Design, synthesis and pharmacological evaluation of novel pyrazolo[3,4-b]thieno[2,3-d]pyridine acid derivatives: A new class of anti-inflammatory and anti-platelet agents

Article abstract of DOI:10.1016/S0960-894X(01)00651-5

A series of pyrazolo[3,4-b]thieno[2,3-d]pyridine alkanoic acid derivatives has been synthesized and evaluated as thromboxane synthetase inhibitors and leukotriene D₄ receptor antagonists. The glutaric acid derivative LASSBio341 (6) was shown to be active in arachidonic acid-induced platelet aggregation (IC₅₀=0.14 μM) and inhibition of the contraction of guinea pig tracheal strip induced with LTD₄ (IC₅₀=43.7 μM), displaying still in vivo anti-inflammatory profile.

Full text of DOI:10.1016/S0960-894X(01)00651-5

Bioorganic & Medicinal Chemistry Letters 12 (2002) 9–12
Design, Synthesis and Pharmacological Evaluation of Novel
Pyrazolo[3,4-b]thieno[2,3-d]pyridine Acid Derivatives:
A New Class of Anti-inflammatory and Anti-platelet Agents
Carla R. Cardoso,b Fernanda C. F. de Brito,a Kelli C. M. da Silva,a
Ana L. P. de Miranda,a Carlos A. M. Fragaa,b and Eliezer J. Barreiroa,b,
*
a
´
LASSBio, Faculdade de Farmacia, Universidade Federal do Rio de Janeiro, PO Box 68006, Rio de Janeiro, 21944-970,
RJ, Brazil
Instituto de Quımica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, 21949-900, RJ, Brazil
b
´
Received 6 April 2001; accepted 20 June 2001
Abstract—A series of pyrazolo[3,4-b]thieno[2,3-d]pyridine alkanoic acid derivatives has been synthesized and evaluated as throm-
boxane synthetase inhibitors and leukotriene D4 receptor antagonists. The glutaric acid derivative LASSBio341 (6) was shown to be
active in arachidonic acid-induced platelet aggregation (IC50=0.14 mM) and inhibition of the contraction of guinea pig tracheal
stripinduced with LTD 4 (IC50=43.7 mM), displaying still in vivo anti-inflammatory profile. # 2001 Elsevier Science Ltd. All rights
reserved.
Introduction
block the effects of LTD4 on the cys-LT receptor (cys-
LTant.).11 The former approach, in fact, has found
application in therapeutics, as illustrated by ozagrel (1),
an important anti-thrombotic, anti-asthmatic agent that
acts as a TXSi (Fig. 1).10,12 On the other hand, the
development of potent cys-LT antagonists, for example,
zafirlukast13,14 (2) and montelukast15 (3), represents an
important therapeutical innovation for the treatment of
the inflammatory symptoms of bronchial asthma.
Asthma is a complex, chronic inflammatory disease of
airways, which affects more than 100 million people
worldwide, making it a serious global health problem.1
This disorder involves multiple inflammatory mediators
that are released from mast cells as a consequence of
immunological response in the airways.2 Both leuko-
triene D4 (LTD4) and thromboxane A2 (TXA2) are
autacoids derived from arachidonic acid (AA) metabo-
lism implicated in the pathogenesis of asthma.3ꢀ5 LTD4
is a potent constrictor of airway smooth muscle6 and act
on the vasculature to produce vasodilatation and
increase vascular permeability,7 processes that are likely
relevant to the recruitment of leukocytes to an inflam-
matory response. These changes promoted by LTD4
and other cys-LTs induces mucous secretion and
decrease of the airway caliber, inherent to asthma.8 On
the other hand, TXA2 is a potent vasoconstrictor,
broncoconstrictor, and platelet-aggregating agent.9
Therefore, considerable effort has been devoted to
modulate the actions of these eicosanoids through the
design of TXS-inhibitors (TXSi)10 or alternatively,
As part of a research program for the design, synthesis,
and pharmacological evaluation of new lead candidates
useful for asthma treatment, the preparation of a new
series of pyrazolo[3,4-b]thieno[2,3-d]pyridine alkanoic
acid derivatives 4–7 has been carried out.16 The synth-
esis of these new compounds representing a structural
hybridization of ozagrel (1) and the quinolinic deriva-
tive 3 (Fig. 1) and envisioned as new anti-asthmatic
candidates possessing dual action, cys-LT receptor
antagonists and TXA2 synthetase inhibitors, is the
subject of this paper.
Chemistry
These new derivatives 4–7 were synthesized by the route
illustrated in Scheme 1. The approach used for the
*Corresponding author. Fax:+55-21-2733890; e-mail: eliezer@
pharma.ufrij.br
0960-894X/02/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00651-5

10
C. R. Cardoso et al. / Bioorg. Med. Chem. Lett. 12 (2002) 9–12
Scheme 1. Synthesis of new pyrazolo[3,4-b]yhieno[2,3-d]pyridine deri-
vatives 4–7.
Figure 1. Design concept of new tricyclic TXSi/LTant.
construction of the functionalized pyrazolo[3,4-
b]thieno[2,3-d]pyridine nucleus employed the previously
described one-pot transformation,17,18 starting from the
chloroester 8.19 Thus, this material was subjected to
heteroatomic nucleophilic displacement of chlorine
atom at C-4 of 8 with the lithium salt of 4-chloro-
toluenethiol, to give the thioether intermediate I,
which underwent intramolecular Claisen condensa-
tion, promoted by excess of LDA present in the
reaction medium, to afford in 75% yield the
Table 2. Effect of zafirlukast (2) and LASSBio341 (6) on LTD4 (100
nM) induced contraction of isolated guinea pig tracheal strip
Compd
IC50 (M)a
Zafirlukastb (2)
LASSBio341c (6)
1.03 (ꢂ0.06)ꢁ10ꢀ9
43.7 (ꢂ 1.9)ꢁ10ꢀ6
aValues are means of five independent experiments, standard deviation
is given in parentheses; Concentration range.
b0.1 nM–100 mM.
c10 M–300 mM.
Table 1. Anti-inflammatory effects of pyrazolo[3,4-b]thieno[2,3-d]pyridine acid derivatives 4–7 and indomethacin in the carrageenan-induced rat
pleurisy24
Compd
Dosea (mmol/Kg) nb Cell number (ꢁ106 cells/cavity) % Inhibitionc Exsudate volume (mL) Á Exsudate volume (mL)
Vehicle control
Indomethacin
4
5
6
7
—
10
10
4
7
4
30.7ꢂ1.1
23.3ꢂ2.2
21.5ꢂ1.8
17.8ꢂ1.8
19.6ꢂ1.4
21.9ꢂ2.0
—
3.25ꢂ0.04
3.21ꢂ0.05
3.22ꢂ0.02
3.16ꢂ0.04
3.05ꢂ0.03
3.10ꢂ0.06
—
100
100
100
100
100
24.1*
30.0*
42.0*
36.2*
28.7*
ꢀ0.04 n.s.d
ꢀ0.03 n.s.d
ꢀ0.09 n.s.d
ꢀ0.20*
7
ꢀ0.15 n.s.d
*p< 0.05 (Student’s t-test). Results are expressed as meanꢂSEM.
aAll compounds were administered po.
bn=number of animals.
cPercentage of inhibition obtained by comparison with control group.
dn.s.=not significant.

C. R. Cardoso et al. / Bioorg. Med. Chem. Lett. 12 (2002) 9–12
11
Table 3. Effects of pyrazolo[3,4-b]thieno[2,3-d]pyridine acid derivatives 4–7 and indomethacin on in vitro platelet aggregation of citrated rabbit
platelet-rich plasma induced by arachidonic acid and collagen2
Compd
Ca (mM)
Arachidonic acid (200 mM)
Collagen (5 mg/mL)
Aggregation (slope)
nb
Aggregation (%)
Inhibitionc (%)
nb
Inhibitionc (%)
Control
Indomethacin
4
5
LASSBio341 (6)
7
—
10
100
100
100
100
5
4
3
4
5
5
68.3ꢂ3.3
0.0ꢂ0.0
58.9ꢂ3.8
56.6ꢂ4.7
0.0ꢂ0.0
60.5ꢂ3.4
—
100*
4
4
4
4
5
4
8.8ꢂ0.6
0.4ꢂ0.1
7.5ꢂ1.3
5.8ꢂ0.9
0.0ꢂ0.0
4.7ꢂ1.3
—
95.4*
13.8 n.s.d
17.1 n.s.d
100*
14.8 n.s.d
34.1*
100*
11.4 n.s.d
46.6*
*p<0.05 compared to appropriate control (Student’s t-test). Results are expressed as meanꢂSEM.
aC=final concentration.
bn=number of independent experiments carried out in triplicate.
cPercentage of inhibition obtained by comparison with control group.
dn.s.=not significant.
key-intermediate, 2-(40-chlorophenyl)-3-hydroxy-6-phenyl-
8-methylpyrazolo[3,4-b]thieno[2,3-d]pyridine (9) (Scheme
1).
Zafirlukast (2) and compound 6, named LASSBio341,
inhibited in a concentration-dependent manner, the con-
traction of tracheal strips induced by 100 nM of LTD4,
with IC50 values of 1.03nM and 43.7mM, respectively,
confirming its actions as cys-LT receptor antagonists.
The next stepin the synthesis of the new derivatives
4–7, consisted in O-alkylation or O-acylation of the
pseudophenolic hydroxyl group of compound 9, which
was effected by treatment with 5 equiv of potassium
carbonate in acetone, followed by the addition of the
appropriate electrophilic species.20 Employing this proce-
dure, methyl esters 10 and 11 were obtained with methyl
5-iodovalerate and methyl 6-iodohexanoate, which was
followed by mild hydrolysis with lithium hydroxyde21 in
THF to give acids 3 and 4 in 27% and 45% yield,
respectively22 (Scheme 1). Compounds 6 and 7 were
obtained in 50 and 57% yield, respectively, with glutaric
anhydride and 4-bromomethylphenylacetic acid.22
Neither of the two substances was effective against tra-
cheal contraction induced by histamine and U-46619.
To evaluate the expected dual profile of these new deri-
vatives 4–7 they were assayed for in vitro platelet
aggregation induced by AA, collagen, and U-46619
(Table 3). Compounds 5 and 7 inhibited significantly the
collagen-induced aggregation, with 34.1 and 46.6%
inhibition, respectively. On the other hand, LASS-
Bio341 (6), at 100 mM concentration, strongly inhibited
the platelet aggregation induced by both AA and col-
lagen by 100%. DMSO, used as vehicle, had neither
pro- nor anti-platelet aggregation activity.
Results and Discussion
Therefore these results lead us to study the concentra-
tion–response of LASSBio341 (6), which inhibited the
platelet aggregation induced by 200 mM of AA, with an
IC50 of 0.14 mM. When compound 6 was tested, at
100 mM, against increasing concentrations of AA-
induced aggregation it presents a competitive inhibi-
tion behavior. At 500 mM AA-induced aggregation,
the new lead-compound 6 presented an inhibition at
the same magnitude of ozagrel27 (1) (61ꢁ50%,
respectively) (data not shown). None of the compounds
inhibited significantly U-46619-induced aggregation,
suggesting that they did not act at the TXA2 receptor
level.
The dual cys-LTant/TXSi profile of the new pyr-
azolo[3,4-b]thieno[2,3-d]pyridine acid derivatives 4–7
was initially evaluated by using carrageenan-induced
pleurisy23 and arachidonic acid-induced platelet aggre-
gation24 models.
The results obtained for the anti-inflammatory activity
of the pyrazolo[3,4-b]thieno[2,3-d]pyridine acid deriva-
tives 4–7 are compiled in Table 1.
All compounds, when administered orally at a dose of
100 mmol/kg, were able to inhibit significantly cellular
migration. Compounds 5 and 6 were the most active,
with inhibitions of 42.0 and 36.2%, respectively. Only
compound 6 was able to inhibit significantly exsudation
and it showed no activity on the carrageenan-induced rat
paw edema (data not given). Otherwise, all compounds
were more active than indomethacin and they did not
induce any gastric toxicity when orally administered.
Concluding Remarks
In summary, we are able to design the new aza-hetero-
cyclic glutaric acid derivative (6, LASSBio341), as a
hybrid of ozagrel (1) and montelukast (2), possessing
the adequate structural requirements to inhibit the con-
traction of guinea-pig tracheal strip induced by LTD4
and promote efficient inhibition of platelet aggregation
induced by AA, strongly suggesting that the dual profile
observed is due to cys-LT receptor blockage and TXS
inhibition, representing an useful profile for asthma
treatment.28
In order to correlate the in vivo anti-chemoatractant
and anti-edematogenic profile of the compound 6
observed in rat pleurisy model with an anti-leukotriene
action,25,26 we performed its in vitro evaluation in
LTD4-induced contraction of guinea-pig tracheal
strip,27 using zafirlukast (2) as standard (Table 2).

12
C. R. Cardoso et al. / Bioorg. Med. Chem. Lett. 12 (2002) 9–12
Yee, Y. K.; Brown, F. J.; Hesp, B.; Giles, R. E. Am. Rev.
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Acknowledgements
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16. Taken, in part, from M.Sc. Thesis of C. R. Cardoso,
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Instituto de Quımica, Universidade Federal do Rio de Janeiro,
Rio de Janeiro, Brazil, 1997.
17. Carvalho, A. S.; Fraga, C. A. M.; Barreiro, E. J. J. Het-
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Thanks are due to FAPERJ (BR.), CNPq (BR., grants
#50.0033/96-5, #460200/00-3 and fellowships to
F.C.F.B., A.L.P.M., C.A.M.F., E.J.B.), CAPES (BR.,
fellowshipto C.R.R.) and FUJB (BR.) for financial
support. We are very grateful to Analytical Center of
NPPN (UFRJ—BR.) for spectroscopic facilities. We
indebted to Prof. Andrew E. Greene (University Joseph
Fourier—FR.) for suggestions and language criticism.
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