The synthesis, biological evaluation and structure-activity relationship of 2-phenylaminomethylene-cyclohexane-1,3-diones as specific anti-tuberculosis agents

Article abstract of DOI:10.1039/c7md00350a

The present study utilised whole cell based phenotypic screening of thousands of diverse small molecules against Mycobacterium tuberculosis H37Rv (M. tuberculosis) and identified the cyclohexane-1,3-dione-based structures 5 and 6 as hits. The selected hit molecules were used for further synthesis and a library of 37 compounds under four families was synthesized for lead generation. Evaluation of the library against M. tuberculosis lead to the identification of three lead antituberculosis agents (37, 39 and 41). The most potential compound, 2-(((2-hydroxyphenyl)amino)methylene)-5,5-dimethylcyclohexane-1,3-dione (39) showed an MIC of 2.5 μg mL^-1, which falls in the range of MICs values found for the known antituberculosis drugs ethambutol, streptomycin and levofloxacin. Additionally, this compound proved to be non-toxic (<20% inhibition at 50 μM concentration) against four human cell lines. Like first line antituberculosis drugs (isoniazid, rifampicin and pyrazinamide) this compound lacks activity against general Gram positive and Gram negative bacteria and even against M. smegmatis; thereby reflecting its highly specific antituberculosis activity.

Full text of DOI:10.1039/c7md00350a

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DOI: 10.1039/C7MD00350A
Synthesis, Biological Evaluation and StructureꢀActivity Relationship of 2ꢀ
Phenylaminomethyleneꢀcyclohexaneꢀ1,3ꢀdiones as Specific Antiꢀtuberculosis Agents
Muzafar Ahmad Rathera, b,#, Ali Mohd Lone c, #, Bisma Teli c,d, Zubair Shanib Bhata, d
,
Paramjeet singhe Mubashir Maqboola, Bashir Ahmad Shairgojrayc, Mohd Jamal Dard,e,
Shajrul Aminb, Syed Khalid Yousufc,d*, Bilal A. Bhatc,d* Zahoor Ahmada,d*
Running title: Phenylaminomethyleneꢀcyclohexaneꢀ1,3ꢀdiones
a Clinical Microbiology and PK/PD Division, CSIRꢀIndian Institute of Integrative Medicine,
Sanatnagar, Srinagar, Indiaꢀ190005
b Department of Biochemistry, University of Kashmir, Hazratbal Srinagar, Indiaꢀ190006
c
Medicinal Chemistry Division, CSIRꢀIndian Institute of Integrative Medicine, Sanatnagar,
Srinagar, Indiaꢀ190005
dAcademy of Scientific & Innovative Research (AcSIR), India
eCancer Pharmacology Division, CSIRꢀIndian Institute of Integrative Medicine, Canal Road,
Jammu, Jammu & Kashmir, Indiaꢀ180001
# equal contribution
*Corresponding authors mailing address:
Medicinal Chemistry Division, Indian Institute of Integrative Medicine, Sanatnagar Srinagar,
India. Fax: +91ꢀ194244133; Tel: +91ꢀ1942431253/55, Eꢀmail: khalidiiim@gmail.com;
bilal@iiim.ac.in
Clinical Microbiology PK/PD/ Laboratory, Indian Institute of Integrative Medicine, Sanatnagar
Srinagar. ZIP: 190005, Tel: +91ꢀ194ꢀ2431253/55, Fax: +91ꢀ1942441331, Cell: +91ꢀ
9906593222, Email: zahoorap@iiim.ac.in

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Abstract
The present study utilised whole cell based phenotypic screening of thousands of diverse
small molecules against Mycobacterium tuberculosis H37Rv (M.tuberculosis) and identified
cyclohexaneꢀ1,3ꢀdione based structures 5 and 6 as hits. The selected hit molecules were used
for further synthesis and a library of 37 compounds under four families was synthesized for
lead generation. Evaluation of the library against M. tuberculosis lead to the identification of
three lead antituberculosis agents (37, 39 and 41). The most potential compound, 2ꢀ(((2ꢀ
hydroxyphenyl)amino)methylene)ꢀ5,5ꢀdimethylcyclohexaneꢀ1,3ꢀdione (39) showed an MIC
of 2.5 µg/mL that falls in the range of MICs values of known antituberculosis drugs
ethambutol, streptomycin and levofloxacin. Additionally, this compound proved to be
nontoxic (< 20% inhibition at 50 ꢁM concentration) against four human cell lines. Like first
line antituberculosis drugs (isoniazid, rifampicin and pyrazinamide) this compound lacks
activity against general gram positive and gram negative bacteria and even against M.
smegmatis; thereby reflecting its highly specific antituberculosis activity.
Keywords: Tuberculosis, Phenotypic screening, Structureꢀactivity relationship, Gram
positive bacteria, Gram negative bacteria, Cyclohexaneꢀ1,3ꢀdiones

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Tuberculosis (TB) continues to be the most deadly infectious disease globally since
prehistoric times.1 In 2015, 10.4 million people fell ill with TB and 1.8 million died from the
disease (including 0.4 million among people with HIV).2 It is estimated that oneꢀthird of the
world’s population harbours Mycobacterium tuberculosis in latent state and is at the risk of
developing active TB and therefore becoming infectious reservoir for the rest.2 The HIV
epidemic, one of the major obstacles to achieve global control of the disease has dramatically
increased the risk of developing active TB and added further complexity to the treatment of
TB.3 Increase in the emergence and global spread of drug resistant forms of M. tuberculosis
isolates is another challenge in curbing TB.2 The above facts and figures of human morbidity
and mortality clearly pin point the failure of current strategies used against TB and are the
real factors underlining the need for development of new antiꢀTB drugs. Furthermore, many
of the drug candidates in the TB drug pipeline are either derivatives of existing drugs or they
target the same cellular processes as the existing drugs in use.4 It is noteworthy that the
chances of the successful development of analogues/derivatives of existing drugs is
significantly reduced due to crossꢀresistance as has been observed with rifamycins and
quinolones.5 Thus, there is the pressing need to develop antiꢀTB agents based on new
chemical entities (NCEs) capable of killing drug sensitive, drug resistant and persistent forms
of M. tuberculosis to counter the TB pandemic. Extensive drug discovery efforts in the past
decade that started with screening of millions of small molecules have identified only a few
promising antituberculosis drug candidates that have either received clinical approval or are
now in advanced clinical trials.6, Diarylquinoline,
Bedaquiline (TMC207)7 (1);
nitroimidazole, PAꢀ8248 (2); nitrodihydroimidazole analogue, Delamanid (OPCꢀ67683)9 (3)
and a diamine, SQꢀ10910 (4) are some of the representative examples (figure 1).
Figure 1. Representative compounds that are either under clinical trials or have
acclaimed clinical use against tuberculosis

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Bedaquiline has recently received approval by the US Food and Drug Administration (USꢀ
FDA) and by the European Medicines Agency (EMA) as a part of combination regimen for
treating pulmonary MDRꢀTB in adults.11 Similarly Delamanid, has also received approval
from European Commission against pulmonary MDRꢀTB in adult patients in whom effective
regimens are not available owing to resistance or tolerability. 12
Keeping in view the need of new antituberculosis drugs and some tangible success of whole
cell based phenotypic screening as an approach in identifying new antituberculosis agents the
presented study utilized the same technique on thousands of small molecules to identify a hit
compound against M. tuberculosis13 A diverse library, consisting of 20,000 compounds
procured from ChemBridge (San Diego, USA) was screened in our Institute to identify the
hit/s
with
significant
antiꢀTB
potential.14
Our
study
identified
2ꢀ(((2ꢀ
hydroxyphenyl)thio)methylene)ꢀ cyclohexaneꢀ1,3ꢀdiones (5 and 6, figure 2) as the two hits.
Structuralꢀactivity relationship guided synthesis was carried out using these hit compounds to
generate a diverse library of molecules that were screened against M. tuberculosis Three
molecules were observed to exhibit significant antituberculosis potential (37, 39 & 41, table
1) and most potent compound 39 was studied further.
Initial screening and synthesis of the active compounds
All compounds were assessed for their potential to inhibit M. tuberculosis H37Rv growth
in Middlebrook 7H9 broth with supplements as described earlier.15 Minimal inhibitory
concentrations (at which no visible growth was observed, MICs) of standard antituberculosis
drugs rifampicin, isoniazid, ethambutol, streptomycin and levofloxacin were 0.078, 0.313,
1.56, 1.25 and 2.5 µg/mL respectively. These results are in well agreement with established
literature.16 Our initial phenotypic screening of small synthetic molecules identified two
compounds (5 and 6, figure 2) as potential antituberculosis hits. Though these compounds
exhibited some activity against M. tuberculosis but this activity was not significant enough to
proceed with them as such for further antituberculosis drug discovery as their MIC values
ranged between 40ꢀ80 µg/mL; which is significantly higher than standard drugs. However
this antiꢀTB activity was a clue to synthesize a diverse library of molecules around this
scaffold in order to find a better lead structure/s for further studies.

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Figure 2. Two hit molecules from phenotypic screening
Initially, we synthesized a selection of 2ꢀalkylꢀaminomethyleneꢀcyclohexaneꢀ1,3ꢀdione based
compounds (family A, table 1, entries 1ꢀ5) by condensation reaction of 1,3ꢀcylohexadiones
and various aliphatic amines in presence of triethylorthoformate (scheme 1). It is to mention
that all these compounds were obtained in excellent yields and their structures were
confirmed by various spectroscopic techniques. When these compounds were evaluated
against M. tuberculosis, it was observed that this synthetic approach totally diminished
activity of parent compounds and MIC values went up and indeed were found to be >80
µg/mL (highest experimental concentration). Therefore, new synthesis was designed to
produce 2ꢀphenylaminomethyleneꢀcyclopentaneꢀ1,3ꢀdione based compounds from 1,3ꢀ
cylopentadione and substitutedꢀanilines (family B, table 1, entries 6ꢀ8) in presence of
triethylorthoformate. Like familyꢀA, compounds of familyꢀB were also screened against M.
tuberculosis and this approach also led to the loss of activity (MIC ˃ 80 µg/mL). These
synthetic approaches indicated that 1,3ꢀcylohexanedione part in parental structures is critical
for activity therefore in next synthetic strategy this was preserved like parental hits 5 and 6.
Variations were made only on the right part of the scaffold with various substituted anilines
in order to produce a series of 2ꢀphenylaminomethyleneꢀcyclohexaneꢀ1,3ꢀdiones (family C,
table 1, entries 9ꢀ34). When this family was screened against M. tuberculosis, it was observed
that most of the compounds also had no activity (MIC > 80 µg/mL) but few of the
compounds from this family exhibited relatively better activity and specially two compounds,
2ꢀ(((2ꢀhydroxyphenyl)amino)methylene)ꢀcyclohexaneꢀ1,3ꢀdione
(37)
and
2ꢀ(((2ꢀ
hydroxyphenyl)amino)methylene)ꢀ5,5ꢀdimethylcyclohexaneꢀ1,3ꢀdione (39) showed excellent
activity with MIC values 5ꢀ10 and 2.5 µg/mL respectively. Indeed MIC of 39 was found in
the MIC ranges of standard antituberculosis drugs ethambutol, levofloxacin and
streptomycin. It is worth to mention that these results demonstrate about 32 fold activity
enhancement of the parent hits owing to the above structure activity guided synthesis. Further
by these observations it is clear that by changing the substituent at Cꢀ5 position of 1,3ꢀ

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cyclohexadione ring changes its MIC values considerably. Based on this fact, we next sought
to synthesize 5ꢀarylꢀ2ꢀphenylaminomethyleneꢀcyclohexaneꢀ1,3ꢀdione based compounds
(family D, table 1, entries 35ꢀ37). It was observed that this structure activity guided synthesis
also resulted in improvement of antituberculosis activity compared to parents hits 5 and 6,
especially
one
of
the
compounds,
2ꢀ(((2ꢀhydroxyphenyl)amino)methylene)ꢀ5ꢀ
phenylcyclohexaneꢀ1,3ꢀdione (41) exhibited significant MIC value of 5ꢀ10 µg/mL (table 1).
Scheme 1. Synthesis of 2ꢀalkyl and arylꢀaminomethyleneꢀcycloalkaneꢀ1,3ꢀdiones
Table 1: Library of 2ꢀalkyl/arylꢀaminomethylene cycloalkaneꢀ 1,3ꢀdiones and their MIC
values against Mycobacterium tuberculosis H37Rv.
Entry
1
Family
Compounda
MIC (µg/ml)
against
M. tuberculosisb
>80
>80
>80
>80
>80
7 (74)c
8 (72)
2
3
4
5
A
2ꢀalkylꢀaminomethyleneꢀ
cyclohexaneꢀ
1,3ꢀdiones
9 (75)
10 (71)
11 (71)

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>80
>80
>80
6
7
8
12 (78)
13 (76)
B
2ꢀphenylaminomethyleneꢀ
cyclopentaneꢀ
1,3ꢀdiones
14 (78)
15 (78)
9
80
80
10
16 (77)
17 (82)
18 (83)
19 (74)
20 (85)
11
12
13
14
15
40ꢀ80
>80
>80
>80
>80
C
2ꢀphenylaminomethyleneꢀ
cyclohexaneꢀ1,3ꢀ
diones
21 (78)
>80
>80
16
17
22 (84)
23 (82)

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>80
>80
18
24 (87)
19
20
25 (79)
>80
>80
26 (76)
27 (76)
21
22
>80
28
(80)
>80
>80
>80
>80
23
24
25
26
29 (70)
30 (74)
31 (70)
32 (72)
33 (79)
34 (82)
35 (80)
>80
>80
>80
>80
27
28
29
30
C
2ꢀphenylaminomethyleneꢀ
cyclohexaneꢀ1,3ꢀ
diones

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36 (80)
31
32
33
5ꢀ10
20
37 (91)
38 (78)
2.5
39 (90)
40d (78)
10ꢀ20
34
35
36
5ꢀ10
41 (74)
42 (72)
43 (75)
D
5ꢀarylꢀ2ꢀphenylaminomethyleneꢀ
cyclohexaneꢀ
1,3ꢀdiones
10ꢀ20
20
37
aAll compounds were synthesized using scheme 1
bMIC of isoniazid, rifampicin, ethambutol, streptomycin and levofloxacin were 0.313, 0.078,
1.25, 1.56 and 2.5 µg/mL respectively
cFigures in parenthesis represent percentage yields after isolation of pure compounds
dCompound 40 is an inseparable mixture of E:Z isomers approximately in 1:1 ratio based on
NMR studies
Structural activity relationship (SAR)
Based on the above observations, we arrived at the SAR depicted in figure 3 below: (a)
Presence of 1,3ꢀcylohexadione motif in left part and oꢀhydroxyl aryl ring in right part of the
scaffold is crucial for antiꢀTB activity (compounds 5, 6, 37ꢀ43). By replacing 1,3ꢀ
cylohexadione motif with 1,3ꢀcyclopentadione motif, a drastic reduction in activity was
observed (compound 12); (b) Presence of 5,5ꢀgemꢀdimethyl substitution at Cꢀ5 in 1,3ꢀ
cylohexadione ring (left part of the scaffold) seems to be optimum for significant MIC values

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(compound 39). Compounds with no substitution at Cꢀ5 position (37) or having any other
substitutions (compounds 38, 41ꢀ43) are comparatively lesser active than compound 39. By
changing the position of gemꢀ dimethyl group to Cꢀ4 position of 1,3ꢀcylohexadione motif also
diminishes the antiꢀTB potential substantially (40); (c) It was interesting to note that
compounds containing Sꢀlinkage (5, 6) exhibit lesser activity than compounds containing ꢀ
NHꢀ linkage (37 and 39) even if the other framework is preserved. This is possibly due to the
hydrogen bonding between ꢀNHꢀ and one of the carbonyl groups of 1,3ꢀdione that imparts
rigidity in the structure; (d) Finally it was also observed that compounds containing aliphatic
right part are not active at all (compounds 7ꢀ11), while as those containing aromatic motif
with proper substitutions are generally active (compounds 33ꢀ39). Among these compounds,
compound containing 5,5ꢀgemꢀdimethyl substitution on 1,2ꢀcyclohexadione ring connected
through ꢀNHꢀlinkage with oꢀhydroxylꢀaryl ring (i,e compound 39) was the most active entity
found in this study. Compounds containing Cꢀ5 aryl substitution (compound 41) also look
promising for future explorations. Based on this study and keeping in consideration the
significant MIC value of compound 39 (2.5 µg/mL), it was studied further towards the
advancement of tuberculosis drug discovery.
Figure 3. SAR of 2ꢀalkyl/arylꢀaminomethylene cycloalkaneꢀ1,3ꢀdiones against M.
tuberculosis
Antibacterial activity spectrum of compound 39
Historically M.tuberculosis has been resistant to antibiotics and that is the reason why
antibiotic development against tuberculosis has been so challenging and indeed bacterial
diseases that appeared much latter than TB do not exist anymore owing to proper antibiotic
development. Surprisingly antibiotics that are active against TB and together constitute
standard first line antituberculosis regimen those include INH, RIF and PZA are either totally

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inactive or are poorly active against other bacteria. Indeed RIF and INH have been shown to
exhibit almost no activity even against a fast growing nonꢀvirulent mycobacterial strain,
Mycobacterium smegmatis i.e. these drugs exhibit 500 to 1000 fold higher MIC against this
strain compared to M.tuberculosis.17 Keeping in view this fact we evaluated test compound
against a panel of gram positive and gram negative bacteria as well as against M. smegmatis
to evaluate its spectrum of activity (table 2) as described earlier.18 Briefly, All the bacterial
strains were challenged with the test compound at the concentrations ranging between 0.25
to 128 µg/ml. Standard drug levofloxacin (LVX) was used as control in a concentration range
between 0.019 to 10 µg/ml. MIC was determined in Muller Hinton Broth and the results were
read after 24 h of incubation at 37 °C. The test compound (39) was not active at all against
most of the gram negative bacterial strains tested (Escherichia coli, Klebsilla pneumonia and
Pseudomonas aeruginosa) as MIC was not observed even up to the highest tested
concentration (128 µg/mL). However test compound exhibited poor activity against E. coli
with MIC 32ꢀ64 µg/mL. Similarly test compound did not exhibit any activity against gram
positive bacterial strains (Staphylococcus aureus, Staphylococcus epidermidis, Bacillus
subtilis, Micrococcus luteus and Enterococcus faecalis) up to highest tested concentration
(128µg/mL). Indeed when test compound was evaluated against a non virulent mycobacterial
strain M. smegmatis it did not also exhibit any activity (MIC ˃ 80 µg/mL) (table 2). Thus
compound 39 like standard antituberculosis drugs INH, RIF and PZA is specifically active
against M.tuberculosis. This finding is also of crucial significance as TB treatment at the best
is even for six months therefore highly specific TB drugs are expected to affect least normal
flora otherwise vice versa observations are expected.
Table 2: Antibacterial Activity of 2ꢀ(((2ꢀhydroxyphenyl)amino)methylene)ꢀ5,5ꢀ
dimethylcyclohexaneꢀ1,3ꢀdione (compound 39) against gram positive bacteria, gram
negative bacteria and non pathogenic mycobacteria.
Type of bacteria
Bacterial strain
MIC (µg/ml)
Test Compound
>128
LVXa
0.078ꢀ0.156
Gram positive
Staphylococcus aureus
(ATCC 25923)
Staphylococcus epidermidis
(ATCC 12228)
Bacillus subtilis
(ATCC 11774)
Micrococcus luteus
(ATCC 10240)
>128
>128
128
0.039ꢀ0.078
0.39
0.078
Enterococcus faecalis
(ATCC 51299)
Escherichia coli
>128
32ꢀ64
0.156
Gram negative
0.078

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(10536)
Klebsilla pneumonia
(ATCC BAAꢀ2146)
Pseudomonas aeruginosa
(ATCC 10145)
>128
>128
>80
0.625ꢀ1.25
0.312ꢀ0.625
0.156
Mycobacteria
M. smegmatis
(ATCC 607)
M. tuberculosis
2.5
2.5
H37Rv (ATCC 25177)
RIF and INH have been reported to show 500 and 1000 fold higher MIC values against M.
smegmatis (100 and 25 µg/mL) compared to those of M. tuberculosis (0.2 and 0.02µg/mL)
respectively.17 aLVX = Levofloxacin, used as a reference antibiotic. Data are from triplicate
sets from three independent experiments
Cytotoxicity evaluation of lead compounds 37-43
One of the critical most considerations for any molecule to proceed successfully during
drug discovery process is its ability to differentiate between bacterial and mammalian cells as
its by this virtue activity and toxicity get seperated.19 We therefore studied the cytotoxic
potential of the lead compounds (37ꢀ43) against a human embryonic kidney cell line 293
(HEKꢀ293) and three cancer cell lines; breast (MCFꢀ7), human colon (HCTꢀ116) and human
prostate (PCꢀ3) cell lines by the MTT cell viability assay at 1ꢀ50 ꢁM.20 MCFꢀ7, HCTꢀ116
and PC3 cells were obtained from National Cancer Institute (NCI), USA, and HEKꢀ293 cells
were purchased from National Centre For Cell Science (NCCS), Pune, India. It was observed
that overall all compounds had little effect on all four cell lines from 1ꢀ40 ꢁM (<20% growth
inhibition approximately). Results at 50 ꢁM of all compounds against four cell lines are
presented in table 3. All the compounds showed ˂ 50% growth inhibition of tested cell lines
at this concentration. With the exception of compound 40 that inhibited the growth of HEKꢀ
293 and HCTꢀ116 by 64.12% and 60.70% respectively and compound 41 inhibited 52.79 %
growth of HEKꢀ293 cells. Compound 39 (best lead) was observed to be nontoxic against all
tested cell lines with <20% inhibition at 50 ꢁM concentration. This compound was further
tested upto 80 ꢁM against HEKꢀ293 (normal human cell line) and only <20% inhibition was
observed. Considering that fact that its MIC is 2.5µg/ml against M.tuberculosis on one hand
and it does not affect human cells even upto 50 ꢁM on the other hand this compound has
significantly suitable therapeutic index and thus merits in vivo evaluation.
Table 3: Cytotoxicity of compounds (37ꢀ43) at 50 ꢁM concentration expressed as
percentage growth inhibition of four human cell lines:
Compound HEKꢀ293
MCFꢀ7
14.52
16.6
HCTꢀ116
10.57
35.70
PCꢀ3
29.51
8.20
28.27
6.08
37
38

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9.03
15.06
20.22
60.70
n.d
42.75
28.65
10.13
41.22
20.36
26.06
18.55
39
40
41
42
43
64.12
52.79
46.44
44.04
n.da
16.52
2.92
23.37
a n.d = not determined
In conclusion, the present study describes synthesis and evaluation of a library of 2ꢀ
phenylaminomethyleneꢀcyclohexaneꢀ1, 3ꢀdiones against M. tuberculosis. This study
identified "2ꢀ(((2ꢀhydroxyphenyl) amino) methylene)ꢀ5, 5ꢀdimethylcyclohexaneꢀ1, 3ꢀdione"
as potential lead molecule whose MIC matches to those of standard antiꢀTB drugs
ethambutol, levofloxacin and streptomycin. Further, its activity is highly selective against M.
tuberculosis compared to other microbes and it proved to be nontoxic on human cell lines.
Structureꢀactivity relationship data is also presented for future studies. To the best of our
knowledge this study introduced a new chemical entity with potential that is worth for
exploration to be a future drug candidate for TB.
Acknowledgments
Authors are highly thankful to Director IIIM, Ram A. Vishwakarma for support and
encouragement. SKY is thankful to DST for INSPIRE faculty award (IFꢀCHꢀ18) and research
grant under budget head GAPꢀ1179. BT and BAB wish to thank SERB for research support
under budget head SR/WOSꢀA/CSꢀ54/2016. This work was supported by funding from
Council of Scientific and Industrial Research Govt. of India (MLPꢀ6010 and BSCꢀ203) and
by Ramalingaswami Fellowship grant from Department of Biotechnology Govt. of India
(GAPꢀ1160) to ZA. IIIM publication No: IIIM/2170/2017
Conflict of Interest
The authors declare no competing interests.
References and notes
1. Ahmad Z, Makaya HN, Grosset JH. History of TB Drug Discovery: Early Evaluation
Studies and Lessons Learnt from Them. Antituberculosis Chemotherapy. Progress in
Respiratory Research. Basel Karger. 2011;40: 1ꢀ8.
2. WHO
Global
tuberculosis
report.
2016.
http://reliefweb.int/sites/reliefweb.int/files/resources/gtbr2016_main_text.pdf.
3. Lalloo UG, Pillay S, Mngqibisa R, et al. British Journal of Respirology. 2016;21:
1166ꢀ1172.

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DOI: 10.1039/C7MD00350A