Second generation modifiers of colistin resistance show enhanced activity and lower inherent toxicity

Article abstract of DOI:10.1016/j.tet.2015.09.019

We recently reported a 2-aminoimidazole-based antibiotic adjuvant that reverses colistin resistance in two species of Gram-negative bacteria. Mechanistic studies in Acinetobacter baumannii demonstrated that this compound downregulated the PmrAB two-compon

Full text of DOI:10.1016/j.tet.2015.09.019

Tetrahedron xxx (2015) 1e5
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Second generation modifiers of colistin resistance show enhanced
activity and lower inherent toxicity
Christopher M. Brackett a, Robert E. Furlani a, Ryan G. Anderson a,
Aparna Krishnamurthy a, Roberta J. Melander a, Samuel M. Moskowitz b,
,
Robert K. Ernst c, Christian Melander a
*
a Department of Chemistry, North Carolina State University, Raleigh, NC 27695-8024, USA
b Department of Pediatrics, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114, USA
c Department of Microbial Pathogenesis, University of Maryland e Baltimore, Baltimore, MD 21201, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
We recently reported a 2-aminoimidazole-based antibiotic adjuvant that reverses colistin resistance in
two species of Gram-negative bacteria. Mechanistic studies in Acinetobacter baumannii demonstrated
that this compound downregulated the PmrAB two-component system and abolished a lipid A modi-
fication that is required for colistin resistance. We now report the synthesis and evaluation of two
separate libraries of substituted 2-aminoimidazole analogues based on this parent compound. From
these libraries, a new small molecule was identified that lowers the minimum inhibitory concentration of
colistin by up to 32-fold greater than the parent compound while also displaying less inherent bacterial
toxicity, thereby minimizing the likelihood of resistance evolution.
Received 19 August 2015
Accepted 7 September 2015
Available online xxx
Keywords:
Antibiotic adjuvant
2-Aminoimidazole
Colistin
ESKAPE pathogens
Acinetobacter baumannii
Pseudomonas aeruginosa
Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction
increased as reliance upon colistin therapy has escalated.5 The
mechanistic basis of colistin resistance is thought to occur pre-
Antibiotic resistant organisms represent a major threat to global
health. The Center for Disease Control and Prevention (CDC) esti-
mates that two million people acquire antibiotic-resistant in-
fections each year, of which 23,000 are fatal.1 The main culprits
behind these infections are referred to as the ESKAPE pathogens
(Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumo-
niae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enter-
obacter species).2 The severity of the problem of multi-drug
resistant (MDR) bacteria has been significantly exacerbated by
the fact that there have only been two new classes of antibiotics
introduced to the clinic in the past two decades, daptomycin and
linezolid.3 More concerning, these two classes of antibiotics are
exclusively active towards Gram-positive bacteria, which leaves
four of the ESKAPE pathogens untreated. As the well of clinically
relevant antibiotics runs dry, the polymyxin antibiotic colistin has
dominantly through modification of lipid A6; however, the two-
component system (TCS) signaling that drives these modifications
has recently been shown to activate additional mechanisms that
are also required for resistance.7 Our group has been focused on
combating the inevitable development of antibiotic resistant bac-
teria by developing compounds capable of disrupting the mecha-
nisms through which these organisms express resistance.8e12 We
recently established that the 2-aminoimidazole (2-AI) compound 1
is capable of reversing colistin resistance in multiple primary
clinical isolates of two of the four Gram-negative ESKAPE patho-
gens: K. pneumoniae, and A. baumannii (Fig. 1).11 Against several
strains of both bacteria, the minimum inhibitory concentration
(MIC) of colistin was lowered from 512 to ꢀ4 (in some cases as low
as 0.25) mg/mL in the presence of 30 mM (8.4 mg/mL) 1. Mechanistic
studies revealed that treatment of colistin-resistant A. baumannii
with 1 led to downregulation of the PmrAB two-component system
while mass spectrometry demonstrated reversal of the phosphoe-
thanolamine modification of lipid A responsible for colistin re-
sistance in A. baumannii.
become
a last line of defense against MDR Gram-negative
infections.4
Unfortunately the frequency of colistin resistant strains of
Gram-negative bacteria that have been observed in the clinic has
Despite this unprecedented activity, we noted that compound 1
itself harbored some inherent toxicity to the bacteria in the ab-
sence of colistin. Given that this toxicity may lead to an
* Corresponding author. E-mail address: ccmeland@ncsu.edu (C. Melander).
http://dx.doi.org/10.1016/j.tet.2015.09.019
0040-4020/Ó 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Brackett, C. M.; et al., Tetrahedron (2015), http://dx.doi.org/10.1016/j.tet.2015.09.019

2
C.M. Brackett et al. / Tetrahedron xxx (2015) 1e5
Fig. 1. Structures of compound 1 and 2.
accelerated rate of resistance evolution, we wondered whether we
could augment the activity of 1 while decreasing inherent toxicity
through analogue synthesis. In this regard, our group has also
recently developed several 2-AIs based upon compound 2 (Fig. 1)
hexylbenzoyl chloride was reacted with diazomethane, and the
resulting diazoketone was subjected to standard ArndteEistert
conditions (silver benzoate in methanol).15 The homologated ester,
3 underwent diazotransfer reaction, accomplished by reaction
with para-acetamidobenzenesulfonyl azide (p-ABSA) in the pres-
ence of DBU, to yield diazoketone 4.16 Analog diversity was then
introduced via a Ru-catalyzed NeH insertion reaction.17 Conver-
sion of the ester to the N-methoxy-N-methylamide (Weinreb
amide) proceeded without the need for protection of the newly
installed amine. Finally, reduction of the Weinreb amide to the
corresponding aldehyde using diisobutylaluminum hydride
(DIBAL-H), followed by cyclization with cyanamide afforded the
1,5-2AI derivatives 7aee.10
that are capable of reversing
b-lactam resistance in methicillin-
resistant S. aureus (MRSA).10,13,14 In these studies, we were able
to modify adjuvant activity through imprinting either a 1,4- or 1,5-
substitution pattern on the 2-AI ring. Specifically, compound 2 was
able to lower the MIC of oxacillin against MRSA four-fold at 25 mM,
while from the library of 1,5 substituted derivatives of compound
2, a compound emerged that is capable of lowering the MIC of
oxacillin against MRSA up to 512-fold at 5 m
M.10 Inspired by these
results, we set out to determine whether imparting either a 1,4- or
1,5-substitution pattern upon the 2-AI of 1 would deliver com-
pounds with augmented activity and reduced inherent toxicity.
Herein we report the synthesis of both 1,5- and 1,4-substituted
analogues of 1, as well as the evaluation of their biological activ-
ity in terms of colistin resistance suppression. Moreover, we report
a compound capable of lowering the MIC of colistin against re-
sistant strains of both A. baumannii and P. aeruginosa to a greater
degree than compound 1.
Our pilot library of 1,5 2-AIs was evaluated for the ability to
break resistance to colistin against the colistin-resistant strains of A.
baumannii that we employed in our previous study.11 These strains,
obtained from the Walter Reed Army Institute of Research (WRAIR),
have colistin MICs significantly higher (512e1024
Clinical and Laboratory Standards Institute (CLSI) defined threshold
g/mL).18 As is common practice
mg/mL) than the
for resistance for A. baumannii (ꢁ4
m
for evaluating adjuvant activity of our 2-AIs, we first established the
intrinsic antibiotic activity of our library alone. Whereas the parent
compound 1 has an MIC of 100
of our library had MICs of ꢁ200
m
M against all strains, all members
2. Results and discussion
m
M. We then determined the MIC of
colistin against two strains of A. baumannii in the presence of 30
2.1. Synthesis and biological evaluation of 1,5 2-AIs
and 60
stitution pattern essentially eradicated activity against A. bau-
mannii in the context of colistin resensitization. At 30
compounds 7aee only reduced the colistin MIC four-fold, from 512
to 128 g/mL, whereas the parent compound at the same concen-
tration was able to lower the MIC to 4 g/mL.
mM of each compound (Table 1). Surprisingly, the 1,5 sub-
As we had the most success in our previous MRSA studies with
the 1,5-substitution pattern where the introduced appendage was
an aromatic substituent, we chose to initially evaluate a pilot li-
brary of five aryl-1,5-substituted 2-AIs that were synthesized
according to Scheme 1. Briefly, commercially available 4-
m
M
m
m
Scheme 1. Synthesis of 1,5-substituted 2-aminoimidazoles. a) i. CH2N2, rt, 1 h. ii. AcOH, rt, 1 h. b) AgOBz, Et3N, MeOH, rt, 16 h. c) p-ABSA, DBU, MeCN, rt, 16 h. d) [Ru(p-cymene)Cl2]2,
ReNH2, CH2Cl2, rt, 1 h. e) HN(OCH3)CH3, i-PrMgCl, THF, ꢂ40 ꢃC, 8 h. f) i. DIBAL-H, THF ꢂ78 ꢃC, 1 h. ii. EtOH/H2O, pH 4.3, H2NCN, 95 ꢃC, 2 h. iii. MeOH/HCl.
Please cite this article in press as: Brackett, C. M.; et al., Tetrahedron (2015), http://dx.doi.org/10.1016/j.tet.2015.09.019

C.M. Brackett et al. / Tetrahedron xxx (2015) 1e5
3
Table 1
Table 2
Antibiotic activity and antibiotic resensitization activity of 1,5 2-AI library against
Antibiotic activity and antibiotic resensitization activity of 1,4 2-AI library against
two strains of A. baumannii
two strains of A. baumannii
Compound
MIC (
mM)
Concentration
A. baumannii strain
Compound
MIC (
m
M)
Concentration
A. baumannii strain
tested (
m
M)
tested (mM)
3941
4112
3941
4112
Colistin MIC (
m
g/mL)
Colistin MIC (
m
g/mL)
1
7a
100
>200
30
60
30
60
30
60
30
60
30
60
30
4
128
512
128
512
128
512
128
512
128
512
4
128
512
128
512
128
512
128
512
128
512
1
9a
100
>200
30
60
30
60
30
60
30
60
30
4
8
16
8
16
8
64
1
2
4
8
16
8
32
16
64
2
7b
7c
7d
7e
>200
>200
>200
200
9b
9c
9d
>200
>200
>200
4
strains, the initial lead 9d was able to lower colistin MICs to
0.5e4 g/mL at 30 M.
m
m
With the initial library results indicating that an alkyl group as
opposed to an aryl group was better tolerated, five more com-
pounds primarily focused on alkyl groups were synthesized, with
the structures being shown in Fig. 2. Compounds 9gei along with
the original 9d lead delivered compounds with side chains of var-
ious lengths while 9e afforded a branched alkyl side chain. In ad-
dition, since compounds 9b and 9c have a phenyl ring with an
increasing number of methylene units, compound 9f was synthe-
sized to further quantify the activity trend in this series of com-
pounds. Compounds 9eei were evaluated identically to the other
1,4 2-AIs, and the colistin resensitization results against A. bau-
mannii strains 3941 and 4112 are shown in Table 3, while results
against all strains can be found in Supplementary data.
2.2. Synthesis and biological evaluation of 1,4 2-AIs
Given the failure of the 1,5-substitution pattern to enhance ac-
tivity, we elected to evaluate the potential of 1,4-substitutions in
the context of compound 1. Similar to the approach taken for the 1,5
2-AI derivatives, a pilot library of four 1,4 2-AIs was synthesized
according to previous methods developed in our group,13 and is
summarized in Scheme 2. Briefly, commercially available 4-
hexylbenzoyl chloride was reacted with diazomethane and
quenched with concentrated hydrobromic acid to yield the corre-
sponding a-bromoketone, 8. This intermediate was reacted with
various primary amines, the pH was then lowered to 4.3 through
addition of 1M HCl, at which point cyanamide was added and the
reaction was heated to reflux for 3 h to yield the 1,4 2-AI derivatives
9aed.13
From the second generation library, the isopropyl and ethyl
analogues 9e and 9i, emerged as the most active compounds, ex-
ceeding the activity of compound 1 and any members of the initial
Scheme 2. Synthesis of 1,4-substituted 2-aminoimidazoles. a) i. CH2N2, 0 ꢃC, 1 h. ii. HBr, 0 ꢃC, 1 h. b) i. RNH2, EtOH, rt, 30 min ii. NH2CN, pH 4.3, EtOH, 95 ꢃC. iii. MeOH/HCl.
We evaluated this initial library of 1,4-substituted 2-AIs in the
same manner as our 1,5 library, first assessing their intrinsic anti-
biotic activity alone, and then their ability to restore activity of
colistin towards A. baumannii at sub-MIC levels. As observed with
the 1,5-substituted 2-AIs, the 1,4 substitution pattern drastically
lowered antibiotic activity as compared to the parent compound,
with the MICs all being above 200
listin against two strains of A. baumannii was determined in the
presence of 60 and 30 M of each compound (Table 2). In the case of
compounds 9aec the substitution led to a decrease in activity,
however in the case of 9d at 30 M, the MIC of colistin was in line
mM. Once again, the MIC of co-
m
m
with the parent compound. Encouraged by these initial results, the
pilot library was screened against three additional strains of colistin
resistant A. baumannii (Supplementary data). Against all five
Fig. 2. Structure of second library of 1,4 2-aminoimidazoles.
Please cite this article in press as: Brackett, C. M.; et al., Tetrahedron (2015), http://dx.doi.org/10.1016/j.tet.2015.09.019

4
C.M. Brackett et al. / Tetrahedron xxx (2015) 1e5
Table 3
phosphoethanolamine modification that drives colistin resistance
in A. baumannii.
Encouraged by the results against A. baumannii, we then eval-
uated the spectrum of activity of our library by testing whether we
could reverse colistin resistance in both clinical and laboratory
strains of P. aeruginosa.19,20 These strains have MICs ranging from
Antibiotic activity and antibiotic resensitization activity of second-generation 1,4 2-
AI library against two strains of A. baumannii
Compound
MIC (
mM)
Concentration
tested (mM)
A. baumannii strain
3941
4112
Colistin MIC (
m
g/mL)
64 to >1024
m
g/mL (Table 4), all of which are above the CLSI
9e
9f
>200
>200
>200
>200
>200
60
30
60
30
60
30
60
30
60
30
0.5
0.5
2
0.5
0.5
1
threshold for colistin resistance for P. aeruginosa (ꢁ8
m
g/mL).18 The
MICs of our entire library of 1,4 2-AIs as well as the parent com-
pound were then determined against all six strains, and in all cases
2
2
9g
9h
9i
64
128
4
128
128
4
the MICs were above the highest concentration tested (200 mM). All
nine compounds were then evaluated for their ability to lower the
MIC of colistin across the six strains of P. aeruginosa at an initial
concentration of 50 mM. Select results from the initial screening are
16
0.5
0.5
8
0.5
0.5
shown in Table 4 (full results in Supplementary data).
As was the case for A. baumannii, compound 9e emerged as the
lead compound, lowering the colistin MIC to at or below the re-
sistance threshold for all six strains, while compound 9i was also
more active than the parent against several strains. In addition, at
50 mM, 9e lowered the colistin MIC further than the parent com-
pound in four of the six strains, and had identical levels of resen-
sitization against the other two strains.
After identifying 9e as the lead compound, we investigated the
dose-response activity of the most active compound, 9e in com-
parison to compound 1 (Fig. 3). This was accomplished by de-
termining the colistin MIC against each strain in the presence of 25
library and lowering the MIC of colistin against all five strains of A.
baumannii to 0.5 g/mL, constituting up to a 1024-fold reduction in
m
MIC. Both compounds lowered the MIC of colistin further than
compound 1 while remaining significantly less toxic alone. In ad-
dition, compound 9f was nearly as active as 9e and 9i, and still more
active than the parent. Following the series of 9b, and 9c, com-
pound 9f has a two-methylene spacer between the 2-AI and the
benzene ring. With compound 9c being less active than 9b, we
expected 9f to follow the trend, so it was surprising when it was
one of our most active compounds.
With more active compounds in hand, we next wanted to verify
that, as we observed with compound 1, we were reversing lipid A
modification. To this end, A. baumannii 4106 was grown in the
presence of 30 mM 9e for 16 h. Bacteria were then collected and
subjected to mass spectrometry-based analysis of their lipid A
to 100
concentration to 100
to 50 M. Lowering the concentration to 25
m
M of either compound. In both cases, increasing compound
M led to a decrease in colistin MIC compared
M led to a dramatic
m
m
m
decrease in activity for compound 1, which was unable to lower the
MIC of colistin below the CLSI threshold in any of the strains tested.
On the other hand, compound 9e was able to effect significant
levels of antibiotic repotentiation at 25 mM (Fig. 3).
We then quantified activity by evaluating the effect of our lead
compound 9e against both A. baumannii and P. aeruginosa as
a function of time by constructing time kill curves. We chose to use
4106 as the representative strain for A. baumannii, as the previous
study with 1 also used this strain,11 while 1133 was chosen as the
representative strain for P. aeruginosa due to its high colistin re-
sistance and sensitivity to 1 and 9e. Initially, we investigated the
effect 9e had on bacterial growth in the absence of antibiotic. When
fraction. As we observed with compound 1, 2-AI 9e reversed the
Table 4
Select resensitization activity of 1,4 library at 50 mM against colistin resistant strains
of P. aeruginosa
P. aeruginosa
strain
Colistin
MIC
Colistin
Colistin
Colistin
Colistin
MICþ1
MICþ9d
MICþ9e
MICþ9i
(m
g/mL)
(
m
g/mL)
(
m
g/mL)
(m
g/mL)
(
mg/mL)
A. baumannii was grown in the presence of 30 mM 9e, there was
a 1.52 and 1.09 log reduction in CFUs/mL at the 2 and 4 h time
points, but growth was identical to the control afterwards. This is in
contrast to the more pronounced effect the same concentration of 1
has on bacterial growth, where at the same time points and con-
centration there is a 2.25 and 3.41 log reduction in CFUs/mL, re-
1016
1018
1029
1033
1109
1133
>1024
1024
64
64
512
128
8
8
8
64
8
64
16
8
16
16
128
8
4
1
8
2
8
32
8
2
8
4
>1024
8
spectively. Our previous study established that at
8 h the
Fig. 3. Dose response colistin resensitization data for compounds 1 and 9e against colistin resistant strains of P. aeruginosa. Dashed line represents the CLSI breakpoint.
Please cite this article in press as: Brackett, C. M.; et al., Tetrahedron (2015), http://dx.doi.org/10.1016/j.tet.2015.09.019

C.M. Brackett et al. / Tetrahedron xxx (2015) 1e5
5
3. Conclusion
Using compound 1 as inspiration we synthesized two distinct
libraries and evaluated their ability to reverse colistin resistance in
multiple strains of A. baumannii. From these studies, compound 9e
was identified as the lead compound and was able to lower the
colistin MIC up to 1024-fold, an eight-fold improvement over
compound 1. Encouraged by this study, we then turned to P. aeru-
ginosa, another bacterial pathogen for which colistin resistance has
been reported. Compound 9e once again emerged as the most ac-
tive compound, capable of lowering the colistin MIC up to 256-fold,
a 32-fold improvement over compound 1. Time-dependent studies
showed that 9e has less effect on bacterial growth than 1 at iden-
tical concentrations at early times points against A. baumannii,
while also leading to an enhanced adjuvant effect thereby further
decoupling intrinsic microbicidal activity from MIC suppression.
Efforts to further tune the activity of 9e and evaluate adjuvant ac-
tivity in vivo are currently underway.
Fig. 4. Time kill curve for combination of 9e and colistin against A. baumannii strain
4106. Black¼untreated control, blue¼30 M 9eþ0.125 g/mL colistin, green¼30
9eþ0.5 g/mL colistin, red¼30 M 9eþ2 g/mL colistin.
m
m
m
mM
m
m
Acknowledgements
The authors would like to thank the National Institutes of Health
(GM055769 to C.M, AI067653 to S.M.M) and the Cystic Fibrosis
Foundation (MOSKOW13P0 to S.M.M.) for their generous support of
this work.
Supplementary data
Supplementary data associated with this article can be found in
the online version, at http://dx.doi.org/10.1016/j.tet.2015.09.019.
References and notes
1. CDC, 2013.
2. Boucher, H. W.; Talbot, G. H.; Bradley, J. S.; Edwards, J. E.; Gilbert, D.; Rice, L. B.;
Scheld, M.; Spellberg, B.; Bartlett, J. Clin. Infect. Dis. 2009, 48, 1e12.
3. Coates, A. R.; Halls, G.; Hu, Y. Br. J. Pharmacol. 2011, 163, 184e194.
4. Falagas, M. E.; Kasiakou, S. K. Clin. Infect. Dis. 2005, 40, 1333e1341.
5. Olaitan, A. O.; Morand, S.; Rolain, J. M. Front. Microbiol. 2014, 5, 643.
6. Beceiro, A.; Llobet, E.; Aranda, J.; Bengoechea, J. A.; Doumith, M.; Hornsey, M.;
Dhanji, H.; Chart, H.; Bou, G.; Livermore, D. M.; Woodford, N. Antimicrob. Agents
Chemother. 2011, 55, 3370e3379.
Fig. 5. Time kill curve for combination of 9e or 1 and colistin against P. aeruginosa
strain 1133. Black¼untreated control, blue solid line¼50
m
M 9eþ16
m
g/mL colistin, red
solid line¼50
m
M 1þ16
m
g/mL colistin. Blue dotted line¼50
m
M 9eþ4
mg/mL colistin red
dashed line¼50
m
M 1þ4
m
g/mL colistin.
7. Gutu, A. D.; Rodgers, N. S.; Park, J.; Moskowitz, S. M. Antimicrob. Agents Che-
mother. 2015, 59, 5377e5387.
8. Brackett, C. M.; Melander, R. J.; An, I. H.; Krishnamurthy, A.; Thompson, R. J.;
Cavanagh, J.; Melander, C. J. Med. Chem. 2014, 57, 7450e7458.
9. Worthington, R. J.; Bunders, C. A.; Reed, C. S.; Melander, C. ACS Med. Chem. Lett.
2012, 3, 357e361.
combination of 30
m
M compound 1 and 2
m
g/mL colistin effected
a 2.4 log reduction in CFU/mL as compared to the control. With the
same concentration of 9e and colistin, at the same time point, there
is a 4.11 log reduction in CFU/mL, further highlighting the improved
efficacy of compound 9e (Fig. 4).
10. Harris, T. L.; Worthington, R. J.; Melander, C. Angew. Chem., Int. Ed. 2012, 51,
11254e11257.
11. Harris, T. L.; Worthington, R. J.; Hittle, L. E.; Zurawski, D. V.; Ernst, R. K.; Mel-
ander, C. ACS Chem. Biol. 2014, 9, 122e127.
12. Rogers, S. A.; Huigens, R. W., 3rd; Cavanagh, J.; Melander, C. Antimicrob. Agents
Chemother. 2010, 54, 2112e2118.
13. Furlani, R. E.; Yeagley, A. A.; Melander, C. Eur. J. Med. Chem. 2013, 62, 59e70.
14. Su, Z.; Yeagley, A. A.; Su, R.; Peng, L.; Melander, C. ChemMedChem 2012, 7,
2030e2039.
15. Kumar, K. S. A.; Chattopadhyay, S. RSC Adv. 2015, 5, 19455e19464.
16. Gao, L.; Hwang, G. S.; Ryu do, H. J. Am. Chem. Soc. 2011, 133, 20708e20711.
17. Su, Z.; Peng, L.; Melander, C. Tetrahedron Lett. 2012, 53, 1204e1206.
18. Institute, C. a. L. S., 2012, 32.
19. Moskowitz, S. M.; Brannon, M. K.; Dasgupta, N.; Pier, M.; Sgambati, N.; Miller,
A. K.; Selgrade, S. E.; Miller, S. I.; Denton, M.; Conway, S. P.; Johansen, H. K.;
Hoiby, N. Antimicrob. Agents Chemother. 2012, 56, 1019e1030.
20. Miller, A. K.; Brannon, M. K.; Stevens, L.; Johansen, H. K.; Selgrade, S. E.; Miller,
S. I.; Hoiby, N.; Moskowitz, S. M. Antimicrob. Agents Chemother. 2011, 55,
5761e5769.
Growth of P. aeruginosa was unaffected by the presence of either
compound 1 or 9e at their active concentration of 50
tionally, P. aeruginosa growth was monitored in the presence of
colistin alone, revealing that at concentrations below 32 g/mL
growth was unaffected. After establishing the lack of toxicity of
both compounds towards P. aeruginosa we next turned our atten-
tion towards combinations of colistin and either compound. After
4 h the combination of 50
resulted in a modest 1.51 log reduction in CFU/mL as compared to
the control, whereas 50 M 9e and only 4 g/mL colistin was able to
produce the same level of reduction in CFU/mL. At the same time
point, the combination of 16 g/mL colistin and 50 M 9e caused
a 3.81 log reduction CFU/mL (Fig. 5).
mM. Addi-
m
mM compound 1 and 16 mg/mL colistin
m
m
m
m
Please cite this article in press as: Brackett, C. M.; et al., Tetrahedron (2015), http://dx.doi.org/10.1016/j.tet.2015.09.019