Antimicrobial activity of tryptanthrins in escherichia coli

Article abstract of DOI:10.1021/jm901847f

Tryptanthrins have potential therapeutic activity against a wide variety of pathogenic organisms, although little is known about their mechanism. Activity against Escherichia coli, however, has not been examined. The effects of tryptanthrin (indolo[2,1-b]quinazolin-6,12-dione) and nine derivatives on growth, survival, and mutagenesis in E. coli were examined. Analogues with a nitrogen atom at the 4-position of tryptanthrin stopped log phase growth of E. coli cultures at concentrations as low as 5 μM. Tryptanthrins decreased viability during incubation with cells in buffer by factors of 10^-2 to <10^-6 at 0.2-40 μM. Derivatives with an oxime group at the 6-position exhibited the greatest bactericidal activity. Most tryptanthrins were not mutagenic in several independent assays, although the 4-aza and 4 aza-8-fluoro derivatives increased frameshift mutations about 22- and 4-fold, respectively. Given the structure of trypanthrins, binding to DNA may occur by intercalation. From analysis using a sensitive linking number assay, several tryptanthrins, especially the 4-aza and 6-oximo derivatives, intercalate into DNA.

Full text of DOI:10.1021/jm901847f

3558 J. Med. Chem. 2010, 53, 3558–3565
DOI: 10.1021/jm901847f
Antimicrobial Activity of Tryptanthrins in Escherichia coli
Pooja P. Bandekar,† Keir Alekseii Roopnarine,† Virali J. Parekh,† Thomas R. Mitchell,†
Mark J. Novak,‡ and Richard R. Sinden*,†
‡
†Department of Biological Sciences and Department of Chemistry, Florida Institute of Technology,
150 West University Boulevard, Melbourne, Florida 32901
Received December 14, 2009
Tryptanthrins have potential therapeutic activity against a wide variety of pathogenic organisms, although
little is known about their mechanism. Activity against Escherichia coli, however, has not been examined.
The effects of tryptanthrin (indolo[2,1-b]quinazolin-6,12-dione) and nine derivatives on growth, survival,
and mutagenesis in E. coli were examined. Analogues with a nitrogen atom at the 4-position of tryptanthrin
stopped log phase growth of E. coli cultures at concentrations as low as 5 μM. Tryptanthrins decreased
viability during incubation with cells in buffer by factors of 10-2 to <10-6 at 0.2-40 μM. Derivatives with
an oxime group at the 6-position exhibited the greatest bactericidal activity. Most tryptanthrins were not
mutagenic in several independent assays, although the 4-aza and 4 aza-8-fluoro derivatives increased
frameshift mutations about 22- and 4-fold, respectively. Given the structure of trypanthrins, binding to
DNA may occur by intercalation. From analysis using a sensitive linking number assay, several tryptan-
thrins, especially the 4-aza and 6-oximo derivatives, intercalate into DNA.
Introduction
leishmania6 or the observed antibacterial or antifungal acti-
vity.1,22 In the case of inhibition of MRD1, tryptanthrin may
act by down-regulating gene expression.15 From the very
diversecollection of targetprokaryotic andeukaryoticorgani-
sms and the wide range of reported biological effects, tryptan-
thrins likely act via multiple biochemical pathways.
Indolo[2,1-b]quinazolin-6,12-dione (tryptanthrin, Figure 1)
and itsanalogues havegenerated interestas a class of potential
therapeutic agents because of their ease of synthesis, stability,
and broad spectrum of activity against pathogenic organisms.
Historically, plant extracts containing this alkaloid have been
used as a remedy for athlete’s foot1 and as anti-inflammatory
and fever-reducing agents.2 Tryptanthrins possess antibac-
terial activity against Bacillus subtilis3 and Mycobacterium
tuberculosis;4,5 antifungal activity against various Trichophy-
ton, Microsporum, and Epidermophyron species;1 and activity
against growth of Leishmania donovani,6 Trypanosoma brucei,7
and Plasmodium falciparum.8,9 In mammalian cells, tryptan-
thrins are reported to possess cytotoxic activity against many
cancer cell lines.10 They can decrease the activity of cyclooxy-
genase-2 (COX-2)11,12 and inhibit the expression of nitric
oxide synthase and prostaglandin E(2) in cells.13 Hepatocyte
growth factor in human fibroblasts14 and the multidrug
resistance gene MDR1 in breast cancer cells15 are inhibited
by tryptanthrins. In mice, interleukin-2 and interferon-γ have
been reported to be inhibited by treatment with these com-
pounds,2,16 and in certain systems tryptanthrins may possess
antitumor activity.17,18
Although IC50 values into the low ng/mL range have been
demonstrated against a wide variety of pathogenic organisms,
very little insight has been gained into the mechanism(s) of
action of tryptanthrins at the cellular and molecular levels. To
our knowledge, the only published hypothesis of any detail
concerning tryptanthrins’ mechanism of action involved hemin
and hemozoin binding as a factor in antimalarial activity.19-21
However, this would not account for the toxicity toward other
organisms that cause tuberculosis,4 trypanosomiasis,7 and
With regard to mechanisms of toxicity from a broad pers-
pective, it is not currently known if tryptanthrins act as
mutagens or inhibitors of key metabolic pathway(s) or if they
act physically, such as by disrupting membranes. It is also
unclear if tryptanthrins act as static agents, preventing growth
and replication, or if they can act as direct lethal agents.
A priori, the planar structures of tryptanthrins suggest that
they could bind to DNA by intercalation, or they may form
covalent adducts to nucleobases. Either action could lead to
mutagenesis or the inhibitionof DNA synthesis, RNA synthe-
sis, or gene expression. To begin to address the broad issue of
how tryptanthrins might affect survival and mutagenesis, we
have examined their effects in E. coli, with which no prior
reports are available. E. coli is a well-established model for the
study of xenobiotics on genetic material that can be extra-
polated to other organisms. The genetic tools available for
E. coli should facilitate future mechanistic studies of tryptan-
thrin action. Presented herein is the first analysis of the effects
of tryptanthrin and several analogues (Figure 1) on the growth,
survival, and mutagenesis of E. coli. In addition, we demon-
strate that certain tryptanthrins alter the twist of the DNA
helix consistent with their binding to DNA by intercalation.
Results
Effect of Tryptanthrins on Growth of E. coli and Viability in
LB Media. E. coli strain AS19 was grown in LBa media at 37 °C
*To whom correspondence should be addressed. Phone: 321-674-
8576. Fax: 321-674-7238. E-mail: rsinden@fit.edu.
a Abbreviations: LB, Luria-Bertaini medum; AMP, ampicillin; CAP,
chloramphenicol; RIF, rifampicin.
r
pubs.acs.org/jmc
Published on Web 04/07/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9 3559
Tryptanthrins 3, 4, and 7 failed to exhibit a significant
effect on growth. As the 6-oxime derivatives are photolyti-
cally unstable, these compounds (3, 4, 7, and 8) may initially
bind to DNA by intercalation, and following subsequent
photoactivation, they may react with DNA to form covalent
adducts. This possibility was tested in two ways. First,
following incubation of cells for several hours in the presence
of the tryptanthrin (conditions that stop growth), samples
were exposed to 360 nm light, and then growth was resumed.
In these experiments, no inhibition of growth was observed
following 360 nm light exposure for cells treated with
tryptanthrins 3, 4, and 7 (data not shown). As a control in
this experiment, cells were also treated with tryptanthrin 8,
which resulted in growth inhibition. Exposure of cells to
360 nm light, following addition of tryptanthrin 8, resulted
in similar growth inhibition kinetics without irradiation. In
other experiments, tryptanthrins were exposed to light in
DMSO, prior to addition to cells; however, no light-dependent
effect was observed (data not shown).
Effect of Tryptanthrins on E. coli Viability during Incuba-
tion in M9 Buffer. To assess the bactericidal activity of
tryptanthrins on E. coli, cells were grown overnight in LB,
adjusted to 105-106 cells/mL in M9 buffer, and incubated in
M9 buffer for 2-3 days in the presence of different concen-
trations of tryptanthrins. At roughly 12 h intervals, cells were
sampled and plated to measure viability. Representative data
from at least four to eight repetitions of each experiment are
shown in Figure 4 for tryptanthrins 1-8. Data are presented
as surviving fraction of cells relative to untreated cells incu-
bated in M9 buffer.
Several aspects of these experiments are significant. First,
most tryptanthrins resulted in a significant decrease in via-
bility of 10-4-10-6 by 3 days of incubation in the presence of
10 μg/mL tryptanthrin. Tryptanthrins 2, 5, and 8, which
caused a cessation in growth in LB media, were effective
bactericidal agents at concentrations lower than 10 μg/mL.
Tryptanthrin 2 was equally effective at concentrations from
0.05 to 10 μg/mL, although the reduction in survival was less
than for some other analogues. The activities of tryptan-
thrins 9 and 10 at 0.4 and 10 μg/mL were similar to those
for tryptanthrin 2. Tryptanthrin 4 was very effective at
both inhibiting growth and killing. Significantly, tryptan-
thrins that failed to show substantial growth inhibition in
LB media (tryptanthrins 3, 4, and 7) were effective, at the
higher concentrations, in killing cells during incubation in
M9 buffer.
Mutagenic Potential of Tryptanthrins in E. coli. Several
selection systems were utilized to test for the potential
induction of mutations by tryptanthrin treatment. AS19
containing plasmid pBRF14C (with a 106 bp inverted repeat
in the chloramphenicol acetyl transferase gene) was used as a
system to test for induction of DNA secondary structure
mutations (deletion of inverted repeats).23,24 In addition, the
frequency of rifampicin resistant mutations, which detects a
more general array of mutations,25 was measured. Cells were
treated with 10 μg/mL tryptanthrins in M9 buffer to reduce
survival by about 10-3. Following this, about 103-104 cells
were inoculated into 10 mL of LB, and the culture was grown
overnight at 37 °C with shaking. Cells were plated to measure
viability, CAP-resistant revertants, and RIF-resistant rever-
tants. In seven independent repetitions of this experiment no
consistent increases in mutation frequencies were observed
for the deletion of the inverted repeat or reversion to rifam-
picin resistance. Thus, tryptanthrins do not possess, at least
Figure 1. Tryptanthrin structure. The molecular structures of the
10 tryptanthrin derivatives are shown. The numbers indicate the
notation used throughout.
in the presence of various concentrations of tryptanthrins
(Figure 1) to assess their effect on growth. The effects of various
concentrations of tryptanthrin 5 on culture growth are shown
in Figure 2. A concentration of 0.01 μg/mL had no effect on
growth, while 0.1 μg/mL had a small effect (Figure 2A). At
1 μg/mL, tryptanthrin 5 stopped growth within 4 h and little
change in the OD650 occurred over the next 20 h of incubation
(Figure 2A). Concentrations of 2.5-20 μg/mL still required
about 4 h to stop growth (Figure 2, B). Following 20-24 h of
incubation, cell viability was reduced to about 15% in cells
treated with 10 μg/mL tryptanthrin 5 (data not shown).
Results of individual treatment of AS19 with seven other
tryptanthrins at 10 μg/mL are shown in Figure 2C, D. A range
of effectiveness in stopping growth was evident. Tryptan-
thrins 3, 4, and 7 had only a slight effect on growth. Tryptan-
thrins 1 and 6 slowed growth and reduced the OD650 end
point by about 2-fold, while tryptanthrins 2, 5, and 8 caused
cessation of growth several hours after addition. Results of
Figure 2 are representative of multiple repetitions of these
experiments. Tryptanthrins 9 and 10 showed weak growth
inhibition (as 3, 4, and 7) (data not shown).
From dose dependence experiments (similar to that shown
in Figure 2), estimations of the concentration that reduced
the rate of growth by half can be determined. For this, the
OD650 4 h after the tryptanthrin addition, at which point
control cells were in late log phase growth, was plotted against
the concentration of tryptanthrin added. Representative
data are shown in Figure 3 for tryptanthrin 8. For all tryptan-
thrins that resulted in a measurable reduction in OD650, con-
centrations of 0.6-2 μg/mL were effective in reducing growth
by half (Table 1).

3560 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9
Bandekar et al.
Figure 2. Effect of 4-aza-8-fluorotryptanthrin on growth of E. coli AS19. (A, B) The effect of tryptanthrin 5 (4-aza-8-fluorotryptanthrin) on
growth of E. coli AS19 in LB is shown at concentrations indicated. A culture is started from an overnight, tryptanthrin is added, and the culture
is incubated at 37 °C with shaking at 200 rpm. OD650 values were determined at various times of growth: (A) no drug (black circle), 0.01 μg/mL
(gray diamond), 0.1 μg/mL (gray box), 1.0 μg/mL (black down-triangle); (B) no drug (black circle), 20 μg/mL (gray diamond), 10 μg/mL (gray
box), 2.5 μg/mL (black down-triangle). Panels A and B are representative of more than six repetitions of this experiment. (C, D) The effects of
tryptanthrins 1-8 on growth were compared at 10 μg/mL: (C) no drug (black circle), tryptanthrin 1 (black down-triangle), tryptanthrin 2 (gray
box), tryptanthrin 3 (gray diamond), tryptanthrin 4 (black up-triangle); (D) no drug (black circle), tryptanthrin 5 (black down-triangle),
tryptanthrin 6 (gray box), tryptanthrin 7 (gray diamond), tryptanthrin 8 (black up-triangle). The growth curves in panels C and D. are
representative of data from three to six repetitions of each tryptanthrin analyzed.
Table 1. Tryptanthrin Concentrations That Reduce the Rate for Growth
by 50%
tryptanthrin
concentration (μg/mL)a
1
2
2.25 ( 0.25
1.3 ( 0.2
NRb
NRb
3
4
5
0.6 ( 0.1
0.6 ( 0.1
NRb
6
7
8
1.5 ( 0.1
9
10
NRb
NRb
a The concentration shown is that required to reduce the OD650 to half
that in untreated cells at 4 h after tryptanthrin addition. Concentrations
represent the average of two to three independent experiments. b NR: no
results due to minimal effect of these tryptanthrins on growth inhibition.
Figure 3. Dose dependence of growth inhibition. To determine
a concentration where the rate of growth is reduced by 50%, the
OD650 at 4 h after tryptanthrin 8 addition, when growth is in
exponential phase, is plotted against tryptanthrin concentration.
These values for all tryptanthrins that lead to growth inhibition are
listed in Table 1.
However, the 4-aza and 4 aza-8-fluoro derivatives (tryptanthrins
2 and 5, respectively) increased frameshift mutations about
22- and 4-fold, respectively, in both the þG and -G reporter
strains (data not shown).
under the conditions of this experiment, a general mutagenic
potential or an increase in the frequency of deletion of inver-
ted repeats.
Tryptanthrins Alter DNA Supercoiling. Intercalating drugs
bind to DNA and alter the helical twist, resulting in DNA
unwinding by about 28° per drug bound.27 The analysis of
DNA topology, therefore, provides a very sensitive assay for
intercalation. To assess intercalation or other binding that
alters the twist of DNA, covalently closed circular DNA is
treated with a topoisomerase in the presence of a suspected
intercalating drug and analyzed by agarose gel electrophor-
esis. Negative supercoils remaining after relaxation indi-
cate that drug binding altered the twist of DNA. DNA,
with no drug present, was relaxed in the presence of Topo I
(Figure 5A, lane 2, sample C). In the presence of tryptanthrin
6 at 100 μg/mL, DNA also appeared to be completely relaxed,
Given that certain intercalating agents are generally
thought to induce frameshift mutations, a specific Lacþ
reversion mutagenesis assay that tests for frameshift muta-
tions26 was also applied for several of these tryptanthrins.
Strains WAR176 and WAR177 that report þG and -G
frameshifts, respectively, were tested. Cells were grown, from
overnight cultures, for 10 generations in MM Gly media in
the presence of 5 or 10 μg/mL tryptanthrins. Cell growth and
numbers of Lacþ revertants were measured to determine
mutation frequencies. Growth in the presence of most tryp-
tanthrins did not induce frameshift mutations in this system.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9 3561
Figure 4. Survival during exposure to tryptanthrins in M9 buffer. Tryptanthrins were added to a suspension of AS19 at 105-106 cells/mL, and
incubation at 37 °C was continued for up to 72 h. Samples were removed at various times, and viability was determined by plating on LB þ
AMP plates. Surviving fraction is that relative to cells incubated in M9 buffer without tryptanthrin addition. Data shown are for duplicate
samples treated with tryptanthrins 1-8 in panels A-H, respectively. For all panels treatment was with 10 μg/mL (black circle, gray circle), 2 μg/mL
(dark-gray triangle, light-gray triangle), 0.4 μg/mL (black box, gray box), 0.05 μg/mL (dark-gray diamond, light-gray diamond). Data are
representative of four to eight independent experiments.
suggesting that minimal binding occurred. The sample contain-
ing tryptanthrin 2, however, remained highly supercoiled,
indicative of minimal supercoil relaxation presumably due to
substantial tryptanthrin binding (lane 4). Tryptanthrins 1, 3, 4,
and 5 (lanes 3, 5, 6, 7) showed various levels of negative
supercoiling indicative of different levels of binding. To demon-
strate that the tryptanthrins were not inhibiting Topo I activity,
the DNA was first relaxed with topoisomerase I, tryptanthrins
were then added, and the reactions were allowed to continue.
The DNA samples would remain relaxed if a drug inhibited the
enzyme. As evident in Figure 5A, lanes 10-14, the distribution
of topoisomers was similar to that seen in lanes 3-7. This shows
that topoisomerase altered the linking number of relaxed DNA
in the presence of tryptanthrins. These results demonstrate that
tryptanthrins do not inhibit human DNA topoisomerase I, and
the enzyme relaxed positive and negative supercoils in the pre-
sence of drug.
To better resolve the distribution of supercoils, samples were
separated on an agarose gel containing chloroquine to unwind
about six to eight supercoils (or introduce about six to eight
positive supercoils in relaxed DNA). The multiple bands of the
relaxed topoisomer samples are thereby separated, facilitating
comparison and analysis (Figure 5B). Lanes 1, 6, and 13 con-
tain control DNA relaxed in the absence of tryptanthrins.
Tryptanthrins 6-10 showed only a small linking number devi-
ation, evident as a series of bands 1-2 superhelical turns higher
in the gel. Tryptanthrins 1, 4, and 5 showed a significant shift in
the linking number pattern. Tryptanthrins 2 and 3 exhibited a
very large change in linking number, indicative of a high level of
drug intercalation (Figure 5B, lanes 3 and 4).

3562 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9
Bandekar et al.
by Topo I decreased. This is consistent with an increase in
bound tryptanthrins with increasing concentration. The num-
bers of drugs bound per plasmid are calculated from the
number of supercoils restrained and a value of 28° unwinding
per drug, as described in Materials and Methods. Data were
analyzed by linear regression to estimate the number of drugs
bound at 100 μg/mL (0.4 mM). At 0.4 mM, 33 molecules of
tryptanthrin 2 were bound per 1000 bp, or 1 drug per 30.3 bp.
From all our analyses, the number of tryptanthrin molecules
bound per 1000 bp at 100 μg/mL are shown in Table 2 for all
analogues.
Discussion
Tryptanthrins have been evaluated as a class of therapeutic
agents. Interest in these compounds stems from their stability,
ease of synthesis, and reports of therapeutic application
against pathogenic agents, including bacteria, fungi, and acti-
vity against cancer cells. However, the mechanism(s) of action
of tryptanthrins remain unknown at the cellular or molecular
level. To begin understanding the molecular mechanisms
involved in the activity of tryptanthrins, we have initiated a
study utilizing E. coli as a model system to evaluate the
bacteriostatic, bactericidal, and potential mutagenic proper-
ties of a series of tryptanthrin analogues. Ten tryptanthrin
analogues were utilized; three contained an oxime group in
place of the 6-keto moiety. The reason for the oxime substitu-
tion was 2-fold. First, the oxime analogues are fluorescent and
appear photolytically unstable, opening up the possibility of
future fluorescent microscopy studies. Second, it is known
that oximes in general tend to be toxic toward single cell orga-
nisms, resulting in the possible addition of a second pharma-
cophore into this class of molecules.10
Several features of this analysis are worth noting (Table 2).
First, at 6-40 μM, some tryptanthrins showed bacteriostatic
activity in that they stopped growth, with some decrease in
viability, while other analogues did not. Second, several
tryptanthrins exhibited significant bactericidal activity on
prolonged exposure in M9 buffer. Third, most derivatives
were not particularly mutagenic, even following treatment
that decreased survival. However, two derivatives, including
tryptanthrin 2 that was the strongest DNA intercalator,
mildly elevated the frequency of frameshift mutations. From
the lack of a general mutator effect, it is assumed that these
compounds are not forming covalent adducts with DNA.
Fourth, significantly, several of these derivatives bound to
DNA, resulting in the unwinding of negative supercoils, a
hallmark of DNA intercalating drugs.
Figure 5. DNA unwinding by tryptanthrins. Plasmid pGEM DNA
was incubated with human DNA topoisomerase I in the presence of
tryptanthrins and the mobility of the reaction products resolved by
agarose gel electrophoresis. (A) Standard agarose gel. Lanes 1, 9,
and 15 contain purified supercoiled DNA. In lanes 2-8, DNA was
relaxed with topoisomerase in the presence of 100 μg/mL of the
tryptanthrins indicated above the lanes. In lanes 10-14, DNA was
relaxed by topoisomerase treatment before addition of the tryptan-
thrin. This plasmid preparation contained both monomer and
dimer plasmids, indicated to the left of the gel. The positions of
migration of supercoiled (Sc) and relaxed (Rel) plasmid monomers
are indicated at the right of the gel. (B) This agarose gel contained
2 μg/mL chloroquine in the gel and buffer, which relaxes about eight
negative supercoils, facilitating analysis of the center of the topoiso-
mer distributions.
The results presented indicate two general features with
regard to a structure-activity correlation. In terms of bacter-
iostatic activity, most tryptanthrins containing the nitrogen
heteroatom in the 4-position (tryptanthrins 2, 5, 7, and 8)
increasedtheir potencyrelativetothoseanaloguesthat didnot
contain the heteroatom. The tryptanthrin analogues that
exhibited the highest bactericidal activity (tryptanthrins 3
and 4) contained the oxime functionality at the 6-position.
Tryptanthrin 8, the 1,4-diaza derivative, was also a very
effective bactericidal agent. Tryptanthrin 7 contained both
the 6-oxime and the 4-position nitrogen heteroatom. It exhi-
bited poor bacteriostatic activity but reasonable bactericidal
activity, suggesting that the oxime may have masked the
activity of the heteroatom. By comparison of tryptanthrins
3 and 4 at 2 μg/mL, the 8-fluoro moiety appeared to have
increased the bactericidal effectiveness, as compound 4 was
more effective in killing than compound 3. The 8-bromo
Figure 6. Dose dependence of DNA unwinding. The extent of
DNA unwinding was determined from gels as shown in Figure 5
and as described under Materials and Methods. Data are presented
as tryptanthrins bound per 1000 bp. Two independent experiments
for tryptanthrin 2 are shown (black up-triangle, black down-
triangle). Single analyses are shown for tryptanthrins 3 (black box)
and 5 (black circle). Lines are least-squares fits to data for tryptan-
thrin 2 (solid line), tryptanthrin 3 (long dashed line), and tryptan-
thrin 5 (short dashed line).
Dose dependence unwinding analyses for tryptanthrins 2,
3, and 5 are shown in Figure 6. As the concentration of try-
ptanthrin increased, the number of negative supercoils relaxed

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9 3563
Table 2. Summary of Tryptanthrin Activities
bactericidal activity
DNA intercalation
derivative
growth inhibitiona
μg/mL
survivalb
(drug/kbp at 100 μg/mL)c
1, tryptanthrin
2, 4-azatryptanthrin
3, 6-oximotryptanthrin
þ
2
2 ꢀ 10-3
5 ꢀ 10-3
10-2-10-4
2 ꢀ 10-6
2 ꢀ 10-6
2 ꢀ 10-2
1 ꢀ 10-3
10-3-10-5
3 ꢀ 10-2
10-3-10-4
10-2-10-3
3 ꢀ 10-4
5 ꢀ 10-6
∼10
33
þþ
(
0.05
2
25
10
2
4, 8-fluoro-6-oximotryptanthrin
5, 4-aza-8-fluorotryptanthrin
(
þþ
∼10
0.4
2
7.3
10
2
6, 8-nitrotryptanthrin
þ
<5
10
0.05
10
0.4
0.4
10
0.4
10
7, 4-aza-6-oximotryptanthrin
(
∼7
8, 1,4-diazatryptanthrin
9, 8-bromotryptanthrin
þþ
<5
<5
(
10-2
10, 8-fluorotryptanthrin
(
<5
10-2
a Growth inhibition at 10 μg/mL: þþ, growth inhibition by 4 h after tryptanthrin addition; þ, growth slowed by 4 h with a 2- to 4-fold reduction in
OD650; (, mild effect of growth inhibition. b Survival is the surviving fraction of cells after 60-72 h of exposure to tryptanthrins at the concentration
indicated. c DNA intercalation represents the tryptanthrins bound by intercalation per 1000 bp at 100 μg/mL. This value is calculated as described in
Materials and Methods.
(tryptanthrin 9) and 8-flouro (tryptanthrin 10) derivatives
were less effective bacteriostatic agents than the parent com-
pound (Table 2), and the bactericidal activity was similar to
the 4-aza derivative, tryptanthrin 2. In the present study,
compound 8, the 1,4 diaza derivative, was overall the most
effective antagonist to E. coli when taking into account the
abilitytoinhibitgrowth as astatic agent and to reducesurvival
of the organism. In summary, these results indicate that the
tryptanthrins may have multiple mechanisms of action that
depend upon modification of the parent tryptanthrin 1.
Various theories have been suggested as mechanisms of
action for tryptanthrins, but at present, little is known. The
planar conformation of tryptanthrins in solution or as a solid
may suggest action as DNA intercalators,28,29 which we con-
firmed here. In light of recently published results that indicate
that tryptanthrins have the potential to assume enantiomeric
conformations upon binding to a substrate in vivo,28,29 the
conformational and electronic behavior of these molecules
may be more complex than initially thought. These com-
pounds are presumably not forming covalent adducts with
DNA, as this would also likely lead to large increase in muta-
tion rate. Our results show that tryptanthrins 2 and 3, the
4-aza and 6-oximo derivatives, strongly unwind DNA super-
coils, consistent with intercalation into the DNA double helix.
Several other derivatives (tryptanthrins 1, 4, 5, and 7) show
lower levels of unwinding, while the remaining analogues
show a minimal effect. At present, no strong correlation exists
between DNA binding and the measured biological effects.
Although tryptanthrins have a broad spectrum of activity
against different organisms, no single analogue is extremely
potent toward all the different types of organisms. For exam-
ple, the most potent analogue against malaria may have very
poor activity against tuberculosis.5,8,9 This suggests different
mechanisms of action at the molecular level and/or the
involvement of different receptors or biochemical pathways
among the different organisms. E. coli is certainly an excellent
model for understanding many basics of DNA interaction;
inhibition of DNA, RNA, or protein biosynthesis; or inhibi-
tion of basic metabolic pathways. However, it is conceivable
that certain toxicological effects involving higher eukaryotic
organisms may not be evident in this system. Experiments are
in progress to increase the activity of tryptanthrins in E. coli
and to understand the mechanisms of action in various
biochemical pathways.
Materials and Methods
Bacterial Strains and Media. E. coli AS19, containing plasmid
pBRF14C,23 was used for the bacteriostatic and bactericidal
analyses. AS19 was selected for permeability to actinomycin,30
which makes it more susceptible to uptake of small molecules.31
Strains WAR176 and WAR177, containing the þG and -G
frameshift reporters (CC107 and CC108),26 respectively, were
used for the frameshift mutation assay. Cells were grown in
Luria-Bertani medium (LB) (10.0 g of tryptone, 5.0 g of yeast
extract, and 10.0 g of NaCl per liter of H2O). LB plates
contained 15.0 g of agar per liter. M9 buffer contained 1 g of
NH4Cl, 5.8 g of Na2HPO4, and 3 g of KH2PO4 per liter of H2O.
Ampicillin plates (LB þ AMP) contained 30 μg/mL ampicillin.
Rifampicin and chloramphenicol plates (LB þ RIF, LB þ CHL)
contained 100 μg/mL rifampicin and 25 μg/mL chlorampheni-
col, respectively.
Tryptanthrin Derivatives. The tryptanthrin derivatives are
shown in Figure 1. Reaction of commercially available isatins
with the appropriate isatoic anhydride, according to the proto-
col of Mitscher,32 afforded the series of tryptanthrin analogues
as crystalline solids. The oximes (3, 4, 7, and 8) were prepared by
reaction of the corresponding tryptanthrin with hydroxylamine
hydrochloride.33 The oximes were synthesized and purified by
recrystallization in the dark. The tryptanthrins utilized in this
study were dissolved at 1-2 mg/mL (3-8 mM) in dimethyl
sulfoxide (DMSO) and stored at -80 °C. Tryptanthrins 1-10
are known compounds. Structural identities were confirmed via
high resolution DART mass spectroscopy and NMR, while
purity of 95% or greater was attained via multiple recrystalliza-
tions (minimum of 3). Melting points were compared to those
previously reported.
Effect of Tryptanthrins on Cell Growth and Survival. To
determine the effect of tryptanthrins on growth, a colony of
E. coli AS19 harboring pBRF14C was picked from an LB þ
AMP plate, inoculated into LB medium containing 30 μg/mL
ampicillin, and grown overnight at 37 °C without shaking.

3564 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9
Bandekar et al.
Ten millimeter aliquots of a culture in LB, adjusted to OD650
=
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0.05-0.1, were placed in 125 mL flasks. Tryptanthrins were then
added from stock solutions in DMSO (from 0.01 to 20 μg/mL),
and the cultures were grown with shaking at 200 rpm at 37 °C.
During growth, samples were taken at routine intervals, and the
OD650 was measured. For viability measurement, after appro-
priate dilutions in M9 buffer, about 200-400 viable cells were
plated on LB þ AMP plates. Plates were incubated for 24 h at
37 °C before counting. Cell viability was determined as the
number of cells/mL following tryptanthrin treatment divided by
the number of cells/mL without treatment.
For measurement of survival in M9 buffer, an overnight
culture was diluted to 105-106 cells/mL, tryptanthrins were added
to a final concentration of 0.2-40 μM, and the cultures were
incubated for up to 3 days at 37 °C. At various times, appropriate
dilutions of cells in M9 buffer were plated (200-400 cells per
plate) on LB þ AMP plates. Viable cells were counted after 24 h.
Measurement of the Mutagenic Activity of Tryptanthrins in
E. coli. The mutagenic potential of tryptanthrins on E.coli AS19
was assessed using two different mutational assays: rifampicin
resistance25 and the deletion of a 106-bp inverted repeat from the
chloramphenicol acetyltransferase gene in pBRF14C,22 restor-
ing chloramphenicol resistance in E. coli strain AS19. For this,
an overnight culture was diluted in M9 buffer and treated with
tryptanthrins as described above for the measurements of
survival in M9 buffer. The next day survival measurements were
made and 0.5 mL of the cultures was inoculated into 10 mL of
LB þ AMP and grown with shaking at 100 rpm for 24 h. Cells
from this culture were pelleted and resuspended in 1 mL of M9
buffer. Appropriate dilutions were plated on LB þ AMP plates
(200-400 cells per plate) to determine viable cell counts, and
0.1-0.2 mL of the resuspension was plated on multiple LB þ
CHL or LB þ RIF plates to ascertain numbers of CHL or RIF
revertants, respectively. CHL and RIF revertants were counted
after 48 h of incubation at 37 °C.
A second assay for mutagenesis involved measurement of þG
or G frameshift mutations resulting in Lacþ revertants using the
frameshift reporter strains of Cupples et al.26 For this analysis,
cells were grown for 10 generations in the presence of tryptan-
thrin analogues before plating for viable cells on glucose plates
and for Lacþ revertants on lactose plates, as described.26
Alteration of DNA Supercoiling by Tryptanthrin. Plasmid
DNA (0.25 μg of pGEM-3Z, 2743 bp), purified by alkaline lysis
followed by purification from a CsCl-ethidium bromide gra-
dient,24 was incubated with human topoisomerase I (TopoGen,
Inc.), in 25 μL of Topo reaction buffer (10 mM Tris, pH 7.9, 150 mM
NaCl, 0.1 mM spermidine, and 5% glycerol, plus 1 U of topoiso-
merase I) in the presence of tryptanthrins (10-100 μg/mL) for
2 h at 37 °C. In some cases DNA was relaxed by treatment with
topoisomerase I 1 h prior to addition of the tryptanthrins,
followed by an additional 1-2 h of incubation. The reaction
was stopped by the addition of 5 μL of 5% sarkosyl, 25%
glycerol with bromophenol blue dye (0.005%). Then 8-10 μL
aliquots were loaded onto 1.5% agarose gels in Topo TAE
buffer (40 mM Tris-acetate, 5 mM sodium acetate, 1 mM EDTA,
pH 8.3) with or without 2 μg/mL chloroquine (Sigma). DNAs
were separated by electrophoresis at 50 V for 3-4 h. Gels were
then stained with ethidium bromide (0.5 μg/mL) for 15 min and
photographed. The distribution of topoisomers were analyzed
using Kodak 1D image analysis software.
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