Structure–Function Studies on IMD-0354 Identifies Highly Active Colistin Adjuvants

Article abstract of DOI:10.1002/cmdc.201900560

Infections caused by multidrug-resistant (MDR) bacteria, particularly Gram-negative bacteria, are an escalating global health threat. Often clinicians are forced to administer the last-resort antibiotic colistin; however, colistin resistance is becoming increasingly prevalent, giving rise to the potential for a situation in which there are no treatment options for MDR Gram-negative infections. The development of adjuvants that circumvent bacterial resistance mechanisms is a promising orthogonal approach to the development of new antibiotics. We recently disclosed that the known IKK-β inhibitor IMD-0354 potently suppresses colistin resistance in several Gram-negative strains. In this study, we explore the structure–activity relationship (SAR) between the IMD-0354 scaffold and colistin resistance suppression, and identify several compounds with more potent activity than the parent against highly colistin-resistant strains of Acinetobacter baumannii and Klebsiella pneumoniae.

Full text of DOI:10.1002/cmdc.201900560

Accepted Article
Title: Structure Function Studies on IMD 0354 Identifies Highly Active
Colistin Adjuvants
Authors: Ansley Nemeth, Akash Basak, Alexander Weig, Santiana
Marrujo, William Barker, Leigh Jania, Tyler Hendricks, Ashley
Sullivan, Patrick O'Connor, Roberta Melander, Beverly Koller,
and Christian Corey Melander
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to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemMedChem 10.1002/cmdc.201900560
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Structure Function Studies on IMD-0354 Identifies Highly Active
Colistin Adjuvants.
Ansley M. Nemeth[a], Akash K. Basak#[a], Alexander W. Weig#[a], Santiana A. Marrujo[a], William T.
Barker[a], Leigh A. Jania[b], Tyler A. Hendricks[a], Ashley E. Sullivan[a], Patrick M. O’Connor[a], Roberta J.
Melander[a], Beverly H. Koller[b], and Christian Melander[a]*
[a]
[b]
A. M. Nemeth, Dr. A. K. Basak, A. W. Weig, S. A. Marrujo, W. T. Barker, T. A. Hendricks, A. E. Sullivan, P. M. O’Connor, Prof. R. J. Melander, Prof. C.
Melander*
Department of Chemistry and Biochemistry
University of Notre Dame
Notre Dame, IN 46556
*E-mail: cmelande@nd.edu
L. A. Jania, Prof. B. H. Koller
Department of Genetics
University of North Carolina at Chapel Hill
Chapel Hill, NC 27599
#These authors contributed equally to this work.
Supporting information for this article is given via a link at the end of the document
Abstract: Infections caused by multi-drug resistant (MDR)
bacteria, particularly Gram-negative bacteria, are an escalating
global health threat. Often clinicians are forced to administer the
last resort antibiotic colistin, however colistin resistance is
becoming increasingly prevalent, giving rise to the potential for a
situation in which there are no treatment options for MDR Gram-
negative infections. The development of adjuvants that circumvent
As a result of this current antibiotic landscape, clinicians
have become more reliant upon polymyxin antibiotics for the
treatment of MDR Gram-negative bacterial infections.[6]
Polymyxins are cationic cyclic peptides whose bactericidal
activity is driven by physical disruption of the Gram-negative
bacterial membrane.[7] As an antibiotic for human use, colistin
was introduced into the clinic in 1955 but rapidly fell out of favor
due to nephrotoxicity,[8] and was replaced by antibiotics with
better safety profiles. However, with limited therapeutic options
currently available, coupled with a historically low incidence of
resistance due to limited use, colistin is now typically viewed
as the antibiotic of last resort for the treatment of MDR Gram-
negative bacterial infections.[9]
As colistin treatment has become more common, there
has been a corresponding upsurge in colistin resistance
observed in the clinic over the past decade. Clinical resistance
to colistin, and other polymyxins, is driven by cationic
modification of the lipid A component of lipopolysaccharide
(LPS) that decorates the outer membrane of Gram-negative
bacteria.[10] In A. baumannii, lipid A is typically modified with
phosphoethanolamine, while in K. pneumoniae it is typically
modified with aminoarabinose.[11] In either case, modification
of lipid A serves to decrease the overall net negative charge of
LPS, which, in turn, impacts the affinity of colistin for the outer
membrane. Historically, colistin resistance has typically been
mediated by chromosomal mutations in genes encoding two-
component systems (such as PmrAB in A. baumannii) that,
combined with colistin’s limited use, meant dissemination of
colistin resistant isolates was slow. The situation changed
dramatically with the emergence of the plasmid-borne colistin
resistance genes mcr-1-9[12] that have raised the possibility of
rapid dissemination of colistin resistance into the general
pathogen pool via horizontal gene transfer, which would
ultimately deprive clinicians of this last resort antibiotic.
bacterial resistance mechanisms is
a promising orthogonal
approach to the development of new antibiotics. We recently
disclosed that the known IKK-β inhibitor IMD-0354 potently
suppresses colistin resistance in several Gram-negative strains. In
this report, we explore the structure-activity relationship (SAR)
between the IMD-0354 scaffold and colistin resistance
suppression, and identify several compounds with more potent
activity than the parent against highly colistin resistant strains of
Acinetobacter baumannii and Klebsiella pneumoniae.
Introduction
The threat to modern medicine caused by rising antibiotic
resistance cannot be understated.[1] Of all bacteria, the
ESKAPE pathogens (Enterococcus faecium, Staphylococcus
aureus, Klebsiella pneumoniae, Acinetobacter baumannii,
Pseudomonas aeruginosa, and Enterobacter species) are
considered to pose the highest threats to human health.[2]
Despite escalating incidence of infections that stem from multi-
drug resistant (MDR) strains of these pathogens, the antibiotic
discovery pipeline to counter these threats still remains
significantly underpopulated.[3] In the case of the Gram-positive
members of this group (E. faecium and S. aureus), limited
success has been achieved with the introduction of linezolid
and daptomycin over the last 20 years.[4] For Gram-negative
bacteria, however, the last class of novel antibiotics to be
introduced into the clinic was the fluoroquinolone class in the
1960s.[5]
1
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With the rising incidence of MDR bacterial infections
colistin resistant Gram-negative bacterial strains: A. baumannii
4106 (AB4106) and K. pneumoniae B9 (KPB9). In the absence
of adjuvant, AB4106 returned a colistin MIC of 2048 µg/mL
while KPB9 returned a colistin MIC of 512 µg/mL. The clinical
breakpoint for colistin susceptibility against these bacterial
species is 2 µg/mL. We have previously established that at 5
µM, IMD-0354 reduces the colistin MIC against AB4106 to 2
µg/mL (1024-fold) while it reduces the colistin MIC against
KPB9 to 0.5 µg/mL (1024-fold). To compare directly to IMD-
0354, all analogues were thus screened for activity at 5 µM,
with any compound displaying an equal or two-fold variation in
MIC labeled as equipotent.
coupled with a depleted antibiotic pipeline, research into
alternatives to traditional antibiotics is warranted. One such
approach is the development of small molecule antibiotic
adjuvants.[13] Such molecules typically possess little stand-
alone microbicidal activity, but instead target antibiotic
resistance mechanisms. When paired with the appropriate
antibiotic, these antibiotic/adjuvant combinations provide a
powerful treatment for MDR bacteria. In the context of colistin
resistance, we have identified a number of adjuvants capable
of reversing colistin resistance in bacterial strains that contain
chromosomally-encoded resistance mechanisms as well as
those that harbor the plasmid-borne mcr-1 gene (Compounds
A-E Figure 1).[14, 15] Recently, we reported that the non-toxic
salicylamide IMD-0354 E, a well-known inhibitor of IKK-β,
reversed lipid A modification and potently suppressed colistin
resistance in A. baumannii, K. pneumoniae, and Escherichia
coli, reducing the minimum inhibitory concentration (MIC) of
colistin upwards of 4096-fold at 5 µM (1.9 µg/mL).[15] Given this
potency, we became interested in delineating the structural
parameters of this scaffold that drive colistin potentiation.
Herein, we report our initial exploration into the structure
activity relationship (SAR) of this scaffold and the identification
of 35 compounds with equivalent or superior activity to IMD-
0354 against both the representative strains used in the initial
screen.
Scheme 1: General synthetic route and routes to
compounds 2, 13 and 36. Reagents and conditions: (a) PCl3,
toluene, reflux, 16h; (b) CH3I, K2CO3, acetone, rt, 16h; (c)
phenyl boronic acid, PdCl2(PPh3), K2CO3, THF, reflux, 48h; (d)
SnCl2, HCl/EtOH, 70 ºC, 12h.
The activity of the lead compounds is summarized in
Table 1. Full data can be found in Table S1 in the supporting
information. All new analogues exhibited standalone MICs of
≥100 µM, with the exception of compound 60, which exhibited
MICs of 50 µM against both strains, compound 25 which
exhibited an MIC of 50 µM against AB 4106, compounds 18
and 62, which exhibited an MIC of 50 µM against KP B9, and
Figure 1. Previously identified compounds that break
colistin resistance
Results and Discussion
All IMD-0354 analogues, with the following exceptions were
synthesized through a one-step PCl3 mediated condensation
reaction between a derivatized aniline and corresponding
benzoic (salicylic) acid.[16] Compound 2 (Figure 2) was
synthesized by methylating the phenolic hydroxyl group of
IMD-0354 with methyl iodide in the presence of potassium
carbonate.[17] The bis-phenyl derivative 13 (Figure 2) was
synthesized from the iodo derivative 10 via a Suzuki coupling
with
phenylboronic
acid
in
the
presence
of
bis(triphenylphosphine)palladium chloride. Finally, aniline 36
was synthesized via a tin (II) chloride mediated reduction of
nitro derivative 35 (Scheme 1).
The first compound set we prepared varied the identity of
the
benzoic
acid
while
retaining
the
3,5-bis-
trifluoromethylaniline component of IMD-0354 (Figure 2). All
derivatives were screened against two representative, highly
Figure 2. Structures of analogues with derivatized benzoic
acid moieties.
2
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compounds 9 and 42, which exhibited MICs of 25 µM against
KP B9.
trifluoromethyl analogue 15 and the dihydroxylated derivative
16. We found that 15 exhibited equipotent activity to IMD-0354
(1 and 0.25 µg/mL against AB4106 and KPB9 respectively),
while compound 14 was four-fold less active than 15 (yet still
considered equipotent to IMD-0354). Finally, we noted that 16
was less active against KPB9 (2 µg/mL) and essentially
inactive against AB4106. Of the 16 analogues prepared in this
subset, we found nine had equipotent or improved activity
against both strains in comparison to IMD-0354, while the
phenyl derivative had equivalent activity against AB4106.
Table 1. Colistin MICs against AB 4106 and KP B9 in the
presence of lead IMD-0354 analogues
aAll compounds screened at 5 µM. bMIC of colistin alone is
2048 µg/mL. cMIC of colistin alone is 512 µg/mL.
Compounda
AB 4106 MIC (µg/mL)
(fold reduction)
KP B9 MIC (µg/mL)
(fold reduction)
IMD-0354
9
2 (1024)b
2 (1024)
0.5 (4096)
1 (2048)
2 (1024)
2 (1024)
0.5 (4096)
1 (2048)
1 (2048)
1 (2048)
1 (2048)
2 (1024)
1 (2048)
4 (512)
0.5 (1024)c
0.25 (2048)
0.5 (1024)
0.25 (2048)
0.25 (2048)
0.25 (2048)
0.5 (1024)
0.25 (2048)
0.25 (2048)
0.5 (1024)
0.25 (2048)
0.125 (4096)
0.25 (2048)
0.25 (2048)
0.125 (4096)
0.5 (1024)
0.5 (1024)
0.5 (1024)
0.5 (1024)
10
15
22
23
24
25
26
29
37
43
49
50
51
52
55
56
1 (2048)
1 (2048)
1 (2048)
1 (2048)
1 (2048)
60
We first tested the necessity for the phenolic hydroxyl
moiety by either complete removal (1) or methylation (2). Both
modifications abrogated activity. Next, activity as a function of
position of the phenolic hydroxyl group was probed through
compounds 3 and 4. Shifting this hydroxyl group from the ortho
to meta (in relationship to the amide, 3) resulted in a compound
with essentially equipotent activity to IMD-0354, lowering the
colistin MIC to 4 µg/mL against AB4106 and 0.5 µg/mL against
KPB9. Further rotation of the hydroxyl group to the para
position (4), abolished activity. The impact that the chloro
substituent had upon activity was probed by changing its
relative position, deletion, or substitution with other halogens
(5-10). Moving the position of the chlorine (5), deletion of the
chlorine (6), or replacement of the chlorine with either fluorine
(8), bromine (9), or iodine (10) was tolerated and delivered
compounds with essentially equipotent activity to IMD-0354.
Interestingly, the iodo derivative, 10, displayed superior activity
to IMD-0354 and reduced the AB4106 colistin MIC to 0.5
µg/mL while it retained equipotent activity against KPB9
(reduced colistin MIC to 0.5 µg/mL). Removal of the chlorine
in tandem with moving the phenolic hydroxyl (7) was not
tolerated and resulted in a derivative devoid of activity. We
further probed the impact substitution of the chloro group had
upon activity by testing the activity of the methoxy analogue 11.
This substitution resulted in a significant impact on activity and
returned colistin MICs of 8 µg/mL (AB4106) and 16 µg/mL
(KPB9). Steric isosteres at the halogen position were
constructed for both IMD-0354 (12) and the iodo derivative 10
(analogue 13). The methyl derivative returned similar colistin
MICs to IMD-0354 (4 and 0.5 µg/mL against AB4106 and
KPB9 respectively), while the phenyl derivative maintained
activity against AB4106 (4 µg/mL) but lost some activity
against KPB9 (4 µg/mL). The last three derivatives of this
subset prepared were the dichloro analogue 14, the
Figure 3. Structures of analogues with derivatized aniline
moieties.
Next we probed the structural constraints of the aniline
segment of IMD-0354 by fixing the identity of the 5-
chlorosalicylic acid and varying the substituents on the aniline
ring (Figure 3). We moved the position of the trifluoromethyl
substituents from a 3,5 orientation to a 2,5- (17) and a 2,4- (18)
substitution pattern. Both compounds retained activity against
KPB9 and AB4106 equivalent to IMD-0354. The requirement
for the bis-trifluromethyl groups was probed by preparing the
corresponding 3,5-dimethoxy and 3,5-dimethyl analogues 19
and 20. Although the dimethoxy derivative was bereft of
activity, the dimethyl analogue returned colistin MICs of 2 and
1 µg/mL against AB4106 and KPB9 respectively.
Previous studies with 2-aminoimidazle-based adjuvants
have demonstrated that compounds containing 3,5-
dihalogenated benzene rings display various activities;
therefore we next studied replacement of the 3,5-bis-
trifluoromethyl groups with difluoro, dichloro, and dibromo
substituents (21-23). All three analogues were equipotent,
suppressing colistin MICs to 2 and 0.25 µg/mL (AB4106 and
KPB9). Addition of either a bromine or chlorine at the 2-
position of the bis-trifluoroaniline scaffold (24 and 25) again
yielded potent compounds returning colistin MICs of 1 and 0.25
µg/mL
against
AB4106
and
KPB9
respectively.
Monotrifluoromethyl derivatives 26-30 were then prepared to
study the impact retaining one of the trifluoromethyl
substituents had upon activity.
The para-trifluoromethyl
derivative 26, as well as the fluorinated analogues 28 and 29,
all returned activity equivalent to IMD-0354. Placement of
thetrifluoromethyl ortho to the amide (27) suppressed activity
such that the colistin AB4106 MIC was only reduced to 8 µg/mL
while the KPB9 colistin MIC was 1 µg/mL. Pairing of the
3
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Figure 4. Structures of hybrid analogues
the exception of the 3,5-dimethyl derivatives (44, 48, and 53)
and the trifluoro derivative 42, all analogues prepared showed
activity essentially equal to IMD-0354 when the halide identity
on the salicylic acid building block was varied. Derivatives that
contained a 3,5-dihaloaniline building block and either a
hydroxyl trifluoromethyl or dichloro salicylic acid fragment were
also equally active to IMD-0354 (54-56 and 62), while again all
of the corresponding 3,5-dimethyl analogues were less active
(57-59). The 2,5-di-trifluoromethyl aniline coupled with the
dichloro salicylic acid fragment (61) also returned equipotent
activity. Overall, 13 analogues from this last set were identified
to have activity levels similar to IMD-0354 with a clear trend
that incorporation of a 3,5-dimethyl substitution pattern was
detrimental to activity.
trifluoromethyl with a para-methyl was detrimental to activity
(30, 64/4 µg/mL against AB4106/KPB9) while the all methyl
analogue 31 also showed minimal activity (64/16 µg/mL
against AB4106/KPB9). Replacement of one of these methyl
groups with a bromo substituent, compound 32, only improved
activity against KPB9 (8 µg/mL), while analogues containing
an ortho bromo substituent (33 and 34) also showed
diminished activity in comparison to IMD-0354 (16/2 and
>64/>64 µg/mL against AB4106/KPB9 for 33 and 34
respectively)
We tested further expansion of functional group
tolerance on the aniline ring by incorporation of a nitro (35) or
an amine (36) substituent in tandem with a para-fluoro
substituent. Neither compound displayed significant activity
with the exception of 35 against KPB9, which returned an MIC
of 1 µg/mL. Next, we focused efforts on monosubstituted
aniline building blocks and assembled compounds 37-40. The
and meta-chloro derivative (40) was active, returning MICs of
2/0.5 µg/mL against AB4106/KPB9, as was the para-bromo
derivative 37 (MICs 1/0.25 against AB4106/KPB9), while the
ortho-chloro derivative 39 was less active. Mirroring the results
from other analogues in this series, incorporation of the
electron donating methoxy group abolished activity (38).
Finally, we prepared analogue 41 to test the effects of
combining the structural components of two active analogues
(22 and 37). This analogue however, showed diminished
activity, ca. 4-8 fold over 22 and 37. Overall, we generated 25
derivatives by varying the identity of the aniline substituent, of
which 12 showed activity at least equivalent to IMD-0354.
The last compound set we prepared tested the impact of
mixing structural elements from both of the previous libraries.
The 21 analogues synthesized are depicted in Figure 4. With
Table 2. Dose response activity of lead compounds
against KP B9
Compound
IMD-0354
Concentration (µM)
Colistin MIC (µg/mL)
(fold reduction)
5
3
1
0.5 (1024)
0.5 (1024)
4 (128)
23
29
51
52
5
3
1
0.5
0.25
0.25 (2048)
0.5 (1024)
0.5 (1024)
1 (512)
4 (128)
5
3
1
0.5
0.25
0.5 (1024)
0.5 (1024)
0.5 (1024)
0.5 (1024)
1 (512)
5
3
1
0.5
0.25
0.125 (4096)
0.25 (2048)
0.5 (1024)
1 (512)
16 (32)
5
3
1
0.5 (1024)
0.5 (1024)
1 (512)
0.5
0.25
1 (512)
16 (32)
4
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Table 3. Activity of lead compounds against colistin resistant isolate panel aAll compounds tested at 5 µM
Colistin MIC (µg/mL) (fold reduction)
Compound
KP
AB
KP C3
KP A5
AB 3941
AB 3942
AB 4112
AB 4119
F2210291mcr-1
17978mcr-1
-
128
2048
16
1024
>2048
2048
1024
64
IMD-0354
0.5a
8
0.5
2
2
2
4
0.5
(256)
(256)
(32)
(512)
(≥2048)
(1024)
(256)
(128)
23
29
51
52
0.5
(256)
0.5
(4096)
0.125
(128)
2
2
2
1
0.5
(128)
(512)
(≥2048)
(1024)
(1024)
1
1
0.25
(64)
1
1
1
2
0.125
(512)
(128)
(2048)
(1024)
(≥4096)
(2048)
(512)
0.25
(8192)
0.25
(64)
1
1
1
0.5
(2048)
0.125
(512)
0.25 (512)
(1024)
(≥4096)
(2048)
0.5
(256)
1
0.25
(64)
1
1
1
2
0.25
(256)
(2048)
(1024)
(≥4096)
(2048)
(512)
Through screening all compounds for activity against
AB4106 and KPB9, we identified 37 salicylamide derivatives
that we deemed were worthy of further investigation. To further
vet activity, we tested whether compound activity was retained
against multiple colistin resistant isolates (Table 2 and Table
S2 SI). We chose two additional highly colistin resistant K.
pneumoniae strains, KPC3 and KPA5, which have stand-alone
colistin MICs of 128 and 2048 µg/mL, respectively, as well as
studies, compound 22 showed significant potentiation of
colistin activity.
Compounds were also tested against four additional A.
baumannii strains AB3941, AB3942, AB4112, and AB4119, as
well as an A. baumannii strain harboring a plasmid containing
the mcr-1 gene (AB17978mcr-1). The colistin MICs of these five
A. baumannii strains are 1024, >2048, 2048, 1024, and 64
µg/mL respectively. All compounds were screened for their
ability to potentiate colistin at 5 µM and compared to IMD-0354
(Table 3).
a
K. pneumoniae strain harboring the mcr-1 gene
(KPF221029mcr-1, colistin MIC = 16 µg/mL).
With the exception of KPA5 and AB4119, IMD-0354
suppresses the colistin MIC of all other isolates to at or below
the clinical breakpoint level (2 µg/mL). Against KPA5, IMD-
0354 suppresses the colistin MIC to 8 µg/mL and against
AB4119 it suppresses it to 4 µg/mL. Of the compounds
screened, 15 outperformed IMD-0354 and suppressed the
colistin MIC against all strains to at or below breakpoint levels.
To further probe activity, we performed a dose response study
with these compounds in comparison to IMD-0354 (Table 2
and Tables S3 and S4 in the supporting information) against
AB4106 and KPB9. At 3 µM, IMD-0354 suppresses the colistin
MIC to 4 µg/mL (AB4106) and 0.5 µg/mL (KPB9), while at 1
µM, it returns colistin MICs of 8 and 4 µg/mL against AB4106
and KPB9. Against AB4106, none of the compounds returned
more potent activity than IMD-0354, although 22 compounds
performed equally well (MIC within two-fold of that observed
with IMD-0354). The most active compounds, 22, 24, 25, 29,
and 51 reduce the colistin MIC to 4 µg/mL at 1 µM. Against
KPB9, however, 21 analogues outperformed IMD-0354 and
returned colistin to at or below breakpoint levels at 1 µM, while
25 derivatives had equipotent activity to IMD-0354. Of the 10
compounds that had superior activity to IMD-0354, four
analogues (23, 29, 51 and 52) retained the ability to break
colistin resistance at 500 nM, and 29 reduced the colistin MIC
below breakpoint levels at 250 nM. All four lead compounds
were active against the isolate panel and equally active against
AB4106 in comparison to IMD-0354.
1.00E+10
1.00E+09
1.00E+08
1.00E+07
1.00E+06
1.00E+05
1.00E+04
1.00E+03
1.00E+02
1.00E+01
1.00E+00
0
4
8
12
Time / hr
16
20
24
1.00E+10
1.00E+09
1.00E+08
1.00E+07
1.00E+06
1.00E+05
1.00E+04
1.00E+03
1.00E+02
1.00E+01
1.00E+00
0
4
8
12
Time / hr
16
20
24
To further quantify activity, and to confirm the non-toxic
nature of the adjuvants, we constructed time kill curves for
colistin and compound 22 as a representative lead compound,
against both AB4106 and KPB9 (Figure 5). Mirroring the MIC
Figure 5. Time kill curves for compound 22 and colistin.
Top: AB 4106. Bottom: KP B9. Blue: Untreated bacteria.
Red: 5 µM 22. Orange 5 µM 22 + 0.5 µg/mL colistin.
Green: 5 µM 22 + 4 µg/mL colistin. Purple: 5 µM 22 + 32
µg/mL colistin. Data ± SD from at least four replicates.
5
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General procedure for synthesis of N-aryl-2-hydroxybenzamides
To a stirring solution of salicylic acid (1 equiv.) in toluene under nitrogen
atmosphere, was added phosphorus trichloride (0.5 equiv.) dropwise
and heated to reflux. Substituted aniline (0.9 equiv.) was added in
portions over 10 minutes and the reaction mixture was refluxed
overnight. The reaction was checked for completion by TLC, then
cooled and ethyl acetate (100 mL) added. The suspension was
washed with 2N HCl (3x30 mL) followed by brine (30 mL) and then
aqueous sodium bicarbonate (3x30 mL). The organic layer was
washed with brine (30 mL) then dried using anhydrous sodium sulfate,
evaporated and purified via flash chromatography using 1:2 to 1:1
DCM/hexanes to obtain a white solid. Compounds were tested without
further purification from stock solutions in biological grade DMSO.
Compounds 1, 2, 5, 6, 8-15, 17-28, 30-34, 37-40, 42, 43, 45-51, 53, 56,
and 62. have been previously reported and characterization for these
compounds can be found in the supporting
Finally, we tested the cytotoxicity of six compounds (21,
22, 23, 25, 26, and 37) in the absence and presence of 1 µg/mL
colistin of the six compounds, 22, 23, 26, and 37 showed no
measurable toxicity up to 200 µM. One compound, 21, showed
comparable toxicity to IMD-0354 (CT50 = 210 µM) while
another (25) was ca. two-fold more toxic than IMD-0354 (CT50
= 110 µM). None of the compounds showed additional toxicity
in the presence of 1 µg/mL colistin.
Conclusions
In conclusion, we have presented an initial SAR study on the
salicylamide lead IMD-0354. A number of structural trends
were elucidated, with the most prominent being that the
salicylic OH is required for activity (although the position may
vary), while the halogen identity is flexible. On the aniline
fragment, the trifluoromethyl groups can be exchanged with
halogens, while electron donating groups are generally not
tolerated. Through these studies, we have managed to identify
a number of analogues with activity similar to or exceeding
IMD-0354. Four of these leads (23, 29, 51, and 52) show
activity in the nanomolar range, which marks them as some of
the most potent colistin adjuvants identified to date. As a
potential treatment for colistin resistant infections, IMD-0354 is
hampered by solubility and its inhibition of inflammation
through targeting IKK- β. To this end, we are currently
conducting additional SAR studies on this scaffold in efforts to
develop analogues that retain their colistin adjuvant activity,
yet are more soluble in aqueous media with muted effect on
IKK-β.
information.
Novel compound characterization
N-(3,5-bis(trifluoromethyl)phenyl)-3-chloro-5-hydroxybenzamide
(3) The title compound was synthesized following the general
procedure for synthesis of N-aryl-2-hydroxybenzamides to afford 3 as
a white solid (52 mg, 39%). 1H NMR (400 MHz, d3-Acetonitrile): 9.11 (s,
1H), 8.41 – 8.30 (m, 2H), 7.75 (s, 1H), 7.46 (t, J = 1.4, 0.7 Hz, 1H), 7.30
(t, J = 1.6 Hz, 1H), 7.08 (t, J = 2.0 Hz, 1H). 13C NMR (100 MHz, d3-
Acetonitrile): 164.5, 157.8, 140.1, 136.6, 134.3, 131.1 (q, J = 33.3 Hz),
123.1 (q, J = 271.8 Hz), 119.8, 119.8, 118.7, 118.5, 113.1. UV(λmax
nm): 265; IRvmax (cm-1): 3324, 1654, 1573, 914; HRMS (ESI): calcd for
C15H9ClF6NO2 [M+H]+: 384.0221, found: 384.0213
N-(3,5-bis(trifluoromethyl)phenyl)-3-chloro-4-hydroxybenzamide
(4) The title compound was synthesized following the general
procedure for synthesis of N-aryl-2-hydroxybenzamides to afford 4 as
a white solid (76 mg, 14%). 1H NMR (400 MHz, d6-DMSO): 11.11 (s,
1H), 10.66 (s, 1H), 8.50 (s, 2H), 8.09 (d, J = 2.3 Hz, 1H), 7.84 (dd, J =
8.6, 2.3 Hz, 1H), 7.80 (s, 1H), 7.11 (d, J = 8.5 Hz, 1H). 13C NMR (100
MHz, d6-DMSO): 164.6, 156.8, 141.2, 130.7 (q, J = 32.7 Hz), 129.7,
128.6, 125.3, 123.4 (q, J = 272.8 Hz), 119.8 (d, J = 4.2 Hz), 119.7,
116.4, 116.2 (m) . UV(λmax nm): 272; IRvmax (cm-1): 3174, 1643, 1118,
681; HRMS (ESI): calcd for C15H9ClF6NO2[M+H]+: 384.0221, found:
384.0194
Experimental Section
General Chemistry Experimental All reactions were carried out under
an atmosphere of nitrogen using anhydrous solvents unless otherwise
specified. All chemical reagents for synthesis were used without further
purification. Analytical thin layer chromatography (TLC) was performed
using 250 μm Silica Gel 60 F254 pre-coated plates (EMD Chemicals
Inc.). Flash column chromatography was performed using 230-400
Mesh 60Å Silica Gel from Sorbent Technologies. NMR spectra were
recorded using broadband probes on a Bruker AVANCE III HD 400
Nanobay (400 MHz for 1H and 100 MHz for 13C). All spectra are
presented using MestReNova (Mnova) software and 1H NMR are
typically displayed from 12 to -0.7 ppm without the use of the signal
suppression function. Spectra were obtained in the following solvents
(reference peaks also included for 1H and 13C NMRs): CDCl3 (1H NMR:
7.26 ppm; 13C NMR: 77.23 ppm), d6-DMSO (1H NMR: 2.50 ppm; 13C
NMR: 39.52 ppm) and d4-MeOD (1H NMR: 3.31 ppm; 13C NMR: 49.00
ppm). All NMR experiments were performed at room temperature.
Chemical shift values (δ) are reported in parts per million (ppm) for all
1H NMR and 13C NMR spectra. 1H NMR multiplicities are reported as:
s = singlet, d = doublet, t = triplet, q = quartet, p = pentet, m = multiplet,
br = broad. High-resolution mass spectra were obtained for all new
compounds from the mass spectrometry and proteomics facility at
N-(3,5-bis(trifluoromethyl)phenyl)-3-hydroxybenzamide (7) The
title compound was synthesized following the general procedure for
synthesis of N-aryl-2-hydroxybenzamides to afford 7 as a white solid
(34 mg, 23%). 1H NMR (400 MHz, d6-DMSO): 10.76 (s, 1H), 9.86 (s,
1H), 8.53 (d, J = 1.8 Hz, 2H), 7.81 (s, 1H), 7.43 (dt, J = 7.6, 1.3 Hz, 1H),
7.40 – 7.33 (m, 2H), 7.03 (ddd, J = 7.9, 2.4, 1.1 Hz, 1H). 13C NMR (100
MHz, d6-DMSO): 13C NMR (100 MHz, d6-DMSO): 166.3, 157.5, 141.2,
135.3, 130.6 (q, J = 32.8 Hz), 129.7, 123.3 (d, J = 272.6 Hz), 119.8 (q,
J = 3.4 Hz), 119.3, 118.3, 116.4 (q, J = 7.3 Hz), 114.6. UV (λmax nm):
274; IR vmax (cm-1): 3080, 1564, 1509, 1220; HRMS (ESI): calcd for
C14H13ClNO3 [M+H]+: 278.0578, found: 278.0596
N-(3,5-bis(trifluoromethyl)phenyl)-2,5-dihydroxybenzamide (16)
The title compound was synthesized following the general procedure
for synthesis of N-aryl-2-hydroxybenzamides to afford 16 as a white
solid (70 mg, 26%). 1H NMR (400 MHz, d6-DMSO): 11.04 (s, 1H), 8.41
(s, 1H), 7.82 (s, 1H), 7.17 (t, J = 8.2 Hz, 1H), 6.43 (d, J = 8.2 Hz, 2H).
13C NMR (100 MHz, d6-DMSO): 167.8, 158.3, 140.3, 132.9, 130.8 (q, J
= 32.8 Hz), 123.3 (q, J = 272.8 Hz), 120.4, 120.4, 116.8, (q, J = 4.2 Hz),
116.7, 107.5, 107.1. UV(λmax nm): 275; IRvmax (cm-1): 3325, 1644, 1445,
974. HRMS (ESI): calcd for C15H10F6NO3 [M+H]+: 366.0559, found:
366.0541
university of Notre Dame performed on
a
Bruker-TOF-ESI
spectrometer in positive module using direct infusion in 9:1 acetonitrile:
water. IR spectra were recorded on Bruker Alpha II FTIR spectrometer.
UV data was taken using a Thermo Scientific, Genesys 10 UV scanning
spectrometer.
6
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201900560
ChemMedChem
FULL PAPER
5-chloro-N-(3-fluoro-4-(trifluoromethyl)phenyl)-2-
N-(3,5-dibromophenyl)-2-hydroxy-5-iodobenzamide (52) The title
compound was synthesized following the general procedure for
synthesis of N-aryl-2-hydroxybenzamides to afford 52 as a white solid
(53 mg, 13%). 1H NMR (400 MHz, d6-DMSO): 11.46 (s, 1H), 10.59 (s,
1H), 8.08 (d, J = 2.3 Hz, 1H), 8.00 (d, J = 1.7 Hz, 2H), 7.71 (dd, J = 8.7,
2.3 Hz, 1H), 7.59 (t, J = 1.7 Hz, 1H), 6.83 (d, J = 8.6 Hz, 1H) . 13C NMR
(100 MHz, d6-DMSO): 165.1, 158.6, 141.5, 141.2, 137.2, 128.4, 122.3,
121.7, 120.3, 79.9. UV(λmax nm): 236; IRvmax (cm-1): 3079, 1636, 1109,
664; HRMS (ESI): calcd for C13H9Br2INO2[M+H]+: 495.8039, found:
495.8059
hydroxybenzamide (29) The title compound was synthesized
following the general procedure for synthesis of N-aryl-2-
hydroxybenzamides to afford 29 as a white solid (21 mg, 9%). 1H NMR
(400 MHz, d6-DMSO): 11.44 (s, 1H), 10.86 – 10.81 (m, 1H), 7.96 (d, J
= 13.4 Hz, 1H), 7.79 (dd, J = 15.3, 5.8 Hz, 2H), 7.67 (d, J = 8.8 Hz, 1H),
7.48 (d, J = 8.8 Hz, 1H), 7.04 (d, J = 8.8 Hz, 1H) . 13C NMR (100 MHz,
d6-DMSO): 165.1, 159.1 (d, J = 249.5 Hz), 155.9, 144.2 (d, J = 11.3
Hz), 133.1, 128.8, 127.9 (qd, J = 6.8, 4.5 Hz),122.9, 122.8 (q, J = 271.0
Hz), 120.8, 119.0, 115.9 (d, J = 3.1 Hz), 111.3 (dd, J = 32.7, 12.5 Hz),
107.8 (d, J = 25.4 Hz). UV(λmax nm): 268; IRvmax (cm-1): 2924, 1606,
1122, 695; HRMS (ESI): calcd for C14H9ClF4NO2 [M+H]+:334.0252,
found: 334.0232
N-(3,5-difluorophenyl)-2-hydroxy-4-(trifluoromethyl)benzamide
(54) The title compound was synthesized following the general
procedure for synthesis of N-aryl-2-hydroxybenzamides to afford 54 as
a white solid (191 mg, 50%).1H NMR (400 MHz, d6-DMSO): 11.54 (s,
1H), 10.70 (s, 1H), 7.88 (d, J = 7.9 Hz, 1H), 7.53 – 7.45 (m, 2H), 7.29
(d, J = 8.5 Hz, 2H), 7.00 (tt, J = 9.4, 2.4 Hz, 1H) . 13C NMR (100 MHz,
d6-DMSO): 164.9, 162.5 (dd, J = 243.2, 15.3 Hz), 156.5, 141.0 (t, J =
13.7 Hz), 132.5 (q, J = 31.8 Hz), 130.8, 124.5, 123.6 (q, J = 272.7 Hz),
115.5 (q, J = 3.8 Hz), 113.3 (q, J = 3.9 Hz), 103.0 (dd, J = 20.8, 8.9 Hz),
99.2 (t, J = 26.2 Hz) . UV(λmax nm): 268; IRvmax (cm-1): 3173, 1620, 1113,
841; HRMS (ESI): calcd for C14H9F5NO2 [M+H]+: 318.0548, found:
318.0536.
5-chloro-N-(4-fluoro-3-nitrophenyl)-2-hydroxybenzamide (35) The
title compound was synthesized following the general procedure for
synthesis of N-aryl-2-hydroxybenzamides to afford 35 as a white solid
(67 mg, 45%). 1H NMR (400 MHz, d6-DMSO): 11.51 (s, 1H), 10.76 (s,
1H), 8.66 (dd, J = 6.9, 2.7 Hz, 1H), 8.05 (dt, J = 9.2, 3.3 Hz, 1H), 7.87
(d, J = 2.7 Hz, 1H), 7.62 (dd, J = 11.2, 9.1 Hz, 1H), 7.48 (dd, J = 8.8,
2.7 Hz, 1H), 7.03 (d, J = 8.8 Hz, 1H). 13C NMR (100 MHz, d6-DMSO):
165.2, 156.4, 150.9 (d, J = 259.3 Hz), 136.4 (d, J = 7.6 Hz), 135.0 (d, J
= 3.4 Hz), 133.1, 128.5, 128.0 (d, J = 8.3 Hz), 122.7, 120.2, 119.1,
118.9 (d, J = 21.8 Hz), 117.1 (d, J = 3.0 Hz). UV(λmax nm): 254 ; IRvmax
(cm-1): 3291, 1620, 1334, 932; HRMS (ESI): calcd for C13H9ClFN2O4
[M+H]+: 311.0229, found: 311.0241
N-(3,5-dichlorophenyl)-2-hydroxy-4-(trifluoromethyl)benzamide
(55) The title compound was synthesized following the general
procedure for synthesis of N-aryl-2-hydroxybenzamides to afford 55 as
a white solid (239 mg, 56%). 1H NMR (400 MHz, d6-DMSO): 11.54 (s,
1H), 10.65 (s, 1H), 7.89 (d, J = 7.9 Hz, 1H), 7.84 (d, J = 1.9 Hz, 2H),
7.37 (t, J = 2.0 Hz, 1H), 7.29 (d, J = 8.7 Hz, 2H) . 13C NMR (100 MHz,
d6-DMSO): 165.0, 156.6, 140.8, 134.1, 132.6 (q, J = 31.9 Hz), 130.8,
124.3, 123.6 (q, J = 272.8 Hz), 123.3, 118.3, 115.5 (q, J = 3.7 Hz),
113.3 (q, J = 4.0 Hz). UV(λmax nm): 270; IRvmax (cm-1): 3081, 1640, 1110,
664; HRMS (ESI): calcd for C14H9Cl2F3NO2[M+H]+: 349.9957, found:
349.9935
N-(3-amino-4-fluorophenyl)-5-chloro-2-hydroxybenzamide (36) To
a solution of 5-chloro-N-(4-fluoro-3-nitrophenyl)-2-hydroxybenzamide
35 (1 equiv.) in anhydrous ethanol (0.1 M) was added tin (II) chloride (2
equiv.). The reaction mixture was heated to 70 °C for 12 h, cooled, and
concentrated under reduced pressure. The residue was taken in a
mixture of 100 mL ethyl acetate and 100 mL saturated aqueous sodium
bicarbonate solution and the organic layer was washed with brine, dried
over anhydrous sodium sulfate, filtered, and concentrated under
reduced pressure, and then purified via flash chromatography using
10:1 DCM/methanol to obtain compound 36 as a white solid (112 mg,
71%). 1H NMR (400 MHz, d6-DMSO): 11.94 (s, 1H), 10.22 (s, 1H), 7.95
(d, J = 2.7 Hz, 1H), 7.45 (dd, J = 8.8, 2.7 Hz, 1H), 7.20 (dd, J = 8.4, 2.6
Hz, 1H), 7.00 (d, J = 9.0 Hz, 1H), 6.97 – 6.91 (m, 1H), 6.77 (ddd, J =
8.7, 4.1, 2.6 Hz, 1H), 5.24 (s, 2H). 13C NMR (100 MHz, d6-DMSO):
165.2, 157.5, 148.0 (d, J = 234.7 Hz), 136.9 (d, J = 13.7 Hz), 134.8 (d,
J = 2.3 Hz), 133.4, 128.7, 123.1, 119.9, 119.6, 115.1 (d, J = 19.3 Hz),
109.3 (d, J = 4.4 Hz), 108.9 (d, J = 6.5 Hz). UV(λmax nm): 274; IRvmax
(cm-1): 3351, 1651, 1128, 933; HRMS (ESI): calcd for C21H14F6NO2
[M+H]+: 281.04876, found: 281.0488
N-(3,5-dimethylphenyl)-2-hydroxy-4-(trifluoromethyl)benzamide
(57) The title compound was synthesized following the general
procedure for synthesis of N-aryl-2-hydroxybenzamides to afford 57 as
a white solid (25 mg, 14%). 1H NMR (400 MHz, d6-DMSO): 11.91 (s,
1H), 10.32 (s, 1H), 8.02 (d, J = 8.0 Hz, 1H), 7.34 (s, 2H), 7.29 (d, J =
9.3 Hz, 2H), 6.79 (s, 1H), 2.27 (s, 6H) . 13C NMR (100 MHz, d6-DMSO):
164.6, 157.4, 138.1, 137.9, 132.6 (q, J = 32.0 Hz), 130.7, 125.9, 123.6
(q, J = 272.8 Hz), 123.4, 118.3, 115.4 (q, J = 3.6 Hz), 113.6 (q, J = 3.8
Hz), 21.1 . UV(λmax nm): 302; IRvmax (cm-1): 2588, 1620, 1119, 681;
HRMS (ESI): calcd for C16H15F3NO2 [M+H]+: 310.1049, found:
310.1046
N-(4-bromo-3,5-dichlorophenyl)-5-chloro-2-hydroxybenzamide
(41) The title compound was following the general procedure for
synthesis of N-aryl-2-hydroxybenzamides to afford 41 as a white solid
(53 mg, 19%). 1H NMR (400 MHz, d6-DMSO): 11.43 (s, 1H), 10.60 (s,
1H), 8.03 (s, 2H), 7.81 (d, J = 2.7 Hz, 1H), 7.48 (dd, J = 8.8, 2.7 Hz,
1H), 7.03 (d, J = 8.8 Hz, 1H) . 13C NMR (100 MHz, d6-DMSO): 165.1,
156.0, 139.1, 135.1, 133.2, 128.7, 122.8, 120.5, 120.2, 119.0, 116.1 .
UV(λmax nm): 248; IRvmax (cm-1): 3149, 1626, 1208, 655; HRMS (ESI):
calcd for C13H8BrCl3NO2 [M+H]+: 393.8799, found: 393.8808
3-chloro-N-(3,5-dimethylphenyl)-2-hydroxybenzamide (58) The title
compound was synthesized following the general procedure for
synthesis of N-aryl-2-hydroxybenzamides to afford 58 as a white solid
(45 mg, 27%). 1H NMR (400 MHz, d6-DMSO): 12.9 (s, 1H), 10.5 (s, 1H),
8.0 (dd, J = 8.1, 1.5 Hz, 1H), 7.7 (dd, J = 7.9, 1.4 Hz, 1H), 7.3 (s, 2H),
7.0 (t, J = 8.0 Hz, 1H), 6.8 (s, 1H), 2.3 (s, 6H). 13C NMR (100 MHz, d6-
DMSO): 167.8, 156.2, 137.8, 137.2, 134.0, 126.8, 126.4, 121.3, 119.6,
119.1, 117.2, 21.0. UV(λmax nm): 271 ; IRvmax (cm-1): 3380, 1614, 1549,
1117; HRMS (ESI): calcd for C15H15ClNO2 [M+H]+: 276.0786, found:
276.0767.
N-(3,5-dimethylphenyl)-5-fluoro-2-hydroxybenzamide (44) The title
compound was synthesized following the general procedure for
synthesis of N-aryl-2-hydroxybenzamides to afford 44 as a white solid
(58 mg, 43%). 1H NMR (400 MHz, d6-DMSO): 11.7 (s, 1H), 10.3 (s, 1H),
7.8 (dd, J = 9.8, 3.2 Hz, 1H), 7.3 (d, J = 1.4 Hz, 2H), 7.3 – 7.3 (m, 1H),
7.0 (dd, J = 9.0, 4.6 Hz, 1H), 6.8 (s, 1H), 2.3 (s, 6H). 13C NMR (100
MHz, d6-DMSO): 165.3 (d, J = 2.5 Hz), 155.3 (d, J = 235.0 Hz), 154.9,
138.3 (d, J = 2.8 Hz), 126.3, 121.0, 120.8, 119.1 (d, J = 7.6 Hz), 119.0,
118.9 (d, J = 6.9 Hz), 115.2 (d, J = 24.4 Hz), 21.5. UV(λmax nm): 271;
IRvmax (cm-1): 3086, 1634, 1578, 912; HRMS (ESI): calcd for
C15H15FNO2 [M+H]+: 260.1081, found: 260.1064
3,5-dichloro-N-(3,5-dimethylphenyl)-2-hydroxybenzamide (59) The
title compound was synthesized following the general procedure for
synthesis of N-aryl-2-hydroxybenzamides to afford 59 as a white solid
(65 mg, 44%). 1H NMR (400 MHz, d6-DMSO): 12.9 (s, 1H), 10.7 (s, 1H),
8.1 (t, J = 2.9, 1.5 Hz, 1H), 7.8 (d, J = 2.6 Hz, 1H), 7.3 (s, 2H), 6.8 (s,
1H), 2.3 (s, 6H). 13C NMR (100 MHz, d6-DMSO): 166.4, 155.5, 137.8,
137.2, 133.0, 126.4, 122.7, 121.8, 119.3, 118.3, 118.2, 21.0. UV(λmax
nm): 265; IRvmax (cm-1): 3234, 1632, 1572, 1168. HRMS (ESI): calcd
for C15H14Cl2NO2 [M+H]+: 310.0396, found: 310.0385.
7
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201900560
ChemMedChem
FULL PAPER
3,5-dichloro-N-(3,5-dibromophenyl)-2-hydroxybenzamide (60) The
title compound was synthesized following the general procedure for
synthesis of N-aryl-2-hydroxybenzamides to afford 60 as a white solid
(25 mg, 19%). 1H NMR (400 MHz, d6-DMSO): 14.75 (s, 1H), 7.92 (d, J
= 1.7 Hz, 2H), 7.64 (d, J = 3.0 Hz, 1H), 7.44 (t, J = 1.7 Hz, 1H), 7.34 (d,
J = 3.0 Hz, 1H). 13C NMR (100 MHz, d6-DMSO): 166.3, 163.9, 143.0,
131.7, 127.5, 127.4, 126.1, 122.9, 121.3, 119.3, 113.3. UV(λmax nm):
278; IRvmax (cm-1): 3293, 1640, 1579, 145;. HRMS (ESI): calcd for
C13H8Br2Cl2NO2 [M+H]+: 437.8293, found: 437.8261.
wells were mixed 6-7 times, then, 100 µL was withdrawn and
transferred to row two. Row two wells were mixed 6-7 times followed
by a 100 µL transfer from row two to row three. This procedure was
used to serially dilute the rest of the rows of the microtiter plate,
excluding the last prefilled row, which was used to measure growth in
the presence of compound alone. Plates were then sealed with GLAD
Press’n Seal and incubated under stationary conditions at 37 °C. After
16 hours, the plates were removed, and MIC values were measured by
recording the OD600 of each well. MIC values were determined as the
minimum concentration required to achieve 90% growth inhibition
compared to growth in untreated wells.
N-(2,5-bis(trifluoromethyl)phenyl)-3,5-dichloro-2-
hydroxybenzamide (61) The title compound was synthesized
following the general procedure for synthesis of N-aryl-2-
hydroxybenzamides to afford 61 as a white solid (21 mg, 12%). 1H NMR
(400 MHz, d3-Acetonitrile): 9.0 (s, 1H), 7.8 (d, J = 8.3 Hz, 1H), 7.7 (d, J
= 3.0 Hz, 1H), 7.5 – 7.4 (m, 1H), 7.3 (d, J = 3.0 Hz, 1H). 13C NMR (100
MHz, d3-Acetonitrile): 167.6, 166.4, 140.3, 134.5, 134.2, 132.3, 128.7,
128.0 (q, J = 5.8 Hz), 127.2, 124.8 (q, J = 271.9 Hz), 124.6 (q, J = 271.9
Hz), 121.8 (q, J = 4.1 Hz), 119.9, 119.4 (q, J = 3.8 Hz), 113.3. UV(λmax
nm): 273; IRvmax (cm-1): 3291, 1640, 1542, 1120; HRMS (ESI): calcd
for C15H7Cl2F6NO2Na [M+Na]+: 439.9650, found: 439.9659.
Time-kill Curves Strains were cultured overnight in CAMHB and
subcultured to 1.04 x 106 CFU/mL for AB 4106 and 1.96 x 106 CFU/mL
for KP B9 in fresh CAMHB. The subculture was then transferred to
culture tubes in 3 mL aliquots, which were dosed with adjuvant, colistin,
or adjuvant plus colistin. One aliquot was not dosed to serve as a
control. All samples were then incubated at 37 °C with shaking. At 2, 4,
6, 8, and 24-hour time points, 100 µL was taken from each sample and
ten-fold diluted in CAMHB up to 7 times. 100 µL of diluted culture was
plated on LB (Lennox) agar and incubated at 37 ˚C overnight. The total
number of bacterial colonies on each plate was determined using a
SphereFlash colony counter (NEUTEC Group Inc.)
General Biological Experimental
Cell line Toxicity 4T1 cells (ATCC Manassas, VA) were plated at a
density of 1 x 104 cells/well in 96-well plates in Roswell Park Memorial
Institute Media 1640 (RPMI) (Gibco, Gaithersburg, MD) supplemented
with 10% Fetal Bovine Serum (Gibco), 2 mM GlutaMAX (Gibco) and 50
µM 2-mercaptoethanol (Sigma Aldrich, St. Louis, MO) and incubated at
37°C under a 5% CO2 atmosphere in the dark for 18 h. Cell cultures
were treated with serial dilutions of compounds in the presence or
absence of 1 µg/mL colistin (3 replicates per condition) and incubated
for an additional 18 hours. The following control conditions were used:
media only (blank), 1% Triton X100 (0% cell viability), 0.5% DMSO
(100% cell viability). Each condition was then treated with 10% volume
Bacterial strains, media, and antibiotics A. baumannii strains 4106,
4112, 4119, 3941 and 3942 were obtained from Walter Reed Army
Institute for Research (WRAIR). A. baumannii strain 17978mcr-1 and K.
pneumoniae strains B9, A5, C3 and F2210291mcr-1 were obtained from
Professor Robert Ernst at The University of Maryland, Baltimore. Stock
cultures were stored in 25% glycerol and maintained at -80 °C. Prior to
use, colonies were grown on LB (Lennox) agar. CAMHB was
purchased from BD Diagnostics. Colistin sulfate salt was purchased
from Sigma Aldrich (Cat# C4461). All assays were run in duplicate and
repeated at least two separate times.
of
a
5
mg/mL solution of 3-(4,5-dimethylthiazol- 2-yl)-2,5-
diphenyltetrazolium bromide (MTT) (Sigma Aldrich) in sterile filtered 1X
phosphate buffered saline (PBS) and incubated for 2 h. at 37º C in 5%
CO2, after which the media was aspirated and the resulting formazan
crystals were resuspended in 100 µL acidified (4 mM HCl) isopropanol.
The 96-well plate was then read at 540 nm on a FLUOstar Optima
(BMG Labtech Cary, NC) microplate reader. Cell viability was
calculated as a percentage using the two previously mentioned controls.
Broth microdilution method for the determination of minimum
inhibitory concentrations Bacteria were cultured for 4 to 6 hours in
CAMHB and subcultured to 1.04 x 106 CFU/mL for AB 4106 and 1.96
x 106 CFU/mL for KP B9 in fresh CAMHB. To aliquots (1 mL) was
added compound from stock solutions in DMSO, such that the
compound concentration equaled the highest concentration tested.
Samples were then dispensed (200 μL) into the first row of a 96-well
microtiter plate in which all but the final row of subsequent wells were
prefilled with 100 μL of the untreated bacterial subculture. The final row
was filled with media to act as a sterility control and blank. Row one
wells were mixed 6-7 times, then, 100 µL was withdrawn and
transferred to row two. Row two wells were mixed 6-7 times followed
by a 100 µL transfer from row two to row three. This procedure was
used to serially dilute the rest of the rows of the microtiter plate,
excluding the last prefilled row, which was used to measure growth in
the absence of compound. Plates were then sealed with GLAD Press’n
Seal and incubated under stationary conditions at 37 °C. After 16 hours,
the plates were removed, and MIC values were measured by recording
the OD600 of each well. MIC values were determined as the minimum
concentration required to achieve 90% growth inhibition compared to
growth in untreated wells.
Acknowledgements
The authors thank the NIH (1R01AI136904) for funding.
Keywords: antibiotic resistance • adjuvant • colistin • Gram-
negative •
References:
[1]
[2]
J. O'Neill, 2014.
H. W. Boucher, G. H. Talbot, J. S. Bradley, J. E. Edwards,
D. Gilbert, L. B. Rice, M. Scheld, B. Spellberg, J. Bartlett, Clin
Infect Dis 2009, 48(1), 1-12.
[3]
[4]
Z. Kmietowicz, BMJ 2017, 358, j4339.
D. L. Stevens, D. Herr, H. Lampiris, J. L. Hunt, D. H. Batts,
Broth microdilution method for measurement of colistin
potentiation Bacteria were cultured for 4 to 6 hours in CAMHB and
diluted to 1.04 x 106 CFU/mL for AB 4106 and 1.96 x 106 CFU/mL for
KP B9 in fresh CAMHB. To aliquots (4 mL) was added compound from
stock solutions in DMSO. One aliquot was not dosed to allow
measurement of the colistin MIC in the absence of compound. A 1 mL
aliquot of each sample was dosed with colistin, and from this 200 μL
was dispensed into the first row of a 96-well microtiter plate in which all
but the final row of subsequent wells was prefilled with 100 μL of the
corresponding compound dosed bacterial suspension The final row
was filled with media to act as a sterility control and blank. Row one
B. Hafkin, Clin Infect Dis 2002, 34(11), 1481-1490; V. G. Fowler,
H. W. Boucher, G. R. Corey, E. Abrutyn, A. W. Karchmer, M. E.
Rupp, D. P. Levine, H. F. Chambers, F. P. Tally, G. A. Vigliani, C.
H. Cabell, A. S. Link, I. DeMeyer, S. G. Filler, M. Zervos, P. Cook,
J. Parsonnet, J. M. Bernstein, C. S. Price, G. N. Forrest, G.
Fätkenheuer, M. Gareca, S. J. Rehm, H. R. Brodt, A. Tice, S. E.
Cosgrove, S. a. E. a. B. S. Group, N Engl J Med 2006, 355(7),
653-665.
8
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FULL PAPER
[5]
M. F. Richter, P. J. Hergenrother, Reaction: Broad-
Spectrum Antibiotics, a Call for Chemists, Vol. 3, Chem 2017, pp.
8-14.
[6]
R. L. Nation, J. Li, O. Cars, W. Couet, M. N. Dudley, K. S.
Kaye, J. W. Mouton, D. L. Paterson, V. H. Tam, U.
Theuretzbacher, B. T. Tsuji, J. D. Turnidge, Lancet Infect Dis
2015, 15(2), 225-234.
[7]
J. M. Rolain, Expert Rev Anti Infect Ther 2012, 10(8), 917-934.
[8] G. Ozkan, S. Ulusoy, A. Orem, M. Alkanat, S. Mungan, E.
S. Biswas, J. M. Brunel, J. C. Dubus, M. Reynaud-Gaubert,
Yulug, F. B. Yucesan, Antimicrob Agents Chemother 2013, 57(8),
3463-3469.
[9]
N. Petrosillo, F. Taglietti, G. Granata, J Clin Med 2019,
8(7).
[10] M. R. Pelletier, L. G. Casella, J. W. Jones, M. D. Adams, D.
V. Zurawski, K. R. Hazlett, Y. Doi, R. K. Ernst, Antimicrob Agents
Chemother 2013, 57(10), 4831-4840.
[11] A. O. Olaitan, S. Morand, J. M. Rolain, Front Microbiol
2014, 5, 643.
[12] A. M. Caliendo, D. N. Gilbert, C. C. Ginocchio, K. E.
Hanson, L. May, T. C. Quinn, F. C. Tenover, D. Alland, A. J.
Blaschke, R. A. Bonomo, K. C. Carroll, M. J. Ferraro, L. R.
Hirschhorn, W. P. Joseph, T. Karchmer, A. T. MacIntyre, L. B.
Reller, A. F. Jackson, A. Infectious Diseases Society of, Clin
Infect Dis 2013, 57 Suppl 3, S139-170.
[13] R. J. Melander, C. Melander, ACS Infect Dis 2017, 3(8),
559-563; G. D. Wright, Trends Microbiol 2016, 24(11), 862-871.
[14] W. M. Huggins, W. T. Barker, J. T. Baker, N. A. Hahn, R. J.
Melander, C. Melander, ACS Med Chem Lett 2018, 9(7), 702-
707; W. T. Barker, C. E. Chandler, R. J. Melander, R. K. Ernst, C.
Melander, Bioorg Med Chem 2019, 27(9), 1776-1788; B. M.
Minrovic, V. B. Hubble, W. T. Barker, L. A. Jania, R. J. Melander,
B. H. Koller, C. Melander, ACS Med Chem Lett 2019, 10(5), 828-
833; C. M. Brackett, R. E. Furlani, R. G. Anderson, A.
Krishnamurthy, R. J. Melander, S. M. Moskowitz, R. K. Ernst, C.
Melander, Tetrahedron 2016, 72(25), 3549-3553.
[15] W. T. Barker, A. M. Nemeth, S. M. Brackett, A. K. Basak,
C. E. Chandler, L. A. Jania, W. J. Zuercher, R. J. Melander, B. H.
Koller, R. K. Ernst, C. Melander, ACS Infect Dis 2019.
[16] S. Kang, H. J. Min, M. S. Kang, M. G. Jung, S. Kim, Bioorg
Med Chem Lett 2013, 23(6), 1748-1751.
[17] E. A. Lindsey, R. J. Worthington, C. Alcaraz, C. Melander,
Org Biomol Chem 2012, 10(13), 2552-2561.
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Entry for the Table of Contents
We report a structure-activity relationship (SAR) study on the recently identified colistin adjuvant IMD-0354, and describe several
compounds with more potent activity than the parent including one analogue that suppresses the colistin minimum inhibitory
concentration (MIC) below the breakpoint level against a highly resistant strain of Klebsiella pneumoniae at 250 nM.
10
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