Note: Descriptions are shown in the official language in which they were submitted.
~ W096/04900 2 1 9 5 7 7 9 PCT~S95/10317
C~",~ c~ n and method for ~,~ut~ iùl~ a~ainst dru~ da,~e"de"~.
The present invention relates to treatments for
substance dependence.
Substance ~rprn~rnre is a major problem, both for
individuals suffering from it, and for society at large. At
the individual level, the condition is characterized by a
need for repeated, and often increasing doses of a substance.
At the societal level, the condition is associated, with some
substances, with increased levels of crime, inr1n~inr theft
and crimes of violence, as sufferers seek to obtain supplies
of the substance.
Individuals dependent on a snh~t~nrP find that they have
to continue taking the substance, even though it produces
harmful effects in them. They may become tolerant to the
substance, which means that they need to take greatly
increased amounts, for example ten times the amount they
originally took, in order to achieve the same effect.
Withdrawal of the substance brings about a variety of
undesirable behavioral and physiological changes, inrl~ing
craving for the substance, anxiety and irritability.
In the ~ n central nervous system (CNS), the
transmission of nerve impulses is controlled by the
interaction between a neurotransmitter, that is released
by a sending neuron, and a surface receptor on a
receiving neuron, causing excitation of this receiving
neuron.
L-Glutamate, which is the most ~hnn~nt
neurotranSmitter in the CNS, mediates the major
excitatory pathway in mammals, and is referred to as an
excitatory amino acid (EAA). The receptors that respond
to glntr~-to are called excitatory amino acid receptors
~ (EAA receptors). See Watkins & ~vans, Ann. Rev.
Pharmacol. ~oxicol., 21, 165 (1981); Mrn~rh~n, Bridges,
and Cotman, Ann. ~ev. Pharmacol. Toxicol., 29, 365
(1989); Watkins, Krogsgaard-Larsen, and Honore, Trans.
Pharm. Sci., 11, 25 (1990). The excitatory amino acids
PC~IUS 9 5 ~ 1~ 31 7
~ X-9329D 2 ~ ~ 5 7 7 9 IPEA]US 1 ~ AUG 1996
are of great physi~logi~Al importance, playing a role in
a variety of physiological processes, such as long-term
potentiation (l~Arn;ng and memory), the development of
synaptic plasticity, motor control, respiration,
cardiovascular regulation, emotional states and sensory
perception.
Excitatory amino acid receptors are classified into
two general types. Receptors that are directly coupled
to the opening of cation rhAnn~l R in the cell membrane
of the neurons are termed "ionotropic." This type of
receptor has been subdivided into at least three
subtypes, which are defined by the depolarizing actions
of the selective agonists N-methyl-D-aspartate (NMDA),
~-amino-3-hydroxy-5-methylicnYA~ol~-4-propionic acid
(AMPA), and kainic acid (KA). The second general type
of receptor is the G-protein or second messenger-linked
"metabotropic" excitatory amino acid receptor. This
second type is coupled to multiple second messenger
systems that lead to ~nhAn~e~ pho9rhn; nnc; ti~e
hydrolysis, activation of phosphnlirAce D, increases or
decreases in cAMP formation, and changes in channel
function. Schoepp and Conn, Trends in Pharmacol. Sci.,
14, 13 (1993). Both types of receptors appear not only
to mediate normal synaptic transmission along excitatory
pathways, but also participate in the ~;fi~Ation of
synaptic connections during development and throughout
life. Schoepp, Bockaert, and S1A~CZ~k, Trends in
Phar~acol. Sci., 11, 508 (1990); McDonald and Johnson,
Brain Research Reviews, 15, 41 (1990).
The metabotropic glutamate receptors are a highly
heteL.g~ ~ q family of glutamate receptors that are linked
to multiple second-messenger pathways. Generally, these
L~c~L~L~ function to modulate the presynaptic release of
glutamate, and the postsynaptic sensitivity of the neuronal
cell to glutamate excitation. The metabotropic glutamate
receptors (mGluR) have been phArr--olngically divided into
two subtypes. One group of receptors is positively coupled
_ _ _ _ _ _ , .. .. . . . .. . _ . _
~ W096/04900 ~l 9~779 PCT~S95110317
to phospholipase C, which causes hydrolysis of cellular
phosphoinositides (PI). This first group are termed PI-
linked metabotropic glutamate receptors. The second group of
receptors is negatively coupled to adenyl cyclase, which
prevents the forskolin-stimulated a,- 11 At~ i nn of cyclic
adenosine monophosphate (cAMP). Schoepp and Conn, Trends
Pharmacol. Sci., 14, 13 (1993). Receptors within this second
group are termed cA~P-linked metabotropic glutamate
receptors.
There are many different substances on which individuals
may become ~pPn~nt. These include opiates,
h~n7n~iA7epines, nicotine, cocaine and ethanol.
Nicotine dependence. which is induced through smoking,
affects hundreds of millions of people around the world. For
many, it leads to illness and premature death. Stopping
smoking (smoking cessation) may evoke a range of symptoms in
~pPn~nt individuals, inrlu~ing craving, depression,
anxiety, difficulty in concentrating and weight gain.
A variety of treatments are available for smoking
~c~Atin~, ;nrlll~ing counseling, hypnosis, aversion
~nn~ i t; ~n i ng, relaxation training, acupuncture, and nicotine
replacement therapy. However, in spite of the avAilAhility
of these tr~A ~, and the widespread knowledge of the
harmful side effect of smoking, many smokers fail to give up
smoking. There is therefore a need for new treatments for
smoking cessation.
senzO~iA7~pinP dependence, such as diazepam dependence,
arises through the use of the benzodiazepines as
pharmaceuticals to treat other disorders. The dependence-
in~ ing properties of the bPn7o~iA7epines limits their
therapeutic use. Withdrawal produces symptoms such as
anxiety, irritability, inq~ iA and impaired uu"c~"~L~tion.
There is therefore a need for new treatments for the
treatment of benzodiazepine withdrawal.
Animal models for the treatment of nicotine and diazepam
withdrawal have been described in Helton et ~1_;
Psychopharmacology (1993), 113:205-210 and RA -A~n ~ ~1_;
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ . _ .
WO 96/04900 2 1 9 5 7 7 9 PCI/US95/10317--
N~UL~'L~OLL 5, 154-156 (lg93). ~hese models can be used to
measure the ability of a test compound to inhibit the
increased startle response in an animal ~rat) following
withdrawal of nicotine or diazepam.
It has now been found that a , _ fl which is an
agonist that acts selectively at negativelY coupled cAME-
linked rihntropic glutamate receptors is capable of
reducing startle response in rats following the cessation of
chronic nicotire or fliA7ep-~m exposure. It is believed that
this finding portends that any agonist that acts at
negatively coupled cAMP-linked metabotropic glutamate
receptors will be useful for treating substance withdrawal,
and indeed may be useful for providing protection against
substance dependence.
According-to one aspect, therefore, the present
invention provides the use of an agonist which acts at
negatively coupled cANP-linked metabotropic glutamate
receptors for protecting a warm blooded ma~mal from
dependence on a-substance.
It has ~een found that a novel compound which is an
agonist that acts selectively at negatively coupled cAMP-
linked metabotropic glutamate receptors, ~+)-2-
aminobicyclo[3.1.0]hexane-2,6-dicarboxylic acid is effective
in the rat startle models for nicotine and diazepam
withdrawal. Accordingly it is believed that any compound
which acts as an agonist at negatively coupled cAMP-linked
metabotropic glutamate raceptors, especially any agonist
which acts selectively, will be useful for the treatment of
the withdrawal or CPqsati nn of these and other dependence-
producing substances. Furthermore, since withdrawal isinrir~tPly related to flPpPnflPn~e and to tolerance, it is
believed that an agonist which acts at negatively coupled
cAME~-linked metabotropic glutamate receptors will also be
useful for the treatment of substance dependence and
tolerance, and indeed generally to protect a warm blooded
mammal from flPpPnflPn~e on a flPpPnflPnnP-producing substance.
~ W096/04900 2 1 9 5 7 7 q PCT~S9~10317
The ~PpPn~Pnre-producing substance may be, for example,
an opiate, benzodiazepine, nicotine, cocaine or ethanol.
According to another aspect, the present invention
provides the use of an agonist which acts at negatively
coupled cAMP-linked metabotropic glutamate receptors for the
treatment of drug tolerance, withdrawal or cessation.
According to yet another aspect, the present invention
provides the use of an agonist which acts at negatively
coupled cA~P-linked metabotropic glutamate receptors for the
treatment of smoking cPq~ti nn .
The particular dose of agonist administered will of
course be ~PtPrminpd by the particular circumstances
surrounding the case, ;nrln~inr the activity of the
particular agonist administered, the route of
administration, the particular condition being treated,
and similar considerations. The agonist can be
administered by a variety of routes inr1ll~ing oral,
rectal, trAnc~Prr-l, subcutaneous, intravenous,
intramuscular, or intranasal routes. Alternatively, the
agonist may be administered by continuous infusion. A
typical daily dose will contain from about O.OOl mg/kg
to about lO0 mg/kg of the agonist. Preferably, daily
doses will be about 0.05 mg/kg to about 50 mg/kg, more
preferably from about O.l mg/kg to about 20 mg/kg.
According to preferrea aspect, the present
invention provides the use of an agonist as defined
hereinabove for use in a method of protecting a warm-
blooded mammal from ~PpPn~Pnre on a dependence-producing
pharmaceutical, for example, a bPn7o~iA7~r;np such as
~;~7Pp~m. In this method, the agonist may be
administered before said phArr--Putical is first
administered, after said phArr--Plltir~l has been
administered or after said phArr-rPnt;rAl has been
withdrawn, or it may be co-administered with said
pharmaceutical.
Agonists which act at negatively coupled cANP-
linked metabotropic glutamate receptors may be
PCTIUS 9 5 / 1~ 3
~-g329D ~ 9~7~q IPEAIUS lb AUG 1996
identified using the following ~Yp~ri L. Firstly, the
affinity of a test compound for metabotropic glutamate
receptors may be demonstrated by the selective
disrlA~ L of (15,3R)-1-aminocyclopentane-1,3-
~io~rh~ylic acid-sensitive [3H]glutamate binding to rat
brain cell membranes. The binding of [3H]glutamate
([3H]Glu) is conducted with crude membranes of rat
forebrain as described by Schoepp and True. Schoepp and
True, Neuroscience Lett., 145, 100-104 (1992); Wright,
McDonald, and Schoepp, J. Neurochem., 63, 938-945
(1994). The affinity of a test ~ _od for the
receptor may be expressed as the concentration of the
test compound that inhibits 50i binding (ICso), or the
percent dis~l~, L of [3H]Glu at a 10 yM or 100 yM
concentration of the formula I c ~. In this test,
the ICso for (+)-2-aminobicyclo[3.1.0]hexane-2,6-
~;o~rh~xylic acid was found to be 0.18 yM.
The ability of a test . _nd to act as an agonist
at negatively coupled cAMP-linked metabotropic receptors
may be measured using the following method. Test
c, ullds are tested for their ability to decrease
forskolin-stimulated cAMP formation in the rat
h;rporP ,us and the rat cerebral cortex, using the
~-~cedul~s described in Schoepp and Johnson. Schoepp
and Johnson, Neurochem. Int., 22, 277-283 (1993). In
this test, (+)-2-~min~h;~yclo[3~l.o]hexane-2~6-
~ie~rh~ylic acid was found to give the result shown in
Table II below.
Table II. Inhibition of Forskolin-6timulated cAMP
F. i~
ECso (yM)
Rat cerebral cortex .055 + .017
Rat hi,p~- ~ .036 1 .015
-9329D ~ ~ 1996
21 ~5779
The ability of negatively coupled cAMP-linked
metabotropic receptor agonists to protect a warm blooded
ma_mal from the effects of drug withdrawal or cessation
may be demonstrated using an auditory startle model. In
5 this model, animals are dosed with a drug (nicotine or
~;?70p~m), then dosing is discontinued. This cessation
of drug dosing elicits an increased startle response to
auditory stimuli. Test compounds are then administered
to animals to determine whether they are capable of
10 attenuating the increased startle response.
Long Evans rats t200-400 g; Harlan Sprague Dawley,
Columbus, IN) were individually housed in a controlled
environment on a 12 hour light-dark cycle and given free
access to food (Purina Rodent Chow) and water. Rats
15 were anesthot;7ed with isoflurane and Alzet osmotic
pumps (Alza Corporation) were implanted su~cutaneously.
Test c- 1 was dissolved in a vehicle of
purified water and neutralized with 5N NaOH to a pH of
7-8 when appl;~hlo. Diazepam (Sigma Chom;~l Company,
20 St. ~ouis, MO) was su~pon~od in a vehicle consisting of
40% PEG 300, 10% EtOH, 2% benzyl alcohol, 1% Tween 80,
and 47% purified water. Nicotine (Research Pio~hom;~
Inc., Natick, MA) was dissolved in saline. Control
animals received the respective vehicle.
25 Nicotine withdrawal: Pumps were filled to deliver
nicotine (6mg/kg/day s.c.), diazepam (lOmg/kg/day s.c.),
test compound (0,1,3,1Omg/kg s.c.) or vehicle. Twelve
days following subcutaneous implantation of pumps, rats
were anesthetized with isoflurane and the pumps were
30 removed. During withdrawal (following pump removal),
the auditory startle response (peak amplitude, Vmax) of
individual rats was recorded using San Diego Instruments
startle rhl ~ (San Diego, CA). Startle sessions
consisted of a 5-minute adaptation period at a
35 background noise level of 70+2dBA immediately followed
by 25 presentations of auditory stimuli (120+2d~A noise,
50ms duration) presented at 8-second intervals. Peak
W096l04900 21 9577~ PCT~S9S/10317 -
startle amplitudes were then averaged for~all 25
presPnt~t; nnc of stimuli for each session and all data
are preseAted here as overall session means. Auditory
startle rPcpnn~; ng was evaluated daily on withdrawal
days 1,2,3,4 or 5. saseline startle rpcpnn~ing was
evaluated prior to pump removal on day 12.
Auditory startle rPcpnn~ing was significantly
increased through the first three days following
cPcqstinn of chronic nicotine exposure when compared to
control rats receiving water. Rats given a rep~
dose of nicotine at doses of 0.03 mg/kg, i.p. or higher
did not display the same heightened startle response
seen for aAimals with no nicotine rPpl~n~ ~nt.
Pretreatment with ~+)-2-aminobicyclo[3.1.0]hexane-2,6-
dicarboxylic acid produced a dose-dependent hlonk~P Of
the withdrawal-induced increase in startle responding as
well. A significant ~ttPnn~tinn of the heightened
startle was apparent at 3mg/kg, p.o. dose of the
compound when compared to nicotine coAErols (ED50=0-7
mg/kg i.p.).
D~7f~m Withdrawal: Auditory startle r~Qpnn~ing was
qi~ni~in~ntly increased through the first four days
following cessation of chronic dia_epam exposure when
compared to control rats receiving vehicle Replacement
doses of 3 and lO mg/kg, i.p. ~;~7ap~m did not block the
increased start~le rPqpnn~;ng and in some ;nqt~nnPR
further iAcreased reactivity indicating toleraAce. Rats
which received 30 mg/kg, i.p. ~;~7Pp~m rep~ daily
60 minutes before ev~ln~tinn of startle roqpnn~;ng, did
not show increased reactivity following ~;~7Pp~m
C~qq~ti nn on days 1 through ~ when I ed to the
~i~7Pp~m control. Pretreatment with (+)-2-
innhinyc1O[3~l~o] hexane-2,6-dicarboxylic acid blocked
the expected increase in startle rpcpnn~i ng which
followed cessation of diazepam exposure. 3Oses of 0.1
aAd 0.3 mg/kg, p.o. of the c _ ~ significaAtly
~ W096/04900 2 1 9 5 7 7 9 PCT~S95/10317
~ttonn~ted enhanced startle when compared to control
rPcprm~;n!J ~ED50=0.1 mg/kg, p.o. ) .
(+)-2-Aminobicyclo[3.1.0]hexane-2,6-dicarboxylic
acid may be prepared by reacting carbethoxYmethyl
dimethy1c-~1f~nillm bromide with 2-cyclopenten-1-one in
the presence of a base, such as 1,8-
diazabicyclo[5.4.0]undec-7-ene to afford ethyl 2-
oxobicyclo[3.l~o]hexane-6-carboxylate. This ester may
then be reacted with an aqueous solution of potassium
cyanide or sodium cyanide and . illm carbonate to
produce an int~L -~;Ate hydantoin, (the sucherer-sergs
reaction), which is then hydrolysed using sodium
hydroxide, to afford a diastereomeric mixture of diethyl
2-aminobicyclo[3.1.0]hexane-2,6-~;c~rh~ylates. The
desired diastereomer may be obtained by crystallization
wi'th oxalic acid. This diastereomer may then be
resolved by forming a crystalline salt with (+)-di-p-
toluoyl-D-tartaric acid and recovering (-)-diethyl Z-
aminobicyclo[3.1.0]hexane-2,6-dicarboxylate. Xydrolysis
of this diester using aqueous sodium hydroxide gives
(+)-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylic acid.
Alternatively, the ethyl 2-oxobicyclo[3.1.0]hexane-6-
carboxylic acid may be hydrolysed using sodium hydroxide
to give 2-oxobicyclo[3.1.0]hexane-6-carboxylic acid.
This acid may then be resolved by formin~ a crystalline
salt with (S)-1-phenylethylamine and recovering (+)-2-
oxobicyclo[3.1.0]hexane-6-carboxylic acid. This acid
may then be converted into (+)-2-aminobicyclo[3.1.0]
hexane-2,6-dicarboxylic acid by reaction with an aqueous
solution of potassium cyanide or sodium cyanide and
ammonium carbonate to produce an intermediate hydantoin
(the sucherer-sergs reaction) followed by hydrolysis of
the hydantoin using sodium hydroxide. This procedure
may also be modified by performing the resolution step
on the hydantoin rather than on the 2-
oxobicyclo[3.1.0]hexane-6-carboxylic acid. In this
W096/04900 2 1 9 5 7 7 ~ PCT~S9~10317 -
case, (~ phenylethylamine has been found to be a
suitable resolving agent.
The agonists are preferably formulated prior to
administration in , ~ in~t;nn with one or more
pharm--Pllt;r~lly-acceptable carriers, ~ lPntq, or
~ripi~ntq. The pharmaceutical formulations are
prepared by known procedures using well-known and
readily availa~le ingredients. In making the
compositions, the active ingredient will usually be
mixed with a carrier, or diluted by a carrier, or
enclosed within a carrier, and may be in the form of a
capsule, sachet, paper, or other nnnt~inPr, When the
carrier serves as a diluent, it may be a solid, semi-
solid, or liriuid material which acts as a 'vehicle,
~nipi~nt, or medium for t-h-e active ingredient. The
compositions can be in the form of tablets, pills,
powders, lozenges, sachets, cachets, elixirs,
suspensions, emulsions, solutions, syrups, aerosols,
ni ' c cnnt~;ning, for example, up to 10% by weight
of active compound, so~t and hard gelatin capsules,
suppositories, sterile iniectable Snl--tinnq, dermal
patch, snhcllt~n~ous implant, and sterile packaged
powders.
Some examples o~ snitRhl~ carriers, excipients, and
diluents include lactose, dextrose, sucrose, sorbitol,
mannitol, starches, gum, acacia, calcium phnsph~te~
alginates, trAgar~nth, gelatin, calcium silicate,
microcrystalline cel1lllose, polyvinylpyrrolidone,
cellulose, water syrup, methyl cellulose, methyl and
propyl hydroxyhPn7oat~q, talc, r~gn~qinm stearate,
stearic acid, and mineral oil. The f~ lat;nnq can
additionally include lubricating agents, wetting agents
(surfactants), emulsifying and suspending agents,
preserving agents, sweetening agents, or flavoring
agents. Compositions may be f~ l~t~ 50 as to provide
r~uick, sustained, or delayed release of the active
~ W096/~900 2 ~ ~5779 P~ 17
ingredient after administration to the patient by
employing procedures well known in the art.
The compositions are preferably formulated in a
unit dosage form, each dosage crntAininr from about 1 mg
to about 500 mg, more preferably about 5 mg to about 200
mg of the active ingredient. The term ~unit dosage
form'~ refers to a physically discrete unit suitable as
unitary dosages for human subjects and other mammals,
each unit rnntA;ninr a predetrrmin~d quantity of active
material calculated to produce the desired therapeutic
effect, in association with a suitable ph~rrace1ltir~
carrier, diluent, or excipient. The following
formulation examples are illustrative only and are not --
int~n~ to limit the scope of the invention in any way.
2~ 95779
W096/04900 PCT~S95/10317 -
Formulation
~ard gelatin capsules are prepared using the ~ollowing
ingredients:
Quantity
(mg/capsule)
2-Aminobicyclo[3.1.0]hexane-2,6-
dicarboxylic Acid 250
Starch, dried 200
~agnesium stearate 10
Total 460 mg
20The above ingredients are mixed and filled into
hard gelatin capsules in 460 mg quantities.
Formulation 2
A tablet is prepared using the ingredients below:
ouantity
30~mg/tablet)
2-~mi nnh; cyclo[3.1.0]hexane-2,6-
dicarboxylic Acid 250
35 rPl l n l nse, microcrystalline400
Silicon dioxide, fumed 10
Stearic acid . 5
Total 665 mg
The c ~ Pntc are blended and compressed to form
tablets each weighing 665 mg.
~ W096104900 2 1 9 ~ 7 7 9 PCT~S95/10317
Formulation 3
An aerosol solution is prepared ~ntAining the following
, ~ntc:
. .
Weight
2-Aminobicyclo[3.1.0]hexane-2,6-
~iCArh~xylic Acid 0.25
Ethanol 29 . 75
Propellant 22 70 . 00
~chlorodifluoromethane)
Total 100.00
The active compound is mixed with ethanol and the
mixture added to a portion of the PropPl 1 Anr 22, cooled
to -30~C and transferred to a filling device. The
re~uired amount is then fed to a stainless steel
r~ntAinPr and diluted with the 1 ;n~Pr of the
propellant. The valve units are then fitted to the
rrlr~ t A; n Pr,
Formulation 4
Tahlets each containing 60 mg of active ingredient are
made as follows:
2 -Aminobicyclo[3.1.0]hexane-2,6-
~;~Arh~xylic Acid 60 mg
Starch 45 mg
Microcrystalline ~Plll,loqe 35 mg
Polyvinylpyrrolidone 4 mg
Sodium carboxymethyl starch 4.5 mg
- MAgnPq;nm stearate 0.5 mg
Talc ~ m~
Total 150 mg
2~ 9577~
W096/04900 PCT~Sg~l03l7
The active ingredient, starch, and cellulose are
passed through a No. 45 mesh U.S. sieve and mixed
thoroughly. The solution of polyvinylpyrrolidone is
mixed with the resultant powders which are then passed
through a No. 14 mesh U.S. sieve. The granules so
produced are dried at 50~C and passed through a No. 18
mesh U.s~ sieve. The sodium carboxymethyl starch,
magnesium stearate, and talc, previously passed through
a No. 60 mesh U.S. sieve, are then added to the granules
which, after mixing, are compressed on a tablet machine
to yield tablets each weighing 150 mg.
FormulatioA 5
Capsules each ~nr~ining 80 mg of active ingredient are
made as follows:
2-~min~hi~yclo[3,1.0]hexane-2,6-
dicarboxylic Acid 80 mg
Starch 59 mg
Microcrystalline cellulose 59 mg
M~gn~qinm stearate 2 ma
Total 200 mg
The active_ingredient, cellulose, starch, and
magnesium stearate are blended, passed through a No. 45
sieve, and filled into hard gelatin capsules in 200 mg
quantities.
Formulation 6
Suppositories each c~nr~;n;ng 225 mg of active
ingredient may be made as follows:
2-Pmin~h;cyclo[3.1.0]hexane-2,6-
dicarboxylic Acid 225 mg
Saturated fatty acid glycerides2.000 ma
Total 2,225 mg
~ W096/04900 2 ~ q ~ 7 7 ~ r~
The active ingredient is passed through a No. 60
mesh U.S. sieve and suspended in the saturated fatty
acid glycerides previously melted using the minimum heat
n~cecq~ry. The mixture is then poured into a
Suppository mold of nominal 2 g capacity and allowed to
cool.
Formulation 7
Suspensions each ~nnt~;ning 50 mg of active ingredient
per 5 ml dose are made as follows:
2-Aminobicyclo[3.1.0]hexane-2,6-
dicarboxylic Acid 50 mg
Sodium carboxymethyl cellulose 50 mg
Syrup 1.25 ml
senzoic acid solution 0.10 ml
Flavor q.v.
Color q.v.
Purified water to total 5 ml
The ~~l;~ nt iS passed through a No. 45 mesh U.S.
sieve and mixed with the sodium carboxymethyl cellulose
and syrup to form a smooth paste. The benzoic acid
solution, flavor and color are diluted with some of the
water and added, with stirring. Sufficient water is
then added to produce the required volume.
Formulation 8
An inLldv~llols f~ l~r;on may be prepared as follows:
2-P~~;nnbi-~yclo[3.1.0]hexane-2,6-
dicarboxylic Acid 100 mg
Mannitol 100 mg
5 N Sodium hydroxide 200 ml
Purified water to total 5 ml
The following ~ _lPq further illustrate methods
for their synthesis of (+)-2-aminobicyclo[3.1.0]hexane-
. _ .. .. .... . .. . _ _ _ _ _ _ . . .
~1 95779
Wo96/04soo ~I/U~,3~ 7 -
2,6-dicarboxylic acid. The Examples are not intended to
be limiting to the scope of the invention in any
respect, and should not be so construed All
experiments were run under a positive pressure of dry
nitrogen or argon. All solvents and reagents were
purchased from commercial sources and used as received,
unless otherwise indicated. Dry tetrahydrofuran (THF)
was rhti~in Pd by disti~ ti~n from sodium or sodium
benzophenone ketyl prior to use. Proton nuclear
magnetic resonance tl~ N~R) spectra were nhtilirrd on a
GE QE-300 spectrometer at 300.15 M~z, a Bruker AM-500
i~e~LL~ ?trr at 500 M~z, or a Bruker AC-200P
spe~LL, -trr at 200 M~z. Free atom bombardment mass
spectroscopy (FAsMS) was performed on a VG ZAB-2SE
instrume t. Field desorption mass spectroscopy tFDMS)
was performed using either a VG 70SE or a Varian MAT 731
in~LLI . Optical rotations were measured with a
Perkin-Elmer 2G1 polarimeter. Chromatographic
separation on a Waters Prep 500 LC was generally carried
out using a linear gradient o~ the solvents in~ t~ in
the text. The reactions were generally monitored for
completion using thin layer chromatography (~LC~. Thin
layer chromatography was performed using E. Merck
Kieselgel 60 F254 plates, 5 cm x ~LO cm, 0.25 mm
thirkn~c8 Spots were detected using a , ,ini~tir,n of
W and chemical detection (plates dipped in a ceric
i ;nm molybdate solution [75 g of i inm molybdate
and 4 g of cerium (IV~ sulfate in 500 mL of 10% aqueous
sulfuric acid] and then heated on a hot plate). Flash
chromatography was performed as described by Still, et
al. Still, Kahn, and Mitra, J. Org. C~em., 43, 2923
(1978). ~ l analyses for carbon, hydrogen, and
nitrogen were ~=rtrrm;ned on a Control F~li, '
Corporation 440 r,l. ti~l Analyzer, or were performed by
the Universidad Complutense Analytical Centre (Facultad
de Farmacia, Madrid, Spain). Melting points were
~etQrmin~d in open glass capillaries on a Gallenkamp hot
.. , ,, .. . , . , .. ... ... .. .. . _ . . . .. . . _ . _ _
21 9577~
096/04900 PCT~S95/10317
air bath melting point apparatus or a shchi melting
point apparatus, and are uncorrected.
Preparation
5~ArhPt~n~ymethyl Dimethylsulfonium sromide
A solution of ethyl bromoacetate (265g) and
dimethyl sulfide (114g) in acetone ~500m3) was stirred
at room temperature. ~fter three days, the title
~ ~ ~ was isolated by filtration of the reaction
mixture. Melting point 88-90~C.
3xample
(lSR,5Rs,6SR) Ethyl 2-Oxobicyclo[3.1.0]h OE ane-6-
15carboxylate
A suspension of carbethoxymethyl diethylsulfonium
bromide (~5.5g) in toluene (350mL) was treated with 1,8-
~;A7~hi~yclo[5.4.0]undec-7-ene (30.2g). The resulting
mixture was stirred at room temperature. After one
hour, the reaction mixture was treated with 2-
cyclopenten-l-one (19.57g). After an additional 18
hours, the reaction mixture was added to a 1 N
hydrochloric acid/sodium chloride solution. The
resulting mixture was OE tracted with diethyl ether. The
~ ;nP~ ether extracts were dried over r-gn~q;~lm
sulfate, filtered, and ~nn~Pntrated in vacuo. The
residue was purified using silica-gel ~l1L~ tn~raPhY,
eluting with a linear gradient of 10% ethyl
acetate/hexanes to 50% ethyl acetate/h OE anes, to give
22.81g of the title compound. Melting point: 36-38 C.
FDMS: m/z = 168 (M+).
Analysis calculated for CgH12O3: C, 64.27; H, 7.19.
Found: C, 64.54; H, 7.11.
7 7 ~
W096!04900 ~CTNS95/l0317
18
Exampl e 2
(lSR,2RS,SRs,65R) Diethyl 2-P~;nnhiryclo[3.1.0]-
hexane-2,6-dicarboxylate and
(lSR,25R,5RS,6SR) Diethyl 2-~minnh;ryclo[3.1.0]-
hexane-2,6-~;rArhn~ylate
A solution of the compound prepared as described in
Example 1 (22. 81g) in ethanol (200mL) was treated with
an aqueous solution of potassium cyanide (9.71g) and
i1lm carbonate (21.2g) in water (200mL). The
resulting mixture was heated to about 50 C. After about
18 hours, the reaction mixture was allowed to cool to
room temperature and treated with sodium hydroxide
(16.2g). The resulting mixture was heated to reflux.
After about 18 hours, the reaction mixture was allowed
to cool to room temperature, then cooled to O C. The pH
of the cold mixture was adjusted to pH 1 by the A~;t;nn
of concentrated hydrochloric acid. This mixture was
concentrated to dryness in vacuo. The residue was
dissolved in ethanol, cooled to O C, and treated with
thionyl chloride~(80.6g). ~he resulting mixture was
heated to reflux. After about 48 hours, the reaction
was concentrated to dryness i~ vacuo. ~he residue was
25 treated with 1 N sodium hydroxide, and the resulting
mixture extracted with diethyl ether. The - ' in~d
ether extracts were dried over potassium rArhnnAre,
filtered, and concentrated in vac~o to give 24.6g of a
mixture of the title compounds.
~xample 3
(lSR, 2SR, 5RS, 6SR) Diethyl 2-Aminobicyclo[3~l~o]
hexane-2,6-~;cArbn~ylate
A solution of the _ ~c prepared as described
in Example 2 (20.71g) in ethyl acetate (200mL) was
treated with a solution of oxalic acid ~15.46g) in
~ WO9~/04900 2 1 q57 7~ PCT~S95/10317
19
ethanol (5omr~). The resulting mixture was stirred at
room temperature. After one hour, the reaction mixture
was treated with additional ethanol (50m~). After 18
hours, the mixture was filtered, and the filtrate was
evaporated to dryness in vacuo. The residue was treated
with 1 N sodium hydroxide, and the resulting mixture
extracted with diethyl ether. The , ';nP~ ether
extracts were washed with brine, dried over potassium
carbonate, filtered, and concentrated in vacuo. The
residue was purified by silica-gel chromatography,
eluting with methylene chloride:5% i inm
hydroxide/--~hi~nnl (97:3), to give 15.41g of the title
compound.
PDMS: m/z = 242 (M+H).
Analysis calculated for C12Hl9N~4: C, 59.74; H~ 7.94;
N, 5.81. Found: C, 59.78; H, 8.13; N, 5.77.
Exam~le 4
20(-)-Diethyl 2-~m; nnh; ~yclo[3.1.0]hexane-2,6-
dicarboxylate
~ solution of the racemic mixture of compounds
prepared as described in Example 3 (6.56g) in ethyl
acetate (lOOmL~ was treated with a solution of (+~-di-p-
toluoyl-D-tartaric acid (12.0g) in ethyl acetate
(loomL). After standing overnight at room temperature,
the crystalline solid was removed by filtration and
dried to give 14.7g. Additional crystalline solid was
obtained by cooling the filtrate to O C. The combined
crystalline solids were dissolved in hot ethyl acetate,
cnntiq;n;n~ enough 2- propanol for complete dissolution.
~After cooling to O C, the crystalline solid was isolated
by filtration, to give 2.3g of a solid having an
~ni~nt; iC excess of ~ 95%. The freebase form was
oht~;no~ by partitioning the salt between a~ueous sodium
h;~arh~ni~te and ethyl acetate. The organic phase was
2~ q~77~
w096/04900 PCT~59~10317
separated, dried over potassium carbonate, filtered, and
c~nrpntrRtp~ in vaCuo to give .77g of the title
compound.
EDMS: m/z = 242 (M+H).
Optical rotation: aD = -5.15- (c = 1, EtOH).
Analysis calculated for C12H19N~4: C~ 59-74; H~ 7-g4;
N, 5.81. Found: C, 59.68; H, 8.13; N, 5.58.
~xample 5
(+)-2-~minnhicyclo[3.1.0]hexane-2,6-dicarboxylic Acid
A solution of the compound prepared as described in
Example 4 (0.69g~ in tetrahydrofuran (lOm~) was treated
with 1 N sodium hydroxide (lOm~1, and the resulting
mixture vigorously stirred at room te-m~perature~ After
several days, the title compound was i crl ~tP~ by anion-
exchange chromatography (sio-Rad AG1-X8), eluting with
50% acetic acid/water, to give 0.53g of the title
1 _ln~
FDMS: m/z = 186 tM+H).
Optical rotation: aD = 21.32- (c = 1, 1 N HC1).
Analysis calculated for CgH11N04-1.25H20: C, 46.26; E~,
6.55; N, 6.74. Found: C, 46.68; ~, 6.47; N, 6.49.
Example 6
2-Oxobicyclor3.1.0]hexane-6-carboxylic acid
A mixture of 60~g of (lSR,5RS,6SR) ethyl 2-oxobicyclo[3.1.0]
hexane-6-carboxylate and 300 ml of 1 N sodium hydroxide was
stirred at 25-30~C. After 2.5 hours, concentrated
hydrochloric acid was added to adjust the pH to 0.8-1.2. The
resulting solution was extracted with ethyl acetate. The
extracts were dried over r~gnPq;llm sulfate, filtered, and
concentrated to give 49.1 g (98%) of the crude material.
~ W096/04900 2 1 9 5 7 7 9 pCT~S9~10317
21
RecrysrAl1;~atinn from 100 ml of ethyl acetate ~ave the title
c ~, mp 123.5-128~C.
S: m/z = 140 (M+)
Analysis calculated for C7HgO3 C, 60.00; H, 5.75. Found:
C, 60.14; H, 5.79.
Example 7
2-Oxobicyclo[3.1.0]hexane-6-carboxylic acid salt with (S)-1-
phenylethylamine
A solution of 14 g of the compound prepared in Example 6 in
140 ml of 25% ethanol in ethyl acetate was ~ -in~ with (S)-
1-phenylethylamine (1 eq.l. After stirring overnight, the
precipitated salt was isolated by filtration and dried to
give 11.87 g (45.4%) of the desired salt. Conversion of the
salt to the partially resolved 2-oxobicyclo[3.1.0]hexane-6-
carboxylic acid by the method of Example 8 and analysis
indicated that the salt was 68% ee. The ~n~nti~ iC excess
was determined by conversion to the methyl ester with
diazomethane followed by chiral ~PLC on a Chiralpak AS column
at 40~C eluted with 10% isopropanol/90% hexane at 1 ml/min
with detection at 210 nm.
Exam~le 8
(+)-2-Oxobicyclo[3.1.0]hexane-6-carboxylic acid
A mixture of 1.31 g of the product of Example 7 and 10 ml of
lN hydrochloric acid was stirred for 5 minutes and extracted
with ethyl acetate. The extracts were dried over sodium
sulfate, filtered, and concentrated to give 0.61 g of the
title compound, mp 110-115~C. The product was detPrmi n~d to
be 68% ee by chiral HPLC ~method of Example 7).
FDMS: m/z = 141 (M+H)
Optical Rotation: ~ D = 49.85~
21 ~5779
W096/04900 ~ ,J/l0
Fxam~le 9
~-)-2-Spiro-5'-hy~Antn;nh;cyclo[3.1.0]hexane-6-carboxylic
acid
A solution of the ~ dL ~d as described in Example 8
~68~ ee, 1 eq.), potasgium cyanide ~1.25 eg.), and , ;nm
ri~rhnnAte ~2.5 eq) were combined and stirred in ethanol/water
at 25~C for 40 hours. The mixture was Ac;~;f;~ with 6N
hydrochloric acid, ~nnr~ntrated, diluted with water, and
filtered to give a 79% yield of a 90:10 mixture of
diastereomers, mp 286-290~C. ~he diastereomeric mixture was
recrystAl1;7P~ ~rom isopropanol/water to give in 48% yield
the title ~- ~ in 100% diastereomeric and 100%
enantiomeric purity (~nAnt;~ ic ratio ~t~rm;ne~ by chiral
HPLC on a 4.6 x 150 mm Chiralcel OD-H column, eluted with 15
~ isopropanol/85% hexane at 1 ml/min at 40~C with detection
at 220 nm; diastereomeric ration determined by HPLC on a
Zorbax ss-phenyl column at 40~C with elution with 90:10
buffer~acetonitrile eluted at 2 ml/min with ~t~t;nn at 220
nm ~buffer = 0_1 ~ dibasic sodium phns~hAt~ monohydrate
adjusted to pH 2 1 with phosphoric acid).
FDMS: m/z = 211 ~M + H)
Optical Rotation: a D = - 25.98~
Analysis calculated for C9HlON204: C, 51.43: H, 4.79; N,
13.33. Found: C, 51.38; H, 4.80; N, 13.26.
~xample 10
Ethyl 2-spiro-5l-hydantoinbicyclo[3~l~o]hexane-6-carboxylate
A mixture of 5.~5 g of ethyl 2-oxobicyclo[3.1.0]hexane-6-
carboxylate, 2.15 g of potassium cyanide, 5.77 g of i ;~lm
~ArhnnAte~ 30 ml of 2s-3 ethanol, and 12 ml of water was
stirred at 35~C until the reaction was complete hy HPLC.
After 15 hours, the reaction mixture was cooled to 0~C and 33
~ W096/04900 219~7~9 ~"~ r~l7
23
ml of-water was added to the mixture. After 2 hours at 0~C,
the precipitate was isolated by filtration and dried to give
5.23 g (73~) of the title compound, mp 217-220~C.
S FDMS: m/z = 238.1 (~+)
~ Analytical r~1r~ tPd for C11H14N2O4: C, 55.46; H, 5.92;
N, 11.76. Found: C, 55.74; H, 5.88; N, 11.50.
Exam~le 11
2-Spiro-5'-hy~nto;rhicyclo[3.1.0]hexane-6-carboxylic acid
A mixture of 16.32 g of the product of Example 10 and 137 ml
of 2N NaOH was stirred at 25~C. After 1 hour, concentrated
hydrochloric acid was added to adjust the pH to 1Ø The
resultiny precipitate was isolated by filtration and dried to
give 13.70 g (95%) of the title compound, mp 277-279~C.
FD~S: m/z = 210.1 ~+)
Analysis r~lcl~latPd for C9~10N2~4: C, 51.43; H, 4.79; N,
13.33. Found: C, 51.70; ~, 4.93; N, 13.43.
Exam~le 12
2-Spiro-5~-hy~nto;nh;cyclo[3.1.0]hexane-6-carboxylic acid,
(S)-l-phenyle t hylamine salt
A mixture of 1.05 g of the product of Example 11 and 16.6 ml
of a 1.6 : 1 solution of acetone : water was stirred at 25~C
while adding 1.53 g of R-(+)-1-phenylethylamine. The mixture
was stirred for 2 hours at room temperature. The crystals
were filtered, rinsed with acetone, and dried to give 0.74 g
(45~) of the title compound, mp 205-212~C.
Optical Rotation: ~ D = -31.88~ (c = 1, methanol)
21 ~779
W096/04900 pcTNss~llo3l7 -
24
~xa~ple 13
~ 2-Spiro-5 ' -hy~An t n i nh; cyclo[3.1.0]hexane-6-carboxylic
acid
A mixture of 0.74 g of the product of Example 12 and 10 ml of
water was stirred at 25~C while the pH was adjusted from 6.81
to 1.0 using lN HCl. The reaction mixture was stirred for 1
hour and the product was rgll ~rt~d by filtration and dried to
give 0.35 g ~75~ of the til-le , __ ~, mp 310~C (decomp).
FDMS: 210.1 (M+)
Optical Rotation: a D = -24.22~ (c = 1, methanol)
Analysis calculated for CgHloN2O4: C, 51.43: H, 4.80;
N,13.33. Found: C, 51.67; H, 4.87; N, 13.61.
Rxa~le 74
(+)-2-Aminobicyclo[3.1.0]hexane-2,6-dicarboxylic Acid
A solution of 184 g of (-)-2-spiro-5~-hydantoinbicyclo[3.1.0]
hexane-6-carboxylic acid and 1750 ml of 3N NaOH was heated at
reflux until the reaction was complete by HPLC. After 28
hours, the solution was cgoled to room temperature and
filtered through glass paper to remove trace amounts of
insoluble material. The p~ of the sslnt;nn was adjusted to
3.0 using concentrated ~Cl. ~he reaction mixture was stirred
1 hour at room temperature and two hours at 0~C. The
precipitated product was collected by filtration, washed with
170 ml of cold water ar,d dried to give 152.5 grams (86~) of
the title compound.
FDMS: m/z = 186.1 (~+1)
optical rotation: a D = 23.18~ (c = 1, lN ~Cl)
