Synthesis and Bioactivity Investigation of the Individual Components of Cyclic Lipopeptide Antibiotics

Article abstract of DOI:10.1021/acs.jmedchem.7b01367

In this paper, 26 natural polymyxin components and a new derivative S₂ were synthesized, and their differences in efficacy and toxicity have been investigated. Almost all of the synthesized components showed strong activity against both susceptible and resistant strains of E. coli, K. pneumoniae, P. aeruginosa, and A. baumannii. The toxicities were obviously different between the components. Only some of the components were tested for toxicity in vivo. Compounds E₂, E₂-Val, A₂, M₂, D₂, and S₂ showed obviously lower renal cytotoxicity and acute toxicity than polymyxins B and E. The in vivo nephrotoxicity of E₂, M₂, and S₂ was similar to that of polymyxin E. Compound S₂, with four positive charges, was especially interesting as it possessed both increased efficacy and decreased toxicity. The SAR and toxicity studies indicated that further structural modification could concentrate on polymyxin S. The results also indicated that S₂ could be a new drug candidate.

Full text of DOI:10.1021/acs.jmedchem.7b01367

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Article
The Synthesis and Bioactivity Investigation of the
Individual Components of Cyclic Lipopeptide Antibiotics
A-Long Cui, Xin-Xin Hu, Yan Gao, Jie Jin, Hong Yi, Xiu-Kun Wang, Tong-Ying Nie, Yang
Chen, Qi-Yang He, Huifang Guo, Jian-Dong Jiang, Xue-Fu You, and Zhuo-Rong Li
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01367 • Publication Date (Web): 07 Feb 2018
Downloaded from http://pubs.acs.org on February 8, 2018
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The Synthesis and Bioactivity Investigation of the
Individual Components of Cyclic Lipopeptide
Antibiotics
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‡
‡
A-Long Cui , Xin-Xin Hu , Yan Gao, Jie Jin, Hong Yi, Xiu-Kun Wang, Tong-Ying Nie, Yang
Chen, Qi-Yang He, Hui-Fang Guo, Jian-Dong Jiang, Xue-Fu You*, Zhuo-Rong Li*
Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union
Medical College, Beijing 100050, China
KEYWORDS: individual polymyxin component, synthesize, bioactivity evaluation, SAR and
toxicity
ABSTRACT: In this paper, twentyꢀsix natural polymyxin components and a new derivative S2
were synthesized, and their differences in efficacy and toxicity have been investigated. Almost
all of the synthesized components showed strong activity against both susceptible and resistant
strains of E. coli, K. pneumoniae, P. aeruginosa, and A. baumannii. The toxicities were
obviously different between the components. Only some of the components were tested for
toxicity in vivo. Compounds E , E ꢀVal, A , M , D , and S showed obviously lower renal
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cytotoxicity and acute toxicity than polymyxins B and E. The in vivo nephrotoxicity of E , M ,
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and S were similar to that of polymyxin E. Compound S , with four positive charges, was
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especially interesting as it possessed both increased efficacy and decreased toxicity. The SAR
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and toxicity studies indicated that further structural modification could concentrate on polymyxin
S. The results also indicated that S could be a new drug candidate.
2
INTRODUCTION
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In February 2017, the World Health Organization published its first list of antibioticꢀresistant
critical pathogens and the "critical group" including carbapenemꢀresistant A. baumannii, P.
aeruginosa and various Enterobacteriaceae (such as E. coli, K. pneumoniae, etc.). These bacteria
are dangerous and can cause deadly bloodstream infections and pneumonia. Polymyxins were
first discovered in 1947 and subsequently were used to treat infections caused by Gramꢀnegative
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ꢀ3
bacteria. Unfortunately, their initial application in the 1970’s was limited because of the
occurrence of serious side effects, especially nephrotoxicity. The incidence of acute kidney
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injury induced by polymyxins occurred in up to 40ꢀ60% of patients. Due to the lack of new
antibiotics, polymyxins are now being employed as a lastꢀresort therapy against infections
caused by multidrugꢀresistant Gramꢀnegative bacteria, especially E. coli, K. pneumoniae, A.
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baumannii, and P. aeruginosa. Therefore, further studies are urgently needed to better balance
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their efficacy and nephrotoxicity in clinical application. Some recent studies have already
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demonstrated that improved dosing regimens are possible.
Polymyxins are cyclic lipopeptide antibiotics manufactured via fermentation from different
strains of Paenibacillus polymyxa, and the obtained lipopeptides are chemically unresolved
mixtures that containing multiple structurally similar constituents. There are more than eleven
distinct types of polymyxins that have been identified to date from the different fermentation
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products, including polymyxins A, B, C, D, E, F, K, M, P, S, and T.
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Currently, polymyxin B sulfate and colistimethate sodium (prodrug of colistin) are clinically
available for the treatment of infections caused by Gramꢀnegative superbugs. The clinically used
polymyxins are cationic antimicrobial lipopeptides that possess a cyclic heptapeptide backbone
with three positive charges, a linear tripeptide segment with two positive charges, and a
hydrophobic fatty acyl chain. They have a common primary sequence, the differences being at
the fatty acyl group and the amino acids occupying positions 6 and 7. A nomenclature for the
polymyxins employs a subscript number for different acyl groups and different letters for
different amino acids composition. For polymyxin B, the major components are polymyxins B1
and B , whereas for polymyxin E (colistin), they are polymyxins E (colistin A) and E (colistin
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B) (Table 1). Orwa et al isolated and characterized the individual components of commercial
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polymyxins B and E.
Polymyxins B , B , B , B , B , B and B ꢀIle were isolated and
1 2 3 4 5 6 1
characterized from polymyxin B, but the stereochemistry of the fatty acyl chain of B was
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unidentified. Polymyxins E , E , E , E , E , E ꢀIle, E ꢀNva, E ꢀVal, E ꢀIle, E ꢀNva, E ꢀVal and
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E8ꢀIle were isolated and characterized from polymyxin E, whereas the stereochemistry of the
fatty acyl chain of E ꢀIle was unidentified. In the case of polymyxin B, a total of 39 possible
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components were speculated in the fermentation products using liquid chromatography coupled
to mass spectrometry, with only six of them having defined chemical structures. As for
polymyxin E, a total of 36 possible components were speculated, with only eleven of them
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having determined chemical structures (Table 1).
Since the available polymyxins B and E are both complex mixtures, there has been concern
about whether these individual constituents have identical pharmacological effects. The
percentages of these major and minor components of polymyxins B and E can vary between
different fermentation products. Different components and contents may not exhibit an
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equivalent pharmacological activity and toxic propensity.
Tam et al were the first to purify
and investigate the in vitro potency of five individual polymyxin B components, including
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polymyxins B , B , B , B , and B ꢀIle. Kassamali et al obtained and evaluated the in vitro
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potencies of four polymyxin B components individually and in combination, including B , B , B
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and B ꢀIle. Roberts et al purified and investigated the in vitro and in vivo antimicrobial
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activities and renal toxicities of four major components of polymyxins B and E, including B , B ,
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E , and E . Polymyxins B and E showed a significantly higher (>3ꢀfold) in vitro cytotoxicity
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to HKꢀ2 cells than polymyxins B and E , respectively. Sakura et al estimated the antimicrobial
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activities of the synthetic polymyxins B , B , B , B , B , B and B ꢀIle. Kline et al synthesized
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and evaluated the in vitro antimicrobial activities of polymyxins E , E , and E ꢀIle. However,
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too little is understood about the pharmacological differences between the individual
constituents, especially their toxicity differences. Therefore, we have synthesized and evaluated
the antimicrobial activities and toxicities of the components of polymyxins B and E, including
B , B , B , B , B , B ꢀIle, E , E , E , E , E , E ꢀIle, E ꢀNva, E ꢀVal, E ꢀIle, E ꢀNva and E ꢀVal.
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In addition to polymyxins B and E, we have also included the components of the polymyxins
A, D, M, S, and T in our studies. Jones et al reported the chemical basis for the classification of
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polymyxins A, B, C, D, and E, while Brownlee et al reported the chemotherapy and
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pharmacology of polymyxins A, B, C, D, and E.
Polymyxin B and polymyxin E were
introduced into clinical medicine due to their lower renal toxicity compared with polymyxin A
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and polymyxin D.
The amino acid configurations in polymyxins C and F were
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undetermined.
The structure of the hydroxy fatty acyl chain of polymyxin K was also
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unidentified.
Moreover, Niu et al concluded that polymyxins P and P from Paenibacillus
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polymyxa Mꢀ1 contained the same fatty acyl chain, which is consistent with the data reported by
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Kimura et al.
Polymyxins A and M were initially considered identical, but ultimately proven
Shoji et al reported the differences in the structure and the pharmacology
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to be different.
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between polymyxins S and T (containing T ).
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Therefore, polymyxins A, D, M, S, and T have known structures, but almost all of the
antimicrobial activities or toxicities were tested using unspecified mixtures. Thus, we have also
synthesized and studied the antimicrobial activities and toxicities of polymyxin A, D, M, S, and
T individual components, including A , A , D , D , M , M , S , T , and T as well as a new
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derivative S2.
All of the synthesized individual components were initially evaluated for their in vitro
antibacterial activity and renal toxicity. A subset of the different lipopeptide components was
then examined to determine the acute toxicity and nephrotoxicity in ICR mice. In addition, we
have summarized the SAR and structureꢀtoxicity relationship (STR) of polymyxins based on
natural individual components.
Table 1. Chemical Structures of the Individual Components of Polymyxins
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polymyxins fatty acid (R1)
position 3
amino
position 6
amino
position 7
amino
position 10
amino
acid (R2)
LꢀDab
LꢀDab
LꢀDab
LꢀDab
LꢀDab
LꢀDab
acid (R3)
DꢀPhe
DꢀPhe
DꢀPhe
DꢀPhe
DꢀPhe
DꢀPhe
acid (R4)
LꢀLeu
LꢀLeu
LꢀLeu
LꢀLeu
LꢀLeu
LꢀLeu
acid (R5)
LꢀThr
LꢀThr
LꢀThr
LꢀThr
LꢀThr
LꢀThr
a
B1
B2
B3
B4
B5
B6
6ꢀMOA
6ꢀMHA
OA
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a
a
a
a
b
HA
NA
3ꢀOHꢀ6ꢀ
MOA
a
B ꢀIle
6ꢀMOA
6ꢀMOA
6ꢀMHA
OA
LꢀDab
LꢀDab
LꢀDab
LꢀDab
LꢀDab
LꢀDab
LꢀDab
LꢀDab
LꢀDab
LꢀDab
LꢀDab
LꢀDab
LꢀDab
DꢀDab
DꢀDab
LꢀDab
LꢀDab
DꢀPhe
DꢀLeu
DꢀLeu
DꢀLeu
DꢀLeu
DꢀLeu
DꢀLeu
DꢀLeu
DꢀLeu
DꢀLeu
DꢀLeu
DꢀLeu
DꢀLeu
DꢀLeu
DꢀLeu
DꢀLeu
DꢀLeu
LꢀIle
LꢀLeu
LꢀLeu
LꢀLeu
LꢀLeu
LꢀLeu
LꢀIle
LꢀThr
LꢀThr
LꢀThr
LꢀThr
LꢀThr
LꢀThr
LꢀThr
LꢀThr
LꢀThr
LꢀThr
LꢀThr
LꢀThr
LꢀThr
LꢀThr
LꢀThr
LꢀThr
LꢀThr
1
a
E1
a
E2
a
E3
a
E4
HA
a
E7
7ꢀMOA
6ꢀMOA
6ꢀMOA
6ꢀMOA
6ꢀMHA
6ꢀMHA
6ꢀMHA
7ꢀMNA
6ꢀMOA
6ꢀMHA
6ꢀMOA
6ꢀMHA
a
E1ꢀIle
a
E1ꢀNva
LꢀNva
LꢀVal
LꢀIle
a
E1ꢀVal
a
E2ꢀIle
a
E2ꢀNva
LꢀNva
LꢀVal
LꢀIle
a
E2ꢀVal
b
E8ꢀIle
a
A1
LꢀThr
LꢀThr
LꢀThr
LꢀThr
a
A2
a
M1
a
M2
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a
a
D1
D2
6ꢀMOA
6ꢀMHA
6ꢀMOA
6ꢀMHA
6ꢀMOA
6ꢀMHA
DꢀSer
DꢀSer
DꢀSer
DꢀSer
LꢀDab
LꢀDab
DꢀLeu
DꢀLeu
DꢀPhe
DꢀPhe
DꢀPhe
DꢀPhe
LꢀThr
LꢀThr
LꢀThr
LꢀThr
LꢀLeu
LꢀLeu
LꢀThr
LꢀThr
LꢀThr
LꢀThr
LꢀLeu
a
S1
a,c
S2
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a
T1
T2
a
LꢀLeu
a
b
c
The synthesized compound. Chemical structure of an undetermined compound. New
compound. Dab, 2,4ꢀdiaminobutyric acid; LꢀVal, Lꢀvaline; 6ꢀMOA, (S)ꢀ6ꢀmethyloctanoic acid; 6ꢀ
MHA, 6ꢀmethylheptanoic acid; OA, octanoic acid; HA, heptanoic acid; NA, nonanoic acid; 3ꢀ
OHꢀ6ꢀMOA, 3ꢀhydroxyꢀ6ꢀmethyloctanoic acid; 7ꢀMOA, 7ꢀmethyloctanoic acid; 7ꢀMNA, 7ꢀ
methylnonanoic acid.
RESULTS AND DISCUSSION
Chemistry. We used the recently reported method employing onꢀresin cyclization to synthesize
3
3
the target compounds (Scheme 4). The first step was the Oꢀallyl ester protection of the Cꢀ
terminal amino acid FmocꢀLꢀDab(Boc)ꢀOH (Scheme 1). The (S)ꢀ6ꢀmethyloctanoic acid was
3
4
synthesized according to the literature (Scheme 2). The 7ꢀmethyloctanoic acid was synthesized
from 7ꢀmethylꢀ7ꢀoctenoic acid (Scheme 3). The sideꢀchain amine of FmocꢀLꢀDabꢀOAll was
anchored on the 2ꢀchlorotrityl chloride resin (2CTC resin) in the presence of DIPEA in DCM.
The linear peptide was synthesized via standard solidꢀphase peptide synthesis using a
consecutive Fmoc group deprotection strategy followed by an amino acid coupling reaction. In
addition, a Dde group was employed as the protecting group for the sideꢀchain amino of Dab4
due to its stability in the Fmoc strategy. After capping the peptide with a fatty acid and removing
the Dde group, FmocꢀLꢀThr(tBu)ꢀOH or FmocꢀLꢀLeuꢀOH was coupled to the sideꢀchain amino of
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Dab4. Subsequently, onꢀresin cyclization was conducted following the deprotection of the allyl
group on Dab9 and Fmoc group on Thr10, followed by cyclization using PyAOP, HOAt and
NMM in DMF at room temperature. Finally, the cyclic peptide was cleaved from the resin using
0
1
2
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4
5
6
7
8
9
0
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7
8
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7
8
9
0
a solution of TFA:TIS:H O (95:2.5:2.5). The crude peptide was purified by using semiꢀ
2
preparative HPLC to afford the polymyxin TFA salt, and the total yield was approximately 30%
based on the initial resin with a purity >95%. Polymyxin S is a new compound not reported in
2
the literature.
Scheme 1. Synthetic Route of FmocꢀLꢀDabꢀOAll
Reagents and conditions: (a) NaHCO , H O, allyl bromide, Aliquat 336, DCM; (b) CF COOH,
3
2
3
DCM.
Scheme 2. Synthetic Route of (S)ꢀ6ꢀmethyloctanoic acid
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Reagents and conditions: TEMPO, NaClO, NaClO , CH CN, H O.
2
3
2
Scheme 3. Synthetic Route of 7ꢀmethyloctanoic acid
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
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5
6
0
1
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7
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
Reagents and conditions: H , Pd/C, MeOH.
2
Scheme 4. Synthetic Route of the Individual Components of Polymyxins
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
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3
3
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0
1
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7
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9
0
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9
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9
0
Reagents and conditions: (a) FmocꢀLꢀDabꢀOAll, DIPEA, DCM, 2 h, MeOH; (b) (i) 20%
piperidine in DMF, 5 min and 10 min; (ii) appropriate amino acid, HCTU, DIPEA, DMF, 1 h;
(
iii) corresponding fatty acid, HCTU, DIPEA, DMF, 1 h; (c) 3% hydrazine in DMF, 2×30 min;
d) FmocꢀLꢀThr(tBu)ꢀOH or FmocꢀLꢀLeuꢀOH, HCTU, DIPEA, DMF, 1 h; (e) (i) PhSiH3,
(
Pd(PPh ) , 50% DCM/50% DMF, 2 h; (ii) 20% piperidine in DMF, 5 min and 10 min; (f)
3 4
PyAOP, HOAt, NMM, DMF, 3 h; (g) TFA:TIS:H O (95:2.5:2.5), 2 h; followed by reverse phase
2
HPLC purification.
The in vitro antimicrobial activity. The in vitro antimicrobial activities of individual polymyxin
components were assessed against a panel of the key Gramꢀnegative pathogens (Table 2). Drugꢀ
susceptible and drugꢀresistant ATCC reference strains and clinical isolates were used, including
E. coli, K. pneumoniae, A. baumannii, and P. aeruginosa. Overall, almost all of the individual
components showed effective activity against the tested Gramꢀnegative pathogens. Additionally,
all of the polymyxin B individual components had comparable MICs (0.25ꢀ2 µg/mL) against the
tested Gramꢀnegative bacteria compared with clinically used polymyxin B (0.5ꢀ2 µg/mL), except
B (2ꢀ4 µg/mL). Meanwhile, all of the polymyxin E individual components had comparable
5
MICs (0.25ꢀ2 µg/mL) with that of clinically used polymyxin E (0.5ꢀ1 µg/mL). These results
suggested that the fatty acyl chain length of the natural components appeared to be C7ꢀC9, as
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1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
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5
5
5
5
5
5
5
5
5
6
component B with a longer fatty acyl chain displayed a noticeably reduced antimicrobial
5
activity (approximately 2ꢀfold). Additionally, whether position 6 was occupied by DꢀPhe or Dꢀ
Leu, or Leuꢀ7 was substituted with other hydrophobic amino acids such as LꢀIle7, LꢀNva7 and Lꢀ
Val7, did not affect the antimicrobial potency. Polymyxins M (≤0.03ꢀ1 µg/mL) and M (0.25ꢀ
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
0
.5 µg/mL) showed noticeably enhanced antimicrobial activities (approximately 2ꢀfold) against
1
E. coli, K. pneumoniae, and P. aeruginosa compared with polymyxins E (0.5ꢀ2 µg/mL) and E2
(
0.5ꢀ2 µg/mL). Meanwhile, polymyxins A (0.5ꢀ1 µg/mL) and A (0.25ꢀ1 µg/mL) showed
1 2
similar antimicrobial activities in comparison with M (≤0.03ꢀ1 µg/mL) and M (0.25ꢀ1 µg/mL).
1
2
These results suggested that the substitution of LꢀLeu7 with a more polar amino acid such as Lꢀ
Thr could noticeably increase the activity, and the configuration of Dab3 would not affect the
antimicrobial activity. Polymyxins D (0.12ꢀ1 µg/mL), D (0.12ꢀ0.5 µg/mL), S (0.25ꢀ1 µg/mL),
1
2
1
and S (0.25ꢀ0.5 µg/mL) with four positive charges showed similar antimicrobial potencies
2
against E. coli and K. pneumoniae but noticeably reduced potencies (2ꢀ4 µg/mL) against P.
aeruginosa compared with polymyxins A and M. Additionally, polymyxins D (0.06 µg/mL), D2
1
(
0.12 µg/mL), S (0.12 µg/mL), and S (0.25 µg/mL) showed noticeably enhanced antimicrobial
1 2
activity (≥2ꢀfold) against A. baumannii compared with polymyxins A and M. These results
showed that the substitution of the positive charge of LꢀDab3 or DꢀDab3 with a polar amino acid
such as DꢀSer3 could noticeably enhance the antimicrobial activity against A. baumannii but
noticeably decrease the antimicrobial activity against P. aeruginosa. Meanwhile, charges of five
Dab residues seemed to be more important for the antimicrobial potency against P. aeruginosa.
Polymyxins T (2ꢀ8 µg/mL) and T (4ꢀ8 µg/mL), with an LꢀLeu10 residue, showed noticeably
1
2
decreased antimicrobial potencies (approximately 4ꢀfold) compared with polymyxins B (0.25ꢀ2
1
µg/mL) and B (0.5ꢀ2 µg/mL). The results indicated that the substitution of LꢀThr10 with a more
2
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hydrophobic amino acid such as LꢀLeu could noticeably reduce the antimicrobial activity against
all of the tested Gramꢀnegative bacteria.
The in vitro nephrotoxicity. We assessed the renal cell cytotoxicity (CC ) of the synthesized
5
0
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
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4
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6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
individual components against immortalized Vero kidney cells of the African green monkey
Table 2). The renal toxicities of these different types of individual components were obviously
(
different. Polymyxin E (154±13 µg/mL) showed noticeably reduced renal cytotoxicity (P<0.01)
compared with polymyxin B (103±13 µg/mL). Polymyxins B (159±14 µg/mL) and B (160±14
2
4
µg/mL) displayed noticeably reduced toxicities (approximately 2ꢀfold) compared with
polymyxins B (71±6 µg/mL) and B (86±8 µg/mL). However, polymyxin B (33±3 µg/mL)
1
3
5
showed significantly increased renal cytotoxicity (>4ꢀfold) compared with polymyxin B2.
Polymyxins E (244±28 µg/mL) and E (216±16 µg/mL) also displayed noticeably reduced
2
4
toxicities (>2ꢀfold) to Vero cells compared with polymyxins E (95±8 µg/mL) and E (87±7
1
7
µg/mL). Polymyxins E ꢀIle, E ꢀNva, and E ꢀVal showed comparable toxicities with polymyxin
2
2
2
E2. These results indicated that the polymyxin fatty acyl chain length of C7 showed lower renal
cytotoxicity than a chain length of C8 or C9. Additionally, Leuꢀ7 was substituted with other
hydrophobic amino acids such as LꢀIle7, LꢀNva7 and LꢀVal7 did not remarkably affect the renal
cytotoxicity. Polymyxins M (276±17 µg/mL) and A (288±23 µg/mL) showed similar renal
2
2
cytotoxicities compared with polymyxin E . These results suggested that the substitution of Lꢀ
2
Leu7 with a more polar amino acid such as LꢀThr and the configuration of Dab3 would not affect
the renal cytotoxicity. Polymyxins D (318±26 µg/mL) and S (>500 µg/mL) with four positive
2
2
charges showed decreased renal cytotoxicities compared with polymyxins M and A . These
2
2
results showed that the substitution of the positive charges of LꢀDab3 or DꢀDab3 with a polar
amino acid such as DꢀSer3 could decrease the renal cytotoxicity. However, polymyxins T (17±2
1
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
µg/mL) and T (30±3 µg/mL) showed significantly increased toxicities (>4ꢀfold) compared with
2
polymyxins B (71±6 µg/mL) and B (159±14 µg/mL), respectively. These results indicated that
1
2
the substitution of LꢀThr10 with a more hydrophobic amino acid such as LꢀLeu10 could
significantly increase the renal cytotoxicity.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 2. Minimum Inhibitory Concentrations (MICs) and Cytotoxicity (CC ) of the Individual
50
Components of Polymyxins
b
compou
MICs (µg/mL)
CC50 (µg/mL)
nd
E. coli
K. pneumoniae
P.
aerugin bauman
osa nii
A.
Vero cells P value, vs
a
ATCC 14ꢀ11
ATCC ATCC ATCC ATCC
E
B
2
5922
700603 BAAꢀ
27853
19606
ESBLs
2146
ESBLs
NDMꢀ1
B
1
0.5
1
0.5
0.5
0.5
0.5
1
1
1
1
0.5
0.25
1
2
1
2
2
1
1
4
2
2
2
1
1
0.5
1
103±13
154±13
71±6
ꢀ
ꢀ
ꢀ
ꢀ
E
B1
B2
B3
B4
B5
2
0.25
0.5
0.5
0.5
2
<0.01 <0.01
0.653 <0.01
<0.01 0.147
0.571 <0.01
<0.01 <0.01
<0.01 <0.05
<0.01 0.454
<0.01 <0.01
0.793 <0.05
<0.01 <0.01
0.5
0.5
0.5
2
1
159±14
86±8
1
1
0.5
2
1
0.5
4
160±14
33±3
2
B ꢀIle
1
1
2
2
1
1
75±6
E1
E2
E3
E4
1
0.5
0.5
0.5
0.5
1
1
0.5
0.5
0.5
2
95±8
0.5
0.5
1
1
1
244±28
150±18
216±16
1
0.5
0.5
0.5
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7
E7
1
1
1
0.5
1
1
1
2
2
1
1
87±7
176±15
109±9
131±9
199±13
168±12
225±17
145±12
288±23
96±9
<0.01 0.086
0.115 <0.01
<0.01 0.434
0.060 <0.05
<0.05 <0.01
0.225 <0.01
<0.01 <0.01
0.427 <0.01
<0.01 <0.01
<0.01 0.444
<0.01 <0.01
<0.05 <0.01
<0.01 <0.01
0.686 <0.05
<0.01 <0.01
<0.01 <0.01
<0.01 <0.01
E1ꢀIle
1
E1ꢀNva
E1ꢀVal
E2ꢀIle
E2ꢀNva
E2ꢀVal
A1
1
1
2
1
1
1
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0.5
1
0.5
1
0.5
1
2
0.5
1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
0.5
0.5
0.5
0.5
1
2
0.5
0.5
0.5
0.5
0.25
0.5
0.12
0.25
0.25
0.25
4
0.25
0.25
1
0.5
0.5
1
1
0.5
1
2
1
1
A2
0.25
≤0.03
0.25
0.25
0.12
0.5
1
0.5
0.5
0.5
0.5
0.5
0.5
0.5
4
1
0.5
0.5
1
M1
0.25
0.5
1
1
M2
0.5
2
276±18
189±11
318±26
160±23
>500
D1
0.06
0.12
0.12
0.25
2
D2
0.5
1
4
S1
4
S2
0.25
4
0.5
8
2
T1
4
17±2
T2
8
4
4
4
4
4
30±3
a
b
Clinical isolate. 50% cytotoxicity concentration. ESBLs, extendedꢀspectrum βꢀlactamases;
NDMꢀ1, New Delhi Metalloꢀbetaꢀlactamaseꢀ1. The MICs were the representative results of at
least two independent experiments, in the case of different results for the two experiments, a
third experiment was conducted, and the confirmed results were given. The CC values were
5
0
indicated as mean ± SD values and calculated from three independent experiments, each
performed in triplicate. Statistical analysis was performed with the Student's tꢀtest using SPSS
1
6.0.
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1
1
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2
2
2
2
2
2
2
2
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6
Acute toxicity. All animal experiments were performed in full compliance with the protocols
approved by the Animal Ethics Committee of the Institute of Medicinal Biotechnology (Beijing,
China). The acute toxicity using ICR mice was evaluated after the intravenous injection of
varying doses of components into four to five groups (weight, 18 to 22 g; n=10 mice per group; 5
male, 5 female) for one compound. The LD values (Table 3) were calculated according to the
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
0
Bliss test protocol. Oneꢀway analysis of variance (ANOVA) was used for comparisons of LD50
values. Nearꢀlethal doses caused immediate signs of the pallor of the ears, convulsions, muscular
incoordination, and respiratory distress. Death from respiratory failure occurred within 5 min
with lethal doses. The different types of these lipopeptide components with similar or improved
antibacterial activity were examined to determine their acute toxicities. In summary, all of the
tested individual components displayed lower acute toxicity than polymyxin B mixtures and
polymyxin E mixtures. Polymyxins B (LD , 12.6 mg/kg), E (LD , 11.3 mg/kg), E ꢀIle (LD ,
2
50
2
50
2
50
12.9 mg/kg), E ꢀNva (LD , 12.4 mg/kg), and E ꢀVal (LD , 13.2 mg/kg) displayed significantly
2
50
2
50
reduced acute toxicities (P<0.01) compared with polymyxins B (LD , 6.9 mg/kg) and E (LD ,
5
0
50
8
.5 mg/kg), respectively. These results indicated that the individual synthetic components were
less toxic than the natural mixtures used clinically. In addition, the differences in the acute
toxicities between mixtures and individual components were probably associated with
impurities. Additionally, whether position 6 was occupied by DꢀPhe or DꢀLeu, or Leuꢀ7 was
substituted with other hydrophobic amino acids such as LꢀIle7, LꢀNva7 and LꢀVal7, did not affect
the acute toxicity. Polymyxins M (LD , 11.4 mg/kg) and M (LD , 13.0 mg/kg) showed the
1
50
2
50
similar acute toxicities compared with polymyxin E . Polymyxin A (LD , 12.6 mg/kg) showed
2
2
50
a similar acute toxicity compared with polymyxin M . These results suggest that a fatty acyl
2
chain length of C7ꢀC8 of the natural components, the configuration of Dab3, and the substitution
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of LꢀLeu7 with a more polar amino acid such as LꢀThr would not affect the acute toxicity.
Additionally, polymyxins D (LD , 20.5 mg/kg) and S (LD , 17.0 mg/kg) with four positive
2
50
2
50
charges showed significantly reduced acute toxicities (P<0.01) compared with polymyxins A2
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
and M . These results suggest that the substitution of the positive charges of Dab3 or DꢀDab3
2
with polar amino acid such as DꢀSer3 could significantly reduce the acute toxicity.
Table 3. Acute Toxicity (LD ) of the Individual Components of Polymyxins
50
compound
Acute toxicity
LD value (mg/kg)
95% Confidence
interval (mg/kg)
P value, vs
5
0
E
B
B
E
6.9
6.5−7.3
ꢀ
ꢀ
8.5
8.0−9.0
ꢀ
ꢀ
B2
12.6
11.3
12.9
12.4
13.2
12.6
11.4
13.0
20.5
17.0
11.9−13.5
10.8−11.8
11.9−13.9
11.7−13.1
12.5−14.0
11.9−13.5
10.8−12.1
12.0−14.0
18.8−22.4
15.8−18.2
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
E2
E2ꢀIle
E2ꢀNva
E2ꢀVal
A2
M1
M2
D2
S2
The LD50 values were calculated according to the Bliss test protocol. Oneꢀway analysis of
variance (ANOVA) was used for comparisons of LD values.
5
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The in vivo nephrotoxicity. The in vivo renal toxicities of the different types of these major
lipopeptide components with similar or improved antibacterial activity and cytotoxicity were
3
5ꢀ36
examined in a mouse nephrotoxicity model reported by Yousef et al.
The ICR mice (weight,
0
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1
8 to 22 g; n=8 mice per group; 4 male, 4 female) were intravenously administered the
component at a dose of 2.5 mg/kg twice daily for 7 days (an accumulated dose of 35 mg/kg),
followed by histological examination (Table 4, Figure 1). In preliminary studies, histological
examination of the kidneys from the mice injected with polymyxins A (SQS +1), B (SQS +1),
2
2
and D (SQS +1) showed mild damage compared with those injected with polymyxin E and the
2
saline controls. Meanwhile, polymyxins E (SQS 0→+1), M (SQS 0→+1), and S (SQS 0→+1)
2
2
2
showed no significant change or mild damage compared with the saline control.
Table 4. Histological Examination Results of the Individual Components of Polymyxins
a
compound
n
8
8
8
8
8
8
8
8
SQS kidney histology score
Polymyxin E
0→+1
0
saline
B2
+1
E2
0→+1
+1
A2
M2
D2
0→+1
+1
S2
0→+1
a
SQS 0=no significant change; SQS +1=mild damage; SQS +2=mild to moderate damage.
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Figure 1. Representative histological images of: (A) mouse kidney from the saline control group
showing no histological damage; (B), (C), (D), (E), (F), and (G) mice kidneys after exposure to
polymyxins A , B , D , E , M , and S with grade 1 lesions (SQS=+1), showing tubular dilatation,
2
2
2
2
2
2
prominent nuclei, and a few pale tubular casts; (H) mouse kidney after exposure to the
polymyxin E control group with grade 1 lesions (SQS=+1). Original magnification: x200 for all
panels.
CONCLUSIONS
In this paper, a systematic study on the synthetic preparation and biological activity of
different components of polymyxins was reported for the first time. Twentyꢀsix reported natural
individual components, including B , B , B , B , B , B ꢀIle, E , E , E , E , E , E ꢀIle, E ꢀNva, E ꢀ
1
2
3
4
5
1
1
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4
7
1
1
1
Val, E ꢀIle, E ꢀNva, E ꢀVal, A , A , D , D , M , M , S , T , T , and a new derivative S were
2
2
2
1
2
1
2
1
2
1
1
2
2
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synthesized, and the differences in efficacy and toxicity between the individual components were
investigated.
The antimicrobial activity was tested against ATCC reference strains and clinical isolates.
Almost all of the synthesized individual components of polymyxins showed very strong
antimicrobial activity against E. coli, K. pneumoniae, P. aeruginosa and A. baumannii, except
for polymyxins B (NA), T (LꢀLeu10), and T (LꢀLeu10). Additionally, polymyxins A , A , M
1
0
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0
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1
2
and M with LꢀThr7 showed much stronger activity against E. coli, K. pneumoniae, and P.
2
aeruginosa than the controls polymyxins B and E. Polymyxins D , D , S , and S with DꢀSer3
1
2
1
2
showed noticeably enhanced antimicrobial activity against E. coli, K. pneumoniae, and A.
baumannii, but noticeably reduced potency against P. aeruginosa compared with the controls.
The increased hydrophobicity at the fatty acyl or position 7, position 10 amino acid residues
could decrease the in vitro activity. The renal cytotoxicities of E , E , E ꢀVal, A , M , D , and S
2
2
4
2
2
2
2
were significantly lower (P<0.01) than those of polymyxins B and E. In contrast, the
cytotoxicities of B , T , and T were significantly higher (P<0.01) than those of the controls. The
5
1
2
increased hydrophobicity at the fatty acyl or position 10 amino acid residue could increase the
renal cytotoxicity. The different types of these major lipopeptide components (B , E , A , M , D ,
2
2
2
2
2
and S ) and other different types of these components (E ꢀIle, E ꢀNva, E ꢀVal, and M ) with
2
2
2
2
1
equal or better antibacterial activity were examined to determine their acute toxicities in ICR
mice. The acute toxicities of all the tested compounds were significantly lower (P<0.01) than
those of the controls polymyxins B (6.9 mg/kg) and E (8.5 mg/kg). The different types of these
major lipopeptide components (B , E , A , M , D , and S ) with equal or better antibacterial
2
2
2
2
2
2
activity and lower cytotoxicity were examined to determine the nephrotoxicity in ICR mice. The
in vivo renal toxicities of polymyxins E , M , and S in ICR mice showed no significant change
2
2
2
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Journal of Medicinal Chemistry
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or mild damage compared with the saline control. Polymyxin individual components with
reduced renal cytotoxicities showed reduced acute toxicities compared with polymyxins B and E,
especially polymyxins D and S . However, differences in their in vivo renal toxicity were
2
2
1
1
1
1
1
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0
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observed, and further studies are urgently needed to investigate this phenomenon.
Overall trends of SAR and STR can be summarized according to the in vitro activity, renal
cytotoxicity and acute toxicity. A fatty acyl chain length of C9 (B ) with high hydrophobicity
5
noticeably reduced the antimicrobial activity compared with fatty acyl chain lengths of C7ꢀC8
(
B , B , B , B ), and the fatty acyl chain lengths of C8ꢀC9 (B , B , B ) obviously increased the
1 2 3 4 1 3 5
renal cytotoxicity compared with a chain length of C7 (B , B ). Additionally, whether position 6
2
4
was occupied by DꢀPhe (B) or DꢀLeu (E), or Leuꢀ7 was substituted with other hydrophobic amino
acids such as LꢀIle7, LꢀNva7 and LꢀVal7, did not substantially affect the antimicrobial potency
and acute toxicity. However, the substitution of LꢀLeu7 (E) with a more polar amino acid LꢀThr7
(M) noticeably increased the activity but did not affect the renal cytotoxicity and acute toxicity.
The configuration of Dab3 (A, M) did not affect the antimicrobial potency, renal cytotoxicity and
acute toxicity. Substitution of the positive charges of Dab3 or DꢀDab3 with a polar amino acid Dꢀ
Ser3 (D, S) enhanced the antimicrobial activity against E. coli, K. pneumoniae, and A. baumannii
but noticeably decreased the antimicrobial activity against P. aeruginosa. Additionally, the
individual components with four positive charges (D, S) decreased the renal cytotoxicity and
acute toxicity. However, the five positive charges of Dab residues were very important for the
potency against P. aeruginosa. The substitution of LꢀThr10 with a more hydrophobic amino acid
LꢀLeu10 (T) significantly reduced the antimicrobial activity and increased renal cytotoxicity.
The results of this study showed that there were obvious differences in biological activity
between the individual components, especially differences in toxicity. More importantly, this
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research found that the new compound S with four positive charges displayed a much more
2
potent activity against E. coli, K. pneumoniae, and A. baumannii as well as a noticeably reduced
toxicity compared with polymyxin B or E. This discovery that components with lower positive
charges could simultaneously increase the efficacy and decrease the toxicity is very exciting. In
addition, polymyxins A , M , and D also showed stronger activities and lower renal
0
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cytotoxicities than the control. These results indicate that S could be a new drug candidate with
2
similar or better antimicrobial potency and lower toxicity than the current clinical polymyxins B
and E. The initial SAR and STR studies indicate that further structural modifications could
concentrate on polymyxin S with four positive charges rather than the previous lead compound
polymyxin B with five positive charges.
EXPERIMENTAL SECTION
Synthetic materials and methods
Materials. The protected amino acids and HCTU were purchased from GL Biochem
(
Shanghai, China). PyAOP, HOAt, and Pd(PPh ) were purchased from Meryer (Shanghai,
3 4
China). Acetonitrile (HPLC grade), TFA (HPLC grade), allyl bromide, Aliquat 336, TEMPO,
S)ꢀ(+)ꢀ6ꢀmethylꢀ1ꢀoctanol, 7ꢀmethylꢀ7ꢀoctenoic acid, NMM, TIS, PhSiH , and DIPEA were
(
3
purchased from J&K Scientific (Beijing, China). Piperidine, diethyl ether anhydrous, hydrazine
hydrate solution (40%), NaHCO , AcOEt, PE, MeOH, and DCM were purchased from Beijing
3
Chemical Works (Beijing, China). DMF was purchased from Beijing Bomaijie (Beijing, China).
2CTC resin (loading=0.53 mmol/g) was purchased from Tianjin Nankaihecheng (Tianjin,
China).
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FmocꢀLꢀDabꢀOAll. FmocꢀLꢀDab(Boc)ꢀOH (38.7 g, 87.8 mmol) was added to water (180 mL)
containing NaHCO (7.4 g, 87.8 mmol). Aliquat 336 (10 g, 23 mmol) and allyl bromide (38 mL,
3
439 mmol) were dissolved in DCM (180 mL). The mixture was then added slowly into the
FmocꢀLꢀDab(Boc)ꢀOH aqueous solution. The reaction was stirred overnight at room temperature.
Subsequently, 180 mL water was added, and the aqueous phase was extracted by DCM (3×100
1
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0
mL). After drying over anhydrous Na SO and concentration under reduced pressure, FmocꢀLꢀ
2
4
Dab(Boc)ꢀOAll was obtained without purification. Then, the obtained residue was dissolved in
0% TFA/DCM (200 mL) and stirred under room temperature for 2 h. Finally, the organic
3
solvent was removed, and the FmocꢀLꢀDabꢀOAll was purified by flash chromatography
1
(DCM:MeOH=20:1) with an 89.8% yield (30 g) as a white powder. H NMR (400 MHz,
DMSO) δ (ppm): 8.12 (s, 2H), 7.98 (d, J = 7.9 Hz, 1H), 7.90 (d, J = 7.5 Hz, 2H), 7.72 (dd, J =
7
.3, 2.5 Hz, 2H), 7.43 (t, J = 7.4 Hz, 2H), 7.38 – 7.30 (m, 2H), 5.90 (ddt, J = 17.2, 10.5, 5.3 Hz,
1H), 5.32 (dd, J = 17.3, 1.6 Hz, 1H), 5.21 (dd, J = 10.5, 1.4 Hz, 1H), 4.61 (d, J = 5.0 Hz, 2H),
1
3
4
.39 – 4.28 (m, 2H), 4.28 – 4.18 (m, 2H), 2.92 – 2.78 (m, 2H), 2.17 – 1.85 (m, 2H). C NMR
(
101 MHz, DMSO) δ (ppm): 171.11, 156.08, 143.74, 143.70, 140.73, 132.24, 127.67, 127.09,
+
1
25.20, 125.18, 120.15, 117.79, 65.75, 65.06, 51.55, 46.59, 35.97, 28.46. LCꢀMS (ESI): [M+H]
81, calcd 381.
3
(S)ꢀ6ꢀMethyloctanoic Acid. Sodium hypochlorite (8 wt % in water, 15 mL) and a solution of
NaClO (5.2 g, 57.6 mmol) in water (15 mL) were added to a mixture of (S)ꢀ(+)ꢀ6ꢀmethylꢀ1ꢀ
2
octanol (4.15 g, 28.8 mmol), TEMPO (0.45 g, 2.88 mmol), aqueous NaH PO (0.67 M, 150 mL),
2
4
and acetonitrile (150 mL). The mixture was stirred for 12 h at room temperature, and a saturated
aqueous solution of Na S O (60 mL) was added. The resulting mixture was extracted with
2
2
3
AcOEt (2×100 mL), and the combined extracts were washed with 1 N HCl (50 mL), dried over
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anhydrous Na SO , filtered, and concentrated under reduced pressure. The obtained residue was
2
4
purified by flash chromatography (AcOEt:PE=1:10) with a 92% yield (4.2 g) as a colorless oil.
2
0
1
[α]D +8.4° (c=1.0, CHCl ). H NMR (400 MHz, CDCl ) δ (ppm): 11.64 (br s, 1H), 2.35 (t, J =
3
3
0
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.5 Hz, 2H), 1.68 – 1.55 (m, 2H), 1.39 – 1.25 (m, 5H), 1.20 – 1.05 (m, 2H), 0.87 – 0.84 (m, 6H).
13
C NMR (101 MHz, CDCl ) δ (ppm): 180.71, 36.20, 34.25, 34.22, 29.46, 26.60, 25.03, 19.15,
3
ꢀ
1
1.39. LCꢀMS (ESI): [MꢀH] 157, calcd 157.
7
ꢀMethyloctanoic Acid. 7ꢀMethylꢀ7ꢀoctenoic acid (0.81 g, 5.2 mmol) was dissolved in MeOH
(20 mL), and palladium on carbon (10%, 200 mg) was added to the vessel. The hydrogenation
vessel was subjected to H (50 psi) at room temperature for 2 h. The resulting mixture was
2
filtered and concentrated under reduced pressure. The obtained residue was dried under vacuum
1
at room temperature for 1 h to give the compound (0.81 g, 98% yield) as a colorless oil. H NMR
(
400 MHz, CDCl ) δ (ppm): 11.50 (s, 1H), 2.39 (t, J = 7.5 Hz, 2H), 1.73 – 1.63 (m, 2H), 1.56
3
(dp, J = 13.2, 6.7 Hz, 1H), 1.41 – 1.29 (m, 4H), 1.27 – 1.16 (m, 2H), 0.90 (d, J = 6.6 Hz, 6H).
1
3
C NMR (101 MHz, CDCl ) δ (ppm): 180.57, 38.76, 34.16, 29.32, 27.93, 27.01, 24.72, 22.61.
3
ꢀ
LCꢀMS (ESI): [MꢀH] 157, calcd 157.
Synthesis of polymyxins A to T . Step 1: 0.5 mmol 2CTC resin (0.95 g, 0.53 mmol/g) was
1
2
swelled in 50% DCM/50% DMF for 5 min before use. Then, FmocꢀLꢀDabꢀOAll (2 mmol, 4 eq.),
DIPEA(4 mmol, 8 eq.) and DCM (8 mL) were added to react with the resin for 2 h. The resin
was capped with 0.5 mL MeOH to quench the remaining 2ꢀchlorotrityl chloride, and compound
1 was obtained.
Step 2: Linear peptide chains were assembled by standard solidꢀphase synthesis using a
CS136XT CSBio Peptide Synthesizer (CSBio, America). The Fmoc group was removed by
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treatment with 20% piperidine in DMF twice (5 min, 10 min), followed by washing with DMF
and DCM. Generally, the subsequent coupling was carried out using a solution of 4 eq. of the
appropriate amino acid or corresponding fatty acid, 4 eq. of HCTU, and 8 eq. of DIPEA in DMF
at room temperature after Fmoc removal. Each coupling step required 1 h, and the resin was
washed by DMF and DCM. The peptide was capped with the corresponding fatty acid to achieve
compound 2.
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Step 3: The Dde group was removed by treatment with 3% hydrazine hydrate in DMF for 30
min two times, and compound 3 was obtained.
Step 4: FmocꢀLꢀThr(tBu)ꢀOH or FmocꢀLꢀLeuꢀOH was coupled to the sideꢀchain amino of
Dab4 to achieve compound 4. The subsequent coupling was carried out using a solution of 4 eq.
of the appropriate amino acid, 4 eq. of HCTU, and 8 eq. of DIPEA in DMF at room temperature
for 1 h after the removal of the Dde group.
Step 5: The Fmoc group was removed by 20% piperidine in DMF twice (5 min, 10 min), and
the allyl group was removed by treatment with PhSiH (7 eq.) and Pd(PPh ) (0.2 eq.) in 50%
3
3 4
DCM/50% DMF for 2 h. After deprotection, the resin was washed with 0.5% sodium
diethyldithiocarbamate in DMF and DMF several times, and compound 5 was obtained.
Step 6: The final cyclization step was carried out with 4 eq. of PyAOP, 4 eq. of HOAt and 8
eq. of NMM for 3 h, and compound 6 was obtained.
Step 7: The cleavage reagent TFA:TIS:H O (95:2.5:2.5) was added into the dry resin
2
prewashed with DMF and DCM, and the cleavage was carried out for 2 h. The crude peptides
were obtained by precipitation with cold diethyl ether and then purification by using semiꢀ
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preparative HPLC to afford the polymyxin TFA salt 7. Total yields were approximately 30%
based on the initial 2CTC resin.
Purification and Analyses. Analytical HPLC was performed on an Agilent Technologies
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200 Series Instrument. The peptides were analyzed using an Eclipse XDBꢀC18 (4.6×150 mm, 5
µm) under a 214 nm UV detector. The gradient mode was 10ꢀ70% solvent B over 25 min, 70ꢀ
0% solvent B over 0.5 min, and 10% solvent B over 4.5 min at a flow rate of 0.8 mL/min
solvent A was 0.08% TFA/water and solvent B was 0.08% TFA/acetonitrile). The semiꢀ
1
(
preparative HPLC was performed on a Shimadzu Prominence HPLC LCꢀ20AT system. The
peptides were purified by using a Shimꢀpack PREPꢀODS (20×250 mm ID, 10 µm) under a 214
nm UV detector. The gradient was 10ꢀ70% solvent B over 30 min at a flow rate of 6.0 mL/min
(solvent A was 0.08% TFA/water and solvent B was 0.08% TFA/acetonitrile). The synthesized
peptide was purified by using semiꢀpreparative HPLC to afford the polymyxin TFA salt with a
purity >95%. LCꢀMS analysis was carried out with a Shimadzu LCꢀMS 2020 system using an
electrospray ionization source (ESI) and a singleꢀquadrupole mass analyzer. A Shim–pack VPꢀ
ODS (2.0×150 mm, 5 µm) was used with a gradient of 10ꢀ90% solvent B over 5 min at a flow
rate of 0.5 mL/min (solvent A was 0.1% formic acid in water and solvent B was 0.1% formic
acid in acetonitrile). Mass spectra were acquired in the negative or positive ion mode with a scan
range of m/z 100ꢀ800. The optical rotations of individual components were measured by a
Rudolph Research Analytical Autopol IV. High resolution mass spectra (HRMS) were taken on a
LTQ Orbitrap XL instrument (Thermo Scientific, USA). All purified peptides were analyzed
using a Bruker 500 MHz spectrometer. Small molecule intermediates were analyzed with a
Bruker 400 MHz spectrometer. (Bruker Bioscience, USA).
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Minimum Inhibitory Concentrations Determination. Minimum inhibitory concentrations
(
MICs) of these individual components were determined by the serial twoꢀfold agar dilution
method as described by the Clinical and Laboratory Standards Institute. Bacteria used in this
study were standard isolates from ATCC or clinical isolates of China. Bacteria were cultured in
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MuellerꢀHinton broth, and the inoculum was about 10 colony forming units (cfu)/spot. The final
concentrations of these components were ranged from 0.03ꢀ128 µg/mL. Culture plates were
incubated at 35 °C for 18 h and the MICs were then recorded. The MIC was defined as the
lowest concentration of the antimicrobial agent at which visible growth of the bacteria was
prevented. The MICs were the representative results of at least two independent experiments, in
the case of different results for the two experiments, a third experiment was conducted, and the
confirmed results were given.
Cytotoxicity assay. Cytotoxicity to Vero cells was determined using the MTT method. In
3
brief, Vero cells were seeded at 2x10 cells/well into 96ꢀwell plates and incubated for 24 h at 37
°
C, 5% CO . Then, compounds were added into each well. After incubation for 72 h, MTT was
2
added to the medium (final to 50 µL) and incubated for 2 h. After removing the culture medium,
00 µL of dimethyl sulfoxide was added. The plates were read using an enzymeꢀlinked immuneꢀ
2
sorbent assay plate reader at 570 nm. The viability of untreated cells was set as 100% and the
viability in other groups was calculated by comparing the optical density reading with the
control. Cell Cytotoxicity (CC ) was calculated with GraphPad Prism 5. The results were
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expressed as the mean ± SD from three independent experiments, each performed in triplicate.
Polymyxins B and E were used as positive control.
Acute toxicity to mice. The acute toxicity was evaluated by intravenous administration of
varying doses of individual polymyxin components as well as polymyxins B and E into ICR
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mice. The ICR mice (weight, 18 to 22 g; age=4 weeks; n=10 mice per group; 5 male, 5 female)
were purchased from Vital River Laboratories (Beijing, China). Four to five doses (four to five
groups) were used for each compound. Survival was monitored daily for 7 days, and the amount
of test compounds per kg of mouse body weight that constituted a lethal dose for 50% of the
animals (LD ) was calculated. The LD values were calculated according to the Bliss test
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protocol. All animal experiments were performed in full compliance with the protocols approved
by the Animal Ethics Committee of the Institute of Medicinal Biotechnology (Beijing, China).
Renal toxicity to mice. Solutions of individual polymyxin components and polymyxin E in
saline (0.25 mg/mL) were stored at 4 °C before use. The ICR mice (weight, 18 to 22 g; age=4
weeks) were purchased from Vital River Laboratories (Beijing, China). The mice (n=8 mice per
group; 4 male, 4 female) were intravenously administered the component at a dose of 2.5 mg/kg
twice daily (8 h apart) for 7 days until an accumulated dose of 35 mg was achieved. Two hours
after the last dose, the mice were sacrificed. Their kidneys were collected and placed in 10%
formalin in a 10 mL plastic tube. The formalinꢀfixed kidneys were dehydrated and embedded in
paraffin wax. The sections (5 mm) were counterstained with hematoxylin and eosin (H&E) and
examined using a light microscope. The whole sections were examined by a pathologist (P. A.
H.) who was blinded to the treatment groups. The SQS of each group was calculated according
35ꢀ36
the grades and the percentages of the kidney slices affected.
All animal experiments were
performed in full compliance with the protocols approved by the Animal Ethics Committee of
the Institute of Medicinal Biotechnology (Beijing, China).
Characterization of synthetic polymyxins A to T . Compound A . Yield 30%, white solid.
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+
Molecular Formula C H O N . HRMS: [M+H] 1157.73788, calcd 1157.73647. HPLC: t =
5
1
96 14 16
R
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10.16 min, purity 97.3%. [α]D ꢀ30.5° (c=0.15, 12% AcOH). H NMR (500 MHz, D O) δ (ppm):
2
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.57 – 4.47 (m, 3H), 4.47 – 4.40 (m, 3H), 4.36 (td, J = 11.6, 5.2 Hz, 3H), 4.32 – 4.20 (m, 3H),
.18 (d, J = 5.0 Hz, 1H), 3.46 – 3.35 (m, 1H), 3.23 – 2.98 (m, 11H), 2.34 (t, J = 7.3 Hz, 2H), 2.32
4
– 1.95 (m, 11H), 1.91 (dt, J = 14.1, 6.5 Hz, 1H), 1.75 – 1.51 (m, 5H), 1.38 – 1.25 (m, 5H), 1.24 –
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.16 (m, 9H), 1.12 (dt, J = 14.1, 7.0 Hz, 2H), 0.97 (d, J = 5.6 Hz, 3H), 0.92 (d, J = 5.5 Hz, 3H),
.83 (t, J = 7.0 Hz, 6H).
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Compound A . Yield 31%, white solid. Molecular Formula C H O N . HRMS: [M+H]
2
50 94 14 16
2
5
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143.72282, calcd 1143.72082. HPLC: t = 9.28 min, purity 95.8%. [α]D ꢀ33.1° (c=0.15, 12%
R
1
AcOH). H NMR (500 MHz, D O) δ (ppm): 4.55 – 4.31 (m, 9H), 4.30 – 4.22 (m, 3H), 4.16 (d, J
2
=
5.0 Hz, 1H), 3.47 – 3.33 (m, 1H), 3.25 – 2.98 (m, 11H), 2.39 – 1.95 (m, 13H), 1.95 – 1.85 (m,
1H), 1.74 – 1.46 (m, 6H), 1.30 (dt, J = 15.4, 7.5 Hz, 2H), 1.24 – 1.15 (m, 11H), 0.97 (d, J = 5.8
Hz, 3H), 0.92 (d, J = 5.7 Hz, 3H), 0.84 (d, J = 6.6 Hz, 6H).
+
Compound B . Yield 32%, white solid. Molecular Formula C H O N . HRMS: [M+H]
1
56 98 13 16
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1203.75736, calcd 1203.75720. HPLC: t = 11.56 min, purity 97.7%. [α]D ꢀ60.0° (c=0.15, 12%
R
1
AcOH). H NMR (500 MHz, D O) δ (ppm): 7.41 (t, J = 7.2 Hz, 2H), 7.38 – 7.33 (m, 1H), 7.28
2
(d, J = 7.1 Hz, 2H), 4.58 (t, J = 8.3 Hz, 1H), 4.55 – 4.47 (m, 3H), 4.37 (d, J = 4.3 Hz, 1H), 4.33 –
4.19 (m, 7H), 3.40 – 3.31 (m, 1H), 3.23 – 3.04 (m, 11H), 2.93 – 2.85 (m, 1H), 2.85 – 2.75 (m,
1H), 2.34 (t, J = 7.3 Hz, 2H), 2.29 – 1.86 (m, 12H), 1.66 – 1.55 (m, 2H), 1.50 (ddd, J = 13.5, 9.4,
3.9 Hz, 1H), 1.41 (dd, J = 18.3, 7.3 Hz, 1H), 1.37 – 1.25 (m, 5H), 1.22 (t, J = 6.4 Hz, 6H), 1.12
(td, J = 13.1, 7.2 Hz, 2H), 0.84 (t, J = 6.9 Hz, 7H), 0.77 (d, J= 5.0 Hz, 3H), 0.70 (d, J = 5.7 Hz,
3H).
+
Compound B . Yield 32%, white solid. Molecular Formula C H O N . HRMS: [M+H]
2
55 96 13 16
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1
189.74287, calcd 1189.74155. HPLC: t = 10.87 min, purity 98.3%. [α]D ꢀ62.6° (c=0.15, 12%
R
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