Discovery and structure optimization of a series of isatin derivatives as Mycobacterium tuberculosis chorismate mutase inhibitors

Article abstract of DOI:10.1111/cbdd.12265

In this study, the crystal structure of the Mycobacterium tuberculosis (MTB) enzyme chorismate mutase (CM) bound to transition state analogue (PDB: 2FP2) was used as a framework for virtual screening of the BITS-Pilani in-house database (2500 compounds) to identify new scaffold. We identified isatin as novel small molecule MTB CM inhibitors; further twenty-four isatin derivatives were synthesized and evaluated in vitro for their ability to inhibit MTB CM, and activity against M. tuberculosis as steps towards the derivation of structure-activity relationships (SAR) and lead optimization. Compound 3-(4-nitrobenzylidene)indolin-2-one, 24 emerged as the most promising lead with an IC₅₀ of 1.01 ± 0.22 μm for purified CM and MIC of 23.5 μm for M. tuberculosis, with little or no cytotoxicity. 2D interaction profile of compounds 1, 7 and 25 with the active site residues.

Full text of DOI:10.1111/cbdd.12265

Chem Biol Drug Des 2014; 83: 498–506
Research Letter
Discovery and Structure Optimization of a Series of
Isatin Derivatives as Mycobacterium tuberculosis
Chorismate Mutase Inhibitors
drugs have been developed for nearly 40 years since the
introduction of rifampicin in 1965. It is thus necessary to
search for new and more effective antimycobacterial
agents with novel mechanisms of action to combat the
emergence of drug resistance and to shorten the duration
of therapy (1). Chorismate occupies a central place in the
shikimate pathway for the biosynthesis of the aromatic
amino acids. Chorismate mutase (CM) (EC 5.4.99.5) catal-
yses the Claisen rearrangement of chorismate to prephen-
ate via an endo-oxabicyclic transition state (Figure 1), the
first committed step in the biosynthesis of tyrosine and
phenylalanine in bacteria (2). Mycobacterium tuberculosis
possess two chorismate mutases belonging to different
subclasses (3), the secreted enzyme (*MtCM, c-subclass)
and the housekeeping enzyme (MtCM, d-subclass),
whereas the former is primarily involved in directing the
protein from the cytoplasm to the pseudoperiplasmic
space and may have some role in host–pathogen interac-
tion. It is the secreted enzyme (usually referred to as
*MtCM) that is likely related to pathogenicity and virulence
of MTB (4–8). As vertebrates do not possess the shikimate
pathway for the biosynthesis of aromatic compounds and
lacks CM activity, this enzyme represents a prime target
for the development of antibacterials. In spite of the tre-
mendous opportunity that MTB CM offers as a target for
the development of potential antimicrobial agents, very lit-
tle has been achieved in this regard. A structure-based
discovery programme by Agrawal et al. has earlier identi-
fied four molecules as potential inhibitors of M. tuberculo-
sis chorismate mutase (MtCM) with low Ki values (9). The
most promising analogue in the study (4-(3,4-dimethoxy-
phenethylamino)-3-nitro-5-sulfamoylbenzoic acid) exhibited
a Ki of 5.7 Æ 1.2 lM. But later an in vitro (8) and in vivo
(10) evaluation of a library of molecules designed and syn-
thesized based on Agrawal claims proved to be com-
pletely ineffective, with Munack et al. (8) recently
discrediting Agrawal’s work in his study, as the reported
lead molecules proved to be inactive. Although the recent
research efforts from Pal and his co-workers identifying a
series of N-aryl substituted thieno[2,3-d]pyrimidin-4(3H)-
ones (11), 3–chloroquinoxaline (12), fused triazinone (13)
and 2-aminochromenes (14) derivatives as potential
*MtCM inhibitors with IC50 values in the lower micromolar
range are encouraging, MTB CM inhibitors with Ki/IC50 val-
ues significantly below the lM range still await discovery.
Variam U. Jeankumar, Reshma Alokam,
Jonnalagadda P. Sridevi, Priyanka Suryadevara,
Siddharth S. Matikonda, Santosh Peddi,
Seedarala Sahithi, Mallika Alvala,
Perumal Yogeeswari and Dharmarajan Sriram*
Department of Pharmacy, Birla Institute of Technology &
Science-Pilani, Hyderabad Campus, Shameerpet, R.R.
District, Hyderabad, Andhra Pradesh 500078, India
*Corresponding author: Dharmarajan Sriram,
drdsriram@yahoo.com
In this study, the crystal structure of the Mycobacterium
tuberculosis (MTB) enzyme chorismate mutase (CM)
bound to transition state analogue (PDB: 2FP2) was
used as a framework for virtual screening of the BITS-
Pilani in-house database (2500 compounds) to identify
new scaffold. We identified isatin as novel small mole-
cule MTB CM inhibitors; further twenty-four isatin deriv-
atives were synthesized and evaluated in vitro for their
ability to inhibit MTB CM, and activity against M.
tuberculosis as steps towards the derivation of
structure–activity relationships (SAR) and lead optimiza-
tion. Compound 3-(4-nitrobenzylidene)indolin-2-one, 24
emerged as the most promising lead with an IC50 of
1.01 Æ 0.22 lM for purified CM and MIC of 23.5 lM for
M. tuberculosis, with little or no cytotoxicity.
Key words: chorismate mutase, isatin, Mycobacterium tuber-
culosis, tuberculosis
Abbreviations: e-pharmacophore, energy-based pharmaco-
phore; MIC, minimum inhibitory concentration; MTB CM,
Mycobacterium tuberculosis chorismate mutase.
Received 26 June 2013, revised 25 October 2013 and
accepted for publication 4 November 2013
Tuberculosis (TB), an infection caused by Mycobacterium
tuberculosis (MTB), has become a major global health
concern. Presently, MTB kills approximately 1.6 million
people annually and has infected one-third of the world
population, rendering TB the second most lethal infectious
disease after HIV/AIDS.a With 400 000 new cases annu-
ally, multiple drug-resistant TB is emerging rapidly, calling
urgently for new anti-TB therapeutics. No truly new TB
498
ª 2014 John Wiley & Sons A/S. doi: 10.1111/cbdd.12265

Isatin Derivatives as MTB CM Inhibitors
Figure 1: Chorismate mutase
reaction scheme.
This study describes the in silico discovery of isatin as
novel small molecule inhibitor of MTB chorismate mutase,
and further, a library of its derivatives was synthesized and
was evaluated in vitro for their ability to inhibit MTB CM
enzyme and whole-cell MTB as steps towards the deriva-
tion of structure–activity relationships.
generation and molecular docking approach has been
described thoroughly in the experimental section of sup-
porting information (Appendix S1). Important interactions
as evidenced from docking studies for isatin were associ-
ated with Arg 134 and Lys 60. Such interactions were pre-
dicted to be crucial for enzyme activity, diagrammatic
representation of which in comparison with that of transi-
tion state analogue is given in Figure 3. In the enzyme inhi-
bition studies, isatin showed excellent MTB CM inhibition
with IC50 of 1.08 Æ 0.1 lM. To investigate the potential of
this lead, a series of substituted isatin derivatives were
synthesized and evaluated for their ability to inhibit MTB
CM enzyme, to explore the structure-activity relationship
(SAR) as well as to evaluate their antimycobacterial
potency.
Rational inhibitor design relies both on mechanistic and
structural information about the target enzyme. The mech-
anism of the uncatalysed MTB CM reaction is known to
involve a transition state with a chair-like conformation,
Figure 1. In fact, the endo-oxabicyclic dicarboxylic acid
(15), a good geometric mimic of this transition state, is
currently the best inhibitor of a broad range of chorismate
mutases consistent with the hypothesis that the enzymatic
process proceeds through a corresponding bicyclic transi-
tion state (16). In this study, an energy-based pharmaco-
phore model (e-pharmacophore), developed using
chorismate mutase-transition state analogue (CM-TSA)
complex as a template, was explored to carry out a high-
throughput virtual screening of our in-house (BITS-Pilani)
small molecule database (2500 compounds). Glide XP
A library was designed with the goal of obtaining a lead
series with tractable SAR and potencies better than the
previously identified virtual screening hit, isatin 1. The
isatin derivatives were synthesized from isatin by a simple
and straightforward route as delineated in Schemes 1, 2
and 3. It was decided to first explore the N-1 position of
isatin by introducing various alkyl, aryl and amide deriva-
tives to understand its importance in activity determina-
tion. Alkylation of isatin with corresponding alkyl iodides
using Cs2CO3 as base in acetonitrile gave the desired
€
(extra precision) module of Schrodinger 9.2 was utilized for
docking of the molecules on to the active site pocket of
MTB CM, and isatin was identified as a potential lead (Fig-
ure 2). The methodology employed in e-pharmacophore
Figure 2: Reported Mycobacterium tuberculosis chorismate mutase inhibitors.
Chem Biol Drug Des 2014; 83: 498–506
499

Jeankumar et al.
(25). All the reactions went smoothly giving the desired
products in good yield. To understand the role of various
substituents in phenyl core of isatin, various aryl and het-
eroaryl/amino groups were introduced at the 5th position
of isatin core. The introduction of the aryl functionality
was brought about by palladium catalysed cross-coupling
reaction as described by Martinez et al. (26). Buchwald–
Hartwing cross coupling was utilized to synthesize the
N-aryl derivatives (27–30). Two condensation products
24–25 were also synthesized via Knoevenagel condensa-
tion of the corresponding aldehydes (31,32) with oxindole,
which in turn was prepared by the one-pot Wolff–Kishner
like reduction in isatin with hydrazine hydrate (33,34). All
the synthesized compounds (Table 1) were characterized
by 1H & 13C NMR, LC-MS and purity checked by ele-
mental analysis.
A
B
The characterized isatin derivatives were then subjected to
MTB CM enzyme inhibition study by adapting a previously
reported protocol (5) to a 96-well plate format, as detailed
in the experimental section of supporting information
(Appendix S1) and the IC50 values reported in Table 1.
The original hit compound isatin
1 showed IC50 of
1.08 Æ 0.1 lM. Of twenty-four isatin derivatives synthe-
sized, three molecules exhibited promising potency com-
parable with that of the initial lead isatin with IC50 of
0.99 Æ 0.14 lM for 1-acetyl isatin, 7, 1.01 Æ 0.22 lM for
3-(4-nitrobenzylidene)indolin-2-one, 24, and 0.93 Æ 0.1 lM
for 3-((5-nitrothiophen-2-yl)methylene)indolin-2-one, 25.
Dose–response curves were plotted for the more potent
inhibitors 1, 7, 24, 25 and the positive control carvacrol,
using graphpad prism software by taking log (inhibitor con-
centration) on X-axis and response (% inhibition) on Y-axis
as shown in Figure 4. To analyse the interaction pattern of
the active molecule reported from assay in the active site
of protein, molecular docking approach was adopted. The
compounds were docked to the protein active site using
extra precision mode (XP) of Glide module and their dock-
ing score, and interaction pattern was compared with that
of their in vitro activity, as steps towards establishing a
structure–activity relationship.
Figure 3: View of key interactions among the residues in the
active site of MTB CM. Amino acids are represented in green (C),
blue (N), red (O) and hydrogen (H); ligand molecules are
represented with yellow. (C) coloured atoms. Polar contacts are in
blue dashes. (A) Interaction of TSA, (B) interaction of isatin.
alkylated derivatives, compounds 2–5 in good yield
(17,18). The Chan–Lam methodology (19,20) was suc-
cessful in achieving the N-phenyl derivative 6. N-Acetyl
derivative 7 was prepared by acetylating with acetic
anhydride under refluxing conditions (21), and pivaloyl
group 8 was introduced by treatment with pivaloyl cho-
ride using triethylamine as base in tetrahydrofuran. We
then focused on the carbonyl group at C-3 position,
which was further modified into their corresponding
semicarbazone, thiosemicarbazone and oxime derivatives
(22–24) 9–10 & 15, by refluxing with semicarbazide, thi-
osemicarbazide and hydroxylamine hydrochloride, respec-
tively, in ethanol. A similar strategy was also employed in
the case of the N-acetyl isatin as well to give the N-ace-
tyl analogue 11–12. Schiff bases 13–14 were prepared
by refluxing isatin with the corresponding amines in etha-
nol in the presence of catalytic amount of acetic acid
The lead compound isatin, which exhibited an in vitro
activity of about 1.08 Æ 0.10 lM, was found to be asso-
ciated in five prominent hydrogen bonds with the active
site residues. The oxygen atom at R1 position was found
to be bonded with Arg 72 and Arg 134. The hydrogen
on nitrogen atom (N-1) was found to be in polar contact
with the carboxyl oxygen of Glu 109, and the carbonyl
oxygen next to nitrogen atom was found associated with
Lys 60 and Arg 134. Various substitutions on isatin
nucleus gave compounds with a wide range of activity.
With respect to structure–activity relationship, introduction
of alkyl group at N-1 position of isatin made the com-
pounds 2–5 less active (IC50 ranging from 1.96 Æ 0.43 to
96.56 Æ 5.2 lM) and increasing chain length decreased
the activity drastically (N-methyl > N-ethyl > N-isopro-
pyl > N-allyl). This decline in activity may be attributed to
500
Chem Biol Drug Des 2014; 83: 498–506

Isatin Derivatives as MTB CM Inhibitors
Scheme 1: Synthetic protocol adopted to attain compounds 2–15; modifications explored at N-1 and C-3 positions.
Scheme 2: Synthetic protocol
adopted to attain compounds
16–23; modifications explored at
C-5 position.
Scheme 3: Synthetic protocol
adopted to attain compounds
24–25; modifications explored at
C-3 position.
the loss of polar contact of compounds with Glu 109. N-
Phenyl isatin showed a moderate IC50 of 23.3 Æ 2.0 lM.
Introduction of simple acetyl group made the compound
(7) equally active (0.99 Æ 0.14 lM) or slightly more potent
than isatin, Figure 5, whereas bulky acyl derivative like
9–10 and its N-acyl derivatives 11–12 brought about a
decline in activity, indicating the importance of third ke-
tone group, which forms the hydrogen bond with Arg 72
and Arg134 of MTB CM. Schiff bases of isatin derivatives
13–14 with 2-amino-
pivaloyl derivative
8
reduced the activity many fold
5-nitrothiazole and 2-amino-6-nitrobenzothiazole made
the compounds completely inactive (>100 lM), suggesting
that the substitution of aromatic bulkier groups at R1
(41.0 Æ 3.6 lM). Replacement of oxygen on isatin at R1
position with semicarbazones and thiosemicarbazones
Chem Biol Drug Des 2014; 83: 498–506
501

Jeankumar et al.
Table 1: Biological activities of isatin derivatives
R1
R2
O
N
R
Cytotoxicityc % inhibition at
Compound
ID
R
R1
R2
M.P (°C)
IC50 (lM)a
1.08 Æ 0.10
MIC (lM)b
50 lM
100 lM
1
H
O
H
H
H
H
H
H
H
H
H
H
H
H
199–200
133–134
86–88
340.13
310.60
285.71
132.28
267.38
28.02
39.53 Æ 0.04
28.1 Æ 0.12
14.06 Æ 0.10
13.93 Æ 0.02
39.89 Æ 0.15
9.77 Æ 0.04
17.31 Æ 0.05
27.81 Æ 0.02
29.97 Æ 0.01
30.07 Æ 0.14
25.91 Æ 0.02
22.99 Æ 0.10
47.10 Æ 0.03
38.6 Æ 0.06
31.6 Æ 0.06
25.80 Æ 0.08
64.90 Æ 0.24
29.09 Æ 0.13
24.62 Æ 0.02
38.57 Æ 0.06
31.71 Æ 0.05
40.36 Æ 0.21
38.6 Æ 0.01
32.31 Æ 0.04
2
CH3–
O
1.96 Æ 0.43
22.70 Æ 3.12
75.12 Æ 5.20
96.56 Æ 5.20
23.30 Æ 2.00
0.99 Æ 0.14
41.00 Æ 3.60
66.90 Æ 5.80
54.09 Æ 2.40
30.50 Æ 2.60
25.00 Æ 3.40
3
C2H5–
O
4
(CH3)2CH–
CH2=CHCH2–
C6H5–
O
76–77
5
O
89–90
6
O
139–141
141–142
157–158
232–234
266–268
247–249
235–237
7
CH3CO–
(CH3)3CCO–
H
O
228.30
125.63
60.96
8
O
9
NH2NHC(S)N=
NH2NHC(O)N=
NH2NHC(S)N=
NH2NHC(O)N=
10
11
12
H
132.28
50.59
CH3CO–
CH3CO–
108.24
O2N
S
13
14
H
H
H
H
191–193
124–126
>100.00
91.24
77.16
0.05 Æ 0.15
23.27 Æ 0.20
36.58 Æ 0.07
N
N
O2N
S
N
>100.00
29.53 Æ 0.07
N
15
16
17
18
H
H
H
H
HON=
H
227–229
208–211
223–225
156–159
98.14 Æ 6.20
77.16
112.10
85.64
25.22 Æ 0.06
0.46 Æ 0.18
25.10 Æ 0.14
26.74 Æ 0.08
42.62 Æ 0.08
32.76 Æ 0.02
32.43 Æ 0.10
31.95 Æ 0.09
O
O
O
C6H5–
37.10 Æ 2.50
95.70
2,3-(Cl)2C6H3–
2,5-(CH3)2C6H3–
36.50
119.20
19
20
21
22
23
H
H
H
H
H
O
O
O
O
O
109–111
133–135
131–133
154–156
146–149
39.90 Æ 2.80
31.90 Æ 2.60
55.70 Æ 1.80
25.60 Æ 1.50
28.80 Æ 1.20
108.22
102.04
66.50
31.61 Æ 0.01
23.32 Æ 0.01
60.58 Æ 0.04
29.97 Æ 0.07
21.22 Æ 0.01
42.56 Æ 0.03
31.18 Æ 0.02
87.10 Æ 0.01
40.98 Æ 0.04
33.27 Æ 0.07
108.69
38.46
502
Chem Biol Drug Des 2014; 83: 498–506

Isatin Derivatives as MTB CM Inhibitors
Table 1: continued
Cytotoxicityc % inhibition at
Compound
ID
R
R1
R2
M.P (°C)
223–225
IC50 (lM)a
1.01 Æ 0.22
MIC (lM)b
50 lM
100 lM
24
H
4-NO2-C6H4CH=
H
23.50
19.97 Æ 0.05
24.17 Æ 0.02
25
H
H
234–236
0.93 Æ 0.10
183.83
18.03 Æ 0.01
25.57 Æ 0.05
C
H
O2N
S
26
27
28
Isoniazid
nd
nd
nd
nd
0.66
0.23
nd
nd
nd
nd
nd
nd
Rifampicin
Ethambutol
nd
nd
15.31
nd, not determined.
aMTB chorismate mutase.
bMinimum inhibitory concentration against Mycobacterium tuberculosis H37Rv.
cRAW 264.7 cell line.
Figure 4: Dose–response curve with error bars (black colour) for the positive control carvacrol and potent inhibitors compound 1, 7, 24
and 25.
Chem Biol Drug Des 2014; 83: 498–506
503

Jeankumar et al.
Figure 5: 2D interaction profile of compounds 1, 7 and 25 with the active site residues. GLH 109 indicates the protonated glutamate
residue. The pink dashed lines representing hydrogen bonding with the side chains of the residues. Amino acids are represented in green
(hydrophobic), blue (polar), red (negatively charged) and purple (positively charged).
position depletes the activity. These substitutions on isatin
ring completely changed the orientation of the compound
at its active site, moving it slightly out of the pocket, fac-
ing towards the solvent. Similarly, 3-(hydroxyimino)indolin-
2-one 15 also showed poor activity. We modified the
phenyl ring of isatin by introducing various aryl groups at
the C-5 position via Suzuki coupling and amino group via
Buchwald–Hartwig cross-coupling reactions. Two of the
5-phenyl-substituted isatins 16 and 18 and two of 5-
amino-substituted isatins 22 and 23 showed moderate
activity. Compounds with substitution at R2 position of
isatin with piperazine derivatives 19–21 were found to be
less active, with activity ranging between 31 and 55 lM,
suggesting the unsuitability of piperazine substitution. This
may be probably attributed to the orientation of these
compounds towards the solvent as observed earlier. Sur-
prisingly, two of the 3-substituted isatin derivatives like 3-
(4-nitrobenzylidene)indolin-2-one 24 and 3-((5-nitrothio-
phen-2-yl)methylene)indolin-2-one 25 showed very potent
activity with compound 25 in lower micromolar range.
These substitutions were found to be suitable as the nitro
group was involved in key interaction with Arg 49 and
Thr 105 (Figure 5). In conclusion, only few substitutions
such as acetyl group on N-1 nitrogen 7 and small arylid-
enes at R1 position 24 and 25 were found to be favour-
able, and their activity supports the same.
synthesized were found to be more potent than isatin as
antitubercular compounds with MIC’s range from 23.5 to
310.60 lM. Seven compounds 6, 9, 11, 17, 21, 23 and 24
inhibited MTB with an MIC <100 lM, and the compound
24 was found to be most potent derivative against MTB
with an MIC of 23.5 lM. The LogP of compound 24 was
also very high (2.68) when compared to the lead molecule
isatin. When compared to first line antitubercular drugs
such as isoniazid (MIC: 0.66 lM), rifampicin (MIC: 0.23 lM)
and ethambutol (MIC: 15.31 lM), compound 24 was found
to be less active.
The safety profile of all the compounds were also
accessed by testing in vitro cytotoxicity against RAW
264.7 cells at 50 and 100 lM concentrations by (4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide
(MTT) assay (36). Mouse macrophage RAW 264.7 cells
were selected for this study, as MTB generally reside in
macrophage cells of humans. Percentage inhibitions of
cells are reported in Table 1. The entire tested com-
pound demonstrated good safety profile with very low
toxicity towards the macrophage and showed good
selectivity index (CC50/MIC), indicating the suitability of
these compounds for further drug development. The
most promising anti-TB compound 24 showed 24.17%
inhibition at 100 lM.
The compounds were further screened for their in vitro
antimycobacterial activity against M. tuberculosis H37Rv
by microplate alamar blue assay method (35). The lead
compound isatin 1, which showed excellent enzyme inhibi-
tion activity in the in vitro enzyme assay, did not show
promising activity in MTB cell line, exhibiting an MIC of
340.13 lM. The lack of translation of strong enzyme inhibi-
tion potency to antitubercular activity can be probably
attributed to the inability of these molecules to penetrate
the mycobacterial cell wall as evident from a lower LogP
value of 0.34. All the twenty-four isatin derivatives
In this study, a structure-based e-pharmacophore model-
ling has been employed to identify small molecule inhibi-
tors of Mycobacterium tuberculosis (MTB) chorismate
mutase (CM) enzyme. Isatin 1 was identified as a poten-
tial lead with an IC50 of 1.08 Æ 0.10 lM. Hit expansion of
compound 1 by chemical synthesis leads to a more
potent inhibitor, compound 3-(4-nitrobenzylidene)indolin-2-
one 24 with promising in vitro activity against MTB CM
enzyme and whole-cell MTB. Structural elements were
identified that provide the basis for further optimization of
the new inhibitors.
504
Chem Biol Drug Des 2014; 83: 498–506

Isatin Derivatives as MTB CM Inhibitors
Reddy C.M., Misra P., Pal M. (2012) AlCl3 induced
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Acknowledgment
This work was supported by a fund from the Aditya Birla
Science & Technology Company Limited Mumbai, India.
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Supporting Information
Additional Supporting Information may be found in the
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Appendix S1 Methodology used for docking, experimen-
tal and spectral details of all the synthesized compounds
and the protocol used for biological evaluation.
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anti-tyrosine kinase activity of 3-(substituted-benzylid-
506
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