U.S. patent application number 16/778960 was filed with the patent office on 2020-09-24 for (r)-3-((3s,4s)-3-fluoro-4-(4-hydroxyphenyl)piperidin-1-yl)-1-(4-methylbenz- yl)pyrrolidin-2-one and its prodrugs for the treatment of psychiatric disorders.
The applicant listed for this patent is Bristol-Myers Squibb Company. Invention is credited to John E. Macor, Lorin A. Thompson, III.
Application Number | 20200297742 16/778960 |
Document ID | / |
Family ID | 1000004882077 |
Filed Date | 2020-09-24 |
View All Diagrams
United States Patent
Application |
20200297742 |
Kind Code |
A1 |
Thompson, III; Lorin A. ; et
al. |
September 24, 2020 |
(R)-3-((3S,4S)-3-FLUORO-4-(4-HYDROXYPHENYL)PIPERIDIN-1-YL)-1-(4-METHYLBENZ-
YL)PYRROLIDIN-2-ONE AND ITS PRODRUGS FOR THE TREATMENT OF
PSYCHIATRIC DISORDERS
Abstract
The disclosure generally relates to compounds of formula I,
including their salts, as well as compositions and methods of using
the compounds. The compounds are ligands for the NR2B NMDA receptor
and may be useful for the treatment of various disorders of the
central nervous system. ##STR00001##
Inventors: |
Thompson, III; Lorin A.;
(Cohasset, MA) ; Macor; John E.; (Washington
Crossing, PA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Bristol-Myers Squibb Company |
Princeton |
NJ |
US |
|
|
Family ID: |
1000004882077 |
Appl. No.: |
16/778960 |
Filed: |
January 31, 2020 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07F 9/59 20130101; C07F
9/65583 20130101; A61K 31/454 20130101; C07D 401/04 20130101; A61K
31/675 20130101 |
International
Class: |
A61K 31/675 20060101
A61K031/675; C07F 9/59 20060101 C07F009/59; C07D 401/04 20060101
C07D401/04; C07F 9/6558 20060101 C07F009/6558; A61K 31/454 20060101
A61K031/454 |
Claims
1. A pharmaceutical composition comprising the compound
4-((3S,4S)-3-fluoro-1-((R)-1-(4-methylbenzyl)-2-oxopyrrolidin-3-yl)piperi-
din-4-yl)phenyl dihydrogen phosphate or a pharmaceutically
acceptable salt thereof and a pharmaceutically acceptable carrier.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This Continuation application claims the benefit of Ser. No.
16/225,631 filed on Dec. 19, 2018, now pending, which is a
Continuation application which claims priority to U.S. Ser. No.
15/971,049 filed May 4, 2018, now abandoned, which is a
Continuation application which claims priority to U.S. Ser. No.
15/846,914 filed Dec. 19, 2017, now abandoned, which is a
Continuation application which claims priority to U.S. Ser. No.
15/679,847 filed Aug. 17, 2017, now abandoned, which is a
Continuation application which claims priority to U.S. Ser. No.
15/490,558 filed Apr. 18, 2017, now abandoned, which is a
Continuation application which claims priority to U.S. Ser. No.
15/357,102 filed Nov. 21, 2016, now abandoned, which is a
Continuation application which claims priority to U.S. Ser. No.
14/881,932 filed Oct. 13, 2015, now abandoned, which is a
Continuation application which claims the priority of U.S. Ser. No.
14/591,372 filed Jan. 7, 2015, now U.S. Pat. No. 9,187,506, which
is a Non-Provisional application which claims the benefit of
Provisional application U.S. Ser. No. 61/925,405 filed Jan. 9,
2014, now expired, hereby incorporated by reference in their
entireties.
BACKGROUND OF THE INVENTION
[0002] The disclosure generally relates to compounds of formula I,
including their salts, as well as compositions and methods of using
the compounds. The compounds are ligands for the NR2B NMDA receptor
and may be useful for the treatment of various disorders of the
central nervous system.
[0003] N-Methyl-D-aspartate (NMDA) receptors are ion channels which
are gated by the binding of glutamate, an excitatory
neurotransmitter in the central nervous system. They are thought to
play a key role in the development of a number of neurological
diseases, including depression, neuropathic pain, Alzheimer's
disease, and Parkinson's disease. Functional NMDA receptors are
tetrameric structures primarily composed of two NR1 and two NR2
subunits. The NR2 subunit is further subdivided into four
individual subtypes: NR2A, NR2B, NR2C, and NR2D, which are
differentially distributed throughout the brain. Antagonists or
allosteric modulators of NMDA receptors, in particular NR2B
subunit-containing channels, have been investigated as therapeutic
agents for the treatment of major depressive disorder (G. Sanacora,
2008, Nature Rev. Drug Disc. 7: 426-437).
[0004] The NR2B receptor contains additional ligand binding sites
in addition to that for glutamate. Non-selective NMDA antagonists
such as Ketamine are pore blockers, interfering with the transport
of Ca.sup.++ through the channel. Ketamine has demonstrated rapid
and enduring antidepressant properties in human clinical trials as
an i.v. drug. Additionally, efficacy was maintained with repeated,
intermittent infusions of Ketamine (Zarate et al., 2006, Arch. Gen.
Psychiatry 63: 856-864). This class of drugs, though, has limited
therapeutic value because of its CNS side effects, including
dissociative effects.
[0005] An allosteric, non-competitive binding site has also been
identified in the N-terminal domain of NR2B. Agents which bind
selectively at this site, such as Traxoprodil, exhibited a
sustained antidepressant response and improved side effect profile
in human clinical trials as an i.v. drug (Preskorn et al., 2008, J.
Clin. Psychopharmacol., 28: 631-637, and F. S. Menniti, et al.,
1998, CNS Drug Reviews, 4, 4, 307-322). However, development of
drugs from this class has been hindered by low bioavailability,
poor pharmacokinetics, and lack of selectivity against other
pharmacological targets including the hERG ion channel. Blockade of
the hERG ion channel can lead to cardiac arrythmias, including the
potentially fatal Torsades de pointe, thus selectivity against this
channel is critical. Thus, in the treatment of major depressive
disorder, there remains an unmet clinical need for the development
of effective NR2B-selective negative allosteric modulators which
have a favorable tolerability profile.
[0006] NR2B receptor antagonists have been disclosed in PCT
publication WO 2009/006437.
[0007] The invention provides technical advantages, for example,
the compounds are novel and are ligands for the NR2B receptor and
may be useful for the treatment of various disorders of the central
nervous system. Additionally, the compounds provide advantages for
pharmaceutical uses, for example, with regard to one or more of
their mechanism of action, binding, inhibition efficacy, target
selectivity, solubility, safety profiles, or bioavailability.
DESCRIPTION OF THE INVENTION
[0008] One aspect of the invention is a compound of formula I
##STR00002##
where: Ar.sup.1 is phenyl or indanyl and is substituted with 0-3
substituents selected from cyano, halo, alkyl, haloalkyl, and
haloalkoxy; Ar.sup.2 is phenyl substituted with 1 OR substituent
and also substituted with 0-3 substituents selected from cyano,
halo, alkyl, haloalkyl, and haloalkoxy; R is a prodrug moiety
selected from the group consisting of alkyl esters, amino acid
esters, alkoxy esters, phosphonic acids, phosphonic alkyl esters,
alkoxyphosphononate acid, alkoxyphosphonate alkyl esters, alkyl
carabamates, amino acid carbamates, alkyl phosporamidates, aryl
phosphoramidates, and sulfamates; X is a bond or C.sub.1-C.sub.3
alkylene; n is 1 or 2; and ring A is azetidinyl, pyrrolidinyl,
piperidinyl, piperazinyl, homopiperidinyl, or homopiperazinyl and
is substituted with 0-4 substituents selected from halo, alkyl,
hydroxy, or alkoxy; or a pharmaceutically acceptable salt
thereof.
[0009] Another aspect of the invention is a compound of the
formula
##STR00003##
where R is a prodrug moiety selected from the group consisting of
alkyl esters, amino acid esters, alkoxy esters, phosphonic acids,
phosphonic alkyl esters, alkoxyphosphononate acid,
alkoxyphosphonate alkyl esters, alkyl carabamates, amino acid
carbamates, alkyl phosporamidates, aryl phosphoramidates, and
sulfamates; or a pharmaceutically acceptable salt thereof.
Synthetic Methods
[0010] Compounds of Formula I may be made by methods known in the
art including those described below and including variations within
the skill of the art. Some reagents and intermediates are known in
the art. Other reagents and intermediates can be made by methods
known in the art using readily available materials. The variables
(e.g. numbered "R" substituents) used to describe the synthesis of
the compounds are intended only to illustrate how to make the
compounds and are not to be confused with variables used in the
claims or in other sections of the specification. The following
methods are for illustrative purposes and are not intended to limit
the scope of the invention. The schemes encompass reasonable
variations known in the art.
[0011] Scheme 1 shows an effective synthesis of example 1,
(R)-3-((3S,4S)-3-fluoro-4-(4-hydroxyphenyl)piperidin-1-yl)-1-(4-methylben-
zyl)pyrrolidin-2-one. Hydroxylactam 1 is available commercially in
optically pure form. It can be protected and N-alkylated to form
lactam 4. Deprotection and activation of the hydroxyl group with
methanesulfonylchloride leads to the lactam 5. Separately, compound
6 can be prepared by the Suzuki coupling reaction between
commercial 4-benzyloxybromobenzene and commercial tert-butyl
4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5,6-dihydropyridine-1(2H)-
-carboxylate. Treatment of 6 with in-situ prepared borane followed
by oxidation results in formation of the trans racemic alcohol 7.
The alcohol 7 can be separated in to the individual enantiomers,
and the phenol can be unmasked using hydrogenation under standard
conditions to the prepare the substituted phenol 8. Fluorination
with de-oxofluor reagent provides selectively the trans aryl
fluoride 9, and deprotection of the Boc group with hydrochloric
acid provides the piperidine as the hydrochloride salt. Simple
extraction under basic conditions provides the piperidine 10 as the
freebase. Careful reaction of the piperidine 10 with the lactam 5
under mildly basic conditions provides
(R)-3-((3S,4S)-3-fluoro-4-(4-hydroxyphenyl)piperidin-1-yl)-1-(4-methylben-
zyl)pyrrolidin-2-one, the title compound of example 1.
##STR00004## ##STR00005##
[0012] The compound of example 1 can be transformed into a variety
of prodrugs using methods known in the art. Thus, according to
scheme 2, treatment of the phenol with POCl.sub.3, pyridine, and
DMAP followed by aqueous hydrolysis provides example 2, the
dihydrogen phosphate ester of example 1.
##STR00006##
[0013] Similarly, reaction of the compound of example 1 with a
Boc-protected amino acid using a variety of methods known in the
art, but preferably using dicyclohexylcarbodiimide and
4-dimethylaminopyridine provides the ester 11.
[0014] Cleavage of the Boc group in acid, preferably HCl, provides
the esters which include the compounds of examples 3 and 4.
##STR00007##
[0015] In a similar manner, Boc-protected aspartic acid tert-butyl
ester (12) can be coupled through the unprotected sidechain to the
compound of example 1 to provide the ester 13. Deprotection with
HCl again provides the compound of example 5.
##STR00008##
DESCRIPTION OF SPECIFIC EMBODIMENTS
[0016] Abbreviations used in the schemes generally follow
conventions used in the art. Chemical abbreviations used in the
specification and examples are defined as follows: "NaHMDS" for
sodium bis(trimethylsilyl)amide; "DMF" for N,N-dimethylformamide;
"MeOH" for methanol; "NBS" for N-bromosuccinimide; "Ar" for aryl;
"TFA" for trifluoroacetic acid; "LAH" for lithium aluminum hydride;
"BOC" for t-butoxycarbonyl, "DMSO" for dimethylsulfoxide; "h" for
hours; "EtOAc" for ethyl acetate; "THF" for tetrahydrofuran; "EDTA"
for ethylenediaminetetraacetic acid; "Et.sub.2O" for diethyl ether;
"DMAP" for 4-dimethylaminopyridine; "DCE" for 1,2-dichloroethane;
"ACN" for acetonitrile; "DME" for 1,2-dimethoxyethane; "HOBt" for
1-hydroxybenzotriazole hydrate; "DIEA" for diisopropylethylamine,
"Nf" for CF.sub.3(CF.sub.2).sub.3SO.sub.2--; and "TMOF" for
trimethylorthoformate.
[0017] Abbreviations as used herein, are defined as follows:
"1.times." for once, "2.times." for twice, "3.times." for thrice,
".degree. C." for degrees Celsius, "eq" for equivalent or
equivalents, "g" for gram or grams, "mg" for milligram or
milligrams, "L" for liter or liters, "mL" for milliliter or
milliliters, "L" for microliter or microliters, "N" for normal, "M"
for molar, "mmol" for millimole or millimoles, "min" for minute or
minutes, "h" for hour or hours, "rt" for room temperature, "RT" for
retention time, "atm" for atmosphere, "psi" for pounds per square
inch, "conc." for concentrate, "sat" or "satd." for saturated, "MW"
for molecular weight, "mp" for melting point, "ee" for enantiomeric
excess, "MS" or "Mass Spec" for mass spectrometry, "ESI" for
electrospray ionization mass spectroscopy, "HR" for high
resolution, "HRMS" for high resolution mass spectrometry, "LCMS"
for liquid chromatography mass spectrometry, "HPLC" for high
pressure liquid chromatography, "RP HPLC" for reverse phase HPLC,
"DCM" for dichloromethane, "TLC" or "tlc" for thin layer
chromatography, "SFC" for supercritical fluid chromatography, "NMR"
for nuclear magnetic resonance spectroscopy, "1H" for proton, "Q"
for delta, "s" for singlet, "d" for doublet, "t" for triplet, "q"
for quartet, "m" for multiplet, "br" for broad, "Hz" for hertz, and
"R", "S", "E", and "Z" are stereochemical designations familiar to
one skilled in the art.
[0018] LC/MS data were acquired using the following conditions:
[0019] Conditions A: Ascentis C18 50.times.2.1 mm, 2.7 .mu.m column
using a 1 mL/min flowrate gradient of 0-100% B over 1.7 minutes
followed by 1.3 minutes at 100% B. Solvent A: 10 mM NH4COOH in
water:acetonitrile (98:2); solvent B=10 mM NH4COOH in
water:acetonitrile (2:98).
[0020] Conditions B: Phenomenex C18 2.0.times.50 mm, 5 .mu.m column
using a 0.8 mL/min flowrate gradient of 0-100% B over 4 minutes.
Solvent A=10% MeOH/90% water/0.1% TFA, Solvent B=90% MeOH/10%
water/0.1% TFA.
SYNTHESIS OF INTERMEDIATES
Intermediate A. tert-Butyl
4-(4-(benzyloxy)phenyl)-5,6-dihydropyridine-1(2H)-carboxylate
##STR00009##
[0022] A solution of commercial 1-(benzyloxy)-4-bromobenzene (104
g, 395 mmol) and commercial tert-butyl
4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5,6-dihydropyridine-1(2H)-
-carboxylate (147 g, 474 mmol) in 1100 mL of acetonitrile was
purged with nitrogen for 2 min. Water (1100 mL) was added, followed
by sodium carbonate (126 g, 1186 mmol) and
tetrakis(triphenylphosphine)palladium (27.4 g, 23.7 mmol). The
reaction mixture was purged with nitrogen for 5 min, and then
heated to 90.degree. C. and stirred for 16 h. The reaction mixture
was then allowed to cool to rt and diluted with 1 L of ethyl
acetate. The layers were separated, and the aqueous layer was
extracted with two additional 250 mL portions of ethyl acetate. The
organic layers were combined, washed with 200 mL of brine, dried
over sodium sulfate, and evaporated in vacuo to provide an
off-white solid. The product was purified by silica gel
chromatography eluting with 6% ethyl acetate in petroleum ether to
provide 129 g (88%) of the desired product. LC/MS RT (conditions
A)=2.732 min, (M-H)+=364.0. .sup.1H NMR (300 MHz, chloroform-d)
.delta. 7.49-7.30 (m, 5H), 7.27 (d, J=10.7 Hz, 2H), 6.99-6.87 (m,
2H), 6.03-5.87 (m, 1H), 5.07 (s, 2H), 4.05 (d, J=2.6 Hz, 2H), 3.62
(t, J=5.7 Hz, 2H), 2.49 (br. s., 2H), 1.49 (s, 9H).
Intermediate B. (+/-)-rel-(3S,4S)-tert-Butyl
4-(4-(benzyloxy)phenyl)-3-hydroxypiperidine-1-carboxylate
##STR00010##
[0024] Sodium borohydride (15.5 g, 410 mmol) was dissolved in THF,
and the solution was chilled to 0.degree. C. Boron trifluoride
etherate (52.3 mL, 424 mmol) was added to the solution and the
mixture was allowed to warm to rt and stirred for 30 min. Then a
solution of tert-butyl
4-(4-(benzyloxy)phenyl)-5,6-dihydropyridine-1(2H)-carboxylate (50
g, 137 mmol, intermediate A) in 500 mL of THF was added and the
reaction mixture was stirred for 2 h at rt. A 100 mL portion of
water was then added slowly to the mixture (Caution: effervescence
is observed). The mixture was diluted with 100 mL of ethanol, and
sodium hydroxide (228 mL, 10% solution in water, 0.684 mol) and
hydrogen peroxide (20.5 mL, 0.684 mol) were added. The reaction
mixture was heated to reflux temperature and stirred for 16 h. The
mixture was cooled to 10.degree. C. and diluted with 1 L of DCM.
Then the pH was adjusted to 7 with 1.5 L of 1.5 N HCl. The layers
were then separated, and the aqueous layer was extracted with an
addition two 500 mL portions of DCM. The organic layers were
combined, washed with 2.times.1 L of water and 200 mL of brine,
dried over sodium sulfate, and evaporated in vacuo to provide an
off-white solid. The solid was triturated with 500 mL of pet ether,
and isolated by filtration to yield 46.5 grams of product (88%,
99.0% purity by HPLC). LC/MS RT (conditions A)=2.372 min,
(M+H).sup.+=382.0. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta.
7.47-7.42 (m, 2H), 7.42-7.36 (m, 2H), 7.36-7.28 (m, 1H), 7.14 (d,
J=9.0 Hz, 2H), 6.92 (d, J=9.0 Hz, 2H), 5.07 (s, 2H), 4.74 (d, J=5.5
Hz, 1H), 4.10 (br. s., 1H), 3.94 (br. s., 1H), 3.46-3.35 (m, 1H),
2.47-2.31 (m, 2H), 1.70-1.61 (m, 1H), 1.55-1.45 (m, 2H), 1.42 (s,
9H).
Intermediate C. (3S,4S)-tert-Butyl
4-(4-(benzyloxy)phenyl)-3-hydroxypiperidine-1-carboxylate
##STR00011##
[0026] Racemic rel-(3S,4S)-tert-butyl
4-(4-(benzyloxy)phenyl)-3-hydroxypiperidine-1-carboxylate (112 g,
intermediate B) was separated into the individual enantiomers using
preparative supercritical fluid chromatography under the following
conditions: A Thar SFC-250 instrument was utilized with a
Lux-Cellulose-2 (250.times.21 mm), 5 .mu.m column eluting with 60%
CO.sub.2 and 40% of a solution of 0.3% diethylamine in methanol at
a flow rate of 100.0 g/min. Sample was injected at 74 mg/mL.
Analytical SFC was carried out on Lux-Cellulose-2 (250.times.4.6
mm), 5 m column eluting with 55% CO.sub.2 and 45% of a solution of
0.3% diethylamine in methanol at a flow rate of 3.0 g/min. The
recovery was 50.0 g of peak 1 with a retention time of 2.49
minutes, which corresponds to the desired (3S,4S)-tert-butyl
4-(4-(benzyloxy)phenyl)-3-hydroxypiperidine-1-carboxylate.
Analytical data matched those from the racemate.
Intermediate D. (3S,4S)-tert-Butyl
3-hydroxy-4-(4-hydroxyphenyl)piperidine-1-carboxylate
##STR00012##
[0028] A solution of (3S,4S)-tert-butyl
4-(4-(benzyloxy)phenyl)-3-hydroxypiperidine-1-carboxylate (26 g,
67.8 mmol, intermediate C) in 260 mL of methanol was treated with
1.6 grams of 10% palladium on carbon (13.6 mmol) in a pressure
bottle. Hydrogen at 50 psi was introduced, and the reaction mixture
was stirred for 16 h. The mixture was filtered through celite and
concentrated to a crude product (18.9 g, 64.4 mmol) which was
sufficiently pure to carry forward without further purification.
LC/MS RT (conditions B)=2.970 min, (M+H with loss of
t-butyl).sup.+=238.0. .sup.1H NMR (400 MHz, DMSO-d.sub.6) 9.10 (br.
s., 1H), 7.01 (d, J=8.5 Hz, 2H), 6.65 (s, 2H), 4.70 (d, J=5.0 Hz,
1H), 4.09 (br. s., 1H), 3.93 (br. s., 1H), 3.17 (s, 2H), 2.79-2.63
(m, 1H), 2.34 (br. s., 1H), 1.68-1.57 (m, 1H), 1.44 (br. s., 1H),
1.42 (s, 9H).
Intermediate E. (3S,4S)-tert-Butyl
3-fluoro-4-(4-hydroxyphenyl)piperidine-1-carboxylate
##STR00013##
[0030] A solution of (3S,4S)-tert-butyl
3-hydroxy-4-(4-hydroxyphenyl)piperidine-1-carboxylate (15.5 g, 61.4
mmol, intermediate D) in 270 mL of acetonitrile was chilled to
0.degree. C. To the stirred solution was added
bis(2-methoxyethyl)aminosulfur trifluoride 50% solution in toluene
(Deoxo-fluor, 58.4 mL, 159 mmol) dropwise via addition funnel over
65 min. After the addition, the reaction mixture was stirred for 30
min at 0.degree. C. and then allowed to come to rt and stirred for
an additional 2 h. A saturated ammonium chloride solution (150 mL)
was then added, and the mixture was extracted with two 150 mL
portions of DCM. The organic layers were combined, dried over
sodium sulfate, and concentrated to afford the crude product. The
product was purified by silica gel chromatography (1.5 kg of
silica) eluting with a gradient of 0-15% acetone in hexanes to
afford 11.9 g (75%) of the desired (3S,4S)-tert-butyl
3-fluoro-4-(4-hydroxyphenyl)piperidine-1-carboxylate. LC/MS RT
(conditions B)=3.295 min, (M+H with loss of t-butyl and elimination
of fluorine).sup.+=220.0. .sup.1H NMR (400 MHz, chloroform-d)
.delta. 7.15 (d, J=8.6 Hz, 2H), 6.83 (dt, J=8.6, 2.0 Hz, 2H),
4.59-4.48 (m, 1H), 4.47-4.37 (m, 1H), 4.23-4.12 (m, 1H), 2.88-2.68
(m, 3H), 1.96-1.84 (m, 1H), 1.80-1.66 (m, 1H), 1.51 (s, 9H).
Intermediate F. 4-((3S,4S)-3-Fluoropiperidin-4-yl)phenol
##STR00014##
[0032] A solution of (3S,4S)-tert-butyl
3-fluoro-4-(4-hydroxyphenyl)piperidine-1-carboxylate (12.0 g, 40.6
mmol, intermediate E) in anhydrous dioxane (80 mL) was treated with
HCl (4 M in 1,4-dioxane, 40.6 mL, 162 mmol). The reaction mixture
was allowed to stir at rt for 6 h and then evaporated in vacuo to
provide the HCl salt of the desired product. Without further
isolation, the HCl salt was suspended in CHCl.sub.3 and 80 mL of a
satd. NaHCO.sub.3 solution was added. The organic layer was
separated, and the aqueous layer was extracted with CHCl.sub.3
(2.times.100 mL). The organic layers were combined, dried over
Na.sub.2SO.sub.4 and concentrated to give the title compound (7.1
g, 36.4 mmol, 90%). LC/MS RT (conditions B)=1.008 min, LC/MS
(M+H).sup.+=196.2.
Intermediate G.
(S)-3-((tert-Butyldimethylsilyl)oxy)pyrrolidin-2-one
##STR00015##
[0034] A stirred solution of commercial
(S)-3-hydroxypyrrolidin-2-one (5 g, 49.5 mmol) in DCM (198 ml) was
treated with DMAP (0.199 g, 1.632 mmol), imidazole (6.73 g, 99
mmol), and TBDMS-Cl (8.94 g, 59.3 mmol). The reaction mixture was
stirred at rt for 16 h, and then was washed with a satd.
NaHCO.sub.3 solution. The organic layer was concentrated and the
crude reaction product was purified by silica gel chromatography
eluting with 50% ethyl acetate in petroleum ether. The desired
product was isolated as a white solid (8.1 g, 76%). LC/MS
(M+H).sup.+=216.2. .sup.1H NMR (400 MHz, chloroform-d) .delta. 6.40
(br. s., 1H), 4.26 (t, J=7.8 Hz, 1H), 3.42-3.34 (m, 1H), 3.29-3.21
(m, 1H), 2.36 (dtd, J=12.7, 7.3, 3.3 Hz, 1H), 2.07-1.96 (m, 1H),
0.91 (s, 9H), 0.15 (d, J=7.0 Hz, 6H).
Intermediate H.
(S)-3-((tert-Butyldimethylsilyl)oxy)-1-(4-methylbenzyl)pyrrolidin-2-one
##STR00016##
[0036] (S)-3-((tert-butyldimethylsilyl)oxy)pyrrolidin-2-one (5 g,
23.22 mmol, intermediate G) was dissolved in anhydrous THF (46.4
ml) and the reaction mixture was cooled to 0.degree. C. under a
nitrogen atmosphere. Sodium hydride (1.393 g, 34.8 mmol) was then
added in one portion and the reaction mixture was allowed to stir
for 5 min before the dropwise addition of
1-(bromomethyl)-4-methylbenzene (5.37 g, 29.0 mmol) in anhydrous
THF (46.4 ml). The reaction was allowed to stir at 0.degree. C. for
5 min, then the cooling bath was removed and mixture was allowed to
warm to rt overnight. The reaction was cautiously quenched with
water (100 mL) and then extracted with ethyl acetate (3.times.100
mL). The combined organic layers were then washed with brine (200
mL) and dried (MgSO.sub.4). Evaporation of the solvent in vacuo
gave the crude product (9.6 g, oil) which was then purified by
silica gel chromatography (330 g of silica) eluting with a gradient
of 0% to 20% ethyl acetate in hexanes to provide 6.53 g (88%) of
the desired product. LC/MS (Conditions B), RT=4.320 min,
(M+H).sup.+=320.3. .sup.1H NMR (400 MHz, chloroform-d) .delta. 7.15
(s, 4H), 4.42 (s, 2H), 4.37 (t, J=7.6 Hz, 1H), 3.32-3.18 (m, 1H),
3.10 (dt, J=9.7, 7.5 Hz, 1H), 2.36 (s, 3H), 2.29 (dtd, J=12.6, 7.6,
3.1 Hz, 1H), 1.97-1.84 (m, 1H), 0.95 (s, 9H), 0.20 (d, J=10.3 Hz,
6H).
Intermediate I.
(S)-3-Hydroxy-1-(4-methylbenzyl)pyrrolidin-2-one
##STR00017##
[0038] HCl (4 M in 1,4-dioxane, 25.5 ml, 102 mmol) was added in one
portion to a solution of
(S)-3-((tert-butyldimethylsilyl)oxy)-1-(4-methylbenzyl)pyrrolidin-2-one
(6.53 g, 20.44 mmol, intermediate H) in anhydrous DCM (20.4 mL) at
rt. A slight exotherm was noted. The reaction mixture was allowed
to stir at rt for 2 h and then evaporated in vacuo. The residue was
taken up in DCM (100 mL) and washed with a satd. sodium bicarbonate
solution (100 mL) and brine (50 mL), and then the solution was
dried over MgSO.sub.4 and concentrated to a residue. The crude
product was purified by silica gel chromatography (120 g of silica)
eluting with a gradient of 40% to 100% ethyl acetate in hexanes to
provide 3.73 g (89%) of the desired product. LC/MS (Conditions B),
RT=2.338 min, (M+H).sup.+=206.2. .sup.1H NMR (400 MHz,
chloroform-d) .delta. 7.26-7.02 (m, 4H), 4.43 (d, J=3.5 Hz, 2H),
4.41-4.37 (m, 1H), 3.66 (d, J=2.6 Hz, 1H), 3.34-3.05 (m, 2H), 2.41
(dddd, J=12.8, 8.4, 6.6, 2.2 Hz, 1H), 2.34 (s, 3H), 1.93 (dq,
J=12.8, 8.8 Hz, 1H).
Intermediate J. (S)-1-(4-methylbenzyl)-2-oxopyrrolidin-3-yl
methanesulfonate
##STR00018##
[0040] Triethylamine (0.509 ml, 3.65 mmol) was added to a cooled
solution of (S)-3-hydroxy-1-(4-methylbenzyl)pyrrolidin-2-one (0.5
g, 2.436 mmol, intermediate I) in anhydrous DCM (12.18 ml) at
0.degree. C. under a nitrogen atmosphere. Methanesulfonyl chloride
(0.198 ml, 2.56 mmol) was then added dropwise and the reaction was
allowed to stir at 0.degree. C. for 15 min before quenching with a
satd. sodium bicarbonate solution (10 mL). The mixture was allowed
to warm to rt and the aqueous layer was separated and extracted
with DCM (2.times.). The combined organic layers were dried over
MgSO.sub.4 and evaporated in vacuo to give a white solid (0.73 g)
which was then purified by silica gel chromatography (40 g of
silica) eluting with a gradient of 0% to 50% ethyl acetate in
hexanes to provide 0.63 g (91%) of the desired product as a white
solid.
Intermediate K.
(S)-3-(tert-Butyldimethylsilyloxy)-1-(4-(difluoromethyl)benzyl)pyrrolidin-
-2-one
##STR00019##
[0042] A 60% dispersion of sodium hydride in mineral oil (232 mg,
5.31 mmol) was added to a stirred solution of
(S)-3-((tert-butyldimethylsilyl)oxy)pyrrolidin-2-one (762 mg, 3.54
mmol, intermediate G) in THF (7 mL) at 0.degree. C. After 15 min, a
solution of 1-(bromomethyl)-4-(difluoromethyl)benzene (980 mg, 4.43
mmol) in THF (7 mL) was added to the reaction mixture. The
resulting mixture was stirred at room temperature for 6 h. The
reaction was carefully quenched with several grams of ice pellets.
The resulting mixture was extracted with EtOAc. The combined
organic layers were washed with water, dried over sodium sulfate,
filtered and concentrated in vacuo. The crude reaction mixture was
purified using silica gel column chromatography (0-30%
EtOAc/hexanes) to afford the desired product (440 mg, 35% yield) as
a white solid: LCMS (M+H).sup.+ 356.3; .sup.1H NMR (500 MHz,
chloroform-d) .delta. 7.49 (d, J=8.1 Hz, 2H), 7.35 (d, J=7.9 Hz,
2H), 6.65 (br. t, J=1.0 Hz, 1H), 4.56-4.44 (m, 2H), 4.38 (t, J=7.5
Hz, 1H), 3.27 (ddd, J=9.7, 8.7, 3.4 Hz, 1H), 3.13 (dt, J=9.7, 7.4
Hz, 1H), 2.36-2.27 (m, 1H), 1.98-1.90 (m, 1H), 0.96 (br. s., 9H),
0.22-0.20 (m, 3H), 0.20-0.18 (m, 3H).
Intermediate L.
(S)-1-(4-(Difluoromethyl)benzyl)-3-hydroxypyrrolidin-2-one
##STR00020##
[0044] A solution of 4 M HCl in dioxane (0.62 mL, 2.5 mmol) was
added to a stirred solution of
(S)-3-((tert-butyldimethylsilyl)oxy)-1-(4-(difluoromethyl)benzyl)pyrrolid-
in-2-one (440 mg, 1.24 mmol, intermediate K) in dichloromethane
(1.24 mL) at rt. The reaction mixture was stirred for 2 h. The
reaction mixture was concentrated in vacuo to afford the desired
product (368 mg, quantitative yield): LC-MS (M+H).sup.+ 242.1.
Intermediate M.
(S)-1-(4-(Difluoromethyl)benzyl)-2-oxopyrrolidin-3-yl
methanesulfonate
##STR00021##
[0046] Triethylamine (0.319 mL, 2.29 mmol) and methanesulfonyl
chloride (0.131 mL, 1.68 mmol) was added to a stirred solution of
(S)-1-(4-(difluoromethyl)benzyl)-3-hydroxypyrrolidin-2-one (368 mg,
1.53 mmol, intermediate L) in dichloromethane (7.63 mL) at
0.degree. C. The reaction mixture was stirred at 0.degree. C. for 1
h. The resulting mixture was diluted with water and the aqueous
mixture was extracted with dichloromethane. The combined organic
layers were washed with 10% sodium bicarbonate solution, dried over
sodium sulfate, filtered, and concentrated in vacuo. The crude
material was purified using silica gel column chromatography
(0-100% EtOAc). The pure fractions were combined and concentrated
in vacuo to afford the desired product (322 mg, 66% yield) as a
white solid: LC-MS (M+H).sup.+ 320.1; .sup.1H NMR (500 MHz,
chloroform-d) .delta. 7.53 (d, J=7.9 Hz, 2H), 7.38-7.33 (m, 2H),
6.67 (br. t, J=1.0 Hz, 1H), 5.27 (dd, J=8.2, 7.5 Hz, 1H), 4.60-4.49
(m, 2H), 3.41-3.35 (m, 1H), 3.33 (s, 3H), 3.27 (dt, J=9.9, 7.3 Hz,
1H), 2.64-2.55 (m, 1H), 2.27 (ddt, J=13.9, 8.9, 7.1 Hz, 1H).
Intermediate N. tert-Butyl
4-hydroxy-4-(4-methoxyphenyl)piperidine-1-carboxylate
##STR00022##
[0048] A mixture of commercial tert-butyl
4-oxopiperidine-1-carboxylate (2.0 g, 10.0 mmol) and diethyl ether
(30 ml) was cooled to 0.degree. C. To this mixture was added
dropwise a solution of (4-methoxyphenyl)magnesium bromide (0.5 M in
diethyl ether, 30 ml, 15 mmol). After complete addition, the
reaction mixture was allowed to warm to rt and stirred for 2 h. It
was then slowly quenched with 150 ml of ice cold water and then the
resulting mixture was extracted with 3.times.150 ml of DCM. The
organic layers were combined, dried, filtered, and concentrated
under vacuum. The crude product was purified by silica gel column
chromatography (30:70 ethyl acetate:hexane) to provide the desired
product (3.0 g, 100% yield): LC-MS (ES-API): m/z 305.5 (M-H).sup.+;
.sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 7.37 (q, J=1.0 Hz, 2H),
6.86 (q, J=1.0 Hz, 2H), 4.94 (s, 1H), 3.82 (d, J=11.5 Hz, 2H), 3.73
(s, 3H), 3.13 (br. s, 2H), 1.75 (td, J=12.9, 4.8 Hz, 2H), 1.56 (d,
J=12.3 Hz, 2H), 1.41 (s, 9H).
Intermediate O. 4-(4-Methoxyphenyl)-1,2,3,6-tetrahydropyridine
hydrochloride
##STR00023##
[0050] A mixture of tert-butyl
4-hydroxy-4-(4-methoxyphenyl)piperidine-1-carboxylate (700 mg, 2.27
mmol, intermediate N) and HCl in dioxane (4.0 ml, 16 mmol) was
stirred at rt for 3 h. The crude mass was concentrated under vacuum
and the solid residue was washed with 3.times.10 ml of DCM to
remove non-polar impurities. The desired salt was collected as a
fine solid (480 mg, 93%). LCMS (ES-API) m/z 190.2 (M+H).sup.+;
.sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 7.37 (d, J=9.0 Hz, 2H),
6.98 (d, J=9.0 Hz, 2H), 6.08-5.98 (m, 1H), 5.11 (s, 1H), 3.97 (br.
s., 1H), 3.52 (s, 1H), 3.32 (s, 3H), 2.47-2.37 (m, 1H).
Intermediate P. 4-(4-Methoxyphenyl)piperidine hydrochloride
##STR00024##
[0052] To a stirred solution of
4-(4-methoxyphenyl)-1,2,3,6-tetrahydropyridine, HCl (3.00 g, 13.3
mmol, intermediate O) in methanol (20 mL) was added 10% palladium
on carbon (1.4 g) and the reaction mixture was stirred at 20 psi of
hydrogen for 12 h. The reaction mixture was filtered through a pad
of celite, which was washed with ethyl acetate, and the combined
organic fractions were concentrated to obtain a white solid (2.0 g,
70% yield): LCMS (ES-API), m/z 192.1 (M+H).sup.+; .sup.1H NMR (300
MHz, DMSO-d.sub.6) .delta. 9.13-8.36 (m, 2H), 7.14 (d, J=8.7 Hz,
2H), 6.90 (d, J=8.7 Hz, 2H), 3.73 (s, 3H), 3.07-2.87 (m, 4H),
2.87-2.65 (m, 4H).
Intermediate Q. 2,4-Dibromo-N-(4-fluorobenzyl)butanamide
##STR00025##
[0054] TEA (8.91 mL, 63.9 mmol) and 2,4-dibromobutanoyl chloride
(5.07 mL, 38.4 mmol) were sequentionall added to solution of
commercial (4-fluorophenyl)methanamine (4.0 g, 32.0 mmol) in
diethyl ether (15 mL) at 0.degree. C. The reaction mixture was
allowed to warm to rt and stir for an additional 24 h. The reaction
mixture was filtered. The solids were washed with diethyl ether.
The filtrate was concentrated in vacuo to afford a crude mixture
containing 2,4-dibromo-N-(4-fluorobenzyl)butanamide (8.0 g, 71%
yield): LCMS (ES-API), m/z 354, 356 (M+H).sup.+.
Intermediate R. 3-Bromo-1-(4-fluorobenzyl)pyrrolidin-2-one
##STR00026##
[0056] A 60% dispersion of NaH in mineral oil (1.70 g, 42.5 mmol)
was added to a stirred solution of
2,4-dibromo-N-(4-fluorobenzyl)butanamide (10.0 g, 28.3 mmol,
intermediate Q) in THF (25 mL) at 0.degree. C. The reaction mixture
was allowed to warm to rt and stir for and additional 2 h. The
reaction mixture was carefully quenched with ice and diluted with
water. The resulting mixture was extracted with EtOAc. The combined
organic layers were washed with water and then brine solution. The
organic layer was over sodium sulfate, filtered, and concentrated
in vacuo. The crude product was purified using silica gel column
chromatography (10% EtOAc/hexanes) to afford the desired product
(5.90 g, 64% yield): LCMS (ES-API), m/z 272.4, 274.3 (M+H).sup.+;
.sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. ppm 2.12-2.27 (m, 1H)
2.56-2.68 (m, 1H) 3.27 (dd, J=7.78, 3.26 Hz, 2H) 4.29-4.38 (m, 1H)
4.40-4.57 (m, 1H) 4.73 (dd, J=7.03, 3.01 Hz, 1H) 7.04-7.35 (m,
4H).
Intermediate S.
1-(4-Fluorobenzyl)-3-(4-(4-methoxyphenyl)piperidin-1-yl)pyrrolidin-2-one
##STR00027##
[0058] TEA (0.768 mL, 5.51 mmol) was added to a stirred solution of
3-bromo-1-(4-fluorobenzyl)pyrrolidin-2-one (0.3 g, 1.10 mmol,
intermediate R) and 4-(4-methoxyphenyl)piperidine hydrochloride
(0.276 g, 1.213 mmol, intermediate P) in acetonitrile (10 mL). The
reaction mixture was sealed and heated in a chemistry microwave at
100.degree. C. for 1 h. The reaction mixture was cooled to rt and
concentrated in vacuo. The residue was diluted with EtOAc. The
organic mixture was washed with water and brine solution. The
organic layer was dried over sodium sulfate, filtered and
concentrated in vacuo to afford a crude mixture containing
1-(4-fluorobenzyl)-3-(4-(4-methoxyphenyl)piperidin-1-yl)pyrrolidin-2-one
(0.35 g, 83% yield): LCMS (ES-API), m/z 383.2 (M+H).
Example 1.
(R)-3-((3S,4S)-3-fluoro-4-(4-hydroxyphenyl)piperidin-1-yl)-1-(4-
-methylbenzyl)pyrrolidin-2-one
##STR00028##
[0060] A solution of 4-((3S,4S)-3-fluoropiperidin-4-yl)phenol (7.10
g, 36.4 mmol, intermediate F) and DIEA (16 mL, 92 mmol) in 100 mL
of acetonitrile was heated to 80.degree. C. This solution was
treated dropwise with a solution of
(S)-1-(4-methylbenzyl)-2-oxopyrrolidin-3-yl methanesulfonate (10.5
g, 37.0 mmol, intermediate J) in acetonitrile (80 mL) over a period
of 4 hours. After the addition was completed, the reaction mixture
was stirred at 80.degree. C. for 16 h. The reaction mixture was
then allowed to cool to rt, and the volume was reduced by rotary
evaporation to 80 mL. A satd. NH.sub.4Cl solution (100 mL) was then
added, and the layers were separated. The aqueous layer was
extracted with DCM (2.times.100 mL) and the organic layers were
combined, dried over Na.sub.2SO.sub.4 and concentrated in vacuo to
give a crude product. The crude product was purified by silica gel
chromatography (750 g of silica gel) eluting with a gradient of 0%
to 20% of solvent B in solvent A, where Solvent B=20% methanol/DCM
and solvent A=DCM. Fractions containing the product were combined.
Evaporation of the solvents gave 9.3 grams of the desired product
with 97% purity by LC/MS analysis (conditions B). The product thus
obtained (8.5 g) was slurried in acetone:hexane (1:5, 200 mL) and
the solid product was isolated by filtration and air dried. Careful
SFC analysis showed the presence of a 2.1% impurity in the product.
Using a Cell4 0.46.times.25 cm 5 .mu.m column and eluting with 45%
methanol in CO.sub.2 at 3 mL/min, the desired product eluted at
3.800 minutes and the undesired impurity eluted at 4.848 minutes.
The product was then further purified by SFC Chromatography using a
Cell4 3.times.25 cm 5 .mu.m column at 150 mL/min injecting 1.5 mL
of a 80 mg/mL solution. Concentration of the active fractions
provided 7.82 grams (20.4 mmol, 56%) of >99.7% pure example 1 as
a white powder. LC/MS (Conditions B), RT=2.512 min,
(M+H).sup.+=383.3. .sup.19F NMR .delta. -182.83. .sup.1H NMR (400
MHz, chloroform-d) .delta. 7.20-7.08 (m, 6H), 6.98-6.78 (m, 2H),
5.68 (s, 1H), 4.77-4.54 (m, 1H), 4.53-4.34 (m, 2H), 3.68 (t, J=8.8
Hz, 1H), 3.41-3.29 (m, 1H), 3.28-3.09 (m, 2H), 2.82 (d, J=10.8 Hz,
1H), 2.74-2.54 (m, 2H), 2.47 (td, J=9.9, 3.6 Hz, 1H), 2.34 (s, 3H),
2.19-1.94 (m, 2H), 1.92-1.80 (m, 2H). .sup.13C NMR (101 MHz,
chloroform-d) .delta. 172.4, 154.9, 137.5, 133.3, 133.0, 129.5,
128.7, 128.3, 115.5, 92.6, 90.8, 65.0, 54.4, 54.2, 48.7, 48.0,
47.8, 46.8, 43.7, 31.7, 31.6, 21.1, 19.6.
Example 2.
4-((3S,4S)-3-Fluoro-1-((R)-1-(4-methylbenzyl)-2-oxopyrrolidin-3-
-yl)piperidin-4-yl)phenyl dihydrogen phosphate
##STR00029##
[0062] To a suspension of
(R)-3-((3S,4S)-3-fluoro-4-(4-hydroxyphenyl)piperidin-1-yl)-1-(4-methylben-
zyl)pyrrolidin-2-one (100 mg, 0.261 mmol, example 1) in 10 mL of
dichloromethane was added pyridine (0.106 mL, 1.31 mmol) and DMAP
(160 mg, 1.31 mmol). The reaction mixture was chilled to
-20.degree. C. To the chilled solution was added POCl.sub.3 (0.122
mL, 1.31 mmol) dropwise, and then the reaction mixture was allowed
to warm to rt and stirred for 1 h. Water (10 mL) was added and the
mixture was stirred for 1.5 h. The layers were then separated and
the organic layer was dried over Na.sub.2SO.sub.4 and evaporated to
dryness. The crude product was purified by HPLC on a Symmetry C8
(300.times.17 mm) 7 mM column eluting with a gradient of 20% B to
50% B over 7 minutes at 15 mL/min where solvent A=10 mM ammonium
acetate in water pH 4.5 and solvent B=acetonitrile. The product
RT=2.2 min. The desired product (5.8 mg, 4.7%) was isolated from
the appropriate fractions by lyophilization as a white solid. LCMS
(Conditions A) RT=1.720 min, (M+H).sup.+=463.2. .sup.1H NMR (400
MHz, methanol-d4) .delta. 7.29-7.16 (m, 8H), 4.74 (br. s., 1H),
4.61-4.34 (m, 2H), 4.01 (t, J=8.3 Hz, 1H), 3.82-3.62 (m, 1H), 3.35
(m, 2H), 3.05 (br. s., 2H), 2.79 (br. s., 2H), 2.34 (s, 4H), 2.18
(br. s., 1H), 2.02-1.87 (m, 1H), 1.83 (br. s., 1H). .sup.19F NMR
(376 MHz, methanol-d4) .delta. -185.143. .sup.31P NMR (162 MHz,
methanol-d4) .delta. -4.260.
Example 3.
(S)-4-((3S,4S)-3-Fluoro-1-((R)-1-(4-methylbenzyl)-2-oxopyrrolid-
in-3-yl)piperidin-4-yl)phenyl 2-amino-3-methylbutanoate
hydrochloride
##STR00030##
[0063] Step 3A.
(S)-4-((3S,4S)-3-Fluoro-1-((R)-1-(4-methylbenzyl)-2-oxopyrrolidin-3-yl)pi-
peridin-4-yl)phenyl
2-((tert-butoxycarbonyl)amino)-3-methylbutanoate
##STR00031##
[0065] To a solution of
(R)-3-((3S,4S)-3-fluoro-4-(4-hydroxyphenyl)piperidin-1-yl)-1-(4-methylben-
zyl)pyrrolidin-2-one (0.02 g, 0.052 mmol, example 1) in DCM (3 mL)
was added (S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanoic acid
(0.059 g, 0.272 mmol) followed by DCC (0.032 g, 0.157 mmol) and
DMAP (6.39 mg, 0.052 mmol). The reaction mixture was stirred at
room temperature for 18 h. Water (10 mL) was then added, and the
layers were separated. The aqueous layer was extracted with DCM
(3.times.10 mL) and the organic layers were combined, dried over
Na.sub.2SO.sub.4, and concentrated to a crude product. The crude
product was purified by preparative TLC eluting with 35% ethyl
acetate in petroleum ether to provide the purified product
(S)-4-((3S,4S)-3-fluoro-1-((R)-1-(4-methylbenzyl)-2-oxopyrrolidin-3-yl)pi-
peridin-4-yl)phenyl
2-((tert-butoxycarbonyl)amino)-3-methylbutanoate (27 mg, 79%).
LC/MS (Conditions A) RT=2.523 min, (M+H).sup.+=582.2. .sup.1H NMR
(400 MHz, methanol-d.sub.4) .delta. 7.36 (d, J=8.5 Hz, 2H), 7.18
(s, 4H), 7.08 (d, J=8.5 Hz, 2H), 4.80-4.59 (m, J=10.0, 10.0, 5.0
Hz, 1H), 4.51 (d, J=15.0 Hz, 1H), 4.39 (d, J=15.0 Hz, 1H), 4.23
(dd, J=8.3, 6.3 Hz, 1H), 3.72 (t, J=8.8 Hz, 1H), 3.56-3.40 (m, 1H),
3.32-3.22 (m, 2H), 2.86-2.61 (m, 2H), 2.47 (td, J=10.0, 5.0 Hz,
1H), 2.34 (s, 3H), 2.32-2.23 (m, 1H), 2.22-2.01 (m, 2H), 1.88 (dd,
J=9.5, 4.0 Hz, 2H), 1.74 (dt, J=13.4, 3.8 Hz, 1H), 1.50 (s, 9H),
1.42-1.30 (m, 1H), 1.09 (dd, J=10.0, 7.0 Hz, 6H). .sup.19F NMR (376
MHz, methanol-d4) .delta. -184.32.
Step 3B.
(S)-4-((3S,4S)-3-Fluoro-1-((R)-1-(4-methylbenzyl)-2-oxopyrrolidin-
-3-yl)piperidin-4-yl)phenyl 2-amino-3-methylbutanoate
hydrochloride
##STR00032##
[0067] To a solution of
(S)-4-((3S,4S)-3-fluoro-1-((R)-1-(4-methylbenzyl)-2-oxopyrrolidin-3-yl)pi-
peridin-4-yl)phenyl
2-((tert-butoxycarbonyl)amino)-3-methylbutanoate (0.025 g, 0.043
mmol) in DCM (1.5 mL) at -20.degree. C. was added HCl in diethyl
ether (2.5 ml, 2.50 mmol, 1.0 M). The reaction mixture was slowly
warmed to rt over 10 min and then allowed to stir at rt for 19 h.
The solvent was then removed in vacuo to provide a pale yellow
semisolid. The crude product was then purified by RP-HPLC on a
Sunfire C18 (250.times.20 mm) 5 .mu.m column using a gradient of
10% solvent B to 75% solvent B over 12 minutes at 15 mL/min where
solvent A=0.05% HCl in water and solvent B=acetonitrile. Active
fractions were concentrated by lyophilization to provide 10.2 mg
(44%) of
(S)-4-((3S,4S)-3-fluoro-1-((R)-1-(4-methylbenzyl)-2-oxopyrrolidin-3-yl)pi-
peridin-4-yl)phenyl 2-amino-3-methylbutanoate hydrochloride, the
titled compound of example 2 as an off-white solid. LC-MS (Method
A) RT=2.20 min, (M+H).sup.+=482.2. .sup.1H NMR: (400 MHz, DMSO-d6)
.delta. ppm 8.64-8.78 (m, 3H) 7.38-7.46 (m, 2H) 7.23 (d, J=8.53 Hz,
2H) 7.18 (s, 4H) 5.04-5.26 (m, 1H) 4.42 (d, J=9.54 Hz, 3H)
4.17-4.23 (m, 2H) 3.29-3.40 (m, 4H) 3.19-3.28 (m, 2H) 2.30 (s, 6H)
2.04-2.19 (m, 2H) 1.11 (dd, J=12.55, 7.03 Hz, 6H). .sup.19F NMR
(376 MHz, DMSO-d6) .delta. -183.904.
Example 4.
(S)-4-((3S,4S)-3-Fluoro-1-((R)-1-(4-methylbenzyl)-2-oxopyrrolid-
in-3-yl)piperidin-4-yl)phenyl 2-aminopropanoate hydrochloride
##STR00033##
[0068] Step 4A.
(S)-4-((3S,4S)-3-Fluoro-1-((R)-1-(4-methylbenzyl)-2-oxopyrrolidin-3-yl)pi-
peridin-4-yl)phenyl 2-((tert-butoxycarbonyl)amino)propanoate
##STR00034##
[0070] To a solution of
(R)-3-((3S,4S)-3-fluoro-4-(4-hydroxyphenyl)piperidin-1-yl)-1-(4-methylben-
zyl)pyrrolidin-2-one (0.03 g, 0.078 mmol, example 1) in DCM (5 mL)
was added (S)-2-((tert-butoxycarbonyl)amino)propanoic acid (0.077
g, 0.408 mmol) followed by DCC (0.049 g, 0.235 mmol) and DMAP (9.58
mg, 0.078 mmol). The reaction mixture was stirred at rt for 18 h.
Water (15 mL) was then added, and the layers were separated. The
aqueous layer was extracted with DCM (3.times.15 mL) and the
organic layers were combined, dried over Na.sub.2SO.sub.4, and
concentrated to a crude product. The crude product was purified by
preparative TLC eluting with 20% ethyl acetate in petroleum ether
to provide the purified product
(S)-4-((3S,4S)-3-fluoro-1-((R)-1-(4-methylbenzyl)-2-oxopyrrolidin-3-yl)pi-
peridin-4-yl)phenyl 2-((tert-butoxycarbonyl)amino)propanoate (0.032
g, 0.058 mmol, 74% yield) as off-white semi solid. LC-MS (Method A)
RT=2.40 min, (M+H).sup.+=554.2. .sup.1H NMR (400 MHz, DMSO-d.sub.6)
.delta. 7.51 (d, J=7.0 Hz, 1H), 7.44-7.32 (m, J=8.5 Hz, 2H),
7.21-7.09 (m, 4H), 7.07-6.98 (m, J=8.5 Hz, 2H), 4.62 (d, J=4.5 Hz,
1H), 4.39 (d, J=15.1 Hz, 1H), 4.30 (d, J=15.1 Hz, 1H), 4.27-4.17
(m, 1H), 3.58 (t, J=8.5 Hz, 1H), 3.50-3.40 (m, 1H), 3.22-3.07 (m,
2H), 2.81-2.64 (m, 3H), 2.39-2.31 (m, 1H), 2.29 (s, 3H), 2.17-2.04
(m, 1H), 1.93 (dd, J=12.8, 8.3 Hz, 1H), 1.78 (br. s., 1H),
1.74-1.59 (m, 1H), 1.41 (s, 9H), 1.39 (d, J=2.5 Hz, 3H). .sup.19F
NMR (376 MHz, DMSO-d6) .delta. -180.172.
Step 4B.
(S)-4-((3S,4S)-3-Fluoro-1-((R)-1-(4-methylbenzyl)-2-oxopyrrolidin-
-3-yl)piperidin-4-yl)phenyl 2-aminopropanoate hydrochloride
##STR00035##
[0072] To a solution of
(S)-4-((3S,4S)-3-fluoro-1-((R)-1-(4-methylbenzyl)-2-oxopyrrolidin-3-yl)pi-
peridin-4-yl)phenyl 2-((tert-butoxycarbonyl)amino)propanoate (0.032
g, 0.058 mmol) in DCM (2 mL) at -20.degree. C. was added HCl in
diethyl ether (2.0 ml, 2.0 mmol, 1.0 M). The reaction mixture was
slowly warmed to rt over 10 min and then allowed to stir at rt for
19 h. The solvent was then removed in vacuo to provide a pale
yellow semisolid. The crude product was then purified by RP-HPLC on
a Kinetex C18 (250.times.20 mm) 5 .mu.m column using a gradient of
10% solvent B to 40% solvent B over 7 minutes at 15 mL/min where
solvent A=0.05% HCl in water and solvent B=acetonitrile. Active
fractions were concentrated by lyophilization to provide 4.7 mg
(16%) of
(S)-4-((3S,4S)-3-fluoro-1-((R)-1-(4-methylbenzyl)-2-oxopyrrolidin-3-yl)pi-
peridin-4-yl)phenyl 2-aminopropanoate hydrochloride, the titled
compound of example 4 as an off-white solid. LC-MS (Method A)
RT=1.762 min, (M+H).sup.+=454. .sup.1H NMR (400 MHz, DMSO-d.sub.6)
.delta. 7.41 (d, J=9.0 Hz, 2H), 7.24-7.10 (m, 6H), 5.10-4.85 (m,
1H), 4.45-4.22 (m, 4H), 4.04-3.94 (m, 1H), 3.34-3.18 (m, 4H), 3.06
(d, J=12.0 Hz, 2H), 2.43-2.31 (m, 1H), 2.27 (s, 3H), 2.24-2.14 (m,
1H), 2.13-2.01 (m, 1H), 2.01-1.85 (m, 1H), 1.58 (d, J=7.0 Hz, 3H).
.sup.19F NMR (376 MHz, DMSO-d6) .delta. 183.778.
Example 5.
(S)-2-Amino-4-(4-((3S,4S)-3-fluoro-1-((R)-1-(4-methylbenzyl)-2--
oxopyrrolidin-3-yl)piperidin-4-yl)phenoxy)-4-oxobutanoic Acid
hydrochloride
##STR00036##
[0073] Step 5A. (S)-1-tert-Butyl
4-(4-((3S,4S)-3-fluoro-1-((R)-1-(4-methylbenzyl)-2-oxopyrrolidin-3-yl)pip-
eridin-4-yl)phenyl) 2-((tert-butoxycarbonyl)amino)succinate
##STR00037##
[0075] To a solution of
(R)-3-((3S,4S)-3-fluoro-4-(4-hydroxyphenyl)piperidin-1-yl)-1-(4-methylben-
zyl)pyrrolidin-2-one (0.03 g, 0.078 mmol) in DCM (5 mL) was added
(S)-4-(tert-butoxy)-3-((tert-butoxycarbonyl)amino)-4-oxobutanoic
acid (0.118 g, 0.408 mmol) followed by DCC (0.049 g, 0.235 mmol)
and DMAP (9.58 mg, 0.078 mmol). The reaction was stirred at rt for
18 hours. Water (15 mL) was then added, and the layers were
separated. The aqueous layer was extracted with DCM (3.times.15 mL)
and the organic layers were combined, dried over Na.sub.2SO.sub.4,
and concentrated to a crude product. The crude product was purified
by preparative TLC eluting with 25% ethyl acetate in petroleum
ether to provide the purified product (37 mg, 68%) as an off-white
semi solid. LC-MS (Method A) RT=2.55 min, (M+H).sup.+=654.4.
.sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 7.46-7.34 (m, 2H),
7.20-6.98 (m, 6H), 4.82-4.53 (m, 1H), 4.43-4.24 (m, 2H), 4.11 (d,
J=14.1 Hz, 1H), 3.58 (t, J=8.8 Hz, 1H), 3.48-3.39 (m, 1H),
3.22-3.07 (m, 3H), 3.02 (dd, J=16.1, 6.5 Hz, 1H), 2.87 (dd, J=15.8,
7.8 Hz, 1H), 2.78-2.63 (m, 2H), 2.38-2.31 (m, 1H), 2.29 (s, 3H),
2.17-2.03 (m, 1H), 1.98-1.86 (m, 1H), 1.78 (br. s., 1H), 1.74-1.58
(m, 1H), 1.42 (s, 9H), 1.39 (s, 9H). .sup.19F NMR (376 MHz,
DMSO-d6) .delta. -180.707.
Step 5B.
(S)-2-amino-4-(4-((3S,4S)-3-fluoro-1-((R)-1-(4-methylbenzyl)-2-ox-
opyrrolidin-3-yl)piperidin-4-yl)phenoxy)-4-oxobutanoic Acid
hydrochloride
##STR00038##
[0077] To a solution of (S)-1-tert-butyl
4-(4-((3S,4S)-3-fluoro-1-((R)-1-(4-methylbenzyl)-2-oxopyrrolidin-3-yl)pip-
eridin-4-yl)phenyl) 2-((tert-butoxycarbonyl)amino)succinate (0.032
g, 0.049 mmol) in DCM (2 mL) at -20.degree. C. was added HCl in
diethyl ether (2.0 ml, 2.0 mmol, 1.0 M). The reaction mixture was
slowly warmed to rt over 10 min and then allowed to stir at rt for
19 h. The solvent was then removed in vacuo to provide a pale
yellow semisolid. The crude product was then purified by RP-HPLC on
a YMC Triart C18 (150.times.19 mm) 5 .mu.m column using a gradient
of 10% solvent B to 40% solvent B over 7 minutes at 15 mL/min where
solvent A=0.05% HCl in water and solvent B=acetonitrile. Active
fractions were concentrated by lyophilization to provide 17 mg
(57%) of
(S)-2-amino-4-(4-((3S,4S)-3-fluoro-1-((R)-1-(4-methylbenzyl)-2-oxopyrroli-
din-3-yl)piperidin-4-yl)phenoxy)-4-oxobutanoic acid hydrochloride,
the titled compound of example 5 as an off-white solid. LC-MS
(Method A) RT=1.808 min, (M+H).sup.+=498.2 .sup.1H NMR (400 MHz,
DMSO-d.sub.6) .delta. 7.36 (d, J=8.5 Hz, 2H), 7.21-7.03 (m, 8H),
6.75 (d, J=8.5 Hz, 1H), 4.95-4.72 (m, 1H), 4.42-4.24 (m, 3H), 4.10
(t, J=5.3 Hz, 1H), 4.07-4.01 (m, 1H), 3.93-3.84 (m, 1H), 3.83-3.68
(m, 1H), 3.33-3.10 (m, 5H), 3.04 (br. s., 1H), 2.93 (br. s., 1H),
2.89-2.71 (m, 2H), 2.26 (s, 3H), 1.96 (br. s., 1H), 1.82 (br. s.,
1H). .sup.19F NMR (376 MHz, DMSO-d6) .delta. -180.707.
Example 6.
(R)-1-(4-(Difluoromethyl)benzyl)-3-((3S,4S)-3-fluoro-4-(4-hydro-
xyphenyl)piperidin-1-yl)pyrrolidin-2-one
##STR00039##
[0079] A solution of
(S)-1-(4-(difluoromethyl)benzyl)-2-oxopyrrolidin-3-yl
methanesulfonate (500 mg, 1.57 mmol, intermediate M) in 5.0 mL of
acetonitrile was added dropwise over 1.5 h to a stirred mixture of
4-((3S,4S)-3-fluoropiperidin-4-yl)phenol, hydrochloride (363 mg,
1.57 mmol, intermediate F) and N,N-diisopropylethylamine (1.09 mL,
6.26 mmol) in 5.0 mL of acetonitrile maintained at 85.degree. C.
After complete addition, the reaction mixture was stirred at
85.degree. C. for 16 h. The reaction mixture was concentrated in
vacuo. The residue was purified using silica gel column
chromatography (0-100% EtOAc/hexanes) to afford a diasteromeric
mixture (partial epimerization of the lactam stereocenter) of
1-(4-(difluoromethyl)benzyl)-3-((3S,4S)-3-fluoro-4-(4-hydroxyphenyl)pi-
peridin-1-yl)pyrrolidin-2-one (235 mg, 35% yield). A sample of the
diastereomeric mixture (780 mg) was separated by preparative chiral
SFC (column=Lux Cellulose-2 (21.times.250 mm, 5 .mu.m); isocratic
solvent=20% methanol (with 15 mM ammonia)/80% CO.sub.2;
temp=35.degree. C.; flow rate=60 mL/min; injection volumn=1.0 mL
(.about.20 mg/mL in MeOH) stacked @ 13 min intervals; .lamda.=210
nM; Peak 1=19.6 min, Peak 2=24.5 min) to afford the titled
compounds of example 6 (Peak-1, 389 mg) and
(S)-1-(4-(difluoromethyl)benzyl)-3-((3S,4S)-3-fluoro-4-(4-hydroxyphenyl)p-
iperidin-1-yl)pyrrolidin-2-one (Peak 2, 242 mg). Data for Example
6: LC-MS m/z 419.3 (M+H.sup.+); .sup.1H NMR (500 MHz, chloroform-d)
.delta. 7.50 (d, J=7.9 Hz, 2H), 7.34 (d, J=7.9 Hz, 2H), 7.15 (d,
J=8.5 Hz, 2H), 6.91-6.80 (m, 2H), 6.65 (t, J=56.4 Hz, 1H), 4.96 (s,
1H), 4.77-4.43 (m, 3H), 3.68 (t, J=8.8 Hz, 1H), 3.42-3.33 (m, 1H),
3.29-3.14 (m, 2H), 2.85 (d, J=10.4 Hz, 1H), 2.78-2.69 (m, 1H),
2.69-2.57 (m, 1H), 2.48 (td, J=9.9, 4.9 Hz, 1H), 2.21-2.11 (m, 1H),
2.04 (dq, J=13.0, 8.6 Hz, 1H), 1.94-1.82 (m, 2H)). The relative and
absolute configuration of Example 114, P-1 was confirmed by single
crystal X-ray analysis.
Example 7.
4-((3S,4S)-3-fluoro-1-((R)-1-(4-methylbenzyl)-2-oxopyrrolidin-3-
-yl)piperidin-4-yl)phenyl dihydrogen phosphate
##STR00040##
[0081] To a suspension of
(R)-1-(4-(difluoromethyl)benzyl)-3-((3S,4S)-3-fluoro-4-(4-hydroxyphenyl)p-
iperidin-1-yl)pyrrolidin-2-one (100 mg, 0.239 mmol, from example 6)
in dichloromethane (10 mL) was added triethylamine (0.233 ml, 1.67
mmol) at -20.degree. C. To the chilled solution was added
POCl.sub.3 (0.111 ml, 1.20 mmol) at -20.degree. C., and then the
reaction mixture was stirred for 2-3 hours at -20.degree. C. Water
(10 mL) was added and the mixture was stirred for 1.5 h. The
mixture was extracted with dichloromethane. The organic layers was
dried over sodium sulfate, filtered, and concentrated in vacuo. The
crude product was purified by reverse phase preparatory HPLC on a
LUNA C8 (250 mm.times.19 mm ID) 5 .mu.m column eluting with a
gradient of solvent A=10 mM ammonium acetate in water pH 4.5 and
solvent B=acetonitrile.
[0082] The titled compound of example 7 (21 mg, 18%) was isolated
from the appropriate fractions by lyophilization as a white solid.
LCMS (M+H).sup.+=499.2; .sup.1H NMR (400 MHz, METHANOL-d4) .delta.
ppm 7.55 (d, J=8.03 Hz, 2H) 7.41 (d, J=8.03 Hz, 2H) 7.21 (s, 4H)
6.62-6.92 (m, 1H) 4.51-4.64 (m, 3H) 3.76 (t, J=8.78 Hz, 1H)
3.43-3.51 (m, 1H) 3.36 (d, J=6.02 Hz, 1H) 3.26-3.30 (m, 1H) 2.81
(br. s., 1H) 2.70-2.78 (m, 1H) 2.59-2.69 (m, 1H) 2.48 (td, J=9.91,
4.77 Hz, 1H) 2.17-2.27 (m, 1H) 2.06-2.15 (m, 1H) 1.80-1.89 (m,
2H).
Example 8.
(R)-1-(4-Fluorobenzyl)-3-(4-(4-hydroxyphenyl)piperidin-1-yl)pyr-
rolidin-2-one
##STR00041##
[0084] To a solution of
1-(4-fluorobenzyl)-3-(4-(4-methoxyphenyl)piperidin-1-yl)pyrrolidin-2-one
(3 g, 7.9 mmol, intermediate S) in dry dichloromethane (100 mL)
under a N.sub.2 atmosphere at -78.degree. C. was added 1 M
borontribromide in dichloromethane (39 mL, 39 mmol) and the
resulting mixture was allowed to warm up to room temperature over 3
h, with stirring. The reaction was quenched with water (30 mL) and
the organic layer was separated, washed with water and brine, and
concentrated. The crude product was purified by flash
chromatography on silica gel using 15% EtOAc in petroleum ether to
yield racemic
1-(4-fluorobenzyl)-3-(4-(4-hydroxyphenyl)piperidin-1-yl)pyrrolidin-2-one
(2.1 g, 73% yield); .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. ppm
1.49-1.74 (m, 4H) 1.90-2.11 (m, 2H) 2.24-2.42 (m, 2H) 2.65-2.80 (m,
2H) 2.99-3.23 (m, 3H) 3.40-3.54 (m, 1H) 4.27-4.46 (m, 2H) 6.61-6.70
(m, 2H) 6.95-7.04 (m, 2H) 7.17-7.31 (m, 4H) 9.10-9.16 (m, 1H). LCMS
(ES-API) 369.2 m/z (M+H).sup.+. A portion of the racemate (40 mg)
was separated via SFC on a Chiralpak-IA 250 mm.times.4.6 mm, 5 m
column eluting with 35% solvent B, where solvent A=CO.sub.2 and
solvent B=0.3% DEA in methanol at a total flow of 3 mL/min. Peak 1
showed a RT of 4.35 min (11 mg) and Peak 2 showed a RT of 6.29 min
(13 mg). Data for example 8 (Peak 2): LC/MS (M+H).sup.+=369.2;
.sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. ppm 1.47-1.59 (m, 1H)
1.65-1.75 (m, 1H) 1.84-1.96 (m, 1H) 2.03-2.12 (m, 1H) 2.24-2.43 (m,
1H) 2.63-2.72 (m, 2H) 2.72-2.85 (m, 2H) 2.96-3.05 (m, 2H) 3.09-3.23
(m, 2H) 3.41-3.54 (m, 1H) 4.23-4.50 (m, 2H) 6.58-6.71 (m, 2H)
6.96-7.10 (m, 2H) 7.15-7.21 (m, 2H) 7.26-7.34 (m, 2H) 9.06-9.19 (m,
1H).
Example 9.
(R)-4-(1-(1-(4-fluorobenzyl)-2-oxopyrrolidin-3-yl)piperidin-4-y-
l)phenyl dihydrogen phosphate
##STR00042##
[0086] Phosphorus oxychloride (1.27 mL, 13.6 mmol) was added to a
round bottom flask charged with THF (10 mL). The solution was
cooled below 0.degree. C. using an ice/methanol bath. A suspension
of
(R)-1-(4-fluorobenzyl)-3-(4-(4-hydroxyphenyl)piperidin-1-yl)pyrrolidin-2--
one (1.00 g, 2.71 mmol, example 8) in THF (18 mL) was added. After
5 min, triethylamine (0.946 mL, 6.79 mmol) was added slowly at a
bath temperature below 5.degree. C. The reaction mixture was
stirred at 0.degree. C. for 90 min. A solution of 1 N aqueous
sodium hydroxide (8.69 mL, 8.69 mmol) was added dropwise. The pH
was measured to be .about.0. The mixture was allowed to warm to rt
and stir for 3 h. The crude reaction mixture was concentrated in
vacuo at <30.degree. C. to afford a clear solution. The solution
was triturated with 1 N aqueous NaOH to pH .about.1. The mixture
was cooled in an ice bath. A semi-solid crashed out. All liquid was
decanted off. The semi-solid was suspended in 90% ethanol and then
a collected by vacuum filtration. The product was presumed to be
the HCl salt of
(R)-4-(1-(1-(4-fluorobenzyl)-2-oxopyrrolidin-3-yl)piperidin-4-yl)phenyl
dihydrogen phosphate (560 mg, 42%). A solution of 25% sodium
methoxide in methanol (250 mg, 1.16 mmol) was added to a slurry of
(R)-4-(1-(1-(4-fluorobenzyl)-2-oxopyrrolidin-3-yl)piperidin-4-yl)phenyl
dihydrogen phosphate, HCl (560 mg, 1.16 mmol) in methanol. The
mixture was stirred until clear and then concentrated in vacuo. The
residue was dissolved in 90% ethanol/water and chilled in the
freezer. The solid precipitate was collected using vacuum
filtration. The solid was dried under high vacuum to afford the
titled compound of example 9 (230 mg, 19% yield): LC/MS
(M+H).sup.+=449.2; .sup.1H NMR (500 MHz, methanol-d.sub.4) .delta.
7.35-7.25 (m, 2H), 7.21-7.11 (m, 4H), 7.11-7.03 (m, 2H), 4.55-4.35
(m, 2H), 3.61 (t, J=8.8 Hz, 1H), 3.30-3.20 (m, 2H), 3.19-3.11 (m,
1H), 2.92-2.84 (m, 1H), 2.75 (td, J=11.1, 3.5 Hz, 1H), 2.55-2.41
(m, 2H), 2.23-2.13 (m, 1H), 2.12-2.00 (m, 1H), 1.84-1.70 (m, 4H);
.sup.31P NMR (202 MHz, methanol-d.sub.4) .delta. ppm -3.38.
Biological Methods
[0087] Radioligand Binding Assay.
[0088] Binding experiments to determine binding to NR2B-subtype
NMDA receptors were performed on forebrains of 8-10 weeks old male
Sprague Dawley rats (Harlan, Netherlands) using .sup.3H Ro 25-6981
(Mutel V; Buchy D; Klingelschmidt A; Messer J; Bleuel Z; Kemp J A;
Richards J G. Journal of Neurochemistry, 1998, 70(5):2147-2155.
Rats were decapitated without anesthesia using a Guillotine
(approved by animal ethics committee) and the harvested brains were
snap-frozen and stored at -80.degree. C. for 3-6 months for
membrane preparation.
[0089] For membrane preparation, rat forebrains were thawed on ice
for 20 minutes in homogenization buffer composed of 50 mM
KH.sub.2PO.sub.4 (pH adjusted to 7.4 with KOH), 1 mM EDTA, 0.005%
Triton X 100 and protease inhibitor cocktail (Sigma Aldrich).
Thawed brains were homogenized using a Dounce homogenizer and
centrifuged at 48000.times.g for 20 min. The pellet was resuspended
in cold buffer and homogenized again using a Dounce homogenizer.
Subsequently, the homogenate was aliquoted, snap-frozen and stored
at -80.degree. C. for not more than 3-4 months.
[0090] To perform the competition binding assay, thawed membrane
homogenate was added to each well of a 96-well plate (20
.mu.g/well). The experimental compounds were serially diluted in
100% DMSO and added to each row of the assay plate to achieve
desired compound concentrations, keeping the DMSO concentration in
the assay plate at 1.33% of the final reaction volume. Next,
.sup.3H Ro 25-6981 (4 nM) was added to the assay plate. After
incubation for 1 hr at room temperature, the membrane bound
radioligand was harvested on to GF/B filter plates (treated with
0.5% PEI for 1 hr at room temperature). The filter plates were
dried at 50.degree. C. for 20 mins, incubated with microscint 20
for 10 minutes and finally, the counts were read on TopCount
(Perkin Elmer). Non-specific binding was determined using MK-0657
(the preparation of this compound is described as example 1 in WO
2004 108705 (40 .mu.M). CPM values were converted to % inhibition
and the concentration response curves were plotted using custom
made software. Each experiment was repeated at least twice to
obtain the final binding K.sub.i values for experimental compounds.
Using this assay, the compound of example 1 showed a binding Ki of
4 nM, the compound of example 6 showed a binding Ki of 4 nM, the
compound of example 8 showed a binding Ki of 1.4 nM.
[0091] Ex Vivo Occupancy Assay.
[0092] This assay demonstrates that the compound of example 1
occupies brain-resident NR2B-subtype receptors in animals after
dosing. 7-9 weeks old male CD-1 mice were dosed intravenously in a
vehicle consisting of 10% dimethylacetamide, 40% PEG-400, 30%
hydroxypropyl betacyclodextrin, and 30% water with experimental
compounds and the forebrains were harvested 15 minutes post-dosing
by decapitation. The brain samples were immediately snap-frozen and
stored at -80.degree. C. On the following day, the dosed brain
samples were thawed on ice for 15-20 minutes followed by
homogenization using Polytron for 10 seconds in cold homogenization
buffer composed of 50 mM KH.sub.2PO.sub.4 (pH adjusted to 7.4 with
KOH), 1 mM EDTA, 0.005% Triton X 100 and protease inhibitor
cocktail (Sigma Aldrich). The crude homogenates were further
homogenized using a Dounce homogenizer and the homogenized membrane
aliquots from all animals were flash-frozen and stored at
-80.degree. C. until further use. The whole homogenization process
was performed on ice.
[0093] For determining occupancy, the membrane homogenates were
first thawed on ice and then needle-homogenized using a 25 gauge
needle. The homogenized membrane (6.4 mg/ml) was added to a 96-well
plate followed by addition of .sup.3H Ro 25-6981 (6 nM). The
reaction mixture was incubated for 5 minutes on a shaker at
4.degree. C. and then harvested onto GF/B filter plates (treated
with 0.5% PEI for 1 hr at room temperature). The filter plates were
dried at 50.degree. C. for 20 mins, incubated with microscint 20
for 10 minutes and read on TopCount (Perkin Elmer). Each dose or
compound group consisted of 4-5 animals. The control group of
animals was dosed with vehicle alone. Membrane from each animal was
added in triplicates to the assay plate. Non-specific binding was
determined using 10 .mu.M Ro 25-6981 added to the wells containing
membrane homogenates from vehicle-dosed animals. Specific
counts/minute was converted to % occupancy at each dose of a
compound for each animal using the following equation:
% Occupancy ( animal A ) = 100 - ( specific CPM of animal A Average
CPM from control group .times. 100 ) ##EQU00001##
Using this procedure, the compound of example 1 showed 95% NR2B
receptor occupancy after a 3 mg/Kg i.v. dose. Drug levels were
determined by mass spectroscopy in the usual manner. Drug levels in
the blood plasma were 1106 nM in at this dose, and drug levels in
the homogonized brain tissue were 1984 nM. The compound of example
6 showed 97% NR2B receptor occupancy after a 3 mg/Kg i.v. dose.
Drug levels in the blood plasma were 1800 nM in at this dose, and
drug levels in the homogonized brain tissue were 2200 nM. The
compound of example 8 showed 96% NR2B receptor occupancy after a 3
mg/Kg i.v. dose. Drug levels in the blood plasma were 570 nM at
this dose, and drug levels in the homogonized brain tissue were 900
nM.
[0094] hERG Electrophysiology Assay.
[0095] The experimental compounds were assessed for hERG activity
on HEK 293 cells stably expressing hERG channels using patch clamp
technique. Coverslips plated with hERG expressing cells were placed
in the experimental chamber and were perfused with a solution
composed of (in mM): 140 NaCl, 4 KCl, 1.8 CaCl.sub.2, 1 MgCl.sub.2,
10 Glucose, 10 HEPES (pH 7.4, NaOH) at room temperature.
Borosilicate patch pipettes had tip resistances of 2-4 Mohms when
filled with an internal solution containing: 130 KCl, 1 MgCl.sub.2,
1 CaCl.sub.2, 10 EGTA, 10 HEPES, 5 ATP-K.sub.2 (pH 7.2, KOH). The
cells were clamped at -80 mV in whole cell configuration using an
Axopatch 200B (Axon instruments, Union City, Calif.) patch clamp
amplifier controlled by pClamp (Axon instruments) software. Upon
formation of a gigaseal, the following voltage protocol was
repeatedly (0.05 Hz) applied to record tail currents:
depolarization step from -80 mV to + 20 mV for 2 seconds followed
by a hyperpolarization step to -65 mV (3 seconds) to elicit tail
currents and then, back to the holding potential. Compounds were
applied after stabilization of tail current. First, tail currents
were recorded in presence of extracellular solution alone (control)
and subsequently, in extracellular solution containing increasing
compound concentrations. Each compound concentration was applied
for 2-5 minutes. The percentage inhibition at each concentration
was calculated as reduction in peak tail current with respect to
the peak tail current recorded in the presence of control solution.
Data analysis was performed in custom made software. The percent
inhibitions at different concentrations were plotted to obtain a
concentration response curve, which was subsequently fitted with a
four parameter equation to calculate the hERG IC.sub.50 value.
Using this procedure, the compound of example 1 is a poor inhibitor
of the hERG channel, with an IC.sub.50=28 .mu.M. The compound of
example 6 is a poor inhibitor of the hERG channel, with an
IC.sub.50=13.5 .mu.M.
[0096] Mouse Forced Swim Test (mFST).
[0097] Forced Swim Test (FST) is an animal model used to assess
antidepressant compounds in preclinical studies. The FST was
performed similar to the method of Porsolt et al. with
modifications (Porsolt R D, Bertin A, Jalfre M. Behavioral despair
in mice: a primary screening test for antidepressants. Arch Int
Pharmacodyn Ther 1977; 229:327-36). In this paradigm, mice are
forced to swim in an inescapable cylinder filled with water. Under
these conditions, mice will initially try to escape and eventually
develop immobility behavior; this behavior is interpreted as a
passive stress-coping strategy or depression-like behavior. Swim
tanks were positioned inside a box made of plastic. Each tank was
separated from each other by opaque plastic sheets to the height of
cylinders. Three mice were subjected to test at a time. Swim
sessions were conducted for 6 min by placing mice in individual
glass cylinders (46 cm height.times.20 cm diameter) containing
water (20-cm deep, maintained at 24-25.degree. C.). At this water
level, the mouse tail does not touch the bottom of the container.
The mouse was judged to be immobile whenever it remained floating
passively without struggling in the water and only making those
movements necessary to keep its nose/head above the water and to
keep it afloat. The duration of immobility was evaluated during the
total 6 min of the test and expressed as duration (sec) of
immobility. Each mouse was tested only once. At the end of each
session, mice were dried with a dry cloth and returned to their
home cage placed on a thermal blanket to prevent hypothermia. Water
was replaced after each trial. All testing sessions were recorded
with a video camera (Sony Handicam, Model: DCR-HC38E; PAL) and
scoring was done using the Forced Swim Scan, Version 2.0 software
(Clever Systems Inc., Reston, Va., USA; see Hayashi E, Shimamura M,
Kuratani K, Kinoshita M, Hara H. Automated experimental system
capturing three behavioral components during murine forced swim
test. Life Sci. 2011 Feb. 28; 88(9-10):411-7 and Yuan P, Tragon T,
Xia M, Leclair C A, Skoumbourdis A P, Zheng W, Thomas C J, Huang R,
Austin C P, Chen G, Guitart X. Phosphodiesterase 4 inhibitors
enhance sexual pleasure-seeking activity in rodents. Pharmacol
Biochem Behav. 2011; 98(3):349-55). For NCE testing: Test compound
was administered in mice 15 min before swim session by i.v. route
and immobility time was recorded for next 6 min. At the end of FST,
the mouse were euthanized by rapid decapitation method and plasma
and brain samples were collected and stored under -80.degree. C.
till further analysis. In the mouse forced swim assay, the compound
of example 1 was dosed intravenously in a vehicle of 30%
hydroxypropyl betacyclodextrin/70% citrate buffer pH 4 at a 5 mL/Kg
dosing volume. The compound of example 1 demonstrated a
statistically significant decrease in immobility time at 1 mg/Kg
under these conditions. Drug levels were 268+/-128 nM in the plasma
and 749+/-215 nM in the brain at this dose. The NR2B receptor
occupancy was determined as reported above and was determined to be
73%. The compound of example 6 demonstrated a statistically
significant decrease in immobility time at 1 mg/Kg under these same
conditions. Drug levels were 360 nM in the plasma. The NR2B
receptor occupancy was determined to be 79%.
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