Synthesis and tautomerism of substituted pyrazolo[4,3-c]pyrazoles

Article abstract of DOI:10.1002/ejoc.201300606

Fused five-membered nitrogen heterocycles comprise a very important group of compounds frequently utilized in pharmaceutical applications. In this study, we report the first systematic synthesis of substituted pyrazolo[4,3-c]pyrazoles and three regioisomers of their N-methyl derivatives. All compounds were fully characterized by NMR spectroscopy in solution and selected compounds also were studied by X-ray diffraction in the solid state. ¹H, ¹³C, and ¹⁵N NMR spectroscopic data for all isomers were interpreted by DFT calculations of nuclear shielding constants and indirect spin-spin coupling constants. The N-methyl isomers were used in the following steps as model compounds to investigate a potential N1-H/N4-H, N2-H/N4-H, and N1-H/N5-H tautomerism of 3,6-substituted pyrazolo[4,3-c]pyrazoles by using low-temperature NMR spectroscopy. All bases were shown to occur predominantly in the N1-H/N4-H tautomeric form and the structure of minor form was governed by the substituents at positions 3 and 6. Stabilities of individual tautomeric forms are calculated by DFT methods and discussed. A relationship between the tautomeric populations and the ratios among N-methyl isomers obtained upon methylation of selected bases in solution are investigated. The stability of various tautomeric forms of substituted pyrazolo[4,3-c]pyrazoles characterized by low-temperature NMR spectroscopy can be qualitatively explained by electronic and steric effects of substituents in positions 3 and 6 in individual tautomers, as revealed by the DFT calculations. Copyright

Full text of DOI:10.1002/ejoc.201300606

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DOI: 10.1002/ejoc.201300606
Synthesis and Tautomerism of Substituted Pyrazolo[4,3-c]pyrazoles
[b,d]
Shivaji S. Kadam,[a,b,c,d] Lukás Maier,
Ioannis Kostakis,[c] Nicole Pouli,[c]
ˇ
[a]
[b]
[c]
[a,b,d]
ˇ
ˇ
Jaromír Tousek, Marek Necas, Panagiotis Marakos,* and Radek Marek*
Keywords: Nitrogen heterocycles / Tautomerism / Low-temperature NMR spectroscopy / Electronic and steric effects /
DFT calculations
Fused five-membered nitrogen heterocycles comprise a very
important group of compounds frequently utilized in phar-
maceutical applications. In this study, we report the first sys-
tematic synthesis of substituted pyrazolo[4,3-c]pyrazoles and
three regioisomers of their N-methyl derivatives. All com-
pounds were fully characterized by NMR spectroscopy in
solution and selected compounds also were studied by X-ray
following steps as model compounds to investigate a poten-
tial N1-H/N4-H, N2-H/N4-H, and N1-H/N5-H tautomerism
of 3,6-substituted pyrazolo[4,3-c]pyrazoles by using low-tem-
perature NMR spectroscopy. All bases were shown to occur
predominantly in the N1-H/N4-H tautomeric form and the
structure of minor form was governed by the substituents at
positions 3 and 6. Stabilities of individual tautomeric forms
diffraction in the solid state. 1H, 13C, and 15N NMR spectro- are calculated by DFT methods and discussed. A relationship
scopic data for all isomers were interpreted by DFT calcula-
tions of nuclear shielding constants and indirect spin–spin
coupling constants. The N-methyl isomers were used in the
between the tautomeric populations and the ratios among N-
methyl isomers obtained upon methylation of selected bases
in solution are investigated.
1. Introduction
cological activities. Furthermore, they are valuable products
and semi-products for the synthesis of biologically and
pharmacologically active compounds, including pyrazole-
containing antibiotics as well as dyestuffs.[4]
Fused five-membered heterocycles derived from pyrrole,
pyrazole, and imidazole, comprise a biologically important
group of compounds with a broad range of pharmaceutical
applications. Fused pyrazoles, specifically pyrazolopyrimid-
ines, are very important purine isosteres capable of in-
hibiting crucial cellular enzymes.[1] A number of variously
substituted pyrazolopyridines have been shown to be potent
inhibitors of phosphodiesterases, matrix metalloproteinases,
glycogen synthase kinase-3, and cyclin-dependent kinases.[2]
The tetrahydropyrrolo[3,4-c]pyrazoles represent a class of
compounds designed to target the ATP binding site of pro-
tein kinases.[3] The pyrazolo[4,3-c]pyrazoles form an inter-
esting class of compounds since they can also be viewed as
isosteric purine derivatives with potential pharma-
Generally, five-membered heterocycles containing several
nitrogen atoms can occur in a number of tautomeric forms.
The tautomerism of isolated nucleic acid bases and several
purine derivatives has been investigated extensively since
tautomeric equilibria need to be considered in any study of
their binding modes with biological targets.[5] The taut-
omerism of purines has been characterized by various spec-
troscopic methods such as UV,[6] IR,[7] NMR[8] and mod-
elled using quantum mechanical methods.[9] NMR spec-
troscopy is a particularly useful method for investigating
various phenomena involving purines and related hetero-
cycles.[10] 13C NMR chemical shifts were used to delineate
protonation sites of purine ions,[11,12] to study differences
between the 13C NMR chemical shifts of N-methylated pur-
ine isomers,[13] and to study the tautomeric populations.[14]
Because the proton transfer occurs at the nitrogen atoms,
15N NMR spectroscopy is an extremely powerful tool for
investigating this phenomenon.[15]
[a] CEITEC – Central European Institute of Technology, Masaryk
University,
Kamenice 5/A4, 62500 Brno, Czech Republic
E-mail: rmarek@chemi.muni.cz
http://www.ceitec.eu
[b] Department of Chemistry, Faculty of Science, Masaryk
University,
Kamenice 5, 62500 Brno, Czech Republic
[c] School of Pharmacy, Department of Pharmaceutical
Chemistry, National and Kapodistrian University of Athens,
Panepistimiopolis Zografou, 15771 Athens, Greece
E-mail: marakos@pharm.uoa.gr
Substituted pyrazolo[3,4-c]pyrazole can occur in five tau-
tomeric forms but only three are notably stable and were
considered in previous studies.[16] The N-phenyl-substituted
pyrazolo[3,4-c]pyrazole analogs, which occur in two iso-
meric forms, have been studied using 13C NMR spec-
troscopy.[16] We were previously involved in the systematic
study of various pyrazolopyridines,[17] focusing on their
http://en.uoa.gr
[d] National Center for Biomolecular Research, Faculty of Science,
Masaryk University,
Kamenice 5/A4, 62500 Brno, Czech Republic
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201300606.
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possible tautomeric forms. As a continuation of our investi- as model compounds to assist in assigning NMR reso-
gation, we report here the first systematic preparation and nances to the individual tautomeric forms observed at low
physicochemical study of a series of derivatives belonging to temperatures. The trends in NMR chemical shift patterns
the closely related pyrazolo[4,3-c]pyrazole scaffold[4,18] (see for the individual isomers and tautomers are rationalized
Scheme 1).
using DFT methods. In addition, structures of several com-
pounds are characterized by single-crystal X-ray diffrac-
tion. The stability of individual tautomers is evaluated and
discussed.
2. Results and Discussion
2.1. Synthesis
The reaction sequences leading to target compounds of
interest are outlined in Scheme 2 and Scheme 3. We used
ethyl oxalate as a starting material which, upon reaction
with the suitable ketones (4-methyl-2-pentanone or acet-
one), provided corresponding diketo esters[20] 3 and 4. Con-
densation of the above mentioned esters with hydrazine in
boiling ethanol,[21,22] followed by hydrolysis of correspond-
ing esters 5 and 6, and subsequent nitration of resulting
pyrazoles[4] 7 and 8 with fuming HNO3/H2SO4, afforded
carboxylic acids[12–23] 9 and 10. Acids were converted into
Scheme 1. Three tautomeric forms N1-H/N4-H, N2-H/N4-H, and
N1-H/N5-H of pyrazolo[4,3-c]pyrazoles considered in this paper.
Pyrazolo[4,3-c]pyrazole derivatives can occur, in prin- the corresponding ethyl esters, which were subjected to a
ciple in, three major tautomeric forms, namely N1-H/N4- catalytic hydrogenation over Pd/C, followed by acetylation
H, N2-H/N4-H, or N1-H/N5-H. The corresponding pop- to furnish diacetamides[24] 17 and 18. These compounds
ulations of the individual tautomers are influenced by tem- were treated with isoamyl nitrite and, via a N-nitroso re-
perature, solvent, and substitution pattern which modulates arrangement,[25] afforded the diacetyl pyrazolopyrazoles,
electron density distribution within the molecule.[19] In this which upon basic hydrolysis resulted in pyrazolo[4,3-c]pyr-
contribution we investigate the tautomerism of selected azoles[8] 21 and 22 (for the full synthetic procedure, see
compounds by using low-temperature NMR spectroscopy. Scheme S2 in Supporting Information). The synthesis of
Isomers of N-methylated pyrazolo[4,3-c]pyrazoles are used chloro-substituted pyrazolopyrazole 31 was effected
Scheme 2. Synthesis of substituted pyrazolopyrazoles 21 and 22 from ethyl oxalate, and 31 from ethyl acetoacetate.
Scheme 3. Synthesis of N-methylated pyrazolo[4,3-c]pyrazole analogues and thermal decarboxylation of the pyrazolo[4,3-c]pyrazole 21.
Reagents and conditions: a) NaH, CH3I, DMF; b) EtOH, NaOH; c) Diphenyl ether, 180 °C, 4 h; d) EtOH, H2SO4, reflux, 15 h.
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Synthesis and Tautomerism of Substituted Pyrazolo[4,3-c]pyrazoles
through condensation of ethyl acetoacetate (23) with hy-
drazine, followed by conversion of the resulting hydroxypyr-
azole to the corresponding chloropyrazole; nitration fol-
lowed by reduction afforded intermediate pyrazole diacet-
amide 29. This compound was converted into target deriva-
tive 31 proceeding through a N-nitroso rearrangement and
deacetylation of the intermediate bisacetylated pyrazolo-
pyrazole (for the full synthetic procedure, see Scheme S3 in
Supporting Information).
The preparation of corresponding N-methylated pyr-
azolopyrazoles 35a–b was effected using compound 21,
which was treated with iodomethane in the presence of
NaH and then, upon esterification of resulting acids 32a–
b, furnished target compounds 35a–b (see Scheme 3). Only
methyl isomers 32a and 32b were isolated from the reaction
mixture obtained from methylation of 21, whereas no traces
of the third possible isomer (32c) were detected upon the
chromatographic purification of the reaction mixture. Con-
versely, analogous methylation of pyrazole carboxylate 22
provided N-methylated derivatives 33a–c, which were then
subjected to esterification furnishing carboxylates 36a–c.
Finally, compounds 34a–c were prepared by methylation of
pyrazolopyrazole 31 (see Scheme 3). Synthesis of pyrazol-
opyrazole 38 (see Scheme 3) was accomplished through ini-
tial basic hydrolysis of ester 21 to provide carboxylic acid
37 which was then thermally decarboxylated. Decarboxyl-
ation of 32b provided corresponding decarboxylated ana-
logue [3-isopropyl-1,5-dimethyl-1,5-dihydropyrazolo[4,3-c]-
pyrazole (39b)] (see Scheme S4 in Supporting Information).
2.2. Structure and NMR Parameters of N-Methyl
Derivatives of Pyrazolo[4,3-c]pyrazoles
Generally speaking, substituted pyrazolo[4,3-c]pyrazoles
can occur in five tautomeric forms but out of these only
three display much in the way of stability (see Scheme 1).
To investigate the tautomerism of pyrazolo[4,3-c]pyrazoles
by NMR spectroscopy in solution and to facilitate the as-
signment of NMR resonances at low temperature (see Sup-
porting Information), we first synthesized model N-methyl
derivatives a, b, and c shown in Scheme 3. We employed X-
ray diffraction, NMR spectroscopy, and mass spectrometry
to unequivocally characterize the structures of individual
compounds. Crystallization of three N-methyl analogs from
dichloromethane yielded single crystals suitable for the X-
ray diffraction analysis. The molecular structures of 32a
and 36c determined by X-ray diffraction are shown in Fig-
ure 1 (for 34a, see Figure S1).
Figure 1. The molecular structures of compounds 32a (top) and
36c (bottom) obtained from single-crystal X-ray diffraction.
1H, 13C, and 15N NMR signals for all model N-methyl
The assignment of 13C NMR chemical shifts was based
compounds were assigned using two-dimensional NMR on the 1H-13C chemical shift correlations in 2D NMR spec-
spectroscopy (see Experimental Section). The selected 13C tra and characteristic values of H-13C coupling constants
1
and 15N NMR chemical shifts for synthesized isomers of for atom H-6 in compounds 34 and 36, the hydrogen atoms
N-methylated pyrazolo[4,3-c]pyrazoles are summarized in of the isopropyl group in position 6 in 35, and atom H-3 in
Table 1 and characteristic indirect spin–spin coupling con- 39b. The 13C NMR signals of atoms C-3 or C-6 were easily
1
stants are summarized in Table 2 (for additional H, 13C, assigned by single-bond interactions with H-3 or H-6,
and 15N NMR spectroscopic data, see Supporting Infor- respectively. The signals of the bridgehead atoms C-3a and
mation).
C-6a correlated with atom H-6 (and with H-3 for 39b) and
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Table 1. 13C NMR and 15N NMR chemical shifts (δ in ppm) for three N-methylpyrazolo[4,3-c]pyrazoles 34a–36c (in CDCl3 at 303 K)
and the differences calculated between individual isomers.
Table 2. Selected JH-C and JH-N coupling constants [Hz] for com- scopic data for compounds 34a–36c in Tables 1 and 2 indi-
pounds 34, 36c, and 39b (in CDCl3 at 303 K).
cate that carefully selected NMR chemical shifts and indi-
rect nuclear spin–spin coupling constants can be employed
as a tool for determining either substitution (isomers) or
protonation (tautomers) sites. The 13C NMR chemical
shifts of C-3, C-3a, C-6, and C-6a as well as nitrogen atoms
N-1, N-2, N-4, and N-5 are remarkably influenced by the
positions of the N-methyl groups (compare Δδ for a vs. b,
and a vs. c in Table 1). In all cases, the 13C NMR reso-
nances of atoms C-3, C-3a, C-6, and C-6a are significantly
[a] Not observed. [b] Not resolved, J Ͻ 2 Hz. [c] JH3-C3a. [d] JH3-C6a
.
deshielded (Δδ in Table 1, 6–18 ppm) if the neighboring
nitrogen is bare (bearing a lone pair of electrons). For ex-
ample, C-3 is deshielded in forms a and c as compared to
form b, where the shielding effect of neighboring N2-R
group is pronounced. A similar trend can be found for all
carbons neighboring nitrogen atoms. The 15N NMR chemi-
cal shifts of N-1, N-2, N-4, and N-5 are significantly affec-
ted by the presence or absence of the methyl group on the
nitrogen atom (see Table 1). It has been known for a long
time that alkylation or protonation of a nitrogen atom (=N-
type) results in significant shielding by about
100 ppm.[8,15,29] For example, the 15N NMR chemical shift
of N-1 is more than 100 ppm larger for isomer b as com-
pared to those for a and c. The deshielding effect of the
[e] JH3-N2
.
the specific couplings H6-C3a and H6-C6a were used to
assign the C-3a and C-6a resonances unambiguously (vide
infra).[26] The assignment of 15N resonances was based on
the characteristic 1H-15N chemical shift correlations ob-
served in 2D NMR experiments for H-6 atom in com-
pounds 34 and 36, for hydrogen atoms of the isopropyl
group in 35, and atom H-3 in compound 39b.
Employment of various NMR parameters to investigate
the tautomerism in purine derivatives has been demon-
strated several times.[27,28] Analogously, the NMR spectro-
Table 3. Experimental and DFT-calculated 13C and 15N NMR chemical shifts (δ in ppm) for compounds 34a–c (in CDCl3 at 303 K) and
the differences in NMR chemical shifts (Δδ) calculated between individual isomers.
[a] Geometry optimization: B3LYP/6-31G*; shielding constants: B3LYP/6-311G** (CHCl3).
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