Synthesis and in vitro evaluation of substituted 3-cinnamoyl-4-hydroxy-pyran-2-one (CHP) in pursuit of new potential antituberculosis agents

Article abstract of DOI:10.1039/c7md00366h

Tuberculosis is an ever-evolving infectious disease that urgently needs new drugs. In the search for new antituberculosis agents, a library of 3-cinnamoyl-4-hydroxy-6-methyl-2H-pyran-2-ones (CHPs) (2a-2y) was synthesized and evaluated against a standard virulent laboratory strain of Mycobacterium tuberculosis H37Rv. Out of 25 compounds, 11, 5, 7 and 2 (2a and 2u) showed least, moderate, good and appreciable activities, respectively, based on minimum inhibitory concentrations (MICs). Both 2a and 2u exhibited an MIC value of 4 μg ml^-1, which was close to those of standard antituberculosis drugs ethambutol, streptomycin and levofloxacin. Neither 2a nor 2u showed any activity against Gram-positive or Gram-negative bacteria and even against non-tuberculous mycobacterium, i.e. Mycobacterium smegmatis. Thus, like the antituberculosis drugs rifampicin, isoniazid and pretomanid, they are highly TB specific. All the pyrone-based chalcones showed no recognizable level of cytotoxicity against normal human kidney cell line (HEK-293) up to 80 μM concentration and 11 exhibited an IC₅₀ ≤ 100 μM (highest tested concentration). On further investigation, both 2a and 2u proved to be nontoxic against four human cell lines but 2a proved to be a better choice as it did not reach IC₅₀ even at 100 μM (highest tested concentration) while the IC₅₀ of 2u was around 80 μM. In conclusion, our results demonstrate that 2a is specific against M. tuberculosis with no appreciable toxicity; its activity matches that of some clinically approved antituberculosis drugs and it therefore merits further evaluation.

Full text of DOI:10.1039/c7md00366h

View Article Online
View Journal
MedChemComm
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: Z. S. Bhat, H. Ul
Lahc, M. A. A. Rather, M. Maqbool, T. Ara, S. K. Yousuf and Z. A. Parry, Med. Chem. Commun., 2017, DOI:
10.1039/C7MD00366H.
This is an Accepted Manuscript, which has been through the
Volume 7 Number 1 January 2016 Pages 1–204
Royal Society of Chemistry peer review process and has been
accepted for publication.
MedChemComm
Accepted Manuscripts are published online shortly after
acceptance, before technical editing, formatting and proof reading.
Broadening the field of opportunity for medicinal chemists
www.rsc.org/medchemcomm
Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. We will replace this Accepted Manuscript with the edited
and formatted Advance Article as soon as it is available.
You can find more information about Accepted Manuscripts in the
author guidelines.
Please note that technical editing may introduce minor changes
to the text and/or graphics, which may alter content. The journal’s
Themed issue: Antibiotic Resistance
ISSN 2040-2503
standard Terms & Conditions and the ethical guidelines, outlined
in our author and reviewer resource centre, still apply. In no
event shall the Royal Society of Chemistry be held responsible
for any errors or omissions in this Accepted Manuscript or any
consequences arising from the use of any information it contains.
rsc.li/medchemcomm

Page 1 of 16
MedChemComm
View Article Online
DOI: 10.1039/C7MD00366H
Synthesis and in vitro evaluation of substituted 3ꢀCinnamoylꢀ4ꢀhydroxyꢀpyranꢀ2ꢀone
(CHP) in pursuit of new potential antituberculosis agents
Zubair Shanib Bhata,b,†, Hafiz Ul Lahc,†, Muzafar Ahmad Rathera, Mubashir Maqboola,
Tabassum Arad, Zahoor Ahmada,b*and Syed Khalid Yousufb,c*
Running title: Pyrones against Tuberculosis
aClinical Microbiology and PK/PD Division, CSIRꢀIndian Institute of Integrative Medicine,
Sanatnagar, Srinagar, Indiaꢀ190005
bAcademy of Scientific and Innovative Research, Indian Institute of Integrative Medicine
(CSIR), Sanatnagar Srinagar, Jammu and Kashmir 190005, India.
cMedicinal Chemistry Division, CSIRꢀIndian Institute of Integrative Medicine, Sanatnagar,
Srinagar, Indiaꢀ190005
dNational Institute of technologyꢀSrinagarꢀ,Jammu, Jammu & Kashmir, Indiaꢀ190006
#equal contribution
*Corresponding authors mailing address:
Medicinal Chemistry Division, Indian Institute of Integrative Medicine, Sanatnagar
Srinagar. ZIP: 190005, Tel:, Fax: , Cell: Email: khalidiiim@gmail.com
Clinical Microbiology and PK/PD/ Laboratory, Indian Institute of Integrative Medicine, Sanatnagar
Srinagar. ZIP: 190005, Tel: +91ꢀ194ꢀ2431253/55, Fax: +91ꢀ1942441331, Cell: +91ꢀ9906593222,
Email: zahoorap@iiim.ac.in

MedChemComm
Page 2 of 16
View Article Online
DOI: 10.1039/C7MD00366H
Abstract
Tuberculosis is an ever evolving infectious disease that demands urgent need for new drugs. In
search for new antituberculosis agents, a library of 3-Cinnamoyl-4-hydroxy-6-methyl-2H-pyran-
2-ones (CHPs) (2aꢀ2y) was synthesised and evaluated against standard virulent laboratory strain
of Mycobacterium tuberculosis H37Rv. Out of 25 compounds 11, 5, 7 and 2 (2a and 2u) showed
least, moderate, good and appreciable activities based on minimum inhibitory concentrations
(MICs). Both 2a and 2u exhibited an MIC value of 4µg/ml that was close to those of standard
antituberculosis drugs ethambutol, streptomycin and levofloxacin. Neither 2a nor 2u showed any
activity either against gram positive or gram negative bacteria and even against non tuberculous
mycobacterium i.e. Mycobacterium smegmatis; thus like antituberculosis drugs rifampicin,
isoniazid and pretomanid they are highly TB specific. All the pyrone based chalcones showed no
recognizable level of cytotoxicity against normal human kidney cell line (HEKꢀ293) up to 80
µM concentration and 11 exhibited an IC50 ≤ 100 µM (highest tested concentration). On further
investigation, both 2a and 2u proved to be non toxic against four human cell lines but 2a proved
to be better choice as it did not reach IC50 even at 100 µM (highest tested concentration) while
IC50 of 2u was around 80 µM. In conclusion our results demonstrate that 2a is specifically
against M.tuberculosis with no appreciable toxicity and its activity matches with few clinically
approved antituberculosis drugs and therefore it merits further evaluation.
Key Words: Pyrone, Chalcones, Cinnamoyl pyrone, Mycobacterium tuberculosis, Tuberculosis
drug discovery, Structure activity relationship

Page 3 of 16
MedChemComm
View Article Online
DOI: 10.1039/C7MD00366H
Tuberculosis (TB) is an old but ever evolving infectious disease that still poses a huge threat to
the human global population1. About one third of the world’s population is harbouring the
causative agent Mycobacterium tuberculosis (M.tuberculosis) as asymptomatic latent TB
infection (LTI)2. The chances of reactivation to active TB disease are higher in HIV infected and
other immune compromised patients3. HIVꢀTB coꢀinfection is a deadly setback as multitude of
complications makes the concurrent treatment of HIVꢀTB more complicated. These include
drugꢀdrug interactions4, overlapping toxicity5, nonꢀadherence6 and TBꢀassociated immune
reconstitution inflammatory syndrome (IRIS)5. Another major challenge is the drug resistance.
Although current firstꢀline antiꢀTB drug regimen can achieve more than 95% efficacy, this is
often reduced owing to the emergence of drug resistant strains of M.tuberculosis7. This demands
urgent need for new drugs8. Considering the failure rates during discovery phase and clinical
trials, the search of new compounds with low cytotoxicity and better efficacy is a daunting
scientific challenge9. Therefore it seems intellectually wise to take forward the core scaffolds that
have already acclaimed some clinical relevance in humans.
In this direction, pyran-2-one (2P) represents the core structural subunit of a number of
biologically significant natural and synthetic compounds10. On one hand, pyran-2-one based
scaffolds have received FDA approval against HIV and on the other hand have displayed
appreciable level of antiꢀTB activity. (+)Calanolide A isolated from Calophyllum lanigerum11 is
a first pyran-2-one based antiꢀHIV agent that significantly inhibited the growth of M.tuberculosis
H37Rv12. In particular, 4-hydroxy-pyran-2-one (4ꢀHP) represents the active component of
naturally isolated antiꢀTB agents like (Pseudopyronine B13 and Myxopyronin14) and clinically
approved antiꢀHIV agent like Tipranavir15 (Fig. 1).
H
OH
O
O
O
O
H
O
N
O
O
Pseudopyronine B
O
Myxopyronin (Myx)
CH3
CH3
O
H3C
CF3
OH
O
H
N
S
O
O
O
O
O
O
H3C
OH
Tipranavir
CH3
(+)-Calanolide A
Fig. 1: 2ꢀPyrone based antiꢀtuberculosis and antiꢀHIV agents.

MedChemComm
Page 4 of 16
View Article Online
DOI: 10.1039/C7MD00366H
Another class of compounds that are of great interest to medicinal chemists are chalcones (1,3-
diaryl-2-propene-1-ones) due to their simple chemistry, ease of synthesis and broad spectrum of
pharmacological activities against major human diseases like cancer16, inflammation17, malaria18,
HIV19–21 and TB22–28.The antiꢀTB activity of chalcones is attributed to their lipophilic nature that
allows them to easily penetrate the mycobacterial cell wall 29,30
.
Interestingly, a class of Cinnamoyl pyrone (CP) based compounds exists that contains both
pyrone moiety (4ꢀHP scaffold) as aromatic ring A and cinnamoyl moiety as ring B, referred to as
3-Cinnamoyl-4-hydroxy-6-methyl-2H-pyran-2-ones (CHPs). Till date, the antiꢀHIV31 and
anticancer32 potential of CHPs is documented but no report about their antiꢀtuberculosis potential
exists so far. Considering the fact that both pyrones and chalcones have shown some antiꢀTB
potential and have attained some clinical relevance on one hand and the need for new
antituberculosis drug on the other hand this study was aimed at synthesising CHPs and exploiting
them for TB drug discovery. To the best of our knowledge the results of this study show for the
first time promising antituberculosis potential of CHPs against M.tuberculosis.
A general synthesis of 3ꢀcinnamoylꢀ4ꢀhydroxyꢀ6ꢀmethylꢀ2Hꢀpyranꢀ2ꢀone derivatives 2aꢀ2y is
exhibited in Scheme 1.
OH
O
OH
O
O
O
DCC (1 equiv.)
DMAP (0.25 equiv)
+
CH3COOH
Toluene
reflux
O
4ꢀhydroxyꢀ6ꢀmethylꢀpyranꢀ2ꢀone
3ꢀacetylꢀ4ꢀhydroxyꢀ6ꢀmethylꢀpyranꢀ2ꢀone
(1)
(2)
OH
O
Piperidine
Ethanol
R
RCHO
2
+
Aldehyde
O
O
Cinnamoyl hydroxy pyrones
(2aꢀy)
Scheme 1: Synthesis of 3ꢀcinnamoylꢀ4ꢀhydroxyꢀ6ꢀmethylꢀ2Hꢀpyranꢀ2ꢀone derivatives.
3ꢀacetylꢀ4ꢀhydroxyꢀ6ꢀmethylꢀpyranꢀ2ꢀone (2) was synthesized from 4ꢀhydroxyꢀ6ꢀmethylꢀpyranꢀ
2ꢀone (1) using acetic acid, DCC and DMAP in toluene under reflux in almost quantitative yield.
The targeted cinnamoyl pyrones (2aꢀy) were synthesized by reacting 3ꢀacetylꢀ4ꢀhydroxyꢀ6ꢀ
methylꢀpyranꢀ2ꢀone (2) with specific benzaldehyde in ethanol using catalytic piperidine under
reflux in good to excellent yield (Table 1). Structure of all the synthesized compounds was

Page 5 of 16
MedChemComm
View Article Online
DOI: 10.1039/C7MD00366H
characterized by using 1H and 13C NMR. For example the 1H NMR spectrum of 2a exhibited two
doublet signals at 8.30 ppm (d, J = 15.7 Hz, 1H) and 7.94 ppm (d, J = 15.8 Hz, 1H),
corresponding to β and αꢀH of enone C=C respectively. The peaks of 7.67 ppm (dd, J = 6.5, 2.8
Hz, 2H), 7.39 ppm (dd, J = 11.3, 7.7 Hz, 3H) are attributed to five aromatic benzene CꢀH. The
proton of C5ꢀH of pyrone ring appeared as singlet at 5.94 ppm. Similarly C6ꢀmethyl group
showed a broad singlet at 2.26 ppm.
Table 1: Library α-pyrone based substituted 3-cinnamoyl-4-hydroxy-6-methyl-2-pyrone derivatives (2a-
2y) and their MIC values against Mycobacterium tuberculosis H37Rv and M.smegmatis.
OH
O
R
O
O
Cinnamoyl hydroxy pyrones
(2aꢀy)
S.No
Code
%Yield
Structure
(R)
M.tuberculosis*
M.Smegmatis**
MIC (µg/ml)
MIC (µg/ml)
1
2
2a
85
4
>128
>128
2b
81
79
80
8
CH3
3
4
2c
256
64
>128
>128
OC2H5
2d
OH
O
5
6
2e
2f
78
82
16-32
32-64
>128
>128
S
OCH3
7
8
2g
2h
77
80
32-64
>128
>128
CF3
8
CF3
9
2i
2j
81
83
256
256
>128
>128
10
CF3

MedChemComm
Page 6 of 16
View Article Online
DOI: 10.1039/C7MD00366H
OCH3
11
12
2k
2l
79
78
256
4-8
>128
>128
CF3
S
Br
Br
13
14
2m
2n
80
77
4-8
>128
>128
8-16
Br
S.No
15
Code
2o
%Yield
Structure
(R)
M.tuberculosis*
M.Smegmatis**
MIC (µg/ml)
MIC (µg/ml)
81
82
8-16
>128
OCH3
16
2p
32
>128
OCH3
17
18
2q
2r
79
82
32
16
>128
>128
Cl
OH
19
2s
82
8-16
>128
OH
OC2H5
OCH3
20
21
2t
78
83
8-16
>128
>128
2u
4
22
2v
80
4-8
>128
>128
OCH3
OCH3
23
24
2w
2x
81
83
8
8
OCH3
OCH3
OCH3
>128
OCH3
OCH3

Page 7 of 16
MedChemComm
View Article Online
DOI: 10.1039/C7MD00366H
25
2y
80
32
>128
OH
OCH3
*Antituberculosis drugs RIF, INH, EMB, STR and LVX showed MICs values of 0.078, 0.312,
2.5, 1.25 and 2.5µg/mL µg/ml respectively against M. tuberculosis. ** LVX served as positive
control against M. smegmatis as RIF and INH have been reported to show 500 and 1000 fold
higher MIC values against M. smegmatis (100 and 25 mg/ml) compared to those of M.
tuberculosis (0.2 and 0.02µg/mL) respectively and therefore can’t act as controls. LVX exhibited
an MIC of 2.5 µg/ml against M. smegmatis.
Antimicrobial susceptibility testing (AST) was done by determining minimum inhibitory
concentration (MIC) of antimicrobial agents and test compounds by 7H9 broth microdilution
assay as discussed previously with slight modifications33. Briefly, Middlebrook 7H9 broth with
10% ADC (albumin, dextrose and catalase) containing the CHP based test compounds in
concentration range (0.25ꢀ128.0 µg/ml) were added in duplicate in 96 well titer plates (Nest
Biotech, China). Each well was inoculated with 50 µl of appropriately diluted mid log phase
M.tuberculosis H37Rv culture corresponding to ~1×105 CFU/ml. Isoniazid (INH), Rifampicin
(RIF), ethambutol (EMB), streptomycin (STR) and levofloxacin (LVX) were used as positive
controls and drug free broth served as negative control respectively.
Original CFU were verified by plating serial 10 fold dilutions of the inoculum onto Middlebrook
7H11 agar plates that were incubated at 37°C and read after four weeks. The minimum inhibitory
concentration (MIC) was taken as the lowest concentration (µg/ml) of an antibiotic that inhibits
the visible growth after two weeks of incubation. MIC against M. smegmatis was done as above
but reading was taken only after 72 hrs owing to its fast growing nature.
MICs of standard antituberculosis drugs RIF, INH, EMB, STR and LVX were found to be 0.078,
0.312, 2.5, 1.25 and 2.5µg/mL respectively against M. tuberculosis H37Rv. MIC results of all the
3ꢀcinnamoylꢀ4ꢀhydroxyꢀ6ꢀmethylꢀ2Hꢀpyranꢀ2ꢀone (CHP’s) derivatives (2aꢀ2y) against M.
tuberculosis are presented in Table 1. Among 25 derivatives 11 did not show any attractive
activity i.e. their MIC values were ≥ 32 µg/mL. Five compounds (2n, 2o, 2r, 2s and 2t) showed
moderate antituberculosis activity with MIC values ranging from 8ꢀ16 µg/ml (Table 1). Seven
compounds (2b, 2h, 2l, 2m, 2v, 2w and 2x) showed good antiꢀTB potential reflected by their
MIC values ranging from 4ꢀ8 µg/ml (Table 1). Two compounds (2a and 2u) displayed excellent
antituberculosis activity against M.tuberculosis with an MIC value of 4 µg/ml (Table 1). This

MedChemComm
Page 8 of 16
View Article Online
DOI: 10.1039/C7MD00366H
MIC value is close to those of standard antituberculosis drugs EMB, STR and LVX, thereby
reflecting promising antituberculosis potential of these two compounds that strongly merits
further evaluation. This significant antituberculosis potential can be due to 2ꢀpyrone based
polyketides which represent a diverse class of secondary metabolites with crucial roles in
M.tuberculosis. Indeed a putative chalcone synthase (PKS11) and a triꢀketide as well as tetraꢀ
ketide pyrone synthase (PKS18) in M.tuberculosis have been characterized that synthesize a
unique cyclic 5ꢀmethylꢀ6ꢀalkylꢀ4ꢀhydroxyꢀ2ꢀpyrone (MAHP) via various intermediates. These
small polyketide molecules are finally incorporated in M.tuberculosis cell wall where they are
perceived to regulate permeability 34ꢀ35. It is one of the ways by which pyrone based molecules
attack M. tuberculosis. Additionally pyrones have been shown to inhibit M. tuberculosis
translation like rifampicin but even in rifampicin resistant strains also. So there are experimental
indications how these molecules might work against M. tuberculosis. Further reasons for this
antiꢀTB activity include their chalcones side which owing to its lipophilic nature easily
penetrates mycobacterial cell wall and hence causes the damage 29ꢀ30. It is to be noted none of
the molecule exhibit appreciable activity against M. smegmatis (Table 1).
Based on our experimental results, the structureꢀactivity relationship (SAR) of this class of antiꢀ
TB agents with respect to benzene ring can be summarised as follows. 2a being the most potent
molecule, it can be concluded that substitution of any sort in the benzene ring leads to decrease
in activity. Though –OC2H5 group is well tolerated but the position of the same is very important
since its presence at para position proved to be detrimental for activity. Though the presence of
halogens and hydroxyl groups is tolerated, electron withdrawing groups like ꢀCF3 totally proved
unfavourable for activity. In general presence of alkyl and alkoxy groups also decrease the
potency of the molecules. Additionally replacement of phenyl ring with heterocyclic rings like
furan and thiophene also decreased antituberculosis activity.
Since first line antituberculosis drugs are specifically active against M.tuberculosis only and
exhibit very poor activity against other microbes, we therefore evaluated the most two potant
CHPs (2a and 2u) against four Gram positive bacteria (Staphylococcus aureus (ATCC 25923),
Staphylococcus epidermidis (ATCC 12228), Micrococcus luteus (ATCC 10240), and Bacillus
subtilis (ATCC 11774)) and four Gram negative (Escherichia coli (ATCC 10536), Enterococcus
faecalis (ATCC 51299), Pseudomonas aeruginosa (ATCC 10145), Klebsilla pneumonia (ATCC
BAAꢀ2146)). Strains were revived according to written instructions, platted onto Muller Hinton

Page 9 of 16
MedChemComm
View Article Online
DOI: 10.1039/C7MD00366H
Agar (MHA) and incubated at 37 °C for 24 h. The MIC was determined by previously described
method with minor modification using MH broth and reading was taken after 24 h of incubation
at 37 °C36. The second line antiꢀTB drug levofloxacin (LVX) served as positive control owing to
its broad spectrum of activity. LVX exhibited MICs values of 0.078ꢀ0.156, 0.078, 0.039ꢀ0.078,
0.39, 0.078, 0.312ꢀ0.625, 0.156 and 0.625ꢀ1.25µg/ml against S. aureus, B. subtilis, M. luteus, S.
epidermidis, E. coli, P. Aeruginosa, E. faecalis and K. Pneumonia respectively; these MIC
values of LVX are well in agreement with reported ones (Table 2). MIC values of 2a and 2u
observed in this study are presented in Table 2. None of them exhibited any significant activity
against gram positive bacteria: S. aureus and B. subtilis, S. epidermidis and M. luteus gram
negative bacteria: E. coli, E. faecalis, K. pneumonia and P. Aeruginosa (Table 2). Full visual
turbidity in 96ꢀwell plate was observed even up to the highest tested concentration i.e. 128 µg/ml
for all compounds against each bacteria. Our results clearly show that 2a and 2u exhibit unique
activity specifically against M.tuberculosis. This observation is of great significance for
developing CHP based antituberculosis drug as it is less likely to interfere with normal human
flora.
Table 2: Activity of lead compounds (2a and 2u) against Gram negative and Gram positive
bacteria
Type of
bacteria
Bacterial
strain
MIC (µg/ml)
2a
2u
LVX a
0.078ꢀ0.156
0.039ꢀ0.078
0.078
S. aureus
S. epidermidis
M. luteus
B. subtilis
E.coli
E. faecalis
P. aeruginosa
K. pneumonia
>128
>128
>128
>128
>128
>128
>128
128
>128
>128
>128
>128
>128
>128
>128
128
Gram positive
Gram negative
0.39
0.078
0.156
0.312ꢀ0.625
0.625ꢀ1.25
a LVX means standard broad spectrum antibacterial as well as antiꢀTB drug that served as positive control
Among the critical factors that allow any molecule to proceed successfully during drug discovery
process is its selective toxicity to words pathogenic microbes and non/less toxicity on
mammalian cells as it’s by this character activity and toxicity get separated and therapeutic index
becomes acceptable. We therefore evaluated cytotoxicity of CHP based derivatives in
concentrations ranging from 1 to 80 µM (1µM, 20µM, 40µM and 80µM) against normal human

MedChemComm
Page 10 of 16
View Article Online
DOI: 10.1039/C7MD00366H
kidney (HEKꢀ293) cell line which was purchased from National Centre for Cell Science
(NCCS), Pune, India. This was done on the basis of percentage growth inhibition assessed by
MTT assay. Over all compounds showed lesser growth inhibition of HEKꢀ293 cells up to 80 µM
concentration and thus CHPs were found to be non toxic at concentrations that are usually high
end concentrations for such studies. We therefore evaluated all compounds at much higher
concentration i.e. at 100 µM. 11 compounds (2x, 2w, 2v, 2u, 2q, 2p, 2n, 2j, 2k, 2l, and 2m)
inhibited HEK cell growth by 50 or >50 % at this highest tested concentration and thus exhibited
an IC50 value of 100 µM. Five compounds (2b, 2i, 2r, 2t and 2y) showed 45ꢀ <50% inhibition
at 100 µM, thus though their IC50 is > 100 µM but will be close to this value. Nine compounds
did not even inhibit 45% growth at highest experimental concentration and thus their 1C50 is
clearly > 100 µM, demonstrating them to be safe for human cells.
Toxicity of the two most potent compounds with respect to antituberculosis activity 2a and 2u
(MIC = 4 µg/ml) was further evaluated at a concentration range (0.0ꢀ 100 µM) against four
human cell lines those include HEKꢀ293, breast cancer (MCFꢀ7), colon cancer (HCTꢀ116) and
prostate cancer (PCꢀ3) by double dilution method using MTT cell viability assay. MCFꢀ7, HCTꢀ
116 and PC3 cells were obtained from National Cancer Institute (NCI), USA. The results of this
study as mean± standard deviation of percentage growth inhibition are presented in Figure 2 iꢀ
iv. Both these compounds 2a and 2u showed some dose dependent growth inhibition response
against all cell lines (Figure 2 iꢀiv). 2a at the highest experimental concentration inhibited <
50% (38ꢀ44%) growth of three cell lines HEKꢀ293, MCFꢀ7 and PC3; inhibited 50% of growth of
HCTꢀ116 cell line at the same concentration (Figure 2 iꢀiv). In contrast 2u inhibited 67ꢀ70%
growth of all the cell lines tested at the same concentration and its IC50 appeared to be 80 µM
against all cell lines (Figure 2 iꢀiv). Therefore 2a displayed crystal clear advantage over 2u with
respect to toxicity profile. Further for 2a MIC is 4 µg/ml against M.tuberculosis and only 40ꢀ
50% inhibition was observed at 100 µM (25.6µg/ml); this demonstrates its broad therapeutic
window. From antituberculosis activity to cytotoxic studies, it is crystal clear that compound 2a
is the choice for future studies.

Page 11 of 16
MedChemComm
View Article Online
DOI: 10.1039/C7MD00366H
Figure 2: Cytotoxicity profile of 3ꢀcinnamoylꢀ4ꢀhydroxyꢀ6ꢀmethylꢀ2Hꢀpyranꢀ2ꢀones (2a and
2u) expressed as percentage growth inhibition.
MCF-7
HEK-293
80
80
2a
2a
60
2u
60
2u
40
40
20
20
0
0
Concentration ꢀM
Concentration ꢀM
i.
ii.
HCT-116
PC-3
80
60
40
20
0
80
60
40
20
0
2a
2u
2a
2u
Concentration ꢀM
Concentration ꢀM
.
ii.
iv.
i) normal human embryonic kidney (HEKꢀ293); ii) breast cancer (MFCꢀ17); iii) colon cancer
(HCTꢀ116) and iv) prostate cancer (PC3) cell. Values are mean ± SD of three sets
Conclusion
Out of 25 compounds subjected to antimycobacterial susceptibility testing, two compounds
displayed potent antiꢀTB activity (2a and 2u) that matches to those of few known
antituberculosis drugs. 2a and 2u were found to be ineffective against a panel of gramꢀpositive,
gramꢀnegative and even against M. smegmatis, reflecting thereby their specific antituberculosis
activity as reported for some antituberculosis drugs. Further cytotoxicity data favoured
significantly 2a over 2u. The structure activity relationship reveals that substitution of any sort in

MedChemComm
Page 12 of 16
View Article Online
DOI: 10.1039/C7MD00366H
the benzene ring leads to decrease in activity.Undoubtly therefore 2a merits further evaluation
with respect to its antiꢀTB potential.
Acknowledgements
Authors are highly thankful to Director IIIM, Dr. Ram A Vishwakarma for unconditional
support. DSTꢀIndia is acknowledged for funding under INSPIRE faculty programme (project
GAPꢀ1179). Thanks are due to UGCꢀGOI for Fellowship and Academy of Scientific and
Innovative Research (AcSIR) for doctoral programme registration to Zubair Shanib Bhat. 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/2160/2017
Conflict of Interest: All authors declare that there is no conflict of interest.
Ethical approval: This article does not contain any study involving animals

Page 13 of 16
MedChemComm
View Article Online
DOI: 10.1039/C7MD00366H
References
1
2
3
A. Zumla, M. Raviglione, R. Hafner and C. F. von Reyn, N. Engl. J. Med., 2013, 368,
745–55.
E. W. Campion, H. Getahun, A. Matteelli, R. E. Chaisson and M. Raviglione, N. Engl. J.
Med., 2015, 372, 2127–2135.
J. Bruchfeld, M. CorreiaꢀNeves and G. Källenius, Cold Spring Harb. Perspect. Med.,
2015, 5, a017871.
4
5
J. Mannu, P. Jenardhanan and P. P. Mathur, Med. Chem. Res., 2014, 23, 905–917.
E. M. Shankar, R. Vignesh, R. Ellegård, M. Barathan, Y. K. Chong, M. K. Bador, D. V.
Rukumani, N. S. Sabet, A. Kamarulzaman, V. Velu and M. Larsson, Pathog. Dis., 2014,
70, 110–118.
6
P. Naidoo, K. Peltzer, J. Louw, G. Matseke, G. McHunu and B. Tutshana, BMC Public
Health, 2013, 13, 396.
7
8
R. J. O’Brien and P. P. Nunn, Am. J. Respir. Crit. Care Med., 2001, 163, 1055–1058.
World Health Organisation, WHO | Multidrugꢀresistant tuberculosis (MDRꢀTB),
http://www.who.int/tb/challenges/mdr/en/.
9
A. Koul, E. Arnoult, N. Lounis, J. Guillemont and K. Andries, Nature, 2011, 469, 483–90.
10
Z. S. Bhat, M. A. Rather, M. Maqbool, H. U. Lah, S. K. Yousuf and Z. Ahmad, Biomed.
Pharmacother., 2017, 91, 265–277.
11
Y. Kashman, K. R. Gustafson, R. W. Fuller, J. H. Cardellina, J. B. McMahon, M. J.
Currens, R. W. Buckheit, S. H. Hughes, G. M. Cragg and M. R. Boyd, J. Med. Chem.,
1992, 35, 2735–2743.
12
13
14
Z. Q. Xu, W. W. Barrow, W. J. Suling, L. Westbrook, E. Barrow, Y. M. Lin and M. T.
Flavin, Bioorganic Med. Chem., 2004, 12, 1199–1207.
A. C. Giddens, L. Nielsen, H. I. Boshoff, D. Tasdemir, R. Perozzo, M. Kaiser, F. Wang, J.
C. Sacchettini and B. R. Copp, Tetrahedron, 2008, 64, 1242–1249.
H. Irschik, K. Gerth, G. Höfle, W. Kohl and H. Reichenbach, J. Antibiot. (Tokyo)., 1983,
36, 1651–1658.
15
16
17
C. Flexner, G. Bate and P. Kirkpatrick, Nat. Rev. Drug Discov., 2005, 4, 955–956.
D. K. ar Mahapatra, S. K. umar Bharti and V. Asati, Eur. J. Med. Chem., 2015, 98.
S. Bano, K. Javed, S. Ahmad, I. G. Rathish, S. Singh, M. Chaitanya, K. M. Arunasree and
M. S. Alam, Eur. J. Med. Chem., 2013, 65, 51–59.
18
B. Insuasty, J. Ramírez, D. Becerra, C. Echeverry, J. Quiroga, R. Abonia, S. M. Robledo,

MedChemComm
Page 14 of 16
View Article Online
DOI: 10.1039/C7MD00366H
I. D. Vélez, Y. Upegui, J. A. Muñoz, V. Ospina, M. Nogueras and J. Cobo, Eur. J. Med.
Chem., 2015, 93, 401–413.
19
20
21
H. Sharma, S. Patil, T. W. Sanchez, N. Neamati, R. F. Schinazi and J. K. Buolamwini,
Bioorganic Med. Chem., 2011, 19, 2030–2045.
A. Hameed, M. I. Abdullah, E. Ahmed, A. Sharif, A. Irfan and S. Masood, Bioorg. Chem.,
2016, 65, 175–182.
A. L. Cole, S. Hossain, A. M. Cole and O. Phanstiel, Bioorganic Med. Chem., 2016, 24,
2768–2776.
22
23
F. Macaev, Med. Chem. (Los. Angeles)., 2014, 4, 487–493.
T. L. B. Ventura, S. D. Calixto, B. De Azevedo AbrahimꢀVieira, A. M. T. De Souza, M.
V. P. Mello, C. R. Rodrigues, L. S. De Mariz E Miranda, R. O. M. A. De Souza, I. C. R.
Leal, E. B. Lasunskaia and M. F. Muzitano, Molecules, 2015, 20, 8072–8093.
24
25
B. R. Copp, Nat. Prod. Rep., 2003, 20, 535–557.
A. Suksamrarn, A. Chotipong, T. Suavansri, S. Boongird, P. Timsuksai, S. Vimuttipong
and A. Chuaynugul, Arch. Pharm. Res., 2004, 27, 507–511.
26
P. M. Sivakumar, S. P. Seenivasan, V. Kumar and M. Doble, Bioorganic Med. Chem.
Lett., 2007, 17, 1695–1700.
27
28
D. Patel, P. Kumari and N. B. Patel, Med. Chem. Res., 2013, 22, 726–744.
P. M. Sivakumarl, V. Kumar, S. P. Seenivasan, Mohanapriya and M. Doble, Adv. Biomed.
Res., Proc. 7th WSEAS Int. Conf. Math. Biol. Ecol. (MABE ’10), Proc. Int. Conf. Med.
Physiol. (PHYSIOL. ’10), Proc. Int. Conf. Biochem. Med. Chem. (BIOMEDCH ’10), 2010,
168–172.
29
30
31
P. J. Brennan and H. Nikaido, Annu. Rev. Biochem., 1995, 64, 29–63.
J. A. Arnott and S. L. Planey, Expert Opin. Drug Discov, 2012, 7, 863–875.
K. Ramkumar, K. V. Tambov, R. Gundla, A. V. Manaev, V. Yarovenko, V. F. Traven and
N. Neamati, Bioorganic Med. Chem., 2008, 16, 8988–8998.
32
33
34
Q. Y. Lan, Q. L. Liu, J. Cai and A. W. Liu, Int. J. Clin. Exp. Pathol., 2015, 8, 155–163.
Z. Ahmad, S. Tyagi, A. Minkowski, et al. Indian J. Med. Res., 2012,136, 808ꢀ814.
P. Saxena, G. Yadav, D. Mohanty, R. S. Gokhale, J Biol Chem., 2003; 278, 44780–90.

Page 15 of 16
MedChemComm
View Article Online
DOI: 10.1039/C7MD00366H
35
36
K. Gokulan, S. E. O’Leary, W. K. Russell, et al., J Biol Chem., 2013; 288: 16484–94.
M. A. Rather, A. M. Lone, B. Teli, Z. S. Bhat, P. Singh, M. Maqbool, B. A. Shairgojray, J.
M. Dar, S. Amin, K. Y. Syed, and Z. Ahmad,
Press.DOI:10.1039/c7md00350a
MedChemComm., 2017, In

MedChemComm
Page 16 of 16
View Article Online
DOI: 10.1039/C7MD00366H
Graphical Abstract:
OH
O
O
CHO
OH
O
O
O
Piperidine
Ethanol
+
R
O
R
3-acetyl-4-hydroxy-6-
methyl-pyran-2-one
(2)
Aldehyde
Cinnamoyl hydroxy pyrones
(2a-y, 70-85%)
Easy to synthsise and scale up
MIC ranging from 4-256 g/mL
µ
Highly specific against M.tuberculosis
Non-toxic to normal and cancerous
human cell line