Synthesis and antibacterial activity of acylides (3-O-acyl-erythromycin derivatives): A novel class of macrolide antibiotics

Article abstract of DOI:10.1021/jm015566s

Introduction of an acyl group to the 3-O-position of erythromycin A derivatives instead of L-cladinose led to a novel class of macrolide antibiotics that we named "acylides". The 3-O-nitrophenylacetyl derivative TEA0777 showed significantly potent activit

Full text of DOI:10.1021/jm015566s

© Copyright 2001 by the American Chemical Society
Volume 44, Number 24
November 22, 2001
Letters
second-generation macrolide antibiotics have been
launched for clinical use.
Syn th esis a n d An tiba cter ia l Activity of
Acylid es (3-O-Acyl-er yth r om ycin
Der iva tives): A Novel Cla ss of Ma cr olid e
An tibiotics
Tetsuya Tanikawa,*,†,‡ Toshifumi Asaka,†
Masato Kashimura,† Yoko Misawa,† Keiko Suzuki,†
Masakazu Sato,† Kazuya Kameo,†
Shigeo Morimoto,† and Atsushi Nishida‡
Medicinal Research Laboratories, Taisho Pharmaceutical
Co. Ltd., 1-403 Yoshino-cho, Saitama-shi 330-8530, J apan,
and Graduate School of Pharmaceutical Sciences,
Chiba University, 1-33 Yayoi-cho, Inage-ku,
Chiba-shi 263-8522, J apan
Clarithromycin2 (CAM, 6-O-methylerythromycin A)
and azithromycin3 (15-membered aza-macrolide) are
widely prescribed due to their efficacy and safety, but
this has led to rapid increases in the rates of resistance
in bacteria isolated clinically.4 Therefore, we have
sought to identify a next-generation macrolide that
exhibits greater efficacy and safety, has a broader
spectrum of activities, and is particularly effective
against resistant pathogens.
In our search for next-generation macrolide anti-
biotics, we initially focused on the L-cladinosyl moiety
at the 3-O-position of the macrolactone skeleton as a
target: modification of this moiety has not yet been
systemically investigated since it has been considered
to be essential for activity.5 By substituting L-cladinose
with various functional groups, we have obtained some
new classes of macrolide antibiotics, such as 3-oxo
derivatives,6 so-called “ketolides”, and 3-alkoxy,7 3-car-
bamoyloxy,8 3-alkoxycarbonyloxy,9a and acyloxy9 deriva-
tives. In this paper, we describe the synthesis and
biological properties of 3-O-acyl-erythromycin A deriva-
tives, which we named “acylides”, as a novel class of
macrolide antibiotics which show good antibacterial
activities against Gram-positive pathogens including a
macrolides-lincosamides-streptogramin B (MLSB)-resis-
tant strain and an efflux-resistant strain.
Received August 30, 2001
Abstr a ct: Introduction of an acyl group to the 3-O-position
of erythromycin A derivatives instead of L-cladinose led to a
novel class of macrolide antibiotics that we named “acylides”.
The 3-O-nitrophenylacetyl derivative TEA0777 showed sig-
nificantly potent activity against not only erythromycin-
susceptible Gram-positive pathogens but also inducibly mac-
rolides-lincosamides-streptogramin B (MLSB)-resistant Staphylo-
coccus aureus and efflux-resistant Streptococcus pneumoniae.
These results indicated that acylides have potential as next-
generation macrolide antibiotics.
In tr od u ction . Macrolide antibiotics such as eryth-
romycin A have been clinically used for more than 45
years and are considered preferable for the treatment
of upper and lower respiratory tract infections. However,
the first-generation macrolide erythromycin A easily
loses its antibacterial activity under acidic conditions
by degradation, and the degraded products are known
to be responsible for undesirable gastrointestinal side
effects.1 Numerous chemical modifications of erythro-
mycin A, to overcome this acid-instability problem, have
been investigated by many groups. As a result, several
* To whom correspondence should be addressed. Tel: 81-48-669-
3029. Fax: 81-48-652-7254. E-mail: tetsuya.tanikawa@po.rd.taisho.co.jp.
† Taisho Pharmaceutical Co. Ltd.
Ch em istr y. 3-O-Substituted-6-O-methylerythromy-
cin A derivatives were synthesized as follows. Treat-
‡ Chiba University.
10.1021/jm015566s CCC: $20.00 © 2001 American Chemical Society
Published on Web 10/27/2001

4028 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 24
Letters
Sch em e 1a
Ta ble 1. In Vitro Evaluation of
3-O-Substituted-6-O-methyl-erythromycin A Derivatives 3
a
(a) 2 M HCl, rt, 53%; (b) AcCl, DMAP, pyridine, THF, rt, 68%;
(c) MeOH, rt, 98%.
Sch em e 2a
a
(a) Ac2O, Me2CO, rt, 96%; (b) Boc-glycine or Cbz-glycine,
EDC-HCl, DMAP, CH2Cl2, rt, 96-97%; (c) MeOH; (d) HCO2NH4,
10% Pd-C, MeOH, 50%; (e) RCO2H, EDC-HCl, DMAP, CH2Cl2,
rt, 80-98% or PhCH2COCl, DMAP, pyridine, rt, 49%; (f) 1-fluoro-
2-nitrobenzene, NaH, THF, rt, 39%; (g) PhCH2SO2Cl, pyridine,
CH2Cl2, rt, 87%.
ment of clarithromycin with 2 M aqueous hydrochloric
acid at room temperature afforded selective cleavage of
the 3-O-sugar moiety to give 3-OH derivative 1 (Scheme
1).
a
b
S. aureus 209P-J C: erythromycin-susceptible strain. S. au-
reus B1: inducibly MLSB-resistant strain. c S. aureus SR138:
constitutively MLSB-resistant strain encoded by an ermA gene.
Treatment of the alcohol 1 with excess acetyl chloride
in the presence of 4-(dimethylamino)pyridine (DMAP)
in tetrahydrofuran (THF)/pyridine gave 2′,3-diacetate
2. 3-Acetate 3a was easily obtained by selective 2′-O-
deacetylation of 2 in methanol (MeOH) at room tem-
perature. The 2′-acetate 410 was used as a common
intermediate to introduce various functional groups at
the 3-O-position (Scheme 2). Treatment of the alcohol
1 with acetic anhydride in acetone at room temperature
selectively gave 2′-acetate 4 as a sole product.
3-O-Acyl derivatives 3b,d ,e,h ,j,k were prepared in
good yields (65-97%) by acylation of 4 with the corre-
sponding carboxylic acids and 1-[3-(dimethylamino)-
propyl]-3-ethylcarbodiimide hydrochloride (EDC-HCl)
in the presence of DMAP in dichloromethane (CH2Cl2),
followed by selective removal of the 2′-O-acetyl group

Letters
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 24 4029
Ta ble 2. Antibacterial Effects of TEA0777 against Representative Pathogens
MIC (µg/mL)
S. pneumoniae
E. faecalis
E. faecium
H. influenzae
compd
TEA0777
erythromycin A
clarithromycin
IID553a
210b
221c
ATCC29212
ATCC19434
ATCC43095
0.20
0.10
0.05
0.20
1.56
1.56
>100
>100
>100
0.20
1.56
1.56
0.05
3.13
1.56
25
6.25
6.25
a
b
S. pneumoniae IID553: erythromycin-susceptible strain. S. pneumoniae 210: efflux-resistant strain. c S. pneumoniae 221: MLSB-
resistant strain encoded by an erm B gene.
Sch em e 3a
to the J apan Society of Chemotherapy.13 S. aureus 209P-
J C is an erythromycin-susceptible strain, S. aureus B1
is an inducibly MLSB-resistant strain, and S. aureus
SR138 is a constitutively MLSB-resistant strain that is
also encoded by an ermA gene.
Removal of the L-cladinosyl moiety of clarithromycin
resulted in a complete loss of antibacterial activity as
defined (MICs > 100 µg/mL). Simple introduction of an
acetyl group at the 3-O-position did not completely
restore the antibacterial activity, although it did seem
to be slightly effective against the erythromycin-
susceptible strain.
Accordingly, cyanoacetate 3b and aminoacetates 3c-e
were synthesized to investigate the structure-activity
relationship at the acetyl position. Both the electron-
withdrawing group in 3b and the electron-releasing
group in 3c appeared to be effective at increasing the
activity against the erythromycin-susceptible strain.
Carbamates 3d ,e showed greater activities than the
parent acetate 3a ; benzyloxycarbamate 3e was 16-fold
more potent than 3a . In contrast to the weak activity
of THP ether 3f, o-nitrophenyl ether 3g showed potent
antibacterial activity. The results described above sug-
gested that a phenyl group may increase the activity
against the erythromycin-susceptible strain. Therefore,
we tried to introduce a phenyl group at the 3-O-acetyl
group. Phenylacetate 3h showed considerably potent
antibacterial activity against the erythromycin-suscep-
tible strain, as anticipated (MIC 0.78 µg/mL). In marked
contrast, the corresponding sulfonate 3i did not show
any antibacterial activity. Therefore, a carbonyl function
in the 3-O-linkage appeared to have a profound effect
on this activity.
After further investigation, we identified 3-O-(4-
nitrophenyl)acetyl-5-O-desosaminyl-6-O-methylerythro-
nolide 3j (TEA0777), which showed 250-fold greater
activity against the erythromycin-susceptible strain
than that of the parent acetate 3a . In addition, this
acylide showed significantly potent activity against the
inducibly MLSB-resistant strain (0.39 µg/mL). Conver-
sion of the phenylacetyl group (3j) to a corresponding
benzoyl group (3k ) resulted in a drastic decrease in
antibacterial activity. The poor activity is consistent
with the results with 3-O-benzoyl-erythromycin A oxime
derivatives reported by LeMahieu.5 The phenylacetyl
group was a promising mimic for L-cladinose at the 3-O-
position.
a
(a) 2 M HCl, rt, 45%; (b) BnCl, n-Bu4NI, KOH, THF, 62%; (c)
Ac2O, Me2CO, rt, (d) (i) 3,4-dihydro-2H-pyran, p-TsOH-H2O, MS-
4A, CH2Cl2, (ii) MeOH, 56% in three steps; (e) (i) 10% Pd-C,
HCO2H, HCO2NH4, MeOH, 50 °C, (ii) NaHSO3, HCO2H, EtOH,
H2O, reflux, 51% in two steps.
by heating with methanol for 2 h. Acylation of 4 with
the corresponding acyl chlorides or mixed acid anhy-
drides led to reduced yields (36-74%). Glycinate 3c was
prepared by deprotection of the benzyloxycarbonyl group
of 3e by catalytic transfer hydrogenation (CTH). Sul-
fonate 3i was obtained in 40% yield by treatment of the
alcohol 4 with benzylsulfonyl chloride in the presence
of pyridine in CH2Cl2 followed by methanolysis.
o-Nitrophenyl ether 3g was straightforwardly syn-
thesized by the 3-O-substitution reaction of the alcohol
1 using 2-fluoronitrobenzene and sodium hydride in
THF without protection of the 2′-hydroxy group (39%
yield). Tetrahydropyranyl (THP) etherification of the
alcohol 4 was achieved by protection of 9-keto with a
benzyloxime group (Scheme 3), since treatment of 4
under acidic conditions caused degradation to form the
9,12-ketal compound. 3-O-Acetalization of 6 with 3,4-
dihydro-2H-pyran in the presence of p-toluenesulfonic
acid and 4 Å molecular sieves in CH2Cl2 afforded the
desired 3-O-THP. Deprotection of the benzyl group of
the 3-O-THP ether with CTH followed by conversion of
the oxime to a ketone at the 9-position using sodium
hydrogen sulfite and formic acid in aqueous ethanol12
gave the desired ether 3f.
The antibacterial activities of TEA0777 against rep-
resentative pathogens are summarized in Table 2. This
acylide demonstrated potent activity against the eryth-
romycin-susceptible strain of Streptococcus pneumoniae,
like other macrolides. Furthermore, it was also highly
effective against Enterococcus strains and the efflux-
resistant strain of S. pneumoniae.
Resu lts a n d Discu ssion . The 3-O-substituted-6-O-
methylerythromycin A derivatives and clarithromycin
as a reference were tested for in vitro antibacterial
activity against three strains of Staphylococcus aureus.
The activities are reported in Table 1 as minimum
inhibitory concentrations (MICs) determined according

4030 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 24
Letters
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Protection Tests (ED50, mg/kg)
S. aureus Smitha
compd
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erythromycin A
clarithromycin
MIC (µg/mL)
ED50 (95% CLb)
0.39
0.20
0.20
15.4 (11.2-21.1)
56.6 (40.4-79.2)
7.6 (4.9-12.0)
a
b
S. aureus Smith: erythromycin-susceptible strain. CL:
confidence limits.
In Vivo Eva lu a tion . The in vivo efficacies of acylide
TEA0777, erythromycin A, and clarithromycin were
assessed by mouse protection tests, using the erythro-
mycin-susceptible strain of S. aureus Smith. The mice
were inoculated with 5.80 × 107 CFU/mouse intraperi-
toneally, and the macrolides were then administered
orally 1 h after inoculation. The efficacy of each mac-
rolide was reported as the effective drug dosage (ED50)
which gave a survival rate of 50% following lethal
infection over the duration of the trial (Table 3).
Acylide TEA0777 was significantly more active than
erythromycin A and comparable to clarithromycin.
Con clu sion . In summary, a series of acylides (3-O-
acyl-erythromycin A derivatives) were synthesized and
evaluated as a novel class of macrolide antibiotics. By
introducing a phenylacetyl group instead of L-cladinose
at the 3-O-position, the abolished antibacterial activity
could be restored. In particular, the 3-O-(4-nitrophenyl)-
acetyl erythromycin A derivative TEA0777 exhibited
significantly potent antibacterial activity against not
only erythromycin-susceptible Gram-positive pathogens
but also inducibly MLSB-resistant S. aureus and efflux-
resistant S. pneumoniae. It has been demonstrated that
acylides are innovative semisynthetic macrolides that
have potential as next-generation macrolide antibiotics.
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