2,4-Dicarboxy-pyrroles as selective non-competitive mGluR1 antagonists: Further characterization of 3,5-dimethyl pyrrole-2,4-dicarboxylic acid 2-propyl ester 4-(1,2,2-trimethyl-propyl) ester and structure-activity relationships

Article abstract of DOI:10.1016/S0960-894X(03)00396-2

Following the disclosure of 3,5-dimethyl pyrrole-2,4-dicarboxylic acid 2-propyl ester 4-(1,2,2-trimethyl-propyl) ester [3,5-dimethyl PPP] as a potent and selective mGluR1 non-competitive antagonist, we report here further in vivo characterization of this important tool and disclose the investigation of the C-5 position, which led to very potent compounds.

Full text of DOI:10.1016/S0960-894X(03)00396-2

Bioorganic & Medicinal Chemistry Letters 13 (2003) 2113–2118
2,4-Dicarboxy-pyrroles as Selective Non-Competitive mGluR1
Antagonists: Further Characterization of 3,5-Dimethyl
Pyrrole-2,4-dicarboxylic Acid 2-Propyl Ester
4-(1,2,2-Trimethyl-propyl) Ester and
Structure–Activity Relationships
Fabrizio Micheli,* Romano Di Fabio*, Fabio Bordi,y Palmina Cavallini, Paolo Cavanni,
Daniele Donati, Stefania Faedo, Micaela Maffeis, Fabio Maria Sabbatini, Giorgio Tarzia
and Maria Elvira Tranquillini
GlaxoSmithKline Medicine Research Centre, Via Fleming 4, 37135 Verona, Italy
Received 28 February 2003; revised 27 March 2003; accepted 10 April 2003
Abstract—Following the disclosure of 3,5-dimethyl pyrrole-2,4-dicarboxylic acid 2-propyl ester 4-(1,2,2-trimethyl-propyl) ester [3,5-
dimethyl PPP] as a potent and selective mGluR1 non-competitive antagonist, we report here further in vivo characterization of this
important tool and disclose the investigation of the C-5 position, which led to very potent compounds.
# 2003 Elsevier Science Ltd. All rights reserved.
Glutamate, the principal excitatory neurotransmitter in
mammalian brain, is a key mediator for a number of
brain functions and acts through modulation of iono-
tropic (NMDA, AMPA and Kainate)1À5 and metabo-
tropic receptors (mGluRs).6,7
transduction mechanisms: Group I (mGluR1 and
mGluR5), Group II (mGluR2 and mGluR3) and Group
III (mGluR4, mGluR6, mGluR7 and mGluR8). Several
splice variants for some of the different subtypes have
also been reported and competitive and non-competitive
antagonists are available.15,16
The metabotropic receptors are characterized by a large
amino-terminal domain, which includes the Glu binding
site, and they belong to the family C group of G-protein
coupled receptors (GPCR). Through different effectors
and second messengers, they control key functions
within the cell and ion channels.8À12 Finally, they share
very little homology with other GPCR’s.13,14
A number of computational studies were therefore aimed
at modelling the function of these receptors and very
interesting results were recently shown by Pellicciari17 and
by Pin and Acher in agreement with X-ray structures.18À20
We have recently reported21À24 that 3,5-dimethyl pyr-
role-2,4-dicarboxylic acid 2-propyl ester 4-(1,2,2-tri-
methyl-propyl) ester (3,5-dimethyl PPP—1, Fig. 1) is a
potent and selective non-competitive mGluR1 antago-
nist endowed with excellent in vitro (15.8 nM) and in
vivo activity in different animal models of pain. A clear
‘opiate-like’ antinociceptive profile was show for these
derivatives in animal models of pain together with the
more widely recognized activity of the mGluR1
antagonists in chronic/inflammatory models.23
To date, eight mGluRs subtypes have been identified
and named mGluR1–8 according to the succession of
the molecular cloning.
These receptors are divided into three main groups on
the basis of sequence similarity, pharmacology and
*Corresponding authors. Tel.: +39-045-921-8515; fax: +39-045-921-
8196; e-mail: fabrizio.e.micheli@gsk.com (F. Micheli); tel.: +39-045-
921-8879; fax: +39-045-921-8196; e-mail: fabio@gsk.com (R. Di Fabio).
yCurrent address: Sigma-tau R&D,Via Pontina km.30.4, 00040 Pome-
zia (Roma), Italy.
The most potent molecules belonging to this newly
reported class are endowed with nanomolar potency at
the mGluR1 receptor, and they are extremely selective
0960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00396-2

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F.Micheli et al./ Bioorg.Med.Chem.Lett.13 (2003) 2113–2118
Figure 1. The recently discovered pyrrole class.
versus the other receptor subtypes both in the same
group I and groups II and III (> >500-fold).
effects of 3,5-dimethyl PPP 1 were selective to nocicep-
tive transmission. These experiments demonstrated that
1 did not suppress firing evoked by non-noxious
stimulation.
In electrophysiological studies, these compounds
showed a very potent activity in the ‘wind-up’ experi-
ment in vitro on baby rat spinal cord23 strongly sug-
gesting that mGluR1 antagonists could be useful in
blocking the central sensitization process.
As it can be seen from Figure 2A, 3,5-dimethyl PPP 1,
dosed at 2 mg/kg ip in urethane anesthetized animals,
inhibited the stimulus-evoked activity of nociceptive
responses in VPL neuron of the rat. The effect was
potent, and selective for noxious stimulation because it
did not alter the firing of non-nociceptive neurons
responding to touch, as reported in Figure 2B. More-
over, as it can be observed in Figure 3, the analgesic
effect is long lasting and similar to the one obtained
after morphine iv injection.
Clearly, such a result would have a much stronger
impact if a similar effect was also recorded in vivo.
Accordingly, electrophysiological recordings were made
of nociceptive responses of wide dynamic range
(WDR) neurons in the ventroposterolateral (VPL)
nucleus of the thalamus of urethane anesthetized rats,25
as recently reported for the mGluR5 antagonist
MPEP.26,27
These very exciting results, together with the ones
already reported23 strengthen our confidence in the
pyrrole class of mGluR1 antagonists and committed us
to further SAR explorations.
Non-nociceptive mechanosensitive neurons were also
studied in VPL nucleus to determine whether the
Figure 2. (A) Evoked firing of a representative neuron before and after administration of 3,5-dimethyl PPP 1 (2 mg/kg, ip). Top, level of applied
increasing pressure to the rat’s paw in register with the post-stimulus histogram below. Bottom, average of 10 trials of firing rate histograms pre-
drug (in black) compared to post-drug (in gray). (B) Activity of a representative non-nociceptive neuron before and after administration of 3,5-
dimethyl PPP 1 (2 mg/kg, ip). The drug did not reduce response to light brush.

F.Micheli et al./ Bioorg.Med.Chem.Lett.13 (2003) 2113–2118
2115
Figure 3. Effects of 3,5-dimethyl PPP 1 (2 mg/kg ip) and morphine (0.5 mg/kg, iv) on nociceptive VPL neurons in the anesthetized rat. Top: Mean of
10 trials of firing rate histograms before and after 3,5-dimethyl PPP 1 administration recorded at different times. Level of applied pressure to the
rat’s paw is in register with the response. At 60 min post-injection, neuron firing to nociceptive stimulation is evidently decreased. At 300 min post-
injection time, neuron firing is starting to recover. Bottom: Firing rate histograms before and after morphine injection. The effect is very rapid
because the injection was iv and the response recovered completely about 2 h post-morphine administration.
More precisely, we were interested in a better under-
standing of the role of the C-5 position and to enlarge
our exploration of the pyrrole nitrogen.
structure–activity of our template, we also replaced the
t-Bu group with the pinacolyl ester present in 3,5-dime-
thyl PPP 1.
A number of compounds were prepared according to
the synthetic routes described in Scheme 1 starting from
derivative 528 and according to the references there
reported.
As already reported, these pyrrole derivatives are com-
pletely inactive when the C-2 and C-4 carboxylic acids
are left unprotected. These original findings were further
confirmed by the introduction of another carboxylic
group in position C-5, with the free acid being com-
pletely inactive (9, Table 1), whereas its methyl ester
(10) retained an acceptable potency. The introduction of
an oxygenated function (6, 7, 8) was well tolerated,
while the presence of a free amine (12, 13) led to com-
plete inactivity.
As shown in Table 1, we started our exploration from
derivative 2 (Fig. 1), retaining the C-3 methyl group and
the C-4 t-Butyl ester, and replacing the C-5 methyl
group with a number of different functionalities. Subse-
quently, in order to have a better understanding of the

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F.Micheli et al./ Bioorg.Med.Chem.Lett.13 (2003) 2113–2118
Scheme 1. Reaction conditions and references: (a) NCS, 1.1 equiv, DMF, 0 ꢀC; 30% yield (23); (b) POCl3, DMF, CH2Cl2, from 0 ꢀC to rt; 50%
yield. (8); (c) NaOClO, CH3CN, H2O, rt, 70% yield (9); (d) NaCNBH3, R0NH2, THF, 0 ꢀC; 30% yield (12); H2, Pd/C, 1 atm 95% yield (13); (e)
R00NH2, DCC, THF, rt; 80% yield (11); (f) (CF3CO)2O, rt, R000OH, THF; 80% yield (10); (g) NaBH4, 1.1 equiv, MeOH, from 0 ꢀC to rt; 95% yield
(6); (h) CH3COCl, Py, THF, rt; 95% yield (7).
While the introduction of an electronwithdrawing
group (23) lead to a 10-fold decrease in potency, to
our great pleasure, the replacement of C-5 methyl
group with a hydrogen atom led to an increased
potency with respect to the parent derivatives (15,
19).
Nonetheless, the presence of free hydroxy groups on linear
alkylic chains (37) or esteric derivatives (28) was tolerated.
This, in agreement with what already shown for the posi-
tion C-5, seems to point out that the region where these
derivatives bind in the receptor might be rich in lipophilic
residues, which clearly tolerate hydrogen bond acceptors,
but tend to repel protonated/charged basic groups.
To increase the water solubility of our derivatives, hav-
ing gained further potency with the C-5 des-methylated
pyrrole, we also incorporated esters carrying a basic
moiety on the position C-2. As previously reported,21,22
this substituents were poorly tolerated, but in this case
some activity was retained (22, 24, 25). It is also worth
noting that derivative 21, the lipophylic pentafluoro
phenyl ester intermediate to an array of amides, still
retained acceptable potency.
All the active compounds reported in Tables 1 and 2
were tested for their selectivity versus mGluR5,
mGluR2 and mGluR4. As for the derivatives previously
reported,23 all these compounds were endowed with a
selectivity greater than 100-fold with respect to the
above mentioned receptors.
The exploitation of our best tool, 3,5 dimethyl PPP 1,
led to further in vivo confirmation of the importance
of the mGluR1 in nociceptive transmission. In parti-
cular, we demonstrated a clear analgesic effect with no
influence on non-nociceptive transmission and devoid
of sedative effect, and therefore suggesting that
mGluR1 antagonists are able to interfere with the
nociceptive pathway without altering the normal, tac-
tile transmission.
As far as the pattern of substitution of the pyrrole
nitrogen is concerned, a number of derivatives were
simply prepared alkylating the anion of derivative 2
(NaH, 1.1 equiv, DMF, 0 ꢀC) with the appropriate
haloderivative (Bromo or Chloro) in DMF at room
temperature. As already observed in previous
exploration,21À23 small alkyl group substituents (33,
Table 2) were tolerated and still showed a certain
degree of potency, while generally, the introduction of
bulkier group carrying basic amines led to complete
inactivity.
The further exploration of the pyrrole template con-
firmed the poor tolerance of the binding site to both

F.Micheli et al./ Bioorg.Med.Chem.Lett.13 (2003) 2113–2118
2117
Table 1. Potency values of the pyrrole derivatives on r-mGluR1a-
CHO cells using the CDP-DAG accumulation method23
Table 2. Potency values of the pyrrole derivatives on r-mGluR1a-
CHO cells using the CDP-DAG accumulation method23
X
R0
R00
Entry
IC50 (mM)
X
Entry
IC50 (mM)
H
H
H
OE
OH
t-BuO
t-BuO
t-BuO
t-BuO
t-BuO
t-BuO
t-BuO
t-BuO
t
3
4
5
6
7
8
9
10
0.34
N.A.
0.075
7.9
2.7
2
CH2CONMe2
CH2COOAllyl
27
28
N.A.
12.6
n-OPr
n-OPr
n-OPr
n-OPr
n-OPr
n-OPr
CH2OH
CH2Oac
CHO
COOH
COOMe
29
N.A.
N.A.
3.2
CH2COOH
30
31
N.A.
N.A.
n-OPr
t-BuO
11
2.5
CH2NHBn
CH2NH2
COSPy
H
n-OPr
n-OPr
n-OPr
t-BuO
t-BuO
t-BuO
t-BuO
t-BuO
12
13
14
15
N.A.
N.A.
3.2
32
N.A.
0.1
H
t-BuO
16
1.2
Me
BOC
(CH2)3OSi-t-BuPh2
(CH2)2OSi-t-BuPh2
(CH2)3OH
33
34
35
36
37
1
2.3
N.A.
N.A.
5.8
H
H
SPy
t-BuO
t-BuO
17
18
3
0.048
IC50s were measured from at least six-point inhibition curves and they
are the geometric means of at least three independent experiments.
The standard error of the mean was less than 0.05; N.A., not active up
to 100 mM.
H
H
n-OPr
t-BuO
19
20
0.004
0.017
Acknowledgements
H
H
Cl
H
H
H
21
22
23
24
25
26
0.16
0.39
0.16
0.72
0.26
1.45
We would like to thank Dr. Carla Marchioro and co-
workers and Dr. Mahmoud Hamdan and co-workers
for their help and analytical support. We would also like
to thanks Dr. Frank King and Dr. Annarosa Ugolini
for helpful discussions during the preparation of this
manuscript.
n-OPr
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