U.S. patent application number 16/945163 was filed with the patent office on 2020-11-19 for use of (2r, 6r)-hydroxynorketamine, (s)-dehydronorketamine and other stereoisomeric dehydro and hydroxylated metabolites of (r,s)-ketamine in the treatment of depression and neuropathic pain.
The applicant listed for this patent is COOPER HEALTH SYSTEM, THE UNITED STATES OF AMERICA, AS REPRESENTED BY THE SECRETARY, DEPARTMENT OF HEALTH AND HUMAN SERVIC, SRI International, THE UNITED STATES OF AMERICA, AS REPRESENTED BY THE SECRETARY, DEPARTMENT OF HEALTH AND HUMAN SERVIC. Invention is credited to MICHEL BERNIER, MICHAEL E. GOLDBERG, RUIN MOADDEL, MARY J. TANGA, MARC C. TORJMAN, IRVING W. WAINER, CARLOS A. ZARATE.
Application Number | 20200360393 16/945163 |
Document ID | / |
Family ID | 1000004989922 |
Filed Date | 2020-11-19 |
View All Diagrams
United States Patent
Application |
20200360393 |
Kind Code |
A1 |
WAINER; IRVING W. ; et
al. |
November 19, 2020 |
USE OF (2R, 6R)-HYDROXYNORKETAMINE, (S)-DEHYDRONORKETAMINE AND
OTHER STEREOISOMERIC DEHYDRO AND HYDROXYLATED METABOLITES OF
(R,S)-KETAMINE IN THE TREATMENT OF DEPRESSION AND NEUROPATHIC
PAIN
Abstract
The disclosure provides pharmaceutical preparations containing
(2R,6R)-hydroxynorketamine, or (R)- or (S)-dehydronorketamine , or
other stereoisomeric dehydro or hydroxylated ketamine metabolite.
##STR00001## (2R,6R)-Hydroxynorketamine The disclosure also
provides novel ketamine metabolite prodrugs. The disclosure
provides methods of treating, bipolar depression, major depressive
disorder, neuropathic and chronic pain, including complex regional
pain synthetic pain disorder (CRPD) by administering a purified
ketamine metabolite or a ketamine metabolite prodrug directly to
patients in need of such treatment.
Inventors: |
WAINER; IRVING W.;
(Washington, DC) ; MOADDEL; RUIN; (Bel Air,
MD) ; BERNIER; MICHEL; (Pikesville, MD) ;
ZARATE; CARLOS A.; (Germantown, MD) ; TORJMAN; MARC
C.; (Southampton, PA) ; GOLDBERG; MICHAEL E.;
(Philadelphia, PA) ; TANGA; MARY J.; (Los Altos,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE UNITED STATES OF AMERICA, AS REPRESENTED BY THE SECRETARY,
DEPARTMENT OF HEALTH AND HUMAN SERVIC
COOPER HEALTH SYSTEM
SRI International |
Bethesda
Camden
Menlo Park |
MD
NJ
CA |
US
US
US |
|
|
Family ID: |
1000004989922 |
Appl. No.: |
16/945163 |
Filed: |
July 31, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15836396 |
Dec 8, 2017 |
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16945163 |
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15341784 |
Nov 2, 2016 |
9867830 |
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15836396 |
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14351441 |
Apr 11, 2014 |
9650352 |
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PCT/US2012/060256 |
Oct 15, 2012 |
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15341784 |
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61547336 |
Oct 14, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07C 2601/14 20170501;
C07C 229/48 20130101; A61K 31/137 20130101; C07D 295/155 20130101;
C07B 2200/07 20130101; C07C 237/04 20130101; C07D 295/112 20130101;
A61K 31/4453 20130101; C07D 295/108 20130101; C07D 295/15 20130101;
A61K 31/165 20130101; A61K 31/5375 20130101; C07C 2601/16 20170501;
C07C 225/20 20130101; A61K 31/265 20130101; A61K 31/222 20130101;
C07C 237/20 20130101; A61K 31/24 20130101 |
International
Class: |
A61K 31/5375 20060101
A61K031/5375; C07C 225/20 20060101 C07C225/20; C07C 237/04 20060101
C07C237/04; C07D 295/108 20060101 C07D295/108; C07D 295/112
20060101 C07D295/112; C07C 229/48 20060101 C07C229/48; C07C 237/20
20060101 C07C237/20; C07D 295/15 20060101 C07D295/15; C07D 295/155
20060101 C07D295/155; A61K 31/137 20060101 A61K031/137; A61K 31/165
20060101 A61K031/165; A61K 31/222 20060101 A61K031/222; A61K 31/24
20060101 A61K031/24; A61K 31/265 20060101 A61K031/265; A61K 31/4453
20060101 A61K031/4453 |
Goverment Interests
STATEMENT OF GOVERNMENT SUPPORT
[0002] The Intramural Research Program of the National Institute of
Aging and the Nation Institute of Mental Health funded the subject
matter of this disclosure. The United States Government has certain
rights in this application.
Claims
1-36. (canceled)
37. A pharmaceutical composition comprising a compound or a
pharmaceutically acceptable salt thereof, wherein the compound is
##STR00018## together with a pharmaceutically acceptable excipient;
wherein (a) the composition is a solution and contains from about
0.05 mg/ml to about 0.5 mg/ml of the compound; (b) the composition
is an intravenous composition that provides about 0.2 mg to about
500 mg per administration; or (c) the composition is formulated for
oral administration and contains about 0.2 mg to about 500 mg of
the compound.
38. A pharmaceutical composition comprising a compound or
pharmaceutically acceptable salt thereof, where the compound is a
compound of the formula ##STR00019## together with a
pharmaceutically acceptable excipient; wherein (a) the composition
is a solution and contains from about 0.05 mg/ml to about 0.5 mg/ml
of the compound; (b) the composition is an intravenous composition
that provides about 0.2 mg to about 500 mg per administration; or
(c) the composition is formulated for oral administration and
contains about 0.2 mg to about 500 mg of the compound.
39. An intravenous pharmaceutical composition comprising a compound
or pharmaceutically acceptable salt thereof, where the compound is
a compound of the formula ##STR00020## and the intravenous
pharmaceutical composition provides a plasma Cmax of about 0.25
mcg/mL to about 125 mcg/mL
40. The intravenous pharmaceutical composition of claim 39, wherein
the composition provides a plasma Cmax of about 1 mcg/mL to about
50 mcg/mL
41. The pharmaceutical composition of claim 1, wherein the compound
is 2R,6R-hydroxynoreketamine.
42. The pharmaceutical composition of claim 38, wherein the
composition is a solution and contains from about 0.05 mg/ml to
about 0.5 mg/ml of the compound or salt thereof.
43. The pharmaceutical composition of claim 38, wherein the
composition is an intravenous composition that provides about 0.2
mg to about 500 mg of the compound per administration.
44. The pharmaceutical composition of claim 38, wherein the
composition is formulated for oral administration and contains
about 0.2 mg to about 500 mg of the compound or salt thereof.
45. The pharmaceutical composition of claim 38, wherein the
composition is formulated for oral administration and contains
about 10 mg to about 200 mg of the compound or salt thereof.
46. A method of treating bipolar depression, major depressive
disorder, schizophrenia, Alzheimer's dementia, amyotrophic lateral
sclerosis, complex regional pain syndrome (CRPS), chronic pain, or
neuropathic pain in a patient comprising administering an effective
amount of a pharmaceutical composition of claim 37 to the
patient.
47. A method of treating bipolar depression, major depressive
disorder, schizophrenia, Alzheimer's dementia, amyotrophic lateral
sclerosis, complex regional pain syndrome (CRPS), chronic pain, or
neuropathic pain in a patient comprising administering an effective
amount of a pharmaceutical composition of claim 38 to the
patient.
48. A method of treating a patient for bipolar depression, major
depressive disorder, schizophrenia, Alzheimer's dementia,
amyotrophic lateral sclerosis, complex regional pain syndrome
(CRPS), chronic pain, or neuropathic pain comprising (1)
determining that the patient is a ketamine non-responder; and (2)
administering an effective amount of a pharmaceutical composition
of claim 38 to the patient.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application claims priority from U.S. Provisional
Application No. 61/547,336, filed Oct. 14, 2011, which is hereby
incorporated by reference in its entirety.
BACKGROUND
[0003] Ketamine, a drug currently used in human anesthesia and
veterinary medicine, has been shown in clinical studies to be
effective in the treatment of several conditions, including the of
treatment-resistant bipolar depression, major depressive disorder,
neuropathic pain, and chronic pain, including complex regional pain
syndrome (CRPS).
[0004] In the current "ketamine paradigm", ketamine and norketamine
(NK) are considered to be responsible for the antinociceptive
response in CRPS patients. However the routine use of the drug is
hindered by unwanted central nervous system (CNS) effects.
Approximately 30% of patients do not respond to ketamine treatment.
Additionally, ketamine treatment is associated with serious side
effects due to the drug's anesthetic properties and abuse
potential.
[0005] Recent studies have demonstrated that in CRPS patients
receiving a continuous 5-day infusion of (R,S)-ketamine the primary
circulating metabolites were (R,S)-dehydronorketamine (DHNK) and
(2S,6S;2R,6R)-hydroxynorketamine (HNK). The data suggest that
downstream metabolites play a role in ketamine analgesic efficacy,
although little is known about the metabolites' pharmacological
activity.
[0006] The need for therapeutics which exhibits the therapeutic
properties of ketamine with efficacy in a higher percentage of
patients, reduced anesthetic properties and reduced abuse liability
exists. The present disclosure fulfills this need and provides
additional advantages set forth herein.
FIELD OF THE DISCLOSURE
[0007] This disclosure demonstrates that an active agents
responsible for the therapeutic response to ketamine in patients is
primarily due to (2R,6R)-hydroxynorketamine and
(S)-dehydronorketamine, ketamine metabolite. The disclosure
provides pharmaceutical preparations containing ketamine
metabolites and prodrug of ketamine metabolites. The disclosure
also provides novel ketamine prodrugs. The disclosure provides
methods of treating, bipolar depression, neuropathic and chronic
pain, including complex regional pain synthetic pain syndrome
(CRPS) by administering purified (2R,6R)-hydroxynorketamine or
purified (S)-dehydronorketamine or a prodrug of these compounds
directly to patients in need of such treatment.
SUMMARY
[0008] In a first aspect the disclosure provides a pharmaceutical
composition comprising a compound of Formula I
##STR00002##
or a pharmaceutically acceptable salt thereof, together with a
pharmaceutically acceptable excipient. Within Formula I the
variables, e.g. R.sub.1-R.sub.4, carry the definitions set forth
below.
[0009] R.sub.1 is hydrogen, hydroxyl, or a group -A.sub.1B.sub.1
where A.sub.1 is --O--, --O(C.dbd.O)--, --(C.dbd.O)O--,
--O(C.dbd.O)O, --O(C.dbd.O)NR.sub.6--, --OS(O).sub.2--,
--OS(O).sub.3, or --OP(O).sub.3-, and B.sub.1 is C.sub.1-C.sub.8
alkyl, C.sub.2-C.sub.8alkenyl, C.sub.2-C.sub.8alkynyl,
(carbocycle)C.sub.0-C.sub.4alkyl,
(heterocycle)C.sub.o-C.sub.4alkyl, each of which is substituted
with from 0 to 4 substituents independently chosen from halogen,
hydroxyl, amino, cyano, C.sub.1-C.sub.4alkoxy,
C.sub.1-C.sub.6alkylester, mono- and
di-(C.sub.1-C.sub.4alkyl)amino,
(C.sub.3-C.sub.7cycloalkyl)C.sub.o-C.sub.2alkyl, (hetero
cycloalkyl)C.sub.0-C.sub.2alkyl, C.sub.1-C.sub.2haloalkyl, and
C.sub.1-C.sub.2halo alkoxy.
[0010] The six-membered ring to which R.sub.1 is bound contains a
double bond when R.sub.1 is hydrogen and is fully saturated when
R.sub.1 is hydroxyl or -A.sub.1B.sub.1;
[0011] R.sub.2 is hydrogen or -A.sub.2B.sub.2 where A.sub.2 is a
bond, --O(C.dbd.O)--, --(C.dbd.O)O--, --S(O).sub.2--,
--(S.dbd.O)NR.sub.6-, or --(C.dbd.O)NR.sub.6-, B.sub.2 is
C.sub.1-C.sub.8alkyl, C.sub.2-C.sub.8alkenyl,
C.sub.2-C.sub.8alkynyl, C.sub.2-C.sub.6alkanoyl,
(carbocycle)C.sub.0-C.sub.4alkyl,
(heterocycle)C.sub.0-C.sub.4alkyl, or an amino acid or dipeptide
covalently bound to A.sub.2 by its C-terminus, each of which is
substituted with from 0 to 4 substituents independently chosen from
halogen, hydroxyl, amino, cyano, C.sub.1-C.sub.4alkyl,
C.sub.1-C.sub.4alkoxy, C.sub.1-C.sub.6alkylester, mono- and
di-(C.sub.1-C.sub.4alkyl)amino,
(C.sub.3-C.sub.7cycloalkyl)C.sub.0-C.sub.2alkyl,
(heterocycloalkyl)C.sub.0-C.sub.2alkyl, C.sub.1-C.sub.2haloalkyl,
and C.sub.1-C.sub.2haloalkoxy.
[0012] R.sub.3 is hydrogen or C.sub.1-C.sub.6alkyl.
[0013] R.sub.4 and R.sub.5 are 0 or 1 or more substituents
independently chosen from halogen, hydroxyl, amino, cyano,
C.sub.1-C.sub.4alkyl, C.sub.1-C.sub.4alkoxy, mono- and
di-C.sub.1-C.sub.4alkylamino, C.sub.1-C.sub.2haloalkyl, and
C.sub.1-C.sub.2haloalkoxy.
[0014] R.sub.6 is hydrogen or C.sub.1-C.sub.6alkyl.
[0015] Also included are prodrugs of ketamine metabolites,
including prodrugs of all hydroxynorketamine diastereomers,
including (2R,6R)-hydroxynorketamine, (R) and
(S)-dehydronorketamine, and other stereoisomeric dehydro and
hydroxylated ketamine metabolites. These prodrugs carry the
definition set forth above for compounds of Formula I however the
following conditions may apply:
[0016] R.sub.1 is not hydrogen or hydroxyl when R.sub.2 is
hydrogen.
[0017] B.sub.1 is methyl when A.sub.1 is --O-.
[0018] A.sub.1B.sub.1 is not (4-methylphenyl)-S(O).sub.2O-.
[0019] B.sub.1 is not methyl when A.sub.1 is a bond.
[0020] B.sub.2 is not methyl when A.sub.2 is a bond or
--(C.dbd.O)O-.
[0021] In another aspect the disclosure provides a method of
treating bipolar depression, major depressive disorder,
schizophrenia, Alzheimer's dementia, amyotrophic lateral sclerosis,
complex regional pain syndrome (CRPS), chronic pain, or neuropathic
pain comprising administering a pharmaceutical composition
containing an effective amount of a compound of Formula I or a
pharmaceutically acceptable salt thereof, together with a
pharmaceutically acceptable carrier to a patient in need of such
treatment.
[0022] The disclosure also includes a method of treating bipolar
depression, schizophrenia, Alzheimer's dementia, amyotrophic
lateral sclerosis, complex regional pain syndrome (CRPS), chronic
pain, or neuropathic pain comprising administering an effective
amount of isolated hydroxynorketamine diastereomers, such as
(2R,6R)-hydroxynorketamine or isolated (R)- or
(S)-dehydronorketamine to a patient in need of such treatment.
[0023] In yet another aspect the disclosure includes a method of
treating a patient for bipolar depression, complex regional pain
syndrome (CRPS), chronic pain, or neuropathic pain comprising
[0024] (1) determining that the patient is a ketamine
non-responder; and [0025] (2) administering an effective amount of
isolated hydroxynorketamine diastereomer, such as
(2R,6R)-hydroxynorketamine or isolated(R)- or
(S)-dehydronorketamine to the patient.
[0026] Applicants have determined that certain compounds of Formula
I are potent serine racemase inhibitors. The disclosure also
provides a method of inhibiting serine racemase comprising
contacting cells with a concentration of a compound of Formula I
sufficient to inhibit serine racemase in vitro.
DETAILED DESCRIPTION
Terminology
[0027] Compounds disclosed herein are described using standard
nomenclature. Unless defined otherwise, all technical and
scientific terms used herein have the same meaning as is commonly
understood by one of skill in the art to which this disclosure
belongs.
[0028] The terms "a" and "an" do not denote a limitation of
quantity, but rather denote the presence of at least one of the
referenced item.
[0029] Formula I includes all subformulae thereof. For example
Formula I includes compounds of Formulas I and subformulae Formula
II-V and the pharmaceutically acceptable salts, prodrugs and other
derivatives, hydrates, polymorphs, and thereof
[0030] The term "chiral" refers to molecules, which have the
property of non-superimposability of the mirror image partner.
[0031] "Stereoisomers" are compounds, which have identical chemical
constitution, but differ with regard to the arrangement of the
atoms or groups in space.
[0032] A "Diastereomer" is a stereoisomer with two or more centers
of chirality and whose molecules are not mirror images of one
another. Diastereomers have different physical properties, e.g.,
melting points, boiling points, spectral properties, and
reactivities. Mixtures of diastereomers may separate under high
resolution analytical procedures such as electrophoresis,
crystallization in the presence of a resolving agent, or
chromatography, using, for example a chiral HPLC column.
[0033] "Enantiomers" refer to two stereoisomers of a compound,
which are non-superimposable mirror images of one another. A 50:50
mixture of enantiomers is referred to as a racemic mixture or a
racemate, which may occur where there has been no stereoselection
or stereospecificity in a chemical reaction or process.
[0034] Stereochemical definitions and conventions used herein
generally follow S. P. Parker, Ed., McGraw-Hill Dictionary of
Chemical Terms (1984) McGraw-Hill Book Company, New York; and
Eliel, E. and Wilen, S., Stereochemistry of Organic Compounds
(1994) John Wiley & Sons, Inc., New York. Many organic
compounds exist in optically active forms, i.e., they have the
ability to rotate the plane of plane-polarized light. In describing
an optically active compound, the prefixes D and L or R and S are
used to denote the absolute configuration of the molecule about its
chiral center(s). The prefixes d and 1 or (+) and (-) are employed
to designate the sign of rotation of plane-polarized light by the
compound, with (-) or 1 meaning that the compound is levorotatory.
A compound prefixed with (+) or d is dextrorotatory.
[0035] A "racemic mixture" or "racemate" is an equimolar (or 50:50)
mixture of two enantiomeric species, devoid of optical activity. A
racemic mixture may occur where there has been no stereoselection
or stereospecificity in a chemical reaction or process.
[0036] Where a compound exists in various tautomeric forms, the
invention is not limited to any one of the specific tautomers, but
rather includes all tautomeric forms.
[0037] The disclosure includes compounds of Formula I having all
possible isotopes of atoms occurring in the compounds. Isotopes
include those atoms having the same atomic number but different
mass numbers. By way of general example, and without limitation,
isotopes of hydrogen include tritium and deuterium and isotopes of
carbon include .sup.11C, .sup.13C, and .sup.14.sub.C.
[0038] Certain compounds are described herein using a general
formula that includes variables, e.g. R.sub.1-R.sub.4. Unless
otherwise specified, each variable within Formula I is defined
independently of other variables. Thus, if a group is said to be
substituted, e.g. with 0-2 R*, then said group may be substituted
with up to two R* groups and R* at each occurrence is selected
independently from the definition of R*. Also, combinations of
substituents and/or variables are permissible only if such
combinations result in stable compounds.
[0039] The term "substituted", as used herein, means that at least
one hydrogen on the designated atom or group is replaced with a
selection from the indicated group, provided that the designated
atom's normal valence is not exceeded. When the substituent is oxo
(i.e., .dbd.O), then 2 hydrogens on the atom are replaced. When
aromatic moieties are substituted by an oxo group, the aromatic
ring is replaced by the corresponding partially unsaturated ring.
For example a pyridyl group substituted by oxo is a pyridone.
Combinations of substituents and/or variables are permissible only
if such combinations result in stable compounds or useful synthetic
intermediates. A stable compound or stable structure is meant to
imply a compound that is sufficiently robust to survive isolation
from a reaction mixture, and subsequent formulation into an
effective therapeutic agent.
[0040] A dash ("-") that is not between two letters or symbols is
used to indicate a point of attachment for a substituent. For
example, --(CH.sub.2)C.sub.3-C.sub.7cycloalkyl is attached through
carbon of the methylene (CH.sub.2) group.
[0041] "Alkyl" includes both branched and straight chain saturated
aliphatic hydrocarbon groups, having the specified number of carbon
atoms, generally from 1 to about 12 carbon atoms. The term
C.sub.1-C.sub.6alkyl as used herein indicates an alkyl group having
from 1 to about 6 carbon atoms. When C.sub.0-C.sub.n alkyl is used
herein in conjunction with another group, for example,
(phenyl)C.sub.0-C.sub.4 alkyl, the indicated group, in this case
phenyl, is either directly bound by a single covalent bond
(C.sub.0), or attached by an alkyl chain having the specified
number of carbon atoms, in this case from 1 to about 4 carbon
atoms. Examples of alkyl include, but are not limited to, methyl,
ethyl, n-propyl, isopropyl, n-butyl, 3-methylbutyl, t-butyl,
n-pentyl, and sec-pentyl.
[0042] "Alkanoyl" is an alkyl group as defined above, attached
through a keto (--(C.dbd.O)-) bridge. Alkanoyl groups have the
indicated number of carbon atoms, with the carbon of the keto group
being included in the numbered carbon atoms. For example a
C.sub.2alkanoyl group is an acetyl group having the formula
CH.sub.3(C.dbd.O)-.
[0043] "Alkenyl" means straight and branched hydrocarbon chains
comprising one or more unsaturated carbon-carbon bonds, which may
occur in any stable point along the chain. Alkenyl groups described
herein typically have from 2 to about 12 carbons atoms. Preferred
alkenyl groups are lower alkenyl groups, those alkenyl groups
having from 2 to about 8 carbon atoms, e.g. C.sub.2-C.sub.8,
C.sub.2-C.sub.6, and C.sub.2-C.sub.4 alkenyl groups. Examples of
alkenyl groups include ethenyl, propenyl, and butenyl groups.
[0044] "Alkoxy" means an alkyl group, as defined above, with the
indicated number of carbon atoms attached via an oxygen bridge.
Examples of alkoxy include, but are not limited to, methoxy,
ethoxy, 3-hexoxy, and 3-methylpentoxy.
[0045] "Mono- and/or di-alkylamino" indicates secondary or tertiary
alkyl amino groups, wherein the alkyl groups are as defined above
and have the indicated number of carbon atoms. The point of
attachment of the alkylamino group is on the nitrogen. The alkyl
groups are independently chosen. Examples of mono- and
di-alkylamino groups include ethylamino, dimethylamino, and
methyl-propyl-amino. "Mono- and/or dialkylaminoalkyl" groups are
mono- and/or di-alkylamino groups attached through an alkyl linker
having the specified number of carbon atoms, for example a
di-methylaminoethyl group. Tertiary amino substituents may by
designated by nomenclature of the form N--R--N--R', indicating that
the groups R and R' are both attached to a single nitrogen
atom.
[0046] "Alkylester" indicates an alkyl group as defined above
attached through an ester linkage. The ester linkage may be in
either orientation, e.g. a group of the formula --O(C.dbd.O)alkyl
or a group of the formula --(C.dbd.O)Oalkyl.
[0047] A "carbocycle" is a 3 to 8 membered saturated, partially
unsaturated, or aromatic ring containing only carbon ring atoms or
a 6 to 11 membered saturated, partially unsaturated, or aromatic
bicyclic carbocylic ring system containing only carbon ring atoms.
Unless otherwise indicated, the carbocycle may be attached to its
pendant group it substitutes at any carbon atom that results in a
stable structure. When indicated the carbocyclic rings described
herein may be substituted on any available ring carbon if the
resulting compound is stable. Carbocyclic groups include,
cycloalkyl groups, such as cyclopropyl and cyclohexyl; cycloalkenyl
groups, such as cyclohexenyl, bridged cycloalkyl groups; and aryl
groups, such as phenyl.
[0048] "Cycloalkyl" indicates saturated hydrocarbon ring groups,
having the specified number of carbon atoms, usually from 3 to
about 7 ring carbon atoms. Examples of cycloalkyl groups include
cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl.
[0049] The term "heterocycle" indicates a 5- to 8-membered
saturated, partially unsaturated, or aromatic ring containing from
1 to about 4 heteroatoms chosen from N, O, and S, with remaining
ring atoms being carbon, or a 7 to 11 membered bicyclic saturated,
partially unsaturated, or aromatic heterocyclic ring system or a 10
to 15-membered tricyclic ring system, containing at least 1
heteroatom in the multiple ring system chosen from N, O, and S and
containing up to about 4 heteroatoms independently chosen from N,
O, and S in each ring of the multiple ring system. Unless otherwise
indicated, the heterocyclic ring may be attached to the group it
substitutes at any heteroatom or carbon atom that results in a
stable structure. When indicated the heterocyclic rings described
herein may be substituted on carbon or on a nitrogen atom if the
resulting compound is stable. A nitrogen atom in the heterocycle
may optionally be quaternized. It is preferred that the total
number of heteroatoms in a heterocyclic groups is not more than 4
and that the total number of S and O atoms in a heterocyclic group
is not more than 2, more preferably not more than 1. Examples of
heterocyclic groups include, pyridyl, indolyl, pyrimidinyl,
pyridizinyl, pyrazinyl, imidazolyl, oxazolyl, furanyl, thiophenyl,
thiazolyl, triazolyl, tetrazolyl, isoxazolyl, quinolinyl, pyrrolyl,
pyrazolyl, benz[b]thiophenyl, isoquinolinyl, quinazolinyl,
quinoxalinyl, thienyl, isoindolyl, dihydroisoindolyl,
5,6,7,8-tetrahydroisoquinoline, pyridinyl, pyrimidinyl, furanyl,
thienyl, pyrrolyl, pyrazolyl, pyrrolidinyl, morpholinyl,
piperazinyl, piperidinyl, and pyrrolidinyl.
[0050] "5- or 6-membered heteroaryl" indicates a stable 5- to
6-membered monocyclic ring that contains from 1 to 4, or preferably
from 1 to 3, heteroatoms chosen from N, O, and S, with remaining
ring atoms being carbon. When the total number of S and O atoms in
the heteroaryl group exceeds 1, these heteroatoms are not adjacent
to one another. It is preferred that the total number of S and O
atoms in the heteroaryl group is not more than 2. It is
particularly preferred that the total number of S and O atoms in
the heteroaryl group is not more than 1. A nitrogen atom in a
heteroaryl group may optionally be quaternized. When indicated,
such heteroaryl groups may be further substituted with carbon or
non-carbon atoms or groups. Such substitution may include fusion to
a 5 to 7-membered saturated cyclic group that optionally contains 1
or 2 heteroatoms independently chosen from N, O, and S, to form,
for example, a [1,3]dioxolo[4,5-c]pyridyl group. Examples of
heteroaryl groups include, but are not limited to, pyridyl,
indolyl, pyrimidinyl, pyridizinyl, pyrazinyl, imidazolyl, oxazolyl,
furanyl, thiophenyl, thiazolyl, triazolyl, tetrazolyl, isoxazolyl,
quinolinyl, pyrrolyl, pyrazolyl, benz[b]thiophenyl, isoquinolinyl,
quinazolinyl, quinoxalinyl, thienyl, isoindolyl, and
5,6,7,8-tetrahydroisoquinoline.
[0051] "Heterocycloalkyl" means a saturated cyclic group containing
from 1 to about 3 heteroatoms chosen from N, O, and S, with
remaining ring atoms being carbon. Heterocycloalkyl groups have
from 3 to about 8 ring atoms, and more typically have from 5 to 7
ring atoms. Examples of heterocycloalkyl groups include
morpholinyl, piperazinyl, piperidinyl, and pyrrolidinyl groups. A
nitrogen in a heterocycloalkyl group may optionally be
quaternized.
[0052] "Haloalkyl" indicates both branched and straight-chain alkyl
groups having the specified number of carbon atoms, substituted
with 1 or more halogen atoms, generally up to the maximum allowable
number of halogen atoms. Examples of haloalkyl include, but are not
limited to, trifluoromethyl, difluoromethyl, 2-fluoroethyl, and
penta-fluoroethyl.
[0053] "Haloalkoxy" indicates a haloalkyl group as defined above
attached through an oxygen bridge (oxygen of an alcohol
radical).
[0054] "Halo" or "halogen" as used herein refers to fluoro, chloro,
bromo, or iodo.
[0055] An "active agent" means any compound, element, or mixture
that when administered to a patient alone or in combination with
another agent confers, directly or indirectly, a physiological
effect on the patient. When the active agent is a compound, salts,
solvates (including hydrates) of the free compound or salt,
crystalline and non-crystalline forms, as well as various
polymorphs of the compound are included. Compounds may contain one
or more asymmetric elements such as stereogenic centers,
stereogenic axes and the like, e.g. asymmetric carbon atoms, so
that the compounds can exist in different stereoisomeric forms.
These compounds can be, for example, racemates or optically active
forms.
[0056] "Administration" means dispensing a compound or composition
containing the compound for use via any appropriate route, for
example, oral administration, in either solid or liquid dosage
form, inhalation, injection, suppository administration, or
transdermal contact. "Administration" also include applying a
compound or composition containing the compound via any appropriate
route such as via oral administration, in either solid or liquid
dosage form, inhalation, injection, suppository administration, or
transdermal contact. "Administration" does not include in vivo
formation of the compound by a metabolic pathway in a person or
animal who has consumed or been treated with ketamine or
norketamine.
[0057] "Depressive symptoms" include low mood, diminished interest
in activities, psychomotor slowing or agitation, changes in
appetite, poor concentration or indecisiveness, excessive guilt or
feelings of worthlessness, and suicidal ideations may occur in the
context of depressive disorders, bipolar disorders, mood disorders
due to a general medical condition, substance-induced mood
disorders, other unspecified mood disorders, and also may be
present in association with a range of other psychiatric disorders,
including but not limited to psychotic disorders, cognitive
disorders, eating disorders, anxiety disorders and personality
disorders. The longitudinal course of the disorder, the history,
and type of symptoms, and etiologic factors help distinguish the
various forms of mood disorders from each other.
[0058] "Depression symptoms rating scale" refers to any one of a
number of standardized questionnaires, clinical instruments, or
symptom inventories utilized to measure symptoms and symptom
severity in depression. Such rating scales are often used in
clinical studies to define treatment outcomes, based on changes
from the study's entry point(s) to endpoint(s). Such depression
symptoms rating scales include, but are not limited to, The Quick
Inventory of Depressive-Symptomatology Self-Report
(QIDS-SR.sub.16), the 17-Item Hamilton Rating Scale of Depression
(HRSD.sub.17), the 30-Item Inventory of Depressive Symptomatology
(IDS-C.sub.30), or The Montgomery-Asperg Depression Rating Scale
(MADRS). Such ratings scales may involve patient self-report or be
clinician rated. A 50% or greater reduction in a depression ratings
scale score over the course of a clinical trial (starting point to
endpoint) is typically considered a favorable response for most
depression symptoms rating scales. "Remission" in clinical studies
of depression often refers to achieving at, or below, a particular
numerical rating score on a depression symptoms rating scale (for
instance, less than or equal to 7 on the HRSD.sub.17; or less than
or equal to 5 on the QIDS-SR.sub.16; or less than or equal to 10 on
the MADRS).
[0059] A "patient" means any human or non-human animal in need of
medical treatment. Medical treatment can include treatment of an
existing condition, such as a disease or disorder, prophylactic or
preventative treatment, or diagnostic treatment. In some
embodiments the patient is a human patient.
[0060] "Pharmaceutical compositions" are compositions comprising at
least one active agent, such as a compound or salt of Formula I,
and at least one other substance, such as a carrier, excipient, or
diluent.
[0061] The term "carrier" applied to pharmaceutical compositions of
the invention refers to a diluent, excipient, or vehicle with which
an active compound is administered.
[0062] A "pharmaceutically acceptable excipient" means an excipient
that is useful in preparing a pharmaceutical composition that is
generally safe, non-toxic, and neither biologically nor otherwise
undesirable, and includes an excipient that is acceptable for
veterinary use as well as human pharmaceutical use. A
"pharmaceutically acceptable excipient" as used in the present
application includes both one and more than one such excipient.
[0063] "Pharmaceutically acceptable salts" are derivatives of the
disclosed compounds, wherein the parent compound is modified by
making non-toxic acid or base addition salts thereof, and further
refers to pharmaceutically acceptable solvates, including hydrates,
of such compounds and such salts. Examples of pharmaceutically
acceptable salts include, but are not limited to, mineral or
organic acid addition salts of basic residues such as amines;
alkali or organic addition salts of acidic residues such as
carboxylic acids; and the like, and combinations comprising one or
more of the foregoing salts. The pharmaceutically acceptable salts
include non-toxic salts and the quaternary ammonium salts of the
parent compound formed, for example, from non-toxic inorganic or
organic acids. For example, non-toxic acid salts include those
derived from inorganic acids such as hydrochloric, hydrobromic,
sulfuric, sulfamic, phosphoric, nitric and the like; other
acceptable inorganic salts include metal salts such as sodium salt,
potassium salt, cesium salt, and the like; and alkaline earth metal
salts, such as calcium salt, magnesium salt, and the like, and
combinations comprising one or more of the foregoing salts.
[0064] Pharmaceutically acceptable organic salts include salts
prepared from organic acids such as acetic, trifluoroacetic,
propionic, succinic, glycolic, stearic, lactic, malic, tartaric,
citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic,
glutamic, benzoic, salicylic, mesylic, esylic, besylic, sulfanilic,
2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane
disulfonic, oxalic, isethionic, HOOC-(CH.sub.2).sub.n-COOH where n
is 0-4, and the like; organic amine salts such as triethylamine
salt, pyridine salt, picoline salt, ethanolamine salt,
triethanolamine salt, dicyclohexylamine salt,
N,N'-dibenzylethylenediamine salt, and the like; and amino acid
salts such as arginate, asparginate, glutamate, and the like, and
combinations comprising one or more of the foregoing salts.
[0065] "Prodrug" means any compound that becomes compound of the
invention when administered to a mammalian subject, e.g., upon
metabolic processing of the prodrug. Examples of prodrugs include,
but are not limited to, acetate, formate, and benzoate and like
derivatives of functional groups (such as alcohol or amine groups)
in the compounds of the invention.
[0066] The term "therapeutically effective amount" or "effective
amount" means an amount effective, when administered to a human or
non-human patient, to provide any therapeutic benefit. A
therapeutic benefit may be an amelioration of symptoms, e.g., an
amount effective to decrease the symptoms of a depressive disorder
or pain. A therapeutically effective amount of a compound is also
an amount sufficient to provide a significant positive effect on
any indicia of a disease, disorder, or condition e.g. an amount
sufficient to significantly reduce the frequency and severity of
depressive symptoms or pain. A significant effect on an indicia of
a disorder or condition includes a statistically significant in a
standard parametric test of statistical significance such as
Student's T-test, where p<0.05; though the effect need not be
significant in some embodiments.
CHEMICAL DESCRIPTION
Compound Structure
[0067] The structure of (2R, 6R)-hydroxynorketamine is given by
Leung and Baillie (J. Med. Chem., (1986) 29: 2396-2399)
##STR00003##
[0068] This compound is known as (Z)-6-hydroxynorketamine. Its
IUPAC name is (2S,6
S)-2-amino-2-(2-chlorophenyl)-6-hydroxycyclohexanone.
(Z)-6-hydroxynorketamine has the following CAS registration
numbers: 111056-64-5 and 95342-35-1.
[0069] The structure of (5)-dehydronorketamine is
##STR00004##
[0070] Its IUPAC name is
(S)-1-amino-2'-chloro-5,6-dihydro-[1,1'-biphenyl]-2(1H)-one. Other
compounds of this disclosure include: (2S,6S)-hydroxynorketamine,
which has the structure
##STR00005##
[0071] The disclosure includes all stereoisomers of
hydroxynorketamine and dehydronorketamine.
[0072] The disclosure includes novel compounds that are prodrugs of
hydroxynorketamine diastereomers, such as (2R,
6R)-hydroxynorketamine, and (R)- and (S)-dehydronorketamine as well
as prodrugs of stereoisomers of these compounds.
Compound Function
[0073] Without wishing to be bound to any particular theory
applicants believe certain compounds of Formula I exert activity
via serine racemase inhibition. Since nicotinic acetylcholine
receptors (nAChR) have been validated as a target in analgesia
applicants investigated the activity of DHNK at two nAChR subtypes,
.alpha..sub.7 and .alpha..sub.3.beta..sub.4 nAChR. Patch-clamp
technique in a whole-cell configuration was used to examine
functional activity of ketamine, norketamine, DHNK and HNK
metabolites at the .alpha.7 and .alpha.3.beta..sub.4 neuronal
acetylcholinesterase receptors (nAChRs). The activity of these
compounds at the .alpha.3.beta..sub.4 nAChR was investigated using
nicotine-stimulated 86Rb+ efflux assays in HEK293 cells expressing
the .alpha.3.beta.4 nAChR. DHNK was identified as a potent
inhibitor of the .alpha..sub.7 nAChR (IC.sub.50=0.05 .mu.M) while
were determined to be inactive ketamine and norketamine are
inactive. DHNK is inactive at the .alpha..sub.3.beta..sub.4 nAChR
while ketamine is a weak non-competitive inhibitor (IC.sub.50=10
.mu.M).
[0074] Applicants have determined that ketamine inhibits NMDA
(N-methyl-D-aspartic acid) receptors by inhibiting serine racemase
and thereby limiting the concentration of D-serine available to
interact with the NMDA receptor. An indirect inhibition of SR
activity by ketamine or ketamine metabolites through the inhibition
of nAChR activity was hypothesized. PC12 cells were chosen to test
the hypothesis since the cell line expresses active SR and
.alpha..sub.7 and .alpha..sub.3.beta..sub.4 nAChRs. Applicants
determined DHNK is a potent and selective inhibitor of .alpha.7
nAChR that greatly reduces intracellular concentration of D-serine
in 1321N1 astrocytoma cells (IC50=16.37 nM).
[0075] Contrary to the current "ketamine paradigm", it appears that
downstream ketamine metabolites are active and can contribute to
the clinically observed antinociceptive effects of the parent drug.
This is consistent with the results of previous
pharmacokinetic/pharmacodynamic studies indicating that ketamine
and NK exposure alone do not explain ketamine's analgesic
properties. In addition, ketamine's remarkable clinical efficacy in
some CRPS patients may reflect individual differences in the
ability to metabolize ketamine.
Compound Embodiments
[0076] In addition to pharmaceutical compositions containing
compounds of Formula I, as defined in the "SUMMARY" section the
disclosure include prodrugs of hydroxynorketamine diastereomers,
such as (2R, 6R)-hydroxynorketamine in which the variables, e.g.,
R.sub.1-R.sub.6 carry the following definitions. The disclosure
includes compounds of Formula I having any combination of variable
definitions that results in a stable compound.
[0077] Thus the disclosure includes compounds and salts of Formula
I
##STR00006##
in which the following conditions are met.
Subformulae of Formula I
[0078] The disclosure includes compounds and salts of the following
subformulae of Formula I.
##STR00007##
[0079] The disclosure also includes compounds and salts of Formula
I-VI in which the 2-Chlorophenyl is replaced by a 2-fluorophenyl or
2-bromophenyl, each of which is substituted with R.sub.5.
[0080] The disclosure also includes compounds of the formula
##STR00008##
and the pharmaceutically acceptable salts thereof and
pharmaceutical compositions containing these compounds and salts
together with at least one pharmaceutically acceptable carrier.
[0081] In a particular embodiment the disclosure includes compounds
of the formula
##STR00009##
and the pharmaceutically acceptable salts thereof and
pharmaceutical compositions containing one or both of these
compounds or salts thereof and at least one pharmaceutically
acceptable carrier.
Prodrug Compounds
[0082] Also included are prodrug compounds and salts of Formula I,
in which the variables R.sub.1-R.sub.4 carry the following
definitions.
[0083] R.sub.1 is hydrogen, hydroxyl or -A.sub.1B.sub.1 where
A.sub.1 is --O--, --O(C.dbd.O)-, --(C.dbd.O)O--, --O (C.dbd.O)O--,
--O(C.dbd.O)NR.sub.6--, --OS(O).sub.2--, --OS(O).sub.3, or
--OP(O).sub.3-, and B.sub.1 is C.sub.1-C.sub.8alkyl,
C.sub.2-C.sub.8alkenyl, C.sub.2-C.sub.8alkynyl,
(carbocycle)C.sub.0-C.sub.4alkyl or
(heterocycle)C.sub.0-C.sub.4alkyl, each of which is substituted
with from 0 to 4 substituents independently chosen from halogen,
hydroxyl, amino, cyano, C.sub.1-C.sub.4alkyl,
C.sub.1-C.sub.4alkoxy, C.sub.1-C.sub.6alkylester, mono- and
di-(C.sub.1-C.sub.4alkyl)amino,
(C.sub.3-C.sub.7cycloalkyl)C.sub.0-C.sub.2alkyl,
(heterocycloalkyl)C.sub.0-C.sub.2alkyl, C.sub.1-C.sub.2haloalkyl,
and C.sub.1-C.sub.2haloalkoxy; with the proviso that
-A.sub.1B.sub.1 is not (4-methylphenyl)-S(O).sub.2O-.
[0084] The six-membered ring to which R.sub.1 is bound is fully
saturated when R.sub.1 is hydroxyl or -A.sub.1B.sub.1.
[0085] R.sub.2 is hydrogen or -A.sub.2B.sub.2 where A.sub.2 is a
bond, --O(C.dbd.O)--, --(C.dbd.O)O--, --S(O).sub.2--,
--(S.dbd.O)NR.sub.6-, or --(C.dbd.O)NR.sub.6-, B.sub.2 is
C.sub.1-C.sub.8alkyl, C.sub.2-C.sub.8alkenyl,
C.sub.2-C.sub.8alkynyl, C.sub.2-C.sub.6alkanoyl,
(carbocycle)C.sub.0-C.sub.4alkyl,
(heterocycle)C.sub.0-C.sub.4alkyl, or an amino acid or dipeptide
covalently bound to A.sub.2 by its C-terminus, each of which is
substituted with from 0 to 4 substituents independently chosen from
halogen, hydroxyl, amino, cyano, C.sub.1-C.sub.4alkyl,
C.sub.1-C.sub.4alkoxy, C.sub.1-C.sub.6alkylester, mono- and
di-(C.sub.1-C.sub.4alkyl)amino,
(C.sub.3-C.sub.7cycloalkyl)C.sub.0-C.sub.2alkyl,
(heterocycloalkyl)C.sub.0-C.sub.2alkyl, C.sub.1-C.sub.2haloalkyl,
and C.sub.1-C.sub.2haloalkoxy; with the proviso that B.sub.2 is not
methyl when A.sub.2 is a bond or --(C.dbd.O)O-.
[0086] R.sub.1 is not hydrogen or hydroxyl when R.sub.2 is
hydrogen.
[0087] R.sub.3 is hydrogen or C.sub.1-C.sub.6alkyl.
[0088] R.sub.4 and R.sub.5 are 0 or 1 or more substituents
independently chosen from halogen, hydroxyl, amino, cyano,
C.sub.1-C.sub.4alkyl, C.sub.1-C.sub.4alkoxy, mono- and
di-C.sub.1-C.sub.4alkylamino, C.sub.1-C.sub.2haloalkyl, and
C.sub.1-C.sub.2haloalkoxy.
[0089] R.sub.6 is hydrogen or C.sub.1-C.sub.6alkyl.
[0090] Also included are embodiments in which the following
conditions are met. These definitions can also be applied for
methods of treatment and pharmaceutical compositions. The
disclosure includes all combinations of these variables so long as
a stable compound results.
[0091] (1) R.sub.1 is hydrogen or hydroxyl.
[0092] R.sub.3 is hydrogen or methyl.
[0093] R.sub.4 and R.sub.5 are each 0 to 2 substituents
independently chosen from halogen, hydroxyl, amino,
C.sub.1-C.sub.2alkyl, and C.sub.1-C.sub.2alkyl.
[0094] R.sub.6 is hydrogen or methyl.
[0095] (2) R.sub.2 is -A.sub.2B.sub.2 where A.sub.2 is a bond,
--(C.dbd.O)O--, --S(O).sub.2--, --(S.dbd.O)NR.sub.6-, or
--(C.dbd.O)NR.sub.6-, B.sub.2 is C.sub.1-C.sub.6alkyl,
C.sub.2-C.sub.4alkanoyl, (phenyl)C.sub.0-C.sub.2alkyl,
(C.sub.3-C.sub.7cycloalkyl)C.sub.0-C.sub.4alkyl,
(heterocycloalkyl)C.sub.0-C.sub.2alkyl, (5- or 6-membered
heteroaryl)C.sub.o-C.sub.2alkyl, or an amino acid covalently bound
to A.sub.2 by its C-terminus, each of which is substituted with
from 0 to 4 substituents independently chosen from halogen,
hydroxyl, amino, cyano, C.sub.1-C.sub.4alkyl,
C.sub.1-C.sub.4alkoxy, C.sub.1-C.sub.6alkylester, mono- and
di-(C.sub.1-C.sub.4alkyl)amino, C.sub.1-C.sub.2haloalkyl, and
C.sub.1-C.sub.2haloalkoxy.
[0096] (3) A.sub.2 is a bond and B.sub.2 is an amino acid
covalently bound to A.sub.2 by its C-terminus.
[0097] (4) B.sub.2 is
##STR00010##
[0098] (5) A.sub.2 is a bond or --(C.dbd.O)O- and B.sub.2 is
C.sub.2-C.sub.6alkyl, (phenyl)C.sub.0-C.sub.2alkyl, or
(C.sub.3-C.sub.7alkyl)C.sub.0-C.sub.4alkyl, each of which is
substituted with from 0 to 4 substituents independently chosen from
halogen, hydroxyl, amino, cyano, C.sub.1-C.sub.4alkyl,
C.sub.1-C.sub.4alkoxy, and mono- and
di-(C.sub.1-C.sub.4alkyl)amino.
[0099] (6) R.sub.2 is hydrogen.
[0100] R.sub.3 is hydrogen or methyl.
[0101] R.sub.4 and R.sub.5 are each 0 to 2 substituents
independently chosen from halogen, hydroxyl, amino,
C.sub.1-C.sub.2alkyl, and C.sub.1-C.sub.2alkyl.
[0102] R.sub.6 is hydrogen or methyl.
[0103] (7) R.sub.1 is -A.sub.1B.sub.1 where A.sub.1 is
--O(C.dbd.O)- or --O(C.dbd.O)O-, and B.sub.1 is
C.sub.1-C.sub.6alkyl, (phenyl)C.sub.0-C.sub.4alkyl,
(C.sub.3-C.sub.7cycloalkyl)C.sub.0-C.sub.4alkyl, (hetero
cycloalkyl)C.sub.o-C.sub.2alkyl, or (5- or 6-membered
heteroaryl)C.sub.0-C.sub.2alkyl, each of which is substituted with
from 0 to 4 substituents independently chosen from halogen,
hydroxyl, amino, cyano, C.sub.1-C.sub.4alkyl,
C.sub.1-C.sub.4alkoxy, C.sub.1-C.sub.6alkylester, mono- and
di-(C.sub.1-C.sub.4alkyl)amino,
(C.sub.3-C.sub.7cycloalkyl)C.sub.0-C.sub.2alkyl, (hetero
cycloalkyl)C.sub.0-C.sub.2alkyl, C.sub.1-C.sub.2haloalkyl, and
C.sub.1-C.sub.2haloalkoxy. In certain embodiments R.sub.1 is
-A.sub.1B.sub.1 and A.sub.1 is --O- or A.sub.1 is --O
(C.dbd.O)-.
[0104] (8) B.sub.1 is C.sub.1-C.sub.6alkyl,
(phenyl)C.sub.0-C.sub.2alkyl, or
(heterocycloalkyl)C.sub.0-C.sub.2alkyl, each of which is
substituted with from 0 to 2 substituents independently chosen from
halogen, hydroxyl, amino, cyano, C.sub.1-C.sub.4alkyl,
C.sub.1-C.sub.4alkoxy, mono- and di-(C.sub.1-C.sub.4alkyl)amino,
(C.sub.3-C.sub.7cycloalkyl)C.sub.0-C.sub.2alkyl, and
(heterocycloalkyl)C.sub.0-C.sub.2alkyl.
[0105] (9) A compound or salt of Formula IV in which
[0106] R.sub.3 is hydrogen or methyl.
[0107] R.sub.4 and R.sub.5 are each 0 to 2 substituents
independently chosen from halogen, hydroxyl, amino,
C.sub.1-C.sub.2alkyl, and C.sub.1-C.sub.2alkyl.
[0108] R.sub.6 is hydrogen or methyl.
PHARMACEUTICAL COMPOSITIONS
[0109] Compounds disclosed herein can be administered as the neat
chemical, but are preferably administered as a pharmaceutical
composition. Accordingly, the disclosure provides pharmaceutical
compositions comprising a compound or pharmaceutically acceptable
salt of Formula I, together with at least one pharmaceutically
acceptable carrier. The pharmaceutical composition may contain a
compound or salt of Formula I as the only active agent, but is
preferably contains at least one additional active agent. In
certain embodiments the pharmaceutical composition is an oral
dosage form that contains from about 0.1 mg to about 1000 mg, from
about 1 mg to about 500 mg, or from about 10 mg to about 200 mg of
a compound of Formula I and optionally from about 0.1 mg to about
2000 mg, from about 10 mg to about 1000 mg, from about 100 mg to
about 800 mg, or from about 200 mg to about 600 mg of an additional
active agent in a unit dosage form.
[0110] Compounds disclosed herein may be administered orally,
topically, parenterally, by inhalation or spray, sublingually,
transdermally, via buccal administration, rectally, as an
ophthalmic solution, or by other means, in dosage unit formulations
containing conventional pharmaceutically acceptable carriers. The
pharmaceutical composition may be formulated as any
pharmaceutically useful form, e.g., as an aerosol, a cream, a gel,
a pill, a capsule, a tablet, a syrup, a transdermal patch, or an
ophthalmic solution. Some dosage forms, such as tablets and
capsules, are subdivided into suitably sized unit doses containing
appropriate quantities of the active components, e.g., an effective
amount to achieve the desired purpose.
[0111] Carriers include excipients and diluents and must be of
sufficiently high purity and sufficiently low toxicity to render
them suitable for administration to the patient being treated. The
carrier can be inert or it can possess pharmaceutical benefits of
its own. The amount of carrier employed in conjunction with the
compound is sufficient to provide a practical quantity of material
for administration per unit dose of the compound.
[0112] Classes of carriers include, but are not limited to binders,
buffering agents, coloring agents, diluents, disintegrants,
emulsifiers, flavorants, glidants, lubricants, preservatives,
stabilizers, surfactants, tableting agents, and wetting agents.
Some carriers may be listed in more than one class, for example
vegetable oil may be used as a lubricant in some formulations and a
diluent in others. Exemplary pharmaceutically acceptable carriers
include sugars, starches, celluloses, powdered tragacanth, malt,
gelatin; talc, and vegetable oils. Optional active agents may be
included in a pharmaceutical composition, which do not
substantially interfere with the activity of the compound of the
present invention.
[0113] The pharmaceutical compositions can be formulated for oral
administration. Preferred oral dosage forms are formulated for once
a day or twice a day administration. These compositions contain
between 0.1 and 99 weight % (wt. %) of a compound of Formula I and
usually at least about 5 wt. % of a compound of Formula. Some
embodiments contain from about 25 wt. % to about 50 wt. % or from
about 5 wt. % to about 75 wt. % of the compound of Formula.
METHODS OF TREATMENT
[0114] Studies using sub-anesthetic does of (R,S)-Ketamine
demonstrated that this drug is effective in the treatment of
neuropathic and chronic pain, including the treatment of patients
suffering from complex regional pain syndrome (CRPS). The analysis
of the plasma samples obtained from CRPS patients receiving
(R,S)-ketamine as a 5-day continuous infusion revealed that the
primary drug, (R,S)-ketamine, was not primarily responsible for the
therapeutic response This disclosure demonstrates that the active
agents responsible for the therapeutic response to ketamine in
patients are (2R,6R; 2S,6S)-hydroxynorketamine and
(R,S)-dehydronorketamine, which are ketamine metabolites. These
metabolites are produced by a number of liver enzymes identified as
Cytochrome P450s (CYPs) including CYP2A6, CYP2B6, CYP2C9, CYP2D6,
and CYP3A5. The CYPs are polymorphic, which means that they are not
equally active in all humans. Thus it is likely that ketamine's
failure to elicit a therapeutic response in about 30% of treated
patients is due the activity differences of one or more of the
identified CYPs among patients leading to unequal production of
(2R,6R, 2S,6S)-hydroxynorketamine and/or
(5)-dehydronorketamine.
[0115] The essence of the discovery is the identification of a new
compound that can be administered directly to the patient in order
to increase the response rate and to avoid treatment-limiting CNS
side effects produced by (R,S)-ketamine. The CNS effects are
associated with (R,S)-ketamine's activity at the NMDA receptor.
(2R,6R; 2S,6S)-hydroxynorketamine (HNK) is not active at the NMDA
receptor and thus avoids these side effects. Direct administration
of the (2S,6S)-hydroxynorketamine metabolite and/or DHNK has the
advantage of producing a therapeutic response in a greater
percentage of patients than ketamine (2S,6S)-hydroxynorketamine and
DHNK also have a long plasma half-lives and are orally
bioavailable. Thus, oral formulations for daily administration are
included in the disclosure.
[0116] Methods of treatment include administering any one of
(2S,6S)-hydroxynorketamine, (2R,6R)-hydroxynorketamine,
(2S,6R)-hydroxynorketamine, (2R,6S)-hydroxynorketamine,
(S)-dehydronorketamine, and (R)-dehydronorketamine, or any
combination of the foregoing. Methods of treatment also include
administering any of any one of (2S,6S)-hydroxynorketamine,
(2R,6R)-hydroxynorketamine, (2S,6R)-hydroxynorketamine,
(2R,6S)-hydroxynorketamine, (S)-dehydronorketamine, and
(R)-dehydronorketamine in optically pure form.
[0117] The disclosure includes a methods of treating bipolar
depression, major depressive disorder, schizophrenia, Alzheimer's
dementia, amyotrophic lateral sclerosis, complex regional pain
syndrome (CRPS), chronic pain, or neuropathic pain comprising
administering a pharmaceutical composition containing an effective
amount of a compound of Formula I or a pharmaceutically acceptable
salt thereof, together with a pharmaceutically acceptable carrier
to a patient in need of such treatment.
[0118] Methods of treatment include providing certain dosage
amounts of a compound or pharmaceutically acceptable salt of
Formula I to a patient. Dosage levels of each active agent of from
about 0.1 mg to about 140 mg per kilogram of body weight per day
are useful in the treatment of the above-indicated conditions
(about 0.5 mg to about 7 g per patient per day). The amount of
active ingredient that may be combined with the carrier materials
to produce a single unit dosage form will vary depending upon the
patient treated and the particular mode of administration.
[0119] In certain embodiments a therapeutically effect amount is an
amount that provide a plasma Cmax of a compound of Formula I of
about of 0.25 mcg/mL to about 125 mcg/mL, or about 1 mcg/mL to
about 50 mcg/mL. For peripheral indications formulations and
methods that provide a Cmax of about 0.25 mcg/mL to about 25 mcg/mL
are preferred, while for CNS indications, formulations and methods
that provide a plasma Cmax of about 0.25 mcg/mL to about 125 mcg/mL
are preferred. The disclosure also includes IV pharmaceutical
compositions that provide about 0.2 mg to about 500 mg per dose of
a compound of Formula I, for peripheral indications compounds that
provide about 0.5 mg to about 500 mg/dose are preferred.
[0120] The compound or salt of Formula I may be the only active
agent administered or may be administered together with an
additional active agent. For example the compound of Formula I may
administered together with another active agent that is chosen from
any of the following:
[0121] Antidepressants: escitalopram, fluoxetine, paroxetine,
duloxetine, sertraline, citalopram, bupropion, venlafaxine,
duloxetine, naltrexone, mirtazapine, venlafaxine, atomoxetine,
bupropion, doxepin, amitriptyline, clomipramine, nortriptyline,
buspirone, aripiprazole, clozapine, loxapine, olanzapine,
quetiapine, risperidone, ziprasidone, carbamazepine, gabapentin,
lamotrigine, phenytoin, pregabalin, donepezil, galantamine,
memantine, rivastigmine, tramiprosate, or pharmaceutically active
salts or prodrugs thereof, or a combination of the foregoing;
[0122] Schizophrenia Medications: aripiprazole, lurasidone,
asenapine, clozapine, ziprasidone, risperidone, quetiapine,
stelazine, olanzapine, loxapine, flupentioxol, perphenazine,
haloperidol, chlorpromazine, fluphenazine, prolixin, paliperidone;
Alzheimer's Dementia Medications: donepezil, rivastigmine,
galantamine, memantine
[0123] ALS Medications: riluzole
[0124] Pain Medications: acetaminophen, aspirin, NSAIDS, including
Diclofenac, Diflunisal, Etodolac, Fenoprofen, Flurbiprofen,
Ibuprofen, Indomethacin, Ketoprofen, Ketorolac, Meclofenamate,
Mefenamic Acid, Meloxicam, Nabumetone, Naproxen, Oxaprozin,
Phenylbutazone, Piroxicam, Sulindac, Tolmetinopiods, Cox-2
inhibitors such as celcoxib, and narcotic pain medications such as
Buprenorphine, Butorphanol, Codeine, Hydrocodone, Hydromorphone,
Levorphanol, Meperidine, Methadone, Morphine, Nalbuphine,
Oxycodone, Oxymorphone, Pentazocine, Propoxyphene, the central
analgesic tramadol.
[0125] The preceding list of additional active agents is meant to
be exemplary rather than fully inclusive. Additional active agents
not included in the above list may be administered in combination
with a compound of Formula I. The additional active agent will be
dosed according to its approved prescribing information, though in
some embodiments the additional active agent will be dosed at less
the typically prescribed dose and in some instances less than the
minimum approved dose.
[0126] The disclosure includes a method of treating bipolar
depression and major depressive disorder where an effective amount
of the compound is an amount effective to decrease depressive
symptoms, wherein a decrease in depressive symptoms is the
achievement of a 50% or greater reduction of symptoms identified on
a depression symptom rating scale, or a score less than or equal to
7 on the HRSD.sub.17, or less than or equal to 5 on the
QID-SR.sub.16, or less than or equal to 10 on the MADRS.
[0127] The disclosure provides an amount effective to decrease
painful symptoms; wherein a decrease in painful symptom is the
achievement of a 50% or greater reduction of painful symptoms on a
pain rating scale.
EXAMPLES
Example 1
Synthesis of Lysine Conjugates of Norketamine
##STR00011##
[0128] Example 2
Synthesis of Ester Conjugates of 6-Hydroxynorketamine
##STR00012##
[0129] Example 3
Synthesis of (+,-)-(2S,6R/2R,6S)-6-Hydroxynorketamine
##STR00013##
[0131] Synthesis of (+,-)-(Z+E)-6-Bromonorketamine. A sample of
racemic (+,-)-norketamine (free base) (10.0 g, 35.8 mmol) in 50 mL
glacial acetic acid was treated with pyridinium tribromide (16.4 g,
51.3 mmol). The resulting mixture was heated at 130.degree. C. for
1 h using microwaves. The solvent was removed in vacuo and the
crude material dissolved in CHCl.sub.3 and washed with saturated
NaHCO.sub.3, dried (Na.sub.2SO.sub.4) and evaporated in vacuo
leaving 12.4 g of a crude product mixture of diasteriomers (Z+E,
3:1). This was chromatographed using silica gel, eluting with a
varying concentration of CH.sub.2Cl.sub.2/MeOH/Et.sub.3N from
(99.9/0/0.1) to (98.9/1/0.1) to give the pure separated isomers,
(+,-)-(E)-6-bromonorketamine (1.22 g) (9% yield) and
(+,-)-(Z)-6-bromonorketamine (6.6 g) (49% yield).
[0132] Analytical Data for (+,-)-(E)-6-bromonorketamine: .sup.1H
NMR: (300 MHz, CDCl.sub.3): .delta. 7.60 (m, 1H), 7.20-7.10 (m,
3H), 5.17 (dd, 1H, J=12.0 Hz, J=6.6 Hz), 2.72-2.39 (m, 3H),
2.36-2.08 (m, 1H), 1.96-1.82 (m, 2H), 1.69 (br s, 2H). MS: (ESI)
m/z (relative intensity): 302 (77, M+H), 304 (100, M+H).
[0133] Analytical Data for (+,-)-(Z)-6-bromonorketamine: .sup.1H
NMR: (300 MHz, CDCl.sub.3): .delta. 7.62 (dd, 1H, J=1.8 Hz, J=7.5
Hz), 7.45-7.22 (m, 3H), 4.90 (dd, 1H, J=12.0 Hz, J=6.6 Hz),
2.70-2.63 (m, 1H), 2.58-2.41 (m, 2H), 2.24-2.09 (m, 2H), 2.10-1.78
(m, 3H). MS: (ESI) m/z (relative intensity): 302 (79, M+H), 304
(100, M+H). Gradient HPLC: Varian Pursuit C-18 (5 micron),
50.times.2 mm; 0.1% TFA in Water/0.1% TFA in Acetonitrile 95/5 to
5/95 over 5 min then hold; 0.200 mL/min; Total Ion Chromatogram
(TIC); R.sub.t 7.03, product, 95% pure.
[0134] Synthesis of (+,-)-(2S,6R/2R,6S)-6-Hydroxynorketamine. A
sample of racemic (+,-)-(Z)-6-bromonorketamine (2.71 g, 8.96 mmol)
in absolute ethanol (20 mL) was treated with 1M ammonium formate
(pH 6.8) (20 mL). The resulting solution was stirred under Ar for
10 days forming only a trace of product. The solvent was removed in
vacuo and the crude product mixture dissolved in DMSO (10 mL) and
purified by injections onto a preparative HPLC. Gradient HPLC:
Waters Sunfire Prep C18 (10 micron), 150.times.30 mm; 5 mM Ammonium
Formate in Water/Acetonitrile (90/10) for 5 min then (90/10) to
(10/90) over 10 min then hold; 10 mL/min, 270 nm; R.sub.t 9.5 min.
Product fractions were combined and evaporated in vacuo giving
(+,-)-(2S,6R/2R,6S)-6-hydroxynorketamine (25 mg) (1% yield).
[0135] Analytical Data for (+,-)-(E)-6-Hydroxynorketamine: .sup.1H
NMR: (300 MHz, CDCl.sub.3): .delta. 7.62 (dd, 1H, J=1.8 Hz, J=7.5
Hz), 7.45-7.22 (m, 3H), 4.90 (dd, 1H, J=12.0 Hz, J=6.6 Hz),
2.70-2.63 (m, 1H), 2.58-2.41 (m, 2H), 2.24-2.09 (m, 2H), 2.10-1.78
(m, 3H). MS: (ESI) m/z (relative intensity): 240 (100, M+H).
Gradient HPLC: Varian Pursuit C-18 (5 micron), 150.times.4.6 mm; 5
mMAmmonium Formate (pH 7.6)/Acetonitrile 80/20 for 5 min then 80/20
to 20/80 over 10 min then hold; 0.250 mL/min; Total Ion
Chromatogram (TIC); R.sub.t 17.26, product, 95% pure.
Example 4
Additional Purified Hydroxynorketamine and Dehydroxynorketamine
Forms
(S)-5,6-Dehydroxynorketamine
##STR00014##
[0137] .sup.1H NMR: (400 MHz, CD.sub.3OD): d 7.58 (dd, 1H, J=8.0
Hz, J=1.2 Hz), 7.49 (dt, 1H, J=7.6 Hz, J=1.6 Hz), 7.40 (dt, 1H,
J=7.2 Hz, J=1.2 Hz), 7.33 (dd, 1H, J=8.0 Hz, J=1.6 Hz), 7.12-7.07
(m, 1H), 6.38-6.34 (m, 1H), 3.41-3.37 (m, 1H), 2.60-2.51 (m, 1H),
2.36-2.28 (m, 1H), 2.15-2.04 (m, 1H). MS: (ESI) m/z (relative
intensity): 222 (100, M+H). Gradient HPLC: Varian Pursuit C-18 (3
micron), 50.times.2 mm, Acetonitrile/10 mM Ammonium Formate (pH 7)
in Water: (2/98) for 2.5 min then (2/98) to (98/2) over 7.5 min
then hold; 0.200 mL/min, 254 nm; R.sub.t 4.10 min; 95% pure. Chiral
HPLC: Supelco Chiral AGP (5 micron), 100.times.4 mm;
Acetonitrile/50 mM Ammonium Acetate in Water (pH 7.2) (10/90);
0.700 mL/min, 225 nm; R.sub.t 3.370 min; 98.48% pure.
(R)-5,6-Dehydroxynorketamine Hydrochloride
##STR00015##
[0139] .sup.1H NMR: (400 MHz, CD3OD): d 7.58 (dd, 1H, J=8.0 Hz,
J=1.2 Hz), 7.49 (dt, 1H, J=7.6 Hz, J=1.6 Hz), 7.40 (dt, 1H, J=7.2
Hz, J=1.2 Hz), 7.33 (dd, 1H, J=8.0 Hz, J=1.6 Hz), 7.12-7.07 (m,
1H), 6.38-6.34 (m, 1H), 3.41-3.37 (m, 1H), 2.60-2.51 (m, 1H),
2.36-2.28 (m, 1H), 2.15-2.04 (m, 1H). MS: ESI) m/z (relative
intensity): 222 (100, M +H). Gradient HPLC: Varian Pursuit C-18 (3
micron), 50.times.2 mm, Acetonitrile/10 mM Ammonium Formate (pH 7)
in Water: (2/98) for 2.5 min then (2/98) to (98/2) over 7.5 min
then hold; 0.200 mL/min, 254 nm; Rt 4.18 min; 95% pure. Chiral
HPLC: Supelco Chiral AGP (5 micron), 100.times.4 mm;
Acetonitrile/50 mM Ammonium Acetate in Water (pH 7.2) (10/90);
0.700 mL/min, 225 nm; Rt 8.059 min; 95.29% pure.
(2R,6R)-6-Hydroxynorketamine Hydrochloride
##STR00016##
[0141] .sup.1H NMR: (300 MHz, CD.sub.3OD): .delta. 7.90-7.86 (m,
1H), 7.62-7.55 (m, 3H), 4.30 (dd, 1H, J=6.8 Hz, J=11.6 Hz),
3.26-3.18 (m, 1H), 2.33-2.27 (m, 1H), 2.00-1.60 (m, 4H). MS: (ESI)
m/z (relative intensity): 240 (100, M+H). Gradient HPLC: Varian
Pursuit C-18 (5 micron), 150.times.4.6 mm; 5 mM Ammonium Formate in
Water /Acetonitrile (80/20) for 5 min then (80/20) to (20/80) over
10 min then hold; 0.250 mL/min, 270 nm; R.sub.t 13.05; 99% pure.
Chiral HPLC: Supelco Chiral AGP (5 micron), 100.times.4 mm; 100% 50
mM Ammonium Acetate in Water (pH 7.2); 0.800 mL/min, 215 nm;
R.sub.t 4.487 min; 97.68% pure.
(2S,6S)-6-Hydroxynorketamine Hydrochloride
##STR00017##
[0143] .sup.1H NMR: (300 MHz, CD.sub.3OD): d 7.90-7.86 (m, 1H),
7.62-7.55 (m, 3H), 4.30 (dd, 1H, J=6.8 Hz, J=11.6 Hz), 3.26-3.18
(m, 1H), 2.33-2.27 (m, 1H), 2.00-1.60 (m, 4H). MS: (ESI) m/z
(relative intensity): 240 (100, M+H). Gradient HPLC: Varian Pursuit
C-18 (5 micron), 150.times.4.6 mm; 5 mM Ammonium Formate in
Water/Acetonitrile (80/20) for 5 min then (80/20) to (20/80) over
10 min then hold; 0.250 mL/min, 270 nm; R.sub.t 13.05; 99% pure.
Chiral HPLC: Supelco Chiral AGP (5 micron), 100.times.4 mm; 100% 50
mM Ammonium Acetate in Water (pH 7.2); 0.800 mL/min, 215 nm;
R.sub.t 5.574 min; 98.61% pure.
Example 5
Serine Racemase Inhibition Assay
Materials
[0144] D-serine (D-Ser), L-serine (L-Ser), D-alanine (D-Ala),
L-alanine (L-Ala), D-arginine, (D-Arg), L-arginine (L-Arg), glycine
(Gly), D-leucine (D-Leu), L-leucine (L-Leu), D-isoleucine (D-Iso),
L-isoleucine (L-Iso), D-glutamic acid (D-Glu), L-glutamic acid
(L-Glu), D-aspartic acid (D-Asp), L-aspartic acid (L-Asp), L-lysine
(L-Lys), .beta.-cyclodextrin (.beta.-CD),
2-hydroxypropyl-.beta.-cyclodextrin (HP-.beta.-CD), methanol,
acetonitrile (ACN), and fluorescein isothiocyanate (FITC) were
obtained from Sigma-Aldrich (St. Louis, Mo., USA). De-ionized water
was obtained from a Milli-Q system (Millipore, Billerica, Mass.,
USA). All other chemicals used were of analytical grade.
Cell Lines and Cell Culture
[0145] The cell lines selected for this study were PC-12
pheochromocytoma derived from rat adrenal medulla, human-derived
1321N1 astrocytoma, rat-derived C6 glioblastoma, and human-derived
HepG2 hepatocellular carcinoma. All of the cell lines were obtained
from ATCC (Manassas, Va., USA). The PC12 cells were maintained in
RPMI7 1640 with L-Gln supplemented with 10% horse serum (heat
inactivated), 5% FBS, 1% sodium pyruvate solution, 1% HEPES buffer
and 1% penicillin/streptomycin solution. The 1321N1 and C6 cells
were maintained in DMEM with L-Gln supplemented with 10% FBS and 1%
penicillin/streptomycin solution. The HepG2 cells were maintained
in E-MEM supplemented with 1% L-Gln, 10% FBS, 1% sodium pyruvate
solution and 1% penicillin/streptomycin solution.
[0146] Dulbecco's Modified Eagle Medium (DMEM) with glutamine,
Eagle's Minimum Essential Medium (E-MEM), RPMI-1640, trypsin
solution, phosphate-buffered saline, fetal bovine serum (FBS),
sodium pyruvate solution (100 mM), L-glutamine (L-Gln) (200 mM) and
penicillin/streptomycin solution (containing 10,000 units/ml
penicillin and 10,000 .mu.g/ml streptomycin) were obtained from
Quality Biological (Gaithersburg, Md., USA), horse serum (heat
inactivated) was obtained from Biosource (Rockville, Md., USA) and
HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer
(1 M) was obtained from Mediatech Inc. (Manassas, Va., USA).
CE-LIF (Capillary Electrophoresis--Laser Induced Fluorescence)
Analysis
[0147] Instrumentation: The CE separations were performed with a
P/ACE MDQ system equipped with a laser-induced fluorescence
detector (Beckman Instruments, Fullerton, Calif., USA). The
laser-induced fluorescence detection was carried out with
excitation at 488 nm and emission at 520 nm. An uncoated
fused-silica capillary of 50 .mu.m I.D. and effective length of 50
cm was used and the running buffer was composed of 0.5 mM
HP-.beta.-CD solution prepared in borate buffer [80 mM, pH 9.3].
The capillary was conditioned before each analysis by flushing
successively with 0.1 N NaOH, 0.1 N H.sub.3PO.sub.4, H.sub.2O and
running buffer each for 4 min. Samples were injected with pressure
at 0.5 p.s.i. for 1 s and separated using a voltage gradient in
which separation voltage was 15 kV between 0-44 min, followed by 22
kV between 45-60 min. At 60 min, the voltage was reduced to 0 kV
and the column washed for 4 min with 0.1 N NaOH followed by a 4 min
wash with 0.1 N phosphoric acid. The total run time was 68 min.
Quantification was accomplished using area ratios calculated for
FITC-D-Ser with FITCD-Arg as the internal standard, where the
concentration of the internal standard was set at 5 .mu.M.
[0148] Standard solutions: A concentrated stock solution of 0.5 mM
D-Ser in borate buffer [80 mM, pH 9.3] was used to prepare 0.25,
0.5, 1, 2, 4, 10, 20, 40, 80, and 100 .mu.M solutions for the
calibration curve. Standard solutions, 1 mM in borate buffer [80
mM, pH 9.3], of L-Ser, D-Ala, L-Ala, D-Arg, L-Arg, Gly, D-Leu,
L-Leu, D-Iso, L-Iso, D-Glu, L-Glu, D-Asp, L-Asp, L-Lys were also
prepared. A 100 .mu.M solution of D-Arg in H.sub.2O was used as the
internal standard solution.
[0149] Sample preparation: Cells were collected and centrifuged for
5 min at 200.times.g at 4.degree. C. The supernatant was discarded
and the cells were suspended in 1.00 ml of H.sub.2O, 0.050 ml of
the internal standard was added and the resulting mixture vortex
mixed for 1 min. A 4.00 ml aliquot of ACN was added and the
suspension was sonicated for 20 min. The mixture was then
centrifuged for 15 min at 2500.times.g at 4.degree. C., the
supernatant collected and stream dried under nitrogen. The residue
was dissolved in 0.90 ml of borate buffer [80 mM, pH 9.3].
[0150] FITC labeling: FITC solution (3 mg/ml) was prepared in
acetone and stored at -20 .degree. C. until use. For the
derivatization of standard amino acids, a 0.05 ml aliquot of the
internal standard solution was added to a 0.85 ml of the standard
solution and 0.10 ml of FITC solution was added and the resulting
solutions were placed in darkness for 12 h at room temperature.
When cellular extracts were assayed, 0.10 ml of FITC solution was
added to the 0.90 ml samples and the resulting solutions were
placed in darkness for 12 h at room temperature.
Effect of Ketamine Metabolites on Serine Racemase Activity and
Expression
[0151] Cells were seeded on 100.times.20 mm tissue culture plates
and were maintained at 37.degree. C. under humidified 5% CO.sub.2
in air until they reached >70% confluence. The original media
was replaced with media containing sequential concentrations of the
test compounds and the plates were incubated for an additional 36
h. Compound concentrations of 0 to 100 nM were used, e.g. 10 nM, 20
nM, 50 nM, 75 nM and 100 nM.
[0152] The medium was removed, and the cells collected for
analysis. All of the studies were done in triplicate.
Western Blotting
[0153] Cells were lysed with RIPA buffer containing ethylene glycol
tetraacetic acid and ethylenediamine tetraacetic acid (Boston
BioProducts, Ashland, Mass., USA). The lysis buffer contained a
protease inhibitor cocktail composed of
4-(2-aminoethyl)benzenesulfonyl fluoride, pepstatin A, E-64,
bestatin, leupeptin, and aprotinin (Sigma-Aldrich). Protein
concentrations were determined using the bicinchoninic acid (BCA)
reagent obtained from Pierce Biotechnology, Inc. (Rockford, Ill.,
USA). Proteins (20 .mu.g/well) were separated on 4 to 12% precast
gels (Invitrogen, Carlsbad, Calif., USA) using SDS-polyacrylamide
gel electrophoresis under reducing conditions and then they were
electrophoretically transferred onto polyvinylidene fluoride (PVDF)
membrane (Invitrogen). Western blots were performed according to
standard methods which involved blocking in 5% non-fat milk and
incubated with the antibody of interest, followed by incubation
with a secondary antibody conjugated with the enzyme horseradish
peroxidase. The visualization of immunoreactive bands was performed
using the ECL Plus Western Blotting Detection System (GE
Healthcare, NJ, USA). The quantification of bands was done by
volume densitometry using Image software and normalization to
.beta.-actin.
[0154] The primary antibodies for SR were obtained from Santa Cruz
Biotechnology (Santa Cruz, Calif., USA), sc-48741, and Abcam
(Cambridge, Mass., USA), ab45434, and the primary antibody for
.beta.-actin was obtained from Abcam, ab6276. The antibodies were
used at a dilution recommended by the manufacturer.
Statistical Analysis
[0155] Graphpad Prism 4 (GraphPad Software, Inc., La Jolla, Calif.,
USA) running on a personal computer was used to perform all the
statistical data analysis including EC50 and IC50 value
calculations.
[0156] Certain compounds of Formula I discussed in this disclosure
were tested in the above serine racemase assay , for NMDA receptor
inhibition and inhibition of the nicotinic acetylcholine receptor
subtype .alpha..sub.7. The assay for nicotinic acetylcholorine
receptor subtype .alpha..sub.7 is given below in Example 6.
TABLE-US-00001 Compounds NMDAR K.sub.i (.mu.M) IC.sub.50
(.alpha..sub.7) IC.sub.50 (D-Serine) (S)-norKet 2.25 .+-. 0.22
.mu.M Not Determined 50-120 nM (R)-norKet 26.46 .mu.M Not available
40-80 nM (R,S)-norKet Not Determined At 100 nM the Not Determined
inhibition was 46% (S)-DHNK 38.95 .mu.M Not Determined Not
Determined (R)-DHNK 74.55 .mu.M Not Determined Not Determined
(R,S)-DHNK Not Determined ~50 nM 35-50 nM (2S,6S)-HNK 21.19 .mu.M
At 100 nM the 0.1-0.3 nM inhibition was 54% (2R,6R)-HNK .sup.
>100 .mu.M At 100 nM the 0.4-0.7 nM inhibition was 51%
[0157] DHNK was found to be a potent inhibitor of the nACh receptor
a, subtype while ketamine and norketamine are inactive at this
receptor subtype.
EXAMPLE 6
Effect of Compounds on Currents Evoked by Acetylcholine
Receptors
[0158] Since nicotinic acetylcholine receptors (nAChR) have been
validated as a target in analgesia we have investigated the
activity of DHNK (dehydroxynorketamine) at two nAChR subtypes,
.alpha..sub.7 and .alpha..sub.3.beta..sub.4 nAChR. The patch clamp
in a whole cell configuration was used to examine functional
activity of ketamine, norketamine, and the DHNK and HNK metabolites
on currents evoked by 280 micromolar acetylcholine in the
KX.alpha.7R1. The data was obtained at -80 mV and normalized to the
amplitude of current elicited by ACh alone, n=4.
TABLE-US-00002 Mean SE (R,S)-norKet 100 nM 0.548 0.099 1000 nM
0.287 0.075 (R,S)-DHNK 100 nM 0.407 0.053 1000 nM 0.205 0.013
(2S,6S)-HNK 100 nM 0.461 0.06 1000 nM 0.361 0.07 (2R,6R)-HNK 100 nM
0.489 0.077 1000 nM 0.354 0.084
* * * * *