2-Aminobenzimidazoles as antibiofilm agents against Salmonella enterica serovar Typhimurium

Article abstract of DOI:10.1039/C8MD00298C

Serovars within the species Salmonella enterica are some of the most common food and water-borne pathogens worldwide. Some S. enterica serovars have shown a remarkable ability to persist both inside and outside the human body. Salmonella enterica serovar

Full text of DOI:10.1039/C8MD00298C

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Commun., 2018, DOI: 10.1039/C8MD00298C.
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2ꢀAminobenzimidazoles as Antibiofilm Agents Against Salmonella enterica Serovar
Typhimurium
William M. Hugginsa, T. Vu Nguyena, Nicholas A. Hahna, James T. Bakera, Laura G. Kuoǂ,
Darpan Kaurǂ, Roberta J. Melandera, John S. Gunnǂ, and Christian Melander*a
aDepartment of Chemistry, North Carolina State University, 2620 Yarbrough Drive, Raleigh,
North Carolina 27695, United States
ǂDepartment of Microbial Infection and Immunity, Infectious Diseases Institute, The Ohio State
University, Columbus, Ohio, USA
Eꢀmail: ccmeland@ncsu.edu; Tel: 1ꢀ919ꢀ513ꢀ2960
ABSTACT: Serovars within the species Salmonella enterica are some of the most common food
and waterꢀborne pathogens worldwide. Some S. enterica serovars have shown a remarkable
ability to persist both inside and outside the human body. Salmonella enterica serovar Typhi can
cause chronic, asymptomatic infection of the human gallbladder. This organism’s ability to
survive inside the gallbladder centers around its ability to form biofilms on gallstone surfaces.
Currently, chronic carriage of S. Typhi is treated by invasive methods, which are not well suited
to areas where Salmonella carriage is prevalent. Herein, we report 2ꢀaminobenzimidazoles that
inhibit S. enterica serovar Typhimurium (a surrogate for S. Typhi) biofilm formation in low
micromolar concentrations. Modifications to the head, tail, and linker regions of the original hit
compound elucidated new, more effective analogues that inhibit S. Typhimurium biofilm
formation while being nonꢀtoxic to planktonic bacterial growth.
Salmonella species are a frequent cause of food and water borne illness worldwide. They
can cause a variety of disease syndromes, and are normally grouped into typhoidal and nonꢀ
typhoidal species. Salmonella enterica serovar Typhi is the causative agent of enteric fever and
Salmonella enterica serovar Typhiumiurum is typically associated with intestinal distress, or
salmonellosis.1 Typhoidal and nonꢀtyphoidal strains of Salmonella have shown a remarkable
ability to persist in a variety of environments, including harsh environments within the human
body. One of these survival strategies centers around the ability of Salmonella to form biofilms
1

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on gallstones.2 Colonization of gallstones can then lead to chronic carriage of S. Typhi, which
allows for the dissemination of Salmonella through fecal shedding. Biofilms can be up to 1000ꢀ
fold more resistant to antibiotic treatment than their planktonic counterparts, rendering typical
antibiotic treatment regimens ineffective at eradicating chronic carriage of Salmonella.3ꢀ5
Presently, chronic carriage of Salmonella is treated by invasive methods, which are not well
suited to areas where Salmonella is prevalent.6 New treatment options for chronic carriage of
Salmonella are needed, and compounds that perturb Salmonella biofilms could be a viable
treatment option.
Recently, there have been reports of derivatized 2ꢀaminoimidazoles (2ꢀAIs) that inhibit S.
Typhimurium biofilm formation.7ꢀ12 As our group has been deeply involved in exploring the
antibiofilm potential of 2ꢀAIs, we were curious if there were 2ꢀAI derivatives in our library, or
derivatives of other nitrogenꢀdense heterocycles that we have assembled and assayed for their
antibiofilm activities, that possessed activity against S. Typhimurium. As S. Typhi is a humanꢀ
specific pathogen, reliable murine pathogenic serovars such as S. Typhimurium have been used
to model S. Typhi infections, allowing for future in vivo testing.13, 14 We performed an initial
library screen for inhibitors of S. Typhimurium biofilm formation and identified 2ꢀ
aminobenzimidazole (2ꢀABI) compound 1 as one lead compound that returned an IC50 value of
13.1±0.6 µM. Previously, we have shown that 2ꢀABIs display a wide variety of biological
activity including antibiotic activity against MRSA and MDR A. baumannii,15 antibiofilm
activity against Gramꢀpositive bacteria,16 and the ability to potentiate βꢀlactam antibiotic activity
against Mycobacterium smegmatis and M. tuberculosis.17 With this compound in hand, we
decided to probe the structureꢀactivity relationship (SAR) of the 2ꢀABIs against S. Typhimurium
biofilms. Herein we report the results of this SAR study of the 2ꢀABI scaffold, focusing on three
regions: the head region, the linker and the tail region (Figure 1).
Fig 1. Structure of original hit compound 1, regions of modification.
2

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The first region of the molecule we decided to modify was the head region of the 2ꢀABI
using a synthetic scheme previously described by our group (Scheme 1A).18 Briefly, 4ꢀfluoroꢀ3ꢀ
nitroaniline 2 was acylated with 4ꢀpentylbenzoyl chloride in the presence of triethylamine and 4ꢀ
dimethylaminopyrimidine (DMAP) in DCM for 16 hours at room temperature to yield
compound 3. SNAr substitution of compound 3 with commercially available amines in refluxing
ethanol for 16 hours yielded compounds 4aꢀn. Subsequent reduction of the nitro group with
ammonium formate and 10% Pd/C in ethanol at reflux followed by cyclization with cyanogen
bromide in DCM at room temperature yielded 2ꢀABIs 5aꢀn. The unsubstituted 2ꢀABI derivative,
compound 5o, was prepared using a previously published method15, 19
3

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Scheme 1. (A) Synthetic route to compounds 5aꢀn and (B) Synthetic route to compounds 9aꢀe:
(a) 4ꢀpentylbenzoyl chloride, DMAP, Et3N, DCM, 16 h (b) RNH2, EtOH, reflux, 16 h (c)
NH4HCO2, 10% Pd/C, EtOH, reflux, 3 h (d) CNBr, DCM, 16 h (e) HCl, MeOH.
Three head group substitutions, nꢀoctyl (5a), butyl phenyl (5b), and nꢀhexyl (5c)
displayed improved activity (Table 1) over the parent compound.
Isobutyl substitution
(compound 5d) or substitution with tryptamine (5e) returned an IC50 value between 10ꢀ15 µM,
similar to that of parent compound 1 (IC50 values were only quantified for compounds that had
marked improvement over compound 1). Substitution with cyclopentyl (5g) or cyclohexyl rings
(5h) returned slightly higher IC50 values compared to the parent (15ꢀ20 µM). Shortening the
phenyl chain to two methylenes also reduced activity, with compound 5i returning an IC50 value
of 20ꢀ40 µM (supplementary information). Replacement of the phenyl ring on compound 5i
with an imidazole (5j) ring also returned an IC50 value of 20ꢀ40 µM. Alkyl chains with less than
four carbon atoms (5k, 5l) or longer than eight carbon atoms (5m, 5n) reduced or completely
abolished antibiofilm activity. Removal of the head group (compound 5o) from the 2ꢀABI
lowers the IC50 from 13.1±0.6 µM to 20ꢀ40 µM, demonstrating the necessity of the head group
for biofilm inhibitory activity.
After investigating different head group substitutions in the para position relative to the
amide, analogues with the head group substitution meta to the amide were prepared. The
synthetic route to these compounds (Scheme 1B), is identical to that of the para analogues
(Scheme 1A) except the starting material is 3ꢀfluoroꢀ4ꢀnitroaniline 6. Compared to their para
substituted counterparts, the meta analogues did not display a significant increase in activity
(Table 1). Only the butyl analogue 9a (IC50 10ꢀ15 µM) displayed increased activity compared to
butyl para analogue 5f (IC50 15ꢀ20 µM). nꢀHexyl analogue 9b displayed almost identical activity
to the para nꢀhexyl analogue 5c, returning an IC50 of 10ꢀ15 µM. Methyl (9c), ethyl (9d), and
isopropyl (9e) analogues all displayed IC50 values of 20ꢀ40 µM (supplementary information).
4

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Table 1. IC50 values of para 2ꢀABI, compounds 5aꢀh, and meta 2ꢀABI, compounds 9aꢀb. Full
inhibition data can be found in the supplementary information.
Compound
5a
R=
IC50 (µM)
( )7
6.66±0.32
9.59±0.23
10.8±0.56
5b
5c
( )5
13.1±0.6
10ꢀ15
1
5d
5e
10ꢀ15
( )3
15ꢀ20
15ꢀ20
5f
5g
15ꢀ20
5h
( )3
10ꢀ15
10ꢀ15
9a
9b
( )5
5

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Figure 2. Structure of compounds 10aꢀt with various tail substitutions
After modifying the head region of the molecule, substitutions to the tail of the molecule
were made in an effort to improve the antibiofilm ability of the parent compound. Previously,
various 2ꢀABI derivatives with an identical head group to the parent compound 1 and various
tails were prepared (Figure 2).17 As these compounds were readily available in our laboratory,
their antiꢀSalmonella biofilm activity was investigated. Halogenation (10b, 10c and 10d) of the
aromatic ring lowers the IC50 value when compared to a 4ꢀpentylbenzoyl group, all returning
IC50 values of less than 7 µM (Table 2). The 3,5ꢀdichloro analogue 10e displays lower activity
compared to the other halogenated tails, but displays essentially equivalent activity to the parent
compound 1. Addition of a pentoxybenzoyl group (10a) returns the lowest IC50 observed, with
an IC50 of 5.22±0.11 µM. Although substituted benzoyl groups are favored in the most active
compounds, removal of the aromatic ring in favor of a straight alkyl chain is tolerated as
observed with nonanoyl (10h) and decanoyl (10i) tails. 4ꢀbutylbenzoyl (10f), and 4ꢀ
propoxybenzoyl (10g) tails all returned an IC50 value of 10ꢀ15 µM, similar to that of the parent
compound (1) and the 3,5ꢀdichlorobenzoyl compound (10e). Additional analogues were
investigated, but all displayed lower activity in comparison to the parent compound. 4ꢀ
Butoxybenzoyl (10j), 4ꢀpropylbenzoyl (10k), 4ꢀhexylbenzoyl (10l), 4ꢀheptylbenzoyl (10m), and
octanoyl (10n) tails all returned IC50 values of 15ꢀ20 µM. 4ꢀethylbenzoyl (10o) and hexanoyl
(10p) tails returned IC50 values between 20ꢀ40 µM (supplementary information). Lastly,
heptanoyl (10q), 4ꢀoctylbenzoyl (10r), undecanoyl (10s), and tridecanoyl (10t) tails returned
IC50 values of greater than 40 µM.
6

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Table 2. IC50 values of 2ꢀABIs with different tails, compounds 10aꢀn. Full inhibition data
can be found in the supplementary information.
Compound
10a
R=
IC50 (µM)
5.22±0.11
10b
10c
5.38±0.79
5.58±0.21
10d
1
6.30±1.29
13.1±0.6
10ꢀ15
10e
10ꢀ15
10ꢀ15
10f
10g
10ꢀ15
10ꢀ15
10h
10i
7

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15ꢀ20
10j
15ꢀ20
15ꢀ20
10k
10l
10m
15ꢀ20
15ꢀ20
10n
The last region of the 2ꢀABIs that we were interested in modifying was the linker region.
The 2ꢀABIs previously synthesized in our laboratory15, 16ꢀ18 that served as the basis for the 2ꢀ
ABIs used in this study have featured an amide linkage, with the nitrogen in the amide connected
to the 2ꢀABI head of the molecule. Previous studies on other 2ꢀAI natural products have
demonstrated the effect that modification of the amide moiety can have on a compound’s ability
to control bacterial behavior as well as its toxicity in C. elegans.20 With this in mind, we aimed
to synthesize 2ꢀABI derivatives with a reverse amide moiety as well as a urea moiety replacing
the amide. Compound 10d was chosen as the compound for further analog development due to
its predicted reduced metabolic liabilities.
The synthesis of the reverse amide analogue (Scheme 2A) began by reacting 4ꢀfluoroꢀ3ꢀ
nitrobenzoic acid with thionyl chloride in methanol at 0 °C, warming to room temperature
overnight to yield the methyl ester 12. Compound 12 was then reacted with 3ꢀphenylꢀ1ꢀ
propylamine in ethanol at reflux to yield 13. Reduction of the nitro group followed by
cyclization with cyanogen bromide in DCM at room temperature yielded the 2ꢀABI 14. Lastly,
saponification of the methyl ester with sodium hydroxide in 1:1 MeOH/H2O followed by EDC
coupling of the carboxylic acid with 3,4ꢀdichloroaniline delivered the reverse amide 2ꢀABI 15.
8

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Scheme 2. (A) Synthetic route to compound 15 and (B) Synthetic route to compound 20:
reagents and conditions (a) SOCl2, MeOH, 0 °C to rt, 16 h (b) 3ꢀphenylꢀ1ꢀpropylamine, EtOH,
reflux, 16 h (c) NH4HCO2, 10% Pd/C, EtOH, reflux, 3 h (d) CNBr, DCM, 16 h (e) NaOH, 1:1
MeOH:H2O , reflux, 2 h (f) EDC, 3,4ꢀdichloroaniline, Et3N, DMAP, 2:1 DCM:DMF, rt , 16 h (g)
9

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HCl, MeOH (h) Boc2O, DMAP, Et3N, DCM, rt, 16 h (i) AllocꢀCl, Et3N, Sc(OTf)3, DCM, rt, 16
h (j) 30% TFA:DCM, rt, 4 h (k) 3,4ꢀdichloroaniline, triphosgene, Na2CO3, DCMꢀH2O, rt, 16 h
(l) Pd(PPh3)4, NaBH4, EtOH, 0 °C to rt, 1 h (m) 12N HCl, pH 1, rt, 2 h.
Synthesis of the urea analogue (Scheme 2B) began with the Boc protection of 4ꢀfluoroꢀ3ꢀ
nitroaniline, yielding 16. Compound 16 was then reacted with 3ꢀphenylꢀ1ꢀpropylamine to yield
the diaminobenzene 17. Reduction of the nitro group using ammonium formate and 10% Pd/C
in ethanol at reflux followed by cyclization with cyanogen bromide in DCM at room temperature
yielded Bocꢀprotected 2ꢀABI 18. Alloc protection of the 2ꢀABI head proceeded smoothly
utilizing scandium (III) triflate as a Lewisꢀacid catalyst in DCM overnight at room temperature,
yielding alloc protected 2ꢀABI 19. Boc deprotection of compound 20 followed by coupling with
3,4ꢀdichloroaniline using triphosgene yielded the Alloc protected 2ꢀABI urea. Finally, Alloc
deprotection with Pd(PPh3)4 and NaBH4 yielded the target 2ꢀABI urea 20.
While the reverse amide analogue 15 displayed a twoꢀfold greater IC50 (12.6±1.8 µM)
than that of the parent compound 10d, the urea analogue 20 displayed only a slight reduction in
IC50 value from 6.30±1.29 to 7.69±0.25 µM, demonstrating that the linker group of the 2ꢀABIs
can be modified while retaining biofilm inhibitory activity against S. Typhimurium.
Additionally, compounds 10a and 20 showed no toxicity to planktonic bacterial growth at their
IC50 values, indicating that they are nonꢀtoxic biofilm inhibitors (supplementary information).
Conclusions
After the identification of 2ꢀABI compound 1 as a S. Typhimurium biofilm inhibitor,
probing of the SAR of the 2ꢀABIs elucidated six new analogues with IC50 values of less than 10
µM. Utilizing the same paraꢀpentyl benzoyl tail as the parent compound 1, structural
modifications to the head of the 2ꢀABIs in the para position to the amide yielded compounds 5a
and 5b with IC50 values of 6.66±0.32 and 9.59±0.23 µM respectively. Modification of the tail
region yielded compounds 15a (5.22±0.11 µM), 15b (5.38±0.79 µM), 15c (5.58±0.21 µM), and
15d (6.3±1.29 µM) that displayed improved activity. Modification of the linker between the head
and the tail yielded the urea compound 20 with a comparable IC50 (7.69±0.25 µM) to that of the
amide parent compound, 10d (6.30±1.29 µM). Substitution the 2ꢀABI head at the meta position
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to the amide (9aꢀe) and reversal of the amide moiety (15) did not produce an increase in activity.
Compounds 10a and 20 were then shown to be nonꢀtoxic to planktonic bacterial growth at their
IC50 values, demonstrating that they show specific antiꢀS. Typhimurium biofilm inhibitory
activity. Additionally, compounds 10d and 10e and other related 2ꢀABIs17 displayed no toxicity
to G. mellonella at 400 mg/kg (supplementary information), thus making them potential
candidates for future in vivo studies.
Acknowledgements
This work was supported by grants to CM (GM05579) and (JSG AI 116917) from the National
Institutes of Health (NIH).
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