Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
CA 02373559 2002-02-27
DOCKET NO: 70207/56595
EXPRESS MAIL NO:
SEROTONIN TRANSPORT INHIBITORS
FIELD OF THE INVENTION
The present invention relates to compounds that inhibit 5-hydroxytryptamine
reuptake and the use of those compounds on diseases mediated by 5HT receptors.
Compounds that provide such inhibition can be useful, for example, as
therapeutic
anti-depressants.
BACKGROUND OF THE INVENTION
Serotonin (5-hydroxytryptamine) neurotransmission is regulated and
terminated by active transport via the serotonin transporter (SERT). The SERT
is
member of a large superfamily of sodium/chloride dependent transporters that
carry
biogenic amines and other biologically active substrates to the interior of
cells (Amara
SG, Kuhar MJ. 1993. 16:73-93; Blakely RD, et al., 1994. J Exp Biol 196:263-
281).
Structurally related to dopamine and norepinephrine transporters (Nelson N.
1998. J
Neurochem 71:1785-1803), the SERT is the primary site of action of diverse
antidepressant drugs, ranging from tricyclics such as imipramine and
amitriptyline, to
serotonin selective reuptake inhibitors (SSRI's) such as citalopram,
fluoxetine and
sertraline.
Antidepressant drugs delay the removal of extracellular serotonin from the
synapse by blocking serotonin transport, thereby prolonging the duration of
serotonin
receptor activity. The increased availability of serotonin triggers a cascade
of
neuroadaptive processes, which produces symptom relief after two to four
weeks.
Presently known antidepressants also produce certain side effects and may
selectively
alleviate specific symptoms of depression (Nestler EJ. 1998. Biol Psychiatry
44:526-
533). Thus, it is desirable to develop novel antidepressants. The majority of
clinically
approved drugs to treat depression or obsessive-compulsive disorder are high
affinity
inhibitors of serotonin and/or norepinephrine transport. Of these transporter
inhibitors,
1
CA 02373559 2002-02-27
none are tropane analogs, they display low affinity for the dopamine
transporter, and
all contain an amine nitro(yen in their structure.
Over the past decade, a wide array of tropane analogs with high affinity for
the
monoamine transporters have been synthesized in a program to develop cocaine
medications (Madras BK, et al., 1990. Pharmacol Biochem Behav 35:949-953;
Madras BK, et al., 1996. Synapse 24:340-348; Carroll Fl, et al., 1992. J Med
Chem
35:2497-2500; Meltzer PC, et al., 1994. J Med Chem 37:2001-2010; Kozikowski
AP,
et al., 1995. J Med Chem 38:3086-3093; Lomenzo SA, et al., 1997. J Med Chem
40:4406-4414; Davies HM, et al., 1994. J Med Chem 37:1262-1268). The majority
of
these compounds target the dopamine transporter and have not been considered
candidate medications for depression because of stimulant or abuse liability
concerns
(Reith ME, et al., 1986. Biochem Pharmaco135:1123-1129; Ritz MC, et al., 1987.
Science 237:1219-1223; Madras BK, et al., 1989. J Pharmacol Exp Ther 251:131-
141;
Bergman J, et al., 1989. J Pharmacol Exp Ther 251:150-155). Tropane analogs
selective for the serotonin over the dopamine transporter have been reported.
(Blough
BE, et al., 1996. J Med Chem 39:4027-4035; Blough BE, et al., 1997. J Med Chem
40:3861-3864; Smith MP, et al., 1998. J Am Chem Soc 1201:9072-9075; Davies,
HM,
et al., 1996. J Med Chem 39:2554-2558.
Psychotherapeutics drugs, including antidepressants, all incorporate an amine
nitrogen into the structure. In fact, antidepressants presently used have an
aromatic
ring(s) and an amine nitrogen. Although the aromatic ring is an indispensable
component of most drugs acting on biogenic amine receptors or transporters, we
previously demonstrated that an amine nitrogen is not necessary for compounds
to
bind to or block the dopamine transporter (Madras BK, et al., 1996. Synapse
24:340-
348; Meltzer PC, et al., 1997. J Med Chem 40:2661-2673; Meltzer PC, et al.,
1999.
Bioorg Med Chem Lett 9:857-862; Meltzer PC, 2000. J Med Chem 43:2982-2991).
Biological activity of these compounds was retained if the amine nitrogen was
replaced either by an oxygen (oxa) atom or a carbon (carba) atom (Madras BK,
et al.,
1996. Synapse 24:340-348; Madras BK, et al., 1998. Soc for Neurosci Abst
24:113.11,
278p; Madras BK, et al., Addiction Biology 5:351-359, 2000; Meltzer PC, 2000.
J
Med Chem 43:2982-2991).
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CA 02373559 2002-02-27
It would be desirable to have high affinity non-amines with selectivity for
the
serotonin transporter and compounds that inhibit the transport of serotonin.
SUMMARY OF THE INVENTION
The present invention relates to the discovery that tropane compounds lacking
an amine group show surprisingly effective results in treating certain
neuropsychiatric
disorders related to serotonin transport.
Compounds that are useful as therapeutic agents in the methods of the present
invention include non-amine tropane compounds represented by the following
general
structural formula:
R2
(O),,, Ar
11 R2
Ar
R1
!It R2~/
Ar
wherein:
R, = COOCH3, COR3, lower alkyl, lower alkenyl, lower alkynyl, CONHRa, or
COR6;
R2 = is a 6a, 6(3, 7a or 7(3 substituent, which can be selected from H, OH,
OR3,
F, Cl, Br, and NHR3;
X = CH2, CHY, CYYl, CO, 0, S; SO, SO2, or C=CXjY with the C, 0 or S
atom being a member of the ring;
X, = NR3, CH2, CHY, CYY1 CO, 0, S; SO, S02, or NS02R3;
3
~- -------
CA 02373559 2002-02-27
R3= H, (CH2)nC6H4Y, C6H4Y, CHCH2, lower alkyl, lower alkenyI or lower
alkynyl;
Y and Y] = H, Br, Cl, 1, F, OH, OCH3, CF3, NO2, NHZ, CN, NHCOCH3,
N(CH3)2, (CH2)õCH3; COCH3, or C(CH3)3;
R4 = CH3, CH2CH3, or CH3SO2;
R6 = morpholinyl or piperidinyl;
Ar = phenyl-R5, naphthyl-R5, anthracenyl-Rs, phenanthrenyl-R5, or
diphenylmethoxy-R5;
R5 = Br, Cl, I, F, OH, OCH3, CF3, NO2, NH2, CN, NHCOCH3, N(CH3)2,
(CH2)õCH3, COCH3, C(CH3)3 where n= 0-6, 4-F, 4-Cl, 4-I, 2-F, 2-Cl, 2-I, 3-F, 3-
Cl,
3-I, 3,4-diCl, 3,4-diOH, 3,4-diOAc, 3,4-diOCH3, 3-OH-4-Cl, 3-OH-4-F, 3-C1-4-
OH,
3-F-4-OH, lower alkyl, lower alkoxy, lower alkenyl, lower alkynyl, CO(lower
alkyl),
or CO(lower alkoxy);
m=0or1;and
n=0, 1,2,3,4or5.
Preferred compounds have a SERT/DAT selectivity ratio of at least about 3.
Other embodiments have a SERT/DAT selectivity ratio of at least about 8 and
other
preferably at least about 50.
The invention also relates to compounds shown above that have a potency (K;),
or IC50, at the SERT of less than about 500 nM, preferably less than about 100
nM. In
certain preferred embodiments the compounds have a Ki at the SERT less than
about
50 nM, preferably less than about 25 nM and more preferably less than about 15
nM.
Especially preferred compounds have a SERT/DAT selectivity ratio of at least
about 3 and an ICso at the SERT of less than about 500 nM.
The substituents at the 2 and 3 position of the ring can be a- or (3-. Thus,
the
compounds include compounds in the boat and chair conformation. Although R1 is
illustrated in the 2- position, it should be recognized that substitution at
the 4-position
is also included and the position is dependent on the numbering of the tropane
ring.
The compounds of the present invention can be racemic, pure R-enantiomers, or
pure
S-enantiomers. Thus, the structural formulas illustrated herein are intended
to
represent each enantiomer and diastereomer of the illustrated compound.
4
CA 02373559 2002-02-27
The term "lower alkyl" when used herein designates aliphatic saturated
branched or straight chain hydrocarbon monovalent substituents containing from
1 to
about 8 carbon atoms such as methyl, ethyl, isopropyl, n-propyl, n-butyl,
(CH2)õCH3,
C(CH3)3; etc., more preferably I to 4 carbons. The term "lower alkoxy"
designates
lower alkoxy substituents containing from 1 to about 8 carbon atoms such as
methoxy,
ethoxy, isopropoxy, etc., more preferably I to 4 carbon atoms.
The term "lower alkenyl" when used herein designates aliphatic unsaturated
branched or straight chain vinyl hydrocarbon substituents containing from 2 to
about 8
carbon atoms such as allyl, etc., more preferably 2 to 4 carbons. The term
"lower
alkynyl" designates lower alkynyl substituents containing from 2 to about 8
carbon
atoms, more preferably 2 to 4 carbon atoms such as, for example, propyne,
butyne,
etc.
The terms substituted lower alkyl, substituted lower alkoxy, substituted lower
alkenyl and substituted lower alkynyl, when used herein, include corresponding
alkyl,
alkoxy, alkenyl or alkynyl groups substituted with halide, liydroxy,
carboxylic acid, or
carboxamide groups, etc. such as, for example, -CH2OH, -CH2CH2COOH, -
CH2CONH2, -OCH2CH2OH, -OCH2COOH, -OCH2CH2CONH2, etc. As used herein,
the terms lower alkyl, lower alkoxy, lower alkenyl and lower alkynyl are meant
to
include where practical substituted such groups as described above.
When X contains a carbon atom as the ring member, reference to X is
sometimes made herein as a carbon group. Thus, when X is a carbon group, as
that
phrase is used herein, it means that a carbon atom is a ring member at the X
position
(i.e., the 8- position).
The present invention also relates to therapeutic uses of non-amine tropane
analogs. More specifically, the invention relates to methods of treating
patients
having SERT related disorders, comprising administering to the patient a
serotonin
reuptake inhibiting amount of a non-amine compound. Such diseases include, but
are
not limited to, e.g., depression, anxiety, eating disorders and obsessive
compulsive
disorders and other. The methods specificaily include therapies for treating
depression.
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CA 02373559 2002-02-27
More specifically, the invention relates to the use of non-amine topane
compounds, as described further below, for the treatment of these diseases.
Particularly preferred compounds comprise the compounds shown in Figure 1 and
described herein.
The present invention provides pharmaceutical therapeutic compositions
comprising the compounds formulated in a pharmaceutically acceptable carrier
for use
in the present methods.
Further, the invention provides a method for inhibiting 5-hydroxytryptamine
(Serotonin) reuptake of a monoamine transporter by contacting the monoamine
transporter with a 5-hydroxy-tryptamine reuptake inhibiting (5-HT inhibiting)
amount
of a non-amine tropane compound. Inhibition of 5-hydroxy-tryptamine reuptake
of a
serotonin transporter in a mammal is provided in accord with the present
invention by
administering to the mammal a 5-HT inhibiting amount of a non-amine tropane
compound in a pharmaceutically acceptable carrier.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows the chemical structure of novel amines and novel non-amines,
all of which share a tropane backbone.
Figure 2 shows reaction Scheme 1 for the synthesis of 2-carbomethoxy-3-
arylbicyclo[3.2.1 ]octanes.
Figure 3 shows reaction Scheme 2 for the synthesis of 3-aryl-8-
oxabicyclo[3.2.1 ]octanes.
Figure 4 shows reaction Scheme 3 for the resolution of keto ester.
DETAILED DESCRIPTION OF THE INVENTION
The present-invention relates to the use of high affinity serotonin transport
inhibitors, designated non-amines, that contain no amine nitrogen in their
structure.
These tropane analogs, in which the amine nitrogen is replaced by an oxa or
carba
atom (Madras BK, et al., 1996. Synapse 24:340-348; Meltzer PC, et al., 1997. J
Med
Chem 40:2661-2673 Meltzer PC, et al., 1997. J Med Chem 40:2661-2673; Meltzer
6
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PC, et al., 1999. Bioorg Med Chem Lett 9:857-862; Meltzer PC, 2000. J Med Chem
43:2982-2991), are generally described in U.S. Patent No. 5,948,933, which
issued
September 7, 1999.
The present invention relates to the use of specific compounds that bind to
the
SERT to treat neuropsychiatric disorders, wherein the compound is a non-amine
and
blocks serotonin transport. Certain preferred compounds have a high
selectivity for
the SERT versus the DAT as described herein.
The compounds used in the methods of the present invention inhibit serotonin
uptake and have the following structural formulas:
Compounds that are useful as therapeutic agents in the methods of the present
invention include compounds represented by the following general structural
formula:
I R2
(O)m Ar
!L R2
Ar
Rti
III R2~1
Ar
wherein:
R, = COOCH3, COR3, lower alkyl, lower alkenyl, lower alkynyl, CONHR4, or
COR6;
R2 = is a 6a, 6p, 7a or 7[3 substituent, which can be selected from H, OH,
OR3,
F, Cl, Br, and NHR3;
X= CH2, CHY, CYYI, CO, 0, S; SO, SO2, or C=CXiY with the C, 0 or S
atom being a member of the ring;
7
CA 02373559 2002-02-27
Xi = NR3, CH2, CHY, CYY] CO, O, S; SO, SO2, or NSO2R3;
R3= H, (CH2)õC6H4Y, C6H4Y, CHCH2, lower alkyl, lower alkenyl or lower
alkynyl;
Y and Y, = H, Br, Cl, 1, F, OH, OCH3, CF3, NO2, NH2, CN, NHCOCH3,
N(CH3)2, (CH2),,CH3, COCH3, or C(CH3)3;
R4 = CH3, CH2CH3, or CH3SO2;
R6 = morpholinyl or piperidinyl;
Ar = phenyl-R5, naphthyl-R5, anthracenyl-R5, phenanthrenyl-R5, or
diphenylmethoxy-R5;
R5 = Br, Cl, I. F, OH, OCH3, CF3, NO2, NH2, CN, NHCOCH3, N(CH3)2,
(CH2)nCH3, COCH3, C(CH3)3 where n= 0-6, 4-F, 4-Cl, 4-I, 2-F, 2-Cl, 2-I, 3-F, 3-
Cl,
3-1, 3,4-diCl, 3,4-diOH, 3,4-diOAc, 3,4-diOCH3, 3-OH-4-Cl, 3-OH-4-F, 3-CI-4-
OH,
3-F-4-OH, lower alkyl, lower alkoxy, lower alkenyl, lower alkynyl, CO(lower
alkyl),
or CO(lower alkoxy);
m=0orl;and
n=0,1,2,3,4or5.
Preferred compounds have a SERT/DAT selectivity ratio of at least about 3.
Other embodiments have a SERT/DAT selectivity ratio of at least about 8 and
other
preferably at least about 50.
The invention also relates to compounds shown above that have a potency (K;),
or IC50, at the SERT of less than about 500 nM, preferably less than about 100
nM. In
certain preferred embodiments the compounds have a K; at the SERT more less
than
about 50 nM, preferably less than about 25 nM and more preferably less than
about 15
nM.
Especially preferred compounds have a SERT/DAT selectivity ratio of at least
about 3 and a potency (Ki) at the SERT of less than about 500 nM.
In another preferred embodiment of the present invention, preferred 8-
oxatropanes and 8-carbatropanes include those having alkenyl and alkynyl
groups on
the 3-aryl ring, particularly of the 2-COOCH3 tropanes, to enhance potency at
the
SERT. Particularly preferred examples of such compounds have the following
formula:
8
CA 02373559 2002-02-27
Ri
IV R2~~ R7
Ra
v R2 R7
Fts
VI R2\/ Rr
R$
where X is an oxygen or a carbon group such as, for example, CH2, CHY, CYYj,
CO,
or C=CXIY where XI, Y and Y, are defined above, and R7 is a lower alkenyl or
lower
alkynyl group having from about 2 to about 8 carbon atoms. Particularly
preferred
lower alkenyl and lower alkynyl groups are ethenyl, propenyl, butenyl,
propynyl,
butynyl and methyipropynyl. R8 is H or Br, Cl, I, F, OH, OCH3, CF3, NO2, NH2,
CN,
NHCOCH3, N(CH3)2, (CH2)õCH3, COCH3, C(CH3)3 where n= 0-6.
The compounds are prepared as racemates and individual enantiomers. These
compounds can be prepared either as free bases or as pharmacologically active
salts
thereof such as hydrochloride, tartrate, sulfate, naphthalene-1,5-disulfonate
or the like.
In certain preferred compounds, when R2 is not H, i.e., the compounds are 6 or
7
substituted compounds, the compounds have the 1 S conformation. In other
preferred
compounds, when R2 is H, the compounds preferably have the R conformation.
Figure 1 shows the chemical structure of amines and non-amines, all of which
share a tropane backbone. The dichlorophenyl substituted non-amines are
derived
from an amine (aza, 0-401) in which the amine nitrogen is replaced with a
oxygen
(oxa, 0-1072) or carbon (carba, 0-1391). The naphthyl substituted non-amines
are
derived from diastereomeric amines (aza, 0-1229, 0-1228) in which the amine
nitrogen is replaced with a carbon (carba, 0-1669, 0-1670). Compounds 0-1585
and
9
CA 02373559 2002-02-27
0-1577 are propynylphenyl derivatives. 0-1738 and 0-1739 are isopropenylphenyl
analogs of tropanes.
Examples of preferred compounds for use in the methods of the repent
invention include, but are not lirriited to: 0-1229: N-methyl-2(3-carbomethoxy-
3(3-(2'-
naphthyl)-8-azabicyclo[3.2.1]octane; 0-1228: N-methyl-2(3--carbomethoxy-3 -(2'-
naphthyl)-8-azabicyclo[3.2.1 ]octane; 0-1072: 2-(3-carbomethoxy-3-[3-(3,4-
dichlorophenyl)-8-oxabicyclo[3.2.1 ]octane; 0-1391: 2-(3-carbomethoxy-3-(3-
(3,4-
dichlorophenyl)bicyclo[3.2.1 ]octane; 0-1577: 2(3-carbomethoxy-3P-(4'-
propynylphenyl))-8-oxabicyclo[3.2.1 ]octane; 0-1585: 2(3-carbomethoxy-3a-(4'-
propynylphenyl)-8-oxabicyclo[3.2.1 ]octane; 0-1669: 2 (3-carbomethoxy-3 j3-(2-
naphthyl)-8-bicyclo[3.2.1 ]octane; 0-1670: 2(3-carbomethoxy-3a-(2-naphthyl)-8-
:
bicyclo[3.2:1]octane; 0-1738: 2(3-carbomethoxy-3a-(4-isopropenylphenyl)-8-
oxabicyclo[3.2.1 ]octane; 0-1739: 2(3-carbomethoxy-3(3-(4-isopropenylphenyl)-8-
oxabicyclo[3.2.1 ]octane; 0-1809: 2(3-carbomethoxy-3(3-(4-isopropenylphenyl)-8-
oxabicyclo[3.2.1 ]octane. The synthesis of these compounds and other is shown
in
Figures 2-4 and described below in the Examples.
The compounds described herein provide a broad array of molecules including
compounds that bind to the SERT with very high affinity. Selectivity for
inhibition of
the SERT versus the DAT is another property of tropanes of considerable
relevance
for development of medications for treating SERT related disorders. Preferred
compounds for the present methods exhibit the desired target:non-target
(SERT:DAT)
specificity.. The serotonin transporter is detectable in the striatum, the
brain region
with the highest density of dopamine neurons and in brain regions surrounding
the
striatum. It is necessary to determine whether the candidate compound is more
potent
at the serotonin than the dopamine transporter. If more selective (e.g., >10-
fold), the
compound will provide effective treatment modality for the SERT. Therefore, a
measure of probe affinity of the serotonin transport is conducted by assays
paralleling
the. dopamine transporter assays. As described below, (3H)Citalopram is used
to
radiolabel binding sites on the serotonin transporter and competition studies
are
CA 02373559 2002-02-27
conducted with the candidate compound at various concentrations in order to
generate
an IC50 value.
Whether oxa or carba based, non-amines of the present invention bound to
[3H]citalopram labeled sites and blocked [3H]serotonin transport in the low
nanomolar
range. These results are comparable or better than those of some conventional
antidepressants. For example, 0-1809, is 99-fold more selective for the
serotonin
transporter over the dopamine transporter.
The non-amines had varying affinities and serotonin:dopamine transporter
selectivities, as measured in monkey brain tissue (Table 2). As described
above,
preferred compounds for use in the methods of the present invention have a
SERT/DAT selectivity ratio of at least about 3. Other embodiments have a
SERT/DAT selectivity ratio of at least about 8 and other preferably at least
about 50.
Examples of preferred serotonin transporter-selective non-amines include 0-
1809, 0-
1739, 0-1577, 0-1738 and 0-1585.
Affinity, i.e., binding to the SERT, is another characteristic that is useful
for
selecting useful compounds.. The oxa (0-1072) or carba (0-1391) analogs of 0-
401, a
high affinity amine that is relatively non=selective for the dopamine over the
serotonin
transporter, displayed similar high affinity and non-selective binding to the
dopamine
and serotonin transporters. The compounds of the present invention have an
affinity,
also known as potency, IC50 or K;, at the SERT of less than about 500 nM,
preferably
less than about 50 nM. In certain preferred embodiments the compounds have a
Ki at
the SERT more preferably less than about 25 nM and more preferably less than
about
15 nM.
For example, the non-amines 0-1072, 0-1391, 0-1809, 0-1669, 0-1739
blocked [3H]serotonin transport in the low nanomolar range, comparable to the
potencies of the conventional amine antidepressants, imipramine, fluoxetine
and
amitriptyline (see Tables 1-4). Amine antidepressants displayed lower
potencies for
blocking serotonin transport than for binding to [3H]citalopram labeled sites.
Initially
reported in 1997 (Owens MJ, et al., 1997. J Pharmacol Exp Ther 283:1305-1322),
this
discrepancy between drug binding potencies and blockade of serotonin transport
was
not observed if [125I]RTI-55 was used to label the serotonin transporter
(Eshleman AJ,
11
CA 02373559 2002-02-27
et al., 1999. J Pharmacol Exp Ther 289:877-885), but was even more prominent
with
[3H]paroxetine as a probe for the serotonin transporter (Kuhar MJ, et al.,
1999. Drug
Alcohol Depend 56:9-15). The potencies of non-amines for blocking the
serotonin
transporter are comparable to conventional and widely used amine-based
antidepressants.
Using the combination of selectivity (SERT/DAT ratio) and potency (IC50)
information for these compounds, one of ordinary skill in the art can readily
select the
appropriate compound for the desired application, e.g., treatment of SERT
related
disorders.
Substituents on the aromatic ring of non-amines enhanced SERT affinities by
10 to 1,000-fold. The increases were greater than those observed with
corresponding
monoamines. Certain preferred compounds for use in the present invention are
non-
amines with substituted aromatic rings at the 3 position.
The compounds and pharmaceutical preparations of the present invention can
be used to inhibit the serotonin reuptake by the serotonin transporter. The
pharmaceutical compositions, preferably comprise the compounds of the present
invention in a pharmaceutically acceptable carrier. Pharmaceutically
acceptable carriers
are well known to those skilled in the art. An exemplary pharmaceutical
composition is
a therapeutically effective amount of a compound of the invention optionally
included
in a pharmaceutically-acceptable and compatible carrier. The term
"pharmaceutically-
acceptable and compatible carrier" as used herein, and described more fully
below,
refers to e.g., one or more compatible solid or liquid filler diluents or
encapsulating
substances that are suitable for administration to a human or other animal.
The route
.of administration can be varied but is principally selected from intravenous,
nasal and
oral routes. For parenteral administration, e.g., it will typically be
injected in a sterile
aqueous or non-aqueous solution, suspension or emulsion in association with a
pharmaceutically-acceptable parenteral carrier such as physiological saline.
The term "therapeutically-effective amount" is that amount of the present
pharmaceutical compositions which produces a desired result or exerts a
desired
influence on the particular condition being treated. Various concentrations
may be
used in preparing compositions incorporating the same ingredient to provide
for
12
CA 02373559 2002-02-27
variations in the age of the patient to be treated, the severity of the
condition, the
duration of the treatment and the mode of administration. An effective dose of
the
compound is administered to a patient based on IC50 values determined in
vitro. The
route of administration can be varied but is principally selected from
intravenous,
nasal and oral routes. The effective dose can vary depending upon the mode of
administration as is well known in the art.
The term "compatible", as used herein, means that the components of the
phannaceutical compositions are capable of being commingled with the compounds
of
the present invention, and with each other, in a manner such that there is no
interaction
that would substantially impair the desired pharmaceutical efficacy.
The dose of the pharmaceutical compositions of the invention will vary
depending on the subject and upon particular route of administration used. The
pharmaceutical compositions can also be administered to a subject according to
a
variety of well-characterized protocols. In a preferred embodiment, the
pharmaceutical
composition is a liquid composition in pyrogen-free, sterilized container or
vial. The
containercan be unit dose or multidose.
This invention will be illustrated further by the following examples. These
examples are not intended to limit the scope of the claimed invention in any
manner.
The Examples provide suitable methods for preparing compounds of the present
invention. However, those skilled in the art may make compounds of the present
invention by any other suitable means. As is well known to those skilled in
the art;
other substituents can be provided for the illustrated compounds by suitable
modification of the reactants.
EXAMPLES
Materials
The following drugs were obtained from the sources listed: (-)cocaine
hydrochloride
(National Institute on Drug Abuse, Bethesda, MD); mazindol base (Sandoz Inc.,
East
Hanover, NJ); citalopram hydrobromide and talsupram hydrochloride~(Lundbeck
A/S,
Copenhagen, Denmark); dopamine hydrochloride, (-)-norepinephrine bitartrate
and
serotonin creatinine sulfate (Sigma Chemical Co., St. Louis, MO); RTI-55
methyl
13
CA 02373559 2008-05-29
ester tartrate (F. Ivy Carroll, Research Triangle Institute, Research Triangle
Park. NC);
imipramine (Ciba Pharmaceuticals, Summit, NJ); sertraline (McNeil
Pharmaceutical,
Raritan, NJ); fluoxetine hydrochloride (Eli Lilly, Indianapolis, IN and
Sigma/Research
Biochemicals, Natick, MA); amitriptyline and desipramine (Merck Sharp and
Dohme
Ltd., Rahway, NJ). Amine and non-amine drugs, designated with the prefix 0-,
were
synthesized by Organix Inc., (Wobum, MA) in accordance with the methods
described
in Patent No. 5,948,933, Meltzer, et a], J. Med. Chem. 40, 2661-2673, 1997 and
Reference: Meltzer et a], J. Med. Chem. 43, 2982-2991, 2000. Examples of
preferred
structures are shown in Figure 1.
CHEMIC.AI. SYNTIIESIS:
A. SYNTHESIS OF 2-CARBOMETHOXY-3-ARYLBICYCLO
[3.2.1 ]OCTANES.
Scheme I (Figure 2) shows the Synthesis of 2-carbomethoxy-3-arylbicyclo
[3.2.1]octanes. All compounds are racemates (1RI1S). NMR spectra were recorded
in
im
CDCI3 on a JEOL 300 NMR spectrometer operating at 300.53 MHz for iH, and 75.58
MHz for 13C. TMS was used as intemal standard. Melting points are uncorrected
and
were measured on a Gallenkamp melting point apparatus. Thin layer
chromatography
(TLC) was carried out on Baker Si250F plates. Visualization was accomplished
with
either UV exposure or treatment with phosphomolybdic acid (PMA). Flash
chromatography was can-ied out on Baker Silica Gel 40mM. Elemental analyses
were
performed by Atlantic Microlab, Atlanta, GA. All reactions were conducted
under an
inert (N2) atmosphere. [3H]WIN 35,428 (2R-carbomethoxy-3 0-(4-fluorophenyl)-N-
[3H]methyltropane, 79.4-87.0 Cilmmol) and [31-H]citalopram (86.8 Ci/mmol) were
purchased from DuPont-New England Nuclear (Boston, MA). A Beckman 1801
scintillation counter was used for scintillation spectrometry. 0.1 % Bovine
serum
albumin was purchased from Sigma Chemicals. (R)-(-)-Cocaine hydrochloride for
the
pharmacological studies was donated by the National Institute on Drug Abuse
[NIDA].
14
CA 02373559 2002-02-27
2-Carbomethoxy-bicyclo[3.2.ljoctan-3-one (3).
To bicyclo[3.2. I ]octan-3-one, 227 (6.42 g, 51.7 mmol) in THF (75 mL),
lithium diisopropyl amide (31 mL, 62 mmol) in THF (125 mL) was added dropwise
at
-78 C. The mixture was stirred at -78 C for I h and methyl cyanoforrriate
(4.9 mL,
62 mmol) was added. The cooling bath was removed and the reaction allowed to
warm
to room temperature. After stirring for 2 h, saturated aqueous NaCI (32 mL)
was
added and about half of THF removed on a rotary evaporator. The remaining
solvent
was extracted with ether (3 x 150 mL) and the dried (Na2SO4) ether layer
concentrated to dryness. The residue was purified by flash chromatography
(eluent:
10% EtOAc/hexanes) to afford 7.85 g (83%) of 3 as a colorless oil: Rf0.56 (10%
EtOAc/hexanes); I H-NMR (75:15:10 mixture of 2-en-3-ol, 2-(x, and 2-
0-carbomethoxy-3-keto tautomers) S 11.87 (s, 1 H); 3.73 (s, 3H), 3.70 (s,
0.6H), 3.69
(s, 0.4H), 3.42 (m, 0.2H), 3.19 (m, 0.13H), 2.93 (m, 1 H), 2.81 (m, 0.13H),
2.71 (m,
0.21-1), 2.65 (ddd, 0.13H, J= 18, 4, 2 Hz), 2.55 (ddd, 1.2H, J= 18, 4, 2 Hz),
2.41 (m,
I H), 2.34 (m, 0.2H), 2.29 (m, 0.13H), 2.03 (dd, 1 H, J= 18, 2 Hz), 1.65-1.95
(m,
4.4H), 1.30-1.55 (m, 3.6H). 13C-NMR (only the signals corresponding to the 2-
en-3-ol
tautomer are reported) 8 172.00, 171.19, 105.38, 51.33, 40.19, 35.92, 35.58,
32.91
(2C), 29.90.
2-Carbomethoxy-3-{[(trifluoromethyl)sulfonyl]oxy)-bicyclo[3.2.1]-2-octene (4).
To 2-carbomethoxy-bicyclo[3.2.1]octan-3-one, 3(6.0 g, 3.29 mmol) in THF
(120 mL), sodium bis(trimethylsilyl) amide (1.OM solution in THF, 49.4 mL) was
added dropwise at -78 C. After stirring-for 30 min, N-phenyltrifluoromethane
sulfonimide (17.6 g, 4.94 mmol) was added in one portion. After 10 min, the
cooling
bath was removed and the reaction mixture stirred overnight. Water (100 mL)
was
added to the reaction mixture and extracted with diethyl ether (3 x 150 mL).
The dried
(Na2SO4) ether layers were concentrated to dryness on a rotary evaporator. The
residue was purified by flash chromatography (eluent: 20% EtOAc/hexanes) to
afford
7.8 g (75%) of 4 as a colorless oil: Rf 0.56 (20% EtOAc/hexanes); 1H-NMR 8
3.79 (s,
~--
CA 02373559 2008-05-29
3H), 3.10 (m, I H), 2.71 (dd, 1 H, J= 18, 5 Hz), 2.52 (m. I H), 2.17 (dd, 1 H,
J= 18,2
Hz), 1.75-2.05 (m, 3H), 1.45-1.70 (m, 3H). 13C-NMR S 164.44, 151.02, 129.04,
118.23 (q, J= 32011z), 52.05, 39.68 (d, J= I Hz), 36.61, 35.34, 34.51, 33.31,
30.01.
2-Carbomethoxy-3-(3,4-dichlorophenyl)-bicycfo[3.2.11-2-octene (5a).
2-Carbomethoxy-3-{ [(ts-ifluoromethyl)sulfonyl]oxy) -bicyclo[3.2.1 ]-2-octene,
4 (2.0 g, 63.6 mmol), 3, 4-dichlorophenyl boronic acid (1.58 g, 82.7 mmol),
tris(dibenzylideneacetone)dipalladium(0) (0.29 g, 0.32 mmol), Na2CO3 (2M
solution,
6.4 mL) and diethoxymethane (32 mL) were combined and refluxed at 95 C for
4h.
Tris(dibenzylideneacetone) dipalladium(0) (1.45 g) was then added in five
equal
portions at 4h interval. The reaction mixture was cooled to room temperature,
filtered
through Celite, and washed with ether (200 mL). The ether solution was then
washed
with saturated aqueous NaCI (100 mL). The dried (Na2SO4) ether layer was
removed
on a rotary evaporator. The residue was purified by flash chromatography
(eluent:
10% EtOAc/hexanes) to afford 1.38 g (69%) of 5a as a colorless oil: Rf0.50
(10%
EtOAc/hexanes); I H-NMR 8 7.34 (d, 1 H, J= 8 Hz), 7.17 (d, 1 H, J = 2 Hz),
6.90 (dd,
I I-i, J= 8, 2 Hz) 3.49 (s, 3H), 3.00 (bt, 1 H, J= 5 Hz), 2.64 (ddd, 1 H, J=
19, 4, 2 Hz),
2.44 (m, IH), 2.14 (dd, IH, J= 19, 1 Hz), 1.75-2.05 (m, 3H), 1.45-1.70 (m,
3H). 13C-
NMR 8 168.54, 142.60, 142.13, 135.55, 132.07, 130.95, 130.00, 128.80, 126.45,
51.48, 44.52, 37.13, 35.65, 34.86, 33.24, 30.76. Anal. (C16H1602C32) C, H, Cl.
2-Carbomethoxy-3-naphthyl-bicyclo[3.21]-2-octene (5b).
2-Carbomethoxy-3-{ [(trifluoromethyl)sulfonyl]oxy} -bicyclo [3.2. I ]-2-
octene,
4(0.50 g, 1.60 mmol), 2-naphthaleneboronic acid (0.36 g, 2.08 mmol),
tris(dibenzylideneacetone) dipalladium(0) (0.07 g, 0.08 mmol) Na2CO3 (2M
solution,
1.6 mL) and diethoxymethane (8 mL) were combined and refluxed at 95 C
overnight.
TM
The reaction mixture was cooled to room temperature, filtered through Celite
and
washed with ether (100 mL). The ether solution was then extracted with
saturated
aqueous NaCI (50 mL). The dried (Na2SO4) ether layer was removed on a rotary
evaporator. The residue was purified by flash chromatography (eluent: 10%
16
CA 02373559 2002-02-27
EtOAc/hexanes) to afford 0.25 g (54%) of 5b as a colorless oil: Rf0.41 (10%
EtOAc/hexanes); iH-NMR 6 7.83 (m, 3H), 7.62 (d, 1H, J= 1 Hz), 7.48 (m, 2H),
7.28
(dd, 1 H, J= 9, 2 Hz), 3.44 (s, 3H), 3.14 (bt, I H, J= 5 Hz), 2.84 (ddd, 1 H,
J= 19, 4, 1
Hz), 2.53 (m, I H), 2.38 (bd, 1 H, J= 19 Hz), 1.80-2.20 (m, 4H), 1.60-1.75 (m,
2H).
13C-NMR S 169.17, 143.83, 139.85, 134.55, 133.04, 132.33, 127_76, 127:47,
127.21,
125.83, 125.54 (2), 124.86, 51.04, 44.35, 37.16, 35.54, 34.82, 33.21, 30.60.
Anal.
(C2oH2002) C, H. -
2-Carbomethoxy-3-(4-fluorophenyl)-bicyclo[3.2.1]-2-octene (5c).
Compound 5c was obtained from with 4-fluorophenylboronic acid as
described for 5a: A colorless oil was obtained (73%): Rf0.5 (10%
EtOAc/hexanes);
1 H-NMR 6 6.95-7.10 (m, 4H), 3.46 (s, 3H), 3.00 (t, 1 H, J= 5 Hz), 2.67 (dd, 1
H, J=
19, 4 Hz), 2.46 (m, 1 H), 2.20 (bd, 1 H, J= 19 Hz), 1.50-2.05 (m, 6H). 13C-NMR
S
169.10, 161.86 (d, J= 245 Hz), 143.05, 138.32, 134.69, 128.30 (d, 1= 8 Hz),
114.81
(d, J= 21 Hz), 51.18, 44.53, 37.15, 35.55, 34.86, 33.21, 30.65. Anal.
(CI6HJ702F) C,
H.
2-Carbomethoxy-3-phenyl-bicyclo[3.2.1]-2-octene (5d).
Compound 5d was prepared from with phenylboronic acid as described for 5a:
A colorless oil was obtained (75%): Rf 0.5 (10% EtOAc/hexanes); 1H-NMR S 7.27
(m, 3H), 7.08 (m, 2H), 3.43 (s, 3H), 3.00 (bt, 1 H, J= 5), 2.70 (ddd, 1 H, J=
19, 4, 1
Hz), 2.46 (m, I H), 2.23 (bd, I H, J= 19 Hz) 1.80-2.05 (m, 3H), 1.73 (d, 1 H,
J= 1 l
Hz), 1.50-1.65 (m, 2H). 13C-NMR 8 169.31, 144.03, 142.46, 134.28, 127.88,
126.95,
126.61, 51.11, 44.37, 37.17, 35.60, 34.90, 33.26, 30.66. Anal. (C16H1802) C,
H.
2((x, (3)-Carbomethoxy-3(a, (3)-(4-fluorophenyl)bicyclo[3.2.1]octane (6c).
Magnesium (47 mg, 1.90 mmol) was added into 2-carbomethoxy-3-(4-
fluorophenyl)-bicyclo[3.2.1 ]-2-octene, 5c (50 mg, 0.19 mmol) in methanol (2
mL).
After 1 h, additional magnesium (47 mg, 1.90 mmol) was added and stirred for 4
h.
IN HCI (4 mL) was added dropwise and stirred for I h. The reaction mixture was
17
...,~...--.
CA 02373559 2002-02-27
extracted with ether (3 x 20 mL) dried over Na2SO4 and the ether layer removed
on a
rotary evaporator. The residue was purified by flash chromatography (eluent:
10%
EtOAc/hexanes) to afford 42 mg (84%) of 6c as a colorless oil: Rf0.42 (10%
EtOAc/hexanes). Anal. (C16H19FO2) C, H.
2(a, (3)-Carbomethoxy-3(a, (3)-phenylbicyclo[3.2.1]octane (6d).
Compound 6d was prepared from 5d with magnesium as described for 6c. A
colorless oil was obtained (48%): Rf0.42 (10% EtOAc/hexanes); Anal. (C16H2002)
C,
H.
2(3-Carbomethoxy-3R-(3,4-dichlorophenyl)-bicyclo[3.2.1]octane (7a),
2a-Carbomethoxy-3(3-(3,4-dichlorophenyl)-bicyclo13.2.1]octane (8a),
2(3-Carbomethoxy-3a-(3,4-dichlorophenyl)-bicyclo[3.2.1 ] octane (9a), and
2a-Carbomethoxy-3a-(3,4-dichlorophenyl)-bicyclo[3.2.1]octane (l0a).
To 2-carbomethoxy-3-(3,4-dichlorophenyl)-bicyclo[3.2.1]-2-octene, 5a (1.38
g, 4.43 mmol) in methanol (50 mL) at -78 C, SmI2 (0.1M in THF, 237 mL) was
added dropwise via an addition funnel. After completing the addition, the
green
mixture was stirred for 4h at -78 C and quenched with TFA (20 mL) in ether
(60
mL). H20 (50 mL) was added and extracted with ether (3 x 200 mL). The dried
(Na2SO4) ether layer was concentrated to dryness. The residue was purified by
flash
chromatography (eluent: 20% EtOAc/hexanes) to afford a mixture of isomers 6a
(1 g,
72%). The isomers were separated by gravity column chromatography (eluent: 10-
50% toluene/hexanes) to afford 65 mg of 7a as a white solid (mp 81.1-81.4 C),
280mg of 8a as a white solid (mp 65.3-65.6 C), 58 mg of 9a as a white solid
(mp
82.8-83.3 C) and 42mg of l0a as a white solid (mp 83.2-83.8 C). 7a: 1H-NMR 6
7.31 (d, 1 H, J= 2 Hz), 7.30 (d, 1 H, J= 8 Hz), 7.08 (ddd, IH, J= 8, 2, 1 Hz),
3.44 (s,
3H), 2.98 (ddd, 1 H, J=13, 6, 6 Hz), 2.84 (dd, 1 H, J= 6, 6 Hz), 2.53 (m, 1
H), 2.42 (m,
1H), 2.31 (ddd, 1H, J= 13, 13, 2 Hz), 1.45-2.00 (m, 5H), 1.85 (bd, 1H, J= 12
Hz),
1.28 (ddd, 1H, J= 12, 6, 6 Hz). 13C-NMR 6 173.24, 144.03, 131.86, 129.77,
129.75,
129.63, 126.95, 52.15, 51.05, 38.37, 35.91, 34.54, 33.24, 32.95, 29.59, 28.03
. Anal.
18
CA 02373559 2002-02-27
(C 1 6H 1 8C1202) C, H, Cl. 8a: I H-NMR S 7.31 (d, 1 H, J= 8 Hz), 7.3 0(d; 1
H, J= 2 Hz)
7.06 (dd, 1 H, J= 8, 2 Hz), 3.51 (s, 3H), 3.10 (ddd, I H, J= 12, 12, 6 Hz),
2.66 (dd, I H,
J= 12,2 Hz), 2.48 (m, IH), 2.32 (m, IH), 1.87 (m, IH), 1.45-1.80 (m, 7H). 13C-
NMR
S 173.94, 144.94, 132.11, 130.18, 129.95, 129.61, 127.18, 52.82, 51.40, 40.83,
39.26,
38.70, 38.28, 34.95, 28.60, 25.14. Anal. (C16HI8C1202) C, H, Cl. 9a: iH-NMR
7.30
(d, 1 H, J= 8 Hz), 7.25 (d, 1 H, J= 2 Hz) 7.01 (dd, 1 H, J= 8, 2 Hz), 3.54 (s,
3H), 3.03
(ddd, 1 H, J= 12, 12, 8 Hz), 2.36 (d, 1 H, J= 12 Hz), 2.30-2.40 (m, 2H), 2.24
(ddd, 1 H,
J= 12, 8, 8 Hz), 1.94 (m, 1 H), 1.92 (bd, 1 H, J= 12 Hz), 1.74 (m, 1 H), 1:56
(m, 1 H),
1.44 (m, 1 H), 1.20 (dd, 1 H, J= 12, 12 Hz), 1.10 (ddd, 1 H, J= 12, 4, 4 Hz).
13C-NMR
S 175.68, 145.19, 132.14, 130.18, 130.05, 129.70, 127.30, 55.93, 51.60, 38.92,
36.99,
36.70, 33.44, 32.89, 31.76, 29.83. Anal. (C16H18C1202) C, H, Cl. 10a: 1H-NMR 6
7.31 (dd, 1 H, J= 2, 1 Hz), 7.2 8 (d, 1 H, J= 8 Hz), 7.07 (ddd, 1 H, J= 8, 2,
1 Hz), 3.45
(s, 3H), 3.31 (dd, 1 H, J= 6, 6 Hz), 3.11 (ddd, I H, J= 12, 6, 6 Hz), 2.64 (m,
1 H), 2.37
(m, 1 H), 2.16 (ddd, 1 H, J= 12, 6, 6 Hz), 1.95 (bdd, 1 H, J=12, 12 Hz), 1.82
(bd, 1 H, J
= 12 Hz), 1.73 (m, 1 H), 1.50-1.65 (m, 2H), 1.42 (m, 114), 1.28 (ddd, 1 H, J=
12, 4, 4
Hz). 13C-NMR S 173.70, 144.18, 131.76, 129.88, 129.61, 129.48, 127.04, 50:89,
50.11, 35.23, 34.49, 34.47, 33.54, 32.31, 32.02, 27.02. Anal. (C16H1 8C1202)
C, H; Cl.
2(3-Carbomethoxy-3(3-naphthyl-bicyclo(3.2.1]octane (7b),
2a-Carbomethoxy-3[3-naphthyl-bicyclo13.2.1]octane (8b),
2[3-Carbomethoxy-3a-naphthyl-bicyclo[3.21]octane (9b) and
2a-Carbomethoxy-3a-naphthyl-bicyclo[3.2.1]octane (IOb).
To 2-carbomethoxy-3-naphthyl-bicyclo [3:2.1 ]-2-octene, 5b (0.75 g, 2.57
mmol) in methanol (30 mL) at -78 C, SmI2 (0.1M in THF, 200 mL) was added
dropwise via an addition funnel. After completing the addition, the green
mixture was
stirred for 4h at -78 C and quenched with TFA (10 mL) in ether (30 mL). H20
(25
mL) was added and extracted with ether (3 x 100 mL). The dried (Na2SO4) ether
layer
was concentrated to dryness. The residue was purified by flash chromatography
(eluent: 10% EtOAc/hexanes) to afford a mixture of isomers, 6b (0.48 g, 64%).
The
isomers were separated by gravity column chromatography (eluent: 40-80%
19
CA 02373559 2002-02-27
toluene/hexanes) to afford 20 mg of 7b as a white solid (mp 78.8-79.1 C), 30
mg of
8b as a white solid (mp 71.3-71.7 C), 50 mg of 9b as a wliite solid (mp 71.1-
71.4 C),
and 10b as a white solid (mp 91.1-91.3 C). 7b: IH-NMR 6 7.77 (m, 2H), 7.73
(d, 1H,
J= 9 Hz), 7.68 (bs, 1 H), 7.41 (m, 3 H), 3.32 (s, 3H), 3.22 (ddd, 1 H, J= 12,
6, 6 Hz),
3.00 (dd, 1 H, J= 6, 4 Hz), 2.56 (m, 1 H), 2.54 (m, 1 H), 2.48 (m, 1 H), 2.00
(bd, 1 H, J=
12 Hz), 1.92 (m, 1 H), 1.55-1.85 (m, 4H); 1.32 (ddd, 1 H, J= 12, 6, 6 Hz). 13C-
NMR S
173.76, 141.10, 133.49, 132.16, 127.90, 127.52, 127.42, 126.45, 125.98,
125.75,
125.26, 52.55, 50.96, 38.60, 36.83, 34.85, 33.58, 33.16, 29.92, 28.29. Anal.
(C20H2202) C, H. 8b: 1 H-NMR S 7.76(m, 3H), 7.66 (bd, 1 H, J= 2 Hz), 7.40 (m,
3H),
3.43 (s, 3H), 3.31 (ddd, 1 H, J= 12, 12, 6 Hz), 2.88 (dd, 1 H, J= 12, 2 Hz),
2.51 (m,
IH), 2.35(m, 1H), 2.00 (m, IH), 1.50-1.85 (m, 7H). 13C-NMR S 174.56, 142.13,
133.66, 132.41, 127.99, 127.77, 127.61, 126.44, 126.12, 125.85, 125.32, 53.14,
51.41,
41.27, 39.60, 39.18, 39.00, 35.33, 28.93, 25.39. Anal. (C20H2202) C, H. 9b: I
H-NMR
S 7.77(m, 3H), 7.63 (bs, I H), 7.44 (m, 2H), 7.34 (dd, 1 H, J= 9, 2 Hz), 3.48
(s, 3H),
3.26 (ddd, IH, J= 11, 11, 7 Hz), 2.58 (d, 1H, J= 11 Hz) 2.35-2.45 (m, 2H), 232
(ddd,
1 H, J= 12, 7, 7 Hz), 2.05 (bd, 1 H, J= 12 Hz), 1.96 (m, 1 H), 1.78 (m, 1 H),
1.66 (m,
1 H), 1.52 (m, 1 H), 1.40 (dd, 1 H, J= 12, 12 Hz), 1.15 (ddd, 1 H, J= 12, 4, 4
Hz). 13 C-
NMR S 176.34, 142.24,133.55, 132.32, 128.03, 127.71, 127.61, 126.35, 126.26,
125.90, 125.35, 56.10, 51.58, 39.24, 37.59, 37.25, 33.65, 33.08, 32.07, 30.09.
Anal.
(C20H2202) C, H.
2a-Carbomethoxy-3a-naphthyl-bicyclo(3.2.1]octane (10b).
Compound 5b (200 mg, 0.68 mmol) was hydrogenated under pressure (50 psi)
in the presence of Pd-C (10% w/w 118 mg) in methanol (40 mL) ovemight. The
resulting mixture was filtered through Celite and the methanol evaporated. The
crude
residue (180 mg) contained a mixture of products and was purified by gravity
column
chromatography 6n flash silica (eluent: 40 - 8-% toluene/hexanes) to afford 85
mg of
a white solid. Recrystallization from ethanol gave analytically pure lOb (70
mg), mp
91.1-91.3 C; IH-NMR S 7.75 (m, 3H), 7.70 (bs, IH), 7.40 (m, 3H), 3.35-3.45 (m,
2H),
3.36 (s, 3H), 2.70 (m, 1 H), 2.40 (m, 1 H), 2.29 (ddd, 1 H, J= 12, 7, 7 Hz),
2.20 (ddd,
CA 02373559 2002-02-27
1 H, J= 12, 12, 2 Hz), 1.89 (bd, I H, J= 12 Hz), 1.50-180 (m, 3H),1.45 (m, 1
H), 1.35
(ddd, 1H, J= 12, 4,4 Hz). 13C-NMR S 174.35, 141.37, 133.36, 131.88, 127.90,
127.45, 127.29, 126.87, 125.75, 125.72, 125.24, 52.55, 50.96, 38.60, 36.83,
34.85,
33.58, 33.16, 29.92, 28.29. Anal. (C20H2202) C, H.
B. SYNTHESIS OF 3-ARY-8-OXABICYCLO [3.2.1 ]OCTANES.
'Schemes 2 and 3(Figures 3 and 4) show the synthesis of 3-ary-8-oxabicyclo
[3.2.1 ]octanes.
(IR,IS)-2-Carbomethoxy-3-{[(trifluoromethyl)sulfonyl]oxy}-8-oxabiryclo[3.2.11-
2-octene (14).
Sodium bis(trimethylsilyl)amide (1.0 M solution in THF, 45 mL) was added
dropwise to 2-carbomethoxy-8-oxabicyclo[3.2.1] octanone, 1325 (7.12 g, 38.65
mmol)
in THF (100 mL) at -70 C under nitrogen. After stirring for 30 min, N-
phenyltrifluoromethanesulfonimide (15.19 g, 42.52 mmol) was added as a solid
at -70
C. The reaction was allowed to warm to room temperature and was then stirred
overnight. The volatiles were removed on a rotary evaporator. The residue was
dissolved in CH2C12 (200 mL) and washed with H20 (100 mL) and brine (100 mL).
The dried (IYlgSO4) CH2C12 layer was concentrated to dryness on a rotary
evaporator.
The residue was purified by flash chromatography (eluent: 5%-10%
EtOAc/hexanes)
to afford 9.62 g (79%) of 14 as a pale yellow oil: 1H-NMR (CDC13, 100 MHz): b
5.0-
5.1 (m, IH), 4.6-4.8 (m, IH), 3.83 (s, 3H), 3.0 (dd, IH, J = 5, 8 Hz), 1.7-
2.35 (m, 5H).
General Procedure for Synthesis of the 2-Octenes: (1R,1S)-2-Carbomethoxy-3-
phenyl-8-oxabicyclo[3.2.1]-2-octene (15a).
2-Carbomethoxy-3-{ [(trifluoromethyl)sulfonyl]oxy}-8-oxabicyclo[3.2.1 ]-2-
octene, 14 (2.0 g, 6.32 mmol), phenyl boronic acid (1.02 g, 8.36 mmol),
diethoxymethane (20 mL), LiCI (578 mg, 13.6 mmol), tris(dibenzylideneacetone)
dipalladium(0) (247 mg, 0.25 mmol) and Na2CO3 (2 M solution, 6.1 mL) were
combined and heated at reflux for 1 h. The mixture was cooled to room
temperature,
21
CA 02373559 2002-02-27
filtered through Celite and washed with ether (100 mI,). The mixture was
basified
with NH40H and washed with brine. The dried (MgSO4) ether layer was
concentrated
to dryness. The residue was purified by flash chromatography (eluent: 10%
EtOAc/hexanes) to afford 1.28 g(82%) of 15a as a light brown viscous oil:
Rf0.26
(20% EtOAc/hexanes); I H-NMR (CDC13, 100 MHz): S 7.1-7.5 (m, 5H), 4.95-5.1 (m,
IH), 4.55-4.75 (m, 1H), 3.52 (s, 3H), 2.95 (dd, 1H, J= 5, 18 Hz), 1.7-2.2 (m,
5H).
Anal. (C15H1603) C, H.
(1R,1S)-2-Carbomethoxy-3-(4-fluorophenyl)-8-oxabicyclo[3.2.1]-2-octene (15b).
Compound 15b was prepared from 14 with 4-fluorophenylboronic acid as
described for 15a. A light brown viscous oil was obtained (88%): RfO.19 (20%
EtOAc/hexanes); 1 H-NMR (CDC13, 100 MHz): S 7.0-7.2 (m, 4H), 4.95-5.05 (m, 1
H),,
4.55-4.75 (m, I H), 3.52 (s, 3H), 2.95 (dd, l H, J= 5, 18 Hz), 1.7-2.3 (m,
5H). Anal.
(C15H1503F) C, H=
(1R,IS)-2-Carbomethoxy-3-(4-chloro.phenyl)-8-oxabicyclo[3.2.1]-2-octene (15c).
Compound 15c was prepared from 14 with 4-chlorophenylboronic acid as
described for 15a. A light brown viscous oil was obtained (92%): Rf0.23 (20%
EtOAc/hexanes); 1 H-NMR (CDC13, 100 MHz): 8 7.0-7.4 (m, 4H), 4.95-5.1 (m, 1
H),
4.55-4.75 (m, IH), 3.52 (s, 3H), 2.95 (dd, 1 H, J= 5, 18 Hz), 1.7-2.2 (m, 5H).
Anal.
(C15H1503CI) C, H, Cl.
(IR,IS)-2-Carbomethoxy-3-(4-bromophenyl)-$-oxabicyclo[3.2.1)-2-octene (15d).
Compound 15d was prepared from 14 with 4-bromophenylboronic acid as
described for 15a. A clear viscous oil was obtained (41%): Rf0.39 (20%
EtOAc/hexanes); I H-NMR (CDC13, 100 MHz): 6 7.48 (d, 2H, J = 9 Hz), 6.97 (d,
2H,
9 Hz), 4.95-5.1 (m, 1 H), 4.5-4.75 (m, 1 H), 3.52 (s, 3H), 2.95 (dd, 1 H, J=
5, 18 Hz),
1.65-2.4 (m, 5H). Anal. (C15H15O3Br) C, H, Br.
22
CA 02373559 2002-02-27
(1R,IS)-2-Carbom eth oxy-3-(3,4-dichlorophenyl)-8-oxabicycio [3.2.1 ] -2-
octene
(15f).
Compound 15f was prepared from 14 with 3,4-chlorophenylboronic acid as
described for 15a. A light brown viscous oil was obtained (97%): Rf0.45 (30%
EtOAc/hexanes); 1 H-NMR (CDC13, 100 MHz): S 7.4 (d, 1 H, J = 10 Hz); 7.23 (d,
IH,
J = 2 Hz), 6.95 (dd, 1 H, J= 2, 10 Hz), 4.95-5.1 (m, 1 H), 4.55-4.75 (m, 1 H),
3.52 (s,
3H), 2.95 (dd, IH, J = 5, 18 Hz), 1.6-2.3 (m, 5H). Anal. (C15H1403C12) C, H,
Cl.
(IR)-2-Carbomethoxy-3-(3,4-dichlorophenyl)-8-oxabicyclo[3.2.1]-2-octene (15g).
Compound 15g was prepared from (1R)-14 with 3,4-chiorophenylboronic acid
as described for 15a. A light brown viscous oil was obtained (94%): Rf0.45
(30%
EtOAc/hexanes); I H-NMR (CDCI;, 100 MHz): identical to 5f above.
(IS)-2-Carbomethoxy-3-(3,4-dichlorophenyl)-8-oxabicyclo[3.2.1]-2-octene (15h).
Compound 15h was prepared from (IS)- 14 with 3,4-chlorophenylboronic acid
as described for 15a. A clear viscous oil was obtained (80%): Rf0.45 (30%
EtOAc/hexanes); I H-NMR (CDC13, 100 MHz): identical to 15f above.
General Procedure for Synthesis of the Octanes: (IR,I.S)-2(3-Carbomethoxy-3(3-
phenyl-8-oxabicyclo[3.2.1]octane (16a) and (IR,IS)-2[i-Carbomethoxy-3a-phenyl-
8-oxabicyclo[3.2.11 octane (17a).
To 2-carbomethoxy-3-phenyl-8-oxabicyclo[3-.2.1]-2-octene, 15a (1.17 g, 4.8
mmol) in THF (10 mL) at -70 C under N2 was added Sm12 (0.1 M in THF, 215 mL,
21.5 mmol). After the mixture was stirred for 30 min, MeOH (anhydrous, 25 mL)
was
added. The mixture was stirred at -70 C for a further 2 h. The mixture was
quenched
with TFA (5 mL) and H20 (100 mL). After warming to 0 C, NH4OH was added to
attain pH 11 and the mixture was then stirred for 30 min. The mixture was
filtered
through Celite and washed with ether (400 mL) and then saturated with Na2S2O3.
The ether layer was washed with brine. The dried (MgSO4) ether layer was
concentrated to dryness. The isomers were separated by gravity column
23
CA 02373559 2002-02-27
chromatography (eluent: 10% EtOAc/hexanes) to afford 270 mg (23%) of 16a as a
white solid: mp 102.5-104 C; Rf 0.30 (30% EtOAc/hexanes); and 789 mg (67%) of
17a as a white solid: mp 96.5-98 C; Rf0.37 (30% EtOAc/hexanes); (1.6a): 1H-
NMR
(CDC13, 100 MHz): S 7.25 (br s, 5H), 4.55-4.8 (m, 2H), 3.48 (s, 3H), 3.25
(ddd, 1H, J
= 5, 5, 14 Hz), 2.6-3.0 (m, 2H), 1.5-2.3 (m, 5H). Anal. (C15H1803) C, H.
(17a): 1H-
NMR (CDC13, 100 MHz): S 7.25 (br s, 5H), 4.4-4.65 (m, 2H), 3.58 (s, 3H), 3.25
(ddd,
1 H, J= 7, 11, 11 Hz), 2.52 (dd, 1 H, J= 2, 11 Hz), 1.6-2.5 (m, 5H), 1.41
(ddd, 1 H, J=
2, 11, 14 Hz). Anal. (C15H1803) C, H.
(IR,IS')-2(3-Carbomethoxy-3[3-(4-fluorophenyl)-8-oxabicyclo[3.2.1]octane (16b)
and (IR,IS)-2j3-Carbomethoxy-3a-(4-fluorophenyl)-8-oxabicyclo [3.2.1]octane
(17b).
Compounds 16b and 17b were prepared from 15b as described for compounds
16a and 17a. Compound 16b was obtained (22%) as a white solid: mp 118-120.5
C;
Rf0.27 (30% EtOAc/hexanes); and 17b (62%) as a white solid: mp 58-60 C;
Rf0.36
(30% EtOAc/hexanes). (16b): 1H-NMR (CDC13, 400 MHz): 6 7.15-7.25 (m, 2H), 6.9-
7.0 (m, 2H), 4.6-4.7 (m, 2H), 3.48 (s, 3H), 3.17 (ddd, 1 H, J = 5, 5, 13 Hz),
2.78 (d, 1 H,
J = 5 Hz), 2.73 (ddd, 1H, J 4, 13, 13 Hz), 1.7-2.2 (m, 4H), 1.5-1.65 (m, 1H).
Anal.
(C15H1703F) C; H. (17b): 1H-NMR (CDC13, 400 MHz): S 7.1-7.2 (m, 2H), 6.9-7.0
(m, 2h), 4.5-4.8 (m, 2H), 3.55 (s, 3H), 3.20 (ddd, 1H, J = 7, 11, 11 Hz), 2.44
(dd, IH, J
= 2, 11 Hz), 2.38 (ddd, I H, J = 7, 9, 13 Hz), 1.9-2.2 (m, 2H), 1.76 (ddd, 1H,
J = 5, 9,
13 Hz), 1.6-1.7 (m, 1 H), 1.32 (ddd, 1 H, J= 2, 11, 13 Hz). Anal. (C 15H 1703
F) C, H.
(IR,IS)-2(3-Carbomethoxy-3(3-(4-chlorophenyl)-8-oxabicyclo(3.2.1]octane (16c)
and (1R,1S)-2(3-Carbomethoxy-3a-(4-chlorophenyl)-8-oxabicyclo (3.2.1] octane
(17c).
Compounds 16c and 17c were prepared from 15c as described for compounds
16a and 17a. Compound 16c was obtained (19%) as a white solid: mp 116-117 C;
Rf
0.27 (30% EtOAc/hexanes); and 17c (51%) as a white solid: mp 89-90 C; Rf0.32
24
= CA 02373559 2002-02-27
(30% EtOAc/hexanes). (16c): 1H-NMR (CDC13, 100 MHz): S 7.1-7.4 (m, 4H), 4.55-
4.8 (m, 2H), 3.55 (s, 3H), 3.20 (ddd, 1 H, J= 5, 5, 12 Hz), 2.55-2.95 (m, 2H),
1.5-2.3
(m, 5H). Anal. (CI5H1703C1) C, H, Cl. (17c): 1H-NIVIR (CDC13, 100 MHz): b 7.1-
7.4 (m, 4H), 4.4-4.65 (m, 2H), 3.58 (s, 3H), 3.05-3.45 (m, 1 H), 1.2-2.6 (m,
7H). Anal.
(C15H17O3C1) C, H, Cl.
(IR,IS)-2p-Carbomethoxy-3oc-(4-bromophenyl)-8-oxabicyclo[3.2.1]octane (16d)
and (IR,IS)-20-Carbomethoxy-3(3-(4-bromophenyl)-8-oxabicyclo(3.2.1]octane
(17d).
Compounds 16d and 17d were prepared from 15d as described for compounds
16a and 17a except no TFA was used when quenching. Compound 16d was obtained
(47%) as a white solid: mp 113-115 C; Rf 0.29 (30% EtOAc/hexanes); and 17d
(32%) as a white solid: mp 96-98 C; Rf0:38 (30% EtOAc/hexanes). (16d): 1H-NMR
(CDC13, 100 MHz): 8 7.45 (d, 2H, J = 9 Hz), 7.15 (d, 2H, J = 9 Hz), 4.6-4.8
(m, 2H),
3.5 (s, 3H), 3.0-3.4 (m, IH), 2.55-2.9 (m, 2H), 1.5-2.4 (m, 5H). Anal.
C15H17O3Br.
(17d): 1H-NMR (CDC13, 100 MHz): S 7.45 (d, 2H, J = 10 Hz), 7.1 (d, 2H, J = 10
Hz),
4.4-4.6 (m, 2H), 3.53 (s, 3H), 3.20 (ddd, IH, J = 6, 11, 11 Hz), 1.6-2.6 (m,
6H), 1.35
(ddd, 1H, J = 2, 11, 13 Hz). Anal. (C15H17O3Br) C, H, Br.
(1R,1S)-2[1-Carbomethoxy-3[3-(3,4-dichlorophenyl)-8-oxabicyclo[3.2.1] octane
(16f) and (IR,IS)-2(3-Carbomethoxy-3a-(3,4-dichlorophenyl)-S-
oxabicyclo[3.2.1]octane (17f).
Compounds 16f and 17f were prepared from 15f as described for compounds
16a and 7a. Compound 16f was obtained (14%) as a white solid: mp. 132-133.5
C;
Rf0:31 (30% EtOAc/hexanes); and 17f (55%) as a white solid: mp. 88.5-90 C; Rf
0.33 (30% EtOAc/hexanes). (6f): 1H-NMR (CDC13, 100 MHz): 8 7.0-7.5 (m, 3H),
4.55-4.85 (m, 2H), 3.55 (s, 3H), 3.20 (ddd, 1 H, J = 5, 5, 11 Hz), 2.55-2.95
(m, 2H),
1.45-2.35 (m, 5H). Anal. (C15H1603C12) C; H, Cl. (17f): 1H-NMR (CDC13, 100
MHz): 6 7.0-7.5 (m, 3H), 4.4-4.65 (m, 2H), 3.60 (s, 3H), 3.20 (ddd, IH, J = 7,
11, 11
CA 02373559 2002-02-27
Hz), 1.5-2.5 (m, 6H), 1.30 (ddd, 1H, J= 2, 11, 13 Hz). Anal. (C15H1603C12) C.
H,
Cl.
(IR)-2[3-Carbomethoxy-3(3-(3,4-dichlorophenyl)-8-oxabicyclo[3.2.1] octane
(16g)
and
(1R)-2(3-Carbomethoxy-3a-(3,4-dichlorophenyl)-8-oxabicyclo[3.2.11 octane
(17g).
Compounds 16g and 17g were prepared from (IR)-15f as described for
compounds 16a and 17a. Compound 16g was obtained (13%) as a white solid: mp
121-122 C; Rf0.31 (30% EtOAc/hexanes); and 17g (45%) as a white solid: mp
103.5-104.5 C; [a]21D= -79 (c = 1, MeOH); Rf0.33 (30% EtOAc/hexanes). (16g
and
17g): 1 H-NMR (CDC13, 100 MHz): identical to 16f and 17f above. Anal.
(C15H1603C12) C, H, Cl.
(IS)-2[3-Carbomethoxy-3(3-(3,4-dichlorophenyl)-8-oxabicyclo[3.2.1] octane
(16h)
and (IS)-2[i-Carbomethoxy-3a-(3,4-dichlorophenyl)-8-oxabicyclo[3.2.1]octane
(17h). C
Compounds 16h and 17h were prepared from (IS)-15f as described for
compounds 16a and 17a. Compound 16h was obtained (11%) as a white solid: mp
121-122 C; Rf0.31 (30% EtOAc/hexanes); and 17h (45%) as a white solid: mp 103-
104 C; [a]21D= +76 (c = 1, MeOH); Rf0.33 (30% EtOAc/hexanes). (16b and 17h):
1H-NMR (CDC13, 100 MHz): identical to 16f and 17f above. Anal. (C15H1603C12)
C,H,CI.
Synthesis of (1R,IS)-2-Carbomethoxy-3-(4-iodophenyl)-8-oxabicyclo [3.2.1]-2-
octene,15e: (1R,1S)-2-Carbomethoxy-3-(4-tributylstannylphenyl)-8-
oxabicyclo [3.2.1.1-2-octene.
2-Carbomethoxy-3-(4-bromophenyl)-8-oxabicyclo { 3.2.1 ]-2-octene, 15d (200
mg, 0.62 mmol), tetrakis(triphenylphosphine)palladium(0) (13 mg, 0.011 mmol)
and
bis(tributyltin) (0.74 mL, 1.46 nzmol) in toluene (4 mL) were degassed by
bubbling N2
26
CA 02373559 2002-02-27
through the solution for 10 min. The mixture was then heated at reflux for 6
h.
CH202 (10 mL) was added and the mixture was filtered through Celite. The
filtrate
was concentrated to dryness. The residue was purified sequentially by flash
chromotography (eluent: 30% EtOAc/hexanes) and preparative TLC (eluent: 5%-10%
EtOAc/hexanes) to afford 206 mg (62%) of the title compound as a clear viscous
oil:
Rf0.31 (59% EtOAc/hexanes); I H-NMR (CDCI3, 100 MHz): S 7.43 (d, 2H, J = 7
Hz),
7.05 (d, 2H, J= 7 Hz), 4.95-5.1 (m, IH), 4.55-4.75 (m, 1H), 3.50 (s, 3H), 2.95
(dd,
1 H, J = 5, 18 Hz), 0.7-2.3 (m, 32H).
(IR,IS)-2-Carbomethoxy-3-(4-iodophenyl)-8-oxabicyclo[3.2.1 ]-2-octene (15e).
2-Carbomethoxy-3-(4-tributylstannylphenyl)-8-oxabicyclo[3.2.1 ]-2-octene
(206 mg, 0.39 mmol) from above, in THF (anhydrous, 5mL) was degassed by
bubbling N2 for 10 min. N-Iodosuccinimide (96 mg, 0.43 mmol) was added. The
reaction mixture was stirred at room temperature for I h and concentrated to
dryness.
The residue was dissolved in ether (10 mL), washed with saturated NaHCO3 and
brine. The dried (MgSO4) ether layer was concentrated to dryness. The residue
was
purified by flash chromotography (eluent: 10% EtOAc/hexanes) and preparative
TLC
(eluent: 30% EtOAc/hexanes) to afford 128 mg (90%) of 15e as a pale yellow
viscous
oil: Rf0.49 (30% EtOAc/hexanes); IH-NMR (CDC13; 100 MHz): 8 7.68 (d, 2H, J =
10 Hz), 6.85 (d, 2H, J = 10 Hz), 4.95-5.05 (m, 1 H), 4.55-4.75 (m, 1 H), 3.54
(s, 3H),
2.95 (dd, 1H, J= 5, 18 Hz), 1.55-2.40 (m, 5H). Anal. (CI5H15031) C, H, I.
Synthesis of (1R,1S)-20-Carbomethoxy-3a-(4-iodophenyl)-8-
oxabicyclo[3.2.1]octane (17e): (IR,IS)-2(3-Carbomethoxy-3a-(4-
tributylstannylphenyl)-8-oxabicyclo[3.2.11 octane.
The title compound was prepared from 17d, as described above for
stannylation of 15d. A clear viscous oil (41%) was obtained: Rf 0.48 (30%
EtOAc/hexanes); 1 H-NMR (CDC13, 100 MHz): 8 7.4 (d, 2H, J = 7 Hz), 7.2 (d, 2H,
J
7 Hz), 4.4-4.6 (rn, 2H), 3.60 (s, 3H), 3.25 (ddd, 1 H, J = 6, 10, 10 Hz), 0.7-
2.65 (m,
34H).
27
CA 02373559 2002-02-27
(IR,IS)-2(3-Carbomethoxy-3a-(4-iodophenyl)-8-oxabicyclo[3.2.1]octane (17e).
Compound 17e was prepared from the above stannyl compound as described
for 15e from 2(3-carbomethoxy-3a-(4-tributylstannylphenyl)-8-oxabicyclo[3.2.1]
octane (85%). A white solid was obtained: mp 124-126 C; Rj0.36 (30%
EtOAc/hexanes); 1H-NMR (CDC13, 100 MHz): 8 7.6 (d, 2H, J = 9 Hz), 6.97 (d, 2H,
J
= 9 Hz), 4.35-4.65 (m, 2H), 3.6 (s, 3H), 3.2 (ddd, IH, J= 6, 11, 11 Hz), 1.5-
2.6 (m,
6H), 1.3 5(ddd, IH, J = 2, 11, 13 Hz). Anal. (C 15H 1703I) C, H, I.
Synthesis of (IR,IS)-2[3-Carbomethoxy-3[3-(4-iodophenyl)-8-
oxabicyclo[3.2.1]octane (16e): (1R,1S)-2[3-Carbomethoxy.-3(3-(4-nitrophenyl)-8-
oxabicyclo[3.2.1]octane.
To 2[3-carbomethoxy-3(3-phenyl-8-oxabicyclo[3.2.1]octane, 16a (112 mg,
0.45 mmol) in CH3CN (anhydrous, 5 mL) at -5 C was added NO2BF4 (83 mg, 0.63
mmol). The reaction mixture was stirred at -5 C for 3 h. A small amount of
ice was
added and the mixture was stirred at -25 C for 15 min. The CH3CN was removed,
the
melted ice was extracted with ether. The combined ether extract and CH3CN
solution
were concentrated to dryness. The residue was dissolved in ether (50 mL),
washed
with saturated NaHCO3 and brine. The dried (MgSO4) ether layer was
concentrated to
dryness. The residue was purified by flash chromatography (eluent: 10%-20%
EtOAc/hexanes) to afford 75.6 mg (57%) of the title 4-nitro-compound: Rf0.19
(30%
EtOAc/hexanes); iH-NMR (CDCI3, 100 MHz) 8 8.2 (d, 2H, J= 10 Hz), 7.42 (d, 2H,
J
= 10 Hz), 4.6-4.85 (m, 2H), 3.54 (s, 3H), 3.15-3.45 (m, 1 H), 2.6-3.0 (m, 2H),
1.7-2.4
(m, 5H).
(IR,l.S)-2(3-Carbomethoxy-3[3-(4-aminophenyl)-8-oxabicyclo[3.2.1] octane.
2(3-Carbomethoxy-3[3-(4-nitrophenyl)-8-oxabicyclo[3.2.1 ]octane (75.6 mg,
0.026 mmol) in MeOH ( 20 mL) was hydrogenated overnight at room temperature
using Raney Ni (50%) as catalyst. The reaction mixture was filtered through
Celite,
washed with MeOH and concentrated to dryness. The residue was purified by
flash
28
CA 02373559 2002-02-27
chromatography (eluent: 20%-30% EtOAc/hexanes) to afford 43 mg (75%) of the
title
4-amino-compound: Rf0.22 (50% EtOAc/hexanes); IH-NMR (CDC13, 100 MHz) 6
7.05 (d, 2H, J = 9 Hz), 6.62 (d, 2H, J = 9 Hz), 4.55-4.7 (m, 2H), 3.58 (br s,
2H), 3.50
(s, 3H), 3.0-3.3 (m, 1 H), 2.5-2.9 (m, 2H), 1.4-2.3 (m, 5H).
(1R,I,S)-2(3-Carbomethoxy-3(3-(4-iodophenyl)-8-oxabicyclo[3.2.1]octane (16e).
To 2[3-carbomethoxy-3(3-(4-aminophenyl)-8-oxabicyclo[3.2.l]octane (26 mg,
0.099 mmol) in CH212 (2 mL) under N2 was added isoamyl nitrite (0.17 mL, 0.126
mmol). The reaction mixture was stirred at room temperature for 1 h then at 55
C for
3 h. CH212 was removed under reduced pressure. The residue was purified by
flash
chromatography (eluent: 10% EtOAc/hexanes) to afford 15 mg (60%) of 6e as a
white
solid: mp 119-120.5 C; Rf0.25 (30% EtOAc/hexanes); I H-NMR (CDC13, 100 MHz)
S 7.65 (d, 2H, J = 9 Hz), 7.00 (d, 2H, J = 9 Hz), 4.6-4.8 (m, 2H), 3.52 (s,
3H), 3.05-3.3
(m 1H), 2.55-2.9 (m, 2H), 1.5-2.3 (m, 5H).
(1R,1S)-2-Carbomethoxy-3-(4-acetylphenyi)-8-oxabicyclo[3.2.1J-2-octene (15i).
Compound 15i was prepared from 14 with 4-acetylphenylboronic acid as
described for 15a. A light yellow solid was obtained (64%): m.p. 120-121 C; Rf
0.22
(30% EtOAc/hexanes); 1HNMR 6 (CDC13, 300 MHz): 7.93 (d, 2H), 7.20 (d, 2H),
5.02 (d, IH), 4.66 (t, IH), 3.52 (s, 3H), 2.96 (dd, 1 H), 2.60 (s, 3H), 2.29-
2.06 (m, 4H),
1.83-1.73 (m, 1H). Anal. (C17H1804) C, H.
(1R,1S)-2-Carbomethoxy-3-(4-isopropylphenyl)-8-oxabicyclo [3.2.1 ]-2-octene
(15j)=
Compound 15j was prepared from 14 with 4-isopropylphenylboronic acid as
described for 15a. A light yellow oil was obtained (80%): Rf 0.46 (30%
EtOAc/hexanes); 1 HNMR (CDC13, 300 MHz): S 7.17 (d, 2H), 7.04 (d, 2H), 4.99
(d,
1 H), 4.63 (t, 1 H), 3.51 (s, 3H), 3.02-2.85 (m, 2H), 2.26-2.04 (m, 4H), 1.83-
1.73 (m,
1 H), 1.23 (d, 6H). Anal. (C 18H2203) C, H.
29
CA 02373559 2002-02-27
(IR,1S)-2-Carbomethoxy-3-(4-isopropenylphenyl)-S-oxabicyclo [3.2.1 ]-2-octene
(15k).
Compound 15k was prepared from 15i as follows:
Methyltriphenylphosphonium bromide (0.35 g, 1.0 mmol) was dissolved in
anhydrous
THF (5 mL) under N2 and cooled to -78 C. r}Butyllithium (0.46 mL, 2.5 M in
THF,
1.15 mmol) was added slowly. This mixture was stirred for 20 min at -78 C. (1
R, 1 S)-
2-Carbomethoxy-3-(4-acetylphenyl)-8-oxabicyclo[3.2.1 ]-2-octene (5i, 0.22 g,
0.77
mmol) in THF (2 mL) cooled at 0 C was added via canula. The resulting mixture
was
allowed to warm up to room temperature and was stirred at room temperature for
23 h.
The reaction was.quenched by water (25 mL). This was extracted with ether (40
mL).
The ether extract was washed with brine, dried over sodium sulfate and
evaporated to
dryness. The residue was purified by flash column chromatography to afford 118
mg
white solid (54%): Rf 0.45 (30% EtOAc/hexanes); 1HNMR (CDC13, 300 MHz):
8 7.43 (d, 2H), 7.09 (d, 2H), 5.40 (s, 1 H), 5.09 (s, 1 H), 5.01 (d, 1 H),
4.65 (t, 1 H), 3.54
(s, 3H), 2.96 (dd, 1 H), 2.26-2.05 (m, 7H), 1.83-1.74 (m, I H). Anal. (C 1
8H2003) C, H.
(IR,I S)-2-Carbomethoxy-3-(4-propynylphenyl)-8-oxabicyclo [3.2.11-2-octene
(151).
Compound 151 was prepared from 15m and propynyltributyltin as follows:
(1 R, I S)-2-Carbomethoxy-3-(4-bromophenyl)-8-oxabicyclo[3.2.1]-2-octene (15m;
0.5
g, 1.55 mmol) and propynyltributyltin (0.62 g, 1.88 mrr.mol) were combined in
anhydrous toluene (40 mL) and N2 was bubbled through for 10 min. This solution
was
added via canula to a flask charged with
tetrakis(triphenylphosphine)palladium(0)
(0.18 g, 0.16 mmol) protected under N2. The resulting solution was heated
at.reflux
for 5 h. The reaction solution was cooled to room temperature and diluted with
ether
(50 mL). This was filtered through celite. The filtrate was evaporated to
dryness. The
residue was purified by flash column chromatography to afford 181 mg off-white
solid
(42%). Recrystalization of this slightly impure product (99 mg) from
EtOAc/hexanes
afforded a pure product (white solid, 62 mg): m.p. 93.5-94 C; F¾' 0.40 (30%
EtOAc/hexanes); 1HNMR (CDC13, 300 MHz): S 7.33 (d, 2H), 7.02 (d, 2H), 4.99 (d,
CA 02373559 2002-02-27
1 H), 4.63 (t, 1 H), 3.48 (s, 3H), 2.96 (dd, 1 H), 2.25-2.06 (m, 4H), 2.04 (s,
3H), 1.82-
1.71 (m, 1H). Anal. (C18H1803) C, H.
(IR,IS)-2(3-Carbomethoxy-30-(4-propynylphenyl)-8-oxabicyclo[3.2.1]octane (161)
and (IR,IS)-2(3-Carbomethoxy-3a-(4-propynylphenyl)-8-oxabicyclo[3.2.1]octane
(171).
Compounds 161 and 171 were prepared from 151 as described for compounds
16a and 17a. Compound 161 was obtained (52%) as a white solid: m.p. 142-143 C;
Ff
0.27 (30% EtOAc/hexanes); and compound 171 (13%) as a white solid: m.p. 96.5-
97.5
C; Rf 0.40 (30% EtOAc/hexanes). (161):1HNMR (CDC13, 300 MHz): S 7.30 (d, 2H),
7.15 (d, 2H), 4.70-4:62 (m, 2H), 3.48 (s, 3H), 3.24-3.13 (m, 1H), 2.84-2.70
(m, 2H),
2.20-1.74 (m, 4H), 2.03 (s, 3H), 1:64-1.57 (ni, 1H). Anal. (C18H2003) C, H.
(171):1HNMR (CDC13, 300 MHz): 6 7.30 (d, 2H), 7.13 (d, 2H), 4.54-4.42 (m, 2H),
3.56 (s, 3H); 3.32-3.16 (m, 1H), 2.50 (d, 1H), 2.45-2.32 (m, 1H), 2.18-1.92
(m, 2H),
l5 2.03 (s, 3H), 1.80-1.58 (m, 2H), 1.45-1.31 (m, 1H). Anal. (C18H2003) C, H.
(1R,1S)-2[3-Carbomethoxy-3[3-(4-isopropylphenyl)-8-oxabicyclo[3.2.1]octane
(16j)
and (1R,IS)-2(3-Carbomethoxy-3a-(4-isopropylphenyl)-8-oxabicyclo[3.2.1]octane
(17j)-
Compounds 16j and 17j were prepared from 15j as described for compounds
16a and 17a. Compound 16j was obtained (45%) as a light yellow oil: Rf 0.25
(30%
EtOAc/hexanes); and compound 17j (8.5%) as a light yellow oil: Rf 0.40 (30%
EtOAc/hexanes). (16j):1 HNMR (CDC13, 300 MHz): 8 7.18-7.15 (m, 4H), 4.70-4.62
(m, 2H), 3.49 (s, 3H), 3.23-3.14 (m, 1H), 2.91-2.72 (m, 3H), 2.20-1.74 (m,
4H), 1.64-
1.57 (m, 1 H), 1.22 (d, 6H): Anal. (C 1 8H2403) C, H. (17j):1 HNMR (CDC13, 300
MHz): S 7.13 (s, 4H), 4.54-4.46 (m, 2H), 3.58 (s, 3H), 3.29-3.19 (m, 1H), 2.91-
2.80
(m, 1H), 2.55-2.51 (m, 1H), 2.47-2.35 (m, 1H); 2.19-1.61 (m, 4H), 1.46-1.37
(m, 1H),
1.23 (d, 6H). Anal. (C 18H2403) C, H.
31
CA 02373559 2002-02-27
(I R,I S)-2 (3-Carbometh oxy-3 (3-(4-isopropenylphenyl)-8-oxabicyclo [3.2.1 ]
octane
(16k) and (1R,1.S)-2(3-Carbomethoxy-3a-(4-isopropenylphenyl)-8-
oxabicyclo[3.2.1]octane (17k).
Compounds 16k and 17k were prepared from 15k as described for compounds
16a and 17a. Compound 16k was obtained (54%) as a light yellow solid: m.p.
72.3-
73.3 C; Rf 0.31 (30% EtOAc/hexanes); and compound 17k (24%) as a off-white
solid: m.p. 87-88 C; Rl' 0.40 (30% EtOAc/hexanes). (16k):1 HNMR (CDC13, 300
MHz): b 7.40 (d, 2H), 7.21 (d, 2H), 5.35 (s, 1 H), 5.04 (s, I H), 4.70-4.62
(m, 2H), 3.51
(s, 3H), 3.26-3.17 (m, 1H), 2.89-2.73 (m, 2H), 2.22-1.77 (m, 4H), 2.13 (s,
3H), 1.69-
1.60 (m, 1H). Anal. (C18H2203) C, H. (17k):IHNMR (CDC13, 300 MHz):
S 7.40 (S, 2H), 7.19 (s, 2H), 5.35 (s, 1H), 5:05 (s, 1H), 4.56-4.47 (m, 2H),
3.60 (s. 3H),
3.33-3.22 (m, 1 H), 2.54 (dd, 1 H), 2.46-2.37 (rn, 1 H), 2.22-1.94 (m, 2H),
2.13 (s, 3H),
1.83-1.58 (m, 2H), 1.47-1.38 (m, IH). Anal. (C18H2203) C, H.
(1R)-2-Carbomethoxy-8-oxabicyclo[3.2.1]octa-2-ene-3-(1'S)-camphanate
[(IR,1 'S)-19].
(1R, IS)-2-Carbomethoxy-8-oxabicyclo[3.2.1 ]octan -'3-one,13 (7.4 g, 40.1
mmol) was dissolved in anhydrous THF (200 mL) and cooled to -78 C. To this
solution was added butyl lithium (17.6 mL of a 2.5 M solution, 44.1 mmol); the
color
changed to yellow orange. After 15 min. at -78 C (S)-(-)-camphanic chloride
(9.6 g,
44.1 mrnol) was added in one portion and then the cooling bath was removed.
After 5
min. saturated Na2CO3 was added (300 mL) and ether (300 mL). The layers were
separated and the ether phase washed with brine (100 mL) and dried (MgSO4).
Filtration followed by evaporation gave the crude reaction product (14 g).
Purification
by column chromatography (Si02, 400 g, eluent: 30% ethyl acetate in hexanes)
gave
the diastereomeric mixture, 18 (8.29 g, 57%). 1H-NMR of the diagnostic
camphanate
methyls were 8 (IS,1'S) 1.04 (s, 3H), 1.11 (s, 3H), 1.14 (s, 3H); (1R,1'S)
1.06 (s, 3H)
1.14 (s, 6H). This mixture, 18, was recrystallized eight times from methylene
chloride/hexanes and gave white crystals of the pure title compound (1R,1'S)-
19
(2.25 g, 54%): mp 168.9-169 C; Rf0.25 (30% EtOAc/hexanes); 1H-NMR (CDCI3,
32
CA 02373559 2008-05-29
100 MHz) S 5.04 (br. s, 1 H), 5.55-5. 7 5(m, l H), 3.71 (s, 3H), 2.90 (dd, 3=
5, 18 Hz),
1.6-2.7 (m, 9 H), 1.14 (s, 6 H), 1.06 (s, 3H), Anal. (C I 9H24O7) C, H.
(IS)-2-Carbomethoxy-8-oxabicyclo[3.2.1)octa-2-ene-3-(I'R)-camphanate
[(IS,1'R)- 19).
T7te title compound was obtained as folloxvs: Hydrolysis (LiOH) of the
residual
mother liquor obtained from recrystallization of (IR,1'S)-19 above gave an
enriched
mixture (IS: 60% ee) of (JR, IS)-2-carbomethoxy-8-oxabicyclo[3.2.1 ]octan-3-
one,13.
Reaction with (R)-(+)-camphanic chloride then gave the camphanate (2.79 g,
72%).
Recrystallization twice from methvlene chloride/hexanes gave 1.29 g(92 /a) of
the
pure (IS,1'R)-19 diastereomer. This had the same physical and chemical
properties as
the above ester (IR,I'S)- 19. Anal. (C19H2407) C, H.
(IR)-2-Carbometboxy-8-oxabicyclo[3.2.1)octan-3-one [(IR)- 131.
( IR)-2-Carbomethoxy-8-oxabicyclo[3.2.1 ]octa-2-ene-3-(S)-camphanate, (JR,
I'S)-19 (1.76 g, 4.8 mmol) was dissolved in THF (15 mL) and then methanol (5
mL)
and water (5 mL) were added. The resulting solution was cooled in an ice bath
and
lithium hydroxide (325 mg, 7.7 mmol) was added in one portion. After 20 min no
(IR,
I'S)-19 remained. The solution was neutralized vvith I M hydrochloric acid.
Ether
(200 mL) was then added and the ethereal solution was washed with brine and
dried
(MgSO4). Evaporation gave 1.38 g of the crude reaction product. This was
chromatographed (Si02, 50 g, eluent: 20% ether in hexanes) to yield 833 mg
(94%) of
the pure title compound. 1H-I`'MR and TLC were identical with the racemic
ketone
TM
13. Under chiral HPLC conditions (Chiralcel OC column, eluept: 10% isopropanol
in
hexanes I mL/min.) tR (IS)- 13 = 6.99 min (1.78 %); tR (IR)- 13 = 10.92 min
(98.21
%, ee = 96.4%).
(1.5)-2-Carbometboxy-8-oxabicyclo[3.2.Iloctan-3-one [(IS)-13).
The title compound was obtained upon LiOH hydrolysis, as described above,
from (1S")-2-Carbomethoxy-8-oxabicyclo[3.2.1 ]octa-2-ene-3-(1 R)-camphanate:
0.78
g, 86%. 1 H-NMR and TLC were identical with the racemic ketone 13. Under
chiral
33
CA 02373559 2008-05-29
HPLC conditions (Chiralcel OC column, eluent: 10% isopropanol in hexanes, I
mL/min) tR (IS)- 13 = 6.87 (100 %, ee > 98%); tR (IR)- 13 = not present.
PHARMOCOLOGICAL AND BIOLOGICAL DATA
EXAMPLE 1: Stable Expression of SERT in HEK-293 cells
The human serotonin transporter vector construct was tranfected as a lipid
TM
suspension with Lipofectamine (Life Technologies, Inc., Gaithersburg, MD) into
human embryonic kidney cells (HEK-293, American Type Culture Collection,
Rockville, MD). Cells were plated 24 hours before transfection in Dulbecco's
Modified Eagle growth medium supplemented with 10% fetal bovine serum, 100
U/mi
penicillin and 100 g/mi streptomycin, 1% non-essential amino acids (Gibco-
BRL,
Tm
Grand Island, NY) in 100 mm Falcon tissue culture dishes (VWR, S. Plainfield,
NJ).
The human serotonin transporter cDNA (courtesy of Dr. R.D. Blakely, Vanderbilt
University, TN) was subcloned into (-)peDNA3. I(Invitrogen, San Diego, CA),
which
contained an antibiotic resistance gene. The cDNA expression vector construct
TM
encoding SERT (10 g) was diluted in 1 ml of serum free medium (Opti-Mem 1,
Life
Technologies, Inc. Rockville, MD). The lipofectamine reagent (30 l) was
diluted in I
ml of serum free medium separately from the DNA to avoid precipitation. The
combined solution was added to 8 ml of serum free medium and incubated for 30
min
to allow formation of DNA-liposome complexes. At about 50% confluence, the
cells
were incubated for 5 hours with the transfection mixture and the medium was
changed. Cells were incubated at 37 C in a humidified 5% COz incubator for 72
hours
and then selected for more than two weeks with 600 glmt Geneticin (G418, Life
Technologies, Inc., Rockville, MD). The resistant cells were divided and
individual
foci were harvested using cloning rings (Bel-Art Products, Pequannock, NJ) and
trypsin (Life Technologies, Inc., Rockville, MD). Multiple cell lines were
tested for
[3H]serotonin transport. The clone that displayed the highest serotonin
transport was
chosen for this study and is referred to as SERT. Thereafter, the selecting
antibiotic
geneticin sulfate (250 }ig/mI) was used continuously for culture of cells
expressing the
serotonin transporter.
34
CA 02373559 2002-02-27
[3HJSerotonin Transport: Cell preparation
Low passage number cells (<25 passage cycle) at 80-90% confluency in 145
mm dishes(Greiner Meditech, Bel Air, MD) were used to measure [3H]serotonin
transport. The medium was removed by aspiration, and cells were washed with
Tris-
Hepes buffer, pH 7.4 at 25 C (Tris base: 5 mM; Hepes: 8.5 mM; NaCI: 120 mM;
KCI: 5.4 mM; CaC12:1.2 mM; MgSO4: 1.2 mM; and glucose: 10 mM), supplemented
with pargyline (100 M). The cells were harvested, centrifuged at 1000g for 5
min,
washed twice with the Tris-Hepes buffer and diluted to 250,000 and 1,250,000
cells/ml for stable and transient cell lines, respectively.
EXAMPLE 2:. [3H]Serotonin transport: Pharmacology
The whole-cell suspension (0.2 ml; 5-16 g of protein) was pre-incubated with
various dilutions of each drug (0.2 ml; 10'" to 10-5M) for 15 min. Compounds
to be
tested were dissolved in ethanol (50 l), hydrochloric acid (10 l; 2N) and
water to
achieve a concentration of 1 mM. Subsequent dilutions were made directly in
assay
buffer. Non-amines were dissolved in 30% to 50% ethanol and buffer to achieve
a
concentration of 1 mM. The first dilution used (10"5 M) contained less than
1.75%
ethanol. [3H]Serotonin transport was initiated by the addition of
[3H]serotonin (0.2 ml)
diluted with unlabeled serotonin to yield a final serotonin concentration of
20 nM.
Transport proceeded for 10 min at 25 C and was terminated as previously
described or
by centrifugation of the cells. Nonspecific transport was defined as transport
in the
presence of 10 M fluoxetine, and these data were subtracted from total counts
to
yield specific accumulation of [3H]serotonin. Each concentration of drug was
assayed
in triplicate and each value is the mean S.E. of 2-5 independent
experiments. Protein
concentrations were determined by Bradford assay (Bio-Rad, Richmond, CA).
EXAMPLE 3: Non-amine affinity and transporter selectivity in primate striatum
Initial screening procedures for non-amines were conducted in primate
striatum, a standard source of brain transporters for drug screening and for
generating
structure-activity relationships for this series (Table 1). [''H]Citalopram
binding
CA 02373559 2002-02-27
studies were conducted with moiikey brain tissue (4 mg/ml). Various dilution
of drugs
(0.2 ml; 10'12 to 10"3M) were incubated with I iiM [3H]citalopram (0.2 ml; ;:z
80
Ci/mmol; DuPont-NEN, Boston, MA) for 2 hours, at 4 C. The binding experiments
were terminated by rapid filtration and radioactivity measured as described
above.
Nonspecific binding was defined by 10 M fluoxetine, and these data were
subtracted
from total counts to yield specific [3H]citalopram binding. The experiments
were
performed in triplicate and each value is the mean S.E. of 2-5 independent
experiments. Analysis of [3H]citalopram competition assays was performed with
EBDA and LIGAND computer programs (Elsevier-Biosoft, Cambridge, U.K.).
Table I shows affinities of amines (0-401, 0-1228, 0-1229) and non-amines
to compete with a selective high affinity serotonin transporter ligand,
[3H]citalopram,
and with a selective high affinity dopamine transporter ligand, [3H]CFT, in
monkey
striatum homogenates. Competition assays were performed with [3H]citalopram (1
nM, SERT) or with [3H]CFT (1 nM, DAT) and 8-12 concentrations of tested non-
amines, each conducted in triplicate as described in Materials and Methods.
Values
were determined by fitting data to the equation for competitive binding with
EBDA
computer program. Data represent the mean S.D. of two or more experiments.
Table 1.
Serotonin transporter Dopamine transporter SERT/DAT
(SERT) (DAT) Selectivity
Ligand IC50 (nM) IC50 (nM)
0-401 2.47 0.14 1.09 0.02 0.4
0-1229 2.19 f 0.18 0.49 t 0.04 0.2
0-1072 4.66 t 1.24 3.88 f 0.93 0.8
0-1228 5.95 :1:1.37 0.57 0.34 0.1
(R)0-1809a 10.2 2.1 1,010 246 99
(RS)0-1739 19.8 t 0.75 1,020 42 52
0-1391 33.9~1.85 13.5t0.9 0.4
0-1577 72.4b 545 f 76 8
0-1669 72.5 12.6 27.1 0.1 0.4
0-1670 77.7 f 13.5 21.2 t 4.1 0.3
0-1585 152 26.5 446 f 94 3
0-1738 158 f 23 912 4- 100 6
0-1809 is the active enantiomer of 0-1739
bn=1
36
CA 02373559 2002-02-27
Non-amines competed with [3H]citalopram binding sites with affinities (range:
4.66 - 158 nM, Table 1), comparable to values for conventional amine nitrogen-
containing antidepressants. Some compounds, including the 2(3,3[3
dichlorophenyl
series, were relatively non-selective for the serotonin over the dopamine
transporter
(0-1072, 0-1391, 0-1669, 0-1670) whereas others were 52-, 99-fold (0-1809, 0-
1739) or moderately selective (0-1738, 0-1585). Within the dichloro series
(Fig. 1),
0-1072, the oxa analog of 0-40I retained high affinity for the serotonin (4.66
1.24
nM) and dopamine transporters (3.88 0.93 nM) compared with the progenitor
amine
0-401 (Table 1). Substitution of the 8-oxa with an 8-carba (0-1391) reduced
potency
for the SERT by 7-fold to 33.9 1.85 n1V1. Nevertheless, the results indicate
that
putative aza-driven ionic bonding or oxa-driven hydrogen bonding are not
necessary
for high affinity binding to the serotonin transporter.
Among the most potent novel compounds tested were the naphthyl
monoamines (0-1228, 0-1229; 5.95 1.37 and 2.19 0.18 nM, respectively) but
the
corresponding carba-based non-amines 0-1669 and 0-1670 displayed lower
affinities
(72.5 12.6; 77.7 13.5 nM) than the parent monoamines. The 4-
isopropenylphenyl
non-amines were potent ((R)-0-1809: 10.2 2.1 nM; (RS)-0-1739: 19.8 0.75)
and
selective for the serotonin over the dopamine transporter. In contrast, the
oxa
diastereoisomeric propynyl pair 0-1577 and 0-1585 displayed modest affinity
and
selectivity for the serotonin over the dopamine transporter, lower than the
corresponding dichloroaryl or 4'-isopropenylphenyl analogs. With the exception
of the
highly lipophilic non-amines 0-1669 and 0-1670, the affinities of compounds
for
inhibiting [3H]citalopram labeled sites in monkey striatum and in HEK cells
transfected with the human hSERT were similar (Tables 1, 2).
Table 2 shows affinities of novel amines and non-amines at [3H]citalopram
binding sites and [3H]serotonin transport in HEK-293 cells stably or
transiently
transfected with the human SERT. Non-amines are listed in accordance with
their rank
order of potency. Competition assays were performed with [3H]citalopram (1 nM)
or
with [3H]serotonin (20 nM) and 8-12 concentrations of tested non-amines, each
conducted in triplicate as described above. Values were determined by fitting
data to
37
CA 02373559 2002-02-27
the equation for competitive binding with EBDA computer program. Data
represent
the mean S.E.M. of n independent experiments and are expressed as Ki values
[Ki
=IC50!(I+c/ Kd or Km)]_
Table 2.
[HJcitalopram binding [ H]serotonin transport
Ligand Ki Ki
(nM) (nM)
0-1229 1.42 0.78 0.14t0.07
0-1228 3.26 0.26 4.57 0.37
0-1809 9.6 f 1.3 16 + 4
0-1391 10.0f4.30. 11.1 t7.44
0-1739 17.7 6.3 33t2
0-1577 32.6 f 7.88 63.0 16.8
0-1585 76.1 f10.5 56.7t10.4
0-1738 218 ~ 45.1 376 +74
0-1669 289 150 28 ~ 2
0-1670 303t8.7 425~332
To determine the effects of non-amines and monoamines on [3H]serotonin
transport, we initially characterized [3H]serotonin transport in hSERT cells
with
clinically relevant antidepressants (see above). Compounds that inhibited
[3H]serotonin transport produced monophasic inhibition curves with Hill
coefficients
close to unity (data not shown). Correlation analysis of the potencies of
amines for
inhibiting [3H]serotonin transport and competing with [3H]citalopram binding
sites
yielded a low and insignificant Pearson correlation coefficient (r2: 0.63; p:
0.21). This
poor correlation was attributable to the 2-17 fold higher affinities of
antidepressant
drugs and phenyltropane analogs for [3H]citalopram binding sites compared with
their
potencies for inhibiting [3H]serotonin transport (data not shown).
In hSERT cells, non-amine aÃfinities. for [3H]citalopram binding sites and
[3H]serotonin transport were measured and compared with conventional amine
38
CA 02373559 2008-05-29
antidepressants. In these experiments the density of [3H]citalopram binding
sites, B,,.,
ranged from 1419 to 2800 finol/mg of protein (data not shown). Non-amines were
selected on the basis of severaI addressable comparisons, including affinity,
enantioselectivity, serotonin: dopamine transporter selectivity and amines
(aza) vs oxa
or carba non-amines (Fig. 1).
Carba or oxa non-amines inhibited [3H]serotonin transport into HEK-293 cells
stably expressing the hSERT in a concentration-dependent and saturable manner.
The
most potent non-amines, 0-1391, 0-1072, 0-1809, blocked serotonin transport in
the
10-20 nM range (Table 2).
The present invention has been described in detail, including the preferred
embodiments thereof. However, it will be appreciated that those skilled in the
art,
upon consideration of the present disclosure, may make modifications and/or
improvements of this invention and still be within the scope and spirit of
this invention
as set forth in the following claims.
39
