Second-Generation Meridianin Analogues Inhibit the Formation of Mycobacterium smegmatis Biofilms and Sensitize Polymyxin-Resistant Gram-Negative Bacteria to Colistin

Article abstract of DOI:10.1002/cmdc.202000438

Drug-resistant bacteria are rapidly becoming a significant problem across the globe. One element that factors into this crisis is the role played by bacterial biofilms in the recalcitrance of some infections to the effects of conventional antibiotics. Bacteria within a biofilm are highly tolerant of both antibiotic treatment and host immune responses. Biofilms are implicated in many chronic infections, including tuberculosis, in which they can act as bacterial reservoirs, requiring an arduous antibiotic regimen to eradicate the infection. A separate, compounding problem is that antibiotics once seen as last-resort drugs, such as the polymyxin colistin, are now seeing more frequent usage as resistance to front-line drugs in Gram-negative bacteria becomes more prevalent. The increased use of such antibiotics inevitably leads to an increased frequency of resistance. Drugs that inhibit biofilms and/or act as adjuvants to overcome resistance to existing antibiotics will potentially be an important component of future approaches to antibacterial treatment. We have previously demonstrated that analogues of the meridianin natural product family possess adjuvant and antibiofilm activities. In this study, we explore structural variation of the lead molecule from previous studies, and identify compounds showing both improved biofilm inhibition potency and synergy with colistin.

Full text of DOI:10.1002/cmdc.202000438

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Second-Generation Meridianin Analogues Inhibit the
Formation of Mycobacterium smegmatis Biofilms and
Sensitize Polymyxin-Resistant Gram-Negative Bacteria to
Colistin
Michael J. Zeiler,[a] Roberta J. Melander,[a] and Christian Melander*[a]
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Drug-resistant bacteria are rapidly becoming a significant
problem across the globe. One element that factors into this
crisis is the role played by bacterial biofilms in the recalcitrance
of some infections to the effects of conventional antibiotics.
Bacteria within a biofilm are highly tolerant of both antibiotic
treatment and host immune responses. Biofilms are implicated
in many chronic infections, including tuberculosis, in which
they can act as bacterial reservoirs, requiring an arduous
antibiotic regimen to eradicate the infection. A separate,
compounding problem is that antibiotics once seen as last-
resort drugs, such as the polymyxin colistin, are now seeing
more frequent usage as resistance to front-line drugs in Gram-
negative bacteria becomes more prevalent. The increased use
of such antibiotics inevitably leads to an increased frequency of
resistance. Drugs that inhibit biofilms and/or act as adjuvants to
overcome resistance to existing antibiotics will potentially be an
important component of future approaches to antibacterial
treatment. We have previously demonstrated that analogues of
the meridianin natural product family possess adjuvant and
antibiofilm activities. In this study, we explore structural
variation of the lead molecule from previous studies, and
identify compounds showing both improved biofilm inhibition
potency and synergy with colistin.
Introduction
pounds that target non-essential bacterial functions in order to
potentiate the activity of antibiotics. By targeting non-essential
pathways, adjuvants potentially exert reduced selective pres-
sure on bacteria, thus slowing the development of resistance.
Overall, adjuvants hold the potential to enable the use of old
antibiotics previously rendered obsolete by resistance and
provide an avenue to help resolve the current problem of a
limited antibiotic pipeline.
Another important mechanism that is frequently associated
with increased drug tolerance is the formation of biofilms.
Bacterial biofilms are defined as a surface attached community
of bacteria encased in an extracellular matrix of biomolecules
(polysaccharides, proteins, DNA, and lipids). Bacteria in a biofilm
can exhibit a 1000-fold increase in drug tolerance compared to
those in a free-floating (planktonic) state.[4]. Multiple factors
contribute to this tolerance. The matrix itself acts as a physical
barrier between the bacteria and the outside environment,
including some antibacterial drugs and the host immune
response. Furthermore, many cells within the biofilm have
reduced metabolic activity, which decreases the efficacy of
antibiotics that target cell reproduction and growth.[4] In
addition, the increased cellular density within biofilms increases
the frequency of horizontal gene transfer (HGT). Increased HGT
rates lead to increased acquisition and spread of antibiotic
resistance.
Antibiotic resistance has become one of the largest threats to
the medical community.[1] In 2019, the Centers for Disease
Control and Prevention (CDC) reported 2.8 million cases of
drug-resistant infections, 35000 of which resulted in death.[2] As
resistance to an antibiotic increases past its breakpoint, defined
by the Clinical and Laboratory Standards Institute (CLSI) as the
minimum inhibitory concentration (MIC) of the antibiotic
corresponding to therapeutically achievable levels, effective
in vivo concentrations can no longer be achieved, and the
infection becomes untreatable with that antibiotic. As a
consequence of its well-documented side effects, colistin is
typically prescribed as a last-resort drug to treat infections
caused by multidrug-resistant (MDR) Gram-negative bacteria.[3]
However, resistance to colistin has been observed worldwide,
highlighting the need for alternative approaches to combat
MDR Gram-negative infections. With the output of new anti-
biotics unable to keep up with the rapid development of
resistance, orthogonal strategies are being explored to combat
this problem.
One of these approaches involves the use of antibiotic
adjuvants. This approach involves circumventing bacterial
defense mechanisms in order to re-sensitize bacteria to
approved antibiotics. Adjuvants are non-microbicidal com-
It has been postulated that latent reservoirs of Mycobacte-
rium tuberculosis, the causative agent of tuberculosis (TB), exist
in a biofilm-like state within infected individuals. For example, it
has been demonstrated that M. tuberculosis can form biofilm-
like communities on cellular debris (which mimics the environ-
ment of lung lesions in TB patients) and subsequently exhibit
[a] M. J. Zeiler, Prof. R. J. Melander, Prof. C. Melander
Department of Chemistry and Biochemistry
University of Notre Dame, Notre Dame, IN 46556 (USA)
E-mail: cmelande@nd.edu
Supporting information for this article is available on the WWW under
https://doi.org/10.1002/cmdc.202000438
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isoniazid tolerance.[5] The World Health Organization (WHO)
attributed 1.5 million deaths to TB in 2019 alone.[6] Treatment of
drug-susceptible TB is a long process that lasts a minimum of
six months where patients undergo antibiotic combination
therapy with isoniazid, rifampicin, and pyrazinamide.[7] Chal-
steric and electronic effects of this ring on activity. We also
determined activity based on the position of the bromide, and
the relative orientation of the 2-aminopyrimidine (2-AP).
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lenges with this regimen include substantial drug toxicity and Results and Discussion
ensuring patient compliance. The lengthy regimen is due in
part to the necessity of eradicating latent reservoirs to avoid
disease rebound.[5] MDR TB infections are caused by strains that
have acquired genetic resistance to both of the front-line drugs:
isoniazid and rifampicin, in addition to drug tolerance due to
the formation of biofilms. With the frequency of MDR-TB cases
increasing, the CDC has marked MDR-TB as a serious problem in
the coming decades.[2] Though challenging, targeting bacteria
in a biofilm state is one approach for combatting TB and could
potentially shorten the treatment regimen, which would
increase patient compliance, and in turn slow resistance
acquisition.
Due to the pathogenicity of M. tuberculosis, Mycobacterium
smegmatis is often used as a model bacterium because it is
non-pathogenic and faster growing. In addition, M. smegmatis
shares over 2000 genetic homologues with M. tuberculosis, such
as cell-wall composition and many cellular functions.[7] We have
previously demonstrated that activity against M. smegmatis
often translates to activity against M. tuberculosis.[8,9]
The pilot library based on lead analogue 2 was synthesized
according to previously reported methods (Scheme 1).[11] 5-
Bromo indole was acylated with acetyl chloride to afford the
corresponding C3-acylated indole, which was then protected
with p-toluene sulfonyl chloride (TsCl) to give the tosyl-
protected indole. 6-Bromo indole underwent the same reaction
sequence to afford the corresponding tosyl-protected indole.
The tosyl-protected indoles were reacted with N,N-dimeth-
ylformamide dimethyl acetal (DMF-DMA) to generate their
corresponding vinylogous amide products (3a and 3b), which
were cyclized with the appropriate benzylguanidine derivatives
to produce the 2-aminopyrimidine final products 4a–n. Varia-
tion of the benzylguanidines was achieved by reacting S-methyl
isothiourea hemisulfate with commercially available benzyl-
amines to form benzylguanidine sulfates that were then used in
the formation of the 2-aminopyrimidine rings.[12]
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Further derivatization of lead analogue 2 was achieved with
attachment of the 2-AP at C5 rather than C4 (Scheme 2). To
access these derivatives, 5-bromo indole was halogenated at C3
Figure 1. Meridianin D and lead analogue 2.
Recently, we reported that analogues of the marine natural
product meridianin D, 1 (Figure 1), inhibit and disperse biofilms
of M. smegmatis.[10] Lead compound 2 was shown to be superior
to 1 and demonstrated the ability to inhibit biofilms at low
micromolar concentrations as well as the ability to disperse
preformed biofilms. Separately, lead compound 2 displayed the
ability to re-sensitize Gram-negative bacteria to colistin.[11]
Probing this activity, we wanted to create a pilot library that
diversified the structure of our lead compound 2 in order to
potentially improve its potency both against mycobacterial
biofilms and as an adjuvant for colistin against Gram-negative
bacteria. Our studies focused on the pendent phenyl ring, by
adding electron-withdrawing (EWG) and electron-donating
(EDG) groups with various substitution patterns to probe the
Scheme 1. Synthesis of C4-substituted derivatives: a) acetyl chloride, SnCl4,
toluene; b) TsCl, DIPEA, DMAP, CH2Cl2; c) DMF-DMA, toluene; d) S-methyl
isothiourea, H2O, EtOH; e) benzylguanidine, K2CO3, 2-methoxyethanol; f) HCl,
MeOH
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methyl ester as its corresponding hydrochloride salt. The
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5
pyrimidines were then coupled to the tosyl-protected indole (5)
using Suzuki-Miyaura cross-coupling conditions.[13] Deprotection
of the tosylates was achieved by treatment with cesium
carbonate to yield desired final products 6a–e.[15]
With the pilot library in hand (Figure 2), we began testing
all 20 compounds for anti-biofilm activity in M. smegmatis. The
library was tested for microbicidal activity and proved to be
nontoxic, with most compounds possessing MIC’s�200 μM. All
compounds were tested in a crystal violet reporter assay to
determine their IC50 values (Table 1). The parent compound, 2,
returned an IC50 of 6.9 μM. Compound 4h was discovered to be
the most active biofilm inhibitor, with an IC50 value of 4.5 μM,
followed by an IC50 of 5.6 μM for compound 4i. In general,
compounds containing an EWG on the C4 position of the
phenyl ring (4b, 4c, 4h, 4j) proved to be more active than
those with an EDG (Table 1). For example, compounds 4d, and
4f, which incorporated EDGs, exhibited higher IC50 values than
the parent compound 2. In addition, increasing the steric bulk
of the substituent (i.e., installation of naphthyl (4o)) diminished
activity substantially, displaying an IC50 value of >20 μM
(Table 1).
Changing the attachment position between the indole and
the pyrimidine to the C5 position on the 2-AP (compound 6a)
from the C4 position (compound 2) conferred no significant
change in activity. However, the incorporation of substituents
on the benzyl groups (compounds 6b–6e) diminished biofilm
inhibition activity compared to the unsubstituted compound
6a (Table 1).
It was previously reported that meridianin D 1 and parent
compound 2 demonstrated adjuvant activity with colistin in
Gram-negative bacteria.[11] Therefore, all 20 compounds from
this library were tested with colistin against polymyxin-resistant
strains of Klebsiella pneumoniae (KP) and Acinetobacter bauman-
nii (AB). Compounds were first tested individually to ensure
they had no microbicidal effects on planktonic cells, by record-
ing their MIC values. All compounds proved to be non-
microbicidal alone with MIC values greater than 200 μM
(Table S2 in the Supporting Information). The library was then
assayed at subinhibitory concentrations for their ability to
potentiate colistin against the resistant KP and AB strains.
Several compounds showed adjuvant activity at 60 μM, with
several possessing potency either greater than or comparable
to the parent compound 2 (Table 2). Analogues 4l and 4g
demonstrated the greatest activity, lowering the colistin MIC
from 1024 to 2 and 4 μg/mL, respectively, in A. baumannii 4106
and from 512 to 0.5 μg/mL for both in K. pneumoniae B9
(Table 2). Inspired by the significant activity of the C3-methyl-
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Scheme 2. Synthesis of C5-substituted analogues. a) NIS, TEA, CH2Cl2; b) TsCl,
DIPEA, DMAP, CH2Cl2; c) 2-fluoro-5-bromo pyrimidine, TEA, DMF; d) B2Pin2,
KOAc, Pd(dppf)Cl2, dioxane; e) Pd(PPh3)4, K2CO3, toluene, EtOH; f) Cs2CO3,
THF, MeOH.
using N-iodosuccinimide to form the corresponding C3-iodo
indole, which was then protected with TsCl as described
previously to afford compound 5.[13] The 2-benzylamino-5-
pyrimidinyl boronic acid pinacol esters (Bpin) were synthesized
by treating the desired, commercially available benzylamines
with 2-fluoro 5-bromo pyrimidine followed by a Miyaura
borylation to install a Bpin group[14] In the case of the methyl
ester derivative, 6c, the ester used was synthesized by a Fischer
esterification of 4-aminomethyl benzoic acid to afford the
Figure 2. Pilot library of lead analogue 2.
Table 1. Biofilm inhibition values in M. smegmatis.
Compound
IC50 [μM]
Compound
IC50 [μM]
2
6.9�0.8
16.6�1.8
17.1�5.8
8.5�1.4
19.1�1.9
5.9�1.3
14�5
4j
4k
4l
4m
6a
6b
6c
6d
6e
>20
>20
4a
4b
4c
4d
4e
4f
4g
4h
4i
Table 2. Colistin MIC values [μg/mL] with 4n, 4h and 4p at 60 μM.
10.4�4.4
>20
Compound
A .baumannii 4106
K. pneumoniae B9
7.9�2.3
>20
>20
>20
>20
–
2
4l
4g
4n
1024
2
2
4
512
0.5
2
0.5
>16
18�3.5
4.5�1.7
5.6�2.1
>32
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analogues for both mycobacterial biofilm inhibition, including
against M. tuberculosis, and as adjuvants for colistin. Addition-
ally, future work will be conducted to improve water solubility,
as this area could hinder future development.
Table 3. Dose-response of 2 and 4g for colistin MIC values [μg/mL].
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[Cpd] μM
A. baumannii 4106
K. pneumoniae B9
2
4g
2
4g
60
30
15
10
7.5
5
2
2
2
8
32
>32
4
4
4
4
4
8
0.5
0.5
1
4
8
0.5
0.5
0.5
0.5
1
Experimental Section
>16
2
Chemistry experimental: All reagents were purchased from com-
mercially available sources without further purification. Flash
chromatography was performed using 60 Å mesh standard grade
silica gel from Sorbetch. NMR solvents were obtained from Cam-
bridge Isotope Labs and used as is. All 1H NMR (400 MHz) were
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substituted analogue (4l), we wanted to see if creating
symmetry on the phenyl ring would affect adjuvant activity by
synthesizing the 3,5-dimethyl-substituted analogue 4n. Com-
pound 4n, however, showed negligible activity, with a colistin
MIC of �16 μg/mL at 60 μM for both AB and KP strains.
recorded at 25 C on a Bruker Avance spectrometer. All 13C NMR
°
°
(101 and 201 MHz) spectra were also recorded at 25 C on Bruker
Avance spectrometers. Chemical shifts (δ) are given in parts per
million (ppm) relative to the respective NMR solvent. All high-
resolution mass spectrometry measurements were made in the
Mass Spectrometry and Proteomics Facility at the University of
Notre Dame. Infrared spectra were obtained on a FT/IR-4100
spectrophotometer (νmax in cmÀ 1). UV absorbance was recorded on
a Genesys 10 scanning UV/visible spectrophotometer (λmax in nm).
The parent compound 2 was unable to bring the colistin
MIC below the breakpoint when dosed at lower concentrations.
The addition of a CF3 group, however, to the para position of
the phenyl ring (4g) improved the dose-response activity of the
molecule (Table 3). While having comparable activity to 2 at
60 μM, 4g was able to maintain the colistin MIC at 4 μg/mL
down to 7.5 μM in A. baumannii 4106. Dose-response was
improved further in K. pneumoniae B9, holding colistin below or
at its breakpoint (2 μg/mL) down to 5 μM, effectively making
the colistin-resistant strain susceptible and once again poten-
tially treatable with colistin. It is worth noting that shifting the
CF3 substitution from a para orientation (4g) to a meta
orientation (4j) made the molecule significantly less active, with
a colistin MIC of >32 μg/mL in A. baumannii 4106 and >16 μg/
ml in K. pneumoniae B9 (Table S3).
General Synthetic Procedures
Benzyl guanidine formation: The appropriately substituted benzyl-
amine (1 g, 1 equiv) was added to a stirring solution of S-methyl
isothiourea hemi-sulfate (1 equiv) in H2O/EtOH (1:1, 2 M to benzyl-
°
amine), and the reaction mixture was heated at 80 C under a
condenser overnight. The mixture was cooled to room temperature.
Diethyl ether was added to the mixture until a white solid was
°
observed, then the mixture was placed in a refrigerator at 5 C for
1 h. The resulting white solid was filtered and washed further with
diethyl ether to afford the substituted benzylguanidine sulfates as a
white solid (80%). Used without further purification.
General procedure for acylation of indole: Indole (5 g, 1 equiv)
was dissolved in toluene (0.2 M) then acetyl chloride (2 equiv) was
°
Conclusion
added. Tin(IV) chloride (2 equiv) was added slowly at 0 C. The
reaction mixture was warmed slowly to room temperature and
stirred for 2 h. TLC in hexanes/EtOAc (1:1) showed absence of
starting material. The reaction was quenched with saturated
sodium bicarbonate to neutral pH then extracted with EtOAc. The
organic layer was washed with water then brine (2x) and dried over
magnesium sulfate. Solvent was removed under reduced pressure.
The residue was purified over silica gel with hexanes/EtOAc (4:1).
The resulting solid was washed with cold MeOH to afford the
product. NMR taken in CDCl3.
In conclusion, structure-activity relationship (SAR) studies were
performed on a previously reported meridianin analogue 2,
generating several analogues with enhanced activities. Mem-
bers of the synthesized library showed improved ability to
inhibit biofilm formation in M. smegmatis, relevant for the
potential application for the treatment of TB. In general,
installation of halogens at the para position of the phenyl ring
led to increased activity, where halogens displayed the greatest
biofilm inhibiting activity. Electron-donating groups, with the
exception of para-methoxy, had deleterious effects on biofilm
inhibition activity. In addition, several compounds were able to
potentiate colistin against two clinically relevant, colistin-
resistant Gram-negative bacterial strains, A. baumannii 4106 and
K. pneumoniae B9. It was shown that taking an active meta-
methylated phenyl head group and introducing symmetry
through a 3,5-dimethyl substitution pattern effectively abol-
ished colistin potentiation activity. However, 4g, which intro-
duced a CF3 group onto the para position of the phenyl ring,
lowered the colistin MIC to its breakpoint at an adjuvant
concentration of 5 μM, thus exceeding the activity of the
previous lead analogue 2. Further SAR studies are planned to
implement these preliminary findings in creating more potent
N-Tosyl protection of indole: 3-Acetyl Indole (4.8 g, 1 equiv), p-
toluenesulfonyl chloride (1.5 equiv), DIPEA (2 equiv), and DMAP
(0.05 equiv) were stirred in CH2Cl2 (0.2 M to indole) at room
temperature overnight. The reaction mixture was washed with
water and brine (2x) and dried over magnesium sulfate, then
solvent was removed under reduced pressure. The residue was
purified over silica gel with hexanes/EtOAc (7:1) to afford a white
solid.
Vinylogous amide formation: N-Tosyl 3-acetyl indole (6.6 g,
1 equiv) was dissolved in toluene (0.4 M to indole) followed by
addition of DMF-DMA (1,1-dimethoxy-N,N-dimethylmethanamine)
°
(2 equiv) and heated at 90 C overnight. After cooling the reaction
to room temperature reaction was diluted with EtOAc then washed
with water and brine (2x) and dried over magnesium sulfate.
Solvent was removed under reduced pressure and the residue was
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purified over silica gel with hexanes/EtOAc (1:1) to afford the
product as a bright yellow solid. NMR taken in CDCl3.
mixed with water and extracted 3× with EtOAc. The organic layer
was washed with water and brine (2x), dried over magnesium
sulfate and solvent was removed under reduced pressure. Residue
was purified over silica gel with hexanes/EtOAc (6:1–2:1) to afford
the product as a white solid.
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2-Amino pyrimidine formation: Vinylogous amide (600 mg,
1 equiv), the appropriate benzyl-guanidine sulfate (2 equiv) and
potassium carbonate (2.1 equiv) were dissolved in 2-methoxy
°
ethanol (22 mM to vinylogous amide) and stirred at 130 C over-
N-Tosyl deprotection: N-Tosyl-protected indole (571 mg, 1 equiv)
and Cs2CO3 (3 equiv) were dissolved in THF/MeOH (3:1) (20 mM to
night. After cooling to room temperature, reaction was diluted with
EtOAc and washed with water and brine (2×), dried over
magnesium sulfate, and solvent was removed under reduced
pressure. The resulting residue was purified over silica gel with
hexanes/EtOAc (1:1). The resulting off-white solid was washed
multiple times with both diethyl ether and warm hexanes.
°
indole) and heated to 40 C overnight. Solvent was removed under
reduced pressure followed by addition of water. Mixture was
extracted with EtOAc 3x and the organic layer was washed with
water and brine (2×), dried over magnesium sulfate and the solvent
was removed under reduced pressure. Residue was purified over
silica gel with hexanes/EtOAc (2:1) to afford the deprotected
product.
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C-3 Iodination of indole: To a stirring solution of 5-Br Indole (1.5 g,
1 equiv) in CH2Cl2 (0.2 M to indole) was added TEA (1 equiv). After
30 min, N-iodosuccinimide (1 equiv) was added. The reaction
mixture was stirred at room temperature overnight then quenched
with saturated sodium thiosulfate, washed with water and brine
(2x), dried over magnesium sulfate and solvent was removed under
reduced pressure. The resulting residue was purified over silica gel
with hexanes/EtOAc (7:1) to afford the product as a light lavender-
colored solid.
4a: N-Benzyl-4-(6-bromo-1H-indol-3-yl)pyrimidin-2-amine: Pro-
duced according to the general method for 2-amino pyrimidine
1
formation (42 mg, 8%). Tan solid. H NMR (400 MHz, [D4]methanol):
δ=8.20 (s, 1H), 8.10 (d, J=5.5 Hz, 1H), 8.01 (s, 1H), 7.56 (d, J=
1.8 Hz, 1H), 7.42 (d, J=7.6 Hz, 2H), 7.32 (t, J=7.7 Hz, 2H), 7.22 (t, J=
7.3 Hz, 1H), 7.14 (d, J=8.7 Hz, 1H), 7.00 (d, J=5.5 Hz, 1H), 4.71 (s,
2H). 13C NMR (201 MHz, DMSO): δ=162.7, 162.5, 157.6, 141.3, 138.3,
138.2, 129.8, 128.6, 127.4, 126.9, 124.8, 123.5, 115.2, 114.9, 114.3,
105.7, 44.7. IRνmax (cmÀ 1): 3412, 3240, 1543, 514 HRMS (ESI) for
C19H15BrN4 calculated [M+H]+ 379.0553, found 379.0558. UV (λmax
nm) 224.
Fischer esterification of 4-(aminomethyl) benzoic acid: To a
stirring mixture of 4-(aminomethyl) benzoic acid (1 g, 1 equiv.) in
MeOH (0.2M to benzoic acid) was added slowly conc. HCl (1 equiv).
°
The resulting solution was then refluxed at 85 C for 16 h. The
reaction was cooled to room temperature and the MeOH was
removed under vacuum repeatedly to remove any excess HCl.
Product afforded as a white solid (95%) and used without any
further purification.
4b:
4-(6-Bromo-1H-indol-3-yl)-N-(4-chlorobenzyl)pyrimidin-2-
amine: Produced according to the general method for 2-amino
pyrimidine formation (35 mg, 6%). Off-white solid. 1H NMR
(400 MHz, [D4]methanol): δ=8.16 (s, 1H), 8.11 (d, J=5.5 Hz, 1H),
8.01 (s, 1H), 7.57 (d, J=1.8 Hz, 1H), 7.40 (d, J=8.4 Hz, 2H), 7.35–7.28
(m, 2H), 7.15 (d, J=8.7 Hz, 1H), 7.01 (d, J=5.5 Hz, 1H), 4.68 (s, 2H).
13C NMR (101 MHz, DMSO): δ=162.7, 162.6, 157.6, 140.4, 138.3,
131.4, 129.8, 129.2, 128.6, 124.8, 124.4, 123.5, 115.1, 114.9, 114.3,
105.9, 44.1. IRνmax (cmÀ 1): 3414, 3220, 2992, 829 HRMS (ESI) for
C19H14BrClN4 calculated [M+H]+ 413.0163, found 413.0172. UV (λmax
nm) 220.
Nucleophilic aromatic substitution of 2-fluoro-5-bromopyrimi-
dine: To a stirred solution of 2-fluoro 5-bromo pyrimidine (1 g,
1 equiv), and DIPEA (4 equiv) in DMF (0.3 M to pyrimidine) was
added slowly the appropriate benzylamine (2 equiv), and the
°
reaction mixture was heated to 85 C for 1 h. After the mixture had
cooled to room temperature, water was added until precipitate was
°
observed. The flask was placed in a refrigerator at 5 C for 1 h. The
solid was filtered and washed with excess water to remove
remaining DMF then dried under vacuum. Crude product used
without further characterization.
4c:
4-(6-Bromo-1H-indol-3-yl)-N-(4-fluorobenzyl)pyrimidin-2-
amine: Produced according to the general method for 2-amino
pyrimidine formation (44 mg, 8%). Tan solid. 1H NMR (400 MHz, [D6]
DMSO): δ=11.80 (s, 1H), 8.24 (d, J=2.8 Hz, 1H), 8.16 (d, J=5.3 Hz,
2H), 7.69–7.57 (m, 2H), 7.41 (dd, J=8.3, 5.5 Hz, 2H), 7.27–7.06 (m,
2H), 7.03 (d, J=5.3 Hz, 1H), 4.56 (s, 2H). 13C NMR (201 MHz, [D6]
DMSO): δ=162.7, 162.5, 161.5 (d, J=241.3 Hz), 157.5, 138.3, 137.4
(d, J=3.5 Hz), 129.8, 129.2, 124.8, 124.2, 123.5, 115.3 (d, J=21.2 Hz),
115.2, 114.9, 114.3, 105.8, 43.9. IRνmax (cmÀ 1): 3415, 3221, 2934, 1214
HRMS (ESI) for C19H14BrFN4 calculated [M+H]+ 397.0459, found
397.0470. UV (λmax nm) 202.
Miyaura borylation: To a flame-dried RBF was added the appro-
priate 2-amino pyrimidine (1 g, 1 equiv), B2Pin2 (1.1 equiv) and
KOAc (3 equiv). The flask was evacuated under vacuum and
backfilled with argon. This was repeated 3x. Anhydrous dioxane
(0.2 M to pyrimidine) was added and stirred for 5 min. Argon was
then bubbled through for 15 min before the addition of Pd(dppf)
Cl2 (0.05 equiv). Following addition of catalyst, argon was further
bubbled through for another 5 min. The mixture was refluxed
°
under argon at 115 C for 4 h which after completion had taken on
4d:
4-(5-Bromo-1H-indol-3-yl)-N-(4-methylbenzyl)pyrimidin-2-
a black color. EtOAc was added, sonicated, and filtered. The organic
layer was washed with water and brine and run through a plug of
silica with EtOAc. After removal of solvent the remaining solid was
columned over silica in hexanes with 0–50% EtOAc to remove any
remaining baseline impurities, affording a solid which was washed
with EtOH three times to afford the product as a fine, white solid
(50%). Used without further purification.
amine: Produced according to the general method for 2-amino
pyrimidine formation (47 mg, 9%). Tan solid. 1H NMR (400 MHz, [D4]
methanol): δ=8.67 (d, J=1.9 Hz, 1H), 8.08 (d, J=5.5 Hz, 1H), 8.04 (s,
1H), 7.37–7.30 (m, 3H), 7.27 (dd, J=8.7, 1.9 Hz, 1H), 7.12 (d, J=
7.7 Hz, 2H), 6.98 (d, J=5.5 Hz, 1H), 4.68 (s, 2H), 2.29 (s, 3H). 13C NMR
(201 MHz, DMSO): δ=162.8, 162.5, 157.7, 138.2, 136.9, 135.8, 130.3,
129.3, 129.3, 129.2, 127.5, 127.3, 125.06, 114.3, 113.7, 105.5, 44.3,
21.1. IRνmax (cmÀ 1): 3394, 3165, 2924, 1525 HRMS (ESI) for C20H17BrN4
calculated [M+H]+ 393.0709, found 393.0730. UV (λmax nm) 220.
Suzuki cross-coupling of indole and pyrimidine: 3-Iodoindole
(800 mg, 1 equiv), boronic acid pinacol ester (1.1 equiv) and Pd
(PPh3)4 (0.05 equiv) were added to an RBF. The flask was placed
under vacuum and backflushed with argon 3x. Toluene/EtOH (3:1,
40 mM to indole) was added followed by potassium carbonate
(2 equiv, 2 M in water). The resulting mixture was degassed with
4e: 4-(5-Bromo-1H-indol-3-yl)-N-(4-methoxybenzyl)pyrimidin-2-
amine: Produced according to the general method for 2-amino
pyrimidine formation (30 mg, 5%). Tan solid. 1H NMR (400 MHz, [D4]
methanol): δ=8.69 (d, J=2.0 Hz, 1H), 8.08 (dd, J=5.5, 1.5 Hz, 1H),
8.04 (d, J=2.4 Hz, 1H), 7.43–7.22 (m, 4H), 6.98 (dd, J=5.5, 2.3 Hz,
1H), 6.90–6.82 (m, 2H), 4.65 (s, 2H), 3.75 (d, J=1.4 Hz, 3H). 13C NMR
°
argon for 15 min, then heated to 90 C overnight. Following
completion, the solvent was removed under reduced pressure and
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(201 MHz, DMSO): δ=162.8, 162.7, 162.5, 158.4, 157.7, 136.2, 133.2,
130.3, 130.1, 128.6, 127.5, 125.0, 114.3, 114.1, 113.7, 105.4, 55.4,
43.9. IRνmax (cmÀ 1): 3415, 3119, 2927, 1217 HRMS (ESI) for
C20H17BrN4O calculated [M+H]+ 409.0658, found 409.0650. UV (λmax
nm) 222.
2H), 3.73 (s, 3H). 13C NMR (201 MHz, [D6]DMSO): δ=168.0, 159.9,
154.4, 144.6, 140.0, 136.8, 136.7, 130.2, 127.2, 126.4, 124.8, 119.5,
115.5, 115.2, 113.0, 112.9, 112.8, 105.4, 55.5, 44.6. HRMS (ESI) for
C20H17BrN4O calculated [M+H]+ 409.0658, found 409.0659. UV (λmax
nm) 220. Rf: 0.15 (hexanes/ethyl acetate 1:2).
1
2
3
4
5
6
7
8
4f:
N-(4-Aminobenzyl)-4-(5-bromo-1H-indol-3-yl)pyrimidin-2-
4l:
4-(5-Bromo-1H-indol-3-yl)-N-(3-methylbenzyl)pyrimidin-2-
amine: Produced according to the modified method for 2-amino
pyrimidine formation. Purified over silica using 3% methanol
saturated with ammonia in CH2Cl2 (33 mg, 6%). Tan solid. H NMR
amine: Produced according to the general method for 2-amino
pyrimidine formation (49 mg, 9%). Tan solid. 1H NMR (400 MHz, [D4]
methanol): δ=8.68 (d, J=2.0 Hz, 1H), 8.09 (d, J=5.5 Hz, 1H), 8.05 (s,
1H), 7.37–7.15 (m, 5H), 7.03 (d, J=7.4 Hz, 1H), 6.99 (d, J=5.5 Hz,
1H), 4.70 (s, 2H), 2.31 (s, 3H). 13C NMR (201 MHz, DMSO): δ=162.8,
162.5, 157.7, 141.2, 137.6, 136.2, 130.3, 130.2, 128.6, 128.0, 127.6,
127.5, 127.4, 125.0, 124.5, 114.3, 113.7, 105.5, 44.5, 21.6. HRMS (ESI)
for C20H17BrN4 calculated [M+H]+ 393.0709, found 393.0713. UV
(λmax nm) 224. Rf: 0.2 (hexanes/ethyl acetate 1:2).
1
(400 MHz, [D4]methanol): δ=8.71 (d, J=1.9 Hz, 1H), 8.07 (d, J=
5.5 Hz, 1H), 8.04 (s, 1H), 7.34 (d, J=8.6 Hz, 1H), 7.27 (dd, J=8.7,
2.0 Hz, 1H), 7.25–7.15 (m, 2H), 6.97 (d, J=5.5 Hz, 1H), 6.81–6.64 (m,
2H), 4.59 (s, 2H). 13C NMR (101 MHz, DMSO): δ=162.8, 162.5, 157.6,
147.7, 136.2, 136.0, 130.2, 130.1, 128.5, 128.2, 127.5, 125.1, 114.3,
114.2, 113.9, 113.7, 105.3, 44.3. IRνmax (cmÀ 1): 3394, 3386, 3019, 2920
HRMS (ESI) for C19H16BrN5 calculated [M+H]+ 394.0662, found
394.0669. UV (λmax nm) 222.
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4m: 4-(5-Bromo-1H-indol-3-yl)-N-(naphthalen-1-ylmethyl)pyrimi-
din-2-amine: Produced according to the general method for 2-
amino pyrimidine formation (33 mg, 6%). Tan solid. 1H NMR
(400 MHz, [D4]methanol): δ=8.57 (s, 1H), 8.22 (dt, J=410.8, 8.9,
8.2 Hz, 1H), 8.14 (d, J=5.5 Hz, 1H), 8.07 (s, 1H), 7.97–7.88 (m, 1H),
7.79 (d, J=8.3 Hz, 1H), 7.62–7.47 (m, 3H), 7.43 (dd, J=8.2, 7.1 Hz,
1H), 7.31 (d, J=8.6 Hz, 1H), 7.21 (dd, J=356.6, 12.6 Hz, 1H), 7.03 (d,
J=5.5 Hz, 1H), 5.22 (s, 2H). 13C NMR (101 MHz, DMSO): δ=162.4,
162.4, 162.2, 157.0, 135.7, 135.5, 133.4, 131.0, 129.9, 129.7, 128.5,
127.0, 127.0, 126.1, 125.7, 125.4, 124.6, 123.4, 113.8, 113.3, 113.2,
105.3, 42.2. IRνmax (cmÀ 1): 3087, 3036, 2907, 2879 HRMS (ESI) for
C23H17BrN4 calculated [M+H]+ 429.0709, found 429.0710. UV (λmax
nm) 224.
4g: 4-(5-Bromo-1H-indol-3-yl)-N-(4-(trifluoromethyl)benzyl)pyri-
midin-2-amine: Produced according to the general method for 2-
amino pyrimidine formation (45 mg, 7%). Tan solid. 1H NMR
(400 MHz, [D4]methanol): δ=8.56 (s, 1H), 8.10 (d, J=5.5 Hz, 1H),
8.04 (s, 1H), 7.61 (d, J=2.2 Hz, 4H), 7.32 (d, J=8.6 Hz, 1H), 7.25 (dd,
J=8.6, 2.0 Hz, 1H), 7.01 (d, J=5.5 Hz, 1H), 4.81 (s, 2H). 13C NMR
(201 MHz, [D6]DMSO): δ=162.7, 162.4, 157.9, 146.3, 136.2, 130.4,
130.3, 127.8, 127.7, 127.5 (d, J=19.2 Hz), 127.4, 125.6, 125.1, 124.9
(q, J=272.0 Hz), 114.3, 113.7, 105.9, 44.3. IRνmax (cmÀ 1): 3251, 3088,
2908, 1326 HRMS (ESI) for C20H14BrF3N4 calculated [M+H]+
447.0427, found 447.0420. UV (λmax nm) 220.
4h:
4-(5-Bromo-1H-indol-3-yl)-N-(4-chlorobenzyl)pyrimidin-2-
4n: 4-(5-Bromo-1H-indol-3-yl)-N-(3,5-dimethylbenzyl)pyrimidin-2-
amine: Produced according to the general method for 2-amino
pyrimidine formation (45 mg, 8%). White solid. H NMR (400 MHz,
amine: Produced according to the general method for 2-amino
pyrimidine formation (34 mg, 6%). Tan solid. 1H NMR (400 MHz, [D4]
methanol): δ=8.61 (s, 1H), 8.10 (d, J=5.5 Hz, 1H), 8.05 (s, 1H), 7.43
(d, J=8.2 Hz, 2H), 7.37–7.23 (m, 4H), 7.00 (d, J=5.5 Hz, 1H), 4.71 (s,
2H). 13C NMR (201 MHz, DMSO): δ=162.7, 162.5, 157.9, 140.4, 136.2,
131.4, 130.4, 129.3, 129.0, 128.6, 127.4, 125.1, 124.8, 114.3, 113.7,
105.7, 43.9. IRνmax (cmÀ 1): 3411, 3015, 2925, 791 HRMS (ESI) for
C19H14BrClN4 calculated [M+H]+ 413.0163, found 413.0169. UV (λmax
nm) 218.
1
[D4]methanol): δ=8.70 (d, J=2.0 Hz, 1H), 8.09 (d, J=5.5 Hz, 1H),
8.05 (s, 1H), 7.34 (d, J=8.6 Hz, 1H), 7.27 (dd, J=8.7, 2.0 Hz, 1H), 7.06
(s, 2H), 6.99 (d, J=5.5 Hz, 1H), 6.85 (s, 1H), 4.66 (s, 2H), 2.27 (s, 6H).
13C NMR (101 MHz, DMSO): δ=162.8, 162.5, 157.5, 141.2, 137.5,
136.2, 130.3, 130.2, 128.4, 127.5, 125.1, 124.7, 114.3, 113.8, 113.7,
105.4, 44.5, 21.5. HRMS (ESI) for C21H16BrN4 calculated [M+H]+
407.0866, found 407.0865. UV (λmax nm) 200. Rf: 0.2 (hexanes/ethyl
acetate 1:1).
4i:
4-(5-Bromo-1H-indol-3-yl)-N-(4-bromobenzyl)pyrimidin-2-
amine: Produced according to the general method for 2-amino
pyrimidine formation (37 mg, 6%). Tan solid. 1H NMR (400 MHz, [D4]
methanol): δ=8.60 (s, 1H), 8.09 (d, J=5.4 Hz, 1H), 8.03 (s, 1H), 7.48–
7.23 (m, 6H), 6.99 (d, J=5.5 Hz, 1H), 4.68 (s, 2H). 13C NMR (101 MHz,
DMSO): δ=162.7, 157.5, 140.8, 136.2, 131.8, 131.5, 130.4, 129.5,
127.4, 125.1, 124.8, 119.8, 114.3, 113.8, 113.7, 105.7, 44.0. IRνmax
(cmÀ 1): 3420, 3103, 2919, 657 HRMS (ESI) for C19H14Br2N4 calculated
[M+H]+ 456.9658, found 456.9659. UV (λmax nm) 226.
6a: N-Benzyl-5-(5-bromo-1H-indol-3-yl)pyrimidin-2-amine: Pro-
duced according to the general Tosyl deprotection (380 mg, 94%).
1
White solid. H NMR (400 MHz, [D4]methanol): δ=8.53 (s, 2H), 7.80
(d, J=1.8 Hz, 1H), 7.48 (s, 1H), 7.43–7.19 (m, 7H), 4.65 (s, 2H). 13C
NMR (101 MHz, DMSO): δ=160.8, 155.8, 140.6, 135.3, 128.2, 127.0,
126.7, 126.5, 124.1, 124.0, 121.0, 117.8, 113.9, 112.2, 109.9, 44.0.
IRνmax (cmÀ 1): 3221, 3206, 2935, 1457 HRMS (ESI) for C19H15BrN4
calculated [M+H]+ 379.0553, found 379.0556. UV (λmax nm) 206.
4j: 4-(5-Bromo-1H-indol-3-yl)-N-(3-(trifluoromethyl)benzyl)pyrimi-
din-2-amine: Produced according to the general method for 2-
amino pyrimidine formation (39 mg, 7%). Tan solid. 1H NMR
(400 MHz, [D4]methanol): δ=8.59 (s, 1H), 8.11 (d, J=5.4 Hz, 1H),
8.03 (s, 1H), 7.76–7.68 (m, 2H), 7.57–7.44 (m, 2H), 7.32 (d, J=8.6 Hz,
1H), 7.25 (dd, J=8.6, 2.0 Hz, 1H), 7.00 (d, J=5.5 Hz, 1H), 4.80 (s, 2H).
13C NMR (101 MHz, [D6]DMSO): δ=162.7, 157.9, 142.9, 136.2, 131.4,
130.4, 129.7, 129.5, 129.4, 127.4, 126.8, 125.1, 124.8 (q, J=272.2 Hz),
123.7 (d, J=4.1 Hz), 122.8, 114.3, 113.7, 105.9, 44.2. IRνmax (cmÀ 1):
3134, 3075, 2917, 1328 HRMS (ESI) for C20H14BrF3N4 calculated [M+
H]+ 447.0427, found 447.0421. UV (λmax nm) 220.
6b:
4-(((5-(5-Bromo-1H-indol-3-yl)pyrimidin-2yl)amino)methyl)
benzonitrile: Produced according to the general Tosyl deprotection
(361 mg, 95%). White solid. H NMR (400 MHz, [D4]methanol): δ=
1
8.54 (s, 2H), 7.79 (d, J=1.8 Hz, 1H), 7.74–7.64 (m, 2H), 7.56 (d, J=
8.1 Hz, 2H), 7.48 (s, 1H), 7.36 (d, J=8.7 Hz, 1H), 7.26 (dd, J=8.7,
1.8 Hz, 1H), 4.72 (s, 2H). 13C NMR (201 MHz, DMSO): δ=161.1, 156.3,
147.2, 135.8, 132.7, 128.2, 127.2, 124.6, 124.5, 121.4, 119.5, 118.6,
114.3, 112.7, 110.3, 109.7, 44.4. IRνmax (cmÀ 1): 3278, 3087, 2228, 1457
HRMS (ESI) for C20H14BrN5 calculated [M+H]+ 404.0505, found
404.0511. UV (λmax nm) 228.
6c: Methyl 4-(((5-(5-bromo-1H-indol-3-yl)pyrimidin-2-yl)amino)
methyl)benzoate: Produced according to the general Tosyl depro-
tection (303 mg, 96%). White solid. 1H NMR (400 MHz, [D4]
methanol): δ=8.54 (s, 2H), 8.04–7.95 (m, 2H), 7.80 (d, J=1.8 Hz,
1H), 7.58–7.46 (m, 3H), 7.36 (d, J=8.6 Hz, 1H), 7.26 (dd, J=8.6,
1.9 Hz, 1H), 4.72 (s, 2H), 3.89 (s, 3H). 13C NMR (201 MHz, DMSO): δ=
4k: 4-(5-Bromo-1H-indol-3-yl)-N-(3-methoxybenzyl)pyrimidin-2-
amine: Produced according to the general method for 2-amino
pyrimidine formation (43 mg, 8%). Tan solid. 1H NMR (400 MHz, [D4]
methanol): δ=8.68 (d, J=2.0 Hz, 1H), 8.10 (d, J=5.5 Hz, 1H), 8.05 (s,
1H), 7.39–7.16 (m, 3H), 7.10–6.96 (m, 3H), 6.83–6.71 (m, 1H), 4.71 (s,
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166.6, 161.2, 156.3, 146.9, 135.8, 129.7, 128.4, 127.6, 127.2, 124.6,
124.5, 121.4, 118.5, 114.3, 112.6, 110.3, 52.5, 44.4. IRνmax (cmÀ 1): 3152,
2986, 1705, 1281 HRMS (ESI) for C21H17BrN4O2 calculated [M+H]+
437.0608, found 437.0608. UV (λmax nm) 232.
compound was added from 100 mM stock samples in DMSO, such
that the compound concentration equaled the highest concen-
tration tested (200 μM). Samples were then aliquoted (200 μL) into
the first row of wells of a 96-well plate, with all remaining wells
being filled with 100 μL of initial bacterial subculture. Row 1 wells
were mixed five times before 100 μL was transferred to the
following row (row 2). Row 2 was then mixed five times, and 100 μL
was transferred to row 3. This process provided a serial dilution of
the compound and was continued until the final row had been
mixed. Plates were covered with Glad Press n’ Seal and were
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4
5
6
7
8
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6d:
5-(5-Bromo-1H-indol-3-yl)-N-(4-chlorobenzyl)pyrimidin-2-
amine: Produced according to the general Tosyl deprotection
1
(345 mg, 92%). White solid. H NMR (400 MHz, [D4]methanol): δ=
8.53 (s, 2H), 7.80 (d, J=1.8 Hz, 1H), 7.48 (s, 1H), 7.42–7.23 (m, 6H),
4.63 (s, 2H). 13C NMR (101 MHz, DMSO): δ=160.7, 155.8, 139.7,
135.3, 131.0, 128.9, 128.1, 126.7, 124.1, 124.1, 121.0, 118.0, 113.9,
112.2, 109.9, 43.5. IRνmax (cmÀ 1): 3421, 3104, 3005, 624 HRMS (ESI) for
C19H14BrClN4 calculated [M+H]+ 413.0163, found 413.0153. UV (λmax
nm) 220.
°
incubated under stationary conditions at 37 C for 48 h. After 48 h,
the wells were stained with 10 μL of alamarBlue (Thermo-Fisher)
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°
and were incubated under stationary conditions at 37 C for
8 hours. After 8 h, the wells with viable bacteria were visualized as
pink and the wells with no viable bacteria were visualized as blue.
MIC values were recorded as the lowest concentration at which no
viable bacteria were observed.
6e:
5-(5-Bromo-1H-indol-3-yl)-N-(4-fluorobenzyl)pyrimidin-2-
amine: Produced according to the general Tosyl deprotection
(320 mg, 86%). White solid. H NMR (400 MHz, [D4]methanol): δ=
1
8.54 (s, 2H), 7.81 (d, J=1.9 Hz, 1H), 7.49 (s, 1H), 7.46–7.33 (m, 3H),
7.28 (dd, J=8.6, 1.9 Hz, 1H), 7.11–6.99 (m, 2H), 4.63 (s, 2H). 13C NMR
(201 MHz, [D6]DMSO): δ=161.80 (d, J=242.3 Hz), 156.0, 135.8,
135.3, 129.8, 129.8 (d, J=8.0 Hz), 126.8, 125.8, 124.8, 121.5, 118.6,
115.6 (d, J=21.2 Hz), 114.5, 113.0, 108.2, 44.0. IRνmax (cmÀ 1): 3469,
3165, 2919, 1213 HRMS (ESI) for C19H14BrFN4 calculated [M+H]+
397.0459, found 397.0472. UV (λmax nm) 228.
Broth microdilution method for antibiotic re-sensitization: Over-
night cultures of bacteria in cation adjusted Mueller-Hinton Broth
(CAMHB) were subcultured to 5×105 CFU/mL in CAMHB. Aliquots
(5 mL) were placed in culture tubes and dosed with compound
from stock solutions to give concentrations of 30% of the MIC of
the compound against the particular bacterial strain. One milliliter
of the resulting solution was placed in a separate culture and dosed
with antibiotic at the highest concentration to be tested. Bacteria
treated with antibiotic alone were used as the control. Row 1 of a
96-well plate was filled with 200 μL of the antibiotic/adjuvant, and
rows 2–8 were filled with 100 μL each of the remaining 4 mL of
bacterial subculture. Row 1 was then mixed five times, and 100 μL
was transferred to row 2, which was then mixed five times before
being transferred to row 3. This process was repeated until all rows
had been mixed, except for row 8, which would have only
compound, to serve as a control. The 96-well plate was then
covered in Glad Press n’ Seal and incubated under stationary
General biological procedures: M. smegmatis strain (ATCC 700084,
mc2155) was obtained from ATCC (Manassas, VA). A. baumannii
strain 4106 was obtained from Walter Reed Army Institute of
Research, K. pneumoniae strain B9 was obtained from Prof. Robert
Ernst at The University of Maryland, Baltimore. Stock cultures were
stored as glycerol stocks (25% v/v glycerol and 7H9 (BD cat#
271310), ADC (BD cat# 211887), Tween 80 (M. smegmatis) or 25% v/
°
v glycerol and LB Gram-negative strains) and maintained at À 80 C.
M. smegmatis was cultured in 7H9 and plated on 7H10 agar (OADC,
glycerol) Gram-negative strains were plated on LB (Lennox) agar.
M9 minimal salts media was purchased from BD Diagnostics (cat#
248510). Cation adjusted mueller hinton broth (CAMHB) was
purchased from BD Diagnostics (cat# 212322). Colistin sulfate was
purchased from Sigma (cat# C4461). 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 10
°
conditions at 37 C for 16 h. MIC values were determined as the
lowest concentration at which no bacterial growth was observed;
fold reductions were determined by comparison to control lane.
Biofilm inhibition assay: M. smegmatis was grown in 7H9 (ADC,
0.5% Tween 80) for 48 h and this culture was used to inoculate
Difco M9 minimal salts medium (OD600 =0.01) supplemented with
glucose (20% Sigma-Aldrich, 2 mL per 100 mL), MgSO4 (1 M, 200 μL
per 100 mL), and CaCl2 (1 M, 10 μL per 100 mL). 100 μL per well of
the subculture was aliquoted into the center two columns of a 96-
well PVC microtiter plate. Columns 1 and 12 served as negative
control wells. Then compound from DMSO stock solutions was
added to aliquots of the subculture to give the desired concen-
tration to be tested and aliquoted (100 μL per well) into the
remaining wells of the 96-well PVC microtiter plate. Sample plates
were then wrapped in GLAD Press n’Seal and were incubated under
°
or 100 mM stock solutions and stored at À 20 C.
Broth microdilution method for MIC determination (A. baumannii,
K. pneumoniae): Overnight cultures of bacterial strain were
subcultured to 5×105 CFU/mL in cation-adjusted Mueller–Hinton
medium. The resulting bacterial suspension was aliquoted (1.0 mL)
into culture tubes. Samples were prepared from these culture tubes
containing either 200 μmol of specified antibiotic or no test
compound as a control. Samples were then aliquoted (200 μL) into
the first row of wells of a 96-well microtiter plate in which
subsequent wells were prefilled with 100 μL of Mueller–Hinton
medium based 5×105 CFU/mL bacterial subculture. Using the
multichannel pipettor set at 100 μL, row 1 wells were mixed 8–10
times. Then, 100 μL were withdrawn and transferred to row two.
Row 2 wells were mixed 8–10 times followed by a 100 μL transfer
from row 2 to row 3. This procedure was used to serial dilute the
rest of the rows of the microtiter plate. The microtiter plate sample
was then covered with a microtiter plate lid and then placed in a
covered plastic container. The chamber was incubated under
°
stationary conditions for 48 h at 37 C. After incubation, the
medium was discarded, and the plates were washed thoroughly
with water. The wells were stained with 110 μL of a 0.1% solution
of crystal violet and then left at ambient temperature for 30 min.
Crystal violet solution was discarded and plates washed thoroughly
with water. 200 μL of 95% ethanol was added to each well, and the
plates left covered at ambient temperature for 10 min. 125 μL of
the ethanol solution was transferred to a polystyrene microtiter
plate. Biofilm inhibition was quantified by measuring the OD540 of
each well. The values obtained from the two negative control lanes
were subtracted from the OD540 of the other columns.
°
stationary conditions at 37 C. After 16 h, the lid was removed, and
MIC values were recorded.
Broth microdilution method for MIC determination of M.
smegmatis Cultures: (ATCC 700084, mc2155; 48 h) were stand-
ardized to 5×105 CFU/mL in 7H9 broth supplemented with ADC
enrichment. Aliquots (1 mL) were placed in culture tubes, and
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doi.org/10.1002/cmdc.202000438
ChemMedChem
[5] D. F. Ackart, L. Hascall-Dove, S. M. Caceres, N. M. Kirk, B. K. Podell, C.
Acknowledgements
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The authors would like to thank the National Institutes of Health
(AI106733 and AI136904) for funding.
[6] World Health Organization, Global Tuberculosis Report, 2019.
[7] K. E. Cox, C. Melander, MedChemComm 2019, 10, 1177–1179.
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Rep. 2019, 9, 1513.
[9] A. B. Jeon, A. Obregon-Henao, D. F. Ackart, B. K. Podell, J. M. Belardinelli,
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B. K. Johnson, R. B. Abramovitch, R. J. Basaraba, PLoS One 2017, 12,
e0180925.
Conflict of Interest
Dr. Christian Melander is the co-founder of Agile Sciences Inc.,
which focuses on harnessing small molecules to combat
biofilms and antibiotic resistance.
[10] S. M. Brackett, K. E. Cox, S. L. Barlock, W. M. Huggins, D. F. Ackart, R. J.
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Keywords: Adjuvants · antibiotic resistance · biofilms · colistin ·
Gram-negative bacteria
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Manuscript received: June 18, 2020
Accepted manuscript online: July 14, 2020
Version of record online: ■■■, ■■■■
[4] R. J. Worthington, J. J. Richards, C. Melander, Org. Biomol. Chem. 2012,
10, 7457–74.
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Providing backup: Structure-activity
relationship studies were performed
on an analogue of the natural
product meridianin D. Members of
the library exhibited increased inhibi-
tory activity against M. smegmatis
biofilms as well as the ability to po-
tentiate colistin against two clinically
relevant polymyxin-resistant bacterial
strains.
M. J. Zeiler, Prof. R. J. Melander, Prof. C.
Melander*
1 – 9
Second-Generation Meridianin
Analogues Inhibit the Formation of
Mycobacterium smegmatis Biofilms
and Sensitize Polymyxin-Resistant
Gram-Negative Bacteria to Colistin
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