From pyrroles to 1-oxo-2,3,4,9-tetrahydro-1H-β-carbolines: A new class of orally bioavailable mGluR1 antagonists

Article abstract of DOI:10.1016/j.bmcl.2007.01.055

Exploiting the SAR of the known pyrrole derivatives, a new class of mGluR1 antagonists was designed by replacement of the pyrrole core with an indole scaffold and consequent cyclization of the C-2 position into a tricyclic β-carboline template. The appropriate exploration of the position C-6 with a combination of H-bond acceptor groups coupled with bulky/lipophilic moieties led to the discovery of a new series of mGluR1 antagonists. These compounds exhibited a non-competitive behavior, excellent pharmacokinetic properties, and good in vivo activity in animal models of acute and chronic pain, after oral administration.

Full text of DOI:10.1016/j.bmcl.2007.01.055

Bioorganic & Medicinal Chemistry Letters 17 (2007) 2254–2259
From pyrroles to 1-oxo-2,3,4,9-tetrahydro-1H-b-carbolines:
A new class of orally bioavailable mGluR1 antagonists
Romano Di Fabio,* Fabrizio Micheli, Giuseppe Alvaro, Paolo Cavanni, Daniele Donati,
Tatiana Gagliardi, Gabriele Fontana, Riccardo Giovannini, Micaela Maffeis,
Anna Mingardi, Maria Elvira Tranquillini and Giovanni Vitulli
GlaxoSmithkline Medicine Research Centre, Via Fleming 4, 37135 Verona, Italy
Received 9 November 2006; revised 16 January 2007; accepted 19 January 2007
Available online 25 January 2007
Abstract—Exploiting the SAR of the known pyrrole derivatives, a new class of mGluR1 antagonists was designed by replacement of
the pyrrole core with an indole scaffold and consequent cyclization of the C-2 position into a tricyclic b-carboline template. The
appropriate exploration of the position C-6 with a combination of H-bond acceptor groups coupled with bulky/lipophilic moieties
led to the discovery of a new series of mGluR1 antagonists. These compounds exhibited a non-competitive behavior, excellent phar-
macokinetic properties, and good in vivo activity in animal models of acute and chronic pain, after oral administration.
Ó 2007 Elsevier Ltd. All rights reserved.
Glutamate is the major excitatory neurotransmitter
present in the mammalian brain and spinal cord. Its
action mediates several key brain functions, including
cognition and memory.1 In addition, glutamate has been
hypothesized to play a critical role in the pathophysiol-
ogy of a variety of neurological and psychiatric diseases
such as: acute stroke, traumatic brain injury, neuropath-
ic pain, Huntington’s disease, schizophrenia, depression,
dependence, and addiction.2 Two different families of
glutamate receptors have been identified, namely iono-
tropic glutamate receptors3,4 (NMDA, AMPA, and
kainate) and metabotropic receptors5,6 (mGluRs).
Ionotropic receptors are ligand-gated cation channels
responsible for fast excitatory neurotransmission and
neuronal plasticity. Metabotropic receptors belong to
the family of 7 TM class C G-protein coupled receptors
(GPCRs) and control both glutamate release and post-
synaptic excitability.
data, shed new light on the mechanism of interaction of
these receptors with both agonists and antagonists.8,9
To date, eight different mGluR subtypes have been iden-
tified, named mGluR1–8. They can be subdivided into
three groups based on their similarity sequence, pharma-
cology, and transduction mechanisms, namely group I
(mGluR1 and mGluR5), group II (mGluR2 and
mGluR3), and group III (mGluR4, mGluR6, mGluR7,
and mGluR8). Our interest in this area was mainly
devoted to the discovery of antagonists of the mGluR1
receptor subtype.10 Pyrrole derivatives 1–4 (Fig. 1) have
O
O
O
O
O
O
H
N
H
1
N
O
O
H
These receptors are characterized by the presence of a
large amino-terminal domain containing a bilobal-struc-
tured Venus fly trap.7 Recent molecular modeling stud-
ies, as well as receptor models based on crystallographic
2
O
O
O
O
H
O
OMe
N
H
N
H
N
N
O
R
Keywords: mGluR1 antagonists; Metabotropic receptors; Excitatory
amino acids; Glutamate; Acute and chronic pain.
H
4
3
*
Corresponding author. Tel.: +390458218879; fax: +390458218196;
e-mail: rdf26781@gsk.com
Figure 1. Series of pyrrole mGluR1 antagonists previously described.
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.01.055

R. D. Fabio et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2254–2259
Table 1. Potency results on mGluR1 receptor
2255
modify
cyclize
2
O
O
R
N
R
6
1
5
1
4
H
N
R
H
N
R
O
3
R
N
R
2
N
N
2
O
O
H
N
O
H
N
aromatic
ring
N
H
N
H
H
1
H
O
O
6-12
13-14
A
R1
R2
a
IC50 (nM)
Entry
Figure 2. Proposed evolution of the pyrrole scaffold.
1
6
—
—
H
16
(CH3)3CHCH3
(CH3)3CHCH3
1-Adamantyl
2-Adamantyl
nab
120
71
been discovered11–16 as potent and selective compounds,
active in different in vivo animal models of acute and
chronic pain.17
7
CH3
H
8
9
H
93
10
11
12
13
14
Bicycle(2.2.1)hept-2-ylc
CH2-1-adamantyl
C6H5
H
226
312
1040
71
H
As part of a broad chemical strategy aimed toward the
discovery of new classes of orally bioavailable mGluR1
receptor antagonists, an attempt to modify the pyrrole
scaffold was performed removing simultaneously the
flexible/lipophilic side chain present in the top region
of the pyrrole molecules. To this end, b-carboline deriv-
atives of type A, shown in Figure 2, were designed as a
potential structural evolution of the pyrrole compounds.
In particular, this scaffold, as depicted in Figure 2,
resulted from the replacement of the pyrrole nucleus
by the corresponding indole, along with the cyclization
of the C-2 amido moiety and the C-3 methyl group.
Then, once identified the core template, we managed
to maximize the in vitro activity varying the nature of
the R group present at the position C-6. From the pre-
vious SAR elements gathered on the pyrrole series, it
was known that the north-western region of these new
molecules could have been highly sensitive to the effect
of the substituents. In particular, in the pyrrole deriva-
tives, the combination of a H-bond acceptor and
bulky/lipophilic groups was essential to maximize their
in vitro potency.
H
1-Adamantyl
NH-1-adamantyl
H
H
4660
a Values are means of three experiments.
b na, activity > 10 lM.
c compound obtained as a mixture of endo/exo isomers.
Table 2. Potency results on mGluR1 receptor
H
N
O
R
O
N
R'
H
a
IC50 (nM)
Entry
R
H
R0
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
H
H
H
H
H
H
H
na
na
CH3
(CH3)3CHCH3
239
4070
104
5560
380
167
2850
3980
1670
na
C6H5
CH2C6H5
CH2C6H11
CH(CH3)C6H5
CH2C6H5
b
To explore this hypothesis two appropriate series of
amides and a series of ether derivatives shown in Tables
1 and 2, respectively, were studied, targeting specifically
the substitution at the position C-6.
CH3
—
—
—
—
—
—
—
—
na
na
As far as the synthesis of amides 6–12 reported in Table 1
is concerned, they have been obtained in good yield as
depicted in Scheme 1, from the known 6-carboxyethyl
b-carboline intermediate 5a18 by sequential hydrolysis
of the ethyl ester, activation of the carboxyl group as
the pentafluorophenylester, and amidation reaction in
good to excellent yield (41–100%) with either primary
or secondary amines. Compounds 13 and 14 were
prepared from the nitro derivative 5b19,20 in two steps,
by reduction of the nitro group to the corresponding
amine (CuCl, KBH4, CH3OH)21 in 61% yield, followed
by an amidation reaction with 1-adamantyl acyl
chloride or formation of the corresponding urea in the
presence of the 1-adamantyl isocyanate, respectively.
683
1020
—
a Values are means of three experiments; na, activity > 10 lM.
b Racemate.
reader (FLIPR).22 From the results summarized in
Table 1 (entries 6–12), it was evident that the 1,2,2-tri-
methylpropyl derivatives (entries 6 and 7) were signifi-
cantly less active than the corresponding pyrrole
analogues,11 suggesting a different SAR operating in this
new series. In this case, only the tertiary amide 7 gave
encouraging in vitro activity (IC50 = 120 nM), whereas
compound 6 was found to be inactive (IC50 > 1 lM).
These compounds were characterized in terms of in vitro
affinity on rat mGluR1a CHO cells, measuring intracel-
lular [Ca2+]i mobilization by a fluorescent imaging plate
The 1-adamantyl 8 and the 2-adamantyl 9 derivatives
were the most potent compounds identified (IC50
=

2256
R. D. Fabio et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2254–2259
O
O
Compounds 15–22 were then evaluated in the in vitro
assays. As shown in Table 2, both the unsubstituted
phenol derivative 15 and the methoxy derivative 16
were found inactive, further reinforcing the hypothesis
regarding the need for the presence of a lipophilic/
bulky group in this region of the molecule. Conversely,
the corresponding 1,2,2-trimethylpropyl ether 17 gave
a positive sign of activity (IC50 = 239 nM), whereas
the introduction of an aromatic ring directly linked
to the oxygen atom led to a poorly active molecule
(entry 18, IC50 = 4070 nM). Notably, the benzyl deriv-
ative 19 exhibited the best in vitro potency
(IC50 = 104 nM) as far as this subclass of ether com-
pounds is concerned.
1
H
H
N
N
R
a,b,c
EtO
N
R
2
O
O
N
N
H
H
6-12
5a
2
R
H
1
N
O2N
H
N
R
N
c,d
O
N
O
O
N
H
H
5b
13-14
Scheme 1. Reagents and conditions: (a) LiOH, EtOH/H2O 8:2, 60 °C,
3 h; (b) C6F5OCOCF3, pyridine, DMF, rt, 2 h; (c) R1R2NH, DMF,
80 °C, 1–4 h; (d) CuCl, KBH4 CH3OH, rt, 30 min; (e) 1-adamantyl
acyl chloride, pyridine, rt, 1 h or 1-adamantyl isocyanate, THF, rt,
12 h.
The replacement of the phenyl ring by the correspond-
ing cyclohexyl group caused a significant reduction of
the in vitro affinity (entries 20 and 19: IC50 = 5560 nM
and 104 nM, respectively). Moreover, the introduction
of a methyl group at the benzylic position (entry 21)
gave an activity 3–4 times lower than the compound
19 (IC50 = 380 nM and 104 nM, respectively). Finally,
the introduction of a methyl at the position C-7 can be
considered almost neutral as far as the in vitro activity
is concerned. (entries 22 and 19: IC50 = 167 nM and
104 nM, respectively).
71 nM and 93 nM, respectively), confirming the need for
the presence, similar to pyrroles, of a lipophilic/bulky
substituent in the north-eastern region of the molecule
to maximize the in vitro activity.11 Notably, these com-
pounds displayed the same non-competitive antagonism
behavior already observed for the pyrrole derivatives.11
The introduction of a methylene spacer (entry 11) result-
ed in a significant drop in potency (IC50 = 312 nM) with
respect to compound 8. The presence of an aromatic
substituent was less tolerated; the phenylamide deriva-
tive 12 was found in fact to be significantly less active
(IC50 = 1040 nM) than the corresponding adamantyl
analogues. Finally, the inverse adamantyl amide 13
exhibited the same in vitro potency as compound 8
(IC50 = 71 nM), whilst the corresponding urea derivative
14 was less active, probably due to the presence of an
additional H-bond donor, a polar moiety not tolerated
in this region of the molecule.
Following these interesting findings, a wider synthetic
program was undertaken to explore in more detail the
SAR associated to the b-carboline scaffold. To this
end, the series of modified analogues 23–30, shown in
Figure 3, were synthesized. As depicted in Scheme 3,
compound 25 was smoothly prepared from derivative
19 by reduction with Mg in methanol at reflux for
1 h.23 The amino analogues 26 and 27 were obtained
as a 1:1 mixture by simple reduction of compound 19
with BH3ÆTHF, followed by smooth separation by flash
chromatography (AcOEt/CH3OH/NH4OH 80:19:1).
The five-membered ring derivative 29 was synthesized
in three steps, in good total yield, from the known alde-
hyde derivative 31,24 by sequential reductive amination,
hydrolysis of the ethyl ester, and final cyclization reac-
tion. Compound 30 was synthesized as previously
reported.24
To enlarge the scope of this exploration, maintaining the
presence of H-bond acceptor group, a series of C-6 substi-
tuted ether derivatives was prepared. To this end, com-
pounds 15–22, reported in Table 2, were synthesized.
In particular, 16, 17, 18, 19, 21, and 22 were obtained in
good yield following the classical synthetic approach for
the preparation of substituted b-carboline compounds,19
whereas analogue 20 was prepared, as shown in Scheme 2,
by direct alkylation of the phenol derivative 15, easily
obtained by debenzylation reaction of compound 19.
As shown in Table 2, a significant reduction in
potency was observed for both the C-5 and C-7
benzyloxy derivatives 23 and 24 with respect to C-6
analogue 19. In addition, both the amino derivative
25 and 26 were inactive. The five-membered ring 29
and compound 30 exhibited reduced in vitro potency
compared to compound 19 (IC50 = 683 nM and
1020 nM, respectively vs 104 nM). Finally, com-
pounds 26, 27 and 28 were found to be inactive
(IC50 > 10 lM).
As expected, this alkylation reaction occurred in low
yield, due to the intrinsic difficulty in controlling the par-
allel alkylation of the indole moiety.
H
N
PhCH2O
H
N
HO
In summary, from the exploration performed, it seems
that the following pharmacophoric features should be
present to maximize the in vitro potency of this class
of mGluR1 antagonists:25
a
b
20
O
N
O
N
H
H
19
15
(a) a N-unsubstituted d-lactam moiety acting as
H-bond acceptor and/or donor;
Scheme 2. Reagents and conditions: (a) HCO2NH4, Pd/C 5%, MeOH,
reflux, 1 h; (b) RX, K2CO3, CH3CN, 80 °C, 24 h.

R. D. Fabio et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2254–2259
2257
OCH2Ph
H
H
N
H
H
N
N
PhCh2O
N
PhCH2O
O
O
O
N
N
N
N
PhCH2O
PhCH2O
H
H
H
H
25
24
23
26
H
N
H
N
N
H
N
PhCH2O
PhCh2O
PhCh2O
O
N
O
N
O
N
N
H
H
H
H
27
28
30
29
Figure 3. Structural modifications of the b-carboline core.
H
H
N
N
PhCH2O
PhCH2O
H
N
PhCH2O
b
+
O
N
N
N
H
H
H
19
26
27
a
H
N
PhCH2O
O
N
H
25
CHO
COOEt
NH2
COOH
H
O
PhCH2O
PhCH2O
c,d
e
N
PhCH2O
N
N
N
H
H
H
31
32
29
Scheme 3. Reagents and conditions: (a) Mg, CH3OH, reflux, 1 h; (b) BH3. THF, reflux, 4 h; (c) CH3CO2NH4, (CH3O)3BHNa, dichoroethane, rt,
24 h; (d) LiOH, THF/H2O 1:1, reflux, 8 h; (e) EDC, DMAP, N-methyl morpholine, rt, 36 h.
(b) a H-bond acceptor group (ether or amide moiety)
at the position C-6;
(c) an appropriate bulky/lipophilic group in the north-
eastern region of the molecule.
substituted analogue 22, a compound specifically
synthesized to shield the metabolically labile benzyl
position,28 exhibited a considerable improvement of
the pharmacokinetic profile both in terms of oral bio-
availability (F = 36%) and maximum exposure in plasma
(Cmax = 264 ng/ml).
Based on these results, the most potent compounds26
identified 8, 13, and 19 were characterized in terms of
in vivo pharmacokinetic in rats. As shown in Table 3,
compound 8 showed encouraging signals of oral bio-
availability (F = 12%). Conversely, 13 exhibited a poor
in vivo pharmacokinetics profile despite the comparable
Clp (52 ml/min/kg and 54 ml/min/kg for compounds 13
and 8, respectively), accounting for a solubility-limited
absorption process. As far as compound 19 is con-
cerned, the oral bioavailability was variable and highly
dependent on the oral dose used.27 Conversely, the
Compound 22 also showed an excellent level of brain
penetration (B/P = 6.1), assessed 1 h after dosing at
3 mg/kg iv.
According to these results, compound 22 was charac-
terized in vivo in the formalin test in mice, a model of
sustained inflammatory pain.29 As shown in Figure 4,
when 22 was given po at 10 mg/kg and 30 mg/kg,
30 min before the injection of formalin into the left
hind paw, a significant reduction30 of the nociceptive
behavior was observed only in the LP of the study.
Conversely, at a dose 30 mg/kg of 22, a significant
suppression30 of the nociception in both the EP and
LP was observed,31,32 further confirming the broad
spectrum analgesic potential associated with the
mGluR1 antagonists.33
Table 3. Comparison of the PK profiles
Compound
8
13
19
22
36
264
F%
Cmax
12
37
—
—
9
145
a
a Maximum concentration in plasma after oral administration (ng/ml).

2258
R. D. Fabio et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2254–2259
3. Meldrum, B. In Excitatory Amino Acid Antagonists;
Blackwell: Oxford, England, 1991.
4. Collingridge, G. L.; Watkins, J. C. In The NMDA
Receptor, 2nd ed.; IRL: Oxford, England, 1994.
5. Knoepfel, T.; Kuhn, R.; Allgeier, H. J. Med. Chem. 1995,
38, 1417.
6. Pin, J.-P.; Acher, F. Current Drug Targets-CNS Neurol.
Disord. 2002, 1, 297.
7. Bertrand, H. O.; Bessis, A. S.; Pin, J. P.; Acher, F. J. Med.
Chem. 2002, 45, 3171.
EP
LP
200
150
100
400
350
200
*
**
**
8. Costantino, G.; Macchiarulo, A.; Pellicciari, R. J. Med.
Chem. 1999, 42, 2816.
50
0
100
0
9. Bessis, A. S.; Bertrand, H.-O.; Galvez, T.; De Colle, C.;
Pin, J. P.; Acher, F. Protein Sci. 2000, 9, 2200.
10. For a recent review on the different series of m-GluR1
antagonists identified to date, see: Sabbatini, F.; Micheli,
F. Expert Opin. Ther. Patent 2004, 14, 1593.
11. Micheli, F.; Di Fabio, R.; Cavanni, P.; Rimland, J. M.;
Capelli, A. M.; Chiamulera, C.; Corsi, M.; Corti, C.;
Donati, D.; Feriani, A.; Ferraguti, F.; Maffeis, M.;
Missio, A.; Ratti, E.; Paio, A.; Pachera, R.; Quartaroli,
M.; Reggiani, A.; Sabbatini, F. M.; Trist, D. G.;
Ugolini, A.; Vitulli, G. Bioorg. Med. Chem. 2003, 11,
171.
12. Micheli, F.; Di Fabio, R.; Bordi, F.; Cavallini, P.;
Cavanni, P.; Donati, D.; Faedo, S.; Maffeis, M.; Sabba-
tini, F. M.; Tarzia, G.; Tranquillini, M. E. Bioorg. Med.
Chem. Lett. 2003, 13, 2113.
13. Micheli, F.; Di Fabio, R.; Benedetti, R.; Capelli, A. M.;
Cavallini, P.; Cavanni, P.; Davalli, S.; Donati, D.; Feriani,
A.; Gehanne, S.; Hamdan, M.; Maffeis, M.; Sabbatini, F.
M.; Tranquillini, M. E.; Viziano, M. V. A. Farmaco 2004,
59, 175.
14. Micheli, F.; Cavanni, P.; Di Fabio, R.; Marchioro, C.;
Donati, D.; Faedo, S.; Maffeis, M.; Sabbatini, F. M.;
Tranquillini, M. E. Bioorg. Med. Chem. Lett. 2006, 16,
1342.
15. For the replacement of the pyrrole nucleus by alterna-
tive heterocycles, see: Fabrizio, M.; Di Fabio, R.;
Cavanni, P.; Donati, D.; Faeedo, F.; Gehanne, S.;
Maffeis, M.; Marchioro, C.; Sabbatini, F. M.; Tarzia,
G.; Tranquillini, M. E.; Viziano, M. Farmaco 2003, 58,
1005.
Vehicle
10
30 mg/kg
Vehicle
10
30 mg/kg
Figure 4. Formalin test in mice. EP (early phase): 0–5 min after 20 ll
1% formalin; LP (late phase): 20–60 min after 20 ll 1% formalin;
*p < 0.05, **p < 0.01; n = 8–19.
Therefore, despite the limited in vitro potency, compound
22 showedgood in vivo activity, dueto the combination of
the good oral bioavailability, excellent brain penetration,
and relatively high fraction unbound in plasma.34
In conclusion, C-6 substituted b-carboline derivatives
were identified as new mGluR1 antagonists. These com-
pounds were rationally designed based on the known
pharmacophore model available for the pyrrole deriva-
tives previously explored in house. In particular, the
appropriate exploration of the C-6 position of this class
of b-carboline derivatives enabled the design of com-
pounds exhibiting good in vitro potency and similar
non-competitive antagonism behavior as the pyrrole
derivatives. Among the different compounds prepared,
the benzyloxy derivative 22 was identified as the most
balanced compound in terms of in vitro activity and
pharmacokinetic properties. According to these positive
characteristics the compound was tested in vivo and a
good analgesic activity both in the EP and LP of the for-
malin test in mice was observed, further confirming the
therapeutic potential of the mGluR1 antagonists in
acute and chronic pain.
16. For a wide exploration of the C-2 amide moiety of
compound 3, see: Micheli, F.; Di Fabio, R.; Cavallini, P.;
Donati, D.; Hamdam, M.; Sabbatini, F. M.; Messeri, T.
Farmaco 2004, 59, 119.
17. Jessel, T. M.; Kelly, D. D. In Principles of Neural
Science; Kandel, E. R., Schwartz, J. H., Jessell, T. M.,
Acknowledgments
Eds.; Appleton
385–399.
18. Cegla, M. T.; Boksa, J.; Chojnacka-Wojcik, E.; Misztal, S.
Pharmazie 1996, 51, 932.
19. Abramovitch, R. A. J. Chem. Soc. 1956, 4593.
20. Abramovitch, R. A.; Shapiro, D. J. Chem. Soc. 1956,
4589.
21. Yun, He; He, Zhao; Xinfu, Pan; Wang, Shaofei. Synth.
Commun. 1989, 19, 3047.
& Lange: Norwalk, CT, 1991; pp
The authors thank Dr. Carla Marchioro and her
coworkers for the NMR and IR spectra and Dr.
Handam and his coworkers for the MS spectra. Special
thanks are due to Dr. Stefania Faedo and Dr. Palmira
Cavallini for the in vitro binding studies and to Dr.
Letizia Bettelini and Gabriella Maraia for the in vivo
characterization in the formalin model in mice. Finally,
as far as the PK studies are concerned, thanks are due to
Dr. Franca Pugnaghi and Dr. Dino Montanari for the
support received.
22. For a detail description of the intracellular calcium
determination using FLIPR technology, see Ref. Micheli
et al. (2003), biology section, p 179.
23. Jorgensen, M. R.; Olsen, C. A.; Mellor, I. R.; Usherwood,
P. N. R.; Witt, M.; Franzyk, H.; Jaroszewski, J. W.
J. Med. Chem. 2005, 48, 56.
References and notes
24. Monge, A.; Parrado, P.; Font, M.; Fernandez-Alvarez, E.
J. Med. Chem. 1987, 30, 1029.
25. From the exploration performed to date, it is evident that
the presence of the ether group present at the C-6 position,
probably acting as H-bond acceptor, is critical for
1. Wroblewski, J. T.; Danysz, W. Annu. Rev. Pharmacol.
Toxicol. 1989, 29, 441.
2. Danysz, W.; Parsons, C. G.; Bresnik, I.; Quack, G. Drug
News Perspect. 1998, 11, 523.

R. D. Fabio et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2254–2259
2259
maximizing the in vitro potency of this subclass of
O
S
-
H
N
O
O
b-carboline derivatives. To prove this hypothesis, a limited
series of C-6 carbon linked derivatives (aryl heterocycle,
hetero–aryl, alkyl–aryl, alkenyl–aryl, and alkynil–aryl
derivatives) was prepared by cross-coupling reaction
starting from the 6-Br and/or 6-OTf b-carboline deriva-
tives (R. Di Fabio and R. Giovannini, unpublished
results). These compounds showed a significant drop of
the in vitro potency versus compound 19. The only one
exhibiting residual sign of activity was the (E)-2-(4-
fluorophenyl)ethenyl derivative (IC50 = 549 nM), in which
the double bound is able to mimic somehow the electron
content of the ether moiety. Final results of this explora-
tion will be reported in due course.
O
O
N
H
33
29. For a detailed description of the experimental procedure
used for the in vivo in the formalin model in mice, see:
Quartaroli, M.; Carignani, C.; Dal Forno, G.; Mugnaini,
M.; Ugolini, A.; Arban, R.; Bettelini, L.; Maraia, G.;
Belardetti, F.; Reggiani, A.; Trist, D. G.; Ratti, E.; Di
Fabio, R.; Corsi, M. J. Pharmacol. Exp. Ther. 1999, 290,
158, and references herein enclosed.
30. EP: vehicle mean = 121.7 s, SD = 20.7 s, SE = 4.7 s;
10 mg/kg mean = 114.4 s, SD = 40.2 s, SE = 10.1 s;
30 mg/kg mean = 83.5 s, SD = 13.6 s, SE = 4.8 s. LP: vehi-
cle mean = 415.6 s, SD = 167.0 s, SE = 38.3 s; 10 mg/kg
mean = 299.7 s, SD = 139.7 s, SE = 34.9 s; 30 mg/kg
mean = 227.6 s, SD = 96.3 s, SE = 34.0 s.
31. The exposure in plasma of compound 22 in mice was
measured at the end of the formalin test, 2 h after the oral
administration of the compound at 10 mg/kg dose. The
average maximum concentration observed was 495 ng/ml,
confirming that the animals were successfully exposed to
the compound during the in vivo test.
32. Compound 22 displayed the same non-competitive antag-
onism behavior seen for the corresponding amide deriv-
atives previously described, along with good receptor
selectivity over the mGluR5 receptor.
26. Compound 8: 1H NMR (300 MHz): DMSO d 11.77 (s,
1H); 8.10 (s, 1H); 7.66–7.46 (m, 3H); 7.34 (d, 1H); 3.51 (m,
2H); 2.94 (t, 2H); 2.08–2.05 (m, 9H); 1.65 (m, 6H).Com-
1
pound 13: H NMR (300 MHz): DMSO d 11.47 (s, 1H);
8.99 (s, 1H); 7.86 (d, 1H); 7.50 (s, 1H); 7.36 (dd, 1H); 7.26
(d, 1H); 3.48 (td, 2H); 2.85 (t, 2H); 2.0 (s, 3H); 1.90 (s, 6H);
1.69 (s, 6H).Compound 19: H NMR (300 MHz): DMSO
1
d 11.41 (s, 1H); 7.49 (s, 1H); 7.46 (d, 1H); 7.38 (t, 2H); 7.31
(t, 1H); 7.27 (d, 1H); 7.16 (d, 1H); 6.93 (dd, 1H); 5.09 (s,
1
2H); 3.48 (m, 2H); 2.86 (t, 2H).Compound 22: H-NMR
(300 MHz): DMSO d 11.29 (s, 1H); 7.48 (d, 2H); 7.39 (m,
3H); 7.31 (t, 1H); 7.16–7.11 (ds, 2H); 5.10 (s, 2H); 3.46 (m,
2H); 2.85 (t, 2H); 2.28 (s, 3H).
27. The oral bioavilability was 0% to 9% at 1 mg/kg po and
5 mg/kg po, respectively. At the latter dose an encouraging
concentration of compound in plasma was observed
(Cmax = 145 ng/ml). This result was explained in terms of
progressive saturation of the first-pass metabolism at the
higher dose.
33. This research complied with national legislation and with
company policy on the Care and Use of Animals and with
related codes of practice.
28. As expected, the oxidation of the benzyl position was
the main route of metabolism of this subclass of
compounds. Compound 33 was in fact the most
abundant metabolite excreted in urine in the 0–4 h
interval after oral dosing, roughly accounting for the
40% of the amount of parent compound 22
administered.
34. The free fraction in rat plasma was 3.4%. For a recent
paper on the importance of the free fraction in plasma/
brain, see: Liu, X.; Smith, B. J.; Chen, C.; Callegari, E.;
Becker, S. L.; Chen, X.; Cianfrogna, J.; Doran, A. C.;
Doran, S. D.; Gibbs, J. P.; Hosea, N.; Liu, J.; Nelson, F.
R.; Szewc, M. A.; Van Deusen, J. Drug Metab. Dispos.
2006, 34, 1443, and references herein enclosed.