Design, synthesis and antibacterial activity of isatin derivatives as FtsZ inhibitors

Article abstract of DOI:10.1016/j.molstruc.2016.03.036

Seven isatin derivatives have been designed, and their chemical structures were characterized by single crystal X-ray diffraction studies, ¹H NMR, MS, and elemental analysis. Structural stabilization followed by intramolecular as well as intermolecular H-bonds makes these molecules as perfect examples in molecular recognition with self-complementary donor and acceptor units within a single molecule. These compounds were evaluated for antimicrobial activities. Docking simulations have been performed to position compounds into the FtsZ active site to determine their probable binding models. All of the compounds exhibited better antibacterial activities. Interestingly, compound 5c and 5d exhibited better antibacterial activities with IC₅₀ values of 0.03 and 0.05 μmol/mL against Staphylococcus aureus, respectively. Compound 5g displays antibacterial activity with IC₅₀ values of 0.672 and 0.830 μmol/mL against Escherichia coli and Pseudomonas aeruginosa, respectively.

Full text of DOI:10.1016/j.molstruc.2016.03.036

Accepted Manuscript
Design, synthesis and antibacterial activity of Isatin derivatives as FtsZ inhibitors
Zhi-Min Lian, Juan Sun, Hai-Liang Zhu
PII:
S0022-2860(16)30231-9
10.1016/j.molstruc.2016.03.036
MOLSTR 22347
DOI:
Reference:
To appear in: Journal of Molecular Structure
Received Date: 29 December 2015
Revised Date: 21 February 2016
Accepted Date: 14 March 2016
Please cite this article as: Z.-M. Lian, J. Sun, H.-L. Zhu, Design, synthesis and antibacterial
activity of Isatin derivatives as FtsZ inhibitors, Journal of Molecular Structure (2016), doi: 10.1016/
j.molstruc.2016.03.036.
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ACCEPTED MANUSCRIPT
Design, synthesis and antibacterial activity of Isatin derivatives
as FtsZ inhibitors
Zhi-Min Lian, Juan Sun*, Hai-Liang Zhu*
School of Life Sciences, Shandong University of Technology,
Shandong 255049, People’s Republic of China
Abstract: Seven isatin derivatives have been designed, and their chemical structures
1
were characterized by single crystal X-ray diffraction studies, H NMR, MS, and
elemental analysis. Structural stabilization followed by intramolecular as well as
intermolecular H-bonds makes these molecules as perfect examples in molecular
recognition with self-complementary donor and acceptor units within a single
molecule. These compounds were evaluated for antimicrobial activities. Docking
simulations have been performed to position compounds into the FtsZ active site to
determine their probable binding models. All of the compounds exhibited better
antibacterial activities. Interestingly, compound 5c and 5d exhibited better
antibacterial activities with IC50 values of 0.03 and 0.05 µmol/mL against
Staphylococcus aureus, respectively. Compound 5g displays antibacterial activity with
IC50 values of 0.672 and 0.830 µmol/mL against Escherichia coli and Pseudomonas
aeruginosa, respectively.
Keywords: Isatin derivatives, Schiff base, Isatin, Antibacterial activity, FtsZ
inhibitors
1. Introduction
Filamentous temperature-sensitive protein Z (FtsZ) , the major prokaryotic cell
division protein, serves as an extremely important component in the bacteria cell
devision [1,2]. The inhibition of FtsZ assembly can restrain bacterial cellular fission,
further leading to bacterial apoptosis [3-5]. Based on the current therapeutic value and

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future potential of FtsZ inhibitors, the discovery of new classes of compounds that
inhibit the FtsZ are justified. As a continuous work of our previous work[6], we have
researched a series of isatin derivatives as potential FtsZ inhibitors. Isatin
(1H-indol-2,3-dione) analogues, the importance of indole back bone, have exhibited a
multiplicity of biological activities, which include antirotic [7], antifungal [8,9],
anti-angiogenic [10], antineoplastic [11], anti-convulsant [12], anti-HIV [13] and
antiparkinsonian activity [14]. Schiff bases have shown numerous of biological
activities such as antifungal [15-18], anticancer [19,20], antibacterial [21,22] and
phytocidal [23] activities. Owing to the broad spectrum chemotherapeutic properties
of isatin and schiff bases, it maybe appear as potential antimicrobial agent.
Simultaneously, the structure of isatin derivatives have been performed to position
compounds into the FtsZ active site to determine their probable binding model vias
Docking simulations. Therefore, we combined two scaffolds and explored their
possibilities to inhibit the bacteria cell devision and curb the related diseases.
2. Experimental
2.1 Chemistry
All chemicals and reagents used in the current study were of analytical grade.
The reactions were monitored by thin layer chro-matography (TLC) on Merck
pre-coated silica GF254 plates. Melting points (uncorrected) were determined on a
Digital Melting Point apparatus (Shenguang., Shanghai, China). ESI mass spectra
1
were obtained on a Mariner System 5304 mass spectrometer, and H NMR spectra
were collected on a Bruker DPX400 spectrometer at room temperature with TMS and
solvent signals allotted as internal standards. Chemical shifts are reported in ppm (δ).
Elemental analyses were performed on a CHN-O-Rapid instrument, and were within
±0.4% of the theoretical values.
The synthetic route to target compounds (5a – 5g) is shown in Scheme 1.
2.1.1 Synthesis of substituted benzohydrazide. (4a-4g)
Substituted benzoic acid (1.0 mmol) was dissolved in thionyl chloride and
refluxed for 2 h, then the solvent was removed under reduced pressure to obtain the
white solid. The white solid in methanol was added concentrated sulfuric acid (1 mL)

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and the mixture was refluxed for 4 h, the solvent was removed to obtain crude solid.
The crude solid was extracted with ethyl acetate and water. The solvents were
evaporated to afford the pure product. Finally, the pure product was dissolved in
ethanol, and the hydrazine hydrate was added. The mixture was refluxed for 9 h, and
the solvent was removed under reduced pressure to obtain substituted
benzohydrazide .
2.1.2 General method of synthesis of benzohydrazide derivatives. (5a -5g)
Substituted benzohydrazide (1 mmol) and isatin (1 mmol) in dichloromethane
were refluxed for 10-12 h at room temperature, then the reaction mixture was
concentrated under reduced pressure, recrystallized from ethanol to give
benzohydrazide derivatives 5a-5g.
2.1.2.1 N'-(2-oxoindolin-3-ylidene)benzohydrazide (5a)
1
Mp: 294.8-295.4 °C. Yield: 75%. H NMR (400 MHz, CDCl3) δ (ppm):
6.97-6.99 (d, J = 8.0Hz, 1H), 7.11-7.15 (m, 1H), 7.39-7.43 (m, 1H), 7.61-7.65 (m, 3H),
7.69-7.72 (m, 1H), 7.90-7.92 (d, J = 8.0Hz, 2H), 11.40 (s, 1H), 13.96 (s, 1H). MS
(ESI): 266 (C15H12N3O2, [M+H]+). Anal. Calcd. (%) for C15H11N3O2: C, 67.92; H,
4.18; N, 15.84; Found (%) : C, 67.70; H, 4.22; N, 15.76.
2.1.2.2. 2-Fluoro-N'-(2-oxoindolin-3-ylidene)benzohydrazide (5b)
1
Mp: 302.0-302.2 °C. Yield: 75%. H NMR (400 MHz, CDCl3) δ (ppm):
6.94-6.96 (d, J = 8.0Hz, 1H), 7.10-7.15 (m, 2H), 7.39-7.42 (m, 1H), 7.60-7.63 (m,
2H), 7.71-7.73 (m, 1H), 7.90-7.92 (m, 1H), 10.88 (s, 1H), 11.83 (s, 1H). MS (ESI):
284 (C15H11FN3O2, [M+H]+). Anal. Calcd. (%) for C15H10FN3O2: C, 63.60; H, 3.56; N,
14.83; Found (%) : C, 63.66; H, 3.49; N, 14.87.
2.1.2.3. 3-Chloro-N'-(2-oxoindolin-3-ylidene)benzohydrazide (5c)
1
Mp: 286.7-286.8 °C. Yield: 80%. H NMR (400 MHz, CDCl3) δ (ppm):
6.92-6.94 (d, J = 8.0 Hz,1H), 7.08-7.12 (m, 1H), 7.40-7.44 (m, 1H), 7.60-7.64 (m,
1H), 7.72-7.75 (m, 1H), 7.91-7.95 (m, 2H) 8.01 (s, 1H), 10.86 (s, 1H), 11.79 (s, 1H).
MS (ESI): 301 (C15H11ClN3O2, [M+H]+). Anal. Calcd. (%) for C15H10ClN3O2: C,
60.11; H, 3.36;N, 14.02; Found (%) : C, 59.96; H, 3.50;N, 14.15.
2.1.2.4. 3-Bromo-N'-(2-oxoindolin-3-ylidene)benzohydrazide (5d)

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1
Mp: 280.8-280.9 °C. Yield: 75%. H NMR (400 MHz, CDCl3) δ (ppm):
6.90-6.92 (d, J = 8.0 Hz,1H), 7.05-7.11 (m, 2H), 7.40-7.52 (m, 1H), 7.63-7.71 (m,
1H), 7.72-7.75 (m, 1H), 7.91-7.95 (m, 1H), 8.01-8.05 (m, 1H), 10.86 (s, 1H), 11.79
(s, 1H). MS (ESI): 344 (C15H11BrN3O2, [M+H]+). Anal. Calcd. (%) for
C15H10BrN3O2: C, 52.35; H, 2.93;N, 12.21; Found (%) : C, 52.38; H, 2.90;N, 12.27.
2.1.2.5. 3-Methyl-N'-(2-oxoindolin-3-ylidene)benzohydrazide (5e)
Mp: 288.9-289.5 °C. Yield: 85%. 1H NMR (400 MHz, CDCl3) δ (ppm): 3.354 (s,
3H), 6.92-6.95 (m, 1H), 7.33-7.46(m, 2H), 7.62-7.72 (m, 2H), 7.81-7.85 (m, 2H),
7.92-7.96 (m, 1H), 10.87 (s, 1H), 11.77 (s, 1H). MS (ESI): 280 (C16H14N3O2,
[M+H]+). Anal. Calcd. (%) for C16H13N3O2: C, 68.81; H, 4.69; N, 15.05; Found (%) :
C, 68.75; H, 4.73; N, 15.12.
2.1.2.6. 2-Bromo-N'-(2-oxoindolin-3-ylidene)benzohydrazide (5f)
1
Mp: 244.3-244.9 °C. Yield: 70%. H NMR (400 MHz, CDCl3) δ (ppm):
6.89-6.91 (d, J = 8.0 Hz, 1H), 7.03-7.07 (m, 1H), 7.38-7.62 (m, 4H), 7.73-7.75 (d, J =
8.0 Hz, 1H), 8.01-8.19 (m, 1H), 10.82 (s, 1H), 11.81 (s, 1H). MS (ESI): 345
(C15H11BrN3O2, [M+H]+). Anal. Calcd. (%) for C15H10BrN3O2: C, 52.35; H, 2.93; N,
12.21; Found (%) : C, 52.49; H, 2.82; N, 12.29.
2.1.2.7. 3-Fluoro-N'-(2-oxoindolin-3-ylidene)benzohydrazide (5g)
1
Mp: 294.9-295.8 °C. Yield: 80%. H NMR (400 MHz, CDCl3) δ (ppm):
6.92-6.94 (d, J = 8.0Hz, 1H), 7.09-7.12 (m, 1H), 7.41-7.44 (m, 1H), 7.52-7.55 (m,
1H), 7.62-7.67 (m, 1H), 7.78-7.84 (m, 2H), 7.94-7.96 (d, J = 8.0 Hz, 1H), 10.87 (s,
1H), 11.77 (s, 1H). MS (ESI): 284 (C15H11FN3O2, [M+H]+). Anal. Calcd. (%) for
C15H10FN3O2: C, 63.60; H, 3.56; N, 14.83; Found (%) : C, 63.83; H, 3.46; N, 14.94.
2.2 X-ray crystallography
Single crystal X-ray diffraction data was collected on a Bruker D-8 venture
diffractometer at room temperature (293 K). The X-ray generator was operated at 50
KV and 35 mA using Mo Kα radiation (λ = 0.71073 Å). The data was collected using
SMART software package. The data were reduced by SAINT-PLUS, an empirical
absorption correction was applied using the package SADABS and XPREP were used
to determine the space group. The crystal structure was solved by direct methods

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using SIR92 and refined by full-matrix least-squares method using SHELXL97
[24,25]. All non-hydrogen atoms were refined anisotropically and hydrogen atoms
have been refined in the riding mode on their carrier atoms wherever applicable.
2.3 Molecular docking study
Automated docking studies were carried out using Discovery Studio (version 3.1)
as implemented through the graphical user interface DS-CDocker protocal.
The three-dimensional structures of the aforementioned compounds were
constructed using Chem. 3D ultra 11.0 software [Chemical Structure Drawing
Standard; Cambridge Soft corporation, USA (2009)], then they were energetically
minimized by using MOPAC with 100 iterations and minimum RMS gradient of 0.10.
The Gasteiger-Hückel charges of ligands were assigned. The crystal structures of FtsZ
protein (PDB code: 2VAM, 2VAW, 3VO8,1S1S) [26] complex were retrieved from
the RCSB Protein Data Bank. All bound waters and ligands were eliminated from the
protein and the polar hydrogens and the Kollman-united charges were added to the
proteins.
2.4 Antibacterial assay
The antibacterial activity of the synthesized compounds was tested against
Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa and Staphylococcus
aureus using LB medium (Luria-Bertani medium: yeast extract 5.0 g, peptone 10.0 g,
sodium chloride 5.0 g, distilled water 1000 mL). The IC50 of the test compounds was
determined
by
a
colorimetric
method
using
the
dye
MTT
(3-(4,5-dimethylth-iazol-2-yl)-2,5-diphenyl tet-razoliumbromide), and the IC50 values
are presented in Table 1. Also included was the activity of reference compound
Kanamycin and Chloromycetin under identical conditions for comparison. A stock
solution of the synthesized compound (1000 µmol/mL) in DMSO was prepared and
graded quantities of the test compounds were incorporated in specified quantity of
sterilized liquid LB medium. A specified quantity of the medium containing the
compound was poured into microtitration plates. Suspension of the microorganism
was prepared to contain approximately 105 cfu/mL and applied to microtitration plates

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with serially diluted compounds in DMSO to be tested and incubated at 37ꢀfor 24 h.
After 10 ꢀL of PBS containing 4 mg of MTT/mL was added to each well. Incubation
was continued at room temperature for 4-5 h. The content of each well was removed,
and 150 ꢀL of DMSO was added to extract the dye. The optical density (OD) was
measured with an ELISA plate reader at 492 nm.
3. Results
3.1 Crystal structures of compounds 5a-5g
Crystals of seven compounds were obtained from ethanol solution. Fig. 1 shows
a perspective view of the monomeric unit with the atomic numbering scheme, Fig. 2
depicts the intra-molecular and inter-molecular hydrogen bonds. Crystallographic data,
details of data collection and structure refinement parameters are listed in Table 2 and
Table 3. The hydrogen bond lengths and bond angles are given in Table 4 and Table 5.
Single crystals of 5a (0.32 mm × 0.27 mm ×0.25 mm), 5b (0.28 mm×0.26
mm×0.25 mm), 5c (0.30 mm×0.25 mm×0.25 mm), 5d (0.31 mm×0.27 mm×0.25
mm), 5e (0.35 mm×0.27 mm×0.25 mm), 5f (0.21 mm×0.26 mm×0.25 mm) and
5g (0.30 mm×0.26 mm×0.25 mm) were mounted on a D-8 venture diffractometer
equipped with graphite-monochromated MoKa (k = 0.71073 Å) radiation.
For 5a, a total of 11,254 reflections were collected, of which 2,241 were unique
with Rint = 0.032 and 1,734 observed reflections with I > 2σ (I) were used in the
succeeding structure calculations. The final cycle of refinement of fullmatrix
least-squares was converged to R = 0.0386 and wR = 0.1017. The highest and lowest
residual peaks in the final difference Fourier map are 0.17 and -0.16 e/Å3,
respectively.
For 5b, a total of 13,051 reflections were collected, of which 3,013 were unique
with Rint = 0.026 and 2,182 observed reflections with I > 2σ (I) were used in the
succeeding structure calculations. The final cycle of refinement of fullmatrix
least-squares was converged to R = 0.0498 and wR = 0.1378. The highest and lowest
residual peaks in the final difference Fourier map are 0.39 and -0.36 e/Å3,
respectively.
For 5c, a total of 12,182 reflections were collected, of which 2,396 were unique

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with Rint = 0.118 and 1,187 observed reflections with I > 2σ (I) were used in the
succeeding structure calculations. The final cycle of refinement of fullmatrix
least-squares was converged to R = 0.0569 and wR = 0.1062. The highest and lowest
residual peaks in the final difference Fourier map are 0.18 and -0.22 e/Å3,
respectively.
For 5d, a total of 23,057 reflections were collected, of which 2,428 were unique
with Rint = 0.047 and 1,960 observed reflections with I > 2σ (I) were used in the
succeeding structure calculations. The final cycle of refinement of fullmatrix
least-squares was converged to R = 0.0338 and wR = 0.0859. The highest and lowest
residual peaks in the final difference Fourier map are 0.51 and -0.62 e/Å3,
respectively.
For 5e, a total of 12,263 reflections were collected, of which 2,426 were unique
with Rint = 0.056 and 1,536 observed reflections with I > 2σ (I) were used in the
succeeding structure calculations. The final cycle of refinement of fullmatrix
least-squares was converged to R = 0.0494 and wR = 0.11400. The highest and lowest
residual peaks in the final difference Fourier map are 0.19 and -0.15 e/Å3,
respectively.
For 5f, a total of 10,866 reflections were collected, of which 4,981 were unique
with Rint = 0.045 and 3,295 observed reflections with I > 2σ (I) were used in the
succeeding structure calculations. The final cycle of refinement of fullmatrix
least-squares was converged to R = 0.0605 and wR =0.1305. The highest and lowest
residual peaks in the final difference Fourier map are 0.62 and -0.54 e/Å3,
respectively.
For 5g, a total of 13,518 reflections were collected, of which 3,053 were unique
with Rint = 0.029 and 2,177 observed reflections with I > 2σ (I) were used in the
succeeding structure calculations. The final cycle of refinement of fullmatrix
least-squares was converged to R =0.0507 and wR = 0.1402. The highest and lowest
residual peaks in the final difference Fourier map are 0.33 and -0.39 e/Å3,
respectively.
In the crystal structure of compound 5a, there are two benzene rings and one

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5-Membered Ring in the molecule. C(10), C(11), C(12), C(13), C(14) and C(15) form
the first plane, defined as plane I; C(7), C(8), N(1), C(4) and C(5) forms the second
plane, defined as plane II; Similarly, C(1), C(2), C(3), C(4), C(5) and C(6) forms the
third plane, defined as plane III. The dihedral angle between plane I and plane II is
14.113(57)°; The dihedral angle between plane I and plane III is 14.086(57)°.
In the crystal structure of compound 5b, there are two benzene rings and one
5-Membered Ring in the molecule. C(10), C(11), C(12), C(13), C(14) and C(15) form
the first plane, defined as plane I; C(7), C(8), N(1), C(4) and C(5) forms the second
plane, defined as plane II; Similarly, C(1), C(2), C(3), C(4), C(5) and C(6) forms the
third plane, defined as plane III. The dihedral angle between plane I and plane II is
5.087(50)°; The dihedral angle between plane I and plane III is 5.099(54)°.
In the crystal structure of compound 5c, there are two benzene rings and one
5-Membered Ring in the molecule. C(10), C(11), C(12), C(13), C(14) and C(15) form
the first plane, defined as plane I; C(7), C(8), N(1), C(4) and C(5) forms the second
plane, defined as plane II; Similarly, C(1), C(2), C(3), C(4), C(5) and C(6) forms the
third plane, defined as plane III. The dihedral angle between plane I and plane II is
4.930(114)°; The dihedral angle between plane I and plane III is 4.349(95)°.
In the crystal structure of compound 5d, there are two benzene rings and one
5-Membered Ring in the molecule. C(10), C(11), C(12), C(13), C(14) and C(15) form
the first plane, defined as plane I; C(7), C(8), N(1), C(4) and C(5) forms the second
plane, defined as plane II; Similarly, C(1), C(2), C(3), C(4), C(5) and C(6) forms the
third plane, defined as plane III. The dihedral angle between plane I and plane II is
12.477(105)°; The dihedral angle between plane I and plane III is 12.932(99)°.
In the crystal structure of compound 5e, there are two benzene rings and one
5-Membered Ring in the molecule. C(10), C(11), C(12), C(13), C(14) and C(15) form
the first plane, defined as plane I; C(7), C(8), N(1), C(4) and C(5) forms the second
plane, defined as plane II; Similarly, C(1), C(2), C(3), C(4), C(5) and C(6) forms the
third plane, defined as plane III. The dihedral angle between plane I and plane II is
6.119(70)°; The dihedral angle between plane I and plane III is 5.359(65)°.
In the crystal structure of compound 5f, there are two benzene rings and one

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5-Membered Ring in the molecule. C(10), C(11), C(12), C(13), C(14) and C(15) form
the first plane, defined as plane I; C(7), C(8), N(1), C(4) and C(5) forms the second
plane, defined as plane II; Similarly, C(1), C(2), C(3), C(4), C(5) and C(6) forms the
third plane, defined as plane III. The dihedral angle between plane I and plane II is
53.37(25)°; The dihedral angle between plane I and plane III is 54.394(209)°.
In the crystal structure of compound 5g, there are two benzene rings and one
5-Membered Ring in the molecule. C(10), C(11), C(12), C(13), C(14) and C(15) form
the first plane, defined as plane I; C(7), C(8), N(1), C(4) and C(5) forms the second
plane, defined as plane II; Similarly, C(1), C(2), C(3), C(4), C(5) and C(6) forms the
third plane, defined as plane III. The dihedral angle between plane I and plane II is
14.424(57)°; The dihedral angle between plane I and plane III is 15.130(59)°.
3.2 Antibacterial activities
The IC50 of compounds 5a-5g against these bacterial strains are tested by MTT
method. Based on the data obtained, we found that Compounds 5a-5g possess high
selectivity to exhibit better antibacterial activity; 5c and 5d exhibit better antibacterial
activity with IC50 values of 0.03 and 0.05 µmol/mL against Staphylococcus aureus,
respectively; compound 5g displays anti-gram-negative bacterial activity with IC50
values of 0.672 and 0.830 µmol/mL against Escherichia coli and Pseudomonas
aeruginosa, respectively.
3.3 Experimental protocol of docking study
The pdb file about the crystal structures of the FtsZ were obtained from the
RCSB protein data bank (http://www.pdb.org). The molecular docking procedure was
performed by using CDOCKER protocol for receptoreligand interactions section of
Discovery Studio 3.1 (Accelrys Software Inc, San Diego, CA). Initially both the
ligands and the receptor were pretreated. For ligand preparation, the 3D structures of
all our synthesized compounds were generated with ChemBioOffice 2010 and
optimized with MMFF94 method. For enzyme preparation, the hydrogen atoms were
added with the pH of the protein in the range of 7.25-7.65.
Molecular docking of the synthesized compounds were performed on the binding
model based on the FtsZ protein complex structures. Take FtsZ protein complex

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(2VAM.pdb) as an example, The binding poses of active compounds are selected
through CDOCKER INTERACTION ENERGY [27]. Docking algorithm utilized:
CDOCKER algorithm; definition of binding site: 39.172, -4.425, 8.93; radius: 9;
scoring function: CDOC-KER interaction energy; rigid receptor: PDB code 2VAM;
flexible ligand docking: YES; cluster analysis of docking poses: ten optimal poses
were retained.
The binding model of compounds was depicted in Fig. 3 . In the binding model,
compounds 5a-5g were bound to the FtsZ with hydrogen interaction bonds, π-cation
interaction. Compound 5b and 5d were primely bound to the Gly108, Thr109, Gly107,
Thr111, Gly110, Gly104 with H-bond interactions, and it had π-cation interactions
with Arg143, respectively. Compound 5a, 5c, 5e and 5f were bound to the Gly108,
Thr109, Thr111, Gly110, Gly104 with H-bond interactions, and they also had π-cation
interactions with Arg143, respectively. Besides, compound 5g was bound to the Ala71,
Gly108, Thr109, Gly110 with H-bond interactions, and it had π-cation interactions
with Arg143.
The binding models with other protein complex were showed in the supplement
files.
4. Discussion and Conclusion
In conclusion, a series of isatin derivatives 5a-5g were synthesized, molecular
docked and tested for their inhibitory activities against E. coli, P. aeruginosa, B.
subtilis and S. aureus. Compared to Vanillin derivatives, isatin derivatives exhibit
potent antibacterial activity. According to the data presented in Fig. 3, the binding
model of isatin derivatives were nicely bound to the FtsZ with hydrogen interaction
bonds and π-cation interactions. Especially, compound 5b and 5d were primely bound
to six amino acids with H-bond interactions, and bound to the Arg143 with π-cation
interactions. Besides, compound 5g was bound to four amino acids with H-bond
interactions, and it had π-cation interactions to the Arg143. Furthermore, the docking
studies of compounds reveals that isatin as one of isatin derivatives scaffolds was
primely bound to the Gly110, Thr111, Gly104 with H-bond interactions. Thus, the
introduction of hydrogen bonds makes the corresponding compounds possess high

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selectivity antibacterial activities. Moreover, the Gly110, Thr111, Gly104, Arg143
play a crucial role as well. From the theoretical perspective, we could conclude that
the title compounds were appropriately positioned into the FtsZ active site.
However, according to the data presented in Table 1, compounds 5a-5g possess
high selectivity to exhibit better antibacterial activity; compound 5g displays
antibacterial activity against Escherichia coli and Pseudomonas aeruginosa; 5c and
5d exhibit better antibacterial activity against Staphylococcus aureus. From the close
inspection of the data, the activities of the title compounds are inconsistent with the
theoretical results. Firstly, we considered that the variability and modification of the
structures maybe accountant for this phenomenon. Compound 5g having
meta-fluorine substituent exhibited potent anti-gram-positive bacterial activity.
Meanwhile, a comparison of the substitution on the benzene ring was demonstrated as
follows: ortho-fluorine-substituted derivatives (5b, 5f, 5e and 5a) had better
antibacterial activities against Escherichia coli. Interestingly, compound 5c and
compound 5d containing meta-substituted displayed better potent antibacterial
activities against Staphylococcus aureus among the synthesized compounds. Given
the title compounds having different substituent exhibited highly selectivity
antibacterial activities, the key to discover isatin derivatives with antibacterial activity
lies in a detailed understanding of the FtsZ active sites. Second, there possibly exist
other mechamisms. We will further study mechamisms to expound this phenomenon.
Furthermore, the study of mechanisms may also provide new insights to research the
FtsZ inhibition ability of this kind of compounds, and modify these structures to treat
the related diseases.
References
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Fig 1. Molecular structures of the title compounds with atomic numbering
scheme.
Compound 5a
Compound 5b
Compound 5c
Compound 5d
Compound 5e
Compound 5f
Compound 5g

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Fig 2. Crystal packing of the title compounds.
Compound 5a
Compound 5b
Compound 5c
Compound 5d
Compound 5e
Compound 5f
Compound 5g

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Fig.3. 2D molecular docking modeling with 2VAM.
Compound 5a
Compound 5b
Compound 5d
Compound 5f
Compound 5c
Compound 5e
Compound 5g

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Table 1. Inhibitory activity (IC50 ) of the synthetic compounds
against microbes.
IC50 ( µmol/mL)
Compound
5a
Bacillus
subtilis
ꢁ50
Staphylococcus Escherichia Pseudomonas
aureus
coli
aeruginosa
ꢁ50
6.6137
ꢁ50
ꢁ50
ꢁ50
ꢁ50
ꢁ50
ꢁ50
ꢁ50
1.346
44.86
0.030
0.050
ꢁ50
44.86
ꢁ50
0.1803
1.112
ꢁ50
ꢁ50
13.25
ꢁ50
ꢁ50
ꢁ50
0. 830
5b
5c
5d
4.861
2.481
0.672
5e
5f
5g
Penicillin
0.631
-
-
-
Chloromycetin -
0.023
0.130

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Table 2. Crystallographic data, details of data collection and structure
refinement parameters.
Compound
5a
5b
5c
5d
Empirical formula
C15H11N3O2
265.27
C15H10FN3O2 C15H10ClN3O2 C15H10BrN3O2
Formula weight
283.26
299.71
344.16
Crystal system
Monoclinic
P2(1)/c
Monoclinic
P2(1)/c
Monoclinic
P2(1)/c
Orthorhombic
Pbca
Space group
a (Å)
8.9368(8)
12.1151(10)
11.7820(1)
90.00
8.9532(6)
12.1563(9)
11.9863(9)
90.00
4.6583(8)
25.221(4)
11.5041(19)
90.00
13.0250(8)
10.1415(6)
20.6903(13)
90.00
b (Å)
c (Å)
α (°)
β (°)
95.219(3)
90.00
99.153(2)
90.00
93.531(5)
90.00
90.00
γ (°)
90.00
V (Å)
1270.35(19)
4
1287.95(16)
4
1349.0(4)
4
2733.0(3)
8
Z
D calc/g cm-3
1.387
1.461
1.476
1.673
θ Range (°)
2.4, 25.0
552.0
2.85,27.70
584.0
2.40, 25.03
616.0
2.51, 25.05
1376.0
F(000)
Reflections collected/unique
11254/2241
13051/ 3013
2182/0/181
0.110
12182/ 2396
1187/0/190
0.291
23057/ 2428
1960/0/190
3.016
Data/restraints/parameters
1734/0/181
Absorption coefficient (mm -1 ) 0.095
R1/ WR2 [I > 2 σ (I)]
R1/ WR2 (all date)
GOOF
0.0386/0.1017 0.0498/0.1378 0.0569/0.1062 0.0338/0.0859
0.0574/0.1017 0.0735/0.1378 0.1574/0.1062 0.0476/0.0859
1.02
1.034
0.978
1.037

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Table 3. Crystallographic data, details of data collection and structure
refinement parameters.
Compound
5e
5f
5g
Empirical formula
C16H13N3O2
279.29
C15H10BrN3O2 C15H10FN3O2
Formula weight
344.16
283.26
Crystal system
Monoclinic
P2(1)/c
Triclinic
P-1
Monoclinic
P2(1)/c
Space group
a (Å)
4.6754(4)
25.274(2)
11.6188(10)
90.00
7.9158(15)
8.8743(17)
20.784(4)
80.338(5)
89.476(5)
73.219(5)
1376.8(5)
4
8.7258(9)
12.7898(13)
11.8132(12)
90.00
b (Å)
c (Å)
α (°)
β (°)
93.203(3)
90.00
96.693(3)
90.00
γ (°)
V (Å)
1370.8(2)
4
1309.4(2)
4
Z
D calc/g cm-3
1.353
1.660
1.437
θ Range (°)
2.38, 25.00
594
2.69, 24.43
688.0
2.36, 27.29
584.0
F(000)
Reflections collected/unique
Data/restraints/parameters
12263/2426
1536 /0/191
10866/ 4981
3295/0/379
2.993
13518/ 3053
2177 /0/190
0.108
Absorption coefficient (mm -1 ) 0.092
R1/ WR2 [I > 2 σ (I)]
R1/ WR2 (all date)
GOOF
0.0494/0.1140 0.0605/0.1305 0.0507/0.1402
0.1044/0.1140 0.1035/0.1305 0.0779/0.1402
1.036
1.072
1.027

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Table 4. Hydrogen Bond Lengths (Å) and Bond Angles (°)
Compound
D–H...A
d(D–H) d(H...A) d(D...A) ꢂDHA
Inter
Inter
Inter
Intra
Inter
Inter
Inter
Intra
Intra
Intra
Inter
Inter
Inter
Intra
Intra
Inter
Inter
Inter
Intra
Inter
Inter
Inter
Inter
Intra
N(1)--H(1) ..O(2)
N(1)--H(1) ..N(2)
0.86
0.86
2.18
2.53
2.58
2.02
2.18
2.60
2.55
2.08
2.03
2.41
2.22
2.49
2.50
2.01
2.46
2.07
2.58
2.9217(18) 145
3.2744(18) 145
5a
C(14)--H(14) ..O(1) 0.93
3.401(2)
2.7146(17) 137
2.915(2) 144
3.3603(19) 147
3.259(2) 133
147
N(3)--H(3) ..O(1)
N(1)--H(1) ..O(2)
N(1) --H(1) ..N(2)
0.86
0.86
0.86
5b
C(13) --H(13)..O(1) 0.93
N(3) --H(3) ..O(1) 0.86
2.7479(19) 134
2.6945(19) 133
N(3) --H(3) ..F(1) 0.86
C(15) --H(15)..O(2) 0.93
2.739(2)
2.984(3)
3.223(3)
3.180(4)
2.696(3)
2.776(4)
2.821(3)
3.245(3)
3.275(3)
2.683(3)
2.945(2)
3.246(2)
3.238(3)
2.788(3)
2.698(2)
101
147
143
130
137
100
146
128
169
138
147
144
132
100
137
N(1) --H(1) ..O(2)
N(1) --H(1) ..N(2)
C(6) --H(6) ..O(1)
N(3) --H(3) ..O(1)
0.86
0.86
0.93
0.86
5c
C(15) --H(15)..O(2) 0.93
N(1) --H(1) ..O(2) 0.86
5d
5e
C(3) --H(3A) ..O(2) 0.93
0.93
C(6) --H(6) ..O(1)
2.36
1.98
N(3) --H(3) ..O(1)
N(1) --H(1) ..O(2)
0.86
0.86
2.19
2.51
2.54
2.48
2.01
N(1) -H(1) ..N(2) 0.86
C(6) --H(6) ..O(1)
C(15) --H(15)..O(2)
N(3) --H(3) ..O(1)
0.93
0.93
0.86

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Table 5. Hydrogen Bond Lengths (Å) and Bond Angles (°)
Compound
D–H...A
d(D–H) d(H...A) d(D...A) ꢂDHA
Inter
Inter
Inter
Intra
Intra
Inter
Inter
Inter
Inter
N(1) --H(1) ..O(2)
N(6) --H(6) ..O(3)
C(16)--H(16) ..O(1)
N(3) --H(3) ..O(1)
N(5) --H(5) ..O(4)
N(1) --H(1) ..O(2)
N(1)--H(1)..N(2)
C(2)--H(2)..F(1)
C(12) --H(12)..O(1)
C(13) --H(13)..F(1)
N(3) --H(3)..O(1)
0.86
0.86
2.00
1.98
2.55
2.03
2.09
2.23
2.52
2.49
2.46
2.50
2.02
2.812(7)
2.775(7)
3.215(7)
2.723(7)
2.765(7)
2.969(2)
157
153
128
137
135
144
5f
0.93
0.86
0.86
0.86
5g
0.86
0.93
0.93
0.93
0.86
3.2703(19) 147
3.389(3)
3.337(2)
3.319(3)
163
157
146
Inter
Intra
2.7101(18) 137
Scheme 1. Synthesis route of the title compounds.
Reagents and conditions: (a) thionyl chloride, reflux; (b) Concentrated sulfuric
acid, methanol, reflux; (b) hydrazine hydrate, ethanol, reflux; (d) isatin,
dichloromethane, reflux.

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1. The Crystal structures of Isatin derivatives have not been reported so far.
2. The biological activities of the title compounds are also evaluated for antibacterial
activity.
3. Docking simulations have been performed to position compounds into the FtsZ active
site .