Meridianin D Analogues Display Antibiofilm Activity against MRSA and Increase Colistin Efficacy in Gram-Negative Bacteria

Article abstract of DOI:10.1021/acsmedchemlett.8b00161

In the last 30 years, development of new classes of antibiotics has slowed, increasing the necessity for new options to treat multidrug resistant bacterial infections. Development of antibiotic adjuvants that increase the effectiveness of currently available antibiotics is a promising alternative approach to classical antibiotic development. Reports of the ability of the natural product meridianin D to modulate bacterial behavior have been rare. Herein, we describe the ability of meridianin D to inhibit biofilm formation of methicillin-resistant Staphylococcus aureus (MRSA) and to increase the potency of colistin against colistin-resistant and sensitive Gram-negative bacteria. Analogues were identified that are capable of inhibiting and dispersing MRSA biofilms and lowering the colistin MIC to below the CLSI breakpoint against Acinetobacter baumannii, Klebsiella pneumoniae, and Escherichia coli.

Full text of DOI:10.1021/acsmedchemlett.8b00161

Subscriber access provided by UNIV OF CAMBRIDGE
Letter
Meridianin D analogues display antibiofilm activity against
MRSA and in-crease colistin efficacy in Gram-negative bacteria
William M. Huggins, William T Barker, James T Baker,
Nicholas A Hahn, Roberta J. Melander, and Christian Melander
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.8b00161 • Publication Date (Web): 25 May 2018
Downloaded from http://pubs.acs.org on May 27, 2018
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 7
ACS Medicinal Chemistry Letters
1
2
3
4
5
6
7
8
9
Meridianin D analogues display antibiofilm activity against MRSA, in-
crease colistin efficacy in Gram-negative bacteria
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
William M. Hugginsa, William T. Barkera, James T. Bakera, Nicholas A. Hahna, Roberta J. Melandera, and Chris-
tian Melandera*
Department of Chemistry, North Carolina State University, 2620 Yarbrough Drive, Raleigh, NC 27695, USAa
KEYWORDS: antibiotic resistance, antibiotics, biofilms, antibiotic repotentiation, antibiotic adjuvants
ABSTRACT: In the last 30 years, development of new classes of antibiotics has slowed, increasing the necessity for new options to treat mul-
ti-drug resistant bacterial infections. Development of antibiotic adjuvants that increase the effectiveness of currently available antibiotics is a
promising alternative approach to classical antibiotic development. Reports of the ability of the natural product meridianin D to modulate
bacterial behavior have been rare. Herein, we describe the ability of meridianin D to inhibit biofilm formation of methicillin-resistant Staphy-
lococcus aureus (MRSA) and increase the potency of colistin against colistin-resistant and sensitive Gram-negative bacteria. Analogues were
identified that are capable of inhibiting and dispersing MRSA biofilms and lowering the colistin MIC to below the CLSI breakpoint against
Acinetobacter baumannii, Klebsiella pneumoniae, and Escherichia coli.
Antibiotic resistance is quickly becoming one of the larg-
est threats to human health. Without the development of new
strategies to defeat antibiotic resistance it is predicted that 10 mil-
lion people will die from multi-drug resistant (MDR) bacterial
infections by 2050.1 Compounding the problem, resistant isolates
to the two classes of antibiotics introduced most recently in the
clinic, cyclic lipopeptides and oxazolidinones, were observed within
five years of clinical use.2 An alternative strategy to new antibiotics
is to develop adjuvants that intercept the pathways responsible for
resistance to clinically relevant antibiotics. Bacteria are capable of
avoiding antibiotic treatment in many ways, including biofilm for-
mation. Biofilms are highly organized surface-associated communi-
ties that are encased in an extra-cellular polymeric substance (EPS).
Bacteria within a biofilm are upwards of 1000-fold more resistant to
antibiotic treatment and reach a higher cell density than their
planktonic counterparts, increasing the chances of horizontal gene
transfer.3,4 In many cases, bacteria also evade antibiotic treatment
by acquiring resistance elements in small gene vectors. One exam-
ple is the spread of the plasmid-borne mobile colistin resistance-1
(mcr-1)5,6 gene, which likely evolved from overuse of colistin as a
food additive in animal husbandry. Selective pressure has also been
applied by the resurgence of colistin treatment clinically as an anti-
biotic of last resort in MDR Gram-negative bacterial infections.7
The emergence of these colistin resistant strains brings us closer to
a post-antibiotic world., exemplified when a MDR strain of Klebsiel-
la pneumoniae was observed clinically in 2016 that was resistant to
all clinically available antibiotics.8
were first reported in 1998 after being isolated from the marine
invertebrate Aplidium meridianum near the South Georgia Islands.9
The family of meridianins and their respective derivatives have
shown diverse biological activities including kinase inhibition10,11
,
adipogenesis inhibition12, antitumor activity13, and anti-malarial
activity.14 Reports of antibacterial activity of these compounds,
however, have been scarce and fragmentary. These reports have
been limited to antimicrobial activity against Staphylococcus
aureus14, Mycobacterium tuberculosis15 and an unidentified sympatric
marine Antarctic bacterium.16 Despite these limited reports, we
posited that the meridianins and their analogues would possess the
ability to control bacterial behavior based on the shared structural
features of the meridianins with the desformylflustrabromine
(dFBr)17, 2-aminopyrimidine (2-AP)18, and oroidin19,20 analogues
that were previously shown by our group to possess antibiofilm
activity against methicillin-resistant S. aureus (MRSA) (2 and 3)
and the ability to lower the colistin minimum inhibitory concentra-
tion (MIC) against Gram-negative bacterial pathogens carrying the
mcr-1 plasmid (4) (Figure 1). Herein, we report the antibiofilm and
antibiotic activity of analogues based on the meridianin scaffold
against S. aureus. Moreover, we report the ability of meridianin
analogues to lower the MIC of colistin against both colistin-
sensitive and resistant strains of Gram-negative bacteria.
H2N
R
N
HN
O
H2N
N
N
N
H
N
HN
Cl
N
H
R
N
H
Br
Cl
N
Br
H
Natural products from marine sponges have long been a
rich source of molecules that display a myriad of biological activi-
ties. The meridianins are one such example of a family of structur-
ally related marine natural products. These secondary metabolites
1
2
3
4
Figure 1. Structures of meridianin
formylflustramine analogues (compound 2), 2-AP analogues
D (compound 1), des-
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 2 of 7
(compound 3), and oroidin analogue (compound 4) previously
shown to control bacterial behavior.
chloride or commercially available substituted guanidine deriva-
tives yielded compounds 1, 9a-o.
1
2
Compounds were first assessed for their ability to inhibit
MRSA biofilm formation. Meridianin D (1) returned an IC50 value
of 87.4 4.0 µM (Table 1), where the IC50 value is defined as the
concentration at which a compound inhibits 50% of biofilm for-
mation. This result confirmed our hypothesis that the meridianin
natural products would be capable of inhibiting MRSA biofilm
formation. The 4-, 5-, and 7-bromo analogues were assayed to
investigate the effect that the substitution of the bromine atom had
on the compound’s antibiofilm activity. The 4-bromo (compound
9k) and 7-bromo (compound 9j) analogues displayed reduced
antibiofilm activity, with IC50 values of >100 and 99.8 15.2 µM
respectively. The 5-bromo analogue, compound 9a, displayed
increased biofilm inhibitory activity with an IC50 value of 17.9 2.2
µM.
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
After identifying the 5-bromo and 6-bromo analogues as
the most active derivatives, various substitutions on the indole ring
were prepared while preserving the 2-AP ring to probe the promis-
cuity of the indole substitution. Of these analogues, the debromo
analogue 9o, 5-methyl 9n, 5-fluoro 9l and 6-fluoro 9m analogues
all displayed no antibiofilm activity with IC50 values of >100 µM.
The 6-chloro analogue 8i displayed comparable activity to merid-
ianin D while the 5-chloro analogue 9h showed reduced activity
compared to the 5-bromo analogue (8b). Finally, the 6-iodo ana-
logue 9f displayed increased activity compared to meridianin D,
with an IC50 of 42.5 8.1 µM and the 5-iodo analogue 9g displayed
reduced activity compared to the 5-bromo analogue (9a), with an
IC50 of 49.3 5.1 µM.
Upon identifying compound 9a as the most active com-
pound from this series, the effect of alkylation of the exocyclic
amine of the 2-AP ring on the antibiofilm activity was investigated.
Cyclization with a substituted guanidine in place of guanidine hy-
drochloride in the final step of the synthesis yielded methyl (com-
pound 9e), ethyl (compound 9d), dimethyl (compound 9c) and
benzyl (compound 9b) analogues. Methyl (9e) and ethyl (9d)
substitutions on the 2-AP ring decreased activity, delivering com-
pounds with IC50’s of 28.9 2.3 and 24.6 0.7 µM respectively. The-
se substitutions also made these analogues more toxic to planktonic
bacterial growth with MICs of 100 µM for the methyl and 50 µM
for the ethyl, compared to 200 µM for the unsubstituted parent.
Dimethyl substitution (9c) of the 2-AP was still less active than the
unsubstituted 2-AP but showed similar activity compared to the
mono methyl substituted analogue, with an IC50 of 23.4 1.8 µM.
Placement of a benzyl group at the exocyclic amine of the 2-AP
ring, compound 9b, significantly increased the activity of the com-
pound, lowering the IC50 to 9.62 1.4 µM. The reduction in IC50 for
the benzyl analogue was coupled with a significant increase in tox-
icity, reducing the MIC from 200 µM for compound 9a to 6.25µM
for the benzyl substituted analogue 9b. Observing a significant
decrease in MIC, the antibiotic activity of compound 9b was ex-
plored against a small panel of Gram-positive pathogens. The ben-
zylated analogue returned an MIC value of 6.25 µM (2.60 µg/mL)
against two additional S. aureus isolates and an MIC of 25 µM (10.4
µg/mL) against a strain of vancomycin resistant Enterococcus faeci-
um (VRE). No significant difference in compound MIC value was
observed when compounds 9b, 9d, 9n and 9o were tested in the
presence or absence of 0.01% triton X-100 against MRSA 43300.
Scheme 1. Synthetic route to compounds 1, 9a-o (A) and 13a-m
(B): (a) Acetyl chloride, SnCl4, toluene, 0 °C to rt, 2 h; (b) p-
toluenesulfonyl chloride, triethylamine, 4-dimethylaminopyridine,
DCM, rt, 16 h; (c) DMF-DMA, 110 °C, 3 h; (d) Guanidine deriva-
tive, K2CO3, 2-methoxyethanol, reflux, 16 h (e) MeOH/HCl (f)
Dimethyl sulfate or R2X, 50% NaOH, DCM, 16 h; (g) 1-bromo-2-
methylpropane, K2CO3, acetone, reflux, 16 h; (h) pyrrolidine,
DMA, 80 °C, 1 h (i) DMF, 110 °C, 4 h.
To begin the structure-activity relationship (SAR) study
of the meridianin molecules, we synthesized meridianin D 1 to
establish its biological activity. Attempts to synthesize meridianin
D following the procedure described by Jiang et al.21 were unsuc-
cessful as problems described by Simon et al.22 were encountered.
Therefore, we applied the synthetic approach outlined by
Bredereck to access meridianin D and analogues (Scheme 1A).11, 22-
24
To begin, commercially available substituted indole derivatives
were acylated at the 3-position using acetyl chloride and tin chlo-
ride in toluene to yield compounds 6a-l. The indolic nitrogen of
compounds 6a-l was subsequently protected using p-
toluenesulfonyl chloride (TsCl), triethylamine, and 4-
dimethylaminopyrimidine (DMAP) in DCM. Next, the enami-
none derivatives were prepared by reacting compounds 7a-l with
DMF/dimethylformamide-dimethylacetal (DMF-DMA) at 110° C
for 4 hours. Cyclization and deprotection of the enaminone in 2-
methoxyethanol using potassium carbonate and guanidine hydro-
ACS Paragon Plus Environment

Page 3 of 7
ACS Medicinal Chemistry Letters
Table 1. IC50 and MIC values for compounds 1, 9a-g, 13a-f. All
values displayed against MRSA 43300. Full biofilm inhibition re-
sults can be found in the supporting information.
pyl derivative, compound 13a, displayed increased activity with an
IC50 of 34.3 6.8 µM. The butyl derivative, compound 13j, dis-
played no antibiofilm activity with an IC50 of >100 µM. Interesting-
ly, alkylation of the indole nitrogen of the 5-bromo analogues fol-
lowed a different activity trend than the 6-bromo analogues. Sub-
stitution with a methyl group, compound 13g, and n-pentyl group,
compound 13h, abolished antibiofilm activity (IC50’s >100 µM).
Ethyl and n-propyl derivatives, compounds 13e and 13f respective-
ly, showed decreased activity compared to the 5-bromo derivative
with a free indolic nitrogen with IC50 values of 66.2 7.6 µM and
69.3 3.6 µM respectively. The n-butyl derivative, compound 13b,
displayed an IC50 of 38.1 2.8 µM, but was more toxic than other
analogues with an MIC of 50 µM indicating that it may be acting
via a toxic mechanism to prevent biofilm formation. Branching of
the alkyl chains with isobutyl substitutions on both the 5-bromo
and 6-bromo analogues, compounds 13k and 13l respectively,
abrogated antibiofilm activity with both N-isobutyl derivatives both
displaying IC50 values of >100 µM. Next, the 6-iodo analogue,
compound 9f, was alkylated off the indolic nitrogen with a propyl
group to yield compound 13m. Again, it was observed that the
indolic alkyl substituent and halogen substitution of the indole did
not correlate to each other as compound 13m displayed no antibio-
film activity. Finally, concurrent alkylation of the indolic nitrogen
and the exocyclic 2-AP nitrogen, compound 13n, abolished all
antibiofilm activity.
1
2
3
4
5
6
7
8
9
MIC
Compound
R1
R2
R3
IC50 (µM)
(µM)
>200
200
6.25
100
50
1
6-Br
5-Br
5-Br
5-Br
5-Br
5-Br
6-I
H
H
H
H
H
Me
H
H
H
H
H
H
H
H
H
H
87.4 4.0
17.9 2.2
9.62 1.4
23.4 1.8
24.6 0.7
28.9 2.3
42.5 8.1
49.3 5.1
34.3 6.8
38.1 2.8
59.6 2.3
64.0 8.8
66.2 7.6
69.3 3.6
9a
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
9b
Bn
Me
Et
9c
9d
9e
Me
H
100
>200
200
200
50
9f
9g
5-I
H
13a
13b
13c
13d
13e
13f
6-Br
5-Br
6-Br
6-Br
5-Br
5-Br
n-Pr
n-Bu
Me
Et
With a panel of 2-AP analogues in hand, we turned our
200
200
200
100
interests towards replacement of the 2-AP ring with
a 2-
aminoimidazole (2-AI). Previously, 2-AIs have shown excellent
antibiofilm activity against a wide variety of pathogenic bacteria,
including MRSA.25-27 The synthesis of the 2-AI analogues proceed-
ed with the acylation of a substituted indoles 5a and 5l with chloro-
acetyl chloride in toluene (Scheme 2). The indolic nitrogen of
compounds 14a-b then were Boc protected to yield compounds
15a-b. Cyclization of the α-chloroketone with Boc guanidine and
sodium iodide in DMF yielded compounds 16a-b. Subsequent Boc
deprotection using TFA in DCM at 0° C for 16 hours delivered the
2-AI derivatives 17a-b. Interestingly, both compounds 17a-b were
toxic to planktonic bacterial growth at 60 µM under the conditions
of the biofilm inhibition assay and displayed no antibiofilm activity
below this concentration. Both compounds did display moderate
antimicrobial activity returning MICs of 25 µM (7.8 µg/mL)
against our test MRSA strain.
Et
Pr
With compounds 1, 9.5, and 9f established as leads dis-
playing minimal inherent toxicity, the effect of alkylation of the
indole nitrogen on antibiofilm activity was next interrogated. Syn-
thesis of compounds 13a-m was adapted from a previous disclo-
sure by Simon et al. (Scheme 1B).22 Acylation of a substituted in-
dole at the 3 position proceeded as previously reported. Com-
pounds l and 6a-b were then alkylated using dimethyl sulfate or the
desired alkyl halide with tetrabutylammonium bromide as a phase
transfer catalyst in a biphasic mixture of dichloromethane and 50%
NaOH. Alkylation with isobutylbromide failed under these condi-
tions and required compounds to be refluxed in acetone with iso-
butyl bromide and potassium carbonate to yield the desired prod-
uct. Compounds 10a-k, 11a-b were then transformed into enami-
nones 12a-m by stirring a mixture of DMA with pyrrolidine for 1
hour at 80° C followed by the addition of a solution of the appro-
priate n-alkylated acetyl indole (compounds 10a-k, 11a-b) dis-
solved in DMF and stirring the reaction at 110° C for 3 hours. Fi-
nally, cyclization of the enaminone with guanidine hydrochloride
or commercially available substituted guanidine derivatives pro-
ceeded in 2-methoxyethanol at reflux with potassium carbonate for
16 hours to yield compounds 13a-m. Methylation of the indolic
nitrogen of the 6-bromo derivative, compound 13c, improved the
IC50 value to 59.6 2.3 µM (Table 1) from 87.4 4.0 µM (com-
pound 1). Ethylation of the indole nitrogen, compound 13d, dis-
played no improvement compared to compound 13c, but the pro-
Scheme 2. Synthetic route to compounds 17a-b: (a) (i) Chloroa-
cetylchloride, toluene, 60 °C, 2 h (ii) MeOH, H2O, rt, 1 h (b) Boc-
anhydride, 4-dimethylaminopyridine, THF, rt,
4 h (c) Boc-
guanidine, sodium iodide, DMF, rt, 48 h (d) 30% Trifluoroacetic
acid, DCM, 0 °C to rt, 16 h (e) MeOH/HCl.
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Noting that various meridianin derivatives were capable
Page 4 of 7
ure 1). This combination successfully disarmed colistin resistance
in multiple strains carrying the mcr-1 plasmid-borne resistance
gene. To our knowledge, there is no precedent for indole-
containing compounds directly modulating polymyxin defense
pathways in Gram-negative pathogens. Holistically, both com-
pound 4 and meridianin D (compound 1) are small, indole-derived
compounds with an additional nitrogenous heterocyclic append-
age. Given their semblance, we postulated that compound 1 and its
analogues potentially had the ability to modulate colistin resistance
in Gram-negative bacteria.
1
2
3
4
5
6
7
8
of inhibiting MRSA biofilm formation, we next investigated wheth-
er they were capable of dispersing pre-formed MRSA biofilms.
Interestingly, the alkylated 2-AP analogues displayed the greatest
activity against pre-formed MRSA biofilms, with the methyl (com-
pound 9e) and ethyl (compound 9d) derivatives displaying EC50
values, the concentration at which a compound disperses 50% of a
preformed biofilm, of 73.1 2.4 and 75.8 5.8 µM respectively (Ta-
ble 2). It does not appear that the ability to disperse pre-formed
biofilms is related to increased toxicity, because the more toxic
compound 9b displayed only 26.2% dispersion at 80 µM. Interest-
ingly, dimethylation of the exocyclic amine (compound 9c) and
alkyl substituents on the indolic nitrogen (compounds 13a-b) did
not impart the ability to disperse pre-formed biofilms with all dis-
playing EC50 values of greater than 160 µM. Compound 9a re-
turned an EC50 value of 138.4 15.2 µM, demonstrating its ability to
inhibit and disperse MRSA biofilms.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 3. Colistin potentiation by select compounds against Gram-
negative bacteria, colistin MIC(fold reductions). Concentrations
are shown in µg/mL.
Colistin
Colistin+ Colistin+ Colistin+
MIC
1
9d
13f
(µg/mL)
After confirming the ability of multiple analogues of me-
ridianin D to disperse pre-formed MRSA biofilms, the most active
compounds (9a, d, and e) were tested for synergy with vancomycin
at a concentration in which vancomycin does not affect pre-formed
biofilms. This concentration was determined to be 19.0 µg/mL
(supplemental information) against MRSA 43300, which is 19
times its MIC. Accordingly, each compound was tested with 19
µg/mL of vancomycin to investigate whether the compounds
would show a synergistic effect with vancomycin and disperse pre-
formed biofilms at a lower concentration. Compound 9a displayed
a 33% reduction in EC50 value (Table 3) when combined with a
non-active concentration of vancomycin against pre-formed bio-
films. Other compounds tested that were capable of dispersing pre-
formed MRSA biofilms did not display a significant reduction in
EC50 value when combined with vancomycin.
E. coli
ATCC
8
4 (2)
0.5 (16)
2 (4)
25922mcr-1
E. coli
0.0625
(8)
0.03125
(16)
ATCC
0.5
16
0.5 (0)
25922parent
A. bau-
0.125
(128)
mannii
0.5 (32)
4 (4)
17978mcr-1
A. bau-
0.0625
(16)
0.0625
(16)
mannii
1
0.5 (2)
16 (64)
16 (64)
17978parent
A. bau-
1024
512
2 (512)
1 (512)
16 (64)
1 (512)
mannii 4106
K. pneu-
moniae B9
Table 2. EC50 values with and without vancomycin for active anti-
biofilm analogues. All values displayed against MRSA 43300
A. bau-
mannii 5075
0.0625
(16)
0.0078
(128)
1
1
0.25 (4)
0.5 (2)
EC50 (µM)+ van-
Compound
EC50 (µM)
comycin
92.1 17.5
71.4+6.0
66.5 7.5
>100
K. pneu-
moniae
0.0625
(16)
0.0625
(16)
9a
9e
138.4 15.2
73.1 2.4
75.8 5.8
101.5 0.5
101.0 3.9
>80
ATCC
2146NDM-1
9d
To this end, Acinetobacter baumannii ATCC 17978mcr-1
was chosen as a test strain.28 As summarized in table 3, we indeed
found activity with multiple meridianin D analogues demonstrating
synergy with colistin in a diverse panel of bacterial isolates com-
prised of both colistin-resistant and colistin-sensitive A. baumannii,
K. pneumoniae, and Escherichia coli. Of note, across all strains, all
analogues with the exception of compounds 13a, 17a, and 17b had
no inherent toxicity, with MICs of >200 µM. Accordingly, all ana-
logues were dosed at 60 µM unless otherwise noted (Supporting
Information).
17a
17b
9b
>100
>80
9c
>160
N/A
13a
13b
>160
N/A
Cross referencing activity in these eleven strains, limited
activity is seen in compounds with varied indole halogenation while
alkylation of the indolic nitrogen increased activity in colistin-
sensitive isolates. Alkylation of the exocyclic amine of the 2-AP
delivered compounds with enhanced synergistic activity against
colistin resistant strains while retaining synergy in colistin-sensitive
>160
N/A
In a recent disclosure,19 synergy was found in concomi-
tant treatment with colistin (polymyxin E) and compound 4 (Fig-
ACS Paragon Plus Environment

Page 5 of 7
ACS Medicinal Chemistry Letters
strains, with compound 9d being a nearly universal modulator.
Compound 9d outperforms all other analogues in both A. bau-
mannii 17978mcr-1 and E. coli 25922mcr-1 with 128 and 16-fold reduc-
tions in colistin MIC, respectively.
resistant primary clinical isolates of chromosomally encoded re-
sistance, strains of E. coli and A. baumannii harboring the mcr-1
gene, and a strain of K. pneumoniae carrying the New Dehli metallo-
beta-lactamase-1 (NDM-1) gene. Compounds 13a, 13b, 13f, and
13k which bear an alkylation on the indolic nitrogen displayed
equivalent or greater potentiation of colistin activity against some
strains, but did not display the broad-spectrum potentiation of
colistin activity of compound 9d. Mechanistic studies and addi-
tional structural modifications in an effort to increase activity of the
analogues are currently underway.
1
2
3
4
5
6
7
8
A. baumannii 3941/4106 and K. pneumoniae C3/B9 are
primary clinical isolates that contain genomically encoded colistin
resistance. Such strains typically possess much higher colistin MICs
than their mcr-1 counterparts. All four strains return colistin MICs
of 512 µg/mL or greater, well exceeding the 4 µg/mL Clinical &
Laboratory Standards Institute (CLSI) breakpoint. Compounds 9b
and 9d recover a breakpoint MIC in A. baumannii 3941, while
compound 9d delivers a 512-fold reduction in A. baumannii 4106,
reducing the MIC to 1 µg/mL. Colistin sensitivity is also re-
established in K. pneumoniae C3 by compounds 9b, 9d, and 9e, all
producing 512-fold reductions, and this same activity is seen in K.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ASSOCIATED CONTENT
Supporting Information
Compound characterization for all novel compounds, H and 13C
NMR spectra, biofilm, colistin repotentiation and full in-vitro test-
ing data. This material is available free of charge via the Internet at
http://pubs.acs.org.
1
pneumoniae B9 with compounds 9d, and 9e, and 13f.
No
significant difference in colistin MIC value was observed when
compounds 9b, 9d, 9n and 9o were tested in the presence or ab-
sence of 0.01% triton X-100 against A. baumannii 4106 and K.
pneumoniae B9.
AUTHOR INFORMATION
Corresponding Author
*(C.M.) E-mail: ccmeland@ncsu.edu. Phone: (919) 513-2960.
A select cohort of analogues were capable of increasing
colistin sensitivity in strains with no inherent resistance to the anti-
biotic. In the parent A. baumannii 17978 strain, 9d is equipotent
with 9b, 13f, 13b, and 13k, all generating a 16-fold reduction in
colistin MIC. A 16-fold reduction in parent strain E. coli 25922 is
observed by 13b, 13f, and 13k, while 9d shows an 8-fold reduction.
A total of six compounds (compounds 9b, 9d, 9e, 13a, 13b, and
13k) produced a colistin MIC of 0.0625 µg/mL (16-fold reduc-
tion) in A. baumannii ATCC 19606, while a more impressive 128-
fold reduction (0.0078 µg/mL) was achieved with compound 13f
in A. baumannii ATCC 5075. Compound 13f was equipotent with
compound 9d in K. pneumoniae ATCC 2146NDM-1, returning a final
MIC of 0.0625 µg/mL (16-fold reduction).
Author Contributions
The manuscript was written through contributions of all authors. All
authors have given approval to the final version of the manuscript.
Funding Sources
The authors thank the National Institutes of Health (GM055769) for
funding.
REFERENCES
(1)
Review on Antimicrobial resistance 2014.
(2) Clatworthy, A. E.; Pierson, E.; Hung, D. T., Targeting
O'Neill, J., The Review on Antimicrobial resistance. The
virulence: a new paradigm for antimicrobial therapy. Nat. Chem.
Biol. 2007, 3 (9), 541-8.
In conclusion, after identifying the potential of the natu-
ral product meridianin D (compound 1) to control MRSA biofilm
formation, a panel of analogues were synthesized in an effort to
augment activity. Structural modification of the meridianin D core
delivered molecules that modified both Gram-positive and Gram-
negative bacterial defense mechanisms. In many cases, modifica-
tion of key positions of the scaffold amended bacterial behavior in
divergent manners. Of note, compound 9a inhibited and dispersed
MRSA biofilms as a stand-alone treatment. Furthermore, the EC50
of compound 9a was reduced by 33.4% in concomitant treatment
with vancomycin dosed at levels that do not perturb pre-formed
MRSA biofilms. Alkylation of the exocyclic amine of compound 9a
with ethyl (9d) or methyl (9e) groups delivered analogues with
increased ability to disperse MRSA biofilms; however, they do so
with a tandem increase in toxicity and diminished synergy with
vancomycin. Benzylated analogue 9b was the most potent MRSA
biofilm inhibitor with an IC50 of 9.62 1.4 µM, but did not disperse
pre-formed MRSA biofilms. Interestingly, compound 9b also re-
turned modest antimicrobial activity against a small panel of Gram-
positive bacteria. Furthermore, we established that certain merid-
ianin analogues increase the efficacy of colistin against Gram-
negative bacteria. Compound 9d, bearing an ethyl substitution on
the exocyclic nitrogen of the 2-AP ring, displayed the greatest range
of colistin synergy across a panel of colistin-sensitive and colistin-
resistant strains of Gram-negative bacteria which included highly
(3)
bacteria in biofilms. Lancet 2001, 358, 135-138.
(4) Donlan, R. M.; Costerton, J. W., Biofilms: Survival
Stewart, P. S.; Consterton, J. W., Antibiotic resistance of
Mechanisms of Clinically Relevant Microorganisms. Clinical
Microbiology Reviews 2002, 15 (2), 167-193.
(5)
Liu, Y.-Y.; Wang, Y.; Walsh, T. R.; Yi, L.-X.; Zhang, R.;
Spencer, J.; Doi, Y.; Tian, G.; Dong, B.; Huang, X.; Yu, L.-F.; Gu,
D.; Ren, H.; Chen, X.; Lv, L.; He, D.; Zhou, H.; Liang, Z.; Liu, J.-
H.; Shen, J., Emergence of plasmid-mediated colistin resistance
mechanism MCR-1 in animals and human beings in China: a
microbiological and molecular biological study. Lancet Infect. Dis.
2015, 16 (2), 161-168.
(6)
Feng, Y., Transferability of MCR-1/2 Polymyxin
Resistance: Complex Dissemination and Genetic Mechanism. ACS
Infect. Dis. 2018, 4 (3), 291-300.
(7)
Falagas, M. E.; Kasiakou, S. K.; Saravolatz, L. D., Colistin:
The Revival of Polymyxins for the Management of Multidrug-
Resistant Gram-Negative Bacterial Infections. Clin. Infect. Dis.
2005, 40 (9), 1333-1341.
(8)
Chen, L.; Todd, R.; Kiehlbauch, J.; Walters, M.; Kallen, A.,
Notes from the Field: Pan-Resistant New Delhi Metallo-Beta-
Lactamase-Producing Klebsiella pneumoniae — Washoe County,
Nevada, 2016. MMWR Morb Mortal Wkly Rep 2017, 66 (1), 33.
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 6 of 7
(9)
Franco, L. H.; Joffé, E. B. d. K.; Puricelli, L.; Tantian, M.;
Melander, C., Small molecule adjuvants that suppress both
chromosomal and mcr-1 encoded colistin-resistance and amplify
colistin efficacy in polymyxin-susceptible bacteria. Bioorg. Med.
Chem. 2017, 25 (20), 5749-5753.
(20) Richards, J. J.; Ballard, T. E.; Huigens, R. W., 3rd; Melander,
C., Synthesis and screening of an oroidin library against
Pseudomonas aeruginosa biofilms. ChemBioChem 2008, 9 (8), 1267-
79.
(21) Jiang, B.; Yang, C.-G., Synthesis of indolylpyrimidines via
cross-coupling of indolylboronic acid with chloropyrimidines:
facile synthesis of meridianin D. Heterocycles 2000, 53 (7), 1489-
1498.
1
2
3
4
5
6
7
8
Seldes, A. M.; Palermo, J. A., Indole Alkaloids from the Tunicate
Aplidium meridianum. J. Nat. Prod. 1998, 61, 1130-1132.
(10) Gompel, M.; Leost, M.; De Kier Joffe, E. B.; Puricelli, L.;
Franco, L. H.; Palermo, J.; Meijer, L., Meridianins, a new family of
protein kinase inhibitors isolated from the ascidian Aplidium
meridianum. Bioorg. Med. Chem. Lett. 2004, 14 (7), 1703-7.
(11) Giraud, F.; Alves, G.; Debiton, E.; Nauton, L.; Thery, V.;
Durieu, E.; Ferandin, Y.; Lozach, O.; Meijer, L.; Anizon, F.; Pereira,
E.; Moreau, P., Synthesis, protein kinase inhibitory potencies, and
in vitro antiproliferative activities of meridianin derivatives. J. Med.
Chem. 2011, 54 (13), 4474-89.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(12) Park, Y. K.; Lee, T. Y.; Choi, J. S.; Hong, V. S.; Lee, J.; Park,
J. W.; Jang, B. C., Inhibition of adipogenesis and leptin production
in 3T3-L1 adipocytes by a derivative of meridianin C. Biochem.
Biophys. Res. Commun. 2014, 452 (4), 1078-83.
(13) Radwan, M. A.; El-Sherbiny, M., Synthesis and antitumor
activity of indolylpyrimidines: marine natural product meridianin
D analogues. Bioorg. Med. Chem. 2007, 15 (3), 1206-11.
(14) Bharate, S. B.; Yadav, R. R.; Khan, S. I.; Tekwani, B. L.;
Jacob, M. R.; Khan, I. A.; Vishwakarma, R. A., Meridianin G and its
analogs as antimalarial agents. MedChemComm 2013, 4 (6), 1042.
(15) Yadav, R. R.; Khan, S. I.; Singh, S.; Khan, I. A.;
Vishwakarma, R. A.; Bharate, S. B., Synthesis, antimalarial and
antitubercular activities of meridianin derivatives. Eur. J. Med.
Chem. 2015, 98, 160-9.
(16) Nunez-Pons, L.; Carbone, M.; Vazquez, J.; Rodriguez, J.;
Nieto, R. M.; Varela, M. M.; Gavagnin, M.; Avila, C., Natural
products from Antarctic colonial ascidians of the genera Aplidium
and Synoicum: variability and defensive role. Mar. Drugs 2012, 10
(8), 1741-64.
(17) Bunders, C. A.; Minvielle, M. J.; Worthington, R. J.; Ortiz,
M.; Cavanagh, J.; Melander, C., Intercepting bacterial indole
signaling with flustramine derivatives. J. Am. Chem. Soc. 2011, 133
(50), 20160-3.
(22) Simon, G.; Couthon-Gourves, H.; Haelters, J.-P.; Corbel,
B.; Kervarec, N.; Michaud, F.; Meijer, L., Towards the Syntheses of
N-H and N-Alkylated Derivatives of Meridianins. J. Heterocycl.
Chem. 2007, 44.
(23) Fresneda, P. M.; Molina, P.; Bleda, J. A., Synthesis of the
indole alkaloids meridianins from the tunicate Aplidium
meridianum. Tetrahedron 2001, 57, 2355-2363.
(24) Bredereck, H.; Effenberger, F.; Botsch, H.; Rehn, H.,
Umsetzungen von
vinylogen Carbonsaureamiden zu
Heterocyclen. Ber. Dtsch. Chem. Ges. 1964, 98 (4), 1081-1086.
(25) Su, Z.; Yeagley, A. A.; Su, R.; Peng, L.; Melander, C.,
Structural studies on 4,5-disubstituted 2-aminoimidazole-based
biofilm modulators that suppress bacterial resistance to beta-
lactams. ChemMedChem 2012, 7 (11), 2030-9.
(26) Furlani, R. E.; Yeagley, A. A.; Melander, C., A flexible
approach to 1,4-di-substituted 2-aminoimidazoles that inhibit and
disperse biofilms and potentiate the effects of beta-lactams against
multi-drug resistant bacteria. Eur. J. Med. Chem. 2013, 62, 59-70.
(27) Yeagley, A. A.; Su, Z.; McCullough, K. D.; Worthington, R.
J.; Melander, C., N-substituted 2-aminoimidazole inhibitors of
MRSA biofilm formation accessed through direct 1,3-bis(tert-
butoxycarbonyl)guanidine cyclization. Org. Biomol. Chem. 2013,
11 (1), 130-7.
(18) Lindsey, E. A.; Brackett, C. M.; Mullikin, T.; Alcaraz, C.;
Melander, C., The discovery of N-1 substituted 2-
aminobenzimidazoles as zinc-dependent S. aureus biofilm
inhibitors. MedChemComm 2012, 3 (11), 1462-1465.
(19) Barker, W. T.; Martin, S. E.; Chandler, C. E.; Nguyen, T. V.;
Harris, T. L.; Goodell, C.; Melander, R. J.; Doi, Y.; Ernst, R. K.;
(28) Liu, Y. Y.; Chandler, C. E.; Leung, L. M.; McElheny, C. L.;
Mettus, R. T.; Shanks, R. M. Q.; Liu, J. H.; Goodlett, D. R.; Ernst,
R. K.; Doi, Y., Structural Modification of Lipopolysaccharide
Conferred by mcr-1 in Gram-Negative ESKAPE Pathogens.
Antimicrob. Agents Chemother. 2017, 61 (6).
ACS Paragon Plus Environment

Page 7 of 7
ACS Medicinal Chemistry Letters
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Insert Table of Contents artwork here
7
ACS Paragon Plus Environment