Synthesis of New Oxindoles and Determination of Their Antibacterial Properties

Article abstract of DOI:10.1155/2020/8021920

A versatile method for the synthesis of new oxindoles was developed by the reaction between substituted isatins and 5-aminopyrazoles. The reaction was carried out at room temperature in ethanol using p-toluenesulfonic acid as the catalyst. The products we

Full text of DOI:10.1155/2020/8021920

Hindawi
Heteroatom Chemistry
Volume 2020, Article ID 8021920, 9 pages
https://doi.org/10.1155/2020/8021920
Research Article
Synthesis of New Oxindoles and Determination of Their
Antibacterial Properties
l
1
1
2
Pablo E. Romo, Braulio Insuasty, Rodrigo Abonia, Mar ı´ a del Pilar Crespo ,
1
and Jairo Quiroga
1Research Group of Heterocyclic Compounds (GICH), Department of Chemistry, Universidad del Valle, A. A. 25360, Cali,
Colombia
2
Department of Microbiology, Universidad del Valle, A. A. 25360, Cali, Colombia
Correspondence should be addressed to Jairo Quiroga; jairo.quiroga@correounivalle.edu.co
Received 21 June 2019; Accepted 6 January 2020; Published 8 February 2020
Academic Editor: Xing-Hai Liu
Copyright © 2020 Pablo E. Romo et al. +is is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
A versatile method for the synthesis of new oxindoles was developed by the reaction between substituted isatins and 5-ami-
nopyrazoles. +e reaction was carried out at room temperature in ethanol using p-toluenesulfonic acid as the catalyst. +e
products were obtained with acceptable to excellent yields (44–96%). Structures of the new compounds were unambiguously
established by spectroscopic and analytical techniques. +e antibacterial activity was determined by microdilution assays.
Compounds 3b, 3e, and 3g showed antistaphylococcal activity, particularly compound 3e displayed a potent activity against the
vancomycin intermediate Staphylococcus aureus (VISA). Compounds 3i, 3j, and 3o inhibited Neisseria gonorrhoeae growth.
via an InCl -catalyzed tricomponent reaction between aryl-
1
. Introduction
3
hydrazines, 3-aminocrotonitrile, and isatin under reflux in
°
Nitrogen-containing heterocycles are present in many rec-
ognized drugs [1]; thus, synthetic chemists are increasingly
motivated to discover new methods for the rapid construction
of pharmacologically important nitrogen-based drug-like
heterocyclic compounds [2]. Isatin- and pyrazole-based com-
pounds are nowadays a huge nest of research that correlate with
the biological activity that they show, such as antitumoral [3–6],
antimicrobial [7–9], anti-inflammatory [8], and antimicrobial
against multidrug-resistant cells [10], among others [11–16].
Previously, we have described different strategies to obtain
pyrazole-based compounds and the evaluation of their bio-
logical activity as antifungal, antibacterial, and antitumor agents
showing that some these pyrazole-derivatives were bioactive as
antimicrobials [17–22]. Combination of isatin and pyrazole
moieties could be a good alternative to trigger a better biological
activity into a hybrid structure. Isatin-pyrazole Baylis-Hillman-
type adducts have been obtained as intermediates in the
synthesis of tricyclic-fused heterocycles [23–25]. Balamurugan
et al. [26] described the synthesis of 3-(4-pyrazolyl)-oxindoles
water at 100 C. Also, Yang et al. synthesized 3-(4-pyrazolyl)-
oxindoles by a reaction of isatin derivatives and 5-amino-
pyrazoles under reflux in water [27].
+e antimicrobial activity of isatin derivatives has also
been reported [28]. +ey have been described as a versatile
scaffold used as synthetic building blocks for diverse
heterocyclic compounds, some of them with substantial
antimicrobial activity. Isatin-N-Mannich bases of isatin-3-
thiosemicarbazones have shown activity against several
viruses and M. tuberculosis at the micromolar level [29].
Likewise, Ugale et al. reported that some derivatives of N′-
(5 or 7 substituted-2-oxoindolin-3-ylidene) benzofuran-2-
carbohydrazides exhibited antibacterial and antifungal
activity. +ey found that combining benzofuran and isatin
moieties may modulate the biological activity of these
compounds [30]. Moreover, in an effort to fight against
bacterial resistance, fluoroquinolone-isatin hybrids have
been tested during the last decades showing potential anti-
HIV, anti-bacterial, and anti-M. tuberculosis activity [31].

2
Heteroatom Chemistry
Recently, +akur et al. described that isatin-based gluco-
conjugated oxindoles had potent activity against Plasmo-
dium falciparum with a good selectivity index [32]. Pervez
et al. reported some isatin-derived bis-Schiff bases as
promising cytotoxic agents and urease inhibitors. Although
in other research they evaluated several isatin-3-hydra-
zonothiazolines as urease inhibitors, despite some good
Table 1: Synthesis of 3-(4-pyrazolyl)oxindoles 3.
Entry
3a
R
1
R
2
R
3
Yield (%)
H
H
H
Bn
H
H
69
62
51
96
70
48
66
45
48
62
51
55
52
44
47
72
3
3
3
3
3
3
3
3
b
c
d
e
f
g
h
i
H
H
5-F
H
Hexyl
Bn
Me
Hexyl
Bn
Bn
H
H
5-Cl
results, substituents like 5-OCF and 5-F over the isatin
3
H
5-Cl
ring decreased the activity in various cases [33, 34]. In a
more recent research, Pervez et al. reported several thio-
semicarbazone-isatin derivatives with important activity as
a urease inhibitors with lower IC (0.87 to 11.23 μM) than
H
5-Cl
4-Cl
4-F
5-Cl
5-F
5
0
3j
3k
3l
4-Cl
3,5-di-Cl
3,5-di-Cl
3,5-di-Cl
4-Cl
3,5-di-Cl
H
Hexyl
Bn
5-Cl
thiourea (IC � 22.3 μM) [35–37]. According to the
5-Cl
5
0
abovementioned background about this kind of products,
we have designed a series of isatin-pyrazole hybrids that
could be effective as antimicrobials. In this sense, we ex-
amined the potential inhibitory activity of these novel
compounds against several relevant drug-sensitive and
Hexyl
Bn
5-Cl
3
3
3
3
m
5-F
n
o
p
Bn
5,7-di-Cl
5,7-di-Cl
5,7-di-Cl
Bn
Bn
-
resistant bacteria.
protonation of isatin and formation of intermediate (I), and
then the 5-aminopyrazole 1 performed the addition over
C-3, affording the intermediate (II). Subsequently, a tau-
tomeric process afforded the isolated products 3.
2
. Results and Discussion
2
.1. Chemistry. In this work, we focused on the synthesis of
highly substituted 3-(4-pyrazolyl)oxindoles 3 bearing lipo-
philic groups like hexyl and benzyl over the nitrogen atom of
isatin, owing to several studies about the relationship of the
lipophilic character of compounds and their biological ac-
tivities [38, 39]. Another structural modification undertaken
in order to improve the biological activity corresponded to
the introduction of different halides such as chlorine and
fluorine in the aryl ring, oriented to the known halogen bond
effect [40–42], which is characterized for showing σ-hole
interactions, that could increase the possibility for im-
proving the activity of the studied compounds.
2.2. Antibacterial Studies. All the isatin derivatives 3 were
screened for the in vitro antibacterial activity against Gram-
positive and Gram-negative bacteria. Wildtype and multi-
resistant strains were included as follows: methicillin-sus-
ceptible Staphylococcus aureus ATCC 25923 (MSSA),
methicillin-resistant Staphylococcus aureus ATCC 43300
(MRSA), vancomycin-intermediate Staphylococcus aureus
(VISA), Escherichia coli ATCC 25922, carbapenemase-
positive Klebsiellapneumoniae BAA 1705, Klebsiella pneu-
moniae ATCC 700603 (extended spectrum beta lactamase,
ESBL positive), Pseudomonas aeruginosa ATCC 27853, and
Neisseria gonorrhoeae ATCC 49226. S. aureus, K. pneu-
moniae, and P. aeruginosa are part of the ESKAPE pathogens
which are bacteria causing nosocomial infections. +e
minimum inhibitory concentration (MIC) is determined
and reported in Table 2.
N. gonorrhoeae and VISA have been identified by the
World Health Organization as priority pathogens as re-
sistance has been developed against the first-line treatment
and other commonly used antibiotics [43]. +ree com-
pounds (3b, 3e, and 3f) showed antistaphylococcal activity
with MICs ranging from 3.9 μg/mL to 250 μg/mL. Com-
pound 3e (the benzyl-chloro-hydroxyl indolin) induced
substantial growth inhibition of the VISA strain. +is
compound also showed discrete growth inhibition of the
other two Staphylococcus strains (MRSA and MSSA). SAR
analysis revealed that the 1-benzyl-5-chloro-substitution in
the pyrazol-indolin seemed to confer to activity against the
VISA strain whereas the 1-hexyl instead of the 1-benzyl
improved growth inhibition of MSSA. Figure 1. VISA
growth inhibition was improved 4 times in 3e compared
with 3b analog suggesting that the activity is clearly en-
hanced by 5-chloro substitution at the isatin core. Two
analogue compounds 3i (the benzyl-fluoro-hydroxyl
In a first attempt, we carried out the reaction between 3-
methyl-1-phenyl-1H-pyrazol-5-amine (1a) and isatin (2a)
using p-toluenesulfonic acid (PTSA) as the catalyst in eth-
anol at room temperature (Scheme 1). +e reaction was
monitored by thin layer chromatography (TLC). After
3
0 min, a white solid formed, it was isolated by filtration
under vacuum, and washed with ethanol. +e product 3a, so
obtained, did not require further purification with a yield of
6
9%. Spectroscopic and analytical data (NMR, FT-IR, EI-MS
and elemental analysis) of compound 3a matched with the
information supplied for the same structure in references
1
[
24–27]. +e main H-NMR signals for 3a correspond to the
NH protons at 5.28 ppm as a broad singlet, and the hy-
2
droxyl proton as a singlet at 6.61 ppm.
In order to evaluate the versatility and scope of this procedure,
we performed the reaction, under the same reaction conditions,
starting from substituted aminopyrazoles 1a-d and isatins 2a-i
(
Scheme 2), leading to the formation of diverse 3-(4-pyrazolyl)
oxindole derivatives, 3a-p (Scheme 2, Table 1).
Despite the acceptable-to-good reaction yields (44–96%)
obtained in our approach, in comparison with the procedure
described by Yang et al. [27], remarkably, our methodology
required less reaction time and less consumption of energy.
A possible mechanistic route for the described reaction is
outlined in Scheme 3. +e procedure should involve the

Heteroatom Chemistry
3
Table 2: In vitro antibacterial activity (MIC values, μg/mL).
S. aureus ATCC 25923 μg/mL S. aureus ATCC 43300 μg/mL VISA μg/mL
Compound
N. gonorrhoeae ATCC 49226 μg/mL
3
a
>1000
>1000
>1000
>1000
3
b
1000
1000
62.5
>1000
3
c
>1000
>1000
>1000
>1000
3
d
>1000
>1000
>1000
>1000
3
e
250
250
3.9
>1000
3
f
>1000
>1000
>1000
>1000
3
g
125
1000
>1000
>1000
>1000
3
h
1000
1000
>1000
3
i
>1000-
>1000
>1000
15.6
3
j
>1000
>1000
>1000
15.6
3
k
1000
1000
1000
>1000
3
l
>1000
>1000
>1000
>1000
3
m
>1000
>1000
>1000
>1000
3
n
>1000
>1000
>1000
>1000
3
3
o
p
>1000
>1000
0.4
>1000
>1000
>1000
>1000
>1000
>1000
Gentamicin
Penicillin
Tetracycline
0.25
0.2
0.05
0.1
indolin) and 3j (choro-hexyl-hydroxyl indolin with a
chlorophenyl substitution in the pyrazole ring) were active
against N. gonorrhoeae (MICs 15.6 μg/mL), and the benzyl-
dichlorohydroxyindolin 3o showed discrete inhibition of
bacterial growth (MIC 250 μg/mL), Figure 1. +e 4-fluoro or
and the limited therapeutic options for penicillin-resistant
N. gonorrhoeae. None of the active compounds showed
relevant hemolytic activity suggesting low membrane in-
teractions and toxicity.
4-chloro substituent at phenyl moiety of the pyrazole ring
4
. Experiment
and the C-5 of the benzyl or hexyl-isatin seemed to be
important for gonococcal inhibition. +e presence of elec-
tron withdrawing substituents at C-5 of the isatin core has
been previously associated with biological activity here, as
well as moieties at N-1 may also modulate the activity [29].
4.1. General Information. Reagents and solvents used were
obtained from commercial sources. +e progress of the
reactions was monitored by TLC with 0.2 μm precoated
plates of silica gel 60GF254 (Merck). Melting points were
measured using a Stuart SMP3 melting point apparatus. IR
spectra were obtained with a Shimadzu IRAffinity-1 with
+
e ability of the biologically active isatin-pyrazole
compounds 3 to induce hemolysis in human red blood cells
ranged from 0 to 7%. None of these compounds seemed to
be active against E. coli and drug-resistant K. pneumoniae or
P. aeruginosa.
1
13
ATR probe. +e H and C-NMR spectra were recorded in a
BRUKER DPX 400 spectrophotometer operating at 400 and
100 MHz, respectively, using dimethyl sulfoxide-d as the
6
+
e most active compounds are highlighted in bold.
solvent. +e mass spectra were obtained on a SHIMADZU-
GCMS-QP2010 spectrometer operating at 70 eV.
MIC values >1 mg/mL were considered as not relevant or
not active.
4
.2. General Procedure for Preparation of 3-(4-Pyrazolyl)-
oxindoles 3a-p. An equimolar mixture of 5-aminopyrazoles
and the corresponding isatin 2, in the presence of 10 mol%
3
. Conclusions
1
In summary, we synthesized a series of 3-(4-pyrazolyl)-
oxindoles 3 by the reaction of 5-aminopyrazoles 1 with
highly substituted isatins 2 using PTSA as catalyst in mild
conditions, room temperature, short reaction times, and in
acceptable-to-excellent yields (44–96%). Some compounds
of this series showed antistaphylococcal and antigonococcal
activity; the benzyl substitution at N-1 and chloro substit-
uent at C-5 of isatin seemed to promote activity against
vancomycin-intermediate Staphylococcus aureus (VISA),
whereas the hexyl at N-1 increased the activity against
methicillin-susceptible Staphylococcus aureus (MSSA). +e
active compounds might be a starting point to design new
series of analogs potentially active against VISA and N.
gonorrhoeae. +is finding is particularly relevant because of
the rising of infections caused by drug-resistant S. aureus
of p-toluenesulfonic acid in 1 mL of ethanol, was stirred at
room temperature for 1 h. +e solid formed was filtered
under vacuum and washed with ethanol. Products 3 did not
require further purification.
4
.3. Characterization Data of the Compounds 3
4.3.1. 3-(5-Amino-3-methyl-1-phenyl-1H-pyrazol-4-yl)-3-hydrox-
°
yindolin-2-one 3a. White solid, Yield: 69%. M.p.: 235–236 C
−
1
°
°
(236 C [26], 218–220 C [27]). FT-IR (cm ): 3437, 3360,
1
3069, 1711. H NMR (400 MHz, DMSO-d ) δ (ppm): 1.45 (s,
6
3 H, 3-CH ), 5.28 (br. s., 2 H, NH ), 6.61 (s, 1 H, OH), 6.86
3
2
(d, J � 7.61 Hz, 1 H, Ar-H), 6.99 (t, J � 7.80 Hz, 1 H, Ar-H),
7.22–7.33 (m, 3 H, Ar-H), 7.46 (t, J � 7.90 Hz, 2 H, Ar-H),

4
Heteroatom Chemistry
HN
N
N
O
O
O
Cl
OH
OH
OH
N
N
N
NH2
NH2
NH2
N
N
N
3
a
3b
3e
MSSA MIC: >1mg/mL
VISA MIC: >1mg/mL
MSSA MIC: >1mg/mL
VISA MIC: 62.5μg/mL
MSSA MIC: 25μg/mL
VISA MIC: 3.9μg/mL
MRSA MIC: 250μg/mL
N
O
N
O
N
Cl
Cl
O
Cl
OH
OH
OH
N
NH2
N
N
NH2
NH2
N
N
N
3
f
3g
3j
Cl
MSSA MIC: >1mg/mL
VISA MIC: >1mg/mL
N. gonorrhoeae MIC: >500µg/mL
MSSA MIC: 125µg/mL
VISA MIC: >1mg/mL
N.gonorrhoeae MIC: 15.6µg/mL
N
O
F
OH
N
NH2
N
3
i
F
N.gonorrhoeae MIC: 15.6µg/mL
Figure 1: SAR of isatin-pyrazole compounds.
7.57 (d, J � 7.61 Hz, 2 H, Ar-H), 10.34 (s, 1 H, NH). 13C
HN
N
O
NMR (100 MHz, DMSO-d ) δ (ppm): 13.2 (3-CH ), 75.0
6 3
H
N
PTSA (10 mol%)
EtOH, rt
OH
(C), 100.1 (C), 110.2 (CH), 122.4 (C), 123.2 (CH), 125.5
(CH), 126.5 (CH), 129.6 (CH), 129.9 (C), 133.3 (C), 136.4
(C), 139.5 (C), 142.2 (C), 145.1 (C), 146.6 (C), 178.6 (C). EI-
N
O
NH2
+
N
N
NH2
O
2
a
ꢀ
MS (20 eV) m/z: 320 (29, M ), 304 (33), 291 (23), 275 (34),
1
73 (100), 120 (80), 92 (55), 69 (31). Anal. Calcd. For
1
a
3
a
C H N O : C: 67.49; H: 5.03; N: 17.49. Found: C: 67.51;
1
8
16
4
2
Scheme 1: Synthesis of the 3-(4-pyrazolyl)oxindole 3a.
H: 5.01; N: 17.43.

Heteroatom Chemistry
5
ꢀ
R2
N
404 (23, M ), 388 (24), 310 (22), 173 (100), 161 (62), 132 (98), 119
41), 69 (36). Anal. Calcd. For C H N O : C: 71.26; H: 6.98; N:
R3
(
2
4 28 4 2
O
N
R2
N
13.85. Found: C: 71.35; H: 6.91; N: 13.72.
OH
N
+
PTSA (10 mol%)
EtOH, rt
NH2
N
O
R3
NH2
N
O
2
a-i
4.3.5. 3-(5-Amino-3-methyl-1-phenyl-1H-pyrazol-4-yl)-1-benzyl-
-chloro-3-hydroxyindolin-2-one 3e. Yellow pale solid, Yield:
R1
5
7
R1
− 1
1
a-d
3a-p
°
0%. M. p.: 208–210 C. FT-IR (cm ): 3447, 3362, 3065,
1
Scheme 2: Established approach for synthesis of diverse 3-(4-
pyrazolyl)oxindoles 3a-p.
2359, 1720. H NMR (400 MHz, DMSO-d ) δ (ppm): 1.33 (s,
6
3 H, 3-CH ), 4.85 (d, J � 15.61 Hz, 1 H, N-CHH), 4.94 (d,
3
J � 15.80 Hz, 1 H, N-CHH), 5.37 (s, 2 H, NH ), 6.99 (d,
2
J � 8.59 Hz, 1 H, Ar-H), 7.06 (s, 1 H, OH), 7.25–7.39 (m, 8 H,
Ar-H), 7.47 (t, J � 8.00 Hz, 2 H, Ar-H), 7.53–7.57 (m, 2 H,
4
.3.2. 3-(5-Amino-3-methyl-1-phenyl-1H-pyrazol-4-yl)-1-benzyl-
3
-hydroxyindolin-2-one 3b. Yellow pale solid, Yield: 62%. M.
Ar-H). 13C NMR (100 MHz, DMSO-d
) δ (ppm):13.2 (3-
6
−
1
1
°
p.:191–193 C. FT-IR (cm ): 3451,3366, 3067,1715. H NMR
400 MHz, DMSO-d6) δ (ppm):1.29 (s, 3 H, 3-CH ), 4.85 (d, J:
CH ), 43.4 (CH ), 74.7 (C), 99.1 (C), 100.0 (C), 111.5 (C),
3
2
(
3
1
1
1
23.4 (CH), 125.2 (CH), 126.8 (CH), 127.5 (C), 128.0 (CH),
28.1 (CH), 129.1 (CH), 129.7 (CH), 129.8 (CH), 134.6 (C),
1
5.80 Hz, 1 H, CHH), 4.93 (d, J: 15.80 Hz, 1 H, CHH), 5.36
(
s, 2 H, NH ), 6.88 (s, 1 H, OH), 6.95 (d, J � 7.80 Hz, 1 H, Ar-
2
36.3 (C), 139.1 (C), 141.4 (C), 144.7 (C), 146.9 (C), 176.6
H) 7.06 (t, J � 7.80 Hz, 1 H, Ar-H), 7.24–7.42 (m, 8 H, Ar-H),
ꢀ
(
C).EI-MS (40 eV) m/z: 444 (35, M ), 391 (31), 271 (21), 173
7
.47 (t, J � 7.90 Hz, 2 H, Ar-H), 7.57 (d, J � 7.80 Hz, 2 H, Ar-
13
(71), 91 (100), 77 (44). Anal. Calcd. For C25
H21ClN O : C:
4 2
H). C NMR (100 MHz, DMSO-d ) δ (ppm): 13.2 (3-CH ),
6
3
6
7.49; H: 4.76; N: 12.59. Found: C: 67.41; H: 4.84; N: 12.48.
4
3.3 (CH ), 74.8 (C), 99.7 (C), 109.8 (CH), 123.3 (CH), 125.3
2
(
(
CH), 126.7 (CH), 128.0 (CH), 129.1 (CH), 129.6 (CH), 130.0
CH), 132.5 (C), 136.7 (C), 139.3 (C), 142.7 (C), 145.1 (C),
ꢀ
4.3.6. 3-(5-Amino-3-methyl-1-phenyl-1H-pyrazol-4-yl)-5-chloro-
-hydroxy-1-methylindolin-2-one 3f. White solid, Yield: 48%.
1
46.8 (C), 177.0 (C). EI-MS (20 eV) m/z: 410 (15, M ), 394
3
(
22), 237 (64), 173 (100), 146 (95), 91 (53). Anal. Calcd. For
− 1
°
M. p.:199–200 C. FT-IR (cm ):3399, 3308, 3063, 2785, 2359,
C H N O : C: 73.15; H: 5.40; N: 13.65. Found: C: 73.10; H:
1
2
5
22 4 2
1
3
721. H NMR (400 MHz, DMSO-d ) δ (ppm): 1.41 (s, 3 H,
-CH ), 3.15 (s, 3 H, N-CH ), 5.34 (s, 2 H, NH ), 6.89 (s, 1 H,
6
5
.45; N: 13.73.
3
3
2
OH), 7.10 (d, J � 8.39 Hz, 1 H, Ar-H), 7.27–7.37 (m, 2 H, Ar-
H), 7.39–7.51 (m, 3 H, Ar-H), 7.54 (d, J � 7.80 Hz, 2 H, Ar-
4
.3.3. 3-(5-Amino-3-methyl-1-phenyl-1H-pyrazol-4-yl)-5-fluoro-
13
H). C NMR (100 MHz, DMSO-d ) δ (ppm): 13.1 (3-CH ),
6
3
3
2
3
1
6
-hydroxyindolin-2-one 3c. White solid, Yield: 51%. M. p.:
−
1
26.6 (N-CH ), 74.7 (C), 99.3 (C), 110.9 (CH), 123.4 (CH),
°
°
3
27–229 C (212–214 C [27]). FT-IR (cm ): 3391, 3308,
1
124.8 (CH),126.8 (CH), 127.3 (C), 129.7 (CH), 129.8 (C),
073, 2359, 1709. H NMR (400 MHz, DMSO-d ) δ (ppm):
6
1
34.7 (C), 139.2 (C), 142.2 (C), 144.7 (C), 146.8 (C), 174.8
.49 (s, 3 H, 3-CH ), 5.30 (s, 2 H, NH ), 6.76 (s, 1 H, OH),
3
2
ꢀ
(
C), 176.4 (C). EI-MS (20 eV) m/z: 368 (32, M ), 352 (16),
.87 (dd, J � 8.39, 4.29 Hz, 1 H, Ar-H), 7.06–7.16 (m, 2 H, Ar-
3
22 (24), 195 (29), 173 (100), 138 (48), 81 (39), 69 (45). Anal.
H), 7.27–7.33 (m, 1 H, Ar-H), 7.43–7.50 (m, 2 H, Ar-H),
1
3
Calcd. For C H ClN O : C: 61.88; H: 4.65; N:15.19. Found:
19 17 4 2
7
.54–7.59 (m, 2 H, Ar-H), 10.39 (s, 1 H, NH). C NMR
C: 61.81; H: 4.77; N: 15.27.
(
(
(
100 MHz, DMSO-d ) δ (ppm):13.1 (3-CH ), 75.2 (C), 99.6
6
3
C), 123.3 (CH), 126.7 (CH) 129.6 (CH), 138.2 (C), 139.3
C), 144.9 (C), 146.7 (C), 160.5 (C), 178.6 (C). EI-MS (20 eV)
ꢀ
4.3.7. 3-(5-Amino-3-methyl-1-phenyl-1H-pyrazol-4-yl)-5-chloro-
m/z: 338 (43, M ), 322 (29), 310 (22), 296 (33), 293 (54), 236
1
6
1
-hexyl-3-hydroxyindolin-2-one 3g. Brown pale solid, Yield:
(
32), 173 (100), 138 (72), 82 (69), 57 (88). Anal. Calcd. For
−
1
°
6%. M. p.: 184–187 C. FT-IR (cm ):3451, 3360, 2916, 2361,
C H FN O : C: 63.90; H: 4.47; N: 16.56. Found: C: 63.85;
1
8
15
4
2
1
728. H NMR (400 MHz, DMSO-d ) δ (ppm): 0.83 (t,
6
H: 4.38; N: 16.50.
J � 6.92 Hz, 3 H, CH ), 1.18–1.35 (m, 6 H, 3 × CH ), 1.41 (s, 3
3
2
H, 3-CH ), 1.51–1.63 (m, 2 H, CH ), 3.59–3.73 (m, 2 H,
3
2
4
.3.4. 3-(5-Amino-3-methyl-1-phenyl-1H-pyrazol-4-yl)-1-hexyl-3-
CH
2
), 5.34 (s, 2 H, NH
2
), 6.89 (s, 1 H, OH), 7.12 (d,
hydroxyindolin-2-one 3d. Yellow pale solid, Yield: 96%. M. p.:
J � 8.39 Hz, 1 H, Ar-H), 7.28–7.35 (m, 2 H, Ar-H), 7.41 (dd,
−
1
1
°
J � 8.39, 2.15 Hz, 1 H, Ar-H), 7.47 (t, J � 7.80 Hz, 2 H, Ar-H),
1
59–161 C. FT-IR (cm ): 3451, 3356, 2928, 2359, 1719. H
13
7
.55 (d, J � 7.80 Hz, 2 H, Ar-H). C NMR (100 MHz,
NMR (400 MHz, DMSO-d ) δ (ppm): 0.83 (t, J � 6.94 Hz, 3 H,
6
DMSO-d ) δ (ppm): 13.2 (3-CH ), 14.4 (CH ), 22.5 (CH ),
CH ), 1.19–1.34 (m, 6 H, 3 × CH ), 1.36 (s, 3 H, 3-CH ), 1.53–1.65
6
3
3
2
3
2
3
2
9
1
1
6.4 (CH ), 27.3 (CH ), 31.4 (CH ), 39.9 (CH ), 74.6 (C),
2 2 2 2
(
m, 2 H, CH ), 3.57–3.73 (m, 2 H, CH ), 5.33 (s, 2 H, NH ), 6.71
2
2
2
9.3 (C), 111.1 (CH), 123.4 (CH), 125.1 (CH), 126.8 (CH),
27.1 (C), 129.6 (CH), 129.9 (CH), 134.7 (C), 139.2 (C),
(
s, 1 H, OH), 7.02–7.11 (m, 2 H, Ar-H), 7.26–7.38 (m, 3 H, Ar-H),
7
.47 (t, J � 7.81 Hz, 2 H, Ar-H), 7.55 (d, J � 7.61 Hz, 2 H, Ar-
1
3
41.8 (C), 144.6 (C), 146.9 (C), 176.3 (C).EI-MS (45 eV) m/
H). C NMR (100 MHz, DMSO-d ) δ (ppm): 13.1 (3-CH ), 14.4
6
3
ꢀ
(
CH ), 22.5 (CH ), 26.5 (CH ), 27.5 (CH ), 31.4 (CH ), 39.7
z: 438 (8, M ), 422 (8), 385 (8), 265 (7), 195 (19), 173 (52),
3
2
2
2
2
(
CH ), 74.7 (C), 99.8 (C),109.3 (CH), 123.0 (CH), 123.2 (CH),
166 (39), 86 (60), 43 (100). Anal. Calcd. For C24H27ClN O :
4 2
C: 65.67; H: 6.20; N: 12.76. Found: C: 65.57; H: 6.22; N:
12.64.
2
1
25.3 (CH), 126.6 (CH), 129.6 (CH), 130.1 (CH) 132.6 (C), 139.3
(
C), 143.0(C), 145.0 (C), 146.8 (C), 176.7 (C). EI-MS (20 eV) m/z:

6
Heteroatom Chemistry
R2
N
O
R3
O
2a-i
R2
N
R2
N
PTSA
O
R3
R3
PTSA–
O
H
R2
OH
OH
–
H+
N
N
NH2
+
N
N
N
O
R3
NH2
NH2
N
N
HO
I
R1
II
1a-d
3
Scheme 3: Suggested mechanistic pathway for the synthesis of 3-(4-pyrazolyl)-oxindoles 3.
−
1
°
4
.3.8. 3-(5-Amino-1-(4-chlorophenyl)-3-methyl-1H-pyrazol-4-yl)-
Yield: 62%. M. p.: 169–170 C. FT-IR (cm ): 3464, 3437,
1
1
-benzyl-5-chloro-3-hydroxy-indolin-2-one 3h. White solid,
3360, 2960, 2360, 1715. H NMR (400 MHz, CDCl -d) δ
3
−
1
°
Yield: 45%. M. p.: 207–209 C. FT-IR (cm ): 3466, 3435,
(ppm): 0.87 (t, J � 6.92 Hz, 3 H, CH ), 1.22–1.41 (m, 6 H,
3
1
3
358, 3067, 2922, 2359, 1717. H NMR (400 MHz, DMSO-
3 × CH ), 1.61 (s, 3 H, 3-CH ), 1.63–1.70 (m, 2 H, CH ),
2
3
2
d ) δ (ppm): 1.33 (s, 3 H, 3-CH ), 4.85 (d, J � 15.61 Hz, 1
3.52–3.62 (m, 1 H,N-CHH), 3.67–3.76 (m, 1 H,N-CHH),
6
3
H,CHH), 4.94 (d, J � 15.61 Hz, 1 H, CHH), 5.43 (s, 2 H,
4.04 (br. s., 1 H, OH) 4.62 (br. s., 2 H, NH ), 6.80 (d,
2
NH ), 7.00 (d, J � 8.98 Hz, 1 H, Ar-H), 7.04 (s, 1 H, OH),
J � 8.20 Hz, 1 H, Ar-H), 7.31 (dd, J � 8.39, 2.15 Hz, 1 H,
2
7
7
.25–7.41 (m, 7 H, Ar-H), 7.52 (d, J � 8.78 Hz, 2 H, Ar-H),
Ar-H), 7.40–7.44 (m, 3 H, Ar-H), 7.44–7.48 (m, 2 H, Ar-
13
13
.60 (d, J � 8.78 Hz, 2 H, Ar-H). C NMR (100 MHz,
H). C NMR (100 MHz, CDCl -d) δ (ppm): 13.2 (3-CH ),
3
3
DMSO-d ) δ (ppm): 13.2 (3-CH ), 43.4 (CH ), 74.7 (C), 99.6
14.0 (CH ), 22.5 (CH ), 26.6 (CH ), 27.2(CH ), 31.4(CH ),
6
3
2
3 2 2 2 2
(
(
(
C), 111.5 (CH), 124.9 (CH), 125.2 (CH), 127.5 (C), 128.0
CH), 128.1 (CH), 129.1 (CH), 129.6 (CH), 129.8 (CH), 130.9
40.5(N-CH ), 74.9 (C), 99.5 (C), 109.9 (CH), 124.9 (C), 125.3
2
(CH), 125.9 (CH), 128.7 (C), 129.5 (C), 129.7 (CH), 130.0
(CH), 132.5 (C), 133.1 (C), 136.6 (C), 141.1 (C), 145.5 (C),
C), 134.5 (C), 136.3 (C), 138.1 (C), 141.5 (C), 145.2 (C),
ꢀ
ꢀ
1
47.1 (C), 176.5 (C). EI-MS (45 eV) m/z: 478 (4, M ), 271
145.6 (C), 176.5 (C). EI-MS (20 eV) m/z: 472 (4, M ), 456 (7),
(
17), 207 (31), 180 (54), 91 (100). Anal.Calcd. For
265 (24), 207 (100), 195 (49), 166 (57), 153 (34), 43 (28).
Anal. Calcd. For C H Cl N O : C: 60.89; H: 5.54; N: 11.84.
C H Cl N O : C: 62.64; H: 4.21; N: 11.69. Found: C: 62.71;
2
5
20
2
4
2
24 26
2 4 2
H: 4.24; N: 11.77.
Found: C: 60.77; H: 5.66; N: 11.75.
4
.3.9. 3-(5-Amino-1-(4-fluorophenyl)-3-methyl-1H-pyrazol-4-
4.3.11. 3-(5-Amino-1-(3,5-dichlorophenyl)-3-methyl-1H-pyr-
yl)-1-benzyl-5-fluoro-3-hydroxy-indolin-2-one 3i. White
azol-4-yl)-1-benzyl-5-chloro-3-hydroxy-indolin-2-one 3k.
−
1
− 1
°
°
solid, Yield: 48%. M. p.: 224–226 C. .FT-IR (cm ): 3431,
White solid, Yield: 51%. M. p.: 214–216 C. FT-IR (cm ):
1
1
3
352, 3090, 2359, 1711. H NMR (400 MHz, DMSO-d ) δ
3474, 3374, 3069, 2361, 1748. H NMR (400 MHz,
6
(
ppm): 1.33 (s, 3 H, 3-CH ), 4.85 (d, J � 15.80 Hz, 1 H,
DMSO-d ) δ (ppm): 1.33 (s, 3 H, 3-CH ), 4.85 (d,
3
6
3
CHH), 4.92 (d, J � 15.60 Hz, 1 H, CHH), 5.34 (s, 2 H, NH ),
J � 15.61 Hz, 1 H, N-CHH), 4.94 (d, J � 15.61 Hz, 1 H,
2
6
7
2
.96 (dd, J � 8.49, 4.19 Hz, 1 H, Ar-H), 6.99 (s, 1 H, OH),
.13 (td, J � 9.02, 2.63 Hz, 1 H, Ar-H), 7.21 (dd, J � 7.80,
.54 Hz, 1 H, Ar-H), 7.25–7.40 (m, 7 H, Ar-H), 7.57 (dd,
N-CHH), 5.63 (s, 2 H, NH ), 7.01 (d, J � 8.59 Hz, 1 H, Ar-H),
2
7.10 (s, 1 H, OH), 7.25–7.40 (m, 7 H, Ar-H), 7.51 (t,
1
3
J � 1.76 Hz, 1 H, Ar-H), 7.67 (d, J � 1.95 Hz, 2 H, Ar-H). C
1
3
J � 8.88, 4.98 Hz, 2 H, Ar-H). C NMR (100 MHz, DMSO-
NMR (100 MHz, DMSO-d ) δ (ppm): 13.2 (3-CH ), 43.5
6
3
d ) δ (ppm): 13.2 (3-CH ), 43.4 (CH ), 74.9 (C), 99.3 (C),
(CH ), 74.6 (C), 100.4 (C), 111.6 (CH), 121.1 (CH), 125.3
6
3
2
2
1
10.9 (CH), 113.1 (CH), 116.1 (CH), 116.2 (CH), 116.5
(CH), 125.7 (CH), 127.5 (C), 128.1 (CH), 129.1 (CH), 129.9
(CH), 134.3 (C), 134.9 (C), 136.3 (C), 141.4 (C), 141.5 (C),
146.3 (C), 147.7 (C), 176.4 (C). EI-MS (20 eV) m/z: 514 (2,
(
CH), 125.6 (CH), 125.7 (CH), 128.0 (CH), 129.1 (CH),
34.3(C), 135.6(C), 136.5 (C), 138.8 (C), 144.8 (C), 147.0
C), 158.0 (C), 159.5 (C), 160.4 (C), 161.9 (C), 176.9(C). EI-
1
ꢀ
(
M ), 498 (11), 368 (14), 271 (46), 241 (45), 180 (71), 91 (100).
ꢀ
MS (20 eV) m/z: 446 (64, M ), 430 (18), 400 (25), 255 (51),
98 (69), 191 (97), 164 (100), 91 (94). Anal. Calcd. For
C H F N O : C: 67.26; H: 4.52; N: 12.55. Found: C: 67.33;
Anal. Calcd. For C H Cl N O : C: 58.44; H: 3.73; N: 10.90.
25
19
3 4 2
1
Found: C: 58.36; H: 3.81; N: 10.83.
2
5
20
2
4
2
H: 4.50; N: 12.51.
4
.3.12. 3-(5-Amino-1-(3,5-dichlorophenyl)-3-methyl-1H-
pyrazol-4-yl)-5-chloro-1-hexyl-3-hydroxy-indolin-2-one
−
1
°
4.3.10. 3-(5-Amino-1-(4-chlorophenyl)-3-methyl-1H-pyrazol-
4-yl)-5-chloro-1-hexyl-3-hydroxyindolin-2-one 3j. White solid,
3l. White solid, Yield: 55%. M. p.:181–183 C. FT-IR (cm ):
3466, 3362, 2916, 2360,1728. H NMR (400 MHz, DMSO-d )
1
6

Heteroatom Chemistry
7
δ (ppm): 0.83 (t, J � 7.02 Hz, 3 H, CH ), 1.20–1.35 (m, 6 H,
DMSO-d ) δ (ppm): 1.45 (s, 3 H, 3-CH ), 5.18 (d,
3
6
3
3
3
7
× CH ), 1.41 (s, 3 H, CH ), 1.52–1.63 (m, 2 H, CH ),
J � 16.70 Hz, 1 H, N-CHH), 5.26 (d, J � 16.70 Hz, 1 H,
2
3
2
.56–3.73 (m, 2 H, CH ),5.59 (s, 2 H, NH ), 6.94 (s, 1 H, OH),
N-CHH), 5.63 (s, 2 H, NH ), 7.23–7.29 (m, 3 H, Ar-H), 7.30
2
2
2
.13 (d, J � 8.39 Hz, 1 H, Ar-H), 7.32 (d, J � 2.15 Hz, 1 H, Ar-
(s, 1 H, OH), 7.31–7.36 (m, 2 H, Ar-H), 7.40 (d, J � 2.15 Hz, 1
H), 7.41 (dd, J � 8.29, 2.24 Hz, 1 H, Ar-H), 7.51 (t, J � 1.85 Hz,
H, Ar-H), 7.50 (d, J � 1.95 Hz, 1 H, Ar-H), 7.52 (t,
1
3
13
1
H, Ar-H), 7.66 (d, J � 1.76 Hz, 2 H, Ar-H). C NMR
J � 1.76 Hz, 1 H, Ar-H), 7.66 (d, J � 1.76 Hz, 2 H, Ar-H). C
(
(
(
(
100 MHz, DMSO-d ) δ (ppm):13.2 (3-CH ), 14.3 (CH ), 22.5
NMR (100 MHz, DMSO-d ) δ (ppm): 13.4 (3-CH ), 44.8
6
3
3
6
3
CH ), 26.4 (CH ), 27.3 (CH ), 31.4 (CH ),40.0 (CH ), 74.5
(CH ), 74.1 (C), 99.9 (C), 115.7 (C), 121.3 (CH), 124.6 (CH),
2
2
2
2
2
2
C), 100.5 (C), 111.1 (CH), 121.1 (CH), 125.1 (CH), 125.7
CH), 127.2 (C), 130.0 (CH), 134.3 (C), 134.9 (C), 141.4 (C),
125.9 (CH), 126.7 (CH), 127.7 (CH), 128.3 (C), 129.0 (CH),
131.4 (CH), 134.9 (C), 136.9 (C), 137.7 (C), 141.3 (C), 146.1
ꢀ
1
5
41.8 (C), 146.2 (C), 147.6 (C), 176.1 (C). EI-MS (45 eV) m/z:
(C), 147.8 (C), 177.2 (C). EI-MS (20 eV) m/z: 548 (1, M ),
ꢀ
08 (6, M ), 492 (8), 265 (16), 241 (50), 195 (46), 166 (95), 111
532 (3), 305 (19), 241 (30), 214 (35), 91 (100). Anal. Calcd.
For C H Cl N O : C: 54.77; H: 3.31; N: 10.22. Found: C:
(
4
47), 43 (100). Anal. Calcd. For C H Cl N O : C: 56.76; H:
24 25 3 4 2
25 18
4 4 2
.96; N: 11.03. Found: C: 56.81; H: 4.88; N: 11.14.
54.70; H: 3.26; N: 10.17.
4
.3.13. 3-(5-Amino-1-(3,5-dichlorophenyl)-3-methyl-1H-pyr-
4
.3.16. 3-(5-Amino-3-methyl-1-phenyl-1H-pyrazol-4-yl)-1-ben-
azol-4-yl)-1-benzyl-5-fluoro-3-hydroxy-indolin-2-one 3m.
zyl-5,7-dichloro-3-hydroxyindolin-2-one 3p. White solid,
°
− 1
Brown pale solid, Yield: 52%. M. p.: 231–233 C. FT-IR
°
Yield: 72%. M. p.: 211–213 C. FT-IR (cm ): 3429, 3341,
−
1
1
(
(
cm ): 3215, 2361, 1722. H NMR (400 MHz, DMSO-d ) δ
1
6
3
1
5
071, 2818, 1730. H NMR (400 MHz, DMSO-d ) δ (ppm):
6
ppm): 1.32 (s, 3 H, CH3), 4.85 (d, J � 15.60 Hz 1 H,
.46 (s, 3 H, 3-CH ), 5.19 (d, J � 16, 76 Hz, 1 H, N-CHH),
3
N-CHH), 4.92 (d, J � 15.60 Hz, 1 H, N-CHH), 6.98 (dd,
J � 8.59, 4.10 Hz, 1 H, Ar-H), 7.11 (s, 1 H, OH), 7.13–7.17 (m,
.27 (d, J � 16, 76 Hz, 1 H, N-CHH), 5.36 (s, 2 H, NH ), 7.20
2
(
s, 1 H, OH), 7.23–7.29 (m, 3 H, Ar-H), 7.30–7.37 (m, 3 H,
1
H, Ar-H), 7.21 (dd, J � 7.80, 2.54 Hz, 1 H, Ar-H), 7.23–7.30
Ar-H), 7.43 (d, J � 2.15 Hz, 1 H, Ar-H), 7.45–7.51 (m, 3 H,
(
m, 2 H, Ar-H), 7.31–7.40 (m, 5 H, Ar-H, NH ), 7.50–7.54
13
2
Ar-H), 7.53–7.57 (m, 2 H, Ar-H). C NMR (100 MHz,
13
(
m, 1 H, Ar-H), 7.67 (d, J � 1.76 Hz, 2 H, ArH). C NMR
DMSO-d ) δ (ppm): 13.5 (3-CH ), 44.8 (CH ),74.2 (C), 98.2
6
3
2
(
100 MHz, DMSO-d ) δ (ppm): 13.1 (3-CH ), 43.5 (CH ),
6
3
2
(
(
(
C), 115.6 (C), 123.5 (CH), 124.6 (CH), 126.7 (CH), 126.9
CH), 127.7 (CH), 128.2 (C), 129.0 (CH), 129.7 (CH), 131.3
7
1
1
4.8 (C), 100.6 (C), 111.0 (CH), 113.2 (CH), 116.3 (CH),
21.2 (CH), 125.8 (CH), 128.1 (CH), 129.1 (CH), 133.9 (C),
CH), 137.7 (C), 137.8 (C), 144.6 (C), 147.0 (C), 177.4 (C).
34.9 (C), 136.4 (C), 138.8 (C), 141.2 (C), 146.3 (C), 147.8
ꢀ
EI-MS (20 eV) m/z: 548 (1, M ), 532 (3), 305 (19), 241 (30),
14 (35), 91 (100).Anal. Calcd. For C H Cl N O : C: 62.64;
ꢀ
(
C), 176.6 (C). EI-MS (45 eV) m/z: 496 (2, M ), 480 (5), 255
2
25 20 2 4 2
(
21), 241 (16), 198 (21), 164 (45), 108 (22), 91 (100). Anal.
H: 4.21; N: 11.69. Found: C: 62.59; H: 4.27; N: 11.63.
Calcd. For C H Cl FN O : C: 60.37; H: 3.85; N: 11.27.
2
5
19
2
4 2
Found: C: 60.43; H: 3.77; N: 11.20.
4
.4. Antibacterial Activity. Stock solutions (100 mg/mL) of
compounds 3 were prepared in dimethyl sulfoxide (DMSO)
and diluted to a final screening concentration of 500 μg/mL.
An initial screening of bacterial inhibition was performed by
the agar diffusion method. In brief, sterile Mueller Hinton
agar (MHA, BBL) was prepared in Petri dishes and inoc-
ulated with a bacterial suspension prepared in trypticase soy
4
4
.3.14. 3-(5-Amino-1-(4-chlorophenyl)-3-methyl-1H-pyrazol-
-yl)-1-benzyl-5,7-dichloro-3-hydroxy-indolin-2-one 3n.
−
1
°
White solid, Yield: 44%. M. p.: 205–207 C. FT-IR (cm ):
1
3
447, 3331, 3065, 2359, 1726. H NMR (400 MHz,
DMSO-d ) δ (ppm): 1.45 (s, 3 H, 3-CH ), 5.18 (d,
6
3
J � 16.56 Hz, 1 H, N-CHH), 5.26 (d, J � 16.56 Hz 1 H,
8
broth and adjusted to 1.5 ×10 colony-forming unit CFU/
N-CHH), 5.44 (s, 2 H, NH ), 7.22 (s, 1 H, OH), 7.24–7.30 (m,
2
mL (i.e., 0.08–0.1 OD at 600 nm) [44]. Wells (6 mm in di-
ameter) were punched in the agar, and 10 μL of each
compound (stock solution) were filled into each well. Di-
methyl sulfoxide and trypticase soy broth were included as
negative controls (i.e., no inhibition of bacterial growth).
Gentamicin (Sigma-Aldrich) and trimethoprim sulfame-
thoxazole were included as positive controls of growth in-
hibition. Derivatives 3 showing growth inhibition were
tested at least twice before being selected for microdilution
testing. For N. gonorrhoeae, the agar diffusion method was
also used in the screening process with some modifications.
3
H, Ar-H), 7.30–7.37 (m, 2 H, Ar-H), 7.42 (d, J � 1.95 Hz, 1
H, Ar-H), 7.50 (d, J � 2.15 Hz, 1 H, Ar-H), 7.52 (d,
13
J � 8.78 Hz, 2 H, Ar-H), 7.60 (d, J � 8.78 Hz, 2 H, Ar-H). C
NMR (100 MHz, DMSO-d ) δ (ppm): 13.5 (3-CH ), 44.8
6
3
(
CH ), 74.2 (C), 99.1 (C), 115.7 (C), 124.6 (CH), 125.1 (CH),
2
1
1
1
26.7 (CH), 127.7 (CH), 128.2 (C), 129.0 (CH), 129.6 (CH),
31.0 (C), 131.3 (CH), 137.2 (C), 137.7 (2 × C), 138.0 (C),
45.1 (C), 147.2 (C), 177.3 (C). EI-MS (45 eV) m/z: 515 (3,
ꢀ
M ), 496 (4), 305 (16), 214 (34), 207 (29), 91 (100), 65 (20).
Anal. Calcd. For C H Cl N O : C: 58.44; H: 3.73; N: 10.90.
25
19
3 4 2
Found: C: 58.37; H: 3.66; N: 10.82.
For this method, 200 μL of a bacterial suspension
8
(
1.5 ×10 CFU/mL) was inoculated in gonococcal (GC) agar
4
.3.15. 3-(5-Amino-1-(3,5-dichlorophenyl)-3-methyl-1H-pyr-
(BBL) supplemented with 1% isovitalex (BBL), and then the
azol-4-yl)-1-benzyl-5,7-dichloro-3-hydroxyindolin-2-one 3o.
compounds were added to the wells as mentioned above and
−
1
°
°
incubated at 35–36.5 C in 5% CO atmosphere for 48 h.
White solid, Yield: 47%. M. p.: 208–210 C. FT-IR (cm ):
3
2
1
474,3374, 3067, 2920, 2359, 1732. H NMR (400 MHz,
Penicillin and ceftriaxone (BBL) were used as controls [45].

8
Heteroatom Chemistry
4
.4.1. Microdilution Test. Minimum inhibitory concentra-
References
tion (MIC) was determined in those compounds with visible
and reproducible antibacterial inhibition by the screening
test. Bacterial suspensions were adjusted with Mueller
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5
5
Hinton broth (MHB) to a concentration of 5 ×10 –8 ×10
[
2] A. Padwa, “Heterocycles as vehicles for synthesis,” A Critical
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44]. Stock solutions of the novel compounds were diluted in
MHB containing 5% DMSO and 0.1% Tween 80 [46] and
added to 90 μL of the bacterial inoculum. +e microplates
were incubated for 24 h at 35–37 C. MICs defined the lowest
[
°
concentration with visible inhibition of bacterial growth [44]
and/or detection using resazurin (125 μg/mL). Gentamicin
and tetracycline (Sigma-Aldrich) were included as control of
inhibition growth; MHB and DMSO were used as a negative
control. Experiments were performed in duplicate.
[
[
4] R. Lin, G. Chiu, Y. Yu et al., “Design, synthesis, and evaluation
of 3,4-disubstituted pyrazole analogues as anti-tumor CDK
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5] J. B. Shi, W. J. Tang, X. B. Qi, R. Li, and X. H. Liu, “Novel
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