Chemical scaffolds with structural similarities to siderophores of nonribosomal peptide-polyketide origin as novel antimicrobials against Mycobacterium tuberculosis and Yersinia pestis

Article abstract of DOI:10.1016/j.bmcl.2011.08.052

Mycobacterium tuberculosis (Mtb) and Yersinia pestis (Yp) produce siderophores with scaffolds of nonribosomal peptide-polyketide origin. Compounds with structural similarities to these siderophores were synthesized and evaluated as antimicrobials against Mtb and Yp under iron-limiting conditions mimicking the iron scarcity these pathogens encounter in the host and under standard iron-rich conditions. Several new antimicrobials were identified, including some with increased potency in the iron-limiting condition. Our study illustrates the possibility of screening compound libraries in both iron-rich and iron-limiting conditions to identify antimicrobials that may selectively target iron scarcity-adapted bacteria and highlights the usefulness of building combinatorial libraries of compounds having scaffolds with structural similarities to siderophores to feed into antimicrobial screening programs.

Full text of DOI:10.1016/j.bmcl.2011.08.052

Bioorganic & Medicinal Chemistry Letters 21 (2011) 6533–6537
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Chemical scaffolds with structural similarities to siderophores of nonribosomal
peptide–polyketide origin as novel antimicrobials against Mycobacterium
tuberculosis and Yersinia pestis
Julian A. Ferreras a, , Akash Gupta b,à, Neal D. Amin a,§, Arijit Basu c, Barij N. Sinha c, Stefan Worgall d,
b,
Venkatesan Jayaprakash c, , Luis E.N. Quadri
⇑
⇑
a Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, NY 10021, USA
b Department of Biology, Brooklyn College, City University of New York, 2900 Bedford Avenue, Brooklyn, NY 11210, USA
c Department of Pharmaceutical Sciences, Birla Institute of Technology, Mesra, Ranchi 835 215, India
d Department of Pediatrics and Genetic Medicine, Weill Medical College of Cornell University, New York, NY 10021, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Mycobacterium tuberculosis (Mtb) and Yersinia pestis (Yp) produce siderophores with scaffolds of nonrib-
osomal peptide–polyketide origin. Compounds with structural similarities to these siderophores were
synthesized and evaluated as antimicrobials against Mtb and Yp under iron-limiting conditions mimick-
ing the iron scarcity these pathogens encounter in the host and under standard iron-rich conditions. Sev-
eral new antimicrobials were identified, including some with increased potency in the iron-limiting
condition. Our study illustrates the possibility of screening compound libraries in both iron-rich and
iron-limiting conditions to identify antimicrobials that may selectively target iron scarcity-adapted bac-
teria and highlights the usefulness of building combinatorial libraries of compounds having scaffolds
with structural similarities to siderophores to feed into antimicrobial screening programs.
Published by Elsevier Ltd.
Received 30 June 2011
Revised 9 August 2011
Accepted 11 August 2011
Available online 26 August 2011
Keywords:
Nonribosomal peptide
Polyketide
Siderophore
Mycobacterium tuberculosis
Yersinia pestis
Antimicrobial compound
Pyrazoline
Hydroxyhydrazine
Mycobacterium tuberculosis (Mtb), the causative agent of tuber-
culosis, and Yersinia pestis (Yp), the etiologic agent of plague, are
bacterial pathogens with serious impacts on global public health.
Multidrug-resistant (MDR) tuberculosis is an emerging pandemic,
and the more recent emergence of extensively drug-resistant
(XDR) tuberculosis poses a new global threat.1 Plague is a re-
emerging disease for which the documented occurrence of MDR
Yp strains and self-transferable Yp plasmids conferring antibiotic
resistance raises concerns about future plague control.2 These grim
scenarios underscore the need for expanding the anti-tuberculosis
and anti-plague drug armamentarium.
scavenge Fe3+ from their microenvironments and transport it into
the cell.3 The Mtb siderophore (mycobactin/carboxymycobactin)
and the Yp siderophore (yersiniabactin) are based on substituted
Many bacteria utilize secreted, small (<1000 Da) Fe3+-chelating
compounds (compds) (Kd < 10ꢀ25 M) called siderophores to
⇑
Corresponding authors. Tel.: +91 651 2275843 (V.J.); +1 718 951 5000x6254
(L.E.N.Q.).
E-mail addresses: venkatesanj@bitmesra.ac.in (V. Jayaprakash), LQuadri@
brooklyn.cuny.edu (L.E.N. Quadri).
Present address: Department of Genetics, Universidad Nacional de Misiones,
Posadas (3300), Misiones, Argentina.
à
Present address: Yale University School of Medicine, New Haven, CT 06510, USA.
Present address: University of California San Diego, School of Medicine, La Jolla,
§
Figure 1. Structures of M. tuberculosis and Y. pestis siderophores.
CA 92093, USA.
0960-894X/$ - see front matter Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2011.08.052

6534
J. A. Ferreras et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6533–6537
scaffolds of nonribosomal peptide–polyketide origin (Fig. 1).4 Stud-
ies in cellular and animal models of infection have established the
relevance of the mycobactin/carboxymycobactin and yersiniabac-
tin siderophore systems in these pathogens.5 The siderophores
are believed to facilitate iron scavenging inside the host, where free
iron is scarce (10ꢀ25 to 10ꢀ15 M) and the pathogens experience and
must adapt to iron-limiting conditions.6 These observations sug-
gest that the Mtb and Yp siderophore systems represent potential
in vivo conditionally essential target candidates for the develop-
ment of alternative therapeutics against tuberculosis and plague.7
We hypothesize that screening compds with structural features
resembling Mtb and Yp siderophores for growth inhibitory activity
against these pathogens may lead to the discovery of novel antimi-
crobial scaffolds. Such novel antimicrobials could illuminate
alternative paths to drug development and/or be useful as small-
molecule tools to assist in the elucidation of new target candidates
for drug development. Compds with structural features resembling
Mtb and Yp siderophores may impair the siderophore systems
(e.g., by inhibiting siderophore biosynthesis or transport) and halt
bacterial growth in the host’s iron-limiting environments. Alterna-
tively, these compds might gain access to the intracellular environ-
ment using siderophore transport systems and inhibit essential
functions unrelated to iron acquisition. Consistent with these
views, we have recently demonstrated potent antimicrobial activ-
ity against Mtb and Yp for novel diaryl-carbothioamide-pyrazoline
derivatives with structural features resembling the hydroxy-
phenyl-oxazoline/thiazoline-containing half of Mtb and Yp
siderophores.8
In an effort to identify additional novel inhibitors of Mtb and Yp
growth, we synthesized and evaluated the antimicrobial activity of
new 3,5-diaryl-substituted pyrazoline (DAP) derivatives (compds
1–22,9a Table 1). In addition, we synthesized and tested the activity
of a group of (2E)-2-benzylidene-N-hydroxyhydrazine carbo(ox/
thio/oximid)-amide (BHHC) derivatives (compds 23–32,9b Table 2)
with hydroxyphenyl-cap functionalities resembling that of the sid-
erophores. The compds were tested for growth inhibitory activity
against Mtb and Yp in iron-limiting media, which mimic the
iron-scarcity condition that the pathogens encounter in the host,
and in standard iron-rich media.10a We also assessed selected
compds for mode of action (bactericidal or bacteriostatic) in iron-
limiting media and for cytotoxicity toward mammalian cells.10b
Testing against Mtb revealed that 17 compds (1, 2, 5, 8–15, 24,
Table 1
3.5-Diarvl-substituted pyrazoline (DAP) derivatives (1–22)
27–31) had IC50s and MICs (3–222 lM range, Table 3) within the
concentration series tested in the iron-limiting medium, GASTD.
Of these 17 compds, 15 (1, 2, 5, 9–12, 14, 15, 24, 27–31) also had
determinable IC50s and MICs (2–132 lM range) in the iron-rich
medium, GASTD+Fe. Examination of IC50GASTD+Fe/IC50GASTD and
MICGASTD+Fe/MICGASTD ratios revealed that the inhibitors had no
noteworthy increased potency in the iron-limiting medium within
the concentration series tested. This suggests that interference
with iron acquisition, or any other bacterial process differentially
required for growth under the iron-limiting condition, is not a
property that significantly contributes to the compds’ antimicro-
bial activity against Mtb. Interestingly, 14 is 5-fold more potent
against Mtb cultured in GASTD+Fe, as judged by MIC values. This
phenomenon might suggest that the mechanism(s) of action of
14 against Mtb might be potentiated by an elevated production
of cytotoxic hydroxyl radicals originated through increased levels
Compound
R
R0
R
1
2
–H
–OH
–OH
–H
–H
–H
3
–H
–OH
–H
4
–OH
–H
5
–OH
–H
6
–OH
–H
Table 2
(2E)-2-Benzylidene-N-hydroxyhydrazine carbo(ox/thio/oximid)-amide (BHHC) deriv-
atives (23–32)
7
–OH
–H
8
–OH
–H
9
–OH
–CH3
–OH
–H
10
11
12
13
14
15
16
17
18
–H
–H
–OH
–H
–OH
–H
Compound
R
R1
R2
R3
–OH
–H
23
24
25
26
27
28
29
30
31
–H
–OH
–H
–OH
–H
–OH
–H
–OH
–H
–OH
–H
–OH
–H
–OH
–OH
–H
–H
–H
–CH,
–CH3
–H
–H
–CH3
–H
@O
@O
@O
@O
@S
@S
@S
–NH
–NH
–Oh
–H
–OH
–H
–OH
–H
–OH
–H
–OH
–H
19
20
21
22
–H
–OH
–H
–OH
–H
32
–OH
–H
–OH

J. A. Ferreras et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6533–6537
6535
Table 3
Antimicrobial activity against M. tuberculosis
a
b
Compound
IC50
(l
M)
Ratio
MIC90
GASTD+Fe
(
lM)
Ratio
Mode of actionc
GASTD+Fe
GASTD
GASTD
DAP series
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
18
37
>16
>8
64
>32
>32
40
50
7
23
26
32
22
8
>4
98
25
46
>31
>8
77
>63
54
42
53
7
44
31
37
47
8
>8
84
1
1
59
66
1
1
BC (2 ꢁ MIC)
125
>16
>8
104
>63
>31
>31
125
16
66
76
>125
31
16
125
>32
>8
208
>63
>63
125
125
16
BC (I ꢁ MIC)
nd
nd
1
nd
nd
1
1
1
1
1
1
0.5
1
nd
1
nd
nd
nd
nd
nd
nd
nd
0.5
nd
nd
nd
1
1
1
1
>l
nd
nd
BC (2 ꢁ MIC)
nd
nd
BC (1 ꢁ MIC)
BC (2 ꢁ MIC)
BC (2 ꢁ MIC)
BC (2 ꢁ MIC)d
BC (2 ꢁ MIC)d
BC (2 ꢁ MIC)
BC (2 ꢁ MIC)
BC (2 ꢁ MIC)
nd
nd
nd
nd
nd
66
125
125
125
16
0.2
1
>4
>8
nd
nd
nd
nd
nd
nd
nd
17
18
19
20
21
22
>500
>63
>31
>31
>31
>31
>500
>63
>63
>63
>63
>63
>16
>16
>3I
>31
>3I
>63
>63
>63
>63
>63
nd
nd
BHHC series
23
24
25
26
27
28
29
30
>16
40
>8
>500
2
33
4
15
43
>8
>16
97
>8
>500
3
31
4
11
43
20
nd
0.4
nd
nd
1
1
1
1
1
>16
97
>8
>500
6
59
9
26
132
>8
>16
146
>8
>500
7
59
10
222
222
>31
0.2
nd
1
nd
nd
1
1
1
1
1
nd
BC (2 ꢁ MIC)
nd
nd
BC (4 ꢁ MIC)
BC (2 ꢁ MIC)
BC (2 ꢁ MIC)
BC (2 ꢁ MIC)
BC (2 ꢁ MIC)
nd
31
32
Isoniazid
nd
1
nd
1
0.09
0.07
0.2
BC (2 ꢁ MIC)
a
IC50 values were calculated from sigmoidal curves fitted to triplicate sets of dose–response data.
MIC90 values are means of triplicates. Ratio, GASTD+Fe/GASTD. All values >l, <l, >0.5, and 60.5 were rounded to the nearest whole number, to 1, and to one significant
b
digit, respectively.
c
Mode of action was evaluated in GASTD in duplicate. The concentration at which each compound was tested for mode of action is indicated between parentheses.
Some bactericidal activity detected, yet below the 99% killing criterion set for defining bactericidal mode of action. BS, bacteriostatic; BC, bactericidal; nd, not determined.
d
of Fenton reaction in the iron-rich medium. Such a potentiation
would be in line with recent findings of Collins and co-workers
regarding antibiotic-induced cell death.16 Alternatively, 14 might
inhibit an oxidative stress protection function(s) more critically
needed in the iron-rich medium. Testing for cytotoxicity at the
2–4 lM, MIC = 6–10 lM, Table 3). These two compds displayed
no significant cytotoxicity in mammalian cells at their respective
MIC values determined against Mtb (Supplementary data,
Fig. S1). Gratifyingly, 27, 29 and other active compds in this series
displayed bactericidal mode of action against Mtb at concentra-
tions of 1.7–4 ꢁ MICGASTD (Table 3).
MIC against Mtb (125 lM) revealed that 14 had no significant cyto-
toxicity at short cell–compd contact time (4 h), yet cell viability
was reduced by 70% relative to untreated controls after prolonged
contact time (24 h) (Supplementary data, Fig. S1).
Testing against Yp revealed that 14 compds (1, 2, 7–9, 11, 13,
14, 19, 27–29, 31, 32; Table 1) reached IC50s (0.04–181 lM range,
Table 4) within the concentration series tested in the iron-limiting
Among the active DAP derivatives, 10 and 15 were the most po-
medium, PMHD. Nine of these compds (1, 11, 13, 14, 27–29, 31, 32)
tent against Mtb (IC50 = 7–4
two compds, only 10 displayed significant cytotoxicity at the MIC
against Mtb (16 M). Compd 10 had a modest impact on cell via-
l
M, MIC = 16
l
M, Table 3). Of these
also reached MICs (0.2–388
had determinable activity in the iron-rich medium, PMHD+Fe
(29: IC50 = 156 M, MIC = 233 M; 30: IC50 = 305 M). Interest-
lM range) in PMHD. Only 29 and 30
l
l
l
l
bility, which was reduced only by 24% after 24 h of cell–compd
contact (Supplementary data, Fig. S1). Encouragingly, these and
most other inhibitors in the DAP derivatives series examined for
mode of action against Mtb were bactericidal (>99% killing relative
to inoculum) at concentrations of 1–2 ꢁ MICGASTD (Table 1). This
finding is significant since bactericidal activity is a desirable prop-
erty in any early lead compd evaluated for antibacterial drug
development programs. It is worth noting that the only two comp-
ds (11 and 12) defined as bacteriostatic in Table 1 showed signifi-
cant bactericidal activity, yet below the 99% killing criterion set in
this study for defining bactericidal mode of action. Among the
compds of the BHHC derivatives series with defined IC50 and MIC
ingly, examination of the IC50PMHD+Fe/IC50PMHD ratios revealed a
number of inhibitors (7, 11, 13, 14, 19, 27, 29, 31, 32) with in-
creased potency (>3-fold) against Yp cultured under iron scarcity.
Compd 32, with >100-fold and >20-fold higher potency in PMHD
based on IC50 and MIC values, respectively, stood out in this group.
The higher potency of these compds in the iron-limiting medium
raises the possibility that interference with an iron acquisition
function, or other function more critically required for growth un-
der the iron-limiting condition, is a property that significantly con-
tributes to the compds’ antimicrobial activity against Yp. One of
the possible mechanisms of action of these compds could be
related to iron-binding properties. An iron-binding ability strong
enough to outcompete the powerful iron chelating capacity of
values, 27 and 29 were the most active against Mtb (IC50
=

6536
J. A. Ferreras et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6533–6537
Table 4
Antimicrobial activity against Y. pestis
a
b
Compound
PMHD+Fe
DAP series
IC50
(
lM)
Ratio
MIC90
PMHD+Fe
(lM)
Ratio
Mode of actionc
PMHD
PMHD
1
2
3
4
5
6
7
8
>8
9
20
nd
>2
nd
nd
nd
nd
>4
>2
nd
nd
>9
nd
>4
>13
nd
nd
nd
nd
>4
nd
nd
nd
>8
31
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
>l
nd
>1
>3
nd
nd
nd
nd
nd
nd
nd
nd
BC (1 ꢁ MIC)
>31
>31
>16
>250
>31
>31
>31
>63
>31
>63
>63
>16
>63
>16
>8
>31
>31
>16
>250
>31
>3l
>31
>63
>31
>63
>63
>16
>63
>16
>8
>31
>31
>16
>250
>31
>31
>31
>250
>63
42
>125
16
21
>16
>8
>31
>3l
nd
>31
>16
>250
>31
8
nd
nd
ml
nd
nd
nd
nd
nd
13
9
181
>62
7
>125
4
5
>16
>8
>31
>31
8
>16
>31
>31
10
11
12
13
14
15
16
17
18
19
20
21
22
BC (1 ꢁ MIC)
nd
BC (2 ꢁ MIC)
BC (2 ꢁ MIC)
nd
nd
nd
nd
nd
nd
nd
nd
>63
>16
>31
>31
>16
>16
>63
>16
>31
>31
>16
>16
>31
>16
>31
>31
BHHC series
23
24
25
26
27
28
29
30
>4
>250
>2
>500
>59
>233
156
305
>500
>4
>16
>250
>4
>500
4
137
61
70
nd
nd
nd
nd
>15
>2
>3
4
>4
>250
>2
>500
>59
>233
233
>500
>500
>4
>16
>250
>4
>500
15
388
116
250
>500
0.2
nd
nd
nd
nd
>4
nd
2
>2
nd
>20
2
nd
nd
nd
nd
BC (2 ꢁ MIC)
BC (1 ꢁ MIC)
BC (2 ꢁ MIC)
BC (2 ꢁ MIC)
nd
31
32
>500
0.04
1
nd
>100
1
BC (2 ꢁ MIC)
Streptomycin
1
2
1
BC (2 ꢁ MIC)
a
IC50 values were calculated from sigmoidal curves fitted to triplicate sets of dose response data.
MIC90 values are means of triplicates. Ratio, PMHD+Fe/PMHD. All values >1, <l, >0.5, and 60.5 were rounded to the nearest whole number, to 1 and to one significant digit,
b
respectively.
c
Mode of action was evaluated in PMHD in duplicate The concentration at which each compound was tested for mode of action is indicated between parentheses. nd, not
determined; US, bacteriostatic; BC, bactericidal.
the bacterial siderophore could lead to sequestration of the traces
of iron in the iron-limiting medium, thus reducing further iron
bioavailability and producing a stronger antimicrobial activity un-
der the iron-scarcity condition. Alternatively, it is possible that
these compds gain intracellular access using the iron-scarcity-
unregulated yersiniabactin’s uptake system,5e therefore reducing
IC50 and MIC values in PMHD.
of action against Yp in PMHD at concentrations of 1–2 ꢁ MICPMHD
.
Under the conditions tested, 29 displayed bactericidal activity,
whereas 27, 28, 31, and 32 were bacteriostatic.
In sum, 20 of 32 compds synthesized and evaluated herein have
detectable antimicrobial against Mtb and/or Yp in at least one con-
dition, iron scarcity or iron sufficient. To our knowledge, these are
novel scaffolds not previously shown to have antimicrobial proper-
ties. Most active compds identified herein have comparable po-
tency in the low and high iron conditions. This finding suggests
that their pharmacological targets are likely to be essential bacte-
rial functions required under both these conditions. In line with
our aforementioned hypothesis, however, some of our compds
have higher potency under the iron-limiting condition. Under this
condition, bacteria depend on siderophores for efficient iron scav-
enging and engage an adaptive response to tailor their physiology
to iron scarcity, thus exposing novel potential in vivo conditional
target candidates.7a Some of these antimicrobials may impair sid-
erophore system functioning as discussed above, a property that
would result in bacteriostatic activity conditional to environmental
iron scarcity (e.g., as seen with 27 and 32 against Yp). Overall, our
study illustrates the possibility of screening compd libraries in
both iron-sufficient and iron-limiting conditions to identify anti-
microbials that may selectively target iron scarcity-adapted bacte-
ria and highlights the usefulness of building combinatorial libraries
of compds having scaffolds with structural similarities to
siderophores to feed antimicrobial screening programs.
Compds 13 (IC50PMHD = 4
lM, MICPMHD = 16
lM) and 14
(IC50PMHD = 5 M, MICPMHD = 21
l
lM) were the most potent among
the active DAP derivatives with detectable activity against Yp
(Table 4). These two compds displayed no significant cytotoxicity
when they were evaluated at their respective MIC values deter-
mined against Yp (Supplementary data, Fig. S1). Testing of these
and two other active DAP derivatives (1, 11) for mode of action
against Yp revealed that the four compds were bacteriostatic at
concentrations of 1–2 ꢁ MICPMHD (Table 4). Among the BHHC
derivatives with defined IC50 and MIC values in at least one condi-
tion (iron limiting or iron rich), 32 stood out due to its remarkable
potency (IC50PMHD = 0.04 lM, MICPMHD = 0.2 lM) against Yp cul-
tured under iron scarcity. Encouragingly, 32 displayed no signifi-
cant cytotoxicity when evaluated in cytotoxicity assays at the
MIC determined against Yp (Supplementary data, Fig. S1). Notably,
the activity of 32 was significantly higher than that of streptomy-
cin (IC50 and MIC ꢂ1
lM), a bactericidal drug used to treat plague
and included herein as an anti-Yp activity reference. Five compds
(27-29, 31, 32) of the BHHC derivatives series were tested for mode

J. A. Ferreras et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6533–6537
6537
18, respectively. Similarly, reaction of sulphonyl chlorides with 1 and 2 in THF
provided 19–22. Compds 23–32 were synthesized as outlined in Scheme 3
(Supplementary data). (b) Compds 23–26 were prepared as reported.14 N-
hydroxy semicarbazides were prepared by reaction of hydroxylamine,
phenylchloroformate and hydrazine hydrate (80%) in moist ether. The N-
hydroxy semicarbazides were then condensed with appropriate aldehyde/
ketone to yield 23–26. Methyl hydrazinecarbodithioate was prepared as
reported15 and condensed with appropriate aldehyde/ketone to produce 27–
29. Compds 30 and 31 were prepared by a strategy similar to that used for 13
Acknowledgments
This work was supported by NIH Grant 1R01AI075092-01A1
(LENQ). LENQ acknowledges the endowment support from Carol
and Larry Zicklin. We are grateful to the Sophisticated Analytical
Instrument Facility (CDRI, Lucknow, India) for providing spectral
data.
and 14.
A slight excess of methyl iodide followed by hydroxylamine to
thiosemicarbazones of appropriate aldehydes provided 30 and 31. Compds 32
was synthesized as we described earlier.15a Reaction of 27 with N-
hydroxypiperidine-4-carboxamide provided 32. The intermediates were
characterized by elemental analysis and FT-IR spectra. Final compds were
characterized through 1H NMR and FAB-MS spectral data. Synthetic
procedures, physicochemical and spectral characteristics of 1–32 are
presented in Supplementary data.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.08.052.
10. (a) Antimicrobial activity was determined in dose–response experiments using
96-well-plate-based, twofold-microdilution assays as reported.7b,8,17 Virulent
Mtb H37Rv was grown in iron-limiting GASTD medium and GASTD
References and notes
supplemented with 100
2082.1+ was grown in iron-limiting PMHD medium and PMHD
lM
FeCl3 (GASTD+Fe).7b,17 Avirulent Yp KIM6-
1. (a) Dye, C. Lancet 2006, 367, 938; (b) Gandhi, N. R.; Moll, A.; Sturm, A. W.;
Pawinski, R.; Govender, T.; Lalloo, U.; Zeller, K.; Andrews, J.; Friedland, G. Lancet
2006, 368, 1575; (c) Zignol, M.; Hosseini, M. S.; Wright, A.; Weezenbeek, C. L.;
Nunn, P.; Watt, C. J.; Williams, B. G.; Dye, C. J. Infect. Dis. 2006, 194, 479.
2. (a) Galimand, M.; Guiyoule, A.; Gerbaud, G.; Rasoamanana, B.; Chanteau, S.;
Carniel, E.; Courvalin, P. N. Engl. J. Med. 1997, 337, 677; (b) Guiyoule, A.;
Gerbaud, G.; Buchrieser, C.; Galimand, M.; Rahalison, L.; Chanteau, S.;
Courvalin, P.; Carniel, E. Emerg. Infect. Dis. 2001, 7, 43.
supplemented with 100 lM
FeCl3 (PMHD+Fe).7b,17 Cultures were started
(OD600 = 0.001) from deferrated culture stocks prepared as reported.7b
Growth was assessed as OD600 after incubation at 37 °C (Mtb: 10 days,
stationary condition; Yp: 26 h, 200 rpm) using a Spectra Max Plus reader
(Molecular Dynamics). Compds were evaluated at up to the highest
concentration permitted by their solubility, with
a 500 lM upper testing
3. (a) Ratledge, C.; Dover, L. G. Annu. Rev. Microbiol. 2000, 54, 881; Drechsel, H.;
Winkelmann, G. Iron chelation and siderophores. In Transition metals in
microbial metabolism; Winkelmann, G., Carrano, C. J., Eds.; Harwood
Accademic: Amsterdam, 1997; p 1; (c) Neilands, J. B. J. Biol. Chem. 1995, 270,
26723; (d) Wooldridge, K. G.; Williams, P. H. FEMS Microbiol. Rev. 1993, 12, 325;
(e) Wandersman, C.; Delepelaire, P. Annu. Rev. Microbiol. 2004, 58, 611.
4. Quadri, L. E. N. Mol. Microbiol. 2000, 37, 1.
5. (a) De Voss, J. J.; Rutter, K.; Schroeder, B. G.; Su, H.; Zhu, Y.; Barry, C. E., III Proc.
Natl. Acad. Sci. U.S.A. 2000, 97, 1252; (b) Rodriguez, G. M.; Smith, I. J. Bacteriol.
2006, 188, 424; (c) Bearden, S. W.; Fetherston, J. D.; Perry, R. D. Infect. Immun.
1997, 65, 1659; (d) Fetherston, J. D.; Bertolino, V. J.; Perry, R. D. Mol. Microbiol.
1999, 32, 289; (e) Fetherston, J. D.; Kirillina, O.; Bobrov, A. G.; Paulley, J. T.;
Perry, R. D. Infect. Immun. 2010, 78, 2045.
6. (a) Jurado, R. L. Clin. Infect. Dis. 1997, 25, 888; (b) Ward, C. G.; Bullen, J. J.;
Rogers, H. J. J. Trauma. 1996, 41, 356.
7. (a) Quadri, L. E. N. Infect. Disord. Drug Targets 2007, 7, 230; (b) Ferreras, J. A.;
Ryu, J. S.; Di Lello, F.; Tan, D. S.; Quadri, L. E. Nat. Chem. Biol. 2005, 1, 29.
8. Stirrett, K. L.; Ferreras, J. A.; Jayaprakash, V.; Sinha, B. N.; Ren, T.; Quadri, L. E.
Bioorg. Med. Chem. Lett. 2008, 18, 2662.
limit, and added as DMSO solutions. DMSO was kept at 0.5% in treated and
DMSO-control cultures. IC50s were calculated from sigmoidal curves fitted to
triplicate dose–response data using KaleidaGraph (Synergy Software) as
reported.7b MIC90s were calculated as the lowest concentration tested that
inhibited growth by P90% relative to DMSO controls. Compds were tested at
up to the maximum multiple of the MIC permitted by their solubility. (b) After
bacterial cultures were treated with compds and incubated as noted above, the
mode of action was evaluated by enumerating CFU/mL after plating serial 10-
fold dilutions of duplicate Mtb and Yp cultures on plates of Middlebrook
7H1118 and plates of TBA,17 respectively. Plates were incubated at 37 °C for
30 days for Mtb and at 30 °C for 3 days for Yp before colony counting.
Cytotoxicity was assessed against HeLa cells in a 96-well plate platform using a
standard ATPLite™ reagent-based cytotoxicity assay (Perkin-Elmer) as
previously reported.8
11. (a) Azarifar, D.; Shaabanzadeh, M. Molecules 2002, 7, 885; (b) Gökhan, N.;
Yesßilada, A.; Uçar, G.; Erol, K.; Bilgin, A. A. Arch. Pharm. Pharm. Med. Chem. 2003,
336, 362; Rao, Y. K.; Fang, S. H.; Tzeng, Y. M. Bioorg. Med. Chem. 2004, 2679, 12.
12. Tai, A. W.; Lien, E. J.; Moore, E. C.; Chun, Y.; Roberts, J. D. J. Med. Chem. 1983, 6,
1326.
9. (a) Compds 1–14 were synthesized from appropriate 20-hydroxy chalcone
derivatives as outlined in Scheme 1 (Supplementary data). Compds 9 and 10
were prepared as we reported earlier.11 1N-acetyl pyrazolines (3 and 4) were
obtained by refluxing appropriate 20-hydroxy chalcones with hydrazine
hydrate (80%) in acetic acid.12 Similarly, 5 and 6 as well as 11 and 12 were
obtained by refluxing appropriate 20-hydroxy chalcones with semicarbazide
hydrochloride/aminoguanidine bicarbonate in methanol for 4–6 h. Compds 7
and 8 were synthesized from 1 and 2, respectively. Ethyl chloroformate was
added to a solution of pyrazolines (1 and 2) in methanol with constant stirring
at room temperature. Triethylamine was added to neutralize the acid liberated.
Hydroxylamine in methanol was then added with stirring at room temperature
for 2 h to obtain 7 and 8. Compds 13 and 14 were synthesized from 9 and 9a,
respectively. A slight excess of methyl iodide was added to 9 and 9a dropwise
with stirring (<10 °C, 1 h). Hydroxylamine in methanol was then added and
stirring continued (22 °C, 30 min) to obtain 13 and 14. Compds 15–22 were
synthesized from appropriate 20-hydroxy chalcone derivatives as outlined in
Scheme 2 (Supplementary data). Compds 15 and 16 were prepared in similar
13. Sharma, T. C.; Saksena, V.; Reddy, N. J. Acta Chim. Acad. Sci. Hungar. 1977, 93,
415.
14. Klayman, D. L.; Bartosevich, J. F.; Griffin, T. S.; Mason, C. J.; Scovill, J. P. J. Med.
Chem. 1979, 22, 855.
15. (a) Bhadaliya, C.; Bunha, M.; Jagrat, M.; Sinha, B. N.; Saiko, P.; Graser, G.;
Szekeres, T.; Raman, G.; Rajendran, P.; Moorthy, D.; Basu, A.; Venkatesan, J.
Bioorg. Med. Chem. Lett. 2010, 20, 3906; (b) Ren, S.; Wang, R.; Komatsu, K.;
Bonaz-Krause, P.; Zyrianov, Y.; McKenna, C. E.; Csipke, C.; Tokes, Z. A.; Lien, E. J.
J. Med. Chem. 2002, 45, 410.
16. (a) Dwyer, D. J.; Kohanski, M. A.; Hayete, B.; Collins, J. J. Mol. Syst. Biol. 2007, 3,
91; (b) Kohanski, M. A.; Dwyer, D. J.; Collins, J. J. Nat. Rev. Microbiol. 2010, 8,
423; (c) Kohanski, M. A.; Dwyer, D. J.; Hayete, B.; Lawrence, C. A.; Collins, J. J.
Cell 2007, 130, 797.
17. Stirrett, K. L.; Ferreras, J. A.; Rossi, S. M.; Moy, R. L.; Fonseca, F. V.; Quadri, L. E.
BMC Microbiol. 2008, 8, 122.
18. (a) Chavadi, S. S.; Edupuganti, U. R.; Vergnolle, O.; Fatima, I.; Singh, S. M.; Soll,
C. E.; Quadri, L. E. J. Biol. Chem. 2011, 286, 24616; (b) Ferreras, J. A.; Stirrett, K.
L.; Lu, X.; Ryu, J. S.; Soll, C. E.; Tan, D. S.; Quadri, L. E. Chem. Biol. 2008, 15, 51.
manner to
isoniazide. Compds 17–22 were prepared from
earlier.13 Reaction of benzoyl chloride with 1 and 2 in pyridine provided 17 and
5
and
6
and by replacing semicarbazide hydrochloride with
1
and as we reported
2