1H and 13C NMR spectral assignment of 29 N′-(3-([1,1′-biphenyl]-4-yl)-1-phenyl-1H-pyrazol-4-yl)acylhydrazones

Full text of DOI:10.1002/mrc.5113

Received: 14 September 2020
DOI: 10.1002/mrc.5113
Revised: 21 October 2020
Accepted: 28 October 2020
L E T T E R - S P E C T R A L A S S I G N M E N T
1H and 13C NMR spectral assignment of 29 N0-(3-([1,10-
biphenyl]-4-yl)-1-phenyl-1H-pyrazol-4-yl)acylhydrazones
Jihyun Park1 | Seunghyun Ahn2 | Youngshim Lee1 | Dongsoo Koh2
Yoongho Lim1
|
1Division of Bioscience and Biotechnology, BMIC, Konkuk University, Seoul, South Korea
2Department of Applied Chemistry, Dongduk Women's University, Seoul, South Korea
Correspondence
Yoongho Lim, Division of Bioscience and Biotechnology, BMIC, Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul 05029, South Korea.
Email: yoongho@konkuk.ac.kr
Funding information
Konkuk University
1 | INTRODUCTION
2 | EXPERIMENTAL
2.1 | Syntheses
Doxorubicin (Figure 1a) is an anthracycline drug that
has been used for chemotherapy since 1974.[1] In light
of its safety and effectiveness for various cancers, sev-
eral derivatives of doxorubicin have been synthe-
Phenylhydrazine was reacted with biphenylacetophenone
in the presence of a catalytic amount of glacial acetic acid
to obtain methyl biphenyl hydrazine (I). According to lit-
erature methods,[10] biphenyl hydrazine (I) was trans-
formed into pyrazolo-carbaldehyde (III) via a dialdehyde
intermediate (II) by the Vilsmeier–Haack reaction.
Pyrazolo-carbaldehyde (III) was functionalized with
benzoylhydrazide derivatives (IV) to afford a series of
pyrazolo-benzohydrazide compounds (V) used in this
study. The typical procedure for derivative 1 (Table 1) fol-
lows. Phenylhydrazine (3 mmol, 0.295 ml, d: 1.098 g/ml)
and 4-acetylbiphenyl (3 mmol, 589 mg) were dissolved in
20 ml of ethanol, and the reaction mixture was refluxed
for 3 h in the presence of a catalytic amount of acetic
acid. The reaction mixture was cooled to room tempera-
ture, and the obtained precipitate was filtered and
washed with ethanol to afford methyl biphenyl hydrazine
(I) in 85% yield as a pale-yellow solid. DMF (6 ml) and
POCl3 (0.6 ml) were stirred at 0ꢀC for 1 h to form the
Vilsmeier reagent; biphenyl hydrazine (I, 2 mmol,
570 mg) was added to it and heated at 60ꢀC for 2 h. After
the complete disappearance of biphenyl hydrazine (I)
(confirmed by TLC), the reaction mixture was poured
into 200 ml of ice water to give a precipitate of compound
III. The dried powder of compound III (0.5 mmol,
sized.[2,3] Aldoxorubicin (Figure 1b), which is
a
prodrug of doxorubicin, is the 6-maleimidocap
roylhydrazone of doxorubicin.[4] It can be prepared by
the addition of 6-maleimidocaproylhydrazide to the
ketone of the 1-hydroxypropan-2-one group in doxoru-
bicin. A box marked in Figure 1b indicates the N-
acylhydrazone moiety in aldoxorubicin. Nifuroxazide
(Figure 1c), which has been used to treat diarrhea,
colitis, and cancer, also contains the N-acylhydrazone
moiety.[5] The antimicrobial compound nitrofurazone
(Figure 1d)[6] and the hemostatic anti-inflammatory
agent carbazochrome (Figure 1e)[7] also have the N-
acylhydrazone moiety. Furthermore, several com-
pounds containing
a pyrazole moiety have been
reported to show anticancer activity.[8,9] Based on these
considerations, we designed and synthesized 29 com-
pounds containing both the N-acylhydrazone and
pyrazole moieties (Figure 1f). The compounds were
characterized by NMR spectroscopy and high-
resolution mass spectrometry. The spectral data pres-
ented herein can be used for the characterization of
synthetic or natural compounds that contain N-
acylhydrazone and pyrazole moieties.
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© 2020 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/mrc
Magn Reson Chem. 2021;59:648–662.

PARK ET AL.
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FIGURE 1 Structures of (a) doxorubicin, (b) aldoxorubicin, (c) nifuroxazide, (d) nitrofurazone, (e) carbazochrome, and (f)N0-(3-([1,10-
biphenyl]-4-yl)-1-phenyl-1H-pyrazol-4-yl)acylhydrazone
150 mg) and 3-anisic hydrazide (0.5 mmol, 85 mg) were
added to 10 ml of ethanol in the presence of a catalytic
amount of glacial acetic acid, and the reaction mixture
was refluxed at 75ꢀC for 4 h. The resulting mixture was
cooled to room temperature, and the obtained precipitate
washed with ethanol to give derivative 1 in 55% yield.
The synthetic procedure for N0-(3-([1,10-biphenyl]-4-yl)-
1-phenyl-1H-pyrazol-4-yl)acylhydrazone derivatives is
provided as Scheme 1.
were dissolved in pyridine-d5. Their concentrations were
adjusted to approximately 50 mM and transferred into
2.5-mm NMR tubes. All the NMR data were collected on
an Avance 400 spectrometer system (9.4 T; Bruker,
Karlsruhe, Germany) at room temperature, and the
chemical shifts were referenced to TMS. The relaxation
delay, 90ꢀ pulse, spectral width, number of data points,
1
and digital resolution for the H NMR experiments were
1 s, 11.6 μs, 5,500 Hz, 32 K, and 0.34 Hz/point, respec-
tively. The same parameters for the 13C NMR experiments
were 3 s, 15.0 μs, 21,000 Hz, 64 K, and 0.64 Hz/point,
respectively. For the two-dimensional (2D) experiments,
including COSY, TOCSY, HMQC, HMBC, and NOESY,
2 K × 256 (t2 × t1) data points were acquired.[11] The num-
ber of scans per increment was 16 for all 2D experiments.
2.2 | NMR spectra
All the derivatives listed in Table 1 were dissolved in
dimethylsulfoxide-d6, except 4, 17, 22, 25, and 27, which

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TABLE 1 Structures and names of N0-(3-([1,10-biphenyl]-4-yl)-1-phenyl-1H-pyrazol-4-yl)acylhydrazone derivatives and their high-
resolution mass spectroscopic data
Derivative
1
R1
R2
Mass (calc/found)
Name
3-Methoxyphenyl
Phenyl
472.1899/473.1986
(E)-N0-((3-([1,10-Biphenyl]-4-yl)-1-phenyl-1H-pyrazol-4-yl)
methylene)-3-methoxybenzohydrazide
2
4-Methoxyphenyl
3-Fluorophenyl
4-Fluorophenyl
2-Chlorophenyl
3-Bromophenyl
4-Bromophenyl
3-Pyridinyl
Phenyl
472.1899/473.1964
460.1699/461.1795
460.1699/461.1788
476.1404/477.1501
520.0899/521.0978
520.0899/521.0987
443.1746/444.1822
443.1746/444.1806
442.1794/443.1855
458.1743/459.1805
502.2005/503.2107
502.2005/503.2085
490.1805/491.1842
490.1805/491.1895
506.1510/505.1418
473.1852/474.1949
472.1899/ND
(E)-N0-((3-([1,10-Biphenyl]-4-yl)-1-phenyl-1H-pyrazol-4-yl)
methylene)-4-methoxybenzohydrazide
3
Phenyl
(E)-N0-((3-([1,10-Biphenyl]-4-yl)-1-phenyl-1H-pyrazol-4-yl)
methylene)-3-fluorobenzohydrazide
(E)-N0-((3-([1,10-Biphenyl]-4-yl)-1-phenyl-1H-pyrazol-4-yl)
methylene)-4-fluorobenzohydrazide
(E)-N0-((3-([1,10-Biphenyl]-4-yl)-1-phenyl-1H-pyrazol-4-yl)
methylene)-2-chlorobenzohydrazide
(E)-N0-((3-([1,10-Biphenyl]-4-yl)-1-phenyl-1H-pyrazol-4-yl)
methylene)-3-bromobenzohydrazide
(E)-N0-((3-([1,10-Biphenyl]-4-yl)-1-phenyl-1H-pyrazol-4-yl)
methylene)-4-bromobenzohydrazide
(E)-N0-((3-([1,10-Biphenyl]-4-yl)-1-phenyl-1H-pyrazol-4-yl)
methylene)nicotinohydrazide
(E)-N0-((3-([1,10-Biphenyl]-4-yl)-1-phenyl-1H-pyrazol-4-yl)
methylene)isonicotinohydrazide
(E)-N0-((3-([1,10-Biphenyl]-4-yl)-1-phenyl-1H-pyrazol-4-yl)
methylene)benzohydrazide
(E)-N0-((3-([1,10-Biphenyl]-4-yl)-1-phenyl-1H-pyrazol-4-yl)
methylene)-3-hydroxybenzohydrazide
(E)-N0-((3-([1,10-Biphenyl]-4-yl)-1-(4-methoxyphenyl)-1H-
pyrazol-4-yl)methylene)-3-methoxybenzohydrazide
(E)-N0-((3-([1,10-Biphenyl]-4-yl)-1-(4-methoxyphenyl)-1H-
pyrazol-4-yl)methylene)-4-methoxybenzohydrazide
(E)-N0-((3-([1,10-Biphenyl]-4-yl)-1-(4-methoxyphenyl)-1H-
pyrazol-4-yl)methylene)-3-fluorobenzohydrazide
(E)-N0-((3-([1,10-Biphenyl]-4-yl)-1-(4-methoxyphenyl)-1H-
pyrazol-4-yl)methylene)-4-fluorobenzohydrazide
(E)-N0-((3-([1,10-Biphenyl]-4-yl)-1-(4-methoxyphenyl)-1H-
pyrazol-4-yl)methylene)-2-chlorobenzohydrazide
(E)-N0-((3-([1,10-Biphenyl]-4-yl)-1-(4-methoxyphenyl)-1H-
pyrazol-4-yl)methylene)nicotinohydrazide
(E)-N0-((3-([1,10-Biphenyl]-4-yl)-1-(4-methoxyphenyl)-1H-
pyrazol-4-yl)methylene)benzohydrazide
(E)-N0-((3-([1,10-Biphenyl]-4-yl)-1-(4-methoxyphenyl)-1H-
pyrazol-4-yl)methylene)-3-hydroxybenzohydrazide
(E)-N0-((3-([1,10-Biphenyl]-4-yl)-1-(4-fluorophenyl)-1H-
pyrazol-4-yl)methylene)-3-methoxybenzohydrazide
(E)-N0-((3-([1,10-Biphenyl]-4-yl)-1-(4-fluorophenyl)-1H-
pyrazol-4-yl)methylene)-4-methoxybenzohydrazide
(E)-N0-((3-([1,10-Biphenyl]-4-yl)-1-(4-fluorophenyl)-1H-
pyrazol-4-yl)methylene)-3-fluorobenzohydrazide
(E)-N0-((3-([1,10-Biphenyl]-4-yl)-1-(4-fluorophenyl)-1H-
pyrazol-4-yl)methylene)-2-chlorobenzohydrazide
(E)-N0-((3-([1,10-Biphenyl]-4-yl)-1-(4-fluorophenyl)-1H-
pyrazol-4-yl)methylene)-3-bromobenzohydrazide
(E)-N0-((3-([1,10-Biphenyl]-4-yl)-1-(4-fluorophenyl)-1H-
pyrazol-4-yl)methylene)nicotinohydrazide
(E)-N0-((3-([1,10-Biphenyl]-4-yl)-1-(4-fluorophenyl)-1H-
pyrazol-4-yl)methylene)isonicotinohydrazide
4
Phenyl
5
Phenyl
6
Phenyl
7
Phenyl
8
Phenyl
9
4-Pyridinyl
Phenyl
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
Phenyl
Phenyl
3-Hydroxyphenyl
3-Methoxyphenyl
4-Methoxyphenyl
3-Fluorophenyl
4-Fluorophenyl
2-Chlorophenyl
3-Pyridinyl
Phenyl
4-Methoxyphenyl
4-Methoxyphenyl
4-Methoxyphenyl
4-Methoxyphenyl
4-Methoxyphenyl
4-Methoxyphenyl
4-Methoxyphenyl
4-Methoxyphenyl
4-Fluorophenyl
4-Fluorophenyl
4-Fluorophenyl
4-Fluorophenyl
4-Fluorophenyl
4-Fluorophenyl
4-Fluorophenyl
4-Fluorophenyl
4-Fluorophenyl
Phenyl
3-Hydoxyphenyl
3-Methoxyphenyl
4-Methoxyphenyl
3-Fluorophenyl
2-Chlorophenyl
3-Bromophenyl
3-Pyridinyl
488.1848/489.1898
490.1805/491.1874
490.1805/491.1897
478.1605/479.1662
494.1310/495.1358
538.0805/539.0876
461.1652/462.1701
461.1652/462.1772
460.1699/461.1738
476.1649/477.1739
4-Pyridinyl
Phenyl
(E)-N0-((3-([1,10-Biphenyl]-4-yl)-1-(4-fluorophenyl)-1H-
pyrazol-4-yl)methylene)benzohydrazide
(E)-N0-((3-([1,10-Biphenyl]-4-yl)-1-(4-fluorophenyl)-1H-
pyrazol-4-yl)methylene)-3-hydroxybenzohydrazide
3-Hydroxyphenyl

PARK ET AL.
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TABLE 1 (Continued)
Derivative
29
R1
R2
Mass (calc/found)
Name
4-Bromophenyl
4-Methoxyphenyl
550.1004/551.1115
(E)-N0-((3-([1,10-Biphenyl]-4-yl)-1-(4-methoxyphenyl)-1H-
pyrazol-4-yl)methylene)-4-bromobenzohydrazide
Note: The calculated mass data denote the exact mass. All the experimental mass data were found in the [M + H]+ mode, except for derivative 16,
which was found in the [M − H]− mode. ND denotes “not detected.”
SCHEME 1 Synthetic
procedure forN0-(3-([1,10-
biphenyl]-4-yl)-1-phenyl-1H-
pyrazol-4-yl)acylhydrazone
derivatives
The long-range coupling time for HMBC was 70 ms, and
the mixing times for TOCSY and NOESY were 400 ms and
1 s, respectively. The NMR data were processed using the
NMRPipe program[12] and analyzed using the SPARKY
3 program developed by T. D. Goddard and D. G. Kneller
(University of California at San Francisco).
2.3 | General experimental procedures
To confirm the structures of the 29 N0-(3-([1,10-biphenyl]-
4-yl)-1-phenyl-1H-pyrazol-4-yl)acylhydrazone derivatives
determined from the NMR assignments, ultra-
performance liquid chromatography-hybrid quadrupole-

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time-of-flight mass spectrometry (QTOF-MS) was used
on a Waters Acquity UPLC system (Waters Corp.,
Milford, MA) with the help of Prof. C. H. Lee at Konkuk
University, South Korea.[13]
3 | RESULTS AND DISCUSSION
The 29 derivatives have a common structure, where
only substituents on the R1 and R2 phenyl groups varied;
as a result, NMR assignments of all the N0-(3-([1,10-
biphenyl]-4-yl)-1-phenyl-1H-pyrazol-4-yl)acylhydrazones
were performed similarly. Derivative 1, (E)-N0-((3-([1,10-
biphenyl]-4-yl)-1-phenyl-1H-pyrazol-4-yl)methylene)-3-
methoxybenzohydrazide, was chosen as a representative
among these. The 13C NMR spectrum of derivative 1 rev-
ealed 24 peaks including a methoxy, a carbonyl, and
1
22 aromatic hydrocarbons. Based on the H–13C HMQC
spectrum, the 22 peaks were found to consist of 8 quater-
nary carbons and 14 aromatic methines. The carbonyl
carbon at 162.5 ppm was correlated with two 1H peaks at
8.66 ppm (s) and 7.51 ppm (dd, J = 7.7, 1.8 Hz) in the
1H–13C HMBC spectrum; the two proton peaks were
assigned as H-Py6 and H-6000 based on their splitting pat-
terns, respectively. The other carbons and protons in the
R1 phenyl group were easily assigned using COSY and
FIGURE 2 Important correlations obtained from the HMBC
spectrum of derivative 1
1
HMBC experiments. The H peak of H-6000 was correlated
with both C-2000 (112.9 ppm) and C-4000 (117.0 ppm) in the
HMBC spectrum. In the COSY spectrum, H-4000
(7.16 ppm) showed a cross peak with H-5000 (7.51 ppm).
The H-5000 and methoxy protons at 3.82 ppm showed a
correlation with the 13C peak at 159.2 ppm in the HMBC
spectrum, implying that it could be attributed to C-3000.
Two proton peaks at 8.66 and 9.01 ppm were long range
coupled to the 13C peak at 117.3 ppm. Because the former
proton was H-Py6, the latter proton and this carbon were
assigned H-Py5 and C-Py4, respectively. H-Py5 also
showed a correlation with the quaternary carbon peak at
151.4 ppm; the latter was assigned as C-Py3 based on an
additional HMBC connection with the 1H peak at
7.84 ppm (d, J = 8.4 Hz). The aromatic protons at 7.84
and 7.86 ppm had identical coupling constants (8.4 Hz)
and showed double intensities; thus, they were assigned
as H-30 and H-20, respectively. It was necessary to com-
pare HMBC correlations (between (H-20 and C-40) and
(H-30 and C-10)) to assign these two 1H signals. The
important correlations obtained from the HMBC spectra
of derivative 1 are shown in Figure 2. N0-(3-([1,10-Biphe-
nyl]-4-yl)-1-phenyl-1H-pyrazol-4-yl)acylhydrazones con-
tain three phenyl groups including N-phenyl, biphenyl,
and phenyl group attached to acylhydrazone. To distin-
guish their chemical shifts, the NOESY experiment was
performed for derivative 28. The NOE cross peaks
FIGURE 3 The important correlations obtained from the
COSY (dot lines) and NOESY (arrow lines) spectra of derivative 28

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between H-Py6 (8.65 ppm) and H-2000 (7.33 ppm),
hydroxyl group (9.75 ppm) and NH (11.69 ppm), and H-
Py5 (8.99 ppm) and H-2 (8.06 ppm) were observed. The
important correlations obtained from the COSY and
NOESY spectra of derivative 28 are shown in Figure 3.
The remained protons except N-phenyl group and phenyl
group attached to acylhydrazone can be assigned biphe-
nyl group. All the other derivatives have identical struc-
tures and differed only in the R1 and R2 phenyl groups;
NMR assignments were performed similarly. The com-
plete NMR assignments for all the derivatives are detailed
in Tables 2 and 3. The spectra collected from NMR spec-
troscopy and high-resolution mass spectrometry and
tables assigned based on the 1D and 2D experiments are
provided as Supporting Information.
PEER REVIEW
The peer review history for this article is available at
https://publons.com/publon/10.1002/mrc.5113.
ORCID
Yoongho Lim https://orcid.org/0000-0002-8945-3027
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The R1 group alone differed in derivatives 1 to 11,
whereas R2 (phenyl ring) was constant. R2 was a 400-
methoxyphenyl group in derivatives 12 to 19 and 29,
whereas it was a 400-fluorophenyl group in derivatives 20
to 28. Derivative 1 has a methoxy substituent at C-3000 of R1
(meta position), whereas derivative 2 has a methoxy sub-
stituent at C-4000 (para position). The change in position of
1
the methoxy group leads to slight changes in the H and
13C chemical shifts of the common structure. For example,
the chemical shifts of C-1000 and C O were shifted to
higher frequency when the methoxy group was at the meta
position, compared with the values for methoxy at the
1
para position. The H signals of the amine also showed a
similar trend. The 1H and 13C signals of Py6 also shifted to
higher frequency, even though the Py6 is not close to the
methoxy group of R1. This was likely due to the resonance
effect. This phenomenon was also observed in other deriv-
ative pairs (12 and 13, 20 and 21) that differed only in the
position of the methoxy group. In contrast, the substitution
of the R2 group with methoxy or fluoride at the para posi-
tion had a negligible effect on the chemical shifts of the
common structure. Compounds containing pyrazoline
moiety, one of the most useful groups in medicinal chem-
istry, have been known to show anti-inflammatory, anti-
[13] H. Jung, S. Ahn, Y. Jung, D. Koh, Y. Lim, Magn. Reson. Chem.
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SUPPORTING INFORMATION
Additional supporting information may be found online
in the Supporting Information section at the end of this
article.
tumor,
antiviral,
and
inhibitory
effects
on
phosphodiesterase and alcohol dehydrogenase.[14] Like-
wise, various compounds with N-acylhydrazone moiety
have been developed as antibacterial and hemostatic
agents and for the treatment of colitis and diarrhea.[15]
Therefore, the spectral data reported here can be useful to
characterize synthetic or natural compounds containing
N-acylhydrazone and pyrazole moieties.
How to cite this article: Park J, Ahn S, Lee Y,
Koh D, Lim Y. 1H and 13C NMR spectral
assignment of 29 N0-(3-([1,10-biphenyl]-4-yl)-1-
phenyl-1H-pyrazol-4-yl)acylhydrazones. Magn
Reson Chem. 2021;59:648–662. https://doi.org/10.
1002/mrc.5113
ACKNOWLEDGMENTS
This work is supported by Konkuk University in 2018.
J. Park, S. Ahn, and Y. Lee contributed equally to
this work.