Synthesis and characterization of the atropisomeric relationships of a substituted N-phenyl-bipyrazole derivative with anti-inflammatory properties

Article abstract of DOI:10.1002/chir.22016

This work describes the atropisomeric relationships of 3-methyl-5-(3- methyl-5-phenyl-1H-pyrazol-1-yl)-1-phenyl-1H-pyrazol-4-amine (2d), which belongs to series 4-aminobipyrazole derivatives designed as anti-inflammatory agents. The ¹H nuclear magnetic resonance spectra obtained in the presence of a chiral lanthanide shift salt associated to chiral high-performance liquid chromatography analysis, X-ray diffraction, and molecular modeling tools confirmed that ortho bis-functionalized bipyrazole 2d exists as a mixture of aR,aS-atropisomers. These results provide useful information to understand the pharmacological profile of this derivative and of other 4-aminobipyrazole analogs. Copyright

Full text of DOI:10.1002/chir.22016

CHIRALITY (2012)
Synthesis and Characterization of the Atropisomeric Relationships
of a Substituted N-Phenyl-Bipyrazole Derivative with
Anti-inflammatory Properties
MARCIA P. VELOSO,1,2,3 NELILMA C. ROMEIRO,4 GILBERTO M.S. SILVA,1,5,6 HÉLIO DE M. ALVES,1 ANTONIO C. DORIGUETTO,7
JAVIER ELLENA,8 ANA L. P. MIRANDA,1,5 ELIEZER J. BARREIRO1,2,5 AND CARLOS A.M. FRAGA1,2,5
*
1Laboratório de Avaliação e Síntese de Substâncias Bioativas (LASSBio), Faculdade de Farmácia, Universidade Federal do Rio de Janeiro,
Rio de Janeiro, RJ, Brazil
2Programa de Pós-Graduação em Química, Instituto de Química, Universidade Federal do Rio de Janeiro, RJ, Brazil
3Faculdade de Ciências Farmacêuticas, Universidade Federal de Alfenas, Alfenas, MG, Brazil
4Universidade Federal do Rio de Janeiro, Macaé, RJ, Brazil
5Programa de Pós-Graduação em Farmacologia e Química Medicinal, Instituto de Ciências Biomédicas,
Universidade Federal do Rio de Janeiro, RJ, Brazil
6Instituto de Pesquisa Clínica Evandro Chagas, FIOCRUZ, Rio de Janeiro, RJ, Brazil
7Instituto de Ciências Exatas, Universidade Federal de Alfenas, Alfenas, MG, Brazil
8Instituto de Física de São Carlos, Universidade de São Paulo, São Carlos, SP, Brazil
ABSTRACT
This work describes the atropisomeric relationships of 3-methyl-5-(3-methyl-5-
phenyl-1H-pyrazol-1-yl)-1-phenyl-1H-pyrazol-4-amine (2d), which belongs to series 4-aminobipyra-
zole derivatives designed as anti-inflammatory agents. The 1H nuclear magnetic resonance spectra
obtained in the presence of a chiral lanthanide shift salt associated to chiral high-performance liquid
chromatography analysis, X-ray diffraction, and molecular modeling tools confirmed that ortho
bis-functionalized bipyrazole 2d exists as a mixture of aR,aS-atropisomers. These results provide
useful information to understand the pharmacological profile of this derivative and of other 4-amino-
bipyrazole analogs. Chirality 00:00–00, 2012. © 2012 Wiley Periodicals, Inc.
KEY WORDS: bipyrazole; atropisomerism; axial chirality; nonsteroidal anti-inflammatory drugs;
celecoxib; COX inhibitors
INTRODUCTION
MATERIALS AND METHODS
Chemistry
Cyclooxygenase-2 (COX-2) is an enzyme involved in
the production of prostaglandins associated with many
important physiological and pathological states,1 such as
inflammation.2 Selective COX-2 inhibitors,3 for example,
celecoxib (1),4 have been shown to be potent anti-
inflammatory agents with fewer side effects on the gastro-
intestinal (GI) tract and renal system than those of
currently marketed nonselective nonsteroidal anti-
inflammatory drugs,5,6 in spite of some authors having
described the cardiovascular side effects of other drugs
belonging to this therapeutic class.7
In this context, as part of an ongoing research program
designed to identify new anti-inflammatory drug candi-
dates, a new series of N-phenylbipyrazole derivatives
2a-e was designed (Fig. 1), synthesized, and pharmaco-
logically assayed.8 Based on these studies, we were able
to identify the new anti-inflammatory prototype LASSBio-455
Melting points were determined with a Quimis 340M apparatus
(Quimis, São Paulo, SP, Brazil) and are uncorrected. 1H and 13C nuclear
magnetic resonance (NMR) spectra were determined in deuterochloro-
form containing approximately 1% tetramethylsilane as an internal stan-
dard with Brucker DRX200 spectrometer (Bruker, Billerica, MA, USA)
at 200 and 50 MHz, respectively. Splitting patterns are as follows: s, sin-
glet; d, doublet; br, broad; m, multiplet. 1H and 13C NMR assignments
given for each compound were confirmed by heteronuclear multiple
quantum correlation (HMQC) and heteronuclear multiple bon coherence
(HMBC) experiments. Infrared (IR) spectra were obtained with a Nicolet-
550 Magna spectrophotomer (Thermo Fisher Scientific Inc., West Palm
Beach, Florida, USA) using KBr pellets. The mass spectra (MS) were
obtained on GC/VG Micromass 12 spectrometer (VG Micromass Ltd.,
Manchester, UK) at 70 eV. Ultraviolet (UV) spectra were determined in
a methanol solution on a Beckmann DU-70 spectrophotometer (Beck-
mann Coulter, Brea, CA, USA). Microanalysis data were obtained with a
Perkin–Elmer 240 analyzer using a Perkin–Elmer AD-4 balance (Perkin-
Elmer, Waltham, Massachusetts, USA). Before concentration under re-
duced pressure, all organic extracts were dried over anhydrous sodium
sulfate powder. The progress of all reactions was monitored by thin-layer
chromatography (TLC) performed on 2.0 ꢀ 6.0-cm aluminum sheets
precoated with silica gel 60 (HF-254, Merck) to a thickness of 0.25 mm.
The developed chromatograms were viewed under UV light at 254 nm.
(2c), which displayed
a moderate anti-edematogenic
profile (Table S1, see Supplementary Material) without
presenting GI toxicity. In addition, we have anticipated
that the derivative 2d could exist as a pair of atropisomers
because of the presence of axial chirality resultant from
the torsional barrier of the dihedral angle θ formed by
N1–C5–N1–C5 atoms. Even for non-natural N,C-coupled
heterobiaryl derivatives, atropisomerism studies have
rarely been described in literature.9,10
Additional Supporting Information may be found in the online version of
this article.
*Correspondence to: Carlos Alberto Manssour Fraga, LASSBio, Faculty of
Pharmacy, Federal University of Rio de Janeiro, PO Box 68023, 21941-902
Rio de Janeiro, RJ, Brazil. E-mail: cmfraga@ccsdecania.ufrj.br
Received for publication 24 July 2011; Accepted 18 January 2012
DOI: 10.1002/chir.22016
Therefore, this article describes the synthesis and charac-
terization of the atropisomeric relationships of the N-phenyl-
bipyrazole derivative 2d (Fig. 1), including the resolution
and stereochemical assignment of its enantiomers.
© 2012 Wiley Periodicals, Inc.
Published online in Wiley Online Library
(wileyonlinelibrary.com).

VELOSO ET AL.
Fig. 1. New anti-inflammatory N-phenylbipyrazole derivatives (2a–e).
For column chromatography, Merck silica gel (70–230 mesh) was used.
(2:1) was heated at reflux for 1 h. The hot mixture was filtered through
Celite and concentrated in vacuum. The residue was diluted with H2O
and extracted with EtOAc (5 ꢀ 30 ml). The EtOAc layer was dried over
anhydrous Na2SO4 and concentrated to give the corresponding amino de-
rivative 2d obtained in 98% yield, as clear orange oil. 1H NMR (300 MHz,
CDCl3) d 2.3 (s, 3H, 3-CH3 Py), 2.4 (s, 3H, 3-CH3 Py), 3.0 (br, 2H, NH2 Py),
6.3 (s, 1H, 4-HPy), 6.7 (d, 2H, J = 8 Hz, 200-H5Ph, 600-H5Ph), 6.9 (d, 2H,
J = 8 Hz, 200-H1Ph, 600-H1Ph), 7.1 (m, 6H, 300-H1Ph, 400-H1Ph, 500-H1Ph, 300-H5
Ph, 400-H5Ph, 500-H5Ph). 13C NMR (75 MHz, CDCl3) d 11.3 (3-CH3 Py), 13.6
(3-CH3 Py), 106.5 (4-CPy), 121.4 (200-C1Ph, 600-C1Ph), 125.4 (5-CPy), 125.9
(4-CPy, 400-C1Ph), 127.0 (200-C5Ph, 600-C5Ph), 128.1 (300-C5Ph, 500-C5Ph), 128.4
(300-C1Ph, 500-C1Ph), 129.6 (100-C5Ph), 138.2 (100-C1Ph), 139.2 (3-CPy), 146.6
(5-CPy), 151.0 (3-CPy); IR (KBr) cm-1: 3388, 3295, 3206, and 3129 ( N-H),
3068 ( C-Harom), 2962 and 2927 ( C-Haliph), 1642 and 1595 (d N-H), 1502
( CC); UV (MeOH), l, nm (log e): 210 (4.22), 246 (4.12); Anal. Calc. for
Solvents used in the reactions were dried, redistilled before use, and
stored over +3–4 Å of molecular sieves. Reaction mixture was generally
stirred under a dry nitrogen atmosphere.
Preparation of 1-(3-Methyl-4-nitro-1-phenyl-1H-pyrazol-5-yl)
hydrazine (4)
To a solution of 5-chloro-3-methyl-4-nitro-1-phenyl-1H-pyrazole (3)
(2.3 g, 9.6 mmol) in ethanol (15 ml), maintained under reflux, was added
slowly 85% aq. hydrazine hydrate (1.9 ml), and the resulting mixture
was heated at reflux for an additional 15 min. The reaction mixture was
cooled, and the precipitate that formed was removed by filtration and
crystallized from ethanol to give 4 (1.5 g, 68%), as an orange solid, mp.
150–151 ꢁC; 1H NMR (200 MHz, CDCl3) d 2.5 (s, 3H, CH3 Py), 3.5
(br, 2H, NHNH2 Py), 7.5 (m, 5H, PhenylPy), 8.0 (br, 1H, NHNH2 Py); 13C
C20H19N5: C, 72.93; H, 5.81; N, 21.26. Found: C, 73.12; H, 5.78; N, 21.10.
NMR (50 MHz, CDCl3) d 14.6 (3-CH3 Py), 118.8 (4-CPy), 125.2 (2-CPh
,
A thorough description of the 4-nitrobipyrazole (6a, 6b, 6c, and 6e)
and 4-aminobipyrazole (2a, 2b, 2c, and 2e) synthesis can be found in
the Supplementary Material.
6-CPh), 128.7 (4-CPh), 129.3 (3-CPh, 5-CPh, 140.0 (1-CPh), 146.6 (5-CPy),
148.7 (3-CPy); IR (KBr) cmꢂ1: 3348, 3331, 3233, and 3198 ( N–H), 3060
( C–Harom), 2995 and 2939 ( C–Haliph), 1649 and 1594 (d N–H), 1535
(d NO); MS (m/z): 233 (4%, M+), 215 (43%), 170 (13%), 129 (53%), 104
(16%), 77 (100%); Anal. Calc. for C10H11N5O2: C, 51.50; H, 4.75; N, 30.03.
Found: C, 51.67; H, 4.58; N, 29.99.
Preparation of 1-(3-Methyl-5-(3-methyl-5-phenyl-1H-
pyrazol-1-yl)-1-phenyl-1H-pyrazol-4-yl)-3-((R)-1-
(naphthalen-2-yl)ethyl)urea (7a,b)
Preparation of 3-Methyl-1-(3-methyl-4-nitro-1-phenyl-1H-
pyrazol-5-yl)-5-phenyl-1H-pyrazole (6d)
A mixture of 3-methyl-5-(3-methyl-5-phenyl-1H-pyrazol-1-yl)-1-phenyl-
1H-pyrazol-4-amine (2d) (3 mmol), (R)-(ꢂ)-ethyl-1-(1-naphthyl)-isocya-
nate (0.59 g, 3 mmol), and triethylamine (0.91 g, 9 mmol) dissolved in
5 ml of tetrahydrofuran (THF) was stirred at room temperature on N2 at-
mosphere for 15 min. The THF was removed under reduced pressure,
and the reaction mixture was poured in water and the precipitate
collected by filtration and dried to give 96% of urea derivative 7a,b
as a brown solid, mp 139–140 ꢁC. 1H NMR (300 MHz, CDCl3) d 1.3
(dd, 3H, J = 5.1 Hz, CH-CH3 [a]), 1.6 (dd, 3H, J = 5.0 Hz, CH-CH3 [b]), 2.1
(s, 6H, 3-CH3-Py [a + b]), 2.2 (s, 3H, 3-CH3-Py [a]), 2.4 (s, 3H, 3-CH3-Py
[b]), 3.7 (q, 1H, J= 5.1Hz, CH-CH3 [b]), 4.1 (q, 1H, J=5.1Hz, CH-CH3 [b]),
5.7 (s, 1H, 4-H-Py [a]), 5.8 (s, 1H, 4-H-Py [b]), 6.1 (br, 1H, NH2-Py [a]), 6.2
(br, 2H, NH2-Py [b] + NHCO [a]), 6.7 (br, 1H, NHCO [b]), 7.1 (m, 17H,
200-H-[5-Ph]–600-H-[5-Ph], 200-H-[1-Ph]–600-H-[1-Ph], 1-H-Naph–7-H-Naph [a]),
7.3 (m, 17H, 200-H-[5-Ph]–600-H-[5-Ph], 200-H-[1-Ph]–600-H-[1-Ph], 1-H-Naph–7-
H-Naph [b]); 13C NMR (75 MHz, CDCl3) d 12.0 and 12.5 (3-CH3-Py), 13.5
and 13.7 (3-CH3-Py), 24.1 and 25.0 (CHCH3), 47.7 and 49.6 (CHCH3), 107.3
and 107.5 (400-C-Py), 109.5 109.0 (4-C-Py), 124.0 124.3 (8-Naph), 124.1 124.5
(200-C-[1-Ph]–600-C-[1-Ph]), 124.4 124.7 (7-Naph), 125.1 125.5 (3-Naph), 125.3
125.8 (4-Naph), 126.2 126.7 (5-Naph), 126.5 126.9 (200-C-[5-Ph]–600-C-[5-Ph]),
126.9 127.1 (1-Naph), 127.6 127.8 (6-Naph), 128.1 128.5 (400-C-[5-Ph]), 128.7
129.0 (5-C-Py), 129.1 129.4 (300-C-[1-Ph]–500-C-[1-Ph]), 129.9 130.2 (300-C-[5-
Ph]–500-C-[5-Ph]), 130.5 130.7 (4-C-[1-Ph]), 131.5 131.9 (100-C-[5-Ph]), 135.0
135.3 (100-C-[1-Ph]), 139.6 140.2 (2-Naph), 142.6 142.9 (5-C-Py), 145.6 145.7
A mixture of hydrazine derivative 4 (1.0 g, 4.3 mmol) and benzoylace-
tone (5d) (4.3 mmol) in ethanol (10 ml) containing hydrochloric acid
(1.0 ml) was heated at reflux for 1 h. The reaction mixture was poured
onto cold water, producing a precipitate that was filtered and air-dried.
The precipitate was crystallized from 50% aq. ethanol, yielding the desired
bipyrazole derivative 6d in 90% yield as pink crystals, mp. 113–114 ꢁC; 1H
NMR (200 MHz, CDCl3) d 2.4 (s, 3H, 3-CH3 Py), 2.6 (s, 3H, 3-CH3 Py), 6.3
(s, 1H, 4-HPy), 6.8 (d, 2H, J = 8 Hz, 200-H5Ph, 600-H5Ph), 6.9 (d, 2H, J = 8 Hz,
200-H1Ph, 600-H1Ph), 7.3 (m, 6H, 300-H5Ph, 400-H5Ph, 500-H5Ph, 300-H1Ph, 400-
H1Ph, 500-H1-Ph). 13C NMR (50 MHz, CDCl3) d 14.0 (3-CH3 Py), 14.6
(3-CH3 Py), 107.9 (4-CPy), 123.5 (200-C1Ph , 600-C1Ph), 126.2 (4-CPy), 127.4
(200-C[5Ph, 600-C5Ph), 128.7 (300-C1Ph, 500-C1Ph), 129.0 (400-C1Ph), 129.1 (400-C5
Ph), 129.3 (300-C5Ph–500-C5Ph), 129.6 (100-C1Ph), 135.4 (5-CPy), 137.0 (100-C5
Ph), 146.8 (3-CPy), 147.8 (5-CPy), 153.1 (3-CPy). IR (KBr) cm-1: 3060 and
3003 ( C-Harom); 2962 and 2923 ( C-Haliph); 1603 and 1586 (d N-H); 1599
( CC); 1435 and 1364 (d NO). Anal. Calc. for C20H17N5O2: C, 66.84; H,
4.77; N, 19.49. Found: C, 67.03; H, 4.88; N, 19.55.
Preparation of 3-Methyl-5-(3-methyl-5-phenyl-1H-
pyrazol-1-yl)-1-phenyl-1H-pyrazol-4-amine (2d)
A mixture of nitro-bipyrazole derivative 6d (3.7 mmol), iron powder
(0.01 g, 20.8 mmol), and NH4Cl (0.1 g, 2.2 mmol) in 7.3 ml de EtOH:H2O
Chirality DOI 10.1002/chir

ATROPISOMERIC RELATIONSHIPS OF AN ANTI-INFLAMMATORY N-PHENYL-BIPYRAZOLE DERIVATIVE
(3-C-Py), 148.3 148.4 (3-C-Py); 151.6 152.0 (CO). Anal. Calc. for C33H30N6O:
C, 75.26; H, 5.74; N, 15.96. Found: C, 75.49; H, 5.72; N, 16.03.
TABLE 2. Crystal data and structure refinement for atropiso-
meric bipyrazole derivative 2d
High-Performance Liquid Chromatography Analysis
Empirical formula
C20H19N5
A Lachrom high-performance liquid chromatography (HPLC) system
Merck equipped with a model D7000 interface, an L-7100 pump, an
L-7450A diode array detector (DAD), and an L-7612 solvent degasser was
used. The injections were performed manually with a Rheodyne injector
(IDEX Health & Science, Oak Harbor, WA, USA) valve equipped with a
20-ml sample loop. Data were analyzed using Multi-Hauser Mixed Standard
software supported on a Compaq Pentium II 250MHz computer. A
Lichosorb (N. 738342) RP-18 column (250mmꢀ 4 mmꢀ 5 mm) was coupled
to a Lichrocart 25-4 HPLC guard column cartridge. The chromatographic
analyses were run with acetonitrile and water (adjusted to pH 3 with
trifluoroacetic acid (TFA) 0.1%) gradients. The sample elution was carried
out using a liner gradient of acetonitrile/water (taken to pH 3 with 0.1% of
trifluoroacetic acid ) from 20:80 to 80:20 over 20min, and then to 100% ace-
tonitrile over 1 min, followed by isocratic elution with acetonitrile for 5 min.
The mobile phase was returned to its initial proportion and the column was
equilibrated for 5 min. The analysis time was about 15min, and the flow rate
was constant at 1.0ml/min. UV detection was performed using DAD in the
range 210–350 nm to resolve most of the constituents. Integration para-
meters were used from 5 to 30min to avoid interference of the solvents.
For chiral HPLC analysis, we have used a Regis (S,S) Whelk-O 1, 5 mm,
100 Å (25 cm ꢀ 4.6 mm i.d.) column, and the samples were eluted with a
mixture of n-hexane/ethyl acetate (60:40). UV detection was performed
at 250 nm.
Formula weight
Temperature
Wavelength
Crystal system
Space group
Unit cell dimensions
329.40
120(2) K
0.71073 Å
Monoclinic
P21/c
a = 9.0950(4) Å, b = 10.4050(5) Å
c = 18.8545(9), b = 99.485(3)
1759.9(1) Å3
Volume
Z
Density (calculated)
Absorption coefficient
Crystal size
Theta range for data collection
Reflections collected
Independent reflections
Completeness to θ = 25.00ꢁ
Refinement method
Data/restraints/parameters
Goodness-of-fit on F2
Final R indices [I>2s(I)]
R indices (all data)
Extinction coefficient
Largest diff. peak and hole
4
1.243 Mg/m3
0.077 mmꢂ1
0.20 ꢀ 0.08 ꢀ 0.05 mm3
2.94ꢁ to 25.00ꢁ
10 221
3086 [R(int) = 0.0482]
99.4%
Full-matrix least-squares on F2
3086/0/235
1.028
R1 = 0.0498, wR2 = 0.1291
R1 = 0.0620, wR2 = 0.1402
0.046(7)
0.345 and ꢂ0.278 e.Åꢂ3
The solvents used were of HPLC grade purchased from Tedia
(Tedia Brazil, Rio de Janeiro, RJ, Brazil). Solvents were filtered
through a Millipore filter (0.45 mm) and degassed in an ultrasonic
bath (Thornton model T28220) for 10 min before use.
RESULTS AND DISCUSSION
Chemistry
X-Ray Experiment
The planned synthetic route to achieve the new derivative
2d and other analogs exploited as raw material 5-chloro-3-
methyl-4-nitro-1-phenylpyrazole 320 previously used in our
research group in the synthesis of bioactive antiplatelet21
and anticholinesterase22 agents (Scheme 1). This easily
accessible compound was used to obtain the corresponding
hydrazine derivative 4, in 68% yield, by nucleophilic displace-
ment of the chlorine atom at C-5 of 3 with hydrazine hydrate.
The pyrazolyl-hydrazine derivative 4 was subsequently used
in the regioselective construction of the second pyrazole ring
A well-shaped single crystal was chosen for the X-ray experiment. The
measurement was made at 120 K on an Enraf–Nonius Kappa-CCD diffrac-
tometer (Bruker AXS, Inc., Madison, WI, USA) with graphite monochro-
mated Mo Ka. Data were collected up to 50ꢁ in 2θ, with a redundancy of
4. The final unit cell parameters were based on all reflections. The temper-
ature was controlled using an Oxford Cryosystem low temperature device.
Data collection was made using the COLLECT software;11 integration and
scaling of the reflections were performed with the HKL Denzo–Scalepack
software system.12 Absorption corrections were carried out using the
multiscan method.13 The structures were solved with SHELXS-97.14 The
model was refined using SHELXL-97.15 The H atoms of the phenyl and
methyl groups were positioned stereochemically and were refined with
fixed individual displacement parameters [Uiso(H) = 1.2Ueq(Caromatic) or
1.5Ueq(Cmethoxy)] using the SHELXL riding model with C–H length
ranging from 0.93 to 0.98 Å. The hydrogen bond to N(1) atom (Fig. 6)
was found in successive difference Fourier maps. Its fractional coordinates
and isotropic thermal displacement were allowed to vary during the refine-
ment. WINGX software was used to analyze and prepare the data for
publication.16 Crystal data, data collection procedures, structure determi-
nation methods, and refinement results are summarized in Table 2.
Crystallographic data for the structural analysis for the complexes
discussed here have been deposited at the Cambridge Crystallographic
Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK, and are available
on request, quoting the deposition number CCDC 817208.
Computational Methods
In this work, semi-empirical optimizations were carried out using the
AM117 method with the Spartan for Linux ’08 software package.18 AM1
results were used as input for the ab initio molecular orbital calculations,
which were carried out using Spartan ’08. Restricted Hartree–Fock
calculations with the split-valence 6-31G* basis set, which includes a set
of d-type polarization functions on all nonhydrogen atoms, were used.19
Single point energy calculations at HF/6-31G* level were used to evaluate
the order of stability of atropisomers. Conformational analysis of com-
pound 1a has been undertaken in Spartan ’082 using the dihedral defined
by C1–C2–N3–N4 (Fig. 7), which has been rotated systematically by 10ꢁ.
Scheme 1. Synthetic route used for the preparation of bipyrazole
derivative 2a–e.
Chirality DOI 10.1002/chir

VELOSO ET AL.
present at C-5 of 2a–e, through its condensation with the
singlet signals in d 2.39 and 2.29 ppm, respectively (Fig. 2A).
These results are in agreement with the hypothesis that 2d
exists as a mixture of enantiomers that are able to form
diastereomeric complexes with the chiral lanthanide salt.
Moreover, it can be observed that hydrogens of C-3 attached
methyl group showed a major difference in the chemical shift
(Table 1), after the subsequent increase of the molar concen-
appropriate 1,3-dicarbonyl compound (5), furnishing, respec-
tively, the desired 4-nitro-1,5-bipyrazole derivative 6a–e, in
yields ranging from 80% to 95%. Compounds 6a–e presented
structural patterns in agreement with what has been previ-
ously described.20 Finally, the introduction of the pharma-
cophoric amino subunit at C-4 of the central heteroaromatic
ring of the target compound 2a–e was achieved in 93–95%
yield by using activated iron powder in aqueous ethanol
at reflux to reduce the nitro group of the corresponding
derivative 6a–e.23
1
tration of the H-NMR shift salt, because of a great stereo-
spatial influence in the rotation of sigma bond between the
two pyrazole rings, confirming the existence of the atropi-
somers of 2d.
In addition, to confirm the NMR results previously
evidenced, we performed an HPLC analysis of the atropiso-
meric mixture of 2d using a chiral stationary phase supported
in an analytical (S,S)-Whelk-O 1 column,25 using a mixture of
n-hexane/AcOEt (60:40) as eluent. Under those conditions,
we were able to detect two UV absorptions at 250 nm with
retention time of 2.96 and 6.80min, corresponding to the
enantiomeric pair of atropisomers of 2d (Fig. 3). Despite that
we have separated the atropisomers of 2d by using this
analytical procedure, under the experimental conditions
developed, we were not able to obtain sufficient amount of the
pure atropisomers to perform the determination of their biolog-
ical activity, even after several consecutive runs. The obtained
samples contained at least 5% of each other atropisomer,
probably because of their partial configurational instability.
Determination of the Atropisomerism Using Nuclear
Magnetic Resonance and High-Performance Liquid
Chromatography as Tools
The characterization of the atropisomerism of N-phenylbi-
pyrazole derivative 2d began with the analysis of its
1H-NMR chemical shifts using different concentrations
(0.005 to 0.1 M) of ytterbium tris[3-(heptafluoropropylhydrox-
ymethylene)-(+)-camphorate] as chiral lanthanide salt,16,24
which can be coordinated with the amino group at C-4, pro-
moting different stereoelectronic effects in functional groups
of the atropisomers of 2d. In fact, we were able to identify a
clear duplication of the signals referent to the hydrogens of
the two methyl groups at C-3 and C-3 of 2d, which appeared,
1
in the H-NMR spectrum without addition of (+)-Yb(hfc)3, as
Fig. 2. 1H-NMR spectra of the bipyrazole derivative 2d with variant concentrations of Yb(thc)3. (A) No addiction; (B) addition of Yb(thc)3 (0.005 M); (C) addition
of Yb(thc)3 (0.010 M); (D) addition of Yb(thc)3 (0.025 M); (E) addition of Yb(thc)3 (0.050 M); (F) addition of Yb(thc)3 (0.1 M) (Table 1).
Chirality DOI 10.1002/chir

ATROPISOMERIC RELATIONSHIPS OF AN ANTI-INFLAMMATORY N-PHENYL-BIPYRAZOLE DERIVATIVE
TABLE 1. Chemical shifts of the methyl hydrogens of the derivative 2d in the absence or in the presence of (+)-Yb(thc)3
CH3-3-C
CH3-3-C
Concentration
Yb(thc)3 (M)1
Entry
dA (ppm)
dB (ppm)
d2
dA (ppm)
dB (ppm)
d
1
2
3
4
5
6
0
2.39
0
2.29
0
0.005
0.010
0.025
0.050
0.100
2.83
3.09
3.32
3.65
3.82
2.97
3.40
3.76
4.32
4.60
0.14
0.40
0.44
0.67
0.78
3.13
3.64
3.99
4.52
4.78
3.17
3.64
4.07
4.71
5.02
0.04
0.00
0.08
0.19
0.24
1All experiments were made at room temperature in a Brucker DRX200 spectrometer operated at 200 MHz.
2d = dB ꢂ dA.
Fig. 3. Chiral HPLC analysis of atropisomeric mixture of N-phenylbipyrazole
derivative 2d.
Scheme 2. Synthesis of diastereomeric bipyrazole urea derivatives 7a,b.
In spite of our previous results having strongly indicated
the presence of a mixture of the atropisomers of the
N-phenyl-bipyrazole derivative 2d, we decided to promote
its derivatization to the corresponding mixture of diastereo-
meric ureidic derivatives 7a,b through the reaction with
(R)-(ꢂ)-ethyl-1-(1-naphthyl)-isocyanate and triethylamine in
THF (Scheme 2). The diastereomeric mixture of the
R,M and R,P urea derivatives 7a and 7b was obtained in
96% yield and characterized by the analysis of its 1H-
and 13C-NMR spectra (see Materials and methods section),
which presented duplicated signals relative to the hydrogen
at the chiral center of 7a,b, in agreement with the presence
of two diastereomers. Moreover, reversed-phase HPLC
analysis of the mixture 7a,b confirmed this evidence,
through the presence of two peaks with retention times of
9.81 and 10.72 min (Fig. 4), after elution with a mixture of
acetonitrile/water adjusted to pH 3.
X-Ray Analysis
The crystal structure of 2d was determined in space group
P21/c (Table 2). This is an important result taking into
account that the initial motivation to perform the X-ray diffrac-
tion study arose from the possible atropisomerism of 2d, as
identified by NMR and HPLC techniques. Considering the
restrictions on the formation of chiral and achiral crystal
structures from chiral or achiral molecules,26,27 an enantio-
merically pure chiral molecule is not allowed to crystallize
in a centrosymmetric space group. On the other hand, race-
mates of chiral molecule are permitted to crystallize either
in achiral or chiral structures. Therefore, because 2d is a
chiral molecule crystallized in a centrosymmetric space
group (P21/c), it is a crystalline racemate in which the two
atropisomers (P/M: 50/50) are present in equal amounts in
Chirality DOI 10.1002/chir

VELOSO ET AL.
Fig. 6. ORTEP-3 view showing the intermolecular contact of a pair of atro-
Fig. 4. Reversed-phase HPLC analysis of the diastereomeric mixture of
N-phenylbipyrazole urea derivatives 7a,b.
pisomers of 2d thought hydrogen bonds. The displacement ellipsoids are
shown at the 50% probability level. Hydrogen bonds are indicated by double
dashed lines. Symmetry codes: i = -x + 2, -y + 1, -z + 1.
a well-defined arrangement within the P21/c lattice confirm-
ing the NMR and HPLC results. Figure 5 is an ORTEP-328
representation of the atropisomers that make up the crystallo-
graphic asymmetric, which was arbitrarily chosen to be
the aS-atropisomer. It is important to note that aS- and
aR-atropisomers form dimers related by the inversion
symmetry and linked by two intermolecular symmetrically
dependent hydrogen bonds (Fig. 6). These hydrogen bonds
take place between the pyrazole nitrogen atom and the
terminal amino group (N(1)–H(1a). . .N(5)i, symmetry code
as in Figure 6. Considering the aS-atropisomer, the intramo-
lecular geometry consists of four non-coplanar rings: two phe-
nyl rings and two pyrazole ones. All of them are individually
planar, including all the first neighbor atoms linked to them.
Considering the non-H atoms, the largest deviations from
the least-squares plane through the rings are 0.014(2), 0.002
(2), -0.006(1), and 0.004(2) Å, for A, B, C, and D, respectively
(Fig. 5). The main intramolecular geometric parameters are
given in Table S2 (see Supplementary Material). The two phe-
nyl rings show the expected geometry with bond lengths and
bond angles equivalents. On the other hand, the rings B and
C present significant differences, mainly to N—N bonds: The
difference between N(2)—N(3) and N(4)—N(5) is approxi-
mately 0.16 Å. These differences can be explained in terms
of the electronic effects promoted by the amino group
attached to pyrazole ring B. It is important to analyze the
intramolecular geometry in terms of the dihedral angles
between the rings. The angles formed between the least-
square planes through A and B, B and C, and C and D rings
are 34.2(1)ꢁ, 74.1(1)ꢁ, and 41.3(1)ꢁ, respectively. Considering
the aR-atropisomer of 2d in the racemic crystal, these values
will be, as expected, -34.2(1)ꢁ, -74.1(1)ꢁ, and ꢂ41.3(1)ꢁ.
Analyzing the intermolecular structure of 2d, the dimers
formed by pair of atropisomers are stacked along the direc-
tion through intermolecular nonclassic hydrogen bonds of
the type H . . . p-aryl involving the amine group hydrogen
(donor) and the p acceptor of the D ring (Figure S1, see
Supplementary Material). That means the H1b . . . p-aryl(ring D)
hydrogen bonds contact the dimers through a translational
symmetry along a axis. The final hydrogen bond geometry is
shown in Table S3 (see Supplementary Material). In order to
obtain the most realistic intermolecular geometry, the posi-
tional parameters of the two H atoms connected to the N atoms
were not constrained during the refinements performed here.
Our experimental data show that the dihedral angles between
the NH2 groups and the pyrazole ring plane are 59.82ꢁ and
-10.64ꢁ for the H(1a)–N(1)–C(2)–C(3) and H(1b)–N(1)–C(2)–
C(1) dihedral angles, respectively. Therefore, it is clear that
the ideal planar geometry of the amino group is extremely
affected by the packing conditions. Our experimental data show
that the dihedral angles between the NH2 groups and the pyra-
zole ring plane are 59.82 and -10.64ꢁ for the H(1a)–N(1)–C(2)–
C(3) and H(1b)–(N1)–(C2)–(C1) dihedral angles, respectively.
Molecular Modeling
This study was undertaken to investigate rotational barrier
energies around the dihedral angle (θ) defined in Figure 7
using HF/6-31G* level for single point energy calculations.
The relative energies using θ = 180º as a starting point
(kcal/mol) for this calculation have been recorded, and the
analysis of the plot of relative energy versus dihedral angle
(Fig. 7) allowed us to identify two atropisomers of compound
2d, in which the dihedral angle is approximately ꢃ74ꢁ
(Fig. 8). Both are separated by an energy barrier of approxi-
mately 41.11 kcal/mol (Fig. 7). Therefore, the calculations
undertaken in this work support the existence of a high
energy rotational barrier around the studied dihedral
angle (θ), corroborating the existence of atropisomeric spe-
cies of bipyrazole derivative 2d. It is important to emphasize
that the angles formed between the least-square planes
through B and C rings in the X-ray analysis of the atropiso-
meric pair of 2d, that is, 74.1(1)o and ꢂ74.1(1)o (Fig. 5), are
Fig. 5. ORTEP-3 view of 2d (aS-atropisomer), showing the atom and ring
labeling. The displacement ellipsoids are shown at the 50% probability level.
Chirality DOI 10.1002/chir

ATROPISOMERIC RELATIONSHIPS OF AN ANTI-INFLAMMATORY N-PHENYL-BIPYRAZOLE DERIVATIVE
Fig. 7. Plot of relative energies versus dihedral angle (θ) of bipyrazole derivative 2d.
3. Rodrigues CR, Veloso MP, Fraga CAM, Miranda ALP, Barreiro EJ. Selec-
tive PGHS-2 inhibitors: a rational approach for treatment of the inflamma-
tion. Curr Med Chem 2002;9:849–867.
4. Penning TD, Talley JJ, Bertneshaw SR, Carten JS, Collins PW, Docter S,
Graneto MJ, Lee LF, Malecha JW, Miyashiro JM, Rogers RS, Rogier DJ,
Yu SS, Anderson GD, Burton EG, Cogburn JN, Gregory SA, Koboldt
CM, Perkins WE, Seibert K, Veenhuizen AW, Zhang YY, Isakson PC.
Synthesis and biological evaluation of the 1,5-diarylpyrazole class of
cyclooxygenase-2 inhibitors: identification of 4-[5-(4-methylphenyl)-3-
(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide (SC-58635, Cele-
coxib). J Med Chem 1997;40:1347–1365.
5. Wu K. Biochemical pharmacology of nonsteroidal anti-inflammatory
drugs. Biochem Pharmacol 1998;55:543–547.
6. Wallace JL, Chin BC. New generation NSAIDs. The benefits without the
risks? Drugs Today 1997;33:371–378.
Fig. 8. Representation of more stable conformations of bipyrazole
atropisomers 2d.
7. Davies NM, Jamali F. COX-2 inhibitors and cardiac toxicity: getting to the
heart of the matter. J Pharm Pharmaceut Sci 2004;7:332–336.
8. Veloso MP. PhD Thesis, Chemistry Institute, Federal University of Rio de
Janeiro, Rio de Janeiro, Brazil, 2000.
in a perfect agreement with the dihedral angles obtained by
molecular modeling studies.
9. Santos AR, Pinheiro AC, Sodero ACR, Cunha AS, Padilha MC, Sousa PM,
Fontes SP, Veloso MP, Fraga CAM. Atropisomerism: the effect of the
axial chirality in bioactive compounds. Quim Nova 2007;30:125–135.
10. LaPlante SR, Edwards PJ, Fader LD, Jakalian A, Hucke O. Revealing atropi-
somer axial chirality in drug discovery. ChemMedChem 2011;6:505–513.
11. Enraf-Nonius. COLLECT. Nonius BV, Delft, The Netherlands; 1997–2000.
12. Otwinowski Z, Minor W. Processing of X-ray diffraction data collected in
oscillation mode. In: Carter Jr. CW, Sweet RM, editors. Methods in enzy-
mology. New York: Academic Press; 1997. p. 307–326.
CONCLUSIONS
We were able to confirm, by using different analytical
techniques and molecular modeling tools, that the ortho
bis-functionalized bipyrazole 2d exists as a mixture of aR,
aS-atropisomers. These results provide useful information to
understand the pharmacological profile of this derivative
and of several other 4-aminobipyrazole analogs.
13. Blessing RH. An empirical correction for absorption anisotropy, Acta
ACKNOWLEDGMENTS
Cryst 1995;A51:33–38.
Special thanks are due to CAPES (BR.), CNPq (BR.), FINEP
(BR.), and FAPERJ (BR.) for financial support and fellowships.
14. Sheldrick GM. SHELXL-97. software for crystal structures analysis.
Göttingen, Germany: Univ. of Göttingen; 1997.
15. Sheldrick GM. SHELXS-97. software for crystal structure resolution.
Göttingen, Germany: University of Göttingen; 1997.
LITERATURE CITED
1. Smith WL, Meade EA, DeWitt DL. Pharmacology of prostaglandin endo-
peroxide synthase isozymes-1 and -2. Ann N Y Acad Sci 1994;714:136–142.
16. Parker D. NMR determination of enantiomeric purity. Chem Rev
1991;91:1441–1457.
2. Smith WL, Lecomte M, Laneuville O, Breuer DK, DeWitt DL. Differential
inhibition of human prostaglandin endoperoxide H synthases-1 and -2 by
aspirin and other nonsteroidal anti-inflammatory drugs. Eur J Med Chem
1995;30(Suppl):417–427.
17. Dewar MJS, Zoebisch EG, Healy EF, Stewart JJP. Development and
use of quantum mechanical molecular models. 76. AM1: a new gen-
eral purpose quantum mechanical molecular model. J Am Chem Soc
1985;107:3902–3909.
Chirality DOI 10.1002/chir

VELOSO ET AL.
18. Spartan ’08; Wavefunction, Inc. 18401 Von Karman Avenue, Suite 370.
23. Lages AS, Silva KCM, Miranda ALP, Fraga CAM, Barreiro EJ. Synthesis
and pharmacological evaluation of new flosulide analogues, synthesized
from natural safrole. Bioorg Med Chem Lett 1998;8:183–188.
Irvine, California 92612, USA.
19. Hariharan PC, Pople JA. The influence of polarization functions
on molecular orbital hydrogenation energies. Theor Chim Acta
1973;28:213–222.
24. Rothchild R. NMR methods for determination of enantiomeric excess.
Enantiomer 2000;5:457–471.
25. Wolf C, Pranatharthiharan L, Volpe EC. A convenient method for the
determination of the absolute configuration of chiral amines. J Org Chem
2003;68:3287–3290.
26. Cianci M, Helliwell JR, Helliwell M, Kaucic V, Logar NZ, Mali G,
Tusar NN. Anomalous scattering in structural chemistry and biology.
Crystallogr Rev 2005;11:245–335.
20. Khan MA, Freitas ACC. Hetarylpyrazoles. IV. Synthesis and reactions of
1,5-bipyrazoles. J Heterocyclic Chem 1983;20:277–279.
21. Silveira AFB, Paulo LG, Miranda ALP, Rocha SO, Freitas ACC, Barreiro
EJ. New pyrazolylhydrazone derivatives as inhibitors of platelet aggrega-
tion. J Pharm Pharmacol 1993;45:646–649.
22. Barreiro EJ, Camara CA, Verli H, Brazil-Más L, Castro NG, Cintra WM,
Aracava Y, Rodrigues CR, Fraga CAM. Design, synthesis, and pharmaco-
logical profile of novel fused pyrazolo[4,3-d]pyridine and pyrazolo[3,4-b]
[1,8] naphthyridine isosteres: a new class of potent and selective acetyl-
cholinesterase inhibitors. J Med Chem 2003;46:1144–1152.
27. Flack HD. Chiral and achiral crystal structures. Helv Chim Acta
2003;86:905–921.
28. Farrugia LJ. ORTEP-3 for Windows—a version of ORTEP-III with a graph-
ical user interface (GUI). J Appl Cryst 1997;30:565.
Chirality DOI 10.1002/chir