Crystal structure determination of N- and O-alkylated tautomers of 1-(2-pyridinyl)-5-hydroxypyrazole

Article abstract of DOI:10.1016/j.molstruc.2020.128272

The crystal structure determination of tautomeric products produced by the alkylation of 1-(2-pyridinyl)-3-phenyl-4-propyl-1H-5-hydroxypyrazole 2 was investigated. Treatment of 2 with isopropyloxycarbonyloxymethyl iodide and potassium carbonate under phas

Full text of DOI:10.1016/j.molstruc.2020.128272

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Crystal structure determination of N- and O-alkylated tautomers of 1-(2-pyridinyl)-5-
hydroxypyrazole
Venkata S. Sadu, Yoon Mi Choi, Koteswara R. Kamma, Chong-Hyeak Kim, Yun Soo
Bae, Kee-In Lee
PII:
S0022-2860(20)30597-4
DOI:
https://doi.org/10.1016/j.molstruc.2020.128272
MOLSTR 128272
Reference:
To appear in: Journal of Molecular Structure
Received Date: 28 January 2020
Revised Date: 13 April 2020
Accepted Date: 14 April 2020
Please cite this article as: V.S. Sadu, Y.M. Choi, K.R. Kamma, C.-H. Kim, Y.S. Bae, K.-I. Lee, Crystal
structure determination of N- and O-alkylated tautomers of 1-(2-pyridinyl)-5-hydroxypyrazole, Journal of
Molecular Structure (2020), doi: https://doi.org/10.1016/j.molstruc.2020.128272.
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Crystal structure determination of N- and O-alkylated tautomers of 1-(2-pyridinyl)-5-
hydroxypyrazole
Venkata S. Sadua1, Yoon Mi Choia2, Koteswara R. Kammab, Chong-Hyeak Kima2, Yun Soo
Baec,* and Kee-In Leea1,b*
aGreen Chemistry Division1and Chemical Analysis Center2, Korea Research Institute of
Chemical Technology, Daejon 34144, Korea
bMajor of Green Chemistry and Environmental Biotechnology, University of Science &
Technology, Daejon 34113, Korea
cDepartment of Life Science, Ewha Womans University, Seoul 03760, Korea
Abstract
The crystal structure determination of tautomeric products produced by the alkylation of 1-
(2-pyridinyl)-3-phenyl-4-propyl-1H-5-hydroxypyrazole 2 was investigated. Treatment of 2
with isopropyloxycarbonyloxymethyl iodide and potassium carbonate under phase-transfer
conditions affords two major products out of three possible O-, N-, and C-alkylated
tautomers. The tautomeric structures of O-alkylated 3a and N-alkylated 3c were elucidated
by means of NMR spectroscopic investigations and confirmed by single crystal X-ray
analysis. The single crystal structures of alkylated compounds provide clear difference
between the tautomeric pyrazole and pyrazolone ring systems in terms of bond lengths and
torsional angles, moreover, conformational changes between two tautomers.
Keywords: 1-(2-Pyridinyl)-5-hydroxypyrazole, N-Alkylation, O-Alkylation, Tautomerism,
13C NMR, Crystal structure.

1. Introduction
Pyrazoles are interesting structural units which are frequently found in natural products to
pharmaceuticals. Such heterocyclic frameworks serve as important core structures
possessing a broad spectrum of biological activities. The representative examples
of drugs containing the moieties include celecoxib, rimonabant, metamizole, antipyrene, and
sildenafil [1-3]. Moreover, there has been considerable interest in their capability to
prototropic tautomerism between 5-pyrazolones and 5-hydroxypyrazoles [4-6]. We thus
focused on peripheral modifications of pyrazole scaffold through exploiting the pyrazolone-
pyrazole tautomerization [7-9].
From our recent efforts to identify lead compounds targeting NADPH oxidase (Nox)
enzymes for the modulation of the excessive oxidase damage induced by reactive oxygen
species (ROS) production, we found that 1-(2-pyridnyl)pyrazole 1 block the RANKL-
dependent cell signaling cascade leading to reduced differentiation of osteoclast cells [10].
Further structure-activity relationships revealed enhanced physicochemical properties by the
introduction of an alkyl group onto the parent molecule (e.g., 2, Figure 1). We next
investigated a prodrug 3a likely to optimize the physicochemical properties and to improve
pharmacological and toxicological profile.
Figure 1. Peripheral modification of 1-(2-pyridinyl)pyrazole scaffold.

2. Experimental
2-1. General
All solvents and reagents were purchased from commercial sources and used as received
without further purification, unless otherwise stated. Reactions were monitored by thin layer
chromatography carried out on S-2 0.25 mm E. Merck silica gel plates (60F-254) using UV
light as the visualizing agent and an acidic mixture of anisaldehyde or a ninhydrin solution in
ethanol and heat as developing agents. E. Merck silica gel (60, particle size 0.040-0.063 mm)
was used for flash column chromatography. All yields were calculated from isolated products.
Melting points were recorded on Electrothermal IA9200 apparatus and are uncorrected. All
1
NMR spectra were recorded on either Bruker AV-500 or AV-300 instruments. H and 13C
NMR spectra were referenced internally to the residual undeuterated chloroform (δH = 7.26
ppm and δC = 77.0 ppm). The NMR data were analyzed using MNova 10.0 processing
software (Mestrelab Research). The following abbreviations are used to designate
multiplicities: s = singlet, d = doublet, t = triplet, td = triplet of doublets, sept = septet, m =
multiplet, br s = broad singlet, ddd = doublet of doublets of doublets. Chemical shifts are
reported in ppm and coupling constants are in Hertz (Hz). High resolution mass spectra using
Electronic Ionization (HRMS-EI) were recorded on Joel JMS-700 mass spectrometer.
2-2. Crystallographic data
Single-crystal X-ray diffractions were measured on a Bruker APEX-II CCD diffractometer
equipped with a monochromatic Mo-Kα radiation (λ = 0.71073 Å). The data were collected
at low temperature of 100K by the φ–ω scan method. The collected data were integrated by
using Bruker-SAINT software and an absorption correction wasn’t applied. The structure
were solved and refined through the least-squares method with SHELXT and SHELXL

program, respectively. All the non-hydrogen atoms were refined anisotropically and
hydrogen atoms were placed in calculated positions. Table 1 presented the crystallographic
data and structural refinements. Structural information was deposited at the Cambridge
Crystallographic Data Center (CCDC reference numbers are 1902060 and 1902059 for 3a
and 3c, respectively). Crystallographic data of 1-(2-pyridinyl)-3-phenyl-5-hydroxypyrazole
(1, CCDC 805262) [9] have been obtained free of charge from the Cambridge
Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif
2-3. Synthesis of 1-(2-pyridinyl)-3-phenyl-4-propyl-1H-5-hydroxypyrazole (2) [10]
In a flame-dried flask, 2-hydrazinopyridine (3.67g, 33.6 mmol) and ethyl 2-
benzoylpentanoate (8.04 g, 34.3 mmol) were mixed under neat conditions and then heated to
160 oC for 12h under argon atmosphere. Upon completion, the reaction mixture was cooled to
room temperature. The crude product was purified by silica gel column chromatography
(hexane/EtOAc = 8/2) to afford the title compound as a white solid, which then recrystallized
with hexane/Et2O to give colorless needles (8.16 g, 87%); mp: 68.6- 69.4 oC; 1H NMR (300
MHz, CDCl3): δH 12.53 (brs, 1H), 8.26-8.23 (ddd, J = 0.7, 1.6, 5.0 Hz, 1H ), 7.99 (d, J = 8.8
Hz, 1H), 7.86-7.70 (td, J = 1.8, 7.4 Hz, 1H), 7.71 (dd, J = 1.3, 7.9 Hz, 2H), 7.46-7.34 (m,
3H), 7.13-7.08 (ddd, J = 1.0, 5.2, 7.3 Hz, 1H), 2.54 (t, J = 7.8 Hz, 2H), 1.60 (septet, J = 7.3
Hz, 2H), 0.94 (t, J = 7.4 Hz, 3H); 13C NMR (125 MHz, CDCl3): δC 154.5, 154.0, 152.4,
145.2, 139.8, 134.1, 128.5, 128.1, 127.8, 119.7, 112.2. 100.2, 24.3, 23.0, 14.0; HRMS (EI)
m/z [M+H]+ calc for C17H17N3O, 279.1372; observed: 279.1371.
2-4. Synthesis of N- and O-alkylated tautomers (3a and 3c)
The title compounds were synthesized from the reaction of 1-(2-pyridinyl)-3-phenyl-4-
propyl-5-hydroxypyrazole (2, 438 mg, 1.56 mmol) and isopropyloxycarbonyloxymethyl

iodide (497 mg, 2.03 mmol) with potassium carbonate (650 mg, 4.70 mmol) in the presence
of tetrabutylammonium hydrogen sulfate (532 mg, 1.56 mmol) in dichloromethane (7.5 mL)
and water (7.5 mL) for 12 hr at ambient temperature. The organic layer was separated and the
aqueous layer was extracted with dichloromethane (2 x 5 mL). The combined organic layers
were dried over anhydrous magnesium sulfate and filtered, and then the solvents were
removed. The residue was purified by column chromatography on silica gel (hexane/ethyl
acetate
isopropyloxycarbonyloxymethyloxypyrazole
isopropyloxycarbonyloxymethyl-2-(2-pyridiyl)-4-propyl-5-phenyl-1,2-dihydro-3H-pyrazol-
=
10/1)
to
give
1-(2-pyridinyl)-3-phenyl-4-propyl-5-
(3a,
360 mg, 58%) and 1-
3-one (3c, 210 mg, 34% yield), respectively. Single crystals from these two regioisomers
suitable for X-ray diffraction were prepared by slow evaporation of a solution in ethyl acetate
at room temperature.
2-4-1.
1-(2-Pyridinyl)-3-phenyl-4-propyl-5-isopropyloxycarbonyloxymethyloxypyrazole
(3a). mp 97-98 °C; 1H NMR (500 MHz, CDCl3): δH 8.49 (d, 1H, J = 4.7 Hz), 7.91 (d, 1H, J
= 8.0 Hz), 7.83-7.80 (m, 1H), 7.75-7.73 (m, 2H), 7.46 (t, 2H, J = 7.25 Hz), 7.41-7.38 (m,
1H), 7.22-7.20 (m, 1H), 5.87 (s, 2H), 4.95-4.90 (m, 1H), 2.61 (t, 2H, J = 7.8 Hz), 1.60-1.55
(m, 2H), 1.32 (d, 6H, J = 6.2 Hz), 0.94 (t, 3H, J = 7.3 Hz) ppm; 13C NMR (125 MHz,
CDCl3): δC 153.6, 152.1, 150.9, 149.3, 148.0, 138.3, 134.0, 128.4, 128.0, 127.7, 121.3, 116.0,
110.0, 92.7, 72.8, 24.7, 23.0, 21.7, 14.1 ppm; HRMS (EI) m/z [M+H]+ calcd for C22H25N3O4,
395.1845, found 395.1844; and the crystallographic data of 3a are summarized in Table 1.
2-4-2. 1-Isopropyloxycarbonyloxymethyl-2-(2-pyridiyl)-4-propyl-5-phenyl-1,2-dihydro-3H-
pyrazol-3-one (3c).

mp 83-84 °C; 1H NMR (500 MHz, CDCl3): δH 8.51-8.49 (m, 1H), 8.19 (d, 1H, J = 8.5 Hz),
7.86-7.82 (m, 1H), 7.61-7.59 (m, 2H), 7.54-7.51 (m, 3H), 7.18-7.16 (m,1H), 5.70 (s, 2H),
4.67-4.62 (m, 1H), 2.38 (t, 2H, J = 7.5 Hz), 1.63-1.55 (m, 2H), 1.17 (d, 6H, J = 6.2 Hz), 0.88
(t, 3H, J = 7.0 Hz) ppm; 13C NMR (125 MHz, CDCl3): δC 166.4, 154.9, 153.3, 149.0, 147.9,
138.2, 130.3, 129.4, 128.9, 128.5, 120.5, 116.6, 116.1, 73.6, 72.5, 24.7, 21.9, 21.5, 13.9 ppm;
HRMS (EI) m/z [M+H]+ calcd for C22H25N3O4, 395.1845, found 395.1845; and the
crystallographic data of 3c are summarized in Table 1.
Table 1. Crystallographic data of 3a and 3c.
Compound
3a
3c
Empirical formula
Formula weight
Crystal system
Space group
C22H25N3O4
395.45
C22H25N3O4
395.45
Monoclinic
P2(1)/c
Monoclinic
P2(1)/c
Unit cell dimensions
a = 12.8907(3) Å
b = 10.7224(2) Å
c = 15.8982(3) Å
β = 112.5900(10)°
a = 10.2804(4) Å
b = 13.4480(6) Å
c = 14.9828(6) Å
β = 99.181(2)°
2028.84(7) Å3
1.295 Mg/m3
2044.85(15) Å3
1.285 Mg/m3
Volume
Density (calculated)
Reflections collected
Final R indices
5067
18751
a
b
a
b
R1 = 0.0399, wR2 = 0.0940
R1 = 0.0391, wR2 = 0.1019
[I>2sigma(I)]
R indices (all data)
R1 = 0.0454, wR2 = 0.1073
R1 = 0.0539, wR2 = 0.1048
2 2
a R1 = ||Fo| − |Fc||/Σ|Fo|. b wR2 = [Σ[w(Fo2 − Fc2)2]/Σ[w(Fo ) ]]1/2.

3. Results and Discussion
Prodrugs are chemically modified derivatives of drug-like molecules that are used to improve
the charge and lipophilicity in favor of pharmacokinetic and toxicological properties of the
parent drug [11]. Apparently, alkoxycarbonyloxymethyl prodrugs are soft alkyl derivatives of
active phenolic drugs that are used to overcome the bioavailability problems related to
stability and solubility. Indeed, the alkyloxycarbonyloxymethyl prodrugs have been exploited
as novel permeation-enhancing derivatives of phenolic, antiviral, and nucleoside phosphate
prodrugs [12-14]. During the reaction of 2 and isopropyloxycarbonyloxymethyl iodide with
potassium carbonate under phase-transfer conditions, we identified two products having
exactly the same molecular weight (Scheme 1).
Scheme 1. Preparation of O- and N-alkylated tautomers
The pyrazolone tautomerism is an everlasting issue in pyrazole chemistry and thus it has been
the subject of a considerable number of investigations. For example, 5-pyrazolones are of
particular interest due to their ability to exist in the tautomeric equilibria between the OH (A),
CH (B), and NH form (C) as illustrated in Scheme 2. Therefore the ratio of products is highly
depending on not only the tautomeric contributions but also the nature of reagents and
solvents used both in acylation [15,16] and alkylation of tautomeric pyrazolones [17-19].

Since the products share essentially the same pattern, the structures should be assigned based
1
13
on careful analysis of chemical shifts of H and C NMR favorably with NOEs and long-
range INEPT experiments [18,19], but the most convincingly with X-ray crystallography [4-
6]. Here we report the structural assignments based on X-ray crystal structure determination
since three possible O-, N-, and C-alkylated tautomers have quite similar spectroscopic
properties.
Scheme 2. Tautomeric contributions of 5-pyrazolone and possible products distribution
The 1H and 13C NMR of the crude revealed the presence of two isomeric forms in the ratio of
1.7:1. Most signals of the two products are duplicated and they have exactly the same
molecular weight. The significant difference between 3a and 3c, however, was that the
singlets of a methylene group of the carbonate resonated at 5.87 and 5.70 in 1H, and 92.7 and
13
73.6 ppm in C NMR, respectively. The downfield shifting of the methylene group clearly
shows that it is bonded to a heteroatom, and implying the presence of OH and NH forms

rather than CH form. In 13C NMR study, it has been reported that pyrazolone carbonyl of the
CH form resonates at 170 ppm in contrast to the upfield resonances of 155 ppm and 160 ppm
corresponding to OH and NH forms, respectively [20,21]. Moreover, the differentiation
between the pyrazolone carbonyl and the carbonate carbon is not justified as the latter nearly
centers at 155 ppm in all the three forms [22].
Even though the presence of pyrazolone carbonyl group of 3c resonating at 166.4 ppm
13
somewhat assures the presence of N-alkylated product, the C NMR study does not provide
crucial features to distinguish between the three tautomers since they are sets of isomeric
protonation states with the same carbon atoms. To clarify this structural ambiguity, then the
structure determination by X-ray crystallography should be investigated and the ORTEP
drawings are shown in Figure 2 and 3. It is noteworthy, since the 5-pyrazolone tautomerism
involves the relocation of proton in which single and double bonds are interconvertible, that
the single crystal structures of alkylated compounds are of great help in structural information
between the tautomeric ring systems.

Figure 2. Crystal structure of 3a. Thermal ellipsoids are shown at the 50% probability level.

Figure 3. Crystal structure of 3c. Thermal ellipsoids are shown at the 50% probability level.
X-Ray diffraction analysis revealed that 3a belongs to the monoclinic space group P2(1)/c
with a = 12.8907(3) Å, b = 10.7224(2) Å, c = 15.8982(3) Å, α = 90°, β = 112.5900(10)°, γ =
90° and V = 2028.84(7) Å3. The molecular structure of 3a comprises a near coplanar tetra-
substituted pyrazole ring with an r.m.s deviation of the fitted atoms being 0.002 Å. All bond
distances in the pyrazole ring show partial double bond character, which suggests a

delocalized π-electronic system throughout the ring. The bond lengths and bond angles in the
pyrazole ring are within the normal ranges reported in the literatures [4-6]. The bond length
of C(3)-O(1), 1.3585(14) Å is little longer than that of the parent compound. This is expected
that the C-O bond of 1 attains a partial double bond character because of the keto-enol
tautomerization. The bond distances of N(2)-C(1) and C(2)-C(3) are consistent with
the average of the length of C=N and C=C double bonds, respectively. However, the C(1)-
C(2) bond length of 1.4193(16) Å is apparently longer than a double bond. The X-ray crystal
analysis of 3a is consistent with the OH tautomer (Table 2).
The molecular structure of 3c indicated that the crystal belongs to the monoclinic space group
P2(1)/c with a = 10.2804(4) Å, b = 13.4480(6) Å, c = 14.9828(6) Å, α = 90°, β = 99.181(2)°,
γ = 90° and V = 2044.85(15) Å3, and comprises near planar tri-substituted pyrazolone ring
with an r.m.s deviation of the fitted atoms being 0.005 Å. The C(1)-C(2) bond length is
1.3513(14) Å is consistent with that of C=C double bond and the C(2)-C(3) is 1.4561(14) Å
is corresponding to C-C single bond length. The length of N(2)-C(1) is 1.4118(13) Å, and in
similar line with C-N single bond of amines. The 1.2273(12) Å of C(3)-O(1) clearly show a
C=O double character. All these three bond lengths clearly confirms the presence of the NH
tautomer (Table 2).
Table 2. Comparison of bond length (Å)
Descriptiona
N(1)-N(2)
N(2)-C(1)
C(1)-C(2)
C(2)-C(3)
1b
3a
3c
1.3782(15)
1.3261(18)
1.4130(2)
1.3560(2)
1.3687(14)
1.3348(15)
1.4193(16)
1.3673(17)
1.4159(12)
1.4118(13)
1.3513(14)
1.4561(14)

C(3)-O(1)
C(3)-N(1)
1.3384(16)
1.3750(18)
1.3585(14)
1.3791(15)
1.2273(12)
1.3992(12)
a For a comparative discussion of X-ray crystal structures, the numbering of atoms is given based on the pyrazole
skeleton (see: Fig. 2). b Data taken from CCDC 805262.
The molecules 1 and 2 exclusively exist in the enol form as seeing that the X-ray crystal
structure of 1 clearly shows that the carbonyl oxygen and pyridine nitrogen adopt an almost
syn-periplanar arrangement that is capable of accommodating intramolecular hydrogen
bonding [9, 23, 24]. The dihedral angles of 3a supporting the planarity of pyrazole ring is
given in Table 3. The dihedrals of O(1)-C(3)-C(2)-C(15) and C(9)-C(1)-C(2)-C(15) of 3a are
nearly coplanar with the angles of -4.5(2)o and 6.2(2)o, respectively, regarding to the plane
defined by the pyrazole ring. However, crystal structure of 3a does not exhibit a defined
structure within two flexible alkyl chains. The dihedral angle of C(3)-N(1)-C(4)-N(3)
suggests that 3a still adopt an almost syn-periplanar arrangement. While 3c also shows the
dihedrals of O(1)-C(3)-C(2)-C(15) and C(9)-C(1)-C(2)-C(15) are nearly coplanar to the
pyrazole ring, the dihedral of C(9)-C(1)-N(2)-C(18) is displaced out of plane by 53.12(13)o
with pyrimidalization of the nitrogen atom. The torsion angle of C(3)-N(1)-C(4)-N(3) of 3c
changes remarkably with 143.49(10)° contrary to that of 3a. This clearly suggests that the
pyridine nitrogen and the pyrazolone ketone adopt an anti-periplanar arrangement probably
due to the close proximity and the repulsion between them. The results of X-ray single-
crystal analysis is consistent with those of NMR study.
Table 3. Comparison of dihedral angle (o)
Descriptiona
1b
3a
3c
C(3)-N(1)-C(4)-N(3)
2.3(2)
-13.08(18)
143.49(10)

O(1)-C(3)-C(2)-(15)
C(9)-C(1)-C(2)-(15)
C(9)-C(1)-N(2)-(18)
-
-
-
-4.5(2)
6.2(2)
-
0.34(17)
3.94(19)
53.12(13)
a For a comparative discussion of X-ray crystal structures, the numbering of atoms is given based on the pyrazole
skeleton (see: Fig. 2 and 3). b Data taken from CCDC 805262.
In conclusion, two prodrug-type tautomers have been isolated from the reaction of 1-(2-
pyridinyl)-3-phenyl-4-propyl-1H-5-hydroxypyrazole 2 with isopropyloxycarbonyloxymethyl
iodide and potassium carbonate under phase-transfer conditions, among three possible O-, N-,
and C-alkylated products. The two tautomeric products have been identified as O-and N-
alkylated ones by NMR and mass spectroscopic analysis. However, the differentiation was
still demanding as the pyrazolone carbonyl and carbonate peaks are considerably overlap
with a number of aromatic carbons, we thus have confirmed the structures by single crystal
X-ray crystallography. Finally, the tautomeric structures of O-alkylated 3c and N-alkylated 3c
were elucidated by means of NMR spectroscopic investigations and confirmed by single
crystal X-ray analysis. The single crystal structures of alkylated compounds provide much
structural difference between the tautomeric ring systems can be specified in terms of bond
lengths and torsional angles. It is noteworthy that the orientation of the pyridine nitrogen of
the OH form is syn-periplanar, whereas anti-periplanar to the keto functionality in the NH-
form.
4. Supplementary Materials
Atomic coordinates and crystallographic parameters of 3a (CCDC 1902060) and 3c (CCDC
1902059) have been deposited at the Cambridge Crystallographic Data Centre. These data
can be obtained free of charge from the Cambridge Crystallographic Data Centre via

www.ccdc.cam.ac.uk/data_request/cif.
Acknowledgments
This work was supported by the Institutional Research Program (SI-1911-20) of Korea
Research Institute of Chemical Technology, Aging project (2017M3A9D8062955 to YSB)
and Stem Cell grant (2017M3A9B3061850 to YSB) funded by Ministry of Science and ICT.
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Highlights
• Alkylation of 5-hydroxypyrazole produces O- and N-alkylated tautomers.
• Two tautomeric structures were elucidated by single crystal X-ray diffractions.
• The O-alkylated tautomer exists in pyrazole, whereas N-tautomer in pyrazolone ring.
• Conformational changes have been observed between two tautomers.

Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests: