Inhibition of mycobacterial growth by plumbagin derivatives

Article abstract of DOI:10.1111/j.1747-0285.2010.00987.x

Electron transport and respiratory pathways are active in both latent and rapidly growing mycobacteria and remain conserved in all mycobacterial species. In mycobacteria, menaquinone is the sole electron carrier responsible for electron transport. Menaquinone biosynthesis pathway is found to be essential for the growth of mycobacteria. Structural analogs of the substrate or product of this pathway are found to be inhibitory for the growth of Mycobacterium smegmatis and M. tuberculosis. Several plumbagin [5-hydroxy-2-methyl-1, 4-naphthaquinone] derivatives have been analyzed for their inhibitory effects of which butyrate plumbagin was found to be most effective on M. smegmatis mc ²155, whereas crotonate plumbagin showed greater activity on M. tuberculosis H37Rv. Effect on electron transport and respiration was demonstrated by butyrate plumbagin inhibiting oxygen consumption in M. smegmatis. Structural modifications of these molecules can further be improved upon to generate new molecules against mycobacteria.

Full text of DOI:10.1111/j.1747-0285.2010.00987.x

ª 2010 John Wiley & Sons A/S
doi: 10.1111/j.1747-0285.2010.00987.x
Chem Biol Drug Des 2010; 76: 34–42
Research Article
Inhibition of Mycobacterial Growth by Plumbagin
Derivatives
Ritta Mathew1,*,†, Anil K. Kruthiventi1,†
Jalli V. Prasad1, Sadula P. Kumar1,
,
essential pathways, showing details of structural and metabolic
requirements for mycobacteria, were identified (2). These genes are
found to be essential for the normal growth of this organism and
provide useful information in the development of more specific anti-
microbial inhibitors. Among these, the pathways for electron trans-
port and respiration are involved in both latent and rapidly growing
mycobacteria (3). The respiratory system of mycobacteria has both
aerobic and anaerobic components (1), enabling them to survive in
the unfavorable environment of the host. The recent study on the
respiratory chain of mycobacteria as a rich source of antimycobacte-
rial drug target (4–8) is the driving force behind this work.
Garlapati Srinu1 and Dipankar Chatterji1,2
1Institute of Life Sciences, University of Hyderabad campus,
Gachibowli, Hyderabad-500 046, India
2Molecular biophysics unit, Indian Institute of Science, Bangalore
560 012, India
*Corresponding author: Ritta Mathew, mathewritta@gmail.com
Both authors have equal contribution.
Electron transport and respiratory pathways are
active in both latent and rapidly growing myco-
bacteria and remain conserved in all mycobacte-
rial species. In mycobacteria, menaquinone is the
sole electron carrier responsible for electron
transport. Menaquinone biosynthesis pathway is
found to be essential for the growth of mycobac-
teria. Structural analogs of the substrate or
product of this pathway are found to be inhibitory
for the growth of Mycobacterium smegmatis and
M. tuberculosis. Several plumbagin [5-hydroxy-
2-methyl-1, 4-naphthaquinone] derivatives have
been analyzed for their inhibitory effects of which
butyrate plumbagin was found to be most effec-
tive on M. smegmatis mc2155, whereas crotonate
plumbagin showed greater activity on M. tubercu-
losis H37Rv. Effect on electron transport and res-
piration was demonstrated by butyrate plumbagin
inhibiting oxygen consumption in M. smegmatis.
Structural modifications of these molecules can
further be improved upon to generate new mole-
cules against mycobacteria.
Quinones shuttle electrons between the membrane-bound protein
moieties in the electron transport chain. In mammalian cells, the
membrane-soluble quinone is ubiquinone (coenzyme Q), whereas in
prokaryotes, in addition to or instead of ubiquinone, bacteria utilize
menaquinone (vitamin K2) (9). Menaquinone is the sole quinone in
mycobacteria (10–13). In humans, although vitamin K2 plays an
important role in the blood-clotting mechanism (14), the main source
of it is diet or intestinal bacteria (15). Hence, menaquinone biosyn-
thesis pathway is considered as an attractive target for developing
novel antimycobacterial inhibitors (2,16–19) as it is considered as an
essential pathway for the viability of mycobacteria (20).
Menaquinone biosynthetic pathway has been extensively studied in
E. coli (10,21–24). The genome of M. tuberculosis contains homo-
logs of most of the E. coli men genes (2), whereas some of the
genes in ubiquinone pathway are absent. The proposed menaqui-
none pathway from E. coli (25) is shown in Figure 1, where choris-
mate is converted into menaquinone through a series of reactions
catalyzed by several enzymes.
Structural analogs of the substrate or product of intermediate steps
in the biosynthetic pathway are well known to function as inhibitors
of the selected pathway (26). This approach motivated us to use
naphthoquinone derivatives to analyze their effect on mycobacterial
growth. In the menaquinone biosynthetic pathway, the 5th interme-
diate product DHNA (1,4-dihydroxy-2-naphthoate) has a naphthoqui-
none skeleton. Plumbagin (Figure 2A), which is already reported in
the literature (27) as a naturally occurring naphthoquinone, is known
to have antimicrobial activity (28–32).
Key words: FIC, inhibition, M. smegmatis, Menaquinone, MIC,
plumbagin
Received 17 July 2009, revised 18 December 2009 and 1 April 2010
and accepted for publication 2 April 2010
Inhibition of Gram-positive bacteria by different chemical molecules,
which are selective for specific metabolic pathways, is a subject of
active investigation since last decade. These studies gain additional
importance, keeping in mind the necessity of specific inhibitors
toward the growth of Mycobacterium tuberculosis, the main
causative agent for TB. With the sequencing of M. tuberculosis
genome (1), a wide arena opened up for scientists working on erad-
icating this most successful pathogen. Later, most of the genes and
The 1, 4-diketone quinone motif (Figure 3A) has strong inhibitory
activity on microbial growth. 1,2-naphthoquinone (Figure 3B) is
found to be eightfold less active than 1,4-naphthoquinone (Fig-
ure 3C) (31). Study on the antimycobacterial activity of naturally
occurring phenolic compounds indicated that compounds without
34

Inhibition of Mycobacterial Growth by Plumbagin Derivatives
Figure 1: Schematic representation of menaquinone biosynthetic pathway in E. coli, adapted from the work of Truglio et al. (25).
two hydroxyl groups on the second ring (5,8-hydroxy), is found to
be twofold more effective than its derivative, which has only one
hydroxyl (5-hydroxy) (31).
It should be mentioned here that electron transport inhibitors have
not been developed against M. smegmatis. As M. smegmatis is
considered as a model organism for M. tuberculosis (33), the focus
of this work is to analyze the effect of different plumbagin deriva-
tives on their growth. However, we have also tested the effect of
these molecules on the growth of M. tuberculosis, and the results
show similar trend as that with M. smegmatis.
Figure 2: Plumbagin and its derivatives (A) plumbagin (HNQ); R=
H, (B) acetyl plumbagin (AHNQ); R=CO-CH3, (C) benzoate plumbagin
(BHNQ); R=CO-Ph, (D) butyrate plumbagin (ByHNQ); R=CO-CH2-CH2-
Materials and Methods
CH3, (E) cinnamate plumbagin (CHNQ); R=CO-CH=CH-Ph, (F) croto-
Synthesis of plumbagin derivatives
nate plumbagin (CrHNQ); R=CO-CH=CH-CH3, (G) iodobenzoate
All starting chemicals, acetyl chloride, propionoyl chloride, plumba-
plumbagin (IHNQ); R=CO-Ph-I, (H) levulinoate plumbagin (LHNQ);
gin, cinnamic acid, butyric acid, levulinic acid, crotonoic acid,
R=CO-CH2-CH2-CO-CH3, (I) propionate plumbagin (PHNQ); R=CO-CH2-
benzoic acid, and 2-iodobenzoic acid were purchased from Sigma
CH3.
Aldrich, USA ⁄ Merck, USA ⁄ Spectrochem, India. Reactions were mon-
this quinone structure had much lower activity. Benzil (Figure 3D),
where the diketone motif itself was retained yet moved off of the
aromatic ring, shows a decrease in activity indicating that the
ketone groups themselves are not toxic. Tran and coworkers found
that adding a second conjugated ring mildly improved activity [in
M. smegmatis minimal inhibitory concentration (MIC) of benzoqui-
none (Figure 3E) is 463 lM, MIC of naphthoquinone (Figure 3C) is
316 lM], adding a third ring strongly decreased activity (anthraqui-
none (Figure 3F) MIC is 30 700 lM). The presence of hydroxyl
groups on the second ring had a major effect on potency. Naph-
thazarin (5,8-dihydroxy-1,4-naphthoquinone) (Figure 3G), which has
itored by thin-layer chromatography (Merck, Kieselgel 60 PF254 TLC
plates); TLC spots were visualized under ultraviolet light.
Method A: esterification using dicyclohexyl-
carbodiimide (DCC), 4-dimethylaminopyridine
(DMAP)/pyridine (Scheme 1)
General procedure
The solution of R-acid (2.0 eq) in 10 mL of dichloromethane (DCM)
was cooled to 0 ꢀC. DCC (77 mg, 1.5 eq) and pyridine (0.168 mL,
Chem Biol Drug Des 2010; 76: 34–42
35

Mathew et al.
A
B
C
D
E
F
G
Figure 3: (A) 1,4-diketone
quinone (B) 1,2-naphthoquinone
(C) 1,4-naphthoquinone (D) benzil
(E) benzoquinone (F) anthraquinone
(G) naphthazarin.
0 ꢀC and stirred for 5 min at the same temperature. Freshly pre-
pared acid chloride (2.0 eq) (described earlier) was added dropwise
to this reaction mixture at 0 ꢀC and was stirred for 3 h at room
temperature. This was diluted with dichloromethane and washed
with water and brine solution. The dried organic layer on column
chromatography yielded the desired product.
Plumbagin (HNQ) [5-hydroxy 2-methyl 1,4
naphthoquinone] (C11H8O3) (Figure 2A)
Plumbagin was purchased from Sigma Aldrich. (400 MHz, CDCl3), dH
ppm: 12.0 (1H, OH), 8.09–8.05 (1H, m), 7.74–7.70 (1H, t,
J = 8.0 Hz), 7.36–7.34 (1H, d, J = 8.0 Hz), 6.70 (1H, s), 2.16 (3H, s).
Scheme 1: Esterification using dicyclohexylcarbodiimide (DCC),
4-dimethylaminopyridine (DMAP) ⁄ pyridine (Method A).
2.0 eq) were added to this, followed by plumbagin (Figure 2A)
(47 mg, 1.0 eq) and catalytic amount of DMAP (0.1 eq). The reaction
mixture was stirred overnight at room temperature and was diluted
with DCM and washed with water and brine solution. The dried
organic layer on column chromatography yielded the desired product.
Acetyl plumbagin (AHNQ) [5-O-acetyl 2-methyl
1,4 naphthoquinone] (C13H10O4) (Figure 2B)
This was synthesized using Method B. Rf 0.35 (SiO2, hexane: EtOAc,
100:20); tmax (KBr) cm)1 1765, 1735, 1655, 1630, 1590; (400 MHz,
CDCl3), dH ppm: 8.09–8.05 (1H, m), 7.74–7.70 (1H, t, J = 8.0 Hz),
7.36–7.34 (1H, d, J = 8.0 Hz), 6.70 (1H, s), 2.438 (3H, s), 2.16 (3H,
s); m ⁄ z 230 (M+).
Method B: esterification using acid chloride/
pyridine (Scheme 2)
General procedure: preparation of acid chloride
Acid (2.0 eq) in 10-mL DCM was cooled to 0 ꢀC, and thionyl chlo-
ride (2.2 eq) was added to it. The solution was stirred from room
temperature to 50 ꢀC for 3 h. After conversion to acid chloride,
reaction mixture was concentrated on rotavapour and dried on high
vacuum. To the stirred solution of plumbagin (Figure 2A) (47 mg,
1.0 eq) in 10 mL of DCM, pyridine (0.252 mL, 3.0 eq) was added at
Benzoate plumbagin (BHNQ) [5-O-benzoyl
2-methyl 1,4 naphthoquinone] (C18H12O4)
(Figure 2C)
This was synthesized using Method B. Rf 0.40 (SiO2, hexane: EtOAc,
100:20); tmax (KBr) cm)1 1737, 1662, 1626, 1594; (400 MHz,
CDCl3), dH ppm: 8.26–8.10 (3H, m), 7.80–7.47 (5H, m), 6.70 (1H, s),
2.16 (3H, s); m ⁄ z 292 (M+).
Butyrate plumbagin (ByHNQ) [5-O-butyroyl
2-methyl 1,4 naphthoquinone] (C15H14O4)
(Figure 2D)
This was synthesized using Method A. Rf 0.370 (SiO2, hexane:
EtOAc, 100:20); tmax (KBr) cm)1 1752, 1665, 1627, 1595;
(400 MHz, CDCl3), dH ppm: 8.09-8.05 (1H, m), 7.74-7.70 (1H, t,
J = 8.0 Hz), 7.36-7.34 (1H, d, J = 8.0 Hz), 6.70 (1H, s), 2.73–2.69
(2H, t, J = 7.6 Hz), 2.16 (3H, s), 1.89–1.80 (2H, m), 1.1-1.07 (3H, t,
J = 7.6 Hz); m ⁄ z 258 (M+).
Scheme 2: Esterification using acid chloride ⁄ pyridine (Method
B).
36
Chem Biol Drug Des 2010; 76: 34–42

Inhibition of Mycobacterial Growth by Plumbagin Derivatives
Propionate plumbagin (PHNQ) [5-O-propionoyl
2-methyl 1,4 naphthoquinone] (C14H12O4)
(Figure 2I)
This was synthesized using Method B. Rf 0.33 (SiO2, hexane: EtOAc,
100:20); tmax (KBr) cm)11765, 1739, 1659, 1630, 1592; (400 MHz,
CDCl3), dH ppm: 8.09–8.05(1H, m), 7.74–7.70 (1H, t, J = 8.0 Hz),
7.36-7.34 (1H, d, J = 8.0 Hz), 6.70 (1H, s), 2.79-2.741 (2H, q,
J = 7.6 Hz), 2.16 (3H, s), 1.34–1.30 (3H, t, J = 7.6 Hz); m ⁄ z 244
(M+).
Mycobacterial growth inhibitory assay
The wild-type strain of Mycobacterium smegmatis mc2155 was
grown in Middlebrook 7H9 (MB7H9; Difco, USA) medium supple-
Figure 4: Diospyrin, R1 = R2 = H; diospyrin derivatives:-D-1:
R1 = CH3, R2 = H; D-2: R1 = CH2-CH3, R2 = H; D-3: R1 = CH3, R2 =
-NH-CO-CH3.
mented with 0.05% Tween 80 and glucose. Both 0.02% glucose
(starvation) and 2% glucose (high concentration) concentrations were
studied in this assay (34). The mycobacterial cultures were grown,
and along with it, the synthetic derivative molecules of plumbagin
were added into the medium (35). Different concentrations of these
derivative molecules were dissolved in a final concentration of 1%
dimethylsulfoxide (DMSO). Cultures without inhibitor molecules and
DMSO were considered as positive control. Also, cultures were
grown in the presence of DMSO alone to examine whether there is
any inhibitory effect of DMSO. Optical densities were measured at
600 nm for more than 100 h, and growth rates were determined.
Each experiment was performed in triplicates.
Cinnamate plumbagin (CHNQ) [5-O-cinnamoyl
2-methyl 1,4 naphthoquinone] (C20H14O4)
(Figure 2E)
This was synthesized using Method A. Rf 0.45 (SiO2, hexane: EtOAc,
100:20); tmax (KBr) cm)1 3080, 2956, 1733, 1694, 1662, 1637;
(400 MHz, CDCl3), dH ppm: 8.10-8.08 (1H, d, J = 6.4 Hz), 7.95-7.91
(1H, d, J = 16.0 Hz), 7.77–7.61 (3H, m) 7.45–7.20 (4H, m), 6.77–6.73
(1H, d, J = 12.4 Hz), 6.71 (1H, s), 2.16 (3H, s); m ⁄ z 318 (M+).
Determination of minimal inhibitory
Crotonate plumbagin (CrHNQ) [5-O-crotonoyl
2-methyl 1,4 naphthoquinone] (C15H12O4)
(Figure 2F)
This was synthesized using Method B. Rf 0.47 (SiO2, hexane: EtOAc,
100:20); tmax (KBr) cm)1 1736, 1726, 1660, 1627; (400 MHz,
CDCl3), dH ppm: 8.09–8.05 (1H, m), 7.74–7.70 (1H, t, J = 8.0 Hz),
7.36–7.34 (1H, d, J = 8.0 Hz), 7.20 (1H, m), 6.70 (1H, s), 6.19–6.15
(1H, dd, J = 12.4 Hz & 1.6 Hz), 2.16 (3H, s), 2.03–2.01 (3H, dd,
J = 5.2 Hz & 1.6 Hz); m ⁄ z 256 (M+).
concentration (MIC)
Minimal inhibitory concentration is the lowest concentration of an
antimicrobial that will inhibit the visible growth of an organism. In
this study, MIC for each plumbagin derivative was determined from
the respective growth curves of M. smegmatis mc2155.
Mycobacterium tuberculosis H37Rv were grown in MB7H9 medium
supplemented with 0.2% glycerol, 0.25% Tween 80% and 10%
albumin dextrose catalase. Minimal inhibitory concentrations in
M. tuberculosis H37Rv were determined by resazurin microplate
assay. Using a multichannel pipette, 200 lL of M. tuberculosis cul-
tures was added into the wells along with different concentrations
of plumbagin derivatives. Some wells served as inhibitor-free media
or inoculum controls. The plates were packed in gas-permeable
polythene bags and were incubated at 37 ꢀC for 5 days. Thereafter,
40 lL of freshly prepared resazurin (20 mg ⁄ 100 mL) reagent and
10% Tween 80 were added. The wells were observed after 24 and
48 h for a color change from blue to pink. A blue color in the well
was interpreted as no growth, and pink color was scored as growth
(36).
Iodobenzoate plumbagin (IHNQ) [5-O-(2-
iodobenzoyl) 2-methyl 1,4 naphthoquinone]
(C18H11IO4) (Figure 2G)
This was synthesized using Method A. Rf 0.47 (SiO2, hexane: EtOAc,
100:20); tmax (KBr) cm)1 1740, 1668, 1629, 1590; (400 MHz,
CDCl3), dH ppm: 8.36–8.34 (1H, dd, J = 6.4 Hz), 8.13-8.07 (2H, m),
7.81–7.77 (1H, t, J = 8.0 Hz), 7.57-7.52 (2H, m), 7.28 (1H, m) 6.72
(1H, s), 2.18 (3H, s); m ⁄ z 418 (M+).
Levulinoate plumbagin (LHNQ) [5-O-levuloyl
2-methyl 1,4 naphthoquinone] (C16H14O5)
(Figure 2H)
This was synthesized using Method A. Rf 0.33 (SiO2, hexane: EtOAc,
100:20); tmax (KBr) cm)1 1758, 1717, 1668, 1657, 1627, 1594,
1577, 1536; (400 MHz, CDCl3), dH ppm: 8.07-8.05 (1H, m), 7.74–7.70
(1H, t, J = 8.0 Hz), 7.4–7.37 (1H, d, J = 8.0 Hz), 6.70 (1H, s), 3.03–
2.932 (4H, m), 2.23 (3H, s) 2.16 (3H, s); m ⁄ z 286 (M+).
Biofilm inhibition assay
Biofilms were grown as described by (37), and their quantification
was performed according to O'Toole and coworkers (38).
M. smegmatis cultures were grown in 96-well polystyrene plates in
Sauton's medium, and at each time-point, samples (cultures with or
without derivative molecules) were removed from the wells and
incubated with 1% crystal violet for 45 min. Later the dye was
Chem Biol Drug Des 2010; 76: 34–42
37

Mathew et al.
A
B
Figure 5: (A) Growth curve of M. smegmatis in 0 lg ⁄ ml, 0 lg ⁄ ml + 1% D (DMSO), 0.5, 1, 2, 2.5, 3, 4, 5, 6,7, 8 and 9 lg ⁄ mL of butyrate
plumbagin (BHNQ) in 0.02% glucose. (B) Growth curve of M. smegmatis in 0 lg ⁄ mL, 0 lg ⁄ mL + 1% D (DMSO), 0.5, 1, 2, 2.5, 3, 4, 5, 6, 7, 8
and 9 lg ⁄ mL of butyrate plumbagin (BHNQ) in 2% glucose.
washed off with water, and 80% ethanol was added. Optical densi-
ties were measured at 590 nm using a microplate reader (39).
bagin were considered as positive control. After incubation for more
than 100 h, 0.01% methylene blue was added and was further incu-
bated at 37 ꢀC for 12 h. Rate of oxygen consumption was determined
by decolorization of methylene blue as described by Boshoff et al. (42).
Fractional inhibitory concentration (FIC) assay
FIC is the coefficient indicating whether the combined inhibitory
effects of molecules are synergistic, additive, indifferent or antago-
nistic (40). This assay was carried out along with rifampicin, which
is the most potent antitubercular drug in the market (41). Both
plumbagin and butyrate plumbagin were incorporated separately
along with rifampicin to the M. smegmatis cultures as mentioned
earlier, and inhibitory concentrations were determined.
Results and Discussions
Design and synthesis
Plumbagin and its derivatives (Figure 2A–I) were chosen for our work
because a naphthoquinone moiety is the substrate ⁄ product of the
last three steps in the biosynthesis of menaquinone (Figure 1). Also,
plumbagin, which is a naphthoquinone, has a basic structural scaffold
similar to 1, 4-dihydroxy-2-naphthoate (DHNA) of this pathway.
Oxygen-consumption assay
M. smegmatis were grown as mentioned previously in the presence
and absence of butyrate plumbagin. Cultures without butyrate plum-
In our attempt to understand the effect of derivatization of the
naphthoquinone moiety on the bactericidal action, we started by
38
Chem Biol Drug Des 2010; 76: 34–42

Inhibition of Mycobacterial Growth by Plumbagin Derivatives
Figure 6: IC50 of butyrate
plumbagin in 0.02% and 2%
glucose.
first derivatizing the phenolic group, because modification of pheno-
lic functionality was found to affect the activity. For example, diosp-
yrin (Figure 4), a known dinaphthoquinone isolated from Euclea
natalensis, exhibits mild antitubercular activity with an MIC of
100 mg ⁄ L. Its semi-synthetic ether derivatives (D-1 & D-2) (Figure 4)
were found to be less active than the methylether aminoacetate
derivative D-3 (Figure 4) (MIC of 10–50 mg ⁄ L) against both wild-
type M. tuberculosis H37Rv and a range of drug-resistant strains
(43).
Table 1: IC50 data for plumbagin and its derivatives
0.02%
Glucose Standard 2% Glucose Standard
Sl. No. Chemical molecules
IC50 (lM) deviation IC50 (lM)
deviation
1.
2.
3.
4.
5.
6.
7.
8.
9.
Plumbagin
Acetyl Plumbagin
9.13
19.39
14.44
9.06
–
18.00
–
–
0.074
0.083
0.041
0.003
–
0.016
–
–
9.19
18.53
10.54
8.26
–
17.61
–
–
0.007
0.052
0.015
0.064
–
0.013
–
–
Benzoate Plumbagin
Butyrate Plumbagin
Cinnamate Plumbagin
Crotonate Plumbagin
Iodobenzoate Plumbagin
Levulinoate Plumbagin
Effect of plumbagin derivatives on mycobacterial
growth
Propionate Plumbagin 13.58
0.009
13.79
0.017
Figures 5A and B show a representative growth curve for the inhibi-
tion of M. smegmatis in 0.02% and 2% glucose-containing media
at different concentrations of butyrate plumbagin. Growth of
M. smegmatis in different butyrate plumbagin concentrations
showed the maximum inhibition among all the derivatives synthe-
sized. Although the nature of inhibition appears to be the same in
both the glucose concentrations, certain interesting features, none-
theless, became apparent like cells showed no growth till 40 h at
medium inhibitor concentrations, whereas cells grew well in lower
concentrations as though there were no inhibitors at all. However,
cell density increases at further time-points for the medium inhibitor
concentrations and reaches saturation, similar to the positive con-
trol. This raises a possibility of bacterial adaptation to the inhibitors
at medium inhibitor concentrations. However, no growth of
M. smegmatis was observed when the concentration of the inhibi-
tor was further increased to lethal levels.
– stands for no inhibition.
behavior and the concentration of the inhibitors were different in
each case. Chromatographic analysis of the M. smegmatis culture
filtrate, which showed inhibition even after 100 h, did not show
any traces of plumbagin, indicating that the ester derivatives of
plumbagin (Figure 2B–I) did not hydrolyze under the experimental
conditions.
Unlike the growth curve observed in rifampicin, there was no signif-
icant difference between starvation (0.02%) and high (2%) glucose
concentrations in these derivatives. Table 2 shows the MIC values
of the different plumbagin derivatives for both M. smegmatis and
M. tuberculosis. When the M. smegmatis cultures with these MIC
values were further subjected to 2% glucose with 0.05% Tween
80, restoration of growth occurs. However, such was not the case
when cultures were taken from the media with higher concentration
(concentration above the MIC value: plumbagin – 15 lM, acetyl
plumbagin – 35 lM, benzoate plumbagin – 24 lM, butyrate plumba-
gin – 19 lM, crotonate plumbagin – 23 lM, propionate plumbagin
– 20 lM) of inhibitor. No growth was observed beyond this concen-
tration even when grown for more than 200 h.
From Figure 5A,B, one can derive the IC50 value, which is the
amount of inhibitor required for 50% inhibition on the growth of an
organism and is depicted in Figure 6. Among the eight different
derivatives of plumbagin (Figure 2B–I), only five of them had inhibi-
tory effect on M. smegmatis mc2155 (Table 1).
Interestingly, the growth curves showed adaptation of M. smegma-
tis to the inhibitors present in the media, so the growth was moni-
tored for more than 100 h. It should be mentioned here that the
first-line TB drug, rifampicin shows a monotonous inhibitory growth
pattern (data not shown), while all the plumbagin derivatives
showed adaptive response, although the time required to show this
We observed that there were no inhibitory effects of these deriva-
tives on biofilm formation by M. smegmatis (data not shown).
Hence, these molecules appear to have effect only on planktonic
cultures.
Chem Biol Drug Des 2010; 76: 34–42
39

Mathew et al.
Table 2: Minimal inhibitory concentration (lM) data for plumba-
gin and its derivatives
treatment with butyrate plumbagin inhibited oxygen consumption in
M. smegmatis. This may imply that there is effect of this inhibitor
molecule on electron transport and respiration as decolorization of
methylene blue which is a well-known redox dye has been reported
to unambiguously demonstrate effects on respiration (42).
M. smegmatis M. smegmatis
mc2155 0.02% mc2155 2%
M. tuberculosis
Compound
Glucose
Glucose
H37Rv
Plumbagin
Acetyl Plumbagin
13.3
28.2
20.5
15.5
–
19.5
–
–
13.3
30.4
20.5
15.5
–
19.5
–
–
21.3
34.8
27.4
77.4
NT
15.6
NT
NT
These studies were extensively carried out in M. smegmatis, as it
allowed us to manipulate different growth conditions because of its
faster growth rate and non-pathogenicity. These results were infor-
mative while testing these inhibitors on the pathogenic counterpart
M. tuberculosis, as they share the major metabolic pathways and
have related homologs with the virulence genes (33,44,45).
Benzoate Plumbagin
Butyrate Plumbagin
Cinnamate Plumbagin
Crotonate Plumbagin
Iodobenzoate Plumbagin
Levulinoate Plumbagin
Propionate Plumbagin
16.4
16.4
16.4
Conclusion
– stands for no inhibition.
NT, not tested.
To design an effective derivative of naphthaquinone moiety for anti-
mycobacterial effect, we started by first derivatizing the phenolic
group of plumbagin. Among the different derivatives of plumbagin,
five of them had inhibitory effect on M. smegmatis mc2155 of
which butyrate plumbagin was the most effective one, whereas on
M. tuberculosis, H37Rv crotonate plumbagin showed greater activ-
ity. Moreover, butyrate plumbagin demonstrated effect on electron
transport and respiration by inhibiting oxygen consumption in
M. smegmatis. Future work will concentrate on the diversity of oxy-
gen containing functional groups on the aromatic ring and also
derivatizing positions C-2 and C-3 of the quinonoid moiety. Struc-
tural modifications of these molecules can further be improved to
generate better molecules against both rapidly multiplying and
latent mycobacteria.
Fractional inhibitory concentration is the coefficient indicating
whether the combined inhibitory effects of molecules are synergis-
tic, additive, indifferent or antagonistic. FIC = A+B; where A = (MIC
of combination X + Y) ⁄ (MIC of drug X alone) and B = (MIC of com-
bination X + Y) ⁄ (MIC of drug Y alone). FIC is interpreted as syner-
gistic when FIC £ 0.5; additive when FIC > 0.5–1.0; indifferent
when FIC > 1.0 - £ 4.0 and antagonistic when FIC ‡ 4. When
experimentally calculated, the FIC of rifampicin with plumbagin or
butyrate plumbagin was indifferent in both starvation (0.02%) and
high (2%) glucose concentrations for M. smegmatis.
Oxygen-consumption assay showed decolorization of methylene blue
in the control and also in the concentrations below the MIC of
butyrate plumbagin (Figure 7). These results demonstrated that
Acknowledgments
The financial and facility support of ILS, DBT (DC) and CSIR-OSDD
are acknowledged. The authors RM & AK thank Prof. Javed Iqbal,
Director, ILS for his constant support and encouragement. The
authors acknowledge with thanks Dr. Santanu Datta (Astra Zeneca)
who helped us with the MIC studies on M. tuberculosis.
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