Full text of DOI:10.1039/d0md00238k

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RESEARCH ARTICLE
A scaffold hopping strategy to generate new aryl-
2-amino pyrimidine MRSA biofilm inhibitors†
Cite this: DOI: 10.1039/d0md00238k
Alexander W. Weig,a Samantha L. Barlock,a Patrick M. O'Connor,a
Orry M. Marciano,a Richard Smith,b Robert K. Ernst,b
a
Roberta J. Melandera and Christian Melander
*
Infections that stem from bacterial biofilms are difficult to eradicate. Within a biofilm state, bacteria are
upwards of 1000-fold more resistant to conventional antibiotics, necessitating the development of
alternative approaches to treat biofilm-based infections. One such approach is the development of small
molecule adjuvants that can inhibit/disrupt bacterial biofilms. When such molecules are paired with
conventional antibiotics, these dual treatments present a combination approach to eradicate biofilm-based
infections. Previously, we have demonstrated that small molecules containing either a 2-amino pyrimidine
(2-AP) or a 2-aminoimidazole (2-AI) heterocycle are potent anti-biofilm agents. Herein, we now report a
scaffold hopping strategy to generate new aryl 2-AP analogs that inhibit biofilm formation by methicillin-
resistant Staphylococcus aureus (MRSA). These molecules also suppress colistin resistance in colistin
resistant Klebsiella pneumoniae, lowering the minimum inhibitory concentration (MIC) by 32-fold.
Received 9th July 2020,
Accepted 15th November 2020
DOI: 10.1039/d0md00238k
rsc.li/medchem
(planktonic) counterparts. Such factors include protection
imparted by the biofilm matrix itself, higher cell density, and
Introduction
Enterococcus faecium, Staphylococcus aureus, Klebsiella
pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa,
and the Enterobacter species are opportunistic bacteria
dubbed the ESKAPE pathogens and are notorious for their
impact on human health.1 Multi-drug resistant isolates of
these pathogens pose some of the most serious health
threats, as a result of a diminishing number of treatment
options.1 Of the ESKAPE pathogens, methicillin resistant S.
aureus (MRSA) remains one of the highest in terms of
infections and mortality, and is responsible for approximately
80 000 severe infections, which result in over 10 000 deaths
annually in the United States alone.2
varying metabolic rates of cells within the biofilm.3–6
Biofilms are typically present in chronic, difficult/impossible
to treat infections including lung infections of cystic fibrosis
(CF) patients, infections of indwelling medical devices, as
well as chronic and diabetic wounds. For this reason,
inhibiting biofilm formation and/or dispersing preformed
biofilms is one approach to aid in combatting these
recalcitrant infections.
Although the development of small molecules with anti-
biofilm activity is an attractive approach to fighting chronic
bacterial infections, there are still relatively few chemical
scaffolds that have been developed that are effective at
modulating the bacterial biofilm life cycle.7 Our group,
among others, has disclosed a handful of structures with
such activities.8 Many of our earlier reported scaffolds
focused on functionalized 2-aminoimidazoles (2-AIs), seen in
compound 1 (Fig. 1), to harness structural similarities to
marine alkaloid natural products that display anti-biofilm
activity against marine biofilms (commonly referred to as
Outside of acquired resistance mechanisms, one way in
which many bacteria evade the action of antibiotics is by
adopting
a
biofilm phenotype. Biofilms are surface-
associated communities of microorganisms surrounded by
an extracellular matrix made up of biomolecules called the
extracellular polymeric substance (EPS). As a result of several
factors, bacterial cells within a biofilm exhibit up to 1000-
fold increased tolerance to antibiotics than their free-floating
a Department of Chemistry and Biochemistry, University of Notre Dame, Notre
Dame, IN 46556, USA. E-mail: cmelande@nd.edu
b Department of Microbial Pathogenesis, University of Maryland-Baltimore,
Baltimore, MD 21201, USA
† Electronic supplementary information (ESI) available: Synthetic methods,
compound characterization, and assays methods. See DOI: 10.1039/d0md00238k
Fig.
1 Previous biofilm life cycle modulating molecules and new
scaffold design.
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microfouling). Analogues of 1 were shown to inhibit the
formation of Escherichia coli and P. aeruginosa biofilms.9
Later
studies
demonstrated
that
derivatized
2-aminopyrimidines (2-AP), as seen in compounds 2 and 3
(Fig. 1) inhibited the formation of MRSA biofilms10,11 In an
initial study, compound 2 was the most active derivative,
displaying an IC50 of 72 μM (where the IC50 is defined as the
concentration at which a compound inhibits 50% of biofilm
formation compared to untreated bacteria).10 Recently, we
posited that the meridianins, a family of natural products
that contain a 2-AP subunit, possessed anti-biofilm activity.
To test this hypothesis, we synthesized a family of meridianin
D derivatives and discovered that compound 3 (Fig. 1)11
inhibited the formation of MRSA biofilms and surprisingly
suppressed colistin resistance in colistin resistant Gram-
negative bacteria. Given the activity of these 2-AP analogs, we
hypothesized that a scaffold hopping approach centered
around replacing the 2-AI of 1 with a 2-AP would deliver
compounds with anti-biofilm activity and/or the ability to
suppress colistin resistance. By replacing the 2-AI with the
2-AP we remove a hydrogen bond donating group and
increase the size of the ring. Additionally, we change the
placement and identity of the aromatic rings attached to the
2-AP which proved effective on compound 3. In order to
probe the SAR of this proposed class of aryl 2-AP analogs, we
synthesized a library of compounds utilizing six distinct
orientations: ortho, meta, and para substituted phenyl core
with either C-4 or C-5 attachment to the pyrimidine head
group (4, Fig. 1).
Scheme 2 Reagents and condition for synthesis of compound 14: (a)
Boc-anhydride, DMAP, DCM, 18 h; (b) H2, Pd/carbon, rt, 16 h; (c)
3,5-dichlorobenzoyl chloride, K3PO4, dry THF, 0 °C to rt, 10 h; (d) TFA,
DCM (e)HCl/MeOH.
reacted with DMF/dimethylformamide-dimethylacetal (DMF-
DMA) to generate their corresponding vinylogous amide,
which underwent cyclization with guanidine hydrochloride to
afford the C-4 substituted 2-AP nitro intermediates 6a–c.
Reduction of the nitro group followed by coupling with the
appropriate derivatized benzoyl chloride in the presence of
tri-basic potassium phosphate allowed access to the aryl 2-AP
analogs. After purification, each 2-AP analog (8a–h) was then
converted to the corresponding HCl salt for biological testing.
C-5 linked derivatives were synthesized using the route
depicted in Scheme 1B. Each boronic acid (10a–c) was cross-
coupled with 2-amino-5-bromopyrimidine to afford 2-AP
derivatives 11a–c. Reduction, coupling, and conversion to the
HCl salt as above generated target analogs 13a–e.
While this synthetic route was used for the majority of
analogues, initially we attempted to mask the reactivity of the
exocyclic pyrimidine amine using Boc protection. Although this
ultimately proved unnecessary, five analogues (compounds 14,
and 15a–d) were synthesized via this lengthier route that
involved protection and deprotection steps. To synthesize the
C-4 linked 2-AP with a 3,5-dichlorobenzoyl tail (14, Scheme 2),
intermediate 6c was di-Boc protected using Boc-anhydride and
DMAP. Following Boc-protection, reduction, acylation, and
purification proceeded smoothly. The intermediate 2-AP was
then deprotected using TFA in DCM and converted to its
corresponding HCl salt for testing.
Results and discussion
Synthesis
The synthetic approach to access C-4 linked 2-AP derivatives
is outlined in Scheme 1A. Each acetophenone (5a–c) was
The four remaining compounds were accessed using the
synthetic approach summarized in Scheme 3. 2-Amino-5-
bromopyrimidine (9) was Boc protected using Boc anhydride
and pyridine. Following Boc protection, a Suzuki reaction
was carried out as before with either 2- or 3-nitroboronic
Scheme 1 Reagents and conditions for general synthesis: A: (a) DMF-
DMA, 110 °C 4 h; (b) guanidine hydrochloride, 2-methoxyethanol,
reflux, 16 h; (c) H2, Pd/carbon, rt, 16 h; (d) ArCOCl, K3PO4, dry THF, 0
°C to rt, 10 h; (e) HCl/MeOH; B: (a) PdCl2(PPH3)2, Na2CO3, dry THF,
reflux 20 h; (b) H2, Pd/carbon, rt, 16 h; (c) ArCOCl, K3PO4, dry THF, 0
°C to rt, 10 h; (d) HCl/MeOH.
Scheme 3 Reagents and condition for synthesis of compounds 15a–d:
(a) Boc-anhydride, pyridine 18
h (b) boronic acid, PdCl2(PPh3)2,
Na2CO3, dry THF, reflux 20 h; (c) H2, Pd/carbon, rt, 16 h; (d)
corresponding benzoyl chloride, K3PO4, dry THF, 0 °C to rt, 10 h; (e)
TFA, DCM (f) HCl/MeOH.
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Table 1 IC50's of aryl 2-APs against MRSA 43300 biofilms
Compound
8a
8b
8c
8d
8e
8f
IC50 (μM)
>200
>200
>200
>50
26.4 4.9
>200
Compound
8g
8h
13a
13b
13c
13d
IC50 (μM)
17.4 6.4
>50
>200
>200
>200
43 8.3
Compound
13e
14
15a
15b
15c
15d
IC50 (μM)
>100
41 12
>200
>100
>200
69 16
acid. With 2-nitro boronic acid, we noted that both Boc
groups remained intact after cross-coupling, while reaction
with 3-nitroboronic acid resulted in cleavage of one of the
Boc groups. Following reduction of the nitro-groups to their
corresponding anilines, acylation was carried out as before.
Finally, each 2-AP analog was deprotected using TFA in DCM,
that, following salt exchange, delivered compounds 15a–d
(Scheme 3).
are active against multiple MRSA strains. Following this, we
sought to determine whether these compounds were
specifically inhibiting biofilm formation, and not simply
inhibiting planktonic growth under the conditions of the
biofilm assay. To that end, we constructed time kill curves
using strain 43 300 as our test bacterium and compared
growth in the absence and presence of compounds 8e and 8g
at their IC50 concentration (Fig. S1†). Growth curves in the
absence and presence of either compound were similar,
establishing that these compounds are not toxic to
planktonic bacteria and could potentially demonstrate a
lower frequency of resistance.
Biological evaluation
All compounds were first evaluated for their ability to inhibit
MRSA biofilms. Initially, each compound was tested at 200
μM against MRSA (strain ATCC 43300) and inhibition was
measured using a crystal violet assay as previously reported.12
Compounds that exhibited greater than 50% inhibition at
200 μM were then subjected to a dose response assay to
determine their IC50 values (Table 1).
From the 18-compound pilot library, only five analogues
had IC50's less than 100 μM (Table 1). Two of these analogues
contained the 3,5-dichloro tail while the other three
compounds possessed the 3,5-dibromo tail. The highest
activity was observed when the 2-AP was substituted at C-4
(versus the C-5) and the amide linker was positioned meta to
the 2-AP (8e and 8g). These two compounds displayed IC50's
of 26.4 4.9 μM (8e) and 17.4 6.4 μM (8g). Compounds 14,
Next, we screened our compounds for their ability to
disperse preformed biofilms. None of the 18 compounds
demonstrated any dispersion activity against MRSA ATCC
43300 at a concentration of 100 μM.
Due to the activity of the meta 3,5-dichloro (8e) and the
meta 3,5-dibromo (8g) analogues, we elected to construct and
screen a hybrid analogue containing one chloro and one
bromo substituent. To access this derivative, we acylated
compound 7b with 3-bromo-5-chlorobenzoic acid (16) under
standard EDC coupling conditions. Purification and
conversion to the corresponding HCl salt as previously
described yielded compound 17 (Scheme 4). With this
compound in hand, we first assessed the MIC of the
compound and determined it to be >200 μM. We then
evaluated its biofilm inhibition properties and determined
that its IC50 was 27.6 9.6 μM, which is within the error of
both 8e and 8g.
Finally, having previously shown that analogs of the
2-aminopyrimidine-containing marine sponge natural
product meridianin D potentiated colistin in Gram-negative
bacteria, we probed whether these compounds also
potentiated colistin. To that end, we first determined the
minimum inhibitory concentration (MIC) of each compound
against A. baumannii (AB 4106) and K. pneumoniae (KP B9),
both of which have chromosomally encoded colistin
resistance. All 18 compounds registered an MIC of ≥200 μM.
We then screened each compound at 60 μM in combination
13d and 15d all displayed moderate activity. Analog 14 (IC50
=
41 12 μM) possessed a 3,5-dichloro tail placed at the para
position of the phenyl core and substituted at C-4 of the
2-AP. Compound 13d (IC50 = 43 8.3 μM) had a 3,5-dibromo
tail placed at the meta position of the phenyl core and
substituted at C-5 of the 2-AP. Finally, 15d (IC50 = 69 16
μM) had the 3,5-dibromo tail placed at the ortho position of
the phenyl core and substituted at C-5 of the 2-AP.
We next tested the two most active compounds, 8e and 8g,
for their anti-biofilm activity against additional MRSA strains,
as well as a methicillin susceptible S. aureus strain. Both
compounds had comparable IC50's across each of the four
strains tested (Table 2) demonstrating that these compounds
Table 2 IC50's of lead aryl 2-AP analogues against panel of S. aureus strains biofilms
Compound
MRSA 43300 (μM)
MRSA BAA-44 (μM)
MRSA 33591 (μM)
S. aureus 6538 (μM)
8e
8g
26.4 4.9
17.4 6.4
29.5 2.1
14.1 2.2
26.6 4.1
20.2 3.0
24.9 4.2
15.6 6.4
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would be little to no lysis of eukaryotic cells when dosed at
a lower concentration.
Conclusions
In conclusion, we have employed a scaffold hopping strategy
to develop compounds with anti-biofilm activity against
MRSA. While the lead compound 8g shows comparable
inhibitory activity with previously reported compounds, none
of the disclosed compounds were able to disperse preformed
biofilms. The lead compound 8g also suppressed resistance
to the polymyxin antibiotic colistin in K. pneumoniae,
encouraging future structural derivatizations that could
increase this activity. We determined that the specific
connectivity between the 2-AP head, the phenyl core, and the
aryl tail has a significant effect on activity, and studies to
diversify this scaffold further are underway.
Scheme 4 Reagents and condition for synthesis of compound 17: (a)
EDC, DMAP, DCM, 18 h (b) HCl/MeOH.
with colistin to determine the effect on colistin activity. The
MIC of colistin was 1024 and 512 μg mL−1 against AB 4106
and KP B9, respectively, as previously reported.11,13
None of the compounds lowered the MIC of colistin
against AB 4106 below 64 μg mL−1 at 60 μM. However, one
compound from this pilot library, compound 8g, did lower
the MIC of colistin from 512 to 16 μg mL−1 against KP B9, a
32-fold reduction. Interestingly, compound 8g was also the
most active MRSA biofilm inhibitor out of the 18-compound
library. With antibiotic-adjuvants, we look for adjuvants that
can lower the MIC of an antibiotic to its breakpoint against a
bacterial strain. The colistin breakpoint in AB 4106 and KP
B9 is 2 μg mL−1; therefore, further diversification of this
scaffold may allow for a new class of molecules that can
potentiate colistin activity.
To determine if 8g exhibits synergy with colistin, we
performed checkerboard assays with compound 8g and
colistin in KP B9, and observed a fractional inhibitory
concentration index (FICI) of ≤0.09 (Table S3†). We also
performed the checkerboard assay in two additional strains
of K. pneumoniae; A5, an additional highly colistin resistant
clinical isolate, and F2210219mcr-1, an engineered strain
harboring a plasmid containing the mcr-1 gene.14 In both
strains, compound 8g exhibited synergy with colistin,
returning FICI values of ≤0.31 and ≤0.19 for A5 and
F2210219mcr-1 respectively (Table S3†).
The authors would like to thank the National Institutes of
Health (AI136904 and DE022350) for support.
Conflicts of interest
Dr. C. Melander is
a co-founder of Agile Sciences, a
biotechnology company seeking to commercialize antibiotic
adjuvants.
Notes and references
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7 J. J. Richards and C. Melander, ChemBioChem, 2009, 10(14),
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8 Z. Yan, M. Huang, C. Melander and B. V. Kjellerup, J. Appl.
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9 C. A. Bunders, J. J. Richards and C. Melander, Bioorg. Med.
Chem. Lett., 2010, 20(12), 3797–3800.
Resistance to colistin typically involves modification of the
lipid A anchor of the lipopolysaccharide (LPS) of Gram-
negative bacteria. To begin probing the mechanism of action
for this decrease in colistin resistance we analyzed lipid A
extracted from KP B9 grown in the absence and presence of
8g via mass spectrometry. No change in the lipid
substitution pattern was noted between the two samples,
indicating that these compounds potentiate colistin through
A
10 E. A. Lindsey, R. J. Worthington, C. Alcaraz and C. Melander,
Org. Biomol. Chem., 2012, 10(13), 2552–2561.
11 W. M. Huggins, W. T. Barker, J. T. Baker, N. A. Hahn, R. J.
Melander and C. Melander, ACS Med. Chem. Lett., 2018, 9(7),
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12 G. A. O'Toole and R. Kolter, Mol. Microbiol., 1998, 30(2), 295–304.
13 A. M. Nemeth, A. K. Basak, A. W. Weig, S. A. Marrujo, W. T.
Barker, L. A. Jania, T. A. Hendricks, A. E. Sullivan, P. M.
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a
mechanism not dependent upon reversing lipid A
modification. Further investigation into the mechanism of
action is ongoing.
Finally, we tested the hemolytic activity of compounds
8g and 8e by performing
a
hemolysis assay using
defibrinated sheep's blood challenged with either
compound. Triton-X (1%) used as the 100% lysis marker,
and phosphate buffered saline as the 0% lysis marker.
Compounds 8e and 8g only lysed 5% and 3.4% of cells
respectively when dosed at 200 μM, a concentration higher
than the IC50 of either compound suggesting that there
14 Y. Y. Liu, C. E. Chandler, L. M. Leung, C. L. McElheny, R. T.
Mettus, R. M. Shanks, J. H. Liu, D. R. Goodlett, R. K. Ernst
and Y. Doi, Antimicrob. Agents Chemother., 2017, 61(6),
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