Design, synthesis, and anti-Helicobacter pylori activity of erythromycin A (E)-9-oxime ether derivatives

Article abstract of DOI:10.1016/j.bmcl.2005.10.045

The synthesis and anti-Helicobacter pylori (H. pylori) activity evaluation of a new series of erythromycin A (E)-9-oxime ether derivatives are described. These compounds exhibited comparable in vitro anti-H. pylori activity and improved acid stability com

Full text of DOI:10.1016/j.bmcl.2005.10.045

Bioorganic & Medicinal Chemistry Letters 16 (2006) 569–572
Design, synthesis, and anti-Helicobacter pylori activity
of erythromycin A (E)-9-oxime ether derivatives
Ghilsoo Nam,a Tae Won Kang,b Jung Hyu Shinc and Kyung Il Choia,*
aBiochemicals Research Center, Korea Institute of Science and Technology, PO Box 131, Cheongryang, Seoul, Republic of Korea
bChong Kun Dang Corporation, CKD Research Institute, Chonan PO Box 74, 330-831 Chonan, Republic of Korea
cSchool of Chemistry and Molecular Engineering, Seoul National University, Seoul 151-742, Republic of Korea
Received 22 August 2005; revised 30 September 2005; accepted 17 October 2005
Available online 4 November 2005
Abstract—The synthesis and anti-Helicobacter pylori (H. pylori) activity evaluation of a new series of erythromycin A (E)-9-oxime
ether derivatives are described. These compounds exhibited comparable in vitro anti-H. pylori activity and improved acid stability
compared to the reference compound clarithromycin.
ꢀ 2005 Elsevier Ltd. All rights reserved.
The Gram-negative bacterium Helicobacter pylori can
infect the stomach in childhood and cause lifelong
chronic gastritis, which can lead to peptic ulcer disease
and gastric cancer.1 Although a number of novel and
potent anti-H. pylori agents including antibiotics, pro-
ton-pump inhibitors, and H2-receptor antagonists have
been developed, their application to the infection is very
limited. Only a small number of multi-drug therapies
such as proton-pump inhibitors or H2-receptor antago-
nists with antibiotics have been used for eradication of
H. pylori.2,3 Dual treatments combining clarithromycin,
a potent anti-H. pylori agent derived from erythromycin,
with other agents were most popular because of their
powerful efficacy in eradicating H. pylori.4 The major
problem associated with clarithromycin, however, is its
acid instability, leading to degradation in the stomach.5
Another erythromycin-derived antibiotic roxithromycin
is known to have good pharmacokinetic character ow-
ing to its stability under gastric acid condition. From
the structural points of view, clarithromycin has a meth-
oxy group at the C-6 position, and roxithromycin has an
oxime methoxyethoxymethyl (MEM) ether group at the
C-9 position. These two functional groups are known to
affect their conformation and increase their stability in
acidic solution.6 Based on these structural information,
a new series of analogues I and II that combines the
structural characteristics of both clarithromycin and
roxithromycin was designed (Fig. 1). It was expected
that the compounds I and II would show synergistic
effects such as comparable anti-H. pylori activity to
and higher acid stability than clarithromycin.
O
O
OCH3
O
N
OCH3
HO
OH
HO
NMe2
OH
HO
NMe2
OH
OH
HO
O
O
O
O
O
O
O
O
MeO
MeO
O
O
OH
O
O
Clarithromycin
Roxithromycin
O
O
OCH3
NMe2
N
O
O
OCH3
NMe2
9
11
HO
N
OCH3
9
OH
HO
6
11
OCH3
O
HO
O
OH
6
O
HO
1
O
O
O
O
MeO
1
O
O
O
OH
O
Keywords: Anti-Helicobacter pylori; Erythromycin; Oxime ether; MIC;
Acid stability.
l
II
*
Corresponding author. Tel.: +82 2 958 5166; fax: +82 2 958
5189; e-mail: kichoi@kist.re.kr
Figure 1. Erythromycin A derivatives.
0960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.10.045

570
G. Nam et al. / Bioorg. Med. Chem. Lett. 16 (2006) 569–572
Herein, the synthesis and anti-H. pylori activity of new
erythromycin A (E)-oxime ether derivatives represented
by the structures I and II, and their stability under acidic
condition are described.
the best result was obtained by using 2.0 equiv potassi-
um hydroxide and 2.5 equiv methyl iodide at 0 ꢁC
(Table 1). Removal of the trimethylsilyl group with eth-
anolic formic acid gave 6-O-methylated compounds 14
and 15 in good yields.
Erythromycin A (E)-oxime ether derivatives were
synthesized via several stages. The first stage was the
9-oxime ether formation to yield the compounds 4 and
5 (Scheme 1). It could be performed via two reaction
routes. One was the direct condensation of the corre-
sponding alkoxyamines 8 and 9 with the 9-carbonyl
group of the erythromycin A, and the other was the for-
mation of the known compound oxime 27 followed by
the oxime ether formation with the corresponding
alkyl chlorides 3. The alkyl chlorides 3 and the alkoxy-
amines 8 and 9 could easily be prepared from dimeth-
oxybenzenes by general synthetic methods illustrated
in Scheme 2.
Further reaction of 14 and 15 was carried out to pro-
duce ketolide derivatives 20 and 21 (Scheme 3). Hydro-
lysis of the cladinose with 2 N hydrochloric acid
followed by acetylation of the 20-hydroxy group with
acetic anhydride gave 3-hydroxy intermediates 16 and
17. Oxidation of the 3-hydroxy group was efficiently per-
formed with JohnÕs reagent, and deprotection of the 20-
acetyl group with methanol gave the corresponding
ketolides 20 (51%) and 21 (55%). All new compounds
were purified by flash column chromatography and
1
13
characterized by H NMR, C NMR, and MS.9
The erythromycin A (E)-9-oxime ether derivatives 4, 5,
14, 15, 20, and 21 were tested for in vitro antimicrobial
activity against H. pylori.10 The activity results are sum-
marized in Table 2.
The second stage was the selective methylation of 6-hy-
droxy group (Scheme 3). After silylation of the hydroxy
groups on the sugar rings of the compounds 4 and 5, the
resulting compounds 10 and 11 were subjected to meth-
ylation using methyl iodide and potassium hydroxide in
a mixed solvent of DMSO and THF. A number of pre-
vious studies on selective methylation had already been
reported discussing the difficulty in controlling such
reactions.8 Upon tuning the equivalents of the reagents,
All of the compounds obtained by introducing guaia-
cylmethyl (GAM) or p-anisylmethyl (PAM) moiety at
the 9-oxime group and a methyl group at the 6-hydroxy
group exhibited potent in vitro activity with the MICs
ranging from 0.02 to 0.04 lg/ml, which were equivalent
OH
N
OH
HO
NMe2
O
OH
HO
O
O
O
MeO
O
b
a
OH
2
O
R
O
N
O
OH
HO
NMe2
OH
HO
OH
HO
O
NMe2
OH
O
HO
O
O
O
c
O
O
MeO
O
O
MeO
OH
O
O
OH
O
4 R = Guaiacyl methyl (GAM)
5 R = p-Anisyl methyl (PAM)
1 Erythromycin A
H3CO
O
PAM =
GAM =
O
OCH3
Scheme 1. Reagents and conditions: (a) hydroxylamine hydrochloride, TEA, MeOH, reflux, 68%; (b) 3 (GAM-Cl or PAM-Cl), K2CO3, DMF/ether
(1/3), 88%; (c) 8 (GAM-ONH2) or 9 (PAM-ONH2), MeOH, 43%.
O
OCH3
OCH3
a
c
b
O
O
N
O
O
NH2.HCl
R
Cl
R
O
H3CO
O
6 R = 2-methoxyphenyl
7 R = 4-methoxyphenyl 9 R = 4-methoxyphenyl
8 R = 2-methoxyphenyl
3
Scheme 2. Reagents and conditions: (a) PCl5, benzoyl peroxide, CCl4, reflux, 82%; (b) N-hydroxysuccinimide, K2CO3, DMF, 66%; (c) i—hydrazine,
EtOH, reflux; ii—HCl, 63%.

G. Nam et al. / Bioorg. Med. Chem. Lett. 16 (2006) 569–572
571
O
O
R
R
N
N
OH
TMSO
OCH3
TMSO
HO
HO
NMe2
NMe2
a
4 R = GAM
5 R = PAM
OH
OH
b
O
O
O
O
O
O
O
O
MeO
MeO
O
O
OTMS
OTMS
O
O
12 R = GAM (74%)
13 R = PAM (73%)
10 R = GAM (98%)
11 R = PAM (98%)
O
R
O
R
N
N
OCH3
HO
OCH3
HO
c
HO
NMe2
d
NMe2
e
OH
OH
HO
O
O
O
O
O
O
O
O
OH
MeO
O
OH
O
16 R = GAM (82%)
17 R = PAM (81%)
14 R = GAM (91%)
15 R = PAM (92%)
O
N
R
O
R
N
OCH3
AcO
OCH3
HO
HO
OH
HO
NMe2
NMe2
f
OH
O
O
O
O
O
O
O
OH
O
O
20 R = GAM (51%)
21 R = PAM (55%)
18 R = GAM (82%)
19 R = PAM (81%)
Scheme 3. Reagents and conditions: (a) HMDS, NH4Cl, DMF, 50 ꢁC; (b) methyl iodide, KOH, DMSO/THF (1/1); (c) formic acid, 50% aq EtOH;
(d) 2 N HCl, rt; (e) Ac2O, K2CO3, acetone; (f) JohnÕs reagent, acetone, rt.
Table 1. Optimization of selective methylation conditions for the compounds 10 and 11
Reactant
KOH (equiv)
CH3I (equiv)
Temp (ꢁC)
Time (h)
Area % of HPLCa
6-OCH3
6-OH
Di-OCH3
10
10
10
10
10
10
11
11
1.1
1.1
1.1
1.1
1.5
2.0
2.0
2.0
1.3
1.3
1.3
2.0
2.0
2.5
2.5
2.5
ꢀ5
0
2.0
2.0
2.0
5.0
4.0
2.0
0.5
2.0
88.6
86.8
81.3
15.4
19.3
0.5
11.4
13.1
16.7
28.4
80.6
94.5
85.8
90.3
—
—
5
2.0
59.5
0
0
0
—
0
14.2
7.6
0
2.1
a Waters 2487 Model. Detector; UV = 210 nm. Eluent; MeOH/H2O (0.04% ethanolamine) = 96/4.
Table 2. MICs of the compounds against Helicobacter pylori
NCTC10638
affected by the position of the methoxy group on the
phenyl ring.
Compound
H. pylori NCTC10638
MIC (lg/ml)
The acid stability of the new erythromycin A (E)-9-
oxime ether derivatives was examined at pH 1 by HPLC
analysis.11 The results are summarized in Table 3.
4
0.03
0.02
0.02
0.03
0.04
0.02
0.02
5
14
15
20
Except for the oxime compounds 4 and 5, all the other
compounds were more stable than the reference CAM.
The compounds 14 and 15 remained intact 15 times
more than CAM after 1 h at pH 1. The ketolide deriva-
tives 20 and 21 were far more stable than CAM. Over
60% of the compounds remained unaffected after 3 h.
The excellent acid stability of the ketolides could be
21
Clarithromycin
to the reference clarithromycin (CAM). The inhibitory
activity of the compounds against H. pylori was little

572
G. Nam et al. / Bioorg. Med. Chem. Lett. 16 (2006) 569–572
Table 3. HPLC area percent of residual compounds at pH 1
Time (h)
Area % of HPLCa
4
5
14
15
20
21
CAM
SMb
0c
92.32
82.42
29.21
2.60
96.33
68.17
23.63
6.51
—
96.11
67.00
56.04
46.09
26.01
15.98
81.94
74.03
56.59
46.61
28.31
16.17
95.75
80.91
73.53
69.31
65.43
60.78
96.21
82.46
76.84
69.27
65.17
61.03
92.39
81.95
29.92
3.12
1.10
—
0.5
1.0
1.5
3.0
1.00
1.00
—
a Eluent: MeOH (650)/.067 M KH2PO4 (350) pH 4.0; column: Shiseido, CAPCELL PAK, RP-18C, 5 lm, 4.6 · 250 mm; UV detector wavelength:
210 nm, 245 nm.
b SM means the purity of the compounds checked by HPLC.
c Checked right after the complete dissolution of the samples in pH 1 buffer solution (0.2 M KCl/0.2 M HCl).
CH3), 1.07–1.27 (m, 25H), 1.34 (s, 3H, 18-CH3), 1.55 (m,
5H), 1.99 (m, 2H), 2.77 (s, 6H, N(CH3)2), 2.34 (m, 2H, 200-
Hex), 2.65 (dd, J = 13.6 Hz, 6.48 Hz, 1H, 400-H), 2.88 (m,
1H, 30-H), 3.01 (t, J = 9.14 Hz, 1H), 7.3 (s, 1H), 3.21 (dd,
J = 10.1 Hz, 7.3 Hz, 1H, 20-H), 3.31 (s, 3H, 300-OCH3),
3.40–3.50 (m, 2H, 5-H, 8-H), 3.62 (m, 1H, 50-H), 3.77 (s,
3H), 3.98 (m, 2H, 500-H, 3-H), 4.25 (s, 1H, 11-H), 4.37 (d,
1H, J = 7.21 Hz, 10-H), 4.89 (d, 1H, J = 7.50 Hz, 100H),
5.15 (dd, 2H, J = 10.8 Hz, 1.59 Hz, 13-CH2), 5.52 (dd, 2H,
J = 27.9 Hz, 7.19 Hz, ph-O-CH2O), 6.83 (m, 3H), 6.95 (m,
2H). 13C NMR (CDCl3, 75.5 MHz): d 9.52, 11.04, 14.93,
16.53, 16.62, 18.97, 21.50, 21.77, 21.88, 27.17, 27.51, 29.02,
33.59, 35.43, 37.95, 39.32, 40.65, 65.79, 65.86, 69.19. 70.58,
71.33, 73.04, 74.66, 75.31, 77.23, 80.44, 83.70, 95.86, 96.67,
103.42, 115.00, 115.05, 118.82, 150.80, 155.47, 174.31,
175.57. MS (FAB) 886+ (M++1).
understood by the absence of the acid-labile 6-cladinose
moiety in their structures.
In summary, a new series of erythromycin A 9-(E)-
oxime ether derivatives 4, 5, 14, 15, 20, and 21 was de-
signed and synthesized. The compounds demonstrated
potent anti-H. pylori activity and good stability under
acidic condition. This series of compounds, especially
the ketolides 20 and 21, seems to be a good starting
point for the development of novel and effective anti-
H. pylori agents.
Acknowledgment
1
Spectral data for 15: H NMR (CDCl3, 300 MHz): d 0.74
This work was financially supported by the MOST of
Korea (2N23250).
(m, 6H), 1.19 (m, 40H), 1.74 (m, 2H), 2.12 (s, 6H,
N(CH3)2), 2.41 (m, 1H), 2.78 (s, 3H, 6-OCH3), 3.30 (m,
2H), 3.47 (m, 3H), 3.60 (s, 3H, ph-OCH3), 3.84 (m, 1H),
4.18 (m, 1H), 4.25 (m, 1H), 4.76 (br s, 1H), 4.93 (d,
J = 10.4 Hz, 1H), 5.37 (m, 2H), 6.64 (m, 4H). 13C NMR
(CDCl3, 75.5 MHz): d 8.20, 9.76, 14.17, 14.60, 17.72,
19.19, 19.66, 21.00, 21.30, 26.23, 27.72, 32.34, 33.99, 36.51,
38.10, 39.39, 44.16, 48.60, 49.97, 54.75, 64.62, 64.73, 66.90,
67.76, 68.92, 70.11, 71.78, 73.10, 75.93, 77.07, 77.41, 77.71,
79.41, 95.13, 101.84, 113.72, 116.94, 150.36, 153.86,
171.75, 174.80. MS (FAB): 972+ (M+).
References and notes
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6. Gharbi-Benarous, J.; Delaforge, M.; Jankowski, C. K.;
Girault, J. P. J. Med. Chem. 1991, 34, 1117.
1
Spectral data for 21: H NMR (CDCl3, 300 MHz): d 0.82
(t, J = 7.07 Hz, 3H), 0.92 (d, J = 6.71 Hz, 3H), 1.13 (d,
J = 6.68 Hz, 3H), 1.22 (m, 25H), 2.01 (m, 1H), 2.26 (s, 6H,
N(CH3)2), 2.52 (s, 3H, 6-OCH3), 2.57 (d, J = 9.89 Hz, 2H),
3.15 (m, 2H), 3.53 (m, 3H), 3.75 (s, 3H, ph-OCH3), 3.81
(m, 2H), 4.26 (m, 3H), 5.15 (d, J = 9.91 Hz, 1H), 5.52 (dd,
J = 20.52 Hz, 7.58 Hz, 2H), 6.77 (dd, J = 37.87 Hz,
8.81 Hz, 4H). 13C NMR (CDCl3, 75.5 MHz):d 11.19,
14.82, 15.00, 15.52, 16.73, 18.95, 20.18, 21.76, 22.01, 27.57,
28.77, 33.78, 38.47, 40.77, 47.00, 50.16, 51.42, 56.17, 66.35,
69.96, 70.45, 70.83, 74.26, 78.19, 78.56, 95.71, 103.95,
114.96, 118.02, 151.65, 155.22, 169.90, 172.55, 205.96. MS
(FAB): 739+ (M+).
7. (a) Djokic, S.; Tamburasev, Z. Tetrahedron Lett. 1967,
1645; (b) G.B Patent 1,100,504, 1966; (c) Chantot, J. P.;
Bryskier, A.; Gase, J. C. J. Antibiot. 1986, 39, 660; (d)
`
Gase, J. C.; Gouin dÕAmbrieres, S.; Lutz, A.; Chantot, J.
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Kawashima, Y.; Morimoto, S.; Matsunaga, T.; Kashim-
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Waddell, S. T.; Santorelli, G. M.; Graham, A.; Blizzard, T.
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Goto, H.; Kayashima, Y.; Kashimura, M.; Morimoto, S.;
Osawa, E. J. Chem. Soc., Perkin Trans. 2 1993, 1647.
9. Spectral data for 5: 1H NMR (CDCl3, 300 MHz): d 0.84 (t,
J = 7.24 Hz, 3H, 15-CH3), 0.97 (d, J = 6.88 Hz, 3H, 19-
10. The strain is H. pylori NCTC10630 obtained from the
Seoul National University medical center.
11. Percent of remaining compounds after stirring the corre-
sponding compound at pH 1 was analyzed. The sample
(1 mg) was dissolved in 1 ml of pH 1 buffer solution (0.2 M
KCl/0.2 M HCl) and stirred. After proper time periods,
10 ll aliquots were sampled, filtered through a membrane
filter, and injected for HPLC analysis. Elution solvent was
0.067 M phosphate buffer solution in methanol, which was
adjusted to pH 4.0. Reverse-phase 18C column was used
and the dual detector wavelength was 210 and 245 nm.