4-SUBSTITUTED-3-BENZYLOXY-BICYCLO[3.1.0]HEXANE COMPOUNDS AS
mGluR 2/3 ANTAGONISTS
Glutamate is the major excitatory neurotransmitter in the brain and is involved in
a wide variety of physiological processes mediated through no less than 11 distinct
receptors, each with its own pharmacology. Metabotropic Glutamate
Receptor subtypes 2 and 3 (known as mGlu2 and mGlu3) are often grouped together as
Group II mGlu receptors based on their sequence homology, similar second messenger
coupling, and similar pharmacological characteristics. Antagonists of mGlu2/3 receptors
have exhibited significant pharmacological effects in animal models for depressive
disorders and disorders of excessive sleepiness. As such, mGlu2/3 antagonists are
deemed to be useful in the treatment of depressive disorders such as major depressive
disorder (MDD), unipolar depression, dysthymia, and/or cyclothymia, and/or useful in the
treatment of disorders of excessive sleepiness, such as excessive daytime sleepiness
(EDS), hypersomnia associated with obstructive sleep apnea or narcolepsy, circadian
rhythm sleep disorders (including, but not limited to shift work sleep disorder, jet lag
disorder, delayed sleep phase disorder, advanced phase sleep disorder, and non-24 hour
sleep-wake syndrome), idiopathic hypersomnolance and/or excessive sleepiness
associated with non-restorative sleep (NRS).
US 5,916,920 describes certain 3-monosubstituted bicyclo[3.1.0]hexane
compounds as metabotropic glutamate receptor modulators useful for treating a variety of
disorders including as antidepressant agents. US 7,157,594 describes various 3-
monosubstituted bicyclo[3.1.0]hexane compounds as Group II mGlu receptor antagonists
for use in treating various disorders including depressive symptoms. US 2007/0021394
Al describes various 3-monosubstituted bicyclo[3.1.0]hexane compounds as Group II
mGlu receptor antagonists and prodrugs thereof for use in treating various disorders
including depression.
The present invention provides a family of 4-substituted-3-phenylsulfanylmethylbicyclo[
3. 1.0]hexane compounds with high antagonist potency for the mGlu2 and mGlu3
receptors. The compounds of the present invention are also selective for the mGlu2 and
mGlu3 receptors, particular as against other mGlu receptors. Certain compounds have
also demonstrated through animal models that the compounds of the present invention
may be useful for the treatment of depressive disorders (which may include major
depressive disorder (MDD), unipolar depression, dysthymia, and/or cyclothymia) and
disorders of excessive sleepiness (which may include excessive daytime sleepiness
(EDS), hypersomnia associated with obstructive sleep apnea or narcolepsy, circadian
rhythm sleep disorders (including, but not limited to shift work sleep disorder, jet lag
disorder, delayed sleep phase disorder, advanced phase sleep disorder, and non-24 hour
sleep-wake syndrome), idiopathic hypersomnolance and/or excessive sleepiness
associated with non-restorative sleep (NRS)). The antidepressant-like and wakepromoting
effects of this mechanism also predict impact on symptoms of depressive
disorders such as fatigue that are otherwise difficult to treat with existing antidepressants.
The present invention rovides compounds of Formula I :
I
where R1 and R2 are each independently hydrogen, C1-C3 alkoxycarbonyloxymethyl, Ci-
C 3 alkylcarbonyloxymethyl, or C3-6 cycloalkylcarbonyloxymethyl;
R is independently at each occurance methyl, fluoro, or chloro;
R4 is hydroxyl, methylcarbonylamino, hydroxymethylcarbonylamino,
methoxycarbonylamino, imidazol-2-ylsulfanyl, thiazol-2-ylsulfanyl, l,2,4-triazol-3-
ylsulfanyl, 1-methyl- l,2,4-triazol-3-ylsulfanyl, or 1-methyl- l,2,4-triazol-5-ylsulfanyl;
and
n is 1 of 2;
or a pharmaceutically acceptable salt thereof.
It is a feature of the present invention that compounds of Formula I wherein R1
and R2 are both hydrogen (the di-acid compounds) are the therapeutically active
compounds in vivo, whereas compounds where R1 or R2 or both are other than hydrogen
are prodrugs of their therapeutically active di-acid analogs. The compounds where R1 or
R2 or both are other than hydrogen are hydrolyzed in vivo to provide the therapeutically
active di-acid analog. The prodrug compounds when administered orally, particularly diester
prodrugs, provide improved bioavailability of the di-acid metabolite compared to
oral administration of the di-acid compounds (R1 and R2 both hydrogen), but the di-acid
compounds provide better activities when administered intravenously, intramuscularly or
subcutaneously.
In another aspect of the invention there is provided a pharmaceutical composition
comprising a compound of Formula I or a pharmaceutically acceptable salt thereof, in
combination with at least one pharmaceutically acceptable carrier, diluent, or excipient.
Furthermore, this aspect of the invention provides a pharmaceutical composition adapted
for the treatment of depressive disorders, as for example major depressive disorder,
unipolar depression, dysthymia, and/or cyclothymia, comprising a compound of Formula
I or a pharmaceutically acceptable salt thereof, in combination with one or more
pharmaceutically acceptable excipients, carriers, or diluents thereof.
A further embodiment of this aspect of the invention provides a pharmaceutical
composition comprising a compound according to Formula I, or pharmaceutically
acceptable salt thereof, in combination with at least one pharmaceutically acceptable
carrier, exciepient or diluents, and optionally other therapeutic ingredients. In a yet
further embodiment of this aspect of the invention, the pharmaceutical composition
further comprises a second therapeutic agent which is a drug useful in the treatment of
depressive disorders, as for example a serotonin reuptake inhibitor, as for example
fluoxetine and/or citalopram.
In yet another embodiment of this aspect of the invention there is provided a
pharmaceutical composition adapted for the treatment of disorders of excessive
sleepiness, as for example, excessive daytime sleepiness (EDS), hypersomnia associated
with obstructive sleep apnea or narcolepsy, circadian rhythm sleep disorders (including,
but not limited to shift work sleep disorder, jet lag disorder, delayed sleep phase disorder,
advanced phase sleep disorder, and non-24 hour sleep-wake syndrome), idiopathic
hypersomnolance and/or excessive sleepiness associated with non-restorative sleep
( RS), comprising a compound of Formula I or a pharmaceutically acceptable salt
thereof, in combination with one or more pharmaceutically acceptable excipients,
carriers, or diluents thereof.
The present invention also provides a method of treating depressive disorders, as
for example major depressive disorder (MDD), unipolar depression, dysthymia, and/or
cyclothymia, in a mammal comprising administering to a mammal in need of such
treatment an effective amount of a compound of Formula I or a pharmaceutically
acceptable salt thereof. In another embodiment of this aspect of the invention, the method
further comprises administering in simultaneous, separate or sequential combination, a
second therapeutic agent which is a drug useful in the treatment of depressive disorders,
as for example a serotonin reuptake inhibitor, as for example fluoxetine and/or
citalopram.
Other embodiments of the invention provide methods of treating disorders of
excessive sleepiness comprising administering to a mammal in need of such treatment an
effective amount of a compound of Formula I, or a pharmaceutically acceptable salt
thereof. In other embodiments of this aspect of the invention, the excessive sleepiness is
due to any one or more of the following: excessive daytime sleepiness (EDS),
hypersomnia associated with obstructive sleep apnea or narcolepsy, circadian rhythm
sleep disorders (including, but not limited to shift work sleep disorder, jet lag disorder,
delayed sleep phase disorder, advanced phase sleep disorder, and non-24 hour sleep-wake
syndrome), idiopathic hypersomnolance or excessive sleepiness associated with nonrestorative
sleep (NRS).
In one particular embodiment of these methods of treatment, the mammal is a
human.
This invention also provides a compound of Formula I or a pharmaceutically
acceptable salt thereof for use in therapy. Within this aspect, the invention provides a
compound of Formula I, or a pharmaceutically acceptable salt thereof, for use in the
treatment of depressive disorders. In further embodiments, the depressive disorder is any
one of major depressive disorder (MDD), unipolar depression, dysthymia, and/or
cyclothymia. In another embodiment of this aspect of the invention, the invention
provides a compound according to Formula I, or a pharmaceutically acceptable salt
thereof, for use in simultaneous, separate or sequential combination with a serotonin
reuptake inhibitor, as for example fluoxetine and/or citalopram, in the treatment of
depressive disorders.
Further, this aspect of the invention includes a compound of Formula I, or a
pharmaceutically acceptable salt thereof, for use in the treatment of disorders of excessive
sleepiness. In particular embodiments of this aspect of the invention, the excessive
sleepiness is due to any one or more of the following: excessive daytime sleepiness
(EDS), hypersomnia associated with obstructive sleep apnea or narcolepsy, circadian
rhythm sleep disorders (including, but not limited to shift work sleep disorder, jet lag
disorder, delayed sleep phase disorder, advanced phase sleep disorder, and non-24 hour
sleep-wake syndrome), idiopathic hypersomnolance or excessive sleepiness associated
with non-restorative sleep ( RS).
One particular embodiment of this aspect of the inventions, the uses are in
mammals, particular humans.
Another aspect of this invention provides the use of a compound of Formula I, or
a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the
treatment of depressive disorders, as for example major depressive disorder (MDD),
unipolar depression, dysthymia, and/or cyclothymia. Another embodiment of this aspect
of the invention provides the use of a compound of Formula I, or a pharmaceutically
acceptable salt thereof, and a second therapeutic agent useful in the treatment of
depressive disorders, as for example a serotonin reuptake inhibitor, as for example
fluoxetine and/or citalopram, in the manufacture of a medicament for the treatment of
depressive disorders. Another embodiment of the invention provides the use of a
compound of Formula I, or a pharmaceutically acceptable salt thereof, in the manufacture
of a medicament for the treatment of disorders of excessive sleepiness. In particular
embodiments of this aspect of the invention, the medicament is for the treatment of any
one or more of the following: excessive daytime sleepiness (EDS), hypersomnia
associated with obstructive sleep apnea or narcolepsy, circadian rhythm sleep disorders
(including, but not limited to shift work sleep disorder, jet lag disorder, delayed sleep
phase disorder, advanced phase sleep disorder, and non-24 hour sleep-wake syndrome),
idiopathic hypersomnolance or excessive sleepiness associated with non-restorative sleep
(NRS).
Compounds of this invention have basic and acidic moieties, and accordingly
react with a number of organic and inorganic acids and bases to form pharmaceutically
acceptable salts. Pharmaceutically acceptable salts of each of the compounds of the
present invention are contemplated within the scope of the present application. The term
"pharmaceutically acceptable salt" as used herein, refers to any salt of a compound of the
invention that is substantially non-toxic to living organisms. Such salts include those
listed in Journal of Pharmaceutical Science, 66, 2-19 (1977), which are known to the
skilled artisan.
Preferred classes of compounds of the present invention are compounds wherein:
1) R1 and R2 are both hydrogen;
2) R1 or R2 or both are other than hydrogen;
3) R1 and R2 are both other than hydrogen;
4) R1 and R2 are the same and are other than hydrogen;
5) R1 and R2 are each isopropoxycarbonyloxymethyl;
6) n is 2;
7) n is 2, both R groups are chloro, and the chloro groups are at the phenyl 3-
and 4-positions;
8) R4 is hydroxyl.
It will be understood that further preferred compounds are those combining the
above preferred selections for a given substituents with preferred selections of other
substituents. Examples of such combinations include, but are not limited to the following
preferred classes of compounds:
9) preferred compounds of any one of paragraphs 1-5 (preferred selections for R1
and R2) wherein n is 2, both R groups are chloro, and the chloro groups are at the phenyl
3- and 4-positions (paragraph 7);
10) preferred compounds of any one of paragraphs 1-5 (preferred selections for R1
and R2) wherein R4 is hydroxyl (paragraph 8);
11) preferred compounds of any one of paragraphs 1-5 (preferred selections for
R1 and R2) wherein n is 2, both R3 groups are chloro, and the chloro groups are at the
phenyl 3- and 4-positions (paragraph 7), and where R4 is hydroxyl (paragraph 8).
Specific preferred compounds of the invention are those described in the examples,
including their free bases and pharmaceutically acceptable salts thereof.
Certain preferred compounds are:
(lS,2R,3S,4S,5R,6R)-2-amino-3-[(3,4-dichlorobenzyl)oxy]-4-hydroxybicyclo
[3.1.0]hexane-2,6-dicarboxylic acid or a pharmaceutically acceptable salt thereof;
and
bis(isopropoxycarbonyloxymethyl)(lR,2S,3S,4R,5S,6R)-4-amino-3-[(3,4-
dichlorophenyl)methoxy]-2-hydroxy-bicyclo[3. 1.0]hexane-4,6-dicarboxylate or a
pharmaceutically acceptable salt thereof (i.e. the compounds of Examples 1, 10
and 15, and alternative pharmaceutically acceptable salts thereof).
Abbreviations used herein are defined as follows:
"BSA" means bovine serum albumin.
"DCG IV" means (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine.
"DMEM means Dulbecco's Minimum Eagle's Medium.
"DMSO" means dimethyl sulfoxide.
"DPBS" means Dulbecco's Phosphate Buffered Saline.
"EDTA" means ethylene diamine tetraacetic acid.
"GTP" means guanosine triphosphate.
"HBSS" means Hank's Buffered Salt Solution.
"HEPES" means 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid.
"HPLC" means high-pressure liquid chromatography.
"IBMX" means 3-isobutyl-l-methylxanthine
"IC50" means the concentration at which 50% of the maximum inhibition is
achieved.
"i.v." means intravenous or intravenously
"i.p." means intraperitoneal.
"L-AP-4" means L-(+)-2-amino-4-phosphonobutyric acid.
"LC/MS" means liquid chromatography followed by mass spectroscopy.
"mFST" means mouse forced swim test; an animal model for antidepressant
activity.
'MS" means mass spectroscopy.
'MS (ES+)" means mass spectroscopy using electrospray ionization.
'NMR" means nuclear magnetic resonance
'p.o." means per os, by mouth.
'?Bu" means a tertiary-butyl moiety.
General Chemistry
The compounds of the present invention can be prepared according to the
following synthetic schemes by methods well known and appreciated in the art. Suitable
reaction conditions for the steps of these schemes are well known in the art and
appropriate substitutions of solvents and co-reagents are within the skill of the art.
Likewise, it will be appreciated by those skilled in the art that synthetic intermediates
may be isolated and/or purified by various well known techniques as needed or desired,
and that frequently, it will be possible to use various intermediates directly in subsequent
synthetic steps with little or no purification. Furthermore, the skilled artisan will
appreciate that in some circumstances, the order in which moieties are introduced is not
critical. The particular order of steps required to produce the compounds of Formula I is
dependent upon the particular compound being synthesized, the starting compound, and
the relative liability of the substituted moieties, as is well appreciated by the skilled
chemist. All substituents, unless otherwise indicated, are as previously defined, and all
reagents are well known and appreciated in the art.
Prodrug compounds 1 may be prepared as illustrated in Scheme I where R1, R2,
R , R4, and n are as previously defined. R1 and R2 are not both hydrogen in compound .
Scheme I
The compounds can be made by chemistry illustrated in Scheme I . Compound 4
is reacted with an amino protecting reagent such as di-tert-butyldicarbonate under
conditions well known to the skilled artisan to provide the compound 3. When the R1 and
R2 groups are identical in the compound 2, the compound 3 is reacted with sufficient
amount of proper chloro- or iodomethyl alkyl carbonate and appropriate reagents such as
sodium iodide and cesium carbonate in a suitable solvent such as dimethylformamide to
give the desired di-ester compound 2 where R1 and R2 are the same. When R1 and R2 are
different in compound 2, by controlling the amount of first chloromethyl alkyl carbonate
to about one equivalent, the carboxylic acid on the five-membered ring can be converted
to a R2 mono ester first. The R2 mono ester compound can further react with one
equivalent of different chloromethyl alkyl carbonate. The free carboxylic acid group on
the three-membered ring can then be converted to a R1 ester to provide the desired di-ester
with different R1 and R2. To make a R2 mono ester on the five membered ring of the
compound 2, the compound 3 is reacted with about one equivalent of proper chloromethyl
alkyl carbonate and appropriate reagents such as sodium iodide and cesium carbonate in a
suitable solvent such as dimethylformamide to give the desired R2 mono ester compound
2, in which R1 is hydrogen. To make a R1mono ester on the three-membered ring, the
carboxylic acid group on the five-membered ring should be protected first since it is more
reactive. More specifically, the carboxylic acid group on the five-membered ring in
compound 3 can react with alpha-chloro-4-methoxytoluene, sodium iodide and sodium
bicarbonate in a suitable solvent such as dimethylformamide to provide a 4-
methoxylbenzyl mono ester. The free carboxylic acid group on the three-membered ring
of the protected 4-methoxylbenzyl mono ester compound is then reacted with a proper
chloromethyl alkyl carbonate to afford a desired R1 ester on the three-membered ring.
The di-ester is treated with a proper acid such as trifluroacetic acid to deprotect both 4-
methoxylbenzyl and N-tert-butoxycarbonyl group to afford the desired R1mono ester
compound The compound 2, including R2 mono ester and di-ester with same or
different R1 and R2, is then de-protected with a proper acid such as hydrochloric acid in
dioxane to give the desired compound 1 or a pharmaceutically acceptable salt.
9
Active parent compound 4 may be prepared as illustrated in Scheme II.
Compound 9 is reacted with methanesulfonyl chloride and a proper base such as
triethylamine in a suitable solvent such as tetrahydrofuran to give the mesylate compound
6. Compound 6 can react with thiol heterocycles such as lH-l,2,4-triazole-3 -thiol and a
suitable base such as potassium carbonate in a solvent such as dimethylformamide to give
the desired compound 5, in which R4 is a desired thio linked heterocycle. Compound 6
can also react with sodium azide to give an azide intermediate, which is then reduced with
trimethylphosphine in tetrahydrofuran and water to provide an amine. The resulted amine
can further form a desired amide or carbamate with methods well known to skilled
artisans to give compound 5, in which R4 is a desired amide or carbamate group. The
compound 5 is then de-protected with proper acid such as hydrochloric acid or acetic acid
to give the compound 4. When R4 is hydroxyl group in compound 4, the desired active
parent compound 4 can be made directly from compound 7 by removing all protecting
groups with a proper acid such as HC1 in a suitable solvent such as dioxane.
Scheme III
The two key intermediates 7 and 9 can be prepared as illustrated in Scheme III.
Compound 14 (See WO03/1042 17/A2 for synthesis details) is reduced to
compound j_3 with proper reducing reagent such as lithium tri(sec-butyl)borohydride in a
proper solvent such as tetrahydrofuran. Compound 13 is reacted with methanesulfonyl
chloride and a base such as triethyamine in a proper solvent such as tetrahydrofuran to
give mesylate compound 12, which is further reacted with trabutylammonium fluoride or
potassium tert-butoxide in tetrahydrofuran to give compound 11. Compound _ is
reacted with Os0 4, N-methylmorpholine-N-oxide in acetone and water to give a diol
compound 10. Compound 10 is then reacted with properly substituted benzyl halide such
as 3,4-dichlorobenzyl bromide, using tetra-n-butylammonium iodide and silver oxide in
suitable solvent such as dimethylformamide to afford the compound 9. Under Mitsunobu
reaction condition, compound 9 is reacted with 4-nitrobenzoic acid, triphenylphosphine,
and diisopropyl azodicarboxylate in a proper solvent such as tetrahydrofuran to yield the
compound 8. Compound 8 is then reacted with potassium carbonate in a suitable solvent
such as methanol to provide the compound 7.
Preparation 1: Di-tert-butyl ( 1S,2S,4S,5R,6R)-2-[(tert-butoxycarbonyl)amino]-4-
hydroxy-bicyclo[3. 1.0]hexane-2,6-dicarboxylate
Add 1M lithium tri(seobutyl)borohydride in tetrahydrofuran(42.27 mL, 42.27
mmol) dropwise to di-tert-butyl ( l S,2S,5R,6R)-2-[(tert-butoxycarbonyl)amino]-4-
oxobicyclo[3 .1.0]hexane-2,6-dicarboxylate (7. 10 g, 17.25 mmol, see WO03/1042 17/A2
for synthesis details) in tetrahydrofuran(43 1.35 mL) at 0°C and stir. After 90 minutes,
carefully add 1M aqueous sodium bicarbonate (60.07 mL, 62. 11 mmol) followed by
hydrogen peroxide (30% wt, 2.64 mL, 86.27 mmol) at 0°C. The reaction is warmed to
room temperature. After 40 minutes, the reaction is extracted with ethyl acetate, washed
with 10% aqueous citric acid followed by brine, dried over sodium sulfate, filtered, and
concentrated under reduced pressure to give a residue. The residue is purified by flash
chromatography eluting with 5-50% ethyl acetate in hexanes to give the title compound
(6.70 g, 16.20 mmol, 94%).
Preparation 2: Di-tert-butyl (lS,2S,4S,5R,6R)-2-[(tert-butoxycarbonyl)amino]-4-
[(methylsulfonyl)oxy]bicyclo[3. 1.0]hexane-2,6-dicarboxylate
Methanesulfonyl chloride (224.61 ΐ , 2.90 mmol) is added to di- -butyl
( 1S,2S,4S,5R,6R)-2-[(tert-butoxycarbonyl)amino]-4-hydroxy-bicyclo[3. 1.0]hexane-2,6-
dicarboxylate (400 mg, 967 ) and triethylamine (472 L, 3.39 mmol) in
tetrahydrofuran(4.84 mL) at 0°C and stirred. Cooling bath is removed and the reaction is
allowed to warm to room temperature and stirred overnight. Dilute with water and
saturated aqueous sodium bicarbonate then extract with ethyl acetate. The combined
organics are washed with brine, dried over sodium sulfate, filtered, and concentrated to
under reduced pressure to give the title compound (600 mg, 1.22 mmol, 100%).
Preparation 3: Di- -butyl (lS,2S,5R,6S)-2-[(tertbutoxycarbonyl)
amino]bicyclo[3 .1.0]hex-3-ene-2,6-dicarboxylate
Add 1M tetrabutylammonium fluoride in tetrahydrofuran(3.84 mL, 3.84 mmol) to
di-tert-butyl (lS,2S,4S,5R,6R)-2-[(tert-butoxycarbonyl)amino]-4-
[(methylsulfonyl)oxy]bicyclo[3.1.0]hexane-2,6-dicarboxylate (600.00 mg, 1.22 mmol) in
tetrahydrofuran (2.03 mL) and heat at reflux for 3 hours. The reaction is then cooled to
room temperature, diluted with water, and extracted with ethyl acetate, washed with
brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure to give
a residue. The residue is purified by flash chromatography eluting with 5-75% ethyl
acetate in hexanes to give the title compound (0.23 g, 0.58 mmol, 48%).
Preparation 4: Di-tert-butyl ( 1S,2R,3 S,4R,5R,6R)-2-[(tert-butoxycarbonyl)amino]-3,4-
dihydroxybicyclo[3.1.0]hexane-2,6-dicarboxylate
Add Os04 (2.5% ) in 2-methyl-2-propanol (5.48 g, 0.54 mmol) to Nmethylmorpholine-
N-oxide (2.43 g, 17.97 mmol), di-tert-butyl (lS,2S,5R,6S)-2-[(tertbutoxycarbonyl)
amino]bicyclo[3.1.0]hex-3-ene-2,6-dicarboxylate (3.23 g, 8.17 mmol),
acetone (80.66 g, 102.09 mL, 1.39 moles), and water (40.83 mL; 40.83 g, 2.27 moles).
Stir at room temperature. After 5 hours, a saturated, aqueous solution of sodium
thiosulphate (10 mL) is added to the reaction. Concentrate to half volume under reduced
pressure. Dilute the concentrate with water, extract with ethyl acetate (3x50mL). The
combined organic layers are washed with brine, dried over sodium sulfate, filtered, and
concentrated under reduced pressure to give a residue. The residue is purified by flash
chromatography eluting with 5-50% ethyl acetate in hexanes to give the title compound
(2.7g, 6.3 mmol; 77%)
Preparation 5: Di-tert-butyl ( 1S,2R,3 S,4R,5R,6R)-2-[(tert-butoxycarbonyl)amino]-3-
[(3,4-dichlorobenzyl)oxy]-4-hydroxybicyclo[3 .1.0]hexane-2,6-dicarboxylate
Add 3,4-dichlorobenzyl bromide (1.02 mL, 1.02 g, 4.26 mmol) to di- -butyl
(lS,2R,3S,4R,5R,6R)-2-[(tert-butoxycarbonyl)amino]-3,4-
dihydroxybicyclo[3.1.0]hexane-2,6-dicarboxylate (1.22 g, 2.84 mmol), tetra-nbutylammonium
iodide (1.07 g, 2.84 mmol) and silver oxide (987.35 mg, 4.26 mmol) in
dimethylformamide (17mL) at room temperature. Stir overnight. Dilute with diethyl
ether and hexanes (1:1), filter through celite, and then wash with ether and hexanes (1:1).
The filtrate is washed with water, brine, dried over magnesium sulfate, and concentrated
under reduced pressure to give a residue. The residue is purified by flash
chromatography, eluting with 0-30% ethyl acetate: hexanes to give the title compound
(1.7 g, 2.84 mmol, 78%): MS (m/z): 610/612(M+Na).
Preparation 6: Di-tert-butyl ( 1S,2R,3S,4S,5R,6R)-2-[(tert-butoxycarbonyl)amino]-3-
[(3,4-dichlorobenzyl)oxy]-4-{[(4-nitrophenyl)carbonyl]oxy}bicyclo[3.1.0]hexane-2,6-
dicarboxylate
Triphenylphosphine (12.66 g, 48.26 mmol) and 4-nitrobenzoic acid (8.19 g, 48.26
mmol) are added to di- -butyl (lS,2R,3S,4R,5R,6R)-2-[(tert-butoxycarbonyl)amino]-
3-[(3,4-dichlorobenzyl)oxy]-4-hydroxybicyclo[3. 1.0]hexane-2,6-dicarboxylate (14.20 g,
24.13 mmol) and tetrahydrofuran (241.28 mL, 2.97 moles) at room temperature. Cool the
mixture in an ice bath. Diisopropyl azodicarboxylate (9.57 mL, 9.76 g, 48.26 mmol) in
tetrahydrofuran (20mL) is added and the reaction mixture is gradually warmed to room
temperature overnight. Dilute with diethyl ether, and then wash with aqueous sodium
carbonate. Extract the aqueous layer again with diethyl ether. Combine all ether extracts,
wash with water, brine, dry over magnesium sulfate, and concentrate under reduced
pressure to give a residue. The residue is purified by flash chromatography, eluting with
10-100% ethyl acetate in hexanes to give the title compound (9 g, 12.2 mmol, 51%): MS
(m/z): 759 (M+l).
Preparation 7: Di-tert-butyl ( 1S,2R,3S,4S,5R,6R)-2-[(tert-butoxycarbonyl)amino]-3-
[(3,4-dichlorobenzyl)oxy]-4-hydroxybicyclo[3 .1.0]hexane-2,6-dicarboxylate
Potassium carbonate (5.11 g, 36.60 mmol) is added to a solution of di- -butyl
(lS,2R,3S,4S,5R,6R)-2-[(½rt-butoxycarbonyl)amino]-3-[(3,4-dichlorobenzyl)oxy]-4-
{[(4-nitrophenyl)carbonyl]oxy}bicyclo[3.1.0]hexane-2,6-dicarboxylate (9.00 g, 12.20
mmol) and methanol (122.01 mL, 3.01 moles), and then stirred at room temperature.
After 1 hour, dilute the reaction mixture with ethyl acetate, wash with water (75mL),
brine (75mL), dry over sodium sulfate, and concentrate under reduced pressure to give a
residue. The residue is purified by flash chromatography, eluting with 10-60% ethyl
acetate: hexanes to give the title compound (5.3 g, 9.01 mmol, 74%). MS (m/z):
610/612(M+1).
Preparation 8: Di-/er/-butyl ( 1S,2R,3 S,4R,5R,6R)-2-[(/er/-butoxycarbonyl)amino]-3-
[(3,4-dichlorobenzyl)oxy]-4-[(methylsulfonyl)oxy]bicyclo[3.1.0]hexane-2,6-
dicarboxylate
Methanesulfonyl chloride (0.841 mL, 1.25 g, 10.87 mmol) is added to di-tertbutyl
(lS,2R,3S,4R,5R,6R)-2-[(tert-butoxycarbonyl)amino]-3-[(3,4-dichlorobenzyl)oxy]-
4-hydroxybicyclo[3.1.0]hexane-2,6-dicarboxylate (2.00 g, 3.40 mmol), triethylamine
(1.66 mL, 1.20 g, 11.89 mmol), and tetrahydrofuran (33.98 mL, 30.11 g, 417.61 mmol) at
0°C and stirred. Gradually warm the reaction to room temperature and stir overnight.
Dilute the reaction with saturated aqueous sodium bicarbonate then extract with ethyl
acetate (3xl00mL). The combined organic extracts are washed with brine, dried over
magnesium sulfate, filtered, and then concentrated under reduced pressure to yield a
residue. The residue is purified by flash chromatography using eluting with 5-60% ethyl
acetate in hexanes to give the title compound (1.40 g, 2.10 mmol, 62%).
Preparation 9: Di-tert-butyl (lR,2R,3S,4S,5R,6R)-2-[(tert-butoxycarbonyl)amino
[(3 ,4-dichlorobenzyl)oxy] -4-( 1H-1,2,4-triazol-3 -ylsulfanyl)bicyclo [3.1.0]hexanedicarboxylate
Potassium carbonate (1.45 g, 10.50 mmol) is added to lH-l,2,4-triazole-3 -thiol
(424.79 mg, 4.20 mmol) and dimethylformamide (10.50 mL, 9.93 g, 135.80 mmol) at
room temperature and stirred. After 5 minutes, di-tert-butyl (lS,2R,3S,4R,5R,6R)-2-
[(tert-butoxycarbonyl)amino]-3-[(3,4-dichlorobenzyl)oxy]-4-
[(methylsulfonyl)oxy]bicyclo[3.1.0]hexane-2,6-dicarboxylate(1.40 g, 2.10 mmol) is
added to the reaction and stirred at 85°C. After 2 days, cool the reaction to room
temperature, dilute with saturated sodium bicarbonate, then extract with ethyl acetate
(3X). The combined organics are washed with water, brine, filtered, and concentrated
under reduced pressure to give a residue. The residue is purified by flash
chromatography eluting with 5-75% ethyl acetate in hexanes to give the title compound
(978.0mg, 1.46 mmol, 69%). MS (m/z): 671/669 (M-l).
Preparation 10 : Di- -butyl (lR,2R,3S,4S,5R,6R)-2-[(tert-butoxycarbonyl)
[(3,4-dichlorobenzyl)oxy]-4-(l,3-thiazol-2-ylsulfanyl)bicyclo[3.1.0]hexane-2,
dicarboxylate
This compound is prepared essentially as described in Preparation 9 by using 2-
mercaptothiazole. Residue is purified by normal phase chromatography to yield 29% of
the title compound. MS (m/z): 465 (M+l).
Preparation 11: Di-tert-butyl (lR,2R,3S,4S,5R,6R)-2-[(tert-butoxycarbonyl)amino]-3-
[(3 ,4-dichlorobenzyl)oxy]-4- [(1-methyl- 1,2,4-triazol-3 -yl)sulfanyl]bicyclo [3 .1.0]hexane-
2,6-dicarboxylate
Dissolve di- -butyl (lR,2R,3S,4S,5R,6R)-2-[(tert-butoxycarbonyl)amino]-3-
[(3,4-dichlorobenzyl)oxy]-4-( 1H-1,2,4-triazol-3 -ylsulfanyl)bicyclo[3 .1.0]hexane-2,6-
dicarboxylate (0.60 g, 0.893 mmol) in anhydrous tetrahydrofuran (3.6 mL) in a flamedried
flask and cool to -78°C. Add potassium ?-butoxide (0.80 g, 0.893 mmol) and stir for
15 minutes. Add methyl iodide (0.127 g, 0.893 mmol, pretreated by filtration through
basic alumina) and allow to stir at -78°C for 15 minutes. Allow the mixture to warm to
room temperature and then stir for 30 minutes at room temperature. Pour crude reaction
mixture into water and adjust pH to 6 with IN aqueous HCl and extract into ethyl acetate.
Dry the organic layer over magnesium sulfate, filter and concentrate under reduced
pressure. Purify by reverse phase flash chromatography (lOOg CI 8 column eluting with
45-95% Acetonitrile / Water (with 0.1% trifluoroacetic acid in both), compound elutes at
89% Acetonitrile). Combine the desired fractions and neutralize with aqueous
bicarbonate. Extract with ethyl acetate, dry the organic layer over magnesium sulfate,
filter and concentrate under reduced pressure to yield the title compound as a white foam
(0.16 g, 0.23 mmol, 26%): MS (m/z): 473/475/477 (M+l).
Preparation 12 : Di- -butyl (lS,2R,3R,4S,5R,6S)-4-azido-2-[(tertbutoxycarbonyl)
amino]-3-[(3,4-dichlorobenzyl)oxy]bicyclo[3.1.0]hexane-2,6-
dicarboxylate
Sodium azide (8.29 g, 127.45 mmol) is added to di-tert-butyl
(lS,2R,3S,4R,5R,6R)-2-[(tert-butoxycarbonyl)amino]-3-[(3,4-dichlorobenzyl)oxy]-4-
[(methylsulfonyl)oxy]bicyclo[3.1.0]hexane-2,6-dicarboxylate (16.99 g, 25.49 mmol) and
15-crown-5 (508.57 , 561.46 mg, 2.55 mmol) in dimethylformamide (254.90 mL,
240.96 g, 3.30 moles) at 85°C and stirred. After 7 days, the reaction is cooled to room
temperature then diluted with water and extracted with ethyl acetate (3X). The combined
organics are washed with water, brine, dried over magnesium sulfate, filtered, and
concentrated under reduced pressure giving a residue. The residue is purified by flash
chromatography eluting 2-60% ethyl acetate in hexanes to yield the title compound
( 11.2g, 18.3mmol, 72%). MS (m/z): 635/637 (M+l).
Preparation 13 : Di- -butyl (lS,2R,3R,4S,5R,6S)-4-amino-2-[(ter/-
butoxycarbonyl)amino]-3-[(3,4-dichlorobenzyl)oxy]bicyclo[3.1.0]hexane-2,6-
dicarboxylate
Add a 1M solution of trimethylphosphine in tetrahydrofuran(19.56 mL, 19.56
mmol) to di-/er/-butyl (lS,2R,3R,4S,5R,6S)-4-azido-2-[(/er/-butoxycarbonyl)amino]-3-
[(3,4-dichlorobenzyl)oxy]bicyclo[3.1.0]hexane-2,6-dicarboxylate (8.00 g, 13.04 mmol) in
tetrahydrofuran(43 .46 mL) and water (130.39 mL) at room temperature and stir
overnight. Saturated aqueous sodium bicarbonate is added to the reaction then extracted
with ethyl acetate (3X). The combined organics are washed with brine, dried over
magnesium sulfate, filtered, and concentrated under reduced pressure giving the title
compound (7.42 g, 12.63 mmol, 97%). MS (m/z): 609/61 1 (M+Na).
Preparation 14 : Di- -butyl (lS,2R,3R,4S,5R,6S)-4-(acetylamino)-2-[(tertbutoxycarbonyl)
amino]-3-[(3,4-dichlorobenzyl)oxy]bicyclo[3.1.0]hexane-2,6-
dicarboxylate
Acetyl chloride (334.31 , 368.75 mg, 4.70 mmol) in dichloromethane (5mL) is
added to a stirring, cooled solution of di- -butyl (lS,2R,3R,4S,5R,6S)-4-amino-2-
[(/er/-butoxycarbonyl)amino]-3-[(3,4-dichlorobenzyl)oxy]bicyclo[3. 1.0]hexane-2,6-
dicarboxylate (2.30 g, 3.91 mmol) and triethylamine (709.32 , 514.97 mg, 5.09 mmol)
in dichloromethane (19.57 mL) at 0°C then stirred. After 90 minutes, dilute with water
and extract with dichloromethane (3X25mL). The combined organic extracts are dried
over magnesium sulfate, filtered, and concentrated under reduced pressure giving the title
compound (2.40 g, 3.81 mmol, 97%): MS (m/z): 653 (M+l).
Preparation 15 : Di-tert-butyl (lS,2R,3R,4S,5R,6S)-2-[(tert-butoxycarbonyl)amino]-3-
[(3,4-dichlorobenzyl)oxy]-4-[(methoxycarbonyl)amino]bicyclo[3.1.0]hexane-2,6-
dicarboxylate
This compound is prepared essentially as described in Preparation 14 by using
methyl chloro formate to yield 88% of the title compound.
Preparation 16: Di-tert-butyl (lR,2R,3S,4S,5R,6R)-2-(tert-butoxycarbonyl
[(3,4-dichlorophenyl)methoxy]-4-[(2-methyl-l,2,4-triazol-3-
yl)sulfanyl]bicyclo[3 .1.0]hexane-2,6-dicarboxylate
Add 2-methyl-lH-l,2,4-triazole-3-thione (0.126 g, 1.09 mmol) and potassium
carbonate (0.35 g, 2.52 mmol) to a solution of di- -butyl (lS,2R,3S,4R,5R,6R)-2-[(tertbutoxycarbonyl)
amino]-3-[(3,4-dichlorobenzyl)oxy]-4-
[(methylsulfonyl)oxy]bicyclo[3.1.0]hexane-2,6-dicarboxylate (0.80 g, 0.84 mmol) in
dimethylformamide (1.68 mL. After heating for 2 days at 80°C, pour the solution into
brine and extract with ethyl acetate three times, dry the combined organic phases with
sodium sulfate, filter and concentrate under reduced pressure. Purify by flash
chromatography eluting with 10% ethyl acetate/CH^C^ to yield the title compound as an
oil (0.576 g, 0.84 mmol, 100%). MS (m/z): 685/687/689 (M+l).
Preparation 17 : Ditert-butyl (lR,2R,3S,4S,5R,6R)-2-(tert-butoxycarbonylamino)-3-[(3,4-
dichlorophenyl)methoxy]-4-(lH-imidazol-2-ylsulfanyl)bicyclo[3.1.0]hexane-2,6-
dicarboxylate
This compound is prepared essentially as described in Preparation 20 by using ditert-
butyl (lS,2R,3S,4R,5R,6R)-2-[(tert-butoxycarbonyl)amino]-3-[(3,4-
dichlorobenzyl)oxy]-4-[(methylsulfonyl)oxy]bicyclo[3.1.0]hexane-2,6-dicarboxylate.
Purify by flash chromatography eluting with 30-50% ethyl acetate: hexanes to yield the
title compound in 38% yield. MS (m/z): 671/673/675 (M+l).
Preparation 18: Diethyl (lS,2R,3R,4S,5R,6S)-4-[(2-acetoxyacetyl)amino]-2-(/'ertbutoxycarbonylamino)-
3-[(3,4-dichlorophenyl)methoxy]bicyclo[3.1.0]hexane-2,6-
dicarboxylate
Add acetoxyacetyl chloride (0.116 mL, 1.08 mmol) to a solution of diethyl
(lS,2R,3R,4S,5R,6S)-4-amino-2-(tert-butoxycarbonylamino)-3-[(3,4-
dichlorophenyl)methoxy]bicyclo[3.1.0]hexane-2,6-dicarboxylate (0.47 g, 0.89 mmol) and
diisoproylethylamine (0.32 mL, 1.83 mmol) in dichloromethane (9 mL) at room
temperature. After 2.5 hours, wash the solution with saturated NaHCC (2x), brine (lx),
dry over MgS0 4, filter, and cone under reduced pressure. Purify by flash
chromatography eluting with a gradient of 0 to 100% ethyl acetate in hexanes over 30
min. to yield the title compound as a white solid (0.43 g, 0.68 mmol, 76%). MS (m/z):
53 1 (M+H-BOC).
Preparation 19 : (lS,2R,3R,4S,5R,6S)-2-(tert-butoxycarbonylamino)-3-[(3,4-
dichlorophenyl)methoxy]-4-[(2-hydroxyacetyl)amino]bicyclo[3.1.0]hexane-2,6-
dicarboxylic acid
Add 1M aqueous lithium hydroxide (4.0 mL, 4.0 mmol) to a solution of diethyl
(lS,2R,3R,4S,5R,6S)-4-[(2-acetoxyacetyl)amino]-2-(tert-butoxycarbonylamino)-3-[(3,4-
dichlorophenyl)methoxy]bicyclo[3.1.0]hexane-2,6-dicarboxylate (0.42 g, 0.67 mmol) in
tetrahydrofuran (7.0 mL) at room temperature and stir overnight. Acidify the reaction to
approximately pH 1 with 5N aqueous HCl. Extract with ethyl acetate (3 x 25 mL). Dry
the combined organic layers, filter, and cone under reduced pressure. HPLC analysis of
crude reaction mixture shows starting material present. Add a 1M aqueous lithium
hydroxide (4.0 mL, 4.0 mmol) to a solution of the crude reaction mixture in
tetrahydrofuran (7.0 mL) and heat at 50 °C. After 17 hours, acidify reaction to
approximately pH 1 with 5N aqueous HCl. Extract with ethyl acetate (3 x 25 mL). Dry
the combined extracts, filter, and cone under reduced pressure. Purify by preparative
reverse-phase high performance liquid chromatography using a gradient of 95% water
(with 0.03% HCl) / 5% acetonitrile to 5% water (with 0.03% HCl) / 95% acetonitrile over
40 min. to provide (lS,2R,3R,4S,5R,6S)-2-(tert-butoxycarbonylamino)-3-[(3,4-
dichlorophenyl)methoxy]-4-[(2 -hydroxyacetyl)amino]bicyclo[3.1.0]hexane-2,6-
dicarboxylic acid (0.181 g, 0.34 mmol, 51%) as a white solid. MS (m/z): 531 (M-H).
Preparation 20: (lS,2R,3S,4S,5R,6R)-2 -[( rt -butoxycarbonyl)amino]-3-[(3,4-
dichlorobenzyl)oxy]-4-hydroxybicyclo[3 .1.0]hexane-2,6-dicarboxylic acid
Dissolve (lS,2R,3S,4S,5R,6R)-2-amino-3-[(3,4-dichlorobenzyl)oxy]-4-
hydroxybicyclo[3.1.0]hexane-2,6-dicarboxylic acid (2g, 5.32 mmol) in dioxane (10. 63
mL), then treat with saturated aqueous sodium bicarbonate (10.63 mL, 6.90 mmol) and
di-tert-butyldicarbonate (5.80 g, 26.58 mmol). Stir the biphasic mixture at room
temperature for 18 days, then concentrate to remove solvent. Dissolve the resulting
residue in acetonitrile (assisting with IN H ), and extract twice to remove di-tertbutyldicarbonate.
Concentrate the acetonitrile layer. Dissolve the resulting residue in
ethyl acetate, then wash twice with IN HC1, wash once with brine, dry over sodium
sulfate, and concentrate to dryness to give the title compound (2.1 g, 4.41 mmol, 83%).
MS (m/z): 474 (M-l).
Preparation 2 1: (lS,2R,3R,4S,5R,6S)-4-(acetylamino)-2-[(tert-butoxycarbonyl)amino]-3-
[(3,4-dichlorobenzyl)oxy]bicyclo[3.1.0]hexane-2,6-dicarboxylic acid
This compound is prepared essentially as described in Preparation 20 by using
(lS,2R,3R,4S,5R,6S)-4-(acetylamino)-2-amino-3-[(3,4-
dichlorobenzyl)oxy]bicyclo[3.1.0]hexane-2,6-dicarboxylic acid to yield the title
compound in 74% yield. MS (m/z): 515/517 (M-l).
Preparation 22: Bis({[(l-methylethoxy)carbonyl]oxy}methyl) (lS,2R,3S,4S,5R,6R)-2-
[(/er/-butoxycarbonyl)amino]-3-[(3,4-dichlorobenzyl)oxy]-4-
hydroxybicyclo[3 .1.0]hexane-2,6-dicarboxylate
To a solution of (lS,2R,3S,4S,5R,6R)-2-[(ter/-butoxycarbonyl)amino]-3-[(3,4-
dichlorobenzyl)oxy]-4-hydroxybicyclo[3.1.0]hexane-2,6-dicarboxylic acid (1.89 g, 3.96
mmol) in dimethylformamide (23.3 mL) is added chloromethyl isopropyl carbonate (1.27
g, 8.33 mmol), sodium iodide (59.4 mg, 0.396 mmol), and cesium carbonate (2.84 g, 8.72
mmol). Stir the mixture overnight at room temperature under nitrogen. Dilute the
reaction mixture with ethyl acetate and water. Separate the layers. Extract the aqueous
layer twice with ethyl acetate. Extract the combined organic layers with brine, dry over
magnesium sulfate, filter and concentrate. Purify the residue by flash chromatography
eluting with 0 to 75% ethyl acetate in hexane to yield the title compound as a foam (940
mg, 1.33 mmol, 33%). MS (m/z): 732 (M+Na).
The compounds of Preparation 23-26 may be prepared essentially as described in
Preparation 22:

Example 1: (lS,2R,3S,4S,5R,6R)-2-Amino-3-[(3,4-dichlorobenzyl)oxy]-4-
hydroxybicyclo[3. 1.0]hexane-2,6-dicarboxylic acid
Di- -butyl (lS,2R,3S,4S,5R,6R)-2-[(tert-butoxycarbonyl)amino]-3-[(3,4-
dichlorobenzyl)oxy]-4-hydroxybicyclo[3.1.0]hexane-2,6-dicarboxylate (5.30 g, 9.01
mmol) is added to water (30.02 mL) and acetic acid (30.02 mL). Heat the reaction at
reflux until the complete consumption of the starting material. Cool and concentrate on a
rotary evaporator. Triturate the concentrate with diethyl ether, collect the precipitate,
then dry in a vacuum oven to give the title compound (3.30 g, 8.77 mmol, 97%). MS
(m/z): 376/378 (M+1).
The compounds of Example 2-3 may be prepared essentially as described in Example 1:
Example 4: (lS,2R,3R,4S,5R,6S)-4-(Acetylamino)-2-amino-3-[(3,4-
dichlorobenzyl)oxy]bicyclo[3.1.0]hexane-2,6-dicarboxylic acid
Di- -butyl ( 1S,2R,3R,4S,5R,6S)-4-(acetylamino)-2-[(tertbutoxycarbonyl)
amino]-3-[(3,4-dichlorobenzyl)oxy]bicyclo[3.1.0]hexane-2,6-
dicarboxylate (2.40 g, 3.81 mmol) is added to acetic acid (7.99 g, 7.62 mL, 133.05
mmol,) and water (3.81 mL, 3.81 g, 2 11.60 mmol) and heated in a microwave to 140°C.
After 15 minutes, cool to room temperature, dilute with water and filter to collect the
solids. Sequentially wash the solids with water (2x50mL) and diethyl ether(2x50mL),
and then dry in a vacuum oven giving the title compound (1.23 g, 2.95 mmol, 77%): MS
(m/z): 417/419 (M+l).
Example 5: (lS,2R,3R,4S,5R,6S)-2-Amino-3-[(3,4-dichlorobenzyl)oxy]-4-
[(methoxycarbonyl)amino]bicyclo[3 .1.0]hexane-2,6-dicarboxylic acid
This compound is prepared essentially as described in Example 4 by using
(lS,2R,3R,4S,5R,6S)-2-amino-3-[(3,4-dichlorobenzyl)oxy]-4-
[(methoxycarbonyl)amino]bicyclo[3.1.0]hexane-2,6-dicarboxylic acid. 81% yield. MS
(m/z): 433/435 (M+l).
Example 6: (lR,2R,3S,4S,5R,6R)-2-amino-3-[(3,4-dichlorobenzyl)oxy]-4-[(l-methyl-lHl,
2,4-triazol-5-yl)sulfanyl]bicyclo[3.1.0]hexane-2,6-dicarboxylic acid hydrochloride
HCI
Add acetic acid (2.0 mL) and water (2.0 mL) to di- -butyl
(lR,2R,3S,4S,5R,6R)-2-(tert-butoxycarbonylamino)-3-[(3,4-dichlorophenyl)methoxy]-4-
[(2-methyl-l,2,4-triazol-3-yl)sulfanyl]bicyclo[3. 1.0]hexane-2,6-dicarboxylate (0.305 g,
0.445 mmol) in a microwave vessel and seal and heat the mixture to 140°C for 25
minutes. Cool and concentrate the mixture to dryness, and then add 5 N hydrochloric
acid (2mL), acetonitrile (2 mL), and transfer to a vial. Lyophilize overnight to yield the
title compound as a white solid (0.227 g, 0.445 mmol; 95%): MS (m/z): 473/475/477
(M+l).
The compounds of Example 7-8 may be prepared essentially as described in Example 6 :
Example 9: (lS,2R,3R,4S,5R,6S)-2-Amino-3-[(3,4-dichlorobenzyl)oxy]-4-
[(hydroxyacetyl)amino]bicyclo[3. 1.0]hexane-2,6-dicarboxylic acid hydrochloride
Add 4N hydrogen chloride (3.0 mL, 12.0 mmol) in 1,4-dioxane to a mixture of
(lS,2R,3R,4S,5R,6S)-2-(tert-butoxycarbonylamino)-3-[(3,4-dichlorophenyl)methoxy]-4-
[(2-hydroxyacetyl)amino]bicyclo[3.1.0]hexane-2,6-dicarboxylic acid (0.032 g, 0.06
mmol) in water (0.3 mL). Wash the solid with acetonitrile and diethylether on a fritted
funnel. Sequentially wash the solid with acetonitrile and diethylether and then further dry
in a vacuum oven overnight at 50 °C to provide the title compound as a white solid (0.018
g, 0.04 mmol, 65%). MS (m/z): 433 (M+H).
Example 10 : Bis({[(l-methylethoxy)carbonyl]oxy}methyl) (lS,2R,3S,4S,5R,6R)-2-
amino-3-[(3,4-dichlorobenzyl)oxy]-4-hydroxybicyclo[3.1.0]hexane-2,6-dicarboxylate
hydrochloride
Add bis({[(l-methylethoxy)carbonyl]oxy}methyl) (lS,2R,3S,4S,5R,6R)-2-[(tertbutoxycarbonyl)
amino]-3-[(3,4-dichlorobenzyl)oxy]-4-hydroxybicyclo[3.1.0]hexane-2,6-
dicarboxylate in a 4.0M solution of hydrogen chloride in dioxane (14.47 mL, 57.87
5 mmol) and stir for 45 minutes. Add ethyl acetate, and concentrate the reaction mixture to
a foam. Triturate the foam with ethyl acetate to to produce a solid. Concentrate and dry
under vacuum at 40 °C overnight to provide the title compound as an off-white solid (1.77
g, 2.75 mmol, 95%): MS (m/z): 608 (M+H).
The compounds of Example 11-14 may be prepared essentially as described in Example
10:
Ex. Yield Physical Data
Chemical Name Structure
o. (%) MS(m/z)
Bis {[(ethoxycarbonyl)oxy] methyl}
(lS,2R,3S,4S,5R,6R)-2-amino-3-[(3,4-
11 dichlorobenzyl)oxy]-4- 97 608(M+1)
hydroxybicyclo[3 .1.0]hexane-2,6-
dicarboxylate hydrochloride
Bis {[(2-methylpropanoyl)oxy] methyl}
(lS,2R,3S,4S,5R,6R)-2-amino-3-[(3,4-
12 dichlorobenzyl)oxy]-4- 73 575/577(M+l)
hydroxybicyclo[3 .1.0]hexane-2,6-
dicarboxylate hydrochloride
Bis {[(cyclopropylcarbonyl)oxy]methyl}
13 (lS,2R,3S,4S,5R,6R)-2-amino-3-[(3,4- 97 572/574(M+l)
dichlorobenzyl)oxy]-4-
hydroxybicyclo[3 .1.0]hexane-2,6-
dicarboxylate hydrochloride
Bis[(acetyloxy)methyl]
¾
(lS,2R,3R,4S,5R,6S)-4-(acetylamino)-2-
amino-3-[(3,4- 82 561/563(M+1)
dichlorobenzyl)oxy]bicyclo[3.1.0]hexane-
2,6-dicarboxylate hydrochloride
Example 15: BisCird-methylethoxvlcarbonvlloxvlmethvn (1S.2R.3S.4S.5R.6RV2-
amino-3-[(3,4-dichlorobenzyl)oxy]-4-hydroxybicyclo[3.1.0]hexane-2,6-dicarboxylate
hydrochloride
Step 1: Di- -butyl (lS,2S,4S,5R,6R)-2-[(tert-butoxycarbonyl)amino]-4-hydroxybicyclo[
3.1.0]hexane-2,6-dicarboxylate
Add 1M lithium tri(sec-butyl)borohydride in tetrahydrofuran(2.67L, 2.67mol)
dropwise to a solution of di-tert-butyl(lS,2S,5R,6R)-2-[(tert-butoxycarbonyl)amino]-4-
oxobicyclo[3.1.0]hexane-2,6-dicarboxylate (lOOOg, 2.43mol) in dry tetrahydrofuran(lOL)
at 0°C under nitrogen. After 2 hours at 0°C, 2M Na2C0 in water (850g, 8.02mol) is
added at 0°C over 2 hours followed by 35% aqueous hydrogen peroxide (740mL,
8.99mol) in water (3.3L). After 40 minutes, the mixture is warmed to ambient
temperature whereupon the organic phase is separated. The organic phase is diluted with
methyl tert-butyl ether (3.5L) and the layers are separated again. The aqueous phase is
extracted with methyl -butyl ether (3.5L). All the combined organic phases are
washed successively with 1M aqueous a2S0 (2.67L), water (2.67L), and brine (2.67L),
dried over sodium sulfate, filtered and concentrated to a volume of 3L. The precipitated
solid is filtered and washed with heptane (1.3L) to obtain the title compound (864.76g).
The mother liquors are concentrated to dryness and the residue is triturated with heptane
(500mL) and filtered to obtain additional title compound(130.64g, total 995.4g, yield
99%). MS (m/z): 436 (M+23).
Step 2: Di-tert-butyl (lS,2S,5R,6S)-2-[(tert-butoxycarbonyl)amino]bicyclo[3. 1.0]hex-3-
ene-2,6-dicarboxylate
Triethylamine (735 mL, 5.30 mol) is added dropwise at 0°C under 2 over 40
minutes to a solution of di-tert-butyl(2S,4S)-2-[(ter?-butoxycarbonyl)amino]-4-
hydroxybicyclo[3.1.0]hexane-2,6-dicarboxylate (995.4g, 2.41mol) in dry tetrahydrofuran
(8.4 L) Methanesulfonyl chloride (373mL, 4.82mol) is added at 0°C over 50 minutes.
The reacation is warmed to 23°C. After 3 hours, the reaction if filtered to remove
triethylamine hydrochloride salt. Wash the filter cake with dry tetrahydrofuran(2x2L).
The filtrate (13 L) is split into two equal parts. To one half of filtrate, add 1M potassium
-butoxide in tetrahydrofuran(3.6 L, 3.6 mol) dropwise to the solution obtained in
previous step (Crude V=6.5 L, 1.2 mol) at 15°C over 2 hours 15 minutes then allow to
warm to 23°C. After 2 hours 30 minutes the reaction is quenched by pouring into 10%
aqueous acetic acid (950mL). Separate the layers and extract the aqueous layer with ethyl
acetate (1.7 L). The organic layers are combined and concentrated in vacuo to obtain a
solid which is dissolved in acetone (1.45 L) and added to water (7.3 L). Stir the resulting
suspension overnight at 23°C. Filter off the precipitate, wash with water (2x1 L), and dry
in the vacuum oven at 40°C to obtain the title compound (450.67g, yield 95% ) . MS
(m/z): 418 (M+23).
Step 3: Di- -butyl (lS,2R,3S,4R,5R,6R)-2-[(tert-butoxycarbonyl)amino]-3,4-
dihydroxybicyclo[3. 1.0]hexane-2,6-dicarboxylate
Osmium tetraoxide (4% wt/wt in water, 231.78 mL, 241.05 g, 37.93 mmol,) is
added at 23°C to a solution of N-methylmorpholine-N-oxide hydrate (101.62 mL; 114.83
g, 424.78 mmol), N-methylmorpholine-N-oxide hydrate ( 116.88 g, 864.74 mmol), 4-
methylmorpholine-4-oxide (198.88 mL; 195.50 g, 1.67 mol), and di-tert-butyl
(lS,2S,5R,6S)-2-[(tert-butoxycarbonyl)amino]bicyclo[3.1.0]hex-3-ene-2,6-dicarboxylate
(600.00 g, 1.52 mol) in acetone (12.00 L) and water (1.80 L). The mixture is stirred at
23°C overnight. Thin layer chromatograph (hexane/ethyl acetate 3:2; stain: PMA)
analysis of the reaction mixture revealed complete consumption of di-tert-butyl
(lS,2S,5R,6S)-2-[(tert-butoxycarbonyl)amino]bicyclo[3.1.0]hex-3-ene-2,6-dicarboxylate.
The reaction is quenched with saturated aqueous Na2 S203 ( 1 L) and extracted with methyl
-butyl ether (15 L). The organic phase is washed with an aqueous solution (1.5 L)
prepared from 37% aqueous HC1 (300 mL) and brine (1.2 L), dried with anhydrous
MgS0 4, filtered, and concentrated in vacuo. The residue is slurried in heptane (1.5 L),
filtered, washed with heptane (2 x 500 mL), and resulting solid. The solid is dried under
vacuum to yield the title compound (450g, 69% yield).
Step 4: Di- -butyl (lS,2R,3S,4R,5R,6R)-2-[(tert-butoxycarbonyl)amino]-3-[(3,4-
dichlorobenzyl)oxy]-4-hydroxybicyclo[3 .1.0]hexane-2,6-dicarboxylate
To a solution of di- -butyl (lS,2R,3S,4R,5R,6R)-2-[(ter/-
butoxycarbonyl)amino]-3,4-dihydroxybicyclo[3. 1.0]hexane-2,6-dicarboxylate (450 g,
1.05 mol) in dichloromethane (4.50 L) at room temperature is sequentially added 3,4-
dichlorobenzyl bromide (144.73 mL, 238.80 g, 995.32 mmol), tetra-N-butylammonium
chloride (58.24 g, 209.54 mmol), and a 50% wt/wt aqueous solution of sodium hydroxide
(829.80 mL, 15.72 mol). After 7 hours, dilute the reaction with water ( 1 L) and separate
the phases. Extract the organic phase with brine (500 mL), dry over anhydrous MgS0 4,
filter and concentrate. Purify the residue by chromatography (2.5 kg of S1O2; eluent:
hexane/ethyl acetate 12:1 to 0 :1) to give the title compound (539 g, 72% yield).
Step 5: Di- -butyl (lS,2R,3S,4S,5R,6R)-2-[(/er/-butoxycarbonyl)amino]-3-[(3,4-
dichlorobenzyl)oxy]-4-{[(4-nitrophenyl)carbonyl]oxy}bicyclo[3.1.0]hexane-2,6-
dicarboxylate
To a solution of di-/er/-butyl (lS,2R,3S,4R,5R,6R)-2-[(/er/-butoxycarbonyl)
amino]-3-[(3,4-dichlorobenzyl)oxy]-4-hydroxybicyclo[3.1.0]hexane-2,6-dicarboxylate
(539.00 g, 751.00 mmol) in tetrahydrofuran (6.76 L) is added triphenylphosphine (393.96
g, 1.50 mol) and 4-nitrobenzoic acid (251.02 g, 1.50 mol). The mixture is cooled to 10°C
whereupon diisopropyl azodicarboxylate (40% v/v in toluene, 744.41 mL, 759.30 g, 1.50
mol) is added dropwise over 15 minutes and allowed to warm to 23°C. Afterl8 hours, the
mixture is diluted with methyl tert-butyl ether (2 L), washed sequentially with aq. sat.
aHC0 (2 x 2 L) and brine ( 1 L), and concentrated in vacuo. The residue is slurried in
heptane (5 L) at 40°C for 30 minutes then cooled to 23°C. The resulting solid is filtered
and washed sequentially with heptane (2 x 500 mL), heptane/ methyl tert-butyl ether (3 x
1 L). The combined filtrates are concentrated in vacuo and residue is purified by
chromatography (2.5 kg of Si02; eluent: hexane/ethyl acetate 95:5 to 75:25) followed by
recrystallization in heptane (10 L/kg) to yield the title compound as a white solid (273g,
49% yield).
Step 6: Di-tert-butyl (lS,2R,3S,4S,5R,6R)-2-[(½rt-butoxycarbonyl)amino]-3-[(3,4-
dichlorobenzyl)oxy]-4-hydroxybicyclo[3 .1.0]hexane-2,6-dicarboxylate
Potassium carbonate (153.45 g, 1.1 1 mol) is added to a suspension of di-tert-butyl
(lS,2R,3S,4S,5R,6R)-2-[(tert-butoxycarbonyl)amino]-3-[(3,4-dichlorobenzyl)oxy]-4-
{[(4-nitrophenyl)carbonyl]oxy}bicyclo[3.1.0]hexane-2,6-dicarboxylate (273.00 g, 370.11
mmol) in methanol (3.28 L) at ambient temperature. After 16 hours, the reaction is
concentrated in vacuo. The residue is dissolved in methyl -butyl ether (2 L), and
washed subsequently with water (0.8 L), brine (0.8 L), dried over MgS0 4, and
concentrated in vacuo to yield the title compound as a white solid (215g, 98% yield).
Step 7: (lS,2R,3S,4S,5R,6R)-2-Amino-3-[(3,4-dichlorobenzyl)oxy]-4-
hydroxybicyclo[3.1.0]hexane-2,6-dicarboxylic acid hydrochloride
A mixture of water (430 mL) and 37% HCI in water (299.9 mL, 3.65 mol) is
added to a suspension of di-tert-butyl (lS,2R,3S,4S,5R,6R)-2-[(/er/-butoxycarbonyl)
amino]-3-[(3,4-dichlorobenzyl)oxy]-4-hydroxybicyclo[3.1.0]hexane-2,6-dicarboxylate
(215 g, 365.3 mmol) in 1,4-dioxane (73.1 mL). The resulting slurry is stirred at 100°C for
4.5 hours, cooled to 25°C and concentrated in vacuo to provide a white solid. Triturate
sequentially with methyl -butyl ether (2 L) and hexanes (2 L) and then filter to collect
the solids. Dry under vacuum for 16 hours. The solid is washed again with methyl tertbutyl
ether (2 x 1 L) and hexane ( 1 L), then dried under vacuum at 50°C overnight to
yield the title compound as a white solid (140.4 g, 93% yield). MS (m/z): 376/378 (M+l).
Step 8: (lS,2R,3S,4S,5R,6R)-2-[(ter/-butoxycarbonyl)amino]-3-[(3,4-
dichlorobenzyl)oxy]-4-hydroxybicyclo[3 .1.0]hexane-2,6-dicarboxylic acid
Triethylamine (212.80 mL, 154.49 g, 1.53 mol) and 2-(tertbutoxycarbonyloxyimino)-
2-phenylacetonitrile (125.33 g, 508.90 mmol) are added
sequentially to a suspension of (lS,2R,3S,4S,5R,6R)-2-amino-3-[(3,4-
dichlorobenzyl)oxy]-4-hydroxybicyclo[3 .1.0]hexane-2,6-dicarboxylic acid hydrochloride
(140 g, 339 mmol) in 1,4-dioxane (203.56 mL), and water (0.6 mL/mmol-pure-LR,
203.56 mL), at ambient temperature. After 4.5 hours, add additional 2-(tertbutoxycarbonyloxyimino)-
2-phenylacetonitrile (25.07 g, 101.78 mmol) and triethylamine
(23.64 mL, 17.17 g, 169.63 mmol) to the reaction and stir at 50°C overnight. Cool to
23°C, dilute with water (500 mL), and wash with methyl tert-butyl ether (6 x 500 mL).
Dilute the aqueous phase with 10% aq. HC1 (300 mL; final pH = 2) and extract with ethyl
acetate (2 x 500 mL). The ethyl acetate extracts are dried over MgS0 4 and concentrated
in vacuo to yield the title compound (148. 3g, 92 % yield).
Step 9: Bis({[(l-methylethoxy)carbonyl]oxy}methyl) (lS,2R,3S,4S,5R,6R)-2-[(tertbutoxycarbonyl)
amino]-3-[(3,4-dichlorobenzyl)oxy]-4-hydroxybicyclo[3.1.0]hexane-2,6-
dicarboxylate
To a solution of bis({[(l-methylethoxy)carbonyl]oxy}methyl)
(lS,2R,3S,4S,5R,6R)-2-[(tert-butoxycarbonyl)amino]-3-[(3,4-dichlorobenzyl)oxy]-4-
hydroxybicyclo[3.1.0]hexane-2,6-dicarboxylate (148.30 g, 311.35 mmol) in
dimethylformamide (2.49 L) at 23°C is sequentially added potassium carbonate (172.12 g,
1.25 mol) , chloromethyl isopropyl carbonate (118.03 mL; 135.73 g, 871.79 mmol) and
sodium iodide (9.33 g, 62.27 mmol). Stir for 18 hours. Dilute with methyl tert-butyl
ether (2 L) and water (2 L). Separated the layers and wash the organic phase sequentially
with water (200 mL) and brine (200 mL). Dry over anhydrous MgS0 4, filter and
concentrate in vacuo. Purify the residue by chromatography (2.5 kg of SiC^; eluent:
hexane /ethyl acetate 3:1 to 1:1) to yield the title compound (167g, 76% yield): MS
(m/z): 608/610 (M-100 (-C0 2tBu)).
Step 10 : Bis({[(l-methylethoxy)carbonyl]oxy}methyl) (lS,2R,3S,4S,5R,6R)-2-[(tertbutoxycarbonyl)
amino]-3-[(3,4-dichlorobenzyl)oxy]-4-hydroxybicyclo[3.1.0]hexane-2,6-
dicarboxylate
Bis( {[(1 -methylethoxy)carbonyl]oxy}methyl) ( 1S,2R,3 S,4S,5R,6R)-2-[(tertbutoxycarbonyl)
amino]-3-[(3,4-dichlorobenzyl)oxy]-4-hydroxybicyclo[3.1.0]hexane-2,6-
dicarboxylate (165.50 g, 233.58 mmol) is treated with 4M HC1 in 1,4-dioxane (1.16 L,
4.63 mol) at 23°C and stir for 2 hours. Volatiles are removed under vacuum. The residue
obtained is slurried in methyl -butyl ether (1200 mL) overnight. The resulting solid is
filtered, washed with methyl -butyl ether (2 x 100 mL), and dried under vacuum at 1
mbar/45°C for 6 hours to yield the title compound as a white solid (134g, 89% yield). MS
(m/z): 608/610 (M+l)
Literature data (Witkin, Jeffrey M., and Eiler, William J .A. (2006), Antagonism of
Metabotropic Glutamate Group II Receptors in the Potential Treatment of Neurological
and Neuropsychiatric Disorders. Drug Development Research vol 67, pg. 757-769; and
Yasuhara, Akito and Chaki, Shigeyuki, (2010) Metabotropic Glutamate Receptors:
Potential Drug Targetsfor Psychiatric Disorders, The Open Medicinal Chemistry
Journal, vol. 4, pg. 20-36.) and data generated in non-clinical animal studies support a
role for mGlu2/3 antagonists in the treatment of depressive disorders and disorders of
excessive sleepiness. Specifically, it is found that mGlu 2/3 receptor antagonists are
effective in rodent models of depressive disorders and promote wakefulness using EEG
monitored rodents without disproportionate or clinically relevant hyperactivity or
overwhelming compensatory hypersomnolence. The increased alertness manifests in
increased attention, improved cognitive performance, and a likelihood of reduced fatigue.
As the previously described disorders represent common co-morbid clinical conditions,
an mGlu2/3 receptor antagonist may be particularly effective in specific patient
populations, such as patients with major depressive disorder, treatment refractory
depression, unipolar depression, dysthymia, and/or cyclothimia, or any disorders of
excessive sleepiness. Disorders of excessive sleepiness may include, but are not limited to
excessive daytime sleepiness (EDS), hypersomnia associated with obstructive sleep apnea
or narcolepsy, circadian rhythm sleep disorders (including, but not limited to shift work
sleep disorder, jet lag disorder, delayed sleep phase disorder, advanced phase sleep
disorder, and non-24 hour sleep-wake syndrome), idiopathic hypersomnolance and
excessive sleepiness associated with non-restorative sleep (NRS)
To further demonstrate the characteristics of the present compounds,
representative compounds may be run in the following in vitro and in vivo assays:
mGlu2 and mGlu3 Receptor cAMP Antagonist Assays
Antagonist activity is assayed in recombinant AV12 cells stably expressing human
mGlu2 or mGlu3 receptors and the rat glutamate transporter EAAT 1 (Excitatory Amino
Acid Transporter 1). The cell lines are maintained by culturing in DMEM with high
glucose and pyridoxine hydrochloride supplemented with 5 % dialyzed fetal bovine
serum (FBS), 1mM sodium pyruvate, 1mM HEPES and 1mM L-glutamine; geneticin
and hygromycin B are used as selection antibiotics. Confluent cultures are grown at 37°C
in an atmosphere containing 6.5% CO2, and passaged biweekly. Cells are harvested
using 0.25% trypsin, suspended in freeze media (FBS with 10% DMSO) at 10 cells/ml,
and aliquots are stored in liquid nitrogen. Twenty-four hours before the assay, cells are
plated at a density of 8,000-10,000 cells per well in a tissue culture treated, 96-well, halfarea
black plates (Costar 3875) in 50 ΐ of DMEM with high glucose and pyridoxine
hydrochloride supplemented with 5 % dialyzed FBS, 1mM sodium pyruvate, 1mM
HEPES, 100 g/ml ampicillin, and 250 (mGlu2) or 125 (mGlu3) of L-glutamine.
Reversal of the inhibition of forskolin-stimulated cAMP production by test
compounds is measured using homogeneous time resolved fluorescence technology
(HTRF; Cisbio cat # 62AM4PEB). The medium is removed and the cells are incubated
with 100 ΐ cAMP stimulation buffer (STIM) for 30 minutes at 37°C. (STIM buffer
contains 500 ml HBSS, 1000 ml DPBS, 0.034 % BSA, 1.67 mM HEPES and 500 
IBMX (Sigma 15879).) Compounds are tested in 10-point concentration response curves
using 3X serial dilution followed by further 40-fold dilution into STIM buffer. DCG IV
(Tocris 0975) serves as the reference agonist. The final reaction mixture contains 1 
(for mGlu2) or 3 (for mGlu3) of forskolin (Sigma F6886), DCG IV at its EC90, and
up to 25 of test compound. Cells are incubated at 37°C for 20 minutes. To measure
the cAMP levels, cAMP-d2 conjugate and anti cAMP-cryptate conjugate in lysis buffer
are incubated with the treated cells at room temperature for 1 hour (mGlu2) or 1.5 hour
(mGlu3). The HTRF signal is detected using an EnVision plate reader (Perkin-Elmer) to
calculate the ratio of fluorescence at 665 to 620 nM. The raw data are converted to
cAMP amount (pmole/well) using a cAMP standard curve generated for each experiment.
Relative IC50 values are calculated from the top-bottom range of the concentration
response curve using a four-parameter logistic curve fitting program (ActivityBase
V5.3.1.22).
FLIPR and cAMP Assays for mGlu Receptor Selectivity
The relative antagonist potencies of the compounds of the invention for the other
human mGlu receptors can be assessed with either a cAMP assay or fluorometric calcium
response assay (see for example Fell et al, JPET (in press)). Briefly, individual AV12
cell lines containing the rat EAAT1 glutamate transporter and stably expressing the
human mGlul, 2, 3, 4, 5, 6, & 8 receptors are used for these studies. The mGlul and 5
receptors are Gq-coupled, so they naturally signal through phospholipase C, producing a
calcium flux response which can be used to measure receptor activation using a
Fluorometric Imaging Plate Reader (FLIPR, Molecular Devices). The cell lines
expressing the mGlu2, 3, 4, and 8 receptor are designed to express the Gal 5 subunit so
that these Gi-coupled receptors will generate a calcium flux response similar to the
mGlul and 5 receptor expressing cell lines. The mGlu6 receptor is tested in a cAMP
format using methods analogous to those developed for mGlu2 and mGlu3 above. These
cell lines are maintained as previously described except that amounts of L-glutamine and
selection agents (geneticin, hygromycin B, zeocin, and blasticidin) may vary depending
on the cell line. Confluent cultures are passaged biweekly.
Intracellular calcium levels are monitored using FLIPR before and after the
addition of test compounds and Fluo-3 AM (Invitrogen) or Calcium 4 (Molecular
Devices) dye, depending on the cell line. Cells are plated 24 hours prior to assay in a
variable concentration of glutamine and a variable density of cells per well, depending on
the cell line. The medium is removed and the cells are incubated with 8 of dye (50 ΐ
per well) for 90 or 120 minutes (depending on cell line) at 25°C. A single-addition
FLIPR assay generating an 11-point concentration response curve for the agonist
glutamate is conducted prior to each experiment to confirm the appropriate sensitivity of
the cells. The results are analyzed using GraphPad Prism v4.03 to calculate the
concentrations of glutamate needed to induce the EC90 (antagonist assay) and EC10
(potentiator assay) responses.
Compounds are tested at each mGlu receptor in a two-addition FLIPR assay using
a 10-point concentration response profile starting at a final concentration of 25 for the
agonist assay and 12.5 for the potentiator and antagonist assays. The first addition
detects any agonist activity, and the second addition consists of 100 ΐ of select
concentrations (depending on cell line) of glutamate in assay buffer generating an EC10 or
EC90 glutamate response. Agonist effects are quantified as percent stimulation induced
by compound alone relative to the maximal glutamate response. Antagonist effects are
quantified by calculating the percent inhibition of the EC90 glutamate response caused by
the compound. Potentiation effects are quantified as percent increase in the presence of
an ECio response in glutamate relative to the ECm ax response. All data are calculated as
relative IC50 or EC50 values using a four-parameter logistic curve fitting program
(ActivityBase v5.3.1.22).
Antagonist activity in mGlu6 cells is measured using cAMP in a method
analogous to that described above for mGlu2 and mGlu3 activity, except that the
reference agonist was L-AP4 (Tocris). To measure mGlu6 agonist activity, the extent to
which the compound inhibits the forskolin-stimulated cAMP production is calculated.
Relative IC50 and EC50 values are calculated from the top-bottom range of the
concentration response curve using a four-parameter logistic curve fitting program
(ActivityBase v5.3.1.22).
Exemplified compounds wherein R1 and R2 are both hydrogen are tested
essentially as described above and are found to have high antagonist potency for the
mGlu2 and mGlu3 receptors. The exemplified compounds wherein R1 and R2 are both
hydrogen are also found to be selective antagonists of the mGlu2 and mGlu3 receptors as
against other mGlu receptor subtypes, having ICso's for the mGlu2 and mGlu3 receptors
less than 70 nM and 50 nM, respectively, while the ICso's for other mGlu receptors tested
are found to be significantly greater. See Table 1.
Table 1. Selectivity data
ND = not determined
Further, certain compounds of the present invention show a lack of significant
activity at other physiologically important receptors such as, but not limited to, the hERG
channel, serotonin receptors (specifically 5-H T 2A and 5-H T 2B), muscarinic receptors
(specifically M2), and iGluR receptors (specifically iGluR5). The compound of example
1 is tested using known assay methods and is found to have no appreciable activity at
these receptors.
Therefore, physiologically relevant doses of the compounds of the invention are
expected to provide substantial inhibition of mGlu2 and mGlu3 receptors in vivo, while
not substantially interacting with other mGlu receptors, or other physiologically relevant
receptors, and thus are expected to provide the desired pharmacology while avoiding
undesired effects associated with off-target activity.
(mFST)
mFST is an established in vivo assay for antidepressant activity (Li et al. J
Pharmacol Exp Ther. 319(l):254-9, 2006.). Mice treated with known clinically effective
antidepressants (selective serotonin reuptake inhibitors and/or tricyclic antidepressants)
exhibit the behavior of reduced time spent immobile after being placed in a water tank, a
behavior associated with despair. The mFST was used to evaluate potential
antidepressant-like activity of novel mGlu2/3 antagonists essentially as described in
previously published methods (see for example, Li et al. J Pharmacol Exp Ther.
19(1):254-9, 2006.). Briefly, male NIH-Swiss mice (Harlan Sprague-Dawley,
Indianapolis, ) weighing between 25-30 g are used. Group housed animals are
removed from the vivarium to the testing area in their own cages and allowed to adapt to
the new environment for at least 1hour before testing. Compounds where R1 and R2 are
both hydrogen are dissolved in water with minimal NaOH added for dissolution and are
administered i.p. Compounds where R1 and/or R2 are other than hydrogen are prepared
on the day of use in 2.0-2.5% N-methyl-pyrrolidinone and then suspended in 1% HEC,
0.25% Tween 80, and 0.05% Dow antifoam, and administered orally. Mice are placed in
a cylinder (diameter: 10 cm; height: 25 cm) filled with 6 cm of water (22-25°C) for 6 min.
The duration of immobility during the last 4 min. of the 6 min. period of the test was
scored. A mouse is recorded as immobile when floating motionless or making only those
movements necessary to keep its head above water.
Representative compounds are tested essentially as described above and are found
to significantly reduce immobilization times in wild type mice. See Table 2. Therefore
compounds of the present invention are expected to have antidepressant activity in vivo.
Table 2. mFST
Example ED60 (mg/kg) Maximal Decrease
( 1-compound/control)* 100%
1 1.0 (i.p.) 59%
2 2.08 (i.p.) 62%
3 8.2 (i.p.) 47%
4 9.5 (i.p.) 38%
5 7.45 (i.p.) 56%
6 7.3 (i.p.) 47%
7 >10 (i.p.) 18%
8 9.1 (i.p.) 41%
9 9.68 (i.p.) 41%
10/15 22.1 (p.o.) 50.5%
11 51.5 (p.o.) 32.9%
12 >28 (p.o.) 7%
13 11.3 (p.o.) 62.8%
In other experiments, mice with receptor deletions (mGlu2 knock-out mice) are
studied; these mice are bred by heterozygote x heterozygote breeding and used as
littermates for -/- and +/+ mouse comparisons (Taconic Farms). The compound of
example 1 (lOmg/kg, i.p., 30 min prior) is found to significantly decrease immobility time
in mGlu2+/+ mice, but not in mGlu2-/- mice. Similarly, the compound of example 10/15
(30 mg/kg, po, 120 min prior) is found to decrease immobility time in mGlu2+/+ mice,
but not in mGlu2-/- mice. These findings further demonstrate that the mGlu2 receptor
contributes to the antidepressant-like effects of the compounds of the invention.
The compounds of the invention may also be tested in combination with other
compounds useful for the treatment of depressive disorders, as for example SSRI's, for
their ability to enhance the antidepressant-like effects over that of either compound alone.
The compound of example 10 (17 mg/kg p.o.) is tested in the mouse forced swim test
alone and in combination with fluoxetine (10 mg/kg, i.p.) and found to significantly
increase the antidepressant-like effect over that of either compound alone as shown in
Table 3, below. Further, testing of brain and plasma levels of the active di-acid moiety of
the compound of example 10 (i.e. the same compound as the freebase of example 1),
show no increase in exposure levels, supporting the finding that the increased
antidepressant-like activity was not due merely to an increase in central exposure to the
compound.
Table 3. mFST with SSRI
Wakefulness and behavioral monitoring in rats: Representative compounds of the present
invention are tested in rats for their ability to increase the amount of time in a state of
wakefulness without undesired effects such as inhibition of REM sleep, waking motor
impairment (disproportionate hyper- or hypolocomotion), and/or rebound hypersomnia.
Test animals are continuously monitored by electro-encephalograms (EEG),
electromyograms (EMG), and motion to measure cumulative time awake, rebound
hypersomnia, and locomotor activity intensity during wakefulness. Methods for such
studies are known in the art (see for example methods described in Edgar DM, Seidel
WF. Modafinil induces wakefulness without intensifying motor activity or subsequent
rebound hypersomnolence in the rat. J Pharmacology &Experimental Therapeutics
1997; 283: 757-769; van Gelder RN, Edgar DM, Dement WC. Real-time automated
sleep scoring: validation of a microcomputer-based system for mice. Sleep 1991, 14: 48-
55; and Gross BA, Walsh CM, Turakhia AA, Booth V, Mashour GA, Poe GR. Opensource
logic-based automated sleep scoring software using electrophysiological
recordings in rats. J Neurosci Methods. 2009; 184(1): 10-8.) Studies are conducted as
follows:
Animal preparation. Adult, male Wistar rats (approximately 270-300 g at time of
surgery) are surgically fitted for chronic recording of EEG, EMG, body temperature, and
motion as follows: Rats are surgically prepared with a cranial implant consisting of four
stainless steel screws for EEG recording (two frontal [3.9 mm anterior from bregma, and
±2.0 mm mediolaterally] and two occipital [6.4 mm posterior from bregma, ±5.5 mm
mediolaterally]), and with two Teflon-coated stainless steel wires for EMG recording
(positioned under the nuchal trapezoid muscles). All leads are soldered to a miniature
connector (Microtech, Boothwyn, PA) prior to surgery. The implant assembly is affixed
to the skull by the combination of the stainless steel EEG recording screws, cyanoacrylate
applied between the implant connector and skull, and dental acrylic. Body temperature
and locomotor activity is monitored via a miniature transmitter (Minimitter PDT4000G,
Philips Respironics, Bend, OR) surgically placed into the abdomen. At least 3 weeks are
allowed for recovery.
Recording environment. Each rat is housed individually within a microisolator cage
modified with an inserted polycarbonate filter-top riser to allow more vertical headroom.
A flexible cable that minimally restricts movement is connected at one end to a
commutator afixed to the cage top and at the other end to the animal's cranial implant.
Each cage is located within separate, ventilated compartments of a stainless steel sleepwake
recording chamber. Food and water are available ad libitum and the ambient
temperature is maintained at about 23±1°C. A 24-hr light-dark cycle (LD 12:12) using
fluorescent light is maintained throughout the study. Relative humidity averages
approximately 50%. Animals are undisturbed for at least 30 hrs before and after each
treatment.
Study design and dosing. Compounds where R1 and R2 are both hydrogen are dissolved
in water with minimal NaOH added for dissolution and are administered i.p in a volume
of 1.0 mL per kg body weight. Compounds where R1 and/or R2 are other than hydrogen
are administer p.o. in a volume of 2 mL per kg body weight in one of two alternative
vehicles: i) 2.5% N-methyl-2-pyrrolidinone in hydroxyethylcellulose; or ii) 10% acacia
with 0.05% Dow Corning® Antifoam in water. The vehicle or one of the compound dose
levels is administered pseudo-randomly such that no rat receives the same treatment
twice, and no rat receives more than two of the 8 treatments in any one study. Each rat is
removed from its cage for about a minute to be weighed and treated. At least 6 days
"washout" period precede and follow each treatment.
Data collection. Sleep and wakefulness discrimination may be automated (e.g., Van
Gelder et al. 1991; Edgar et al. 1997, Winrow et al, 2010; Gross et al, 2009). EEG is
amplified and filtered ( I0,000, bandpass 1-30 Hz), EMG is amplified and integrated
(bandpass 10-100 Hz, RMS integration), and non-specific locomotor activity (LMA) is
monitored simultaneously. Arousal states are classified in 10 second epochs as non-REM
sleep, REM sleep, wakefulness, or theta-dominated wakefulness. Locomotor activity
(LMA) is recorded as counts per minute and is detected by commercially available
telemetry receivers (ER4000, Minimitter, Bend, OR).
Statistical Analysis. Ages and body weights are summarized by mean, minimum and
maximum over the treatment groups. All animals having at least one outcome are
included in the summary results (for example, we include appropriate data from an animal
treatment for which telemetry data are usable but EEG data are not). The post-treatment
observation period is divided into 2 post-dosing intervals (the first 7 hours, and the first
19 hours) where the time of dosing is defined as the start of Hour = 0. The outcomes are
summarized in each period by computing either the mean hourly or the cumulative value
across each period. Each outcome in each period is analyzed by analysis of covariance
using treatment group and treatment date as factors and the corresponding pre-treatment
interval, 24 hrs earlier, as the covariate. Adjusted means and the change from vehicle
means and their corresponding standard errors are summarized for each treatment group.
Adjusted Dunnett's multiple-comparison P-values are shown for each outcome in each
period. Not all outcomes are analyzed in all periods, as shown in Table 1, which thus
affect the experiment-wise type I error rate. As such, no further adjustments are made for
multiple testing.
Determining efficacy. The threshold efficacious dose is estimated as the lowest dose for
which cumulative time awake exceeds 50 minutes relative to vehicle controls across the
first 7 hours post-treatment. A finer determination may be made by conducting
subsequent studies of more closely spaced doses around the efficacious dose.
Determining undesired effects. Two potentially undesired effects in particular are
evaluated: rebound hypersomnolence and intensified motor activity (Edgar DM, Seidel
WF, 1997).
(i) Rebound hypersomnolence may be measured as decreased levels of wakefulness
during the period 8-19 hours after efficacious treatment doses. A biologically significant
decrease is defined as a greater than 50 percent of the cumulative increase during the first
7 hours. Thus, if wakefulness increased by 100 minutes during the first 7 hours, then a
decrease in cumulative wakefulness of 50 minutes or more, relative to vehicle controls,
during the period 8-19 hours after treatment would be deemed biologically significant.
Group mean changes, shown in Table 2, show a lack of rebound hypersomnolence.
(u) Intensified motor activity is defined as an average increase relative to vehicle controls
that exceeds 5 LMA counts per minute of EEG-defined wakefulness at the efficacy
threshold dose, and for which the effect is dose related. Group mean increases in Table 2
were all under 5 counts per minute of wakefulness and are not dose dependent.
Exemplified compounds are tested essentially as described and are found to
promote wakefulness without significant rebound hypersomnia or intensified motor
activity. Exemplified compounds where R1 and R2 are both hydrogen (administered i.p.)
are tested essentially as described and are found to be efficacious at doses of 10 mg/kg or
lower. The compound of Example 10 is tested essentially as described and is found to
have the cumulative time awake profile and locomotor activity intensity as shown in
Table 4.
Table 4.
Cumulative Time Awake first 7 hours
Dose (mg/kg PO) N Mean SE P
60 9 64.7 13.9 <0.0001
30 8 48.8 14.4 0.0019
10 10 20.4 13.4 0.1387
Cumulative Time Awake 8-19 hours
Dose (mg/kg PO) N Mean SE P
60 9 34.7 12.4 0.0087
30 8 25.6 13.6 0.0696
10 10 15.2 12.3 0.2257
Locomotor Activity Intensity (note 1)
standard error of the mean; P = P-value adjusted for multiple contrasts for the
efficacy variable. Unadjusted P values are shown for 'undesired effect'
measures (Cumulative Time Awake 8-19 hours, and Locomotor Activity
Intensity). Cumulative time awake given in minutes. Note 1. Locomotor
activity (LMA) intensity = counts of LMA per minute of EEG-defined
wakefulness, averaged over the first 7 hr post-treatment.
While it is possible to administer compounds employed in the methods of this
invention directly without any formulation, the compounds are usually administered in
the form of pharmaceutical compositions comprising at least one compound of Formula I,
or a pharmaceutically acceptable salt thereof, as an active ingredient and at least one
pharmaceutically acceptable carrier, diluent and/or excipient. These compositions can be
administered by a variety of routes including oral, subcutaneous, intravenous, and
intramuscular. Such pharmaceutical compositions and processes for preparing them are
well known in the art. See, e.g., Remington: The Science and Practice of Pharmacy
(University of the Sciences in Philadelphia, ed., 2 1st ed., Lippincott Williams & Wilkins
Co., 2005). Compounds of Formula I where R1 and/or R2 are other than hydrogen are
preferred for oral administration to improve bioavailability, whereas Compounds of
Formula I where R1 and R2 are both hydrogen are preferred for i.v. or i.p. administration.
The compositions are preferably formulated in a unit dosage form, each dosage
containing from about 1 to about 600 mg, more usually about 30 to about 300 mg, as for
example between about 50 and about 250 mg of the active ingredient. The term "unit
dosage form" refers to physically discrete units suitable as unitary dosages for human
subjects and other mammals, each unit containing a predetermined quantity of active
material calculated to produce the desired therapeutic effect, in association with at least
one suitable pharmaceutically acceptable carrier, diluent and/or excipient.
The compounds of Formula I are generally effective over a wide dosage range.
For example, dosages per day normally fall within the range of about 0.01 to about 10
mg/kg, more usually from about 0.3 to 5.0 mg/kg, and as for example between 0.5 and 3.0
mg/kg of body weight. In some instances dosage levels below the lower limit of the
aforesaid range may be more than adequate, while in other cases still larger doses may be
employed without causing any harmful side effect, and therefore the above dosage range
is not intended to limit the scope of the invention in any way. It will be understood that
the amount of the compound actually administered will be determined by a physician, in
the light of the relevant circumstances, including the condition to be treated, the chosen
route of administration, the actual compound or compounds administered, the age,
weight, and response of the individual patient, and the severity of the patient's symptoms.
We CLAIM:
1. A compound of the formula
where R1 and R2 are each independently hydrogen, C1-C3 alkoxycarbonyloxymethyl, Ci-
C3 alkylcarbonyloxymethyl, or C -6 cycloalkylcarbonyloxymethyl;
R is independently at each occurance methyl, fluoro, or chloro;
R4 is hydroxyl, methylcarbonylamino, hydroxymethylcarbonylamino,
methoxycarbonylamino, imidazol-2-ylsulfanyl, thiazol-2-ylsulfanyl, 1,2,4-
triazolylsulfanyl, 1-methyl- l,2,4-triazol-3-ylsulfanyl, or 1-methyl- l,2,4-triazol-5-
ylsulfanyl; and
n is 1 of 2;
or a pharmaceutically acceptable salt thereof.
2. The compound according to Claim 1 where n is 2, both R groups are chloro, and the
chloro groups are at the phenyl 3- and 4-positions, or a pharmaceutically acceptable salt
thereof.
3. The compound according to either Claim 1 or 2 where R1 and R2 are each hydrogen, or
a pharmaceutically acceptable salt thereof.
4. The compound according to either Claim 1 or 2 where R1 and R2 are both other than
hydrogen, or a pharmaceutically acceptable salt thereof.
5. The compound according to either Claim 1 or 2 where R1 and R2 are the same and are
other than hydrogen, or a pharmaceutically acceptable salt thereof.
6. The compound according to Claim 5 where R1 and R2 are each
isopropyloxycarbonyloxymethyl.
7. The compound according to claim 1which is (lS,2R,3S,4S,5R,6R)-2-amino-3-[(3,4-
dichlorobenzyl)oxy]-4-hydroxybicyclo[3 .1.0]hexane-2,6-dicarboxylic acid or a
pharmaceutically acceptable salt thereof.
8. The compound according to claim 1which is bis(isopropoxycarbonyloxymethyl)
(lR,2S,3S,4R,5S,6R)-4-amino-3-[(3,4-dichlorophenyl)methoxy]-2-hydroxybicyclo[
3 .1.0]hexane-4,6-dicarboxylate or a pharmaceutically acceptable salt thereof.
9. A pharmaceutical composition comprising a compound according to any one of
Claims 1to 8, or a pharmaceutically acceptable salt thereof, and a pharmaceutically
acceptable carrier, excipient or diluent.
10. A compound according to any one of Claims 1to 8, or a pharmaceutically acceptable
salt thereof, for use in therapy.
11. A compound according to any one of Claims 1to 8, or a pharmaceutically acceptable
salt thereof, for use in the treatment of depressive disorders.
12. A compound according to any one of Claims 1to 8, or a pharmaceutically acceptable
salt thereof, for use in the treatment of disorders of excessive sleepiness.
13. The compound for use according to either Claim 11 or 12 in a human.
14. A compound according to any one of Claims 1to 8, or a pharmaceutically acceptable
salt thereof, for use in simultaneous, separate or sequential combination with a serotonin
reuptake inhibitor in the treatment of depressive disorders in a mammal.
15. The compound for use according to Claim 14 where the serotonin reuptake inhibitor
is fluoxetine.
16. The compound for use according to Claim 14 or 15 in a human.
17. A method for treating depressive disorders in a mammal comprising administering to
a mammal in need of such treatment an effective amount of a compound according to
Claim 1, or a pharmaceutically acceptable salt thereof.
18. The method of Claim 17 where the mammal is a human.
19. A method for treating disorders of excessive sleepiness in a mammal comprising
administering to a mammal in need of such treatment an effective amount of a compound
according to Claim 1, or a pharmaceutically acceptable salt thereof.
20. The method of Claim 19 where the mammal is a human.
21. A method for treating depressive disorders in a mammal comprising administering to
a mammal in need of such treatment an effective amount of a compound according to
Claim 1, or a pharmaceutically acceptable salt thereof, in simultaneous, separate or
sequential combination with a serotonin reuptake inhibitor.
22. The method of Claim 2 1 wherein the serotonin reuptake inhibitor is fluoxetine.
23. The method of Claim 21wherein the mammal is a human.
24. The method of Claim 22 wherein the mammal is a human.
25. A pharmaceutical composition comprising a compound according to any one of
claims 1to 8, or a pharmaceutically acceptable salt thereof, in combination with at least
one pharmaceutically acceptable carrier, excipient or diluent, and optionally other
therapeutic ingredients
26. A pharmaceutical composition comprising a compound according to any one of
Claims 1to 8, or a pharmaceutically acceptable salt thereof, and a serotonin reuptake
inhibitor.
27. The pharmaceutical composition of Claim 26 wherein the serotonin reuptake
inhibitor is fluoxetine.