound is as shown.
3. The method of claim 1, wherein the compound is ##STR00150## where the absolute stereochemistry of the compound is as shown.
4. The method of claim 1, wherein the compound is ##STR00151## where the absolute stereochemistry of the compound is as shown.
5. The method of claim 1, wherein the compound is ##STR00152## where the absolute stereochemistry of the compound is as shown.
6. The method of claim 1, wherein the compound is ##STR00153## where the absolute stereochemistry of the compound is as shown.
7. The method of claim 1, wherein the compound is ##STR00154## where the absolute stereochemistry of the compound is as shown.
8. The method of claim 1, wherein the compound is ##STR00155## where the absolute stereochemistry of the compound is as shown.
9. The method of claim 1, wherein the compound is ##STR00156## where the absolute stereochemistry of the compound is as shown.
10. The method of claim 1, wherein the compound is ##STR00157## where the absolute stereochemistry of the compound is as shown.
11. The method of claim 1, wherein the compound is ##STR00158## where the absolute stereochemistry of the compound is as shown.
12. The method of claim 1, wherein the compound is a pharmaceutically acceptable salt of ##STR00159## where the absolute stereochemistry of the compound is as shown.
13. The method of claim 1, wherein the compound is a pharmaceutically acceptable salt of ##STR00160## where the absolute stereochemistry of the compound is as shown.
14. The method of claim 1, wherein the compound is a pharmaceutically acceptable salt of ##STR00161## where the absolute stereochemistry of the compound is as shown.
15. The method of claim 1, wherein the compound is a pharmaceutically acceptable salt of ##STR00162## where the absolute stereochemistry of the compound is as shown.
16. The method of claim 1, wherein the compound is a pharmaceutically acceptable salt of ##STR00163## where the absolute stereochemistry of the compound is as shown.
17. The method of claim 1, wherein the compound is a pharmaceutically acceptable salt of ##STR00164## where the absolute stereochemistry of the compound is as shown.
18. The method of claim 1, wherein the compound is a pharmaceutically acceptable salt of ##STR00165## where the absolute stereochemistry of the compound is as shown.
19. The method of claim 1, wherein the compound is a pharmaceutically acceptable salt of ##STR00166## where the absolute stereochemistry of the compound is as shown.
20. The method of claim 1, wherein the compound is a pharmaceutically acceptable salt of ##STR00167## where the absolute stereochemistry of the compound is as shown.
21. The method of claim 1, wherein the compound is a pharmaceutically acceptable salt of ##STR00168## where the absolute stereochemistry of the compound is as shown.
22. The method of claim 1, wherein the CNS-related condition is Alzheimer's disease.
23. The method of claim 1, wherein the CNS-related condition is Parkinson's disease.
24. The method of claim 1, wherein the CNS-related condition is Huntington's disease.
Description
DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION
(1) The inventors of the present invention, during an on-going exploration of Org-1 analogs for NMDA modulation, a portion of which is described in PCT/US2012/054261, incorporated herein by reference, discovered several specific combination of elements which provides NMDA modulators with comparatively superior properties. For example, as shown in Table 1, compounds bearing a beta-hydrogen at C.sub.5 are disfavored compared to compounds bearing either alpha-hydrogen C.sub.5 or double bond across C.sub.5-C.sub.6 due to loss of potentiation of the NMDA receptor. The removal of the methyl at C.sub.21 also results in significant loss of NMDA potentiation. Disubstitution at C.sub.3 is expected to increase metabolic stability of these compounds and is thus a preferred feature of the invention. Fluorination on the C.sub.17 side chain has been shown to improve potency and limit maximum potentiation of the NMDA receptor when tested as high as 1 μM concentration of compound. A secondary or tertiary terminal alcohol on the C.sub.17 side chain has been shown to improve potency and limit maximum potentiation of the NMDA receptor when tested as high as 1 μM concentration of compound, and is thus a preferred feature of the invention, with a preference for bulkier groups at the terminating end containing 2-3 carbons, or a group comprising fluorine substitution. Such properties are expected limit the risk of inducing glutamate driven neurotoxicity relative to compounds that achieve a greater maximum potentiation of the NMDA receptor. Compounds of the present invention encompass various combinations of these specified features to provide superior NMDA modulators.
(2) Compounds
(3) In one aspect, provided herein are compounds according to Formula (I):
(4) ##STR00008##
and pharmaceutically acceptable salts thereof;
(5) R.sup.1 is substituted or unsubstituted aliphatic;
(6) R.sup.2 is hydrogen, halogen, substituted or unsubstituted C.sub.1-6alkyl, substituted or unsubstituted cyclopropyl, or —OR.sup.A2, wherein R.sup.A2 is hydrogen or substituted or unsubstituted alkyl;
(7) R.sup.3a is hydrogen or —OR.sup.A3, wherein R.sup.A3 is hydrogen or substituted or unsubstituted alkyl, and R.sup.3b is hydrogen; or R.sup.3a and R.sup.3b are joined to form an oxo (═O) group;
(8) R.sup.4 is hydrogen, substituted or unsubstituted alkyl, or halogen;
(9) X is —C(R.sup.X).sub.2— or —O—, wherein R.sup.X is hydrogen or fluorine, or one R.sup.X group and R.sup.5b are joined to form a double bond;
(10) each instance of R.sup.5a and R.sup.5b is independently hydrogen or fluorine;
(11) R.sup.6a is a non-hydrogen group selected from the group consisting of substituted and unsubstituted alkyl, substituted and unsubstituted alkenyl, substituted and unsubstituted alkynyl, substituted and unsubstituted carbocyclyl, substituted and unsubstituted heterocyclyl, substituted and unsubstituted aryl, and substituted and unsubstituted heteroaryl group, wherein the non-hydrogen group is optionally substituted with fluorine; and
(12) R.sup.6b is hydrogen or a substituted or unsubstituted alkyl group optionally substituted with fluorine;
(13) represents a single or double bond, provided if a single bond is present, then the hydrogen at C5 is in the alpha configuration;
(14) and further provided that:
(15) (1) at least one of R.sup.X, R.sup.5a, and R.sup.5b is fluorine; or
(16) (2) at least one of R.sup.6a and R.sup.6b is a non-hydrogen group substituted with a fluorine; or
(17) (3) R.sup.6a is a non-hydrogen group comprising between two and ten carbon atoms.
(18) As generally described herein, compounds wherein the hydrogen at C.sub.5 is provided in the beta configuration demonstrate loss of NMDA potentiation compared to compounds wherein the hydrogen at C.sub.5 is alpha, or wherein a double bond is present at C.sub.5-C.sub.6. Thus, the compound of Formula (I) encompasses only compounds of Formula (I-A) and (I-B):
(19) ##STR00009##
and pharmaceutically acceptable salts thereof.
Group R.sup.1
(20) As generally defined herein, R.sup.1 is substituted or unsubstituted alphatic, i.e., substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, or substituted or unsubstituted carbocyclyl.
(21) In certain embodiments, R.sup.1 is substituted or unsubstituted alkyl, e.g., substituted or unsubstituted C.sub.1-6alkyl, substituted or unsubstituted C.sub.1-2alkyl, substituted or unsubstituted C.sub.2-3alkyl, substituted or unsubstituted C.sub.3-4alkyl, substituted or unsubstituted C.sub.4-5alkyl, or substituted or unsubstituted C.sub.5-6alkyl. Exemplary R.sup.1 C.sub.1-6alkyl groups include, but are not limited to, substituted or unsubstituted methyl (C.sub.1), ethyl (C.sub.2), n-propyl (C.sub.3), isopropyl (C.sub.3), n-butyl (C.sub.4), tert-butyl (C.sub.4), sec-butyl (C.sub.4), iso-butyl (C.sub.4), n-pentyl (C.sub.5), 3-pentanyl (C.sub.5), amyl (C.sub.5), neopentyl (C.sub.5), 3-methyl-2-butanyl (C.sub.5), tertiary amyl (C.sub.5), n-hexyl (C.sub.6), C.sub.1-6 alkyl substituted with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more fluoro groups (e.g., —CF.sub.3, —CH.sub.2F, —CHF.sub.2, difluoroethyl, and 2,2,2-trifluoro-1,1-dimethyl-ethyl), C.sub.1-6 alkyl substituted with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more chloro groups (e.g., —CH.sub.2C1, —CHCl.sub.2), and C.sub.1-6 alkyl substituted with alkoxy groups (e.g., —CH.sub.2OCH.sub.3, —CH.sub.2OCH.sub.2CH.sub.3, —CH.sub.2O-cyclopropyl). In certain embodiments, R.sup.1 is substituted alkyl, e.g., R.sup.1 is haloalkyl, alkoxyalkyl, or aminoalkyl. In certain embodiments, R.sup.1 is Me, Et, n-Pr, n-Bu, i-Bu, fluoromethyl, chloromethyl, difluoromethyl, trifluoromethyl, trifluoroethyl, difluoroethyl, 2,2,2-trifluoro-1,1-dimethyl-ethyl, methoxymethyl, methoxyethyl, or ethoxymethyl.
(22) In certain embodiments, R.sup.1 is unsubstituted C.sub.1-3 alkyl, e.g., R.sup.1 is —CH.sub.3, —CH.sub.2CH.sub.3, or —CH.sub.2CH.sub.2CH.sub.3.
(23) In certain embodiments, R.sup.1 is alkyl substituted with one or more fluorine atoms; e.g., R.sup.1 is —CH.sub.2F, —CHF.sub.2, or —CF.sub.3.
(24) In certain embodiments, R.sup.1 is alkyl substituted with one or more —OR.sup.A1 groups, wherein R.sup.A1 is hydrogen or substituted or unsubstitued alkyl. In certain embodiments, R.sup.1 is —CH.sub.2OR.sup.A1, e.g., wherein R.sup.A1 is hydrogen, —CH.sub.3, —CH.sub.2CH.sub.3, or —CH.sub.2CH.sub.2CH.sub.3.
(25) In certain embodiments, R.sup.1 is substituted or unsubstituted alkenyl, e.g., substituted or unsubstituted C.sub.2-6alkenyl, substituted or unsubstituted C.sub.2-3alkenyl, substituted or unsubstituted C.sub.3-4alkenyl, substituted or unsubstituted C.sub.4-5alkenyl, or substituted or unsubstituted C.sub.5-6alkenyl. In certain embodiments, R.sup.1 is ethenyl (C.sub.2), propenyl (C.sub.3), or butenyl (C.sub.4), unsubstituted or substituted with one or more substituents selected from the group consisting of alkyl, halo, haloalkyl, alkoxyalkyl, or hydroxyl. In certain embodiments, R.sup.1 is ethenyl, propenyl, or butenyl, unsubstituted or substituted with alkyl, halo, haloalkyl, alkoxyalkyl, or hydroxy. In certain embodiments, R.sup.1 is ethenyl.
(26) In certain embodiments, R.sup.1 is substituted or unsubstituted alkynyl, e.g., substituted or unsubstituted C.sub.2-6alkynyl, substituted or unsubstituted C.sub.2-3alkynyl, substituted or unsubstituted C.sub.3-4alkynyl, substituted or unsubstituted C.sub.4-5alkynyl, or substituted or unsubstituted C.sub.5-6alkynyl. Exemplary substituted or unsubstituted R.sup.1 alkynyl groups include, but are not limited to, ethynyl, propynyl, or butynyl, unsubstituted or substituted with alkyl, halo, haloalkyl (e.g., CF.sub.3), alkoxyalkyl, cycloalkyl (e.g., cyclopropyl or cyclobutyl), or hydroxyl. In certain embodiments, R.sup.1 is selected from the group consisting of trifluoroethynyl, cyclopropylethynyl, cyclobutylethynyl, and propynyl, fluoropropynyl, and chloroethynyl. In certain embodiments, R.sup.1 is ethynyl (C.sub.2), propynyl (C.sub.3), or butynyl (C.sub.4), unsubstituted or substituted with one or more substituents selected from the group consisting of substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted carbocyclyl, and substituted or unsubstituted heterocyclyl. In certain embodiments, R.sup.1 is ethynyl (C.sub.2), propynyl (C.sub.3), or butynyl (C.sub.4) substituted with substituted phenyl. In certain embodiments, the phenyl substitutent is further substituted with one or more substituents selected from the group consisting of halo, alkyl, trifluoroalkyl, alkoxy, acyl, amino or amido. In certain embodiments, R.sup.1 is ethynyl (C.sub.2), propynyl (C.sub.3), or butynyl (C.sub.4) substituted with substituted or unsubstituted pyrrolyl, imidazolyl, pyrazolyl, oxazoyl, thiazolyl, isoxazoyl, 1,2,3-triazolyl, 1,2,4-triazolyl, oxadiazolyl, thiadiazolyl, or tetrazolyl.
(27) In certain embodiments, R.sup.1 is ethynyl, propynyl, or butynyl, unsubstituted or substituted with alkyl, halo, haloalkyl, alkoxyalkyl, or hydroxyl. In certain embodiments, R.sup.1 is ethynyl or propynyl, substituted with substituted or unsubstituted aryl. In certain embodiments, R.sup.1 is ethynyl or propynyl, substituted with phenyl unsubstituted or substituted with halo, alkyl, alkoxy, haloalkyl, trihaloalkyl, or acyl. In certain embodiments, R.sup.1 is ethynyl or propynyl, substituted with substituted or unsubstituted carbocyclyl. In certain embodiments, R.sup.3a is ethynyl or propynyl, substituted with substituted or unsubstituted cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl. In certain embodiments, R.sup.1 is ethynyl or propynyl, substituted with substituted or unsubstituted heteroaryl. In certain embodiments, R.sup.1 is ethynyl or propynyl, substituted with substituted or unsubstituted pyridinyl, or pyrimidinyl. In certain embodiments, R.sup.1 is ethynyl or propynyl, substituted with substituted or unsubstituted pyrrolyl, imidazolyl, pyrazolyl, oxazoyl, thiazolyl, isoxazoyl, 1,2,3-triazolyl, 1,2,4-triazolyl, oxadiazolyl, thiadiazolyl, tetrazolyl. In certain embodiments, R.sup.1 is ethynyl or propynyl, substituted with substituted or unsubstituted heterocyclyl. In certain embodiments, R.sup.1 is ethynyl or propynyl, substituted with substituted or unsubstituted pyrrolidinyl, piperidinyl, piperazinyl, or mopholinyl. In certain embodiments, R.sup.1 is propynyl or butynyl, substituted with hydroxyl or alkoxy. In certain embodiments, R.sup.1 is propynyl or butynyl, substituted with methoxy or ethoxy. In certain embodiments, R.sup.1 is ethynyl or propynyl, substituted with chloro. In certain embodiments, R.sup.1 is ethynyl or propynyl, substituted with trifluoromethyl.
(28) In certain embodiments, R.sup.1 is substituted or unsubstituted carbocyclyl, e.g., substituted or unsubstituted C.sub.3-6carbocyclyl, substituted or unsubstituted C.sub.3-4carbocyclyl, substituted or unsubstituted C.sub.4-5 carbocyclyl, or substituted or unsubstituted C.sub.5-6 carbocyclyl. In certain embodiments, R.sup.1 is substituted or unsubstituted cyclopropyl or substituted or unsubstituted cyclobutyl.
(29) Groups R.sup.2, R.sup.3a, R.sup.3b, and R.sup.4
(30) As generally defined herein, R.sup.2 is hydrogen, halogen, substituted or unsubstituted C.sub.1-6alkyl, substituted or unsubstituted cyclopropyl, or —OR.sup.A2, wherein R.sup.A2 is hydrogen or substituted or unsubstituted alkyl. In certain embodiments, R.sup.2 is hydrogen. In certain embodiments, R.sup.2 is halogen, e.g., fluoro, chloro, bromo, or iodo. In certain embodiments, R.sup.2 is fluoro or chloro. In certain embodiments, R.sup.2 is substituted or unsubstituted C.sub.1-6alkyl, e.g., substituted or unsubstituted C.sub.1-2alkyl, substituted or unsubstituted C.sub.2-3alkyl, substituted or unsubstituted C.sub.3-4alkyl, substituted or unsubstituted C.sub.4-5alkyl, or substituted or unsubstituted C.sub.5-6 alkyl. In certain embodiments, R.sup.2 is —CH.sub.3, —CH.sub.2CH.sub.3, —CH.sub.2CH.sub.2CH.sub.3, or cyclopropyl. In certain embodiments, R.sup.2 is —OR.sup.A2. In certain embodiments, R.sup.A2 is hydrogen. In certain embodiments, R.sup.A2 is substituted or unsubstituted alkyl, e.g., substituted or unsubstituted C.sub.1-6alkyl, substituted or unsubstituted C.sub.1-2alkyl, substituted or unsubstituted C.sub.2-3alkyl, substituted or unsubstituted C.sub.3-4alkyl, substituted or unsubstituted C.sub.4-5alkyl, or substituted or unsubstituted C.sub.5-6alkyl. In certain embodiments, R.sup.A2 is hydrogen, —CH.sub.3, —CH.sub.2CH.sub.3, or —CH.sub.2CH.sub.2CH.sub.3, i.e., to provide a group R.sup.2 of formula —OH, —OCH.sub.3, —OCH.sub.2CH.sub.3, or —OCH.sub.2CH.sub.2CH.sub.3. In certain embodiments, R.sup.2 is a non-hydrogen substitutent in the alpha configuration. In certain embodiments, R.sup.2 is a non-hydrogen substituent in the beta configuration.
(31) As generally defined herein, R.sup.3a is hydrogen or —OR.sup.A3, wherein R.sup.A3 is hydrogen or substituted or unsubstituted alkyl, and R.sup.3b is hydrogen; or R.sup.3a and R.sup.3b are joined to form an oxo (═O) group.
(32) In certain embodiments, both R.sup.3a and R.sup.3b are both hydrogen.
(33) In certain embodiments, R.sup.3a and R.sup.3b are joined to form an oxo (═O) group.
(34) In certain embodiments, R.sup.3a is —OR.sup.A3 and R.sup.3b is hydrogen. In certain embodiments, wherein R.sup.3a is —OR.sup.A3, R.sup.3a is in the alpha or beta configuration. In certain embodiments, wherein R.sup.3a is —OR.sup.A3, R.sup.3a is in the alpha configuration. In certain embodiments, wherein R.sup.3a is —OR.sup.A3, R.sup.3a is in the beta configuration. In certain embodiments, R.sup.A3 is hydrogen. In certain embodiments, R.sup.A3 is substituted or unsubstituted alkyl, e.g., substituted or unsubstituted C.sub.1-6alkyl, substituted or unsubstituted C.sub.1-2alkyl, substituted or unsubstituted C.sub.2-3 alkyl, substituted or unsubstituted C.sub.3-4alkyl, substituted or unsubstituted C.sub.4-5alkyl, or substituted or unsubstituted C.sub.5-6alkyl. In certain embodiments, R.sup.A3 is hydrogen, —CH.sub.3, —CH.sub.2CH.sub.3, or —CH.sub.2CH.sub.2CH.sub.3, i.e., to provide a group R.sup.3a of formula —OH, —OCH.sub.3, —OCH.sub.2CH.sub.3, or —OCH.sub.2CH.sub.2CH.sub.3.
(35) As generally defined herein, R.sup.4 is hydrogen, substituted or unsubstituted alkyl, or halogen. In certain embodiments, R.sup.4 is hydrogen. In certain embodiments, R.sup.4 is halogen, e.g., fluoro. In certain embodiments, R.sup.4 is substituted or unsubstituted alkyl, e.g., substituted or unsubstituted C.sub.1-6alkyl, substituted or unsubstituted C.sub.1-2alkyl, substituted or unsubstituted C.sub.2-3alkyl, substituted or unsubstituted C.sub.3-4alkyl, substituted or unsubstituted C.sub.4-5alkyl, or substituted or unsubstituted C.sub.5-6alkyl. In certain embodiments, R.sup.4 is C.sub.1 alkyl, e.g., —CH.sub.3 or —CF.sub.3. In certain embodiments, R.sup.4 is hydrogen, —CH.sub.3, or —F. In certain embodiments, wherein represents a single bond, R.sup.4 is a non-hydrogen substitutent in the alpha configuration. In certain embodiments, wherein
represents a single bond, R.sup.4 is a non-hydrogen substituent in the beta configuration.
(36) Group X, R.sup.5a, R.sup.5b, R.sup.6a, and R.sup.6b
(37) As generally defined herein, X is —C(R.sup.X).sub.2— or —O—, wherein R.sup.X is hydrogen or fluorine, or one R.sup.X group and R.sup.5b are joined to form a double bond; each of R.sup.5a and R.sup.5b is independently hydrogen or fluorine; R.sup.6a is a non-hydrogen group selected from the group consisting of substituted and unsubstituted alkyl, substituted and unsubstituted alkenyl, substituted and unsubstituted alkynyl, substituted and unsubstituted carbocyclyl, substituted and unsubstituted heterocyclyl, substituted and unsubstituted aryl, and substituted and unsubstituted heteroaryl group, wherein the non-hydrogen group is optionally substituted with fluorine; and R.sup.6b is hydrogen or a substituted or unsubstituted alkyl group optionally substituted with fluorine; provided: (1) at least one of R.sup.X, R.sup.5a, and R.sup.5b is fluorine; or (2) at least one of R.sup.6a and R.sup.6b is a non-hydrogen group substituted with fluorine; or (3) R.sup.6a is a non-hydrogen group comprising between two and ten carbon atoms.
(38) In certain embodiments, X is —O—. In certain embodiments, X is —CH.sub.2—. In certain embodiments, X is —CF.sub.2—.
(39) In certain embodiments, at least one of R.sup.5a and R.sup.5b is hydrogen. In certain embodiments, at least one of R.sup.5a and R.sup.5b is fluorine. In certain embodiments, R.sup.5a and R.sup.5b are both hydrogen. In certain embodiments, R.sup.5a and R.sup.5b are both fluorine. In certain embodiments, R.sup.X and R.sup.5b are joined to form a double bond, e.g., cis or trans double bond.
(40) In certain embodiments, R.sup.6a is a non-hydrogen group, as described herein, which is not substituted with fluorine. In certain embodiments, R.sup.6a is substituted or unsubstituted alkyl (e.g., —CH.sub.3, —CH.sub.2CH.sub.3, —CH(CH.sub.3).sub.2), substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, or substituted or unsubstituted carbocyclyl (e.g., isopropanol). In certain embodiments, R.sup.6a is a non-hydrogen group, as described herein, which is substituted with fluorine.
(41) In certain embodiments, R.sup.6a is a non-hydrogen group, as described herein, and R.sup.6b is hydrogen. In certain embodiments, R.sup.6a is a non-hydrogen group, as described herein, and R.sup.6b is a substituted or unsubstituted alkyl group optionally substituted by fluorine. In certain embodiments, R.sup.6b is an alkyl group which is not substituted with fluorine. In certain embodiments, R.sup.6a is an alkyl group which is substituted with fluorine.
(42) In certain embodiments, R.sup.6b is hydrogen. In certain embodiments, R.sup.6b is substituted or unsubstituted alkyl, e.g., substituted or unsubstituted C.sub.1-6alkyl, substituted or unsubstituted C.sub.1-2alkyl, substituted or unsubstituted C.sub.2-3alkyl, substituted or unsubstituted C.sub.3-4alkyl, substituted or unsubstituted C.sub.4-5alkyl, or substituted or unsubstituted C.sub.5-6alkyl, optionally substituted by fluorine. In certain embodiments, R.sup.6b is C.sub.1 alkyl optionally substituted by fluorine, e.g., —CH.sub.3 or —CF.sub.3.
(43) In certain embodiments, R.sup.6a is substituted or unsubstituted alkyl, e.g., substituted or unsubstituted C.sub.1-6alkyl, substituted or unsubstituted C.sub.1-2alkyl, substituted or unsubstituted C.sub.2-3alkyl, substituted or unsubstituted C.sub.3-4alkyl, substituted or unsubstituted C.sub.4-5alkyl, or substituted or unsubstituted C.sub.5-6alkyl. Exemplary R.sup.6a C.sub.1-6alkyl groups include, but are not limited to, substituted or unsubstituted methyl (C.sub.1), substituted or unsubstituted ethyl (C.sub.2), substituted or unsubstituted n-propyl (C.sub.3), substituted or unsubstituted isopropyl (C.sub.3), substituted or unsubstituted n-butyl (C.sub.4), substituted or unsubstituted tert-butyl (C.sub.4), substituted or unsubstituted sec-butyl (C.sub.4), substituted or unsubstituted iso-butyl (C.sub.4), substituted or unsubstituted n-pentyl (C.sub.5), substituted or unsubstituted 3-pentanyl (C.sub.5), substituted or unsubstituted amyl (C.sub.5), substituted or unsubstituted neopentyl (C.sub.5), substituted or unsubstituted 3-methyl-2-butanyl (C.sub.5), substituted or unsubstituted tertiary amyl (C.sub.5), substituted or unsubstituted n-hexyl (C.sub.6). In certain embodiments, R.sup.6a is alkyl, as described above, substituted with one or more fluorines, e.g., 1, 2, 3, 4, or more fluorines. In certain embodiments, R.sup.6a is —CF.sub.3, —CH.sub.2F, —CHF.sub.2, difluoroethyl, or 2,2,2-trifluoro-1,1-dimethyl-ethyl). In certain embodiments, R.sup.6a is alkyl, as described above, substituted with one or more —OR.sup.A6 groups, wherein R.sup.A6 is hydrogen or substituted or unsubstitued alkyl. In certain embodiments, R.sup.6a is —CH.sub.2OR.sup.A6, —CH.sub.2CH.sub.2OR.sup.A6, or —CH.sub.2CH.sub.2CH.sub.2OR.sup.A6, e.g., —CH.sub.2OCH.sub.3, —CH.sub.2CH.sub.2OCH.sub.3, or —CH.sub.2CH.sub.2CH.sub.2OCH.sub.3.
(44) In certain embodiments, R.sup.6a is substituted or unsubstituted alkenyl, e.g., substituted or unsubstituted C.sub.2-6alkenyl, substituted or unsubstituted C.sub.2-3alkenyl, substituted or unsubstituted C.sub.3-4alkenyl, substituted or unsubstituted C.sub.4-5alkenyl, or substituted or unsubstituted C.sub.5-6alkenyl, optionally substituted with fluorine. In certain embodiments, R.sup.6a is substituted or unsubstituted vinyl (C.sub.2) or substituted or unsubstituted allyl (C.sub.3).
(45) In certain embodiments, R.sup.6a is substituted or unsubstituted alkynyl, e.g., substituted or unsubstituted C.sub.2-6alkynyl, substituted or unsubstituted C.sub.2-3alkynyl, substituted or unsubstituted C.sub.3-4alkynyl, substituted or unsubstituted C.sub.4-5alkynyl, or substituted or unsubstituted C.sub.5-6alkynyl, optionally substituted with fluorine. In certain embodiments, R.sup.6a is substituted or unsubstituted ethynyl (C.sub.2) or substituted or unsubstituted propargyl (C.sub.3).
(46) In certain embodiments, R.sup.6a is substituted or unsubstituted carbocyclyl, e.g., substituted or unsubstituted C.sub.3-6carbocyclyl, substituted or unsubstituted C.sub.3-4carbocyclyl, substituted or unsubstituted C.sub.4-5 carbocyclyl, or substituted or unsubstituted C.sub.5-6 carbocyclyl, optionally substituted with fluorine. In certain embodiments, R.sup.6a is substituted or unsubstituted cyclopropyl.
(47) In certain embodiments, R.sup.6a is substituted or unsubstituted heterocyclyl, e.g., substituted or unsubstituted C.sub.3-6 heterocyclyl, substituted or unsubstituted C.sub.3-4 heterocyclyl, substituted or unsubstituted C.sub.4-5 heterocyclyl, or substituted or unsubstituted C.sub.5-6 heterocyclyl, optionally substituted with fluorine.
(48) In certain embodiments, R.sup.6a is substituted or unsubstituted aryl, e.g., substituted or unsubstituted phenyl, optionally substituted with fluorine.
(49) In certain embodiments, R.sup.6a is substituted or unsubstituted heteroaryl, e.g., optionally substituted 5- to 6-membered heteroaryl, optionally substituted with fluorine.
(50) In certain embodiments, R.sup.6a is a non-hydrogen group comprising between two and ten carbon atoms, e.g., between two and nine, two and eight, two and seven, two and six, two and five, two and four, or two and three carbon atoms, inclusive. For example, in certain embodiments, R.sup.6a is substituted or unsubstituted C.sub.2-3 alkyl, substituted or unsubstituted C.sub.2-3 alkenyl, substituted or unsubstituted C.sub.2-3 alkynyl, or substituted or unsubstituted C.sub.3 carbocyclyl.
(51) In certain embodiments, wherein at least one of R.sup.X, R.sup.5a, and R.sup.5b is fluorine; or at least one of R.sup.6a and R.sup.6b is a non-hydrogen group substituted with fluorine; R.sup.6a is substituted or unsubstituted C.sub.1-3 alkyl, substituted or unsubstituted C.sub.1-3 alkenyl, substituted or unsubstituted C.sub.1-3 alkynyl, or substituted or unsubstituted C.sub.3 carbocyclyl.
(52) In certain embodiments, R.sup.6a and R.sup.6b are the same group. In certain embodiments, R.sup.6a and R.sup.6b are different groups, and the carbon to R.sup.6a is attached is in the (S) or (R) configuration. In certain embodiments, the carbon to which R.sup.6a is attached is in the (S) configuration. In certain embodiments, the carbon to which R.sup.6a is attached is in the (R) configuration. In certain embodiments, R.sup.6a is —CF.sub.3 and R.sup.6b is hydrogen or C.sub.1-4 alkyl. In certain embodiments, R.sup.6a is a non-hydrogen group substituted with fluorine, and R.sup.6b is —CH.sub.3. In certain embodiments, R.sup.6a is substituted with one or more —OR.sup.A6 groups, wherein R.sup.A6 is hydrogen or substituted or unsubstitued alkyl. In certain embodiments, R.sup.6a is a substituted or unsubstituted C.sub.2-4 alkyl, substituted or unsubstituted C.sub.2-3 alkenyl, substituted or unsubstituted C.sub.2-3 alkynyl, or substituted or unsubstituted C.sub.3 carbocyclyl, and R.sup.6b is —CH.sub.3. In certain embodiments, R.sup.6a is a unsubstituted C.sub.2-4 alkyl, unsubstituted C.sub.2-3 alkenyl, or unsubstituted C.sub.2-3 alkynyl, or unsubstituted C.sub.3 carbocyclyl, and R.sup.6b is —CH.sub.3. In certain embodiments, R.sup.6a is a non-hydrogen group substituted with fluorine, and R.sup.6b is —CH.sub.3.
(53) Various Combinations of Certain Embodiments
(54) Various combinations of certain embodiments are further contemplated herein.
(55) For example, in certain embodiments, wherein X is —CH.sub.2— and R.sup.5a and R.sup.5b are both hydrogen, provided is a compound of Formula (I-a):
(56) ##STR00010##
or a pharmaceutically acceptable salt thereof. In certain embodiments, R.sup.6a is a non-hydrogen group comprising between two and ten carbon atoms. In certain embodiments, at least one of R.sup.6a and R.sup.6b is a non-hydrogen group substituted with fluorine. In certain embodiments, the carbon to which R.sup.6a is attached is in the (S) configuration. In certain embodiments, the carbon to which R.sup.6a is attached is in the (R) configuration. In certain embodiments, R.sup.6a is methyl (C) optionally substituted with one or more fluorines, e.g., —CH.sub.3 or —CF.sub.3. In certain embodiments, R.sup.6a is substituted or unsubstituted ethyl (C.sub.2), substituted or unsubstituted n-propyl (C.sub.3), or substituted or unsubstituted isopropyl (C.sub.3). In certain embodiments, R.sup.6a is —CH.sub.2OR.sup.A6, —CH.sub.2CH.sub.2OR.sup.A6, or —CH.sub.2CH.sub.2CH.sub.2OR.sup.A6. In certain embodiments, R.sup.6a is substituted or unsubstituted vinyl (C.sub.2) or substituted or unsubstituted allyl (C.sub.3). In certain embodiments, R.sup.6a is substituted or unsubstituted ethynyl (C.sub.2) or substituted or unsubstituted propargyl (C.sub.3). In certain embodiments, R.sup.6a is substituted or unsubstituted cyclopropyl. In certain embodiments, R.sup.6b is hydrogen. In certain embodiments, R.sup.6b is —CH.sub.3 or —CF.sub.3. In certain embodiments, represents a single bond, and the hydrogen at C5 is alpha. In certain embodiments,
represents a double bond. In certain embodiments, R.sup.1 is —CH.sub.3 or —CH.sub.2CH.sub.3. In certain embodiments, R.sup.2 is hydrogen, —OH, —OCH.sub.3, —OCH.sub.2CH.sub.3, —OCH.sub.2CH.sub.2CH.sub.3, —CH.sub.3, —CH.sub.2CH.sub.3, —CH.sub.2CH.sub.2CH.sub.3, cyclopropyl, fluoro, or chloro. In certain embodiments, R.sup.2 is a non-hydrogen substitutent in the alpha configuration. In certain embodiments, R.sup.2 is a non-hydrogen substituent in the beta configuration. In certain embodiments, R.sup.3a and R.sup.3b are both hydrogen. In certain embodiments, R.sup.3a and R.sup.3b are joined to form ═O (oxo). In certain embodiments, R.sup.4 is hydrogen.
(57) In certain embodiments, wherein X is —CH.sub.2— and R.sup.5a and R.sup.5b are both fluorine, provided is a compound of Formula (I-b):
(58) ##STR00011##
or a pharmaceutically acceptable salt thereof. In certain embodiments, R.sup.6a is a non-hydrogen group comprising between two and ten carbon atoms. In certain embodiments, at least one of R.sup.6a and R.sup.6b is a non-hydrogen group substituted with fluorine. In certain embodiments, the carbon to which R.sup.6a is attached is in the (S) configuration. In certain embodiments, the carbon to which R.sup.6a is attached is in the (R) configuration. In certain embodiments, R.sup.6a is methyl (C.sub.1), optionally substituted with one or more fluorines, e.g., —CH.sub.3 or —CF.sub.3. In certain embodiments, R.sup.6a is substituted or unsubstituted ethyl (C.sub.2), substituted or unsubstituted n-propyl (C.sub.3), or substituted or unsubstituted isopropyl (C.sub.3). In certain embodiments, R.sup.6a is —CH.sub.2OR.sup.A6, —CH.sub.2CH.sub.2OR.sup.A6, or —CH.sub.2CH.sub.2CH.sub.2OR.sup.A6. In certain embodiments, R.sup.6a is substituted or unsubstituted vinyl (C.sub.2) or substituted or unsubstituted allyl (C.sub.3). In certain embodiments, R.sup.6a is substituted or unsubstituted ethynyl (C.sub.2) or substituted or unsubstituted propargyl (C.sub.3). In certain embodiments, R.sup.6a is substituted or unsubstituted cyclopropyl. In certain embodiments, R.sup.6b is hydrogen. In certain embodiments, R.sup.6b is —CH.sub.3 or —CF.sub.3. In certain embodiments, represents a single bond, and the hydrogen at C5 is alpha. In certain embodiments,
represents a double bond. In certain embodiments, R.sup.1 is —CH.sub.3 or —CH.sub.2CH.sub.3 In certain embodiments, R.sup.2 is hydrogen, —OH, —OCH.sub.3, —OCH.sub.2CH.sub.3, —OCH.sub.2CH.sub.2CH.sub.3, —CH.sub.3, —CH.sub.2CH.sub.3, —CH.sub.2CH.sub.2CH.sub.3, cyclopropyl, fluoro, or chloro. In certain embodiments, R.sup.2 is a non-hydrogen substitutent in the alpha configuration. In certain embodiments, R.sup.2 is a non-hydrogen substituent in the beta configuration. In certain embodiments, R.sup.3a and R.sup.3b are both hydrogen. In certain embodiments, R.sup.3a and R.sup.3b are joined to form ═O (oxo). In certain embodiments, R.sup.4 is hydrogen.
(59) In certain embodiments, wherein X is —C(R.sup.X).sub.2— and one R.sup.X group and R.sup.5b are joined to form a trans double bond, provided is a compound of Formula (I-c):
(60) ##STR00012##
or a pharmaceutically acceptable salt thereof. In certain embodiments, R.sup.6a is a non-hydrogen group comprising between two and ten carbon atoms. In certain embodiments, at least one of R.sup.6a and R.sup.6b is a non-hydrogen group substituted with fluorine. In certain embodiments, the carbon to which R.sup.6a is attached is in the (S) configuration. In certain embodiments, the carbon to which R.sup.6a is attached is in the (R) configuration. In certain embodiments, R.sup.6a is methyl (C.sub.1) optionally substituted with one or more fluorines, e.g., —CH.sub.3 or —CF.sub.3. In certain embodiments, R.sup.6a is substituted or unsubstituted ethyl (C.sub.2), substituted or unsubstituted n-propyl (C.sub.3), or substituted or unsubstituted isopropyl (C.sub.3). In certain embodiments, R.sup.6a is —CH.sub.2OR.sup.A6, —CH.sub.2CH.sub.2OR.sup.A6, or —CH.sub.2CH.sub.2CH.sub.2OR.sup.A6. In certain embodiments, R.sup.6a is substituted or unsubstituted vinyl (C.sub.2) or substituted or unsubstituted allyl (C.sub.3). In certain embodiments, R.sup.6a is substituted or unsubstituted ethynyl (C.sub.2) or substituted or unsubstituted propargyl (C.sub.3). In certain embodiments, R.sup.6a is substituted or unsubstituted cyclopropyl. In certain embodiments, R.sup.6b is hydrogen. In certain embodiments, R.sup.6b is —CH.sub.3 or —CF.sub.3. In certain embodiments, represents a single bond, and the hydrogen at C5 is alpha. In certain embodiments,
represents a double bond. In certain embodiments, R.sup.1 is —CH.sub.3 or —CH.sub.2CH.sub.3. In certain embodiments, R.sup.2 is hydrogen, —OH, —OCH.sub.3, —OCH.sub.2CH.sub.3, —OCH.sub.2CH.sub.2CH.sub.3, —CH.sub.3, —CH.sub.2CH.sub.3, —CH.sub.2CH.sub.2CH.sub.3, cyclopropyl, fluoro, or chloro. In certain embodiments, R.sup.2 is a non-hydrogen substitutent in the alpha configuration. In certain embodiments, R.sup.2 is a non-hydrogen substituent in the beta configuration. In certain embodiments, R.sup.3a and R.sup.3b are both hydrogen. In certain embodiments, R.sup.3a and R.sup.3b are joined to form ═O (oxo). In certain embodiments, R.sup.4 is hydrogen.
(61) In certain embodiments, the compound of Formula (I) is selected from a compound of Formula (II):
(62) ##STR00013##
or a pharmaceutically acceptable salt thereof. In certain embodiments, R.sup.6a is a non-hydrogen group comprising between two and ten carbon atoms. In certain embodiments, at least one of R.sup.6a and R.sup.6b is a non-hydrogen group substituted with fluorine. In certain embodiments, the carbon to which R.sup.6a is attached is in the (S) configuration. In certain embodiments, the carbon to which R.sup.6a is attached is in the (R) configuration. In certain embodiments, R.sup.6a is methyl (C) optionally substituted with one or more fluorines, e.g., —CH.sub.3 or —CF.sub.3. In certain embodiments, R.sup.6a is substituted or unsubstituted ethyl (C.sub.2), substituted or unsubstituted n-propyl (C.sub.3), or substituted or unsubstituted isopropyl (C.sub.3). In certain embodiments, R.sup.6a is —CH.sub.2R.sup.A6, —CH.sub.2CH.sub.2OR.sup.A6, or —CH.sub.2CH.sub.2CH.sub.2OR.sup.A6. In certain embodiments, R.sup.6a is substituted or unsubstituted vinyl (C.sub.2) or substituted or unsubstituted allyl (C.sub.3). In certain embodiments, R.sup.6a is substituted or unsubstituted ethynyl (C.sub.2) or substituted or unsubstituted propargyl (C.sub.3). In certain embodiments, R.sup.6a is substituted or unsubstituted cyclopropyl. In certain embodiments, R.sup.6b is hydrogen. In certain embodiments, R.sup.6b is —CH.sub.3 or —CF.sub.3. In certain embodiments, represents a single bond, and the hydrogen at C5 is alpha. In certain embodiments,
represents a double bond. In certain embodiments, R.sup.1 is —CH.sub.3 or —CH.sub.2CH.sub.3.
(63) In certain embodiments, the compound of Formula (I) is selected from a compound of Formula (II-A):
(64) ##STR00014##
or a pharmaceutically acceptable salt thereof. In certain embodiments, R.sup.6a is a non-hydrogen group comprising between two and ten carbon atoms. In certain embodiments, at least one of R.sup.6a and R.sup.6b is a non-hydrogen group substituted with fluorine. In certain embodiments, the carbon to which R.sup.6a is attached is in the (S) configuration. In certain embodiments, the carbon to which R.sup.6a is attached is in the (R) configuration. In certain embodiments, R.sup.6a is methyl (C.sub.1) optionally substituted with one or more fluorines, e.g., —CH.sub.3 or —CF.sub.3. In certain embodiments, R.sup.6a is substituted or unsubstituted ethyl (C.sub.2), substituted or unsubstituted n-propyl (C.sub.3), or substituted or unsubstituted isopropyl (C.sub.3). In certain embodiments, R.sup.6a is —CH.sub.2OR.sup.A6, —CH.sub.2CH.sub.2OR.sup.A6, or —CH.sub.2CH.sub.2CH.sub.2OR.sup.A6. In certain embodiments, R.sup.6a is substituted or unsubstituted vinyl (C.sub.2) or substituted or unsubstituted allyl (C.sub.3). In certain embodiments, R.sup.6a is substituted or unsubstituted ethynyl (C.sub.2) or substituted or unsubstituted propargyl (C.sub.3). In certain embodiments, R.sup.6a is substituted or unsubstituted cyclopropyl. In certain embodiments, R.sup.6b is hydrogen. In certain embodiments, R.sup.6b is —CH.sub.3 or —CF.sub.3. In certain embodiments, R.sup.1 is —CH.sub.3 or —CH.sub.2CH.sub.3.
(65) In certain embodiments, the compound of Formula (I) is selected from a compound of Formula (II-B):
(66) ##STR00015##
or a pharmaceutically acceptable salt thereof. In certain embodiments, R.sup.6a is a non-hydrogen group comprising between two and ten carbon atoms. In certain embodiments, at least one of R.sup.6a and R.sup.6b is a non-hydrogen group substituted with fluorine. In certain embodiments, the carbon to which R.sup.6a is attached is in the (S) configuration. In certain embodiments, the carbon to which R.sup.6a is attached is in the (R) configuration. In certain embodiments, R.sup.6a is methyl (C.sub.1) optionally substituted with one or more fluorines, e.g., —CH.sub.3 or —CF.sub.3. In certain embodiments, R.sup.6a is substituted or unsubstituted ethyl (C.sub.2), substituted or unsubstituted n-propyl (C.sub.3), or substituted or unsubstituted isopropyl (C.sub.3). In certain embodiments, R.sup.6a is —CH.sub.2OR.sup.A6, —CH.sub.2CH.sub.2OR.sup.A6, or —CH.sub.2CH.sub.2CH.sub.2OR.sup.A6. In certain embodiments, R.sup.6a is substituted or unsubstituted vinyl (C.sub.2) or substituted or unsubstituted allyl (C.sub.3). In certain embodiments, R.sup.6a is substituted or unsubstituted ethynyl (C.sub.2) or substituted or unsubstituted propargyl (C.sub.3). In certain embodiments, R.sup.6a is substituted or unsubstituted cyclopropyl. In certain embodiments, R.sup.6b is hydrogen. In certain embodiments, R.sup.6b is —CH.sub.3 or —CF.sub.3. In certain embodiments, R.sup.1 is —CH.sub.3 or —CH.sub.2CH.sub.3.
(67) In certain embodiments, a compound of Formula (I) is selected from the group consisting of:
(68) ##STR00016## ##STR00017## ##STR00018## ##STR00019## ##STR00020## ##STR00021## ##STR00022## ##STR00023## ##STR00024## ##STR00025## ##STR00026## ##STR00027## ##STR00028## ##STR00029## ##STR00030## ##STR00031##
and pharmaceutically acceptable salts thereof.
Pharmaceutical Compositions
(69) In another aspect, the invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a effective amount of a compound of Formula (I).
(70) When employed as pharmaceuticals, the compounds provided herein are typically administered in the form of a pharmaceutical composition. Such compositions can be prepared in a manner well known in the pharmaceutical art and comprise at least one active compound.
(71) In one embodiment, with respect to the pharmaceutical composition, the carrier is a parenteral carrier, oral or topical carrier.
(72) The present invention also relates to a compound of Formula (I) or pharmaceutical composition thereof for use as a pharmaceutical or a medicament.
(73) Generally, the compounds provided herein are administered in a therapeutically effective amount. The amount of the compound actually administered will typically be determined by a physician, in the light of the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual compound administered, the age, weight, and response of the individual patient, the severity of the patient's symptoms, and the like.
(74) The pharmaceutical compositions provided herein can be administered by a variety of routes including oral, rectal, transdermal, subcutaneous, intravenous, intramuscular, and intranasal. Depending on the intended route of delivery, the compounds provided herein are preferably formulated as either injectable or oral compositions or as salves, as lotions or as patches all for transdermal administration.
(75) The compositions for oral administration can take the form of bulk liquid solutions or suspensions, or bulk powders. More commonly, however, the compositions are presented in unit dosage forms to facilitate accurate dosing. The term “unit dosage forms” refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient. Typical unit dosage forms include prefilled, premeasured ampules or syringes of the liquid compositions or pills, tablets, capsules or the like in the case of solid compositions. In such compositions, the compound is usually a minor component (from about 0.1 to about 50% by weight or preferably from about 1 to about 40% by weight) with the remainder being various vehicles or carriers and processing aids helpful for forming the desired dosing form.
(76) Liquid forms suitable for oral administration may include a suitable aqueous or nonaqueous vehicle with buffers, suspending and dispensing agents, colorants, flavors and the like. Solid forms may include, for example, any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
(77) Injectable compositions are typically based upon injectable sterile saline or phosphate-buffered saline or other injectable carriers known in the art. As before, the active compound in such compositions is typically a minor component, often being from about 0.05 to 10% by weight with the remainder being the injectable carrier and the like.
(78) Transdermal compositions are typically formulated as a topical ointment or cream containing the active ingredient(s), generally in an amount ranging from about 0.01 to about 20% by weight, preferably from about 0.1 to about 20% by weight, preferably from about 0.1 to about 10% by weight, and more preferably from about 0.5 to about 15% by weight. When formulated as a ointment, the active ingredients will typically be combined with either a paraffinic or a water-miscible ointment base. Alternatively, the active ingredients may be formulated in a cream with, for example an oil-in-water cream base. Such transdermal formulations are well-known in the art and generally include additional ingredients to enhance the dermal penetration of stability of the active ingredients or the formulation. All such known transdermal formulations and ingredients are included within the scope provided herein.
(79) The compounds provided herein can also be administered by a transdermal device. Accordingly, transdermal administration can be accomplished using a patch either of the reservoir or porous membrane type, or of a solid matrix variety.
(80) The above-described components for orally administrable, injectable or topically administrable compositions are merely representative. Other materials as well as processing techniques and the like are set forth in Part 8 of Remington's Pharmaceutical Sciences, 17th edition, 1985, Mack Publishing Company, Easton, Pa., which is incorporated herein by reference.
(81) The above-described components for orally administrable, injectable, or topically administrable compositions are merely representative. Other materials as well as processing techniques and the like are set forth in Part 8 of Remington's The Science and Practice of Pharmacy, 21st edition, 2005, Publisher: Lippincott Williams & Wilkins, which is incorporated herein by reference.
(82) The compounds of this invention can also be administered in sustained release forms or from sustained release drug delivery systems. A description of representative sustained release materials can be found in Remington's Pharmaceutical Sciences.
(83) The present invention also relates to the pharmaceutically acceptable formulations of a compound of Formula (I). In one embodiment, the formulation comprises water. In another embodiment, the formulation comprises a cyclodextrin derivative. The most common cyclodextrins are α—, β—and γ—cyclodextrins consisting of 6, 7 and 8 α-1, 4—linked glucose units, respectively, optionally comprising one or more substituents on the linked sugar moieties, which include, but are not limited to, methylated, hydroxyalkylated, acylated, and sulfoalkylether substitution. In certain embodiments, the cyclodextrin is a sulfoalkyl ether β—cyclodextrin, e.g., for example, sulfobutyl ether β—cyclodextrin, also known as CAPTISOL. See, e.g., U.S. Pat. No. 5,376,645. In certain embodiments, the formulation comprises hexapropyl-β-cyclodextrin. In a more particular embodiment, the formulation comprises hexapropyl-β-cyclodextrin (10-50% in water).
(84) The present invention also relates to the pharmaceutically acceptable acid addition salt of a compound of Formula (I). The acid which may be used to prepare the pharmaceutically acceptable salt is that which forms a non-toxic acid addition salt, i.e., a salt containing pharmacologically acceptable anions such as the hydrochloride, hydroiodide, hydrobromide, nitrate, sulfate, bisulfate, phosphate, acetate, lactate, citrate, tartrate, succinate, maleate, fumarate, benzoate, para-toluenesulfonate, and the like.
(85) The following formulation examples illustrate representative pharmaceutical compositions that may be prepared in accordance with this invention. The present invention, however, is not limited to the following pharmaceutical compositions.
(86) Exemplary Formulation 1—Tablets:
(87) A compound of Formula (I), or pharmaceutically acceptable salt thereof, may be admixed as a dry powder with a dry gelatin binder in an approximate 1:2 weight ratio. A minor amount of magnesium stearate is added as a lubricant. The mixture is formed into 240-270 mg tablets (80-90 mg of active compound per tablet) in a tablet press.
(88) Exemplary Formulation 2—Capsules:
(89) A compound of Formula (I), or pharmaceutically acceptable salt thereof, may be admixed as a dry powder with a starch diluent in an approximate 1:1 weight ratio. The mixture is filled into 250 mg capsules (125 mg of active compound per capsule).
(90) Exemplary Formulation 3—Liquid:
(91) A compound of Formula (I), or pharmaceutically acceptable salt thereof, (125 mg) may be admixed with sucrose (1.75 g) and xanthan gum (4 mg) and the resultant mixture may be blended, passed through a No. 10 mesh U.S. sieve, and then mixed with a previously made solution of microcrystalline cellulose and sodium carboxymethyl cellulose (11:89, 50 mg) in water. Sodium benzoate (10 mg), flavor, and color are diluted with water and added with stirring. Sufficient water may then be added to produce a total volume of 5 mL.
(92) Exemplary Formulation 4—Tablets:
(93) A compound of Formula (I), or pharmaceutically acceptable salt thereof, may be admixed as a dry powder with a dry gelatin binder in an approximate 1:2 weight ratio. A minor amount of magnesium stearate is added as a lubricant. The mixture is formed into 450-900 mg tablets (150-300 mg of active compound) in a tablet press.
(94) Exemplary Formulation 5—Injection:
(95) A compound of Formula (I), or pharmaceutically acceptable salt thereof, may be dissolved or suspended in a buffered sterile saline injectable aqueous medium to a concentration of approximately 5 mg/mL.
(96) Exemplary Formulation 6—Tablets:
(97) A compound of Formula (I), or pharmaceutically acceptable salt thereof, may be admixed as a dry powder with a dry gelatin binder in an approximate 1:2 weight ratio. A minor amount of magnesium stearate is added as a lubricant. The mixture is formed into 90-150 mg tablets (30-50 mg of active compound per tablet) in a tablet press.
(98) Exemplary Formulation 7—Tablets:
(99) v may be admixed as a dry powder with a dry gelatin binder in an approximate 1:2 weight ratio. A minor amount of magnesium stearate is added as a lubricant. The mixture is formed into 30-90 mg tablets (10-30 mg of active compound per tablet) in a tablet press.
(100) Exemplary Formulation 8—Tablets:
(101) A compound of Formula (I), or pharmaceutically acceptable salt thereof, may be admixed as a dry powder with a dry gelatin binder in an approximate 1:2 weight ratio. A minor amount of magnesium stearate is added as a lubricant. The mixture is formed into 0.3-30 mg tablets (0.1-10 mg of active compound per tablet) in a tablet press.
(102) Exemplary Formulation 9—Tablets:
(103) A compound of Formula (I), or pharmaceutically acceptable salt thereof, may be admixed as a dry powder with a dry gelatin binder in an approximate 1:2 weight ratio. A minor amount of magnesium stearate is added as a lubricant. The mixture is formed into 150-240 mg tablets (50-80 mg of active compound per tablet) in a tablet press.
(104) Exemplary Formulation 10—Tablets:
(105) A compound of Formula (I), or pharmaceutically acceptable salt thereof, may be admixed as a dry powder with a dry gelatin binder in an approximate 1:2 weight ratio. A minor amount of magnesium stearate is added as a lubricant. The mixture is formed into 270-450 mg tablets (90-150 mg of active compound per tablet) in a tablet press.
(106) Injection dose levels range from about 0.1 mg/kg/hour to at least 10 mg/kg/hour, all for from about 1 to about 120 hours and especially 24 to 96 hours. A preloading bolus of from about 0.1 mg/kg to about 10 mg/kg or more may also be administered to achieve adequate steady state levels. The maximum total dose is not expected to exceed about 2 g/day for a 40 to 80 kg human patient.
(107) For the prevention and/or treatment of long-term conditions the regimen for treatment usually stretches over many months or years so oral dosing is preferred for patient convenience and tolerance. With oral dosing, one to five and especially two to four and typically three oral doses per day are representative regimens. Using these dosing patterns, each dose provides from about 0.01 to about 20 mg/kg of the compound provided herein, with preferred doses each providing from about 0.1 to about 10 mg/kg, and especially about 1 to about 5 mg/kg.
(108) Transdermal doses are generally selected to provide similar or lower blood levels than are achieved using injection doses.
(109) When used to prevent the onset of a CNS-disorder, the compounds provided herein will be administered to a subject at risk for developing the condition, typically on the advice and under the supervision of a physician, at the dosage levels described above. Subjects at risk for developing a particular condition generally include those that have a family history of the condition, or those who have been identified by genetic testing or screening to be particularly susceptible to developing the condition.
(110) Methods of Treatment and Use
(111) Compounds of Formula (I), and pharmaceutically acceptable salts thereof, as described herein, are generally designed to modulate NMDA function, and therefore to act as neuroactive steroids for the treatment and prevention of CNS-related conditions in a subject. Modulation, as used herein, refers to the inhibition or potentiation of NMDA receptor function. In certain embodiments, the compound of Formula (I), or pharmaceutically acceptable salt thereof, may act as a negative allosteric modulator (NAM) of NMDA, and inhibit NMDA receptor function. In certain embodiments, the compound of Formula (I), or pharmaceutically acceptable salt thereof, may act as positive allosteric modulators (PAM) of NMDA, and potentiate NMDA receptor function.
(112) Exemplary CNS conditions related to NMDA-modulation include, but are not limited to, adjustment disorders, anxiety disorders (including obsessive-compulsive disorder, posttraumatic stress disorder, social phobia, generalized anxiety disorder), cognitive disorders (including Alzheimer's disease and other forms of dementia), dissociative disorders, eating disorders, mood disorders (including depression, bipolar disorder, and dysthymic disorder), schizophrenia or other psychotic disorders (including schizoaffective disorder), sleep disorders (including insomnia), substance abuse-related disorders, personality disorders (including obsessive-compulsive personality disorder), autism spectrum disorders (including those involving mutations to the Shank group of proteins), neurodevelopmental disorders (including Rett syndrome), pain (including acute and chronic pain), seizure disorders (including status epilepticus and monogenic forms of epilepsy such as Dravet's disease, and Tuberous Sclerosis Complex (TSC)), stroke, traumatic brain injury, movement disorders (including Huntington's disease and Parkinson's disease) and tinnitus. In certain embodiments, the compound of Formula (I), or pharmaceutically acceptable salt thereof, can be used to induce sedation or anesthesia. In certain embodiments, the compound of Formula (I), or pharmaceutically acceptable salt thereof, is useful in the treatment or prevention of adjustment disorders, anxiety disorders, cognitive disorders, dissociative disorders, eating disorders, mood disorders, schizophrenia or other psychotic disorders, sleep disorders, substance-related disorders, personality disorders, autism spectrum disorders, neurodevelopmental disorders, pain, seizure disorders, stroke, traumatic brain injury, movement disorders and tinnitus.
(113) In another aspect, provided is a method of treating or preventing brain excitability in a subject susceptible to or afflicted with a condition associated with brain excitability, comprising administering to the subject an effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof.
(114) In yet another aspect, the present invention provides a combination of a compound of Formula (I), or pharmaceutically acceptable salt thereof, and another pharmacologically active agent. The compounds provided herein can be administered as the sole active agent or they can be administered in combination with other agents. Administration in combination can proceed by any technique apparent to those of skill in the art including, for example, separate, sequential, concurrent and alternating administration.
EXAMPLES
(115) In order that the invention described herein may be more fully understood, the following examples are set forth. The synthetic and biological examples described in this application are offered to illustrate the compounds, pharmaceutical compositions and methods provided herein and are not to be construed in any way as limiting their scope.
(116) Materials and Methods
(117) The compounds provided herein can be prepared from readily available starting materials using the following general methods and procedures. It will be appreciated that where typical or preferred process conditions (i.e., reaction temperatures, times, mole ratios of reactants, solvents, pressures, etc.) are given, other process conditions can also be used unless otherwise stated. Optimum reaction conditions may vary with the particular reactants or solvent used, but such conditions can be determined by one skilled in the art by routine optimization.
(118) Additionally, as will be apparent to those skilled in the art, conventional protecting groups may be necessary to prevent certain functional groups from undergoing undesired reactions. The choice of a suitable protecting group for a particular functional group as well as suitable conditions for protection and deprotection are well known in the art. For example, numerous protecting groups, and their introduction and removal, are described in T. W. Greene and P. G. M. Wuts, Protecting Groups in Organic Synthesis, Second Edition, Wiley, New York, 1991, and references cited therein.
(119) The compounds provided herein may be isolated and purified by known standard procedures. Such procedures include (but are not limited to) recrystallization, column chromatography, HPLC, or supercritical fluid chromatography (SFC). The following schemes are presented with details as to the preparation of representative substituted biarylamides that have been listed herein. The compounds provided herein may be prepared from known or commercially available starting materials and reagents by one skilled in the art of organic synthesis. Exemplary chiral columns available for use in the separation/purification of the enantiomers/diastereomers provided herein include, but are not limited to, CHIRALPAK® AD-10, CHIRALCEL® OB, CHIRALCEL® OB-H, CHIRALCEL® OD, CHIRALCEL® OD-H, CHIRALCEL® OF, CHIRALCEL® OG, CHIRALCEL® OJ and CHIRALCEL® OK.
(120) General method for supercritical fluid chromatography (SFC): SFC purification was carried out using a Thar 200 preparative SFC instrument equipped with a ChiralPak AD-10 μM, 200×50 mm ID. The compounds were separated eluting with mixtures of carbon dioxide and methanol or ethanol (e.g., 20-35% methanol or ethanol and 0.1% ammonium hydroxide) at a flow rate of 55-200 mL/min and monitored at 220 nm wavelength.
(121) Single pure isomers were obtained after SFC chromatographic separation, yielding two isomers with a diasteriomeric ratio ≥95:5, as determined by SFC chromatography.
(122) The configuration of the steroid C-24 stereocenter of 1-13 and 1-14, and 2-20 and 2-21 isomers was determined by the Mosher Method (Dale, J. A., Dull, D. L., and Mosher, H. S. (1969) J. Org. Chem. 34, 2543). The C-24 configuration of subsequent derivatives that employed such intermediates, for example 1-15 and 1-17, were assigned accordingly.
(123) For all other single diastereomers, for which the C-24 stereocenter was not determined by the Mosher Method, the first eluting diastereomer from the SFC was tentatively assigned to be attached in the (R) configuration at C-24, whereas the second eluting diastereomer from the SFC was tentatively assigned to be attached in the (S) configuration at C-24. The assignments were not unambiguously confirmed by the Mosher Method or other techniques.
Example 1
(124) ##STR00032## ##STR00033##
(125) Preparation of Compound 1-2.
(126) To a solution of ketone 1-1 (50.0 g, 0.17 mol) and ethylene glycol (62 mL) in toluene (600 mL) was added p-toluenesulfonic acid (1.4 g, 7.28 mmol). The reaction mixture was refluxed overnight with a Dean-Stark trap. The mixture was cooled to room temperature, diluted with ethyl acetate (500 mL), and washed with saturated aqueous sodium bicarbonate (300 mL×2) and brine (300 mL×2). The organic phase was dried over sodium sulfate and concentrated in vacuum to afford crude product 1-2 (64.0 g, 100%) which was directly used in the next step without further purification. .sup.1H NMR: (400 MHz, CDCl3) δ 5.35 (d, J=5.6 Hz, 1H), 3.97-3.82 (m, 4H), 3.59-3.47 (m, 1H), 2.34-2.21 (m, 2H), 2.06-1.94 (m, 2H), 1.90-1.74 (m, 3H), 1.73-1.64 (m, 1H), 1.63-1.33 (m, 10H), 1.32-1.19 (m, 1H), 1.14-1.03 (m, 1H), 1.01 (s, 3H), 0.99-0.93 (m, 1H), 0.86 (s, 3H).
(127) Preparation of Compound 1-3.
(128) To a solution of compound 1-2 (32 g, 96 mmol) in dry CH.sub.2Cl.sub.2 (1200 mL) was added Dess-Martin reagent (81 g, 192 mmol) in portions at 0° C. Then the reaction mixture was stirred at room temperature for 3 h. TLC (petroleum ether:ethyl acetate=3:1) showed the starting material was consumed completely. The mixture was quenched with saturated aqueous NaHCO.sub.3/Na.sub.2S.sub.2O.sub.3=1:3 (1 L). The organic phase was washed with brine (500 mL) and dried over Na.sub.2SO.sub.4, and the solvent was evaporated to afford crude product 1-3 (33.0 g, 100%), which was directly used in the next step without further purification. 1H NMR: (400 MHz, CDCl3) δ 5.34 (d, J=5.2 Hz, 1H), 3.77-4.00 (m, 4H), 3.19-3.39 (m, 1H), 2.83 (dd, J=16.44, 2.13 Hz, 1H), 2.38-2.59 (m, 1H), 2.21-2.37 (m, 1H), 1.95-2.09 (m, 3H), 1.54-1.73 (m, 4H), 1.74-1.90 (m, 2H), 1.37-1.51 (m, 3H), 1.21-1.34 (m, 2H), 1.19 (s, 3H), 0.98-1.12 (m, 1H), 0.83-0.93 (m, 3H).
(129) Preparation of MAD.
(130) To a solution of 2,6-di-tert-butyl-4-methylphenol (40 g, 180 mmol) in toluene (200 mL) was added a solution of AlMe.sub.3 (45 mL, 90 mmol, 2 M in hexane) at room temperature. The resulting mixture was stirred at room temperature for 1 h and used as a solution of MAD in toluene in the next step without any purification.
(131) Preparation of Compound 1-4.
(132) To a solution of MAD (90 mmol, freshly prepared) in toluene (200 mL) was added dropwise a solution of compound 1-3 (10 g, 30 mmol) in toluene (80 mL) at −78° C. during a period of 1 h under nitrogen. Then the reaction mixture was stirred for 30 min, a solution of CH.sub.3MgBr (30 mL, 90 mmol, 1.0 M in toluene) was added dropwise at −78° C. The reaction mixture was warmed to −40° C. and stirred at this temperature for 3 h. TLC (petroleum ether:ethyl acetate=3:1) showed that the starting material was consumed completely. The mixture was poured into saturated aqueous NH.sub.4Cl solution (200 mL) and extracted with EtOAc (150 mL×2). The combined organic phases were dried over Na.sub.2SO.sub.4, and the solvent was evaporated to afford crude product, which was purified by column chromatography on silica gel (eluent: petroleum ether:ethyl acetate=15:1) to give compound 1-4 (4 g, 38%) as white powder. 1H NMR: (400 MHz, CDCl3) δ 5.30 (d, J=5.2 Hz, 1H), 3.75-4.04 (m, 4H), 2.42 (d, J=13.6 Hz, 1H), 1.88-2.12 (m, 3H), 1.73-1.86 (m, 2H), 1.64-1.72 (m, 2H), 1.52-1.63 (m, 4H), 1.35-1.51 (m, 4H), 1.19-1.32 (m, 1H), 1.12-1.18 (m, 1H), 1.10 (s, 3H), 0.99-1.03 (m, 3H), 0.92-0.98 (m, 1H), 0.86 (s, 3H).
(133) Preparation of Compound 1-5.
(134) To a solution of compound 1-4 (6.0 g, 17.3 mmol) in THF (200 mL) was added aqueous HCl solution (35 mL, 1 M) and acetone (35 mL). The reaction mixture was stirred for 20° C. at room temperature. TLC (petroleum ether:ethyl acetate=3:1) indicated that the reaction was complete. Then the reaction mixture was diluted with EtOAc (200 mL), washed with saturated aqueous NaHCO.sub.3 solution (200 mL), dried over Na.sub.2SO.sub.4 and evaporated under reduced pressure to give 1-5 (5.2 g, 99.2%). 1H NMR: (400 MHz, CDCl3) δ 5.27 (d, J=6.8 Hz, 1H), 2.45-2.35 (m, 2H), 2.09-1.84 (m, 4H), 1.82-1.57 (m, 6H), 1.50-1.35 (m, 4H), 1.26-1.08 (m, 4H), 1.05 (s, 3H), 0.95 (s, 3H), 0.86 (s, 3H).
(135) Preparation of Compound 1-6.
(136) To a solution of Ph.sub.3PEtBr (12.25 g, 33.00 mmol) in dry THF (15 mL) was added dropwise a solution of t-BuOK (3.70 g, 33.00 mmol) in dry THF (10 mL) under N.sub.2 at 0° C. The mixture was stirred at room temperature for 1.5 h. Then a solution of 1-5 (1.00 g, 3.31 mmol) in THF (10 mL) was added dropwise and the resulting mixture was stirred at 70° C. for 4 h. TLC (petroleum ether:ethyl acetate=3:1) indicated that the starting material was consumed completely. The reaction was quenched with saturated aqueous NH.sub.4Cl solution (50 mL) and extracted with EtOAc (30 mL×2). The combined organic phases were dried over Na.sub.2SO.sub.4 and concentrated in vacuum. The residue was purified by column chromatography on silica gel (eluent: petroleum ether:ethyl acetate=12:1) to give 1-6 (900 mg, 90.9%) as white powder. 1H NMR: (400 MHz, CDCl3) δ 5.32 (d, J=5.2 Hz, 1H), 5.15-5.12 (m, 1H), 2.44-2.30 (m, 3H), 2.29-2.21 (m, 1H), 2.05-1.97 (m, 2H), 1.81-1.45 (m, 14H), 1.30-1.15 (m, 3H), 1.12 (s, 3H), 1.02 (s, 3H), 0.95-1.01 (m, 1H), 0.90 (s, 3H).
(137) Preparation of Compound 1-7.
(138) To a solution of compound 1-6 (1.00 g, 3.20 mmol) and methyl propiolate (0.67 g, 8.00 mmol) in dry CH.sub.2Cl.sub.2 (15 mL) was added dropwise a solution of Et.sub.2AlCl (12.8 mL, 12.8 mmol, 1 M in toluene) with stirring at 0° C. Then the reaction was warmed to room temperature and stirred for 20 h. TLC (petroleum ether:ethyl acetate=5:1) indicated that the starting material was consumed completely. The mixture was quenched with saturated aqueous NaHCO.sub.3 solution (30 mL) and extracted with CH.sub.2Cl.sub.2 (30 mL×2). The combined organic phases were dried over Na.sub.2SO.sub.4 and concentrated in vacuum. The residue was purified by column chromatography on silica gel (eluent: petroleum ether:ethyl acetate=10:1) to give 1-7 (1.00 g, 78.7%) as white powder. 1H NMR: (400 MHz, CDCl3) δ 6.97-6.91 (m, 1H) 5.82 (d, J=16 Hz, 1H), 5.42-5.41 (m, 1H), 5.32 (d, J=5.2 Hz, 1H), 3.73 (s, 3H), 3.04-3.00 (m, 1H), 2.43 (d, J=12.8 Hz, 1H), 2.11-1.97 (m, 3H), 1.88-1.50 (m, 12H), 1.40-1.20 (m, 3H), 1.21-1.26 (m, 1H), 1.18 (d, J=6.78 Hz, 3H), 1.12 (s, 3H), 1.04 (s, 3H), 0.82 (s, 3H).
(139) Preparation of Compound 1-8.
(140) To a solution of compound 1-7 (1.75 g, 4.4 mmol) in dry THF (20 mL), DIBAL-H (1 M in THF, 22 mL, 22.0 mmol) was added dropwise at −78° C. under nitrogen. The reaction mixture was warmed to 30° C. and then stirred for 2 h at 30° C. The reaction was quenched with addition of H.sub.2O (2 mL), diluted with EtOAc (200 mL) and dried over anhydrous Na.sub.2SO.sub.4, filtered through a pad of celite and the pad was washed with EtOAc (50 mL×3). The combined filtrates were concentrated in vacuum to give the crude product 1-8 (1.6 g, 98%) which was directly used in the next step without further purification.
(141) Preparation of Compound 1-9.
(142) A mixture of 1-8 (1.6 g, 4.3 mmol) and MnO.sub.2 (7.5 g, 86.0 mmol) in CH.sub.2Cl.sub.2 (50 mL) was stirred at 30° C. for 20 h. The reaction mixture was filtered through a pad of celite and the pad was washed with CH.sub.2Cl.sub.2 (50 mL×3). The combined filtrates were concentrated to dryness to give the crude product 1-9 (1.3 g, 82%) which was directly used in the next step without purification. .sup.1H NMR: (400 MHz, CDCl3) δ 9.54 (d, J=7.6 Hz, 1H), 6.84-6.78 (dd, J.sub.1=15.6 Hz, J.sub.2=7.6 Hz, 1H), 5.54-5.49 (dd, J.sub.1=15.6 Hz, J.sub.2=7.6 Hz, 1H), 5.45-5.44 (m, 1H), 5.32 (d, J=5.2 Hz, 1H), 3.19-3.12 (m, 1H), 2.42 (d, J=12.8 Hz, 1H), 2.14-2.08 (m, 1H), 2.00-1.52 (m, 13H), 1.42-1.35 (m, 3H), 1.24 (d, J=6.8 Hz, 3H), 1.12 (s, 3H), 1.05 (s, 3H), 0.80 (s, 3H).
(143) Preparation of Compound 1-10.
(144) To a suspension of 1-9 (600 mg, 1.63 mmol) and CsF (120 mg, 0.82 mmol) in toluene/THF (18 mL, 8/1) was added TMSCF.sub.3 (2.4 mL, 16.3 mmol) and the mixture was stirred for 20° C. at room temperature under nitrogen. TLC (petroleum ether:ethyl acetate=3/1) showed the starting material was consumed completely. A solution of TBAF (6.8 mL, 1 M in THF) was added and the mixture was stirred for 4 h at room temperature. The mixture was diluted with MTBE (200 mL), washed with a saturated NaHCO.sub.3 solution (30 mL×3) and concentrated in vacuum. The residue was purified by column chromatography on silica gel (eluent: petroleum ether:ethyl acetate=12/1) to afford 1-10 (300 mg, 42%) as white solid. .sup.1H NMR: (400 MHz, CDCl3) δ 5.97-5.91 (dd, J.sub.1=15.6 Hz, J.sub.2=7.6 Hz, 1H), 5.54-5.49 (dd, J.sub.1=15.6 Hz, J.sub.2=6.8 Hz, 1H), 5.42-5.38 (m, 1H), 5.30 (d, J=5.2 Hz, 1H), 4.44-4.36 (m, 1H), 2.97-2.94 (m, 1H), 2.42 (d, J=12.0 Hz, 1H), 2.01-1.98 (m, 2H), 1.88-1.64 (m, 6H), 1.40-1.32 (m, 3H), 1.26-1.21 (m, 2H), 1.17 (d, J=6.8 Hz, 3H), 1.12 (s, 3H), 1.05 (s, 3H), 1.00-0.95 (m, 2H), 0.79 (s, 3H).
(145) Preparation of Compound 1-11.
(146) A mixture of 1-10 (40 mg, 0.09 mmol) and 5% Pd/C (10 mg) in EA (10 mL) was hydrogenated for 2 h at 30° C. under 1 atm of hydrogen pressure. The reaction mixture was filtered through a pad of celite and the pad was washed with EA (10 mL×3). The combined filtrates were concentrated. The residue was purified by column chromatography on silica gel (eluent: PE/EA=8/1) to afford 1-11 (20 mg, 50%) as white solid. .sup.1H NMR: (400 MHz, CDCl3) δ 5.31 (d, J=5.2 Hz, 1H), 3.87-3.86 (m, 1H), 2.42 (d, J=12.8 Hz, 1H), 2.15-2.12 (m, 1H), 2.05-1.96 (m, 3H), 1.86-1.41 (m, 16H), 1.38-1.11 (m, 5H), 1.11 (s, 3H), 1.08-1.04 (m, 1H), 1.01 (s, 3H), 0.95 (d, J=6.6 Hz, 3H), 0.69 (s, 3H).
(147) Preparation of Compound 1-13 and 1-14.
(148) 1-13 (120 mg, 40%) and 1-14 (120 mg, 40%) were obtained by SFC purification from 1-10 (300 mg, 0.814 mmol). The configuration of 1-13 and 1-14 was confirmed by Mosher method.
(149) Preparation of Compound 1-15.
(150) A mixture of 1-13 (120 mg, 0.27 mmol) and 5% Pd/C (20 mg) in EtOAc (10 mL) was hydrogenated for 20 h at room temperature under H.sub.2 (1 atm). The reaction mixture was filtered through a pad of celite and the pad was washed with EtOAc (10 mL×3). The combined filtrates were concentrated. The residue was purified by column chromatography on silica gel (eluent: petroleum ether:ethyl acetate=8/1) to afford 1-15 (70 mg, 59%) as white powder. .sup.1H NMR: (400 MHz, CDCl3) δ 5.30 (d, J=5.2 Hz, 1H), 4.00-3.90 (m, 1H), 2.42 (d, J=13.2 Hz, 1H), 2.02-1.29 (m, 18H), 1.28-1.08 (m, 6H), 1.03 (s, 3H), 1.02 (s, 3H), 0.97 (d, J=6.8 Hz, 3H), 0.73 (s, 3H).
(151) Preparation of Compound 1-17.
(152) A mixture of 1-14 (120 mg, 0.27 mmol) and 5% Pd/C (20 mg) in EtOAc (10 mL) was hydrogenated for 20 h at room temperature under H.sub.2 (1 atm). The reaction mixture was filtered through a pad of celite and the pad was washed with EtOAc (10 mL×3). The combined filtrates were concentrated. The residue was purified by column chromatography on silica gel (eluent: petroleum ether:ethyl acetate=8/1) to afford 1-17 (71 mg, 59%) as white powder. .sup.1H NMR: (400 MHz, CDCl3) δ 5.27 (d, J=5.6 Hz, 1H), 4.00-3.90 (m, 1H), 2.42 (d, J=13.2 Hz, 1H), 2.03-1.28 (m, 19H), 1.25-1.03 (m, 5H), 1.03 (s, 3H), 1.02 (s, 3H), 0.97 (d, J=6.4 Hz, 3H), 0.73 (s, 3H).
Example 2
(153) ##STR00034## ##STR00035## ##STR00036##
(154) Preparation of 2-2.
(155) To a solution of MAD (28.87 mmol, freshly prepared) in toluene (20 mL) was added dropwise a solution of 2-1 (4 g, 9.62 mmol) in toluene (20 mL) at −78° C. during a period of 1 h under nitrogen. Then the reaction mixture was stirred for 30 min, a solution of EtMgBr (29 mL, 28.87 mmol, 1.0 M in toluene) was added dropwise at −78° C. The reaction mixture was warmed to −40° C. and stirred at this temperature for 3 hours. TLC (petroleum ether:ethyl acetate=3:1) showed that the starting material was consumed completely. The mixture was poured into aqueous saturated NH.sub.4Cl solution (200 mL) and extracted with EtOAc (150 mL×2). The combined organic phases were dried over Na.sub.2SO.sub.4, and the solvent was evaporated to afford crude product. The crude product was purified by column chromatography on silica gel (eluent: petroleum ether:ethyl acetate=15:1) to give the product 2-2 (2.0 g, 47.6%) as white powder. .sup.1H NMR: (400 MHz, CDCl3) δ 5.28 (d, J=5.2 Hz, 1H), 3.69 (s, 3H), 3.17 (s, 3H), 2.45-2.34 (m, 3H), 2.04-1.95 (m, 3H), 1.94-1.61 (m, 4H), 1.62-1.60 (m, 2H), 1.53-1.26 (m, 10H), 1.19-1.01 (m, 4H), 1.10 (s, 3H), 0.98-0.90 (m, 4H), 0.85 (t, J=6.8 Hz, 3H), 0.68 (s, 3H).
(156) Preparation of 2-3.
(157) To a suspension of LiAlH.sub.4 (852.6 mg, 22.43 mmol) in THF (20 ml) was added 2-2 (2.0 g, 4.48 mmol) at −78° C., then the solution was stirred at −78° C. for 2 hours. The mixture was poured into aqueous saturated NaOH solution (2 mL) and extracted with EtOAc (50 mL×2). The combined organic phases were dried over Na.sub.2SO.sub.4, and the solvent was evaporated to afford crude product. The crude product was purified by column chromatography on silica gel (eluent: petroleum ether:ethyl acetate=20:1) to give the product 2-3 (600 mg, 35%) as white powder. .sup.1H NMR: (400 MHz, CDCl3) δ 9.78 (s, 1H), 5.28 (d, J=5.2 Hz, 1H), 2.51-2.22 (m, 3H), 2.03-1.91 (m, 3H), 1.89-1.73 (m, 3H), 1.67-1.61 (m, 2H), 1.65-1.629 (m, 1H), 1.50-1.21 (m, 10H), 1.19-1.06 (m, 4H), 1.02 (s, 3H), 1.01-0.99 (m, 1H), 0.98-0.93 (m, 4H), 0.87 (t, J=6.8 Hz, 3H), 0.68 (s, 3H).
(158) Preparation of 2-4.
(159) To a mixture of 2-3 (0.3 g, 0.78 mmol) and CsF (0.06 g, 0.39 mmol) in toluene/THF (18 mL, 8/1) was added TMSCF.sub.3 (1.2 mL, 7.8 mmol) and the reaction mixture was stirred at room temperature overnight under nitrogen. TLC (petroleum ether:ethyl acetate=3/1) showed the starting material was consumed completely. A solution of TBAF (7.8 mL, 7.8 mmol, 1 M in THF) was added and the mixture was stirred for 4 h at room temperature. The reaction mixture was diluted with tert-Butyl methyl ether (30 mL), washed with aq. saturated NaHCO.sub.3 solution (10 mL×3) and concentrated in vacuum. The residue was purified by column chromatography on silica gel (eluent: petroleum ether:ethyl acetate=20:1) to afford 2-4 (80 mg, 22%) as white powder. .sup.1H NMR: (400 MHz, CDCl3) δ 5.29 (d, J=5.2 Hz, 1H), 3.87-3.84 (m, 1H), 2.36 (d, J=13.2 Hz, 1H), 2.05-1.95 (m, 3H), 1.86-1.61 (m, 6H), 1.54-1.06 (m, 17H), 1.03 (s, 3H), 1.02-0.91 (m, 5H), 0.85 (t, J=6.8 Hz, 3H), 0.68 (s, 3H).
(160) Preparation of 2-5 and 2-6.
(161) A mixture of 2-4 (0.07 g, 0.15 mmol) and 10% Pd/C (20 mg) in EtOAc (10 mL) was hydrogenated for 36 h at 50° C. under H.sub.2 (50 psi). The reaction mixture was filtered through a pad of celite and the pad was washed with EtOAc (20 mL×3). The combined filtrates were concentrated. The residue was purified by column chromatography on silica gel (eluent: petroleum ether:ethyl acetate=25/1) to give 2-5 (25 mg, 35.7%) and 2-6 (20 mg, 28.6%) as white powder. .sup.1H NMR (2-5): (400 MHz, CDCl3) δ 3.87-3.82 (m, 1H), 2.05-1.94 (m, 2H), 1.86-1.58 (m, 6H), 1.56-1.17 (m, 16H), 1.13-0.96 (m, 6H), 0.93 (d, J=6.8 Hz, 3H), 0.88 (t, J=6.8 Hz, 3H), 0.86-0.84 (m, 1H), 0.83 (s, 3H), 0.67-0.61 (m, 4H). .sup.1H NMR (2-6): (400 MHz, CDCl3) δ 3.83-3.76 (m, 1H), 1.95-1.52 (m, 10H), 1.43-0.98 (m, 22H), 0.89 (s, 3H), 0.88-0.82 (m, 6H), 0.59 (s, 3H).
(162) Preparation of 2-14.
(163) To a solution of MAD (91 mmol, freshly prepared) in toluene (200 mL) was added dropwise a solution of compound 2-13 (10 g, 30 mmol) in toluene (80 mL) at −78° C. during a period of 1 h under nitrogen. Then the reaction mixture was stirred for 30 min, a solution of EtMgBr (91 mL, 91 mmol, 1.0 M THF) was added dropwise at −78° C. The reaction mixture was warmed to −40° C. and stirred at this temperature for 3 h. TLC (petroleum ether:ethyl acetate=3:1) showed that the starting material was consumed completely. The mixture was poured into saturated aqueous NH.sub.4Cl solution (200 mL) and extracted with EtOAc (150 mL×2). The combined organic phases were dried over Na.sub.2SO.sub.4, and the solvent was evaporated to afford crude product, which was purified by column chromatography on silica gel (eluent: petroleum ether:ethyl acetate=15:1) to give compound 2-14 (4 g, 40%) as white powder.
(164) Preparation of 2-15.
(165) To a solution of 2-14 (4.0 g, 111 mmol) in THF (200 mL) was added aqueous HCl solution (35 mL, 1 M) and acetone (35 mL). The reaction mixture was stirred for 20° C. at room temperature. TLC (petroleum ether:ethyl acetate=3:1) indicated that the reaction was complete. Then the reaction mixture was diluted with EtOAc (200 mL), washed with saturated aqueous NaHCO.sub.3 solution (200 mL), dried over Na.sub.2SO.sub.4 and evaporated under reduced pressure to give 2-15 (3 g, 88%) as white solid.
(166) Preparation of 2-16.
(167) To a solution of Ph.sub.3PEtBr (15.8 g, 42.6 mmol) in dry THF (50 mL) was added dropwise a solution of t-BuOK (4.8 g, 42.6 mmol) in dry THF (20 mL) under N.sub.2 at 0° C. The mixture was stirred at room temperature for 1.5 h. Then a solution of 2-15 (2.7 g, 8.5 mmol) in THF (20 mL) was added dropwise and the resulting mixture was stirred at 80° C. for 16 h. TLC (petroleum ether:ethyl acetate=3:1) indicated that the starting material was consumed completely. The reaction was quenched with saturated aqueous NH.sub.4Cl solution (100 mL) and extracted with EtOAc (30 mL×2). The combined organic phases were dried over Na.sub.2SO.sub.4 and concentrated in vacuum. The residue was purified by column chromatography on silica gel (eluent: petroleum ether:ethyl acetate=12:1) to give 2-16 (1.8 g, 64%) as white solid.
(168) Preparation of 2-17.
(169) To a solution of compound 2-16 (1.8 g, 5.5 mmol) and methyl propiolate (1.1 g, 13.7 mmol) in dry CH.sub.2Cl.sub.2 (20 mL) was added dropwise a solution of Et.sub.2AlCl (22 mL, 22 mmol, 1 M in toluene) with stirring at 0° C. Then the reaction was warmed to room temperature and stirred for 20 h. TLC (petroleum ether:ethyl acetate=5:1) indicated that the starting material was consumed completely. The mixture was quenched with saturated aqueous NaHCO.sub.3 solution (30 mL) and extracted with CH.sub.2Cl.sub.2 (30 mL×2). The combined organic phases were dried over Na.sub.2SO.sub.4 and concentrated in vacuum. The residue was purified by column chromatography on silica gel (eluent: petroleum ether:ethyl acetate=10:1) to give 2-17 (2.0 g, 88%) as white powder. 1H NMR: (300 MHz, CDCl3) δ 6.99-6.92 (m, 1H) 5.84 (d, J=10.5 Hz, 1H), 5.45-5.41 (m, 1H), 5.32 (d, J=5.2 Hz, 1H), 3.75 (s, 3H), 3.06-2.99 (m, 1H), 2.38 (d, J=12.6 Hz, 1H), 2.14-1.67 (m, 10H), 1.54-1.25 (m, 7H), 1.21 (d, J=6.8 Hz, 3H), 1.15-0.99 (m, 5H), 0.87 (t, J=7.2 Hz, 3H), 0.80 (s, 3H).
(170) Preparation of 2-18.
(171) To a solution of compound 2-17 (2.2 g, 5.3 mmol) in dry THF (20 mL), DIBAL-H (1 M in THF, 27 mL, 27.0 mmol) was added dropwise at −78° C. under nitrogen. The reaction mixture was warmed to 30° C. and then stirred for 2 h at 30° C. The reaction was quenched with addition of water (3 mL), diluted with EtOAc (200 mL) and dried over anhydrous Na.sub.2SO.sub.4, filtered through a pad of celite and the pad was washed with EtOAc (50 mL×3). The combined filtrates were concentrated in vacuum to give 1.9 g of the crude product, which was directly used in the next step without further purification. A mixture of the crude product (1.9 g, 4.9 mmol) and MnO.sub.2 (8.6 g, 98 mmol) in CH.sub.2Cl.sub.2 (50 mL) was stirred at room temperature for 20 h. The reaction mixture was filtered through a pad of celite and the pad was washed with CH.sub.2Cl.sub.2 (50 mL×3). The combined filtrates were concentrated. The residue was purified by column chromatography on silica gel (eluent: petroleum ether:ethyl acetate=15:1) to give 2-18 (1.5 g, 79%) as white solid. .sup.1H NMR: (400 MHz, CDCl3) δ 9.55-9.53 (m, 1H), 6.84-6.78 (m, 1H), 6.15-6.09 (m, 1H), 5.45-5.41 (m, 1H), 5.30 (d, J=5.2 Hz, 1H), 3.15-3.14 (m, 1H), 2.36 (d, J=13.2 Hz, 1H), 2.10-2.03 (m, 3H), 1.90-1.60 (m, 9H), 1.59-1.27 (m, 7H), 1.24 (d, J=6.8 Hz, 3H), 1.10-1.22 (m, 6H), 0.87-0.83 (m, 4H), 0.80 (s, 3H).
(172) Preparation of 2-19.
(173) To a suspension of 2-18 (1.5 g, 3.92 mmol) and CsF (0.3 g, 1.96 mmol) in toluene/THF (22 mL, 9/1) was added TMSCF.sub.3 (5.8 mL, 39.2 mmol) and the mixture was stirred for 20 h at room temperature under nitrogen. TLC (petroleum ether:ethyl acetate=3/1) showed the starting material was consumed completely. A solution of TBAF (39.2 mL, 39.2 mmol, 1 M in THF) was added and the mixture was stirred for 4 h at room temperature. The mixture was diluted with MTBE (200 mL), washed with a saturated NaHCO.sub.3 solution (30 mL×3) and concentrated in vacuum. The residue was purified by column chromatography on silica gel (eluent: petroleum ether:ethyl acetate=25/1) to afford 2-19 (0.65 g, 37%) as white solid.
(174) Preparation of 2-20 & 2-21.
(175) 2-20 (210 mg, 32%) and 2-21 (210 mg, 32%) were obtained by SFC purification from 2-19 (650 mg, 1.44 mmol). The configuration of 2-20 and 2-21 was confirmed by Mosher method. .sup.1H NMR (2-20): (400 MHz, CDCl3) δ 5.92 (dd, J.sub.1=15.6 Hz, J.sub.2=7.2 Hz, 1H), 5.53 (dd, J.sub.1=15.6 Hz, J.sub.2=7.2 Hz, 1H), 5.40-5.37 (m, 1H), 5.30 (d, J=5.2 Hz, 1H), 4.43-4.40 (m, 1H), 2.95-2.94 (m, 1H), 2.37 (d, J=13.6 Hz, 1H), 2.09-1.98 (m, 4H), 1.87-1.18 (m, 18H), 1.16 (d, J=6.8 Hz, 3H), 1.12-0.97 (m, 6H), 0.85 (t, J=6.8 Hz, 3H), 0.78 (s, 3H). .sup.1H NMR (2-21): (400 MHz, CDCl3) δ 5.95 (dd, J.sub.1=15.6 Hz, J.sub.2=7.2 Hz, 1H), 5.53 (dd, J.sub.1=15.6 Hz, J.sub.2=6.8 Hz, 1H), 5.39-5.36 (m, 1H), 5.30 (d, J=5.2 Hz, 1H), 4.44-4.41 (m, 1H), 2.99-2.92 (m, 1H), 2.37 (d, J=13.2 Hz, 1H), 2.10-1.98 (m, 4H), 1.87-1.25 (m, 18H), 1.16 (d, J=6.8 Hz, 3H), 1.09-0.99 (m, 6H), 0.85 (t, J=7.2 Hz, 3H), 0.80 (s, 3H).
(176) Preparation of 2-7.
(177) A mixture of 2-20 (200 mg, 0.44 mmol) and 5% Pd/C (50 mg) in EtOAc (20 mL) was hydrogenated for 72 h at 30° C. under H.sub.2 (1 atm). The reaction mixture was filtered through a pad of celite and the pad was washed with EtOAc (10 mL×3). The combined filtrates were concentrated. The residue was purified by column chromatography on silica gel (eluent: petroleum ether:ethyl acetate=25/1) to afford crude 2-7, which was purified by pre-HPLC to afford 2-7 (64 mg, 52%) as white powder. .sup.1H NMR (2-7): (400 MHz, CDCl3) δ 5.29 (d, J=4.8 Hz, 1H), 3.90-3.80 (m, 1H), 2.36 (d, J=13.6 Hz, 1H), 2.05-1.60 (m, 11H), 1.53-1.06 (m, 15H), 1.03 (s, 3H), 1.02-0.89 (m, 5H), 0.85 (t, J.sub.1=14.8 Hz, J.sub.2=7.2 Hz, 3H), 0.69 (s, 3H).
(178) Preparation of 2-8.
(179) A mixture of 2-21 (200 mg, 0.44 mmol) and 5% Pd/C (50 mg) in EtOAc (20 mL) was hydrogenated for 72 h at 30° C. under H.sub.2 (1 atm). The reaction mixture was filtered through a pad of celite and the pad was washed with EtOAc (10 mL×3). The combined filtrates were concentrated. The residue was purified by column chromatography on silica gel (eluent: petroleum ether:ethyl acetate=25/1) to afford 2-8 (105 mg, 52%) as white powder. .sup.1H NMR: (400 MHz, CDCl.sub.3) δ 5.29 (d, J=4.8 Hz, 1H), 3.86-3.83 (m, 1H), 2.36 (d, J=13.2 Hz, 1H), 2.05-1.95 (m, 4H), 1.86-1.60 (m, 7H), 1.54-1.08 (m, 15H), 1.03 (s, 3H), 1.01-0.90 (m, 5H), 0.85 (t, J=6.8 Hz, 3H), 0.68 (s, 3H).
(180) Preparation of 2-10 and 2-12.
(181) A mixture of 2-8 (30 mg, 0.067 mmol) and 10% Pd/C (10 mg) in EtOAc (10 mL) was hydrogenated for 20 h at 50° C. under H.sub.2 (50 psi). The reaction mixture was filtered through a pad of celite and the pad was washed with EtOAc (20 mL×3). The combined filtrates were concentrated. The residue was purified by column chromatography on silica gel (eluent: petroleum ether:ethyl acetate=25/1) to give 2-10 (11 mg, 37%) and 2-12 (7 mg, 23%) as white powder. .sup.1H NMR (2-10): (400 MHz, CDCl3) δ 3.85-3.82 (m, 1H), 2.04-1.93 (m, 2H), 1.84-1.59 (m, 6H), 1.56-1.20 (m, 14H), 1.14-0.96 (m, 7H), 0.93 (d, J=6.8 Hz, 3H), 0.88-0.84 (m, 4H), 0.83 (s, 3H) 0.67-0.61 (m, 4H). .sup.1H NMR (2-12): (400 MHz, CDCl3) δ 3.89-3.80 (m, 1H), 2.08-1.93 (m, 2H), 1.91-1.66 (m, 6H), 1.52-1.01 (m, 23H), 0.97 (s, 3H), 0.95-0.90 (m, 6H), 0.66 (s, 3H).
Example 3
(182) ##STR00037##
(183) Preparation of 3-2.
(184) To a suspension of 3-1 (400 mg, 1.035 mmol) and CsF (76 mg) in toluene/THF (20 mL, 8/1) was added TMSCF.sub.3 (1.53 mL, 10.35 mmol) and the mixture was stirred for 20° C. at room temperature under nitrogen. TLC (petroleum ether:ethyl acetate=3/1) showed the starting material was consumed completely. A solution of TBAF (6.8 mL, 1 M in THF) was added and the mixture was stirred for 4 h at room temperature. The mixture was diluted with MTBE (200 mL), washed with aq. saturated NaHCO.sub.3 solution (30 mL×3) and concentrated in vacuum. The residue was purified by column chromatography on silica gel (eluent: petroleum ether:ethyl acetate=20:1) to afford 3-2 (220 mg, 46%) as white solid. 1H NMR (400 MHz, CDCl3) δ 5.31 (d, J=2.0 Hz, 1H), 2.44-2.41 (m, 1H), 2.04-1.96 (m, 3H), 1.81-1.67 (m, 5H), 1.65-1.39 (m, 11H), 1.34-1.32 (m, 3H), 1.31-1.25 (m, 1H), 1.21-1.10 (m, 3H), 1.12-0.98 (m, 4H), 0.96 (s, 3H), 0.98-0.90 (m, 4H), 0.68 (s, 3H.)
(185) Preparation of 3-3 and 3-4.
(186) To a solution of compound 3-2 (220 mg, 0.569 mmol) in EtOAc (10 mL) was added Pd/C (20 mg), then the mixture was stirred under hydrogen (50 psi) at 50° C. overnight. The mixture was filtered through a pad of celite and the filtrate was evaporated under reduced pressure. The residue was purified by column chromatography on silica gel (eluent: petroleum ether:ethyl acetate=20:1) to afford the pure product 3-3 (100 mg, 38.5%) and 3-4 (51 mg, 19.3%) as white powder. .sup.1H NMR (3-3): (400 MHz, CDCl3) δ 2.01-1.95 (m, 1H), 1.89-1.75 (m, 2H), 1.69-1.55 (m, 9H), 1.52-1.43 (m, 5H), 1.32-1.28 (m, 4H), 1.27-1.20 (m, 7H), 1.17-1.08 (m, 4H), 1.06-0.96 (m, 3H), 0.96-0.91 (m, 3H), 0.80 (s, 3H), 0.68-0.49 (m, 4H). .sup.1H NMR (3-4): (400 MHz, CDCl3) δ 2.01-1.95 (m, 1H), 1.89-1.67 (m, 5H), 1.66-1.60 (m, 2H), 1.63-1.36 (m, 8H), 1.35-1.31 (m, 4H), 1.29-1.24 (m, 4H), 1.22 (s, 3H), 1.28-1.06 (m, 6H), 0.96 (s, 3H), 0.95-0.92 (m, 3H), 0.68 (s, 3H).
(187) Preparation of 3-5 and 3-6.
(188) Compound 3-2 (1.2 g, 2.63 mmol) was split by SFC to get Product 3-5 (400 mg) and 3-6 (400 mg) as white powder (total yield: 66.7%). .sup.1H NMR (3-5): (400 MHz, CDCl.sub.3) δ 5.32 (d, J=4.0 Hz, 1H), 2.50-2.40 (m, 1H), 2.08-1.95 (m, 3H), 1.90-0.90 (m, 35H), 0.70 (s, 3H). .sup.1H NMR (3-6): (400 MHz, CDCl.sub.3) δ 5.32 (d, J=4.0 Hz, 1H), 2.50-2.40 (m, 1H), 2.08-1.95 (m, 3H), 1.90-0.92 (m, 35H), 0.70 (s, 3H).
(189) Preparation of 3-7
(190) To a solution of compound 3-6 (300 mg, 0.66 mmol) in EtOAc (8 mL) was added Pd/C (10%, 200 mg) under N.sub.2. The suspension was degassed under vacuum and purged with H.sub.2 several times. Then the mixture was stirred under H.sub.2 (50 psi) at 50° C. for 24 h. The suspension was filtered through a pad of celite and the pad was washed with EtOAc (50 mL×2). The combined filtrates were concentrated to dryness to give the crude product, which was purified by column chromatography on silica gel (petroleum ether:ethyl acetate=20:1) to afford 3-7 (142 mg, 47%) as white solid. .sup.1H NMR: (3-7) (400 MHz, CDCl.sub.3) δ 1.96-1.92 (m, 1H), 1.90-1.75 (m, 1H), 1.70-1.57 (m, 5H), 1.55-1.35 (m, 6H), 1.30-1.20 (m, 12H), 1.20-1.06 (m, 12H), 1.19-0.81 (m, 11H), 0.80 (s, 3H), 0.70-0.60 (m, 4H). .sup.1H NMR: (3-7A) (400 MHz, CDCl.sub.3) δ 1.96-1.92 (m, 1H), 1.90-1.75 (m, 3H), 1.70-1.57 (m, 2H), 1.55-1.25 (m, 13H), 1.21-1.00 (m, 15H), 0.96-0.86 (m, 8H), 0.65 (s, 3H)
(191) Preparation of 3-8
(192) To a solution of compound 3-5 (300 mg, 0.66 mmol) in EtOAc (8 mL) was added Pd/C (10%, 200 mg) under N.sub.2. The suspension was degassed under vacuum and purged with H.sub.2 several times. Then the mixture was stirred under H.sub.2 (50 psi) at 50° C. for 24 h. The suspension was filtered through a pad of celite and the pad was washed with EtOAc (50 mL×2). The combined filtrates were concentrated to dryness to give the crude product, which was purified by column chromatography on silica gel (petroleum ether:ethyl acetate=20:1) to afford 3-8 (141.6 mg, 47%) as white solid. .sup.1H NMR: (3-8) (400 MHz, CDCl.sub.3) δ 1.96-1.92 (m, 1H), 1.90-1.70 (m, 2H), 1.69-1.57 (m, 5H), 1.55-1.20 (m, 18H), 1.19-0.81 (m, 10H), 0.80 (s, 3H), 0.70-0.60 (m, 4H). .sup.1H NMR: (3-8A) (400 MHz, CDCl.sub.3) δ 1.97-1.70 (m, 6H), 1.70-1.57 (m, 2H), 1.50-1.30 (m, 13H), 1.25-1.05 (m, 15H), 1.00-0.86 (m, 7H), 0.65 (s, 3H)
Example 4
(193) ##STR00038## ##STR00039##
(194) Preparation of Compound 4-2.
(195) To a solution of 4-1 (38 g, 101.5 mmol) in THF (400 mL) at room temperature was added HATU (46.3 g, 121.8 mmol), DIPEA (45.9 g, 355.2 mmol). The mixture was stirred for 1 h, and N,O-dimethylhydroxylamine hydrochloride (19.8 g, 203 mmol) was added. The mixture was stirred at room temperature for another 6 h. The reaction mixture was concentrated, poured into water, extracted with EtOAc, washed with water, dried over Na.sub.2SO.sub.4, and concentrated to give crude product. The crude product was purified by column chromatography on silica gel (eluent: PE:EA=3:1) to afford the desired product 4-2 (24 g, 57%) as white solid. .sup.1H NMR: (300 MHz, CDCl3) δ: ppm 5.25 (d, J=5.2 Hz, 1H), 3.59 (s, 3H), 3.46-3.37 (m, 1H), 3.07 (s, 3H), 2.70 (s, 1H), 2.40-2.09 (m, 4H), 1.92-1.63 (m, 6H), 1.44-1.33 (m, 6H), 1.29-1.15 (m, 3H), 1.11-0.93 (m, 5H), 0.90 (s, 3H), 0.85 (d, J=6.4 Hz, 3H), 0.82-0.78 (m, 1H), 0.58 (s, 3H).
(196) Preparation of Compound 4-3.
(197) To a solution of compound 4-2 (14 g, 33.52 mmol, 1.0 eq) in dry CH.sub.2Cl.sub.2 (600 mL) was added Dess-Martin (28 g, 67.04 mmol, 2.0 eq) in portions at 0° C. Then the reaction mixture was stirred at room temperature for 6.5 h. TLC (PE:EA=3:1) showed the starting material was consumed completely. The mixture was quenched with saturated aqueous NaHCO.sub.3/Na.sub.2S.sub.2O.sub.3=1:3 (800 mL). The organic phase was washed with brine (500 mL) and dried over Na.sub.2SO.sub.4, and the solvent was evaporated to afford crude product 4-3 (14.0 g, 100%), which was directly used in the next step without further purification.
(198) Preparation of Compound 4-4.
(199) To a solution of MAD (101 mmol, 3.0 eq) in toluene, freshly prepared by addition of a solution of Me.sub.3Al (50.5 mL, 101.00 mmol, 2 M in hexane) to a stirred solution of 2,6-di-tert-butyl-4-methylphenol (44.4 g, 202 mmol) in toluene (200 mL) followed by stirring for 1 h at room temperature, was added dropwise a solution of 4-3 (14.0 g, 33.7 mmol, 1.0 eq) in toluene (10 mL) at −78° C. under nitrogen. Then the reaction mixture was stirred for 30 min, a solution of MeMgBr (33.7 mL, 101 mmol, 3.0 eq, 3 M in ether) was added dropwise at −78° C. The reaction mixture was warmed to 25° C. and stirred at this temperature for 12 h. TLC (PE:EA=3:1) showed that the starting material was consumed completely. The mixture was poured into aqueous saturated NH.sub.4Cl solution (200 mL) and extracted with EtOAc (200 mL×2). The combined organic phases were dried over Na.sub.2SO.sub.4, and the solvent was evaporated to afford crude product. The crude product was purified by column chromatography on silica gel (eluent: PE:EA=3:1) to give the pure target (7.5 g, 52%) as white powder. .sup.1H NMR: (400 MHz, CDCl3) δ 5.30 (d, J=5.2 Hz, 1H), 3.69 (s, 3H), 3.17 (s, 3H), 2.50-2.30 (m, 3H), 2.05-1.70 (m, 7H), 1.52-1.30 (m, 9H), 1.20-0.90 (m, 15H), 0.68 (s, 3H).
(200) Preparation of Compound 4-5.
(201) To a solution of compound 4-4 (7.5 g, 17.4 mmol, 1.0 eq) in THF (150 mL) was added dropwise a solution of MeMgBr (29 mL, 87 mmol, 5.0 eq, 3 M in THF) at room temperature during a period of 30 min under nitrogen. Then the reaction mixture was stirred at room temperature for 12 h. TLC (PE:EA=1:1) showed that the starting material was consumed completely. The mixture was poured into aqueous saturated NH.sub.4Cl solution (200 mL) and extracted with EtOAc (150 mL×2). The combined organic phases were dried over Na.sub.2SO.sub.4, and the solvent was evaporated to afford crude product. The crude product was purified by column chromatography on silica gel (eluent: PE:EA=4:1) to give the product 4-5 (5.2 g, 77%) as white powder. .sup.1H NMR: (400 MHz, CDCl3) δ 5.30 (d, J=5.2 Hz, 1H), 2.50-2.30 (m, 3H), 2.14 (s, 3H) 2.03-1.93 (m, 3H), 1.87-1.68 (m, 4H), 1.60-1.18 (m, 12H), 1.12 (s, 3H), 1.11-1.03 (m, 1H), 1.01 (s, 3H), 1.00-0.94 (m, 1H), 0.91 (d, J=6.4 Hz, 3H), 0.68 (s, 3H).
(202) Preparation of 4-6.
(203) To a solution of compound 4-5 (300 mg, 0.777 mmol, 1.0 eq) in toluene (5 mL) was added dropwise a solution of EtMgBr (4.5 mL, 4.5 mmol, 6.0 eq, 1 M in THF) at room temperature during a period of 10 min under nitrogen. Then the reaction mixture was stirred at room temperature for 12 h. TLC (PE:EA=3:1) showed that the starting material was consumed completely. The mixture was poured into aqueous saturated NH.sub.4Cl solution (20 mL) and extracted with EtOAc (50 mL×2). The combined organic phases were dried over Na.sub.2SO.sub.4, and the solvent was evaporated to afford crude product. The crude product purified by column chromatography on silica gel (eluent: PE:EA=8:1) to give the product 4-6 (200 mg, 62%) as white powder. .sup.1H NMR: (400 MHz, CDCl3) δ 5.23 (d, J=5.6 Hz, 1H), 2.40-2.30 (m, 1H), 2.00-1.55 (m, 7H), 1.50-1.98 (m, 25H), 0.95 (s, 3H), 0.94-0.80 (m, 8H), 0.62 (s, 3H).
(204) Preparation of 4-7 and 4-8.
(205) To a solution of compound 4-6 (175 mg, 0.42 mmol) in EtOAc (10 mL) was added 10% Pd/C (40 mg) under argon. The suspension was degassed under vacuum and purged with H.sub.2 several times. The mixture was stirred under H.sub.2 (50 Psi) at 50° C. overnight. The suspension was filtered through a pad of celite and the pad was washed with EA (20 mL×3). The combined filtrates were concentrated in vacuum and the residue was purified by column chromatography on silica gel (eluent: PE:EA=8:1) to give 4-7 (84 mg, 48%) and 4-8 (25 mg, 14%) as white powder. .sup.1H NMR (4-7): (400 MHz, CDCl3) δ 1.98-1.92 (m, 1H), 1.87-1.78 (m, 1H), 1.70-1.60 (m, 2H), 1.58-1.20 (m, 21H), 1.20-0.97 (m, 11H), 0.95-0.82 (m, 7H), 0.80 (s, 3H), 0.70-0.61 (m, 4H). .sup.1H NMR (4-8): (400 MHz, CDCl3) δ 2.00-1.78 (m, 4H), 1.68-1.63 (m, 1H), 1.57-1.55 (m, 1H), 1.53-1.35 (m, 10H), 1.32-1.12 (m, 16H), 1.11-0.99 (m, 5H), 0.97 (s, 3H), 0.95-0.83 (m, 6H), 0.67 (s, 3H).
(206) Preparation of 4-9
(207) To a solution of compound 10-12B (80 mg, 0.193 mmol) in EtOAc (20 mL) was added 10% Pd/C (20 mg) under N.sub.2. The suspension was degassed under vacuum and purged with H.sub.2 several times. Then the mixture was stirred under H.sub.2 (50 psi) at 50° C. for 12 hours. The mixture was filtered through a pad of celite and the pad was washed with EtOAc (5 mL×2). The combined filtrates were concentrated to dryness to give the product, which was purified by column chromatography on silica gel (eluent: petroleum ether:ethyl acetate=12:1 to 10:1) to afford the 4-9 (40 mg, 50%) as white powder. .sup.1H NMR (4-9): (400 MHz, CDCl.sub.3) δ 2.02-1.93 (m, 1H), 1.92-1.80 (m, 1H), 1.70-0.85 (m, 41H), 0.82 (s, 3H), 0.67 (s, 3H).
(208) Preparation of 4-10
(209) To a solution of compound 10-12A (80 mg, 0.193 mmol) in EtOAc (20 mL) was added 10% Pd/C (20 mg) under N.sub.2. The suspension was degassed under vacuum and purged with H.sub.2 several times. Then the mixture was stirred under H.sub.2 (50 psi) at 50° C. for 48 hours. The mixture was filtered through a pad of celite and the pad was washed with EtOAc (5 mL×2). The combined filtrates were concentrated to dryness to give the product, which was purified by column chromatography on silica gel (eluent: petroleum ether:ethyl acetate=12:1 to 10:1) to afford the 4-10 (40 mg, 50%) as white powder. .sup.1H NMR (4-10): (400 MHz, CDCl.sub.3) δ 2.02-1.93 (m, 1H), 1.92-1.80 (m, 1H), 1.70-0.85 (m, 41H), 0.82 (s, 3H), 0.67 (s, 3H).
(210) Preparation of 4-11 and 4-12.
(211) 4-11 (100 mg, 15.38%) and 4-12 (90 mg, 13.85%) were obtained by SFC purification from 4-6 (600 mg, 1.55 mmol). .sup.1H NMR (Isomer 1): (400 MHz, CDCl3) δ 5.30 (m, 1H), 2.43-2.40 (d, J=12.4 Hz, 1H), 2.14-1.99 (m, 3H), 1.96-1.68 (m, 3H), 1.68-1.52 (m, 5H), 1.51-1.24 (m, 13H), 1.19-1.09 (m, 8H), 1.02 (s, 3H), 0.96-0.93 (m, 3H), 0.93-0.87 (m, 3H), 0.69 (s, 3H). .sup.1H NMR (Isomer 2): (400 MHz, CDCl3) δ 5.30 (m, 1H), 2.44-2.40 (d, J=14 Hz, 1H), 2.17-1.96 (m, 3H), 1.96-1.67 (m, 3H), 1.67-1.18 (m, 18H), 1.16-1.09 (m, 8H), 1.06 (s, 3H), 0.96-0.93 (m, 3H), 0.93-0.87 (m, 3H), 0.69 (s, 3H).
(212) ##STR00040##
Example 5
(213) Preparation of Compound 5-2.
(214) To a solution of 5-1 (200 mg, 0.52 mmol) in toluene (5 mL) at −78° C. was added n-PrMgBr (1.3 mL, 2 M in THF, 2.6 mmol) dropwise. The mixture was warmed up to room temperature gradually and stirred for 6 h. The reaction mixture was quenched with NH.sub.4Cl aqueous, extracted with EtOAc. The organic layer was dried over chromatography on silica gel (eluent: PE:EA=15:1) to afford 5-2 (130 mg, 58%) as white solid. .sup.1H NMR: (300 MHz, CDCl3) δ: ppm 5.30 (d, J=4.8 Hz, 1H), 2.48-2.38 (m, 1H), 2.02-1.95 (m, 3H), 1.88-1.66 (m, 3H), 1.63-1.52 (m, 5H), 1.52-1.46 (m, 4H), 1.43-1.41 (m, 1H), 1.41-1.35 (m, 4H), 1.30-1.22 (m, 3H), 1.20-1.14 (m, 4H), 1.13-1.08 (m, 4H), 1.03 (s, 3H), 0.95-0.90 (m, 3H), 0.90-0.87 (m, 3H), 0.87-0.85 (m, 1H) 0.68 (s, 3H).
(215) Preparation of 5-3 and 5-4.
(216) To a solution of compound 5-2 (400 mg, 0.93 mmol) in EtOAc (20 mL) was added 10% Pd/C (100 mg). Then the mixture was stirred under hydrogen (50 psi) at 50° C. overnight. The mixture was filtered through a pad of celite and the filtrate was evaporated under reduced pressure. The residue was purified by column chromatography on silica gel (eluent: petroleum ether:ethyl acetate=15:1) to afford the pure product 5-3 (150 mg, 37.3%) and 5-4 (27 mg, 6.7%) as white powder. .sup.1H NMR (5-3): (300 MHz, CDCl3) δ 1.97-1.94 (m, 1H), 1.93-1.77 (m, 1H), 1.67-1.62 (m, 3H), 1.56-1.51 (m, 6H), 1.47-1.30 (m, 11H), 1.24 (s, 6H), 1.20 (s, 1H), 1.13 (s, 5H), 1.09-0.99 (m, 4H), 0.94-0.90 (m, 6H), 0.80 (s, 3H), 0.65 (s, 3H). .sup.1H NMR (5-4): (300 MHz, CDCl3) δ 1.98-1.94 (m, 2H), 1.91-1.78 (m, 5H), 1.65-1.51 (m, 5H), 1.47-1.46 (m, 3H), 1.38-1.35 (m, 9H), 1.32-1.30 (m, 2H), 1.25 (s, 3H), 1.22 (s, 6H), 1.16-1.10 (m, 4H), 1.06-1.04 (m, 4H), 0.98-0.94 (m, 4H), 0.92-0.89 (m, 6H), 0.86-0.83 (m, 1H), 0.64 (s, 3H).
(217) Preparation of 5-5 and 5-6
(218) To a solution of compound 5-1 (1500 mg, 3.88 mmol) in dry THF (30 mL) was added a solution of n-PrMgBr (11.6 mL, 23.3 mmol) dropwise at 0° C. The mixture was stirred at 40° C. for 16 h. TLC (PE/EtOAc=2/1) showed the reaction was complete. Saturated aqueous NH.sub.4Cl (5 mL) was added slowly to quench the reaction. The resulting solution was separated between EtOAc (30 mL×3) and H.sub.2O (30 mL). The combined organic layers were concentrated in vacuum and the residue was purified by silica gel column eluted with PE/EtOAc=10/1 to give the mixture of the diastereomeric pair (1.1 g) as white power. The diastereimeric pair was separated by prep-SFC to give 5-6 (380 mg, 22.8%) as a white solid and 5-5 (385 mg, 23.1%) as a white solid. .sup.1H NMR (5-5): (400 MHz, CDCl.sub.3) δ 5.31-5.30 (m, 1H), 2.44-2.41 (d, 1H, J=12.8 Hz), 2.01-1.96 (m, 3H), 1.86-1.69 (m, 3H), 1.58-1.25 (m, 16H), 1.14-1.08 (m, 11H), 1.06-0.99 (m, 4H), 0.94-0.91 (m, 6H), 0.68 (s, 3H). .sup.1H NMR (5-6): (400 MHz, CDCl.sub.3) δ 5.31-5.30 (m, 1H), 2.44-2.41 (d, 1H, J=12.4 Hz), 2.02-1.96 (m, 3H), 1.87-1.68 (m, 3H), 1.57-1.25 (m, 16H), 1.18-1.08 (m, 10H), 1.02-0.99 (m, 4H), 0.94-0.91 (m, 6H), 0.68 (s, 3H).
(219) Preparation of 5-8
(220) A mixture of 5-6 (200 mg, 0.464 mmol) and Pd/C (100 mg, cat.) in EtOAc (30 mL) was hydrogenated under 50 psi of hydrogen for 48 h at 50° C. The reaction mixture was filtered through a celite pad. The pad was washed with EtOAc (50 mL). The filtrate was concentrated in vacuum and the residue was purified by silica gel column eluted with PE/EtOAc=20/1 to give 5-8 (111.3 mg, 55.4%) as a white solid. 1H NMR (5-8) (400 MHz, CDCl.sub.3), δ (ppm) 1.97-1.94 (d, 1H, J=12.0 Hz), 1.83-1.78 (m, 1H), 1.65-1.61 (m, 3H), 1.50-1.24 (m, 20H), 1.13-1.00 (m, 11H), 0.94-0.85 (m, 7H), 0.80 (s, 3H), 0.68-0.65 (m, 4H). 1H NMR (5-8A) (400 MHz, CDCl.sub.3), δ (ppm) 1.98-1.95 (d, 1H, J=11.2 Hz), 1.88-1.80 (m, 3H), 1.65-1.60 (m, 1H), 1.51-1.47 (m, 1H), 1.40-1.31 (m, 12H), 1.28-1.20 (m, 8H), 1.16-1.01 (m, 11H), 0.96-0.80 (m, 10H), 0.65 (s, 3H).
(221) Preparation of 5-7
(222) A mixture of 5-5 (200 mg, 0.464 mmol) and Pd/C (100 mg, cat.) in EtOAc (30 mL) was hydrogenated under 50 psi of hydrogen for 48 h at 50° C. The reaction mixture was filtered through a celite pad. The pad was washed with EtOAc (50 mL). The filtrate was concentrated in vacuum and the residue was purified by silica gel column eluted with PE/EtOAc=20/1 to give 5-7 (118.5 mg, 59.0%) as a white solid. 1H NMR (5-7) (400 MHz, CDCl.sub.3), δ (ppm) 1.97-1.94 (d, 1H, J=12.8 Hz), 1.88-1.79 (m, 1H), 1.71-1.61 (m, 3H), 1.51-1.24 (m, 20H), 1.13-1.00 (m, 11H), 0.94-0.85 (m, 7H), 0.80 (s, 3H), 0.68-0.65 (m, 4H). 1H NMR (5-7A) (400 MHz, CDCl.sub.3), δ (ppm) 1.98-1.95 (d, 1H, J=11.2 Hz), 1.88-1.79 (m, 3H), 1.65-1.59 (m, 1H), 1.52-1.47 (m, 1H), 1.41-1.31 (m, 11H), 1.27-1.22 (m, 9H), 1.13-1.11 (m, 7H), 1.06-1.01 (m, 4H), 0.96-0.90 (m, 10H), 0.65 (s, 3H).
Example 6
(223) ##STR00041## ##STR00042## ##STR00043##
(224) Preparation of 6-2.
(225) To a solution of 6-1 (150 mg, 0.39 mmole) in THF (4 mL) was added allylmagnesium bromide (2.34 mL, 2.34 mmole, 1M in ether) at −78° C. Then the reaction mixture was warmed to room temperature and stirred for 12 hours. The mixture was quenched with NH.sub.4Cl (20 mL) solution and extracted with EtOAc (10 mL×2). The organic phase was dried by Na.sub.2SO.sub.4 and purified by column chromatography on silica gel (eluent: PE:EA=10:1) to get the 6-2 (100 mg, 59%). .sup.1H NMR: (400 MHz, CDCl.sub.3) δ 5.89-5.82 (m, 1H), 5.31 (d, J=5.2 Hz, 2H), 5.15-5.09 (m, 2H), 2.43-2.40 (m, 1H), 2.22-2.20 (d, J=7.6 Hz, 2H), 2.04-1.96 (m, 3H), 1.95-1.57 (m, 3H), 1.54-1.24 (m, 12H), 1.19-1.11 (m, 5H), 1.09-1.05 (m, 6H), 1.03 (s, 3H), 0.98-0.92 (m, 5H), 0.68 (s, 3H).
(226) Preparation of 6-3.
(227) To a solution of 9-BBN (3.2 mL, 1.6 mmol, 2M in THF) was added dropwise a solution of 6-2 (70 mg, 0.16 mmol) in THF (2 mL) at 0° C. The reaction mixture was heated at 60° C. and stirred for 12 hours. The mixture was cooled to 0° C. and aq. NaOH (10%) solution (2 mL) was added followed by H.sub.2O.sub.2 (30%, 1 mL). The mixture was stirred for 2 hours at 0° C. and then extracted with EtOAc. The combined organic layer was washed with brine, dried over Na.sub.2SO.sub.4 and concentrated to to give crude product. The crude product was purified by column chromatography on silica gel (eluent: petroleum ether:ethyl acetate=2:1) to afford 6-3 (30 mg, 42%) as white solid. .sup.1H NMR: (300 MHz, CDCl3) δ: 5.30 (d, J=5.2 Hz, 1H), 3.68-3.65 (m, 2H), 2.43-2.39 (m, 1H), 2.03-1.80 (m, 6H), 1.79-1.62 (m, 6H), 1.47-1.36 (m, 5H), 1.32-1.25 (m, 7H), 1.17-1.13 (m, 4H), 1.11-1.07 (m, 6H), 10.5-0.98 (m, 4H), 0.94-0.90 (m, 5H), 0.68 (s, 3H).
(228) Preparation of 6-4 and 6-5.
(229) A mixture of 6-1 (1.0 g, 2.59 mmol) and 10% Pd/C (140 mg) in EtOAc (30 mL) was hydrogenated for 16 h at 50° C. under H.sub.2 (50 psi). The reaction mixture was filtered through a pad of celite and the pad was washed with EtOAc (20 mL×3). The combined filtrates were concentrated. The residue was purified by column chromatography on silica gel (eluent: petroleum ether:ethyl acetate=15:1) to afford 6-4 (500 mg, 49.5%) and 6-5 (200 rig, 19.8%) as white solid.
(230) Preparation of 6-6.
(231) To a solution of 6-4 (70 mg, 0.18 mmol) in dry THF (2 mL) at −78° C. was added C.sub.3H.sub.5MgBr (1.1 mL, 1.08 mmol) dropwise under N.sub.2. The mixture was warmed up to room temperature gradually and stirred for 12 h. The reaction was quenched with NH.sub.4Cl aqueous and extracted by EtOAc. The organic layer was dried over Na.sub.2SO.sub.4, filtered and concentrated to give crude product. The crude product was purified by column chromatography on silica gel (eluent: petroleum ether:ethyl acetate=15:1) to afford the pure product 6-6 (40 mg, 51.9%) as white powder. .sup.1H NMR: (300 MHz, CDCl3) δ: ppm 5.92-5.79 (m, 1H), 5.15 (d, J=4.2 Hz, 1H), 5.11 (d, J=13.2 Hz, 1H), 2.21 (d, J=7.5 Hz, 2H), 1.97-1.75 (m, 5H), 1.67-1.34 (m, 19H), 1.30-0.94 (m, 11H), 0.91 (d, J=6.3 Hz, 3H), 0.80 (s, 3H), 0.69-0.61 (m, 4H).
(232) Preparation of 6-7 and 6-8
(233) Compound 6-2 (400 mg, 0.849 mmol) was split by SFC to get 6-7 (96 mg) and 6-8 (162 mg) as white powder (total yield: 65%). .sup.1H NMR (6-7) (400 MHz, CDCl.sub.3), δ 5.90-5.81 (m, 1H), 5.31 (d, J=5.2 Hz, 1H), 5.20-5.09 (m, 2H), 2.45-2.35 (m, 1H), 2.25-2.15 (m, 2H), 2.04-0.90 (m, 36H), 0.68 (s, 3H). 1H NMR (6-8) (400 MHz, CDCl.sub.3), δ 5.90-5.80 (m, 1H), 5.31 (d, J=5.2 Hz, 1H), 5.21-5.09 (m, 2H), 2.45-2.34 (m, 1H), 2.25-2.15 (m, 2H), 2.04-0.89 (m, 36H), 0.68 (s, 3H).
(234) Preparation of 6-6-Bz
(235) To a solution of 6-6 (100 mg, 0.23 mmol) in pyridine (3 mL) was added BzCl (64.4 mg, 0.46 mmol) dropwise at room temperature. Then the reaction mixture was stirred at 40° C. for 12 hours. TLC showed the starting material was consumed completely. The mixture was quenched by saturated aqueous water and extracted with EtOAc. The combined organic phase was washed with 1 M HCl (30 mL) and brine, dried over anhydrous Na.sub.2SO.sub.4 then concentrated in vacuum. The residue was purified by column chromatography on silica gel (petroleum ether:ethyl acetate=80:1) to afford 6-8-Bz (60 mg, 48%) as a white solid.
(236) Preparation of 6-11-Bz
(237) Compound 6-6-Bz (60 mg, 0.11 mmol) was split by SFC to get 6-11-Bz (40 mg, 66%) as a white solid. .sup.1H NMR: (400 MHz, CDCl.sub.3) δ 7.99-7.98 (d, J=7.2 Hz, 2H), 7.53-7.49 (t, J=7.2 Hz, 1H), 7.42-7.38 (t, J=7.2 Hz, 2H), 2.22-2.20 (d, J=7.6 Hz, 2H), 1.98-1.57 (m, 11H), 1.54-1.26 (m, 16H), 1.15 (s, 3H), 1.12-1.10 (m, 6H), 0.92-0.91 (d, J=6.0 Hz, 3H), 0.80 (s, 3H), 0.64-0.60 (m, 4H)
(238) Preparation of 6-11
(239) To a solution of compound 6-11-Bz (40 mg, 0.075 mmol) in a mixture solvent of THF (2 mL) and MeOH (1 mL) was added a solution of LiOH (90 mg, 3.75 mmol) in H.sub.2O (1 mL). The mixture was stirred at 40° C. for 3 days. TLC showed the starting material was consumed completely. The reaction mixture was treated with water and extracted with EtOAc. The combined organic phase was washed with brine, dried over anhydrous Na.sub.2SO.sub.4 then concentrated by vacuum. The residue was purified by column chromatography on silica gel (petroleum ether:ethyl acetate=8:1) to afford 6-11 (23 mg, 71%) as a white solid. .sup.1H NMR: (400 MHz, CDCl.sub.3) δ 5.86-5.84 (m, 1H), 5.13-5.09 (m, 2H), 2.21-2.19 (d, J=7.6 Hz, 2H), 1.84-1.25 (m, 19H), 1.24 (s, 3H), 1.14 (s, 3H), 1.13-1.09 (m, 7H), 0.91-0.90 (d, J=6.8 Hz, 3H), 0.80 (s, 3H), 0.64-0.60 (m, 4H)
Example 7
(240) ##STR00044## ##STR00045##
(241) Preparation of Compound 7-2.
(242) To a solution of 7-1 (193 mg, 0.5 mmol, 1.0 eq) in dry THF (3 mL), n-BuLi (1.6 mL, 4 mmol, 8.0 eq) was added dropwise at −78° C. The resulting mixture was stirred at this temperature for 0.5 h, and then the temperature was allowed to warm to room temperature and stirred at this temperature for another 18 h. TLC (PE/EA=5/1) showed the reaction was complete. The mixture was quenched with saturated aqueous NH.sub.4Cl and extracted with EtOAc (10 mL×3). The combined organic layers were washed with brine (10 mL), dried over sodium sulfate and concentrated in vacuum. The residue was purified by column chromatography on silica gel (eluent: PE:EA=20:1) to give the product 7-2 (85 mg, 38.6%) as white powder. .sup.1H NMR: (400 MHz, CDCl.sub.3) δ 5.31 (d, J=5.2 Hz, 1H), 2.41 (d, J=13.2 Hz, 1H), 2.10-1.95 (m, 3H), 1.94-1.62 (m, 42H), 1.52-1.22 (m, 17H), 1.22-1.20 (m, 1H), 1.15 (s, 3H), 1.10 (s, 3H), 1.05 (s, 3H), 1.04-1.00 (m, 3H), 1.00-0.85 (m, 9H), 0.67 (s, 3H).
(243) Preparation of Compound 7-3.
(244) A mixture of 7-2 (100 mg, 2.59 mmol) and 10% Pd/C (140 mg) in EtOAc (30 mL) was hydrogenated for 16 h at 50° C. under H.sub.2 (50 psi). The reaction mixture was filtered through a pad of celite and the pad was washed with EtOAc (20 mL×3). The combined filtrates were concentrated. The residue was purified by column chromatography on silica gel (eluent: petroleum ether:ethyl acetate=15:1) to afford 7-3 (35 mg, 35%) and 7-3A (19 mg, 19%) as white powder. .sup.1H NMR (7-3): (400 MHz, CDCl3) δ 2.02-1.92 (m, 1H), 1.90-1.77 (m, 1H), 1.70-1.38 (m, 14H), 1.36-1.29 (m, 6H), 1.28-1.20 (m, 8H), 1.20-1.08 (m, 6H), 1.07-0.96 (m, 4H), 0.96-0.84 (m, 7H), 0.82 (s, 3H), 0.70-0.60 (m, 4H). .sup.1H NMR (7-3A): (400 MHz, CDCl3) δ 1.98-1.80 (m, 4H), 1.67-1.48 (m, 6H), 1.45-1.33 (m, 9H), 1.32-1.23 (m, 10H), 1.22-1.18 (m, 4H), 1.17-1.10 (m, 6H), 1.10-0.97 (m, 4H), 0.94 (s, 3H), 0.93-0.87 (m, 6H), 0.64 (s, 3H).
(245) Preparation of 7-4 and 7-5
(246) To a solution of compound 7-1 (1.5 g, 3.88 mmol) in dry THF (15 mL) was added n-BuLi (12.5 mL, 31 mmol, 2.5 M in THF) dropwise at −78° C. The resulting mixture was stirred at this temperature for 0.5 h, and then the temperature was allowed to warm to room temperature and stirred at this temperature for another 18 h. TLC (PE/EA=5/1) showed the reaction was complete. The mixture was quenched with saturated aqueous NH.sub.4Cl and extracted with EtOAc (30 mL×3). The combined organic layers were washed with brine (10 mL), dried over sodium sulfate and concentrated in vacuum. The residue was purified by column chromatography on silica gel (eluent: PE:EA=20:1) to give 7-2 (800 mg, 46.4%) as white powder, which was split by SFC to give 7-4 (207 mg) and 7-5 (360 mg) as white powder. .sup.1H NMR (7-4): (400 MHz, CDCl3) δ 5.38-5.29 (m, 1H), 2.44 (d, 1H, J=12.5 Hz), 2.04-1.69 (m, 6H), 1.57-1.25 (m, 18H), 1.20-0.89 (m, 23H), 0.70 (s, 3H). 1H NMR (7-5): (400 MHz, CDCl3) δ 5.32 (s, 1H), 2.44 (d, 1H, J=12.3 Hz), 2.08-1.68 (m, 6H), 2.55-1.25 (m, 17H), 2.22-0.85 (m, 24H), 0.70 (s, 3H).
(247) Preparation of 7-6
(248) To a solution of 7-4 (0.17 g, 0.38 mmol) in 15 mL EtOH was added Pd/C (100 mg) then the reaction mixture was stirred under hydrogen (50 psi) at 50° C. for 24 h. The resulting solution was filtered and concentrated. The product was purified by column chromatograph on silica gel elude with (PE:EA=20:1) to give 7-6 (40 mg, yield: 23.42%) as white solid. .sup.1H NMR (7-6) (400 MHz, CDCl.sub.3), δ 1.97-1.94 (m, 1H), 1.88-1.76 (m, 1H), 1.71-1.59 (m, 3H), 1.56-1.23 (m, 2111), 1.23-0.86 (m, 1911), 0.81 (s, 311), 0.65 (s, 311).
(249) Preparation of 7-7
(250) To a solution of 7-5 (0.23 g, 0.52 mmol) in 15 mL EtOH was added Pd/C (200 mg), then the reaction mixture was stirred under hydrogen (50 psi) at 50° C. for 24 h. The resulting solution was filtered and concentrated. The product was purified by column chromatograph on silica gel elude with (PE:EA=20:1) to give 7-7 (70 mg, yield: 30.3%) as white solid. H NMR (7-7) (400 MHz, CDCl.sub.3), δ (ppm) 1.99-1.92 (m, 1H), 1.88-1.78 (m, 1H), 1.70-1.52 (m, 6H), 1.46-1.20 (m, 21H). 1.18-0.87 (m, 20H), 0.81 (s, 3H), 0.65 (s, 3H).
Example 8
(251) ##STR00046##
(252) Preparation of 8-2.
(253) To a solution of compound 8-1 (100 mg, 0.25 mmol) in toluene (8 mL) was added dropwise a solution of i-PrMgBr (1.5 mL, 1.5 mmol, 1 M in THF) at room temperature during a period of 10 min under nitrogen. Then the reaction mixture was stirred at room temperature for 12 h. TLC showed that the starting material was consumed completely. The mixture was poured into aqueous saturated NH.sub.4Cl solution (20 mL) and extracted with EtOAc (50 mL×2). The combined organic phases were dried over Na.sub.2SO.sub.4, and the solvent was evaporated to afford crude product. The crude product purified by column chromatography on silica gel (eluent: petroleum ether:ethyl acetate=8:1) to give the product 8-2 (66 mg, 59.46%) as white powder. .sup.1H NMR: (400 MHz, CDCl3) δ 5.30 (d, J=5.2 Hz, 1H), 2.43-2.40 (m, 1H), 2.04-1.55 (m, 3H), 1.88-1.66 (m, 5H), 1.58-1.13 (m, 15H), 1.11 (s, 3H), 1.08 (s, 3H), 1.01 (s, 3H), 0.96-0.90 (m, 6H), 0.90-0.86 (m, 3H), 0.68 (s, 3H).
(254) Preparation of 8-3 and 8-4.
(255) To a solution of compound 8-2 (60 mg, 0.14 mmol) in EtOAc (15 mL) was added 10% Pd/C (20 mg) under argon. The suspension was degassed under vacuum and purged with H.sub.2 several times. The mixture was stirred under H.sub.2 (50 Psi) at 50° C. overnight. The suspension was filtered through a pad of celite and the pad was washed with EA (20 mL×3). The combined filtrates were concentrated in vacuum and the residue was purified by column chromatography on silica gel (eluent: petroleum ether:ethyl acetate=10:1) to give 8-3 (27 mg, 45%) and 8-4 (9 mg, 15%) as white powder. .sup.1H NMR (8-3): (400 MHz, CDCl3) δ 1.97-1.94 (m, 1H), 1.85-1.78 (m, 2H), 1.74-1.42 (m, 12H), 1.48-1.20 (m, 12H), 1.18-1.09 (m, 3H), 1.07 (s, 3H), 1.02-0.98 (m, 2H), 0.93-0.88 (m, 6H), 0.88-0.86 (m, 3H), 0.80 (s, 3H), 0.63 (s, 3H). .sup.1H NMR (8-4): (400 MHz, CDCl3) δ1.98-1.95 (m, 1H), 1.89-1.79 (m, 3H), 1.75-1.54 (m, 7H), 1.48-1.24 (m, 16H), 1.23 (s, 3H), 1.19-1.11 (m, 4H), 1.08 (s, 4H), 0.95 (s, 3H), 0.94-0.88 (m, 6H), 0.88-0.86 (m, 3H), 0.63 (s, 3H).
(256) Preparation of 8-7 and 8-8.
(257) To a solution of compound 8-1 (1500 mg, 3.88 mmol) in dry THF (30 mL) was added a solution of i-PrMgCl (11.6 mL, 23.3 mmol) dropwise at 0° C. The mixture was stirred at 40° C. for 16 h. TLC (PE/EtOAc=2/1) showed the reaction was complete. Saturated aqueous NH.sub.4Cl (5 mL) was added slowly to quench the reaction. The resulting solution was separated between EtOAc (30 mL×3) and H.sub.2O (30 mL). The combined organic layers were concentrated in vacuum and the residue was purified by silica gel column eluted with PE/EtOAc=10/1 to give the mixture of the diastereomeric pair (800 mg) as white power. The diastereimeric pair was separated by prep-SFC to give 8-8 (317 mg, 19.0%) and 8-7 (250 mg, 15.0%) as white solid. .sup.1H NMR (8-8): (400 MHz, CDCl.sub.3) δ 5.30 (s, 1H), 2.42 (d, J=12.4 Hz, 1H), 2.01-1.99 (m, 3H), 1.89-1.65 (m, 4H), 1.59-1.58 (m, 1H), 1.51-1.26 (m, 9H), 1.20-1.05 (m, 12H), 1.04-0.99 (m, 4H), 0.94-0.88 (m, 10H), 0.68 (s, 3H). .sup.1H NMR (8-7): (400 MHz, CDCl.sub.3) δ 5.30 (d, J=3.6 Hz, 1H), 2.42 (d, J=12.4 Hz, 1H), 2.00-1.97 (m, 3H), 1.89-1.68 (m, 4H), 1.58-1.25 (m, 10H), 1.19-1.08 (m, 10H), 1.03-0.98 (m, 4H), 0.95-0.88 (m, 10H), 0.68 (s, 3H).
(258) Preparation of 8-6.
(259) A mixture of 8-8 (200 mg, 0.464 mmol) and Pd/C (100 mg, cat.) in EtOAc (30 mL) was hydrogenated under 50 psi of hydrogen for 48 h at 50° C. The reaction mixture was filtered through a celite pad. The pad was washed with EtOAc (50 mL). The filtrate was concentrated in vacuum and the residue was purified by silica gel column eluted with PE/EtOAc=20/1 to give 8-6 (85.9 mg, 42.8%) as a white solid and 8-6A (17.6 mg, 8.8%) as a white solid. 1H NMR (8-6) (400 MHz, CDCl.sub.3), δ (ppm) 1.97-1.94 (d, 1H, J=12.8 Hz), 1.88-1.79 (m, 1H), 1.71-1.61 (m, 3H), 1.54-1.45 (m, 3H), 1.36-1.19 (m, 13H), 1.16-0.96 (m, 12H), 0.92-0.87 (m, 10H), 0.80 (s, 3H), 0.68-0.65 (m, 4H). 1H NMR (8-6A) (400 MHz, CDCl.sub.3), δ (ppm) 1.98-1.95 (d, 1H, J=10.8 Hz), 1.88-1.79 (m, 3H), 1.71-1.59 (m, 3H), 1.53-1.48 (m, 2H), 1.42-1.31 (m, 6H), 1.27-0.96 (m, 20H), 0.92-0.87 (m, 12H), 0.80 (s, 3H), 0.64 (s, 3H).
(260) Preparation of 8-5
(261) A mixture of 8-7 (150 mg, 0.348 mmol, 1.0 eq) and Pd/C (75 mg, cat.) in EtOAc (20 mL) was hydrogenated under 50 psi of hydrogen for 48 h at 50° C. The reaction mixture was filtered through a celite pad. The pad was washed with EtOAc (50 mL). The filtrate was concentrated in vacuum and the residue was purified by silica gel column eluted with PE/EtOAc=20/1 to give 8-5 (89.0 mg, 44.3%) as a white solid and 8-5A (4.6 mg, 2.3%) as a white solid. 1H NMR (8-5) (400 MHz, CDCl.sub.3), δ (ppm) 1.97-1.94 (d, 1H, J=12.8 Hz), 1.88-1.79 (m, 1H), 1.71-1.61 (m, 3H), 1.54-1.45 (m, 3H), 1.36-1.19 (m, 13H), 1.16-0.96 (m, 12H), 0.92-0.87 (m, 10H), 0.80 (s, 3H), 0.68-0.65 (m, 4H). .sup.1H NMR (8-5A) (400 MHz, CDCl.sub.3), δ (ppm) 1.98-1.95 (d, 1H, J=10.8 Hz), 1.91-1.79 (m, 3H), 1.72-1.64 (m, 2H), 1.54-1.50 (m, 1H), 1.46-1.00 (m, 28H), 0.96-0.87 (m, 12H), 0.64 (s, 3H).
Example 9
(262) ##STR00047##
(263) Preparation of 9-2.
(264) To a solution of compound 9-1 (100 mg, 0.25 mmol) in THF (2 mL) was added dropwise a solution of CyclopropylmagnesiumBromide (2.5 mL, 2.5 mmol, 1 M in THF) at room temperature during a period of 10 min under nitrogen. Then the reaction mixture was stirred at room temperature for 12 h. TLC showed that the starting material was consumed completely. The mixture was poured into aqueous saturated NH.sub.4Cl solution (20 mL) and extracted with EtOAc (50 mL×2). The combined organic phases were dried over Na.sub.2SO.sub.4, and the solvent was evaporated to afford crude product. The crude product purified by column chromatography on silica gel (eluent: petroleum ether:ethyl acetate=10:1) to give the product 9-2 (33 mg, 30%) as white powder. .sup.1H NMR: (400 MHz, CDCl3) δ: 5.31 (d, J=5.2 Hz, 1H), 2.42 (d, J=12.8 Hz, 1H), 2.08-1.93 (m, 3H), 1.90-1.65 (m, 3H), 1.62-1.27 (m, 13H), 1.22-1.08 (m, 11H), 1.01 (s, 3H), 1.00-0.85 (m, 6H), 0.68 (s, 3H), 0.40-0.25 (m, 4H).
(265) Preparation of 9-3 and 9-4
(266) Compound 9-2 (200 mg, 0.46 mmol) was separated by SFC to get 9-3 (90 mg) and 9-4 (100 mg) as white solid, (total yield: 95%). .sup.1H NMR: (9-3) (400 MHz, CDCl.sub.3) δ 5.31-5.30 (m, 1H), 2.44-2.41 (m, 1H), 2.02-1.99 (m, 3H), 1.95-1.60 (m, 3H), 1.50-1.25 (m, 9H), 1.20-1.05 (m, 11H), 1.02-0.93 (m, 11H), 0.68 (s, 3H), 0.35-0.28 (m, 4H). .sup.1H NMR: (9-4) (400 MHz, CDCl.sub.3) δ 5.31-5.30 (m, 1H), 2.44-2.41 (m, 1H), 2.02-1.95 (m, 3H), 1.93-1.60 (m, 3H), 1.50-1.25 (m, 10H), 1.20-1.05 (m, 11H), 1.02-0.93 (m, 11H), 0.68 (s, 3H), 0.36-0.24 (m, 4H)
(267) Preparation of 9-7
(268) To a solution of compound 9-3 (100 mg, 0.23 mmol) in EtOAc (8 mL) was added Pd/C (10%, 200 mg) under N.sub.2. The suspension was degassed under vacuum and purged with H.sub.2 several times. Then the mixture was stirred under H.sub.2 (50 psi) at 50° C. for 24 hours. The suspension was filtered through a pad of celite and the pad was washed with EtOAc (30 mL×2). The combined filtrates were concentrated to dryness to give the crude product, which was purified by column chromatography on silica gel (petroleum ether:ethyl acetate=20:1) to afford 9-7 (27.8 mg, 27.8%) as white solid. .sup.1H NMR: (9-7) (400 MHz, CDCl.sub.3) δ 1.97-1.94 (m, 1H), 1.90-1.80 (m, 1H), 1.64-1.57 (m, 3H), 1.54-1.30 (m, 7H), 1.28-0.85 (m, 25H), 0.80 (s, 3H), 0.65-0.60 (m, 4H), 0.36-0.33 (m, 4H). .sup.1H NMR: (9-7A) (400 MHz, CDCl.sub.3) δ 1.95-1.83 (m, 4H), 1.70-1.57 (m, 1H), 1.45-1.11 (m, 22H), 1.05-0.85 (m, 17H), 0.65 (s, 3H), 0.36-0.34 (m, 4H)
(269) Preparation of 9-8
(270) To a solution of compound 9-4 (100 mg, 0.23 mmol) in EtOAc (8 mL) was added Pd/C (10%, 200 mg) under N.sub.2. The suspension was degassed under vacuum and purged with H.sub.2 several times. Then the mixture was stirred under H.sub.2 (50 psi) at 50° C. for 24 hours. The suspension was filtered through a pad of celite and the pad was washed with EtOAc (30 mL×2). The combined filtrates were concentrated to dryness to give the crude product, which was purified by HPLC to afford 9-8 (18.3 mg, 18%) as white solid. .sup.1H NMR: (9-8) (400 MHz, CDCl.sub.3) δ 1.97-1.94 (m, 1H), 1.90-1.80 (m, 1H), 1.60-1.57 (m, 3H), 1.54-1.20 (m, 16H), 1.19-0.82 (m, 16H), 0.80 (s, 3H), 0.65-0.60 (m, 4H), 0.36-0.28 (m, 4H)
Example 10
(271) ##STR00048## ##STR00049## ##STR00050## ##STR00051## ##STR00052##
(272) Preparation of 10-2.
(273) To a solution of compound 10-1 (100 mg, 0.25 mmol) in toluene (8 mL) was added dropwise a solution of ethynylmagnesium bromide (4 mL, 2.0 mmol, 0.5 M in THF) at room temperature during a period of 10 min under nitrogen. Then the reaction mixture was stirred at 50° C. over night. TLC showed that the starting material was consumed completely. The mixture was poured into aqueous saturated NH.sub.4Cl solution (10 mL) and extracted with EtOAc (25 mL×2). The combined organic phases were dried over Na.sub.2SO.sub.4, and the solvent was evaporated to afford crude product. The crude product purified by column chromatography on silica gel (eluent: petroleum ether:ethyl acetate=10:1) to give the product 10-2 (80 mg, 74.98%) as white powder. .sup.1H NMR: (400 MHz, CDCl3) δ 5.30 (d, J=5.2 Hz, 1H), 2.43-2.40 (m, 2H), 2.06-1.81 (m, 5H), 1.80-1.67 (m, 3H), 1.67-1.59 (m, 2H), 1.49 (s, 3H), 1.48-1.42 (m, 2H), 1.40-1.24 (m, 4H), 1.20-1.13 (m, 2H), 1.10 (s, 3H), 0.96-0.92 (m, 3H), 0.69 (s, 3H).
(274) Preparation of 10-3 and 10-4.
(275) Compound 10-2 (350 mg, 0.849 mmol) was split by SFC to get 10-3 (82 mg) and 10-4 (94 mg) as white powder (total yield: 50%). .sup.1H NMR (.sup.a10-3) (400 MHz, CDCl.sub.3), δ 5.29 (d, J=5.2 Hz, 1H), 2.43-2.40 (m, 2H), 2.05-0.95 (m, 38H), 0.68 (s, 3H). 1H NMR (10-4) (400 MHz, CDCl.sub.3), δ 5.29 (d, J=5.2 Hz, 1H), 2.43-2.40 (m, 2H), 2.05-0.95 (m, 38H), 0.68 (s, 3H).
(276) Preparation of 10-5.
(277) To a solution of compound 10-1 (3.0 g, 7.76 mmol) in a mixture solvent of EtOAc (20 mL) and EtOH (10 mL) was added Pd/C (33%, 1.0 g) under N.sub.2. The suspension was degassed under vacuum and purged with H.sub.2 several times. Then the mixture was stirred under H.sub.2 (50 psi) at 50° C. for 6 days. The suspension was filtered through a pad of celite and the pad was washed with EtOAc (100 mL×3). The combined filtrates were concentrated to dryness to give the crude product, which was purified by column chromatography on silica gel (petroleum ether:ethyl acetate=20:1) to afford 10-5 (1.7 g, 56%) as a white solid. .sup.1H NMR: (400 MHz, CDCl.sub.3) δ 2.48-2.44 (m, 1H), 2.43-2.40 (m, 1H), 2.13 (s, 3H), 1.95-1.25 (m, 20H), 1.23 (s, 3H), 1.22-1.00 (m, 8H), 0.90-0.88 (d, J=6.4 Hz, 3H), 0.80 (s, 3H), 0.63-0.60 (m, 4H)
(278) Preparation of 10-6.
(279) To a solution of 10-5 (550 mg, 1.41 mmol) in dry THF (10 mL) was added ethynylmagnesium bromide (28.2 mL, 14.1 mmol) dropwise at 0° C. under N.sub.2. Then the reaction mixture was stirred at room temperature for 12 hours. TLC showed the starting material was consumed completely. The mixture was quenched by saturated aqueous NH.sub.4Cl (80 mL) and extracted with EtOAc. The organic phase was washed with brine, dried over anhydrous Na.sub.2SO.sub.4 then concentrated by vacuum. The residue was purified by column chromatography on silica gel (petroleum ether:ethyl acetate=15:1) to afford 10-6 (380 mg, 64%) as a white solid. .sup.1H NMR: (400 MHz, CDCl.sub.3) δ 2.42 (s, 1H), 1.97-1.48 (m, 14H), 1.47 (s, 3H), 1.29-1.26 (m, 7H), 1.24 (s, 3H), 1.23-0.94 (m, 7H), 0.93-0.92 (d, J=6.4 Hz, 3H), 0.80 (s, 3H), 0.65-0.62 (m, 4H)
(280) Preparation of 10-6-Bz
(281) To a solution of 10-6 (250 mg, 0.60 mmol) in pyridine (3 mL) was added BzCl (168 mg, 1.2 mmol) dropwise at room temperature. Then the reaction mixture was stirred at 45° C. for 12 hours. TLC showed the starting material was consumed completely. The mixture was quenched by saturated aqueous water and extracted with EtOAc. The combined organic phase was washed with 1 M HCl (20 mL) and brine, dried over anhydrous Na.sub.2SO.sub.4 then concentrated by vacuum. The residue was purified by column chromatography on silica gel (petroleum ether:ethyl acetate=80:1) to 10-6-Bz (200 mg, 64%) as a white solid.
(282) Preparation of 10-8-Bz and 10-9-Bz
(283) Compound 10-6-Bz (200 mg, 0.39 mmol) was split by SFC to afford 10-8-Bz (80 mg, 40%) and 10-9-Bz (70 mg, 35%) as white solid. .sup.1H NMR: (10-8-Bz) (400 MHz, CDCl.sub.3) δ 7.99-7.98 (d, J=7.6 Hz, 2H), 7.51-7.49 (d, J=7.2 Hz, 1H), 7.42-7.38 (t, J=7.2 Hz, 2H), 2.42 (s, 1H), 2.05-1.68 (m, 8H), 1.65 (s, 3H), 1.60-1.49 (m, 7H), 1.48 (s, 3H), 1.45-1.11 (m, 16H), 0.94-0.92 (d, J=6.4 Hz, 3H), 0.87 (s, 3H), 0.66-0.62 (m, 4H). .sup.1H NMR: (10-9-Bz) (400 MHz, CDCl.sub.3) δ 7.99-7.98 (d, J=7.6 Hz, 2H), 7.51-7.49 (d, J=7.2 Hz, 1H), 7.42-7.38 (t, J=7.6 Hz, 2H), 2.43 (s, 1H), 2.05-1.67 (m, 8H), 1.65 (s, 3H), 1.60-1.48 (m, 5H), 1.47 (s, 3H), 1.45-1.20 (m, 11H), 1.19-0.95 (m, 9H), 0.94-0.92 (d, J=6.8 Hz), 0.87 (s, 3H), 0.66-0.62 (m, 4H)
(284) Preparation of 10-8
(285) To a solution of compound 10-8-Bz (80 mg, 0.15 mmol) in a mixture solvent of THF (3 mL) and MeOH (1.5 mL) was added a solution of LiOH (180 mg, 7.5 mmol) in H.sub.2O (1.5 mL). The mixture was stirred at 40° C. for 3 days. TLC showed the starting material was consumed completely. The reaction mixture was treated with water and extracted with EtOAc. The combined organic phase was washed with brine, dried over anhydrous Na.sub.2SO.sub.4 then concentrated by vacuum. The residue was purified by column chromatography on silica gel (petroleum ether:ethyl acetate=8:1) to afford 10-8 (57 mg, 92%) as a white solid. .sup.1H NMR: (400 MHz, CDCl.sub.3) δ 2.42 (s, 1H), 1.93-1.49 (m, 11H), 1.48 (s, 3H), 1.35-1.20 (m, 16H), 1.19-0.94 (m, 5H), 0.93-0.92 (d, J=6.4 Hz, 3H), 0.80 (s, 3H), 0.65-0.62 (m, 4H)
(286) Preparation of 10-9
(287) To a solution of compound 10-9-Bz (70 mg, 0.14 mmol) in a mixture solvent of THF (3 mL) and MeOH (1.5 mL) was added a solution of LiOH (168 mg, 7.0 mmol) in H.sub.2O (1.5 mL). The mixture was stirred at 40° C. for 3 days. TLC showed the starting material was consumed completely. The reaction mixture was treated with water and extracted with EtOAc. The combined organic phase was washed with brine, dried over anhydrous Na.sub.2SO.sub.4 then concentrated by vacuum. The residue was purified by column chromatography on silica gel (petroleum ether:ethyl acetate=8:1) to afford 10-9 (53 mg, 91%) as a white solid. .sup.1H NMR: (400 MHz, CDCl.sub.3) δ 2.42 (s, 1H), 1.93-1.49 (m, 11H), 1.48 (s, 3H), 1.29-0.94 (m, 21H), 0.93-0.92 (d, J=6.4 Hz, 3H), 0.80 (s, 3H), 0.65-0.62 (m, 4H)
(288) Preparation of 10-7
(289) To a solution of 10-5 (550 mg, 1.41 mmol) in dry THF (10 mL) was added vinylmagnesium bromide (9.87 mL, 9.87 mmol) dropwise at 0° C. under N.sub.2. Then the reaction mixture was stirred at room temperature for 12 hours. TLC showed the starting material was consumed completely. The mixture was quenched by saturated aqueous NH.sub.4Cl (30 mL) and extracted with EtOAc. The organic phase was washed with brine, dried over anhydrous Na.sub.2SO.sub.4 then concentrated by vacuum. The residue was purified by column chromatography on silica gel (petroleum ether:ethyl acetate=15:1) to afford 10-7 (300 mg, 51%) as a white solid. .sup.1H NMR: (400 MHz, CDCl.sub.3) δ 5.93-5.86 (m, 1H), 5.20-5.16 (d, J=17.6 Hz, 1H), 5.05-5.02 (d, J=10.8 Hz, 1H), 1.96-193 (m, 1H), 1.60-1.57 (m, 4H), 1.51-1.20 (m, 20H), 1.19-1.00 (m, 8H), 0.91-0.89 (d, J=6 Hz, 3H), 0.80 (s, 3H), 0.64-0.60 (m, 4H)
(290) Preparation of 10-7-Bz.
(291) To a solution of 10-7 (220 mg, 0.53 mmol) in pyridine (3 mL) was added BzCl (150 mg, 1.06 mmol) dropwise at room temperature. Then the reaction mixture was stirred at 40° C. for 12 hours. TLC showed the starting material was consumed completely. The mixture was quenched by saturated aqueous water and extracted with EtOAc. The combined organic phase was washed with 1 M HCl (30 mL) and brine, dried over anhydrous Na.sub.2SO.sub.4 then concentrated by vacuum. The residue was purified by column chromatography on silica gel (petroleum ether:ethyl acetate=80:1) to afford 10-7-Bz (150 mg, 54%) as a white solid.
(292) Preparation of 10-10-Bz and 10-11-Bz
(293) Compound 10-7-Bz (190 mg, 0.37 mmol) was split by SFC to get 10-10-Bz (75 mg, 39%) and 10-11-Bz (70 mg, 37%) as white solid. .sup.1H NMR: (10-10-Bz) (400 MHz, CDCl.sub.3) δ 7.99-7.97 (d, J=7.2 Hz, 1H), 7.51-7.49 (d, J=7.6 Hz, 1H), 7.42-7.38 (t, J=8.0 Hz, 2H), 5.93-5.86 (dd, J.sub.1=11.2 Hz, J.sub.2=17.2, 1H), 5.21-5.16 (d, J=17.6 Hz, 1H), 5.05-5.02 (d, J=10.4 Hz, 1H), 2.05-1.75 (m, 8H), 1.65-1.27 (m, 19H), 1.26 (s, 3H), 1.25-0.93 (m, 10H), 0.91-0.90 (d, 6.0 Hz, 3H), 0.86 (s, 3H), 0.70-0.64 (m, 4H).sup.1H NMR: (10-11-Bz) (400 MHz, CDCl.sub.3) δ 7.99-7.97 (d, J=7.2 Hz, 1H), 7.51-7.49 (d, J=7.6 Hz, 1H), 7.42-7.38 (t, J=8.0 Hz, 2H), 5.93-5.86 (dd, J.sub.1=10.8 Hz, J.sub.2=17.6, 1H), 5.20-5.16 (d, J=17.2 Hz, 1H), 5.05-5.02 (d, J=10.4 Hz, 1H), 2.05-1.75 (m, 8H), 1.65-1.27 (m, 10H), 1.26 (s, 3H), 1.25-0.93 (m, 10H), 0.91-0.90 (d, 6.4 Hz, 3H), 0.86 (s, 3H), 0.70-0.64 (m, 4H)
(294) Preparation of 10-10.
(295) To a solution of compound 10-10-Bz (75 mg, 0.14 mmol) in a mixture solvent of THF (3 mL) and MeOH (1.5 mL) was added a solution of LiOH (168 mg, 7.0 mmol) in H.sub.2O (1.5 mL). The mixture was stirred at 40° C. for 3 days. TLC showed the starting material was consumed completely. The reaction mixture was treated with water and extracted with EtOAc. The combined organic phase was washed with brine, dried over anhydrous Na.sub.2SO.sub.4 then concentrated by vacuum. The residue was purified by column chromatography on silica gel (petroleum ether:ethyl acetate=8:1) to afford 10-10 (55 mg, 94%) as a white solid. .sup.1H NMR: (400 MHz, CDCl.sub.3) δ 1.96-1.92 (m, 1H), 1.90-1.70 (m, 2H), 1.69-1.57 (m, 5H), 1.55-1.20 (m, 18H), 1.19-0.81 (m, 10H), 0.80 (s, 3H), 0.70-0.60 (m, 4H)
(296) Preparation of 10-11-Bz.
(297) To a solution of compound 10-11-Bz (70 mg, 0.13 mmol) in a mixture solvent of THF (3 mL) and MeOH (1.5 mL) was added a solution of LiOH (168 mg, 7.0 mmol) in H.sub.2O (1.5 mL). The mixture was stirred at 40° C. for 3 days. TLC showed the starting material was consumed completely. The reaction mixture was treated with water and extracted with EtOAc. The combined organic phase was washed with brine, dried over anhydrous Na.sub.2SO.sub.4 then concentrated by vacuum. The residue was purified by column chromatography on silica gel (petroleum ether:ethyl acetate=8:1) to afford 10-11 (49 mg, 91%) as a white solid. .sup.1H NMR: (400 MHz, CDCl.sub.3) δ 1.96-1.92 (m, 1H), 1.90-1.70 (m, 2H), 1.69-1.57 (m, 5H), 1.55-1.20 (m, 18H), 1.19-0.81 (m, 10H), 0.80 (s, 3H), 0.70-0.60 (m, 4H)
(298) Preparation of 10-22 and 10-23.
(299) To a solution of 10-14 (550 mg, 1.27 mmol) in THF (10 mL) was added NaH (254 mg, 6.36 mmol) at 0° C., and stirred at the same temperature for 30 minutes. Then CH.sub.3I (127 mg, 0.770 mmol) was added dropwise to the mixture. The reaction was monitored by TLC. After 1 h, 127 mg of CH.sub.3I was added in two portions. After stirring at room temperature for 1.5 h, the reaction mixture was quenched with aqueous NH.sub.4Cl (20 mL), extracted with EtOAc (20 mL×3), dried over Na.sub.2SO.sub.4 and concentrated to give crude product. The crude product was purified by column chromatography on silica gel (petroleum ether/ethyl acetate=15/1) to give 10-14 as a white powder. The diastereomeric pairs (340 mg) were separated by prep-SFC to give 10-22 (130 mg, 22.9%) as a white power and 10-23 (135 mg, 23.8%) as a white power. .sup.1H NMR (10-22): (400 MHz, CDCl.sub.3) δ 5.30 (s, 1H), 3.65-3.53 (m, 2H), 3.35 (s, 3H), 3.04 (br, 1H), 2.44-2.40 (d, 1H, J=13.6 Hz), 2.02-1.95 (m, 3H), 1.86-1.64 (m, 5H), 1.62-1.58 (m, 1H), 1.52-1.23 (m, 9H), 1.17-1.05 (m, 11H), 1.04-0.98 (m, 4H), 0.95-0.93 (d, 4H, J=6.8 Hz), 0.68 (s, 3H). .sup.1H NMR (10-23): (400 MHz, CDCl.sub.3) δ 5.30 (s, 1H), 3.61 (t, 2H, J=6.0 Hz), 3.35 (s, 3H), 3.04 (br, 1H), 2.44-2.40 (d, 1H, J=12.8 Hz), 2.02-1.95 (m, 3H), 1.86-1.64 (m, 5H), 1.57-1.25 (m, 12H), 1.16-0.93 (m, 17H), 0.68 (s, 3H).
(300) Preparation of 10-17.
(301) A mixture of 10-22 (100 mg, 0.224 mmol) and Pd/C (50 mg, cat.) in EtOAc (10 mL) was hydrogenated under 50 psi of hydrogen for 48 h at 50° C. The reaction mixture was filtered through a celite pad. The pad was washed with EtOAc (40 mL). The filtrate was concentrated in vacuum and the residue was purified by silica gel column eluted with PE/EtOAc=15/1 to give 10-17 (68.4 mg, 68.1%) as a white solid. 1H NMR (10-17) (400 MHz, CDCl.sub.3), δ 3.62-3.58 (m, 2H), 3.35 (s, 3H), 3.07 (br, 1H), 1.97-1.93 (d, 1H, J=12.8 Hz), 1.83-1.74 (m, 2H), 1.69-1.55 (m, 5H), 1.50-1.43 (m, 3H), 1.37-1.23 (m, 12H), 1.16-0.97 (m, 10H), 0.93-0.91 (d, 1H, J=6.0 Hz), 0.80 (s, 3H), 0.68-0.64 (m, 3H).
(302) Preparation of 10-19.
(303) A mixture of 10-23 (100 mg, 0.224 mmol) and Pd/C (50 mg, cat.) in EtOAc (10 mL) was hydrogenated under 50 psi of hydrogen for 48 h at 50° C. The reaction mixture was filtered through a celite pad. The pad was washed with EtOAc (40 mL). The filtrate was concentrated in vacuum and the residue was purified by silica gel column eluted with PE/EtOAc=15/1 to give 10-19 (68.6 mg, 68.3%) as a white solid. 1H NMR (10-19) (400 MHz, CDCl.sub.3), δ 3.60 (t, 2H, J=6.0 Hz), 3.35 (s, 3H), 3.07 (br, 1H), 1.97-1.94 (d, 1H, J=12.8 Hz), 1.81-1.57 (m, 6H), 1.54-1.43 (m, 4H), 1.36-1.22 (m, 12H), 1.16-0.97 (m, 10H), 0.92-0.91 (d, 1H, J=6.0 Hz), 0.80 (s, 3H), 0.68-0.61 (m, 3H).
(304) Preparation of 10-7.
(305) To a solution of 10-6 (60 mg, 0.14 mmol) in EtOAc (2 mL) was adde lindlar cat (24 mg). Then the mixture was stirred under hydrogen (1 atm) at room temperature for 1.5 hours. The mixture was filtered through a pad of celite and the filtrate was evaporated under reduced pressure. The residue was purified by column chromatography on silica gel (eluent: petroleum ether:ethyl acetate=10:1) to afford the pure product 10-7 (26 mg, 43.0%) as white powder. .sup.1H NMR: (400 MHz, CDCl3) δ 5.93-5.85 (m, 1H), 5.20-5.16 (d, J=17.2 Hz, 1H), 5.05-5.02 (d, J=10.8 Hz, 1H), 1.96-1.93 (m, 1H), 1.79-1.67 (m, 1H), 1.66-1.57 (m, 4H), 1.55-1.36 (m, 11H), 1.35-1.27 (m, 9H), 1.26-0.97 (m, 8H), 0.96-0.89 (m, 3H), 0.81 (s, 3H), 0.68-0.62 (m, 4H).
(306) Preparation of Compound 10-12.
(307) To a solution of 10-1 (50 mg, 0.13 mmol) in THF (2 mL), vinyl magnesium bromide solution (1 mmol, 1 M in THF, 1 mL) was added drop-wise at −50° C. The reaction mixture was warmed to room temperature and stirred at room temperature for 16 hours. TLC (petroleum ether:ethyl acetate=3:1) showed the reaction was finished, the reaction mixture was quenched with aq. saturated NH.sub.4Cl solution (10 mL) and then extracted with EtOAc (10 mL×3). The combined organic layer was washed with brine (10 mL×2), dried over anhydrous Na.sub.2SO.sub.4 and concentrated in vacuum. The residue was purified by column chromatography on silica gel (eluent: petroleum ether:ethyl acetate=15/1) to afford 10-12 (27 mg, 54%) as white powder. .sup.1H NMR: (400 MHz, CDCl3) δ 5.94-5.86 (m, 1H), 5.30 (d, J=5.2 Hz, 1H), 5.19 (d, J=17.2 Hz, 1H), 5.04 (d, J=10.4 Hz, 1H), 2.42 (d, J=12.8 Hz, 1H), 2.01-1.95 (m, 3H), 1.80-1.61 (m, 4H), 1.56-1.37 (m, 10H), 1.27 (s, 3H), 1.18-1.13 (m, 3H), 1.11 (s, 3H), 1.10-1.04 (m, 3H), 1.01 (s, 3H), 1.00-0.95 (m, 2H), 0.92 (d, J=6.4 Hz, 3H), 0.67 (s, 3H).
(308) Preparation of 10-12A and 10-12B.
(309) Compound 10-12 (350 mg, 0.84 mmol) was split by SFC to give 10-12A (160 mg) and 10-12B (110 mg) as a white solid (total yield: 77%). .sup.1H NMR (10-12-A): (400 MHz, CDCl.sub.3) δ 5.94-5.86 (m, 1H), 5.30 (d, J=5.2 Hz, 1H), 5.19 (d, J=17.2 Hz, 1H), 5.04 (d, J=10.4 Hz, 1H), 2.50-2.40 (m, 1H), 2.05-0.85 (m, 36H), 0.67 (s, 3H). .sup.1H NMR (10-12-B): (400 MHz, CDCl.sub.3) δ 5.94-5.86 (m, 1H), 5.30 (d, J=5.2 Hz, 1H), 5.19 (d, J=17.2 Hz, 1H), 5.04 (d, J=10.4 Hz, 1H), 2.50-2.40 (m, 1H), 2.05-0.85 (m, 36H), 0.67 (s, 3H).
(310) Preparation of Compound 10-13.
(311) To a solution of 10-12 (500 mg, 1.21 mmol) in THF (5 mL) was added 9-BBN (24.2 mL, 12.1 mmol) gradually at 0° C. under N.sub.2 protection. The mixture was stirred at 60° C. for 16 hours. Then the reaction mixture was cooled to 0° C., and 10% aqueous NaOH (10 mL), 30% H.sub.2O.sub.2 (5 mL) was added. The resulting mixture was stirred at 0° C. for 2 hours. The reaction mixture was quenched with aqueous Na.sub.2S.sub.2O.sub.3 (10 mL), extracted with EtOAc (10 mL×3), dried over Na.sub.2SO.sub.4 and concentrated to give crude product. The crude product was purified by pre-HPLC to give 10-13 (100 mg, 19.2%) as white solid. .sup.1H NMR: (300 MHz, CD.sub.3OD) δ 5.32 (d, J=5.2 Hz, 1H), 3.70 (d, J=6.4 Hz, 2H), 2.51-2.35 (m, 1H), 2.14-1.84 (m, 4H), 1.82-1.26 (m, 16H), 1.24-1.10 (m, 7H), 1.08-1.00 (m, 7H), 1.00-0.93 (m, 4H), 0.73 (s, 3H).
(312) Preparation of Compound 10-14.
(313) To a solution of 10-13 (50 mg, 0.11 mmol) in THF (5 mL) was added NaH (13.2 mg, 0.55 mmol) at 0° C., and stirred at the same temperature for 30 minutes. Then CH.sub.3I (78 mg, 0.55 mmol) was added drop-wise to the mixture. The mixture was stirred at room temperature for 1 hour. The reaction mixture was quenched with aqueous NH.sub.4Cl (10 mL), extracted with EtOAc (10 mL×3), dried over Na.sub.2SO.sub.4 and concentrated to give crude product. The crude product was purified by column chromatography on silica gel (petroleum ether:ethyl acetate=5:1) to give 10-14 (13 mg, 25.2%) as white powder. .sup.1H NMR: (300 MHz, CDCl.sub.3) δ 5.23 (d, J=5.2 Hz, 1H), 3.54 (d, J=6.4 Hz, 2H), 3.29 (s, 3H), 2.38-2.34 (m, 1H), 1.95-1.88 (m, 3H), 1.74-1.58 (m, 5H), 1.52-1.19 (m, 14H), 1.10 (s, 3H), 1.09-1.05 (m, 1H), 1.04 (s, 3H), 1.02-0.94 (m, 2H), 0.91 (s, 3H), 0.87 (d, J=6.4 Hz, 3H), 0.61 (s, 3H).
(314) Preparation of 10-20 and 10-21.
(315) The crude product 10-13 was washed with EtOAc (30 mL) to give the diastereomeric pair (900 mg, 53.9%) as a white solid. The mixture (400 mg) was separated by SFC to give 10-20 (30 mg, 4.0%) as a white solid and 10-21 (68 mg, 9.2%) as a white solid. .sup.1H NMR (10-20): (400 MHz, Methaol-d4) δ 5.28 (s, 1H), 3.69 (t, 2H, J=7.2 Hz), 2.42-2.39 (d, 1H, J=11.6 Hz), 2.04-1.90 (m, 5H), 1.78-1.28 (m, 17H), 1.17-1.02 (m, 12H), 0.95-0.93 (d, 4H, J=6.8 Hz), 0.71 (s, 3H). .sup.1H NMR (10-21): (400 MHz, Methaol-d4) δ 5.28 (s, 1H), 3.68 (t, 2H, J=7.2 Hz), 2.42-2.39 (d, 1H, J=11.6 Hz), 2.04-1.90 (m, 5H), 1.78-1.28 (m, 16H), 1.18-0.98 (m, 13H), 0.95-0.93 (d, 4H, J=7.0 Hz), 0.71 (s, 3H).
(316) Preparation of 10-16.
(317) A mixture of 10-20 (20 mg, 0.046 mmol) and Pd/C (20 mg, cat.) in EtOAc (5 mL) was hydrogenated under 50 psi of hydrogen for 48 h at 50° C. The reaction mixture was filtered through a celite pad. The pad was washed with EtOAc (50 mL). The filtrate was concentrated in vacuum and the residue was purified by silica gel column eluted with PE/EtOAc=5/1 to give 10-16 (7.6 mg, 39.3%) as a white solid. 1H NMR (10-16) (400 MHz, Methaol-d4), δ 3.70 (t, 2H, J=7.2 Hz), 2.01-1.98 (d, 1H, J=12.4 Hz), 1.93-1.82 (m, 1H), 1.72-1.57 (m, 5H), 1.53-1.39 (m, 5H), 1.35-0.99 (m, 22H), 0.96-0.94 (d, 4H, J=6.4 Hz), 0.84 (s, 3H), 0.70-0.66 (m, 4H).
(318) Preparation of 10-18.
(319) A mixture of 10-21 (40 mg, 0.092 mmol, 1.0 eq) and Pd/C (20 mg, cat.) in EtOAc (5 mL) was hydrogenated under 50 psi of hydrogen for 48 h at 50° C. The reaction mixture was filtered through a celite pad. The pad was washed with EtOAc (50 mL). The filtrate was concentrated in vacuum and the residue was purified by silica gel column eluted with PE/EtOAc=5/1 to give 10-18 (12.9 mg, 32.1%) as a white solid. 1H NMR (10-18) (400 MHz, Methaol-d4), δ 3.68 (t, 2H, J=7.2 Hz), 1.99-1.96 (d, 1H, J=12.4 Hz), 1.92-1.82 (m, 1H), 1.68-1.58 (m, 5H), 1.52-1.41 (m, 5H), 1.37-0.97 (m, 22H), 0.94-0.92 (d, 4H, J=6.4 Hz), 0.82 (s, 3H), 0.67-0.65 (m, 4H).
Example 11
(320) ##STR00053## ##STR00054##
(321) Preparation of Compound 11-2.
(322) To a solution of crude compound 11-1 (30 g, 77 mmol) in dichloromethane (200 mL) was added imidazole (10.4 g, 154 mmol) and tert-butylchlorodimethylsilane (13.8 g, 92 mmol). The mixture was then stirred at 15° C. for 16 h. The mixture was washed with water, dried over anhydrous Na.sub.2SO.sub.4 and concentrated. The residue was purified by column chromatography on silica gel (petroleum ether:ethyl acetate=150:1 to 80:1) to give crude product of 11-2 (38 g, 98%) as white solid.
(323) Preparation of Compound 11-3.
(324) To a solution of diisopropylamine (34.3 g, 340 mmol) in THF (1 L) was added butyl lithium (136 mL, 340 mmol, 2.5 M in hexane) under nitrogen atmosphere at −78° C. The mixture was then stirred at −78° C. for 10 minutes and then 25° C. for 10 minutes and at last −78° C. for 10 minutes. A solution of crude compound 11-2 (34 g, 68 mmol) in THF (100 mL) was then added and stirred for 1 h at −78° C. To the mixture was then added triethyl phosphite (22.6 g, 136 mmol), the mixture was then stirred under oxygen atmosphere for 3 h at −78° C. and then 16 h at 25° C. To the mixture was then added ammonium chloride (aq.). The organic layer was separated, purified by column chromatography on silica gel (petroleum ether:ethyl acetate=10:1 to 3:1) to give crude product of 11-3 (10 g, 28%) as yellow solid.
(325) Preparation of Compound 11-4.
(326) To a solution of crude 11-3 (10 g, 19 mmol) in dichloromethane (100 mL) was added Dess-Matin reagent (16 g, 38 mmol) at 0° C. under nitrogen atmosphere. The mixture was then stirred at 30° C. for 3 h. To the mixture was then added a mixed solution of sodium bicarbonate and sodium thiosulfate in water. The organic layer was separated, washed with water, dried over anhydrous sodium sulfate, concentrated under vacuum to give crude compound 11-4 as (5.9 g, 59%) white solid.
(327) Preparation of Compound 11-5.
(328) To a solution of crude 11-4 (5.9 g, 11 mmol) in THF (60 mL) was added hydrogen chloride (aq., 6 mL, 6 mmol, 1M). The mixture was stirred at 15° C. for 16 h. To the mixture was then added sodium bicarbonate (aq.). The organic layer was separated, dried over anhydrous sodium sulfate, concentrated under vacuum to give crude 11-5 (3.2 g, yield: 70%) as white solid.
(329) Preparation of Compound 11-6.
(330) To a solution of crude 11-5 (3.2 g, 7.9 mmol) in pyridine (50 mL), acetyl chloride (1.5 g, 19 mmol) was added dropwise at 0° C. as monitored by TLC until the reaction was completed. To the mixture was then added water, concentrated under vacuum. To the residue was added water, extracted with dichloromethane. The organic layer was dried over anhydrous sodium sulfate, purified by column chromatography on silica gel (eluent: petroleum ether:ethyl acetate=100:1) to give crude 11-6 (2.8 g, 79%) as white solid.
(331) Preparation of Compound 11-7.
(332) To a solution of crude 11-6 (2.8 g, 6.3 mmol) in dichloromethane (10 mL) was added diethylaminosulfur trifluoride (8 g, 50 mmol) at 0° C. dropwise. The mixture was then stirred for 16 h at 30° C. The mixture was then added to sodium bicarbonate (aq.). The organic layer was separated, dried over anhydrous sodium sulfate, purified by column chromatography on silica gel (eluent: petroleum ether:ethyl acetate=100:1 to 33:1) to give crude 11-7 (2 g, 68%) as white solid.
(333) Preparation of Compound 11-8.
(334) To a solution of crude 11-7 (2 g, 4.2 mmol) in THF (10 mL) wad added a solution of lithium hydroxide monohydrate (900 mg, 21 mmol) in water (10 mL) and then was added methanol (5 mL). The mixture was then stirred at 30° C. for 16 h. The mixture was then concentrated under vacuum. To the residue was added water, filtered. The solid was washed with water, dried under vacuum to give 11-8 (1.5 g, 85%) as white solid. .sup.1H NMR: (400 MHz, methanol-d4) δ 5.34 (d, J=5.2 Hz, 1H), 3.45-3.35 (m, 1H), 2.30-2.10 (m, 3H), 2.10-1.68 (m, 7H), 1.68-1.44 (m, 6H), 1.35-1.28 (m, 2H), 1.28-1.12 (m, 3H), 1.12-0.98 (m, 8H), 0.74 (s, 3H).
(335) Preparation of Compound 11-9.
(336) To a solution of 11-8 (1 g, 2.4 mmol) in methanol (15 mL) was added hydrogen chloride (5 mL, 4 M in methanol). The mixture was stirred at 30° C. for 15 minutes. Sodium bicarbonate (aq.) was added till pH=7. The mixture was then concentrated under vacuum. To the residue was added water, extracted with ethyl acetate, The organic layer was separated, dried over anhydrous sodium sulfate, concentrated under vacuum, purified by column chromatography on silica gel (eluent: petroleum ether:ethyl acetate=10:1 to 5:1) to give 11-9 (970 mg, 93%) as white solid. .sup.1H NMR: (400 MHz, CDCl3) δ 5.34 (d, J=5.2 Hz, 1H), 3.87 (s, 3H), 3.60-3.48 (m, 1H), 2.32-2.15 (m, 2H), 2.10-1.95 (m, 2H), 1.95-1.70 (m, 5H), 1.65-1.40 (m, 8H), 1.30-0.90 (m, 13H), 0.70 (s, 3H).
(337) Preparation of Compound 11-10.
(338) To a solution of 11-9 (0.97 g, 2.3 mmol) in dichloromethane (50 mL) was added Dess-Matin reagent (2.3 g, 5.4 mmol) at 0° C. under nitrogen atmosphere. The mixture was then stirred at 30° C. for 3 h. To the mixture was then added a mixed solution of sodium bicarbonate and sodium thiosulfate in water. The organic layer was separated, washed with water, dried over anhydrous sodium sulfate, concentrated under vacuum to give crude compound of 11-10 (1 g, 100%) as yellow oil.
(339) Preparation of Compound 11-11 and 11-12.
(340) To a solution of butylated hydroxytoluene (3.1 g, 14.2 mmol) in toluene (20 mL) was added Me.sub.3Al (3.6 mL, 7.2 mmol, 2 M in toluene) at 15° C. The mixture was then stirred at 15° C. for 30 minutes. A solution of 11-11 (0.9 g, 2.4 mmol) in toluene (5 mL) was added at −78° C. The mixture was then stirred at −78° C. for 1 h. methylmagnesium bromide (2.4 mL, 7.2 mmol, 3M in ether) was then added at −78° C. The mixture was then stirred at −78° C. for 1 hour. To the mixture was then added ammonium chloride (aq.), filtered. The organic layer was separated and the aqueous phase was extracted with ethyl acetate. The combined organic layer was dried over anhydrous sodium sulfate, concentrated under vacuum, purified by column chromatography on silica gel (eluent: petroleum ether:ethyl acetate=20:1 to 10:1) to give 240 mg of crude 11-11 (yield: 28%) and 210 mg of crude 11-12 (yield: 25%). .sup.1H NMR (400 MHz, CDCl3): δ 5.33-5.25 (m, 1H), 3.87 (s, 3H), 2.50-0.75 (m, 33H), 0.70 (s, 3H). .sup.1H NMR: (400 MHz, CDCl3) δ 5.35-5.27 (m, 1H), 2.50-2.37 (m, 1H), 2.32 (s, 3H), 2.20-0.75 (m, 32H), 0.70 (s, 3H).
(341) Preparation of Compound 11-13.
(342) To a solution of 11-12 (70 mg, 0.16 mmol) in ethanol (2 mL) was added sodium borohydride (100 mg, 2.6 mmol) at 15° C. The mixture was stirred at 15° C. for 30 minutes. To the mixture was then added ammonium chloride (aq.), concentrated under vacuum. To the residue was added water, extracted with ethyl acetate. The organic layer was separated, dried over anhydrous sodium sulfate, concentrated under vacuum, purified by column chromatography on silica gel (eluent: petroleum ether:ethyl acetate=10:1 to 8:1) to give 11-13 (40 mg, 57%) as white solid. .sup.1H NMR: (400 MHz, methanol-d4) δ 5.35-5.28 (m, 1H), 3.88-3.68 (m, 1H), 2.49-2.37 (m, 1H), 2.18-1.22 (m, 20H), 1.19 (d, J=6.0 Hz, 3H), 1.18-1.14 (m, 1H), 1.11-1.08 (m, 3H), 1.06 (s, 3H), 1.04 (s, 3H), 1.02-0.95 (m, 1H), 0.76 (s, 3H).
(343) Preparation of Compound 11-15 and 11-16.
(344) Diastereomeric mixture 11-13 (30 mg, 0.071 mmol) was split by SFC to get 11-15 (12.2 mg) and 11-16 (14.7 mg) as white powder (total yield: 90%). .sup.1H NMR (11-15): (400 MHz, MeOD) δ 5.32 (d, J=5.2 Hz, 1H), 3.85-3.72 (m, 1H), 2.50-2.40 (m, 1H), 2.20-1.57 (m, 11H), 1.52-0.85 (m, 23H), 0.78 (s, 3H). .sup.1H NMR (11-16): (400 MHz, MeOD) δ 5.32 (d, J=5.2 Hz, 1H), 3.85-3.72 (m, 1H), 2.50-2.40 (m, 1H), 2.20-1.45 (m, 15H), 1.40-0.85 (m, 20H), 0.78 (s, 3H).
(345) Preparation of Compound 11-19.
(346) To a solution of 11-12 (70 mg, 0.16 mmol) in THF (2 mL) was added methylmagnesium bromide (1 mL, 3 mmol, 3M in ether) at −78° C. The mixture was stirred at 15° C. for 30 minutes. To the mixture was then added ammonium chloride (aq.), concentrated under vacuum. To the residue was added water, extracted with ethyl acetate. The organic layer was separated, dried over anhydrous sodium sulfate, concentrated under vacuum, purified by column chromatography on silica gel (eluent: petroleum ether:ethyl acetate=10:1 to 8:1) to give 11-19 (39 mg, 55%) as white solid. .sup.1H NMR: (400 MHz, methanol-d4) δ 5.33-5.28 (m, 1H), 2.48-2.38 (m, 1H), 2.12-1.70 (m, 17H), 1.23 (s, 6H), 1.20-1.12 (m, 3H), 1.10 (d, J=6.4 Hz, 3H), 1.06 (s, 3H), 1.04 (s, 3H), 1.03-0.91 (m, 2H), 0.76 (s, 3H).
Example 12. Preparation of Intermediate 0-9
(347) ##STR00055## ##STR00056##
(348) Preparation of 0-2.
(349) To a solution of compound 0-1 (100 g, 255 mmol, 1.0 eq) in dry MeOH (500 mL) was added concentrated H.sub.2SO.sub.4 (14 mL). The mixture was heated to reflux overnight and then cooled to room temperature. The mixture was quenched with aq. saturated NaHCO.sub.3 solution (0.5 L) and then evaporated to remove MeOH. The residue mixture was extracted with EtOAc (300 mL×3). The combined organic layers were washed with brine (200 mL), dried over Na.sub.2SO.sub.4 and evaporated to give the product (100 g crude, 96%) as off-white powder. .sup.1H NMR: (400 MHz, CDCl3) δ 4.09-4.02 (m, 1H), 3.66 (s, 3H), 3.63-3.58 (m, 1H), 2.39-2.31 (m, 1H), 2.25-2.15 (m, 1H), 1.97-1.91 (m, 1H), 1.91-1.55 (m, 10H), 1.52-1.02 (m, 14H), 0.95-0.88 (m, 6H), 0.62 (s, 3H).
(350) Preparation of 0-3.
(351) To a solution of compound 0-2 (250 g, 615 mmol, 1.0 eq) in dry pyridine (0.8 L) was added a solution of TsCl (352 g, 1844 mmol, 3.0 eq) in dry pyridine (200 mL). The mixture was stirred at room temperature for 18 h. Ice chips were added gradually to the mixture, and the precipitated solid was filtered, washed with aq. 10% HCl solution (400 mL×3) and water (400 mL×2), and then evaporated to dryness to give crude product (500 g, crude) as a off-white powder, which was used to next step directly
(352) Preparation of 0-4.
(353) A mixture of compound 0-3 (250 g crude), CH.sub.3COOK (24 g, 245 mmol, 0.77 eq), water (150 mL) and DMF (900 mL) was heated at reflux for 24 h. The solution was cooled to room temperature, with ice chips added gradually. The precipitated solid was filtered off and washed with water (100 mL×2). The crude solid was purified on silica gel column (PE/EtOAc=8/1) to give compound 0-4 (40 g, yield 34.3% of two steps) as white solid. .sup.1H NMR (400 MHz, CDCl.sub.3) δ 5.32-5.38 (m, 1H), 3.66 (s, 3H), 3.47-3.57 (m, 1H), 2.16-2.41 (m, 4H), 1.93-2.04 (m, 2H), 1.74-1.92 (m, 4H), 1.30-1.59 (m, 9H), 0.90-1.19 (m, 12H), 0.68 (s, 3H)
(354) Preparation of 0-5.
(355) To a solution of compound 0-4 (33 g, 85 mmol, 1.0 eq) in dry CH.sub.2Cl.sub.2 (700 mL) was added Dess-Martin reagent (72 g, 170 mmol, 2.0 eq) in portions at 0° C. Then the reaction mixture was stirred at room temperature for 1 h. TLC (PE:EA=3:1) showed the starting material was consumed completely. The reaction mixture were quenched with a saturated aqueous solution of NaHCO.sub.3/Na.sub.2S.sub.2O.sub.3=1:3 (250 mL). The organic phase was washed with brine (200 mL×2) and dried over Na.sub.2SO.sub.4, and the solvent was evaporated to afford desired product (35 g, crude), which was used in the next step without further purification.
(356) Preparation of 0-6.
(357) To a solution of MAD (0.42 mol, 3.0 eq) in toluene, freshly prepared by addition of a solution of Me.sub.3Al (210 mL, 0.42 mmol, 2 M in hexane) to a stirred solution of 2,6-di-tert-butyl-4-methylphenol (185 g, 0.84 mol) in toluene (200 mL) followed by stirring for 1 h at room temperature, was added dropwise a solution of compound 0-5 (54 g, 0.14 mol, 1.0 eq) in toluene (200 mL) at −78° C. under nitrogen. Then the reaction mixture was stirred for 30 min, a solution of MeMgBr (140 mL, 0.42 mol, 3.0 eq, 3 M in ether) was added dropwise at −78° C. The reaction mixture was warmed to −40° C. and stirred at this temperature for 3 h. TLC (PE:EA=3:1) showed that the starting material was consumed completely. The mixture was poured into aqueous saturated NH.sub.4Cl solution (100 mL) and extracted with EtOAc (300 mL×2). The combined organic phases were dried over Na.sub.2SO.sub.4, and the solvent was evaporated to afford crude product. The crude product was purified on silica gel chromatography eluted with PE:EA=10:1 to give the pure target (30 g, 53%) as white powder. .sup.1H NMR: (400 MHz, CDCl3) δ 5.31-5.29 (m, 1H), 3.66 (s, 3H), 2.39-2.33 (m, 2H), 2.24-2.22 (m, 1H), 1.99-1.95 (m, 3H), 1.85-1.68 (m, 4H), 1.59-1.40 (m, 8H), 1.31-1.26 (m, 2H), 1.17-1.01 (m, 11H), 0.93-0.91 (m, 4H), 0.67 (s, 3H).
(358) Preparation of 0-7.
(359) To a solution of compound 0-6 (30.0 g, 74.51 mmol) in THF/H.sub.2O (800 mL, 1/1) was added LiOH.H.sub.2O (17.51 g, 417.28 mmol). The reaction was stirred at room temperature for 18 h. TLC (PE/EA=2/1) showed that compound 0-6 was consumed completely. The mixture was concentrated in vacuum, diluted with water (2 L), and then acidified to pH=4 with 1 M aqueous HCl. The precipitate was collected by filtration and dried in vacuum to give the product compound 0-7 (33 g, crude) as off-white solid. .sup.1H NMR: (400 MHz, CDCl3) δ 5.31-5.30 (m, 1H), 2.44-2.36 (m, 2H), 2.29-2.24 (m, 1H), 2.01-1.95 (m, 3H), 1.87-1.71 (m, 5H), 1.61-1.56 (m, 2H), 1.50-1.32 (m, 8H), 1.17-1.09 (m, 7H), 1.01 (s, 3H), 0.95-0.93 (m, 4H), 0.68 (s, 3H).
(360) Preparation of 0-8.
(361) A mixture of compound 0-7 (32.0 g, 82.35 mmol), N,O-dimethylhydroxylamine (16.07 g, 164.70 mmol), HATU (37.57 g, 98.82 mmol) and Et.sub.3N (46.0 mL, 329.40 mmol) in 500 mL anhydrous CH.sub.2Cl.sub.2 was stirred for 18 h at room temperature. TLC showed the reaction was completed. Then CH.sub.2Cl.sub.2 was added to the mixture and the resulting solution was washed with water, 1 N HCl aqueous, saturated aqueous NaHCO.sub.3 and brine, dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated, purified by silica gel (PE:EtOAc=10:1 to 3:1) to afford the target compound 0-8 (17.0 g, yield: 47.8%) as off-white solid. .sup.1H NMR: (400 MHz, CDCl3) δ 5.31-5.29 (m, 1H), 3.69 (s, 3H), 3.17 (s, 3H), 3.03 (s, 2H), 2.47-2.29 (m, 3H), 2.04-1.68 (m, 7H), 1.60-1.43 (m, 7H), 1.38-1.30 (m, 2H), 1.20-1.08 (m, 6H), 1.03-0.91 (m, 8H), 0.68 (s, 3H).
(362) Preparation of Key Intermediate 0-9.
(363) To a solution of compound 0-8 (17.0 g, 39.38 mmol) in 300 mL anhydrous THF was added dropwise MeMgBr (65.6 mL, 196.92 mmol, 3 M in ether) under N.sub.2 at 0° C. After the addition was completed, the reaction mixture was stirred for 2 h at room temperature. TLC showed the reaction was completed. Then saturated aqueous NH.sub.4Cl was slowly added to the mixture at 0° C., then the mixture was poured to water, extracted with EtOAc (2*200 mL), the organic layers were washed with brine, dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated, purified on silica gel (PE:EtOAc=20:1 to 6:1) to afford the target compound 0-9 (11.0 g, yield: 72%) as white solid. .sup.1H NMR: (400 MHz, CDCl3) δ 5.31-5.30 (m, 1H), 2.50-2.30 (m, 3H), 2.17 (s, 2H), 2.14 (s, 3H), 2.02-1.94 (m, 3H), 1.88-1.67 (m, 4H), 1.61-1.58 (m, 1H), 1.56-1.49 (m, 5H), 1.47-1.41 (m, 2H), 1.31-1.11 (m, 7H), 1.08-0.91 (m, 8H), 0.68 (s, 3H).
(364) Assay Methods
(365) Compounds of the present invention can be evaluated using various in vitro and in vivo assays described in the literature; examples of which are described below.
(366) The following examples are offered to illustrate the biological activity of the compounds, pharmaceutical compositions, and methods provided herein and are not to be construed in any way as limiting the scope thereof.
(367) NMDA Potentiation
(368) NMDA potentiation was assessed using either whole cell patch clamp of mammalian cells which expressed NMDA receptors, or using two-electrode voltage clamp (TEVC) of Xenopus Laevis oocytes expressing NMDA receptors.
(369) Whole-Cell Patch Clamp of Mammalian Cells
(370) The whole-cell patch-clamp technique was used to investigate the effects of compounds (0.1 mM and 1.0 mM) on the NMDA receptor (GRIN1/GRIN2A subunits) expressed in HEK cells. NMDA/Glycine peak and steady-state currents were recorded from stably transfected cells expressing the NMDA receptor and the modulatory effects of the test items on these currents were investigated. Results are shown on Table 1.
(371) Cells were stably transfected with human GRIN1 (variant NR1-3). These cells were transiently transfected (LIPOFECTAMINE™) with GRIN2A cDNA and CD8 (pLeu) antigene cDNA. About 24-72 hours following transfection 1 μl Dynabeads M-45 CD8 was added to identify successfully transfected cells (Jurman et al., Biotechniques (1994) 17:876-881). Cells were passaged to a confluence of 50-80%. Cells were seeded onto Poly-L-Lysine coated cover slips covered with culture complete medium in a 35 mm culture dish. Confluent clusters of cells are electrically coupled (Pritchett et al., Science (1988), 242:1306-8). Because responses in distant cells are not adequately voltage clamped and because of uncertainties about the extent of coupling (Verdoorn et al., Neuron (1990), 4:919-28), cells were cultivated at a density that enables single cells (without visible connections to neighboring cells) to be measured. Cells were incubated at 37° C. in a humidified atmosphere with 5% CO.sub.2 (rel. humidity about 95%). The cells were continuously maintained in and passaged in sterile culture flasks containing a 1:1 mixture of Dulbecco's modified eagle medium and nutrient mixture F-12 (D-MEM/F-12 1x, liquid, with L-Glutamine) supplemented with 9% fetal bovine serum and 0.9% Penicillin/Streptomycin solution. The complete medium was supplemented with 3.0 μg/ml Puromycin.
(372) Whole cell currents were measured with HEKA EPC-10 amplifiers using PatchMaster software. Cell culture dishes for recordings were placed on the dish holder of the microscope and continuously perfused (1 ml/min) with “bath solution” (NaCl 137 mM, KCl 4 mM, CaCl.sub.2 1.8 mM, MgCl.sub.2 1 mM, HEPES 10 mM, D-Glucose 10 mM, pH (NaOH) 7.4). All solutions applied to cells including the pipette solution were maintained at room temperature (19° C.-30° C.). After formation of a Gigaohm seal between the patch electrodes and transfected individual HEK 293 cells (pipette resistance range: 2.5 MΩ-6.0 MΩ; seal resistance range: >1 GΩ) the cell membrane across the pipette tip was ruptured to assure electrical access to the cell interior (whole-cell patch-configuration). At this point the bath solution is switched to “NMDA bath solution” (NaCl 137 mM, KCl 4 mM, CaCl.sub.2 2.8 mM, HEPES 10 mM, D-Glucose 10 mM, Cremophore 0.02%, pH (NaOH) 7.4). NMDA inward currents were measured upon application of 30 μM NMDA (and 5.0 μM Glycine) to patch-clamped cells (2 applications) for 5 s. The cells were voltage clamped at a holding potential of −80 mV. For the analysis of test articles, NMDA receptors were stimulated by 30 μM NMDA and 5.0 μM Glycine after sequential pre-incubation of increasing concentrations of the test article. Pre-incubation duration was 30 s. Stimulation duration was 5 s Test articles were dissolved in DMSO to form stock solutions of 0.1 mM and 1 mM. Test articles were diluted to 0.1 μM and 1 μM in “NMDA bath solution”. Both concentrations of test articles were tested on each cell. The same concentration was applied at least three times or until the steady state current amplitude was reached. Every day one cell was tested with 50 μM PREGS (positive control) using the same application protocol to test whether cells were successfully transfected with NMDA receptors.
(373) TABLE-US-00001 TABLE 1 NMDA 1a2A NMDA 1a2A (%) Potentiation (%) Potentiation Structure 0.1 uM 1 uM
Oocytes
(374) The Two Electrode Voltage Clamp (TEVC) technique was used to investigate the effects of compounds (10 μM) on the NMDA receptor (GRIN1/GRIN2A) expressed in Xenopus oocytes. Glutamate/Glycine peak and steady-state currents were recorded from oocytes that expressed the NMDA receptor and the modulatory effects of the test items on these currents were investigated. Results are shown on Table 2.
(375) Ovaries were harvested from Xenopus Laevis females that had been deeply anesthetized by cooling at 4° C. and immersion in Tricaine methanesulfonate (MS-222 at a concentration of 150 mg/L) in sodium bicarbonate (300 mg/L). Once anesthetized the animal was decapitated and pitched following the rules of animal rights from the Geneva canton. A small piece of ovary was isolated for immediate preparation while the remaining part was placed at 4° C. in a sterile Barth solution containing in mM NaCl 88, KCl 1, NaHCO.sub.3 2.4, HEPES 10, MgSO.sub.4.7H.sub.2O 0.82, Ca(NO.sub.3).sub.2.4H.sub.2O 0.33, CaCl.sub.2.6H.sub.2O 0.41, at pH 7.4, and supplemented with 20 μg/ml of kanamycin, 100 unit/ml penicillin and 100 ag/ml streptomycin. All recordings were performed at 18° C. and cells were super-fused with medium containing in mM: NaCl 82.5, KCl 2.5, HEPES 5, CaCl.sub.2.2H.sub.2O, 6H.sub.2O 1, pH 7.4.
(376) Oocytes were injected with either cDNAs encoding for the human GRIN1 and GRIN2A subunits, using a proprietary automated injection device (Hogg et al., J. Neurosci. Methods, (2008) 169: 65-75) and receptor expression was assessed using electrophysiology at least two days later. The ratio of cDNA injection for GRIN1 and GRIN2A was 1:1. Electrophysiological recordings were made using an automated process equipped with standard TEVC and data were captured and analyzed using a proprietary data acquisition and analysis software running under Matlab (Mathworks Inc.). The membrane potential of the oocytes was maintained at −80 mV throughout the experiments. To explore the effects of proprietary compounds, currents were evoked by applying 3 μM Glutamate and 10 μM Glycine for 10 s. Oocytes were then washed for 90 s before being exposed to the test article at a concentration of 10 μM for 120 s. Following this, 3 μM Glutamate and 10 μM Glycine were immediately reapplied for 10 s. Potentiation of both the peak current and the steady state current was assessed. For statistical analysis values were computed either with Excel (Microsoft) or Matlab (Mathworks Inc.). To obtain mean measurements with standard deviations, all experiments were carried out using at least three cells.
(377) Glutamate was prepared as a concentrated stock solution (10.sup.−1 M) in water and then diluted in the recording medium to obtain the desired test concentration. Glycine was prepared as a stock solution at 1 M in water. Compounds were prepared as stock solution (10.sup.−2 M) in DMSO and then diluted in the recording medium to obtain the desired test concentration. Residual DMSO did not exceed the concentration of 1% a concentration that has been shown to have no effects on Xenopus oocytes function.
(378) TABLE-US-00002 TABLE 2 Structure % Potentiation at 10 μM
(379) As shown in Table 1, compounds bearing a beta-hydrogen at C.sub.5 are disfavored compared to compounds bearing either alpha-hydrogen C.sub.5 or double bond across C.sub.5-C.sub.6 due to loss of potentiation of the NMDA receptor. This is illustrated by Comparison Compound 5 vs 4-6 and 4-7. The removal of the methyl at C.sub.21 also results in significant loss of NMDA potentiation, for example Comparison Compound 4 lost five fold potentiation compared to Comparison Compound 3 when measured at 0.1 μM concentration. Therefore the compounds in this selection bear both a methyl group in C.sub.21 and either a double bond across C.sub.5-C.sub.6 or an alpha-hydrogen in C.sub.5. In addition, compounds in this selection showed improved potency and limited maximum potentiation of the NMDA receptor when tested as high as 1 μM concentrations of compound (for example Comparison Compound 2 vs 4-6 and 1-11). Such properties are expected limit the risk of inducing glutamate driven neurotoxicity relative to compounds that achieve a greater maximum potentiation of the NMDA receptor.
OTHER EMBODIMENTS
(380) In the claims articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.
(381) Furthermore, the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Where elements are presented as lists, e.g., in Markush group format, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should it be understood that, in general, where the invention, or aspects of the invention, is/are referred to as comprising particular elements and/or features, certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements and/or features. For purposes of simplicity, those embodiments have not been specifically set forth in haec verba herein. It is also noted that the terms “comprising” and “containing” are intended to be open and permits the inclusion of additional elements or steps. Where ranges are given, endpoints are included. Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or sub-range within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.
(382) This application refers to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. If there is a conflict between any of the incorporated references and the instant specification, the specification shall control. In addition, any particular embodiment of the present invention that falls within the prior art may be explicitly excluded from any one or more of the claims. Because such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the invention can be excluded from any claim, for any reason, whether or not related to the existence of prior art.
(383) Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described herein. The scope of the present embodiments described herein is not intended to be limited to the above Description, but rather is as set forth in the appended claims. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present invention, as defined in the following claims.