Synthesis and antibacterial activity of 6-O-(heteroaryl-isoxazolyl)propynyl 2-fluoro ketolides

Article abstract of DOI:10.1016/j.bmcl.2012.06.092

Macrolide antibiotics are widely prescribed for the treatment of respiratory tract infections; however, the increasing prevalence of macrolide-resistant pathogens is a public health concern. Therefore, the development of new macrolide derivatives with act

Full text of DOI:10.1016/j.bmcl.2012.06.092

Bioorganic & Medicinal Chemistry Letters 22 (2012) 5739–5743
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Bioorganic & Medicinal Chemistry Letters
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Synthesis and antibacterial activity of 6-O-(heteroaryl-isoxazolyl)propynyl
2-fluoro ketolides
⇑
Tomohiro Sugimoto , Yoichi Shimazaki, Akira Manaka, Tetsuya Tanikawa, Keiko Suzuki,
Kayoko Nanaumi, Yoshie Kaneda, Yukiko Yamasaki, Hiroyuki Sugiyama
Medicinal Research Laboratories, Taisho Pharmaceutical Co. Ltd, 1-403 Yoshino-cho, Kita-ku, Saitama-shi, Saitama 331-9530, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Macrolide antibiotics are widely prescribed for the treatment of respiratory tract infections; however, the
increasing prevalence of macrolide-resistant pathogens is a public health concern. Therefore, the devel-
opment of new macrolide derivatives with activities against resistant pathogens is urgently needed. A
series of novel 6-O-(heteroaryl-isoxazolyl)propynyl 2-fluoro ketolides has been synthesized from eryth-
romycin A. These compounds have shown very promising in vitro and in vivo antibacterial activities
against key respiratory pathogens including erythromycin-susceptible/resistant strains.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 30 April 2012
Revised 16 June 2012
Accepted 18 June 2012
Available online 6 July 2012
Macrolide antibiotics1–4 (see Fig. 1 for some structures) are a
safe and effective drug class for the treatment of bacterial infec-
tions in the respiratory tract. The first macrolide antibiotic, eryth-
romycin A (EM-A) 1, was commercialized in 1952. Since EM-A
decomposes to antibacterially inactive spiroketal products5 under
acidic conditions in the stomach, its bioavailability is relatively
low and varies interindividually.6 To improve the pharmacokinetic
profile of EM-A caused by its acid instability, an enteric coating is
applied to EM-A tablets and further chemical modifications of
EM-A have been performed.1–4 Second-generation macrolides,
such as clarithromycin7 (CAM) 2 and azithromycin8 (AZM) 3, were
investigated in the 1980s and were eventually launched in the
1990s as a result of these chemical modification efforts. These
macrolide antibiotics have been widely prescribed for more than
five decades. Because of their widespread use, the increasing prev-
alence of macrolide-resistant pathogens among clinical isolates has
become a public health concern.9–12 The major mechanisms of
resistance against Gram-positive pathogens are ribosome methyl-
ation by erm methyltransferase and efflux by macrolide efflux
pumps (mediated by the mef-gene product).13–15
resistance caused by ribosome methylation.18–22 The aryl-alkyl
side chains are attached to different positions of the macrolactone
skeleton (11, 12-cabamate nitrogen for telithromycin and C-6
position for cethromycin). Despite this difference in the attached
positions, the side chains interact with similar sites in domain II
Ketolides are a chemical class of semi-synthetic erythromycin
derivatives, in which the natural C3-cladinose sugar is replaced
by a keto group. The most advanced ketolides are telithromycin
4 and cethromycin 5. These agents are known to be effective
against erythromycin-susceptible and -resistant strains of
Streptococcus pneumoniae, Streptococcus pyogenes, and Haemophilus
influenzae.16,17 These two ketolides have similar structural features,
such as a 3-keto group and a heteroaryl-alkyl side chain. The
3-keto group is important for preventing efflux resistance. The het-
eroaryl-alkyl side chain is believed to play a key role in overcoming
⇑
Corresponding author. Tel.: +81 48 669 3064; fax: +81 48 652 7254.
E-mail address: tomohiro.sugimoto@po.rd.taisho.co.jp (T. Sugimoto).
Figure 1. Structures of macrolide antibiotics.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.06.092

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T. Sugimoto et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5739–5743
of 23S rRNA.23 2-Fluoro ketolides are one of the most successful
modification of ketolides. Introduction of a fluorine atom to the
C-2 position tends to enhance the antibacterial activity of the cor-
responding ketolides both in vitro and in vivo.19,24
Modifications at the C-6 position, such as in cethromycin, are
thought to be a promising approach for improving the antibacterial
activity and pharmacokinetic profile. Most of these studies involv-
ing the modification of side chain linkage produced good activities
against erythromycin-resistant S. pneumoniae.18–22 These results
encouraged us to develop novel 2-fluoro ketolides with in vitro
and in vivo potency against resistant strains. As a result of our
continuing medicinal chemistry efforts, we have identified a novel
series of 2-fluoro ketolides in which the heteroaryl-isoxazolyl
group is attached to the 6-O-propargyl side chain.
The synthesis of novel 6-O-(heteroaryl-isoxazolyl)propynyl 2-
fluoro ketolides is shown in Schemes 1–3. Compound 6 was
prepared from EM-A in 2 steps.25 EM-A was treated with hydrox-
ylamine to produce 9-oxime erythromycin. Subsequent trans-
ketalization with O-isopropyl cyclohexylketal led to cyclohexyl
ketal 6. Intermediate 10 was prepared from ketal 6 using a previ-
ously reported method.26 Briefly, the selective protection of 20,400
hydroxyl groups was achieved using benzoyl anhydride (Bz2O) in
the presence of triethyl amine and 4-dimethylaminopyridine, and
the subsequent selective 6-O-propargylation was achieved by
treatment with propargyl bromide in the presence of potassium
tert-butoxide as the base. Deoximation with sodium nitrate pro-
duced compound 8. The formation of the carbamate functionality
was performed using a three-step, one-pot sequence to yield
compound 9. The cladinose sugar of 9 was removed under acidic
conditions, and the subsequent Corey-Kim oxidation of the 3-hy-
droxyl group produced the 3-ketolide derivative 10 (Scheme 1).
Compound 10 was treated with NaH followed by N-fluoroben-
zene sulfonamide, and the 20-O-benzoyl group was then deprotec-
ted with methanol to yield intermediate 12. Sonogashira coupling
of 12 with iodide reagents 14a–c was achieved using
bis(triphenylphosphino)palladium dichloride in the solvent as
Scheme 2. Reagents and conditions: (a) (PhSO2)2NF, NaH, DMF; (b) MeOH, reflux,
54% for two steps; (c) iodide reagents (14a–c), PdCl2(PPh3)2, Et3N, CH3CN, 60–83%.
Scheme 3. Reagents and conditions: (a) t-BuOK, t-BuNO2, THF, 71%; (b) tributy-
lethynylstannane, NCS, sat. NaHCO3–AcOEt; (c) I2, THF, 17–35%; (d) HONH2–HCl,
pyridine, EtOH; (e) HONH2–HCl, NaOMe, MeOH, 78%; (f) NaNO2, HCl, 68%; (g)
tributylethynylstannane, Et3N, THF; (h) I2, THF, 62% for two steps.
triethylamine and acetonitrile to give 6-O-(heteroaryl-isoxazolyl)
propynyl 2-fluoro ketolides 13a–c(Scheme 2). The stereochemistry
of the fluorine was determined from an X-ray crystal structure of
12 (Fig. 2).27
The preparation of 3-heteroaryl-5-iodoisoxazole reagents 14a–
c is shown in Scheme 3. Generally, isoxazoles are constructed by
the [2+3] cycloaddition of a nitrile oxide to an alkyne. In particular,
5-iodoisoxazoles are synthesized by the electrophilic halogenation
of 5-tributylstannylisoxazole,28–30 prepared by the 1,3-dipolar
cycloaddition of tributylethynylstannane with nitrile oxides. We
applied this methodology to the preparation of 3-heteroaro-
matic-substituted 5-iodoisoxazoles. Isoxazole 14a and 14b were
prepared as follows. The oximation of 3-methyl pyridazine 15 with
Scheme 1. Reagents and conditions: (a) Bz2O, DMAP, Et3N, AcOEt, 73%; (b)
propargylbromide, t-BuOK, THF-DMSO, 61%; (c) NaNO2, HCl, 60%; (d) DBU, CDI,
NH3, t-BuOK, THF, 63%; (e) 2 M HCl, EtOH; (f) dimethylsulfide, NCS, Et3N, THF, 93%
for two steps.

T. Sugimoto et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5739–5743
5741
Figure 2. X-ray crystal structure of 12.
tert-butylnitrate produced oxime 16a. The oximation of aldehyde
18 produced oxime 16b. The formation of isoxazole 17a/b was
smoothly advanced by the addition of NCS to the ethyl acetate
solution of oxime 16a/b, tributylethynylstannane, and aq. NaHCO3
as a base. In this reaction, nitrile oxide was generated in situ from
hydroximoyl chloride, formed by the chlorination of oxime 16a/b
with NCS, and the subsequent [2+3] cycloaddition of the nitrile
oxide with tributylethynylstannane was performed in one-pot to
yield isoxazole 17a/b. The iodination of 17a/b was smoothly re-
acted with iodine in THF to give iodoisoxazole 14a/b.31 14c was
prepared as follows. Aminooxime 20 was prepared from cyanide
19, and the subsequent chlorination of 20 with sodium nitrate in
hydrogen chloride yielded hydroximoyl chloride 21. The 1,3-dipo-
lar cycloaddition of tributylethynylstannane with nitrile oxides
generated from 21, and the subsequent iodination yielded isoxaz-
ole 14c.
The in vitro antibacterial activity of 6-O-(heteroaryl-isoxazol-
yl)propynyl 2-fluoro ketolides 13a–c was evaluated, as shown in
Table 1.
The minimal inhibitory concentrations (MICs) were determined
against selected respiratory pathogens including erythromycin-
susceptible S. pneumoniae ATCC49619, erythromycin-resistant
S. pneumoniae 210 with the mef(A) efflux pump gene coded,
erythromycin-resistant 205 and 1104 with the erm(B) ribosomal
methylase gene coded, erythromycin-resistant S. pyogenes with
the erm(B) gene coded, and Haemophilus influenzae ATCC43095.32
All three ketolides 13a–c were at least four-fold more active
(lower MIC values) than CAM and AZM against erythromycin-sus-
ceptible S. pneumoniae. Furthermore, 13a–c had excellent activities
against mef(A) and erm(B) gene coded erythromycin-resistant S.
pneumoniae. Especially, the MIC values of 13a–c against erm(B)
gene coded erythromycin-resistant S. pneumoniae were dramati-
cally improved, compared with those of second-generation macro-
lides (CAM and AZM). The activities of 13a–c against S. pyogenes
were sufficient but were slightly weaker than the case in S. pneu-
moniae. Compounds 13a–c were two-fold more active than CAM
against H. influenzae, but four-fold less active than AZM.
The in vivo efficacy of 2-fluoro ketolides 13a–c was evaluated in
murine pulmonary infection models,33,34 as shown in Figures 3 and
4. In an erythromycin-resistant S. pneumoniae infection model,
13a–c decreased the lung bacterial count in a dose-dependent
manner. In a H. influenzae infection model, 13a–c decreased the
lung bacterial count in a dose-dependent manner. Compound
13a had the most potent effect at a dose of 50 mg/kg. Although,
13a was four-fold less active in vitro than AZM, its in vivo efficacy
was comparable to that of AZM.
Table 1
In vitro antibacterial activities of 6-O-(heteroaryl-isoxazolyl)propynyl 2-fluoro ketolides
Compound
X
MIC (lg/mL)
S. pneumoniae
S. pyogenes
H. influenzae
ATCC49619 Ery-S
210 mef (A)
205 erm (B)
1104 erm (B)
ATCC43095 Amp-S
CAM (2)
AZM (3)
0.03
0.06
2
2
>128
>128
>128
>128
4
0.5
13a
13b
13c
0.008
0.008
0.008
0.03
0.03
0.03
0.008
0.008
0.008
1
2
2
2
N
N
N
N
NH2
0.06
2
N
N

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T. Sugimoto et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5739–5743
Figure 3. In vivo efficacy of 13a–c against murine pulmonary infection caused by S. pneumoniae 1101 (erm(B) gene coded) in mice. Values are mean S.E. Statistical analysis
was performed using Steel’s test. ⁄p <0.05 versus control.
Figure 4. In vivo efficacy of 13a–c against murine pulmonary infection caused by H. influenzae ATCC43095 in mice. Values are mean S.E. Statistical analysis was performed
using Steel’s test. ⁄p <0.05, ⁄⁄p <0.01 versus control, #p <0.05, ##p <0.01 versus 13a (comparison at 50 mg/kg).
In conclusion, a novel 6-O-(heteroaryl-isoxazolyl)propynyl 2-
fluoro ketolides has been synthesized. These 2-fluoro ketolides
showed very promising in vitro antibacterial activity against key
respiratory pathogens including erythromycin-susceptible/
resistant S. pneumoniae, erythromycin-resistant S. pyogenes and
H. influenzae. These ketolides exhibited good in vivo efficacy
against erm-containing S. pnuemoniae. Especially, piridazinyl
derivative 13a showed most potent efficacy comparable to AZM.
Further exploration of these heteroaryl-isoxazolyl ketolide will
be reported in the future.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.
06.092.
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