Rational design of a novel peripherally-restricted, orally active CB 1 cannabinoid antagonist containing a 2,3-diarylpyrrole motif

Article abstract of DOI:10.1016/j.bmcl.2010.06.017

A new series of 2,3-diarylpyrroles have been prepared and evaluated as CB₁ antagonists. Modulation of the topological polar surface area allowed the identification of high affinity peripherally-restricted CB ₁ antagonists. Compound 1

Full text of DOI:10.1016/j.bmcl.2010.06.017

Bioorganic & Medicinal Chemistry Letters 20 (2010) 4573–4577
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Rational design of a novel peripherally-restricted, orally active CB1
cannabinoid antagonist containing a 2,3-diarylpyrrole motif
Laurent Hortala a, Murielle Rinaldi-Carmona a, Christian Congy a, Laurent Boulu a, Freddy Sadoun b,
Gérard Fabre b, Olivier Finance c, Francis Barth a,
*
a Exploratory Unit, Sanofi-aventis R&D, 371 rue du Professeur Joseph Blayac, 34184 Montpellier, France
b Disposition, Safety and Animal Research, Sanofi-aventis R&D, 371 rue du Professeur Joseph Blayac, 34184 Montpellier, France
c Prospective and Strategic Initiative, Sanofi-aventis R&D, 371 rue du Professeur Joseph Blayac, 34184 Montpellier, France
a r t i c l e i n f o
a b s t r a c t
Article history:
A new series of 2,3-diarylpyrroles have been prepared and evaluated as CB1 antagonists. Modulation of
the topological polar surface area allowed the identification of high affinity peripherally-restricted CB1
antagonists. Compound 11, obtained after further optimization of the metabolic profile displayed very
low brain penetration, yet was able to reverse CP55940-induced gastrointestinal transit inhibition fol-
lowing oral administration.
Received 30 April 2010
Revised 1 June 2010
Accepted 2 June 2010
Available online 8 June 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Peripherally-restricted cannabinoid
antagonist
Pyrrole
CB1 antagonist
CB1 cannabinoid receptor antagonists are a promising approach
in the treatment of metabolic disorders. Their efficacy was recently
demonstrated not only in animal models,1 but also in several
clinical studies targeting different patient populations including
obese dyslipidemic2 and diabetic3 patients. However, rimonabant4
(Fig. 1), the first CB1 antagonist to reach the market, was with-
drawn in 2008 by the European Medicines Agency. The agency
considered that the experience with rimonabant had indicated
that the incidence of serious psychiatric disorders (mainly depres-
sion) may be more common than in the clinical trials and con-
cluded that the benefits of rimonabant no longer outweighed its
risks.
One attractive approach to the development of safer CB1 antag-
onists would be the identification of peripherally-restricted drugs.
While psychiatric side effects are most likely centrally mediated,
several lines of evidence indicate that the mechanism of action of
CB1 antagonists on metabolic disorders involved both central and
peripheral pathways.5,6 CB1 receptors expressed in adipocytes,7
in the liver8 but also in skeletal muscle9 and in pancreatic b-cells10
maybe involved in the effects of CB1 antagonists on body weight,
dyslipidemia, hepatic steatosis, and glucose metabolism.
CB1 antagonist.11 However, it was recently demonstrated that the
compound was actually brain penetrant and acted on body weight
by a non-CB1 mechanism.12
Recently, URB447 ([4-amino-1-[(4-chlorophenyl) methyl]-
2-methyl-5-phenyl-1H-pyrrol-3-yl] phenyl-methanone) was char-
acterized as a mixed CB1 antagonist (IC50 = 313 nM)/CB2 agonist
(IC50 = 41 nM). URB447 was not detected in brain following sys-
temic acute administration (20 mg/kg ip) but effectively reduced
food intake and body weight gain in genetically obese ob/ob mice
following ip administration over a 14 day period.13
More recently a more potent CB1 antagonist (IC50 = 0.19 nM)/
inverse agonist (IC50 = 0.10 nM), (5-(4-chloro-phenyl)-1-(2-chloro-
phenyl)-3-[4-(4-fluoro-phenyl)-4-hydroxy-piperidine-1-carbo-
nyl]-1H-pyrazole-4-carboxamide) was identified with higher
polar surface and lower lipophilicity than rimonabant. A pharma-
cokinetic study demonstrated that in mice, 6 h after oral adminis-
tration a 10-fold higher concentration in plasma than brain was
reached. Interestingly, this compound showed effects on body
weight loss comparable to rimonabant after subchronic oral
administration (10 mg/kg) in the DIO (diet induced-obese) mouse
model. However, some contribution of central effect could not be
definitively ruled out.14
At least three putative CB1 peripherally-restricted antagonists
have been described in the literature. The triazole derivative
LH-21 (5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-3-hexyl-1H-1,2,
4-triazole) was first described as a peripherally acting neutral
However, the need for a potent, selective and orally active
peripherally-restricted CB1 antagonist remains clear.
The 2,3-diarylpyrrole scaffold was chosen as a starting point for
our rational design approach to peripherally-restricted CB1 antag-
onists. This new scaffold was discovered during our extensive
* Corresponding author.
E-mail address: francis.barth@sanofi-aventis.com (F. Barth).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.06.017

4574
L. Hortala et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4573–4577
O
O
N
R
O
X
N
N
NH2
N
H
N
N
Cl
Cl
Cl
Cl
Cl
Cl
compounds 2 to 11
1 SR141716 (rimonabant)
Figure 1. Structure of rimonabant and polar pyrrole bioisostere compounds 2–11.
efforts of scaffold hopping in which the pyrazole ring of SR141716
was replaced by various heterocyclic five-membered rings.15,16
1,5-Diarylpyrroles CB1 antagonists have also been described in
the patent literature.17 However, the 2,3-diarylpyrrole series ap-
peared as an ideal starting point to design CB1 peripherally-re-
stricted antagonists because high potency (sub-nanomolar
activity) could be obtained in the series. This was important be-
cause a loss of affinity was expected to occur during the optimiza-
tion to a less brain-penetrant compound. Moreover the nitrogen
substituent could be easily modulated via different types of N-
alkylation reaction, affording an additional modification point in
the SAR exploration.
Based on several studies conducted over the years, a picture of
the molecular determinants of blood–brain barrier (BBB) perme-
ation is now available. In brief, increasing hydrogen bonding capac-
ity, molecular weight and polar surface area (PSA), introducing
acidic function or decreasing lipophilicity (c log P < 1) are all asso-
ciated with decreased brain penetration.18,19 Unfortunately,
increasing polarity, molecular charge and lowering log P is also
usually associated with a decrease in affinity towards the CB1
receptor. Furthermore several of the factors which decrease
blood–brain barrier permeation also decrease intestinal barrier
permeation and therefore negatively impact oral bioavailability.
We decided to focus on the topological polar surface area (TPSA),
a molecular determinant which can be modulated by simple struc-
tural changes, is easily predicted using molecular polar surface cal-
culation,20 and has been shown to be a dominating determinant for
brain penetration.21
N-Methyl compounds 2, 3, and 4 were synthesized by alkyl-
ation of the pyrrole B with methyl iodine in the presence of potas-
sium carbonate in dichloromethane (DCM) followed by hydrolysis
of the ester group in standard basic condition, acidification and
coupling with 4-substituted-4-carboxamidopiperidine in the pres-
ence of 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium
hexafluorophosphate (HBTU) in DMF. On the opposite, carboxylic
acids 6 and 7 were obtained by conversion of the ester B in the cor-
responding amide as described above, alkylation of the pyrrole
nitrogen by methyl acrylate using catalytic amount of TritonB as
a base for compound 6, or by alkylation with methyl 5-chloro-
2,2-dimethyl valerate in refluxing acetone in the presence of potas-
sium carbonate in the case of 7. Mild hydrolysis conditions of the
esters were required to generate the carboxylic acids in the last
step.
For the introduction of the sulfonyl group, methyl-vinylsulfone
was used as a Michael acceptor in the same conditions as described
for the acrylate reagent above; mild hydrolysis of the ester was
performed with lithium hydroxide in a THF/water mixture at
90 °C followed by the usual coupling conditions to afford com-
pound 8. Similarly, compound 5 was obtained when acrylonitrile
was used as Michael acceptor. To prepare the methylsulfonamides
9–11, the intermediate cyanoester C was selectively reduced to
aminoester using sodium borohydride in presence of Cobalt(II)
chloride hexahydrate.25 The sulfonamides 9–11 were finally ob-
tained by mesylation of the amino group followed by the standard
sequence hydrolysis/acidification/coupling.
In vitro, the affinity of compounds 2–11 for mice brain CB1
receptors was evaluated as described by Rinaldi-Carmona et al.,4
while brain penetration was assessed using the ex vivo binding
in mice described above. The results are reported in Table 1, to-
gether with calculated TPSA.
Starting from the high affinity compound 2 we decided first to
replace the phenyl substituent of the piperidine ring by less lipo-
philic groups (morpholine, piperidinyl)27; those modifications
afforded diversely active compounds. Introduction of a morpholine
ring (compound 3) increased TPSA but reduced the CB1 affinity
(about 50-fold). Interestingly, the piperidinyl analog 4 was even
more potent than compound 2. In line with its similar TPSA, the
compound was brain penetrant too.
Our strategy was based on a conservation of the positions of
three substituents (chlorophenyl, dichlorophenyl, and carbonyl
group) connected around the pyrrole core.
Compound 215 (Table 1) was selected for further modifications
with the objective of increasing TPSA. During our optimization pro-
cess, we routinely used brain ex vivo binding to roughly assess
brain penetration. The compound was administered iv (10 mg/
kg) to mice 30 min before they were killed by decapitation. Then
the inhibition of [3H]-CP55940 binding was performed in homoge-
nized brain (without the cerebellum) as previously described.22 In
this assay, compound 2 inhibited 86% of CP55940 binding, indicat-
ing a good brain penetration, in accordance with its calculated
TPSA of 68 Å2.
In order to further increase TPSA, we decided to introduce addi-
tional heteroatoms in the molecule by substituting the pyrrole
nitrogen with alkylene groups bearing various polar substituents.
Introduction of the ethylcyano substituent afforded a reasonably
potent compound 5 with a moderate brain penetration, in line with
its TPSA of 83 Å2. Introduction of carboxylic acids groups afforded
even more polar compounds (TPSA above 100 Å2) but unfortu-
nately to the detriment of affinity. Interestingly, increasing chain
length and steric bulk (gem-dimethyl group) around the acid func-
tion as in compound 7 does not alter the binding compared to 6.
The compounds 2–11 were synthesized as shown in Scheme 2
from the key intermediate pyrrole B.23 The latter was easily acces-
sible by a Suzuki coupling with 4-iodo-2,3-dihydropyrrole A using
2,4-dichlorophenyl boronic acid followed by a one pot detosylation
and aromatization with two equivalents of 1,8-diaza-bicy-
clo[5.4.0]undecene (DBU) in hot dimethyl formamide (DMF) as
indicated in Scheme 1. The 4-iodo-2,3-pyrrole intermediate A
was accessible from homopropargylic tosylamide according to a
brief and straightforward approach described in the litterature.24

L. Hortala et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4573–4577
4575
Table 1
rCB1 and rCB2 in vitro binding, ex vivo rat brain binding and TPSA by representative 1-[5-(4-Chloro-phenyl)-4-(2,4-dichloro-phenyl)-1H-pyrrole-2-carbonyl]-piperidine-4-
carboxamides
O
R
X
N
NH2
N
O
Cl
Cl
Cl
rCB1 binding IC50a (nM) rCB2 binding % inhibition at 1 Ma Ex vivo brain binding % inhibitionb TPSAc (Å2)
l
Compound
R
X
1
(SR141716)
—
2.5
66%
100%
50
2
3
4
Me
Me
Me
0.7
17%
86%
68
N
N
N
N
N
N
N
N
N
34
42%
48%
18%
14%
52%
26%
16%
27%
39%
32%
96%
39%
nt
81
O
0.2
8
72
5
–(CH2)2CN
83
6
–(CH2)2CO2H
180
160
0.4
1.4
4.3
1.0
109
109
114
126
126
126
7
–(CH2)3C(CH3)2CO2H
–(CH2)2SO2Me
nt
8
19%
18%
33%
28%
9
–(CH2)3NHSO2Me
–(CH2)3NHSO2Me
–(CH2)3NHSO2Me
10
11
F
F
nt, not tested.
a
rCB1 from rat brain synaptosomal membranes, rCB2 from rat spleen, as described in Ref. 4.
At 10 mg/kg, iv. Determined as described in Ref. 22.
b
c
Calculated TPSA using ACDv9 software.26
Introducing the sulfonyl and amino sulfonyl groups further in-
in plasma and brain after systemic administration of compound
11 (10 mg/kg po) to male Swiss CD1 mice. Plasma levels peaked
2 h after administration (Cmax = 570 ng/mL), while the maximal
concentration in brain was obtained after 6 h and did not exceed
6.4 ng/g of tissue. Brain to plasma AUC(0–24h) ratio was 0.01. Com-
bined with the intravenous route (3 mg/kg), the oral bioavailability
of compound 11 was assessed at 37% in Tween 80/DMSO/water
(0.5:10:89.5; v/v/v).
In line, with this low brain penetration, and to the contrary of
rimonabant, compound 11 was also ineffective in reversing
CP55940-induced hypothermia in mice (Table 2).
Compound 11 was also a potent ligand for human recombinant
CB1 receptors expressed in CHO cells (IC50 = 1.4 nM) and was selec-
creased the TPSA to 126 Å2 and afforded the most interesting com-
pounds. Compounds 8 and 9 are both very potent, and display very
low ex vivo brain binding compared to 1.
Oral activity of compound
9 was then assessed in the
intestinal transit test22 (Table 2). Compared to rimonabant, 9
was only moderately active in this assay (Table 2). In vitro
microsome metabolism studies allowed us to pin-point an
important oxidation on the piperidine rings.28 To prevent this
metabolic pathway, we attempted to block the 4 position of
the piperidyl group by introducing gem-dimethyl or gem-di-
fluoro groups.
Compared to analog 9 the respective resulting compounds 10
and 11 have increased microsomal stability while retaining
high affinity and low brain penetration. Both were significantly
more active following oral administration in the intestinal transit
test.
tive versus human and rat CB2 receptors (IC50 > 1 lM). Compound
11 also dose-dependently antagonized CP55940-induced inhibi-
tion of forskolin-stimulated cAMP accumulation in U373-MG
cells29 with an EC50 of 15 nM.
We decided to focus on the most metabolically stable com-
pound 11 to assess brain penetration. Drug levels were measured
Complementary in vitro functional test using CHO cells co-
expressing hCB1 receptor and a cAMP response element (CRE)-

4576
L. Hortala et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4573–4577
luciferase reporter gene30 showed that compound 11 behaved as a
weak CB1 inverse agonist (10% stimulation at 1 M).
l
In conclusion, we presented a successful strategy around a
pyrrole core leading to a series of high affinity analogs with sig-
nificantly increased TPSA compared to rimonabant. Our investi-
CO2Me
Cl
H
N
O
S
CO2Me
O
a)
b)
N
gations of different types of functionalities attached to
a
Cl
variable carbon linker allowed us to identify a sulfonamide
diarylpyrrole 11, presenting a non-brain penetrant CB1 antago-
nist profile with a 100-fold higher exposure in plasma than
brain. In order to assess the potential therapeutic value of the
peripheral compound 11 versus rimonabant, complementary
studies are under investigation in several models of metabolic
disorders.
Cl
I
Cl
B
A
Scheme 1. Reagents and conditions: (a) 1 equiv 2,4-dichlorophenyl boronic acid,
Pd Tetrakis, 3 equiv Na2CO3 aq, toluene/MeOH, 70 °C; (b) 2 equiv DBU, DMF, 110 °C.
O
H
O
R1
O
R1
O
CO2Me
N
N
N
N
N
R2
R2
b,c
Ar5
Ar5
Ar5
Ar4
Ar4
Ar4
7
2, 3, 4
a
j,k
O
CO2Me
H
S
R1
O
N
O
N
H
N
CO2Me
b,c
R2
f
Cl
N
Cl
Ar5
Ar5
Ar4
Ar4
Cl
B
d,e
b',c
O
g
N
O
O
N
S
HO
R1
R1
O
R1
O
O
CO2Me
b,c
N
N
N
N
N
N
R2
R2
N
R2
Ar5
Ar5
Ar5
Ar5
Ar4
Ar4
Ar4
Ar4
C
8
5
6
h,i
O
S
O
S
O
N
O
N
H
H
R1
R2
O
CO Me
b,c
2
N
N
N
Ar5
Ar5
Ar 4
Ar4
9, 10, 11
Scheme 2. Reagents and conditions: (a) 2 equiv MeI, 3 equiv K2CO3, DCM, rt; (b) 10 equiv KOH, MeOH/water, reflux, HCl aq; (c) 1.3 equiv 4-substituted-4-
acetamidopiperidine, DiPEA, HBTU, HOBt, DMF, rt; (d) 5 equiv methyl acrylate, TritonB, dioxane, reflux; (e) 10 equiv KOH, MeOH/water, reflux; HCl aq; (f) 5 equiv methyl
vinylsulfone, TritonB, dioxane,70 °C; (g) 5 equiv acrylonitrile, TritonB, dioxane, reflux; (h) 5 equiv NaBH4, 2 equiv CoCl26H2O, THF/water, 0 °C to rt; (i) 1.1 equiv MsCl, 2 equiv
NEt3, DCM, rt; (j) methyl-5-chloro-2,2-dimethyl valerate, K2CO3, acetone, reflux; (k) 10 equiv LiOH, THF/water, reflux, HCl aq.

L. Hortala et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4573–4577
10. Nakata, M.; Yada, T. Regul. Pept. 2008, 145, 49.
4577
Table 2
11. Pavon, F. J.; Bilbao, A.; Hernandez-Folgado, L.; Cippitelli, A.; Jagerovic, N.;
Abellan, G.; Rodriguez-Franco, M. I.; Serrano, A.; Macias, M.; Gomez, R.;
Navarro, M.; Goya, P.; Rodriguez de Fonseca, F. Neuropharmacology 2006, 51,
358.
Reversion of CP-induced intestinal transit inhibition and hypothermia
Compound
Intestinal transita
DE50 po mg/kg
Hypothermiab
DE50 po mg/kg
12. Chen, R. Z.; Frassetto, A.; Lao, J. Z.; Huang, R. R.; Xiao, J. C.; Clements, M. J.;
Walsh, T. F.; Hale, J. J.; Wang, J.; Tong, X.; Fong, T. M. Eur. J. Pharmacol. 2008,
584, 338.
13. LoVerme, J.; Duranti, A.; Tontini, A.; Spadoni, G.; Mor, M.; Rivara, S.; Stella, N.;
Xu, C.; Tarzia, G.; Piomelli, D. Bioorg. Med. Chem. Lett. 2009, 19, 639.
14. Receveur, J. M.; Murray, A.; Linget, J. M.; Norregaard, P. K.; Cooper, M.; Bjurling,
E.; Nielsen, P. A.; Hogberg, T. Bioorg. Med. Chem. Lett. 2010, 20, 453.
15. Barth, F.; Congy, C.; Hortala, L.; Rinaldi-Carmona, M. Patent Application
WO2006/024777, 2006.
1 (SR141716)
9
10
11
1
0.6
51% at 10 mg/kg
4.1
2.4
1% at 30 mg/kg
0% at 30 mg/kg
0% at 30 mg/kg
nt, not tested.
Reversion of CP55940 (0.15 mg/kg, ip) induced inhibition of intestinal transit.22
Reversion of CP55940 (0.3 mg/kg, ip) induced hypothermia.22
a
b
16. Barth, F. Annu. Rep. Med. Chem. 2005, 40, 104.
17. Berggren, A. I. K. Patent Application WO2004/058249, 2004.
18. Clark, D. E. Annu. Rep. Med. Chem. 2005, 40, 403.
Acknowledgment
19. Gleeson, M. P. J. Med. Chem. 2008, 51, 817.
20. Ertl, P.; Rohde, B.; Selzer, P. J. Med. Chem. 2000, 43, 3714.
21. Kelder, J.; Grootenhuis, P. D. J.; Bayada, D. M.; Delbressine, L. P. C.; Ploemen, J. P.
Pharm. Res. 1999, 16, 1514.
The authors express their gratitude to Dr. D. Aldous for useful
discussions and suggestions.
22. Rinaldi-Carmona, M.; Barth, F.; Congy, C.; Martinez, S.; Oustric, D.; Pério, A.;
Poncelet, M.; Maruani, J.; Arnone, M. l.; Finance, O.; Soubrié, P.; Le Fur, G. J.
Pharmacol. Exp. Ther. 2004, 310, 905.
References and notes
23. Barth, F.; Congy, C.; Hortala, L.; Rinaldi-Carmona, M. Patent Application
WO2008/068423, 2008.
24. Knight, D. W.; Redfern, A. L.; Gilmore, J. J. Chem. Soc., Perkin Trans. 1 2002, 622.
25. Könekamp, T.; Ruiz, A.; Duwenhorst, J.; Schmidt, W.; Borrmann, T.; Stohrer, W.
D.; Montforts, F. P. Chem. Eur. J. 2007, 13, 6595.
26. ACD/Labs’ logD/Solubility Suite v9 software available from Advanced
Chemistry Development, Inc. Available from: http://www.acdlabs.com.
27. Van de Westeringh, C.; Van Daele, P.; Hermans, B.; Van Der Eycken, C.; Boey, J.;
Janssen, P. A. J. J. Med. Chem. 1964, 7, 619.
1. Gary-Bobo, M.; Elachouri, G.; Gallas, J. F.; Janiak, P.; Marini, P.; Ravinet-Trillou,
C.; Chabbert, M.; Cruccioli, N.; Pfersdorff, C.; Roque, C.; Arnone, M.; Croci, T.;
Soubrié, P.; Oury-Donat, F.; Maffrand, J. P.; Scatton, B.; Lacheretz, F.; Le Fur, G.;
Herbert, J. M.; Bensaid, M. Hepatology 2007, 46, 122.
2. Despres, J. P.; Golay, A.; Sjostrom, L. N. Engl. J. Med. 2005, 353, 2121.
3. Scheen, A. J.; Finer, N.; Hollander, P.; Jensen, M. D.; Van Gaal, L. F. Lancet 2006,
368, 1660.
4. Rinaldi-Carmona, M.; Barth, F.; Héaulme, M.; Shire, D.; Calandra, B.; Congy, C.;
Martinez, S.; Maruani, J.; Néliat, G.; Caput, D.; Ferrara, P.; Soubrié, P.; Brelière, J.
C.; Le Fur, G. FEBS Lett. 1994, 350, 240.
5. Cota, D.; Marsicano, G.; Tschöp, M.; Grubler, Y.; Flachskamm, C.; Schubert, M.;
Auer, D.; Yassouridis, A.; Thöne-Reineke, C.; Ortmann, S.; Tomassoni, F.;
Cervino, C.; Nisoli, E.; Linthorst, A. C. E.; Pasquali, R.; Lutz, B.; Stalla, G. K.;
Pagotto, U. J. Clin. Invest. 2003, 112, 423.
6. Nogueiras, R.; Veyrat-Durebex, C.; Suchanek, P. M.; Klein, M.; Tschöp, J.;
Caldwell, C.; Woods, S. C.; Wittmann, G.; Watanabe, M.; Liposits, Z.;
Fekete, C.; Reizes, O.; Rohner-Jeanrenaud, F.; Tschöp, M. H. Diabetes 2008,
57, 2977.
7. Bensaid, M.; Gary-Bobo, M.; Esclangon, A.; Maffrand, J. P.; Le Fur, G.; Oury-
Donat, F.; Soubrié, P. Mol. Pharmacol. 2003, 63, 908.
8. Osei-Hyiaman, D.; Liu, J.; Zhou, L.; Godlewski, G.; Harvey-White, J.; Jeong, W. i.;
Batkai, S.; Marsicano, G.; Lutz, B.; Buettner, C.; Kunos, G. J. Clin. Invest. 2008,
118, 3160.
9. Esposito, I.; Proto, M. C.; Gazzerro, P.; Laezza, C.; Miele, C.; Alberobello, A. T.;
D’Esposito, V.; Beguinot, F.; Formisano, P.; Bifulco, M. Mol. Pharmacol. 2008, 74,
1678.
28. The metabolism of compound
9 was investigated in vitro using hepatic
microsomal fractions prepared from mouse, rat and humans following in house
procedure:
Microsomal proteins concentration = 1 mg/mL, substrate concentration =
5 lM, incubation duration = 10 min. Cytochrome P-450 (CYP) and Flavin-
containing monooxygenases (FMO) cofactor = 1 mM NADPH. Chromatographic
analysis of the supernatant fluids was performed after removal of precipitated
proteins using an HPLC (column:YMC-Pack J sphere H80) coupled with UV
(254 nm) and mass spectrometry detection.
The main metabolic pathway observed was hydroxylation (mouse species) on
the piperidine moiety, di and tetra-dehydrogenation on the bi-piperidinyl
moiety (rat and humans species).
29. Bouaboula, M.; Bourrié, B.; Rinaldi-Carmona, M.; Shire, D.; Le Fur, G.; Casellas,
P. J. Biol. Chem. 1995, 270, 13973.
30. Calandra, B.; Portier, M.; Kerneis, A.; Delpech, M.; Carillon, C.; Le Fur, G.;
Ferrara, P.; Shire, D. Eur. J. Pharmacol. 1999, 374, 445.