13C bis-labeled pyrroles: A tool for the identification of the rat metabolism of 3-methyl pyrrole-2,4-dicarboxylic acid 2-propyl ester 4-(1,2,2-trimethyl-propyl) ester

Article abstract of DOI:10.1016/j.bmcl.2006.11.039

Following the recent disclosure of 3-methyl pyrrole-2,4-dicarboxylic acid 2-propyl ester 4-(1,2,2-trimethyl-propyl) ester as a potent and selective mGluR1 non-competitive antagonist, the use of a doubly ¹³C-labeled analogue to identify, and con

Full text of DOI:10.1016/j.bmcl.2006.11.039

Bioorganic & Medicinal Chemistry Letters 17 (2007) 969–973
13C bis-labeled pyrroles: A tool for the identification of the rat
metabolism of 3-methyl pyrrole-2,4-dicarboxylic acid 2-propyl
ester 4-(1,2,2-trimethyl-propyl) ester
Fabrizio Micheli,a,* Paolo Cavanni,a Romano Di Fabio,a Daniele Donati,a
Mahmoud Hamdan,a Stefano Provera,a Maria Elvira Tranquillinib and Giovanni Vitullic
aGlaxoSmithKline Medicines Research Centre, Via Fleming 4, 37135 Verona, Italy
bGlaxoSmithKline, Gunnels Wood Rd., Stevenage, Herts, SG1 2NY, UK
cSandoz Industrial Product S.p.A., corso Verona 165, 38068 Rovereto (TN), Italy
Received 30 October 2006; revised 13 November 2006; accepted 13 November 2006
Available online 16 November 2006
Abstract—Following the recent disclosure of 3-methyl pyrrole-2,4-dicarboxylic acid 2-propyl ester 4-(1,2,2-trimethyl-propyl) ester as
a potent and selective mGluR1 non-competitive antagonist, the use of a doubly 13C-labeled analogue to identify, and consequently
prevent, metabolically labile positions is reported.
Ó 2006 Elsevier Ltd. All rights reserved.
Glutamate, one of the major excitatory neurotransmit-
ters in mammalian brain, exerts its action through iono-
tropic (specifically NMDA, AMPA, and Kainate)1–5
and metabotropic receptors (mGluRs).6,7
We have recently reported21–23 that 3-methyl pyrrole-
2,4-dicarboxylic acid 2-propyl ester 4-(1,2,2-trimethyl-
propyl) ester (1, Fig. 1) is a potent and selective
non-competitive mGluR1 antagonist endowed with an
excellent in vitro and in vivo activity in different animal
models of pain. Further derivatives, endowed with a
better pharmacokinetic profile in preclinical species
(e.g., 2, Fig. 1), were also recently published.24,25
The metabotropic receptors are characterized by a large
amino-terminal domain and they belong to the family C
of G-protein coupled receptors (GPCR). Through differ-
ent effectors and second messengers they are able to con-
trol crucial functions within the cell;8–12 moreover, their
structures are peculiar and show very little homology
with those of other cloned receptors.13–15 To date, eight
mGluR 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, pharmacolo-
gy, and transduction mechanisms: Group I (mGluR1
and mGluR5), Group II (mGluR2 and mGluR3) and
Group III (mGluR4, mGluR6, mGluR7, and mGluR8).
A number of recent reviews reported the current state of
the art of competitive and non-competitive antagonists
and modulators of these important receptors.16–20
To gain a further understanding of the preclinical profile
of this series avoiding the lengthy and expensive synthe-
sis of the [14C]-labeled molecules—generally used in ad-
vanced development phase—it was decided to apply
13
some stable isotope [ C] labeling to the pyrrole deriva-
tives.27–29 This would greatly facilitate the identification
of major metabolic pathways in vivo and in vitro, with-
out the need for complex and time-consuming purifica-
tion processes.
O
O
O
O
O
OMe
N
H
N
H
N
H
O
O
Keywords: mGluR1; Metabotropic; Glutamate; Metabolism.
*
2
1
Corresponding author. Tel.: +39 045 8218515; fax: +39 045
8218196; e-mail: Fabrizio.E.Micheli@gsk.com
Figure 1. The recently reported mGluR1 antagonist pyrrole class.
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.11.039

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F. Micheli et al. / Bioorg. Med. Chem. Lett. 17 (2007) 969–973
Based on the synthetic route described for 1 and 2,21–23
the introduction of two 13C atoms (25% of the total
amount) into the pyrrole template was decided to gener-
ate a well-identifiable ‘fingerprint’ in the mass and NMR
spectra of both the derivatives and their metabolites.
Both 13C atoms have been positioned on the core scaf-
fold, thus ensuring that the main metabolic path-
ways—more likely to affect groups in C2 and C4—did
still maintain the stable isotopes useful to their identifi-
cation. The detailed synthetic route is reported in
Scheme 1.
lar, the mass spectra allowed the identification of two
hydroxylated derivatives (3 and 4) and two carboxylated
ones (5 and 6).
Further MS–MS experiments on the study of the
decomposition pattern of carboxylate 5 led to hypothet-
ical metabolic route represented in Scheme 3, allowing
the unequivocal attribution of the two derivatives now
identified as compounds 7 and 8 through the direct com-
parison of the spectra available from HPLC–NMR
studies of the biological samples and the synthetic ones.
After oral administration30 of derivatives 1 and 2 to rats,
the chromatographic peaks of parent compounds and
their major metabolites in biofluid extracts were easily
identified by MS and NMR spectroscopy, tracking the
dual 13C fingerprints which therefore allowed a quick
formulation of some working hypothesis on the degra-
dation pattern of the pyrrole template.
As far as derivative 2 is concerned (Scheme 4), after a ra-
pid cleavage of the methoxy group, the corresponding
primary hydroxyl group 9 was readily oxidized to the
corresponding carboxylic acid 10. Also in this case, the
working hypothesis was confirmed through the compar-
ison between the spectra coming from the biological
samples and the synthetic compounds ad hoc prepared.
As far as derivative 1 is concerned, the hypothesized
metabolites (3–6) are depicted in Scheme 2. In particu-
As far as derivative 3 is concerned, the site of hydroxyl-
ation could not be unequivocally assigned, due to the
N
+
COOEt
O
2
C
O
13C
13C
O
O
i
O
ii
75%
25%
O
O
O
+
H
N
H
N
H
13C
13C
H
O
O
25%
75%
O
13C
13C
O
O
O
13C
HO
+
H
HO
iii
25%
75%
13C
O
O
H
O
O
N
H
H
O
N
N
H
H
O
O
+
O
75%
25%
O
H
N
H
O
O
O
13C
13C
v
iv
O
H
O
+
O
O
H
H
H
N
H
N
H
O
F
O
75%
25%
O
O
13C
13C
F
vi
O
O
F
F
O
O
O
H
+
H
N
N
F
F
H
O
H
F
F
F
F
75%
25%
O
O
13C
13C
O
O
+
X
R
X
H
R
N
H
X=O, NH
N
H
O
H
O
75%
25%
Scheme 1. Reagents and conditions: (i) DBU/THF, 50 °C; (ii) H2SO4, 0 °C; (iii) 1—trifluoroacetic anhydride; toluene; 0 °C, 1 h; 2—(S) pinacolyl
alcohol, rt, 4 h; (iv) LiOH (4 equiv), EtOH; H2O, 50 °C; (v) C6F5OCOCF3 (1.2 equiv), DMF, Py (1.2 equiv), rt, 12 h; (vi) RNH2 or ROH, 0–60 °C.

F. Micheli et al. / Bioorg. Med. Chem. Lett. 17 (2007) 969–973
971
O
O
O
HO
HO
O
O
O
O
O
O
H
H
H
N
N
H
1
N
OH
H
H
O
O
O
3
A
O
O
HOOC
O
O
HO
O
O
O
O
O
COOH
O
H
O
N
H
O
H
OH
N
H
H
O
H
N
H
6
COOH
N
O
O
H
O
5
B
4
Scheme 2. Hypothetical metabolic pathway.
OH
O
O
O
O
O
O
O
O
O
O
H
N
H
O
NHAc
H
NHAc
N
O
N
N
H
O
N
7
N
H
H
O
O
12
11
Figure 2. t-Bu group was identified as one metabolically labile position
within the pinacolyl group.
O
HOOC
O
O
O
OHC
O
H
N
O
H
O
H
N
8
H
O
able; the first one was to prevent the t-Bu group
oxidation; the second one was to identify a suitable
replacing group more metabolically stable than the
t-Bu one.
Scheme 3. Identified metabolic pathway.
O
O
O
The second approach is rapidly described below. It was
known,21–26 that appropriate potency could be ob-
tained in the pyrrole template using lipophilic groups
in the C4 position; nonetheless, appropriate stereo-
chemistry was also needed. Accordingly, a number of
non-bornyl derivatives were prepared via trifluoroacetic
mixed anhydride in accordance with previously de-
scribed methods21–26 and their in vitro potency on
r-mGluR1 receptor was evaluated. Data are reported
in Table 1.
O
OMe
N
OH
N
H
H
N
H
2
N
H
O
O
9
O
O
COOH
N
H
N
H
O
10
While a slight improvement in metabolic stability was
achieved through this replacement (data not shown), a
clear decrease in potency was also observed with re-
spect to the nanomolar potency of compound 1,21–26
confirming once more the need for the particular
shape and chirality represented by the pinacolyl sub-
stituent in the pyrrole class for the ideal fit within
the mGluR1 receptor. Nonetheless, these molecules
might constitute the starting point for further
exploration.
Scheme 4. Metabolic pathway for amide 2.
competitive biotransformation on the C2-ester,31 involv-
ing the transient intermediates A and B (Scheme 2).
Consequently, and to allow enough time to study the
metabolic fate of the C4 position, more stable C225,26
substituted amides were prepared and LC/NMR/MS
studies were performed on freeze-dried rat urines col-
lected after oral administration. The NMR data ob-
tained clearly demonstrated that the tert-butyl group
of the pinacolyl ester underwent a metabolic hydroxyl-
ation. In particular, derivative 11 was rapidly converted
to derivative 12 reported in Figure 2.
A double [13C] labeling of the pyrrole template allowed
the rapid identification of the metabolic pathway in pre-
clinical species through NMR/LC, HPLC/MS/MS tech-
niques. Some more stable, but less potent derivatives
were identified and might represent new potential hits
for further exploration.
Two alternative medicinal chemistry strategies to re-
duce the metabolic clearance were therefore avail-

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F. Micheli et al. / Bioorg. Med. Chem. Lett. 17 (2007) 969–973
Table 1. Potency values of the pyrrole derivatives on r-mGluR1a-CHO obtained as previously reported21–26
O
X
R 2
1 R
N
H
O
Compound
X
R1
H
R2
OCH CH CH
pIC50
8.4
2
2
3
1
O
O
2
13
14
15
16
17
18
19
H
NHCH2CH2OMe
OEt
7.5
7.7
6.7
6.8
7.0
6.0
6.5
<5
H
O
H
HN(CH2)4NHAc
HN(CH2)2OMe
OEt
O
O
O
O
O
N
H
Me
Me
Me
H
HN(CH2)4NHAc
HN(CH2)2OMe
HN(CH2)2OMe
IC50s were measured from at least six-point inhibition curves and they are the geometric means of at least three independent experiments. No
selectivity data were generated at this point on these derivatives.
Marchioro, C.; Orlandi, A.; Pozzan, A.; Quaglia, A. M.;
Reggiani, A.; van Amsterdam, F. Arch. Pharm.- Pharm.
Med. Chem. 1999, 332, 73.
Acknowledgment
We thank Prof. Mario Geysen for his contribution to
this project.
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30. The research complied with national legislation and with
company policy on the Care and Use of Animals and with
related codes of practice.
31. The different stability of the C2 esters was previously
reported21,23–25 and is species dependent also in rodents;
moreover, it is sensitive to steric hindrance as expected
(e.g., the C2 n-Pr ester in compound 1 has a T1/2 of 2.8 h in
mice and 0.2 h in rats, while the corresponding C2 t-Bu
ester has a T1/2 > 24 h in mice and >5 h in rats). A detailed
comparison of the PK profile of compounds 1 and 2 is
reported in Ref. 25.