7-Substituted-pyrrolo[3,2-d]pyrimidine-2,4-dione derivatives as antagonists of the transient receptor potential ankyrin 1 (TRPA1) channel: A promising approach for treating pain and inflammation

Article abstract of DOI:10.1016/j.bmc.2012.01.020

The transient receptor potential ankyrin 1 (TRPA1) channel is activated by a series of by-products of oxidative/nitrative stress, produced under inflammatory conditions or in the case of tissue damage, thus generating inflammatory and neuropathic pain and

Full text of DOI:10.1016/j.bmc.2012.01.020

Bioorganic & Medicinal Chemistry 20 (2012) 1690–1698
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Bioorganic & Medicinal Chemistry
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7-Substituted-pyrrolo[3,2-d]pyrimidine-2,4-dione derivatives as antagonists
of the transient receptor potential ankyrin 1 (TRPA1) channel:
A promising approach for treating pain and inflammation
Pier Giovanni Baraldi a, , Romeo Romagnoli a, Giulia Saponaro a, Mojgan Aghazadeh Tabrizi a,
⇑
Stefania Baraldi a, Pamela Pedretti b, Camilla Fusi b, Romina Nassini b, Serena Materazzi b,
Pierangelo Geppetti b, Delia Preti a,
⇑
a Department of Pharmaceutical Sciences, University of Ferrara, Via Fossato di Mortara 17-19, 44121 Ferrara, Italy
b Department of Preclinical and Clinical Pharmacology, University of Florence, Viale Pieraccini 6, 50139 Florence, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
The transient receptor potential ankyrin 1 (TRPA1) channel is activated by a series of by-products of oxi-
dative/nitrative stress, produced under inflammatory conditions or in the case of tissue damage, thus
generating inflammatory and neuropathic pain and neurogenic inflammatory responses. These findings
have identified TRPA1 as an emerging opportunity for the design and synthesis of selective inhibitors
as potential analgesic and antiinflammatory agents. Herein we present the synthesis and functional eval-
uation of a new series of 7-substituted-1,3-dimethyl-1,5-dihydro-pyrrolo[3,2-d]pyrimidine-2,4-dione
derivatives designed as TRPA1 antagonists. A small library of compounds has been built by the introduc-
tion of differently substituted N7-phenylacetamide or N7-[4-(substituted-phenyl)-thiazol-2-yl]-
acetamide chains. All the synthesized compounds were assayed to evaluate their ability to block
acrolein-mediated activation of native human and rat TRPA1 channels employing a fluorometric calcium
imaging assay. Our study led us to the identification of compound 3h which showed considerably
improved potency (IC50 = 400 nM) against human TRPA1 with regard to some of the most representative
antagonists previously reported and integrated in our screening program as reference compounds. In
addition, 3h proved to maintain its efficacy toward rTRPA1, which designates it as a possible candidate
for future evaluation of in vivo efficacy in rodent animal model of inflammatory and neuropathic pain.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 3 November 2011
Revised 9 January 2012
Accepted 11 January 2012
Available online 24 January 2012
Keywords:
TRPA1 channel
Pain
Inflammation
Asthma
TRPA1 antagonists
1. Introduction
different members of the superfamily, and also among the individ-
ual members of each subfamily. TRPA1 channel, first cloned from
The transient receptor potential ankyrin 1 (TRPA1) belongs to
the largest family of TRP ion channels, which exert pleiotropic
functions in a variety of cells and pathophysiological conditions.1
The superfamily of TRP channels in mammals consists of 28 differ-
ent proteins behaving as nonselective Ca2+-permeable cation chan-
human fetal lung fibroblasts,2 is the sole representative of the A
subfamily; it exhibits 14 NH2-terminal ankyrin repeats,3 an unu-
sual structural feature, from which it derives its name.4,5 TRPA1
localizes to primary sensory neurons of the trigeminal (TG), vagal
(VG), dorsal root (DRG) ganglia, and in hair cells.3,5–7 More specif-
ically, TRPA1 co-localizes with the vanilloid, TRPV1, channel in a
subset of somatosensory neurons with non-myelinated or thinly
myelinated fibers and with nociceptive and pain producing func-
tions.3 Most of the TRPA1-expressing neurons contain and release
proinflammatory neuropeptides which mediate nociceptive
responses and neurogenic inflammation.8
Growing evidence has been accumulated in the last 5 years with
respect to the contribution of TRPA1 to nociceptive responses
evoked by a host of xenobiotics,9 including ingredients of various
spicy foods, such as wasabi and mustard oil (allyl isothiocyanate),
cinnamon (cinnamaldehyde), garlic (allicin), and other spices9 or
irritant compounds, such as crotonaldehyde (contained in cigarette
nels with
a
variable permeability ratio (Ca2+/Na+) between
Abbreviations: AITC, allyl isothiocyanate; BSA, bovine serum albumin; CA,
cinnamaldehyde; CHO, Chinese hamster ovary cells; COPD, chronic obstructive
pulmonary disease; DMEM, Dulbecco’s modified eagle medium; DRG, dorsal root
ganglia; EDAC, N-ethyl-N0-(3-dimethylaminopropyl)carbodiimide hydrochloride;
FBS, fetal bovine serum; HBSS, Hanks’ balanced salt solution; HEPES, 4-(2-
hydroxyethyl)-1-piperazineethanesulfonic acid; HOBt, 1-hydroxybenzotriazole
hydrate; IMR90, fetal human lung fibroblasts; NGF, nerve growth factor; ROS,
reactive oxygen species; RNS, reactive nitrogen species; TG, trigeminal ganglia;
TRPA1, transient receptor potential ankyrin 1; TRPV1, transient receptor potential
vanilloid 1; VG, vagal ganglia.
⇑
Corresponding authors. Tel./fax: +39 0532 455921.
E-mail addresses: baraldi@unife.it (P.G. Baraldi), prtdle@unife.it (D. Preti).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2012.01.020

P. G. Baraldi et al. / Bioorg. Med. Chem. 20 (2012) 1690–1698
1691
smoke),6 formaldehyde,10 and general anesthetics.11 This action
accounts for their irritant and pain producing action. Endogenous
TRPA1 activators generally include reactive oxygen species (ROS),
reactive nitrogen species (RNS), and their by-products, and specif-
ically acrolein, 4-hydroxy-2-nonenal, hydrogen peroxide, peroxini-
trite anion, hypochlorite, nitrooleic acid, lipid peroxidation
by-products and cyclopentenone prostaglandins, and isopros-
tanes.9,12–14 All these agents, formed in large amounts and ubiqui-
tously at sites of inflammation or tissue injury, are now identified
as mediators which signal a major pain generating system. The
possibility that by-products of oxidative/nitrative stress, produced
under inflammatory conditions or in case of tissue damage, target
TRPA1, thus contributing to inflammatory and neuropathic pain,
furnished the rational for the design and synthesis of selective
inhibitors as potential analgesic and antiinflammatory agents. De-
spite the fact that in recent years much effort has been dedicated to
the discovery of better and safer analgesics, the medical need for
this type of drugs remains substantially unmet. In particular, com-
pounds capable of targeting both nociceptive and neuropathic pain
are lacking. Thus, the contribution of neuronal TRPA1 in the
orchestration of the inflammatory response and its emerging role
in the mechanism of neuropathic pain indicates TRPA1 antagonists
as novel analgesic drugs. Moreover, there is increasing evidence
that selective inhibitors of TRPA1 channels may be considered po-
tential therapeutic agents in treating respiratory diseases charac-
terized by airways inflammation with beneficial effects in asthma
and COPD, and maybe, chronic cough.15,16
a series of (heteroaryl)alkenone oxime derivatives26, as TRPA1
antagonists. Even though the current status of this research field
shows stimulating pioneering aspects, the applicative value of the
area emerges from the recent announcement by Glenmark Pharma-
ceuticals of the identification of the first TRPA1 selective antagonist
(GRC-17536) planned to enter phase I human clinical trials for the
treatment of pain and respiratory disorders in January 2011.
Herein we present our efforts aimed at the identification of new
TRPA1 antagonists starting from the structure of HC-030031. This
compound, displaying
lM potency in blocking TRPA1, shows a
central xanthine core functionalized at the 7-position with a
phenyl-acetamide chain. In order to improve HC-030031 potency,
we synthesised and tested a new series of 7-substituted-1,3-
dimethyl-1,5-dihydro-pyrrolo[3,2-d]pyrimidine-2,4-dione deriva-
tives (general structures 3–5, Fig. 1) conceived as 9-deaza-analogs
of the xanthine-based reference compounds with general struc-
tures 1 and 2.27 This approach allowed us to indirectly evaluate
the importance of N9 for receptor interaction. Thanks to the optimi-
zation of a helpful synthetic approach (Scheme 1), we were able to
build a small library of compounds introducing an N7-(substi-
tuted)phenylacetamide (in accordance to HC) or an N7-[4-(substi-
tuted-phenyl)-thiazol-2-yl]-acetamide (in accordance to 2) chain.
Different substituents have been introduced at the phenyl moiety
of the N7-phenyl/thiazol-2-yl-acetamide chain such as halogens
or bulky cyclo/arylalkyl groups. During the progress of our work,
Glenmark Pharmaceuticals claimed an analogous series of fused
pyrimidinedione derivatives as TRPA1 modulators.28
In agreement with the hypothesis that TRPA1 blockade may be
beneficial in the treatment of pain, the TRPA1 antagonist 2-(1,3-di-
methyl-2,6-dioxo-1,2,3,6-tetrahydro-purin-7-yl)-N-(4-isopro-
pyl-phenyl)-acetamide 1 developed at Hydra Biosciences (HC-
030031, Fig. 1),10 proved to inhibit mechanical hypersensitivity
caused by both inflammatory and neuropathic pain models.17 In
addition, the N0-(4-sec-butyl-phenyl) analog of 1 (namely Chem-
bridge-5861528) synthesized by ChemBridge Corporation is under
preclinical investigation for the treatment of diabetic neuropathy.18
Different bicycle/tricycle congeners of 1 have recently been devel-
oped at Glenmark Pharmaceuticals as TRPA1 antagonists: substi-
tuted phthalimide,19 imidazo[2,1-b]purine,20 thieno-pyrimidine
dione,21 furo-pyrimidinedione,22 isothiazolo-pyrimidinedione,23
2. Results and discussion
2.1. Synthesis
1,3-Dimethyl-1,5-dihydro-pyrrolo[3,2-d]pyrimidine-2,4-dione
9 (Scheme 1) was prepared in good yield starting from standard
nitration at the 5-position of the commercially available 1,3,6-tri-
methyl-uracil 6 followed by reaction with an excess of triethyl
orthoformate in the presence of morpholine to give the 6-morpho-
lino vinyl derivative 8 and final reductive cyclization with zinc in
acetic acid, according to previously reported procedures.29 Alkyl-
ation at the 7-position with ethyl 2-bromoacetate was performed
in DMF in presence of K2CO3 to obtain the ethyl ester 10 which
was subsequently hydrolyzed using 10% sodium hydroxide to the
corresponding acid intermediate 11. Final compounds with general
structures 3 and 4 were obtained by a coupling reaction of 11 with
(substituted)aryl-, arylalkyl- or cycloalkyl-amines in presence of N-
quinazolinedione24 derivatives. Thanks to
a high-throughput
screening approach, some other molecules have been identified,
such as a small series of trichloro(sulfanyl)ethyl benzamides25 and
ethyl-N0-(3-dimethylaminopropyl)carbodiimide
hydrochloride
(EDAC) and 1-hydroxybenzotriazole hydrate (HOBt) in DMF, at
room temperature. Final derivatives 5a–f were synthesized via an
analogous EDAC/HOBt-mediated condensation of 11 with 4-
(substituted)phenyl-thiazol-2-yl amines which have been conve-
niently prepared from the appropriate commercially available
(substituted)-acetophenones and thiourea.30
O
S
R
O
O
N
N
N
H
O
N
N
H
N
N
N
N
N
N
O
O
2
1, HC030031
2.2. Evaluation of rat and human TRPA1 inhibition
O
Thanks to the identification of hTRPA1 antagonists which
display very low or reverse (agonist) activity against the rat iso-
form of the channel, both the Amgen25 and Abbott31 groups, high-
lighted as TRPA1 is a problematic target as it is subject to a marked
species-specific modulation. Although activity at the human recep-
tor is, in fact, essential in view of future therapeutic perspectives,
the ability to inhibit the rat/mouse receptor is strictly required be-
cause the vast majority of tests for inflammatory, neuropathic and
cancer pain have been developed and validated in rodent species.
In light of this, both human (fetal lung fibroblasts, IMR90) and
rat (primary cultures of rat DRG neurons) cells that constitutively
R'
O
N
N
R
N
N
O
3-5
R = H or CH3
R' = (substituted)aryl, arylalkyl, 4-phenyl-thiazol-2-yl or cycloalkyl moieties
Figure 1. General structures of the newly designed compounds (3–5) and their 9-
aza analogues 1 and 2 reported in literature.

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P. G. Baraldi et al. / Bioorg. Med. Chem. 20 (2012) 1690–1698
O
N
O
N
O
N
NO2
NO2
N
N
N
i
ii
iii
O
O
O
N
O
6
7
8
O
O
O
O
N
O
H
N
R
OR
vi
N
H
N
N
N
N
iv
O
N
O
O
N
N
3a-t
4a-d
5a-f
9
10, R = Et
11, R = H
v
Scheme 1. Reagents and conditions: (i) H2SO4/HNO3, 0 °C, 45’; (ii) Morpholine, HC(OCH2CH3)3, 120 °C, 4h; (iii) Zn, CH3CO2H, 90 °C, 30’; (iv) BrCH2CO2CH2CH3, K2CO3, DMF, rt,
3h; (v) NaOH 10%, CH3OH, rt, 2h; (vi) (substituted)aryl-, arylalkyl- or cycloalkyl- amines EDAC, HOBt, DMF, rt, 12–24 h for 3,4; 4-(substituted)phenyl-thiazol-2-yl amines,
EDAC, HOBt, DMAP, DCE, rt, 8–12 h for 5.
expressed the TRPA1 channel, have been used in the present work
for the in vitro functional screening. We based our research pro-
gram on the evaluation of the modulation of native channels activ-
ity in cells which constitutively express the TRPA1 channel, thus
overcoming problems not infrequently inherent with cloned recep-
tors expressed in recombinant systems that may affect the precise
estimate of potency measurement because of receptor over- or un-
der-expression.
All the synthesized compounds were assayed to evaluate their
ability to block acrolein-mediated activation of human (h) and rat
(r) TRPA1 channels employing a fluorometric calcium imaging as-
say, and the results are shown in Table 1 (hTRPA1) and 2 (rTRPA1).
HC-030031,17 AP18,26 and 4-(3-methoxy-phenyl)-2-thioxo-1,2,3,4-
tetrahydro-indeno[1,2-d]pyrimidin-5-one32 (compound 12 in
tables and figures) were also tested as reference compounds (the
chemical structures of reference compounds are depicted in Fig. 3
of the Supplementary data). The functional data are expressed as
IC50 values and/or percentages of inhibition of channel activity at
with the activity of the 4-t-butyl (3o, IC50 = 3.1
lM), the 4-n-butyl
(3p, IC50 = 1.1 M) and the 4-n-hexyl (3q, IC50 = 1.6) derivatives,
l
confirmed that the presence of a bulky and lipophilic moiety at
the 4-position of the phenyl ring may be of particular importance
for ligand–receptor interaction. Nevertheless, it has to be noted
that 3n, the direct pyrrolopyrimidine analogue of HC030031,
substituted with a 4-isopropyl moiety, proved to be inactive. This
would suggest that the N9 of the xanthine nucleus could be some-
what involved in a favorable interaction with the channel.
While branched and linear alkyl chains were shown to mostly
enhance activity, the 4-cycloalkyl derivative 3r showed a percent-
age of hTRPA1 inhibition lower than 50% at the highest concentra-
tion (30
function (see the 4-N-morpholinyl derivative 3s), did not improve
the activity (hTRPA1 inhibition <50% at 30 M).
lM). The introduction of heteroatoms in the 4-cycloalkyl
l
Compound 3t is the only example of N0-cycloalkyl-acetamide
functionalization of the series. In this derivative, indeed, a cyclo-
alkyl moiety (1-adamantyl) has been directly linked to the nitrogen
of the acetamide side chain. The lack of activity of 3t would indi-
cate the importance of a N-phenyl ring for receptor interaction.
With the synthesis of the N-arylalkyl derivatives 4a–d we intended
to evaluate the effect on TRPA1 channel activity of a methylene (4a,
4d), an ethylene (4b) or a propylene (4c) spacer between the acet-
amide nitrogen and the phenyl ring. Also in this case, the lack of
activity testified the need of an N0-aryl-acetamide moiety on the
N7-side chain.
3 and 30
have been calculated only for compounds with a percentage of
inhibition higher than 50% at 30 M.
lM concentration of antagonist. IC50 values at hTRPA1
l
Differently substituted-phenylacetamide chains have been
introduced at the 7-position of the pyrrolo[3,2-d]pyrimidine-2,
4-dione nucleus (compounds 3a–t, Table 1). The best results have
been obtained in the case of monosubstitution at the 40-position
of the phenyl ring. Among the 4-halo derivatives 3a–d, the 4-
iodo-phenyl substitution appeared particularly effective (3d,
To further extend our research, the N7-[4-(substituted-phenyl)-
thiazol-2-yl]-acetamide derivatives 5a–f have been synthesized as
9-deaza analogues of compounds with general structure 2 (see
Fig. 1) previously reported by Hydra Biosciences.33 As mentioned
above, during the progress of our study, Glenmark Pharmaceuticals
claimed an analogous series of fused pyrimidinedione derivatives
as TRPA1 modulators.28 In these derivatives, the thiazole ring of
the lateral chains behaves as an aromatic spacer between the acet-
amide nitrogen and the terminal phenyl ring. Also in this small
subset, the presence of fluorinated moieties at the 4-position of
the terminal phenyl ring resulted effective to some extent for pro-
moting antagonist activity (see compounds 5b and 5d), while the
presence of a 4-iodine atom determined a substantial loss of activ-
ity (5c). The 3-CF3,4-F-phenyl derivative 5e is the only example of
our series which has also been reported and characterized by Glen-
mark in its patent. The percentages of channel inhibition, obtained
by Glenmark through an in vitro screening performed on a recom-
binant system (human TRPA1 CHO cells), were comparable to
those we obtained with native channels and reported in Table 1.
The pairwise comparison between the activities of analogously
substituted phenylacetamide and (4-phenyl-thiazol-2-yl)-acetam-
ide derivatives resulted in a variable and slight decrease of potency
IC50 = 1.8
l
M). The introduction of a 4-fluoro function determined
M), while
a slight but significant decrease of activity (3a, IC50 = 3.1
l
the 4-chloro substitution produced a marked loss of efficacy (com-
pound 3c showed a percentage of channel inhibition lower than
50% at a concentration of 30 lM). meta-Substitution with an iodine
atom determined a similar decrease of activity if compared to para-
substitution (see compound 3e vs 3d). Also bisubstitution of the
phenyl ring appeared ineffective (compounds 3f and 3g). Analogous
results have been achieved by the introduction of strong electron
donating (3i–k) or withdrawing (3l–m) groups. The bisubstitution
of the acetamide nitrogen determined a significant decrease of effi-
cacy (see compound 3b vs 3a). This would indicate that the free N–
H group could establish a favorable interaction with the receptor.
The efficacy of 4-iodo substitution suggested a possible steric
control around 4-phenyl position, thus we tried to evaluate the ef-
fect of the introduction of hindered halogenated moieties, such as
the –CF3 group (3h), or bulky linear (3p–q), branched (3n–o) or
cycloalkyl (3r) functions. Among these derivatives, the best results
have been obtained with the introduction of a 4-trifluoromethyl
group, which led us to the identification of compound 3h as the
most potent of the whole series (IC50 = 400 nM). This datum, along

P. G. Baraldi et al. / Bioorg. Med. Chem. 20 (2012) 1690–1698
1693
Table 1
In vitro biological activity of the synthesized compounds in the human TRPA1 Ca2+ influx assay in IMR90 fibroblasts
O
O
O
S
R
R
O
N
O
N
O
N
N
H
N
N
R'
N
H
n
N
N
N
N
N
N
R
O
O
O
3a-t
4a-d
5a-f
R
R0
n
hTRPA1a
M)b
% Inhibitionc
R0
IC50
(
l
(30 lM)
HC-03003117
1.8 (1.1–2.8)
2.2 (1.6–3.1)
1.5 (0.9–2.2)
3.1 (1.9–4.9)
73.2 6.4
69.1 7.1
83.1 3.8
53.1 7.9
97.5 0.5
98.9 0.6
98.8 0.9
96.7 1.6
< 50
< 50
82.2 6.2
< 50
< 50
< 50
99.1 2.0
95.6 2.2
<50
<50
<50
<50
<50
89.6 3.1
99.0 0.7
99.0 1.0
<50
<50
<50
AP1826
12d32
3a
3b
3c
4-F-Ph
4-F-Ph
4-Cl-Ph
4-I-Ph
3-I-Ph
2,4-F-Ph
3,4-F-Ph
4-CF3-Ph
4-OCF3-Ph
4-SCF3-Ph
4-OCH3-Ph
4-(SO2NEt2)-Ph
4-(SO2NHBn)-Ph
4-isopropyl-Ph
4-C(CH3)3-Ph
4-n-Butyl-Ph
4-n-Hexyl-Ph
4-Cyclohexyl-Ph
N-Morpholinyl-Ph
H
Me
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
1.8 (0.7–3.9)
61.9 16.4
0.4 (0.3–0.5)
3.7 (2.3–6.2)
99.0 3.0
40.5 9.0
3.1 (1.6–6.0)
1.1 (0.8–1.4)
1.6 (0.9–2.8)
46.0 7.8
93.5 4.2
74.5 6.2
3s
3t
1-Adamantyl
4a
4b
4c
4d
5a
5b
5c
H
H
H
Me
H
4-F
4-I
1
2
3
1
<50
<50
<50
<50
<50
82.8 4.7
<50
4.1 (1.2–8.2)
47.9 8.1
5d
5e
5f
4-CF3
3-CF3,4-F
4-n-Hexyl
2.1 (0.8–6.4)
1.3 (0.6–2.8)
2.6 (1.2–5.6)
66.8 10.6
76.1 9.6
50.9 11.5
99.0 1.0
99.3 1.2
99.0 1.0
a
b
c
Human TRPA1 receptor activated by acrolein (30 lM).
Inhibitory potency is expressed as the molar concentration of antagonist producing 50% of the maximum effect (IC50) with confidence interval (CI).
% of inhibition (mean SEM) at indicated concentrations in at least 25 independent cells and three different experiments.
4-(3-Methoxy-phenyl)-2-thioxo-1,2,3,4-tetrahydro-indeno[1,2-d]pyrimidin-5-one.
d
for the thiazole-spaced compounds (see IC50 values and% of inhibi-
tion of 3a, 3d, 3h and 3q vs those of 5b, 5c, 5d and 5f, respectively).
This effect is particularly significant in the case of 4-iodo- (see 5c
vs 3d) or 4-CF3- (see 5d vs 3h) substitution.
The most potent compounds against human TRPA1 channel
have been screened for their potential to inhibit the activity of
the rat channel subtype (rat DRG neurons). The resulting potencies,
the remaining derivatives of the series 3 and 5 obtained in human
TRPA1 expressing cells have been reported in Fig. 4 of the
Supplementary data). IC50 values from rat DRG neurons have been
calculated for selected compounds (i.e. 3h and 5e, Fig. 2B) and even
though 3h proved to maintain antagonist properties, it resulted
7-fold less potent against the rat isoform of the channel, while
the thiazole derivative 5e showed to be equipotent.
expressed as percentage of inhibition at 3 and 30
l
M, have been
Consistent with our experimental conditions, HC-030031 and
AP18 displayed activities (IC50 and % inhibition) comparable to
those previously reported and measured in different assays,9
whereas compound 1232 resulted sensibly less potent (hTRPA1
reported in Table 2. While the efficacies toward rat and human
TRPA1 at the highest concentration resulted mostly comparable,
a sharp decrease of activity in DRG neurons has been occasionally
detected at a concentration of 3
lM (see compounds 3d, 3o, 5b).
IC50 = 1.5 lM; rTRPA1 IC50 = 3.4 lM) with regard to data previously
Nevertheless, compound 3p, 5e and 5f confirmed their efficacy
against rTRPA1 with percentages of inhibition higher than 80% at
the lowest considered concentration.
Figure 2 depicts the dose-dependent inhibition curves evoked
by 3h, 5e (as representative pyrrolo[3,2-d]pyrimidines), HC-
030031, AP18 and 12 (as reference compounds) in human (A) or
rat (B) TRPA1 (the concentration-dependent inhibition curves of
obtained with hTRPA1 transfected in CHO cells (IC50 = 128 nM).
This discrepancy could be partly related to differences in receptor
expression between recombinant and native systems together
with the employment of acrolein as agonist instead of cinnamalde-
hyde (CA) or allyl isothiocyanate (AITC) used in most of the re-
ported studies.9 Acrolein is an exogenous irritant component of
air pollution and cigarette smoke6, which, unlike CA and AITC, is

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P. G. Baraldi et al. / Bioorg. Med. Chem. 20 (2012) 1690–1698
Table 2
circumstances that may be encountered in vivo. The disadvantage
is that acrolein, a very reactive byproduct of oxidative stress may
act on the channel by still undetermined mechanisms and there-
fore its action at the native TRPA1 may result in a larger variability
in the calcium response.
Inhibitory effect of different concentrations on the calcium response evoked by
acrolein (30 M) in rat DRG neurons
l
rTRPA1a% of inhibitionb
30 lM
3
l
M
Compounds showing activity at the h/rTRPA1 have been also
screened in rat DRG neurons, against capsaicin (0.1 lM) to verify
the selectivity of the antagonism versus TRPV1. In no case was ob-
served a significant inhibition (higher than 10%) on the response
evoked by capsaicin.
HC-03003117
80.7 5.2
45.2 15.1
40.2 15.3
53.1 10.9
12.4 9.3
58.8 11.8
36.8 11.2
10.7 6.6
94.4 3.5
35.5 13.0
3.6 5.2
99.8 0.6
90.1 9.8
99.5 0.9
96.7 1.6
85.4 14.6
99.7 0.8
99.5 0.6
95.8 4.2
97.1 2.1
93.3 4.1
99.2 0.8
80.2 12.6
99.6 0.8
99.4 3.7
AP1826
12c30
3a
3d
3h
3i
3o
3p
3q
5b
5d
3. Conclusions
The treatment of chronic pain, and particularly neuropathic
pain, remains unsatisfactory. This observation underscores the
importance of considering, validating, and pursuing alternative
therapeutic targets. One emerging opportunity in this therapeutic
area is offered by selective blockade of TRPA1 channels. In this
study we described the design, synthesis and functional evaluation
of a new series of molecules capable of inhibiting TRPA1. Most of
the few specific antagonists so far reported suffered from low po-
tency, and the limited number of lead compounds displaying nano-
molar potency have been screened in recombinant systems against
exogenous agonists. Our study, based on the employment of both
human and rat cells that constitutively expressed the investigated
target, led us to the identification of compound 3h (hTRPA1:
58.3 7.6
80.6 6.9
92.4 5.7
5e
5f
a
Rat TRPA1 receptor activated by acrolein (30
% of inhibition (mean SEM) at indicated concentrations in at least 20 inde-
pendent neurons and three different experiments.
4-(3-Methoxy-phenyl)-2-thioxo-1,2,3,4-tetrahydro-indeno[1,2-d]pyrimidin-5-
one.
l
M).
b
c
IC50 = 400 nM, percentage of channel inhibition at 1 lM = 88.67
6.31) as the most potent of the series at the human channel. This
derivative showed a considerably improved potency (sub-micro-
molar) with regard to some of the most representative antagonists
previously reported (AP18, HC-030031 and 12), and included in
our screening program as reference compounds. In addition, 3h
proved to be selective versus TRPV1channel and to maintain a sat-
isfactory rank of efficacy toward rTRPA1, which designates it as a
possible candidate for future optimization in view of potential
evaluation of in vivo efficacy in rodent animal models. It must be
highlighted that the potency of 3h has been assessed against acro-
lein as an endogenous mediator of inflammation. This contributes
to the extension of the potential of such molecules as valuable
tools for the pharmacological characterization of the TRPA1 chan-
nel, and its validation as a target in the therapeutic area of pain,
inflammation, and airways diseases.
4. Experimental section
4.1. Chemistry
4.1.1. Materials and methods
AP18 was purchased from Tocris Bioscience, while HC-0300316
Figure 2. Concentration-dependent inhibition curves and IC50 values of the
selective TRPA1 antagonists, HC-030031 and AP18, and the pyrrolo[3,2-d]pyrimi-
dines derivatives obtained in human TRPA1 (A) or rat TRPA1 (B) constitutive
expressing cells. Inhibitory potency on TRPA1 activation has been obtained testing
and
4-(3-methoxy-phenyl)-2-thioxo-1,2,3,4-tetrahydro-indeno
[1,2-d]pyrimidin-5-one32 were synthesized as previously described.
Reactions progress and product mixtures were routinely monitored
by thin-layer chromatography (TLC) on silica gel (precoated F254
Merck plates) and visualized by UV lamp (254 nm light source) or
with aqueous potassium permanganate. Light petroleum refers to
the fractions boiling at 40–60 °C. Chromatography was performed
on Merck 230–400 mesh silica gel. After extraction from aqueous
phases, theorganic solutions were dried over anhydroussodiumsul-
fate. Melting points for purified products were determined in a glass
capillary on a Stuart Scientific electro thermal apparatus SMP3 and
are uncorrected. 1H NMR data were determined in CDCl3 or
DMSO-d6 solutions with a Varian VXR 200 spectrometer. Peak posi-
tions are given in parts per million (d) downfield from tetramethyl-
silane as internal standard, and J values are given in Hertz. Splitting
patterns are designed as s, singlet; d, doublet; t, triplet; q, quartet; m,
the antagonists at different concentrations (all 0.1–30
lM) against a concentration
of the selective endogenous TRPA1 agonist, acrolein (30
l
M), which induces about
80% of the maximum effect. Each point represents the average of at least 25
independent cells; values are mean SEM.
generated endogenously during lipid peroxidation and contributes
to oxidative stress-related pathophysiologic conditions in cells and
tissues.34 Thus, as a byproduct of oxidative/nitrative stress con-
verging on TRPA1 to alert the sensory system of the presence of
inflammation or tissue damage, acrolein has been chosen as TRPA1
agonist for in vitro screenings of potential antagonists. The advan-
tage of using the present assay system (acrolein targeting the
TRPA1 channel expressed by DRG neurons) is that this mimics real

P. G. Baraldi et al. / Bioorg. Med. Chem. 20 (2012) 1690–1698
1695
multiplet; b, broad. All the detected signals were in accordance with
the proposed structures. Elemental analyses were performed by the
microanalytical laboratory of Dipartimento di Chimica, University of
Ferrara, and were within 0.4% of the theoretical values for C, H, and
N. All final compounds revealed a purity of not less than 95%. The
mass spectra were obtained on a ESI Micromass ZMD 2000 mass
spectrometer.
(d, 1H, J = 2.8), 7.19 (d, 1H, J = 3), 7.38–7.54 (m, 4H). MS (ESI):
[MH]+ = 345.2. Anal. (C17H17FN4O3) C, H, N.
4.1.4.3. N-(4-Chloro-phenyl)-2-(1,3-dimethyl-2,4-dioxo-1,2,3,4-
tetrahydro-pyrrolo[3,2-d]pyrimidin-5-yl)-acetamide (3c). The
product was purified by crystallization with MeOH; white solid;
60% yield; mp 262–263 °C; 1H NMR (200 MHz, DMSO-d6): d (ppm)
3.18 (s, 3H), 3.39 (s, 3H), 5.18 (s, 2H), 7.60 (d, 2H, J = 9), 10.43 (br s,
1H). MS (ESI): [MH]+ = 347.6. Anal. (C16H15ClN4O3) C, H, N.
4.1.2. (1,3-Dimethyl-2,4-dioxo-1,2,3,4-tetrahydro-pyrrolo[3,2-
d]pyrimidin-5-yl)-acetic acid ethyl ester (10)
1,3-Dimethyl-1,5-dihydro-pyrrolo[3,2-d]pyrimidine-2,4-dione
9 (2 g, 11.2 mmol) was dissolved in DMF (15 mL), K2CO3 (3.1 g,
22.4 mmol) was added and the mixture was stirred at room tem-
perature for 10 min. After cooling at 0 °C, a solution of ethyl bromo-
acetate (2.05 g, 12.32 mmol) in DMF (5 mL) was added dropwise
and the reaction was stirred at room temperature for a further
3 h. The solvent was evaporated and the residue partitioned be-
tween EtOAc (15 mL) and water (20 mL). The aqueous phase was
further extracted with EtOAc (2 ꢀ 15 mL) and the organic layers
were dried over anhydrous Na2SO4. Evaporation of the solvent gave
a solid residue which was filtered after suspension with a 1:1 mix-
ture of Et2O/Petroleum ether (15 mL). White solid; 80% yield; mp
171–172 °C; 1H NMR (200 MHz, CDCl3): d (ppm) 1.29 (t, 3H,
J = 7.4), 3.37 (s, 3H), 3.46 (s, 3H), 4.23 (q, 2H, J = 7), 5.10 (s, 2H),
5.99 (d, 1H, J = 3), 6.93 (d, 1H, J = 3). MS (ESI): [MH]+ = 265.3.
4.1.4.4.
2-(1,3-Dimethyl-2,4-dioxo-1,2,3,4-tetrahydro-pyrrol-
o[3,2-d ]pyrimidin-5-yl)-N-(4-iodo-phenyl)-acetamide (3d).
The product was purified by crystallization with DMF/Et2O; white
solid; 52% yield; mp 286–287 °C; 1H NMR (200 MHz, DMSO-d6): d
(ppm) 3.18 (s, 3H), 3.39 (s, 3H), 5.17 (s, 2H), 6.19 (s, 1H), 7.33 (s,
1H), 7.40 (d, 2H, J = 7.4), 7.64 (d, 2H, J = 6.8), 12.00 (br s, 1H). MS
(ESI): [MH]+ = 438.9. Anal. (C16H15IN4O3) C, H, N.
4.1.4.5.
2-(1,3-Dimethyl-2,4-dioxo-1,2,3,4-tetrahydro-pyrrolo
[3,2-d]pyrimidin-5-yl)-N-(3-iodo-phenyl)-acetamide (3e). The
product was purified by crystallization with 1,4-dioxane; white so-
lid; 42% yield; mp 286–287 °C; 1H NMR (200 MHz, DMSO-d6): d
(ppm) 3.18 (s, 3H), 3.39 (s, 3H), 5.17 (s, 2H), 6.20 (d, 1H, J = 2.8),
7.14–7.16 (m, 3H), 7.32 (d, 1H, J = 2.6), 8.09 (s, 1H), 10.40 (br s,
1H). MS (ESI): [MH]+ = 439.1. Anal. (C16H15IN4O3) C, H, N.
4.1.3. (1,3-Dimethyl-2,4-dioxo-1,2,3,4-tetrahydro-pyrrolo[3,2-
d]pyrimidin-5-yl)-acetic acid (11)
4.1.4.6. N-(2,4-Difluoro-phenyl)-2-(1,3-dimethyl-2,4-dioxo-1,2,3
,4-tetrahydro-pyrrolo[3,2-d]pyrimidin-5-yl)-acetamide (3f).
The product was purified by crystallization with EtOH; white solid;
62% yield; mp 196–198 °C; 1H NMR (200 MHz, DMSO-d6): d (ppm)
3.19 (s, 3H), 3.38 (s, 3H), 5.23 (s, 2H), 6.20 (d, 1H, J = 2.8), 6.99–7.16
(m, 1H), 7.22–7.41 (m, 2H), 7.80–8.00 (m, 1H), 10.09 (br s, 1H). MS
(ESI): [MH]+ = 349.1. Anal. (C16H14F2N4O3) C, H, N.
The ester derivative 10 (2 g, 7.55 mmol) was hydrolyzed by
treatment with a mixture of NaOH 10% (20 mL) and MeOH
(20 mL) for 2 h at room temperature under magnetic stirring. The
solution was concentrated to half volume and the product was pre-
cipitated by acidification with HCl 10%, filtered, washed with water
and dried in vacuum over phosphorus pentoxide. White solid; 84%
yield; mp 264–265 °C; 1H NMR (200 MHz, DMSO-d6): d (ppm) 3.19
(s, 3H), 3.37 (s, 3H), 5.04 (s, 2H), 6.17 (d, 1H, J = 3), 7.30 (d, 1H,
J = 3), 13.00 (br s, 1H). MS (ESI): [MH]+ = 337.1.
4.1.4.7. N-(3,4-Difluoro-phenyl)-2-(1,3-dimethyl-2,4-dioxo-1,2,
3,4-tetrahydro-pyrrolo[3,2-d]pyrimidin-5-yl)-acetamide (3g).
The product was purified by crystallization with EtOH; white solid;
67% yield; mp 238–239 °C; 1H NMR (200 MHz, DMSO-d6): d (ppm)
3.18 (s, 3H), 3.39 (s, 3H), 5.18 (s, 2H), 6.21 (d, 1H, J = 2.9), 7.20–7.42
(m, 3H), 7.60–7.80 (m, 1H), 10.54 (br s, 1H). MS (ESI):
[MH]+ = 349.3. Anal. (C16H14F2N4O3) C, H, N.
4.1.4. General procedure for the synthesis of pyrrolo[3,2-
d]pyrimidin derivatives 3a–t and 4a–d
The acetic acid derivative 11 (0.4 mmol) was dissolved in DMF
(3 mL) then EDAC (0.4 mmol) and HOBt (0.4 mmol) were added.
The mixture was stirred at room temperature for 10 min, then
the appropriate (substituted)aryl/arylalkyl/cycloalkyl amine
(0.4 mmol) was added, and the reaction stirred at room tempera-
ture until disappearance of the starting reagents (12–24 h). The
solvent was removed under vacuum to give a residue which was
partitioned between EtOAc and water. The water layer was further
extracted with EtOAc (2 ꢀ 10 mL) and the combined organic layers
were dried over anhydrous Na2SO4. Evaporation of the solvent gave
a crude residue which was purified by crystallization with the
appropriate solvents.
4.1.4.8. 2-(1,3-Dimethyl-2,4-dioxo-1,2,3,4-tetrahydro-pyrrolo[3,
2-d ]pyrimidin-5-yl)-N-(4-trifluoromethyl-phenyl)-acetamide
(3h). The product was purified by crystallization with EtOH;
white solid; 56% yield; mp 266–267 °C; 1H NMR (200 MHz,
DMSO-d6): d (ppm) 3.18 (s, 3H), 3.39 (s, 3H), 5.22 (s, 2H), 6.21 (d,
1H, J = 3), 7.34 (d, 1H, J = 3), 7.66–7.80 (m, 4H), 10.69 (br s, 1H).
MS (ESI): [MH]+ = 381.3. Anal. (C17H15F3N4O3) C, H, N.
4.1.4.9. 2-(1,3-Dimethyl-2,4-dioxo-1,2,3,4-tetrahydro-pyrrolo[3,
2-d ]pyrimidin-5-yl)-N-(4-trifluoromethoxy-phenyl)-acetamide
(3i). The product was purified by crystallization with EtOH; white
solid; 81% yield; mp 281–282 °C; 1H NMR (200 MHz, DMSO-d6): d
(ppm) 3.18 (s, 3H), 3.39 (s, 3H), 5.19 (s, 2H), 6.20 (d, 1H, J = 2.9),
7.30–7.34 (m, 3H), 7.66 (d, 2H, J = 9), 10.54 (br s, 1H). MS (ESI):
[MH]+ = 397.3. Anal. (C17H15F3N4O4) C, H, N.
4.1.4.1.
2-(1,3-Dimethyl-2,4-dioxo-1,2,3,4-tetrahydro-pyrrol-
o[3,2-d ]pyrimidin-5-yl)-N-(4-fluoro-phenyl)-acetamide (3a).
The product was purified by crystallization with EtOH; white solid;
75% yield; mp 217–218 °C; 1H NMR (200 MHz, DMSO-d6): d (ppm)
3.18 (s, 3H), 3.39 (s, 3H), 5.16 (s, 2H), 6.19 (d, 1H, J = 3), 7.10–7.19
(m, 2H), 7.33 (d, 1H, J = 3), 7.55–7.62 (m, 2H), 10.33 (s, 1H). MS
(ESI): [MH]+ = 331.4. Anal. (C16H15FN4O3) C, H, N.
4.1.4.10. 2-(1,3-Dimethyl-2,4-dioxo-1,2,3,4-tetrahydro-pyrrol-
o[3,2-d ]pyrimidin-5-yl)-N-(4-trifluoromethylsulfanyl-phenyl)-
acetamide (3j). The product was purified by crystallization with
EtOH; white solid; 44% yield; mp 298–299 °C; 1H NMR
(200 MHz, DMSO-d6): d (ppm) 3.18 (s, 3H), 3.39 (s, 3H), 5.21 (s,
2H), 6.20 (d, 1H, J = 2.9), 7.33 (d, 1H, J = 2.8), 7.64–7.75 (m, 4H),
10.63 (br s, 1H). MS (ESI): [MH]+ = 413.1. Anal. (C17H15F3N4O3S)
C, H, N.
4.1.4.2.
2-(1,3-Dimethyl-2,4-dioxo-1,2,3,4-tetrahydro-pyrrolo
[3,2-d ]pyrimidin-5-yl)-N-(4-fluoro-phenyl)-N-methyl-acetam-
ide (3b). The product was purified by crystallization with EtOH;
white solid; 55% yield; mp 222–223 °C; 1H NMR (200 MHz, DMSO-
d6): d (ppm) 3.15 (s, 3H), 3.19 (s, 3H), 3.36 (s, 3H), 4.84 (s, 2H), 6.14

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P. G. Baraldi et al. / Bioorg. Med. Chem. 20 (2012) 1690–1698
4.1.4.11. 2-(1,3-Dimethyl-2,4-dioxo-1,2,3,4-tetrahydro-pyrrolo
[3,2-d]pyrimidin-5-yl)-N-(4-methoxy-phenyl)-acetamide (3k).
The product was purified by crystallization with MeOH; white so-
lid; 60% yield; mp 238–239 °C; 1H NMR (200 MHz, DMSO-d6): d
(ppm) 3.18 (s, 3H), 3.38 (s, 3H), 3.71 (s, 3H), 5.14 (s, 2H), 6.18 (d,
1H, J = 3), 6.86 (d, 2H, J = 8.2), 7.32 (d, 1H, J = 3), 7.46 (d, 2H,
J = 8.2), 10.11 (br s, 1H). MS (ESI): [MH]+ = 343.3. Anal.
(C17H18N4O4) C, H, N.
(ppm) 1.10–1.50 (m, 6H), 1.60–1.80 (m, 4H), 2.40–2.49 (m, 1H),
3.18 (s, 3H), 3.39 (s, 3H), 5.15 (s, 2H), 6.18 (d, 1H, J = 3), 7.13 (d,
2H, J = 8.6), 7.32 (d, 1H, J = 2.8), 7.46 (d, 2H, J = 8.4), 10.20 (br s,
1H). MS (ESI): [MH]+ = 395.0. Anal. (C22H26N4O3) C, H, N.
4.1.4.19. 2-(1,3-Dimethyl-2,4-dioxo-1,2,3,4-tetrahydro-pyrrol-
o[3,2-d]pyrimidin-5-yl)-N-(4-morpholin-4-yl-phenyl)-acetam-
ide (3s). The product was purified by crystallization with 1,4-
dioxane; white solid; 50% yield; mp 265–266 °C; 1H NMR
(200 MHz, DMSO-d6): d (ppm) 3.00–3.05 (m, 4H), 3.19 (s, 3H),
3.38 (s, 3H), 3.69–3.74 (m, 4H), 5.14 (s, 2H), 6.18 (d, 1H, J = 2.8),
6.88 (d, 2H, J = 9.2), 7.32 (d, 1H, J = 2.8), 7.43 (d, 2H, J = 9), 10.05
(br s, 1H). MS (ESI): [MH]+ = 398.4. Anal. (C20H23N5O4) C, H, N.
4.1.4.12. N-(4-Diethylsulfamoyl-phenyl)-2-(1,3-dimethyl-2,4-di
oxo-1,2,3,4-tetrahydro-pyrrolo[3,2-d]pyrimidin-5-yl)-acetam-
ide (3l). The product was purified by column chromatography
eluting with EtOAc; white solid; 35% yield; mp 217–218 °C; 1H
NMR (200 MHz, DMSO-d6): d (ppm) 1.02 (t, 6H, J = 7.2), 3.07–
3.18 (m, 7H), 3.39 (s, 3H), 5.21 (s, 2H), 6.21 (d, 1H, J = 2.8), 7.33
(d, 1H, J = 3), 7.70–7.80 (m, 4H), 10.71 (br s, 1H). MS (ESI):
[MH]+ = 448.2. Anal. (C20H25N5O5S) C, H, N.
4.1.4.20. N-Adamantan-1-yl-2-(1,3-dimethyl-2,4-dioxo-1,2,3,4-
tetrahydro-pyrrolo[3,2-d]pyrimidin-5-yl)-acetamide (3t). The
product was purified by column chromatography eluting with a
mixture of EtOAc/Petroleum ether 7:3; white solid; 80% yield;
mp 277–278 °C; 1H NMR (200 MHz, DMSO-d6): d (ppm) 1.60 (br
s, 6H), 1.91 (br s, 6H), 1.99 (br s, 3H), 3.19 (s, 3H), 3.37 (s, 3H),
4.87 (s, 2H), 6.12 (d, 1H, J = 2.8), 7.22 (d, 1H, J = 3), 7.60 (br s,
1H). MS (ESI): [MH]+ = 371.0. Anal. (C22H26N4O3) C, H, N.
4.1.4.13. N-(4-Benzylsulfamoyl-phenyl)-2-(1,3-dimethyl-2,4-dio
xo-1,2,3,4-tetrahydro-pyrrolo[3,2-d]pyrimidin-5-yl)-acetamide
(3m). The product was purified by column chromatography elut-
ing with EtOAc; white solid; 45% yield; mp 273–274 °C; 1H NMR
(200 MHz, DMSO-d6): d (ppm) 3.18 (s, 3H), 3.40 (s, 3H), 3.95 (s,
2H), 5.22 (s, 2H), 6.21 (d, 1H, J = 2.8), 7.18–7.35 (m, 6H), 7.70–
7.80 (m, 4H), 8.10 (br s, 1H), 10.8 (br s, 1H). MS (ESI):
[MH]+ = 482.2. Anal. (C23H23N5O5S) C, H, N.
4.1.4.21.
N-Benzyl-2-(1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahy-
dro-pyrrolo[3,2-d]pyrimidin-5-yl)-acetamide (4a). The product
was purified by crystallization with MeOH; white solid; 61% yield;
mp 213–214 °C; 1H NMR (200 MHz, DMSO-d6): d (ppm) 3.21 (s,
3H), 3.38 (s, 3H), 4.28 (d, 2H, J = 5.8), 5.03 (s, 2H), 6.17 (d, 1H,
J = 3), 7.20–7.40 (m, 6H), 8.51 (br t, 1H). MS (ESI): [MH]+ = 327.6.
Anal. (C17H18N4O3) C, H, N.
4.1.4.14. 2-(1,3-Dimethyl-2,4-dioxo-1,2,3,4-tetrahydro-pyrrolo
[3,2-d
]pyrimidin-5-yl)-N-(4-isopropyl-phenyl)-acetamide
(3n). The product was purified by crystallization with MeOH;
white solid; 74% yield; mp 252–253 °C; 1H NMR (200 MHz,
DMSO-d6): d (ppm) 1.15 (s, 3H), 1.18 (s, 3H), 2.70–2.90 (m, 1H),
3.18 (s, 3H), 3.39 (s, 3H), 5.16 (s, 2H), 6.19 (d, 1H, J = 2.9), 7.16 (d,
2H, J = 8.6), 7.33 (d, 1H, J = 3), 7.47 (d, 2H, J = 8.2), 10.19 (br s, 1H).
MS (ESI): [MH]+ = 355.7. Anal. (C19H22N4O3) C, H, N.
4.1.4.22. 2-(1,3-Dimethyl-2,4-dioxo-1,2,3,4-tetrahydro-pyrrol-
o[3,2-d]pyrimidin-5-yl)-N-phenethyl-acetamide (4b). The prod-
uct was purified by crystallization with EtOH; white solid; 30%
yield; mp 192–193 °C; 1H NMR (200 MHz, DMSO-d6): d (ppm)
2.70 (t, 2H, J = 6.8), 3.20–3.40 (m, 8H), 4.93 (s, 2H), 6.16 (d, 1H,
J = 2.8), 7.17–7.27 (m, 6H), 8.04 (bt, 1H). MS (ESI): [MH]+ = 341.6.
Anal. (C18H20N4O3) C, H, N.
4.1.4.15.
N-(4-tert-Butyl-phenyl)-2-(1,3-dimethyl-2,4-dioxo-
1,2,3,4-tetrahydro-pyrrolo[3,2-d]pyrimidin-5-yl)-acetamide
(3o). TheproductwaspurifiedbycrystallizationwithMeOH;white
solid; 65% yield; mp 238 °C dec; 1H NMR (200 MHz, DMSO-d6): d
(ppm) 1.25 (s, 9H), 3.19 (s, 3H), 3.39 (s, 3H), 5.16 (s, 2H), 6.19 (d,
1H, J = 3), 7.29–7.33 (m, 3H), 7.48 (d, 2H, J = 8.8), 10.18 (br s, 1H).
MS (ESI): [MH]+ = 369.3. Anal. (C20H24N4O3) C, H, N.
4.1.4.23. 2-(1,3-Dimethyl-2,4-dioxo-1,2,3,4-tetrahydro-pyrrolo
[3,2-d ]pyrimidin-5-yl)-N-(3-phenyl-propyl)-acetamide (4c).
The product was purified by crystallization with EtOH; white solid;
44% yield; mp 190–191 °C; 1H NMR (200 MHz, DMSO-d6): d (ppm)
1.65–1.74 (m, 2H), 2.49–2.59 (m, 2H), 3.00–3.10 (m, 2H), 3.19 (s,
3H), 3.37 (s, 3H), 4.95 (s, 2H), 6.16 (d, 1H, J = 3), 7.16–7.30 (m, 6H),
8.04 (bt, 1H). MS (ESI): [MH]+ = 355.7. Anal. (C19H22N4O3) C, H, N.
4.1.4.16. N-(4-Butyl-phenyl)-2-(1,3-dimethyl-2,4-dioxo-1,2,3,4-
tetrahydro-pyrrolo[3,2-d]pyrimidin-5-yl)-acetamide (3p). The
product was purified by crystallization with MeOH; white solid;
60% yield; mp 233–234 °C; 1H NMR (200 MHz, DMSO-d6): d (ppm)
0.87 (t, 3H, J = 7.2), 1.20–1.40 (m, 2H), 1.40–1.60 (m, 2H), 2.48–
2.55 (m, 2H), 3.18 (s, 3H), 3.39 (s, 3H), 5.16 (s, 2H), 6.19 (d, 1H,
J = 2.8), 7.11 (d, 2H, J = 8.6), 7.31 (d, 1H, J = 2.8), 7.46 (d, 2H, J = 8.4),
10.18 (br s, 1H). MS (ESI): [MH]+ = 369.5. Anal. (C20H24N4O3) C, H, N.
4.1.4.24. 2-(1,3-Dimethyl-2,4-dioxo-1,2,3,4-tetrahydro-pyrrolo
[3,2-d ]pyrimidin-5-yl)-N-(4-methyl-benzyl)-acetamide (4d).
The product was purified by crystallization with EtOH; white solid;
60% yield; 236–237 °C; 1H NMR (200 MHz, DMSO-d6): d (ppm) 2.27
(s, 3H), 3.21 (s, 3H), 3.38 (s, 3H), 4.23 (d, 2H, J = 5.6), 5.01 (s, 2H),
6.17 (d, 1H, J = 3), 7.10–7.20 (m, 4H), 7.28 (d, 1H, J = 2.6), 8.45
(bt, 1H). MS (ESI): [MH]+ = 341.6. Anal. (C18H20N4O3) C, H, N.
4.1.4.17. 2-(1,3-Dimethyl-2,4-dioxo-1,2,3,4-tetrahydro-pyrrolo
[3,2-d]pyrimidin-5-yl)-N-(4-hexyl-phenyl)-acetamide (3q). The
product was purified by crystallization with EtOH; white solid;
80% yield; mp 217 °C; 1H NMR (200 MHz, DMSO-d6): d (ppm) 0.84
(t, 3H, J = 7.1), 1.10–1.40 (m, 6H), 1.40–1.60 (m, 2H), 2.48–2.55 (m,
2H), 3.18 (s, 3H), 3.38 (s, 3H), 5.15 (s, 2H), 6.17 (d, 1H, J = 2.6), 7.10
(d, 2H, J = 8.2), 7.31 (d, 1H, J = 2.6), 7.46 (d, 2H, J = 8.2), 10.17 (br s,
1H). MS (ESI): [MH]+ = 397.0. Anal. (C22H28N4O3) C, H, N.
4.1.5. General procedure for the synthesis of pyrrolo[3,2-
d]pyrimidin derivatives 5a–f
The acetic acid derivative 11 (0.4 mmol) was dissolved in DCE
(3 mL), then EDAC (0.48 mmol), HOBt (0.12 mmol) and DMAP
(0.04 mmol) were added. The mixture was stirred at room temper-
ature for 15 min, then the appropriate 4-(substituted)phenyl-thia-
zol-2-yl amine (0.4 mmol) was added and the reaction stirred at
room temperature until disappearance of the starting reagents
(8–12 h). The solvent was removed under vacuum to give a residue
which was partitioned between EtOAc and water. The water layer
was further extracted with EtOAc (2 ꢀ 10 mL) and the combined
4.1.4.18. N-(4-Cyclohexyl-phenyl)-2-(1,3-dimethyl-2,4-dioxo-1,
2,3,4-tetrahydro-pyrrolo[3,2-d]pyrimidin-5-yl)-acetamide
(3r). The product was purified by crystallization with EtOH; white
solid; 82% yield; mp 276–277 °C; 1H NMR (200 MHz, DMSO-d6): d

P. G. Baraldi et al. / Bioorg. Med. Chem. 20 (2012) 1690–1698
1697
organic layers were dried over anhydrous Na2SO4. Evaporation of
the solvent gave a crude residue which was purified by crystalliza-
tion with the opportune solvents.
ity-controlled vivarium (12 h dark/light cycle, free access to food
and water) for at least 48 h before test. Animals were eutha-
nized with a high dose of sodium pentobarbital (200 mg/kg,
intraperitoneal).
4.1.5.1. 2-(1,3-Dimethyl-2,4-dioxo-1,2,3,4-tetrahydro-pyrrolo[3,
2-d]pyrimidin-5-yl)-N-(4-phenyl-thiazol-2-yl)-acetamide (5a).
The product was purified by crystallization with DMF/H2O; white
solid; 30% yield; mp >300 °C; 1H NMR (200 MHz, DMSO-d6): d
(ppm) 3.17 (s, 3H), 3.40 (s, 3H), 5.32 (s, 2H), 6.23 (d, 1H, J = 3),
7.20–7.44 (m, 4H), 7.64 (d, 1H, J = 2.8), 7.89–7.94 (m, 2H), 12.5
(br s, 1H). MS (ESI): [MH]+ = 396.3. Anal. (C19H17N5O3S) C, H, N.
4.2.2. Cell culture and isolation of primary sensory neurons
We used fetal human lung fibroblasts (IMR90; American Type
Culture Collection; Manassas, VA, USA), that constitutively express
the human TRPA1 channel2, and primary culture of rat sensitive
neurons taken from dorsal root ganglion. Fibroblasts were grown
in DMEM added with 10% FBS, 2 mM
L-glutamine, 100 U/ml peni-
cillin, 100 g/ml streptomycin in 95% air, 5% CO2 at 37 °C in a
l
humidified atmosphere. For calcium imaging measurement, cells
were plated on glass coverslips and grown to a 60% of confluence.
For isolation of rat primary sensitive neurons, ganglia were re-
moved, placed in cold HBSS, and transferred to HBSS containing
collagenase type 1A (2 mg/ml), trypsin (1 mg/ml), for 35 min at
37 °C. Ganglia, placed in warmed DMEM complete medium con-
4.1.5.2. 2-(1,3-Dimethyl-2,4-dioxo-1,2,3,4-tetrahydro-pyrrolo[3,
2-d ]pyrimidin-5-yl)-N-[4-(4-fluoro-phenyl)-thiazol-2-yl]-acet-
amide (5b). The product was purified by crystallization with
DMF/H2O; white solid; 35% yield; mp >300 °C; 1H NMR
(200 MHz, DMSO-d6): d (ppm) 3.18 (s, 3H), 3.40 (s, 3H), 5.32 (s,
2H), 6.23 (d, 1H, J = 3), 7.23–7.36 (m, 3H), 7.63 (s, 1H), 7.91–7.98
(m, 2H), 12.60 (br s, 1H). MS (ESI): [MH]+ = 414.3. Anal.
(C19H16FN5O3S) C, H, N.
taining 10% FBS, 10% horse serum, 2 mM
L-glutamine, 100 U/ml
penicillin, and 100 g/ml streptomycin, were dissociated in single
l
cells by several passages through a series of syringe needles (23–
25 G). Medium and ganglia cells were filtered to remove debris
and centrifuged. The pellet was resuspended in DMEM complete
4.1.5.3. 2-(1,3-Dimethyl-2,4-dioxo-1,2,3,4-tetrahydro-pyrrolo[3,
2-d]pyrimidin-5-yl)-N-[4-(4-iodo-phenyl)-thiazol-2-yl]-acetam-
ide (5c). The product was purified by crystallization with DMF/
H2O; white solid; 35% yield; mp >300 °C; 1H NMR (200 MHz,
DMSO-d6): d (ppm) 3.17 (s, 3H), 3.40 (s, 3H), 5.31 (s, 2H), 6.23 (d,
1H, J = 3), 7.35 (d, 1H, J = 3), 7.70–7.83 (m, 5H), 12.67 (br s, 1H).
MS (ESI): [MH]+ = 522.3. Anal. (C19H16IN5O3S) C, H, N.
medium containing mouse-NGF (100 ng/ml) and cytosine-b-
bino-furanoside free base (2.5 M). Neurons were plated on glass
coverslips coated with poly- -lysine (8.3 M) and laminin (5 M)
D-ara-
l
L
l
l
cultured for 2–3 days before calcium experiment.
4.2.3. Calcium imaging measurement
Intracellular calcium [Ca2+
] fluorescence has been measured
i
in single cells as previously reported.6 Plated cells were loaded
with Fura-2, AM-ester (5 M) (Alexis Biochemicals; Lausen, Swit-
4.1.5.4. 2-(1,3-Dimethyl-2,4-dioxo-1,2,3,4-tetrahydro-pyrrolo[3,
2-d ]pyrimidin-5-yl)-N-[4-(4-trifluoromethyl-phenyl)-thiazol-2-
yl]-acetamide (5d). The product was purified by crystallization
with DMF/H2O; white solid; 32% yield; mp >300 °C; 1H NMR
(200 MHz, DMSO-d6): d (ppm) 3.18 (s, 3H), 3.40 (s, 3H), 5.33 (s,
2H), 6.23 (d, 1H, J = 3), 7.35 (d, 1H, J = 3), 7.79–7.88 (m, 3H), 8.12
(d, 2H, J = 8.2) 12.70 (br s, 1H). MS (ESI): [MH]+ = 464.0. Anal.
(C20H16F3N5O3S) C, H, N.
l
zerland) added to the buffer solution (37 °C) containing the fol-
lowing: CaCl2, 1.6 mM; KCl, 5.4 mM; MgSO4, 0.4 mM; NaCl,
135 mM;
D-glucose, 5 mM; HEPES, 10 mM; and BSA, 0.1%, at
pH 7.4. After 40 min, cells were washed and transferred to a
chamber on the stage of a Nikon Eclipse TE2000U microscope
for recording. Cells were excited at 340 and 380 nM to indicate
relative [Ca2+]i changes by the F340/F380 ratio recorded with a
dynamic image analysis system (Laboratory Automation 2.0;
RCSoftware; Florence, Italy). Cells or neurons were exposed to
a selective TRPA1 agonist, acrolein13 in the presence of selective
4.1.5.5. 2-(1,3-Dimethyl-2,4-dioxo-1,2,3,4-tetrahydro-pyrrolo[3,
2-d ]pyrimidin-5-yl)-N-[4-(4-fluoro-3-trifluoromethyl-phenyl)-
thiazol-2-yl]-acetamide (5e). The product was purified by crys-
tallization with DMF/H2O; white solid; 22% yield; mp >300 °C; 1H
NMR (200 MHz, DMSO-d6): d (ppm) 3.16 (s, 3H), 3.41 (s, 3H),
5.31 (s, 2H), 6.22 (d, 1H, J = 3), 7.34 (d, 1H, J = 3), 7.40–7.70 (m,
1H), 7.75–7.90 (m, 1H), 8.10–8.30 (m, 2H), 12.80 (br s, 1H). MS
(ESI): [MH]+ = 482.0. Anal. (C20H15F4N5O3S) C, H, N.
TRPA1 antagonists, HC-030031 or AP18 (both, 0.1–30
pyrrolo[3,2-d]pyrimidines analogues (all 0.1–30 M), or their
maximum vehicle (3% DMSO). In rat DRG neurons, the addition
of capsaicin (0.1 M) has been used to identify capsaicin-sensi-
lM), and
l
l
tive neurons and to verify the selectivity of all compounds. Inhi-
bition lower than ꢁ10% has been considered not effective.
Calcium response has been normalized to the maximum re-
4.1.5.6. 2-(1,3-Dimethyl-2,4-dioxo-1,2,3,4-tetrahydro-pyrrolo[3,
2-d ]pyrimidin-5-yl)-N-[4-(4-hexyl-phenyl)-thiazol-2-yl]-acet-
amide (5f). The product was purified by crystallization with
DMF/H2O; white solid; 42% yield; mp 270–272 °C; 1H NMR
(200 MHz, DMSO-d6): d (ppm) 0.85 (t, 3H, J = 7.2), 1.20–1.40 (m,
6H), 1.42–1.70 (m, 2H), 2.50–2.70 (m, 2H), 3.18 (s, 3H), 3.40 (s,
3H), 5.31 (s, 2H), 6.22 (d, 1H, J = 3), 7.25 (d, 2H, J = 8.4), 7.35 (d,
1H, J = 3), 7.54 (s, 1H), 7.80 (d, 2H, J = 8.4), 12.60 (br s, 1H). MS
(ESI): [MH]+ = 480.1. Anal. (C25H29F4N5O3S) C, H, N.
sponse induced by ionomycin (5
experiment. Results are expressed as % of inhibition of the effect
induced by a concentration of acrolein (30 M) which represents
lM) added at the end of each
l
80% of the maximum effect (EC80) induced by the agonist (See
the concentration–dependent response curves and EC50 values
of acrolein in human fetal lung fibroblasts and primary rat
DRG neurons in Fig. 5 of the Supplementary data). Antagonist
potency is expressed as IC50, that is, the molar concentration
of antagonist producing 50% of the inhibitory measured effect
with confidence interval (CI), calculated fitting data by a sigmoi-
dal regression with variable slope (GraphPad, San Diego, CA,
USA).
4.2. Functional studies
4.2.1. Animals
We used Sprague–Dawley rats (male, 100 g) (Harlan Labora-
tories; Milan, Italy) in compliance with the experimental proce-
dures approved by the Animal Care Committee of the University
of Florence. Animals were housed in a temperature- and humid-
Acknowledgment
We acknowledge the Italian Institute of Technology (IIT) SEED
and Regione Toscana for financial support.

1698
P. G. Baraldi et al. / Bioorg. Med. Chem. 20 (2012) 1690–1698
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