Muraminomicins, novel ester derivatives: in vitro and in vivo antistaphylococcal activity

Article abstract of DOI:10.1038/s41429-019-0235-3

Novel muraminomicin derivatives with antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA) were synthesized by esterification of the hydroxy group on the diazepanone ring of muraminomicin Z₁. Compound 1b (DS14450354)

Full text of DOI:10.1038/s41429-019-0235-3

The Journal of Antibiotics
https://doi.org/10.1038/s41429-019-0235-3
SPECIAL FEATURE: ARTICLE
Muraminomicins, novel ester derivatives: in vitro and in vivo
antistaphylococcal activity
Yoshiko Kagoshima1 Akane Tokumitsu2 Takeshi Masuda3 Eiko Namba4 Harumi Inoue5 Chika Sugihara6
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Mizuka Yokoyama7 Yuko Yamamoto8 Keiko Suzuki1 Kouki Iida8 Akihiro Tamura9 Yoko Fujita9
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Toshio Takatsu9 Toshiyuki Konosu10 Tetsufumi Koga4
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Received: 8 June 2019 / Revised: 9 August 2019 / Accepted: 29 August 2019
© The Author(s), under exclusive licence to the Japan Antibiotics Research Association 2019
Abstract
Novel muraminomicin derivatives with antimicrobial activity against methicillin-resistant Staphylococcus aureus
(MRSA) were synthesized by esterification of the hydroxy group on the diazepanone ring of muraminomicin Z1.
Compound 1b (DS14450354) possessed a diheptoxybenzyl-β-Alanyl-β-Alanyl group and exhibited minimum inhibitory
concentrations (MICs) against MRSA comparable to those against methicillin-susceptible S. aureus (MSSA). The MICs
that inhibited 50 and 90% of the strains were 1 and 2 μg/mL, respectively. Compound 1a (DS60182922) possessed an
aminoethylbenzoyldodecylglycyl moiety and showed bactericidal activity against MSSA Smith. The bactericidal activity
of 1a against MRSA 10925 was comparatively lower, whilst 1b exhibited dose-dependent bactericidal activity against
MRSA 10925. The mutation frequency of 1b was lower than that of 1a. An amino acid substitution (F226I) was
observed in MraY mutants isolated from culture plates containing 1a or 1b. Subcutaneous 1a and 1b administration
showed good therapeutic efficacy in murine systemic infection models with MSSA Smith and MRSA 10925, comparable
to that of vancomycin, suggesting that the novel muraminomicin derivatives may be effective therapeutic agents against
MRSA that warrant further investigation. A scheme for the formulation of the key ester intermediate, requiring no HPLC
preparation, was also established.
Introduction
tissue infections to life-threatening endocarditis, pneumo-
nia, and osteomyelitis [1, 2]. Recently, the number of bac-
teria resistant to existing antimicrobials has increased in
clinical practice and has become a major therapeutic pro-
blem. Methicillin-resistant S. aureus (MRSA) exhibits
resistance to almost all therapeutic β-lactams and other
classes of antibiotics. Several anti-MRSA drugs have been
launched in the past few decades, such as linezolid [3],
daptomycin [4], and ceftaroline [5]; however, vancomycin
Staphylococcus aureus is a leading cause of bacterial
infections worldwide, ranging from minor skin and soft
Supplementary information The online version of this article (https://
doi.org/10.1038/s41429-019-0235-3) contains supplementary
material, which is available to authorized users.
* Tetsufumi Koga
6
Biologic Research Department, Daiichi Sankyo RD Novare Co.,
Ltd., Tokyo, Japan
koga.tetsufumi.zj@daiichisankyo.co.jp
7
1
Medical Science Department, Daiichi Sankyo Co., Ltd.,
Medicinal Chemistry Research Laboratories, Daiichi Sankyo Co.,
Tokyo, Japan
Ltd., Tokyo, Japan
8
2
Process Technology Research Laboratories, Daiichi Sankyo Co.,
Biological Research Laboratories, Sankyo Co., Ltd., Tokyo, Japan
Ltd., Kanagawa, Japan
3
Biologics Technology Research Laboratories, Daiichi Sankyo Co.,
Ltd., Gunma, Japan
9
Organic Synthesis Department, Daiichi Sankyo RD Novare Co.,
Ltd., Tokyo, Japan
4
Vaccine Research Laboratories, Daiichi Sankyo Co., Ltd.,
Tokyo, Japan
10
Research Function, Daiichi Sankyo Co., Ltd., Tokyo, Japan
5
Technology Department, Daiichi Sankyo Biotec Co., Ltd.,
Saitama, Japan

Y. Kagoshima et al.
remains the most important first-line therapy for severe
MRSA infection. Furthermore, the emergence of MRSA
with reduced susceptibility to vancomycin [6] as well as
linezolid [7] and daptomycin resistance [8] has been
reported; therefore, novel drugs to treat antibiotic-resistant
infections and drugs with new mechanisms of action are
urgently needed.
We hypothesized that chemically modifying the
3″′-hydroxyl diazepanone ring of muraminomicin Z1 with
a lipid moiety to modulate its cell membrane permeability
whilst retaining its MraY potency could improve its
antimicrobial potency. Here, we report an efficient semi-
synthetic methodology for obtaining a key 3″′-hydroxyl
muraminomicin Z1 ester precursor by selectively pro-
tecting the muraminomicin core structure and hydrolyzing
the fatty acid side chain, which does not require mur-
aminomicin HPLC purification. By screening a series of
muraminomicin derivatives for therapeutic efficacy in the
murine infection model, we selected DS60182922 (1a)
and DS14450354 (1b), which possess an ami-
noethylbenzoyldedecylglycyl moiety, and a diheptox-
ybenzyl-β-Ala- β-Ala moiety, respectively (Fig. 1). In this
study, we evaluated the in vitro and in vivo activities of
these muraminomicin derivatives against S. aureus.
Bacterial phospho-N-acetylmuramylpentapeptide trans-
locase (MraY: EC 2.7.8.13) is an enzyme involved in the
second stage of the peptidoglycan biosynthetic pathway that
constitutes the bacterial cell-wall [9]. The enzyme is
regarded as a promising target for novel antibiotics since it
is essential for viability [10, 11] and has no counterparts in
mammalian cells. MraY is the target of many naturally
produced nucleoside antibiotics, such as tunicamycin [12],
liposidomycins [13], capuramycins [14], mureidomycins
[15], pacidamycins [16], naspamycins [17], muraymycins
[18], caprazamycins [19], A-102395 [20], A-94964 [21],
and A-90289s [22]. Structure-function studies have been
carried out on these nucleosides [9, 23], finding that the
structures of each family have a specific antibacterial
spectrum. For example, mureidomycin A is active against
Pseudomonas aeruginosa [15, 24], liposidomycin and
capuramycin are active against Mycobacterium spp.
[13, 14], muraymycin B1, tunicamycin, and mur-
aminomicins are active against gram-positive bacteria
[12, 18, 25], and caprazamycin is active against Myco-
bacterium spp. and gram-positive bacteria [19]. A series of
liposidomycin and muraymycin analogs were shown to
exhibit broader spectrums of activity against Escherichia
coli and P. aeruginosa, respectively [26, 27]. Consequently,
the antibacterial spectrum could be a result of effective
uptake into the target organism (compounds that are not
efflux pump substrates) and structural features. Numerous
chemical modification approaches have been reported for
improving and expanding antibacterial activities, including
liposidomycin analogs with simple amines and amides
replacing its diazepanone ring [26], muraymycin derivatives
possessing two functional groups, a lipophilic side chain,
and a guanidine group at the accessory moiety [27], and
caprazamycin derivatives which are 1″′-alkylanilide analogs
of caprazene [28].
Materials and methods
General
1H nuclear magnetic resonance (NMR) spectra were mea-
sured using a Varian Mercury 400 spectrometer at 400 MHz
in CDCl3 or CD3OD, with 0.03% tetramethylsilane as an
internal standard. Mass spectra were obtained using an
Agilent Technologies Agilent 1100 series LC/MS.
Benzhydryl 2-[[(2S,3S,5R)-5-(2,4-dioxopyrimidin-1-
yl)-3-hydroxy-tetrahydrofuran-2-yl]-[4-hydroxy-5-
[[(4-nitrophenyl)methoxycarbonylamino]methyl]
tetrahydrofuran-2-yl]oxy-methyl]-1,4-dimethyl-3-
oxo-6-[(E)-tetradec-2-enoyl]oxy-1,4-diazepane-5-
carboxylate (2)
To a solution of tetrahydrofuran (300 mL), water (100 mL),
partially purified muraminomicin complex obtained by
ethyl acetate extraction following reverse extraction with
0.2 M phosphate buffer (containing muraminomicin F 5.8
g, 4.89 mmol, calculated by HPLC analysis) was added p-
nitrobenzyloxycarbonyl chloride (4.21 g, 19.5 mmol) and
sodium carbonate (2.07 g, 19.5 mmol) at 0 °C. After stir-
ring at 0 °C for 2 h, the reaction mixture was quenched with
aqueous potassium hydrogen sulfate solution and ethyl
acetate extraction was performed three times. The com-
bined organic phases were washed with water three times
and with saturated aqueous sodium chloride solution once,
then dried over sodium sulfate and filtrated. The solvent
was removed under reduced pressure and the resultant
residue was used in the next reaction without further pur-
ification. Diphenyldiazomethane (15.0 g, 75.7 mmol) was
added to a solution of p-nitrobenzyloxycarbonyl in
Muraminomicins are novel liponucleoside antibiotics
isolated from the culture broth of Streptosporangium sp.
SANK 60501, which are composed of a 2-deoxyuridine
moiety, a 5-amino-D-2-deoxyribose moiety, a diazepa-
none ring system, and a fatty acid moiety [25]. Figure 1
shows the principal member of the muraminomicin
family, muraminomicin F, and related minor members,
muraminomicins G and H. Under mild alkaline hydro-
lysis, muraminomicins form the deacyl products, mur-
aminomicin Z1 and Z2, which inhibit MraY but exhibit no
antimicrobial activity in whole-cell assays (Fig. 1) [25].

Muraminomicins, novel ester derivatives: in vitro and in vivo antistaphylococcal activity
Fig. 1 Muraminomicins, deacyl
core structure compounds, and
novel derivatives
dichloromethane (150 mL) at room temperature. After
stirring at room temperature overnight, the solvent was
removed under reduced pressure and the resultant residue
was purified by flash column chromatography on neutral
silica gel (0–10% methanol in ethyl acetate) to produce the
title compound (12.0 g, 45%) as a pale yellow amorphous
substance.
(8H, m), 7.33−7.55 (3H, m), 7.68−7.75 (1H, m), 8.15−8.23
(2H, m), 9.46−9.56 (1H, m). MS (FAB) m/z 1097 (M + H)+.
Benzhydryl 2-[[(2S,3S,5R)-5-(2,4-dioxopyrimidin-1-
yl)-3-[(4-methoxyphenyl)methoxy]tetrahydrofuran-
2-yl]-[4-[(4-methoxyphenyl)methoxy]-5-[[(4-
nitrophenyl)methoxycarbonylamino]methyl]
tetrahydrofuran-2-yl]oxy-methyl]-6-hydroxy-1,4-
dimethyl-3-oxo-1,4-diazepane-5-carboxylate (7)
1H-NMR (400 MHz, CDCl3) δ: 0.85-0.90 (3H, m),
1.20−1.50 (16H, m), 1.80−2.10 (5H, m), 2.15−2.30
(3H, m), 2.40−2.50 (3H, m), 3.08−3.25 (2H, m), 3.25−3.33
(3H, m), 3.60−3.80 (3H, m), 4.00−4.20 (2H, m), 4.25−
4.30 (1H, m), 4.40−4.60 (2H, m), 4.90−5.00 (1H, m),
5.05−5.20 (2H, m), 5.28−5.50 (2H, m), 5.75−5.83 (1H, m),
6.78−6.82 (1H, m), 6.95−7.05 (1H, m), 7.20−7.33
p-Methoxybenzyl trichloroacetimidate (61.4 g, 217 mmol)
and p-toluenesulfonic acid monohydrate (3.50 g, 18.4
mmol) were added to a solution of 2 (12.0 g, 7.07 mmol) in
dichloromethane (300 mL) at 0 °C. After stirring at room

Y. Kagoshima et al.
temperature for 6 h, the reaction was quenched with satu-
rated sodium hydrogen carbonate solution in water and
dichloromethane extraction was performed three times. The
combined organic phases were washed with saturated
aqueous sodium chloride solution once, dried over sodium
sulfate, and filtrated, then the solvent was removed under
reduced pressure and the resultant residue was purified by
flash column chromatography on neutral silica gel
(20–70% ethyl acetate in hexane) to produce the di-
(3H, m), 2.90−3.03 (1H, m), 3.18−3.33 (2H, m), 3.33
(3H, s), 3.50−3.60 (1H, m), 3.75−3.80 (6H, m),
3.39−4.00 (2H, m), 4.12−4.23 (3H, m), 4.30−4.45
(7H, m), 5.50−5.38 (5H, m), 6.70−6.90 (5H, m),
7.08−7.38 (14H, m), 7.42−7.50 (2H, m), 7.60−7.66
(1H, m), 8.15−8.20 (2H, m), 8.42−8.50 (1H, m). MS
(FAB) m/z 1129 (M + H)+.
4-(2-azidoethyl)benzoic acid (9)
methoxybenzyl compound
3 (11.15 g, 81%) as a
pale yellow amorphous substance. Palladium on carbon
(10 wt%, 3.70 g) was added to a solution of 3 (11.1 g, 8.30
mmol) in tetrahydrofuran (70 mL) and water (50 mL) under
an argon atmosphere. The flask was evacuated and purged
with hydrogen gas five times on a hydrogen manifold, then
the mixture was stirred under a hydrogen atmosphere at
room temperature for 4 h. After complete conversion
(monitored by thin layer chromatography), the catalyst was
removed by filtration through celite, which was washed
with tetrahydrofuran and water. The filtrate was used in the
next reaction without further purification. p-Nitrobenzy-
loxycarbonyl chloride (4.21 g, 19.5 mmol) and sodium
carbonate (2.07 g, 19.5 mmol) were added to the filtrate of
4 at 0 °C. After stirring at 0 °C for 0.5 h, the reaction
mixture was quenched with aqueous potassium hydrogen
sulfate solution and ethyl acetate extraction was performed
three times. The combined organic phases were washed
with water three times and with saturated aqueous sodium
chloride solution once, dried over sodium sulfate, and fil-
trated. The solvent was removed under reduced pressure
and the resultant residue was purified by flash column
chromatography on neutral silica gel (0–30% methanol in
ethyl acetate) to produce the carboxylic acid 5 (7.79 g,
80%) as a pale yellow amorphous substance. Potassium
carbonate (4.63 g, 33.5 mmol) was added to a solution of 5
(7.79 g, 6.64 mmol) in methanol (380 mL) at room tem-
perature. After stirring at room temperature for 4 days, the
pH of the reaction mixture was adjusted to 4 using Dowex
50 W (H+ form), then the mixture was filtered. The
remaining Dowex 50 W was washed with 5% aqueous
ammonium solution. The combined filtrate was con-
centrated in vacuo and the resultant residue was used for
the next step without further purification. Diazodiphe-
nylmethane (3.00 g, 15.4 mmol) and acetic acid (0.5 mL)
were added to a solution of the resulting residue of 6 in
dichloromethane (100 mL) at room temperature. After
stirring at room temperature for 0.5 h, the solvent was
removed under reduced pressure and the resultant residue
was purified by flash column chromatography on neutral
silica gel (20–100% ethyl acetate in hexane) to produce the
title compound (2.93 g, 53%) as a colorless solid.
Sodium azide (8.51 g, 131 mmol) was added to a solution of
4-(2-bromoethyl)benzoic acid 8 (20.0 g, 87.3 mmol) in
dimethylformamide (200 mL) at room temperature. After
stirring at 50 °C for 2 h, the reaction mixture was cooled to
room temperature, quenched with water, and ethyl acetate
extraction was performed three times. The combined
organic phases were washed with water three times and
with saturated aqueous sodium chloride solution once, dried
over sodium sulfate, and filtrated. The filtrate was con-
centrated in vacuo to produce an azide compound (15.44 g,
93%) as a colorless solid.
1H-NMR (400 MHz, CDCl3) δ: 2.93 (2H, t, J = 7.0 Hz),
3.55 (2H, t, J = 7.0 Hz), 7.32 (2H, d, J = 9.0 Hz), 8.05 (2H,
d, J = 9.0 Hz). MS (FAB) m/z 192 (M + H)+.
4-[2-[(4-nitrophenyl)methoxycarbonylamino]ethyl]
benzoic acid (10)
Triphenylphosphine (8.51 g, 89 mmol) and water (2.9 mL,
90 mmol) were added to a solution of 9 (15.44 g,
80.8 mmol) in tetrahydofuran (155 mL) at room tem-
perature. After stirring at room temperature for 2 h, the
reaction mixture was filtrated and concentrated in vacuo
to produce an amino compound (31.21 g, 91%) as a col-
orless solid. p-Nitrobenzyl chloride (18.4 g, 80.8 mmol)
and 1 N NaOH solution (160 mL, 160 mmol) were added
to a solution of 4-(2-aminoethyl)benzoic acid (31.21 g,
80.8 mmol) in tetrahydofuran (155 mL) at 0 °C. After
stirring at 0 °C for 2 h, the reaction mixture was washed
with ethyl acetate and the organic phase was extracted
with 1 N NaOH solution once, then the combined water
phases were acidified with saturated KHSO4 solution and
ethyl acetate extraction was performed three times. The
combined organic phases were washed with saturated
aqueous sodium chloride solution once, dried over sodium
sulfate, filtrated, and concentrated in vacuo to produce a
carboxylic acid compound (27.3 g, 92% in two steps) as a
colorless solid.
1H-NMR (DMSO-D6) δ: 2.79 (2H, t, J = 7.0 Hz),
3.22−3.32 (3H, m), 5.13 (2H, s), 7.31 (2H, d, J = 7.8 Hz),
7.52 (2H, d, J = 7.8 Hz), 7.84 (2H, d, J = 7.8 Hz), 8.21
(2H, d, J = 7.8 Hz), 12.81 (1H, s). MS (FAB) m/z 345
(M + H)+.
1H-NMR (400 MHz, CDCl3) δ: 1.78−1.88 (2H, m),
1.95−2.02 (1H, m), 2.03−2.13 (1H, m), 2.35−2.45

Muraminomicins, novel ester derivatives: in vitro and in vivo antistaphylococcal activity
2-[dodecyl-[4-[2-[(4-nitrophenyl)
methoxycarbonylamino]ethyl]benzoyl]amino]acetic
acid (11)
silica gel (20–90% ethyl acetate in hexane) to produce the
title compound (541 mg, containing starting material 11) as
a colorless amorphous substance.
MS ðFABÞ m=z 1680 ðM þ HÞþ:
1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide,
hydro-
chloride (water soluble carbodiimide) (17.0 g, 89 mmol),
dimethylaminopyridine (10.9 g, 89 mmol), and methyl
2-(dodecylamino)acetate (19.1 g, 74 mmol) were added to a
solution of 10 (25.5 g, 74.1 mmol) in dichloromethane
(250 mL) at 0 °C. After stirring at room temperature for 5 h,
the reaction mixture was quenched with water and ethyl
acetate extraction was performed three times. The combined
organic phases were washed with saturated aqueous sodium
chloride solution, dried over sodium sulfate, and filtrated. The
filtrate was concentrated in vacuo and the resultant residue
was purified by flash column chromatography on neutral
silica gel (10–60% ethyl acetate in hexane) to produce the
methyl ester (33.0 g, 76%) as a colorless solid. Potassium
hydroxide solution (1.0 mol/L) in water (8.5 mL, 8.5 mmol)
was added to a solution of methyl ester (1.65 g, 2.83 mmol)
in tetrahydrofuran (20 mL) and water (20 mL) at room tem-
perature. After stirring for 15 min, the reaction mixture was
quenched with saturated aqueous potassium hydrogen sulfate
solution and ethyl acetate extraction was performed three
times. The combined organic phases were washed with brine,
dried over sodium sulfate, and filtrated. The resultant filtrate
was concentrated in vacuo to produce the title compound
(1.58 g, 98%) as a yellow amorphous substance.
6-[2-[[4-(2-aminoethyl)benzoyl]-dodecylamino]
acetyl]oxy-2-[[5-(aminomethyl)-4-hydroxy-
tetrahydrofuran-2-yl]oxy-[(2S,3S,5R)-5-(2,4-
dioxopyrimidin-1-yl)-3-hydroxy-tetrahydrofuran-2-
yl]methyl]-1,4-dimethyl-3-oxo-1,4-diazepane-5-
carboxylic acid (1a)
Water (1 mL) and 2,3-dichloro-5,6-dicyano-p-benzoquinone
(219 mg, 0.87 mmol) were added to a solution of 16a
(541 mg, containing starting material 11) in dichloromethane
(20 mL) at 0 °C. After stirring at room temperature for 5 h, the
reaction mixture was quenched with aqueous sodium hydro-
gen carbonate solution and dichloromethane extraction was
performed three times. The combined organic phases were
washed with saturated sodium chloride solution in water,
dried over sodium sulfate, and filtrated. The filtrate was
concentrated in vacuo and the resultant residue was purified
by flash column chromatography on silica gel (5–10%
methanol in dichloromethane) to produce the diol compound
(232 mg, 67% in two steps) as a colorless amorphous sub-
stance. Palladium on carbon (10 wt%, 250 mg) was added to a
solution of the diol compound (230 mg, 0.161 mmol) in tet-
rahydrofuran (20 mL) under an argon atmosphere. The flask
was evacuated and purged with hydrogen gas five times on a
hydrogen manifold, then the mixture was stirred under a
hydrogen atmosphere at room temperature for 1 h. After
complete conversion (monitored by thin layer chromato-
graphy), the catalyst was removed by filtration through celite,
which was then washed with tetrahydrofuran. The filtrate was
concentrated in vacuo and the resultant residue was purified
by reverse phase column chromatography on C18 silica gel
(0−50% acetonitrile in water) to produce the title compound
(40.5 mg, 27%) as a colorless amorphous substance.
MS ðFABÞ m=z 570 ðM þ HÞþ:
Benzhydryl 2-[[(2S,3S,5R)-5-(2,4-dioxopyrimidin-1-
yl)-3-[(4-methoxyphenyl)methoxy]tetrahydrofuran-
2-yl]-[4-[(4-methoxyphenyl)methoxy]-5-[[(4-
nitrophenyl)methoxycarbonylamino]methyl]
tetrahydrofuran-2-yl]oxy-methyl]-6-[2-[dodecyl-[4-
[2-[(4-nitrophenyl)methoxycarbonylamino]ethyl]
benzoyl]amino]acetyl]oxy-1,4-dimethyl-3-oxo-1,4-
diazepane-5-carboxylate (16a)
1H-NMR (400 MHz, D2O) δ: 1.50 (3H, t, J = 5.9 Hz),
1.68−1.97 (22H, m), 2.12−2.22 (2H, m), 2.60−2.64
(3H, m), 2.77−3.02 (4H, m), 3.04 (3H, s), 3.47−
3.67 (4H, m), 3.69 (2H, s), 3.72−4.04 (5H, m), 4.34−4.61
(2H, m), 4.83−5.07 (3H, m), 6.00−6.21 (2H, m), 6.39−6.44
(1H, m), 6.58−6.63 (1H, m), 7.89−8.04 (4H, m), 8.36−
8.45 (1H, m). MS (FAB) m/z 916 (M + H)+.
Compound 7 (270 mg, 0.239 mmol), 1-ethyl-3-(3-dimethy-
laminopropyl)carbodiimide hydrochloride (366 mg, 1.91
mmol), and N,N-dimethylaminopyridine (116 mg, 0.95
mmol) were added to a solution of 11 (1.09 g, 1.91 mmol)
in dichloromethane (30 mL) at 0 °C. After stirring at 0 °C
for 1 h, the reaction mixture was quenched with aqueous
potassium hydrogen sulfate solution and dichloromethane
extraction was performed three times. The combined
organic phases were washed with saturated sodium chloride
solution in water, dried over sodium sulfate, and filtrated.
The filtrate was concentrated in vacuo and the resultant
residue was purified by flash column chromatography on
(3,4-diheptoxyphenyl)methanamine (13)
Potassium carbonate (79.7 g, 576 mmol) and 1-
bromoheptane (86.53 g, 391 mmol) were added to a solu-
tion of 3,4-dihydroxybenzonitrile 12 (25.1 g, 186 mmol) in
N,N-dimethylformamide (240 mL) at room temperature.

Y. Kagoshima et al.
After stirring at 80 °C for 2 h, the reaction mixture was
cooled to room temperature, quenched with saturated aqu-
eous sodium chloride solution, and ethyl acetate extraction
was performed three times. The combined organic phases
were washed with saturated aqueous sodium chloride
solution, dried over magnesium sulfate, and filtrated.
The filtrate was concentrated in vacuo and the resultant
residue was recrystallized with hexane and ethyl acetate to
produce 3,4-diheptoxybenzonitrile (56.0 g, 91%) as a pale
red solid. The nitrile compound (51.5 g, 155 mmol) dis-
solved in tetrahydrofuran (200 mL) was added to a sus-
pension of lithium aluminum hydride (8.00 g, 210 mmol) in
tetrahydrofuran (300 mL) at 0 °C using a dropping funnel.
After stirring at 60 °C for 20 min, the reaction mixture was
cooled to 0 °C, then water (8 mL), 15% aqueous sodium
hydroxide (8 mL), and water (24 mL) were added succes-
sively at 0 °C. After stirring at room temperature for 40 min,
the reaction mixture was filtered through celite and the filter
cake was washed with ethyl acetate. The filtrate was con-
centrated in vacuo and the resultant residue was purified by
flash column chromatography on silica gel (10–50%
methanol in ethyl acetate) to produce the title compound
(51.5 g, 98%) as a pale red solid.
added to the PNZ-protected compound (43.3 g, 72.2 mmol)
dissolved in 1,4-dioxane (700 mL) and water (300 mL) at
0 °C. After stirring at room temperature for 20 min, the
reaction mixture was quenched with aqueous potassium
hydrogen sulfate solution and ethyl acetate extraction was
performed three times. The combined organic phases were
washed with saturated aqueous sodium chloride solution,
dried over sodium sulfate, and filtrated. The filtrate was
concentrated in vacuo to produce the title compound (39.9 g,
94%) as a colorless solid. The resultant residue was used in
the next reaction without any further purification.
1H-NMR (CD3OD) δ: 0.85−0.90 (6H, m), 1.25−1.50
(12H, m), 1.65−1.78 (4H, m), 2.45−2.57 (2H, m),
3.50−3.58 (2H, m), 3.75−3.91 (2H, m), 3.94 (2H, t, J =
6.6 Hz), 4.46 (2H, d, J = 17.2 Hz), 5.26 (2H, d, J =
11.3 Hz), 6.71−6.87 (3H, m), 7.39−7.48 (1H, m),
7.56−7.65 (1H, m), 8.10−8.26 (2H, m). MS (FAB) m/z 609
(M + Na)+.
3-[3-[(3,4-diheptoxyphenyl)methyl-[(4-nitrophenyl)
methoxycarbonyl]amino]propanoylamino]
propanoic acid (15)
1H-NMR (400 MHz, CDCl3) δ: 0.87 (4H, t, J = 6.8 Hz),
1.24−1.37 (8H, m), 1.39−1.52 (8H, m), 1.74−1.83
(4H, m), 3.77 (2H, s), 3.97 (3H, dt, J = 12.0, 4.7 Hz),
6.76−6.85 (3H, m). MS (FAB) m/z 336 (M + H)+.
β-alanine ethyl ester hydrochloride (11.5 g, 74.8 mmol),
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
hydro-
chloride (16.9 g, 88.4 mmol), and 2-dimethylaminopyridine
(10.8 g, 88.4 mmol) were added to 14 (39.9 g, 72.2 mmol)
dissolved in dichloromethane (400 mL) at 0 °C. After stir-
ring at room temperature for 1 h, the reaction mixture was
quenched with 1 N hydrogen chloride solution and
dichloromethane extraction was performed three times. The
combined organic phases were washed with saturated
sodium hydrogen carbonate solution in water, dried over
sodium sulfate, and filtrated. The filtrate was concentrated
in vacuo and the resultant residue was purified by flash
column chromatography on silica gel (10–80% ethyl acetate
in hexane) to produce the ethyl ester compound (40.2 g,
86%) as a colorless amorphous substance. A solution of 1 N
sodium hydroxide in water (170 mL) was added to the ethyl
ester compound (40.2 g, 58.6 mmol) dissolved in 1,4-diox-
ane (300 mL) at 0 °C. After stirring at room temperature for
20 min, the reaction mixture was quenched with 1 N
hydrogen chloride solution in water and ethyl acetate
extraction was performed three times. The combined
organic phases were washed with saturated aqueous sodium
chloride solution, dried over sodium sulfate, and filtrated.
The filtrate was concentrated in vacuo to produce the title
compound (37.3 g, 97%) as a colorless solid. The resultant
residue was used in the next reaction without any further
purification.
3-[3-[(3,4-diheptoxyphenyl)methyl-[(4-nitrophenyl)
methoxycarbonyl]amino]propanoylamino]
propanoic acid (14)
Methyl acrylate (8.18 g, 45 mmol) was added to a solution of
13 (33.5 g, 100 mmol) in methanol (100 mL) at room tem-
perature. After stirring at room temperature for 24 h, the
reaction mixture was concentrated in vacuo and the resultant
residue was purified by flash column chromatography on
neutral silica gel (20–70% ethyl acetate in hexane) to pro-
duce an amine compound (31.2 g, 74%) as a colorless
amorphous substance. p-Nitrobenzyloxycarbonyl chloride
(15.6 g, 72.5 mmol) and sodium carbonate (7.68 g, 72.5
mmol) were added to amine compound (29.08 g, 69.0 mmol)
dissolved in tetrahydrofuran (200 mL) and water (200 mL) at
0 °C. After stirring at 0 °C for 2 h, the reaction mixture was
quenched with water and ethyl acetate extraction was per-
formed three times. The combined organic phases were
washed with saturated aqueous sodium chloride solution,
dried over sodium sulfate, and filtrated. The filtrate was
concentrated in vacuo and the resultant residue was purified
by flash column chromatography on silica gel (10–50%
ethyl acetate in hexane) to produce a PNZ-protected com-
pound (43.3 g, 94%) as a pale yellow amorphous substance.
A solution of 1 N sodium hydroxide in water (180 mL) was
1H-NMR (CD3OD) δ: 0.83−0.92 (6H, m), 1.20−1.50
(12H, m), 1.63−1.78 (4H, m), 2.36−2.49 (4H, m),
3.31−3.41 (2H, m), 3.50−3.58 (2H, m), 3.74−3.90

Muraminomicins, novel ester derivatives: in vitro and in vivo antistaphylococcal activity
(2H, m), 3.93 (2H, t, J = 6.4 Hz), 4.37−4.47 (2H, m),
5.21−5.31 (2H, m), 6.69−6.87 (3H, m), 7.37−7.48
(1H, m), 7.56−7.66 (1H, m), 8.01−8.26 (2H, m). MS
(FAB) m/z 680 (M + Na)+.
dried over sodium sulfate, and filtrated. The filtrate was
concentrated in vacuo and the resultant residue was purified
by flash column chromatography on silica gel (5–10%
methanol in dichloromethane) to produce a diol compound
(5.50 g, 58%) as a colorless amorphous substance. Palla-
dium on carbon (10 wt%, 1.28 g) was added to the diol
compound (1.81 g, 5.41 mmol) dissolved in tetrahydrofuran
(100 mL) under an argon atmosphere. The flask was evac-
uated and purged with hydrogen gas five times on a
hydrogen manifold, then the mixture was stirred under a
hydrogen atmosphere at room temperature for 2 h. After
complete conversion (monitored by thin layer chromato-
graphy), the catalyst was removed by filtration through
celite, which was washed with tetrahydrofuran. The filtrate
was concentrated in vacuo and the resultant residue was
purified by reverse phase column chromatography on
C18 silica gel (0–40% acetonitrile in water) to produce the
title compound (736 mg, 60%) as a colorless amorphous
substance.
Benzhydryl 6-[3-[3-[(3,4-diheptoxyphenyl)methyl-
[(4-nitrophenyl)methoxycarbonyl]amino]
propanoylamino]propanoyloxy]-2-[[(2S,3S,5R)-5-
(2,4-dioxopyrimidin-1-yl)-3-[(4-methoxyphenyl)
methoxy]tetrahydrofuran-2-yl]-[4-[(4-
methoxyphenyl)methoxy]-5-[[(4-nitrophenyl)
methoxycarbonylamino]methyl]tetrahydrofuran-2-
yl]oxy-methyl]-1,4-dimethyl-3-oxo-1,4-diazepane-5-
carboxylate (16b)
Compound 7 (7.0 g, 6.20 mmol), 1-ethyl-3-(3-dimethyla-
minopropyl)carbodiimide hydrochloride (7.1 g, 37.0 mmol),
and 2-dimethylaminopyridine (1.97 g, 16.1 mmol) were
added to 15 (14.2 g, 21.6 mmol) dissolved in dichlor-
omethane (700 mL) at 0 °C. After stirring at 0 °C for 2 h, the
reaction mixture was quenched with aqueous potassium
hydrogen sulfate solution and dichloromethane extraction
was performed three times. The combined organic phases
were washed with saturated sodium chloride solution in
water, dried over sodium sulfate, and filtrated. The filtrate
was concentrated in vacuo and the resultant residue was
purified by flash column chromatography on silica gel
(10–80% ethyl acetate in dichloromethane) to produce the
title compound (9.58 g, 87%) as a colorless amorphous
substance.
1H-NMR (400 MHz, CD3CN + D2O) δ: 0.84 (6H, t, J =
6.6 Hz), 1.23−1.34 (16H, m), 1.38−1.44 (2H, m),
1.68−1.73 (5H, m), 2.13−2.32 (4H, m), 2.34 (3H, s),
2.48−2.56 (4H, m), 2.85 (1H, dd, J = 13.7, 7.8 Hz),
2.99−3.04 (1H, m), 2.99 (3H, s), 3.07 (2H, t, J = 6.6 Hz),
3.11−3.16 (1H, m), 3.27−3.32 (1H, m), 3.38 (2H, t, J =
6.6 Hz), 3.88 (1H, dd, J = 6.6, 1.7 Hz), 3.93−3.97 (4H, m),
4.00 (2H, s), 4.07−4.16 (2H, m), 4.18−4.21 (1H, m),
4.27−4.32 (1H, m), 4.33−4.37 (1H, m), 5.40−5.43
(1H, m), 5.43−5.45 (1H, m), 5.74 (1H, d, J = 7.8 Hz), 5.95
(1H, dd, J = 6.8, 4.4 Hz), 6.91−6.93 (2H, m), 6.97 (1H, s),
7.73 (1H, d, J = 8.3 Hz). MS (FAB) m/z 1004 (M + H)+.
1H-NMR (400 MHz, DMSO-D6) δ: 0.78−0.87 (6H, m),
1.16−1.30 (10H, m), 1.30−1.43 (4H, m), 1.53−1.70
(4H, m), 1.78−2.45 (4H, m), 2.94−3.50 (7H, m),
3.65−3.75 (6H, m), 3.74−4.19 (8H, m), 4.20−4.41
(4H, m), 4.90−5.53 (9H, m), 6.51−6.93 (8H, m),
7.14−7.27 (15H, m), 7.36−7.65 (6H, m), 7.97−8.24
(6H, m), 11.27 (1H, s). MS (FAB) m/z 1769 (M + H)+.
Bacterial strains
The S. aureus clinical isolates (24 strains of MSSA and
22 strains of MRSA) used in the susceptibility tests were
obtained from the Tokyo Clinical Research Center in Japan.
S. aureus ATCC 29213 was used as the control strain for
the susceptibility test, S. aureus ATCC 6538P was used to
measure spontaneous mutation frequency, and MSSA Smith
was used in the in vitro time-kill studies and the murine
systemic infection model. The strains were obtained from
the American Type Culture Collection, the National Insti-
tute of Infectious Diseases, and Toho University, respec-
tively. Methicillin-resistant S. aureus (MRSA) 10925 was a
clinical isolate obtained from the Tokyo Clinical Research
Center for use in the in vitro time-kill studies and the
murine systemic infection model.
2-[[5-(aminomethyl)-4-hydroxy-tetrahydrofuran-2-
yl]oxy-[(2S,3S,5R)-5-(2,4-dioxopyrimidin-1-yl)-3-
hydroxy-tetrahydrofuran-2-yl]methyl]-6-[3-[3-[(3,4-
diheptoxyphenyl)methylamino]propanoylamino]
propanoyloxy]-1,4-dimethyl-3-oxo-1,4-diazepane-5-
carboxylic acid (1b)
Water (18 mL) and 2,3-dichloro-5,6-dicyano-p-benzoqui-
none (3.69 g, 16.3 mmol) were added to 16b (9.58 g, 5.42
mmol) dissolved in dichloromethane (335 mL) at 0 °C.
After stirring at room temperature for 5 h, the reaction
mixture was quenched with aqueous sodium hydrogen
carbonate solution and dichloromethane extraction was
performed three times. The combined organic phases were
washed with saturated sodium chloride solution in water,
Susceptibility tests
The MICs of 1b and vancomycin against MSSA and MRSA
were determined using a standard microdilution broth

Y. Kagoshima et al.
method [29], with Mueller−Hinton broth (Becton
Dickinson and Company, Sparks, MD) containing 25 mg of
Ca2+ and 12.5 mg of Mg2+ per liter (cation-adjusted
Mueller−Hinton broth: CAMHB) and a 4 × 105 CFU/mL
inoculum. The MIC was defined as the lowest concentration
of the compound that inhibited the visible growth of the
organism on the microdilution plates.
Murine systemic infection model
Mice were intraperitoneally infected with 0.2 mL of bac-
terial suspensions of MSSA Smith and MRSA 10925. The
MRSA 10925 bacterial suspension was mixed with hog
stomach gastric mucin to a final concentration of 5% prior
to inoculation. MSSA Smith and MRSA 10925 were
inoculated at concentrations of 7.4 × 106 – 2.5 × 107 CFU/
mouse and 3.1 × 108 – 4.7 × 108 CFU/mouse, respectively.
Solutions (0.1 mL) of 1a, 1b, and vancomycin were admi-
nistered subcutaneously immediately and 4 h after inocula-
tion. Five doses of serial twofold dilutions were used for
each 50% effective dose (ED50) determination, with seven
mice used for each dose. After inoculation, the mortality of
the mice was recorded daily for 7 days. The ED50 and 95%
confidence intervals were calculated from the survival rates
on the seventh day after inoculation using the probit
method.
In vitro time-kill studies
The bactericidal activity of 1a and vancomycin against
MSSA Smith, and that of 1a, 1b, and vancomycin against
MRSA 10925, were determined using the time-kill method
according to CLSI guideline M26-A [30]. S. aureus was
grown overnight in brain heart infusion medium (BHI), then
the cells (optical density at 620 nm = ~0.10) were diluted
with CAMHB and cultured at 37 °C to yield an initial cell
count of approximately 1 × 105 CFU/mL during exponential
growth. Test compounds were added to the bacterial cul-
tures at concentrations 0.25, 1, 4, 16, and 64 times the MIC,
and incubated at 37 °C. At 0, 1, 2, 6, and 24 h after the
addition of the test compounds, samples were collected
from the culture medium, serially diluted, spread on BHI,
and cultured at 37 °C. The number of colonies growing on
the plate was counted after 24 h. The detection limit was
1 log10 CFU/mL; if no colonies were detected, the value of
1 log10 CFU/mL was adopted.
Results and discussion
Semisynthesis of 3″′-ester precursor of
muraminomicin Z1
In preliminary studies, when the 1″′-ester derivatives of
muraminomicin F were treated under basic conditions, such
as with sodium hydroxide solution, the major product was
muraminomicin Z2; however, when the 1″′-carboxylic acid
free derivatives of crude muraminomicins were treated
under basic conditions, the major product was mur-
aminomicin Z1. The 1″′-carboxylic acid chemical mod-
ification appeared to influence 3″′-fatty acid ester hydrolysis
and elimination; therefore, we developed a scheme for
synthesizing a key intermediate of the 3″′-ester modification
of muraminomicin Z1 (Scheme 1). As described in our
previous paper, culturing SANK 60501 with 0.5% myristic
acid enhances muraminomicin F production and generates
low levels of muraminomicins G and H. All three mur-
aminomicins possessed 3a-methylglutaryl myristyl esters at
their 3″′-hydroxyl diazepanone moieties. Partially purified
muraminomicin complexes were obtained by ethyl acetate
extraction and reverse extraction with 0.2 M phosphate
buffer [25]. Following p-nitrobenzylcarbonate (PNZ) pro-
tection of the 5″-amino group, the complexes were treated
under basic cooling conditions with an ice bath to generate
2,3-didehydro-tetradecanoyl esters via 3a-methylglutaryl
ester elimination. Diphenylmethyl ester protection of the
1″′-carboxylic acid then produced compound 2, which was
easily purified via silica gel column chromatography.
We used the 2,3-didehydro-tetradecanoyl ester to protect
the 3″′-hydroxyl group of the diazepanone ring during
successive modification. Using p-methoxybenzyl (PMB)
Spontaneous mutation frequency measurement
The frequency of spontaneous single-step mutations for 1a and
1b was determined by inoculating cultures (~109 CFU/mL;
100 μL) into Mueller−Hinton agar containing each com-
pound at 2, 4, and 8 times the MIC. Plates were incubated
aerobically at 35 °C for 72 h. The mutation frequency was
calculated as the number of grown colonies on compound-
containing medium per inoculum. The MICs for the parent
and mutant strains were determined using the microdilution
broth method.
A DNA fragment containing the mraY gene was ampli-
fied from the mutant strains by the polymerase chain reac-
tion (PCR) and used for sequence analysis. mraY mutations
(GenBank accession number NC 007795) responsible for
resistance were identified by comparing the nucleotide
sequence with that of the parent strain.
Animals
Male ddY (Japan SLC, Inc., Shizuoka, Japan) specific-
pathogen-free mice were used in this study. All animal
experiments were carried out according to the guidelines
provided by the Institutional Animal Care and Use Com-
mittee of Daiichi Sankyo.

Muraminomicins, novel ester derivatives: in vitro and in vivo antistaphylococcal activity
Scheme 1 Reagents
and conditions:
a p-nitrobenzyloxycarbonyl
chloride, Na2CO3,
tetrahydrofuran (THF), water, 0
°C; b diphenyldiazomethane,
CH2Cl2, room temperature (rt),
45% (2 steps);
c p-methoxybenzyl
trichloroacetimidate,
p-toluenesulfonic acid
monohydrate, CH2Cl2, rt, 81%;
d H2, Pd-C, THF, water, rt;
e p-nitrobenzyloxycarbonyl
chloride, Na2CO3, THF, water,
0 °C, 80% (two steps);
(f) Na2CO3, methanol, rt, then
Dowex 50 W (H+); and
g diphenyldiazomethane,
CH2Cl2, rt, 53% (two steps)
Scheme 2 Reagents and
conditions: a NaN3, DMF,
50 °C, 93%; b
triphenylphosphine, water, THF,
room temperature (rt), 91%;
c p-nitrobenzyloxycarbonyl
chloride, NaOH, THF, water,
0 °C, 91% (two steps); d methyl
2-(dodecylamino)acetate,
1-ethyl-3-(3-
dimethylaminopropyl)
carbodiimide, hydrochloride
(water soluble carbodiimide,
WSC), dimethylaminopyridine
(DMAP), 0 °C; e NaOH,
THF, water, rt, 81%;
f 1-bromoheptane, K2CO3, rt,
91%; g LiAlH4, THF, 0 °C,
98 %; h methyl acrylate,
methanol, rt, 74%;
i p-nitrobenzyloxycarbonyl
chloride, Na2CO3, THF,
water, 0 °C, 94%; j NaOH,
1,4-dioxane, water, rt, 94%;
k β-alanine ethyl ester
hydrochloride, WSC, DMAP,
0 °C, 94%; and l NaOH, 1,4-
dioxane, water, rt, 97%
trichloroacetimidate and p-toluenesulfonic acid (TsOH) to
protect 2 produced PMB ether 3. Hydrogenation selectively
deprotected the diphenylmethyl and PNZ groups to produce
PMB ether 4, with PNZ reprotection yielding carboxylic
acid 5. Methanolysis of compound 5 using sodium carbo-
nate produced alcohol 6, whilst reprotection of the car-
boxylic acid in the diphenylmethyl ester yielded the key
intermediate ester precursor 7. The semisynthesis of

Y. Kagoshima et al.
Scheme 3 Reagents and
conditions: a 11 or 15, WSC,
DMAP, CH2Cl2, 0 °C, 16a not
determined, and 16b 87%; b
2,3-dichloro-5,6-dicyano-p-
benzoquinone, water, CH2Cl2,
0 °C; and c H2, Pd-C, THF,
water, room temperature (rt),
1a 18% (three steps), 1b 35%
(two steps)
compound 7 using partially purified muraminomicin com-
plexes was successfully developed without any HPLC
preparation. Thus, it is possible to increase the processing
speed while facilitating scale-up synthesis.
condensation and hydrolysis produced carboxylic acid 15,
the side chain of 1b. As shown in Scheme 3, the acylation
of ester precursor 7 with carboxylic acids 11 and 15
was readily accomplished using WSCI and 4-
dimethylaminopyridine (DMAP) to obtain esters 16a and
16b, respectively. The deprotection of PMB using 2,3-
dichloro-5,6-dicyano-p-benzoquinone (DDQ) followed by
diphenylmethyl ester hydrogenation and p-nitrobenzylox-
ycarbonate deprotection yielded 1a and 1b.
Muraminomicins possessing new side chains
1a and 1b (Fig. 1) were selected by in vitro and in vivo
screening. Their unique side chains were synthesized as
shown in Scheme 2. Compound 11, the side chain of 1a,
was yielded by treating methyl 2-(dodecylamino)acetate
Antibacterial activity
with
4-[2-[(4-nitrophenyl)methoxycarbonylamino]ethyl]
benzoic acid 10, which was derived from 4-(2-bromoethyl)
benzoic acid 8 via azide replacement, reduction, and pro-
tection, followed by hydrolysis. The alkylation of 3,4-
dihydroxybenznitrile using 1-bromoheptane and potassium
carbonate, followed by reduction of the nitrile group using
lithium aluminum hydride, produced 3,4-diheptox-
ybenzylamine 13. Michael addition using methyl acrylate in
methanol, PNZ protection, and hydrolysis yielded beta-
alanine derivative 14, whilst methyl beta-alanine
The MICs that inhibited 50 and 90% of the strains (MIC50
and MIC90, respectively) of MSSA and MRSA of 1b were 1
and 2 μg/mL, respectively. The MICs of 1b against MSSA
were the same as those against MRSA, suggesting that no
cross resistance exists between methicillin and mur-
aminomicin derivatives. The MIC50 and MIC90 of MSSA
and MRSA of vancomycin were 0.5 and 1 μg/mL, respec-
tively. Moreover, muraminomicin derivatives exhibited
almost same antibacterial activity against vancomycin-

Muraminomicins, novel ester derivatives: in vitro and in vivo antistaphylococcal activity
(a)
(b)10
10
1a
Vancomycin
9
9
8
8
*
*
7
7
6
6
5
5
4
4
3
3
2
2
≤1
≤1
-2
0
2
4
6
24
-2
0
2
4
6
24
Time (h)
Time (h)
(c) 10
(d) 10
(e)10
1a
1b
Vancomycin
9
8
7
6
5
4
3
2
1
9
8
7
6
5
4
3
2
1
9
8
7
6
5
4
3
2
1
*
*
*
-3
0
2
6
24
-3
0
2
6
24
-3
0
2
6
24
Time (h)
Time (h)
Time (h)
Fig. 2 Time-killing curves against MSSA Smith (a, b) and MRSA
10925 (c–e). Arrows with asterisks indicate the addition of the anti-
biotic to the bacterial culture. Symbols: cross, drug-free control; filled
circle, 0.25 times the MIC; filled triangle, MIC; filled square, four
times the MIC; open circle, 16 times the MIC; open triangle, 64 times
the MIC. The dotted line indicates a 3 log10 reduction from the
inoculum size
Table 1 Mutant frequencies of 1a and 1b against S. aureus
ATCC 6538P
resistant enterococci as the susceptible strain (data not
shown).
Compound MIC (μg/mL) Mutant frequency selected at
Bactericidal activity
2 × MIC
4 × MIC
8 × MIC
1a
1b
0.25
2
7.7 × 10−5
1.4 × 10−8 <2.0 × 10−9 <2.0 × 10−9
3.2 × 10−5
3.1 × 10−7
Figure 2a, b shows the time-kill curves for 1a and vanco-
mycin against MSSA Smith. 1a reduced the viable cell
count of MSSA Smith to almost 2 log10 CFU/mL of the
inoculum within 4 h of exposure to the MIC and higher
concentrations; however, regrowth was observed 24 h after
treatment with 1a at the MIC. Concentrations of 1a higher
than the MIC reduced the viable cell count by at least 3
log10 CFU/mL after 24 h. Both 1a and vancomycin killed
MRSA 10925 in a time-dependent manner (Fig. 2c, e);
Spontaneous mutation frequency
The mutation frequencies of 1a at 2, 4, and 8 times the MIC
were 7.7 × 10−5, 3.2 × 10−5, and 3.1 × 10−7, respectively
(Table 1). Susceptibility tests were performed on ten colo-
nies grown on plates containing 1a (Table 2); all colonies
were 4−16-fold less susceptible to 1a. The only mutant that
was 16-fold less susceptible harbored a single mraY gene
mutation which caused a phenylalanine to isoleucine sub-
stitution at the 226 position (F226I) of the MraY protein,
indicating that MraY was the primary target. The
mutation frequencies of 1b at 2, 4, and 8 times the MIC
were 1.4 × 10−8, below the limit of detection, and below the
however, 1b killed MRSA 10925 in
a stronger,
concentration-dependent manner. 1b also reduced the viable
cell count to 3 log10 CFU/mL of the inoculum within 6 h of
exposure to 16 times the MIC (Fig. 2d). The reason for the
difference between the bactericidal activities of 1a and 1b
against MRSA is unclear; however, cell permeability,
membrane cytotoxicity or target protein affinity may be
contributing factors.

Y. Kagoshima et al.
Table 2 1a and vancomycin susceptibility, and mraY mutations in strains cultured with 1a
Selection conc.
Strain
1a
Vancomycin
MraY mutation
N.T.
MIC (μg/mL) ratio
MIC (μg/mL) ratio
S. aureus
0.5
—
0.5
—
ATCC 6538P
2 × MIC
4 × MIC
8 × MIC
1a—#1
1a—#2
1a—#3
1a—#4
1a—#5
1a—#6
1a—#7
1a—#8
1a—#9
1a—#10
2
4
4
4
4
2
4
2
8
2
4
8
8
8
8
4
8
4
16
4
0.5
0.5
0.5
0.5
1
1
1
1
1
2
1
1
1
1
1
None
None
None
None
None
None
None
None
F226I
None
0.5
0.5
0.5
0.5
0.5
Ratio: MIC of resistant strain/MIC of S. aureus ATCC 6538P
N.T. not tested
Table 3 1b, 1a, and vancomycin susceptibility and mraY mutations in strains cultured with 1b
Selection conc.
Strain
1b
1a
Vancomycin
MraY mutation
N.T.
MIC (μg/mL) ratio
MIC (μg/mL) ratio
MIC (μg/mL) ratio
S. aureus
0.5
—
0.5
—
0.5
—
ATCC 6538P
2 × MIC
1b—#1
1b—#2
1b—#3
1b—#4
1b—#5
1b—#6
1b—#7
1b—#8
1b—#9
1b—#10
1b—#11
0.5
1
1
2
2
2
2
2
2
2
1
1
2
0.25
4
0.5
8
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.25
0.5
0.25
0.5
1
N.T.
1
F226I
F226I
F226I
F226I
F226I
F226I
F226I
N.T.
1
4
8
1
1
4
8
1
1
4
8
1
1
4
8
1
1
4
8
1
1
4
8
0.5
1
0.5
0.5
1
0.5
0.5
4
1
1
0.5
1
N.T.
8
F226I
Ratio: MIC of resistant strain/MIC of S. aureus ATCC 6538P
N.T. not tested
limit of detection (Table 1), respectively. Of the 11 colo-
nies, 8 were twofold less susceptible to 1b, eightfold less
susceptible to 1a, and possessed the F226I MraY substitu-
tion (Table 3). The difference between the 1b susceptibility
of these mutants and the parent strain was only twofold, and
was smaller than that of 1a. Both the F226I mutants and the
parent strains were susceptible to vancomycin, suggesting
that there was no cross resistance between muraminomicin
derivatives and vancomycin. No mutation in mraY gene was
identified in the nine 1a-resistant mutants, suggesting the
presence of other mechanisms of resistance such as
impermeability and efflux pumps; however, further studies
are required to clarify this.
Murine systemic infection model
The ED50 and 95% confidence intervals of 1a, 1b, and
vancomycin against systemic murine infection caused by
MSSA Smith and MRSA 10925 are summarized in Table 4.
The therapeutic efficacy of 1a against MSSA Smith and
MRSA 10925 infections was comparable to that of vanco-
mycin. For 1b, five of the seven mice infected by MSSA

Muraminomicins, novel ester derivatives: in vitro and in vivo antistaphylococcal activity
Table 4 Therapeutic efficacies of 1a, 1b, and vancomycin against
murine systemic infection
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1a
1b
Vancomycin
MSSA Smith
MRSA10925
0.88
N.T.
0.75
(0.65–1.42)
N.T.
(Incalculable)
0.79
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Acknowledgements We thank Mr. Yoshikazu Nezu (Specialty Med-
icine Research Laboratory), Dr. Naoyuki Maeda (Biomarker &
Translational Research Department) and Dr. Yasuyuki Abe (Specialty
Medicine Research Laboratory) for the safety evaluation and Dr.
Takahiro Shibayama (Clinical Pharmacology Department) for the PK
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Compliance with ethical standards
18. McDonald LA, et al. Structures of the muraymycins, novel pep-
Conflict of interest The authors declare that they have no conflict of
tidoglycan biosynthesis inhibitors.
J
Am Chem Soc.
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Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
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