Identification of Anti-Mycobacterial Biofilm Agents Based on the 2-Aminoimidazole Scaffold

Article abstract of DOI:10.1002/cmdc.201900033

Tuberculosis (TB) remains a significant global health problem for which new therapeutic options are sorely needed. The ability of the causative agent, Mycobacterium tuberculosis, to reside within host macrophages and form biofilm-like communities contributes to the persistent and drug-tolerant nature of the disease. Compounds that can prevent or reverse the biofilm-like phenotype have the potential to serve alongside TB antibiotics to overcome this tolerance, and decrease treatment duration. Using Mycobacterium smegmatis as a surrogate organism, we report the identification of two new 2-aminoimidazole compounds that inhibit and disperse mycobacterial biofilms, work synergistically with isoniazid and rifampicin to eradicate preformed M. smegmatis biofilms in vitro, are nontoxic toward Galleria mellonella, and exhibit stability in mouse plasma.

Full text of DOI:10.1002/cmdc.201900033

Accepted Article
Title: Identification of novel anti-mycobacterial biofilm agents based
upon the 2-aminoimidazole scaffold
Authors: Christian Corey Melander, T. Vu Nguyen, Bradley M.
Minrovic, and Roberta J. Melander
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To be cited as: ChemMedChem 10.1002/cmdc.201900033
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Identification of novel anti-mycobacterial biofilm agents based
upon the 2-aminoimidazole scaffold
T. Vu Nguyen,[a] Bradley M. Minrovic[b], Roberta J. Melander[b] and Christian Melander[b] *
[a]
[b]
Department of Chemistry, North Carolina State University, Raleigh, NC 27695.
Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556.
*to whom correspondence should be addressed: cmelande@nd.edu
Supporting information for this article is given via a link at the end of the document
Abstract: Tuberculosis (TB) remains a significant global health
problem for which new therapeutic options are sorely needed. The
ability of the causative agent, Mycobacterium tuberculosis, to reside
within host macrophages and form biofilm-like communities
contributes to the persistent and drug tolerant nature of the disease.
Compounds that can prevent or reverse the biofilm-like phenotype
have the potential to serve alongside TB antibiotics to overcome this
tolerance, and reduce treatment duration. Using Mycobacterium
smegmatis as a surrogate organism, we report the identification of
two new 2-aminoimidazole compounds that inhibit and disperse
mycobacterial biofilms, work synergistically with isoniazid and
rifampicin to eradicate preformed M. smegmatis biofilms in vitro, are
non-toxic toward Galleria mellonella, and exhibit stability in mouse
plasma.
evade host immune responses and drug therapy.[14] Targeting M.
tuberculosis biofilm formation therefore represents a potential
strategy to combat TB persistence, whereby compounds that
inhibit and disperse M. tuberculosis biofilms could be
administered alongside antibiotics to overcome tolerance. This
approach has the potential to enable reduced duration of
therapy, and subsequently improve patient compliance.
Our group has previously reported several small molecules
that possess the ability to inhibit and disperse mycobacterial
biofilms (Figure 1),[15] including aryl 2-aminoimidazole (2-AI) 1,
reverse amide 2-aminoimidazole 2, and 2-aminobenzimidazole 3.
Compound 1, which has also previously been shown to have
anti-biofilm and antimicrobial activity in other pathogenic
bacteria,[16] was observed to restore isoniazid susceptibility to M.
tuberculosis expressing drug tolerance in an in vitro model
system that utilizes lysed human leukocytes to mimic the in vivo
microbial communities of virulent M. tuberculosis.[15b]
Introduction
Tuberculosis (TB), caused by infection with Mycobacterium
tuberculosis (Mtb), still reigns as one of deadliest diseases
impacting global health in both developed and especially
developing countries,[1] and is estimated to be responsible for
more than
2
million deaths each year.[2] TB is highly
contagious,[3] and manifestations of the disease include
persistent coughing, extreme weakness or fatigue, weight loss,
and constant chest pain.[4] Although tuberculosis is primarily an
infection of the respiratory system, it also affects the central
nervous system, kidney, brain, and other organs.[5] If left
untreated, TB can lead to organ failure and mortality.[6] Currently,
TB is the leading cause of death from a single infectious agent,[7]
and is the biggest killer of people infected with HIV[8].
Current treatment regimens for drug susceptible TB
consist of six to nine months of combination therapy with the
first-line anti-TB drugs isoniazid, rifampicin, ethambutol, and
pyrazinamide, while infections caused by multi-drug resistant
(MDR) and extensively drug resistant (XDR) strains of M.
tuberculosis require even more protracted regimens involving
additional antibiotics.[9] The prolonged treatment times, and
deleterious side effects of these regimens result in considerable
patient noncompliance, and this is thought to be a significant
underlying factor in the persistence of active TB.[10]
M. tuberculosis has a remarkable propensity to persist in
stressful environments such as those experienced during
prolonged antibiotic treatment.[11] This is thought to be a result of
several factors including: the ability to reside within host
macrophages,[12] and the ability to form multicellular surface
attached communities, known as biofilms.[13] Many studies have
proposed that biofilms play a key role in bacterial persistence
and drug tolerant bacterial infections, and it is thought that M.
tuberculosis uses biofilm formation as a defense strategy to
Figure 1. Structures of previously reported compounds with anti-mycobacterial
biofilm activity.
As in our[15] and other group’s[17] previous reports, we
employed M. smegmatis as a surrogate organism for M.
tuberculosis due to the slow growing nature of M. tuberculosis,
as well as the fact that M. tuberculosis is a BSL3 organism. We
have previously shown that anti-biofilm and antibiotic
potentiation activity against M. smegmatis typically translates to
15b]
activity against M. tuberculosis.[15a,
Using this screening
model, and by performing analogue synthesis upon the scaffold
of compound 1, we have identified two novel derivatives that
possess potent anti-biofilm activity, work synergistically with
isoniazid and rifampicin to eradicate preformed M. smegmatis
biofilms in vitro, are non-toxic against Galleria mellonella, and
exhibit stability in mouse plasma.
Results and Discussion
To delineate the structure activity relationship (SAR) of this class
of compounds in the context of mycobacterial anti-biofilm activity,
compound 1 along with members of a library of previously
reported related 2-AI compounds and several newly synthesized
1
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analogues, were probed for the ability to inhibit and disperse
biofilms formed by M. smegmatis mc2155 (ATCC 700084).
Compound minimum inhibitory concentrations (MICs) were also
determined to give an indication of the toxicity of each analogue
toward planktonic bacteria.
activity of the new compounds, returning an EC50 of 94.9 µM.
The heptyl derivative 9c exhibited IC50 and EC50 values of 23.5
and 130 µM respectively, indicating that for this series, the hexyl
chain of compound 1 was optimal for anti-biofilm activity.
In our initial studies with library stocks, an IC50 value
(concentration required to inhibit 50% biofilm formation) of 8.0
µM was recorded for compound 1, while the dispersion activity
of this compound was found not to be dose responsive, thereby
precluding the determination of an EC50 value (concentration
required to disperse 50% of a pre-formed biofilm). Following
resynthesis of compound 1 for this study, IC50 and EC50 values
of 10.4 and 80.5 µM respectively were obtained (Table 1). The
MIC of compound 1 is 100 µM. We next screened several
previously reported substituted 2-aminoimidazoles,[18] for the
ability to inhibit and disperse M. smegmatis biofilms. Of these,
none of the compounds that contain substituted phenyl moieties
at the 5-position of the 2-AI ring inhibited or dispersed biofilms to
any significant degree (compounds S1-S4 Table S1).
Compounds 4, 5, and 6 (Figure 2), which possess short aliphatic
substituents (ethyl, isopropyl and butyl respectively) at the 31-
position of the 2-AI ring exhibited IC50 values below 30 µM
and/or EC50 values below 100 µM, however all exhibited
reduced activity compared to the parent compound (Table 1).
The most active compound of this series was the ethyl derivative
4, which exhibited IC50 and EC50 values of 15.5 and 92.4 µM
respectively. Larger substituents at this position, including heptyl,
cyclohexyl and phenyl groups, abolished anti-biofilm activity
(compounds S7-S9 Table S1).
Scheme 1. Synthesis of 2-AI analogues with varying substituents at the aryl
tail group. Reagents and Conditions: a) (COCl)2, DCM, DMFcat 0 ˚C, 1 h; b)
CH2N2, Et2O, 0 ˚C, 1 h; c) HBr, 0 ˚C, 20 min: d) Boc-guanidine, DMF, r.t., 48 h;
e) TFA/DCM 1:4, r.t. 2 h; f) HCl/MeOH
Table 1 Biofilm inhibition and dispersion activity of 2-aminoimidazole
derivatives.
Compound
MIC (µM)
100
IC50 (µM)
10.4 ± 0.6
15.5 ± 2.3
16.4 ± 1.1
27.1 ± 4.5
19.8 ± 2.8
13.6 ± 1.2
23.5 ± 0.9
>50a
EC50 (µM)
80.6 ± 2.2
92.4 ± 5.4
90.1 ± 4.3
>200a
1
4
100
5
100
6
200
9a
9b
9c
9d
9e
9f
150
98.1 ± 2.3
94.9 ± 4.2
130 ± 2.3
>200
200
150
150
>200
100
>50
>200
Figure 2. Structures of previously reported compounds 4-6
>50
>200
9g
9h
9i
100
45.0 ± 3.3
29.0 ± 4.3
17.3 ± 1.2
30.1 ± 4.4
27.9 ± 1.8
20.9 ± 2.2
18.7 ± 1.9
12.5 ± 1.3
>50
>200
A series of mono-substituted analogues of compound 1, in
which the para-hexyl substituent on the phenyl ring was varied,
was next explored to probe the SAR of this portion of the
molecule. Previously reported compounds 9a-e,[16a] along with
several novel derivatives (9f-r) containing a variety of para-
substituted phenyl groups were synthesized as outlined in
Scheme 1. Briefly, carboxylic acids 7a-r were converted to the
corresponding α-bromo ketones 8a-r by treatment with oxalyl
chloride followed by reaction with diazomethane and finally
quenching with hydrobromic acid. The α-bromo ketones were
then cyclized with Boc-guanidine and, following immediate Boc-
deprotection with 30% TFA in dichloromethane, were purified
and then treated with HCl/MeOH to afford compounds 9a-r as
HCl salts for biological testing.
Each compound was assayed for its inherent toxicity
toward the bacterium, and no compound was found to have an
MIC less than 100 µM. Compounds 9a-c and 9f-i, which
possess aliphatic chains of increasing length, displayed no
improvement in anti-biofilm activity in comparison to the parent
compound 1, with the most active being pentyl derivative 9b,
which exhibited an IC50 of 13.6 µM. These analogues dispersed
pre-formed biofilms with EC50 values ranging from 94.9 to >200
µM, with the pentyl derivative again exhibiting the greatest
100
150 ± 6.2
152 ± 10
136 ± 5.6
170 ± 8.6
143.0 ± 4.8
105 ± 6.3
88.2 ± 4.3
180 ± 10
>200
200
9j
200
9k
9l
200
100
9m
9n
9o
9p
9q
9r
>200
150
>200
>200
200
>50
>50
>200
150
>50
>200
aHighest concentration tested was 50 µM for IC50 and 200 µM for EC50
.
We next investigated the effect that replacement of the
alkyl chain on the phenyl ring of 1 with an alkoxy chain had upon
anti-biofilm activity. Compounds 9j-p exhibited similar activity to
the corresponding alkyl derivatives, with pentoxy derivative 9n,
2
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which has the same chain length as compound 1, exhibiting the
greatest activity, returning IC50 and EC50 values of 12.5 and 88.2
µM respectively. The introduction of additional oxygen atoms
into the alkyl chain in poly(ethylene glycol) (PEG) derivatives 9o
and 9p resulted in a dramatic loss in activity, with IC50/EC50
values greater than 50 µM and 200 µM respectively. The
introduction of a tert-butyl, azido, phenyl, or bromo substituent
(compounds 9d-e and 9q-r) was not well tolerated as evident by
the inability of these analogues to inhibit and disperse biofilms
below 50 µM and 200 µM respectively.
potent biofilm inhibitors with IC50 values in the range of 8.6-13.5
µM, but were less effective at dispersing biofilms in comparison
to the parent compound 1. Compounds possessing aromatic
substituents at the 4-position (12d-f), displayed a reduced ability
to inhibit or disperse M. smegmatis biofilms (IC50 and EC50
values >50 µM and 150 µM respectively).
As a result of the activity displayed by compound 12g, we
next synthesized three additional 4-trifluoromethyl analogues
12h-j (Scheme 3). The goal was to elucidate if the
trifluoromethyl group would improve biofilm activity for the well-
tolerated alkoxy derivatives. Briefly, the 1-iodobenzene 4-alkoxy
benzene analogues were reacted with n-butylithium and then
subsequently added to the N-Boc trifluoroalanine Weinreb amide
10g to form α-amino ketones 11h-j. Boc deprotection and
subsequent condensation with cyanamide yielded the 4,5-
disubstituted 2-AIs 12h-j. After purification, each compound was
converted to the corresponding HCl salt for biological testing.
Scheme
2 Synthesis of 4,5-substituted 2-AI analogues. Reagents and
Conditions: a) HN(OMe)MeHCl, BOP,Et3N, DCM, 0 ˚C, 1 h; b) 4-hexyl phenyl
lithium, THF, -78 ˚C –r.t. 16 h; c) TFA/DCM, 0 ˚C - r.t. 2 h; d) H2NCN,
H2O/EtOH pH 4.3, 95 ˚C, 3 h; e) HCl/MeOH
Scheme 3 Synthesis of additional trifluoromethyl 2-AI analogues. Reagents
and Conditions: a) 4-alkoxyphenyl lithium, THF, -78 ˚C –r.t. i h; b) TFA/DCM, 0
˚C - r.t. 2 h; c) H2NCN, H2O/EtOH pH 4.3, 95 ˚C, 3 h; d) HCl/MeOH
To further this SAR study, we next investigated the impact
of introducing various substituents at the 5-position of the 2-AI
on the inhibition and dispersion of M. smegmatis biofilms by
The presence of the 4-trifluoromethyl substituent in
compounds 12h-j resulted in increased anti-biofilm activity in
comparison to the corresponding unsubstituted alkoxy
derivatives 7l-n (Table 3). This series followed the same SAR
trend observed for the unsubstituted analogues, with pentoxy
derivative 12j being the most active of this series, and exhibiting
comparable, although slightly reduced activity to 12g, with IC50
and EC50 values of 7.4 µM and 74.2 µM respectively. Finally,
colony count analysis at 48 hours was performed for each
compound at its IC50 to verify a non-bactericidal mode of action.
synthesizing
a series of 4,5-substituted 2-aminoimidazole
derivatives as previously reported16. Briefly, readily available
Boc-protected amino acids were converted to the corresponding
Weinreb amides 10a-g, followed by reaction with 4-hexylphenyl
lithium to form the α-amino ketones 11a-g. Boc-deprotection and
subsequent condensation with cyanamide yielded the 4,5-
disubstituted 2-AIs 12a-g (Scheme 2). After purification, each
compound was converted to the corresponding HCl salt for
biological testing.
Table 3 Biofilm inhibition and dispersion activity of trifluoromethyl 2-AIs
Compound
MIC
200
200
200
IC50
EC50
Table 2 Biofilm inhibition and dispersion activity of 4,5 substituted 2-AIs
12h
17.1 ± 0.9
15.4 ± 1.0
7.4 ± 0.60
119.5 ± 3.0
95.2 ± 5.4
74.2 ± 2.5
Compound
MIC
IC50
8.6 ± 0.5
13.5 ± 1.4
10.1 ± 0.8
>50a
EC50
12i
12a
25
86.5 ± 3.4
94.4 ± 3.8
90.1 ± 4.3
>200a
12j
12b
25
25
12c
We next asked the question whether lead compounds 12g
and 12j would show synergistic effects with conventional anti-
tuberculosis antibiotics upon biofilm dispersion. Studies by the
Ojha Lab have shown that M. tuberculosis and M. smegmatis
can form biofilms that tolerate more than 50 times the MIC of
both isoniazid (INH) and rifampicin (RIF).19-21 These two anti-
tuberculosis drugs were chosen because they are the antibiotics
most commonly administered in the first 6 to 9 months of TB
chemotherapy.22
To study synergistic effects, biofilms were established in
96-well PVC plates for 48 hours. Media and planktonic bacteria
were removed, and the wells were treated with either: media
alone, media plus antibiotic, media plus compound 12g or 12j,
or media plus a combination of compound and antibiotic. Dose
response studies were performed to determine the EC50 of the
100
200
200
>200
12d
>50
185.6 ± 9.3
150.5 ± 10.3
70.1 ± 3.6
12e
>50
12f
6.8 ± 0.9
12g
aHighest concentration tested was 50 µM for IC50 and 200 µM for EC50
.
Many of the 4,5-substituted analogues exhibited both good
inhibition and dispersion activity against M. smegmatis (Table 2),
and this was especially so for the trifluoromethyl derivative 12g,
for which an IC50 value of 6.8 µM and an EC50 value of 70.1 µM
was recorded. Compound 12g was also inherently nontoxic
toward the bacterium (MIC >200 µM). The corresponding 4-
methyl analogue 12a displayed greater toxicity toward the
bacterium (MIC 25 µM), as did the 4-ethyl and 4-ispropyl
analogues 12b and c (MICs 25 µM). All three analogues were
3
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tested compound as a function of antibiotic concentration and
this value was compared to the EC50 of the compound alone.
Neither INH nor (RIF) affected biofilm mass at the
concentrations tested (up to 1024 µg mL-1) when compared to
samples treated with media only. The addition of 256 µg/mL INH
lowered the EC50 of 12g from 70.1 to 25.4 µM, while a similar
effect was seen for RIF (128 µg/mL), which lowered the EC50 to
30.3 µM. Concurrent addition of both antibiotics resulted in an
EC50 value of 20.5 µM, a three-fold reduction (Table 4) See SI
for a full list of concentrations tested). Likewise, the EC50 value
of compound 12j was reduced in the presence of the same
concentrations of INH, RIF, or INH + RIF to 27.5, 26.6 and 17.7
µM respectively, the latter representing a four-fold reduction.
highest concentration tested (500 µM). While the amphipathic
nature of these compounds likely contributes to the observed
moderate hemolytic activty, and may also play a role in the
mechanism of action with regards to anti-biofilm activity, it is
unlikely that the anti-biofilm activity is derived entirely from this
characteristic given the similarly structured compounds in this
library that do not possess antibiotfilm activity. Determination of
the mechnism of action is currently being investigated.
We next evaluated whole organism toxicity of all three
compounds in Galleria melonella, a model organism that has
been employed to predict treatment efficacy and outcome for
mycobacterial infections.[20] All three compounds were dosed at
400 mg/Kg with >93% survival observed for each dose after six
days.
Table
INH/RIF
4
Dispersal enhancement between compounds 12g and 12j and
The stability of the lead compounds was assessed in both
microsomes and plasma. Microsome stability was assessed
using microsomes from liver pooled from (CD-1) male mice,
Phenacetin was used as positive control and was rapidly
degraded with a half-life (t1/2) of 35.4 minutes. The half-life for
Antibiotic
Compound
Antibiotic
EC50 (µM)
Concentration
(µg mL-1)
INH
256
25.4 ± 1.2
30.3 ± 2.6
20.5 ± 2.9
27.5 ± 1.3
26.6 ± 1.5
17.7 ± 1.9
compound
1 was calculated to be 66.0 min, while the
12g
12j
RIF
128
trifluoromethyl derivatives 12g and 12j exhibited similar half-lives
of 58.7 min. and 61.3 min respectively. The stability of the lead
compounds in mouse plasma was next determined. Verapamil
was used as a negative control and showed prolonged stability
in plasma with a half-life >480 minutes. Propanolol was used as
a positive control and was rapidly degraded with a half-life of
20.6 minutes. The parent compound 1 exhibited a t1/2 of 230 min.
in mouse plasma, while the trifluoromethyl compounds 12g and
12j showed improved stability in plasma compared to compound
1, with half-lives of 266 min and 288 min respectively. The
toxicity and stability profiles of the lead compounds indicate their
suitability as viable leads for an in vivo proof-of-concept
assessment of this therapeutic approach.
INH + RIF
INH
256 + 128
256
RIF
128
INH + RIF
256 + 128
After establishing the synergistic effects of compounds 12g
and 12j, we next asked whether the cells being dispersed
displayed the same response to antibiotics as cells grown under
planktonic conditions. Under planktonic growth, incubation with
isoniazid or rifampicin at their MIC of 32 µg mL-1 and 8 µg mL-1,
respectively, resulted in cell death. We have also established
that M. smegmatis biofilms were non-responsive to isoniazid and
rifampicin up to 1024 µg mL-1. To determine whether cells being
dispersed by 12g and 12j showed phenotypic variation, we first Conclusions
established M. smegmatis biofilms over 48 hours. The biofilms
were washed to remove planktonic bacteria then treated with
either compound alone at the EC50 concentration or compound
with isoniazid (256 µg mL-1) plus rifampicin (128 µg mL-1) at its
lowest synergistic EC50 concentration. After 24 hours of
treatment, an aliquot of the culture was serially diluted and
plated to determine the number of colony forming units (CFUs).
The log reduction was calculated by taking the log of the CFUs
from treatment with just INH and RIF minus the log of the CFUs
from compound treatment with INH and RIF. Compound 12g in
combination with isoniazid (256 µg mL-1) plus rifampicin (128 µg
mL-1) gave a log reduction of 2.94 ± 0.28. Combinatorial
treatment with compound 12j similarly resulted in a log reduction
of 2.66 ± 0.37. The log reductions indicate that the dispersed M.
smegmatis cells are drug susceptible.
In summary, using the previously identified compound 1 as a
lead, SAR studies identified trifluoromethyl derivatives 12g and
12j with increased activity, inhibiting the formation of M.
smegmatis biofilms below 10 µM. The compounds exhibited
synergistic biofilm dispersion activity with the frontline TB
antibiotics isoniazid and rifampicin, with 3-4 fold reductions in
EC50 value. The compounds were non-toxic against G.
mellonella, showed plasma stability beyond 4 hours. Current
efforts are focused on further tuning this class of 2-AI molecule
to improve upon its anti-biofilm activity, stability and toxicity
profile, and examining its activity against M. tuberculosis.
Experimental Section
To assess the potential of lead compounds as candidates
for in vivo studies, both the toxicity profiles, and stability were
investigated. To assess eukaryotic toxicity, the hemolytic activity
of 1, 12g, and 12j was first assessed using difibrinated sheep
blood.[19] Compounds 12g and 12j showed some hemolytic
activity, effecting 50% lysis (compared to 1% Triton-X) at
concentrations of 151 µM and 159 µM respectively. These
concentrations are well above (7-8 fold) the EC50 concentrations
observed for synergy with anti-tuberculosis drugs. The parent
compound 1 elicited less than 50% red blood cell lysis at the
General Chemistry Experimental All reagents used for chemical
synthesis were purchased from commercially available sources and used
without further purification. Chromatography was performed using 60 Å
mesh standard grade silica gel from Sorbtech. NMR solvents were
obtained from Cambridge Isotope Labs and used as is. 1H NMR (300
MHz or 400 MHz) and 13C NMR (75 MHz or 100 MHz) spectra were
recorded at 25 °C on Varian Mercury spectrometers. Chemical Shifts (δ)
are given in ppm relative to tetramethylsilane or respective NMR solvent;
coupling constants (J) are in hertz (Hz). Abbreviations used are s =
singlet, bs = broad singlet, d = doublet, dd = doublet of doublets, t =
4
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10.1002/cmdc.201900033
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triplet, dt = doublet of triplets, bt = broad triplet, qt = quartet, m = multiplet,
bm = broad multiplet and br = broad. High and low resolution mass
spectra were obtained at the NCSU Department of Chemistry Mass
Spectroscopy Facility. Infrared Spectra were obtained on an FT/IR-4100
spectrophotometer (νmax in cm-1). UV absorbance was recorded on a
Genesys 10 scanning UV/visible spectrophotometer (λmax in nm). The
purities of the tested compounds were all verified to be >95% by LC-MS
analysis on a Shimadzu LC-MS 2020 with Kinetex, 2.6 mm, C18 50 x
2.10 mm.
7.79 (m, 2H), 7.31 (d, J = 7.9 Hz, 2H), 4.44 (d, J = 0.9 Hz, 1H), 2.72 (q, J
= 7.6 Hz, 2H), 1.26 (td, J = 7.6, 2.2 Hz, 4H). 13C NMR (101 MHz, cdcl3)
δ 191.1, 151.3, 131.8, 129.3, 128.9, 128.5, 128.5, 46.1, 31.1, 29.8, 29.1,
15.2. IR νmax (cm-1) 3087, 2065, 1524, 1510, 1309; UV (λmax nm) 282;
HRMS (ESI) calcd for C10H11BrO (M+) 228.1010, found 228.1029.
2-Bromo-1-(4-propylphenyl)ethan-1-one 8i was synthesized as
described in the general procedure for α-bromo ketone synthesis.
Following purification via flash column chromatography the product was
obtained as a clear oil (75 % yield) 1H NMR (400 MHz, cdcl3) δ 8.13 –
7.72 (m, 2H), 7.50 – 7.23 (m, 2H), 4.44 (s, 2H), 2.65 (dd, J = 8.5, 6.8 Hz,
2H), 1.86 – 1.47 (m, 2H), 0.95 (t, J = 7.3 Hz, 3H). 13C NMR (101 MHz,
cdcl3) δ 191.1, 149.8, 131.8, 129.2, 129.1, 129.1, 128.8, 46.1, 38.2, 31.1,
24.3, 13.9. IR νmax (cm-1) 3208, 1927, 1604, 1590; UV (λmax nm) 274;
HRMS (ESI) calcd for C11H13BrO (M+) 242.1280, found 242.1283.
Compounds 9f, 9c, 9d, 9q, 9e, 9r, 12d, 12e, 12f were previously
synthesized, and characterized.16 Compounds were tested without
further purification from 100 mM stock solutions in biological grade
DMSO. Compounds 1, and 12a-f were synthesized as previously
reported, 13C NMR, and LRMS (ESI) aligned with previously reported
spectral and MS data.16
2-Bromo-1-(4-methoxyphenyl)ethan-1-one 8j was synthesized as
described in the general procedure for α-bromo ketone synthesis.
Following purification via flash column chromatography the product was
obtained as a brown oil (55 % yield) 1H NMR (400 MHz, cdcl3) δ 8.12 –
7.80 (m, 2H), 6.95 (dd, J = 9.1, 0.7 Hz, 2H), 4.40 (d, J = 0.7 Hz, 2H), 3.88
(d, J = 0.7 Hz, 3H). 13C NMR (101 MHz, cdcl3) δ 190.1, 166.5, 164.3,
131.5, 131.1, 127.1, 114.2, 55.7, 30.8. IR νmax (cm-1) 3059, 1804, 1467;
UV (λmax nm) 286; HRMS (ESI) calcd for C9H9BrO2 (M+) 230.0754,
found 230.0753.
General procedure for α-bromo ketone synthesis:10 mmol of the
carboxylic acid was dissolved in dichloromethane and allowed to stir
under an inert atmosphere. Three drops of DMF were added and the
reaction was cooled to 0 °C. Oxalyl chloride (3 equivalents) was added
dropwise and left to stir for one hour. In a diazomethane kit was added a
stir bar, KOH (9 equivalents), 25 mL of ethanol, 25 mL of water was
added to the top of the diazomethane apparatus and was heated to
65 °C and stirred. To a diazomethane addition funnel was added diazald
(3 equivalents) and diethyl ether (100 mL). The diethyl ether/diazald
mixture was added dropwise to the KOH/water/ethanol mixture so that
the diazomethane was generated and distilled over to the collection flask
that was cooled to 0 °C. Once the diazomethane was collected, the
carboxylic acid, oxalyl chloride reaction had reacted for 1 hour and the
mixture was concentrated in vacuo, dissolved in 5 mL of dichloromethane
and added slowly to the flask containing the diazomethane while being
cooled at 0 °C. The reaction mixture was allowed to stir for 1 hour at 0 °C,
followed by addition of concentrated hydrobromic acid (10 equivalents)
and allowed to stir for 20 minutes. Then 100 mL of saturated aqueous
sodium bicarbonate was added to the reaction mixture and allowed to stir
for 30 minutes. The resulting mixture was extracted with ethyl acetate,
washed twice with brine, and concentrated in vacuo and then purified via
column chromatography with 10% ethyl acetate/hexanes solution
providing the α-bromo keto product.
2-Bromo-1-(4-ethoxyphenyl)ethan-1-one 8k was synthesized as
described in the general procedure for α-bromo ketone synthesis.
Following purification via flash column chromatography the product was
obtained as a brown oil (55 % yield) 1H NMR (400 MHz, cdcl3) δ 7.92 (d,
J = 8.9 Hz, 2H), 6.91 (d, J = 8.8 Hz, 2H), 4.38 (s, 2H), 4.21 – 3.98 (m,
2H), 1.42 (t, J = 6.9 Hz, 3H). 13C NMR (101 MHz, cdcl3) δ 190.0, 166.5,
163.7, 131.5, 131.0, 126.8, 114.6, 64.0, 45.9, 31.0, 29.8, 14.8. IR νmax
(cm-1) 2945, 2807, 1623, 1580; UV (λmax nm) 270; HRMS (ESI) calcd for
C10H11BrO2 (M+) 244.2793, found 244.2790.
2-Bromo-1-(4-propoxyphenyl)ethan-1-one 8l was synthesized as
described in the general procedure for α-bromo ketone synthesis.
Following purification via flash column chromatography the product was
obtained as a clear oil (72 % yield). 1H NMR (400 MHz, cdcl3) δ 8.06 –
7.80 (m, 2H), 6.94 (d, J = 9.1 Hz, 2H), 4.39 (s, 2H), 3.99 (t, J = 6.5 Hz,
2H), 1.83 (dt, J = 7.5, 6.6 Hz, 3H), 1.04 (t, J = 7.4 Hz, 4H). 13C NMR (101
MHz, cdcl3) δ 190.0, 189.7, 163.9, 163.9, 131.4, 131.0, 127.0, 126.7,
114.6, 114.6, 69.9, 45.8, 30.9, 22.5, 10.5. IR νmax (cm-1) 3280, 2986,
1542; UV (λmax nm) 292; HRMS (ESI) calcd for C11H12BrO2 (M+)
258.0078, found 258.0080.
General procedure for Boc-guanidine cyclization followed by Boc-
deprotection: The α-bromo ketone (5 mmol) was dissolved in 10 mL of
DMF. Boc guanidine (3 equivalents) was then added to the reaction
mixture it was allowed to stir for 48 hours. Water was then added to the
reaction mixture and it was placed in a seperatory funnel and washed
twice with water and twice with brine, and concentrated in vacuo. The
crude product was then dissolved in a 1:4 mixture of trifluoroacetic
acid/dichloromethane and allowed to stir for 2 hours, then washed with
saturated sodium bicarbonate twice, concentrated in vacuo and purified
via column chromatography (5% methanol saturated ammonia/95%
dichloromethane) to provide the 2-aminoimidazole product. The sample
was then dissolved in dilute HCl/methanol (5 drops HCl/ 10 mL methanol)
and then concentrated in vacuo to provide the HCl salt.
2-Bromo-1-(4-butoxyphenyl)ethan-1-one 8m was synthesized as
described in the general procedure for α-bromo ketone synthesis.
Following purification via flash column chromatography the product was
obtained as a pale yellow oil (56% yield). 1H NMR (400 MHz, CDCl3) δ
7.94 (dt, J = 9.0, 1.1 Hz, 2H), 6.93 (dt, J = 7.8, 0.9 Hz, 2H), 4.38 (d, J =
0.8 Hz, 2H), 4.03 (t, J = 6.5 Hz, 2H), 1.90 – 1.67 (m, 2H), 1.64 – 1.39 (m,
2H), 0.98 (td, J = 7.4, 0.8 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 190.0,
163.9, 163.9, 131.4, 131.0, 126.8, 114.6, 114.6, 68.2, 45.8, 31.2, 30.8,
19.3, 13.9. IR νmax (cm-1) 3168, 1645; UV (λmax nm) 282; HRMS (ESI)
calcd for C12H14BrO2 (M+) 272.0236, found 272.0234.
2-Bromo-1-(p-tolyl)ethan-1-one 8g was synthesized as described in the
general procedure for α-bromo ketone synthesis. Following purification
via flash column chromatography the product was obtained as a white
solid (73 % yield). 1H NMR (400 MHz, CDCl3) δ 7.87 (d, J = 8.2 Hz, 1H),
7.31 – 7.24 (m, 1H), 4.42 (d, J = 0.6 Hz, 1H). 13C NMR (101 MHz, CDCl3)
δ 191.0, 145.1, 131.6, 129.7, 129.6, 129.1, 128.7, 46.0, 31.1, 29.8, 21.8.
IR νmax (cm-1) 3250, 1976, 1608 1514; UV (λmax nm) 280; HRMS (ESI)
calcd for C9H9BrO (M+) 214.0740, found 214.0738.
2-Bromo-1-(4-(pentyloxy)phenyl)ethan-1-one 8n was synthesized as
described in the general procedure for α-bromo ketone synthesis.
Following purification via flash column chromatography the product was
obtained as a light brown solid (76% yield). 1H NMR (400 MHz, CDCl3) δ
7.94 (dd, J = 10.5, 9.0 Hz, 2H), 6.94 (dd, J = 9.0, 1.2 Hz, 2H), 4.40 (s,
2H), 4.02 (td, J = 6.5, 0.9 Hz, 3H), 1.81 (td, J = 6.6, 1.6 Hz, 3H), 1.57 –
1.26 (m, 4H), 0.93 (t, J = 7.1 Hz, 4H). 13C NMR (101 MHz, CDCl3) δ
189.9, 189.6, 163.8, 163.8, 131.3, 130.9, 126.9, 126.7, 114.5, 114.5,
68.4, 45.6, 30.7, 28.7, 28.1, 22.4, 14.0. IR νmax (cm-1) 2930, 1853, 1632,
2-Bromo-1-(4-ethylphenyl)ethan-1-one 8h was synthesized as
described in the general procedure for α-bromo ketone synthesis.
Following purification via flash column chromatography the product was
obtained as a clear oil (64 % yield). 1H NMR (400 MHz, cdcl3) δ 8.08 –
5
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10.1002/cmdc.201900033
ChemMedChem
FULL PAPER
1309; UV (λmax nm) 270; HRMS (ESI) calcd for C13H15BrO2 (M+)
286.0392, found 286.0395
(43%). 1H NMR, 13C NMR, and LRMS (ESI) aligned with previously
reported spectral and MS data.16
2-Bromo-1-(4-(2-methoxyethoxy)phenyl)ethan-1-one
8o
was
4-(4-Methoxyphenyl)-1H-imidazol-2-amine hydrochloride 9j was
synthesized as described in the general procedure for Boc-guanidine
cyclization followed by Boc-deprotection. Following purification via flash
column chromatography the product was obtained as a dark brown solid
(35%). 1H NMR (400 MHz, CD3OD) δ 7.51 – 7.41 (m, 2H), 6.94 – 6.83 (m,
2H), 6.76 (s, 1H), 3.78 (s, 3H). 13C NMR (101 MHz, CD3OD) δ 126.3,
115.0, 111.2, 55.7. IR νmax (cm-1) 3345, 2980; UV (λmax nm) 296;
HRMS (ESI) calcd for C10H11N3O (M+) 190.0923, found 190.0921.
synthesized as described in the general procedure for α-bromo ketone
synthesis. Following purification via flash column chromatography the
product was obtained as white solid (80% yield). 1H NMR (400 MHz,
CDCl3) δ 7.94 (d, J = 8.9 Hz, 2H), 6.97 (d, J = 8.9 Hz, 2H), 4.39 (s, 2H),
4.25 – 4.13 (m, 2H), 3.83 – 3.68 (m, 2H), 3.44 (s, 3H). 13C NMR (101
MHz, CDCl3) δ 190.0, 163.5, 163.5, 131.4, 131.0, 127.5, 127.2, 114.8,
114.7, 70.8, 67.7, 59.4, 45.8, 30.9. IR νmax (cm-1) 3143, 2954, 13,45,
1288; UV (λmax nm) 280; HRMS (ESI) calcd for C11H13BrO3 (M+)
274.0031, found 274.0026.
4-(4-Ethoxyphenyl)-1H-imidazol-2-amine hydrochloride 9k was
synthesized as described in the general procedure for Boc-guanidine
cyclization followed by Boc-deprotection. Following purification via flash
column chromatography the product was obtained as a light red solid
(49%). 1H NMR (400 MHz, CD3OD) δ 7.48 (d, J = 8.9 Hz, 2H), 7.02 –
6.93 (m, 3H), 4.06 (q, J = 7.0 Hz, 2H), 1.39 (t, J = 7.0 Hz, 3H). 13C NMR
(101 MHz, CD3OD) δ 160.7, 149.0, 129.1, 127.1, 121.2, 116.1, 108.4,
64.6, 15.1. IR νmax (cm-1) 3433, 3006, 2807; UV (λmax nm) 248; HRMS
(ESI) calcd for C11H13N3O (M+) 204.1088, found 204.1079
2-Bromo-1-(4-(2-ethoxyethoxy)phenyl)ethan-1-one
8p
was
synthesized as described in the general procedure for α-bromo ketone
synthesis. Following purification via flash column chromatography the
product was obtained as a white solid (78% yield). 1H NMR (400 MHz,
CDCl3) δ 7.93 (d, J = 8.9 Hz, 2H), 6.96 (dd, J = 9.0, 1.4 Hz, 2H), 4.38 (s,
2H), 4.25 – 4.05 (m, 2H), 3.93 – 3.73 (m, 2H), 3.58 (d, J = 7.0 Hz, 2H),
1.22 (t, J = 7.0 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 190.0, 189.7,
163.6, 163.5, 131.4, 131.0, 127.4, 127.1, 114.8, 114.8, 68.7, 67.9, 67.0,
45.8, 30.9, 15.2. IR νmax (cm-1) 3095, 1873, 1652, 1273; UV (λmax nm)
286; HRMS (ESI) calcd for C12H15BrO3 (M+) 288.0184, found 288.0185.
4-(4-Propoxyphenyl)-1H-imidazol-2-amine hydrochloride 9l was
synthesized as described in the general procedure for Boc-guanidine
cyclization followed by Boc-deprotection. Following purification via flash
column chromatography the product was obtained as a light red solid
(54%). 1H NMR (400 MHz, CD3OD) δ 7.47 (d, J = 8.8 Hz, 2H), 7.02 –
6.90 (m, 3H), 3.93 (t, J = 6.4 Hz, 2H), 1.90 – 1.70 (m, 2H), 1.03 (t, J = 7.4
Hz, 3H). 13C NMR (101 MHz, CD3OD) δ 160.9, 148.9, 129.0, 127.1,
121.2, 116.1, 108.3, 70.7, 23.6, 10.8. IR νmax (cm-1) 3392, 2854, 1673,
1345; UV (λmax nm) 280; HRMS (ESI) calcd for C12H15N3O (M+)
218.1276, found 218.1268.
4-(p-Tolyl)-1H-imidazol-2-amine hydrochloride 9g was synthesized as
described in the general procedure for Boc-guanidine cyclization followed
by Boc-deprotection. Following purification via flash column
chromatography the product was obtained as a light red solid (0.83 g,
52%) 1H NMR (400 MHz, CD3OD) δ 7.42 (d, J = 8.2 Hz, 2H), 7.11 (d, J =
8.1 Hz, 3H), 6.82 (s, 1H), 2.30 (s, 3H) ppm; 13C NMR (101 MHz, CD3OD)
δ 136.8, 130.2, 124.9, 21.2 ppm; IR νmax (cm-1) 3323, 2123, 1578; UV
(λmax nm) 286; HRMS (ESI) calcd for C10H11N3 (M+) 173.2187, found
173.2186.
4-(4-Butoxyphenyl)-1H-imidazol-2-amine hydrochloride 9m was
synthesized as described in the general procedure for Boc-guanidine
cyclization followed by Boc-deprotection. Following purification via flash
column chromatography the product was obtained as a light red solid
(68%). 1H NMR (400 MHz, CD3OD) δ 7.48 (d, J = 8.9 Hz, 2H), 7.03 –
6.92 (m, 3H), 4.00 (d, J = 6.4 Hz, 2H), 1.75 (m, 2H), 1.58 – 1.45 (m, 2H),
0.98 (t, J = 7.4 Hz, 3H). 13C NMR (101 MHz, CD3OD) δ 160.9, 148.9,
129.0, 127.1, 121.2, 116.1, 108.3, 68.8, 32.4, 20.3, 14.2. IR νmax (cm-1)
3124, 3076, 2974, 1569; UV (λmax nm) 254; HRMS (ESI) calcd for
C13H17N3O (M+) 232.1444, found 232.1442.
4-(4-Ethylphenyl)-1H-imidazol-2-amine
hydrochloride
9h
was
synthesized as described in the general procedure for Boc-guanidine
cyclization followed by Boc-deprotection. Following purification via flash
column chromatography the product was obtained as a brown solid
(45%). 1H NMR (400 MHz, CD3OD) δ 7.46 (d, J = 8.2 Hz, 2H), 7.28 –
7.06 (m, 2H), 2.60 (q, J = 7.6 Hz, 2H), 1.18 (t, J = 7.6 Hz, 3H). 13C NMR
(101 MHz, CD3OD) δ 152.6, 148.9, 147.2, 146.0, 129.5, 128.9, 126.1,
125.5, 109.1, 15.9, 15.5. IR νmax (cm-1) 3086, 1423, 1367, 1232; UV
(λmax nm) 250; HRMS (ESI) calcd for C11H13N3 (M+) 188.1132, found
188.1139.
4-(4-(Pentyloxy)phenyl)-1H-imidazol-2-amine hydrochloride 9n was
synthesized as described in the general procedure for Boc-guanidine
cyclization followed by Boc-deprotection. Following purification via flash
column chromatography the product was obtained as a light brown sticky
oil (57%). 1H NMR (400 MHz, CD3OD) δ 7.45 (d, J = 8.8 Hz, 2H), 6.85 (d,
J = 8.8 Hz, 2H), 6.74 (s, 1H), 3.94 (t, J = 6.4 Hz, 2H), 1.76 (dd, J = 8.0,
6.3 Hz, 2H), 1.52 – 1.34 (m, 4H), 0.95 (t, J = 7.1 Hz, 3H). 13C NMR (101
MHz, CD3OD) δ 159.1, 151.6, 127.4, 126.2, 115.6, 69.0, 30.2, 29.4, 23.6,
14.4. IR νmax (cm-1) 3473, 3193, 1543, 1123; UV (λmax nm) 270; HRMS
(ESI) calcd for C14H19N3O (M+) 246.1573, found 246.1569.
4-(4-Propylphenyl)-1H-imidazol-2-amine hydrochloride 9i was
synthesized as described in the general procedure for Boc-guanidine
cyclization followed by Boc-deprotection. Following purification via flash
column chromatography the product was obtained as a light solid (52%).
1H NMR (300 MHz, CD3OD) δ 7.48 – 7.39 (m, 2H), 7.10 (d, J = 8.2 Hz,
1H), 6.82 (s, 1H), 2.53 (t, J = 7.6 Hz, 2H), 1.61 (h, J = 7.3 Hz, 2H), 0.92 (t,
J = 7.3 Hz, 2H). 13C NMR (75 MHz, CD3OD) δ 151.6, 141.5, 134.5, 131.9,
129.6, 129.5, 124.8, 112.4, 38.7, 25.7, 14.1. IR νmax (cm-1) 2845, 1723,
1532, 1190; UV (λmax nm) 256; HRMS (ESI) calcd for C12H15N3 (M+)
202.1356, found 202.1350.
4-(4-(2-Methoxyethoxy)phenyl)-1H-imidazol-2-amine hydrochloride
9o was synthesized as described in the general procedure for Boc-
guanidine cyclization followed by Boc-deprotection. Following purification
via flash column chromatography the product was obtained as a white
solid (64%). 1H NMR (400 MHz, CD3OD) δ 7.53 – 7.46 (m, 2H), 7.05 –
6.96 (m, 3H), 4.18 – 4.09 (m, 2H), 3.78 – 3.71 (m, 2H), 3.42 (s, 3H). 13C
NMR (101 MHz, CD3OD) δ 160.5, 149.0, 128.9, 127.1, 121.6, 116.2,
108.5, 72.1, 68.5, 59.2. IR νmax (cm-1) 3213, 2921, 1734; UV (λmax nm)
250; HRMS (ESI) calcd for C12H15N3O2 (M+) 234.1153, found 234.1152.
4-(4-Butylphenyl)-1H-imidazol-2-amine
hydrochloride
9a
was
synthesized as described in the general procedure for Boc-guanidine
cyclization followed by Boc-deprotection. Following purification via flash
column chromatography the product was obtained as a brown oil (60%).
1H NMR, 13C NMR, and LRMS (ESI) aligned with previously reported
spectral and MS data.16
4-(4-Pentylphenyl)-1H-imidazol-2-amine hydrochloride 9b was
synthesized as described in the general procedure for Boc-guanidine
cyclization followed by Boc-deprotection. Following purification via flash
column chromatography the product was obtained as a light yellow solid
4-(4-(2-Ethoxyethoxy)phenyl)-1H-imidazol-2-amine hydrochloride 9p
was synthesized as described in the general procedure for Boc-
guanidine cyclization followed by Boc-deprotection. Following purification
6
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10.1002/cmdc.201900033
ChemMedChem
FULL PAPER
via flash column chromatography the product was obtained as a white
solid (54%). 1H NMR (400 MHz, CD3OD) δ 7.49 (d, J = 8.8 Hz, 2H), 7.04
– 6.99 (m, 3H), 4.18 – 4.09 (m, 2H), 3.83 – 3.74 (m, 2H), 3.60 (q, J = 7.0
Hz, 2H), 1.22 (t, J = 7.1 Hz, 3H). 13C NMR (101 MHz, CD3OD) δ 160.6,
127.1, 121.6, 116.2, 108.5, 70.0, 68.7, 67.7, 15.4. IR νmax (cm-1) 3419,
1734, 1245, 1167; UV (λmax nm) 264; HRMS (ESI) calcd for C13H17N3O2
(M+) 248.1365, found 248.1263.
on a high vacuum pump for one hour. Then, to the round bottom was
added 5 mL of ethanol and 5 mL of water and cyanamide (0.26 g, 6.19
mmol). The pH of the reaction was adjusted to pH 4.3 using 1N HCl. The
reaction was heated at 95 °C for 3 hours. The reaction was allowed to
cool to room temperature and poured into ethyl acetate and washed two
times with sodium bicarbonate and two times with brine. The ethyl
acetate layer was dried over magnesium sulfate and then concentrated in
vacuo. The residue was purified via silica gel column chromatography (0-
5% methanol saturated ammonia in dicholomethane). The purified
product was then dissolved in five drops of concentrated HCl in methanol
to give 4-(4-hexylphenyl)-5-(trifluoromethyl)-1H-imidazol-2-amine as a
pale yellow solid (0.10 g, 53% yield). 1H NMR (400 MHz, CD3OD) δ 7.45
(d, J = 8.2 Hz, 2H), 7.37 – 7.33 (m, 2H), 2.69 (t, J = 7.7 Hz, 2H), 1.68 –
1.60 (m, 2H), 1.32 (d, J = 4.3 Hz, 6H), 0.94 – 0.87 (m, 3H). 13C NMR
(101 MHz, CD3OD) δ 154.4, 149.3, 147.2, 130.1, 129.8 (q J = 1.6 Hz),
124.0, 36.7, 32.8, 32.4, 30.0, 23.6, 14.4. IR νmax (cm-1) 3287, 2987,
1653, 1522, 1197; UV (λmax nm) 258; HRMS (ESI) calcd for C16H22F3N3
(M+) 312.1634, found 312.1630.
tert-Butyl (1,1,1-trifluoro-3-(methoxy(methyl)amino)-3-oxopropan-2-
yl)carbamate 10g 1.50 g (6.16 mmol) of 2-((tert-butoxycarbonyl)amino)-
3,3,3-trifluoropropanoic acid, a stir bar, and 12 mL of dichloromethane
were added to a 100-mL round bottom flask and allowed to stir. To the
mixture was added 2.36
g
(12.33 mmol) of 1-Ethyl-3-(3-
dimethylaminopropyl)carbodiimide hydrochloride and 1.89g (12.33 mmol)
of hydroxybenzotriazole hydrate and 2.58 mL (18.51 mmol) of
trimethylamine and the mixture was allowed to stir for 2 minutes.
Following was added 0.90g (9.25 mmol) of N,O-dimethylhydroxylamine
hydrochloride. The reaction was allowed to stir overnight and the solvent
was then removed in vacuo. The mixture was dissolved in 20 mL of ethyl
acetate and washed with saturated sodium bicarbonate twice and
saturated brine twice. The organic layer was dried over magnesium
sulfate and purified via silica gel column chromatography using 0 to 25%
ethyl acetate in hexanes to give tert-butyl (1,1,1-trifluoro-3-
(methoxy(methyl)amino)-3-oxopropan-2-yl)carbamate as a white solid
(1.3 g, 76% yield). 1H NMR (400 MHz, CDCl3) δ 5.75 – 5.43 (m, 1H), 3.75
(s, 3H), 3.23 (s, 3H), 1.42 (s, 9H). 13C NMR (101 MHz, CDCl3 δ 154.6,
124.3, 81.0, 61.6, 51.1, 50.8, 32.1, 28.1, 28.1. IR νmax (cm-1) 1823, 1139,
1032; melting point 84 – 88 °C; HRMS (ESI) calcd for C10H17F3N2O4 (M+)
287.1164, found 287.1160.
tert-Butyl
(1,1,1-trifluoro-3-oxo-3-(4-propoxyphenyl)propan-2-
yl)carbamate 11h was synthesized as described in the general
procedure for 11g. Following purification via flash column
chromatography the product was obtained as a light yellow solid in 47%
1
yield. H NMR (300 MHz, CDCl3) δ 7.98 (d, J = 8.7 Hz, 2H), 6.96 (d, J =
8.9 Hz, 2H), 5.92 – 5.79 (m, 1H), 4.00 (t, J = 6.5 Hz, 2H), 1.83 (h, J = 7.0
Hz, 2H), 1.45 (s, 9H), 1.04 (t, J = 7.4 Hz, 3H). 13C NMR (101 MHz,
CDCl3) δ 189.0, 166.5, 164.7, 154.8, 141.1, 132.0, 127.1, 124.5, 121.7,
114.7, 81.2, 70.0, 55.2, 54.9, 54.6, 54.3, 28.3, 22.5, 10.5. IR νmax (cm-1)
3520, 3147, 1429, 1278; UV (λmax nm) 228; HRMS (ESI) calcd for
C17H22F3NO4 (M+) 362.1548, found 362.1550.
tert-Butyl
(1,1,1-trifluoro-3-(4-hexylphenyl)-3-oxopropan-2-
yl)carbamate 11g To a flame dried 100-mL round bottom flask under a
nitrogen atmosphere was added 1-bromo-4-hexylbenzene (0.14 mL, 0.70
mmol), a stir bar and 8 mL of dry tetrahydrofuran. The mixture was stirred
at -78 °C and n-butyllitium (0.29 mL, 2.5 M solution in hexanes) was
added dropwise over a two minute period. After another five minutes,
tert-Butyl
(3-(4-butoxyphenyl)-1,1,1-trifluoro-3-oxopropan-2-
yl)carbamate 11i was synthesized as described in the general
procedure for 11g. Following purification via flash column
chromatography the product was obtained as a light yellow solid in 57%
yield. 1H NMR (400 MHz, CDCl3) δ 7.98 (dd, J = 8.9, 2.8 Hz, 2H), 6.94
(dd, J = 6.8, 2.4 Hz, 2H), 6.01 – 5.81 (m, 1H), 4.03 (q, J = 6.7 Hz, 2H),
1.78 (q, J = 6.9 Hz, 2H), 1.44 (s, 9H), 0.96 (t, J = 7.1 Hz, 3H). 13C NMR
(101 MHz, CDCl3) δ 189.0, 164.7, 154.8, 132.0, 127.1, 114.7, 81.2, 68.3,
55.2, 54.6, 54.3, 31.1, 28.2, 19.2, 13.8. IR νmax (cm-1) 3377, 2743, 1604;
UV (λmax nm) 234; HRMS (ESI) calcd for C18H24F3NO4 (M+) 376.1675,
found 376.1676.
tert-butyl
(1,1,1-trifluoro-3-(methoxy(methyl)amino)-3-oxopropan-2-
mL of dry
yl)carbamate (0.1 g, 0.35 mmol) was dissolved in
2
tetrahydrofuran and added dropwise to the reaction mixture over a two
minutes. After a three minute period, the reaction was removed from the -
78 °C bath and allowed to warm to room temperature over an hour period.
Then, the reaction was placed at 0 °C and saturated ammonium chloride
was added dropwise. After stirring for ten minutes, the mixture was
diluted with 20 mL of ethyl acetate and washed twice with water and
twice with brine. The organic layer was dried over sodium sulfate and
then concentrated in vacuo. The resulting residue was then purified via
silica gel column chromatography 0-25% ethyl acetate in hexanes to give
tert-butyl (1,1,1-trifluoro-3-(4-hexylphenyl)-3-oxopropan-2-yl)carbamate
as a light yellow solid (0.80g, 59% yield). 1H NMR (400 MHz, CDCl3) δ
7.89 (d, J = 7.9 Hz, 2H), 7.27 (d, J = 8.1 Hz, 2H), 5.32 (d, J = 8.7 Hz, 2H),
2.65 (t, J = 7.8 Hz, 2H), 1.61 (d, J = 8.5 Hz, 2H), 1.43 (s, 9H), 1.30 (tt, J =
4.5, 2.6 Hz, 4H), 1.06 (m, 2H), 0.88 (t, J = 6.6 Hz, 3H). 13C NMR (101
MHz, CDCl3) δ 199.6, 155.7, 149.4, 128.8, 79.5, 53.4, 43.0, 36.0, 31.6,
31.0, 28.9, 28.3, 25.0, 23.4, 22.5, 21.8, 14.0. IR νmax (cm-1) 3304, 1823,
1256; UV (λmax nm) 204; HRMS (ESI) calcd for C20H28F3NO3 (M+)
388.2042, found 388.2039.
tert-Butyl
(1,1,1-trifluoro-3-oxo-3-(4-(pentyloxy)phenyl)propan-2-
yl)carbamate 11j was synthesized as described in the general
procedure for 11g. Following purification via flash column
chromatography the product was obtained as a white solid in 53% yield.
1H NMR (400 MHz, CDCl3) δ 7.99 (d, J = 9.1 Hz, 2H), 6.96 (d, J = 8.9 Hz,
2H), 5.90 – 5.79 (m, 1H), 4.04 (t, J = 6.6 Hz, 2H), 1.80 (dt, J = 8.0, 6.5 Hz,
2H), 1.46 (s, 11H), 0.93 (t, J = 7.1 Hz, 3H). 13C NMR (101 MHz, CDCl3)
δ 166.5, 164.8, 154.9, 132.1, 127.2, 114.8, 81.3, 68.7, 55.0, 54.7, 28.3,
14.1. IR νmax (cm-1) 3428, 3046, 1420, 1265; UV (λmax nm) 260; HRMS
(ESI) calcd for C19H26F3NO4 (M+) 390.1847, found 390.1849.
4-(4-Propoxyphenyl)-5-(trifluoromethyl)-1H-imidazol-2-amine
12h
was synthesized as described in the general procedure for 12g.
Following purification via flash column chromatography the product was
obtained as a white solid in 41% yield. 1H NMR (400 MHz, DMSO-d6) δ
8.18 (d, J = 7.3 Hz, 2H), 7.13 (d, J = 7.3 Hz, 2H), 6.41 (q, J = 7.3 Hz, 1H),
4.09 (t, 2H), 1.83 – 1.68 (m, 2H), 0.98 (t, J = 6.6 Hz, 3H). 13C NMR (101
MHz, DMSO-d6) δ 165.0, 161.6, 132.8, 126.8, 115.3, 70.2, 22.3, 10.7. IR
νmax (cm-1) 3066, 2875, 1567, 1123; UV (λmax nm) 246; HRMS (ESI)
calcd for C13H14F3N3O (M+) 286.1138, found 286.1139.
4-(4-Hexylphenyl)-5-(trifluoromethyl)-1H-imidazol-2-amine 12g To a 6
dram vial open to air was added tert-butyl (1,1,1-trifluoro-3-(4-
hexylphenyl)-3-oxopropan-2-yl)carbamate (0.24g, 0.62 mmol) in 3.5 mL
of dichloromethane. The mixture was placed in a 0 °C ice bath and 1.5
mL of trifluoroacetic acid was added dropwise. After five minutes, the
reaction was removed from the ice bath and allowed to stir at room
temperature over two hours and monitored by TLC. Then, the solvent
was removed in vacuo and the residue was dissolved in 10 mL of ethyl
acetate and washed three times with saturated sodium bicarbonate. The
ethyl acetate layer was then dried over sodium sulfate and concentrated
in vacuo in a 100 mL round bottom flask. The sticky residue was placed
4-(4-Butoxyphenyl)-5-(trifluoromethyl)-1H-imidazol-2-amine 12i was
synthesized as described in the general procedure for 12g. Following
purification via flash column chromatography the product was obtained
1
as a white solid in 35% yield. H NMR (400 MHz, CD3OD) δ 8.12 (d, J =
7
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201900033
ChemMedChem
FULL PAPER
8.9 Hz, 2H), 7.11 (d, J = 8.9 Hz, 2H), 6.24 (q, J = 7.4 Hz, 1H), 4.13 (t, J =
6.4 Hz, 2H), 1.87 – 1.75 (m, 2H), 1.59 – 1.46 (m, 2H), 1.00 (t, J = 7.4 Hz,
3H). 13C NMR (101 MHz, CD3OD) δ 166.9, 133.5, 127.5, 124.6, 121.8,
116.1, 69.5, 56.3, 56.0, 32.2, 20.2, 14.1. IR νmax (cm-1) 3366, 3048,
1765, 1527; UV (λmax nm) 228; HRMS (ESI) calcd for C14H16F3N3O (M+)
300.1264, found 300.1258.
the plates were left at ambient temperature for 30 min. After 30 min, the
crystal violet was disposed, and the plates were washed thoroughly with
water. 200 µL of 95% ethanol was added to each well, and the plates
were left at ambient temperature for 10 min. 125 µL of the ethanol
solution was transferred to a fresh polystyrene microtiter plate, and the
plate was quantified by measuring the OD540. The percent inhibition was
calculated by comparing the OD540 of the treated wells with the OD540 of
the untreated wells. The first and last column, which had only sterile
media, were used as blanks and those values were subtracted from the
OD540 obtained in the other columns.
4-(4-(Pentyloxy)phenyl)-5-(trifluoromethyl)-1H-imidazol-2-amine 12j
was synthesized as described in the general procedure for 12g.
Following purification via flash column chromatography the product was
obtained as a pale yellow solid in 57% yield. 1H NMR (400 MHz, CD3OD)
δ 7.45 (d, J = 8.7 Hz, 2H), 7.04 (d, J = 8.8 Hz, 2H), 4.03 (t, J = 6.4 Hz,
2H), 1.87 – 1.72 (m, 2H), 1.54 – 1.34 (m, 4H), 0.95 (t, J = 7.1 Hz, 3H).
13C NMR (101 MHz, CDCl3) δ 162.4, 131.3, 131.3, 131.3, 118.5, 115.9,
69.2, 30.0, 29.3, 23.5, 14.4. IR νmax (cm-1) 3143, 2854, 1697; UV (λmax
nm) 264; HRMS (ESI) calcd for C15H18F3N3O (M+) 314.1455, found
314.1457.
General biofilm dispersion assay protocol for M. smegmatis Cultures
were incubated for 48 hr and then subcultured to 0.01 in Difco M9
minimal salts media. 100 µL of the subculture was aliquoted into every
well in columns 2–11 of a 96- well PVC microtiter plate. Columns 1 and
12 were left empty to serve as control wells. Plates were covered with
Glad Press n’ Seal and were incubated under stationary conditions at
37°C for 48 hr. After 48 hr, the media were discarded, and the plates
were washed thoroughly with water. 100 µL of fresh media containing the
appropriate concentration of compound was added to all of the wells in
columns 2–4 and 9–11. 100 µL of sterile media was added to all of the
wells in columns 1 and 12 and columns 5–8. Plates were covered with
Glad Press n’ Seal and were incubated under stationary conditions at
37°C for 24 hr. After 24 hr, media were dis-carded, and the plates were
washed thoroughly with water. 110 µL of a 0.1% aqueous solution of
crystal violet was added to every well, and the plates were left at ambient
temperature for 30 min. After 30 min, the crystal violet was disposed, and
the plates were washed thoroughly with water. 200 µL of 95% ethanol
was added to each well, and the plates were left at ambient temperature
for 10 min. 125 µL of the ethanol solution was transferred to a fresh
polystyrene microtiter plate, and the plate was quantified by measuring
the OD540. The percent dispersion was calculated by comparing the
OD540 of the treated wells with the OD540 of the untreated wells, which
contained only media after biofilm growth. The first and last column,
which had only sterile media, were used as blanks and those values
were subtracted from the OD540 obtained in the other columns.
Biological Methods
Bacterial strains, media, and antibiotics The M. smegmatis strain
(ATCC 700084, mc2155) was obtained from ATCC (Manassas, VA).
Stock cultures were stored in glycerol stock media (50% v/v glycerol and
7H9, ADC, Tween 80) and maintained at -80 °C. Prior to use, colonies
were grown on 7H10 agar (OADC, glyercol) for 2 days, and single
colonies were subcultured in 7H9 (ADC, Tween 80) for 2 days. 7H10,
7H9, OADC, and ADC were purchased from BD Diagnostics. Glycerol
and Tween 80 were purchased from Sigma Aldrich. Ampicillin,
carbenicillin, cefaclor, cefadroxil, cefmetazole, cefotaxime, cefoxitin,
ceftazidime, cephalothin, methicillin, naficillin, and penicillin were
purchased from Sigma Aldrich. Oxacillin was purchased from TCI
America. All assays were run in duplicate and repeated at least two
separate times. All compounds were dissolved as their HCl salts in
molecular biology grade DMSO as 100 mM stock solutions and stored at
-20 °C.
Broth microdilution method for determination of minimum inhibitory
concentrations M. smegmatis was grown in 7H9 (ADC, 0.5% Tween 80)
for 48 h, and this culture was used to inoculate fresh 7H9 (5 × 105 CFU
mL-1, OD600 = 0.006). Aliquots (1 mL) were placed in culture tubes, and
compound was added from 100 mM stock solutions in DMSO, such that
the compound concentration equaled the highest concentration tested.
Antibiotics, from a water stock, were added at the highest concentration
tested to 1 mL aliquots of cultures. Inoculated media not treated with
compound served as the control. Samples were then aliquoted (200 µL)
into the first wells of a 96-well plate, with all remaining wells being filled
with 100 µL of initial bacterial subculture. Row 1 wells were mixed 8
times, before 100 µL was transferred to row 2. Row 2 was then mixed 8
times, and 100 µL was transferred to row 3. This process was repeated
to serially dilute the rest of the rows. One row with bacteria subculture
served as the control. Plates were then covered with GLAD Press n’ Seal
and incubated under stationary conditions at 37 °C. After 48 h, 10 µL of
alamarBlue was added to each well, and the plate was resealed and
incubated under stationary conditions at 37 °C. After 6 h, the minimum
inhibitory concentration (MIC) values were recorded as the lowest
concentration of compound or antibiotic at which no visible growth of
bacteria was observed.
General synergy dispersion assay protocol for M. smegmatis
Cultures were incubated for 48 hr and then subcultured to 0.01 in Difco
M9 minimal salts media. 100 µL of the subculture was aliquoted into
every well in columns 2–11 of a 96- well PVC microtiter plate. Columns 1
and 12 were left empty to serve as control wells. Plates were covered
with Glad Press n’ Seal and were incubated under stationary conditions
at 37°C for 48 hr. After 48 hr, the media were discarded, and the plates
were washed thoroughly with water. 100 µL of fresh media containing the
appropriate concentration of compound and antibiotic was added to all of
the wells in columns 2–4 and 9–11. 100 µL of sterile media was added to
all of the wells in columns 1 and 12 and columns 5–8. Plates were
covered with Glad Press n’ Seal and were incubated under stationary
conditions at 37°C for 24 hr. After 24 hr, media were discarded, and the
plates were washed thoroughly with water. 110 µL of a 0.1% aqueous
solution of crystal violet was added to every well, and the plates were left
at ambient temperature for 30 min. After 30 min, the crystal violet was
disposed, and the plates were washed thoroughly with water. 200 µL of
95% ethanol was added to each well, and the plates were left at ambient
temperature for 10 min. 125 µL of the ethanol solution was transferred to
a fresh polystyrene microtiter plate, and the plate was quantified by
measuring the OD540
. The percent dispersion was calculated by
comparing the OD540 of the treated wells with the OD540 of the untreated
wells, which contained only media after biofilm growth. The first and last
column, which had only sterile media, were used as blanks and those
values were subtracted from the OD540 obtained in the other columns.
General biofilm inhibition assay protocol for M. smegmatis Cultures
were incubated for 48 hr and then subcultured to OD600 = 0.01 in Difco
M9 minimal salts media. 100 µL of subculture containing the appropriate
concentration of compound was added to all of the wells in columns 2–4
and 9–11 of 96- well PVC microtiter plate. Columns 1 and 12 were filled
with 100 µL of sterile M9 minimal salts media to serve as controls. Plates
were covered with Glad Press n’ Seal and were incubated under
stationary conditions at 37°C for 48 hr. After 48 hr, the media were
discarded, and the plates were washed thoroughly with water. 110 µL of
a 0.1% aqueous solution of crystal violet was added to every well, and
Colony counts for dispersion assay protocol for M. smegmatis
Cultures were incubated for 48 hr and then subcultured to 0.01 in Difco
M9 minimal salts media. 100 µL of the subculture was aliquoted into
every well in columns 2–11 of a 96- well PVC microtiter plate. Columns 1
and 12 were left empty to serve as control wells. Plates were covered
8
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201900033
ChemMedChem
FULL PAPER
with Glad Press n’ Seal and were incubated under stationary conditions
at 37°C for 48 hr. After 48 hr, a 50 µL aliquot of the media from selected
wells was serially diluted 10-fold. Then, a 10 µL aliquot was removed
from each aliquot and plated out on a 7H10 + OADC + 0.5% glycerol
round petri dish followed by 48 hours of incubation at 37 °C under
stationary conditions. Viable colonies were quantified through the track-
dilution method. Controls were employed in which only compound was
added and sterile media was added. The number of colonies counted
from the treatment with sterile media was subtracted from the counts of
the other test samples.
The in vitro microsome half-life (t1/2) was calculated using the expression
t1/2=0.693/b, where b is the slope found in the linear fit of the natural
logarithm of the fraction remaining of the parent compound vs. incubation
time.
Plasma stability assay The in vitro stability of the test compounds was
studied in mouse plasma (Sigma 9275). The plasma was diluted to 80%
with 0.05 M PBS (pH 7.4) at 37°C. The reactions were initiated by the
addition of the test compounds to 1 mL of preheated plasma solution to
yield a final concentration of 100 µM. A positive control solution without
the addition of plasma was also included to monitor compound stability
over the course of the experiment. The assays were incubated at 37°C
and shaken at 200 rpm. Samples (50 µl) were taken at 0, 15, 30, 45, 60,
120, 240, 480 min and added to 200 µl acetonitrile in order to
deproteinize the plasma. The samples were subjected to vortex mixing
for 1 min and then centrifugation at 4°C for 15 min at 12,000 rpm. The
clear supernatants were transferred to appropriate LC-MS vials for
analysis. Samples were analyzed using reverse phase liquid
chromatography coupled with low resolution tandem mass spectrometry
(LC-MS/MS) in the laboratory of Dr. Melander at North Carolina State
University Department of Chemistry. A Shimadzu Scientific Instruments
LC system (comprising a solvent degasser, two LC-20A pumps and a
SCL-20A system controller) was coupled to and introduced into the ESI
source of an Expression L CMS Advion mass spectrometer (Advion,
Ithica, NY). Separation was achieved using a Restek Ultra C18 reversed-
phase column (3 µm, 50 x 4.6 mm). For analysis 20µL of sample was
injected. The method used a solvent flow of 0.5 ml/min. Initial gradient
conditions (90% water:10% acetonitrile with 0.1% formic acid) were held
for 2 minutes. From 2 to 5.5 minutes, the mobile phase composition
increased linearly to 10% water:90% acetonitrile with 0.1% formic acid.
From 5.5 to 8 minutes, the mobile phase composition was held at 10%
water:90% acetonitrile with 0.1% formic acid. At 8.01 to 10 min, the
column was re-equilibrated with 90% water:10% acetonitrile with 0.1%
formic acid. HPLC data acquisition and analysis were performed using
LabSolutions software. The settings for the Advion ESI/MS in dual ion
mode are as follows: capillary temperature = 250 °C; capillary voltage =
180 V; ESI voltage = 3500 V; source gas temperature = 200 °C; pirani
pressure = 2.73 e-3 mbar; hexapole bias = 8 V; extraction electrode = 9 V.
Data was acquired using an acquisition method: mass range m/z = 100
to 800; scan time = 632 ms, scan speed (m/z/sec) = 1107. Analysis
performed using Advion Mass Express and Advion Data Express. The
percentage of compound remaining is calculated as the ratio of
[(compound peak area of solution with plasma)/(compound peak
without plasma)] x 100. The in vitro plasma half-life (t1/2) was calculated
using the expression t1/2=0.693/b, where b is the slope found in the linear
fit of the natural logarithm of the fraction remaining of the parent
compound vs. incubation time.
Hemolysis assay Hemolysis assays were performed on mechanically
difibrinated sheep blood (Hemostat Labs: DSB100). Difibrinated blood
(1.5 mL) was placed into a microcentrifuge tube and centrifuged for 10
min at 10,000 rpm. The supernatant was then removed and then the cells
were resuspended in 1 mL of phosphate-buffered saline (PBS). The
suspension was centrifuged, the supernatant was removed and cells
were resuspended two additional times. The final cell suspension was
then diluted 10-fold. Test compounds from DMSO stock solutions were
added to aliquots of the 10-fold suspension dilution of blood. PBS was
used as a negative control and a zero hemolysis marker. Triton X (a 1%
sample) was used as a positive control serving as the 100% lysis marker.
Samples were then placed in an incubator at 37 ˚C while being shaken at
200 rpm for one hour. After one hour, the samples were centrifuged for
10 min at 10,000 rpm. The resulting supernatant was diluted by a factor
of 40 in distilled water. The absorbance of the supernatant at 540 nm
was then measured with a UV spectrometer.
Microsome stability assay A stock solution of compound was added to
a solution of 0.1M phosphate-buffer saline (PBS, pH = 7.4) containing
1mM NADPH to make a final concentration of 100 µM. This solution was
incubated
at
37OC
for
5
minutes
at
which
time
mouse microsomes (pooled CD1 males from Sigma M9441) was added
at a final concentration of 1.0 mg/mL and vortexed gently. A positive
control solution without the addition of microsomes was also included to
monitor compound stability over the course of the experiment. An aliquot
was removed and immediately quenched with the addition of 10x volume
of acetonitrile (HPLC grade). The remaining solution was incubated at 37
˚C while being shaken at 200 rpm. An aliquot was removed at 0, 15, 30,
60, 90, 120, 240, and 480 minutes time points and quenched with
acetonitrile. After quenching, samples were kept on ice and centrifuged
at 10,000 rpm for 5 minutes at 4OC, and the supernatant was transferred
to appropriate LC-MS vials for analysis. Samples were analyzed using
reverse phase liquid chromatography coupled with low resolution tandem
mass spectrometry (LC-MS/MS) in the laboratory of Dr. Melander at
North Carolina State University Department of Chemistry. A Shimadzu
Scientific Instruments LC system (comprising a solvent degasser, two
LC-20A pumps and a SCL-20A system controller) was coupled to and
introduced into the ESI source of an Expression L CMS Advion mass
spectrometer (Advion, Ithica, NY). Separation was achieved using a
Restek Ultra C18 reversed-phase column (3 µm, 50 x 4.6 mm). For
analysis 20µL of sample was injected. The method used a solvent flow of
0.5 ml/min. Initial gradient conditions (90% water:10% acetonitrile with
0.1% formic acid) were held for 2 minutes. From 2 to 5.5 minutes, the
mobile phase composition increased linearly to 10% water:90%
acetonitrile with 0.1% formic acid. From 5.5 to 8 minutes, the mobile
phase composition was held at 10% water:90% acetonitrile with 0.1%
formic acid. At 8.01 to 10 min, the column was re-equilibrated with 90%
water:10% acetonitrile with 0.1% formic acid. HPLC data acquisition and
analysis were performed using LabSolutions software. The settings for
the Advion ESI/MS in dual ion mode are as follows: capillary temperature
= 250 °C; capillary voltage = 180 V; ESI voltage = 3500 V; source gas
temperature = 200 °C; pirani pressure = 2.73 e-3 mbar; hexapole bias = 8
V; extraction electrode = 9 V. Data was acquired using an acquisition
method: mass range m/z = 100 to 800; scan time = 632 ms, scan speed
(m/z/sec) = 1107. Analysis performed using Advion Mass Express and
Advion Data Express. The percentage of compound remaining is
calculated as the ratio of [(compound peak area of solution
G. mellonella toxicity assay G. mellonella larvae (Speedy Worm,
Alexandria, MN) were used within 10 days of shipment from the vendor.
After reception of worms, larvae were kept in the dark at room
temperature for at least 24 h before infection. Larvae weighing between
200 to 300 mg were used in the survival assay. Using a 10- µL glass
syringe (Hamilton, Reno, NV) fitted with a 30 G needle (Exel International,
St. Petersburg, Fl), a 5 µL solution of the desired compound and
concentration were injected into the last left proleg. Injected worms were
left at room temperature in the dark while being assessed at 24 h
intervals over 5 days. Larvae were considered dead if they did not
respond to physical stimuli. Experiment was repeated three times using
10 larvae per experimental group.
Colony count procedure at 48 hour time point for M. smegmatis
Cultures were incubated for 48 hr and then subcultured to an OD600 of
0.01 in 7H9 + ADC + 0.5% Tween 80 media. The resulting bacterial
suspension was aliquoted into 3.0 mL culture tubes. Test compound was
then added to the media at predetermined concentrations. Controls were
employed in which no compound was added to the cultures. Samples
were then placed in a 37 °C incubator with shaking at 200 rpm for 48
with microsomes)/(compound peak without microsomes)]
x
100.
9
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10.1002/cmdc.201900033
ChemMedChem
FULL PAPER
Connell, P. M. Dunman, D. J. Krysan, PLoS One 2015, 10,
e0129234.
hours. At this time point, 100 µL was taken from each culture tube and
diluted serially in 7H9 media. Then 10 µL of was removed from each
serial dilution and plated out on a 7H10 + OADC + glycerol round petri
dish followed by 48 hr incubation at 37 °C and quantification.
[18]
[19]
[20]
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, 3549-3553.
R. W. Huigens, S. Reyes, C. S. Reed, C. Bunders, S. A.
Rogers, A. T. Steinhauer, C. Melander, Bioorganic &
Medicinal Chemistry 2010, 18, 663-674.
Acknowledgements
M. Meir, T. Grosfeld, D. Barkan, Antimicrob Agents
Chemother 2018, 62.
Acknowledgements Text. The authors would like to thank the
National Institutes of Health (AI106733) for support.
Keywords: Biofilm • 2-Aminoimidazole • Tuberculosis •
Mycobacteria • Synergy
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Entry for the Table of Contents
Compounds that can inhibit and disperse mycobacterial biofilms have the potential to serve alongside TB antibiotics to overcome
drug tolerance and reduce treatment duration. Using Mycobacterium smegmatis as a surrogate organism, we have identified two new
2-aminoimidazole compounds that inhibit and disperse mycobacterial biofilms, and work synergistically with isoniazid and rifampicin
to eradicate preformed M. smegmatis biofilms in vitro.
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