Schiff bases of indoline-2,3-dione: Potential novel inhibitors of mycobacterium tuberculosis (Mtb) DNA gyrase

Article abstract of DOI:10.3390/molecules16097864

In the present study a series of Schiff bases of indoline-2,3-dione were synthesized and investigated for their Mtb gyrase inhibitory activity. Promising inhibitory activity was demonstrated with some of these derivatives, which exhibited IC50 values rang

Full text of DOI:10.3390/molecules16097864

Molecules 2011, 16, 7864-7879; doi:10.3390/molecules16097864
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molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Schiff Bases of Indoline-2,3-dione: Potential Novel Inhibitors of
Mycobacterium Tuberculosis (Mtb) DNA Gyrase †
Tarek Aboul-Fadl 1,*, Hatem A. Abdel-Aziz 1, Mohammed K. Abdel-Hamid 2, Tilal Elsaman 1,
Jane Thanassi 3 and Michael J. Pucci 3
1
Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University,
P.O. Box 2457, Riyadh 11451, Saudi Arabia
2
Department of Pharmaceutical Medicinal Chemistry, Faculty of Pharmacy, Assiut University,
Assiut 71526, Egypt
3
Antimicrobial Drug Discovery, Achillion Pharmaceuticals 300 George Street New Haven,
CT 06511, USA
Presented at the 47th International Conference on Medicinal Chemistry (RICT 2011), Lyon-France,
†
6-8 July 2011.
* Author to whom correspondence should be addressed; E-Mail: fadl@ksu.edu.sa;
Tel.: +966-1-467-7341; Fax: +966-1-467-6220.
Received: 27 July 2011; in revised form: 23 August 2011 / Accepted: 7 September 2011 /
Published: 13 September 2011
Abstract: In the present study a series of Schiff bases of indoline-2,3-dione were
synthesized and investigated for their Mtb gyrase inhibitory activity. Promising inhibitory
activity was demonstrated with some of these derivatives, which exhibited IC50 values
ranging from 50–157 μM. The orientation and the ligand-receptor interactions of such
molecules within the Mtb DNA gyrase A subunit active site were investigated applying a
multi-step docking protocol using Molecular Operating Environment (MOE) and
Autodock4 docking software. The results revealed the importance of the isatin moiety and
the connecting side chain for strong interactions with the enzyme active site. Among the
tested compounds the terminal aromatic ring benzofuran showed the best activity.
Promising new leads for developing a novel class of Mtb gyrase inhibitors were obtained
from Schiff bases of indoline-2,3-dione.

Molecules 2011, 16
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Keywords: Schiff bases; Indoline-2,3-dione; microwave irradiation; Mycobacterium
tuberculosis (Mtb); Mtb DNA gyrase; MOE
1. Introduction
Infections caused by mycobacteria are the single largest cause of death worldwide.
Fluoroquinolones have been used with limited success as part of a second-line chemotherapeutic
regime against mycobacterial diseases. With the global emergence of multidrug-resistant tuberculosis
(MDRTB) and extensively drug-resistant tuberculosis (XDRTB) there is an urgent need to develop
new anti-mycobacterials. Design of new inhibitors with greater efficacy against mycobacterial
enzymes would facilitate the development of efficient anti-TB drugs [1]. Mycobacterium tuberculosis
(Mtb), the aetiologic agent of tuberculosis, is unusual in that it possesses only one type II
topoisomerase, DNA gyrase [2]. Consequently, the Mtb DNA gyrase exhibits a different activity
spectrum as compared to other DNA gyrases. Despite the fact that it supercoils DNA with an
efficiency comparable to that of other DNA gyrases it shows enhanced relaxation, DNA cleavage, and
decatenation activities [3].
DNA gyrase consists of two subunits, gyrase A (GyrA) and gyrase B (GyrB), that together form a
functional heterodimer structure A2B2. While the function of the GyrA subunit is primarily the
breakage and reunion of the bacterial DNA, the GyrB subunit possesses an ATP-ase activity. In the
absence of the ATP, DNA gyrase catalyzes only the relaxation of supercoiled DNA but not the
introduction of negative supercoils [4,5].
Quinolones are the only class of the DNA gyrase inhibitors currently used in clinical practice. They
act by inhibiting the GyrA subunit, thus interfering with the DNA cleavage and ligation reactions [6].
The coumarins (e.g., novobiocin) and cyclothialidines (e.g., GR122222X), natural antibiotics from the
Streptomyces organisms, are the most studied inhibitors of the GyrB subunit [7]. Both classes act as
competitive inhibitors of the ATP-binding site on the GyrB subunit, thus inhibiting the ATP-dependent
step in the enzyme catalytic cycle [8]. The inhibitory mechanism has also been characterized with
radiolabelled benzoylcyclothialidine and dihydronovobiocin and by structural analysis [9]. More
recently, GyrB inhibitors from various chemical classes have been reported including indazoles [10],
pyrazoles [11], benzimidazoles [12], phenols [13] and indolinones [14]. Furthermore, gyrase inhibitors
prepared from indoline-2,3-dione (isatin) derivatives have been also reported [15,16], in addition to its
Schiff bases, displaying anti-TB activity [17]. Accordingly isatin was identified as a versatile lead
molecule for further design of potential gyrase inhibitors and the current work is directed to synthesize
and investigate such activity based on this interesting moiety focusing on interactions with the DNA
gyrase A subunit.
2. Results and Discussion
2.1. Chemistry
The targeted hydrazones 3-26 were obtained from the condensation of the appropriate isatin 1a-k
with hydrazide derivatives 2a-k in acidified ethanol for 4 to 6 hours or by using microwave irradiation

Molecules 2011, 16
7866
(MWI) at 110 °C for 5 minutes (Scheme 1). On the other hand, the hydrazide building blocks of
compounds 15-26 were prepared according the reported procedures, either conventionally or by
microwave assisted synthesis [18,19]. Spectral data of the targeted compounds were consistent with
their assigned structures shown in Scheme 1.
Scheme 1. Synthesis of the targeted Schiff Bases.
O
R'
NH
O
N
O
X
X
reflux 4-6h or
NH2
O
O
+
R'
N
H
N
MWI, 110°C, 5 min
N
R
R
3-26
1a-k
2a-k
Compd.
X
R
H
H
R'
3
4
H
Cl
F
5
6
H
H
Me
7
OCF3
NO2
H
H
8
H
9
Ph
N
10
11
12
13
H
CH2Ph
Cl
CH2Ph
CH2Ph
CH2Ph
F
Me
14
H
H
N
15
16
F
H
H
O
H
NO2
O
O
17
18
19
20
Cl
Me
NO2
H
H
H
H
Ph
NO2
21
F
H
O
22
H
Ph
Me
H
N
Me
23
H
CH2Ph

Molecules 2011, 16
7867
Scheme 1. Cont.
Cl
H
N
Me
24
25
Me
H
H
Cl
F
H
F
F
N
OCF3
O
N
26
F
H
Me
N
Et
2.2. Mtb DNA Gyrase Inhibitory Activity
Figure 1. (a) Mtb DNA gyrase supercoiling inhibition by inhibitors and moxifloxacin.
Two-fold increases in inhibitor concentrations ranging from 1.56 to 200 µM were added to
pBR322 relaxed DNA in the presence of Mtb DNA gyrase enzyme. Control lanes are:
Lane 1, DNA with no enzyme or inhibitor and Lane 2, DNA with enzyme but no inhibitor.
IC50 values were calculated by measurement of fluorescence intensity after ethidium
bromide staining of the supercoiling band at the bottom of the lanes. Moxifloxacin was
used as an enzyme inhibitor positive control. (b) IC50 curves of supercoiling (SC)
fluorescence data. DNA fluorescence was quantitated with an Alpha Imager 2200 Analysis
System (Cell Biosciences, Santa Clara, CA, USA) and IC50 values were determined by
nonlinear regression analysis with Graphpad Prism software (Graphpad Software, Inc., San
Diego, CA, USA). IDV = Integrated Density Value.
1a
- + Compound 17
Compound 18 + -
200 µM
1.56 µM 1.56 µM
200 µM
- + Compound 19
Compound 20 + -
.
1.56 3.1 6.25 12.5 25 50 100 200

Molecules 2011, 16
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Figure 1. Cont.
Moxifloxacin + -
0.39 µM
200 µM
1b
Tb gyrase wt SC
Tb gyrase wt SC
25000
20000
15000
10000
5000
0
20000
15000
10000
5000
0
Moxif loxac in
17
18
19
20
0.0
0.5
1.0
1.5
2.0
2.5
-1
0
1
2
3
conc loguM
conc log uM
The isatin Schiff bases synthesized and characterized in this report were tested for their ability to
inhibit supercoiling activity of DNA gyrase. DNA gyrase is the only enzyme known so far having the
ability to introduce negative supercoils in DNA. In all the species of mycobacteria, including Mtb,
DNA gyrase is the sole type II topoisomerase carrying out the reactions of both gyrase and
topoisomerase IV. Since the DNA gyrase from Mtb and M. smegmatis are highly similar at a
functional as well as a protein sequence level and antigenic properties [20], Mtb strain H37RV DNA
gyrase has been used for carrying out the supercoiling assays. The results of gyrase inhibition with
various compounds are presented in Figures 1a and b. The concentration at which enzyme was 50%
inhibited was designated as the IC50. Table 1 shows the IC50 values of the tested compounds which
were found to be much higher compared to the positive control moxifloxacin. Three compounds,
namely 8, 18 and 19, showed better inhibitory activity relative to the other tested compounds. There
were no obvious structure-activity relationships among these three compounds. In addition, the effect
of different substituent variations on the isatin moiety on activity was not clear. Maximum activity was
shown with compound 18, therefore, we propose that it can be utilized as a lead structure for future
chemical optimization studies for novel inhibitors of the Mtb enzyme DNA gyrase.

Molecules 2011, 16
Table 1. Lipophilicity (Clog P) and Mtb wt gyrase inhibitory activity of the synthesized
7869
Schiff bases of isatins.
Compd.
No.
3
Mtb wt gyrase
IC50 (μM) b
>200
Clog P a
0.72
1.4
4
>200
5
6
7
8
0.87
1.22
1.75
0.47
2.64
3.37
4.08
3.51
3.87
0.72
1.54
1.14
3.49
3.28
2.52
4.7
>200
>200
>200
157.3
>200
>200
>200
>200
>200
>200
>200
>200
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
>200 (294)
~50, 56.3
89.9
>200
>200
>200
>200
>200
>200
>200
2.67
5.36
7.29
6.53
6.14
0.27
a Calculated using PC-software package (MacLogP 2.0, BioByte Corp., CA, USA);
b IC50 of the reference drug moxifloxacin is 12.76, 11.23, 12.7 μM.
2.3. Molecular Modeling Study
Investigation of the orientation and the ligand-receptor interactions of such molecules within the
Mtb DNA gyrase active site could provide valuable information to assist in the design of future
inhibitors. This modeling study aims to guide the optimization of the tested compounds toward more
potent inhibitors. With only limited literature data available on the possible active site of Mtb DNA
gyrase, a multi-step docking protocol has been developed using the docking softwares Molecular
Operating Environment (MOE) [21] and Autodock4 [22]. While it is not clear from the biological data
if the tested derivatives inhibit either the GyrA or GyrB subunits, we choose to investigate the GyrA
binding possibility. In this protocol, the recently revealed crystal structure of Mtb DNA gyrase GyrA
N-terminal domain (3ilw) [23] was employed.
The first step was to use the powerful “active site finder” function of the MOE software to nominate
potential docking pockets and this resulted in the identification of 16 potential pockets within the
manipulated 3ilw crystal structure. These pocket sites were refined using preliminary global docking

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of compound 18 where the pocket that shows the highest clustering of docked poses was chosen as the
docking pocket for further studies. This pocket was found to be made of 119 amino acid residues
starting at Arg39 and ending at Tyr276. It is worth noting that this site is distinct but in close proximity
to the pocket recently identified as the active site for fluoroquinolone derivatives [24].
The assigned pocket was used in a more sophisticated docking study using Autodock4 software.
Here the graphical interface of AutoDockTools (ADT 1.5.4) software was used to optimize the enzyme
and the ligands. A grid box size of 50 × 50 × 50 points was built and the box was centered on the
pre-assigned pocket with a grid spacing of 0.375 Å. Four ligands, compounds 8, 17-19, were selected
for their promising activity and docked into Mtb DNA gyrase.
The docking results revealed an average binding energy for the docked compounds in the range
of −8.09 to −3.21 kcal/mol (Table 2). The calculated binding energy trend found to be in rough
agreement with the biological activity. A closer look was obtained by processing the docked poses by
MOE software in order to study the enzyme-ligand interactions in more details.
Table 2. Output for the docking of compounds 8, 17-19.
Compd. No. of poses in Binding energy
Hydrogen bond
residues
Arg98, Asn172, Ser178
Lys49, Ser178
Lys49, Ser178, Gly179
Lys49, Ser178, Gly179
Other
contacts
Lys49
No.
8
major cluster
(kcal/mol)
−7.02
41
35
63
48
17
18
19
−3.21
−8.09
−6.85
-
His52, Arg98
Lys49
It was found that derivatives carrying a benzofuran moiety (compounds 17-19) showed a similar
orientation within the enzyme active site. On the other hand, derivative 8, which carries a pyridine
terminal moiety, was found to have a flip orientation inside the active site. However, this flip
orientation does not prevent its interaction at the active site in the same pattern observed by derivatives
17-19 (Table 2).
It can be noted from Table 2 that residues Lys49, Ser178 and Gly179 showed the most significant
hydrogen bond interactions with the docked ligands. In case of 18 it was found that Lys49 residue,
which is known to show significant interactions with fluoroquinolone-type inhibitors [24], participates
in two hydrogen bond interactions with both the isatin and the exo-cyclic carbonyl groups (Figure 2a).
This type of interaction can be observed for derivatives 17 and 19 as well, but with weaker bonds. In
case of 8 this interaction cannot be observed, however, it is replaced by strong hydrogen bonding with
Ser178. Another interaction that affecting the isatin nucleus that was observed with 18 involves a
strong hydrophobic interaction with His52, a contact that was not noted with other derivatives and may
provide an insight about the hydrophobic requirements for this side of the pocket. Similarly, derivative
18 shows arene-cation interactions with the positively charged Arg98 residue. In the case of 19,
because of the displacement caused by the bulky nitro-group, this interaction was lost and replaced by
a weak arene-cation interaction with Lys49 (Figure 2b).

Molecules 2011, 16
Figure 2. 2D Representation of 18 (2a) and 19 (2b) within the 3ilw active site.
2a
7871
2b
Such interactions cannot be observed for derivative 17, despite the structural similarity with 18 and
19. For reasons that remain unclear, 17 failed to orient into the active pocket showing a steric clash
between the isatin benzo moiety and Met185 residue (Figure 3).

Molecules 2011, 16
7872
Figure 3. Compound 17 (ball and stick) docked into 3ilw active site. Steric clash shown as
a red framework.
The modeling results showed the importance of the isatin moiety and the connecting side chain for
strong interactions with the enzyme active site. However, substituents carried by the isatin ring seem to
also play a crucial role for activity. While only substituents at position 5 of the isatin ring were
attempted, substitution at other positions will be investigated in future studies. In addition, the
benzofuran provided the best activity as the terminal aromatic ring, but we believe that hydrophobic
substituents on this ring may have a positive effect on the inhibitory activity.
Computation of the log P was based on the fragment method developed by Leo contained in a
PC-software package [25]. It was found that there is no obvious relation between the gyrase inhibitory
activity of the tested compounds and their lipophilicity. Clearly the lipophilicity has an influence on
the activity, but it does not solely determine the gyrase activity of these compounds.
3. Conclusions
This work describes the synthesis and investigation of Mtb gyrase inhibitory activity of some Schiff
bases of indoline-2,3-dione. Preliminary biological evaluation demonstrated that some of these
compounds possess notable Mtb gyrase inhibitory activity, suggesting potential for the development of
new class of Mtb gyrase inhibitors. In addition, a pharmacophore based study was performed to
explain the biological activity on structural bases. The modeling results showed the importance of the
isatin moiety and the connecting side chain for strong interactions with the enzyme active site.
However, substituents carried by the isatin ring seem to play a crucial role for activity. While only
substituents at position 5 of the isatin ring were attempted, substitution at other positions will be
investigated in future studies. Furthermore, as terminal aromatic ring the benzofuran moiety provided
the best activity, but we believe that hydrophobic substituents on this ring may have a positive effect
on the inhibitory activity. On the bases of the obtained results, current research is in progress to

Molecules 2011, 16
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improve the Mtb gyrase inhibitory activity/selectivity profile of this chemotype. In addition, further
research will investigate whether these inhibitors are GyrA or GyrB using in vitro methods.
4. Experimental
4.1. General
Isatin building blocks 1(a-f) and the hydrazides 2(a-c) of the designed target Schiff bases were
obtained commercially, however, the other building blocks were synthesized according to the reported
literature [18-21]. All other chemicals used were of commercially available reagent grade and were
used without further purification. Microwave irradiations were carried out using an Explorer-48
microwave reactor (CEM, Matthews, North Carolina, USA). Infrared (IR) Spectra were recorded as
KBr disks using the Perkin Elmer FT–IR Spectrum BX apparatus located at the Research Center,
College of Pharmacy, King Saud University (Riyadh, Saudi Arabia). Melting points were determined on
a Gallenkamp melting point apparatus, and are uncorrected. NMR Spectra were scanned in DMSO-d6
1
13
on a Bruker NMR spectrophotometer operating at 500 MHz for H and 125.76 MHz for C at the
Research Center, College of Pharmacy, King Saud University (Riyadh, Saudi Arabia). Chemical shifts
are expressed in δ-values (ppm) relative to TMS as an internal standard. Coupling constants (J) are
expressed in Hz. D2O was added to confirm the exchangeable protons. Mass spectra were measured on
a Varian MAT CH-5 spectrometer (70 eV) or in Agilent Triple Quadrupole 6410 QQQ LC/MS with
ESI (Electrospray ionization) source. Mtb DNA gyrase inhibitory activity assays were carried out at
Department of Antimicrobial Drug Discovery, Achillion Pharmaceuticals (New Haven, CT, USA).
4.2. General Procedure for the Synthesis of Schiff Bases 3-26:
Method 1. To a mixture of isatin 1a-k (1 mmol) and hydrazide 2a-k (1 mmol) in ethanol (25 mL) a
few drops of glacial acetic acid were added. The reaction mixture was refluxed for 4-6 h, and then
cooled to room temperature. The precipitate was filtered and dried. The crude product was
recrystallized from the appropriate solvent to obtain hydrazone compounds 3-26.
Method 2. A solution of isatin 1a-k (1 mmol) and hydrazide 2a-k (1 mmol) in ethanol (15 mL) was
prepared. Few drops of glacial acetic acid were added and whole reaction mixture was irradiated under
microwave irradiation at 110 °C for 5 minutes. The reaction mixture was cooled. The solid separated
on cooling was filtered, washed with cold ethanol, dried and recrystallised from the appropriate solvent
to obtain hydrazones 3-26.
(Z)-N'-(2-Oxoindolin-3-ylidene)isonicotinohydrazide (3). Yield: 56% (Method 1); Mp: 290–293 °C
(EtOH/DMF); IR ν: 3166, 3057 (2NH), 1702, 1683 (2C=O), 1617 (C=N) cm−1; EI MS m/z (%): 301.2
1
(M+ +1, 37), 299.3 (M+, 1), 44.9 (100); H-NMR δ: 6.93 (1H, d, J = 8.7, C6H), 7.45 (1H, d, J = 2.3,
C4H), 7.79–7.83 (2H, m, C3´H, C5´H), 8.13 (1H, br. s, C7H), 8.82–8.83 (2H, m = 4.6, C2´H, C6´H),
13
10.97 (1H, s, NH), 11.88 (1H, br. s, NH); C-NMR δ: 112.03 (C3a), 116.44 (C7), 120.99 (C3´, C5´),
122.40 (C4), 125.65 (C5), 126.52 (C3, C6), 132.22 (C4´), 140.27 (C7a), 142.88 (C2´, C6´), 150.15 (C=O),
164.29 (C2).

Molecules 2011, 16
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(Z)-N'-(5-Chloro-2-oxoindolin-3-ylidene)isonicotinohydrazide (4). Yield: 74% (Method 2); Mp:
345–348 °C (EtOH/DMF); IR ν: 3166, 3057 (2NH), 1702, 1683 (2C=O), 1617 (C=N) cm−1; EI MS m/z
(%): 301.2 (M+ +1, 37), 299.3 (M+, 1), 44.9 (100); 1H-NMR δ: 6.93 (1H, d, J = 8.7, C6H), 7.45 (1H, d,
J = 2.3, C4H), 7.79–7.83 (2H, m, C3´H, C5´H), 8.13 (1H, br. s, C7H), 8.82–8.83 (2H, m = 4.6, C2´H,
13
C6´H), 10.97 (1H, s, NH), 11.88 (1H, br. s, NH); C-NMR δ: 112.03 (C3a), 116.44 (C7), 120.99 (C3´,
C5´), 122.40 (C4), 125.65 (C5), 126.52 (C3, C6), 132.22 (C4´), 140.27 (C7a), 142.88 (C2´, C6´), 150.15
(C=O), 164.29 (C2).
(Z)-N'-(5-Fluoro-2-oxoindolin-3-ylidene)isonicotinohydrazide (5). Yield: 59% (Method 2); Mp:
346–348 °C (EtOH/DMF); IR ν: 3226, 3057 (2NH), 1709, 1677 (2C=O), 1613(C=N) cm−1; EI MS m/z
1
(%): 284.8 (M+ +202), 283.7 (M+, 20), 44.9 (100); H-NMR δ: 6.92 (1H, br. s, C6H), 7.29 (1H, br. s,
C4H), 7.85–7.94 (3H, m, C3´H, C5´H, C7H), 8.81–8.83 (2H, br. s, C2´H, C6´H), 10.87 (1H, s, NH), 11.94
(1H, s, NH).
(Z)-N'-(5-Methyl-2-oxoindolin-3-ylidene)isonicotinohydrazide (6). Yield: 69% (Method 2); Mp:
319–322 °C (EtOH/DMF); IR ν: 3260 (NH), 1706, 1694 (2C=O), 1626 (C=N) cm−1; EI MS m/z (%):
1
281.5 (M+ +1, 17), 279.5 (M+, 15), 47.8 (100); H-NMR δ: 2.31 (3H, s, CH3), 6.86 (1H, d, J = 8.3,
C7H), 7.22 (1H, d, J = 7.8, C6H), 7.43 (1H, br. s, C4H), 7.78 (2H, d, J = 4.6, C3´H, C5´H), 8.87 (2H, d,
13
J = 4.6, C2´H, C6´H), 10.72 (1H, s, NH), 11.30 (1H, s, NH); C-NMR δ: 20.48 (CH3), 111.10 (C3a),
119.56 (C7), 120.89 (C3´, C5´), 121.27 (C4), 121.51 (C3, C6), 131.99 (C5), 132.63 (C4´), 139.24 (C7a),
140.48 (C2´, C6´), 150.92 (C=O), 162.98 (C2).
(Z)-N'-(2-Oxo-5-(trifluoromethoxy)indolin-3-ylidene)isonicotinohydrazide (7). Yield: 33% (Method 2); Mp:
262–265 °C (EtOH/DMF); IR ν: 3236 (NH), 1712, 1698 (2C=O), 1628 (C=N) cm−1; EI MS m/z (%):
351.6 (M+ +1, 0.6), 350.3 (M+, 4), 40.1 (100); 1H-NMR δ: 7.05–7.07 (1H, m, C7H), 7.41–7.54 (2H, m,
C4H, C6H), 7.71–7.79 (2H, m, C3´H, C5´H), 8.79–8.87 (2H, m, C2´H, C6´H), 11.59 (1H, br. s, NH);
13C-NMR δ: 112.49 (C3a), 114.37 (C7), 120.90 (C3´, C5´), 121.04(CF3), 121.17 (C4), 125.14 (C5),
138.28 (C6), 138.89 (C3), 138.97 (C4´), 141.67 (C2´, C6´), 143.71 (C7a), 150.93 (C=O), 163.04 (C2).
(Z)-N'-(5-Nitro-2-oxoindolin-3-ylidene)isonicotinohydrazide (8). Yield: 70% (Method 2); Mp:
350–353 °C (EtOH/DMF); IR ν: 3328, 3184 (2NH), 1739, 1709 (2C=O), 1606 (C=N) cm−1; EI MS m/z
(%): 313.0 (M+ +2, 2.5), 311.0 (M+, 17), 78.0 (100); 1H-NMR δ: 7.67 (1H, br. s, C7H), 7.71–7.79 (2H,
m, C3´H, C5´H), 8.79–8.87 (2H, m, C2´H, C6´H), 8.31 (2H, br. s, C4H, C6H), 11.48 (1H, br. s, NH),
12.04 (1H, br. s, NH).
(Z)-N'-(2-Oxo-1-phenylindolin-3-ylidene)isonicotinohydrazide (9). Yield: 90% (Method 2); Mp:
201–205 °C; IR ν: 3296 (NH), 1708, 1688 (2C=O), 1613 (C=N) cm−1; EI MS m/z (%): 342.1 (M+, 25),
40.1 (100); 1H-NMR δ: 6.88 (1H, d, J = 8, Cd H), 7.25 (1H, t, J = 8, Ce H), 7.46 (1H, t, J = 7.5, Cf H),
7.53–7.74 (8H, m, Cg H, C3 + 3* H and H of ph), 8.86 (2H, d, J = 3, C2 + 2* H), 13.91 (1H, s, NHN=);
13C-NMR δ: 110.31 (Cc*), 119.12 (Ce), 121.10 (C4-ph), 121.25 (C2-ph+6-ph), 123.87 (C3 + 3*), 126.64
(Cd+g+5-ph), 128.74 (C4-ph), 129.74 (Cf+c), 132.12 (C1-ph), 132.65 (Cg*), 139.01 (C4), 143.89 (C2 + 2*),
150.92 (Cb), 160.55 (C5).

Molecules 2011, 16
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(Z)-N'-(1-Benzyl-2-oxoindolin-3-ylidene)isonicotinohydrazide (10). Yield: 20% (Method 1); Mp:
162–165 °C (EtOH); IR ν: 3224 (NH), 1703, 1676 (2C=O), 1614(C=N) cm−1; EI MS m/z (%): 356.1
1
(M+, 7.5), 40.1 (100); H- NMR δ: 5.03 (2H, s, CH2), 7.31–7.41 (9H, m, Har.), 7.82 (2H, br. s, C3´H,
C5´H), 8.87 (2H, m, C2´H, C6´H); 13C-NMR δ: 42.50 (CH2), 110.64 (C3a), 119.10 (C7), 121.06 (C3´, C5´),
123.54, 127.46, 127.68, 128.72, 132.01, 135.55, 142.93 (Car., C2´, C4´, C6´), 150.89 (C=O), 161.07 (C2).
(Z)-N'-(1-Benzyl-5-chloro-2-oxoindolin-3-ylidene)isonicotinohydrazide (11). Yield: 26% (Method 1); Mp:
167–171 °C (EtOH); IR ν: 3447 (NH), 1701, 1689 (2C=O), 1616 (C=N) cm−1; EI MS m/z (%): 390
1
(M+, 3.2), 47.3 (100); H- NMR δ: 5.03 (2H, s, CH2), 7.31–7.39 (8H, m, Har.), 7.81 (2H, d, J = 7.8,
C3´H, C5´H), 8.86 (2H, d, J = 7.8, C2´H, C6´H); 13C-NMR δ: 43.01 (CH2), 112.15 (C3a), 120.55, 120.96,
121.28, 127.43, 127.72, 128.72, 131.24, 135.31, 138.99, 141.53 (Car.), 150.89 (C=O), 161.50 (C2).
(Z)-N'-(1-Benzyl-5-fluoro-2-oxoindolin-3-ylidene)isonicotinohydrazide (12). Yield: 17% (Method 1);
Mp: 188–192 °C (EtOH); IR ν: 3446 (NH), 1709, 1683 (2C=O), 1625 (C=N) cm−1; EI MS m/z (%):
1
374.8 (M+, 3.2), 62.2 (100); H-NMR δ: 5.03 (2H, s, CH2), 6.91 (1H, dd, J = 8.93, 4.36, C6H),
7.11–7.64 (6H, m, C4H, Har.), 7.71–7.79 (2H, m, C3´H, C5´H), 7.99 (1H, d, J = 7.36, C7H), 8.79–8.87
(2H, m, C2´H, C6´H),11.88 (1H, s, NH).
(Z)-N'-(1-Benzyl-5-methyl-2-oxoindolin-3-ylidene)isonicotinohydrazide (13). Yield: 27% (Method 1);
Mp: 159–162 °C (EtOH); IR ν: 3448 (NH), 1703, 1682 (2C=O), 1623 (C=N) cm−1; EI MS m/z (%):
1
370.0 (M+, 7.5), 44.9 (100); H-NMR δ: 2.31 (3H, s, CH3), 4.99 (2H, s, CH2), 6.98 (1H, d, J = 8.24,
C7H), 7.21–7.40 (6H, m, Har.), 7.50 (1H, br. s, C4H), 7.81–7.85 (2H, m, C3´H, C5´H), 8.87–8.89 (2H, m,
C2´H, C6´H).
(Z)-N'-(2-Oxoindolin-3-ylidene)nicotinohydrazide (14). Yield: 72% (Method 1); Mp: 254–256 °C
(EtOH/DMF); IR ν: 3226 (NH), 1719, 1695 (2C=O), 1620 (C=N) cm−1; EI MS m/z (%): 267.2 (M+ +1, 7),
1
265.9 (M+, 15), 64.0 (100); H-NMR (DMSO-d6) δ: 6.97 (1H, d, J = 7.8, C4H), 7.12 (1H, t, J = 7.8,
C5H), 7.41 (1H, t, J = 7.8, C6H), 7.64–7.67 (2H, m, C7H, C5´H), 8.26 (1H, d, J = 5.1 C4´H), 8.85 (1H,
d, J = 6.1, C6´H), 9.05 (1H, s, C2´), 11.39 (1H, s, NH); 13C-NMR δ: 111.29 (C3a), 119.64 (C7), 121.08,
122.81, 124.05, 128.06, 132.03, 142.60, 148.5 (Car.), 153.12 (C=O), 162.94 (C2).
(Z)-N'-(5-Fluoro-2-oxoindolin-3-ylidene)furan-2-carbhydrazide (15). Yield: 57% (Method 2); Mp:
310–312 °C (EtOH/DMF); IR ν: 3447, 3160 (2NH), 1715, 1667(2C=O), 1611 (C=N) cm−1; EI MS m/z
(%): 272.8 (M+, 6), 40.1 (100); 1H-NMR δ: 6.78–6.79 (1H, m, Har.), 6.89–6.90 (1H, m, Har.), 7.25–7.29
(1H, m, Har.), 7.70 (1H, br. s, Har.), 7.94 (1H, br. s, Har.), 8.07 (1H, s, Har.), 10.87 (1H, s, NH), 11.67
13
(1H, s, NH); C-NMR (DMSO-d6) δ: 111.49 (C4´), 112.47 (C3´), 113.55 (C4), 115.76 (C3a), 118.97
(C6), 119.68 (C7), 135.01, 140.28, 145.26, 147.26 (Car.), 156.15 (C=O), 164.78 (C2).
(Z)-5-Nitro-N'-(2-Oxoindolin-3-ylidene)furan-2-carbohydrazide (16). Yield: 82% (Method 2); Mp:
1
309–11 °C (EtOH/DMF); ESI MS m/z: 299.1 [M − 1]−; H-NMR δ: 6.79 (1H, s, Har.), 6.97–6.99 (1H,
m, Har.), 7.13–7.15 (1H, m, Har.), 7.41–7.43 (1H, m, Har.), 7.64–7.67 (2H, m, Har.), 7.88 (1H, br. s, Har.),
11.38 (1H, s, NH).

Molecules 2011, 16
7876
(Z)-N'-(5-Chloro-2-oxoindolin-3-ylidene)benzofuran-2-carbohydrazide (17). Yield: 85% (Method 2);
Mp: 335–338 °C (EtOH/DMF); ESI MS m/z: 340.1 [M + 1]+; 1H-NMR δ: 6.94 (1H, d, J = 8.08, Har.),
7.38–7.56 (3H, m, Har.), 7.75 (1H, d, J = 8.76, Har.), 7.89 (1H, d, J = 8.04, Har.), 8.09 (1H, s, Har.), 8.19
13
(1H, s, Har.), 11.03 (1H, s, NH), 12.03 (1H, s, NH); C-NMR δ: 112.54 (C3´), 112.61 (C7´), 117.09
(C3a), 121.28, 124.01, 124.63, 126.29, 126.77, 127.51, 128.53, 128.82, 132.01, 132.82, 143.39, 155.08
(Car.), 163.25 (C=O), 165.03 (C2).
(Z)-N'-(5-Methyl-2-oxoindolin-3-ylidene)benzofuran-2-carbohydrazide (18). Yield: 89% (Method 2);
1
Mp: 331–36 °C (EtOH/DMF); ESI MS m/z: 320.2 [M + 1]+; H-NMR δ: 2.32 (CH3), 6.83 (1H, d,
J = 8.08, Har.), 7.21–7.26 (1H, m, Har.), 7.38–7.40 (1H, m, Har.), 7.47–7.59 (2H, m, Har.), 7.76 (1H, t,
13
J = 7.70, Har.), 7.85–7.90 (2H, m, Har.), 10.78 (1H, s, NH), 11.77 (1H, s, NH); C-NMR δ: 21.07
(CH3), 111.09 (C3´), 111.62 (C7´), 112.59 (C3a), 120.27, 122.11, 123.91, 124.60, 127.55, 128.69,
131.32, 132.47, 133.09, 133.88, 141.07, 155.11 (Car.), 163.51 (C=O), 165.30 (C2).
(Z)-N'-(5-Nitro-2-oxoindolin-3-ylidene)benzofuran-2-carbohydrazide (19). Yield: 79% (Method 2);
1
Mp: 367–69 °C (EtOH/DMF); ESI MS m/z: 351.1 [M + 1]+; H-NMR δ: 7.10 (1H, d, J = 8.80, Har.),
7.41 (1H, t, J = 7.70, Har.), 7.58 (1H, t, J = 7.70, Har.), 7.75 (1H, d, J = 8.08, Har.), 7.85–7.92 (2H, m,
13
Har.), 8.07 (1H, s, Har.), 8.29–8.63 (1H, m, Har.), 9.03(1H, s, NH), 11.59 (1H, s, NH).; C-NMR δ:
111.39 (C3´), 112.48 (C7´), 112.62 (C3a), 121.01, 123.96, 124.04, 124.67, 127.50, 128.34, 128.65,
128.93, 142.51, 143.44, 148.35, 150.05, 155.07 (Car.), 163.78 (C=O), 165.58 (C2).
(Z)-N'-(2-Oxo-1-phenylindolin-3-ylidene)benzofuran-2-carbohydrazide (20). Yield: 88% (Method 2);
13
Mp: 235–38 °C (EtOH/DMF); ESI MS m/z: 382.3 [M + 1]+; C-NMR (DMSO-d6) δ: 110.81 (C3´),
112.63 (C7´), 112.69 (C3a), 115.95, 119.83, 121.82, 123.88, 124.37, 124.86, 127.39, 127.52, 128.82,
129.34, 130.28, 130.35, 132.59, 133.36, 144.52, 152.60 (Car.), 155.15 (C2), 161.15 (C=O).
(Z)-N'-(5-Fluoro-2-oxoindolin-3-ylidene)-5-nitrobenzofuran-2-carbhydrazide (21). Yield: 86%
1
(Method 2); Mp: 357–59 °C (EtOH/DMF); ESI MS m/z: 367.1 [M − 1]−; H-NMR δ: 6.92 (1H, dd,
J = 4.40, 8.76, Har.), 6.99 (1H, dd, J = 3.68, 8.08, Har.), 7.25–7.29 (1H, m, Har.), 7.37–7.43 (1H, m,
Har.), 7.49–7.59 (1H, m, Har.), 7.76 (1H, d, J = 8.80, Har.), 8.0–8.12 (1H, m, Har.), 8.25–8.42 (1H, m,
Har.), 10.88 (1H, s, NH), 11.97 (1H, s, NH).
(Z)-N'-(2-oxo-1-phenylindolin-3-ylidene)adamantane-1-carbohydrazide (22). Yield: 56% (Method 2);
1
Mp: 201–203 °C (EtOH/DMF); ESI MS m/z: 400.2 [M + 1]+, 422.2 (M + 23 (Na)]+; H-NMR δ:
1.71–2.03 (15H, m, adamantine 6CH2 and 3CH) 6.84 (1H, br. s, Har.), 7.23–7.68 (8H, m, Har.).
(Z)-N'-(1-Benzyl-2-oxoindolin-3-ylidene)-2-(2,3-dimethylphenylamino)benzohydrazide (23). Yield:
1
37% (Method 1); Mp: 286–288 °C (EtOH); ESI MS m/z: 475.2 [M + 1]+; H-NMR δ: 2.14 (3H, s,
CH3), 2.29 (3H, s, CH3), 5.03 (2H, s, CH2), 6.82–6.89 (2H, m, Har.), 7.01–7.19 (5H, m, Har.), 7.29–7.39
(7H, m, Har.), 7.66–7.70 (2H, m, Har.), 9.23 (1H, s, NH).
(Z)-2-(2,6-Dichloro-3-methylphenylamino)-N'-(5-methyl-2-oxoindolin-3-ylidene)-benzohydrazide (24).
1
Yield: 33% (Method 1); Mp: 283–287 °C (EtOH/DMF); ESI MS m/z: 453.1 [M]+; H-NMR δ: 2.32
(3H, s, CH3), 2.39 (3H, s, CH3), 6.33 (1H, d, J = 8.24, Har.), 6.87 (1H, d, J = 8.24, Har.), 6.94 (1H, t,

Molecules 2011, 16
7877
J = 7.78, Har.), 7.21 (1H, d, J = 8.24, Har.), 7.34–7.41 (2H, m, Har.), 7.44 (1H, s, Har.), 7.52 (1H, d,
13
J = 8.24, Har.), 7.68 (1H, d, J = 8.24, Har.), 9.46 (1H, s, NH), 11.26 (1H, s, NH); C-NMR δ: 20.15
(CH3), 20.57 (CH3), 111.05, 114.44, 118.39, 119.90, 121.28, 128.17, 129.11, 131.89, 132.25, 133.79,
136.56, 140.24, 146.39 (Car.), 163.12 (C=O), 166.01 (C2).
(Z)-N'-(2-Oxo-5-(trifluoromethoxy)indolin-3-ylidene)-2-(3-(trifluoromethyl)phenylamino)-benzo-hydrazide
(25). Yield: 45% (Method 1); Mp: 226 °C (EtOH/DMF); ESI MS m/z: 509.1 [M + 1]+, 531.1 [M + 23
1
(Na)]+; H-NMR δ: 6.99 (1H, d, J = 7.32, Har.), 7.17–7.76 (10H, m, Har.), 9.02 (1H, s, NH), 11.44
(1H, s, NH).
(Z)-1-Ethyl-N'-(5-fluoro-2-oxoindolin-3-ylidene)-7-methyl-4-oxo-1,4-dihydro-1,8-naphthyridine-3-
carbohydrazide (26). Yield: 55% (Method 2); Mp: >360 °C (EtOH/DMF); IR ν: 3070 (2NH),
1
1723–1685 (3C=O), 1608 (2C=N) cm−1; EI MS m/z (%): 392.9 (M+, 5), 40.1 (100); H-NMR δ: 1.58
(3H, t, J = 6.5, N1'CH2CH3), 2.73 (3H, s, C7'CH3), 4.63 (2H, q, J = 7.5, N1'CH2CH3), 6.98 (1H, dd,
J = 8.93, 4.36, C6H), 7.34-7.60 (1H, m, C4H), 8.59 (1H, d, J = 7.5, C6'H), 8.72 (1H, d, J = 7.36, C7H),
8.98 (1H, d, J = 8.5, C5'H), 9.32 (1H, s, C2'H), 10.86 (1H, s, NH).
4.3. Mycobacterium Tuberculosis (Mtb) Gyrase Supercoiling Assay
Relaxed pBR322 DNA (0.1 µg) was incubated with 1 unit of Mtb DNA gyrase for 60 minutes at
37 °C in the following buffer: 2 mM ATP, 4 mM DTT, 25 mM KCl, 6 mM magnesium acetate,
50 µg/mL of molecular grade BSA, 0.1 mg/mL E. coli tRNA, 2mM spermidine, 100 mM potassium
glutamate and 40 mM Tris-HCL pH 7.5 (20 µL). Reactions are stopped with 0.5% SDS,
6 mM EDTA, 5.35% glycerol, and 0.013% bromophenol blue (10 µL). Reactions are then loaded onto
a 1% agarose/ TBE gel to separate supercoiled and relaxed DNA forms and run overnight at 22 volts.
Bands are visualized by staining with 0.5 µg/mL EtBr in 1 × TBE for 40 minutes then washing with
dH2O for 120 minutes. Quantification is done on the AlphaImager 2200 system. DNA gyrase subunits,
GyrA and GyrB, were isolated from Mtb strain H37RV genomic DNA, expressed and purified in pET
overexpression constructs in E coli. One unit of DNA supercoiling activity is defined as the amount
that converts ~50% relaxed DNA, in the standard assay, to supercoiled DNA in 60 minutes at 37 °C.
Acknowledgements
This research project is supported by “NPST program by King Saud University”, Project Number
MED598-02-08.
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Sample Availability: Samples of the compounds are available from Prof. Tarek Aboul-Fadl,
Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, P.O. Box
2457, Riyadh 11451, Saudi Arabia.
© 2011 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/3.0/).