Studies on the acetylation of 3,6-diamino-1H-pyrazolo[3,4-b]pyridine-5- carbonitrile derivatives

Article abstract of DOI:10.1002/jhet.403

(Chemical Equation Presented) The acetylation reaction of the differently substituted 3,6-diamino-1H-pyrazolo[3,4-b]pyridine-5-carbonitrile derivatives 1-6 is reported. The structure of the resulting acetamides has been investigated and confirmed by analy

Full text of DOI:10.1002/jhet.403

July 2010
Studies on the Acetylation of 3,6-Diamino-1H-
pyrazolo[3,4-b]pyridine-5-carbonitrile Derivatives
861
Mourad Chioua, Elena Soriano, Abdelouahid Samadi, and J. Marco-Contelles*
´
Laboratorio de Radicales Libres y Quımica Computacional (IQOG, CSIC), 3, Juan de la Cierva,
28006 Madrid, Spain
*E-mail: iqoc21@iqog.csic.es
Received November 19, 2009
DOI 10.1002/jhet.403
Published online 8 June 2010 in Wiley InterScience (www.interscience.wiley.com).
The acetylation reaction of the differently substituted 3,6-diamino-1H-pyrazolo[3,4-b]pyridine-5-car-
bonitrile derivatives 1–6 is reported. The structure of the resulting acetamides has been investigated and
confirmed by analytical, spectroscopic, and chemical transformations. From these studies, we conclude
that, in general, under mild conditions, and using acetic anhydride, when free, the N(1)H moiety is a
more reactive center respect to the C(3)NH2 and C(6)NH2 groups. This trend is reversed when no steric
hindrance due to presence of a phenyl group at C4 drives the preferred acetylation to C(3)NH2, as it is
evident by comparing the observed results from precursor 1 with 3. When N1 is blocked, the (C3)NH2
group undergoes preferential acetylation over the (C6)NH2 site, which only has been mono (or diacety-
lated) at reflux. Computational analyses based on DFT studies have been extensively used to explain
the observed reactivities.
J. Heterocyclic Chem., 47, 861 (2010).
INTRODUCTION
acetamide 13 from related amide 12 (Chart 3) [8].
Finally, a more complex situation was described for 4,5-
diphenyl-1H-pyrazolo[3,4-c]pyridazine (14) [9] when
using acetyl chloride. Depending on the base used, the
acetylation goes to C(3)NH2 to give compound 15 (for
triethylamine as base) or to 16 (for pyridine as base)
(Chart 3) [9]. Note also that the position of the acetyl
residue at N1 was not unambiguously confirmed by X-
ray analysis neither discussed in depth; however, 4,5-di-
phenyl-1H-pyrazolo[3,4-c]pyridazines have been regio-
selectively N-alkylated at N1 [9], a result that is in good
agreement with similar reaction on 3-amino-1H-pyra-
zolo[3,4-b]quinoxalines [7].
The pyrazolo[3,4-b]pyridine ring system [1] is present
in a number of pharmaceutically important compounds
targeted to inhibit VEGFR/PDGFR kinases [2] or GSK-
3 [3]. In a current project aimed at the synthesis, design,
and biological evaluation of new GSK-3 inhibitors, we
have recently synthesized a series of known and new
3,6-diamino-1H-pyrazolo[3,4-b]pyridines [4] (A) (Chart
1). To carry out basic SAR studies, we decided to
explore the acylation reaction of 3,6-diamino-1H-pyra-
zolo[3,4-b]pyridines (1–6) (Chart 2) to get presumably
more potent inhibitors as previously demonstrated by
other authors [3].
In summary, all these data confirm that a simple reac-
tion such as the acetylation in a complex, polyfunction-
alized, heterocyclic framework can be more complicated
than presumed at first glance [10]. This is in fact what
we have observed in the acetylation of 3,6-diamino-pyr-
azolopyridines 1–6 (Chart 2), and in this work, we
report our results.
In this context, previous reports from other laborato-
ries have described that the acylation (C3H7COCl, pyri-
dine, reflux) of precursor 7 gave the C(3)NH2 acetylated
derivative 8 in 80% yield (Chart 3). Note, however, that
the carbamoylation of the free amino groups on the
fused pyrazole ring system in compound 9 showed a
reversed regioselectivity, providing compound 10 (Chart
3) [5]. In addition, it has been also shown that under
mild conditions, 5-substituted 3-aminopyrazoles are
almost simultaneously acylated at N1 and at C(3)NH2 to
give diacylated derivatives [6]. Substituted 3-amino-1H-
pyrazolo[3,4-b]quinoxalines have been selectively N-
acylated at N1 [7]. Compound 2 has been reported to
give exclusively the C(3)-N-formyl derivative 11 or the
RESULTS AND DISCUSSION
Synthesis, structural analysis, and reactivity. The
synthesis of 3,6-diamino-1H-pyrazolo[3,4-b]pyridine-5-
carbonitrile (1) (Chart 2) proceeded uneventually as
described by reacting 2-amino-6-chloropyridine-3,5-
C
V
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M. Chioua, E. Soriano, A. Samadi, and J. Marco-Contelles
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a second compound that was identical to the compound
isolated when the reaction was carried at out at room
temperature for 5 days and identified as N-(6-amino-5-
cyano-1H-pyrazolo[3,4-b]pyridin-3-yl)acetamide
(18),
isolated in 12% yield (21% taking into account the
recovered starting material); in this reaction, we also iso-
lated diacetamide 19 [14% (24%)] and compound 17
[5% (10%)] (Chart 4). In the 1H NMR spectrum of
monoacetamide 18, we could analyzed only one singlet
for two protons at d 6.95, indicating that the only free
NH2 group was at C6, leaving free the N(1)H (d 12.66),
the acetamido group being thus at C3, as in the 1H
NMR spectrum the signals for NHCOCH3 appeared at d
(NH) 10.69 and d (COCH3) 2.12. These assignments
were confirmed in the HMBC experiment, as the signal
at d 12.66 showed cross-peaks with C3a, C3, and C7a,
while the singlet at d 10.69 showed cross-peaks with
Chart 1. 3-6 Diamino-1H-pyrazolo[3,4-b]pyridines.
dicarbonitrile with hydrazine hydrate [8]. In the HMBC
experiment of pyrazolopyridine 1, we assigned the
chemical shifts for the protons at C(3)NH2 and
C(6)NH2, at d 5.56 and 6.70, as cross-peaks were
observed with the signals at d 99.8 (C3a) and 83.0 (C5),
respectively; in agreement with this, the signal at 99.8
ppm (C3a) showed a cross-peak with the signal at d
11.60, corresponding to N(1)H. Similar chemical shifts
and effects have been observed for protons in the other
related compounds described here, and this trend [d
C(6)NH2 >> d C(3)NH2] has served as diagnostic for
proton assignments and structure determination (see
later).
1
C3a and NHCOCH3. In the H NMR spectrum of com-
pound 19, in addition to similar signals to those
described for compound 18 (see Experimental), no
N(1)H resonance was observed at low field, but a new
singlet appeared for three protons at 2.77 ppm, charac-
teristic for N(1)COCH3 (see compound 16 in Chart 3)
[9]. In summary, we conclude that the acetylation of
3,6-diamino-1H-pyrazolo[3,4-b]pyridine-5-carbonitrile (1)
is relatively complex, and a number of differently substi-
tuted acetamido derivatives were isolated and character-
ized. Under mild reaction conditions, preferent acetyla-
tion at C(3)NH2 followed at N(1) positions was observed;
the acetylation at C(6)NH2 being also possible, at reflux,
to give diacetamide 17 (Chart 4), as the only detected, in
a poor chemical yield. Overall, these facts are in good
agreement with the previous results [5–7] on the acyla-
tion of related substrates (see Chart 3) and strongly point
to the absence of a substituent at C4, the key to favor the
acetylation at C(3)NH2 (see later).
The reaction of compound 1 [8] with acetic anhy-
dride, after 21 h, at 144ꢀC, was complete, but extensive
decomposition was observed by TLC analysis, and only
N,N0-(5-cyano-3a,7a-dihydro-1H-pyrazolo[3,4-b]pyridine-
3,6-diyl)diacetamide (17) could be isolated in 8% yield
(Chart 4). Based on the analytical (MS/elemental analy-
sis) and the NMR spectra, compound 17 was clearly a
1
diacetamide derivative of precursor 1, showing in its H
NMR spectrum two acetyl groups integrating for six pro-
tons (2xNHCOCH3), as a singlet at d 2.12; the singlet at
d 10.96 was assigned to C(3)NHCOCH3 as it showed
cross-peaks in the HMBC spectrum with C3a (d 104.7)
and NHCOCH3 (d 169.2); consequently, the singlet at d
10.79 corresponded to the proton at C(6)NHCOCH3;
finally, the singlet for one proton at d 13.62 was
assigned to N(1)H. In this reaction, we detected traces of
The acetylation of 3,6-diamino-1-methyl-1H-pyra-
zolo[3,4-b]pyridine-5-carbonitrile (2) [8], under mild
conditions, gave only monoacetamide 20 in good yield
1
(Chart 4). In the H MNMR of this compound, we could
Chart 2. Structure of compounds 1–6.
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863
Chart 3. Transformation of compounds 2, 7, 8, 12, and 14.
analyze two singlets for proton at d 10.74 and for two
protons at d 7.08, corresponding to (C3)NHCOCH3 and
(C6)NH2 protons, respectively. In agreement with this
assignment, in the HMBC-NMR experiment of this pyr-
azolopyridine, the NH proton at C(3)NHCOCH3 reson-
ated at d 10.74 and showed cross-peaks with C3a (100.3
ppm) and NHCOCH3 (168.1 ppm), whereas for the pro-
ton at C(6)NH2 (d 7.08) cross-peaks appeared with the
signals at d 86.4 (C5), 158.3 (C6), and 150.7 (C7a). In
addition, a small but evident selective nOe experiments
between the NH proton at d 10.74 [(C3)NHCOCH3] and
H4 (d 8.57) confirmed the position of the acetyl group
on the nitrogen at C3. This result and reactivity is thus
also in good agreement with the reported reactivity of
precursor 2 in the formylation reaction (Chart 3) [8].
The small nOe observed between both protons in the
NHCOCH3 group and H4 in compound 20 could be
rationalized after computational analysis, which showed
that, in fact, rotamer 20b was 7.0 kcal/mol more stable
than conformer 20a (Fig. 1), possibly because of the
lone pair–lone pair electronic repulsion between the car-
bonylic oxygen and the N2 present in conformer 20a.
On the other hand, the calculated chemical shifts for
protons in NHCOCH3 are in good agreement with the
experimental values.
The reaction of 2-amino-6-chloro-4-phenylpyridine-
3,5-dicarbonitrile [11] with hydrazine hydrate [4] gave
3,6-diamino-4-phenyl-1H-pyrazolo[3,4-b]pyridine-5-car-
bonitrile (3) (Chart 2) [12]. In the acetylation of com-
pound 3 with Ac2O, at 0ꢀC, for 20 h (Method A, see
Experimental), a solid was formed; it was filtered,
washed with cold water/EtOH, and recrystallized from
ethanol to give compound 21 in 25% yield (Chart 4).
The analytical and spectroscopic data of this molecule
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M. Chioua, E. Soriano, A. Samadi, and J. Marco-Contelles
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Chart 4. The acetylation reaction of compounds 1–6.
clearly showed that 21 was a monoacetamide, as in the
1H NMR, a methyl group was observed at an anoma-
lous low field (d 2.58) and two broad singlets (7.40 and
4.82 ppm) for two protons, each one corresponding to
the NH2 groups at carbons C6 and C3, respectively.
These data along with the absence of a free NH, or a
NH resonance for a NHCOCH3 moiety, clearly sup-
ported the structure of 21 as 1-acetyl-3,6-diamino-4-
phenyl-1H-pyrazolo[3,4-b]pyridine-5-carbonitrile.This
structure has also been confirmed by the data obtained
in the 13C NMR, HMQC, and HMBC experiments.
To improve the chemical yield, the acetylation reac-
tion was carried out with Ac2O at rt for 5 h or with
AcCl, in pyridine, at 0ꢀC for 5 days, but the yields (21
and 24%, respectively) of compound 21 were not better.
Finally, the acetylation reaction was performed with
Ac2O at reflux for 6 h; after work-up and purification,
peracetylated derivatives 22 (30%) and 23 (40%) were
isolated (Chart 4). The structure of compound 22 was
established as a triacetamide derivative of precursor 3
by its analytical and spectroscopic data. As in the 1H
NMR spectrum, we observed a broad singlet for one
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a monoacetamide bearing the acetyl group at N1, as a
broad singlet appeared at 5.10 ppm integrating for two
protons [C(6)NH2], and the singlet for the acetyl groups
integrating for three protons resonated at d 2.69, a value
that we have found in compound 21 (see earlier) and in
compound 16 [9] (Chart 3).
In view of these results, and as we were interested in
the synthesis of the monoacetamide at C6 in these pyra-
zolopyridines, we considered an alternative synthetic
route based on the acetylation of 2-amino-6-methoxy-4-
phenylpyridine-3,5-dicarbonitrile (27) [13] (Scheme 1).
Carrying out the reaction as reported [13], we obtained
a mixture of monoacetamide 28 (71%) and imide 29
(1%) (Scheme 1) that were easily separated by column
chromatography and submitted to reaction with hydra-
zine hydrate, in DMF at reflux, aiming at the ‘‘one-pot’’
methoxy displacement and simultaneous pyrazolopyri-
dine formation. For compound 28, we obtained com-
pound 27 in 72% yield (Scheme 1). For compound 29,
we isolated and characterized compounds 27, 30, and 3
(Scheme 1). The formation of these compounds is the
result of a series of deacetylation reactions followed by
pyrazolo formation. The structure of compound 30 has
been unequivocally established by comparison of the
reported data in literature [11] and with an authentic
sample prepared in the reaction of DMF [14] with 6-
amino-2-chloro-4-phenylpyridine-3,5-dicarbonitrile (32)
[11,15] (see Experimental), obtained from the reaction
of trimethylorthobenzoate (31) with malononitrile [11]
(see Experimental) (Chart 4).
Figure 1. Conformers for compound 20.
proton at 14.45 ppm, and three acetyl groups [d 2.15
(singlet, one acetyl) and 1.91 (singlet, two acetyl
groups)]; we concluded that N(1) was free, the only
structural problem that remained to be established was
to determine if the NHCOCH3 group was at C3 or at
C6. In the HMBC spectrum, the signal al d 10.98
(NHCOCH3) showed cross-peaks with signals at 100.0,
152.3, and 169.5 (NCOCH3). In the case that the group
NHCOCH3 was at C6, the signals at 100.0 and 152.3
should be assigned to C5 and C6, respectively, the sig-
nal appearing at 106.5 ppm corresponding thus to C3a,
an assignment that seems reasonable, as we have rou-
tinely observed in the compounds investigated here that
in the 13C NMR spectrum d C3a >> C5. To prove this
hypothesis, a series of nOe experiments were carry out.
When the NHCOCH3 was irradiated, only the singlet at
2.15 ppm showed a weak effect; the irradiation of this
signal also showed only a weak effect at 10.98 ppm.
However, the irradiation of the singlet at 1.91 ppm inte-
grating for six protons (two NHCOCH3 groups) pro-
duced a sharp effect on the aromatic protons; the reverse
irradiation also produced the same effect. From these
experiments, we conclude that the two acetamido groups
should be in the same carbon (C3). For compound 23,
Next, we have investigated the acetylation of precur-
sor 5, prepared as usual from compound 6-amino-2-
chloro-4-phenylpyridine-3,5-dicarbonitrile (32) [11] and
N-methylhydrazine (Chart 4). As expected, in the 1H
NMR spectrum of compound 5, a positive nOe effect
between the broad singlet at d 4.36 and the aromatic
protons allowed us to assign this chemical shift for pro-
tons at C(3)NH2, the signal at 6.90 ppm corresponding
to C(6)NH2. The acetylation of pyrazolopyridine 5
(Ac2O, rt, 8 h) provided compound 33 in low yield
1
as in the H MNR spectrum, we observed a broad sin-
glet at d 14.93 for one proton, clearly assigned to
N(1)H, the location of the four acetamido groups present
in the molecule at C3 and C6 was evident.
1
(Chart 4). Not unexpectedly, in the H NMR of com-
The regioselective acetylation at N1 in compound 3,
respect to both NH2 groups at C3 and C6, under mild
conditions, prompted us to investigate the same reaction
on precursor 4, where only two positions at N1 and
C(3)NH2 were available for the acetylation. This com-
pound has been prepared from readily available 2-
chloro-6-methoxy-4-phenylpyridine-3,5-dicarbonitrile
(24) [13] after reaction with hydrazine hydrate; in this
reaction, we isolated also traces of the bis-pyrazolopyri-
dine 25 (Chart 4). When compound 4 was acetylated
with Ac2O (see Experimental) compound 26 was iso-
lated in good yield (84%) (Chart 4). The analytical and
spectroscopic data clearly showed that compound 26 is
pound 33, the singlet for two protons at d 7.12
[(C6)NH2] clearly demonstrated that the acetylation has
taken place in the C(3)NH2 group. Selective nOe experi-
ments also showed the small but evident effect between
the protons at the acetamide group (CONHCH3).
For compound 33 (Fig. 2), rotamer b is 1.8 kcal/mol
more stable than a, possibly because of the electronic
repulsion between the carbonylic oxygen and the N2
present in conformer a, as previously shown for com-
pound 20, although the effect here should be less strong,
because the presence of the phenyl ring prevents a co-
planar arrangement between the amide group and the az-
ole plane, being rotated with a dihedral angle of 58ꢀ.
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M. Chioua, E. Soriano, A. Samadi, and J. Marco-Contelles
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Scheme 1. Synthesis of compounds 28 and 29 and their reaction with hydrazine hydrate.
This fact increases the energy of conformer b and
reduces the energy difference between the two possible
conformers. On the other hand, the calculated and the
experimental chemical shifts for the methyl groups in
NHCOCH3 are in good agreement; the low d chemical
shift observed for this methyl group possibly due to the
shielding effect of the aromatic ring at C4.
All calculations were carried out with Gaussian03
package [17]. All the minima and transition states
involving were fully optimized with the B3LYP hybrid
functional [18]. As the key aspect to account for reactiv-
ity and regioselectivity concerns atoms bearing lone-pair
electrons, we have applied the extended 6-31þG(d,p)
basis set to get reliable structures and energy values.
Then, to optimize computational resources, we have
selected AcCl as acetylated agent instead of Ac2O.
Treatment of precursor 1 with both electrophiles has
shown similar results (see main text). Zero-point ener-
gies and thermal contributions to thermodynamic func-
tions and activation parameters, as well as harmonic fre-
quencies to assess the nature of the stationary points,
were computed at the same level of theory on the
Finally, we have prepared precursor 6 in good yield
in the reaction of 2-amino-6-chloropyridine-3,5-dicarbo-
nitrile (34) with N-phenylhydrazine (Chart 4). After
1
selective nOe experiments in the H NMR spectrum of
this compound, the resonance at d 6.97 was assigned to
the amino group at C3, while the singlet integrating for
two protons at d 6.48 corresponded to (C6)NH2. This
analysis is the reverse that we have observed in the
other precursors investigated in this work and must
ascribed to the presence of a phenyl ring at N2. In fact,
it is well known that the reaction of 2-halogeno-3-cya-
nopyridines with N-arylhydrazines respect to N-alkylhy-
drazines provides 2-aryl-2H-pyrazolo[3,4-b]pyridines
instead of 1-alkyl-4-phenyl-1H-pyrazolo[3,4-b]pyridines
[16]. The acetylation of compound 6 at reflux for 40
min gave the diacetylated derivative 35 in 21% yield
(Chart 4), which showed spectroscopic and analytical
data in good agreement with this structure (see
Experimental).
Computational studies. In view of the results
obtained for pyrazolopyridine 3 (Chart 4), we next
addressed the reaction mechanisms to explain the
observed regioselectivities during the acetylation
reactions.
Figure 2. Conformers for compound 33.
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At the B3LYP/6-31þG(d,p) level, the tautomer 3-2H
is predicted to be less stable than 3-1H by 9.5 and 4.0
kcal/mol in the gas phase and in DMSO, respectively.
According to the Boltzmann distribution, at a tempera-
ture of 298 K, this difference in energy corresponds to a
3-1H:3-2H ratio of >99:<1, with the population of the
minor tautomer below the limit of detection for conven-
tional NMR spectroscopy. Thus, according to the calcu-
lations, tautomer 3-2H is unlikely to coexist with the
other tautomer.
This preference of the 1H tautomer in pyrazolopyri-
dines agrees with structural analysis of 4-aryl-5-cyano-
pyrazolo[3,4-b]pyridines [22], theoretical studies of pyr-
azolo[3,4-b]pyridines bearing a variety of substituents
[23], and crystallographic data of protein–ligand com-
plexes [24], which indeed confirm that in this class of
compounds, the (N1)H forms key H-bonds with the
enzymes (cyclin-dependent kinases).
This picture contrasts with that for the related struc-
tures pyrano[2,3-c]pyrazoles (6-amino-5-cyano-3-methyl-
4-aryl/heteroaryl-2H,4H-dihydropyrano[2,3-c]pyrazoles) as
they exist predominantly in the 2H tautomeric form [25].
To confirm the reliability of our calculations in the predic-
tion of tautomeric equilibria, we have performed further
computations on the 6-amino-5-cyano-3-methyl-4-phenyl-
dihydropyrano[2,3-c]pyrazole ring system [25–27]. Our
results indicate that the 2H tautomer is 3.7 kcal molꢁ1
more stable than the 1H form in the gas phase. These
data are in agreement with the crystallographic results
[25–27], thus supporting our theoretical protocol in the
estimation of the tautomeric equilibrium.
Figure 3. Transition structures for the N-acetylation of 3.
optimized structures. To test the influence of solvation
effects, we have calculated solvation free energies in so-
lution DGsolv for the ground state and transition struc-
tures at the PCM/UAHF/B3LYP/6-31G(d) level [19]
using the previously optimized gas-phase structures.
Combination of these solvation free energies with gas-
phase free energies obtained at B3LYP/6-31þG(d,p)
level yields the relative free energy in solution DGs]ol
compiled in Table 2 in column 4 and Figure 3. Natural
bond orbital (NBO) analyses [20] have been performed
by the module NBO v.3.1 implemented in Gaussian 03
to evaluate the NPA charges at the optimization level.
We have initially carried out the study of the tauto-
meric equilibrium for compound 3. We have focused on
the prototropy tautomerism between the 3-1H and 3-2H
forms involving the azole moiety (Scheme 2) as the
structure with the hydrogen atom attached to the pyridine
nitrogen at the position 7 is energetically unfavorable, as
was expected from valence bond resonance considera-
tions and verified by calculations on this [our calcula-
tions (B3LYP/6-31þG(d,p) reveal a structure 24.5 kcal
molꢁ1 less stable than 3-1H] and related structures [21].
Both structures 3-1H and 3-2H have in common that
the amino groups are partially planarized (out-of-plane
deviation of (C3)N/(C6)N: 29.4ꢀ/10.8ꢀ and 28.9ꢀ/14.2ꢀ
in 3-1H and 3-2H, respectively) as the N electron pairs
are part of the aromatic system, and that the aromatic
ring attached to the C4 position is rotated with respect
to the pyridine plane (with dihedral angles of 57.5 and
59.7ꢀ, respectively) to avoid steric repulsions with sub-
stituents at C3 and C5.
The reactivity of the 3,6-diamino-pyrazolo[3,4-b]pyri-
dines against acetylation merits a careful analysis as
four nucleophilic positions can undergo acetylation:
besides both amino moieties [(C3)N and (C6)N], the pir-
idinic- and pyrrole-type N of the pyrazole (N1, N2).
The electron pair of the pyridinic-type N makes this
position more nucleophilic than the pyrrole-type N,
whose electron pair is part of the aromatic system.
In an attempt to rationalize the regioselectivity, DFT
calculations were performed to determine both the atomic
charges and the HOMO of 3. In general, reactions with
hard (high-lying LUMOs) electrophiles are charged
Scheme 2. Tautomer equilibrium in compound 3.
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Table 1
be mostly the N2-substituted structure that results from
N-acetylation of the major tautomer of 3 (3-1H). In
view of these theoretical results and to support the posi-
tion of the acetyl group at N1/N2, we tried to crystallize
compound 21 as a free base or as its hydrochloride, but
without success; consequently, the location of the acetyl
groups at N1 is a tentative hypothesis that still needs to
be experimentally confirmed.
Calculated NPA charges and HOMO coefficients for the N atoms of 3
prone to undergo attack by the acylating agent.
HOMO
coefficients
NPA charges
3-1H
3-2H
3-1H
3-2H
N1
N2
ꢁ0.397
ꢁ0.340
ꢁ0.861
ꢁ0.829
ꢁ0.327
ꢁ0.380
ꢁ0.861
ꢁ0.837
0.47
0.64
0.58
0.30
0.73
0.34
0.46
0.36
Bases on these data, if the azole positions are blocked
to undergo reaction, the (C3)N should be the preferred
reactive site. This hypothesis agrees with the experimen-
tal observations as (C3)N is the acetylation site found in
20. To further shed light on this result, we have per-
formed calculations for the formation of 20 from the
pyrazolopyridine 2. The HOMO coefficients are parallel
to those found for 3-1H: (C3)N ¼ 0.62, (C6)N ¼ 0.27.
Likewise, the calculated transition structure for the
attacking on the amino group at C3 is 8.3 kcal molꢁ1
more stable than on the amino at C6 (Fig. 4). Accord-
ingly, 20 is acetylated at (C3)N, which is supported by
the experimental evidence.
(C3)N
(C6)N
controlled, whereas reactions with soft (low-lying
LUMOs) electrophiles are under frontier molecular or-
bital (FMO) control. The calculated atomic charges are
not consistent with the experimentally determined regio-
selectivity. Thus, the greatest amount of negative charge
is found at the (C3)N position (atomic charge ¼ ꢁ0.861,
Table 1), while reaction occurs selectively at the azole
moiety. On the other hand, the observed selectivity fits
well with the computed HOMO coefficients on both tau-
tomers 3-1H and 3-2H (Table 1) as the largest value, and
hence providing better overlapping orbital with the elec-
trophile, is situated at the pyridinic-type nitrogen.
In conclusion, in this work, we have described the
acetylation of differently substituted 3,6-diamino-1H-
pyrazolo[3,4-b]pyridine-5-carbonitrile derivatives (1–6.
The structure of the resulting acetamides has been inves-
tigated and confirmed by analytical, spectroscopic, and
chemical transformations. From these studies, we con-
clude that, in general, under mild conditions, and using
acetic anhydride, when free, the N(1)H moiety is the
more reactive center respect to the C(3)NH2 and
C(6)NH2 groups. This trend is reversed when no steric
hindrance due to presence of a phenyl group at C4
drives the preferred acetylation to C(3)NH2, as it is evi-
dent by comparing the observed results from precursor 1
with 3. When N1 is blocked, the (C3)NH2 group under-
goes preferential acetylation over the (C6)NH2 site,
which only has been mono (or diacetylated) at reflux.
On the basis of these data, we have also undertaken a
computational analysis to explain the observed selectiv-
ities during the acetylation reactions. The calculations of
frontier orbital coefficients on the reactants pyrazolopyr-
idines and activation barriers agree with the regiochem-
istry observed. The regioselectivity on the acetylation of
the amino groups can be explained by the availability of
Although orbital properties can account only for the
electronic factors, the steric effects (if exist) are
included in the free energy of activation for the reaction.
From a mechanistic point of view, when the reaction is
carried out in the absence of a deprotonating agent, it
should be expected that the NAH of the azole becomes
acidic enough to be deprotonated only when the pyri-
dinic-type N has been attacked by the acylated agent
[28]. According to this assumption, we have considered
the attack on every N of the neutral structure and have
selected acetyl chloride (see Experimental) as electro-
phile to simulate the acetylation reaction.
The calculated transition structures (Fig. 3) provide
free energy differences that suggest a kinetically favored
attack on the azole (TSN1 and TSN2) rather than on the
amino moieties (TS(C3)N and TS(C6)N) in the gas phase
and in solution (Table 2). In fact, as has been described
earlier, these groups are rather planarized as the lone
pair is partially delocalized in the aromatic system, thus
being less nucleophilic groups than expected. This ob-
servation agrees with the experimental selectivity shown
earlier (Chart 4). In the azole, the kinetically preferred
site for the acetylation, in the gas phase and in solution,
is N2 in the 3-1H form (Table 2), which indeed is the
proposed predominant tautomer. Also, according to
these results, a small amount of the regioisomeric prod-
uct could be formed by attacking at N1 in the 3-2H
form (estimation of the Boltzmann distribution N2:N1
93:7). In summary, these results suggest that 21 should
Table 2
Thermodynamic data (in kcal molꢁ1) in gas phase and in solution for
the potential transition structures for the N-acetylation of 3.
Transition structures
DH]gas
DG]gas
DG]sol
TSN1
TSN2
1.7
0.0
1.6
0.0
1.6
0.0
3.2
7.3
TS(C3)N
TS(C6)N
6.2
11.2
6.5
11.1
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

July 2010
Studies on the Acetylation of 3,6-Diamino-1H-
pyrazolo[3,4-b]pyridine-5-carzbonitrile Derivatives
869
Figure 4. Transition structures for the acetylation of 2 at (C3)N (left) and (C6)N (right). Free-energy differences (in kcal molꢁ1) are shown in the
gas phase and in solution (in parenthesis).
sions, 4096 complex points in t2 and 4 transients for each of
256 time increments, and linear prediction to 512. The data
were zero-filled to 4096 ꢂ 4096 real points. NOESY experi-
ments were acquired with a mix time of 300 ms. Mass spectra
were recorded on a GC/MS spectrometer with an API-ES ioni-
zation source. Elemental analyses were performed at CQO
(CSIC, Spain). TLC was performed on silica F254 and detec-
tion by UV light at 254 nm or by charring with ninhydrin, ani-
saldehyde, or phosphomolybdic-H2SO4 dyeing reagents. Where
anhydrous solvents were needed, they were purified following
the usual procedures. Column chromatography was performed
on silica gel 60 (230 mesh).
the N lone pair. A NBO analysis on 2, 3-1H and the
unsubstituted pyrazole[3,4-b]pyridine (Chart 5, values in
blue) reveals that for the cyano derivatives the lone-pair
orbital at (C6)N is less populated, and hence less prone
to act as nucleophile, than (C3)N because of a higher
delocation on the aromatic system. This induces a
higher planarization of the amino group at C6 (Chart 5,
values in parenthesis). Conversely, the absence of the
electron-withdrawing nitrile substituent allows a more
populated lone-pair orbital at (C6)N, in accordance with
a decreased N-planarization. Therefore, the regioselec-
tivity could be modulated by a careful choice of sub-
stituents [29].
Acetylation of 3,6-diamino-1H-pyrazolo[3,4-b]pyridine-5-
carbonitrile (1)
Method A. A solution of compound 1 [8] (100 mg, 0.57
mmol) in Ac2O (4 mL, 26.52 mmol, 70 equiv) was heated at
144ꢀC for 21 h. The Ac2O in excess was removed, and the
crude submitted to flash chromatography eluting with mixtures
of CH2Cl2/MeOH from 1 to 4% to give N,N0-(5-cyano-3a,7a-
dihydro-1H-pyrazolo[3,4-b]pyridine-3,6-diyl)diacetamide (17)
(13 mg, 8%) [mp 294–296ꢀC; IR (KBr) m 3434, 3306, 32470,
2928, 1685, 1673, 1611, 1584, 1517, 1430, 1395, 1246, 1011
EXPERIMENTAL
Melting points were determined on a microscope type appa-
ratus and are uncorrected. 1H NMR and 13C NMR spectra
were recorded at rt at 300, 400, or 500 MHz and at 75, 100,
or 125 MHz. The assignment of chemical shifts is based on
standard NMR experiments (1H, 13C-DEPT, 1H,1H-COSY,
HSQC, HMBC). In the NMR spectra, values with (*) can be
interchanged. Two-dimensional [1H, 1H] NMR experiments
(NOESY) were carried out with the following parameters: a
delay time of 1 s, a spectral width of 3000 Hz in both dimen-
1
cmꢁ1; H NMR (DMSO-d6, 300 MHz): d 13.62 (s, 1H, N1H),
10.96 (s, 1H, C3NHAc), 10.79 (s, 1H, C6NHAc), 8.89 (s, 1H,
H4), 2.12 (s, 6H, 2xCOCH3); 13C NMR (DMSO-d6, 125
MHz): d 169.2 (2xNCOCH3), 168.4 (C6), 150.8 (C7a), 141.5
(C3),* 141.1 (C4),* 116.9 (CN), 104.7 (C3a), 97.2 (C5), 23.0
Chart 5. NBO analysis on 2,3-1H and the unsubstituted pyrazole[3,4-b]pyridine.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

870
M. Chioua, E. Soriano, A. Samadi, and J. Marco-Contelles
Vol 47
and 22.8 (2xNCOCH3); MS (ES): [M þ 1]þ 259.3, [M þ
Na]þ 281.2, [2M þ Na]þ 539.5. Anal. Calcd. for C11H10N6O2:
C, 51.16; H, 3.90; N, 32.54; found C, 51.29; H, 4.05; N,
32.81.
1H, N(1)H], 7.58–7.50 (m, 5H, aromatic), 6.79 [s, 2H,
C(6)NH2], 4.26 [s, 2H, C(3)NH2]; 13C NMR (DMSO, 75
MHz): d 159.9 (C6), 153.1 (C4), 152.1 (C3), 148.9 (C7a),
134.7 (C10), 130.3 (C40), 129.4 [2C (C20,C60)], 128.9 [2C
(C30,C50)], 118.0 (CN), 98.5 (C3a), 85.1 (C5); MS (ES): [M þ
1]þ 251.3. Anal. Calcd. for C13H10N6: C, 62.39; H, 4.03; N,
33.58; found C, 62.21; H, 4.08; N, 33.58.
Method B. A solution of compound 1 (100 mg, 0.57 mmol)
in Ac2O (4 mL, 26.52 mmol, 70 equiv) was stirred at rt for 5
days. The Ac2O in excess was evaporated. The crude was
washed with water and ethanol to give a solid (84 mg) that
was purified by chromatography eluting with MeOH/CH2Cl2
mixtures (from 1, 2 to 4%) affording compounds 17 [8 mg,
5% (10%)], 18 [15 mg, 12% (21%)], and unreacted precursor
1 (57 mg). The mother liquors were concentrated and recrys-
tallized from ethanol to give compound 19 [20 mg, 14%,
(24%)]. 18: mp 253–256ꢀC; IR (KBr) m 3442, 2219, 1686,
Acetylation of 3,6-diamino-4-phenyl-1H-pyrazolo[3,4-
b]pyridine-5-carbonitrile (3)
Method A. A solution of compound 3 (100 mg, 0.4 mmol)
in Ac2O (2.5 mL, 28 mmol, 70 equiv) was stirred at 0ꢀC for
20 h. Then, the solid was filtered, washed with water/ethanol,
and recrystallized from ethanol to give compound 21 (29 mg,
25%). 21: mp 221–223ꢀC; IR (KBr) m 3471, 3316, 3194, 2212,
1621, 1582, 1406, 1027 cmꢁ1
;
1H NMR (DMSO-d6, 500
1720, 1621, 1590, 1575, 1430, 1382, 1291 cmꢁ1 1H NMR
;
MHz): d 12.66 (s, 1H, NH1), 10.69 [s, 1H, C(3)NHCOCH3],
8.55 [s, 1H, 1CH (H4)], 6.95 (s, 2H, NH2), 2.12 (s, 3H,
(DMSO-d6, 400 MHz): d.7.61–7.50 (m, 5H, Ph), 7.40 (s, 2H,
NH2), 4.82 (s, 2H, NH2), 2.58 (s, 3H, OCCH3); 13C NMR
(DMSO-d6, 75 MHz): d 166.5 (NCOCH3), 160.3 (C6)*, 152.7
(C7a)*, 151.5 (C4), 150.0 (C3), 132.9, 130.1, 129.0, 128.2 (ar-
omatic, C6H5), 116.0 (CN), 101.3 (C3a), 87.6 (C5), 24.5
(NCOCH3); MS (ES): [M þ 1]þ 293.2, [M þ Na]þ 315.2,
[2M þ Na]þ 607.5. Anal. Calcd. for C15H12N6O.1/2H2O: C,
59.79; H, 4.35; N, 27.89; found C, 59.83; H, 4.50; N, 28.30.
Method B. A solution of compound 3 (75 mg, 0.3 mmol) in
Ac2O (2 mL) was refluxed for 6 h to give after column chro-
matography [hexane/ethyl acetate (6/4, 5/5, 4/6)] N-(6-acet-
amido-5-cyano-4-phenyl-1H-pyrazolo[3,4-b]pyridin-3-yl)-N-ace-
tylacetamide (22) (35 mg, 30%) and N,N0-(5-cyano-4-phenyl-
1H-pyrazolo[3,4-b]pyridine-3,6-diyl)bis(N-acetylacetamide) (23)
(50 mg, 40%). 22: mp 183–185ꢀC; IR (KBr) m 3060, 3015,
NCOCH3); 13C NMR (DMSO-d6, 125 MHz):
d 168.2
(NHCOCH3), 158.3 (C6), 152.6 (C7a), 141.7 (C4), 141.1 (C3),
117.7 (CN), 100.2 (C3a), 86.4 (C5), 22.8 (NHCOCH3); MS
(ES): [M þ 1]þ 217.1, [M þ Na]þ 239.3, [2M þ Na]þ 455.1.
Anal. Calcd. for C9H8N6O: C, 50.00; H, 3.73; N, 38.87; found
C, 49.74; H, 3.54; N, 38.66. 19: mp 244–246ꢀC; IR (KBr) m
3471, 3327, 2212, 1671, 1619, 1597, 1426, 1376, 1318, 1141
cmꢁ1 1H NMR (DMSO-d6, 300 MHz): d 11.08 (s, 1H, NH),
;
8.63 (s, 1H, H4), 7.54 (s, 2H, NH2), 2.67 [s, 3H, NCOCH3)],
2.11 (s, 3H, NCOCH3); 13C NMR (DMSO-d6, 125 MHz): d
169.1 [N(3)HCOCH3], 166.9 [N(1)HCOCH3], 159.4 (C6)*,
152.8 (C7a)*, 144.0 (C3), 141.9 (C4), 116.7 (CN), 103.3
(C3a), 88.3 (C5), 24.6 [N(1)HCOCH3], 23.0 [N(3)HCOCH3];
MS (ES): [M þ 1]þ 259.3, [M þ Na]þ 281.2, [2M þ Na]þ
539.5. Anal. Calcd. for C11H10N6O2: C, 51.16; H, 3.90; N,
32.54; found C, 50.98; H, 3.81; N, 32.38.
1
2222, 1746, 1587, 1368, 1231, 1027 cmꢁ1; H NMR (DMSO-
d6, 500 MHz): d 14.46 [s, 1H, N(1)H], 10.98 [s, 1H,
C(3)NHCOCH3)], 7.55–7.34 [m, 5H, aromatic), 2.15
[C(3)NHCOCH3)], 1.91 [s, 6H, 2xC(6)NCOCH3]; 13C NMR
(DMSO-d6, 125 MHz): d 171.6 [2x(C3)NCOCH3], 169.5
[(C(6)NCOCH3], 152.3 (C6), 151.2 (C7a)*, 150.9 (C3)*, 140.1
(C4), 132.4 (C10), 129.9 (C40), 128.6 [2C (C20,C60)], 128.1 [2C
(C30,C50)], 115.4 (CN), 106.5 (C3a), 100.0 (C5), 25.5
[2xC(6)NCOCH3], 23.1 [C(3)NCOCH3]; MS (ES): [M þ 1]þ
377.2, [M þ Na]þ 399.2; [2M þ Na]þ 775.7. Anal. Calcd. for
C19H16N6O3: C, 60.63; H, 4.28; N, 22.33; found C, 60.54; H,
4.39; N, 22.08. 23: mp 137–139ꢀC; IR (KBr) m 3009, 2929,
Acetylation of 3,6-diamino-1-methyl-1H-pyrazolo[3,4-
b]pyridine-5-carbonitrile (2). A solution of compound 2 (100
mg, 0.53 mmol) in Ac2O (2.5 mL, 26.52 mmol, 70 equiv) was
stirred at rt for 3 h. The mixture was cooled at 0ꢀC, the solid
was filtrated, washed with cold ethanol, and recrystallized to
give N-(6-amino-5-cyano-1-methyl-1H-pyrazolo[3,4-b]pyridin-
3-yl)acetamide (20) (100 mg, 82%) as a white solid: mp 246–
249ꢀC; IR (KBr) m 3429, 3332, 3222, 3128, 3086, 2217, 1658,
1617, 1586, 1443, 1276 cmꢁ1 1H NMR (DMSO-d6, 300
;
MHz): d 10.74 (s, 1H, NHCOCH3), 8.57 (s, 1H, H4), 7.08 (s,
2H, C6NH2), 3.70 (s, 3H, NCH3), 2.07 (s, 3H, NHCOCH3);
13C NMR (DMSO-d6, 75 MHz) d 168.1 (CO), 158.3 (C6),
150.7 (C7a), 142.1 (C3), 140.0 (C4), 117.6 (CN), 100.3 (C3a),
86.4 (C5), 32.8 (NCH3), 22.9 (CH3); MS (ES) [M þ 1]þ
231.0; [M þ Na]þ 253.0. Anal. Calcd. for C10H10N6O.1/
2H2O: C, 50.20; H, 4.63; N, 35.13; found C, 50.49; H, 4.61;
N, 34.65.
2855, 2230, 1730, 1589, 1369, 1229, 1029 cmꢁ1 1H NMR
;
(DMSO, 300 MHz) d 14.93 [s, 1H, N(1)H], 7.60–7.43 (m, 5H,
aromatic), 2.35 (s, 6H, 2xNCOCH3), 1.95 (s, 6H, 2xNCOCH3);
MS (ES): [M þ 1]þ 419.2; [M þ Na]þ 441.2; [2M þ Na]þ
859.7. Anal. Calcd. for C21H18N6O: C, 60.28; H, 4.34; N,
20.09; found C, 60.04; H, 4.18; N, 19.95.
Reaction of 2-chloro-6-methoxy-4-phenylpyridine-3,5-
dicarbonitrile (24) hydrazine hydrate. A solution of com-
pound 24 [12] (269 mg, 1 mmol) and hydrazine hydrate (0.1
mL, 2 mmol, 2 equiv) was refluxed in ethanol (20 mL) for 20
h until complete reaction (TLC analysis). The mixture was
cooled at 0ꢀC, water was added, and the solid was filtered,
washed with water, and purified by chromatography eluting
with CH2Cl2/MeOH (from 0.5 to 1%) to give compounds 4-
phenyl-1,7-dihydrodipyrazolo[3,4-b:40,30-e]pyridine-3,5-diamine
(25) (13 mg, 5%) and 3-amino-6-methoxy-4-phenyl-1H-pyra-
zolo[3,4-b]pyridine-5-carbonitrile (4) (95 mg, 58%): mp 248–
250ꢀC; IR (KBr) m 3489, 3391, 3225, 3032, 2938, 2216, 1596,
3,6-Diamino-4-phenyl-1H-pyrazolo[3,4-b]pyridine-5-car-
bonitrile (3). A solution of 2-amino-6-chloro-4-phenylpyri-
dine-3,5-dicarbonitrile [11] (100 mg, 0.39 mmol) and hydra-
zine hydrate (80 lL, 1,57 mmol, 4.0 equiv) in DMF (4 mL,
5mL/mmol) was warmed at 153ꢀC for 1 h until complete reac-
tion (TLC analysis). The mixture was cooled, the solid formed
and filtered, washed with water/ethanol, dried, and purified by
column chromatography eluting with CH2Cl2/MeOH (4%) to
give compound 3 (70 mg, 74%): mp 282–283ꢀC; IR (KBr) m
3498, 3334, 3230, 2931, 1725, 1629, 1571, 1540, 1445, 1269,
1223, 1077 cmꢁ1
;
1H NMR (DMSO, 300 MHz): d 11.88 [s,
1313, 1157 cmꢁ1; H NMR (DMSO-d6, 300 MHz): d 12.63 [s,
1
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

July 2010
Studies on the Acetylation of 3,6-Diamino-1H-
pyrazolo[3,4-b]pyridine-5-carzbonitrile Derivatives
871
1H, N(1)H], 7.61–7.53 (m, 5H, Ph), 4.49 (s, 2H, NH2), 4.01 (s,
3H, OCH3); 13C NMR (DMSO-d6,75 MHz): d 164.0 (C6),
153.4 (C4), 150.9 (C3)*, 149.3 (C7a)*, 133.8, 130.7, 129.6,
129.1 (aromatic, C6H5), 116.5 (CN), 100.3 (C3a), 88.2 (C5),
55.3 (OCH3); MS (ES): [M þ 1]þ 266.0, [M þ Na]þ 288.0,
[2M þ Na]þ 553.3. Anal. Calcd. for C14H11N5O: C, 63.39;
H, 4.18;ꢀN, 26.40; found C, 63.15; H, 4.41; N, 26.37. 25: mp
328–330 C; IR (KBr) m 3428, 3249, 3037, 2948, 2593, 1597,
3,6-Diamino-1-methyl-4-phenyl-1H-pyrazolo[3,4-b]pyri-
dine-5-carbonitrile (5). A mixture of 6-amino-2-chloro-4-
phenylpyridine-3,5-dicarbonitrile (32) (254 mg, 1 mmol) and
methylhydrazine (0.11 mL, 1.1 mmol, 1.1 equiv) in DMF (10
mL) was warmed at 153ꢀC for 5 min. The mixture was cooled
at rt; the solid was filtered and recrystallized from ethanol to
give precursor 5 (211 mg, 80%): mp 279–281ꢀC; IR (KBr) m
3477, 3427, 3379, 3323, 3191, 2201, 1654, 1589, 1573, 1561,
1099 cmꢁ1 1H NMR (DMSO, 300 MHz): d 11.67 (s, 2H, 2
;
1405, 1204 cmꢁ1 1H NMR (DMSO, 300 MHz): d 7.58–7.48
;
NH), 7.63–7.46 (m, 5H, aromatic), 4.24 (s, 4H, 2 NH2); 13C
NMR (DMSO, 75 MHz): d 153.4 (2C, C7a, C8a), 148.1 (2C,
C3, C5), 139.3 (C4), 133.4 (C10), 129.3 (C40), 129.3 [C,
(C20,C60)], 128.8 [C, (C30,C50)], 101.5 (2C (C3a, C4a); MS
(ES): [M þ 1]þ 266.2. Anal. Calcd. for C13H11N7: C, 58.86;
H, 4.18; N, 36.96; found: C, 58.79; H, 4.36; N, 36.72.
(m, 5H, C6H5), 6.90 [s, 2H, C(6)NH2], 4.36 [s, 2H, C(3)NH2],
3.59 (s, 3H, NCH3); 13C NMR (DMSO, 75 MHz): d 158.2
(C6), 151.7 (C4), 150.5 (C7a), 147.5 (C3), 133.8 (C10), 129.7
(C40), 128.8 [2C (C20,C60)], 128.2 [2C (C30,C50)], 117.3 (CN),
98.0 (C3a), 84.3 (C5), 32.4 (NCH3); MS (ES): [2M]þ 528.7,
[2M ꢁ 1]þ 527.7. Anal. Calcd. for C14H12N6: C, 63.62; H,
4.58; N, 31.80; found C, 63.60; H, 4.65; N, 32.04.
1-Acetyl-3-amino-6-methoxy-4-phenyl-1H-pyrazolo[3,4-
b]pyridine-5-carbonitrile (26). A solution of 4 (80 mg, 0.3
mmol) in Ac2O (4 mL, 3.9 mmol, 13 equiv) was stirred at 0ꢀC for
16 h and at rt for 4 h. Alter evaporation of the excess of Ac2O, the
crude was purified by column chromatography, eluting with
CH2Cl2/MeOH 1% to afford compound 26 (63 mg, 84%): mp
236–238ꢀC; IR (KBr) m 3485, 3265, 3181, 2225, 1715, 1624,
N-(6-Amino-5-cyano-1-methyl-4-phenyl-1H-pyrazolo[3,4-
b]pyridin-3-yl)acetamide (33). A solution of compound 5
(100 mg, 0.38 mmol) in Ac2O (2.5 mL, 26.52 mmol, 70 equiv)
was stirred at rt for 8 h. The crude was cooled at 0ꢀC, and the
precipitate was filtered, washed with EtOH, and submitted to
chromatography (AcOEt) to give compound 33 (24 mg, 21%):
mp 249–251ꢀC; IR (KBr) m 3489, 3414, 3330, 3263, 3052,
2218, 1664, 1625, 1590, 1571, 1518, 1444, 1399, 1379, 1259,
1
1589, 1571, 1393, 1349, 1153 cmꢁ1; H NMR (DMSO-d6, 300
MHz): d 7.65–7.57 (m, 5H, Ph), 5.10 (s, 2H, NH2), 4.10 (s, 3H,
OCH3), 2.69 (s, 3H, NCOCH3); 13C NMR (DMSO-d6,75 MHz): d
167.8, 164.9, 153.4, 151.0, 150.7, 132.7, 131.2, 129.8, 129.0 (aro-
matic, C6H5), 115.4 (CN), 104.8 (C3a), 92.5 (C5), 55.6 (OCH3),
25.3 (NCOCH3); MS (ES): [M þ 1]þ 308.3, [M þ Na]þ 370.2,
[2M þ Na]þ 637.5. Anal. Calcd. for C16H13N5O.1/2H2O: C,
60.75; H, 4.46; N, 22.14; found: C, 60.59; H, 4.21; N, 22.07.
Reaction of N-acetyl-N-(3,5-dicyano-6-methoxy-4-phenyl-
pyridin-2-yl)acetamide (29) with hydrazine hydrate. A solu-
tion of compound 29 (50 mg, 0.15 mmol) and hydrazine hydrate
(20 lL, 0.22 mmol, 1.5 equiv) in DMF (5 mL) was refluxed
(153ꢀC) for 30 min until complete reaction. Then, the excess of
DMF was removed, AcOEt was added, and washed with water.
The organic phase was dried, filtered, and evaporated to give a
solid that was submitted to chromatography eluting with (hex-
ane/EtOAc, from 8/2 to 1/1) to give compounds 27 (23 mg,
61%), 30 [11] (2 mg, 6%), and 3 (10 mg, 27%).
1196 cmꢁ1 1H NMR (DMSO-d6, 300 MHz): d 9.49 (s, 1H,
;
NH), 7.58–7.48 (m, 5H, C6H5), 7.12 (s, 2H, NH2), 3.78 (s, 3H,
NCH3), 1.42 (s, 3H, COCH3); 13C NMR (DMSO-d6, 75 MHz):
d 169.1 (NCOCH3), 159.8 (C6), 152.7 (C4), 151.7 (C7a),
139.0 (C3), 133.9 (C10), 129.9 (C40), 129.3 (2C, C20,C60),
128.6 (2C, C30,C50), 117.5 (CN), 102.9 (C3a), 88.3 (C5), 33.8
(NCH3), 22.51 (NCOCH3); MS (ES): [M þ 1]þ 307.1. Anal.
Calcd. for C14H12N6: C, 62.74; H, 4.61; N, 27.44; found C,
62.96; H, 4.68; N, 27.31.
3,6-Diamino-2-phenyl-2H-pyrazolo[3,4-b]pyridine-5-car-
bonitrile (6). A solution of precursor 34 [8] (100 mg, 0.56
mmol) and N-phenylhydrazine (82 lL, 0.84 mmol, 1.5 equiv)
in DMF (2 mL, 5mL/mmol) was warmed at 153ꢀC for 1 h
until complete reaction (TLC analysis). The mixture was
cooled at 0ꢀC; the solid was recovered and submitted to chro-
matography eluting with CH2Cl2/MeOH (from 0.5 to 2%) to
give product 6 (91 mg, 70%): mp 306–308ꢀC; IR (KBr) m
2,6-Diamino-4-phenylpyridine-3,5-dicarbonitrile (30). In a
30-mL glass tube equipped with septa was placed a solution of
3467, 3353, 3299, 3154, 2211, 1620, 1596, 1455, 1343 cmꢁ1
;
1H NMR (DMSO, 300 MHz): d 8.36 [s, 1H, H4), 7.57–7.37
(m, 5H, aromatic), 6.95 [s, 2H, (C3)NH2], 6.47 [s, 2H,
(C6)NH2]; 13C NMR (DMSO, 75 MHz): d 157.9 (C6)*, 156.5
(C7a)*, 143.0 (C3), 140.0 (C4), 138.2 (C10), 129.3 (C30,C50),
127.4 (C40), 123.9 (C20,C60), 118.5 (CN), 96.9 (C3a), 84.6
(C5); MS (CI): m/z 251 [Mþ, 100], 234 [Mþ-NH2, 8], 92 (14),
77(21). Anal. Calcd. for C13H10N6: C, 62.39; H, 4.03; N,
33.58; found C, 62.10; H,4.32; N, 33.31.
6-amino-2-chloro-4-phenylpyridine-3,5-dicarbonitrile
(32)
(0.382 g, 1.5 mmol) in 10 mL of DMF. The reaction mixture
was stirred for 30 s before the irradiation to homogenize the so-
lution and then exposed to MWI 250W at 180ꢀC during 3 min.
After completion showed by TLC (hexane/AcOEt, 3/2), the
reaction mixture was diluted with water, and the precipitate was
filtered and washed with water. The residue was purified by col-
umn chromatography (CH2Cl2/MeOH, 25/1 to 10/1, v/v) to yield
product 30 (155 mg, 44%), which showed spectroscopic data in
good accord with those reported in literature [11].
6-Amino-2-chloro-4-phenylpyridine-3,5-dicarbonitrile (32). To
a solution of trimethylorthobenzoate (31) (1.82 g, 0.01 mol) in
pyridine (5 mL) was added malononitrile (1.32 g, 0.02 mol, 2
equiv). The mixture was heated at 110ꢀC for 7 h. After cool-
ing, concentrated aqueous hydrochloric acid (10 mL) was
added, and the mixture was heated at 100ꢀC for 2.5 h. After
cooling to rt, the mixture was diluted with water and filtered
to afford compound 32 (1.0 g, 40%), which showed spectro-
scopic data in agreement with those reported in literature [11].
N,N0-(5-Cyano-2-phenyl-2H-pyrazolo[3,4-b]pyridine-3,6-
diyl)diacetamide (35). A solution of compound 6 (100 mg,
0.4 mmol) in Ac2O (2.5 mL, 28 mmol, 70 equiv) was heated
at 144ꢀC for 40 min. The mixture was cooled at rt, the solvent
was removed under vacuo, and the crude submitted to chroma-
tography (CH2Cl2/MeOH from 0.1 to 2%) to give product 35
(28 mg, 21%): mp 229–230ꢀC; IR (KBr) m 3467, 3353, 3299,
1
3154, 2211, 1619, 1596, 1454, 1343 cmꢁ1; H NMR (DMSO-
d6, 300 MHz): d 10.82 (s, 1H, NHCOCH3), 10.68 (s, 1H,
NHCOCH3), 8.84 (s, 1H, H4), 7.69–7.57 (m, 5 H, aromatic),
2.12 (s, 3H, NHCOCH3), 2.08 (s, 3H, NHCOCH3); 13C NMR
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

872
M. Chioua, E. Soriano, A. Samadi, and J. Marco-Contelles
Vol 47
[12] Abdelrazek, F. M.; Metwally, N. H.; Sobhy, N. A. Afinidad
2006, 63, 149.
(DMSO-d6, 75 MHz): d 169.8 (NCOCH3), 169.4 (NCOCH3),
154.6 (C6)*, 151.4 (C7a), 141.4 (C4), 137.8 (C10), 131.8 (C3),
129.5 (2C, C30,50), 129.4 (C40), 124.8 (2C, C20,C60), 116.7
(CN), 106.0 (C3a), 100.0 (C5), 22.9 (NCOCH3), 22.7
(NCOCH3); MS (ES): [M þ 1]þ 335.2, [M þ Na]þ 357.2,
[2M þ Na]þ 691.5. Anal. Calcd for C17H14N6O: C, 61.07; H,
4.22; N, 25.14; found C, 60.85; H, 4.36; N, 24.98.
[13] Quintela, J. M.; Soto, J. L. Anal Quim C 1983, 79, 368.
[14] Goswami, S.; Das, N. K. J Heterocycl Chem 2009, 46, 324.
[15] 6-Amino-2-chloro-4-phenylpyridine-3,5-dicarbonitrile (32)
can also be synthesized from 2-amino-3,5-dicyano-5-hydroxy-4-phe-
nylpyridine: Peinador, C.; Veiga, C. M.; Vilar, J.; Quintela, J. M. Het-
erocycles 1994, 38, 1299.
[16] Schmidt, P.; Eichenberger, M.; Wilhelm, M.; Druey, J.
Helv Chim Acta 1959, 42, 763.
Acknowledgments. M. Chioua thanks Instituto de Salud Carlos
III (Ministerio de Salud y Consumo) for a postdoctoral fellow-
ship. A. Samadi thanks CSIC for a I3P postdoctoral contract.
JMC thanks MICINN (SAF2006-08764-C02-01), Comunidad de
Madrid (S/SAL-0275-2006), and Instituto de Salud Carlos III
[Retic RENEVAS (RD06/0026/1002)] for support.
[17] Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G.
E.; Robb, M. A.; Cheeseman, J. R.; Montgomery, J. A., Jr.; Vreven,
T.; Kudin, K. N.; Burant, J. C.; Millam, J. M.; Iyengar, S. S.; Tomasi,
J.; Barone, V.; Mennucci, B.; Cossi, M.; Scalmani, G.; Rega, N.;
Petersson, G. A.; Nakatsuji, H.; Hada, M.; Ehara, M.; Toyota, K.;
Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao,
O.; Nakai, H.; Klene, M.; Li, X.; Knox, J. E.; Hratchian, H. P.; Cross,
J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann,
R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J.
W.; Ayala, P. Y.; Morokuma, K.; Voth, G. A.; Salvador, P.; Dannen-
berg, J. J.; Zakrzewski, V. G.; Dapprich, S.; Daniels, A. D.; Strain, M.
C.; Farkas, O.; Malick, D. K.; Rabuck, A. D.; Raghavachari, K.;
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slowski, J.; Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz, P.;
Komaromi, I.; Martin, R. L.; Fox, D. J.; Keith, T.; Al-Laham, M. A.;
Peng, C. Y.; Nanayakkara, A.; Challacombe, M.; Gill, P. M. W.; John-
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Journal of Heterocyclic Chemistry
DOI 10.1002/jhet