Synthesis and evaluation of isatin-β-thiosemicarbazones as novel agents against antibiotic-resistant Gram-positive bacterial species

Article abstract of DOI:10.1016/j.ejmech.2015.06.047

Methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE) have caused an increasing mortality rate, which means that antibiotic resistance is becoming an important health issue. In the course to screen new agents for r

Full text of DOI:10.1016/j.ejmech.2015.06.047

European Journal of Medicinal Chemistry 101 (2015) 419e430
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Synthesis and evaluation of isatin-
b-thiosemicarbazones as novel
agents against antibiotic-resistant Gram-positive bacterial species
,
,
Xu-Meng Zhang a 1, Hui Guo b 1, Zai-Shun Li a, Fu-Hang Song b, Wei-Min Wang a,
Huan-Qin Dai b **, Li-Xin Zhang b ***, Jian-Guo Wang a
,
,
, *
a State-Key Laboratory and Research Institute of Elemento-Organic Chemistry, Nankai University, Tianjin 300071, China
b CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE) have
caused an increasing mortality rate, which means that antibiotic resistance is becoming an important
health issue. In the course to screen new agents for resistant bacteria, we identified that a series of isatin-
Received 25 February 2015
Received in revised form
19 June 2015
Accepted 23 June 2015
Available online 2 July 2015
b
-thiosemicarbazones (IBTs) could inhibit the growth of MRSA and VRE. This was the first time that the
“familiar” IBT compounds exhibited significant anti Gram-positive pathogen activity. Against a clinical
isolated MRSA strain, 20 of the 51 synthesized compounds showed minimum inhibitory concentration
(MIC) data of 0.78 mg/L and another 12 novel compounds had MICs of 0.39 mg/L. Moreover, these
compounds also inhibited Enterococcus faecalis and VRE at similar levels, indicating that IBTs might have
different mode of action compared with vancomycin. For these IBTs, comparative field analysis (CoMFA)
models were further established to understand the structureeactivity relationships in order to design
new compounds from steric and electrostatic contributions. This work has suggested that IBTs can be
considered as potential lead compounds to discover antibacterial inhibitors to combat drug resistance.
© 2015 Elsevier Masson SAS. All rights reserved.
Keywords:
Methicillin-resistant Staphylococcus aureus
Vancomycin-resistant Enterococcus
Isatin-b-thiosemicarbazone
In vitro activity
Structureeactivity relationships
1. Introduction
resistance has become a public health threat around the world [4].
The emergence of New Delhi metallo-beta-lactamase-1 (NDM-1) in
The antibiotics have contributed enormously to treat bacterial
infection because they decrease the mortality rate significantly [1].
However, with the overuse of antibiotics, bacterial resistance
appeared gradually. In 1961, the first case of methicillin-resistant
Staphylococcus aureus (MRSA) was reported, for which only few
antimicrobial agents were effective [2]. In 1988, vancomycin
resistant Enterococci (VRE) was also identified that increased the
significance of this problem [3]. It is for decades that antimicrobial
2010 indicated that “superbug” era was approaching [5]. Nowadays
MRSA stands for one of the most serious antibiotic-resistant in-
fections and it causes an increasing mortality in hospital.
Currently, there are only a few clinical drugs available for the
treatment of MRSA, including vancomycin, daptomycin, linezolid,
tigecycline and ceftobiprole [6e10]. Nevertheless these clinical
drugs have limitation themselves. Vancomycin and daptomycin
have very complicated chemical structures, and their starting ma-
terials rely heavily on biological fermentation; tigecycline and
ceftobiprole can be synthesized chemically but their synthetic
routes require many steps. On the other hand, these agents have
been reported to display some side effects that cannot be neglected,
including red-man syndrome, muscle toxicity, myelosuppression
and nausea [8], which has hindered their further use. There exists a
great need to discover antibacterial agents with totally new
chemical structures to defeat the increasing bacterial resistance.
Isatin (Fig. 1A) is an important endogenous natural product with
versatile biological activities and it can be found in the mammalian
brain, peripheral tissues and body fluids [11]. This simple com-
pound is also a metabolite by marine bacteria from some shrimp
Abbrevations: MRSA, methicillin-resistant Staphylococcus aureus; VRE, vanco-
mycin-resistant Enterococcus; IBTs, isatin-b-thiosemicarbazones; CoMFA, compar-
ative field analysis; NDM-1, New Delhi metallo-beta-lactamase-1; MAO-B,
monoamine oxidase B; MIC, minimum inhibitory concentration; ppm, parts per
million; DMSO, dimethyl sulfoxide; TMS, tetramethylsilane; IR, infrared; MHA,
Mueller-Hinton agar; MHB, Mueller-Hinton Broth; LOO, leave-one-out; PLS, partial
least squares.
* Corresponding author.
** Corresponding author.
*** Corresponding author.
E-mail addresses: daihq@im.ac.cn (H.-Q. Dai), zhanglixin@im.ac.cn (L.-X. Zhang),
nkwjg@nankai.edu.cn (J.-G. Wang).
1
These two authors contributed equally to this work.
http://dx.doi.org/10.1016/j.ejmech.2015.06.047
0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

420
X.-M. Zhang et al. / European Journal of Medicinal Chemistry 101 (2015) 419e430
with other reported biological activities [19,23]. This study has
provided meaningful information to discover novel lead candidates
of Gram-positive bacteria therapeutics.
2. Results and discussion
2.1. Chemistry of the IBT compounds and crystal structure
The IBT compounds were synthesized by the straightforward
condensation of isatin and thiosemicarbazide, a method which has
been used for over 50 years [24]. Compounds 3-4, 3-6, 3-7, 3-8 and
3-10 have been reported elsewhere [25,26], however their char-
acterization data were not available. Compounds 3-5 and 3-9 have
been characterized by us, but the publication was in Chinese [27].
We therefore list here the analytical data for all the target com-
pounds. Compound 3-1 was recrystallized from ethanol/dichloro-
methane to give yellow crystals suitable for X-ray single-crystal
diffraction. Cif file for 3-1 is also given in the supplemental data.
Generally, the structure of IBT is similar to other reported isatin
derivatives, forming an intramolecular H-bond (O1 and the H atom
linked to N3) [17,23]. Thus, the IBT structures are not E isomers, but
Z isomers. The crystal structure of 3-1 is somewhat interesting in
that it is a duplex crystal. As can be seen, the phenyl ring attached to
N4 atom possesses alternative poses (Fig. 2).
Fig. 1. Structures of isatin (A), indirubin (B), methisazone (C) and NSC73306 (D).
embryos, which makes the embryos remarkably resistant to
infection caused by pathogenic fungus [12]. Isatin was also identi-
fied as a potent inhibitor of human mitochondrial monoamine
oxidase B (MAO-B) [13]. The derivatives of isatin possess a wide
range of biological activities, for instance, antimycobacterial, anti-
HIV, anticancer, anticonvulsant, antiviral, antiinflammatory and
analgesic activities [14e17]. However, there is not any other report
that isatin derivatives are desirable inhibitors of MRSA.
There are some drugs that contain isatin as an active scaffold.
Two representative examples are indirubin (Fig. 1B) and methisa-
zone (Fig. 1C). Indirubin is a bioactive ingredient from the Chinese
traditional medicine Dang Gui Long Hui Wan that has potent
antineoplastic activity such as treating chronic myelogenous leu-
2.2. The in vitro antibacterial activities
We initially synthesized 10 IBT compounds (3-1 to 3-10) and
evaluated their antibacterial activities. The bacterial strains of
S. aureus (ATCC 6538) and Bacillus subtilis (ATCC 6633) were used as
standard Gram-positive strains.
A
clinical isolated strain
kemia [18]. Methisazone, 1-methylisatin
b-thiosemicarbazone, is
(Chaoyang) was used for the anti-MRSA assay. It was interesting
that these compounds exhibited desirable antibacterial activities.
Compound 3-2 displayed the best biological activity, the MICs of
which were 0.78 mg/L, 1.56 mg/L and ꢀ0.78 mg/L for MRSA,
S. aureus and B. subtilis, respectively (Table 1). The other nine
compounds also showed good antimicrobial activities, MICs
ranging from 1.56 mg/L to 25 mg/L for the tested MRSA and
Staphylococcus strains. Except 3-3 and 3-6, all compounds inhibited
the B. subtilis strain completely at a concentration of no more than
1.56 mg/L. In comparison, the MIC values for clinical antibiotic
vancomycin were 1.0 mg/L, 1.0 mg/L and 0.5 mg/L against these
bacterial strains. From a view of the structureeactivity relation-
ships (for MIC against MRSA Chaoyang), a single halogen atom at 7-
position of isatin ring (R1) gives better activity when R2 is a
hydrogen atom. For such a case when R1 is a chlorine atom, the
compound has the best potency (3-2). However, when isatin is
substituted at 5-position (R1), whether there is a halogen or
hydrogen atom at R2 position, the activity becomes weaker.
Therefore 3-2 is regarded as a lead compound for further structural
optimization.
effective against smallpox infection from the 1960s [19]. The
structural optimization of methisazone is still under way although
their precise viral target and mechanism of action is still unknown
[20].
Methisazone has been used as an anti smallpox drug for nearly
50 years, indicating that this type of compounds might be biolog-
ically safe. In 1984, Omar et al. synthesized some isatin
b-thio-
semicarbazone (IBT) compounds and they had not observed their
activity against S. aureus [21]. Recently, Pervez et al. had also syn-
thesized some IBT derivatives and evaluated their antibacterial
activities [22]. The compounds in their study displayed some in-
hibition against human urease, but their activities upon S. aureus
were all very weak even at 10,000 mg/L concentration. Also
recently, Gottesman et al. found that some IBTs were promising
candidates for treating cancer of multidrug resistance by selectively
killing P-glycoprotein-expressing cells (NSC73306, Fig. 1D) [23].
However, Gottesman et al. had not evaluated the antibacterial ac-
tivities for any of their IBT compounds.
In an effort to discover novel anti-MRSA agents, we firstly syn-
thesized 10 (3-1 to 3-10) IBT compounds and evaluated their
antibacterial activity. Excitingly, one compound (3-2) inhibited a
clinical isolated MRSA (Chaoyang) strain significantly, with the
minimum inhibitory concentration (MIC) value of 0.78 mg/L, which
was better than that of vancomycin (1.0 mg/L). This is the first time
that the IBTs possess strong anti-MRSA activity in vitro. Encouraged
by this result, another 41 (3e11 to 3e51) novel IBTs were further
synthesized and 12 of them showed MICs of 0.39 mg/L against the
tested MRSA strain. The second generation of IBTs was subse-
quently tested for their broad-spectrum antibacterial activities
against other antibiotic-resistant isolates. These compounds also
had good efficiency on a tested VRE strain, implying their distinct
mode of action. Obviously, the IBTs are very “old” compounds,
however, their strong anti-MRSA activity is fairly novel compared
Fig. 2. Duplex crystal structure of IBT compound 3-1.

X.-M. Zhang et al. / European Journal of Medicinal Chemistry 101 (2015) 419e430
421
Table 1
Chemical structures of the first batch of isatin b-thiosemicarbazone compounds and their antibacterial activities.
Compd.
R1
R2
MIC (mg/L) for MRSA Chaoyang
MIC (mg/L) for SAa ATCC 6538
MIC (mg/L) for BSa ATCC 6633
3-1
3-2
3-3
3-4
3-5
3-6
3-7
3-8
7-F
7-Cl
5-Cl
5-Br
5-NO2, 7-Br
5-F
H
H
H
H
1.56
0.78
3.12
6.25
3.12
25
3.12
3.12
6.25
12.5
1.0
3.12
1.56
12.5
6.25
1.56
25
3.12
6.25
12.5
6.25
1.0
ꢀ0.78
ꢀ0.78
6.25
ꢀ0.78
ꢀ0.78
25
1.56
1.56
1.56
25
4-F
H
4-F
4-F
H
4-F
4-F
4-F
3-9
3-10
Vancomycin
5-Br, 7-Br
5-NO2
e
0.5
a
SA ¼ Staphylococcus aureus, BS¼Bacillus subtilis.
As far as we know, the isatin
b
-thiosemicarbazone compounds
significant inhibition against other MRSA isolates. Although the
MIC values varied among different MRSA strains (Table 2), com-
pounds 3-11, 3-12, 3-13, 3-15, 3-16, 3-22, 3-29, 3-30, 3-35 and 3-36
had MICs of ꢀ1.56 mg/L. This was in agreement with the data for
MRSA (Chaoyang) strain. For 3-15, 3-16 and 3-35, each of the
compounds had the MIC value of 0.39 mg/L against one clinical
strain. Compound 3-30 had MICs of 0.39 mg/L against two clinical
MRSA strains and compound 3-29 had MIC data of 0.39 mg/L
against three MRSA strains. On the other hand, both 3-34 and 3-49
had MICs >100 mg/L against all the different clinical MRSA strains.
For the control drug, the MIC values for vancomycin against all the
MRSA strains were 1.0 mg/L, and for methicillin, the MICs were
>200 mg/L against all the MRSA strains.
Since the chemical structure of IBT is quite different from that of
vancomycin, this family of novel anti-MRSA inhibitors might have
distinct mode of action. Bearing this in mind, we hence tested the
compounds against Enterococcus faecalis and VRE strains. As can be
seen from Table 2, the MIC of vancomycin for E. faecalis was 2 mg/L
and for VRE-309 was >8 mg/L. Surprisingly, all the IBTs showed
equal or even better inhibition against VRE compared with the
inhibition against E. faecalis. For compounds 3-11, 3-15, 3-16, 3-18,
3-19, 3-27, 3-32, 3-35, 3-36 and 3-37, their MICs for VRE were
1.56 mg/L and their MICs for E. faecalis were 3.12 mg/L. It was
exciting that for compounds 3-29 and 3-30, their MICs against E.
faecalis and VRE were both 1.56 mg/L.
seldom displayed good activities against Gram-positive bacteria. To
study the structureeactivity relationships, we further synthesized
another 41 novel IBT compounds (3-11 to 3-51) based on the
chemical structure of 3-2 (Table S1). These compounds have halides
or methyl substituent at 7-position of the isatin moiety (R1), and
the phenyl ring attached to the thiourea bridge are also substituted
by a single group of halides, methyl or methoxy group (R2). As can
be seen in Table S1, the second generation of the IBT compounds
demonstrated strong activities, indicating that the structural
modifications were successful. The MIC values of compounds 3-11,
3-12, 3-15, 3-16, 3-22, 3-24, 3-29, 3-30, 3-32, 3-35, 3-36 and 3-37
were 0.39 mg/L against MRAS (Chaoyang) strain, twice as potent as
that of 3-2. Compounds 3-13, 3-14, 3-18, 3-19, 3-21, 3-25, 3-26, 3-
27, 3-28, 3-38, 3-39, 3-41, 3-42, 3-44, 3-45, 3-46, 3-48, 3-50 and 3-
51 had equivalent MICs with 3-2, which inhibited MRSA (Chan-
gyang) strain completely at 0.78 mg/L. In comparison with 3-2,
when R1 is a halogen atom at 7-position of isatin ring, another
halogen atom or methyl group at 4-position of the phenyl ring (R2)
provides an improved MIC; however, if such a R2 group is at 3-
position, some compounds have improved inhibition while some
others remain at the same level with 3-2. When R1 is a methyl
group at 7-position and R2 is a halogen atom at 4-position, the
compound exhibits same activity with 3-2. It can also be seen that
when R2 is a methoxy group at 2-position, the compound decreases
its inhibition significantly, although when R2 is a methyl group, the
activity of the compound is similar to 3-2.
The compounds were also screened their inhibition against
Gram-negative Pseudomonas aeruginosa (PAO1) and Klebsiella
pneumoniae (KP-249). However, no compounds could inhibit these
bacteria strains completely even at 100 mg/L concentration. Thus it
seems the IBT compounds are active upon the Gram-positive bac-
teria, whether they are susceptible or resistant strains towards the
clinical online antibiotics methicillin and vancomycin. It is also
interesting that the MICs of the IBT compounds for resistant strains
are sometimes lower than those for the susceptible strains, which
deserves further investigation on their inhibition mechanism.
It was interesting that the anti-MRSA (Chaoyang) activities were
better than the activities against S. aureus ATCC strain. Compounds
3-29 and 3-30 had the best antibacterial activity against S. aureus
ATCC strain, the MICs of which were 0.78 mg/L. However, com-
pounds 3-34, 3-40, 3-43, 3-47 and 3-49 could not inhibit S. aureus
strain completely even at 100 mg/L concentration. The new IBT
compounds were also very potent against B. subtilis, and 30 of the
41 compounds had MIC values of 0.78 mg/L or less.
To exploit the application of these compounds, we further
evaluated the antibacterial activities of the second generation of IBT
compounds together with 3-2 against a series of MRSA strains
(309-4, 6281, 309-8, 6-42, 8-21, 309-3, 309-1, 309-7, 8-24 and 309-
6) from different clinical sources. Besides good inhibition against
the MRSA (Chaoyang) strain, the compounds also displayed
2.3. Three dimensional structureeactivity relationships
Comparative field analysis (CoMFA) is a tool to generate 3D
contour models to quantitatively analyze the structureeactivity
relationships of bioactive compounds by steric and electrostatic

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X.-M. Zhang et al. / European Journal of Medicinal Chemistry 101 (2015) 419e430
Table 2
Selected MIC data (mg/L) of the second generation of IBT compounds against Enterococcus faecalis, Pseudomonas aeruginosa, Klebsiella pneumoniae, clinical isolated VRE and
various MRSA strains.
Compd. R1
R2
E. faecalis VRE-309 PA
KP-249 MRSA
Chaoyang 309-4 6281 309-8 6-42
8-21
309-3 309-1 309-7 8-24
309-6
3-2
7-Cl
H
6.25
3.12
6.25
6.25
3.12
3.12
3.12
3.12
3.12
3.12
3.12
1.56
3.12
3.12
1.56
1.56
1.56
1.56
3.12
1.56
1.56
1.56
1.56
>100
1.56
1.56
1.56
>100
>100
>8.0
ND
>100 >100
>100 >100
>100 >100
>100 >100
>100 >100
>100 >100
>100 >100
>100 >100
>100 >100
>100 >100
>100 >100
>100 >100
>100 >100
>100 >100
>100 >100
>100 >100
>100 >100
>100 >100
>100 >100
0.78
0.39
0.39
0.78
0.39
0.39
0.78
0.78
0.39
0.78
0.39
0.39
0.39
25
1.56
0.78
0.78
1.56
0.78
0.78
1.56
1.56
0.78
0.78
0.39
0.78
0.78
>100
0.78
0.78
0.78
12.5
>100
1.0
1.56
1.56
1.56
1.56
1.56
1.56
3.12
3.12
1.56
1.56
1.56
1.56
3.12
1.56
1.56
0.78
1.56
0.78
0.78
1.56
1.56
0.78
1.56
0.78
0.78
1.56
3.12
0.78
0.78
0.78
0.78
0.78
1.56
1.56
0.78
0.78
0.39
0.39
0.78
3.12
0.78
0.78
0.78
0.78
0.78
1.56
1.56
0.78
1.56
0.78
0.78
1.56
6.25
1.56
0.78
1.56
0.78
0.78
3.12
1.56
0.78
1.56
0.78
0.78
1.56
6.25
0.78
0.78
1.56
0.78
0.78
1.56
1.56
0.78
1.56
0.78
0.78
1.56
>100
0.78
1.56
1.56
12.5
>100
1.0
0.78
0.78
0.78
0.78
0.39
0.39
1.56
0.78
0.78
0.78
0.39
0.39
0.78
>100
0.39
0.78
0.78
6.25
>100
1.0
1.56
1.56
1.56
1.56
1.56
1.56
1.56
1.56
1.56
1.56
0.78
0.78
1.56
6.25
1.56
1.56
1.56
1.56
1.56
3.12
1.56
1.56
3.12
1.56
1.56
1.56
3-11
3-12
3-13
3-15
3-16
3-18
3-19
3-22
3-27
3-29
3-30
3-32
3-34
3-35
3-36
3-37
3-40
3-49
Vanb
Metb
Cipb
7-Cl
7-Cl
7-Cl
7-Br
7-Br
7-CH3 4-Cl
7-CH3 4-F
7-F
7-Cl
7-F
7-F
7-Cl
7-Br
7-Br
7-Br
7-Br
7-I
4-Cl
4-CH3
2-CH3
4-F
4-Cl
3-CH3
3-Cl
3-F
3-Cl
3-F
2-OCH3 >100
3-F
3-Cl
4-Br
2-OCH3 >100
2-OCH3 >100
1.56
3.12
>100 >100
>100 >100 >100
>100 >100
3.12
3.12
3.12
0.39
0.39
0.39
6.25
>100
1.56
1.56
3.12
25
>100 >100
1.0 1.0
>200 >200
ND ND
0.78
1.56
1.56
12.5
0.78
0.78
0.78
6.25
0.78
1.56
1.56
12.5
0.78
1.56
1.56
25
0.78
1.56
1.56
12.5
>100 >100
1.0 1.0
>200 >200
ND ND
1.56
1.56
1.56
25
7-F
>100 >100 >100
1.0 1.0 1.0
>200 >200 >200
ND ND ND
2.0
ND
ND
NDa
ND
1.0
ND
ND
2.0
>200
ND
>200
ND
>200
ND
ND
a
ND ¼ not determined.
b
Van ¼ cancomycin, Met ¼ methicillin, Cip ¼ ciprofloxacin.
contributions [28]. Since the MIC data against MRSA (Chaoyang)
were determined for all the IBTs, they were used to calculate the
biological data D for the molecular simulation. The leave-one-out q2
is 0.601 at the optimum components of 6, and the non-cross-
validated r2 is 0.903, with a standard error of estimate of 0.165
and an F value of 55.963. The steric and electrostatic contributions
are 49.6% and 50.4%, respectively. From Table S3, it can be seen that
except 3-3, the activities of all the other 5 IBTs in the test set were
reasonably predicted, suggesting that the CoMFA model had pre-
dictive ability.
Compound 3-2 was used to illustrate the steric and electrostatic
contour maps. For the steric contour map (Fig. 3A), a bulky group is
favorable for better anti-MRSA activity in the green contour region
and such a group is likely to decrease the inhibition in the yellow
contour space. For the electrostatic contour map (Fig. 3B), in the
blue contour region, an increase in the positive charge will result in
an increase of activity, whereas in the red contour region, negative
charge is favorable to enhance the activity. From the 3D contour
maps, it can be seen a halogen atom is a key structural feature at the
7-position of isatin for desirable anti-MRSA activity of this series of
IBT compounds, from both the steric and the electrostatic re-
quirements. Thus we will design new molecules that have a eCF3 or
eOH group at 7-position of isatin ring to see if more potent bio-
logical activity can be observed. On the other hand, a steric group is
unfavorable at meta-position of the phenyl ring (for the R2 sub-
stituent), this can be explained by the fact that compounds 3-34, 3-
40 and 3-49 all have a eOCH3 group at this position and their ac-
tivities are very weak. The CoMFA contour map has provided
valuable information for further structural optimization of this
family of new anti-MRSA inhibitors.
3. Conclusion
Invention of novel antibiotics is a big challenge owing to the
limited resource from microbial metabolites. With the prevalence
of diseases caused by superbug infection, this task has turned to be
more and more urgent for medicinal research. We present here for
the first time that some novel isatin-b-thiosemicarbazones are
strong inhibitors of Gram-positive bacteria, especially for MRSA
and VRE.
The IBTs studied here were active against the selected Gram-
positive bacteria and non-active against the tested Gram-negative
strains. This “new” family of antibacterial compounds exhibited
desirable activity against dozens of clinical isolated MRSA strains,
and some of them also had potent inhibition against a VRE strain.
This suggests that the IBTs might inhibit the Gram-positive bacteria
through a different mechanism compared with vancomycin, which
deserves further investigation. Further structural optimization is
under way to get compounds with enhanced activity based on the
3D structureeactivity relationships. The present research herein
has provided meaningful information to discover anti-MRSA agents
for potential clinical use.
4. Experimental section
4.1. General synthesis and instruments
The substituted isatins
1
and substituted N-phenyl-
hydrazinecarbothioamide 2 were commercially procured from
Alfa-Aesar, SigmaeAldrich, J&K Chemical or Shanghai Aladdin Re-
agents. The solvents and other materials for the organic synthesis
were purchased from some local chemical suppliers in Tianjin. All
solvents and liquid reagents were dried by standard methods in
advance and distilled before use. Melting points were determined
using an X-4 melting apparatus and were uncorrected. 1H NMR
spectra were obtained using a 300 MHz Bruker AC-P300 spec-
trometer or a 400 MHz Varian Mercury Plus 400 spectrometer. The
From our structureeactivity relationship analysis, a halogen
substitution at 7-position of isatin is a key structural requirement
for IBTs to possess good anti-MRSA activity. Although the com-
pounds reported by Omar and Pervez et al. are very close to our IBT
compounds, they do not have any halogens at this position [21,22].
This might be one reason that the previously reported IBTs do not
have strong activity against S. aureus.
chemical shift values (d) for the NMR spectra were reported as parts

X.-M. Zhang et al. / European Journal of Medicinal Chemistry 101 (2015) 419e430
423
Fig. 3. Three-dimensional contour maps for the CoMFA model of the IBT compounds. Sterically favored and disfavored regions are shown in green and yellow in map A. Electrostatic
favored and disfavored regions are shown in blue and red in map B. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this
article.)
per million (ppm), using deuterated dimethyl sulfoxide (DMSO-d6)
as the solvent and tetramethylsilane (TMS) as an internal reference
standard. The infrared (IR) spectra were recorded with a Bruker
Equinox 55 spectrophotometer in KBr pellets. Elemental analyses
dried to give a crude product. Recrystallization from ethanol gave
pure solid in high yields.
4.2.1. (Z)-2-(7-Fluoro-2-oxoindolin-3-ylidene)-N-
phenylhydrazinecarbothioamide (3-1)
were determined on
a Yanaca CHN Corder MT-3 elemental
Yield 67%; m.p.: 222e224 ꢁC; yellow solid; 1H NMR (DMSO-d6,
analyzer. Mass spectra were recorded on a Thermo Finnigan LCQ
Advantage LC/mass detector instrument. Single-crystal X-ray
diffraction analyses were performed on a Bruker Smart 1000 CCD
diffractometer.
400 MHz),
d 12.82 (s, 1H, NNH), 11.85 (s, 1H, NH), 10.96 (s, 1H,
SCNH), 7.70 (d, 1H, J ¼ 8.0 Hz, Ar1eH4), 7.66 (d, 2H, J ¼ 8.0 Hz,
Ar2eH2 and Ar2eH6), 7.61 (d, 1H, J ¼ 8.4 Hz, Ar1eH6), 7.50 (t, 2H,
J ¼ 8.0 Hz, Ar2eH3 and Ar2eH5), 7.36 (t, 1H, J ¼ 8.4 Hz, Ar2eH4), 7.19
(t, 1H, J ¼ 7.5 Hz, Ar1eH5); Elemental analysis calculated for
4.2. Test compounds preparation
C15H11FN4OS, C: 57.31, H: 3.53, N: 17.82, found C: 56.92, H: 3.64, N:
17.81.
The chemical synthesis route of the target IBT compounds is
illustrated in Scheme 1. The IBTs 3 were synthesized by the reaction
of isatin 1 and N-phenylhydrazinecarbothioamide 2 in an aqueous
solution of ethanol and acetic acid under reflux [22,23]. Generally,
equivalent 1 and 2 was dissolved in a solution of acetic acid/water/
alcohol solution, and then the mixture was refluxed for approxi-
mate 5e6 h. The reaction mixture was subsequently cooled to room
temperature and in most cases a yellow or orange solid precipitated
out. This precipitate was filtered, washed with water, and then
4.2.2. (Z)-2-(7-Chloro-2-oxoindolin-3-ylidene)-N-
phenylhydrazinecarbothioamide (3-2)
Yield 75%; m.p.: 239e241 ꢁC; yellow solid; 1H NMR (DMSO-d6,
300 MHz), d 12.74 (s,1H, NNH),11.71 (s,1H, NH),10.91 (s,1H, SCNH),
7.76 (d, 1H, J ¼ 8.0 Hz, Ar1eH4), 7.60 (d, 2H, J ¼ 8.1 Hz, Ar2eH2 and
Ar2eH6), 7.44 (m, 3H, J ¼ 8.4 Hz, Ar1eH5, Ar2eH3 and Ar2eH5), 7.29
(t, 1H, J ¼ 7.5 Hz, Ar1eH6), 7.14 (t, 1H, J ¼ 6.9 Hz, Ar2eH4); Elemental

424
X.-M. Zhang et al. / European Journal of Medicinal Chemistry 101 (2015) 419e430
Scheme 1. Synthesis route of the target isatin b-thiosemicarbazone (IBT) compounds.
analysis calculated for C15H11ClN4OS, C: 54.46, H: 3.35, N: 16.94,
C), 1149 (C]S), 1029 (NeN); Elemental analysis calculated for
found C: 54.10, H: 3.54, N: 16.66; ESI-MS m/z: 329, [M ꢂ H]ꢂ.
C15H12N4OS, C: 60.79, H: 4.08, N: 18.91, found C: 60.88, H: 4.28, N:
18.75; ESI-MS m/z: 295, [M ꢂ H]ꢂ.
4.2.3. (Z)-2-(5-Chloro-2-oxoindolin-3-ylidene)-N-(4-fluorophenyl)
hydrazinecarbothio amide (3-3)
4.2.8. (Z)-N-(4-Fluorophenyl)-2-(2-oxoindolin-3-ylidene)
hydrazinecarbothioamide (3-8)
Yield 66%; m.p.: 268e270 ꢁC; brown solid; 1H NMR (DMSO-d6,
Yield 69%; m.p.: 242e244 ꢁC; yellow solid; 1H NMR (DMSO-d6,
400 MHz),
d 12.62 (s, 1H, NNH), 11.36 (s, 1H, NH), 10.90 (s, 1H,
SCNH), 7.86 (s, 1H, isatin ArH), 7.62e7.58 (m, 2H, ArH), 7.42e7.39
(m, 1H, isatin ArH), 7.31 (t, 2H, J ¼ 8.8 Hz, ArH), 6.97 (d, 1H,
300 MHz),
d 12.81 (s, 1H, NNH), 11.26 (s, 1H, NH), 10.82 (s, 1H,
SCNH), 7.76 (d, 1H, J ¼ 7.2 Hz, Ar1eH4), 7.63e7.58 (m, 2H, Ar2eH3
and Ar2eH5), 7.38 (t, 1H, J ¼ 7.5 Hz, Ar1eH6), 7.26 (t, 2H, J ¼ 8.7 Hz,
Ar2eH2 and Ar2eH6), 7.11 (t, 1H, J ¼ 7.5 Hz, Ar1eH5), 6.95 (d, 1H,
J ¼ 7.8 Hz, Ar1eH7); Elemental analysis calculated for C15H11FN4OS,
C: 57.31, H: 3.53, N: 17.82, found C:57.03, H: 3.77, N: 18.00; ESI-MS
m/z: 313, [M ꢂ H]ꢂ.
J
C
¼
8.4 Hz, isatin ArH); Elemental analysis calculated for
15H10ClFN4OS, C: 51.65, H: 2.89, N: 16.06, found C: 51.25, H: 3.14,
N: 16.26; ESI-MS m/z: 347, [M ꢂ H]ꢂ.
4.2.4. (Z)-2-(5-Bromo-2-oxoindolin-3-ylidene)-N-
phenylhydrazinecarbothioamide (3-4)
Yield 58%; m.p.: 230e232 ꢁC; yellow solid; 1H NMR (DMSO-d6,
4.2.9. (Z)-2-(5,7-Dibromo-2-oxoindolin-3-ylidene)-N-(4-
fluorophenyl)hydrazinecarbo thioamide (3-9)
400 MHz),
d 12.61 (s, 1H, NNH), 11.36 (s, 1H, NH), 10.91 (s, 1H,
SCNH), 8.02 (s, 1H, isatin ArH), 7.60 (d, 2H, ArH), 7.53 (d, 1H, isatin
ArH), 7.46 (t, 2H, J ¼ 8.8 Hz, ArH), 7.30 (d, 1H, J ¼ 8.4 Hz, isatin ArH),
6.91 (d, 1H, ArH); Elemental analysis calculated for C15H11BrN4OS,
C: 50.16, H: 3.09, N: 15.60, found C: 49.86, H: 3.12, N: 15.26.
Yield 83%; m.p.: 264e265 ꢁC; yellow solid; 1H NMR (DMSO-d6,
300 MHz),
d 12.56 (s, 1H, NNH), 11.70 (s, 1H, NH), 10.95 (s, 1H,
SCNH), 8.01 (s, 1H, isatin ArH), 7.82 (s, 1H, isatin ArH), 7.61e7.57 (m,
2H, ArH), 7.32e7.26 (m, 2H, ArH); Elemental analysis calculated for
C
15H9BrN4O5, C: 38.16, H: 1.92, N: 11.87, found C: 38.47, H: 2.12, N:
4.2.5. (Z)-2-(7-Bromo-5-nitro-2-oxoindolin-3-ylidene)-N-(4-
fluorophenyl)hydrazine carbothioamide (3-5)
12.05; ESI-MS m/z: 472, [M ꢂ H]ꢂ.
Yield 74%; m.p.: 268e269 ꢁC; yellow solid; 1H NMR (DMSO-d6,
4.2.10. (Z)-N-(4-Fluorophenyl)-2-(5-nitro-2-oxoindolin-3-ylidene)
hydrazinecarbothio amide (3-10)
400 MHz), d 12.53 (s,1H, NNH),12.21 (s,1H, NH),11.14 (s,1H, SCNH),
8.69 (d, 1H, J ¼ 2.1 Hz, isatin ArH), 8.46 (d, 1H, J ¼ 2.1 Hz, isatin ArH),
7.60e7.57 (m, 2H, ArH), 7.30 (t, 2H, J ¼ 7.8 Hz, ArH); Elemental
analysis calculated for C15H9BrFN5O3S, C: 41.11, H: 2.07, N: 15.62,
found C: 41.12, H: 2.32, N: 15.62; ESI-MS m/z: 439, [M ꢂ H]ꢂ.
Yield 71%; m.p.: 284e286 ꢁC; orange solid; 1H NMR (DMSO-d6,
400 MHz), d 12.56 (s,1H, NNH),11.87 (s,1H, NH),11.09 (s,1H, SCNH),
8.68 (d, 1H, J ¼ 2.4 Hz, isatin ArH), 8.30 (dd, 1H, J1 ¼ 2.0 Hz,
J2 ¼ 8.4 Hz, isatin ArH), 7.61e7.57 (m, 2H, ArH), 7.29 (t, 2H,
J ¼ 8.4 Hz, ArH), 7.15 (d, 1H, J ¼ 8.8 Hz, isatin ArH); Elemental
analysis calculated for C15H10FN5O3S, C: 50.14, H: 2.81, N: 19.49,
found C: 50.30, H: 2.58, N: 19.66; ESI-MS m/z: 359, [M ꢂ H]ꢂ.
4.2.6. (Z)-2-(5-Fluoro-2-oxoindolin-3-ylidene)-N-(4-fluorophenyl)
hydrazinecarbothio amide (3-6)
Yield 70%; m.p.: 279e280 ꢁC; brown-yellow solid, 1H NMR
(DMSO-d6, 400 MHz),
d 12.69 (s, 1H, NNH), 11.27 (s, 1H, NH), 10.86
(s, 1H, SCNH), 7.62e7.59 (m, 3H, isatin ArH&ArH), 7.28 (t, 2H,
J1 ¼ 8.8 Hz, ArH), 7.24e7.19 (m, 1H, isatin ArH), 6.69e6.93 (m, 1H,
isatin ArH); Elemental analysis calculated for C15H10F2N4OS, C:
54.21, H: 3.03, N: 16.86, found C: 54.55, H: 2.95, N: 16.74; ESI-MS m/
z: 332, [M ꢂ H]ꢂ.
4.2.11. (Z)-2-(7-Chloro-2-oxoindolin-3-ylidene)-N-(4-
chlorophenyl)hydrazinecarbo thioamide (3-11)
Yield 76%; m.p.: 251e253 ꢁC; yellow solid; 1H NMR (DMSO-d6,
400 MHz),
d 12.78 (s, 1H, NNH), 11.73 (s, 1H, NH), 10.93 (s, 1H,
SCNH), 7.74 (d, 1H, J ¼ 7.6 Hz, Ar1eH4), 7.67e7.65 (d, 2H, J ¼ 4.4 Hz,
Ar2eH3 and Ar2eH5), 7.51e7.48 (d, 2H, J ¼ 8.8 Hz, Ar2eH2 and
Ar2eH6), 7.46e7.44 (d, 1H, J ¼ 8.0 Hz, Ar1eH6), 7.16e7.12 (t, 1H,
4.2.7. (Z)-2-(2-Oxoindolin-3-ylidene)-N-
phenylhydrazinecarbothioamide (3-7)
J ¼ 8.0 Hz, Ar1eH5); 13C NMR (DMSO-d6, 101 MHz),
d 176.3, 162.7,
Yield 80%; m.p.: 233e235 ꢁC; orange solid; 1H NMR (DMSO-d6,
139.7, 137.7, 131.8, 131.2, 130.8, 127.6, 123.5, 121.9, 119.8, 118.4, 115.1;
300 MHz),
d
12.79 (s, 1H, NNH), 11.25 (s, 1H, NH), 10.82 (s, 1H,
IR: (KBr, n
/cmꢂ1), 3312 (NNeH), 3175 (CONeH), 3065 (AreH), 1691
SCNH), 7.79e7.35 (m, 6H, 5Ar2eH and Ar1eH4), 7.27 (t, 1H,
J ¼ 7.2 Hz, Ar1eH6), 7.11 (t, 1H, J ¼ 7.5 Hz, Ar1eH5), 6.94 (d, 1H,
(eCONHe), 1618 (C]N), 1589 and 1476 (AreC]C), 1168 (C]S),
1135 (NeN); Elemental analysis calculated for C15H10Cl2N4OS, C:
49.33, H: 2.76, N: 15.34, found C: 49.10, H: 2.95, N: 15.05; ESI-MS m/
z: 362.9, [M ꢂ H]ꢂ.
J ¼ 8.1 Hz, Ar1eH7); IR (KBr,
n
/cmꢂ1): 3299 (NNeH), 3176 (CONeH),
3059 (AreH), 1693 (eCONHe), 1620 (C]N),1541 and 1463 (AreC]

X.-M. Zhang et al. / European Journal of Medicinal Chemistry 101 (2015) 419e430
425
4.2.12. (Z)-2-(7-Chloro-2-oxoindolin-3-ylidene)-N-(p-tolyl)
133.5, 132.0, 130.2, 128.3, 127.3, 123.8, 121.9, 120.3, 103.4; IR: (KBr, n/
hydrazinecarbothioamide (3-12)
cmꢂ1), 3308 (NNeH), 3122 (CONeH), 3056 (AreH), 1696
(eCONHe), 1620 (C]N), 1590 and 1534 (AreC]C), 1240 (C]S),
1158 (NeN); Elemental analysis calculated for C15H10BrClN4OS, C:
43.98, H: 2.46, N: 13.68, found C: 43.69, H: 2.54, N: 13.52; ESI-MS m/
z: 408.9, [M ꢂ H]ꢂ.
Yield 72%; m.p.: 252e254 ꢁC; yellow solid; 1H NMR (DMSO-d6,
400 MHz),
d 12.72 (s, 1H, NNH), 11.70 (s, 1H, NH), 10.83 (s, 1H,
SCNH), 7.77 (d, 1H, J ¼ 7.2 Hz, Ar1eH4), 7.49e7.44 (m, 3H, Ar2eH3,
Ar2eH5 and Ar1eH6), 7.26e7.24 (d, 2H, J ¼ 7.6 Hz, Ar2eH2 and
Ar2eH6), 7.15 (t, 1H, J ¼ 7.6 Hz, Ar1eH5), 2.35 (s, 3H, CH3); 13C NMR
(DMSO-d6, 101 MHz);
d
176.3, 162.7, 139.6, 135.8, 135.4, 131.4, 130.7,
4.2.17. (Z)-2-(7-Methyl-2-oxoindolin-3-ylidene)-N-(p-tolyl)
hydrazinecarbothioamide (3-17)
128.8, 125.5, 123.5, 122.0, 119.8, 115.1, 20.6; IR: (KBr, n
/cmꢂ1), 3287
(NNeH), 3167 (CONeH), 3069 (AreH), 1698 (eCONHe), 1624 (C]
N), 1544 and 1472 (AreC]C), 1294 (C]S), 1140 (NeN); Elemental
analysis calculated for C16H13ClN4OS, C: 55.73, H: 3.80, N: 16.25,
found C: 55.64, H: 3.86, N: 16.08; ESI-MS m/z: 343.1, [M ꢂ H]ꢂ.
Yield 73%; m.p.: 247e249 ꢁC; yellow solid; 1H NMR (DMSO-d6,
400 MHz),
d 12.82 (s, 1H, NNH), 11.32 (s, 1H, NH), 10.74 (s, 1H,
SCNH), 7.63 (d, 1H, J ¼ 7.2 Hz, Ar1eH4), 7.50e7.48 (d, 2H, J ¼ 8.0 Hz,
Ar2eH3 and Ar2eH5), 7.25e7.19 (m, 3H, J ¼ 7.6 Hz, Ar1eH6, Ar2eH2
and Ar2eH6), 7.03 (t, 1H, J ¼ 7.6 Hz, Ar1eH5), 2.34 (s, 3H, Ar2eCH3),
4.2.13. (Z)-2-(7-Chloro-2-oxoindolin-3-ylidene)-N-(o-tolyl)
hydrazinecarbothioamide (3-13)
2.248 (s, 3H, Ar1eCH3); 13C NMR (DMSO-d6, 101 MHz),
d 176.2,
163.1, 141.0, 135.9, 135.3, 132.5, 132.5, 128.8, 125.5, 122.3, 120.4,
Yield 72%; m.p.: 241e243 ꢁC; yellow solid; 1H NMR (DMSO-d6,
119.6, 118.8, 20.6, 15.9; IR: (KBr,
n
/cmꢂ1), 3302 (NNeH), 3164
400 MHz),
d
12.73 (s, 1H, NNH), 11.70 (s, 1H, NH), 10.79 (s, 1H,
(CONeH), 3063 (AreH), 1693 (eCONHe), 1626 (C]N), 1585 and
1478 (AreC]C), 1211 (C]S), 1135 (NeN); Elemental analysis
calculated for C17H16N4OS, C: 62.94, H: 4.97, N: 17.27, found C:
62.70, H: 5.15, N: 17.28; ESI-MS m/z: 323.1, [M ꢂ H]ꢂ.
SCNH), 7.72 (d, 1H, J ¼ 6.8 Hz, Ar1eH4), 7.45 (d, 1H, J ¼ 8.0 Hz,
Ar1eH6), 7.34e7.30 (m, 4H, Ar2eH3, Ar2eH4, Ar2eH5 and Ar1eH5),
2.27 (s, 3H, CH3); 13C NMR (DMSO-d6, 101 MHz),
d
177.1, 162.7, 139.7,
137.3, 135.4,131.4,130.7,130.5,128.3,127.4,126.2,123.5,122.1,119.6,
115.1, 17.6; IR: (KBr,
/cmꢂ1), 3314 (NNeH), 3175 (CONeH), 3143
n
4.2.18. (Z)-N-(4-Chlorophenyl)-2-(7-methyl-2-oxoindolin-3-
ylidene)hydrazinecarbothioamide (3-18)
(AreH), 1692 (eCONHe), 1622 (C]N), 1587 and 1545 (AreC]C),
1295 (C]S), 1193 (NeN); Elemental analysis calculated for
Yield 77%; m.p.: 251e253 ꢁC; yellow solid; 1H NMR (DMSO-d6,
C
16H13ClN4OS, C: 55.73, H: 3.80, N: 16.25, found C: 55.82, H: 3.64, N:
400 MHz), d 12.91 (s, 1H, NNH), 11.35 (s, 1H, NH), 10.86 (s, 1H,
16.28; ESI-MS m/z: 379.0, [M þ Cl]ꢂ.
SCNH), 7.69e7.63 (m, 3H, J ¼ 6.4 Hz, Ar1eH4, Ar2eH3 and Ar2eH5),
7.51e7.43 (m, 2H, J ¼ 7.6 Hz, Ar2eH2 and Ar2eH6), 7.22 (d, 1H
J ¼ 7.6 Hz, Ar1eH6), 7.05 (t, 1H J ¼ 8.0 Hz, Ar1eH5), 2.26 (s, 3H, CH3);
4.2.14. (Z)-2-(7-Chloro-2-oxoindolin-3-ylidene)-N-(m-tolyl)
hydrazinecarbothioamide (3-14)
13C NMR (DMSO-d6, 101 MHz),
d 176.3, 163.1, 141.2, 137.4, 132.9,
Yield 72%; m.p.: 219e221 ꢁC; yellow solid; 1H NMR (DMSO-d6,
132.7, 130.0, 128.2, 127.2, 122.3, 120.5, 119.5, 118.1, 15.8; IR: (KBr, n/
400 MHz),
d
12.72 (s, 1H, NNH), 11.71 (s, 1H, NH), 10.83 (s, 1H,
cmꢂ1), 3310 (NNeH), 3173 (CONeH), 3057 (AreH), 1696
(eCONHe), 1626 (C]N), 1595 and 1539 (AreC]C), 1209 (C]S),
1155 (NeN); Elemental analysis calculated for C16H13ClN4OS, C:
55.73, H: 3.80, N: 16.25, found C: 56.01, H: 4.10, N: 16.46;
HRMS(MALDI) m/z: calculated. 345.0577, found 345.0565,
[M þ H]þ.
SCNH), 7.76 (d, 1H, J ¼ 7.2 Hz, Ar1eH4), 7.63e7.60 (m, 3H, J ¼ 6.8 Hz,
Ar1eH6, Ar2eH2 and Ar2eH6), 7.32 (t, 1H, J ¼ 7.6 Hz, Ar2eH3),
7.15e7.09 (m, 2H, J ¼ 8.4 Hz, Ar1eH5 and Ar2eH4), 2.35 (s, 3H, CH3);
13C NMR (DMSO-d6, 101 MHz),
d 176.1, 162.7, 139.6, 138.2, 137.7,
131.4, 130.7, 128.2, 126.8, 126.0, 123.4, 122.7, 122.0, 119.8, 115.1, 20.9;
IR: (KBr,
n
/cmꢂ1), 3314 (NNeH), 3197 (CONeH), 3148 (AreH), 1693
(eCONHe), 1622 (C]N), 1545 and 1487 (AreC]C), 1248 (C]S),
1172 (NeN); Elemental analysis calculated for C16H13ClN4OS, C:
55.73, H: 3.80, N: 16.25; found C: 55.90, H: 3.68, N: 16.25; ESI-MS
m/z: 343.0, [M ꢂ H]ꢂ.
4.2.19. (Z)-N-(4-Fluorophenyl)-2-(7-methyl-2-oxoindolin-3-
ylidene)hydrazinecarbothioamide (3-19)
Yield 70%; m.p.: 242e244 ꢁC; yellow solid; 1H NMR (DMSO-d6,
400 MHz),
d 12.89 (s, 1H, NNH), 11.33 (s, 1H, NH), 10.84 (s, 1H,
SCNH), 7.68e7.66 (m, 2H, J ¼ 8.0 Hz, Ar2eH3 and Ar2eH5), 7.61 (d,
1H J ¼ 7.2 Hz, Ar1eH4), 7.49e7.48 (m, 2H, J ¼ 4.4 Hz, Ar2eH2 and
Ar2eH6), 7.20 (d, 1H J ¼ 7.2 Hz, Ar1eH6), 7.03 (t, 1H J ¼ 7.6 Hz,
4.2.15. (Z)-2-(7-Bromo-2-oxoindolin-3-ylidene)-N-(4-
fluorophenyl)hydrazinecarbo thioamide (3-15)
Yield 74%; m.p.: 254e256 ꢁC; yellow solid; 1H NMR (DMSO-d6,
Ar1eH5), 2.24 (s, 3H, CH3); 13C NMR (DMSO-d6, 101 MHz),
d 176.3,
400 MHz),
d
12.78 (s, 1H, NNH), 11.59 (s, 1H, NH), 10.93 (s, 1H,
163.1, 141.1, 137.4, 132.9, 132.7, 130.0, 128.2, 127.2, 122.3, 120.5, 119.4,
SCNH), 7.78 (d, 1H, J ¼ 7.6 Hz, Ar1eH4), 7.67e7.65 (d, 2H, J ¼ 8.4 Hz,
Ar2eH3 and Ar2eH5), 7.57 (t, 1H, J ¼ 8.0 Hz, Ar1eH6), 7.51e7.48 (d,
2H, J ¼ 8.4 Hz, Ar2eH2 and Ar2eH6), 7.08 (t, 1H, J ¼ 8.0 Hz, Ar1eH6);
118.8, 15.8; IR: (KBr,
n
/cmꢂ1), 3308 (NNeH), 3165 (CONeH), 3052
(AreH), 1695 (eCONHe), 1625 (C]N), 1572 and 1539 (AreC]C),
1209 (C]S), 1153 (NeN); Elemental analysis calculated for
C16H13FN4OS, C: 58.52, H: 3.99, N: 17.06, found C: 58.75, H: 3.66, N:
13C NMR (DMSO-d6, 101 MHz);
d
176.3, 162.7, 141.4, 137.3, 133.7,
/cmꢂ1
132.0, 130.1, 128.3, 127.3, 123.8, 121.9, 120.2, 103.3; IR: (KBr,
n
)
17.45.
3310 (NNeH), 3129 (CONeH), 3011 (AreH), 1696 (eCONHe), 1621
(C]N), 1590 and 1535 (AreC]C), 1435 (C]S), 1160 (NeN);
Elemental analysis calculated for C15H10BrFN4OS, C: 45.82, H: 2.56,
N: 14.25, found C: 45.59, H: 2.85, N: 14.05.
4.2.20. (Z)-2-(7-Fluoro-2-oxoindolin-3-ylidene)-N-(o-tolyl)
hydrazinecarbothioamide (3-20)
Yield 79%; m.p.: 230e232 ꢁC; yellow solid; 1H NMR (DMSO-d6,
400 MHz),
d 12.75 (s, 1H, NNH), 11.78 (s, 1H, NH), 10.69 (s, 1H,
4.2.16. (Z)-2-(7-Bromo-2-oxoindolin-3-ylidene)-N-(4-
chlorophenyl)hydrazinecarbothioamide (3-16)
SCNH), 7.61 (d, 1H, J ¼ 7.2 Hz, Ar1eH4), 7.33e7.30 (m, 5H, J ¼ 8.0 Hz,
Ar1eH5, Ar1eH6, Ar2eH3, Ar2eH4 and Ar2eH5), 7.14 (m, 1H,
J ¼ 5.2 Hz, Ar1eH5), 2.26 (s, 3H, CH3); 13C NMR (DMSO-d6,101 MHz),
Yield 75%; m.p.: 255e257 ꢁC; yellow solid; 1H NMR (DMSO-d6,
400 MHz),
d
12.79 (s, 1H, NNH), 11.60 (s, 1H, NH), 10.94 (s, 1H,
d 177.1, 162.4, 137.3, 135.4, 130.3, 129.1, 128.3, 127.4, 126.7, 123.2,
SCNH), 7.79 (d, 1H, J ¼ 6.8 Hz, Ar1eH4), 7.68e7.66 (d, 2H, J ¼ 8.0 Hz,
Ar2eH3 and Ar2eH5), 7.58 (d, 1H, J ¼ 7.6 Hz, Ar1eH6), 7.52e7.49 (d,
2H, J ¼ 7.6 Hz Hz, Ar2eH2 and Ar2eH6), 7.10 (t, 1H, J ¼ 7.6 Hz,
123.2, 123.0, 117.9, 117.8, 117.2, 17.6; IR: (KBr, n
/cmꢂ1), 3242 (NNeH),
3176 (CONeH), 3059 (AreH), 1694 (eCONHe), 1646 (C]N), 1541
and 1463 (AreC]C), 1149 (C]S), 1029 (NeN); Elemental analysis
calculated for C16H13FN4OS, C: 58.52, H: 3.99, N: 17.06, found C:
Ar1eH5); 13C NMR (DMSO-d6, 101 MHz),
d 176.3, 162.7, 141.5, 137.3,

426
X.-M. Zhang et al. / European Journal of Medicinal Chemistry 101 (2015) 419e430
58.40, H: 4.03, N: 17.30; ESI-MS m/z: 327.1, [M ꢂ H]ꢂ.
7.33e7.29 (m, 4H, J ¼ 8.4 Hz, Ar1eH5, Ar2eH2, Ar2eH3 and Ar2eH5),
7.07 (t, 1H, J ¼ 7.6 Hz, Ar1eH6), 2.25 (s, 3H, CH3); 13C NMR (DMSO-
4.2.21. (Z)-2-(7-Fluoro-2-oxoindolin-3-ylidene)-N-(4-
fluorophenyl)hydrazinecarbothioamide (3-21)
d6, 101 MHz), d 177.1, 162.7, 141.4, 137.3, 135.4, 133.6, 131.6, 130.3,
128.4, 127.5, 126.2, 123.8, 122.1, 120.1, 103.1, 17.6; IR: (KBr, n
/cmꢂ1),
Yield 78%; m.p.: 241e243 ꢁC; yellow solid; 1H NMR (DMSO-d6,
3310 (NNeH), 3161 (CONeH), 3057 (AreH), 1692 (eCONHe), 1619
(C]N), 1580 and 1472 (AreC]C), 1190 (C]S), 1153 (NeN);
HRMS(MALDI) m/z: calculated for C16H13BrN4OS 386.9915, found
386.9913, [M ꢂ H]ꢂ.
400 MHz),
d 12.78 (s, 1H, NNH), 11.80 (s, 1H, NH), 10.89 (s, 1H,
SCNH), 7.63e7.59 (m, 3H, J ¼ 7.2 Hz, Ar1eH4, Ar2eH3 and Ar2eH5),
7.32e7.25 (m, 3H, J ¼ 9.2 Hz, Ar1eH6, Ar1eH2 and Ar1eH6), 7.14 (m,
1H J ¼ 4.4 Hz, Ar1eH5); 13C NMR (DMSO-d6,101 MHz),
d 176.7,161.2,
162.5, 148.0, 134.7, 131.5, 129.3, 128.0, 123.2, 122.9, 118.0, 117.3,
4.2.26. (Z)-2-(7-Bromo-2-oxoindolin-3-ylidene)-N-(3-
methoxyphenyl)hydrazinecarbothioamide (3-26)
115.2; IR: (KBr,
n
/cmꢂ1), 3294 (NNeH), 3178 (CONeH), 3046
(AreH), 1695 (eCONHe), 1646 (C]N), 1515 and 1479 (AreC]C),
1167 (C]S), 1137 (NeN); Elemental analysis calculated for
Yield 79%; m.p.: 206e208 ꢁC; yellow solid; 1H NMR (DMSO-d6,
400 MHz),
d 12.74 (s, 1H, NNH), 11.58 (s, 1H, NH), 10.85 (s, 1H,
C
15H10FClN4OS, C: 54.21, H: 3.03, N: 16.86, found C: 54.44, H: 3.00,
SCNH), 7.81 (d, 1H, J ¼ 7.2 Hz, Ar1eH4), 7.57 (d, 1H, J ¼ 8.0 Hz,
Ar1eH6), 7.36e7.23 (m, 3H, J ¼ 8.0 Hz, Ar1eH5, Ar2eH2 and
Ar2eH6), 7.08 (t, 1H, J ¼ 8.0 Hz, Ar2eH3), 6.87 (d, 1H, J ¼ 7.6 Hz,
N: 16.57; ESI-MS m/z: 331.0, [M ꢂ H]ꢂ.
4.2.22. (Z)-2-(7-Fluoro-2-oxoindolin-3-ylidene)-N-(m-tolyl)
hydrazinecarbothioamide (3-22)
Ar2eH4), 3.79 (s, 3H, OCH3); 13C NMR (DMSO-d6, 101 MHz),
d 175.9,
162.7, 159.1, 141.4, 139.4, 133.6, 131.7, 129.3, 123.8, 122.0, 120.3, 117.5,
Yield 79%; m.p.: 230e232 ꢁC; yellow solid; 1H NMR (DMSO-d6,
111.6, 111.1, 103.3, 55.2; IR: (KBr,
n
/cmꢂ1), 3246 (NNeH), 3144
400 MHz),
d
12.75 (s, 1H, NNH), 11.81 (s, 1H, NH), 10.85 (s, 1H,
(CONeH), 3065 (AreH), 1698 (eCONHe), 1620 (C]N), 1539 and
1492 (AreC]C), 1156 (C]S), 1129 (NeN); Elemental analysis
calculated for C16H13BrN4O2S, C: 47.42, H: 3.23, N: 13.82, found C:
47.29, H: 3.40, N: 13.68; ESI-MS m/z: 404.9, [M ꢂ H]ꢂ.
SCNH), 7.65 (d, 1H, J ¼ 7.2 Hz, Ar1eH4), 7.45e7.43 (m, 2H, J ¼ 6.8 Hz,
Ar1eH5 and Ar1eH6), 7.34e7.28 (m, 2H, J ¼ 6.8 Hz, Ar2eH3 and
Ar2eH4), 7.16e7.11 (m, 2H, J ¼ 7.2 Hz, Ar2eH2 and Ar2eH6), 2.36 (s,
3H, CH3); 13C NMR (DMSO-d6, 101 MHz),
d 176.1, 162.5, 148.0, 138.2,
137.7, 131.3, 129.2, 128.2, 126.8, 123.2, 123.1, 122.9, 122.7, 118.0, 117.8,
4.2.27. (Z)-2-(7-Chloro-2-oxoindolin-3-ylidene)-N-(3-
chlorophenyl)hydrazinecarbothioamide (3-27)
20.9; IR: (KBr,
n
/cmꢂ1) 3298 (NNeH), 3179 (CONeH), 3048 (AreH),
1695 (eCONHe), 1644 (C]N), 1593 and 1548 (AreC]C), 1247 (C]
S), 1149 (NeN); HRMS(MALDI) m/z: calculated for C16H13FN4OS
329.0872, found 329.0865, [M þ H]þ.
Yield 75%; m.p.: 246e248 ꢁC; yellow solid; 1H NMR (DMSO-d6,
400 MHz),
d 12.79 (s, 1H, NNH), 11.74 (s, 1H, NH), 10.95 (s, 1H,
SCNH), 7.79e7.64 (m, 3H, J ¼ 8.0 Hz, Ar1eH4, Ar1eH6, and Ar2eH4),
7.51e7.35 (m, 3H, J ¼ 7.6 Hz, Ar1eH5, Ar2eH2, and Ar2eH6), 7.15 (t,
4.2.23. (Z)-2-(7-Fluoro-2-oxoindolin-3-ylidene)-N-(3-
methoxyphenyl)hydrazinecarbothioamide (3-23)
1H, J ¼ 8.0 Hz, Ar2eH3); 13C NMR (DMSO-d6, 101 MHz),
d 176.2,
162.7, 139.8, 137.3, 132.4, 131.9, 130.1, 128.3, 127.3, 125.8, 124.0,
Yield 75%; m.p.: 230e232 ꢁC; yellow solid; 1H NMR (DMSO-d6,
123.5, 121.8, 119.8, 115.2; IR: (KBr, n
/cmꢂ1), 3320 (NNeH), 3165
400 MHz),
d
12.79 (s, 1H, NNH), 11.80 (s, 1H, NH), 10.85 (s, 1H,
(CONeH), 3050 (AreH), 1691 (eCONHe), 1621 (C]N), 1586 and
1525 (AreC]C), 1164 (C]S), 1137 (NeN); Elemental analysis
calculated for C15H10Cl2N4OS, C: 49.33, H: 2.76, N: 15.34, found C:
49.75, H: 3.02, N: 15.63; ESI-MS m/z: 363.0, [M ꢂ H]ꢂ.
SCNH), 7.66 (d, 1H, J ¼ 7.6 Hz, Ar1eH4), 7.37e7.29 (m, 3H, J ¼ 6.8 Hz,
Ar1eH5, Ar1eH6 and Ar2eH5), 7.25 (d, 1H, J ¼ 8.0 Hz, Ar2eH6),
7.18e7.12 (m, 1H, J ¼ 8.4 Hz, Ar2eH4), 6.89e6.86 (m, 1H, J ¼ 6.8 Hz,
Ar2eH2), 3.80 (s, 3H, OCH3); 13C NMR (DMSO-d6, 101 MHz),
d 176.0,
162.5, 159.1, 148.1, 145.6, 139.4, 131.5, 129.1, 123.2, 122.9, 118.0, 117.9,
4.2.28. (Z)-2-(7-Chloro-2-oxoindolin-3-ylidene)-N-(3-
methoxyphenyl)hydrazinecarbothioamide (3-28)
117.6, 111.6, 111.1, 55.2; IR: (KBr,
n
/cmꢂ1), 3302 (NNeH), 3162
(CONeH), 3080 (AreH), 1695 (eCONHe), 1595 (C]N), 1541 and
1454 (AreC]C), 1148 (C]S), 1049 (NeN); Elemental analysis
calculated for C16H13FN4O2S, C: 55.80, H: 3.81, N: 16.27, found C:
55.84, H: 4.03, N: 16.13; ESI-MS m/z: 343.1, [M ꢂ H]ꢂ.
Yield 70%; m.p.: 219e221 ꢁC; yellow solid; 1H NMR (DMSO-d6,
400 MHz),
d 12.74 (s, 1H, NNH), 11.72 (s, 1H, NH), 10.86 (s, 1H,
SCNH), 7.77 (d, 1H, J ¼ 7.6 Hz, Ar1eH4), 7.45 (d, 1H, J ¼ 8.0 Hz,
Ar1eH6), 7.34 (t, 1H, J ¼ 8.0 Hz, Ar1eH5), 7.29 (s, 1H, Ar2eH6), 7.24
(d, 1H, J ¼ 7.6 Hz, Ar2eH4), 7.14 (t, 1H, J ¼ 7.6 Hz, Ar2eH3), 6.87 (d,
1H, J ¼ 6.4 Hz, Ar2eH2), 3.79 (s, 3H, OCH3); 13C NMR (DMSO-d6,
4.2.24. (Z)-2-(7-Bromo-2-oxoindolin-3-ylidene)-N-(p-tolyl)
hydrazinecarbothioamide (3-24)
101 MHz), d 176.0, 162.7, 159.1, 139.7, 139.4, 131.5, 130.7, 129.1, 126.5,
Yield 78%; m.p.: 254e256 ꢁC; yellow solid; 1H NMR (DMSO-d6,
121.9, 119.9, 117.7, 115.1, 111.6, 111.1, 55.2; IR: (KBr, n
/cmꢂ1), 3302
400 MHz),
d
12.71 (s, 1H, NNH), 11.57 (s, 1H, NH), 10.84 (s, 1H,
(NNeH), 3179 (CONeH), 3018 (AreH), 1695 (eCONHe), 1624 (C]
N), 1594 and 1542 (AreC]C), 1204 (C]S), 1149 (NeN); Elemental
analysis calculated for C16H13ClN4O2S, C: 53.26, H: 3.63, N: 15.53,
found C: 53.45, H: 3.59, N: 15.34; ESI-MS m/z: 359.0, [M ꢂ H]ꢂ.
SCNH), 7.80 (d, 1H, J ¼ 7.6 Hz, Ar1eH4), 7.57 (d, 1H, J ¼ 8.0 Hz,
Ar1eH6), 7.48e7.46 (d, 2H, J ¼ 8.0 Hz, Ar2eH3 and Ar2eH5),
7.25e7.23 (d, 2H, J ¼ 8.0 Hz, Ar2eH2 and Ar2eH6), 7.07 (t, 1H,
J ¼ 8.0 Hz, Ar1eH5), 2.34 (s, 3H, CH3); 13C NMR (DMSO-d6,101 MHz),
d
176.2, 162.7, 141.3, 135.7, 135.4, 133.6, 131.5, 128.8, 125.5, 123.8,
4.2.29. (Z)-2-(7-Fluoro-2-oxoindolin-3-ylidene)-N-(3-
fluorophenyl)hydrazinecarbothioamide (3-29)
122.0, 120.2, 103.3, 20.6; IR: (KBr, n
/cmꢂ1), 3310 (NNeH), 3125
(CONeH), 3050 (AreH), 1694 (eCONHe), 1595 (C]N), 1535 and
1479 (AreC]C), 1152 (C]S), 1132 (NeN); Elemental analysis
calculated for C16H13BrN4OS, C: 49.37, H: 3.37, N: 14.39, found C:
49.12, H: 3.42, N: 14.16; ESI-MS m/z: 380.9, [M ꢂ H]ꢂ.
Yield 76%; m.p.: 230e232 ꢁC; orange solid; 1H NMR (DMSO-d6,
400 MHz),
d 12.83 (s, 1H, NNH), 11.82 (s, 1H, NH), 10.94 (s, 1H,
SCNH), 7.65e7.63 (m, 2H, J ¼ 7.2 Hz, Ar1eH4 and Ar1eH6), 7.53e7.45
(m, 2H, J ¼ 8.0 Hz, Ar1eH5 and Ar2eH4), 7.31 (t, 1H, J ¼ 8.8 Hz,
Ar2eH5), 7.17e7.12 (m, 2H, J ¼ 4.0 Hz, Ar2eH2 and Ar2eH6); 13C
4.2.25. (Z)-2-(7-Bromo-2-oxoindolin-3-ylidene)-N-(o-tolyl)
hydrazinecarbothioamide (3-25)
NMR (DMSO-d6, 101 MHz), d 176.2, 162.5, 148.1, 145.6, 140.1, 131.8,
129.8, 129.4, 123.2, 122.8, 121.3, 118.2, 117.5, 112.6, 112.4; IR: (KBr, n/
Yield 72%; m.p.: 241e243 ꢁC; yellow solid; 1H NMR (DMSO-d6,
cmꢂ1), 3286 (NNeH), 3160 (CONeH), 3068 (AreH), 1696
(eCONHe), 1644 (C]N), 1602 and 1540 (AreC]C), 1213 (C]S),
1148 (NeN); Elemental analysis calculated for C15H10F2N4OS, C:
400 MHz),
d 12.71 (s, 1H, NNH), 11.57 (s, 1H, NH), 10.79 (s, 1H,
SCNH), 7.75 (d,1H, J ¼ 6.8z, Ar1eH4), 7.57 (d, 1H, J ¼ 7.6 Hz, Ar1eH6),

X.-M. Zhang et al. / European Journal of Medicinal Chemistry 101 (2015) 419e430
427
54.21; H: 3.03; N: 16.86, found C: 54.07, H: 3.27, N: 17.06; ESI-MS m/
z: 331.1, [M ꢂ H]ꢂ.
SCNH), 7.779 (d, 1H, J ¼ 7.6 Hz, Ar1eH4), 7.721 (d, 1H, J ¼ 7.2 Hz,
Ar1eH6), 7.590 (d, 1H, J ¼ 7.6 Hz, Ar2eH5), 7.319 (t, 1H, J ¼ 7.6 Hz,
Ar1eH5), 7.163 (d, 1H, J ¼ 8.0 Hz, Ar2eH5), 7.089 (t, 1H, J ¼ 8.0 Hz,
Ar2eH3), 7.025 (t, 1H, J ¼ 7.6 Hz, Ar2eH4), 3.872 (s, 3H, OCH3); 13C
4.2.30. (Z)-N-(3-Chlorophenyl)-2-(7-fluoro-2-oxoindolin-3-
ylidene)hydrazinecarbothioamide (3-30)
NMR (DMSO-d6, 101 MHz);
d 176.1, 162.7, 153.1, 141.4, 133.7, 131.7,
Yield 69%; m.p.: 210e211 ꢁC; yellow solid; 1H NMR (DMSO-d6,
127.6, 126.9, 126.5, 123.9, 121.9, 120.0, 119.9, 111.9, 103.3, 55.7; IR:
400 MHz),
d
12.82 (s, 1H, NNH), 11.81 (s, 1H, NH), 10.93 (s, 1H,
(KBr,
n
/cmꢂ1), 3294 (NNeH), 3162 (CONeH), 3066 (AreH), 1691
SCNH), 7.79 (d, 1H, J ¼ 7.2 Hz, Ar1eH4), 7.66e7.62 (m, 2H, J ¼ 4.8 Hz,
Ar1eH5 and Ar1eH6), 7.46 (t, 1H, J ¼ 8.0 Hz, Ar2eH3), 7.35e7.27 (m,
3H, J ¼ 6.8 Hz, Ar2eH2, Ar2eH4 and Ar2eH6); 13C NMR (DMSO-d6,
(eCONHe), 1652 (C]N), 1544 and 1479 (AreC]C), 1224 (C]S),
1139 (NeN); Elemental analysis calculated for C16H13BrN4O2S, C:
47.42, H: 3.23, N: 13.82, found C: 47.22, H: 3.35, N: 13.83; ESI-MS m/
z: 405.0, [M ꢂ H]ꢂ.
101 MHz),
d 176.2, 162.4, 148.0, 145.7, 142.2, 139.8, 132.6, 129.9,
125.8, 124.0, 123.2, 120.6, 118.1, 117.4, 115.3; IR: (KBr, n
/cmꢂ1), 3313
(NNeH), 3167 (CONeH), 3077 (AreH), 1692 (eCONHe), 1645 (C]
N), 1589 and 1530 (AreC]C), 1210 (C]S), 1161 (NeN); Elemental
analysis calculated for C15H10FClN4OS, C: 51.65, H: 2.89, N: 16.06,
found C: 51.45, H: 2.68, N: 16.05; ESI-MS m/z: 347.0, [M ꢂ H]ꢂ.
4.2.35. (Z)-2-(7-Bromo-2-oxoindolin-3-ylidene)-N-(3-
fluorophenyl)hydrazinecarbothioamide (3-35)
Yield 76%; m.p.: 243e245 ꢁC; yellow solid; 1H NMR (DMSO-d6,
400 MHz),
d 12.81 (s, 1H, NNH), 11.59 (s, 1H, NH), 10.96 (s, 1H,
SCNH), 7.80 (d, 1H, J ¼ 7.6 Hz, Ar1eH4), 7.64 (d, 1H, J ¼ 6.8 Hz,
Ar1eH6), 7.58 (d, 1H, J ¼ 5.2 Hz, Ar2eH4), 7.53e7.46 (m, 2H,
J ¼ 6.8 Hz, Ar1eH5 and Ar2eH5), 7.16e7.07 (m, 2H, J ¼ 7.6 Hz,
4.2.31. (Z)-2-(7-Fluoro-2-oxoindolin-3-ylidene)-N-(4-
methoxyphenyl)hydrazinecarbothioamide (3-31)
Yield 77%; m.p.: 255e257 ꢁC; yellow solid; 1H NMR (DMSO-d6,
Ar2eH2 and Ar2eH6); 13C NMR (DMSO-d6, 101 MHz),
d 176.2, 162.7,
400 MHz),
d
12.72 (s, 1H, NNH), 11.79 (s, 1H, NH), 10.81 (s, 1H,
141.5,139.9,133.8,132.1,129.9,129.8,123.8,121.9,121.3,120.3,112.6,
SCNH), 7.63 (d, 1H, J ¼ 6.8 Hz, Ar1eH4), 7.47e7.45 (m, 2H, J ¼ 8.0 Hz,
Ar2eH3 and Ar2eH5), 7.30 (t, 1H, J ¼ 9.2 Hz, Ar1eH5), 7.13 (d, 1H,
J ¼ 4.0 Hz, Ar1eH6), 7.00e6.98 (m, 2H, J ¼ 8.0 Hz, Ar2eH2 and
112.4, 103.4; IR: (KBr,
n
/cmꢂ1), 3233 (NNeH), 3132 (CONeH), 3058
(AreH), 1694 (eCONHe), 1638 (C]N), 1548 and 1488 (AreC]C),
1182 (C]S), 1133 (NeN); Elemental analysis calculated for
Ar2eH6), 3.79 (s, 3H, OCH3); 13C NMR (DMSO-d6, 101 MHz),
d
176.5,
C15H10BrFN4OS, C: 45.82, H: 2.56, N: 14.25, found C: 45.65, H: 2.65,
162.5, 157.4, 148.0, 131.1, 129.2, 129.0, 127.2, 123.2, 117.9, 117.9, 117.6,
113.8, 55.2; IR: (KBr,
N: 14.44; ESI-MS m/z: 392.9, [M ꢂ H]ꢂ.
n
/cmꢂ1) 3329 (NNeH), 3189 (CONeH), 3041
(AreH), 1698 (eCONHe), 1643 (C]N), 1597 and 1521 (AreC]C),
1239 (C]S), 1158 (NeN); Elemental analysis calculated for
4.2.36. (Z)-2-(7-Bromo-2-oxoindolin-3-ylidene)-N-(3-
chlorophenyl)hydrazinecarbothioamide (3-36)
C
16H13FN4O2S, C: 55.80, H: 3.81, N: 16.27, found C: 55.57, H: 3.59, N:
Yield 78%; m.p., 232e234 ꢁC; yellow solid; 1H NMR (DMSO-d6,
16.08; ESI-MS m/z: 343.1, [M ꢂ H]ꢂ.
400 MHz), d 12.80 (s, 1H, NNH), 11.60 (s, 1H, NH), 10.95 (s, 1H,
SCNH), 7.79e7.77 (m, 2H, J ¼ 8.0 Hz, Ar1eH4 and Ar2eH6), 7.65 (d,
1H, J ¼ 7.6 Hz, Ar1eH6), 7.58 (d, 1H, J ¼ 8.0 Hz, Ar2eH4), 7.48 (t, 1H,
J ¼ 8.0 Hz, Ar1eH5), 7.35 (d, 1H, J ¼ 8.0 Hz, Ar2eH2), 7.61 (d, 1H,
4.2.32. (Z)-2-(7-Chloro-2-oxoindolin-3-ylidene)-N-(3-
fluorophenyl)hydrazinecarbothioamide (3-32)
Yield 76%; m.p.: 243-245 ꢁC; yellow solid; 1H NMR (DMSO-d6,
J ¼ 8.0 Hz, Ar2eH3), 13C NMR (DMSO-d6, 101 MHz),
d 176.2, 162.8,
400 MHz),
d
12.80 (s, 1H, NNH), 11.73 (s, 1H, NH), 10.95 (s, 1H,
141.5, 139.8, 133.8, 132.6, 132.1, 129.9, 125.9, 125.0, 124.06, 123.8,
SCNH), 7.76 (d, 1H, J ¼ 7.6 Hz, Ar1eH4), 7.63 (d, 1H, J ¼ 11.2 Hz,
Ar1eH6), 7.52e7.45 (m, 3H, J ¼ 7.6 Hz, Ar1eH5, Ar2eH3 and Ar2eH4),
7.17e7.12 (m, 2H, J ¼ 8.0 Hz, Ar2eH2 and Ar2eH6); 13C NMR (DMSO-
121.9, 120.3, 103.4; IR: (KBr, n
/cmꢂ1), 3320 (NNeH), 3222 (CONeH),
3121 (AreH), 1697 (eCONHe), 1618 (C]N), 1587 and 1528 (AreC]
C), 1219 (C]S), 1160 (NeN); Elemental analysis calculated for
d6, 101 MHz),
125.5, 121.9, 121.4, 119.9, 115.2, 112.6; IR: (KBr,
d
176.2, 162.8, 160.3, 140.0, 139.8, 131.9, 130.9, 129.9,
C15H10BrClN4OS, C: 43.98, H: 2.46, N: 13.68, found C: 43.78, H: 2.57,
n
/cmꢂ1), 3282
N: 13.57. ESI-MS m/z: 408.9, [M ꢂ H]ꢂ.
(NNeH), 3136 (CONeH), 3017 (AreH), 1698 (eCONHe), 1623 (C]
N), 1591 and 1530 (AreC]C), 1248 (C]S), 1179 (NeN); Elemental
analysis calculated for C15H10ClFN4OS, C: 51.65, H: 2.89, N: 16.06,
found C: 51.55, H: 3.05, N: 16.01; ESI-MS m/z: 347.0, [M ꢂ H]ꢂ.
4.2.37. (Z)-2-(7-Bromo-2-oxoindolin-3-ylidene)-N-(4-
bromophenyl)hydrazinecarbothioamide (3-37)
Yield 73%; m.p.; 247e249 ꢁC; yellow solid; 1H NMR (DMSO-d6,
400 MHz),
d 12.78 (s, 1H, NNH), 11.59 (s, 1H, NH), 10.92 (s, 1H,
4.2.33. (Z)-2-(7-Chloro-2-oxoindolin-3-ylidene)-N-(4-
methoxyphenyl)hydrazinecarbothioamide (3-33)
SCNH), 7.78 (d, 1H, J ¼ 7.2 Hz, Ar1eH4), 7.66e7.57 (m, 5H, J ¼ 7.6 Hz,
Ar1eH6, Ar2eH2, Ar2eH3, Ar2eH5 and Ar2eH6), 7.08 (t, 1H,
Yield 78%; m.p.: 251e253 ꢁC; yellow solid; 1H NMR (DMSO-d6,
J ¼ 8.0 Hz, Ar1eH5); 13C NMR (DMSO-d6, 101 MHz),
d 176.3, 162.8,
400 MHz),
d
12.69 (s, 1H, NNH), 11.70 (s, 1H, NH), 10.82 (s, 1H,
141.5,137.8,133.8,132.0,131.2,127.6,123.8,121.9,120.2,118.4,103.3;
SCNH), 7.75 (d, 1H, J ¼ 7.6 Hz, Ar1eH4), 7.47e7.44 (m, 3H, J ¼ 7.2 Hz,
Ar1eH5, Ar2eH6 and Ar2eH4), 7.14 (t, 1H, J ¼ 8.0 Hz, Ar2eH3),
7.01e6.98 (m, 2H, J ¼ 9.2 Hz, Ar2eH2 and Ar2eH6), 3.791 (s, 3H,
IR: (KBr, n
/cmꢂ1), 3302 (NNeH), 3240 (CONeH), 3021 (AreH), 1693
(eCONHe), 1626 (C]N), 1585 and 1534 (AreC]C), 1211 (C]S),
1153 (NeN); Elemental analysis calculated for C15H10Br2N4OS, C:
39.67, H: 2.22, N: 12.34, found C: 39.46, H: 2.68, N: 12.25; ESI-MS m/
z, 452.9 [M ꢂ H]ꢂ.
OCH3); 13C NMR (DMSO-d6, 101 MHz),
d 176.5, 162.7, 157.4, 139.6,
131.3, 131.1, 130.6, 127.2, 123.5, 122.0, 119.7, 115.1, 113.5, 55.2; IR:
(KBr,
n
/cmꢂ1), 3330 (NNeH), 3240 (CONeH), 3128 (AreH), 1702
(eCONHe), 1623 (C]N), 1586 and 1518 (AreC]C), 1239 (C]S),
1133 (NeN); Elemental analysis calculated for C16H13ClN4O2S, C:
53.26, H: 3.63, N: 15.53, found C: 53.42, H: 3.90, N: 15.75; ESI-MS m/
z: 378.9, [M þ NH4]þ.
4.2.38. (Z)-2-(7-Bromo-2-oxoindolin-3-ylidene)-N-(4-
methoxyphenyl)hydrazinecarbothioamide (3-38)
Yield 76%; m.p.: 249e251 ꢁC; yellow solid; 1H NMR (DMSO-d6,
400 MHz),
d 12.70 (s, 1H, NNH), 11.59 (s, 1H, NH), 10.81 (s, 1H,
SCNH), 7.80 (d, 1H, J ¼ 6.8 Hz, Ar1eH4), 7.58 (d, 1H, J ¼ 7.6 Hz,
Ar1eH6), 7.49e7.46 (d, 2H, J ¼ 8.0 Hz, Ar2eH3 and Ar2eH5), 7.09 (t,
1H, J ¼ 7.2 Hz, Ar1eH5), 7.01e6.99 (d, 2H, J ¼ 8.0 Hz, Ar2eH2 and
4.2.34. (Z)-2-(7-Bromo-2-oxoindolin-3-ylidene)-N-(2-
methoxyphenyl)hydrazinecarbothioamide (3-34)
Yield 70%; m.p.: 270e272 ꢁC; yellow solid; 1H NMR (DMSO-d6,
Ar2eH6), 3.80 (s, 3H, OCH3); 13C NMR (DMSO-d6, 101 MHz),
d 176.0,
400 MHz);
d
12.746 (s, 1H, NNH), 11.581 (s, 1H, NH), 10.521 (s, 1H,
162.7, 157.4, 141.3, 133.6, 131.4, 131.2, 127.2, 123.8, 122.1, 120.2, 113.6,

428
X.-M. Zhang et al. / European Journal of Medicinal Chemistry 101 (2015) 419e430
103.3, 55.2; IR: (KBr,
(AreH), 1695 (eCONHe), 1612 (C]N), 1595 and 1542 (AreC]C),
1258 (C]S), 1148 (NeN); Elemental analysis calculated for
n
/cmꢂ1), 3302 (NNeH), 3261 (CONeH), 3047
4.2.43. (Z)-2-(7-Iodo-2-oxoindolin-3-ylidene)-N-(3-
methoxyphenyl)hydrazinecarbothioamide (3-43)
Yield 68%; m.p.: 166e168 ꢁC; yellow solid; 1H NMR (DMSO-d6,
C
16H13BrN4O2S, C: 47.42, H: 3.23, N: 13.82, found C: 47.63, H: 3.40,
400 MHz), d 12.75 (s, 1H, NNH), 11.28 (s, 1H, NH), 10.83 (s, 1H,
N: 13.89; ESI-MS m/z: 405.0, [M ꢂ H]ꢂ.
SCNH), 7.81 (d, 1H, J ¼ 6.8 Hz, Ar1eH4), 7.74 (d, 1H, J ¼ 8.0 Hz,
Ar1eH6), 7.36e7.29 (m, 2H, J ¼ 8.0 Hz, Ar1eH5 and Ar2eH6), 7.24 (d,
1H, J ¼ 8.0 Hz, Ar2eH4), 6.93 (t, 1H, J ¼ 8.0 Hz, Ar2eH3), 6.86 (d, 1H,
J ¼ 6.4 Hz, Ar2eH2), 3.79 (s, 3H, OCH3); 13C NMR (DMSO-d6,
4.2.39. (Z)-N-(2-Fluorophenyl)-2-(7-iodo-2-oxoindolin-3-ylidene)
hydrazinecarbothioamide (3-39)
101 MHz), d 176.0, 162.7, 159.1, 145.0, 139.8, 139.4, 132.2, 129.1, 124.1,
Yield 75%; m.p.: 252e254 ꢁC; yellow solid; 1H NMR (DMSO-d6,
121.2, 120.8, 117.5, 111.6, 111.1, 76.5, 56.2; IR: (KBr, n
/cmꢂ1), 3246
400 MHz),
d 12.82 (s, 1H, NNH), 11.29 (s, 1H, NH), 10.77 (s, 1H,
(NNeH), 3133 (CONeH), 3059 (AreH), 1700 (eCONHe), 1610 (C]
N), 1539 and 1477 (AreC]C), 1207 (C]S), 1153 (NeN); Elemental
analysis calculated for C16H13IN4O2S, C: 42.49, H: 2.90, N: 12.39,
found C: 42.65, H: 3.10, N: 12.13; ESI-MS m/z: 379.1, [M þ Cl]ꢂ.
SCNH), 7.75e7.72 (m, 2H, J ¼ 4.8 Hz, Ar1eH4 and Ar2eH6), 7.50 (t,
1H, J ¼ 6.8 Hz, Ar1eH5), 7.43e7.33 (m, 2H, J ¼ 6.8 Hz, Ar2eH2 and
Ar2eH5), 7.28 (t, 1H, J ¼ 7.6 Hz, Ar2eH3), 6.93 (t, 1H, J ¼ 6.8 Hz,
Ar2eH4); 13C NMR (DMSO-d6, 101 MHz),
d 177.9, 162.7, 145.2, 139.9,
132.6, 130.1, 129.0, 126.4, 124.4, 124.2, 121.2, 120.5, 116.1, 115.9, 76.7;
4.2.44. (Z)-2-(7-Iodo-2-oxoindolin-3-ylidene)-N-(m-tolyl)
hydrazinecarbothioamide (3-44)
IR: (KBr,
n
/cmꢂ1), 3319 (NNeH), 3182 (CONeH), 3060 (AreH), 1696
(eCONHe), 1613 (C]N), 1575 and 1542 (AreC]C), 1198 (C]S),
1146 (NeN); HRMS(MALDI) m/z: calculated for C15H10FIN4OS
438.9526, found 438.9526, [M ꢂ H]ꢂ.
Yield 76%; m.p.: 256e258 ꢁC; yellow solid; 1H NMR (DMSO-d6,
400 MHz),
d 12.73 (s, 1H, NNH), 11.28 (s, 1H, NH), 10.82 (s, 1H,
SCNH), 7.81 (d, 1H, J ¼ 7.2 Hz, Ar1eH4), 7.73 (d, 1H, J ¼ 7.6 Hz,
Ar1eH6), 7.44e7.42 (m, 2H, J ¼ 8.0 Hz, Ar2eH4 and Ar2eH6), 7.32 (t,
1H, J ¼ 8.0 Hz, Ar1eH5), 7.10 (d, 1H, J ¼ 7.2 Hz, Ar2eH2), 6.93 (t, 1H,
J ¼ 7.6 Hz, Ar2eH3), 2.35 (s, 3H, CH3); 13C NMR (DMSO-d6,101 MHz),
4.2.40. (Z)-2-(7-Iodo-2-oxoindolin-3-ylidene)-N-(2-
methoxyphenyl)hydrazinecarbothioamide (3-40)
Yield 71%; m.p.: 264e266 ꢁC; yellow solid; 1H NMR (DMSO-d6,
d 176.1, 162.7, 145.0, 139.8, 138.2, 137.7, 132.1, 128.2, 126.8, 126.0,
400 MHz),
d
12.74 (s, 1H, NNH), 11.27 (s, 1H, NH), 10.49 (s, 1H,
124.1, 122.7, 121.3, 120.7, 76.5, 20.9; IR: (KBr, n
/cmꢂ1), 3304 (NNeH),
SCNH), 7.79 (d, 1H, J ¼ 7.2 Hz, Ar1eH4), 7.74e7.70 (m, 2H, J ¼ 8.4 Hz,
Ar1eH6 and Ar2eH5), 7.30 (t, 1H, J ¼ 7.2 Hz, Ar1eH5), 7.15 (d, 1H,
J ¼ 8.0 Hz, Ar2eH2), 7.09 (t, 1H, J ¼ 7.6 Hz, Ar2eH3), 6.93 (t, 1H,
J ¼ 7.6 Hz, Ar2eH4), 3.86 (s, 3H, OCH3); 13C NMR (DMSO-d6,
3225 (CONeH), 3076 (AreH), 1691 (eCONHe), 1610 (C]N), 1547
and 1480 (AreC]C), 1247 (C]S), 1181 (NeN); Elemental analysis
calculated for C16H13IN4OS, C: 44.05, H: 3.00, N: 12.84, Found C:
43.86, H: 3.27, N: 12.73; HRMS(MALDI) m/z: calculated 434.9776,
found 434.9775, [M ꢂ H]ꢂ.
101 MHz), d 176.1, 162.7, 153.0, 145.1, 139.8, 132.1, 127.6, 126.9, 126.4,
124.2, 121.2, 120.3, 120.0, 111.7, 76.6, 55.7; IR: (KBr, n
/cmꢂ1), 3284
(NNeH), 3176 (CONeH), 3056 (AreH), 1689 (eCONHe), 1610 (C]
N), 1544 and 1476 (AreC]C), 1253 (C]S), 1179 (NeN); Elemental
analysis calculated for C16H13IN4O2S, C: 42.49, H: 2.90, N: 12.39,
found C: 42.43, H: 3.05, N: 12.66; ESI-MS m/z: 451.0, [M ꢂ H]ꢂ.
4.2.45. (Z)-N-(4-Chlorophenyl)-2-(7-iodo-2-oxoindolin-3-ylidene)
hydrazinecarbothioamide (3-45)
Yield 74%; m.p.: 258e260 ꢁC; yellow solid, 1H NMR (DMSO-d6,
400 MHz);
d 12.79 (s, 1H, NNH), 11.29 (s, 1H, NH), 10.91 (s, 1H,
SCNH), 7.78 (d, 1H, J ¼ 8.8 Hz, Ar1eH4), 7.74 (d, 1H, J ¼ 7.6 Hz,
Ar1eH6), 7.67e7.65 (m, 2H, J ¼ 8.8 Hz, Ar2eH3 and Ar2eH5),
7.51e7.48 (m, 2H, J ¼ 8.4 Hz, Ar2eH2 and Ar2eH6), 6.93 (t, 1H,
4.2.41. (Z)-2-(7-Iodo-2-oxoindolin-3-ylidene)-N-(o-tolyl)
hydrazinecarbothioamide (3-41)
J ¼ 8.0 Hz, Ar1eH5); 13C NMR (DMSO-d6, 101 MHz),
d 176.3, 162.7,
Yield 74%; m.p.: 241e243 ꢁC; yellow solid; 1H NMR (DMSO-d6,
145.1, 139.9, 137.3, 132.4, 130.1, 128.3, 127.3, 124.1, 121.2, 120.7, 76.5;
400 MHz),
d 12.73 (s, 1H, NNH), 11.27 (s, 1H, NH), 10.78 (s, 1H,
IR: (KBr, n
/cmꢂ1), 3304 (NNeH), 3129 (CONeH), 3060 (AreH), 1696
SCNH), 7.77e7.72 (m, 2H, J ¼ 8.4 Hz, Ar1eH4 and Ar2eH6), 7.34e7.27
(m, 4H, J ¼ 7.6 Hz, Ar1eH5, Ar2eH2, Ar2eH3, and Ar2eH5), 6.93 (t,
1H, J ¼ 7.6 Hz, Ar2eH4), 2.26 (s, 3H, CH3); 13C NMR (DMSO-d6,
(eCONHe), 1612 (C]N), 1591 and 1535 (AreC]C), 1240 (C]S),
1159 (NeN); HRMS(MALDI) m/z: calculated for C15H10IClN4OS
454.9230, found 454.9232, [M ꢂ H]ꢂ.
101 MHz), d 177.1, 162.7, 145.0, 139.7, 137.3, 135.4, 132.1, 130.3, 128.4,
127.4, 126.2, 124.1, 121.3, 120.5, 76.4, 17.6; IR: (KBr, n
/cmꢂ1) 3304
4.2.46. (Z)-N-(4-Bromophenyl)-2-(7-iodo-2-oxoindolin-3-ylidene)
hydrazinecarbothioamide (3-46)
(NNeH), 3240 (CONeH), 3057 (AreH), 1698 (eCONHe), 1611 (C]
N), 1587 and 1541 (AreC]C), 1288 (C]S), 1152 (NeN); Elemental
analysis calculated for C16H13IN4OS, C: 44.05, H: 3.00, N: 12.84;
found C: 44.00, H: 3.05, N: 12.60; ESI-MS m/z: 434.9, [M ꢂ H]ꢂ.
Yield 71%; m.p.: 261e263 ꢁC; yellow solid; 1H NMR (DMSO-d6,
400 MHz),
d 12.80 (s, 1H, NNH), 11.30 (s, 1H, NH), 10.91 (s, 1H,
SCNH), 7.79e7.75 (m, 2H, J ¼ 4.0 Hz, Ar1eH4 and Ar1eH6),
7.63e7.63 (m, 4H, J ¼ 2.4 Hz, Ar2eH2, Ar2eH3, Ar2eH5, and Ar2eH6),
6.97e6.93 (m, 1H, J ¼ 6.8 Hz, Ar1eH5); 13C NMR (DMSO-d6,
4.2.42. (Z)-N-(3-Chlorophenyl)-2-(7-iodo-2-oxoindolin-3-ylidene)
hydrazinecarbothioamide (3-42)
101 MHz); d 176.3, 162.7, 145.1, 139.9, 137.8, 132.5, 131.2, 127.6, 124.1,
Yield 78%; m.p.: 251e253 ꢁC; yellow solid; 1H NMR (DMSO-d6,
121.2, 120.7, 118.4, 75.5; IR: (KBr, n
/cmꢂ1), 3304 (NNeH), 3129
400 MHz),
d
12.81 (s, 1H, NNH), 11.31 (s, 1H, NH), 10.94 (s, 1H,
(CONeH), 3060 (AreH), 1696 (eCONHe), 1612 (C]N), 1591 and
1535 (AreC]C), 1240 (C]S), 1159 (NeN); Elemental analysis
calculated for C15H10IBrN4OS, C: 35.95, H: 2.01, N: 11.18, found C:
36.13, H: 2.14, N: 11.11; ESI-MS m/z: 500.7, [M ꢂ H]ꢂ.
SCNH), 7.80e7.78 (m, 2H, J ¼ 4.4 Hz, Ar1eH4 and Ar2eH6), 7.74 (d,
1H, J ¼ 8.0 Hz, Ar1eH6), 7.66 (d, 1H, J ¼ 8.0 Hz, Ar2eH4), 7.47 (t, 1H,
J ¼ 8.0 Hz, Ar1eH5), 7.35 (d, 1H, J ¼ 8.0 Hz, Ar2eH2), 6.94 (t, 1H,
J ¼ 8.0 Hz, Ar2eH3); 13C NMR (DMSO-d6, 101 MHz),
d 176.2, 162.7,
145.1, 139.9, 139.8, 132.6, 132.4, 129.9, 125.8, 125.0, 124.1, 121.1,
4.2.47. (Z)-2-(7-Iodo-2-oxoindolin-3-ylidene)-N-(4-
methoxyphenyl)hydrazinecarbothioamide (3-47)
120.8, 76.6; IR: (KBr, n
/cmꢂ1), 3311 (NNeH), 3163 (CONeH), 3059
(AreH), 1696 (eCONHe), 1611 (C]N), 1589 and 1541 (AreC]C),
1236 (C]S), 1151 (NeN); Elemental analysis calculated for
Yield 79%; m.p.: 256e258 ꢁC; yellow solid; 1H NMR (DMSO-d6,
400 MHz),
d 12.70 (s, 1H, NNH), 11.26 (s, 1H, NH), 10.78 (s, 1H,
C
15H10IClN4OS, C: 39.45, H: 2.21, N: 12.27, found C: 39.25, H: 2.40,
SCNH), 7.79 (d, 1H, J ¼ 7.2 Hz, Ar1eH4), 7.72 (d, 1H, J ¼ 7.6 Hz,
N: 12.07; ESI-MS m/z: 454.9, [M ꢂ H]ꢂ.
Ar1eH6), 7.48e7.46 (m, 2H, J ¼ 8.4 Hz, Ar2eH3 and Ar2eH5),

X.-M. Zhang et al. / European Journal of Medicinal Chemistry 101 (2015) 419e430
429
7.00e6.98 (m, 2H, J ¼ 8.4 Hz, Ar2eH2 and Ar2eH6), 6.92 (t, 1H,
438.9526, found 438.9529, [M ꢂ H]ꢂ.
J ¼ 7.6 Hz, Ar1eH5), 3.79 (s, 3H, OCH3); 13C NMR (DMSO-d6,
101 MHz), d 176.4, 162.7, 157.4, 144.9, 139.7, 131.9, 131.1, 127.2, 124.1,
4.3. Bacterial strains and in vitro antibacterial susceptibility and
MIC determination
121.3, 120.6, 113.5, 76.4, 55.2; IR: (KBr, n
/cmꢂ1), 3304 (NNeH), 3163
(CONeH), 3058 (AreH), 1695 (eCONHe), 1611 (C]N), 1538 and
1475 (AreC]C), 1248 (C]S), 1134 (NeN); Elemental analysis
calculated for C16H13IN4O2S, C: 42.49, H: 2.90, N: 12.39, found C:
42.70, H: 2.67, N: 12.36; ESI-MS m/z: 218.0, [M ꢂ H]ꢂ.
The Gram-positive bacterial species and strains used in this
study include S. aureus (ATCC 6538), MRSA clinical isolates
(Chaoyang, 309-4, 6281, 309-8, 6-42, 8-21, 309-3, 309-1, 309-7, 8-
24 and 309-6), B. subtilis (ATCC 6633), E. faecalis and VRE (VRE-309).
The Gram-negative bacteria species and strains used in this study
are P. aeruginosa (PAO1) and K. pneumoniae (KP-249). MRSA (309-4,
309-8, 309-3, 309-1, 309-7 and 309-6) and VRE-309 are clinical
isolated strains from the 309th hospital of Chinese People's Liber-
ation Army in Beijing. MRSA (Chaoyang) is a clinical isolated strain
from Chaoyong hospital in Beijing. MRSA (6281, 6-42, 8-21 and 8-
24) are clinical isolated strains from the 306th hospital of Chinese
People's Liberation Army in Beijing. E. faecalis is a clinical isolated
vancomycin-susceptible strain from Frederick Ausubel's lab (Har-
vard Medical School). KP-249 is a clinical isolated K. pneumoniae
strain from 309th hospital of Chinese People's Liberation Army in
Beijing.
The procedure for the antimicrobial bioassay was similar to our
published method [29]. All the IBT compounds were prepared into
4 mg/mL stock solution using sterile dimethyl sulfoxide (DMSO).
The stock solution was serially diluted by sterile DMSO. The mi-
crobes were stored as glycerol stocks in a ꢂ80 ꢁC refrigerator and
streaked onto Mueller-Hinton agar (MHA) for colony growth at
37 ꢁC before use. The antibacterial activities and MICs were deter-
mined in flat bottom, 96-well microtiter plates using a broth
microdilution protocol modified from the Clinical and Laboratory
Standards Institute M7-A6, M-38A and M-27A2 methods. Briefly,
single colonies of bacteria were picked from MHA plate and
adjusted to approximately 105 CFU/mL with Mueller-Hinton Broth
4.2.48. (Z)-2-(7-Iodo-2-oxoindolin-3-ylidene)-N-(p-tolyl)
hydrazinecarbothioamide (3-48)
Yield 74%; m.p.: 260e262 ꢁC; yellow solid; 1H NMR (DMSO-d6,
400 MHz),
d 12.72 (s, 1H, NNH), 11.28 (s, 1H, NH), 10.82 (s, 1H,
SCNH), 7.80 (d, 1H, J ¼ 6.4 Hz, Ar1eH4), 7.73 (d, 1H, J ¼ 7.6 Hz,
Ar1eH6), 7.48e7.46 (m, 2H, J ¼ 7.2 Hz, Ar2eH3 and Ar2eH5),
7.24e7.22 (m, 2H, J ¼ 7.2 Hz, Ar2eH2 and Ar2eH6), 6.93 (t, 1H,
J ¼ 7.2 Hz, Ar1eH5), 2.34 (s, 3H, CH3); 13C NMR (DMSO-d6,101 MHz),
d
176.2, 162.7, 145.0, 139.7, 135.8, 135.4, 132.0, 128.8, 125.5, 124.1,
121.3, 120.7, 76.5, 20.6; IR: (KBr, n
/cmꢂ1); 3306 (NNeH), 3165
(CONeH), 3126 (AreH), 1693 (eCONHe), 1612 (C]N), 1595 and
1536 (AreC]C), 1240 (C]S), 1153 (NeN); Elemental analysis
calculated for C16H13IN4OS C: 44.05, H: 3.00, N: 12.84, found C:
43.94, H: 3.26, N: 12.78; ESI-MS m/z: 434.9, [M ꢂ H]ꢂ.
4.2.49. (Z)-2-(7-Fluoro-2-oxoindolin-3-ylidene)-N-(2-
methoxyphenyl)hydrazinecarbothioamide (3-49)
Yield 77%; m.p.: 236e238 ꢁC; yellow solid; 1H NMR (DMSO-d6,
400 MHz), d 12.76 (s,1H, NNH),11.80 (s,1H, NH),10.51 (s,1H, SCNH),
7.79 (d, 1H, J ¼ 7.6 Hz, Ar1eH4), 7.55 (d, 1H, J ¼ 7.2 Hz, Ar1eH6),
7.33e7.28 (m, 2H, J ¼ 8.4 Hz, Ar1eH5 and Ar2eH5), 7.16e7.10 (m, 2H,
J ¼ 7.2 Hz, Ar2eH2 and Ar2eH3), 7.01 (t,1H, J ¼ 7.2 Hz, Ar2eH4), 3. 86
(s, 3H, OCH3); 13C NMR (DMSO-d6, 101 MHz),
d 176.1, 162.4, 153.0,
148.1,145.8,131.4,129.3,127.6,126.9,123.3,122.8,120.0,118.0,117.0,
111.7, 55.7; IR: (KBr,
n
/cmꢂ1), 3314 (NNeH), 3178 (CONeH), 3077
(MHB) as bacterial suspension. Aliquots 2
of each compound (in DMSO) were added to each row on 96-well
plate containing 78 L of bacteria suspension in each well. The
mL of 2-fold serial dilution
(AreH), 1693 (eCONHe), 1600 (C]N), 1574 and 1483 (AreC]C),
1251 (C]S), 1161 (NeN); HRMS(MALDI) m/z: calculated for
m
16H13FN4O2S 345.0821, found 345.0817, [M þ H]þ.
96-well plate was incubated aerobically at 37 ꢁC for 16 h before
results were recorded. MICs were defined as the lowest concen-
trations of the compounds that can inhibit visible bacterial growth
after 16 h incubation. The MICs were tested twice in triplicate. For
the bioassays, vancomycin was used as a control drug for S. aureus,
MRSA, B. subtilis, E. faecalis and VRE, methicillin was used as a
control drug for MRSA, while ciprofloxacin was used as a control
drug for P. aeruginosa and K. pneumoniae.
C
4.2.50. (Z)-N-(3-Fluorophenyl)-2-(7-iodo-2-oxoindolin-3-ylidene)
hydrazinecarbothioamide (3-50)
Yield 74%; m.p.: 234e236 ꢁC; yellow solid; 1H NMR (DMSO-d6,
400 MHz),
d 12.81 (s, 1H, NNH), 11.29 (s, 1H, NH), 10.92 (s, 1H,
SCNH), 7.79 (d, 1H, J ¼ 7.2 Hz, Ar1eH4), 7.73 (d, 1H, J ¼ 7.6 Hz,
Ar1eH6), 7.63 (d, 1H, J ¼ 10.8 Hz, Ar2eH4), 7.52e7.44 (m, 2H,
J ¼ 8.0 Hz, Ar2eH2 and Ar2eH6), 7.12 (t, 1H, J ¼ 7.2 Hz, Ar1eH5), 6.93
(t, 1H, J ¼ 7.6 Hz, Ar2eH3), 3. 86 (s, 3H, OCH3); 13C NMR (DMSO-d6,
4.4. Comparative field analysis
101 MHz),
d 176.1, 162.7, 160.3, 145.1, 140.0, 139.9, 132.5, 129.9, 124.1,
121.2, 121.1, 120.8, 112.8, 112.1, 76.5; IR: (KBr,
n
/cmꢂ1), 3301 (NNeH),
Chemical structures of the compounds were built within Sybyl
7.3 (Tripos Inc., St Louis, MO) based on the crystal structure of 3-1.
All the molecules were assigned Gasteiger-Hückel charges and
minimized by the Tripos force field when convergence reached
0.001 kcal/mol/Å. The molecules were superimposed using 3-2 as
the template. All the parameters were used the default value within
comparative field analysis (CoMFA) module and the column
filtering was set to 2.0 kcal/mol. Compounds 3-1, 3-3, 3-15, 3-18, 3-
33 and 3-47 were randomly selected into the test set and the other
44 compounds were used in the training set (3-49 was not included
because its MIC could not be determined). The “leave-one-out”
(LOO) cross validation method was applied to determine the opti-
mum number of partial least squares (PLS) components. The bio-
logical activity data were expressed in terms of D using the
following formula
3163 (CONeH), 3055 (AreH), 1693 (eCONHe), 1611 (C]N), 1547
and 1524 (AreC]C), 1219 (C]S), 1147 (NeN); Elemental analysis
calculated for C15H10IFN4OS, C: 40.92, H: 2.29, N: 12.73, found C:
40.90, H: 2.15, N: 12.61; ESI-MS m/z: 438.9, [M ꢂ H]ꢂ.
4.2.51. (Z)-N-(4-Fluorophenyl)-2-(7-iodo-2-oxoindolin-3-ylidene)
hydrazinecarbothioamide (3-51)
Yield 78%; m.p.: 259e261 ꢁC; yellow solid; 1H NMR (DMSO-d6,
400 MHz),
d 12.76 (s, 1H, NNH), 11.29 (s, 1H, NH), 10.89 (s, 1H,
SCNH), 7.79 (d, 1H, J ¼ 7.6 Hz, Ar1eH4), 7.74 (d, 1H, J ¼ 8.0 Hz,
Ar1eH6), 7.62e7.58 (m, 2H, J ¼ 5.6 Hz, Ar2eH3 and Ar2eH5),
7.30e7.26 (m, 2H, J ¼ 4.4 Hz, Ar2eH2 and Ar2eH6), 6.94 (t, 1H,
J ¼ 8.0 Hz, Ar1eH5); 13C NMR (DMSO-d6, 101 MHz),
d 176.7, 162.7,
145.0, 139.8, 134.7, 132.2, 128.0, 127.9, 124.1, 121.2, 120.6, 115.2, 76.5;
IR: (KBr, n
/cmꢂ1), 3247 (NNeH), 3164 (CONeH), 3059 (AreH), 1697
(eCONHe), 1610 (C]N), 1541 and 1478 (AreC]C), 1236 (C]S),
1152 (NeN); HRMS(MALDI) m/z: calculated for C15H10IFN4OS,
D ¼ ꢂlog10½MIC=ðMw ꢃ 1000Þꢄ

430
X.-M. Zhang et al. / European Journal of Medicinal Chemistry 101 (2015) 419e430
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Acknowledgment
This work was supported by the National Basic Research Pro-
gram of China (No. 2013CB734004), the “111” Project of Ministry of
Education of China (No. B06005), China Ocean Mineral Resources R
& D Association Grant (No. DY125-15-T-07), the key project from
Tianjin Natural Science Foundation (No. 12JCZDJC25700) and the
Natural Science Foundation of China (No. 31320103911).
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2015.06.047.
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