Preparation of novel azidopyrazole derivatives with anticipated cytotoxic and antimicrobial activities

Article abstract of DOI:10.1002/jhet.3747

The reactions of azidopyrazole with some of primary amines and active methylene compounds were studied. Structures of the prepared compounds were identified using spectroscopic techniques. The reactions mechanisms were also proposed. The cytotoxic and antimicrobial activities were also examined for the newly synthesized compounds.

Full text of DOI:10.1002/jhet.3747

Received: 21 February 2019
DOI: 10.1002/jhet.3747
Revised: 24 July 2019
Accepted: 1 September 2019
A R T I C L E
Preparation of novel azidopyrazole derivatives with
anticipated cytotoxic and antimicrobial activities
Ewies F. Ewies1
| Marwa El-Hussieny1 | Naglaa F. El-Sayed1
|
Mohamed El-Shazly2,3 | Yu-Cheng Chen4 | Yi-Chang Liu5,6
1Organometallic and Organometalloid
Abstract
Chemistry Department, National
Research Centre, Giza, Egypt
The reactions of azidopyrazole with some of primary amines and active methy-
lene compounds were studied. Structures of the prepared compounds were
identified using spectroscopic techniques. The reactions mechanisms were also
proposed. The cytotoxic and antimicrobial activities were also examined for
the newly synthesized compounds.
2Department of Pharmacognosy and
Natural Products Chemistry, Faculty of
Pharmacy, Ain Shams University, Cairo,
Egypt
3Department of Pharmaceutical Biology,
Faculty of Pharmacy and Biotechnology,
German University in Cairo, Cairo, Egypt
4Program for Cancer Biology and Drug
Discovery, China Medical University and
Academia Sinica, Taichung, Taiwan
5Divisions of Hematology-Oncology,
Department of Internal Medicine,
Kaohsiung Medical University Hospital,
Kaohsiung, Taiwan
6Department of Internal Medicine,
Faculty of Medicine, College of Medicine,
Kaohsiung Medical University,
Kaohsiung, Taiwan
Correspondence
Ewies F. Ewies, Organometallic and
Organometalloid Chemistry Department,
National Research Centre, 33 El-Bohouth
St, (Former El Tahrir) Dokki, PO 12622,
Giza, Egypt.
Email: ewiesfawzy@yahoo.com
Funding information
Kaohsiung Medical University Hospital,
Grant/Award Numbers: KMUH-106M13,
KMUH105-5M13, KMUH104-4M12;
Ministry of Health and Welfare, Grant/
Award Numbers: MOHW107-TDU-B-
212-123006, MOHW106-TDU-B-
212-113006; National Research Centre,
Grant/Award Number: 11090112
J Heterocyclic Chem. 2020;1–13.
wileyonlinelibrary.com/journal/jhet
© 2020 Wiley Periodicals, Inc.
1

2
EWIES ET AL.
1 | INTRODUCTION
1720 cm−1 was absent. In agreement with these findings,
no signal for the aldehydic proton (δH 10.00 ppm) was
detected in the 1H NMR spectra.
The pyrazole ring system is known to evoke wide range
of biological activities.[1–9] The presence of pyrazole
nucleus in many pharmacological agents has proved and
leads to diverse applications in agriculture, technology,
and medicine. Also, pyrazole ring system is one of azole
family that described as anti-inflammatory, antifungal,
antibacterial, anticancer, and antioxidant as well as
antiviral agents.[10,11] In the same manner, aromatic
amines are important reagents for the preparation of
pharmaceuticals, pesticides, additives, dyes, and for
structure-biological activity studies related to food
carcinogens.[12–15] Moreover, azides are important build-
ing blocks due to their potential application in organic
and bioorganic chemical reactions.[16–18] In the light of
this survey and our interest in synthesis of heterocyclic
chemistry,[1,19–23] we have now studied the reactivity of
azidopyrazole carbaldehyde 1 towards certain active
methylenes (2a-e, 3), primary amines (4a-g), and hydra-
zines (4h-k) aiming at preparing new heterocycles of
anticipated anticancer and antimicrobial activities
(Figure 1).
Compound 1 was also reacted at boiling temperature
with ethyl cyanoacetate 2e in methylene chloride in
under basic condition of NaOMe resulting in a mixture of
pyrazol-2-cyanoacrylate derivative 6 in a 55% yield and
pyrazolo[3,4-d][1,2,3]triazine derivatives (7) in a 20%
yield (Scheme 1).
1
Elemental analyses, IR, and H NMR measurements
were compatible with the assigned structures. The IR
spectrum of compound 6 indicated the presence of
absorption bands at 2225 (CN), 2111 (N3), and 1712 cm−1
(C═O). The IR spectrum of compound 7 confirmed the
presence of bands at 3365 (NH), 2184 (CN), 1707 (CO,
ester), and 1668 cm−1 (N═N).15 The 1H NMR spectrum of
6 revealed the presence of an exocyclic ethylenic proton
at δH 7.81, which was absent in the spectrum of
compound 7.
Upon refluxing compound 1 with 2H-indene-1,-
3-dione (3) in ethanol for 1 hour, 2-((5-azido-3-methyl-
1-phenyl-1H-pyrazol-4-yl)methylene)-2H-indene-1,-
3-dione (8) was solely obtained. It showed a band at
2226 cm−1 (N3) in its IR spectrum. A signal at δH
1
8.01 ppm (CH═N) was detected in its H NMR spectrum
2 | RESULTS AND DISCUSSION
as well as two signals at δc 186.6, 188.8 (2 CO) in its 13C
NMR spectrum.
Azidopyrazole carbaldehyde 1 was prepared by the reac-
tion of chloropyrazole carbaldehyde with sodium azide in
DMF at 0ꢀC according to literature procedures.[24]
Condensation of 1 with the active methylene com-
pounds 2a-d proceeded in absolute ethanol at ambient
temperature to yield the respective ethylenes 5a-d
(Scheme 1). The IR spectra of the prepared ethylenes
indicated the presence of an azido functionality around
2140 cm−1 while the aldehydic C═O absorption around
The reaction of compound 1 with the appropriate
hydrazines 4a-c was performed in ethanol at room tem-
perature under stirring. In each case, a mixture of three
products was obtained (cf. 9, 10a-c, and 11a-c, Scheme 2).
The structures of all products were determined with
elemental and spectroscopic analyses (Section 4). As dis-
tinguishing features, the IR spectra of 10a-c and 11a-c
lacked the carbonyl amide absorption, which was
detected at 1667 cm−1 in the spectrum of compound 9.
H3C
CHO
O
O
R1
N
R-NH2
N3
N
R
Ph
1
4a-k
2a-e
3
CH3
a
, R =NH2
a
b
, R = CN
, R = COCH3
R1 = CN
N
R1 = COCH3
R1 = COOEt
b
c
, R =CH3NH
, R = PhNH
N
h
, R =
i
, R = N
S
c
, R = COCH3
d
, R = C6H5
1
d
, R = CH2COCH3 R = COCH3
O
H3C
e
, R = C6H4-Br-4
R1 = COOEt
N
e
, R = CN,
f
, R = C6H4-NH2-4
j
, R =
k
, R =
N
g
, R = C6H4-NH2-2
S
O
H3C
S
N
Ph
FIGURE 1 The reactants

EWIES ET AL.
3
SCHEME 1 Reactions of azidopyrazole (1) with compounds that contain active methylene
SCHEME 2 Reactions of azidopyrazole (1) with
hydrazines 4a-c
On the other hand, unlike 10a-c, compounds 9 and 11a-c
showed strong absorption band around 1660 cm−1 due to
the N═N stretching vibration.[25]
temperature.
A
brown crystalline material was
obtained and named as N0-((5-azido-3-methyl-1-phenyl-
1H-pyrazol-4-yl)methylene)acetohydrazide (12). On the
other hand, under the same experimental conditions,
the reaction of 1 with phenyl hydrazine afforded a
The reaction of compound
1 with hydrazine
hydrate (4a) was conducted in acetic acid at room

4
EWIES ET AL.
SCHEME 3 Formation mechanism[16,26] of
compound 9
mixture of substances including 3-methyl-1-phenyl-
4-((2-phenylhydrazono)methyl)-1H-pyrazol-5-amine
(13), 10c, and 11c.
reflux yielding a mixture of the known Schiff bases 14a
or 14b[27] and their α-amino pyrazoles 15a or 15b in each
case (Scheme 4). The structural assignment of
compounds 14a or 14b and 15a or 15b was based upon
elemental analysis and spectroscopic data (cf. Section 4).
Condensation of 1 with o-phenylenediamine 4g
proceeded in ethanol at room temperature to give a mix-
ture of N-1-((5-azido-3-methyl-1-phenyl-1H-pyrazol-4-yl)
methylene)benzene-1,2-diamine (16) and 2-(5-azido-
3-methyl-1-phenyl-1H-pyrazol-4-yl)-1H-benzo[d]imidaz-
ole (17). On the other hand, under the same conditions,
the reaction of 1 with p-phenylenediamine 4f yielded N-
1-((5-azido-3-methyl-1-phenyl-1H-pyrazol-4-yl)methy-
lene)benzene-1,4-diamine (18) as the sole product
(Scheme 5). Structures of compounds 16-18 were
A possible explanation for the formation of com-
pound 9 from the reaction of 1 with the hydrazine
reagents 4a-c is outlined in Scheme 3. Initial attack by
the anion derived from the azido function on the most
electron deficient center in the formyl group would afford
the dipolar species “A,” which undergoes intramolecular
rearrangement to afford 9. It has been reported that o-
azido-carbaldehyde compounds can be intracyclized with
good yield under the influence of a basic medium.[16,26]
The reaction of compound 1 with the primary amines
4d and 4e was also investigated. Compound 1 reacted
with one molar equivalent of 4d or 4e in ethanol at under
SCHEME 4 Reactions of azidopyrazole (1)
with aniline derivatives 4d,e
SCHEME 5 Reactions of azidopyrazole (1) with o- and p-phenyelendiamine
SCHEME 6 Formation
mechanism of compound 17

EWIES ET AL.
5
SCHEME 7 Reactions of azidopyrazole (1) with some heterocyclic amines (4h-k)
TABLE 1 Cytotoxicity of azide analogs against different
cancer cell lines for 72 h
1
assigned on the basis of their IR, H, 13C NMR, and MS
spectroscopic data (cf. Section 4).
Molt 4
U937
NA
Sup-T1
NA
562
LNcap
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
0.96
Formation of 17 is proposed in terms of molecular
rearrangement of the initially formed 16 to give a
transient intermediate B, which readily undergoes dehy-
drogenation (auto-oxidation) yielding the final product
17. This reaction is similar to the reactions of 3b with car-
bonyl compounds[28] (Scheme 6).
5a
NA
NA
5b
10.45
NA
18.47
4.46
NA
8.41
10.56
NA
6
13.1
9
NA
NA
NA
10a
NA
NA
NA
NA
When compound 1 was reacted with various hetero-
cyclic amines 4h-k in ethanol at room temperature, pure
crystalline products (19a-d) were formed (Scheme 7). The
structures of these compounds were determined based on
their spectroscopic data. An absorption band was
detected in the IR spectra of 19a-d due to the azido func-
tionality around 2100 cm−1. No absorption band of the
10b
11c
NA
NA
19.07
NA
NA
12.18
25.09
13.02
17.24
NA
NA
18.56
10.90
14.20
28.49
NA
12
NA
27.54
11.27
12.43
17.76
NA
15a
NA
16
NA
17
NA
carbonyl group (C═O) was detected around 1700 cm−1
,
19a
NA
NA
NA
which was supported by the absence of the signal of the
1
19b
19c
NA
NA
NA
NA
aldehydic proton at δH 10 ppm in their H NMR spectra
(Scheme 7).
NA
NA
NA
NA
19d
Doxorubicin
13.55
0.001
NA
26.18
0.006
11.52
0.08
0.001
3 | BIOLOGICAL ACTIVITY
3.1 | The cytotoxic activity
Note. NA, not active at 20 μg/mL for 72-h evaluation.
The cytotoxic activity of some of the prepared compounds
was evaluated against Molt 4, U937, Sup-T1, 562, and
LNcap cancer cell lines, and the results are depicted in
Table 1. Among the tested compounds, 3-((5-azido-
3-methyl-1-phenyl-1H-pyrazol-4-yl)methylene)pentane-
2,4-dione (5b) exhibited potent activity against Molt
4, U937, Sup-T1, and 562 cells with IC50 10.45, 18.47,
8.41, and 10.56 μg/mL, respectively after 72 hours. On the
other hand, its analogue, which carries two cyano
groups, 2-((5-azido-3-methyl-1-phenyl-1H-pyrazol-4-yl)
methylene)malononitrile (5a), did not show any activity
(Table 1). The derivative with one cyano and one ethyl
acetate group, ethyl 3-(5-azido-3-methyl-1-phenyl-1H-
pyrazol-4-yl)-2-cyanoacrylate (6), showed activity against
U937 and Sup-T1 cells with IC50 4.46 and 13.1 μg/mL,
respectively. The azido methyl hydrazine derivative
showed activity against Sup-T1 cells with IC50
19.07 μg/mL while the azido hydrazine derivative did not
show any activity (Table 1). The triazine phenyl hydra-
zine derivative, 1-(5-methyl-7-phenyl-3H-pyrazolo[3,4-d]
[1,2,3]triazin-4(7H)-ylidene)-2-phenylhydrazine
(11c),
showed activity against Molt 4 and 562 cells with IC50
12.18 and 18.56 μg/mL. The azido acetohydrazide deriva-
tive,
N0-((5-azido-3-methyl-1-phenyl-1H-pyrazol-4-yl)
methylene)acetohydrazide (12), exhibited activity against
Molt4, Sup-T1, and 562 cells with IC50 25.09, 27.54, and
10.90 μg/mL, respectively (Table 1). The phenyl pyrazol
derivative, 3-methyl-1-phenyl-4-((phenylimino)methyl)-
1H-pyrazol-5-amine (15a), also exhibited activity against
Molt4, Sup-T1, and 562 cells with IC50 13.02, 11.27, and
14.20 μg/mL, respectively. Moreover, the azido phenyl
pyrazol derivative, N-1-((5-azido-3-methyl-1-phenyl-1H-

6
EWIES ET AL.
pyrazol-4-yl)methylene)benzene-1,2-diamine (16), was
active against the same cancer cell lines Molt4, Sup-T1,
and 562 cells with IC50 17.24, 12.43, and 28.49 μg/mL,
respectively (Table 1). A closely similar derivative with
ortho amino group, N-1-((5-azido-3-methyl-1-phenyl-1H-
activity against S. aureus (Table 2). The lack of antimicro-
bial activity suggested that compounds, which were
active against cancer cell lines, were only specific to those
cell lines and did not kill any type of cells. The selectivity
and safety profile of the tested compounds requires
further studies.[31–33]
pyrazol-4-yl)methylene)benzene-1,4-diamine
(18),
showed activity against a single cancel line, Sup-T1, with
IC50 17.76 μg/mL. The azido pyrazol thioxothiazolidin
derivative,
3-((5-azido-3-methyl-1-phenyl-1H-pyrazol-
4 | EXPERIMENTAL
4-yl)methyleneamino)-2-thioxothiazolidin-4-one (19d),
demonstrated activity against Molt4, Sup-T1, and 562 cells
with IC50 13.55, 26.18, and 11.52 μg/mL, respectively
(Table 1).[29,30]
Melting points were determined in open glass capillaries
using Electrothermal IA 9000 series digital melting point
apparatus (Electrothermal, Essex, UK) and are
uncorrected. The IR spectra were measured in KBr pel-
lets with a Perkin-Elmer Infracord Spectrophotometer
1
3.2 | The antimicrobial activity
model 157(Grating). The H and 13C NMR spectra were
recorded in CDCl3 on JEOL-500 MHz Spectrometer, and
the chemical shifts were recorded in δ values relative to
TMS. The mass spectra were performed at 70 eV on a
Shimada GCS-OP 1000 Ex Spectrometer provided with a
data system. Elemental analyses were performed using
Elmenter Varu EL Germany Instrument. The reported
yields are based upon pure materials isolated by column
chromatography. Solvents were dried/purified according
to conventional procedures.
The antimicrobial activity of some of the prepared
compounds was evaluated against Staphylococcus aureus,
Escherichia coli, and Pseudomonas aeruginosa, and the
results are depicted in Table 2. The tested compound
showed no activity except for 17, which showed moderate
TABLE 2 MIC for 16 compounds (N = 3)
MIC, μg/mL
Staphylococcus Escherichia Pseudomonas
4.1 | Reaction of 5-azido-3-methyl-
1-phenyl-1H-pyrazole-4-carbaldehyde (1)
with the active methylene (2a-d)
aureus
NA
128
NA
NA
NA
NA
64
coli
NA
NA
NA
NT
NA
NA
NA
NA
NA
NT
NA
NA
NT
NT
NA
NA
6.4
aeruginosa
NA
NA
NA
NT
5a
5b
6
4.1.1 | General procedure
9
10a
10b
11c
12
NA
NT
In a 25-mL round bottom flak equipped with magnetic
stirrer bar, a mixture of azidopyrazole (1, 0.1 mol) and
0.2 mol of malononitrile (2a), acetylacetone (2b), ethyl
acetoacetate (2c), or acetonyl acetone (2d) in 10-mL abso-
lute ethanol was stirred at room temperature for 1 to
48 hours (TLC monitoring). Few drops of pyridine were
added to the reaction mixture of compound 1 with 2a.
The volatile materials were evaporated, the residual pre-
cipitate washed with petroleum ether, diethyl ether,
dried, and recrystallized from ethanol.
NA
NA
NA
NT
128
NA
NA
32
15a
16
17
NA
NA
NT
18
NA
NA
NA
NA
64
20a
19b
19c
19d
NT
2-((5-Azido-3-methyl-1-phenyl-1H-pyrazol-4-yl)
methylene)malononitrile (5a)
NA
NA
16
Pale yellow crystals; yield 95%; m.p. 209ꢀC to 211ꢀC
Rf = 0.64 (ethyl acetate/cyclohexane = 30/70); IR (KBr):
~ν = 2239 (br, 2CN), 2134 (N3), 1612 (C═C), 1536 (C═N)
cm−1; 1H NMR (500 MHz, CDCl3): δ = 2.65 (s, 3 H, CH3),
7.34 (s, 1 H, ═CH), 7.52-8.32 (m, 5H, CHarom) ppm; 13C
NMR (125 MHz, CDCl3): δ = 13.9 (CH3), 73.7, 156.3
Streptomycina NT
Penicillin Ga
0.64
NT
NT
Abbreviations: MIC, minimum inhibitory concentrations; NA, no activity at
128 μg/mL; NT, no test.
aAs positive control.

EWIES ET AL.
7
(C═C), 114.2, 116.6 (CN), 124.4, 128.2, 129.1, 131.2 (aro-
matic, C-H), 105.9, 138.9 (cyclic C═C), 150.2 (cyclic
C═N) ppm. MS (EI, 70 eV): m/z (%) = 275 (10%) [M]+,
247(15) [M-28]+; Anal. for C14H9N7 (275.27): calcd. C,
61.09; H, 3.30; N, 35.62; found: C, 61.12; H, 3.28;
N, 35.65.
4.2 | Reaction of 5-azido-3-methyl-
1-phenyl-1H-pyrazole-4-carbaldehyde (1)
and ethyl 2-cyanoacetate (2e)
A mixture of compound 1, (0.1 mol) and 0.1 mol of ethyl
2-cyanoacetate (2e), in 20-mL DCM in the presence of
0.1 g NaOMe was stirred in ice bath for 2 hours then
refluxed for 2 hours (TLC monitoring, Rf = 0.31 (ethyl
acetate/petroleum ether (60:80ꢀC) = 30/70)). The reac-
tion mixture was evaporated under reduced pressure, and
the residual material was chromatographed on silica gel
column to give products 6 and 7.
3-((5-Azido-3-methyl-1-phenyl-1H-pyrazol-4-yl)
methylene)pentane-2,4-dione (5b)
Red solid; yield 87%; m.p. 156ꢀC to 158ꢀC Rf = 0.44 (ethyl
acetate/cyclohexane = 30/70); IR (KBr): ~ν = 2147 (N3),
1
1672, 1649 (C═O), 1592 (C═C), 1527 (C═N) cm−1; H
NMR (500 MHz, CDCl3): δ = 2.27, 2.28 (2 s, 6 H, 2CH3),
2.63 (s, 3 H, CH3), 7.45-8.29 (m, 5 H, CHarom), 8.32 (s,
1 H, CH) ppm; 13C NMR (125 MHz, CDCl3): δ = 14.4
(CH3), 29.4 (COCH3), 104.1, 138.7 (cyclic C═C), 124.7,
128.2, 129.7, 134.1 (aromatic, C─H), 148.2, 148.4 (C═C),
150.5 (cyclic C═N), 201.8, 203.5 (C═O) ppm. MS (EI,
70 eV): m/z (%) = 309 (5%) [M]+; Anal. for C16H15N5O2
(309.32): calcd. C, 62.13; H, 4.89; N, 22.64; found: C,
62.16; H, 4.95; N, 22.53.
4.2.1 | Ethyl 3-(5-azido-3-methyl-
1-phenyl-1H-pyrazol-4-yl)-2-cyanoacrylate
(6)
Eluent: petroleum ether (60:80ꢀC) /ethyl acetate (95/5,
v/v). Product 6 was separated as red crystals, yield 55%;
m.p. 120ꢀC to 122ꢀC. IR (KBr): ~ν = 2225 (CN), 2211 (N3),
1
Ethyl 2-((5-azido-3-methyl-1-phenyl-1H-pyrazol-4-yl)
methylene)-3-oxobutanoate (5c)
1712 (C═O), 1619, 1596 (C═C), 1550 (C═N) cm−1; H
NMR (500 MHz, CDCl3): δ = 1.40 (t, 3 H, CH3), 2.67
(s, 3 H, CH3), 4.41 (q, 2 H, CH2), 7.30-7.71 (m, 5 H,
CHarom), 7.81 (s, 1 H, CH) ppm; 13C NMR (125 MHz,
CDCl3): δ = 13.4 (CH3), 15.7 (CH3), 61.9 (CH2), 117.1
(CN), 99.3, 155.1 (C═C), 105.9, 133.7 (cyclic C═C), 124.2,
127.1, 128.2, 129.1, 133.7 (aromatic, C-H), 150.3 (cyclic
C═N), 164.9 (C═O) ppm. MS (EI, 70 eV): m/z (%) = 322
(20%) [M]+; Anal. for C16H14N6O2 (322.32): calcd. C,
59.62; H, 4.38; N, 26.07; found: C, 59.88; H, 4.22; N,
25.99.
Brown crystals, yield 80%; m.p. 165ꢀC to 167ꢀC Rf = 0.62
(ethyl acetate/cyclohexane = 30/70); IR (KBr): ~ν = 2124
(N3), 1674, 1629 (C═O), 1598 (C═C), 1527 (C═N) cm−1;
1H NMR (500 MHz, CDCl3): δ = 1.36 (t, 3 H, CH3), 2.15
(s, 3 H, CH3), 2.45 (s, 3 H, CH3), 4.90 (q, 2 H, CH2),
7.45-7.69 (m, 5 H, CHarom), 9.17 (s, 1 H, CH) ppm; 13C
NMR (125 MHz, CDCl3): δ = 13.9 (CH3), 15.9 (CH3), 29.4
(COCH3), 58.8 (CH2), 106.1, 131.2 (cyclic C═C), 96.4,
138.3 (C═C), 150.7 (cyclic C═N), 125.5, 127.1, 128.2,
129.3, 131.1 (aromatic, C─H), 168.7 (C═O), 198.2 (C═O)
ppm. MS (EI, 70 eV): m/z (%) = 339 (3%) [M]+; Anal. for
C17H17N5O3 (339.35): calcd. C, 60.17; H, 5.05; N, 20.64;
found: C, 60.12; H, 5.10; N, 20.62.
4.2.2 | Ethyl 2-cyano-2-(5-methyl-
7-phenyl-3H-pyrazolo[3,4-d][1,2,3]triazin-
4(7H)-ylidene)acetate (7)
3-((5-Azido-3-methyl-1-phenyl-1H-pyrazol-4-yl)
methylene)hexane-2,5-dione (5d)
Eluent: petroleum ether (60:80ꢀC)/ethyl acetate (80/20,
v/v). Product 7 was separated as yellow crystals, yield
20%; m.p. 150ꢀC to 152ꢀC. IR (KBr): ~ν = 3365 (NH), 2184
(CN), 1707 (C═O), 1668 (N═N), 1604 (C═C), 1585 (C═N)
cm−1; 1H NMR (500 MHz, CDCl3): δ = 1.31 (t, 3 H, CH3),
3.80 (s, 3 H, CH3), 4.28 (q, 2 H, CH2), 6.84-7.53 (m, 5 H,
CHarom), 7.55 (s, 1H, NH, exchangeable with D2O) ppm;
13C NMR (125 MHz, CDCl3): δ = 14.9 (CH3), 15.1 (CH3),
63.2 (CH2), 74.4 (═C─CN), 114.1 (CN), 95.5, 154.5 (cyclic
C═C), 124.4, 124.7, 128.2, 129.3, 138.3 (aromatic, C─H),
146.3 (cyclic C═N), 169.4 (C═O) ppm. MS (EI, 70 eV):
m/z (%) = 322 (30%) [M]+; Anal. for C16H14N6O2
(322.32): calcd. C, 59.62; H, 4.38; N, 26.07; found: C,
59.78; H, 4.24; N, 26.02.
Brown crystals; yield 20%; m.p. 172ꢀC to 173ꢀC Rf = 0.58
(ethyl acetate/cyclohexane = 30/70); IR (KBr): ~ν = 2215
(N3), 1652 (C═O), 1595 (C═C), 1563 (C═N) cm−1; H
1
NMR (500 MHz, CDCl3): δ = 2.61 (s, 3 H, CH3), 3.32
(s, 3 H, CH3), 4.35 (s, 3 H, CH3), 5.13 (s, 2 H, CH2), 6.96
(s, 1 H, CH), 7.17-7.20 (m, 5 H, CHarom) ppm; 13C NMR
(125 MHz, CDCl3): δ = 13.6 (CH3), 23.0, 29.4 (COCH3),
53.4 (CH2), 88.8, 142.6 (C═C), 104.1, 138.6 (cyclic C═C),
150.3 (cyclic C═N), 124.7, 127.1, 128.0, 131.5, 135.6, (aro-
matic, C─H), 181.9, 187.4 (C═O) ppm. MS (EI, 70 eV):
m/z (%) = 323 (2%) [M]+, 281 (2%) [M-42]+; Anal. for
C17H17N5O2 (323.35): calcd. C, 63.15; H, 5.30; N, 21.66;
found: C, 63.21; H, 5.25; N, 21.68.

8
EWIES ET AL.
4.3 | Synthesis of 2-((5-azido-3-methyl-
1-phenyl-1H-pyrazol-4-yl)methylene)-2H-
indene-1,3-dione (8)
2-((5-Azido-3-methyl-1-phenyl-1H-pyrazol-4-yl)
methylene)hydrazine (10a)
Eluent: petroleum ether (60:80ꢀC)/ethyl acetate (80/20,
v/v). Product 10a was separated as colorless crystals, yield
45%; m.p. 217ꢀC to 219ꢀC. IR (KBr): ~ν = 3410, 3277
A mixture of compound 1 (1 mmol) and 1H-indene-
1,3(2H)-dione (3, 1 mmol) in absolute ethanol (20 mL)
was stirred at room temperature for 1 hour. The reac-
tion mixture was evaporated under reduced pressure,
and the residual material was chromatographed on sil-
ica gel column using petroleum ether (60:80ꢀC)/ethyl
acetate (95/5, v/v) as an eluent, to give 8 as colorless
crystals; yield 70%; m.p. 150ꢀC to 152ꢀC Rf = 0.31
(ethyl acetate/petroleum ether (60:80ꢀC) = 20/80); IR
(KBr): ~ν = 2226 (N3), 1626 (C═O), 1620 (C═O),1560
(NH2), 2360 (N3), 1625 (C═N), 1594, 1574 (C═N) cm−1
;
1H NMR (500 MHz, CDCl3): δ = 2.59 (s, 3 H, CH3),
7.31-7.75 (m, 5 H, CHarom), 8.36 (s, 2 H, NH2), 8.69 (s,
1 H, CH) ppm; 13C NMR (125 MHz, CDCl3): δ = 13.9
(CH3), 105.7, 134.1 (cyclic C═C), 125.8, 127.1, 128.2,
129.3, 131.2 (aromatic, C─H), 138.1, 151.7 (2 C═N) ppm.
MS (EI, 70 eV): m/z (%) = 241 (5%) [M]+; Anal. for
C11H11N7 (241.25): calcd. C, 54.76; H, 4.60; N, 40.64;
found: C, 54.68; H, 4.55; N, 40.59.
1
(C═N) cm−1; H NMR (500 MHz, CDCl3): δ = 2.59 (s,
1 H, CH3), 7.21-7.67 (m, 9 H, CHarom), 8.01 (s, 1H, CH)
ppm; 13C NMR (125 MHz, CDCl3): δ = 13.9 (CH3), 124.4,
124.7, 127.1, 128.2, 129.1, 129.2, 133.7, 133.9 (aromatic,
C─H), 104.1, 133.9 (cyclic C═C), 117.3, 143.8 (C═CH),
150.7 (cyclic C═N), 186.6, 188.8 (C═O) ppm. MS (EI,
70 eV): m/z (%) = 355 (2%) [M]+; Anal. for C20H13N5O2
(355.35): calcd. C, 67.60; H, 3.69; N, 19.71; found: C,
67.56; H, 3.73; N, 19.67.
2-((5-Azido-3-methyl-1-phenyl-1H-pyrazol-4-yl)
methylene)-1-methylhydrazine (10b)
Eluent: petroleum ether (60:80ꢀC)/ethyl acetate (80/20,
v/v). Product 10b was separated as yellow crystals,
yield 45%; m.p. 240ꢀC to 242ꢀC. IR (KBr): ~ν = 3333
(NH), 2218 (N3), 1653 (C═C), 1595, 1564 (C═N) cm−1; 1H
NMR (500 MHz, CDCl3): δ = 2.36 (s, 3 H, CH3), 4.51 (s,
3 H, CH3), 6.85 (s, 1H, NH, exchangeable with D2O),
7.26-7.54 (m, 5 H, CHarom), 7.57 (s, 1 H, CH) ppm; 13C
NMR (125 MHz, CDCl3): δ = 13.9 (CH3), 35.7 (CH3),
107.4, 134.3 (C═C), 124.5, 127.1, 128.2, 129.3, 138.1 (aro-
matic, C─H), 141.7, 151.4 (C═N) ppm. MS (EI, 70 eV):
m/z (%) = 255 (5%) [M]+, 198 (100%) [M-58]+; Anal. for
C12H13N7 (255.28): calcd. C, 56.46; H, 5.13; N, 38.41;
found: C, 56.50; H, 5.20; N, 38.39.
4.4 | Reaction of 1 and hydrazines (4a-c)
4.4.1 | General procedure
A mixture of compound 1 (5 mmol) and the appropriate
hydrazines, namely, hydrazine hydrate, methyl hydra-
zine, and/or phenyl hydrazine (10 mmol) in absolute
ethanol (25 mL) was stirred for 1 to 5 hours (TLC moni-
toring, Rf = 0.23 (ethyl acetate/cyclohexane = 30/70)).
The reaction mixture was evaporated under reduced
pressure, and the residual material was chromatographed
on silica gel column to give products 9, 10a-c, and 11a-c.
2-((5-Azido-3-methyl-1-phenyl-1H-pyrazol-4-yl)
methylene)-1-phenylhydrazine (10c)
Eluent: petroleum ether (60:80ꢀC)/ethyl acetate (60/40,
v/v). Product 10c was separated as orange crystals, yield
35%; m.p. 232ꢀC to 234ꢀC. IR (KBr): ~ν = 3291 (NH), 2211
(N3), 1596 (C═C), 1553, 1543 (C═N) cm−1 1H NMR
;
(500 MHz, CDCl3): δ = 2.45 (s, 3 H, CH3), 4.80 (s, 1H,
NH, exchangeable with D2O), 5.95 (s, 1 H, CH), 6.86-7.65
(m, 10 H, CHarom) ppm; 13C NMR (125 MHz, CDCl3):
δ = 13.8 (CH3), 107.4, 133.7 (C═C), 115.8, 116.9, 124.7,
125.8, 127.1, 128.2,129.3, 142.2 (aromatic, C─H), 144.8,
151.4 (C═N) ppm. MS (EI, 70 eV): m/z (%) = 318 (5%)
[M + 1]+; Anal. for C17H15N7 (317.35): calcd. C, 64.34; H,
4.76; N, 30.90; found: C, 64.43; H, 4.55; N, 30.85.
5-Methyl-7-phenyl-3H-pyrazolo[3,4-d][1,2,3]triazin-
4(7H)-one (9)
Eluent: petroleum ether (60:80ꢀC)/ethyl acetate (95/5,
v/v). Product 9 was separated as orange crystals, yield 5%;
m.p. 160ꢀC to 162ꢀC. IR (KBr): ~ν = 3326 (NH), 1672
(C═O), 1665 (N═N), 1619 (C═C), 1596 (C═N) cm−1; H
1
NMR (500 MHz, CDCl3): δ = 2.57 (s, 3 H, CH3), 4.51 (s,
1 H, NH, exchangeable with D2O), 7.49-7.56 (m, 5 H,
CHarom) ppm; 13C NMR (125 MHz, CDCl3): δ = 14.5
(CH3), 96.1, 138.7 (cyclic C═C), 124.4, 124.7, 128.2, 129.3,
129.9 (aromatic, C-H), 148.8 (C═N), 162.0 (C═O) ppm.
MS (EI, 70 eV): m/z (%) = 227 (50%) [M]+; Anal. for
C11H9N5O (227.22): calcd. C, 58.14; H, 3.99; N, 30.82;
found: C, 58.12; H, 4.01; N, 30.74.
1-(5-Methyl-7-phenyl-3H-pyrazolo[3,4-d][1,2,3]triazin-
4(7H)-ylidene)hydrazine (11a)
Eluent: petroleum ether (60:80ꢀC)/ethyl acetate (65/35,
v/v). Product 11a was separated as orange crystals, yield
30%; m.p. 150ꢀC to 152ꢀC. IR (KBr): ~ν = 3432 (NH2),
3346 (NH), 1680 (N═N), 1630 (C═C), 1596 (C═N), 1540
1
(C═N) cm−1; H NMR (500 MHz, CDCl3): δ = 2.00 (s,

EWIES ET AL.
9
1 H, NH, exchangeable with D2O), 2.36 (s, 3 H, CH3),
4.51 (s, 2H, NH2, exchangeable with D2O), 7.26-7.57 (m,
5 H, CHarom) ppm; 13C NMR (125 MHz, CDCl3): δ = 14.5
(CH3), 99.3, 156.2 (cyclic C═C), 124.4, 127.0, 128.2, 129.3,
138.7 (aromatic, C─H), 136.0, 145.6 (2 C═N) ppm. MS
(EI, 70 eV): m/z (%) = 322 (20%) [M]+; Anal. for
C11H11N7 (241.25): calcd. C, 54.76; H, 4.60; N, 40.64;
found: C, 54.75; H, 4.64; N, 40.63.
yield) and 11c (25% yield) were isolated and identified by
comparing their melting points and IR spectra with those
of the previously isolated specimens.
4.5.1 | N0-((5-Azido-3-methyl-1-phenyl-
1H-pyrazol-4-yl)methylene)acetohydrazide
(12)
2-Methyl-1-(5-methyl-7-phenyl-3H-pyrazolo[3,4-d]
[1,2,3]triazin-4(7H)-ylidene)hydrazine (11b)
Brown crystals, 85% yield (acetone); m.p. 140ꢀC to 142ꢀC
Rf
=
0.42
(ethyl
acetate/petroleum
ether
Eluent: petroleum ether (60:80ꢀC)/ethyl acetate (50/50,
v/v). Product 11b was separated as yellow crystals, yield
55%; m.p. 205ꢀC to 207ꢀC. IR (KBr): ~ν = 3380, 3226 (NH),
(60:80ꢀC) = 30/70); IR (KBr): ~ν = 3046 (NH), 2146 (N3),
1628 (C═O), 1591 (C═C), 1541, 1503 (C═N) cm−1; H
1
NMR (500 MHz, CDCl3): δ = 2.39 (s, 3 H, CH3), 2.60 (s,
1H, NH, exchangeable with D2O), 7.46-7.80 (m, 5 H,
CHarom), 8.50 (s, 1H, CH) ppm; 13C NMR (125 MHz,
CDCl3): δ = 13.9 (CH3), 22.9 (COCH3), 107.0, 133.7
(cyclic C═C), 125.8, 127.1, 128.2, 129.3, 138.3 (aromatic,
C─H), 150.7, 151.1 (C═N) ppm. MS (EI, 70 eV): m/z
(%) = 283 (5%) [M]+, 183 (40%)[M-100]; Anal. for
C13H13N7O (283.29): calcd. C, 55.12; H, 4.63; N, 34.61;
found: C, 55.18; H, 4.53; N, 34.45.
1668 (N═N), 1619 (C═C), 1551, 1530 (C═N) cm−1; H
1
NMR (500 MHz, CDCl3): δ = 1.27 (s, 1H, NH, exchange-
able with D2O), 2.39 (s, 3 H, CH3), 2.58, 2.61 (d, 3 H,
CH3), 6.90 (s, 1H, NH), 7.46-7.80 (m, 5 H, CHarom) ppm;
13C NMR (125 MHz, CDCl3): δ = 14.9 (CH3), 36.1 (CH3),
100.0, 156.2 (cyclic C═C), 124.4, 127.1, 128.2, 129.3,
132.5, 138.7 (aromatic, C─H), 145.3 (C═N) ppm. MS (EI,
70 eV): m/z (%) = 257 (40%) [M]+2, 227 (10%) [M-30];
Anal. for C12H13N7 (255.28): calcd. C, 56.46; H, 5.13; N,
38.41; found: C, 56.49; H, 4.99; N, 38.29.
4.5.2 | 3-Methyl-1-phenyl-
4-((2-phenylhydrazono)methyl)-1H-
pyrazol-5-amine (13)
1-(5-Methyl-7-phenyl-3H-pyrazolo[3,4-d][1,2,3]triazin-
4(7H)-ylidene)-2-phenylhydrazine (11c)
Eluent: petroleum ether (60:80ꢀC)/ethyl acetate (60/40,
v/v). Product 11c was separated as orange crystals, yield
45%; m.p. 115ꢀC to 117ꢀC. IR (KBr): ~ν = 3414, 3307 (NH),
Eluent: petroleum ether (60:80ꢀC)/ethyl acetate (50/50,
v/v). Product 13 was separated as yellow crystals, yield
25%; m.p. 156ꢀC to 158ꢀC. IR (KBr): ~ν = 3446 (NH2),
1
1689 (N═N), 1612 (C═C), 1597, 1542 (C═N) cm−1; H
NMR (500 MHz, CDCl3): δ = 2.36 (s, 3 H, CH3), 5.30 (s,
1H, NH, exchangeable with D2O), 7.13-7.69 (m, 10 H,
CHarom) ppm; 13C NMR (125 MHz, CDCl3): δ = 14.2
(CH3), 100.0, 143.8 (cyclic C═C), 115.3, 115.6, 123.8,
127.1, 128.2, 129.3, 138.1 (aromatic, C-H), 145.3, 150.3
(C═N) ppm. MS (EI, 70 eV): m/z (%) = 317 (10%) [M]+;
Anal. for C17H15N7 (317.35): calcd. C, 64.34; H, 4.76; N,
30.90; found: C, 64.32; H, 4.74; N, 30.89.
3322 (NH), 1612 (C═C), 1598, 1583 (C═N) cm−1 1H
;
NMR (500 MHz, CDCl3): δ = 2.29 (s, 3 H, CH3), 2.70 (s,
1H, NH, exchangeable with D2O), 7.20 (s, 2H, NH2,
exchangeable with D2O), 7.28-7.80 (m, 10 H, CHarom),
8.85 (s, 1H, CH) ppm; 13C NMR (125 MHz, CDCl3):
δ = 13.8 (CH3), 98.0, 142.2 (cyclic C═C), 115.7, 125.8,
127.1, 128.2, 129.1, 129.3, 138.7, 144.4 (aromatic, C─H),
144.8, 153.6 (C═N) ppm. MS (EI, 70 eV): m/z (%) = 291
(5%) [M]+; Anal. for C17H17N5 (291.35): calcd. C,
70.08; H, 5.88; N, 24.04; found: C, 70.10; H, 5.90; N,
24.02.
4.5 | Synthesis of compounds 12 and 13
A mixture of compound 1 (5 mmol) hydrazine hydrate
and/or phenyl hydrazine in acetic acid (25 mL) was
stirred for 3 to 5 hours (TLC monitoring, Rf = 0.51 (ethyl
acetate/cyclohexane = 30/70)). The reaction mixture was
poured on ice cold water to give brown precipitate, which
was filtered, washed with cold water, and recrystallized
from acetone to give product 12, and/or the residual
material was chromatographed on silica gel column to
give compounds 10c, 11c, and 13 in case of reacting com-
pound 1 with phenyl hydrazine. Compounds 10c (40%
4.6 | Reaction of 1 with primary amines
(4d,e)
A mixture of compound 1 (5 mmol) and the appropriate
amine, namely, aniline, and/or p-bromo aniline
(10 mmol) in absolute ethanol (25 mL) was refluxed for
2 to 4 hours (TLC monitoring, Rf = 0.44 (ethyl
acetate/petroleum ether (60:80ꢀC) = 50/50)). The reaction
mixture was evaporated under reduced pressure, and the

10
EWIES ET AL.
residual material was chromatographed on silica gel col-
umn to give products 14a,b and 15a,b.
125.2, 125.8, 127.9, 128.2, 129.3, 129.9, 130.3, 138.5 (aro-
matic, C-H), 142.2, 156.3 (C═N) ppm. MS (EI, 70 eV):
m/z (%) = 274 (100%) [M]−2; Anal. for C17H16N4 (276.34):
calcd. C, 73.89; H, 5.84; N, 20.27; found: C, 73.90; H,
5.89; N, 20.30.
4.6.1 | N-((5-Azido-3-methyl-1-phenyl-
1H-pyrazol-4-yl)methylene)benzenamine
(14a)
4.6.4 | 4-((4-Bromophenylimino)methyl)-
3-methyl-1-phenyl-1H-pyrazol-5-amine
(15b)
Eluent: petroleum ether (60:80ꢀC)/ethyl acetate (93/7,
v/v). Product 14a was separated as yellow crystals,
yield 15%; m.p. 68-70 (60:80ꢀC) C (lit. 68ꢀC)15. IR (KBr): ~ν
= 2225 (N3), 1551, 1503 (C═N) cm−1
;
1H NMR
Eluent: petroleum ether (60:80ꢀC)/acetone (90/10, v/v).
Product 15b was separated as golden crystals, yield 85%;
m.p. 177ꢀC to 179ꢀC. IR (KBr): ~ν = 3317 (NH2), 2192
(500 MHz, CDCl3): δ = 2.50 (s, 3 H, CH3), 7.50-8.01 (m,
10 H, CHarom), 8.60 (s, 1 H, CH) ppm; 13C NMR
(125 MHz, CDCl3): δ = 13.9 (CH3), 110.5, 138.7 (C═C),
121.9, 122.6, 124.7, 125.5, 128.2, 129.3, 129.9, 130.4, 140.2
(aromatic, C─H), 151.4, 153.8 (C═N) ppm. MS (EI,
70 eV): m/z (%) = 304 (15%) [M + 2]+, 274 (35); Anal. for
C17H14N6 (302.33): calcd.: C, 67.54; H, 4.67; N, 27.80;
found: C, 67.45; H, 4.72; N, 27.70.
(N3), 1596, 1540 (C═N) cm−1 1H NMR (500 MHz,
;
CDCl3): δ = 1.62 (s, 2H, NH2, exchangeable with D2O),
2.39 (s, 3 H, CH3), 5.94 (s, 1H, CH), 6.81-7.46 (m, 9H,
CHarom) ppm; 13C NMR (125 MHz, CDCl3): δ = 13.1
(CH3), 98.7, 151.9 (cyclic C═C), 119.5, 120.0, 123.8, 124.4,
125.8, 128.2, 129.3, 132.0, 138.6, 142.2 (aromatic, C─H),
154.4, 155.9 (C═N) ppm. MS (EI, 70 eV): m/z (%) = 354
(2%), 356 (3%) [M]+2; Anal. for C17H15BrN4 (355.23):
calcd. C, 57.48; H, 4.26; Br, 22.49; N, 15.77; found: C,
57.50; H, 4.25; Br, 22.50; N, 15.73.
4.6.2 | N-((5-Azido-3-methyl-1-phenyl-
1H-pyrazol-4-yl)methylene)-
4-bromobenzenamine (14b)
Eluent: petroleum ether (60:80ꢀC)/ethyl acetate (90/10,
v/v). Product 14b was separated as yellow crystals,
yield 10%; m.p. 163-165 (60:80ꢀC) C. IR (KBr): ~ν
4.7 | Reaction of 5-azido-3-methyl-
1-phenyl-1H-pyrazole-4-carbaldehyde
(1) with p- and o-phenylenediamine (4g,f)
= 2214 (N3), 1595, 1539 (C═N) cm−1
;
1H NMR
(500 MHz, CDCl3): δ = 2.49 (s, 3 H, CH3), 7.22-8.04 (m,
9 H, CHarom), 9.13 (s, 1 H, CH)ppm; 13C NMR (125 MHz,
CDCl3): δ = 13.9 (CH3), 108.3, 140.7 (C═C), 122.6, 122.7,
123.9, 124.2, 128.2, 129.3, 129.6, 131.2, 138.3 (aromatic,
C─H), 151.4, 154.5 (C═N) ppm. MS (EI, 70 eV): m/z
(%) = 381 (10%), 382 (8%), 383 (4%) [M]+; Anal. for
C17H13BrN6 (381.23): calcd.: C, 53.56; H, 3.44; Br,
20.96; N, 22.04; found: C, 53.55; H, 3.43; Br, 20.99; N,
22.14.
4.7.1 | General procedure
o- or p-Phenylenediamine (4g or 4f) (0.66 g, 5 mmol)
was added to a stirred solution of compound 1 (2.27 g,
10 mmol) in 20-mL ethanol. The mixture was stirred
for 5 to 7 hours (TLC-controlled, Rf = 0.44 (ethyl
acetate/petroleum ether (60:80ꢀC) = 50/50)). The reac-
tion mixture was evaporated under reduced pressure,
and the residual material was chromatographed on
silica gel column to give products 16 and 17 when we
used 4g and give 18 when we used 4f.
4.6.3 | 3-Methyl-1-phenyl-
4-((phenylimino)methyl)-1H-pyrazol-
5-amine (15a)
N-1-((5-Azido-3-methyl-1-phenyl-1H-pyrazol-4-yl)
methylene)benzene-1,2-diamine (16)
Eluent: petroleum ether (60:80ꢀC)/acetone (80/20, v/v).
Product 15a was separated as orange crystals, yield 75%;
m.p. 148ꢀC to 150ꢀC. IR (KBr): ~ν = 3290 (NH2), 2211
Eluent: petroleum ether (60:80ꢀC)/ethyl acetate (95/5,
v/v). Product 16 was separated as orange crystals, yield
5%; m.p. 217ꢀC to 219ꢀC. IR (KBr): ~ν = 3440 (NH2),
(N3), 1596, 1543 (C═N) cm−1 1H NMR (500 MHz,
;
1
CDCl3): δ = 2.39 (s, 3 H, CH3), 2.80 (s, 2H, NH2,
exchangeable with D2O), 6.10 (s, 1H, CH), 6.92-7.75 (m,
10 H, CHarom) ppm; 13C NMR (125 MHz, CDCl3):
δ = 13.9 (CH3), 100.0, 151.6 (cyclic C═C), 121.2, 124.7,
2225 (N3), 1597, 1551 (C═N) cm−1; H NMR (500 MHz,
CDCl3): δ = 1.64 (s, 2H, NH2, exchangeable with D2O),
2.75 (s, 3 H, CH3), 7.28-7.59 (m, 9 H, CHarom), 9.60 (s,
1 H, CH) ppm; 13C NMR (125 MHz, CDCl3): δ = 14.5

EWIES ET AL.
11
(CH3), 108.5, 142.4 (C═C), 117.1, 124.7, 125.8, 127.1,
128.2, 129.3, 129.7, 131.9, 139.8, 145.4 (aromatic, C-H),
151.4, 156.2 (C═N) ppm. MS (EI, 70 eV): m/z (%) = 315
(15%) [M-2H]+, Anal. for C17H15N7 (317.35): calcd. C,
64.34; H, 4.76; N, 30.90; found: C, 64.36; H, 4.75; N,
30.86.
1-((5-Azido-3-methyl-1-phenyl-1H-pyrazol-4-yl)
methylene)-2-(3-methylbenzo[d]thiazol-2(3H)-ylidene)
hydrazine (19a)
Product 19a was separated as yellow crystals, yield 65%;
m.p. 210ꢀC to 212ꢀC. IR (KBr): ~ν = 2225 (N3), 1616
(C═C), 1572, 1545 (C═N) cm−1 1H NMR (500 MHz,
;
CDCl3): δ = 2.34 (s, 3 H, CH3), 3.69 (s, 3 H, CH3),
7.12-8.49 (m, 9 H, CHarom), 8.52 (s, 1 H, CH) ppm; 13C
NMR (125 MHz, CDCl3): δ = 14.2 (CH3), 29.4 (CH3),
109.0, 139.5 (C═C), 112.6, 122.7, 123.1, 124.3, 124.5,
127.1, 128.2, 129.3, 132.5 138.3 (aromatic, C─H), 151.4,
152.5, 166.6 (C═N) ppm. MS (EI, 70 eV): m/z (%) = 388
(2%) [M]+, Anal. for C19H16N8S (388.45): calcd. C,
58.75; H, 4.15; N, 28.85; S, 8.25; found: C, 58.77; H,
4.13; N, 28.90; S, 8.20.
2-(5-Azido-3-methyl-1-phenyl-1H-pyrazol-4-yl)-1H-
benzo[d]imidazole (17)
Eluent: petroleum ether (60:80ꢀC)/ethyl acetate (93/7,
v/v). Product 17 was separated as colorless crystals, yield
85%; m.p. 235ꢀC to 237ꢀC. IR (KBr): ~ν = 3272 (NH), 2237
(N3), 1594, 1543 (C═N) cm−1 1H NMR (500 MHz,
;
CDCl3): δ = 2.90 (s, 3 H, CH3), 7.22-8.04 (m, 9 H,
CHarom), 9.94 (s, 1H, NH, exchangeable with D2O) ppm;
13C NMR (125 MHz, CDCl3): δ = 14.5 (CH3), 90.1, 146.3
(C═C), 115.4, 118.5, 123.1, 124.4, 126.2, 128.9, 137.4,
139.6, 151.4 (aromatic, C-H), 166.1 (C═N) ppm. MS (EI,
70 eV): m/z (%) = 315 (2%) [M]+, Anal. for C17H13N7
(315.33): calcd. C, 64.75; H, 4.16; N, 31.09; found: C,
64.65; H, 4.20; N, 31.10.
N-((5-Azido-3-methyl-1-phenyl-1H-pyrazol-4-yl)
methylene)pyridin-4-amine (19b)
Product 19b was separated as yellow crystals, yield 82%;
m.p. 169ꢀC to 171ꢀC. IR (KBr): ~ν = 2226 (N3), 1615
(C═C), 1628, 1598, 1562 (C═N) cm−1
;
1H NMR
(500 MHz, CDCl3): δ = 2.36 (s, 3 H, CH3), 7.40-8.42 (m,
9 H, CHarom), 9.13 (s, 1 H, CH) ppm; 13C NMR
(125 MHz, CDCl3): δ = 14.3 (CH3), 105.4, 142.4 (C═C),
114.7, 122.9, 123.9, 126.8, 129.4, 137.1, 138.8, 149.2 (aro-
matic, C-H), 149.9, 161.4 (C═N) ppm. MS (EI, 70 eV):
m/z (%) = 303 (10%) [M]+; Anal. for C16H13N7 (303.32):
calcd. C, 63.36; H, 4.32; N, 32.32; found: C, 63.38; H,
4.23; N, 32.35.
N-1-((5-Azido-3-methyl-1-phenyl-1H-pyrazol-4-yl)
methylene)benzene-1,4-diamine (18)
Eluent: petroleum ether (60:80ꢀC)/ethyl acetate (90/10,
v/v). Product 18 was separated as deep red crystals, yield
88%; m.p. 173ꢀC to 175ꢀC. IR (KBr): ~ν = 3433 (NH2),
2141 (N3), 1616 (C═C), 1590, 1545 (C═N) cm−1; 1H NMR
(500 MHz, CDCl3): δ = 2.47 (s, 3 H, CH3), 6.20 (s, 2H,
NH2, exchangeable with D2O), 6.63-8.04 (m, 10 H,
CHarom), 8.78 (s, 1 H, CH) ppm; 13C NMR (125 MHz,
CDCl3): δ = 13.8 (CH3), 109.3, 138.6 (C═C), 118.7, 119.7,
123.1, 124.4, 125.8, 128.2, 129.3, 140.7 (aromatic, C-H),
148.9, 156.2 (C═N) ppm. MS (EI, 70 eV): m/z (%) = 317
(2%) [M]+, Anal. for C17H15N7 (317.35): calcd. C,
64.34; H, 4.76; N, 30.90; found: C, 64.35; H, 4.73;
N, 30.95.
4.9 | Reaction of 1 with heterocyclic
amines (4j,k)
4.9.1 | General procedure
Antipyrine 4j or aminorhodanine 4k (5 mmol) was added
to a stirred solution of compound 1 (2.27 g, 10 mmol) in
20-mL ethanol. The mixture was stirred for 5 to 7 hours
(TLC controlled, Rf = 0.22 (ethyl acetate/petroleum ether
(60:80ꢀC) = 50/50)). The precipitate formed was collected
by filtration and recrystallized from ethanol to give com-
pounds 20c or 20d.
4.8 | Reaction of 1 with heterocyclic
amines (4h-k)
4.8.1 | General procedure
Benzothiazole hydrazine 4h and/or 4-amino pyridine 4i
(5 mmol) was added to a stirred solution of compound
1 (2.27 g, 10 mmol) in 20-mL glacial acetic acid. The
4-((5-Azido-3-methyl-1-phenyl-1H-pyrazol-4-yl)
methyleneamino)-1,2-dihydro-1,5-dimethyl-
2-phenylpyrazol-3-one (19c)
mixture was refluxed for
2
to
5
hours (TLC
Product 19c was separated as yellow crystals, yield 66%;
m.p. 156ꢀC to 158ꢀC. IR (KBr): ~ν = 2207 (N3), 1593, 1548
controlled, Rf = 0.40 (ethyl acetate/petroleum ether
(60:80ꢀC) = 50/50)). The reaction mixture after cooling
was poured on ice water, the precipitate formed was col-
lected by filtration and recrystallized from ethanol to give
compound 19a,b.
1
(C═N) cm−1; H NMR (500 MHz, CDCl3): δ = 2.39 (s,
3 H, CH3), 2.60 (s, 3 H, CH3), 3.28 (s, 3 H, CH3), 7.21-7.67
(m, 10 H, CHarom), 8.00 (s, 1 H, CH) ppm; 13C NMR
(125 MHz, CDCl3): δ = 13.1, 14.3, 39.5 (CH3), 105.4,

12
EWIES ET AL.
142.4, 110.1, 150.2 (C═C), 122.9, 123.9, 126.8, 129.4,
133.1, 139.8 (aromatic, C─H), 149.9 (C═N), 160.8 (C═O),
163.4 (C═N) ppm. MS (EI, 70 eV): m/z (%) = 412 (2%)
[M]+; Anal. for C22H20N8O (412.45): calcd. C, 64.07; H,
4.89; N, 27.17; found: C, 64.07; H, 4.89; N, 27.17.
expressed as a percentage of the control SD established
from n = 4 wells per experiment from three independent
experiments.[29,30]
5.2 | MIC tests
3-((5-Azido-3-methyl-1-phenyl-1H-pyrazol-4-yl)
methyleneamino)-2-thioxothiazolidin-4-one (19d)
Product 19d was separated as red crystals, yield 78%; m.
p. 202ꢀC to 204ꢀC. IR (KBr): ~ν = 2212 (N3), 1673 (C═O),
The minimum inhibitory concentration (MIC) test was
performed by means of broth dilution method in a
96-well microtiter plate (https://www.nature.com/
articles/ncomms13803#t1). Test compound was diluted
with DMSO in the range from 512 to 1 μg/mL and using
Streptomycin and Penicillin G as a standard antibiotic. A
series of two-fold dilution of sample was dispersed in the
columns of wells. Finally, 100 μL of bacterial suspension
prepared equivalent to 0.5 McFarland units with sterile
saline was added to each well and incubated at 37ꢀC for
24 hours and recorded at 600 nm using an ELISA reader
(Anthoslabtec Instrument, Salzburg, Austria).[31–33]
1
1598, 1526 (C═N), 1071 (C═S), 694 (C-S) cm−1; H NMR
(500 MHz, CDCl3): δ = 2.44 (s, 3 H, CH3), 4.13 (s, 2 H,
CH2),7.29-8.08 (m, 9 H, CHarom), 9.32 (s, 1 H, CH) ppm;
13C NMR (125 MHz, CDCl3): δ = 14.3 (CH3), 40.3 (CH2),
105.3, 142.6 (C═C), 123.9, 126.8, 129.4, 137.1, 138.8 (aro-
matic, C─ H), 143.9, 149.4 (C═N), 172.1 (C═O), 197.1
(C═S) ppm. MS (EI, 70 eV): m/z (%) = 357 (10%) [M]+;
Anal. for C14H11N7OS2 (357.41): calcd. C, 47.05; H,
3.10; N, 27.43; S, 17.94; found: C, 47.10; H, 3.08; N,
27.40; S, 17.98.
6 | CONCLUSION
5 | BIOASSAY MATERIALS
A series of azidopyrazole derivatives was prepared by the
The used cell lines and bacteria were obtained from the
American Type Culture Collection (ATCC, Manassas,
VA, USA). RPMI 1640 medium, fetal bovine serum (FBS),
trypan blue, penicillin G, and streptomycin were
obtained from Gibco BRL (Gaithersburg, MD, USA).
Mueller Hinton Broth was obtained for HIMEDIA
reaction
of
5-azido-3-methyl-1-phenyl-1H-pyrazole-
4-carbaldehyde (azidopyrazole) with primary amines and
methylene compounds. The prepared compounds were
obtained in medium to good yields. The reaction mecha-
nism proceeded through the formation of an imine deriv-
ative. The evaluation of the cytotoxic activity of some of
the prepared compounds indicated that 3-((5-azido-
3-methyl-1-phenyl-1H-pyrazol-4-yl)methylene)pentane-
2,4-dione (5b) was the most active compound against
Molt 4, U937, Sup-T1, and 562 cells with IC50 10.45,
18.47, 8.41, and 10.56 μg/mL, respectively, after 72 hours.
The prepared compounds showed weak or no activity
S. aureus, E. coli, and P. aeruginosa, suggesting selective
toxicity towards cancer cell lines.
Laboratories
(India).
3-(4,5-Dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide (MTT), dimethyl sulfox-
ide (DMSO), and doxorubicin were obtained from Sigma-
Aldrich (St Louis, MO, USA).
5.1 | Cytotoxic assay (MTT proliferation
assay)
The cytotoxic activity of the prepared derivatives was
evaluated using MTT proliferation assay. Cells were
seeded at 4 × 104 per well in 96-well culture plates before
treatment with different concentrations of the tested com-
pound. After treatment for 72 hours, the cytotoxicity of
the tested compound was determined using the MTT cell
proliferation assay (thiazolyl blue tetrazolium bromide,
ACKNOWLEDGMENTS
The authors thank the National Research Centre for the
financial support (project no: 11090112) and for the facili-
ties provided and the Kaohsiung Medical University
Hospital (KMUH104-4M12, KMUH105-5M13, and
KMUH-106M13 [Yi-Chang Liu]) Ministry of Health
Sigma-M2128).
Light
absorbance
values
and
Welfare
(MOHW106-TDU-B-212-113006,
(OD = OD570 − OD620) were recorded at 570 and 620 nm
using an ELISA reader (Anthoslabtec Instrument, Salz-
burg, Austria) for calculating the concentration that cau-
sed 50% inhibition (IC50), ie, the cell concentration at
which the light absorbance value of the experimental
group is half that of the control group. These results were
MOHW107-TDU-B-212-123006 [Yi-Chang Liu]) for
financially supporting the work.
ORCID
Ewies F. Ewies https://orcid.org/0000-0002-4832-5766

EWIES ET AL.
13
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