Microwave-assisted synthesis and structure-activity relationships of neuroactive pyrazolo[3,4-b]pyrrolo[3,4-d]pyridine derivatives

Article abstract of DOI:10.1016/j.bmcl.2009.11.038

We described herein the optimization of the synthetic methodology exploited to obtain the pyrazolo[3,4-b]pyrrolo[3,4-d]pyridine sedative prototype 1a and novel analogues designed by successive molecular simplifications. By applying microwave irradiation during the hetero Diels-Alder key-step to obtain the heterotricyclic scaffold, under solvent-free conditions, we were able to obtain the desired compounds in drastically shorter times and better yields. Additionally, in vivo evaluation of the sedative effects of these heterocyclic derivatives showed that 1a and the novel structurally-related analogue 1e were the most efficient compounds to impair the locomotor activity in mice at the dose of 10 μmol/kg.

Full text of DOI:10.1016/j.bmcl.2009.11.038

Bioorganic & Medicinal Chemistry Letters 20 (2010) 74–77
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Microwave-assisted synthesis and structure–activity relationships
of neuroactive pyrazolo[3,4-b]pyrrolo[3,4-d]pyridine derivatives
Nailton M. Nascimento-Júnior a,b, Thaiana C. F. Mendes c,d, Daniella M. Leal c, Claudia Maria N. Corrêa c,
Roberto T. Sudo c, Gisele Zapata-Sudo c, Eliezer J. Barreiro a,b,c,d, Carlos A. M. Fraga a,b,c,d,
*
a Laboratório de Avaliação e Síntese de Substâncias Bioativas (LASSBio), Faculdade de Farmácia, Universidade Federal do Rio de Janeiro, PO Box 68023,
Rio de Janeiro 21941-902, RJ, Brazil
b Programa de Pós-Graduação em Química, Instituto de Química, Universidade Federal do Rio de Janeiro, Rio de Janeiro 21949-9000, RJ, Brazil
c Programa de Desenvolvimento de Fármacos, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro 21949-900, RJ, Brazil
d Programa de Pós-Graduação em Farmacologia e Química Medicinal, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro 21949-000, RJ, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
We described herein the optimization of the synthetic methodology exploited to obtain the pyrazolo[3,4-
b]pyrrolo[3,4-d]pyridine sedative prototype 1a and novel analogues designed by successive molecular
simplifications. By applying microwave irradiation during the hetero Diels-Alder key-step to obtain the
heterotricyclic scaffold, under solvent-free conditions, we were able to obtain the desired compounds
in drastically shorter times and better yields. Additionally, in vivo evaluation of the sedative effects of
these heterocyclic derivatives showed that 1a and the novel structurally-related analogue 1e were the
Received 7 September 2009
Revised 7 November 2009
Accepted 10 November 2009
Available online 16 November 2009
Keywords:
Hetero Diels–Alder
Microwave
most efficient compounds to impair the locomotor activity in mice at the dose of 10 lmol/kg.
Ó 2009 Elsevier Ltd. All rights reserved.
Cycloaddition
Pyrazolo[3,4-b]pyrrolo[3,4-d]pyridine
Sedative agents
Muscarinic agonists
Molecular simplification
Among the most frequently used therapeutic classes, the CNS-
acting drugs correspond to ca. 15% of the total, with worldwide
sales of around U$ 118 billion in 2008.1 In this context, many ef-
forts to develop novel sedative/hypnotic drugs, presenting in-
creased potency and reduced side effects, have been made in
order to assure the availability of suitable alternatives for the treat-
ment of sleep-disorders.2 Previous works from our research group
have described the discovery of two novel sedative and analgesic
agents (1a) and (1b),3 belonging to the pyrazolo[3,4-b]pyrrol-
o[3,4-d]pyridine series, which displayed their CNS actions as mus-
carinic M1 receptor agonists.4 Although these heterotricyclic
compounds presented remarkable neuroactive profile, further pre-
clinical studies were strongly limited by the synthetic approach
used to construct them. The functionalized pyrazolo[3,4-b]pyrrol-
o[3,4-d]pyridine derivatives (1a) and (1b) were prepared in very
low yields (20–35%), after long reaction time (48 h), through hetero
Diels–Alder cycloaddition between the formamidine (2a) and the
corresponding N-phenylmaleimide (3), under conventional heat-
ing,3 using AcOH or DMSO as solvent (Scheme 1).
Hetero Diels–Alder reactions between pyrazolyl-2-azadienes
and nitroalkenes,5 propiolates or maleimides6 using conventional
heating are known to be difficult due to the hard conditions re-
quired and either do not occur or take a long time to produce the
desired product in low yields.3,7
* Corresponding author. Tel.: +55 21 25626503; fax: +55 21 25626478.
E-mail address: cmfraga@ccsdecania.ufrj.br (C.A.M. Fraga).
Scheme 1. Synthesis of heterotricyclic derivatives (1) exploiting hetero Diels–Alder
reaction at the key-step, by using conventional heating.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.11.038

N. M. Nascimento-Júnior et al. / Bioorg. Med. Chem. Lett. 20 (2010) 74–77
75
Figure 1. Design concept of pyrazolo[3,4-b]pyrrolo[3,4-d]pyridine derivatives of series A (1), B (4), and their simplified analogues (5) and (3a).
Alternatively, the use of microwave irradiation under solvent-
free conditions has proved to dramatically improve the process
for obtaining new heterocyclic scaffolds exploiting Diels–Alder
reaction as the key-step.5
Considering this panorama, we described herein the optimiza-
tion of the synthetic route to obtain the pyrazolo[3,4-b]pyrrol-
o[3,4-d]pyridine derivative (1a) and some previously described
analogues (1b–1d) using microwave-assisted synthesis for the het-
ero Diels–Alder step under solvent-free conditions. Thus, we pro-
pose the enlargement of the congeneric series by the
introduction of the isosteric carboxyl group and the electron-
donating methoxy substituent at the W position (series A, Fig. 1).
Moreover, we exploited the developed methodology to synthe-
size a new series of simplified heterocyclic analogues (4a–4f) de-
signed by changing the pyrazole-attached N-phenyl by a N-
methyl group in order to investigate the stereoelectronic and the
lipophilic influences at this position on the sedative profile of pyr-
azolo[3,4-b]pyrrolo[3,4-d]pyridine derivatives (Fig. 1).
On the other hand, the exploitation of successive molecular
simplifications on the structure of sedative prototype (1a) led us
to the design of the 3-pyridinylphthalimide (5) and 4-nitro-
phenylmaleimide (3a) derivatives, as a result of the suppression
of the pyrazole and the pyridine rings (Fig. 1). The comparative
evaluation of the sedative profile displayed by these heterocyclic
derivatives on the locomotor activity in mice8 provided a better
understanding of structure–activity relationships associated with
their CNS actions.
Scheme 2. Synthesis of the functionalized N-phenyl maleimides (3a–3f) and N-
phenyl azaphthlimide (5).
Initially, the six functionalized N-phenylmaleimides (3a–3f)
were prepared starting from maleic anhydride (6) and the corre-
sponding para-substituted anilines (7).
In order to obtain the desired N-phenylmaleimides (3), the dif-
ferent behavior of this reaction, determined by distinct solubility
and nucleophilicity of the exploited anilines, was taken into con-
sideration. Thus, only the N-phenylmaleimides (3a–3d) were pre-
pared, in yields ranging from 63% to 83%, by refluxing a mixture
of (6) and (7) in acetic acid9 (Scheme 2). On the other hand, the at-
tempt to prepare the unsubstituted N-phenylmaleimide (3b) and
the 4-carboxyphenylmaleimide (3f) under the same conditions
led to the formation of the carboxy-amide intermediates (8) or
(9), respectively, accompanied by other undesired subproducts.
To avoid this problem, another two-step methodology was selected
to prepare these two N-phenylmaleimides, using ethyl ether as sol-
vent in the first step, followed by cyclization of the obtained car-
boxy-amide derivatives (8) and (9) after treatment with sodium
acetate in acetic anhydride10 (Scheme 2). Azaphthalimide deriva-
tive (5) was prepared in 23% overall yield (2 steps) through the ini-
tial condensation of 3,4-pyridinedicarboxylic anhydride (10) and
para-nitroaniline (7a) in acetic acid at reflux, followed by cycliza-
tion of the carboxy-amide (11) with AcONa in acetic anhydride
(Scheme 2).
The azadienes (2a) and (2b) were prepared as described previ-
ously,3 in almost quantitative yields through the condensation of
the corresponding pyrazolamines (12) or (13) and DMF dimethyl-
acetal (14) (Scheme 3). The relative configuration (E) at imine dou-
ble bond of azadienes (12) and (13) was determined by 1H NMR,
through the irradiation of the C-4 attached pyrazole hydrogen
and the evidence of a NOE effect at imine hydrogen (Scheme 3).
This configuration is important to assure the formation of hetero
Diels–Alder adduct presenting antiperiplanar orientation between
the N,N-dimethylamino group and the vicinal hydrogen able to

76
N. M. Nascimento-Júnior et al. / Bioorg. Med. Chem. Lett. 20 (2010) 74–77
Scheme 3. Synthesis of pyrazolo[3,4-b]pyrrolo[3,4-d]pyridine derivatives (1a–1f) and (4a–4f) exploiting hetero Diels–Alder reaction under microwave irradiation in solvent-
free conditions.
produce the desired heterocyclic derivatives (1) and (4) after elim-
ination and oxidative aromatization steps.7
Next, in order to optimize the hetero Diels-Alder key-step we
proceeded to the investigation of cycloaddition between (2a) and
(3a) under microwave irradiation and solvent-free conditions,
starting with 80 W, at 50 °C for 30 min.
Table 1
Effects of midazolam, pyrazolo[3,4-b]pyrrolo[3,4-d]pyridine derivatives (1a–1f), (4a–
4f) and the simplified analogues 5 and 3a on the locomotor activity in mice8
Compound
Series
Motor activitya (mov/min)
Control
—
—
A
A
A
A
A
A
B
B
B
B
B
214.4 18.9
79.7 25.1*
87.6 16.2*
266.6 30.6
154.3 24.3
161.7 29.2
108.6 14.9*
174.2 21.8
141.0 25.8
208.6 11.4
133.4 16.3
187.4 23.0
150.6 15.7
128.3 20.6
189.0 16.76
148.0 13.85
Midazolamb
1a
1b
1c
1d
1e
1f
4a
4b
4c
4d
4e
4f
The microwave tube used was exposed to atmospheric oxygen
throughout the experiments to favor the oxidative aromatization
step after Diels–Alder adduct formation.7 Besides these initial con-
ditions, the temperature was systematically increased from 50 to
60, 70, 80 and 90 °C, but (1a) was obtained in highest yield (35%)
at 80 °C. When the temperature was 90 °C and/or the power was
changed to 90 W, degradation of (4) was detected by TLC. After set-
ting the power to 80 W, the temperature at 80 °C and extending
the time of the reaction to 60, 90 and 120 min, (1a) was obtained
in 50%, 80% and 75% yields, respectively, after work-up by washing
with warm MeOH (ca. 60 °C). All other heterocyclic derivatives of
series A (1b–1f) and B (4a–4f) were synthesized following the
same methodology (80 W, 80 °C, 90 min., Scheme 3) and their
structures are in agreement with analytical and spectral data.
The sedation produced by derivatives (1a–1f), (4a–4f) and sim-
plified analogues (5) and (3a) was investigated using locomotor
activity test in mice8 (Table 1). The intraperitoneal administration
B
—
—
5
3a
a
All derivatives were administered ip (10
l
mol/kg) and motor activity was
determined during 40 min after injection. Data are expressed as means of the
movements per minute SEM.
b
Administered at the dose of 2 mg/kg.
*
P <0.05 relative to the control group (DMSO) (statistic test: ANOVA followed by
Dunn’s test).
of a screening dose of 10 lmol/kg for all these compounds revealed
that para-nitro derivative (1a) and the novel para-methoxy deriva-
tive (1e), both from series A, presented the highest sedative
activity, being able to reduce the number of movements/minute
In addition, the compounds of series B, presenting a methyl
group instead of a phenyl subunit attached to the N-1 position of
the pyrazole ring, were able to display no significant sedative
activity. Only derivatives 4c (133.4 16.3 mov/min) and 4f
(128.3 20.6 mov/min) presented slight ability to reduce the
locomotor activity in mice. These results highlight the possible
influence of the phenyl group bonded to the pyrazole ring of the
pyrazolo[3,4-b]pyrrolo[3,4-d]pyridine system to potentiate the
sedative profile of these heterotricyclic derivatives. This behavior
was confirmed through the comparative evaluation of the sedative
activity of four nitro derivatives presenting sequential molecular
*
of treated animals from 214.4 18.9 to 87.6 16.2 and
*
108.6 14.9 mov/min., respectively. These values are statistically
equivalent to the reference drug, midazolam (Table 1). Even
though the nitro and the methoxy groups at the W position
( Fig. 1) exhibit different electronic characteristics, compounds
(1a) and (1e) did not present significant differences in sedative
activity probably due to the capability of these two groups, poten-
tial pharmacophoric points, to be recognized as H-bond acceptors
with complementary residues of occasional target bioreceptors.
This interaction seems to be essential for the effect investigated,
once the corresponding unsubstituted compound (1b) and the
two para-substituted derivatives (1c) and (1d) were not able to af-
fect significantly the motor activity in animals. An exception to this
behavior was observed in carboxyl derivative (1f), which did not
impair the locomotor activity in animals, probably due to pharma-
cokinetic factors resulting from its ionization in vivo. As evidenced
for compound (1a), the CNS effects of (1e) were reversed by the
pre-treatment of animals with atropine, indicating that it could
also act as a muscarinic agonist.4
*
simplifications—1a (87.6 16.2 mov/min), 4a (141.0 25.8 mov/
min),
5 (189.0 16.76 mov/min) and 3a (148.0 13.85 mov/
min)—which have indicated that all subunits contained in the scaf-
fold of series A seem to be important for the observed remarkable
effects of compound (1a) on the locomotor activity test.
As concluding remarks, the optimization of the experimental
conditions exploited to access heterocyclic derivatives presenting
pyrazolo[3,4-b]pyrrolo[3,4-d]pyridine scaffold, through micro-
wave-assisted hetero Diels–Alder reaction, permitted their synthe-
sis in higher yields and shorter times in comparison with the
conventional heating strategy previously described. The molecular

N. M. Nascimento-Júnior et al. / Bioorg. Med. Chem. Lett. 20 (2010) 74–77
77
simplification study confirmed that almost all the subunits present
References and notes
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synthesis of prototype 1a and its analogues possible, enabling the
next steps towards the development of these heterotricyclic deriv-
atives into innovative sedative agents.
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Acknowledgments
The authors thank CAPES (BR), CNPq (BR), FAPERJ (BR), PRONEX
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Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.11.038.