Synthesis, structure, and antibacterial evaluation of new N-substituted-3-amino- 5-oxo-4-phenyl-2,5-dihydro-1 H-pyrazole-1-carbothioamides

Article abstract of DOI:10.1002/hc.20598

Novel N-substituted-3-amino-5-oxo- 4-phenyl-2,5-dihydro-1 H-pyrazole-1-carbothioamide derivatives were synthesized by means of two methods. First is the cyclization reaction of 1- (cyanophenyl)acetyl-4- substituted thiosemicarbazide, and the second one is

Full text of DOI:10.1002/hc.20598

Heteroatom Chemistry
Volume 21, Number 4, 2010
Synthesis, Structure, and Antibacterial
Evaluation of New N-Substituted-3-Amino-
5-oxo-4-phenyl-2,5-dihydro-1H-pyrazole-1-
carbothioamides
Monika Pitucha,1 Liliana Mazur,2 Urszula Kosikowska,3
Anna Pachuta-Stec,1 Anna Malm,3 Łukasz Popiołek,1
and Zofia Rza˛czyn´ska2
1Department of Organic Chemistry, Faculty of Pharmacy, Medical University, Staszica 6,
20-081 Lublin, Poland
2Department of General and Coordination Chemistry, Maria Curie-Słodowska University,
M. Curie-Skłodowska 3, 20-031 Lublin, Poland
3Department of Pharmaceutical Microbiology, Medical University, Chodz´ki 1,
20–093 Lublin Poland
Received 16 July 2009; revised 15 March 2010
and nonnatural products. The cyclization reaction
of suitable linear compounds is one of the most
common and popular methods for preparing these
derivatives. Some asymmetrical ureas have been cy-
clized to produce several heterocycles, such as 1,3,4-
thiadiazoles, 1,2,4-triazoles, and 1,3,5-triazines. The
cyclization of 2,4-disubstituted thiosemicarbazides
has been shown to be an excellent strategy for the
synthesis of pyrazoles [1,2]. Much attention has been
focused on these compounds as antiviral and an-
ticancer agents after the discovery of the pyrazole
C-glycoside pyrazofurin [3]. In addition, pyrazoles
have a broad spectrum of biological activities, such
as inflammatory [4], antipyretic [5], antidepressant
[6], anticonvulsant [7], antifilarial agents [8], and
antibacterial [9,10]. Variously substituted pyrazoles,
like 4-acyl-5-hydroxy-3-methyl-1H-pyrazole (or its
tautomers), are used as the chelating and extract-
ing reagents for many metal ions [11] and as start-
ing materials for the synthesis of biologically active
compounds as well as for the construction of con-
densed heterocyclic systems.
ABSTRACT: Novel N-substituted-3-amino-5-oxo-
4-phenyl-2,5-dihydro-1H-pyrazole-1-carbothioamide
derivatives were synthesized by means of two
methods. First is the cyclization reaction of 1-
(cyanophenyl)acetyl-4-substituted thiosemicarbazide,
and the second one is reaction of cyanophenyl acetic
acid hydrazide with isothiocyanate. Structures of new
compounds were confirmed by elemental analysis, 1H
NMR, and X-ray diffraction analysis. Biological eval-
uation showed that some of them possess promising
ꢀ
C
antibacterial activities.
2010 Wiley Periodicals, Inc.
Heteroatom Chem 21:215–221, 2010; Published online in
Wiley InterScience (www.interscience.wiley.com). DOI
10.1002/hc.20598
INTRODUCTION
Nitrogen heterocycles are of synthesis interest be-
cause they constitute an important class of natural
Correspondence to: Monika Pitucha; e-mail: monika.pitucha@
In view of these observations and in continu-
ation of our research program on the synthesis of
am.lublin.pl.
c
ꢀ
2010 Wiley Periodicals, Inc.
215

216 Pitucha et al.
five-membered heterocyclic compounds [12–14], we
present herein the synthesis, characterization, and
the antibacterial evaluation of representative com-
pounds of N-substituted-3-amino-5-oxo-4-phenyl-
2,5-dihydro-1H-pyrazole-1-carbothioamides.
RESULTS AND DISCUSSION
Chemistry
The synthesis of 1-(cyanophenyl)acetyl-4-substitu-
ted
thiosemicarbazide
and
N-substituted-
FIGURE 1 The molecular structure of compound 3a with
the atom-labeling scheme. The displacement ellipsoids are
drawn at the 50% probability level.
3-amino-5-oxo-4-phenyl-2,5-dihydro-1H-pyrazole-1-
carbothioamide derivatives is outlined in Schemes 1
and 2. Compound 1 was prepared by means of a
known procedure in the reaction of corresponding
ethyl cyanophenyl acetate with 80% hydrazine hy-
drate, at the room temperature, giving cyanophenyl
acetic acid hydrazide 1 [15]. It was used as a key
intermediate for the synthesis of final compounds.
The treatment of compound 1 with ethyl and 4-
methoxyphenyl isothiocyanate gave corresponding
In the case of cyclohexyl isothiocyanate, we
also expected to obtain thiosemicarbazide but
the reaction went quite different. During this
reaction, N-cyclohexyl-3-amino-5-oxo-4-phenyl-2,5-
dihydro-1H-pyrazole-1-carbothioamide 3c was ob-
tained. The reaction led to cyclic compounds with-
out the separation of mediate product (Scheme 2).
The mechanism of the reaction will be further
investigated.
1-(cyanophenyl)acetyl-4-substituted
thiosemicar-
bazide 2a, 2b. The reaction was carried out by
heating substrates in an oil bath. The conditions
of the reactions were established experimentally.
The cyclization of these compounds in alkaline
2a and acid medium 2b gave a new group of
N-substituted-3-amino-5-oxo-4-phenyl-2,5-dihydro-
1H-pyrazole-1-carbothioamide derivatives 3a, 3b
(Scheme 1).
Structure
Figure 1 shows the molecular structure of compound
3a, together with the atomic numbering scheme
O
O
O
NCS
R
C
NH
NH C
NH R
S
C
NH
NH2
C
OC2H5
NH NH
2
2
HC
HC
HC
C H OH
2
5
CN
CN
CN
1
2a,2b
NaOH
HCl
H
N
H2N
NH
C
R
N
C
C
C
O
S
3a,3b
R = C2H5, 4-CH3OC6H4
SCHEME 1
Heteroatom Chemistry DOI 10.1002/hc

Synthesis, Structure, andAntibacterialEvaluationofNew N-Substituted3-Amino-5-oxo-4-phenyl-2,5-dihydro-1H-pyrazole-1-carbothioamides 217
O
O
C
NH
NH2
C
NH
NH C
NH C6H11
NCS
HC
+
HC
C6H11
CN
CN
S
1
H
N
H2N
NH
C
C6H11
N
C
C
C
S
O
3c
SCHEME 2
used. Selected bond distances and torsion angles are
given in Table 1.
Cambridge Structural Database (CSD) [20]. How-
ever, the whole molecule is not planar; the pyra-
zole part forms dihedral angle of 61.3(2)◦ with the
phenyl C1f–C6f ring plane, whereas the ethyl C7
atom is -sc oriented with respect to the thioamide
C1 atom, as indicated by the torsion angle C1–N4–
C6–C7 (Table 1). In the crystal structure, molecules
of 3a are linked by short intermolecular N2–H2/N3–
H3a· · ·O1 and C7–H7c· · ·S1 hydrogen bonding in-
teractions (Table 2) into one-dimensional extended
chains along the a axis (Fig. 2).
Centrosymmetrically related chains are further
stabilized by weak C6–H6a· · ·O1 hydrogen bonds
and aromatic π–π stacking interactions occurring
between adjacent phenyl rings. The details of the
crystal data, X-ray data collection, and refinement
are given in Table 3.
The structural research indicates that the com-
pound 3a adopts the keto-thione form. The main
structural parameters of the molecule are similar
to those found in closely related 5-oxo-1H-pyrazole-
1-carbothioamide derivatives [16–19] in particular;
the S atom of the carbothioamide group is cis ori-
ented with respect to the protonated endocyclic N2
atom. This arrangement enables the formation of
short intramolecular N4-H1n4· · ·O1 hydrogen bond,
which additionally stabilizes the conformation of the
molecule. The central pyrazolone fragment in 3a is
essentially planar, with maximum mean plane devi-
˚
ation of 0.035(4) A for atom N2, and it is coplanar
with the amine N3 atom and the carbothioamide
group, with an angle of 1.4(3)◦ between carboth-
ioamide and pyrazole ring planes. Moreover, the flat
thioamide group is conjugated with pyrazole ring,
as evidenced by shortening of the N1–C5 and N1–
C1 bonds in comparison with average values from
Biological Activity
The newly synthesized compounds were screened
for their antimicrobial activity against 10 reference
strains of aerobic bacteria: 6 Gram-positive (Staphy-
lococcus aureus ATCC 25923, S. aureus ATCC 6538,
S. epidermidis ATCC 12 228, Bacillus subtilis ATCC
◦
˚
TABLE 1 Selected Bond Lengths (A) and Torsion Angles ( )
˚
Bond lengths (A)
N1–N2
N2–C3
C3–C4
C4–C5
N1–C5
N3–C3
1.387 (2)
C5–O1
N1–C1
C1–S1
C1–N4
N4–C6
C6–C7
1.244 (2)
1.381 (2)
1.667 (2)
1.321 (2)
1.452 (3)
1.500 (3)
◦
˚
TABLE 2 Hydrogen-Bonding Geometry (A, )
1.344 (3)
1.387 (2)
1.406 (3)
1.412 (2)
1.333 (3)
D–H
H· · ·A
D· · ·A
<DHA
N4-H1n4· · ·O1
N2-H2· · ·O1i
0.84
0.86
0.86
0.96
0.92
2.06
2.28
2.46
2.83
2.67
2.734 (3)
2.928 (3)
3.119 (3)
3.578 (3)
3.475 (3)
137
133
134
136
141
Torsion angles (◦)
N2–N1–C1–N4 −179.3 (2) S1–C1–N4–C6
N2–N1–C1–S1 1.8 (2) C1–N4–C6–C7
N3-H3a· · ·O1i
C7-H7c· · ·S1ii
C6-H6a· · ·O1iii
−2.3 (3)
−87.1 (2)
N1–C1–N4–C6 178.9 (2)
Symmetry codes: (i) x − 1, y, z; (ii) x + 1, y, −z; (iii) 2 − x, −y, 2 − z
Heteroatom Chemistry DOI 10.1002/hc

218 Pitucha et al.
TABLE 4 Antibacterial activity of compounds 3a and 2b
assessed by agar dilution method (adm) and broth microdilu-
tion method (bmm)
MIC (mg L−1
)
3a
adm bmm
7.82 15.63 62.5
2b
Bacterial strains
adm
bmm
62.5
62.5
125
Staphylococcus aureus
ATCC 25923
S. aureus
ATCC 6538
S. epidermidis
ATCC 12228
Bacillus subtilis
ATCC 6633
B. cereus
ATCC 10876
Micrococcus luteus
ATCC 10240
7.82 31.25
125
7.82
7.82
7.82 62.5
3.91 62.5
62.5
62.5
31.25
7.82 31.25 62.5
7.82 15.63 62.5
Escherichia coli
ATCC 25922
Klebsiella pneumoniae
ATCC 13883
Proteus mirabilis
ATCC 12453
250 62.5
>1000 >1000
250
250
250 >1000 >1000
250 >1000 >1000
FIGURE 2 The packing arrangement of molecules, viewed
along the c axis. Dashed lines indicate hydrogen bonds.
Pseudomonas aeruginosa 1000 1000 >1000 >1000
ATCC 9027
TABLE 3 Crystal Data and Structure Refinement for 3a
6633, B. cereus ATCC 10876, Micrococcus luteus
ATCC 10240) and 4 Gram-negative (Escherichia coli
ATCC 25922, Proteus mirabilis ATCC 12453, Kleb-
siella pneumoniae ATCC 13883, Pseudomonas aerug-
inosa ATCC 9027). The in vitro antimicrobial activ-
ities of substances at concentrations ranging from
3.91 to 1000 mg L−1 were preliminary tested. Using
the agar dilution method, it was found that com-
pound 3a possessed good activity against all tested
Gram-positive bacteria with minimal inhibitory con-
centration (MIC) = 7.82 mg L−1; the growth of Gram-
negative bacteria was inhibited by much higher con-
centration with MIC = 250 mg L−1 to 1000 mg L−1
(Table 4).
Compound 2b also showed good activity against
all Gram-positive bacteria but with MIC values
higher that those noted in the case of 3a compound;
no inhibitory effect of this compound was found
against the tested Gram-negative bacteria (MIC >
1000 mg L−1). Compound 3c (data not shown) inhib-
ited growth of Gram-positive bacteria with MIC =
500–1000 mg L−1, being without the effect on
growth of Gram-negative bacteria (MIC > 1000 mg
L−1). In our experiments, MICs of available an-
tibiotics such as ampicillin, cefuroxim, and gen-
tamicin, which have been extensively used to treat
of bacterial infections, were also estimated. The
range of MICs of ampicillin was 0.03–0.06 mg L−1
Empirical formula
CCDC number
Formula weight
Wavelength
Crystal system
Space group
C12H14N4OS
719663
262.33
0.71073
Triclinic
P-1
Unit cell dimensions
6.276 (3)
˚
a, b, c (A)
10.204 (4)
10.513 (6)
79.01 (5)
78.48 (5)
79.96 (4)
641.1 (5)
2
α, β, γ (◦)
3
˚
V (A )
Z
Density (calculated)
1.359
(g cm−3
)
Crystal size (mm)
0.58 × 0.38 × 0.18
Absorption coefficient
0.246
(mm−1
)
F(000)
Index ranges
276
−8 ≤ h ≤ 8, −14 ≤ k ≤ 14, 0
≤ l ≤ 14
3761 [R(int) = 0.024]
2236
Independent reflections
Reflections with I > 2σ(I )
Data/restrains/parameters
GOOF
3761/0/167
1.002
0.998
completeness to θ
=
max
30.07◦
Final R indices [I > 2σ(I )]
R indices (all data)
R1 = 0.0477, wR2 = 0.1203
R2 = 0.0976, wR2 = 0.1423
−0.37; 0.35
Largest diff. peak and hole
−3
˚
(e A
)
Heteroatom Chemistry DOI 10.1002/hc

Synthesis, Structure, andAntibacterialEvaluationofNew N-Substituted3-Amino-5-oxo-4-phenyl-2,5-dihydro-1H-pyrazole-1-carbothioamides 219
TABLE 5 Antibacterial activity of compound 3a against clin-
ical isolates of Staphylococcus aureus assessed by broth mi-
crodilution method
EXPERIMENTAL
All chemicals were purchased from Merck Co.
or Lancaster (Gdan´sk, Poland) and used without
further purification. Melting points (◦C) were deter-
mined in a Fisher–Johns block and were not cor-
rected. Elemental analysis was made on Perkin–
Elmer 2400 CHN analyzer, and the data were within
Parameter
MIC (mg L−1
)
Range of MIC (number of isolates)
15.63 (2)
31.25 (6)
62.50 (2)
31.25
0.4% of the theoretical values. H and 13C NMR
1
Mode of MIC
spectra were recorded on a Bruker AC 200F instru-
ment (300 MHz) in DMSO-d6 with TMS as internal
standard. IR spectra were recorded in KBr disk using
a Specord IR-75 spectrophotometer. The mass spec-
tra were obtained with an AMD-604 mass spectrom-
eter a 70-eV electron beam. The purity of obtained
compounds was checked by TLC on aluminum oxide
60 F254 plates (Merck) in a CHCl3/C2H5OH (10:1 and
10:2) solvent system with UV or iodine visualization.
for Staphylococcus species, 0.03–62.5 mg L−1 for
the other Gram-positive bacteria, and 7.81–125 mg
L−1 for Gram-negative Enterobacteriaceae rods; the
range of MICs of cefuroxim was 0.049–1.95 mg L−1
for all tested Gram-positive reference strains and
0.049–7.82 mg L−1 for Enterobacteriaceae species;
the range of MICs of gentamicin for Staphylo-
cocci was 0.015–0.06 mg L−1, 0.015–0.49 mg L−1,
for other Gram-positive species, and 0.24–0.98 mg
L−1 for reference Gram-negative rods. The good an-
tibacterial activities of 3a and 2b compounds were
confirmed by using broth microdilution method.
The difference among the MIC values determined by
the two kinds of methods (the agar dilution proce-
dure and broth microdilution test) is in accordance
with the results communicated by other authors
[21–23]. Moreover, it was found that compound 3a
inhibited the growth of 10 clinical isolates of S. au-
reus with MIC ranging from 15.63 to 62.5 mg L−1;
the mode MIC value was 31.25 mg L−1 (Table 5).
The compound 3a appears to be a promising pre-
cursor of agents with antibacterial activity against
Gram-positive bacteria, both pathogenic (includ-
ing Staphylococcus species) as well as opportunistic
(e.g., Micrococcus luteus) [24]. Although this bacte-
rial species is nonpathogenic and usually regarded
as a contaminant, it should be considered as an
emerging nosocomial pathogen in immunocompro-
mised individuals causing recurrent bacterium, sep-
tic arthritis, and pulmonary diseases. The investiga-
tions on that field will be continued.
Synthesis of 1-(Cyanophenyl)acetyl-4-
substituted Thiosemicarbazide (2)
Cyanophenyl acetic acid hydrazide 1 1.7 g (10 mmol)
and appropriate isothiocyanate (10 mmol) were
heated in an oil bath at 110◦C for 20 h. The formed
product was washed with diethyl ether to remove
the unreacted isothiocyanate, dried, and crystallized
from ethanol.
1-(Cyanophenyl)acetyl-4-ethylthiosemicarbazide,
2a. Yield: 73%, mp 304–305◦C. 1H NMR (DMSO-d6)
δ: 1.19 (t, 3H, J = 7.0 Hz, CH3), 3.62 (q, 3H, J = 6.8
CH2), 5.92 (s, 1H, CH), 7.02–7.70 (m, 4H, CHarom),
9.16, 10.49, 11.02 (3s, 3H, 3NH) ppm. 13C NMR
(DMSO-d6) δ: 13.01 (CH3), 85.87 (CH2), 122.45 (CH),
124.68, 126.74 (CHarom), 127.66 (Carom), 132.26 (CN),
153.51 (C O), 166.84 (C S) ppm. IR (KBr) ν: 3486,
3253, 3149 (NH), 2258 (CN), 1647 (C O), 1181
(C S) cm−1. MS (EI) m/z (%): 262 (87) [M+], 175
(100).
1-(Cyanophenyl)acetyl-4-(4-methoxyphenyl)thio-
1
semicarbazide, 2b. Yield: 71%, mp 184–185◦C. H
NMR (DMSO-d6) δ: 3.32 (s, 3H, CH3), 5.00 (s, 1H,
CH), 6.80–7.50 (m, 9H, CHarom), 8.73, 9.49, 10.48 (3s,
3H, 3NH) ppm. 13C NMR (DMSO-d6) δ: 53.75 (CH3),
85.87 (CH), 122.44, 124.70, 126.71 (CHarom), 132.23
(Carom), 139.20 (CN), 153.60 (C O), 166.70 (C S)
ppm. IR (KBr) ν: 3485, 3305, 3146 (NH), 2111 (CN),
1647 (C O), 1153 (C S) cm−1. MS (EI) m/z (%): 324
(6) [M+], 91 (100).
SUMMARY
The synthesis of N-substituted-3-amino-5-oxo-4-
phenyl-2,5-dihydro-1H-pyrazole-1-carbothioamide
derivatives 3a–3c has been described in this paper.
These compounds were obtained from appropriate
thiosemicarbazides and 1-(cyanophenyl)acetic acid
hydrazide. The final compound 3a was confirmed by
X-ray crystallographic studies. Selected compounds
were screened for their in vitro antibacterial activity.
These results showed that some of them possess
promising antibacterial activities.
Synthesis of N-Ethyl-3-amino-5-oxo-4-phenyl-
2,5-dihydro-1H-pyrazole-1-carbothioamide 3a
The thiosemicarbazide derivative 2a (10 mmol) was
dissolved in 2% NaOH (15 mL) and refluxed for 2 h.
Heteroatom Chemistry DOI 10.1002/hc

220 Pitucha et al.
After cooling, the solution was neutralized with 3
M HCl. The solid formed was filtered, dried, and
crystallized from ethanol. Yield: 68%, mp 210–
stability of intensities was monitored by measure-
ments of three standards every 100 reflections. Crys-
tal structure was solved by direct methods using
SHELXS97 [25] and refined by the full-matrix least
squares on F2 using the SHELXL97 [26]. All non-
hydrogen atoms were refined with anisotropic dis-
placement parameters. The hydrogen atom bonded
to N4 was located from a difference Fourier map and
was refined isotropically. The remaining hydrogen
atoms were positioned geometrically and allowed to
ride on their parent atoms, with Uiso(H) = 1.2 Ueq
(C, N).
1
211◦C. H NMR (DMSO-d6) δ: 1.19 (t, 3H, J = 7.0,
CH3), 3.60 (q, 2H, J = 5.0,CH2), 6.74 (s, 2H, NH2),
7.11–7.55 (m, 5H, CHarom), 10.48 (s, 1H, 1H NH),
11.02 (t, 1H, J = 5.0, NH) ppm. 13C NMR (DMSO-
d6) δ: 12.36 (CH3), 84.90 (CH2), 85.95 (C C), 123.51,
124.82, 126.66, 127.32 (CHarom), 130.08 (Carom),
131.91(CN), 152.74 (C O), 160.58 (C S), 173.02
(C NH2) ppm. IR (KBr) ν: 3479, 3311, 3153 (NH),
2122 (CN), 1647 (C O), 1153 (C S) cm−1. MS (EI)
m/z (%): 262 (40) [M+], 175 (100).
Supplementary Material
Synthesis of N-(4-Methoxyphenyl)-3-amino-5-
oxo-4-phenyl-2,5-dihydro-1H-pyrazole-1-
carbothioamide 3b
The CCDC-719663 for 3a contains the supple-
mentary crystallographic data for this paper.
The data can be obtained free of charge at
www.ccdc.cam.ac.uk/conts/retrieving.html or from
the Cambridge Crystallographic Data Centre
(CCDC), 12 Union Road, Cambridge CB2 1EZ, UK;
fax: +44(0) 1223 336 033; e-mail: deposit@ccdc.cam
.ac.uk].
The thiosemicarbazide derivative 2b (10 mmol) was
dissolved in 3 N HCl (15 mL) and refluxed for 2 h.
After cooling, the formed precipitate was filtered
off and then crystallized from ethanol. Yield: 70%,
1
mp 217–218◦C. H NMR (DMSO-d6) δ: 2.51 (s, 3H,
CH3), 6.80 (s, 2H, NH2), 6.95–7.70 (m, 9H, CHarom),
10.55 (s, 1H, NH), 11.42 (t, 1H, J = 6.0, NH) ppm.
13C NMR (DMSO-d6) δ: 14.86 (CH3), 91.40 (C4),
91.40 (C C), 118.41, 129.05, 130.65 (CHarom), 153.72
(Carom), 156.42 (C O), 163.44 (C S), 168.57 (C NH2)
ppm. IR (KBr) ν: 3485, 3321, 3149 (NH), 1653 (C O),
1151 (C S) cm−1. MS (EI) m/z (%): 337 (25) [M+],
175 (100).
Antimicrobial Screening
Fresh bacterial suspensions with an optical density
of 0.5 McFarland standard [150 × 106 CFU (colony
forming units)/mL], were prepared in sterile saline
(0.85% NaCl) and then were diluted 1:100 in the
Mueller–Hinton broth. All stock solutions of the as-
sayed compounds were prepared in dimethyl sulfox-
ide (DMSO) mixed with distilled water (1:1). It was
found that DMSO at the final concentration in the
medium had no influence on growth of the tested mi-
croorganisms. Ampicillin, cefuroxim, and gentam-
icin at the 0.0075–500 mg L−1 concentration were
used as control antibacterial agents. The agar dilu-
tion procedure was performed using the Mueller–
Hinton agar as the base medium to which serial di-
lutions of the tested compounds at concentrations
from 1.96 to 1000 mg L−1 were added. The plates
were poured on the day of testing. 10 μL of each bac-
terial suspension was put onto the Mueller-Hinton
agar, containing the tested compounds; medium
without the compounds was used as a control. The
plates were incubated at 37◦C for 18 h. The MIC
was defined as the lowest concentration of the tested
compounds that completely inhibited visible growth
of bacteria. Antibacterial activity of the compounds
was also determined spectrophotometrically by mi-
crodilution method with the Mueller–Hinton broth,
containing from 0.49 to 1000 mg L−1 of the tested
compounds. Two-fold dilutions of the tested com-
pounds in the medium in the wells of the microdi-
lution plates were prepared, and then each well
Synthesis of N-Cyclohexyl-3-amino-5-oxo-4-phe-
nyl-2,5-dihydro-1H-pyrazole-1-carbothioamide
3c.
Cyanophenyl acetic acid hydrazide 1 1.7 g (10 mmol)
and cyclohexyl isothiocyanate (10 mmol) in 10 mL
of anhydrous diethyl ether was kept at room tem-
perature for 24 h. Then the formed compound was
filtered off, washed with diethyl ether, and crystal-
1
lized from ethanol. Yield: 65%, mp 223–225◦C. H
NMR (DMSO-d6) δ: 1.03–1.79 (m, 10H, 5×CH2),
4.07 (s, 1H, CH), 5.91 (s, 2H, NH2), 76.99–7.61 (m,
5H, CHarom), 9.06 (s, 1H, NH), 9.16 (s, 1H, NH) ppm.
13C NMR (DMSO-d6) δ: 23.22, 23.74, 30.77, 31.02
(5×CH2), 51.03 (CH), 75.38 (C4), 121.83, 122.33,
123.82, 126.67, 127.53 (CHarom), 132.35(Carom),
153.53 (C O), 166.80 (C S), 78.83 (C–NH2) ppm. IR
(KBr) ν: 3484, 3239, 2927 (NH), 1644 (C O), 1177
(C S) cm−1. MS (EI) m/z (%): 314 (5), 175 (100).
Crystallographic Measurement
Single-crystal diffraction data were measured at
room temperature in the ω/2θ mode on the
Oxford Diffraction Xcalibur diffractometer using
the graphite-monochromated Mo Kα radiation. The
Heteroatom Chemistry DOI 10.1002/hc

Synthesis, Structure, andAntibacterialEvaluationofNew N-Substituted3-Amino-5-oxo-4-phenyl-2,5-dihydro-1H-pyrazole-1-carbothioamides 221
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was inoculated with bacterial suspension with an
optical density of 0.5 McFarland standard diluted
1:100 in the Mueller–Hinton broth (200 μL of to-
tal volume per well). Twenty species of bacteria—
the described above 10 reference strains of Gram-
positive or Gram-negative microorganisms and 10
clinical isolates of S. aureus, were used in this pe-
riod of our experiments. After incubation (37◦C for
18 h), the optical density (OD600) measurements were
determined for bacterial culture in broth medium,
and the MIC value were determined by compar-
ison with the growth of control (compound-free)
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