Synthesis and antibacterial activity of tripropeptin C derivatives modified at the carboxyl groups

Full text of DOI:10.1038/ja.2013.128

The Journal of Antibiotics (2014) 67, 265–268
2014 Japan Antibiotics Research Association All rights reserved 0021-8820/14
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&
NOTE
Synthesis and antibacterial activity of tripropeptin C
derivatives modified at the carboxyl groups
Sehei Hirosawa1, Yoshiaki Takahashi1, Hideki Hashizume2, Toshiaki Miyake1 and Yuzuru Akamatsu2
The Journal of Antibiotics (2014) 67, 265–268; doi:10.1038/ja.2013.128; published online 4 December 2013
Keywords: antibacterial activity; MRSA; synthesis; tripropeptin
In recent years, there has been a worrying increase in the number of
In TPPC, there are five hydroxyl groups, two carboxyl groups and a
infections caused by drug-resistant bacteria. Therapeutic agents guanidine group. To investigate the detailed structure–activity rela-
traditionally used to treat these bacterial infections are becoming tionships of TPPC, we have focused our efforts on exploring these
increasingly ineffective.1,2 There is therefore an urgent need for the functional groups. As outlined in Scheme 1, this paper describes the
discovery and development of new antibacterial agents that are role of carboxylic groups for the antibacterial activity. In this initial
effective against multidrug-resistant bacteria, as well as being efforts, the modification of the two carboxyl groups of TPPC in the
structurally different from the existing agents and capable of form of esters and amides and their antibacterial activities are
exerting their inhibitory activity according to a novel mode of action.3 described.
We have screened for novel antibiotics that are active against drug-
The esterification reaction was performed under mild reaction
resistant bacteria from microbial origins. As part of this program, we conditions, where TPPC was treated with an excess of (trimethylsi-
have discovered a structurally analogous mixture of tripropeptins,4–6 lyl)diazomethane (TMSCHN2) in MeOH to give the two separable
which are a group of novel cyclic lipodepsipeptide antibiotics, in the mono-methyl esters 1 and 2 (OCH3 signal at d 3.68 for 1 and at
cultured cells and broth of the soil bacterium Lysobacter sp. strain d 3.53 for 2, refer to Supplementary Information) in 15% and 28%
BMK333–48F3. Tripropeptins consist of a cyclic octapeptide core and yields, respectively, together with two unidentified products. In
a fatty acyl side chain. Based on differences in their fatty acyl side contrast, the reaction of TPPC with diphenyldiazomethane (Ph2CN2,
chains, tripropeptins can be classified into six different components 1.5 equiv. relative to the TPPC) afforded the two mono-diphenyl-
(that is, A–E and Z).
methyl esters 3 (25%) and 4 (18%), together with a 50% recovery of
Its major component, tripropeptin C (TPPC), showed potent TPPC. The extension of the reaction time, as well as an increase in the
antimicrobial activities toward a variety of different Gram-positive charge of Ph2CN2 did not lead to any improvement in the yield of the
pathogens, including methicillin-resistant Staphylococcus aureus corresponding esters. Interestingly, the reaction of the p-toluenesul-
(MRSA), vancomycin-resistant Enterococcus faecalis/faecium (VRE) fonate salt13 of TPPC with a large excess of TMSCHN2 or Ph2CN2
and penicillin-resistant Streptococcus pneumoniae.4,5,7 Plusbacins8,9 gave the bis-methyl ester 5 or bis-diphenylmethyl ester 6 as the major
and empedopeptin,10,11 which are cyclic lipodepsipeptide antibiotics product, respectively. Based on these results, it was suggested that
structurally related to TPPC, have also been reported to exhibit potent TPPC behaved in a similar way to amino acids, in which it existed in
activities toward Gram-positive bacteria. TPPC also showed excellent the zwitterionic form under these reaction conditions.
in vivo therapeutic efficacy in a mouse-MRSA septicemia model when
administered intravenously and its ED50 value was comparable to that by NMR analysis with HMBC and NOESY techniques. Therefore, we
of vancomycin. Furthermore, TPPC exhibited favorable attempted to confirm their structures by amino-acid analysis using
toxicological profile that included no acute toxicity and no 14-day Marfey’s method.14 Using this method, it should be possible to detect
repeated toxicities in mice when administered intravenously.7
threo-b-hydroxy-D-aspartic acid from the methyl ester at the C-4
It was difficult to determine the structures of compounds 1 and 2
a
TPPC inhibited peptidoglycan biosynthesis in a different way than position, but not from the methyl ester at the C-16 position. Thus,
the drugs that currently target peptidoglycan biosynthesis, including compounds 1 and 2 were converted to the corresponding alcohols 12
vancomycin and bacitracin, and showed no cross-resistance to these and 13, respectively, following the reduction of their ester and lactone
drugs.12 Based on its excellent biological properties, TPPC is moieties with NaBH4 as shown in Scheme 2.
considered to be a promising novel class of antibiotic against
MRSA and vancomycin-resistant Enterococcus faecalis/faecium.
Compounds 12, 13 and TPPC were then hydrolyzed in refluxing 6
M aqueous HCl for 20 h to give the corresponding constituent amino
1Institute of Microbial Chemistry (BIKAKEN) Hiyoshi, Kawasaki, Japan and 2Institute of Microbial Chemistry (BIKAKEN), Tokyo, Japan
Correspondence: Dr Y Takahashi, Institute of Microbial Chemistry (BIKAKEN) Hiyoshi, 3-34-17, Ida, Nakahara-ku, Kawasaki, Kanagawa 211-0035, Japan.
E-mail: takashow@bikaken.or.jp
Received 12 June 2013; revised 21 August 2013; accepted 1 November 2013; published online 4 December 2013

Synthesis and antibacterial activity of tripropeptin C derivatives
S Hirosawa et al
266
Scheme 1 The synthetic procedures for preparation of TPPC derivatives modified at the carboxyl groups.
acids. The acidic hydrolysates were treated with 1-fluoro-2,4-dinitro- 12. These results therefore revealed that 1 and 2 were the 16- and
phenyl-5-L-alanine amide to give the corresponding Marfey’s deriva- 4-methyl esters of TPPC, respectively.
tives, which were analyzed by HPLC. The amino-acid derivatives were
The structures of 3 and 4 were determined by converting these
identified through a comparison of their retention times with those of compounds to the corresponding mono-methyl esters. Treatment of the
the Marfey’s derivatives of the authentic amino acids derived from p-toluenesulfonate salts of 3 and 4 with (trimethylsilyl)diazomethane in
TPPC and standard threo-b-hydroxy-L-aspartic acid. As expected, the MeOH, followed by the removal of the diphenylmethyl ester portion
Marfey’s derivative of threo-b-hydroxy-D-aspartic acid was detected using trifluoroacetic acid, gave compounds 2 and 1, respectively.
from 13. In contrast, the threo-b-hydroxy-L-aspartic acid and threo-b- Preparation of amides was achieved by using the p-toluenesulfonate
hydroxy-D-aspartic acid Marfey’s derivatives were not detected from salts of the mono-diphenylmethyl ester 3 or 4. The condensation
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Synthesis and antibacterial activity of tripropeptin C derivatives
S Hirosawa et al
267
Scheme 2 Reductive products (12 and 13) for amino-acid analysis. (Threo-b-hydroxy-D-aspartic acid portion in 13 is highlighted.)
reactions were effectively conducted with the appropriate amines Table 1 Antibacterial activities of ester and amide derivatives of
using 1-hydroxybenzotriazole and N,N0–dicyclohexylcarbodiimide.
Subsequent removal of the protecting group with trifluoroacetic acid
TPPC
MIC (mg mlꢀ1
)
afforded amides 7–11 as the p-toluenesulfonate salts in 52–71% yields
from 3 or 4.
Methicillin-sensitive Staphylococ- Methicillin-resistant Staphylococ-
Table 1 presents the antibacterial activities of the ester and amide
derivatives of TPPC against the clinical isolates of methicillin-sensitive
S. aureus (MSSA, n ¼ 10) and methicillin-resistant S. aureus (MRSA,
n ¼ 10).
cus aureus (MSSA)
cus aureus (MRSA)
(clinical isolates, 10 strains)
(clinical isolates, 10 strains)
The antibacterial activities of the mono-esters 1–4 were less than
that of TPPC. Moreover, the bis-esters 5 and 6 suffered substantial
reductions in their antibacterial activity. TPPC is known to bind to
undecaprenyl pyrophosphate via a calcium ion, with the resulting
complex inhibiting peptidoglycan biosynthesis.12 In contrast,
however, it was not possible for the bis-methyl ester 5 to form a
biologically important complex such as the one formed in the case of
TPPC, as shown in Supplementary Figure S1. Based on these results,
it became clear that the two free carboxyl groups of TPPC were
critical for the binding of TPPC to the target undecaprenyl pyropho-
sphate molecule, and important for the expression and strength of its
antibacterial activity.
Compound
Range
MIC50
MIC90
Range
MIC50
MIC90
1
3.13–6.25
6.25–12.5
1.56–3.13
12.5–50
100
3.13
6.25
3.13
12.5
100
100
25
6.25
12.5
3.13
50
6.25–12.5
12.5
6.25
12.5
3.13
25
12.5
12.5
6.25
50
2
3
3.13–6.25
25–50
4
5
100
100
25
100
100
100
25
100
100
50
6
25–100
12.5–50
50
50–100
6.25–50
25–50
7
8
50
50
50
50
9
3.13–12.5
12.5–50
50
12.5
50
12.5
50
1.56–12.5
6.25–50
25–50
6.25
50
12.5
50
10
11
TPPC
50
50
50
50
In contrast to esters 1–6, amides 7–10, which were synthesized via
the introduction of different amino-acid groups at the C-4 or C-16
position, contained two free carboxyl groups. We were interested in
these compounds because they possessed the two free carboxyl
groups, which may be meaningful and it was expected that these
0.78–1.56
0.78
0.78
0.39–1.56
0.78
1.56
Abbreviation: TPPC, tripropeptin C.
In conclusion, we have made a series of modifications to the
groups would impart high levels of antibacterial activity. Unfortu- carboxyl groups of TPPC, and synthesized the corresponding esters
nately, however, the activities of these compounds greatly reduced and amides as a part of the structure–activity relationship study. The
compared with TPPC. Furthermore, the antibacterial activity of structures of mono-esters obtained were confirmed by amino-acid
amide 11, which had an amino side chain and only one free analysis using Marfey’s method. Unfortunately, all of the modifica-
carboxylic acid group, was much lower than the activities of the tions reported in the current paper led to a reduction in the
corresponding amides 7 and 9, which had the two carboxylic acid antibacterial activity compared with TPPC and therefore highlighted
groups.
the importance of the two free carboxyl groups to the activity of
The Journal of Antibiotics

Synthesis and antibacterial activity of tripropeptin C derivatives
S Hirosawa et al
268
TPPC. As a follow-up to this program, work toward modifying the
terminal guanidine group of the arginine residue in TPPC is currently
underway in our laboratory.
4
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7
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of Microbial Chemistry (BIKAKEN), Hiyoshi, for constructive discussions. We
are also grateful to Kunio Inoue of the Institute of Microbial Chemistry
(BIKAKEN), Tokyo, for determining the spectra of the antibacterial
compounds and Dr Ryuichi Sawa of the Institute of Microbial Chemistry
(BIKAKEN), Tokyo, for the MS experiments.
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Supplementary Information accompanies the paper on The Journal of Antibiotics website (http://www.nature.com/ja)
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