3-Substituted biquinolinium inhibitors of AraC family transcriptional activator VirF from S. flexneri obtained through in situ chemical ionization of 3,4-disubstituted dihydroquinolines

Article abstract of DOI:10.1039/c4ra08384a

During a structure-activity relationship optimization campaign to develop an inhibitor of AraC family transcriptional activators, we discovered an unexpected transformation of a previously reported inhibitor that occurs under the assay conditions. Once pl

Full text of DOI:10.1039/c4ra08384a

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3-Substituted biquinolinium inhibitors of AraC
family transcriptional activator VirF from S. flexneri
obtained through in situ chemical ionization of
3,4-disubstituted dihydroquinolines†
Cite this: RSC Adv., 2014, 4, 39809
Prashi Jain,a Jiaqin Li,b Patrick Porubsky,a Benjamin Neuenswander,c Susan M. Egan,b
d
Jeffrey Aubeacde and Steven Rogers
*
´
During a structure–activity relationship optimization campaign to develop an inhibitor of AraC family
transcriptional activators, we discovered an unexpected transformation of a previously reported inhibitor
that occurs under the assay conditions. Once placed in the assay media, the 3,4-disubstituted
Received 10th July 2014
Accepted 18th August 2014
dihydroquinoline core of the active analogue rapidly undergoes a decomposition reaction to
a
DOI: 10.1039/c4ra08384a
quaternary 3-substituted biquinolinium. Further examination established an SAR for this chemotype while
also demonstrating its resilience to irreversible binding of biologically relevant nucleophiles.
www.rsc.org/advances
inhibition or deletion of AraC family activators has been
demonstrated to ameliorate infections in vitro and in animal
models.8
Introduction
The World Health Organization (WHO) recently published a
warning that microbial resistance to antibiotics has become a
“major threat to public health”.1 A dearth of new FDA approved
antimicrobial medicines coupled with the rapid development of
resistance to available antibiotics is creating a health care void
that necessitates the development of new antimicrobial strate-
gies to combat microbial infectious disease.2 One such strategy
involves targeting non-growth essential virulence factors, which
may avoid triggering resistance mechanism development by not
placing selective pressure on microbes. As an example of this,
the protein members of the AraC family of bacterial transcrip-
tion activators activate expression of genes encoding virulence
factors in various pathogenic bacteria such as Vibrio cholerae
(ToxT),3 enterotoxigenic Escherichia coli (Rns/CfaD) (ETEC),4
Pseudomonas aeruginosa (ExsA)5 and Shigella exneri (VirF),6
while other members of the family regulate bacterial stress
responses or carbon metabolic operons.7 Furthermore,
Our study focuses on VirF, an AraC family transcriptional
activator from Shigella exneri, the causative agent of bacillary
dysentery, or shigellosis.9 This disease affects over 165 million
people worldwide while leading to approximately 1.1 million
deaths annually.10 VirF is necessary for the expression of viru-
lence genes associated with the rst step of S. exneri patho-
genesis, which is rectal, and colonic epithelial cell invasion as
well as the subsequent cell-to-cell dissemination.11 Using cell-
based reporter gene assays with S. exneri and E. coli, coupled
with in vitro DNA-binding assays with puried VirF, we recently
determined that SE-1 (Fig. 1) is an inhibitor of transcription
activation and DNA binding by VirF.12 Furthermore, through
examining mRNA concentrations using real-time quantitative
reverse transcription-PCR (qRT-PCR), it was found that SE-1
reduced the expression of VirF-dependent virulence genes such
as icsS, icsB, ipaB, and virB in S. exneri. This initial study
prompted us to seek analogues of SE-1 with greater potency that
could serve as a lead VirF inhibitor.
In this manuscript, we detail our activity optimization efforts
of SE-1,12 which was originally identied through screening of a
aCenter for Chemical Methodologies and Library Development, The University of
Kansas, 2034 Becker Drive, Lawrence, Kansas, 66047, USA
bDepartment of Molecular Biosciences, University of Kansas, Lawrence, KS, USA
cSpecialized Chemistry Center, The University of Kansas, 2034 Becker Drive, Lawrence,
Kansas 66047, USA
dUniversity of Kansas Center of Biomedical Research Excellence, Center for Cancer
Experimental Therapeutics, 2034 Becker Drive, Lawrence, KS, USA. E-mail:
sarogers@ku.edu
eDepartment of Medicinal Chemistry, University of Kansas, Lawrence, KS, USA
† Electronic supplementary information (ESI) available: Experimental protocols
and structural characterization of target compounds. See DOI:
10.1039/c4ra08384a
Fig. 1 In situ decomposition of SE-1 to biquinolinium 2.
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commercially obtained small molecule library, and show that
SE-1 and related analogues undergo ionization in aqueous
medium to afford a quaternary 3-substituted biquinolinium salt
(Fig. 1). Although the decomposition product is a synthetic
precursor to the 3,4-disubstituted dihydroquinoline core, its
ionization in biological media was unexpected. Thus, we
present evidence here that the biquinolinium salt is the caus-
ative agent responsible for the observed inhibitory activity
toward bacterial AraC family activators, discuss structure–
activity relationships (SARs) associated with this chemotype,
and demonstrate that it is stable in the presence of biologically
relevant nucleophiles.
Scheme 1 Reagents and conditions: (a) PdCl2(PPh3)2, K2CO3, DMF/
H2O, MW, 150 ꢁC; (b) R2Br, MW, 150 ꢁC, 2 h (c) MeNO2, K2CO3, H2O,
dioxane, rt, 4 h.
Results and discussion
active than C4. Briey, various aryl boronic acids were coupled
via Suzuki reaction with 3-bromoquinolines through the treat-
ment with bis(triphenylphosphine)palladium dichloride,
potassium carbonate in a 4 : 1 mixture of dimethylformamide
and water while heating to 150 ꢁC for 30 minutes under
microwave conditions. The resulting 3-substituted quinolines
were subsequently N-substituted via electrophilic attack from
various benzyl and alkyl halides, followed by a nucleophilic
attack at the 4-position of the resulting quinolinium halide
through the treatment with nitromethane under basic condi-
tions in dioxane at room temperature, giving the desired
3,4-disubstituted dihydroquinolines. Both the 3,4-disubstituted
dihydroquinolines and quinolinium halide synthetic interme-
diates were screened for inhibitory activity against VirF.
Initially, seven 3,4-disubstituted dihydroquinolines, three
quinolinium synthetic precursors and a new batch of SE-1 (1)
were synthesized and screened for VirF activator inhibition
(Table 1). Replacing the n-butyl group with a 4-chlorobenzyl (5)
resulted in a signicant loss of activity against VirF activator
inhibition. Replacing the quinoline with other aryl groups such
as 5-methyl pyridine (6), 3-chlorophenyl (7) resulted in
analogues with very modest inhibitory activity, giving IC50
values >100 mM. Diminished activity was also observed when
substituting the nitromethyl of the 1,4-dihydroquinoline with a
phenyl (8) or methyl (11) as well in the same substitution of a
1,2-dihydroquinolines (entries 9 & 10). However, the resynthe-
sized SE-1 (Table 1) repeated the activity observed from the HTS
sample with an IC50 of 11 mM against VirF and >100 mM in the
control strain.
As a rst clue to the instability of these 1,4-dihydroquinoline
cores, we observed that the quaternary amine quinolinium
samples (Table 1, entries 2–4) exhibited the same, if not better
activity against VirF while not affecting the control strain. This
can be observed when comparing Table 1, entries 2 to SE-1 (1),
entries 3 to 5 and entries 4 to E1. Furthermore, the 3,4-disub-
stituted dihydroquinolines (Table 1, SE-1 and entries 5–11)
exhibited notable stability issues during their synthesis because
they readily decomposed if purication attempts were made on
normal phase chromatography with silica gel or reverse phase
chromatography with C18-bonded silica gel. All 3,4-disubsti-
tuted dihydroquinolines had to be puried via recrystallization.
We also noted that upon 1H NMR analysis of various dihy-
droquinoline samples in DMSO-d6 at room temperature two
To gain preliminary SAR information to guide our synthetic
endeavors, we procured and screened 24 commercially available
analogues of SE-1, resulting in three compounds exhibiting
notable activity in comparison to SE-1 in the VirF in vivo dose-
response assay in E. coli (Fig. 2). The assays were performed as
previously described.12 Briey, the analogues were dissolved in
100% dimethyl sulfoxide (DMSO). Bacterial cultures were grown
to an optical density at 600 nm (OD600) of ꢀ0.1 and then mixed
with various concentrations of analogues (nal concentration of
DMSO, 6.2%), induced with 1 mM IPTG (nal concentration) for
3 hours at 37 ꢁC, lysed, and b-galactosidase activity was
measured. Uninduced (no IPTG and no analogues) and unin-
hibited (1 mM IPTG and no analogues) controls were used to
normalize b-galactosidase activity, as described previously.12 To
eliminate from consideration any analogues that nonspeci-
cally inhibited b-galactosidase activity or cell growth, we also
tested these analogues on a control strain that carried a lacZ
reporter fusion (hts-lacZ), with a synthetic promoter that was
repressed by LacI and did not require VirF for activation, as
described previously.12 We found that analogues E1, A2 and C4
inhibited expression of the VirF-activated virB-lacZ fusion to the
same extent as SE-1, with an IC50 of 7 mM, 10 mM and 11 mM
respectively. Other 4-substitutions were assessed such as an
indole, a phenyl ring, a benzimidazole and a napthyl ring in
which all analogues were found to be inactive (full list can be
found in ESI†). Analogues without N-substitution were also
found to not exhibit activator inhibition.
To follow-up on our commercially accessible SAR, a series of
3,4-disubstituted dihydroquinolines were synthesized as shown
in Scheme 1 that was adapted from protocols developed by
Aksenov.13 We focused on the 4-methylnitro substitution
instead of the malonic ester due to potential esterase liability
and instead of the nitrile because E1 was found to be more
Fig. 2 Structures and VirF activator inhibition data for E1 and A2.
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Table 1 First round of synthetic analogues
then every hour on the hour for 48 hours at room temperature.
We found that SE-1 had degraded completely to the corre-
sponding quaternary salt by the 0th time point, or about
30 minutes elapsed time including assay preparation. Given
that the VirF inhibition assays typically run 8–24 hours, the
stability analysis predicted that no detectable concentration of
the 3,4-disubstituted dihydroquinoline species was present
during the assay and that the activity was simply due to the
quinolinium salt. Since most quaternary ammonium salts have
limited pharmaceutical relevance, save topical and oral hygiene
applications due to their hemolytic nature,14 we began exploring
non-quaternary analogues of our most potent VirF activator
inhibitors.
Entry
Structure
VirF IC50 (mM)
Control IC50 (mM)
SE-1/1
—
11
>100
2
11
>100
3
4
5
26
10
80
>100
>100
>100
The 3,4-disubstituted dihydroquinoline core was substituted
with an indole (Table 2, entries 12 and 13), 9-acridone (15) and
4-quinolone (16), resulting in no inhibitory activity towards VirF
or in the control strain assay. Interestingly, we also screened
acridinium (14), pyridinium (17) and triethylammonium (18)
salt analogues that would behave similarly to other cationic
quaternary ammonium chemical detergents such as cetylpyr-
idinium chloride (CPC) to compare the activity proles to our
more active quinolinium compounds. The triethylammonium
analogue exhibited no inhibitory activity in our assays whereas
the acridinium and pyridinium exhibited good to modest, non-
specic activity against both the VirF and control strain, indi-
cating that the activity we see with our quinolinium compounds
is not simply due to detergent effects. This prompted us to
continue optimizing the activity of the quinolinium core.
Various N-substituted and 3-aryl analogues were synthesized
with aim to optimize the quinolinium core (Table 2, entries 19–
24). As seen previously, N-substitution of n-butyl with methyl (4)
gave decreased activity with a 90 mM IC50 value, whereas i-propyl
(19), n-propyl (20), n-hexyl (21) and pent-4-en-1-yl (24) all gave
similar activity to SE-1 with IC50 values of 12 mM, 16 mM, 11 mM
and 11 mM respectively, while not inhibiting the control strain.
Replacing the 3-quinoline substituent with a naphthalene (23)
was also well tolerated and gave an IC50 value of 9 mM against
VirF and >100 mM against the control strain, comparable activity
to the parent compound (Table 1, entry 1). A modest increase in
activity was attained while elongating the N-alkyl chain to
n-nonyl (22); giving an IC50 value of 3 mM against VirF, but
notable selectivity was lost because of control strain inhibition
with an IC50 value of 25 mM. A planktonic bacterial growth curve
analysis of both the VirF and control strains in the presence of
Table 1, entry 6 demonstrated that this compound inhibited the
growth of the bacteria, explaining the increase in activity
6
7
>100
>100
>100
>100
8
No inhibition
>100
No inhibition
>100
9
10
No inhibition
No inhibition
11
>100
>100
days aer the samples had been prepared and analyzed, that a against both strains. This toxic effect on bacterial growth was
signicant reversion to the quinolinium precursor was not seen with the other active compounds.
observed. These observations prompted us to examine (1) the
Given the inherent electrophillic nature of cationic species
latent instability of SE-1 and its analogues, (2) if the observed such quinoliniums, we assessed the stability of some of these
VirF inhibition activity was simply due to the quaternary species analogues towards the thiol based nucleophiles dithiothreitol
and (3) if non-quaternary adducts could be derived that and glutathione to mimic interactions with thiol containing
demonstrated stability and VirF inhibitory activity.
amino acid residues of proteins in a biological system. Briey,
To examine the aqueous stability of the 3,4-disubstituted compounds were dissolved at 10 mM in PBS at pH 7.4 (1% DMSO)
dihydroquinoline analogues, samples were prepared at 10 mM and incubated at room temperature with either no thiol source as
in PBS at pH 7.4 (1% DMSO) and analyzed via auto-sampler by a negative control, 50 mM glutathione (GSH). The mixtures were
RP HPLC/UV/HRMS aer the sample had been prepared and sampled every hour for eight hours for 48 hours and analyzed by
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Table 2 Data
used as a positive control. In both of the quinolinium salts
assayed (entries 2 and 3), no GSH adducts were observed, sug-
gesting these compounds to be relatively stable despite their
expected reactivity, giving this chemotype possible application
toward developing biological probes while suggesting that these
inhibitors may not be acting in a covalent manner.
VirF
IC50 (mM)
Control strain
IC50 (mM)
Entry
12
Structure
No activity
No activity
Conclusions
13
14
No activity
8
No activity
15
Through analysis of SAR data and chemical stability behavior,
we have discovered that our previously reported 3,4-disubsti-
tuted dihydroquinoline inhibitor of AraC transcription activa-
tors,12 readily fragments in assay media to a quinolinium core,
which we ascribe as the active agent against this target. We note
that SE-1 and analogues are available from several commercial
vendors; and the present results suggest that the ionized
precursors may well be responsible for any observed biological
activity for this chemotype. Although we did not increase VirF
activator inhibition potency of this class of compounds, we
expounded the SAR and showed that the activity of the quino-
linium analogues does depend on specic structure and was not
due to generic detergent effects.
15
16
No activity
No activity
No activity
No activity
17
18
>50
>50
Experimental section
All air- and moisture-sensitive reactions were carried out in
ame- or oven-dried glassware under argon atmosphere using
standard gastight syringes, cannula, and septa. Stirring was
achieved with oven-dried magnetic stir bars. Flash column
chromatography was performed with SiO2 from Sorbent Tech-
nology (30930M-25, Silica Gel 60 A, 40ꢂ63 mm) or by using an
automated chromatography instrument with an appropriately
sized column. Thin layer chromatography was performed on
silica gel w/UV254 plates (1624126, sorbent technologies). 1H
and 13C NMR spectra were recorded on instruments operating
at 400 or 500 MHz and 100 or 126 MHz respectively. High-
resolution mass spectrometry (HRMS) spectra were obtained on
an ESI-TOF mass spectrometer. The analytical method utilized a
Waters Aquity BEH C18 column (2.1 ꢃ 50 mm, 1.7 mm) eluting
with a linear gradient of 95% water (modied to pH 9.8 through
addition of NH4OH) to 100% CH3CN at 0.6 mL minꢂ1 ow rate
where purity was determined using UV peak area at 214 nm.
Melting points were determined using an automated apparatus
with digital imaging capability.
No activity
No activity
19
20
21
22
23
12
16
11
3
>100
>100
>100
25
9
>100
General procedure for 4-substituted biquinolines
To a solution of nitromethane (50 mL, 1.0 mmol) in THF (1 mL),
solution of K2CO3 (21.8 mg, 0.15 mmol) in water (0.2 mL) was
added gradually with stirring and the mixture was stirred for 5
min. The 3-(2-quinolyl)quinolinium halide (1a–e) (0.75 mmol)
was added and the mixture stirred for 4 h at 23 ꢁC until a
homogeneous solution was formed. The reaction was moni-
tored by TLC/UPLC. The solution was poured into water (5 mL)
24
11
>100
RP HPLC/UV/HRMS. The masses of potential adducts were and extracted with ethyl acetate (3 ꢃ 5 mL). The organic layers
searched for in the nal samples to determine if any detectable were separated, combined, washed with brine, dried over
adduct formed. Ethacrynic acid, a known Michael acceptor, was Na2SO4, and evaporated in vacuo.
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10-Butyl-40-(nitromethyl)-10,40-dihydro-2,30-biquinoline (SE-
RSC Advances
7.09 (m, 2H), 7.07–6.99 (m, 2H), 6.96 (d, J ¼ 8.0 Hz, 1H), 6.83
(td, J ¼ 7.4, 1.1 Hz, 1H), 4.80 (dd, J ¼ 8.9, 4.1 Hz, 1H), 4.42 (dd,
J ¼ 11.4, 4.1 Hz, 1H), 4.20 (dd, J ¼ 11.4, 8.9 Hz, 1H), 3.81 (ddd,
J ¼ 14.3, 8.2, 5.8 Hz, 1H), 3.61 (ddd, J ¼ 14.8, 8.4, 6.8 Hz, 1H),
1). 1H NMR (400 MHz, chloroform-d) d 7.93 (dd, J ¼ 8.4, 2.3 Hz,
2H), 7.65 (dd, J ¼ 7.9, 1.4 Hz, 1H), 7.58 (ddd, J ¼ 8.5, 6.9, 1.5 Hz,
1H), 7.49 (d, J ¼ 8.8 Hz, 1H), 7.34 (ddd, J ¼ 8.0, 6.9, 1.2 Hz, 1H),
7.29 (s, 1H), 7.24–7.21 (m, 1H), 7.21–7.18 (m, 1H), 6.96 (td, J ¼
7.4, 1.0 Hz, 1H), 6.88 (d, J ¼ 8.5 Hz, 1H), 5.39 (dd, J ¼ 8.2, 4.1 Hz,
1H), 4.70 (dd, J ¼ 10.9, 4.1 Hz, 1H), 4.48 (dd, J ¼ 10.9, 8.2 Hz,
1H), 3.79 (ddd, J ¼ 14.4, 8.5, 5.9 Hz, 1H), 3.55 (ddd, J ¼ 14.9, 8.5,
6.8 Hz, 1H), 1.72 (dqd, J ¼ 15.9, 7.8, 6.0 Hz, 2H), 1.39 (dtd, J ¼
15.1, 7.7, 6.0 Hz, 2H), 0.94 (t, J ¼ 7.3 Hz, 3H). 13C NMR (126
MHz, acetone-d6) d 156.33, 148.99, 139.30, 138.15, 136.21,
130.09, 129.54, 128.90, 128.33, 127.10, 125.71, 123.31, 122.68,
117.46, 113.46, 106.43, 80.95, 51.29, 38.84, 30.90, 20.66, 14.14.
HRMS (ESI) m/z calcd for C23H23N3O2 [M + H]+: 374.1824, found:
374.1856.
1.68–1.57 (m, 2H), 1.39–1.26 (m, 2H), 0.85 (t, J ¼ 7.4 Hz, 3H). 13
C
NMR (126 MHz, acetone-d6) d 141.05, 139.52, 135.22, 134.30,
131.15, 129.85, 129.02, 125.70, 123.92, 122.65, 122.43, 121.45,
113.04, 103.73, 79.85, 50.82, 40.44, 30.88, 20.69, 14.13. HRMS
+
(ESI) m/z calcd for C20H22ClN2O2 [M + H]+: 357.1364, found:
357.1369.
Series of quaternary quinolinium salts
10-Butyl-[2,30-biquinolin]-10-ium bromide (2). 1H NMR (400
MHz, chloroform-d) d 11.33 (d, J ¼ 1.9 Hz, 1H), 10.20 (s, 0H),
9.15 (d, J ¼ 8.6 Hz, 1H), 8.57 (dd, J ¼ 8.2, 1.4 Hz, 1H), 8.46 (d, J ¼
8.6 Hz, 1H), 8.32 (d, J ¼ 8.9 Hz, 1H), 8.25–8.13 (m, 2H), 7.97
(ddd, J ¼ 8.1, 7.0, 0.9 Hz, 1H), 7.90 (d, J ¼ 6.7 Hz, 0H), 7.81 (ddd,
J ¼ 8.5, 6.8, 1.4 Hz, 1H), 7.63 (ddd, J ¼ 8.1, 6.8, 1.2 Hz, 1H), 5.68
(t, J ¼ 7.6 Hz, 2H), 2.24–2.12 (m, 2H), 1.74–1.64 (m, 2H), 1.06 (t, J
¼ 7.3 Hz, 3H). 13C NMR (126 MHz, DMSO) d 150.59, 148.87,
147.35, 144.11, 138.34, 137.17, 136.10, 132.31, 131.52, 130.91,
130.35, 129.65, 129.08, 128.17, 127.86, 127.65, 119.07, 118.87,
10-(4-Chlorobenzyl)-40-(nitromethyl)-10,40-dihydro-2,30-biqui-
noline (5). 1H NMR (400 MHz, acetone-d6) d 8.16 (d, J ¼ 9.0 Hz,
1H), 8.02–7.96 (m, 2H), 7.92 (d, J ¼ 8.9 Hz, 1H), 7.85 (dd, J ¼ 7.9,
1.4 Hz, 1H), 7.71 (ddd, J ¼ 8.5, 6.9, 1.5 Hz, 1H), 7.50–7.35 (m,
6H), 7.21–7.15 (m, 1H), 7.04 (td, J ¼ 7.5, 1.1 Hz, 1H), 6.95 (d, J ¼
8.1 Hz, 1H), 5.56 (dd, J ¼ 7.7, 4.1 Hz, 1H), 5.26–5.08 (m, 2H), 4.93
(dd, J ¼ 11.0, 4.1 Hz, 1H), 4.76 (dd, J ¼ 11.0, 7.7 Hz, 1H). 13C
NMR (126 MHz, acetone-d6) d 156.24, 148.93, 139.33, 138.61,
137.70, 136.35, 133.42, 130.18, 130.17, 129.63, 129.60, 129.24,
128.83, 127.21, 125.92, 123.68, 122.70, 117.52, 114.23, 107.43,
81.31, 54.57, 38.69. HRMS (ESI) m/z calcd for C26H21ClN3O2 [M +
H]+: 442.1244, found: 442.1288.
+
57.68, 31.69, 19.24, 13.50. HRMS (ESI) m/z calcd for C22H21N2
[M]+: 313.1699, found: 313.1695.
10-(4-Chlorobenzyl)-[2,30-biquinolin]-10-ium bromide (3). 1H
NMR (400 MHz, DMSO-d6) d 10.67 (d, J ¼ 2.1 Hz, 1H), 10.24 (s,
1H), 8.79 (d, J ¼ 8.6 Hz, 1H), 8.67 (d, J ¼ 7.8 Hz, 1H), 8.58 (d, J ¼
8.6 Hz, 1H), 8.49 (d, J ¼ 8.9 Hz, 1H), 8.30–8.20 (m, 2H), 8.16 (d, J
¼ 8.1 Hz, 1H), 8.10 (t, J ¼ 7.6 Hz, 1H), 7.95 (ddd, J ¼ 8.4, 6.8, 1.5
Hz, 1H), 7.77 (t, J ¼ 7.6 Hz, 1H), 7.62–7.43 (m, 3H), 6.60 (s, 2H).
13C NMR (126 MHz, DMSO) d 150.58, 150.05, 147.32, 144.94,
138.36, 137.18, 136.21, 133.39, 132.98, 132.64, 131.66, 130.94,
130.46, 129.86, 129.31, 129.06, 129.00, 128.19, 127.90, 127.69,
10-Butyl-10,40-dihydro-[2,30-biquinoline]-40-carbonitrile (C1).
1H NMR (400 MHz, acetone-d6) d 8.17 (d, J ¼ 8.7 Hz, 1H), 8.04–
7.97 (m, 1H), 7.92 (d, J ¼ 8.9 Hz, 1H), 7.90 (s, 1H), 7.86 (dd, J ¼
8.1, 1.5 Hz, 1H), 7.71 (ddd, J ¼ 8.5, 6.9, 1.5 Hz, 1H), 7.61 (dd, J ¼
7.6, 1.5 Hz, 1H), 7.48 (ddd, J ¼ 8.1, 6.9, 1.2 Hz, 1H), 7.40 (ddd, J
¼ 8.6, 7.3, 1.6 Hz, 1H), 7.23 (d, J ¼ 7.6 Hz, 1H), 7.16 (td, J ¼ 7.5,
1.1 Hz, 1H), 5.92 (s, 1H), 4.12–4.00 (m, 1H), 3.91 (ddd, J ¼ 14.9,
8.3, 6.9 Hz, 1H), 1.86–1.74 (m, 2H), 1.52 (dtd, J ¼ 11.9, 7.3, 3.4
Hz, 2H), 1.01 (t, J ¼ 7.4 Hz, 3H). 13C NMR (100 MHz, acetone-d6)
d 155.52, 148.85, 138.62, 137.63, 136.38, 131.21, 130.21, 129.99,
129.55, 128.41, 127.19, 125.96, 123.86, 121.63, 118.39, 117.09,
114.50, 103.64, 51.50, 30.99, 30.50, 20.52, 14.10. HRMS (ESI) m/z
calcd for C23H22N3 [M + H]+: 340.1808, found: 340.1807.
1-Butyl-3-(6-methylpyridin-2-yl)-4-(nitromethyl)-1,4-dihy-
+
119.34, 118.93, 59.79. HRMS (ESI) m/z calcd for C25H18ClN2
[M]+: 381.1153, found: 381.1158.
10-Methyl-[2,30-biquinolin]-10-ium iodide (4). Obtained as a
yellow solid. 1H NMR (400 MHz, DMSO-d6) d 10.40 (s, 1H), 10.12
(s, 1H), 8.76 (d, J ¼ 8.6 Hz, 1H), 8.62 (dd, J ¼ 12.2, 8.4 Hz, 2H),
8.52 (d, J ¼ 8.8 Hz, 1H), 8.36 (ddd, J ¼ 8.7, 7.0, 1.6 Hz, 1H), 8.24
(d, J ¼ 8.5 Hz, 1H), 8.15 (dt, J ¼ 7.8, 3.6 Hz, 2H), 7.95 (ddd, J ¼
8.6, 6.8, 1.6 Hz, 1H), 7.76 (t, J ¼ 7.5 Hz, 1H), 4.83 (s, 3H). HRMS
(ESI) m/z calcd for C19H15N2+ [M]+: 271.1230, found: 271.1226.
1-Nonylpyridin-1-ium bromide (17). 1H NMR (500 MHz,
chloroform-d) d 9.53 (d, J ¼ 5.6 Hz, 2H), 8.54 (t, J ¼ 7.8 Hz, 1H),
8.24–8.07 (m, 2H), 5.02 (t, J ¼ 7.5 Hz, 2H), 2.05 (p, J ¼ 7.5 Hz,
2H), 1.41–1.17 (m, 12H), 0.86 (t, J ¼ 7.0 Hz, 3H). 13C NMR
(126 MHz, CDCl3) d 145.16, 145.12, 128.46, 62.17, 32.06, 31.76,
29.29, 29.14, 29.06, 26.06, 22.61, 14.10. HRMS (ESI) m/z calcd for
1
droquinoline (6). H NMR (400 MHz, acetone-d6) d 7.42 (s, 1H),
7.39 (d, J ¼ 7.7 Hz, 1H), 7.27 (d, J ¼ 8.1 Hz, 1H), 7.15–7.04 (m, 2H),
6.96 (d, J ¼ 8.1 Hz, 1H), 6.84 (dd, J ¼ 7.4, 1.1 Hz, 1H), 6.80 (d, J ¼
7.6 Hz, 1H), 5.04 (dd, J ¼ 8.6, 4.2 Hz, 1H), 4.61 (dd, J ¼ 10.9, 4.1
Hz, 1H), 4.29 (dd, J ¼ 10.9, 8.6 Hz, 1H), 3.82 (ddd, J ¼ 14.2, 8.0, 5.8
Hz, 1H), 3.59 (ddd, J ¼ 14.9, 8.3, 6.9 Hz, 1H), 2.35 (s, 3H), 1.68–
1.52 (m, 2H), 1.38–1.26 (m, 2H), 0.85 (t, J ¼ 7.4 Hz, 3H). 13C NMR
(126 MHz, acetone-d6) d 158.09, 155.89, 150.29, 139.59, 138.35,
137.19, 135.90, 133.94, 130.59, 130.03, 129.55, 128.82, 127.80,
123.21, 122.83, 122.27, 119.55, 118.60, 114.62, 113.16, 105.69,
80.68, 51.02, 39.21, 30.90, 24.76, 20.66, 14.13. HRMS (ESI) m/z
calcd for C20H24N3O2 [M + H]+: 338.1863, found: 338.1855.
1-Butyl-3-(3-chlorophenyl)-4-(nitromethyl)-1,4-dihydroquino-
line (7). 1H NMR (400 MHz, acetone-d6) d 7.45 (t, J ¼ 2.0 Hz, 1H),
7.38 (ddd, J ¼ 8.0, 2.0, 1.0 Hz, 1H), 7.21 (t, J ¼ 8.0 Hz, 1H), 7.19–
C
14H24N+ [M]+: 206.1903, found: 206.1921.
N,N,N-Triethylnonan-1-aminium bromide (18). 1H NMR
(400 MHz, DMSO-d6) d 3.23 (q, J ¼ 7.2 Hz, 6H), 3.11 (dd, J ¼ 7.8,
4.1 Hz, 2H), 1.65–1.49 (m, 2H), 1.34–1.23 (m, 12H), 1.20–1.14
(m, 9H), 0.92–0.81 (m, 3H). 13C NMR (126 MHz, DMSO) d 55.94,
51.89, 45.65, 38.79, 38.66, 31.20, 28.76, 28.48, 25.76, 22.05,
20.87, 13.93, 7.12. HRMS (ESI) m/z calcd for C15H34N+ [M]+:
228.2686, found: 228.2708.
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10-Isopropyl-[2,30-biquinolin]-10-ium iodide (19). 1H NMR
(400 MHz, DMSO-d6) d 10.19 (d, J ¼ 1.9 Hz, 1H), 10.12 (d, J ¼ 1.7
Hz, 1H), 8.82 (d, J ¼ 9.3 Hz, 1H), 8.76 (d, J ¼ 8.6 Hz, 1H), 8.66
(dd, J ¼ 8.3, 1.5 Hz, 1H), 8.58 (d, J ¼ 8.6 Hz, 1H), 8.36 (ddd, J ¼
8.8, 7.1, 1.6 Hz, 1H), 8.27 (d, J ¼ 8.5 Hz, 1H), 8.19–8.10 (m, 3H),
7.94 (ddd, J ¼ 8.4, 6.9, 1.5 Hz, 1H), 7.76 (ddd, J ¼ 8.1, 6.8, 1.2 Hz,
1H), 6.08–5.95 (m, 1H), 1.89 (d, J ¼ 6.5 Hz, 6H). 13C NMR (126
MHz, DMSO) d 150.64, 147.30, 144.00, 138.30, 136.11, 132.32,
131.74, 130.84, 130.25, 129.73, 129.25, 128.67, 128.11, 127.87,
127.07, 124.51, 118.86, 57.53, 31.69, 19.30, 13.51. HRMS (ESI) m/
z calcd for C23H22N+ [M]+: 312.1747, found: 312.1754.
10-(Pent-4-en-1-yl)-[2,30-biquinolin]-10-ium bromide (24).
1
Obtained as a offwhite solid. H NMR (400 MHz, DMSO-d6) d
10.41 (d, J ¼ 2.0 Hz, 1H), 10.17–10.11 (m, 1H), 8.77 (d, J ¼ 8.7 Hz,
1H), 8.68 (dd, J ¼ 16.9, 8.6 Hz, 2H), 8.53 (d, J ¼ 8.6 Hz, 1H), 8.35
(t, J ¼ 7.8 Hz, 1H), 8.25 (d, J ¼ 8.3 Hz, 1H), 8.14 (t, J ¼ 7.7 Hz,
3H), 7.95 (t, J ¼ 7.3 Hz, 1H), 7.77 (dd, J ¼ 8.3, 6.9 Hz, 1H), 6.05–
5.78 (m, 1H), 5.27 (d, J ¼ 7.6 Hz, 2H), 5.18–5.09 (m, 1H), 5.06 (d, J
¼ 10.8 Hz, 1H), 2.35–2.28 (m, 2H), 2.25–2.14 (m, 2H). 13C NMR
(126 MHz, DMSO) d 150.58, 149.21, 149.08, 147.35, 144.16,
138.35, 137.65, 137.18, 137.10, 136.13, 132.28, 131.54, 130.92,
130.36, 129.65, 129.10, 128.17, 127.95, 119.04, 118.85, 115.87,
127.67, 119.03, 118.70, 21.94. HRMS (ESI) m/z calcd for
+
C
21H19N2 [M]+: 299.1543, found: 299.1542.
10-Propyl-[2,30-biquinolin]-10-ium bromide (20). 1H NMR (400
MHz, DMSO-d6) d 10.42 (d, J ¼ 2.0 Hz, 1H), 10.14 (s, 1H), 8.77 (d,
J ¼ 8.7 Hz, 1H), 8.73 (d, J ¼ 8.9 Hz, 1H), 8.66 (d, J ¼ 7.1 Hz, 1H),
8.53 (d, J ¼ 8.7 Hz, 1H), 8.34 (ddd, J ¼ 8.6, 6.9, 1.5 Hz, 1H), 8.25
(d, J ¼ 8.4 Hz, 1H), 8.14 (t, J ¼ 8.0 Hz, 2H), 7.94 (ddd, J ¼ 8.5, 6.9,
1.5 Hz, 1H), 7.76 (t, J ¼ 7.8 Hz, 1H), 5.23 (t, J ¼ 7.7 Hz, 2H), 2.12
(h, J ¼ 7.4 Hz, 2H), 1.09 (t, J ¼ 7.3 Hz, 3H). 13C NMR (126 MHz,
DMSO) d 150.60, 148.90, 147.35, 144.14, 138.35, 137.20, 136.07,
132.27, 131.51, 130.91, 130.36, 129.63, 129.08, 128.17, 127.86,
127.66, 119.10, 118.87, 59.03, 23.17, 10.63. HRMS (ESI) m/z calcd
57.46, 29.89, 28.52. HRMS (ESI) m/z calcd for C23H21N2 [M]+:
+
325.1699, found: 325.1696.
Miscellaneous scaffolds
10-Butyl-40-phenyl-10,40-dihydro-2,30-biquinoline (8).15 1H
NMR (400 MHz, acetone-d6) d 8.22 (d, J ¼ 8.7 Hz, 1H), 8.07 (dd, J
¼ 8.3, 1.0 Hz, 1H), 8.04 (d, J ¼ 8.8 Hz, 1H), 7.88 (dd, J ¼ 8.1, 1.6
Hz, 1H), 7.74 (ddd, J ¼ 8.4, 6.9, 1.5 Hz, 1H), 7.66 (s, 1H), 7.57–
7.49 (m, 3H), 7.25 (dd, J ¼ 7.4, 1.6 Hz, 1H), 7.21–7.07 (m, 4H),
6.72–6.63 (m, 2H), 6.57 (s, 1H), 3.55–3.45 (m, 1H), 3.43–3.28 (m,
1H), 1.78–1.63 (m, 1H), 1.62–1.49 (m, 1H), 1.44 (h, J ¼ 7.3 Hz,
2H), 0.94 (t, J ¼ 7.3 Hz, 3H). 13C NMR (125 MHz, acetone-d6) d
155.95, 148.44, 145.66, 144.60, 136.68, 134.47, 131.35, 130.33,
130.04, 129.76, 128.85, 128.40, 128.08, 128.00, 127.91, 127.15,
127.00, 122.76, 118.88, 116.96, 111.46, 62.76, 49.76, 20.85,
+
for C21H19N2 [M]+: 299.1543, found: 299.1542.
1
10-Hexyl-[2,30-biquinolin]-10-ium bromide (21). H NMR (500
MHz, DMSO-d6) d 10.41 (d, J ¼ 2.0 Hz, 1H), 10.12 (s, 0H), 8.76 (d,
J ¼ 8.6 Hz, 1H), 8.70 (d, J ¼ 9.0 Hz, 1H), 8.65 (d, J ¼ 8.0 Hz, 1H),
8.52 (d, J ¼ 8.6 Hz, 1H), 8.34 (ddd, J ¼ 8.8, 7.0, 1.5 Hz, 1H), 8.24
(d, J ¼ 8.4 Hz, 1H), 8.18–8.09 (m, 2H), 7.94 (ddd, J ¼ 8.4, 6.8, 1.5
Hz, 1H), 7.76 (ddd, J ¼ 8.0, 6.7, 1.2 Hz, 1H), 5.25 (t, J ¼ 7.9 Hz,
2H), 2.07 (p, J ¼ 7.6 Hz, 2H), 1.53 (p, J ¼ 7.3 Hz, 2H), 1.42–1.27
(m, 6H), 0.90 (t, J ¼ 6.9 Hz, 3H). 13C NMR (126 MHz, DMSO) d
150.60, 148.90, 147.36, 144.11, 138.35, 137.16, 136.11, 132.31,
131.52, 130.92, 130.35, 129.64, 129.07, 128.18, 127.87, 127.66,
119.07, 118.88, 57.83, 30.71, 29.70, 25.46, 21.95, 13.86. HRMS
(ESI) m/z calcd for C24H25N2+ [M]+: 341.2012, found: 341.2013.
10-Nonyl-[2,30-biquinolin]-10-ium bromide (22). 1H NMR (400
MHz, DMSO-d6) d 10.42 (d, J ¼ 1.8 Hz, 1H), 10.14–10.11 (d, J ¼
1.54 Hz, 1H), 8.77 (d, J ¼ 8.6 Hz, 1H), 8.70 (d, J ¼ 9.0 Hz, 1H), 8.66
(d, J ¼ 8.3 Hz, 1H), 8.53 (d, J ¼ 8.7 Hz, 1H), 8.34 (ddd, J ¼ 8.7, 7.0,
1.4 Hz, 1H), 8.24 (d, J ¼ 8.5 Hz, 1H), 8.14 (t, J ¼ 8.2 Hz, 2H), 7.94
(ddd, J ¼ 8.4, 6.9, 1.4 Hz, 1H), 7.76 (ddd, J ¼ 8.1, 7.0, 1.1 Hz, 1H),
5.26 (t, J ¼ 7.8 Hz, 2H), 2.08 (p, J ¼ 9.5, 8.6 Hz, 2H), 1.51 (tt, J ¼
9.7, 5.3 Hz, 2H),1.35–1.39 (m, 2H) 1.32–1.18 (m, 6H), 0.85 (t, J ¼
7.1 Hz, 3H). 13C NMR (126 MHz, DMSO) d 150.59, 148.88, 147.35,
144.09, 138.34, 137.15, 136.09, 132.29, 131.51, 130.90, 130.34,
129.63, 129.06, 128.17, 127.86, 127.65, 119.08, 118.88, 57.81,
31.20, 29.70, 28.82, 28.52, 25.77, 22.04, 13.91. HRMS (ESI) m/z
calcd for C27H31N2+ [M]+: 383.2482, found: 383.2464.
+
14.24. HRMS (ESI) m/z calcd for C28H27N2 [M + H]+: 391.2169,
found: 391.2187.
10-Butyl-20-phenyl-10,20-dihydro-2,30-biquinoline (9).15 1H
NMR (400 MHz, acetone-d6) d 8.01 (d, J ¼ 8.5 Hz, 1H), 7.95–7.87
(m, 2H), 7.78–7.72 (m, 2H), 7.62 (ddd, J ¼ 8.4, 6.9, 1.5 Hz, 1H),
7.51 (dd, J ¼ 8.2, 1.4 Hz, 2H), 7.42–7.31 (m, 2H), 7.21–7.08 (m,
4H), 7.06–6.97 (m, 1H), 6.94 (td, J ¼ 7.4, 1.2 Hz, 1H), 5.90 (s, 1H),
4.12–3.99 (m, 1H), 3.87 (dt, J ¼ 14.8, 7.6 Hz, 1H), 1.91–1.79 (m,
2H), 1.63–1.48 (m, 2H), 1.04 (t, J ¼ 7.4 Hz, 3H). HRMS (ESI) m/z
calcd for C28H27N2+ [M + H]+: 391.2169, found: 391.2187.
10-Butyl-20-methyl-10,20-dihydro-2,30-biquinoline
(10).
Synthesized using a reported procedure.15 1H NMR (400 MHz,
acetone-d6) d 8.14 (d, J ¼ 8.9 Hz, 1H), 7.92 (d, J ¼ 8.8 Hz, 1H),
7.87 (d, J ¼ 8.6 Hz, 1H), 7.77 (d, J ¼ 6.7 Hz, 1H), 7.60 (ddd, J ¼
8.7, 7.2, 1.7 Hz, 1H), 7.41 (ddd, J ¼ 8.6, 7.1, 1.1 Hz, 1H), 7.36 (s,
1H), 7.08–6.96 (m, 2H), 6.57 (d, J ¼ 8.2 Hz, 1H), 6.49 (td, J ¼ 7.3,
1.0 Hz, 1H), 5.25 (q, J ¼ 6.2 Hz, 1H), 3.54–3.43 (m, 1H), 3.29–3.11
(m, 1H), 1.63–1.53 (m, 2H), 1.38–1.27 (m, 2H), 1.06 (d, J ¼ 6.3
1-Butyl-3-(naphthalen-2-yl)quinolin-1-ium bromide (23). 1H
NMR (500 MHz, DMSO-d6) d 10.18 (d, J ¼ 2.1 Hz, 1H), 9.80 (d, J
¼ 1.9 Hz, 1H), 8.66 (t, J ¼ 4.5 Hz, 2H), 8.54 (d, J ¼ 6.6 Hz, 0H),
8.29 (ddd, J ¼ 8.8, 7.0, 1.5 Hz, 1H), 8.24 (d, J ¼ 8.7 Hz, 1H), 8.19
(dd, J ¼ 8.5, 1.9 Hz, 1H), 8.14–8.03 (m, 3H), 7.73–7.63 (m, 2H),
5.19 (t, J ¼ 7.8 Hz, 2H), 2.07 (p, J ¼ 7.7 Hz, 1H), 1.53 (h, J ¼ 7.4
Hz, 2H), 0.99 (t, J ¼ 7.3 Hz, 3H). 13C NMR (126 MHz, DMSO) d
148.82, 143.37, 136.27, 135.36, 133.52, 133.09, 132.98, 130.93,
130.80, 130.20, 129.82, 129.26, 128.38, 127.80, 127.54, 127.28,
Hz, 3H), 0.83 (t, J ¼ 7.4 Hz, 3H). HRMS (ESI) m/z calcd for
+
C
23H24N2 [M]+: 328.1939, found: 328.1873.
10-Butyl-40-methyl-10,40-dihydro-2,30-biquinoline (11). 1H
NMR (400 MHz, acetone-d6) d 7.92 (d, J ¼ 9.0 Hz, 1H), 7.77 (dd, J
¼ 8.3, 1.0 Hz, 1H), 7.69–7.61 (m, 2H), 7.50 (ddd, J ¼ 8.4, 6.9, 1.5
Hz, 1H), 7.47 (s, 1H), 7.26 (ddd, J ¼ 8.1, 6.9, 1.2 Hz, 1H), 7.15
(dd, J ¼ 7.4, 1.6 Hz, 1H), 7.04 (ddd, J ¼ 8.6, 7.3, 1.6 Hz, 1H), 6.91–
6.80 (m, 2H), 4.52 (q, J ¼ 6.8 Hz, 1H), 3.87–3.76 (m, 1H), 3.62
(ddd, J ¼ 14.8, 8.2, 7.0 Hz, 1H), 1.72–1.56 (m, 2H), 1.40–1.29 (m,
2H), 1.15 (d, J ¼ 6.8 Hz, 3H), 0.86 (t, J ¼ 7.4 Hz, 3H). 13C NMR
39814 | RSC Adv., 2014, 4, 39809–39816
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(125 MHz, acetone-d6) d 155.79, 148.80, 145.19, 136.74, 136.10, were from three independent experiments with two replicates in
130.87, 130.32, 130.12, 129.54, 128.46, 127.94, 126.91, 126.79, each experiment.
123.49, 118.66, 116.98, 112.44, 54.67, 49.54, 30.92, 20.92, 17.54,
E. coli growth experiments. To test whether the compounds
14.28. HRMS (ESI) m/z calcd for C23H25N2 [M + H]+: 329.2012, in this study had any impact on the growth of the bacterial
found: 329.1991. strains used for in vivo dose-response assays (SME4382 and
+
2-(1-butyl-1H-indol-3-yl)quinoline (12). Synthesized using a SME3359), the growth rate in the presence and absence of the
reported procedure.16 1H NMR (400 MHz, chloroform-d) d 8.87– compounds were compared using a procedure described
8.58 (m, 1H), 8.17 (dd, J ¼ 8.3, 1.1 Hz, 1H), 8.14 (d, J ¼ 8.5 Hz, previously, except the bacterial cultures were grown in TY broth.
1H), 7.87 (s, 1H), 7.85 (d, J ¼ 8.6 Hz, 1H), 7.79 (dd, J ¼ 8.1, 1.4 Briey, the cells were grown at 37 ꢁC in TY broth plus 1 mM
Hz, 1H), 7.71 (ddd, J ¼ 8.4, 6.9, 1.5 Hz, 1H), 7.51–7.47 (m, 1H), IPTG (nal concentration), in 24-well microtiter plate, either
7.47–7.41 (m, 1H), 7.37–7.31 (m, 2H), 4.24 (t, J ¼ 7.1 Hz, 2H), with compounds (44 mM, 6.2% DMSO) or with DMSO (6.2%)
2.01–1.87 (m, 2H), 1.53–1.36 (m, 2H), 1.00 (t, J ¼ 7.4 Hz, 3H). alone, in a PowerWave XS plate reader (BioTek Instruments).
HRMS (ESI) m/z calcd for C21H21N2+ [M + H]+: 301.1699, found:
Stability analysis. Compounds were dissolved at 10 mM in
301.1704.
PBS at pH 7.4 (1% DMSO) and incubated at room temperature
10-Butylacridin-9(10H)-one (15). Synthesized using reported with either no thiol source as a negative control, 50 mM gluta-
procedure.17 1H NMR (400 MHz, chloroform-d) d 8.61 (dd, J ¼ thione (GSH). The mixtures were sampled every hour for eight
8.0, 1.7 Hz, 2H), 7.75 (ddd, J ¼ 8.7, 7.0, 1.7 Hz, 2H), 7.52 (d, J ¼ hours or every 8 hours for 48 hours and analyzed by RP HPLC/
8.7 Hz, 2H), 7.36–7.27 (m, 2H), 4.45–4.22 (m, 2H), 2.01–1.88 (m, UV/HRMS. The analytical RP HPLCUV/HRMS system utilized
2H), 1.62 (h, J ¼ 7.4 Hz, 2H), 1.11 (t, J ¼ 7.4 Hz, 3H). 13C NMR for the analysis was a Waters Acquity system with UV-detection
(100 MHz, CDCl3) d 177.98, 141.82, 133.85, 128.01, 122.52, and mass-detection (Waters LCT Premier). The analytical
121.18, 114.55, 45.98, 29.23, 20.20, 13.86. HRMS (ESI) m/z calcd method conditions included a Waters Acquity HSS T3 C18
for C17H18NO+ [M + H]+: 252.1383, found: 252.1380.
column (2.1 ꢃ 50 mm, 1.8 mm) and elution with a linear
gradient of 99% water to 100% CH3CN at 0.6 mL minꢂ1 ow
rate. Peaks on the 214 nm chromatographs were integrated
using the Waters OpenLynx soware. Absolute areas under the
curve were compared at each time point to determine relative
Antibacterial activity
Bacterial strains, plasmids, and growth conditions. The percent compound remaining. The masses of potential adducts
bacterial strains and plasmids used in this study were described were searched for in the nal samples to determine if
previously. Briey, strain SME4382 carries a single-copy virB- any detectable adduct formed. All samples were prepared in
lacZ reporter fusion in the chromosome and a plasmid duplicate. Ethacrynic acid, a known Michael acceptor, was used
pHG165virF, expressing VirF protein. A control strain SME3359 as a positive control and was tested in both PBS and PBS/
carries a lacZ reporter gene under the control of a synthetic acetonitrile (1/1).
promoter that is repressed by LacI, and a plasmid that expresses
LacI protein. The bacterial cultures for in vivo dose-response
assays were grown in 3-(N-morpholino)propanesulfonic acid
Acknowledgements
(MOPS)-buffered minimal medium. Cultures for growth rate Research reported in this publication was supported by two
experiments were grown in tryptone-yeast extract (TY) broth Institutional Development Awards (IDeA) from the National
(0.8% Difco tryptone, 0.5% Difco yeast extract, and 0.5% NaCl Institute of General Medical Sciences of the National Institutes
[pH 7.0]). Ampicillin (200 mg mLꢂ1), and isopropyl-b-D-thio- of Health (8P30GM103495 and P30GM103326, SME) and the
galactopyranoside (IPTG) were added as indicated. All cultures NIH University of Kansas Chemical Methodology and Library
ꢁ
were grown at 37 C with aeration, unless otherwise noted.
Development Center funded by the NIH Institute of General
In vivo dose-response experiments. Twenty-four commer- Medical Sciences (P50GM069663).
cially available compounds were obtained from Sigma Aldrich
(St. Louis, MO). In vivo dose-response assays were performed as
previously described.12 In brief, each analogue was dissolved in
References and notes
100% dimethyl sulfoxide (DMSO). Bacterial cultures of
SME4382 and SME3359 were grown to an optical density at 600
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induced with 1 mM IPTG (nal concentration) for 3 hours at 37
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duced (no IPTG and no analogues) and uninhibited (1 mM IPTG
and no analogues) controls were included for each of the virB-
lacZ and control strain, and used to normalize b-galactosidase
activity, as described previously. Fiy percent inhibitory
concentrations (IC50) were calculated and graphs were drawn
using Graphpad Prism (GraphPad, La Jolla, CA). All data plotted
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