1-[1-(2-Benzo[b]thiopheneyl)cyclohexyl]piperidine hydrochloride (BTCP) yields two active primary metabolites in vitro: Synthesis, identification from rat liver microsome extracts, and affinity for the neuronal dopamine transporter

Article abstract of DOI:10.1021/jm970078z

1-[1-(2-Benzo[b]thiopheneyl)cyclohexyl]piperidine hydrochloride (BTCP, 1) and cocaine bind to the neuronal dopamine transporter to inhibit dopamine (DA) reuptake. However, on chronic administration, cocaine produces sensitization, but 1 produces tolerance

Full text of DOI:10.1021/jm970078z

J . Med. Chem. 1997, 40, 4019-4025
4019
1-[1-(2-Ben zo[b]th iop h en eyl)cycloh exyl]p ip er id in e Hyd r och lor id e (BTCP ) Yield s
Tw o Active P r im a r y Meta bolites in Vitr o: Syn th esis, Id en tifica tion fr om Ra t
Liver Micr osom e Extr a cts, a n d Affin ity for th e Neu r on a l Dop a m in e Tr a n sp or ter
Carine Deleuze-Masquefa,† Martine Michaud,† J acques Vignon,‡ and J ean-Marc Kamenka*,†
CNRS ERS 155, INSERM U 249, and INSERM U 336, Ecole Nationale Supe´rieure de Chimie, 8, rue de l’Ecole Normale,
34296 Montpellier Cedex 5, France
Received February 5, 1997X
1-[1-(2-Benzo[b]thiopheneyl)cyclohexyl]piperidine hydrochloride (BTCP, 1) and cocaine bind
to the neuronal dopamine transporter to inhibit dopamine (DA) reuptake. However, on chronic
administration, cocaine produces sensitization, but 1 produces tolerance. Because metabolites
of 1 might be responsible for some of its pharmacological properties, we have identified the
primary metabolites of 1 produced by rat liver microsomes and determined their affinities for
the DA transporter. Five monohydroxylated derivatives (3, 5, 9, 10, 14) and two degradation
compounds (15, 16) were identified as metabolites through comparison with synthetic standards
in HPLC and GC systems. Standards were obtained utilizing synthetic schemes previously
used for the synthesis of phencyclidine metabolites. In vitro, two compounds (3, 5) showed a
high affinity for the DA transporter. These active metabolites might be important in the
pharmacology of 1.
In tr od u ction
apparent tolerance. On the other hand, 1 may be very
slowly degraded in mice and rats, leading to a relatively
high circulating concentration. This would be similar
to the situation with cocaine where continued infusion
induces tolerance.20,21 The role for active metabolites
in chronic administration studies is not known. We
therefore decided to study the biotransformations of 1
initially in vitro. This study was designed to identify
the primary metabolites of 1 obtained from rat liver
microsomes and to measure their affinity for the rat
striatal DA transporter labeled with [3H]BTCP. Puta-
tive hydroxylated metabolites of 1 were synthesized
according to those already known in the PCP series and
used for comparative identification (GC and HPLC) of
microsomal metabolites of 1.
1-[1-(2-Benzo[b]thiopheneyl)cyclohexyl]piperidine HCl
(BTCP, 1) is a phencyclidine (PCP) analogue evoking
behavior related to the stimulation of dopaminergic
transmission by a different route to that of dextroam-
phetamine.1 In a drug discrimination paradigm, 1
generalizes in rats trained to discriminate cocaine from
saline.2 It, like cocaine, binds strongly to the neuronal
dopamine (DA) transporter to inhibit neuronal DA
reuptake.3-5 1 is highly selective for the DA reuptake
complex when compared to PCP as it binds very poorly
to the PCP receptor3,6 inside the N-methyl-D-aspartate
(NMDA)-gated Ca2+ channel. Therefore, cocaine and 1
are both potent indirect dopaminergic agonists.
DA uptake inhibition appears to be important in
mediating the rewarding7 and reinforcing8,9 effects of
cocaine, as evidenced by the highly significant correla-
tion between the inhibition of DA uptake and self-
administration.10,11 It has been recognized that the DA
neuronal transporter can bind dopamine and reuptake
blockers on different binding sites which are not com-
pletely overlapping.12-14 This observation led to the
hypothesis that selective ligands could interfere with
the mechanisms which make cocaine a drug of abuse15
and might help to block cocaine dependency in phar-
macotherapeutic interventions.16 In rodents, 1 and
cocaine have similar dose-dependent behavioral and
neurochemical effects after acute administration. Chroni-
cally, however, in mice and rats cocaine induces a
behavioral sensitization while 1 produces tolerance.17,18
These behavioral differences may be accounted for by
the different binding sites for both drugs on the DA
uptake complex.12-14,19 Alternatively, the metabolites
of 1 may also be involved in its pharmacology. The
enzymes responsible for the metabolism of 1 may be
induced during chronic treatment, thus leading to an
Ch em istr y
On the basis of structural similarities, the in vitro
metabolism of 1 should be similar to that of PCP.
Accordingly, structures of the putative metabolites to
be synthesized as probes for identification were chosen
according to known primary PCP metabolites.22,23 Thus,
we prepared monohydroxylated derivatives of 1 bearing
the hydroxyl substitution at either the â and γ position
from the nitrogen atom in the piperidine ring and at
either the â and γ position from the quaternary carbon
atom in the cyclohexyl ring (Figure 1). In this last case
cis- and trans-diastereomers are possible. Their struc-
tures were defined by the relative position of the
hydroxyl group and the piperidine ring in reference to
the plane of the cyclohexyl ring. (In some previous
papers isomers were inappropriately defined relative to
the aromatic moiety, thus cis and trans were reversed.)
Finally, during identification we had to synthesize three
more compounds to fully identify the primary metabo-
lites produced: the isomeric alcohols 15 and 17 and the
corresponding unsaturated compound 16 (Figure 1). In
our previous synthesis in the arylcyclohexylamines
series, we showed that two general synthetic paths could
be used depending on the desired final structures: (i)
structures unsubstituted at the cyclohexyl ring or
* To whom correspondence should be addressed.
† CNRS ERS 155 and INSERM U 249.
‡ INSERM U 336.
X Abstract published in Advance ACS Abstracts, November 15, 1997.
S0022-2623(97)00078-2 CCC: $14.00 © 1997 American Chemical Society

4020 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 25
Deleuze-Masquefa et al.
Sch em e 2
prepare the ketal 7 via the first path (Scheme 1),
regenerate the corresponding ketone, and reduce it by
diborane or by potassium tri-sec-butylborohydride. This
was done to isolate pure samples of cis-9 or trans-10
alcohols respectively after chromatography. The trans-
alcohol 14 could have been obtained by either of the two
paths. We used the azides route synthesis to obtain a
mixture of cis- and trans-alcohols, but we were unable
to achieve a complete separation of this mixture: only
pure samples of the trans-alcohol 14 were isolated.
Structures of isomers were determined by NMR mea-
surements by comparison with those previously ob-
tained for phenyl analogues in the PCP series.33
F igu r e 1. Metabolites obtained from oxidation of 1 by rat liver
microsomes.
Sch em e 1
Resu lts
Id en t ifica t ion of in Vit r o Met a b olit es. The
biotransformation of 1 by rat liver microsomes was
carried out at physiological pH in the presence of a
NADPH-regenerating system. Figure 1 shows the
molecules extracted from the microsomal system and
identified by comparison with synthesized samples.
Metabolites were identified both by means of a HPLC
separation in heptane/2-propanol coupled to a UV or a
MS detector and by means of a GC fitted with a capillary
column (OV 17) coupled to a flame ionization detector
(FID) or a MS detector. Each analysis contained an
internal standard (18: 4-(2-benzo[b]thiopheneyl)-4-pi-
peridinotetrahydropyran) prepared for this purpose and
added to the microsomal medium before metabolite
extraction. A HPLC analysis with a UV detection at
230 nm separated almost all the derivatives with the
exception of 1 and 16 which were eluted at the same
retention time. However, at a 289 nm wavelength
where 1 absorbs less than at 230 nm, we were able to
detect, but not quantify, compound 16. A GC analysis
did not separate all compounds and in particular did
not allow for the quantification of 1 and 16 because 16
is also a thermolysis product of 1. Nevertheless, it
helped to confirm the presence of compounds difficult
to detect by HPLC analysis. Metabolite identification
was confirmed by HPLC and GC analysis coupled to a
MS detector. The retention times measured by HPLC
and GC analysis are reported in Table 1. They repre-
sent all peaks detected in the time window used.
According to our hypothesis, most of the identified
metabolites (3, 5, 9, 10) were similar to those found for
the PCP in vitro metabolism. As for PCP, no peak
corresponding to a biotransformation of the aromatic
ring was detected.22,23 The cis diastereomer of 14 was
obtained in the form of one diastereomer only and (ii)
structures substituted at the cyclohexyl ring obtained
as cis/trans-diastereomeric pairs. The first path is
Bruylant’s reaction from suitable R-amino nitriles and
Grignard reagents (Scheme 1).24,25 The stereospecificity
of this reaction is well-known: it yields only one
diastereomer whose configuration corresponds to an
equatorial attack of the iminium intermediate by the
Grignard reagent.26-28 For example this path would
yield only the cis compound 9 (Figure 1). The second
path stems from the Schmidt and Curtius reactions
adapted to yield diastereomeric mixtures when the
cyclohexyl moiety is substituted (Scheme 2).29-31 More-
over, depending on conditions (time and/or tempera-
ture), this azide synthesis may yield enriched mixtures
of either of the anticipated diastereomers. Final chro-
matography is always needed to separate the iso-
mers.29,30 The common 2-benzo[b]thiopheneyl Grignard
reagent necessary for both pathways was obtained by
reacting MgBr2 with 2-lithiobenzo[b]thiophene.32 As
anticipated, the piperidinol compounds 3 and 5 were
easily obtained via the first path. However cyclohexanol
derivatives were very difficult to obtain in a pure
isomeric form. Two major problems were encountered:
very low yields in the azide pathway and a great
difficulty in resolving isomeric mixtures. Finally, the
simplest method to obtain alcohols 9 and 10 was to

Primary Metabolites of BTCP
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 25 4021
Ta ble 1. Retention Times of Standards and Internal Standard
Ta ble 2. Amounta of Metabolites in the Microsomal Medium
after a 90 min Incubation of 1 (500 µM)
18a
compd HPLC (mn) GC (mn) compd HPLC (mn) GC (mn)
metabolite
amount (pmol)
metabolite
amount (pmol)
1
3
5
9
10
2.6
14.8
7.4
12.0
23.6
14.2
23.9
22.5
23.9
30.5
14
15
16
17
18
9.0
5.9
3.8
6.8
3.5
10
3
5
765 ( 11
615 ( 11
147 ( 2
15
9
14
141 ( 19
91 ( 2
12.8
9.8
4 ( 1.4
a
Mean of three independent determinations (( SEM).
16.3
a
a
Ta ble 3. Affinities (IC50
,
nM) for the Dopamine Uptake
4-(2-Benzo[b]thiopheneyl)-4-piperidinotetrahydropyran.
Complex and Hill Numbers (nH) of 1 and Metabolites
compd
IC50 ( SEM
nH ( SEM
1
3
5
9
10
8.4
1.01
15.9 ( 4
1.75 ( 0.1
53.4 ( 9.9
113 ( 20.5
1.03 ( 0.1
0.92 ( 0.08
0.92 ( 0.06
0.97 ( 0.09
a
IC50: concentration of unlabeled drug that inhibited 50% of
specific [3H]BTCP binding to rat striatal membranes. Each value
is the mean of at least three independent determinations.
consumption of 1 was not quantifiable because it could
not be completely distinguished from 16 after HPLC
separation; in GC it yields in part compound 16 by
thermolysis. Metabolite production is represented by
nonlinear monophasic curves which reach a plateau
after about 90 min (Figure 2). This nonlinear process
probably reflects the formation of secondary metabolites
which may possibly be dihydroxylated derivatives of 1.
These were observable at much longer retention times
F igu r e 2. Effects of incubation time on metabolites production
by rat liver microsomes. Each value is the mean (( SEM) of
three independent determinations.
not detected although it is the sole diastereomer de-
tected in the biotransformation of PCP.22,23,34 Alcohol
15 apparently originates in the biotransformation of the
unsaturated compound 16 since we detected 15 after
incubation of 16 with rat liver microsomes under the
same conditions as for 1. It is interesting to note that
the isomeric alcohol of 15, compound 17, was at no time
detected under the conditions we used. This might be
due to a regiospecific hydration of the double bond of
16 during incubation. The corresponding unsaturated
compound has also been found in PCP-treated mouse
liver microsomes and its production explained either by
a thermolysis during treatment (GC analysis particu-
larly) or by a Cope elimination following a N-oxida-
tion.35,36 In our case, 1 incubated without microsomes
and treated as usual did not yield such an unsaturated
structure (HPLC analysis). As for PCP35 in vitro
metabolization, at no time did we detect a peak which
could be attributed to 1 N-oxide.
Qu a n tifica tion of m eta bolites obta in ed in Vitr o.
To identify of all the putative metabolites and to obtain
good quantification, the dependency of the biotransfor-
mation of 1 on the pH, time, protein content, and
NADPH concentrations was studied in preliminary
experiments. Optimal conditions, i.e., a maximum
number of detectable and measurable metabolites with-
out inhibition of the microsomal system, were found to
be 1 mg/mL microsomal protein, pH ) 7.4, 500 µM 1,
and a NADPH-regenerating system constituted with 10
mM glucose 6-phosphate (G6P), 0.5 mM NADP, 1 unit
of glucose-6-phosphate dehydrogenase (G6PDH). A 500
µM concentration of 1 probably does not comply with
physiological conditions, but at lower concentrations
detection and/or quantification failed and thus linearity
could not be verified. Interestingly, the concentration
we used here is the same as that previously used to
analyze PCP metabolites (0.25 µmol in 0.5 mL).23
Evolution of the biotransformation was checked after
various incubation times (5, 15, 30, 60, 90 min). The
and have not yet been identified (not shown).
A
degradation of metabolizing enzymes, however, cannot
be excluded. After a 90 min incubation time, two major
metabolites, 10 and 3, were produced, three metabolites,
5, 9, and 15, were present but were at least 4 times less
abundant, and a very low amount of 14 was detected
(Table 2).
Biologica l Activity of th e Hyd r oxyla ted Der iva -
tives of 1. The affinities of the alcoholic compounds
identified as in vitro metabolites to bind for the DA
transporter were measured by inhibition of the specific
binding of [3H]BTCP to rat striatum membranes. Due
to their insolubility in an aqueous medium, the affinities
of compounds 15, 16, and 17 for the dopamine trans-
porter were not measured. Nevertheless, such mol-
ecules are very different from 1. Particularly the lack
of a nitrogen atom in their structure may be assumed
to considerably reduce their affinities for [3H]BTCP
binding sites. Measured affinities are reported in Table
3. Relatively large differences were observed in com-
parison with 1: 3, 9, and 10 displayed affinities
respectively 2, 6, and 13 times lower, but 5 displayed a
4 times higher affinity.
Discu ssion
Eight compounds were prepared, seven of which were
identified among the primary metabolites yielded by
incubation of 1 with rat liver microsome preparations.
We were able to quantify five compounds whose struc-
tures correspond to the monooxidation of cyclohexyl or
piperidine moieties of 1. Another compound, 1-(2-benzo-
[b]thiopheneyl)cyclohexanol 15, was quantified which
might be formed by regioselective hydration of 1-(2-
benzo[b]thiopheneyl)cyclohexene (16). Compound 16
yielded 15, but not 17 after incubation of 500 µM 16
with microsomes. It is unlikely that the unsaturated
or the hydroxylated compound would result from a

4022 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 25
Deleuze-Masquefa et al.
0.35 mol) covered with 100 mL of anhydrous ether. Simulta-
neously, a solution of 2-lithiobenzo[b]thiophene was prepared
at -20 °C, in a nitrogen atmosphere, by the careful addition
of a n-butyllithium solution (1.6 M in hexane) (200 mL, 0.32
mol) to a solution of benzo[b]thiophene (43.2 g, 0.32 mol) in
ether. The resulting solution was refluxed for 2 h, cooled to
room temperature and added under nitrogen to the MgBr2
solution. This, after stirring at room temperature for 30 min,
yielded 2-MgBr-benzo[b]thiophene. 2 (22.2 g, 0.106 mol) in
ether was added dropwise to the Grignard reagent solution,
and the resulting mixture was stirred and refluxed for 16 h.
After being cooled to room temperature, the yielded solution
was poured onto an ice-cold saturated solution of NH4Cl in
water, stirred for 30 min, and treated as follows: decantation,
extraction with ether (2 × 200 mL) and CH2Cl2 (200 mL),
washing of the pooled organic phases with 10% HCl (3 × 200
mL), neutralization of the aqueous phase with 20% NH4OH,
extraction with ether (2 × 200 mL) and CH2Cl2 (200 mL), and
washing of the pooled organic phases with water until neutral-
ity was achieved. The dried (Na2SO4) organic layer was
evaporated to dryness under reduced pressure to yield a solid
material purified by crystallization (CH2Cl2) in the form of a
white solid (15.3 g, 46%): mp (HCl) 185-6 °C; 13C NMR
(CDCl3) δ 25.35, 26.31, 32.13, 33.82, 35.77, 48.28, 67.55, 72.68,
124.93, 127.23, 127.67, 128.65, 131.27, 138.79, 141.67, 142.48;
MS 315 (M+, 16), 272 (20), 214 (52), 185 (42), 173 (25), 147
(100), 134 (25), 115 (32). Anal. (C19H26NOSCl) C, H, N.
1-(3-Hyd r oxyp ip er id in o)cycloh exa n eca r bon itr ile (4).
A mixture of cyclohexanone (4.9 g, 0.05 mol), anhydrous
magnesium sulfate (42 g, 0.35 mol), dimethylacetamide (7 g,
0.8 mol), piperidin-3-ol (8 g, 0.082 mol), and acetone cyanhy-
drin (4.7 g, 0.055 mol), treated as described for 2, yielded a
yellow oil (6.75 g, 54%) used without further purification.
1-[1-(2-Ben zo[b]t h iop h en eyl)cycloh exyl]p ip er id in -3-
ol (5). A Grignard reagent obtained as described for 3 from
Mg turnings (4.8 g, 0.2 mol), 1,2-dibromoethane (26 mL, 0.2
mol), and a 1.6 M solution of n-butyllithium in hexane (125
mL, 0.2 mol) and benzo[b]thiophene (27 g, 0.2 mol) was made
to react with the R-amino nitrile 4 (12.5 g, 0.06 mol) to yield
an oily residue. Purification on a column (aluminoxide Merck
II-III) yielded (CH2Cl2) a white solid (3.8 g, 37%): mp (HCl)
165 °C; 13C NMR (CDCl3) δ 20.55, 23.12, 23.91, 31.37, 32.95,
33.38, 46.63, 51.77, 63.85, 70.28, 122.17, 124.50, 125.03,
125.86, 127.75, 136.25, 138.99, 139.97; MS 315 (M+, 18), 272
(21), 215 (54), 214 (43), 185 (30), 173 (21), 147 (100), 134 (18),
115 (24). Anal. (C19H26NOSCl) C, H, N.
1-(1-P ip er id in o)-4,4-(et h ylen ed ioxy)cycloh exa n eca r -
bon itr ile (6). A mixture of 1,4-hexanedione-monoethylene
ketal (20 g, 0.138 mol), anhydrous magnesium sulfate (76 g,
0.638 mol), dimethylacetamide (11.35 g, 0.129 mol), piperidine
(11 g, 0.129 mol), and acetone cyanhydrin (10.71 g, 0.126 mol),
treated as described for 2, gave a white solid (20.2 g, 63%)
which was used without further purification.
1-[1-(2-Ben zo[b]th iop h en eyl)-4,4-(eth ylen ed ioxy)cyclo-
h exyl]p ip er id in e (7). A Grignard reagent obtained as
described for 3 from Mg turnings (8.4 g, 0.35 mol), 1,2-
dibromoethane (43 mL, 0.33 mol), a 1.6 M solution of n-
butyllithium in hexane (200 mL, 0.32 mol), and benzo[b]-
thiophene (43.2 g, 0.32 mol) was made to react with the
R-amino nitrile 6 (22.2 g, 0.106 mol) to yield an oily residue
purified by precipitation in petroleum ether (21.2 g, 53%).
1-[1-(2-Ben zo[b]th iop h en eyl)-4-oxo-cycloh exyl]p ip er i-
d in e (8). Compound 7 (10 g, 0.028 mol) was dissolved in 5%
H2SO4 (100 mL) and stirred for 16 h at room temperature. The
reaction was halted by neutralization with 20% NH4OH, and
the mixture was treated as follows: extraction with CH2Cl2
(3 × 50 mL), washing of the pooled organic phases with 10%
HCl (3 × 50 mL), neutralization of the aqueous phase with
20% NH4OH, extraction with CH2Cl2 (3 × 50 mL), and washing
of the pooled organic phases with water until neutrality was
achieved. The dried (Na2SO4) organic layers were evaporated
under reduced pressure to give an oily residue. This residue
degradation of 1 during incubation, extraction, or HPLC
separation since when 1 was incubated in the usual
fashion but without microsomes, 15 and 16 were not
detected by the HPLC analysis of extracts. Given the
experimental results, the hypothesis that hydration is
a metabolic step is an attractive concept that requires
additional study.
An analogue to compound 14 trans- (pip/OH) 1-[1-(2-
benzo[b]thiopheneyl)-3-hydroxycyclohexyl]piperidine was
not detected at any time in PCP-treated rat liver
microsomes whereas mouse liver microsomes yielded
the corresponding cis structure.22,23,34
Hydroxylation at the 3- or 4-position of the piperidine
ring of 1 yields high-affinity ligands for the DA trans-
porter. Conversely, hydroxylation at the 3- or 4-position
of the cyclohexyl ring decreases the affinity. Indeed, 3
and 5 are high-affinity ligands for the neuronal dopam-
ine transporter, which is different from the situation
with PCP metabolites: all monohydroxylated com-
pounds are low- or very low-affinity ligands for the PCP
receptor, and consequently none of them has been
considered as an active metabolite.33 Experiments in
vivo are needed to evaluate the potential contribution
of 3 and 5 to the pharmacology of 1.
Con clu sion
The identification of two high-affinity ligands for the
DA transporter after metabolism of 1 by rat liver
microsomes leads to the hypothesis that in vivo me-
tabolism could produce the same metabolites. Thus,
considering the very important role which could be
played by highly active metabolites during chronic
administrations, in vivo biotransformations of 1 are
currently being studied in our laboratories.
Exp er im en ta l Section
Melting points were determined with a Bu¨chi-Tottoli ap-
paratus and remain uncorrected. Elemental analyses were
performed at the CNRS Microanalytical Section in Montpellier
on the hydrochloride salts and were within (0.4% of theoreti-
cal values. 13C NMR spectra were obtained on a Brucker AC
200 spectrometer at 50.323 MHz (Brucker Spectrospin, Wis-
sembourg, France) in 5 mm sample tubes in the FT mode. A
spin-echo sequence (J mod) was used for signal assignments.
HPLC/MS data given for synthetic compounds was obtained
from a Shimadzu (Touzart et Matignon, Les Ulis, France)
LC10AT HPLC coupled to a Fisons Trio-1000 mass spectrom-
eter (Fisons Instruments, Les Ulis, France) coupled to a Fisons
Particle Beam Interface LINC; data was treated by Fisons
MassLab system software. For in vitro experiments, com-
pounds were isolated in their hydrosoluble hydrochloride salt
form. The salts were precipitated by bubbling a dry stream
of HCl in an ethereal solution of bases. After filtration, the
solids collected were dried in vacuum.
1-(4-Hyd r oxyp ip er id in o)cycloh exa n eca r bon itr ile (2).
Acetone cyanohydrin (10.71 g, 0.126 mol) was added to a
stirred mixture of cyclohexanone (12.35 g, 0.126 mol), anhy-
drous magnesium sulfate (75 g, 0.63 mol), dimethylacetamide
(16.63 g, 0.189 mol), and 4-hydroxypiperidine (18.9 g, 0.189
mol). The pastelike mixture obtained was heated at 45 °C for
48 h, cooled to room temperature, poured onto ice, and
vigorously stirred for 30 min. The aqueous mixture was
extracted with ether (3 × 200 mL), and the organic phases
were washed with water until neutrality was achieved. The
dried (Na2SO4) organic layer was concentrated to dryness
under reduced pressure to yield a white solid (22.2 g, 85%)
which was used without further purification.
1-[1-(2-Ben zo[b]t h iop h en eyl)cycloh exyl]p ip er id in -4-
ol (3). A MgBr2 solution was prepared by the gradual addition
of 1,2-dibromoethane (43 mL, 0.33 mol) to Mg turnings (8.4 g,
dissolved in a minimum of CH2Cl2 was precipitated by
petroleum ether at 4 °C to produce 8 in the form of a solid
(5.5 g, 58%).

Primary Metabolites of BTCP
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 25 4023
cis- a n d tr a n s- (p ip /OH) 4-(2-Ben zo[b]th iop h en eyl)-4-
p ip er id in ocycloh exa n ols (9 a n d 10). A 1 M diborane
solution in THF (18.2 mL, 0.018 mol) was added to 8 (2.6 g,
0.0083 mol) dissolved in anhydrous THF (20 mL) in a nitrogen
atmosphere. The solution was subsequently stirred at room
temperature for 1 h, then diluted with ethanol and dissolved
in CH2Cl2, and treated as described for 8 to produce a 60/40
mixture eluted on a silica gel chromatography column (silicium
dioxide, SDS, 70-200 µm). This yielded 1.5 g of the isomeric
mixture and 900 mg of the pure cis isomer 9: mp (HCl) 176
°C; 13C NMR (CDCl3) δ 22.38, 23.16, 27.11, 29.67, 47.66, 63.20,
69.41, 122.23, 124.53, 125.11, 125.95, 127.79, 136.35, 138.98,
139.97; MS 315 (M+, 21), 256 (100), 230 (32), 213 (66), 185
crude residue (180 mg). Chromatography on a silica gel
column (silicium dioxide, SDS, 70-200 µm) in ether/methanol
(98:2) yielded the pure trans isomer 14 (40 mg): mp (HCl) 169
°C; 13C NMR (CDCl3) δ 17.33, 22.32, 22.97, 31.16, 32.87, 38.42,
47.21, 47.52, 65.95, 68.36, 122.12, 124.57, 125.03, 125.87,
128.04, 137.54, 138.76, 139.70; MS 315 (M+, 8), 231 (36), 230
(47), 213 (88), 187 (45), 174 (26), 147 (100), 134 (41), 115 (53).
Anal. (C
19H26NOSCl) C, H, N.
1-(2-Ben zo[b]th iop h en eyl)cycloh exa n ol (15). A Grig-
nard reagent obtained as described for 3 from Mg turnings
(4.8 g, 0.2 mol), 1,2-dibromoethane (25 mL, 0.2 mol), a 1.6 M
solution of n-butyllithium in hexane (125 mL, 0.2 mol), and
benzo[b]thiophene (27 g, 0.2 mol) was made to react with
cyclohexanone (10.5 mL, 0.1 mol) to yield a solid residue (43.4
g) purified on silica gel in dichloromethane: mp 88 °C; 13C
NMR (CDCl3) δ 22.15, 25.28, 39.53, 72.34, 118.37, 122.23,
123.27, 123.82, 124.03, 138.94, 139.77, 155.15; MS 232 (M+,
40), 189 (82), 176 (33),161 (42), 147 (35), 134 (100). Anal.
(C14H16OS) C, H.
1-(2-Ben zo[b]th iop h en eyl)cycloh exen e (16). A mixture
(3 mL) of H2SO4/acetic acid (20:80) was added to fused 15 (3.48
g, 0.0015 mol). After 30 s of stirring and immediate cooling,
ether (9 mL) and water (15 mL) were added. The organic
phase was washed with a Na2CO3 solution and water until
neutrality was reached, dried (Na2SO4), and evaporated under
reduced pressure to yield 16 as a white solid (3.23 g, 100%):
mp 90 °C; 13C NMR (CDCl3) δ 22.03, 22.58, 25.78, 26.91,
117.68, 121.91, 123.09, 123.98, 124.06, 126.98, 131.50, 138.24,
140.47, 146.72; MS 214 (M+, 100), 199 (23), 186 (53), 185 (69),
171 (20), 147 (21), 134 (20), 115 (14). Anal. (C14H14S) C, H.
2-(2-Ben zo[b]th iop h en eyl)cycloh exa n ol (17). A 0.77 M
solution of diborane in THF (13 mL, 0.01 mol) was added
dropwise under a nitrogen atmosphere to 16 (2.14 g, 0.01 mol)
dissolved in anhydrous THF. The solution was then stirred
at room temperature for 2 h and the reaction stopped by
addition of 15% H2O2 (2 mL). Separation of the two isomeric
alcohols was achieved on a chromatography column (silicium
dioxide, SDS, 70-200 µm) in a petroleum ether/ether mixture
(70:30) to yield pure 17 (0.92 g, 40%): mp 122 °C; 13C NMR
(CDCl3) δ 24.73, 25.77, 33.79, 34.13, 49.04, 74.79, 121.17,
122.29, 122.99, 123.77, 124.25, 138.90, 139.73, 148.27; MS 232
(M+, 45), 173 (15),161 (42), 148 (48); 147 (100), 134 (27).
Micr osom a l P r ep a r a tion . Rats deprived from food for 24
h were killed by decapitation. Their livers were perfused by
a 10 mM Na2HPO4, 9% NaCl, 1 mM EDTA (pH 7.4) buffer,
removed, washed, and cut up finely with scissors. The liver
tissue was homogenized with a Elvehjem potter (10 strokes,
700 rpm at 4 °C) in three volumes of a 0.25 M sucrose, 10 mM
EDTA, 0.1 mM DTT, 50 mM Na2HPO4/NaH2PO4 (pH 7.4)
buffer. The homogenate was centrifuged at 1000g for 10 min,
the supernatant was centrifuged at 10000g for 20 min, and
the resulting supernatant was centrifuged at 105000g for 60
min. All centrifugation stages were performed at 4 °C. The
pellet was suspended in three volumes of a 100 mM K2HPO4/
KH2PO4, 1 mM DTT, 1 mM EDTA, and 20% glycerol (pH 7.4)
buffer and centrifuged at 105000g for 60 min. The final pellet
was suspended in one volume of the same buffer, the protein
concentration was determined with the Pierce’s reagent37 using
BSA as standard, and the microsomes were stored at -80 °C
until used.
(31), 173 (25), 147 (70), 134 (23), 115 (34). Anal. (C19H26
NOSCl) C, H, N.
-
A 0.5 M solution of potassium tri-sec-butyl borohydride in
THF (165 mL), diluted with anhydrous THF (66 mL), was
added dropwise at -78 °C to 8 (1.1 g, 0.0035 mol) dissolved in
anhydrous THF (16.5 mL) in a nitrogen atmosphere. The
solution was stirred for 5 h at -78 °C and then diluted with
water (11 mL) and ethanol (33 mL), and 6 N NaOH (22 mL)
and 30% H2O2 (22 mL) were added. The resulting aqueous
solution was extracted with CH2Cl2 (2 × 50 mL), and the
pooled organic phases were treated as described for 8 to give
a 20/80 mixture of cis/trans isomers (1 g, 90%). In the same
conditions as above a chromatography on a silica gel column
(silicium dioxide, SDS, 70-200 µm) yielded 600 mg of the
isomeric mixture and 400 mg of the pure trans isomer 10: mp
(HCl) 182 °C; 13C NMR (CDCl3) δ 22.22, 23.05, 30.95, 31.49,
31.61, 48.02, 67.96, 68.72, 122.28, 124.45, 125.22, 126.07,
127.68, 136.14, 139.01, 140.07; MS 315 (M+, 11), 256 (73), 230
(37), 213 (73), 185 (44), 173 (34), 147 (100), 134 (36), 115 (50).
Anal. (C19H26NOSCl) C, H, N.
1-(2-Ben zo[b]th iop h en eyl)-1,3-cycloh exa n ed iols (11). A
Grignard reagent obtained as described for 3 from Mg turnings
(6.21 g, 0.25 mol), 1,2-dibromoethane (31.8 mL, 0.26 mol), a
1.6 M solution of n-butyllithium in hexane (160 mL, 0.26 mol),
and benzo[b]thiophene (35 g, 0.26 mol) was made to react with
3-hydroxycyclohexanone (7.38 g, 0.065 mol) to yield 16.25 g of
an oily residue containing two isomeric diols.
3-Azid o-3-(2-ben zo[b]th iop h en eyl)cycloh exa n ols (12).
Trichloroacetic acid (10.8 g, 0.065 mol) dissolved in CHCl3 (10
mL) was added dropwise to a stirred suspension of sodium
azide (850 mg, 0.013 mol) covered with CHCl3. The solution
was cooled to -20 °C and well stirred until the medium
resembled a jelly; 11 (16.25 g, 0.065 mol) was then dissolved
in CHCl3 (10 mL) and added dropwise, after which the medium
was maintained at -20 °C. It was then vigorously stirred for
3 h and Na2CO3 was added until gas evolvement ceased. The
mixture was filtered, and the solvent was evaporated under
reduced pressure to give 9 g of crude isomeric alcohols in the
form of an oily residue.
cis- a n d tr a n s-3-Am in o-3-(2-ben zo[b]th iop h en eyl)cy-
cloh exa n ols (13). The crude oily azide mixture (3 g, 0.011
mol) dissolved in THF (30 mL) was added dropwise to a stirred
suspension of AlLiH4 (450 mg, 0.011 mol) in THF (50 mL) in
a nitrogen atmosphere. The solution was stirred for 16 h at
room temperature, and then a 5% NaOH (5 mL) solution was
carefully added. The mixture was stirred for 15 min and
filtered on Celite. After evaporation to dryness under reduced
pressure, the residue was dissolved in 10% HCl and rinsed
with ether. The acidic phase was neutralized with 20% NH4-
OH and extracted with CH2Cl2 (3 × 50 mL). The dried
(MgSO4) organic phases were evaporated under reduced pres-
sure to give a brownish oil (500 mg) containing the crude
diastereoisomeric primary amines mixture 13.
tr a n s- (p ip /OH) 1-[2-(Ben zo[b]th iop h en eyl)-3-h yd r oxy-
cycloh exyl]p ip er id in e (14). The crude amines mixture 13
(500 mg, 0.002 mol) in a solution of 1,5-dibromopentane (466
mg, 0.002 mol) and K2CO3 (550 mg, 0.004 mol) in acetonitrile
(10 mL) was refluxed for 48 h. Then hexamethylphosphora-
mide (HMPT) (9 mL) was added and the reflux maintained
for an additional 48 h. After filtration, dissolution in 10% HCl,
and ether extraction (3 × 15 mL), the resulting aqueous phase
was neutralized by 20% NH4OH and extracted by CH2Cl2 (3
× 15 mL). Drying (MgSO4) and solvent evaporation gave a
In cu ba tion a n d Extr a ction . A typical incubation medium
contained 1 mg of microsomal protein, 100 mM potassium
phosphate (pH 7.4) buffer, 10 mM MgCl2, and a NADPH-
generating system (10 mM G6P, 0.5 mM NADP and 1 unit
G6PDH) in a final volume of 1 mL. Incubation mixtures were
preincubated in the presence of a NADPH-generating system
at 37 °C for 15 min. The reaction was initiated by the addition
of 500 µM of 1. Incubations were performed in air at 37 °C in
a shaking bath. Reactions were stopped by adding 1 mL of
0.2 N NaOH and calibrated by adding 100 nmol of 18 as
internal standard in each reaction tube. Extraction of 1, 18,
and metabolites was performed as follows and repeated three
times: addition of 2 mL of ethyl acetate, vortexing for 1 min,
centrifugation at 4000g for 10 min at 4 °C. The organic layers
were pooled, filtered, and evaporated under nitrogen for 12 h

4024 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 25
Deleuze-Masquefa et al.
at 60 °C. The dried residue was solubilized in heptane before
analysis (50 µL for GC analysis, 400 µL for HPLC analysis).
Ack n ow led gm en t. This work was supported by
CNRS, INSERM, and grant MESR 94V0255 (to J .V.).
We would like to thank Patrick Graffin for his as-
sistance in obtaining the HPLC/MS spectra and Brian
Bolger and Daniel Fisher for their careful reading of
the manuscript.
Id en tifica tion , Qu a n tifica tion , a n d Extr a ction yield
of 1, Meta bolites, Syn th etic Com p ou n d s, a n d In ter n a l
Sta n d a r d . Compounds from rat liver microsomal prepara-
tions were recovered by extraction with ethyl acetate. 1,
internal standard 18, and metabolites were generally recov-
ered with a very good yield with the exception of 3. Standard
calibration curves were determined from peak surface analysis
of standards solutions in heptane of increasing amounts of
metabolites (0.3 nmol to >300 nmol) and a fixed amount of 18
(100 nmol). Linear regression lines were obtained with
correlation coefficients varying from 0.991 to 0.996. The
metabolites were identified and quantified through reference
to calibration standards (retention time and peak area). To
determine the extraction yield, 500 nmol of 1 and 100 nmol of
each compound were added to the microsomal preparation
previously stopped by 1 mL of 0.2 N NaOH and then extracted
as described above and analyzed by HPLC separation. The
areas were compared to those obtained after the separation
of 500 nmol of 1 and 100 nmol of each compound in its base
form. The extraction yield did not vary regardless of the
amount of 1 used or regardless of the relative proportion of 1
and synthetic compounds. Finally, the recovery yields (% (
SEM) of synthetic compounds after extraction with ethyl
acetate from microsomal medium were as follows: 1, 100 (0.4);
3, 87 (1); 5, 94 (1.1); 9, 92 (1.2); 10, 98 (1); 14, 91 (1.4); 15, 98
(1.6); 16, 99 (1.5); 17, 96 (1.7); 18, 100 (1.4).
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An a lysis Con d ition s. HP LC system : Shimadzu LC-
10AD pump (Touzart et Matignon, Les Ulis, France); Lichro-
cart Lichrospher 100 NH2 column (5 µm, 250 × 4 mm, 40 °C)
(Merck-Cle´venot, Nogent/Marne, France); Shimadzu SPD-6A
UV spectrophotometric detector (at 230 or 289 nm); heptane/
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GC system : Shimadzu gas chromatograph/FID GC-14B;
Quadrex 007-OV-1701 fused silica capillary column (25 m ×
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