Studies on macrolide antibiotics I. Synthesis and antibacterial activity of erythromycin a 9-O-substituted oxime ether derivatives against Mycobacterium avium complex

Article abstract of DOI:10.1248/cpb.49.1120

A series of erythromycin A 9-O-substituted oxime ether derivatives have been synthesized and evaluated for antibacterial activity against Mycobacterium avium complex (MAC) and Staphylococcus aureus. These compounds possessed stronger in vitro activity aga

Full text of DOI:10.1248/cpb.49.1120

1120
Chem. Pharm. Bull. 49(9) 1120—1127 (2001)
Vol. 49, No. 9
Studies on Macrolide Antibiotics I. Synthesis and Antibacterial Activity
of Erythromycin A 9-O-Substituted Oxime Ether Derivatives against
Mycobacterium avium Complex
Akemi NISHIMOTO,* Ken NARITA, Shinobu OHMOTO, Yoshie TAKAHASHI, Satoshi YOSHIZUMI,
Toshihiko YOSHIDA, Noriyuki KADO, Eichi OKEZAKI, and Hideo KATO
Research and Development Headquaters, Hokuriku Seiyaku Co., Ltd., 37–1–1 Inokuchi, Katsuyama, Fukui 911–8555,
Japan. Received March 19, 2001; accepted May 14, 2001
A series of erythromycin A 9-O-substituted oxime ether derivatives have been synthesized and evaluated
for antibacterial activity against Mycobacterium avium complex (MAC) and Staphylococcus aureus. These com-
pounds possessed stronger in vitro activity against MAC including macrolide-resistant strains than clar-
ithromycin (2), although in vitro antibacterial activities of these compounds were less than that of 2 against
Staphylococcus aureus. Our studies found that several factors contribute to the antibacterial activity against
MAC. The length and spatial orientation of the substituent at 9-position were found to significantly influenced
the anti-MAC activity, especially against macrolide-resistant strains. Of all the compounds prepared, ery-
thromycin A 9-[O-(4-phenylbutyl)oxime] (12q) and erythromycin A 9-[O-(3-phenoxypropyl)oxime] (12t) pos-
sessed 16 times stronger antibacterial activity than 2 against clarithromycin-resistant strains. Surprisingly, the
minimum inhibitory concentrations (MICs) of 12q and 12t against the resistant strains were almost same as
those against the susceptible strains. These results suggest that the erythromycin A 9-O-substituted oxime ether
derivatives would be promising macrolide antibiotics.
Key words macrolide; antibiotic; antibacterial activity; Mycobacterium avium complex; erythromycin A 9-O-substituted oxime
ether
Since erythromycin A (1)1) was discovered in 1952, much
cially the ketolides exhibited excellent activity against not
effort in drug discovery and in searching for a bioactive nat-
ural compound has been directed to the development of these
exquisite macrolide antibiotics bearing potent activity, broad
spectrum, good absorption and lack of serious side effects.
New macrolide antibiotics, such as clarithromycin (2),2) rox-
ithromycin (3),3) azithromycin (4)4) and rokitamycin (5),5)
were consequently developed in the 1980s. Furthermore,
a new class of antibiotics called ketolides, such as
telithromycin (6)6) and ABT-773 (7),7) was discovered in the
1990s. These new macrolide agents with improved pharma-
cokinetic profile and less gastrointestinal side effects, espe-
only common strains but also resistant strains induced by the
previous macrolide antibiotics. In recent days, therapeutic
use of these macrolide agents has expanded into the treat-
ment of respiratory infections by Mycobacterium avium
complex (MAC). The MAC infection has been a serious
worldwide problem and its prevalence has been considered to
be fatal for AIDS patients.8) There have been, however, few
effective agents against MAC infection except 2 or 4.9)
Although a number of the structure–activity relationships
(SAR) of some macrolides against common strains have
been found,10) there have been no systematic studies of
Chart 1
To whom correspondence should be addressed. e-mail: organ-hs@mitene.or.jp
© 2001 Pharmaceutical Society of Japan

September 2001
1121
Chart 2
macrolides against MAC. To discover a novel excellent the 2274-position in 23S rRNA, respectively. These alter-
macrolide bearing good activity against macrolide-resistant ations were identical to those of macrolide-resistant strains
MAC as well as -susceptible MAC, we started to synthesize reported by Nash and Inderlied14b) The same mutation was
various erythromycin A 9-O-substituted oxime ether deriva- also reported in the macrolide-resistant Mycobacterium in-
tives and evaluate them for anti-MAC activity. In this paper, tracellulare.14b) As a result, the strains in this study are
we describe our SAR studies on erythromycin A 9-O-substi- known to be resistant to 2 by the mutation in the 2274-posi-
tuted oxime ether derivatives and several our structural in- tion at 23S rRNA.
sights.
Chemistry The erythromycin A 9-O-substituted oxime Results and Discussion
ether derivatives (12, 13) were basically prepared as shown in
All compounds (12, 13) were evaluated for in vitro an-
Chart 2, according to the literature.11) In the presence of tibacterial activity against Mycobacterium intracellulare
potassium hydroxide or sodium hydride and tetrabutylammo- 20066, Mycobacterium avium 20039, Mycobacterium avium
nium iodide, the reaction of erythromycin A 9-oxime (8)12) 20092, Mycobacterium avium 20096, and Staphylococcus
with various mesylates (10) or bromides (11) in tetrahydrofu- aureus FDA 209P (gram-positive common strain) and their
ran (THF) afforded the 9-O-substituted oxime ether deriva- minimum inhibitory concentrations (MICs, mg/ml) are
tives (12). In a similar manner to the preparation of 12, the 6- shown in Table 3. The data for 1 and 2 are included for com-
O-methylerythromycin A analogue (13t) was derived from parison.
the corresponding 9-oxime (9).13) The physicochemical and
spectral data of these compounds (12d—x, 13t) are listed in FDA 209P With regard to the introduction of alkyl groups
Table 1. into the 9-oxime, the lower alkyl oxime ether derivatives
Antibacterial Activity against Staphylococcus aureus
Alteration of the Target Site Clinically isolated MAC (12a, c, d) showed a similar antibacterial activity to that of 2,
(Mycobacterium intracellulare 20066, Mycobacterium avium but the introduction of more bulky alkyl groups (12e—j) de-
20039, Mycobacterium avium 20092, Mycobacterium avium creased the potency, especially 12g—j which were quite in-
20096) was used in this study. It has been known that resis- active. The quantitative structure–activity analysis of the 6-
tance to macrolides is caused by mutation at several sites in O-methyl erythromycin A 9-O-substituted oxime ether deriv-
the peptidyl transferase loop of 23S rRNA from the clinical atives with the Hansch–Fujita method15) has been studied by
isolated MAC.14) To clarify the target mutation of the strains Kawashima et al.10a) They pointed out that the antibacterial
used in this study, we analyzed the base sequence of each activity against Staphylococcus aureus FDA 209P was signif-
23S rRNA according to the literature.14) The analytical re- icantly correlated with the hydrophobic parameters such as
sults are shown in Table 2. The base sequences of Mycobac- calculated log P. And they concluded that the activity would
terium intracellulare 20066 and Mycobacterium avium be inversely proportional to the calculated log P. A similar
20039 which were considered susceptible to 2 were identified tendency was also observed in our studies.
as those of macrolide-susceptible strains reported by Böttger
Antibacterial Activity against MAC Although most of
and his co-workers14a) and Nash and Inderlied14b) On the the 9-oxime ether derivatives showed good activity against
other hand, the sequences of Mycobacterium avium 20092 susceptible strains, there were not so many compounds with
and Mycobacterium avium 20096 which were resistant to 2 significant activity against resistant strains. Compounds 12q
were altered by A→G and A→C transitional mutations at and 12t exhibited the most potent activity against resistant

1122
Vol. 49, No. 9
Table 1. Physicochemical Data for Erythromycin A 9-O-Substituted Oxime Ether Derivatives
Compd. No.
Yield (%)
85
mp (°C) (Solv.)
Formula
HR-MS m/z Calcd (Found)
804.5347 (Mϩ) (804.5273)
12d
109.5—110.5
(iso-Pr2O)
Amorphous
112—113
C41H76N2O13
a)
12e
12f
62
78
C43H80N2O13
C45H84N2O13
674.4479 (MϪC8H16NO2b)
)
ϩ (674.4460)
686.4843 (MϪC8H16NO3
)
ϩ (686.4842)
(iso-Pr2O–hexane)
75.5—76.5
(H2O–THF)
67.5—68.5
(H2O–THF)
71—72
b)
12g
12h
12i
42
77
38
57
57
C47H88N2O13
C49H92N2O13
C51H96N2O13
C53H100N2O13
C44H80N2O13
715.5234 (Mϩ1ϪC8H16NO3
)
ϩ (715.5217)
a)
758.5418 (MϪC8H16NO2
770.5782 (MϪC8H16NO3
)
ϩ (758.5420)
b)
)
ϩ (770.5774)
(H2O–THF)
77—79.5
(H2O–THF)
115—116.5
(iso-Pr2O)
b)
12j
12k
799.6173 (Mϩ1ϪC8H16NO3
)
ϩ (799.6146)
844.5660 (Mϩ) (844.5695)
b)
12l
12m
47
43
Amorphous
98—100
C45H82N2O13
C46H84N2O13
684.4687 (MϪC8H16NO3b)
)
)
ϩ (684.4674)
ϩ (698.4859)
698.4843 (MϪC8H16NO3
(iso-Pr2O)
109—110
(iso-Pr2O)
99—100
(H2O–Et2O)
91—92
(H2O–Et2O)
94.5—96
b)
12o
12p
12q
12r
93
83
56
54
C45H76N2O13
C46H78N2O13
C47H80N2O13
C48H82N2O13
678.4217 (MϪC8H16NO3
)
)
)
)
ϩ (678.4234)
ϩ (692.4384)
ϩ (722.4486)
ϩ (720.4681)
b)
692.4374 (MϪC8H16NO3
a)
722.4479 (MϪC8H16NO2
b)
720.4687 (MϪC8H16NO3
(iso-Pr2O–AcOEt)
Amorphous
97.5—98
b)
12s
12t
48
59
C45H76N2O14
C46H78N2O14
694.4166 (MϪC8H16NO3b)
)
ϩ (694.4165)
ϩ (708.4328)
708.4323 (MϪC8H16NO3
)
(iso-Pr2O)
91—92
a)
12u
70
C47H80N2O14
738.4429 (MϪC8H16NO2
)
ϩ (738.4451)
(H2O–iso-Pr2O)
Amorphous
Amorphous
Amorphous
105—107
a)
12v
12w
12x
13t
32
65
24
69
C46H79N3O13
C43H74N2O13S
C45H76N2O13S
C47H80N2O14
723.4432 (MϪC8H16NO2b)
)
)
)
)
ϩ (723.4432)
ϩ (684.3788)
ϩ (726.3891)
ϩ (738.4446)
684.3781 (MϪC8H16NO3a)
726.3887 (MϪC8H16NO2a)
738.4429 (MϪC8H16NO2
(iso-Pr2O–heptane)
a) C8H16NO2: desosamine. b) C8H16NO3: -O-desosamine.
Table 2. The Sequence of Part 23S rRNA Gene of MAC
Strain
Propertya)
Partial 23S rRNA gene sequence
M. intracellulare
M. avium
M. avium
20066
20039
20092
20096
Susceptible
Susceptible
Resistant
GCGCGGCAGGACGAAb)AAGACCCCGGGACCT
A
G
C
M. avium
Resistant
a) Susceptible or resistant property for 2 determined by agar dilution method are listed. b) 2274-position in 23S rRNA of MAC.
strains among all compounds, and their length indices of 9- ring. Thus, the length of the 9-substituents plays a key role
substituents, which was calculated by Sterimol calculation for the antibacterial activity against resistant strains.
software,16) were about 10.3 Å. On the contrary, the com-
Although the introduction of a 6-methoxy group enhanced
pounds bearing shorter length, such as 12k—o, tended to de- activity against common strains,2) the antibacterial activity
crease the activity against resistant strains. The inactive com- against resistant strains of 6-O-methoxyerythromycin A 9-O-
pounds against resistant strains actually had somewhat short substituted oxime ether derivative (13t) was 2—4 times less
length of substituents such as lower alkyl (12a—d), cyclo- than that of the parent compound 12t. So the methoxy group
hexylmethyl (12k) and benzyl (12n) at the 9-position. The at position 6 of the 9-oxime ether derivative was not appro-
substituents at the 9-position of 12i, j were not sufficient to priate for antibacterial activity.
show a good antibacterial activity against both the resistant
Molecular Modeling To elucidate factors affecting an-
and susceptible strains. Phenylthioethyl (7.93 Å), phenyl- tibacterial activity in detail, we performed a conformational
propyl (7.91 Å) or phenoxyethyl (7.80 Å) groups at the 9-po- analysis of the compounds (12). The initial conformations
sition were also insufficient, so the compounds (12x, p, s) did were built on the basis of the X-ray structure of 217) using
not show good activity accordingly. And one compound CAChe system.18) We implemented a systematic conforma-
(12w) showed the same potency as another (12o), because a tional analysis around all rotable bonds of the 9-substituents
thiophene ring is considered to be an isostere of a benzene and energy-minimized by -ES force field,19) which excluded

September 2001
1123
Table 3. In Vitro Antibacterial Activities of Erythromycin A 9-O-Substituted Oxime Ether Derivatives
MIC (mg/ml)a)
Compd.
No.
S. aureus
M. intracellulare
M. avium
M. avium
M. avium
FDA209Pb)
20066b)
20039b)
20092c)
20096c)
12a
12b
12c
12d
12e
12f
12g
12h
12i
0.78
3.13
0.39
0.78
6.25
3.13
Ͼ50
Ͼ50
Ͼ50
Ͼ50
1.56
12.5
12.5
6.25
0.78
0.39
3.13
12.5
0.78
0.78
1.56
0.78
1.56
0.39
1.56
0.78
0.78
12.5
6.25
3.13
1.56
3.13
6.25
6.25
50
Ͼ50
Ͼ50
6.25
6.25
6.25
6.25
1.56
1.56
1.56
3.13
3.13
1.56
3.13
1.56
3.13
3.13
3.13
Ͼ50
3.13
3.13
3.13
1.56
0.78
1.56
3.13
12.5
25
Ͼ50
Ͼ50
Ͼ50
Ͼ50
25
25
25
25
Ͼ50
Ͼ50
Ͼ50
Ͼ50
12.5
Ͼ50
Ͼ50
12.5
6.25
6.25
12.5
3.13
12.5
25
Ͼ50
Ͼ50
Ͼ50
Ͼ50
6.25
6.25
6.25
25
Ͼ50
Ͼ50
Ͼ50
50
6.25
Ͼ50
25
12.5
3.13
6.25
25
50
12j
12k
12l
Ͼ50
1.56
3.13
3.13
3.13
1.56
1.56
1.56
3.13
1.56
1.56
1.56
1.56
1.56
1.56
1.56
25
12m
12n
12o
12p
12q
12r
12s
12t
12u
12v
12w
12x
13t
1
3.13
6.25
25
50
25
12.5
Ͼ50
Ͼ50
25
12.5
6.25
Ͼ50
Ͼ50
2
0.78
a) Inoculum size, one loopful of 106 CFU/ml. b) These strains are susceptible to 2. c) These strains are resistant to 2.
Fig. 1. The Minimized Structure of 12d and 12t Calculated by the Molecular Modeling Program CAChe (Version 4.1.1)
the electrostatic energy term and the hydrogen bonding en-
ergy term from the total MM2 force field. This analysis con-
sequently suggested that erythromycin A 9-O-substituted
oxime ether derivatives could be classified into the following
two types: 1) the direction of the substituents at 9-position
was perpendicular to the plane of the aglycon such as the
conformation of 12d (type A, 12a—d), 2) the direction of
the substituents was parallel to the plane of aglycon such as
the conformation of 12t (type B, 12e—x, 13t). The confor-
mations of 12d (type A) and 12t (type B) are shown in Fig.
Fig. 2. NOE Correlations of 12t
1. Interestingly, all compounds (12a—d) classified into type

1124
Vol. 49, No. 9
Table 4. Spectral Data for Erythromycin A 9-O-Substituted Oxime Ether Derivatives
Compd.
No.
IR (KBr)
(cmϪ1
1H-NMR (CDCl3) d (ppm)
MS m/z
)
12d
12e
12f
12g
0.84 (3H, t, Jϭ7.5 Hz), 0.94 (3H, t, Jϭ7.5 Hz), 1.02 (3H, d, Jϭ6.5 Hz), 1.07—1.80 (35H, m), 1.85—2.07 1738
804 (Mϩ)
(2H, m), 2.20 (1H, d, Jϭ10.5 Hz), 2.29 (6H, s), 2.36 (1H, d, Jϭ15.5 Hz), 2.40—2.50 (1H, m), 2.60—
2.70 (1H, m), 2.85—2.95 (1H, m), 3.02 (1H, t, Jϭ9.5 Hz), 3.10 (1H, s), 3.22 (1H, dd, Jϭ10, 7.5 Hz),
3.32 (3H, s), 3.40 (1H, br s), 3.45—3.55 (1H, m), 3.59 (1H, d, Jϭ7.5 Hz), 3.61—3.75 (2H, m), 3.98—
4.08 (4H, m), 4.43 (1H, d, Jϭ7.5 Hz), 4.45 (1H, s), 4.92 (1H, d, Jϭ4.5 Hz), 5.11 (1H, dd, Jϭ11, 2.5 Hz)
0.84 (3H, t, Jϭ7.5 Hz), 0.89 (3H, t, Jϭ7 Hz), 1.00—1.80 (42H, m), 1.87—2.05 (2H, m), 2.21 (1H, d,
Jϭ10.5 Hz), 2.29 (6H, s), 2.36 (1H, d, Jϭ15.5 Hz), 2.39—2.48 (1H, m), 2.60—2.70 (1H, m), 2.85—
2.96 (1H, m), 3.02 (1H, t, Jϭ10 Hz), 3.11 (1H, s), 3.22 (1H, dd, Jϭ10.5, 7.5 Hz), 3.32 (3H, s), 3.39
(1H, s), 3.45—3.54 (1H, m), 3.59 (1H, d, Jϭ7.5 Hz), 3.64—3.75 (2H, m), 3.95—4.10 (4H, m), 4.43
(1H, d, Jϭ6.5 Hz), 4.46 (1H, s), 4.92 (1H, d, Jϭ5 Hz), 5.12 (1H, dd, Jϭ11.5, 2 Hz)
(CϭO)
1738
(CϭO)
832 (Mϩ)
0.84 (3H, t, Jϭ7.5 Hz), 0.88 (3H, t, Jϭ7 Hz), 0.98—1.80 (46H, m), 1.85—2.06 (2H, m), 2.22 (1H, d,
1738
861 (Mϩ1)ϩ
889 (Mϩ1)ϩ
Jϭ10.5 Hz), 2.29 (6H, s), 2.36 (1H, d, Jϭ15.5 Hz), 2.40—2.50 (1H, m), 2.62—2.70 (1H, m), 2.87—2.97 (CϭO)
(1H, m), 3.02 (1H, t, Jϭ10 Hz), 3.11 (1H, s), 3.22 (1H, dd, Jϭ10.5, 7.5 Hz), 3.32 (3H, s), 3.39 (1H, br s),
3.45—3.57 (1H, m), 3.59 (1H, d, Jϭ7.5 Hz), 3.61—3.75 (2H, m), 3.97—4.08 (4H, m), 4.43 (1H, d,
Jϭ7.5 Hz), 4.46 (1H, s), 4.92 (1H, d, Jϭ5 Hz), 5.12 (1H, dd, Jϭ11.5, 2 Hz)
0.84 (3H, t, Jϭ7.5 Hz), 0.88 (3H, t, Jϭ7 Hz), 0.96—1.80 (47H, m), 1.02 (3H, d, Jϭ6.5 Hz), 1.87—2.05
(2H, m), 2.20 (1H, d, Jϭ10.5 Hz), 2.28 (6H, s), 2.36 (1H, d, Jϭ14.5 Hz), 2.38—2.48 (1H, m), 2.60—
2.70 (1H, m), 2.81—2.95 (1H, m), 3.02 (1H, t, Jϭ10 Hz), 3.11 (1H, s), 3.22 (1H, dd, Jϭ10, 7.5 Hz),
3.32 (3H, s), 3.40 (1H, br s), 3.45—3.54 (1H, m), 3.59 (1H, d, Jϭ7.5 Hz), 3.65—3.75 (1H, m), 3.68
(1H, s), 3.95—4.09 (4H, m), 4.43 (1H, d, Jϭ7.5 Hz), 4.45 (1H, s), 4.92 (1H, d, Jϭ5 Hz), 5.11 (1H, dd,
Jϭ11, 2.5 Hz)
1738
(CϭO)
12h
12i
0.84 (3H, t, Jϭ7.5 Hz), 0.88 (3H, t, Jϭ7 Hz), 1.00—1.70 (54H, m), 1.85—2.10 (2H, m), 2.21 (1H, d,
Jϭ10 Hz), 2.29 (6H, s), 2.36 (1H, d, Jϭ15.5 Hz), 2.39—2.50 (1H, m), 2.60—2.70 (1H, m), 2.85—2.95
(1H, m), 3.02 (1H, t, Jϭ10 Hz), 3.10 (1H, s), 3.22 (1H, dd, Jϭ10.5, 7.5 Hz), 3.32 (3H, s), 3.38 (1H,
br s), 3.45—3.55 (1H, m), 3.59 (1H, d, Jϭ7.5 Hz), 3.65—3.75 (2H, m), 3.95—4.08 (4H, m), 4.43 (1H, d,
Jϭ7.5 Hz), 4.45 (1H, s), 4.92 (1H, d, Jϭ5 Hz), 5.08—5.15 (1H, m)
17381
(CϭO)
916 (Mϩ)
0.84 (3H, t, Jϭ7.5 Hz), 0.88 (3H, t, Jϭ6.5 Hz), 1.02 (3H, d, Jϭ7.5 Hz), 1.07—1.80 (55H, m), 1.86—
2.07 (2H, m), 2.21 (1H, d, Jϭ10.5 Hz), 2.29 (6H, s), 2.36 (1H, d, Jϭ15.5 Hz), 2.39—2.48 (1H, m),
2.62—2.70 (1H, m), 2.85—2.95 (1H, m), 3.02 (1H, t, Jϭ10 Hz), 3.11 (1H, s), 3.22 (1H, dd, Jϭ10.5,
7.5 Hz), 3.32 (3H, s), 3.39 (1H, br s), 3.45—3.55 (1H, m), 3.59 (1H, d, Jϭ7.5 Hz), 3.64—3.76 (1H, m),
3.68 (1H, s), 3.94—4.10 (4H, m), 4.43 (1H, d, Jϭ7.5 Hz), 4.46 (1H, s), 4.92 (1H, d, Jϭ5 Hz), 5.11 (1H,
dd, Jϭ11, 2.5 Hz)
1738
(CϭO)
945 (Mϩ1)ϩ
12j
0.84 (3H, t, Jϭ7.5 Hz), 0.88 (3H, t, Jϭ7 Hz), 0.95—1.80 (59H, m), 1.02 (3H, d, Jϭ6.5 Hz), 1.87—2.04
(2H, m), 2.21 (1H, d, Jϭ10.5 Hz), 2.28 (6H, s), 2.36 (1H, d, Jϭ15.5 Hz), 2.39—2.46 (1H, m), 2.61—
2.68 (1H, m), 2.86—2.93 (1H, m), 3.02 (1H, t, Jϭ10 Hz), 3.11 (1H, s), 3.22 (1H, dd, Jϭ10, 7.5 Hz),
3.32 (3H, s), 3.40 (1H, br s), 3.42—3.52 (1H, m), 3.59 (1H, d, Jϭ7.5 Hz), 3.63—3.72 (1H, m), 3.68
1H, s), 3.95—4.08 (4H, m), 4.43 (1H, d, Jϭ7.5 Hz), 4.46 (1H, s), 4.92 (1H, d, Jϭ5 Hz), 5.11 (1H, dd,
Jϭ11, 2 Hz)
1738
(CϭO)
973 (Mϩ1)ϩ
12k
12l
0.84 (3H, t, Jϭ7.5 Hz), 0.92—1.80 (45H, m), 1.87—2.04 (2H, m), 2.21 (1H, d, Jϭ10.5 Hz), 2.29 (6H, s), 1738
844 (Mϩ)
858 (Mϩ)
872 (Mϩ)
2.36 (1H, d, Jϭ14.5 Hz), 2.39—2.47 (1H, m), 2.62—2.68 (1H, m), 2.86—2.95 (1H, m), 3.02 (1H, t,
Jϭ10 Hz), 3.11 (1H, s), 3.22 (1H, dd, Jϭ10.5, 7.5 Hz), 3.32 (3H, s), 3.39 (1H, br s), 3.45—3.54 (1H, m),
3.59 (1H, d, Jϭ8 Hz), 3.65—3.75 (2H, m), 3.77—3.87 (2H, m), 3.98—4.07 (2H, m), 4.43 (1H, d,
Jϭ7.5 Hz), 4.46 (1H, s), 4.92 (1H, d, Jϭ4.5 Hz), 5.11 (1H, dd, Jϭ11, 2 Hz)
(CϭO)
0.84 (3H, t, Jϭ7.5 Hz), 0.88—1.78 (47H, m), 1.86—2.06 (2H, m), 2.20 (1H, d, Jϭ10.5 Hz), 2.29 (6H, s), 1738
2.36 (1H, d, Jϭ14.5 Hz), 2.38—2.47 (1H, m), 2.62—2.69 (1H, m), 2.86—2.96 (1H, m), 3.02 (1H, t,
Jϭ10 Hz), 3.10 (1H, s), 3.22 (1H, dd, Jϭ10, 7.5 Hz), 3.32 (3H, s), 3.40 (1H, s), 3.44—3.54 (1H, m),
3.59 (1H, d, Jϭ7.5 Hz), 3.64—3.74 (2H, m), 3.96—4.09 (4H, m), 4.43 (1H, d, Jϭ7.5 Hz), 4.45 (1H, s),
4.91 (1H, d, Jϭ5 Hz), 5.12 (1H, dd, Jϭ11, 2 Hz)
(CϭO)
12m
12o
12p
12q
0.80—1.81 (49H, m), 0.84 (3H, t, Jϭ7.5 Hz), 1.86—2.06 (2H, m), 2.21 (1H, d, Jϭ10.5 Hz), 2.29 (6H, s), 1738
2.36 (1H, d, Jϭ15.5 Hz), 2.39—2.48 (1H, m), 2.60—2.70 (1H, m), 2.85—2.96 (1H, m), 3.02 (1H, t,
Jϭ9.5 Hz), 3.11 (1H, s), 3.22 (1H, dd, Jϭ10.5, 7.5 Hz), 3.32 (3H, s), 3.40 (1H, s), 3.44—3.54 (1H, m),
3.59 (1H, d, Jϭ7.5 Hz), 3.63—3.75 (2H, m), 3.92—4.09 (4H, m), 4.43 (1H, d, Jϭ7.5 Hz), 4.46 (1H, s),
4.92 (1H, d, Jϭ4.5 Hz), 5.12 (1H, dd, Jϭ11, 2.5 Hz)
(CϭO)
0.85 (3H, t, Jϭ7.5 Hz), 0.98—1.70 (34H, m), 1.88—2.00 (2H, m), 2.20 (1H, d, Jϭ10.5 Hz), 2.28 (6H, s), 1738
679
2.37 (1H, d, Jϭ16 Hz), 2.37—2.46 (1H, m), 2.60—2.69 (1H, m), 2.85—2.99 (3H, m), 3.03 (1H, t,
Jϭ10 Hz), 3.12 (1H, s), 3.20 (1H, dd, Jϭ10.5, 7.5 Hz), 3.32 (3H, s), 3.37 (1H, s), 3.42—3.53 (2H, m),
3.54—3.63 (1H, m), 3.71 (1H, s), 3.95—4.05 (2H, m), 4.28 (2H, t, Jϭ6.5 Hz), 4.39 (1H, d, Jϭ7.5 Hz),
4.46 (1H, s), 4.93 (1H, d, Jϭ5 Hz), 5.14 (1H, dd, Jϭ11, 2.5 Hz), 7.17—7.34 (5H, m)
(CϭO)
(Mϩ1ϪC8H16NO3)ϩ
0.85 (3H, t, Jϭ7.5 Hz), 1.04—1.70 (33H, m), 1.78 (1H, brs), 1.87—2.06 (4H, m), 2.21 (1H, d, Jϭ
10.5 Hz), 2.28 (6H, s), 2.36 (1H, d, Jϭ15.5 Hz), 2.39—2.48 (1H, m), 2.62—2.73 (3H, m), 2.86—2.95
(1H, m), 3.02 (1H, t, Jϭ10 Hz), 3.10 (1H, s), 3.22 (1H, dd, Jϭ10.5, 7.5 Hz), 3.32 (3H, s), 3.41 (1H,
br s), 3.44—3.53 (1H, m), 3.59 (1H, d, Jϭ8 Hz), 3.67—3.78 (2H, m), 3.97—4.09 (4H, m), 4.41 (1H, s),
4.43 (1H, d, Jϭ7.5 Hz), 4.92 (1H, d, Jϭ5 Hz), 5.11 (1H, dd, Jϭ11, 2.5 Hz), 7.13—7.32 (5H, m)
1738
(CϭO)
867 (Mϩ1)ϩ
0.84 (3H, t, Jϭ7.5 Hz), 0.98—1.76 (38H, m), 1.87—2.04 (2H, m), 2.21 (1H, d, Jϭ10.5 Hz), 2.29 (6H, s), 1738
880 (Mϩ)
2.36 (1H, d, Jϭ15.5 Hz), 2.39—2.46 (1H, m), 2.60—2.68 (3H, m), 2.86—2.95 (1H, m), 3.03 (1H, t,
Jϭ10 Hz), 3.10 (1H, s), 3.22 (1H, dd, Jϭ10, 7 Hz), 3.32 (3H, s), 3.41 (1H, br s), 3.45—3.53 (1H, m),
3.58 (1H, d, Jϭ7.5 Hz), 3.63—3.73 (2H, m), 3.97—4.07 (4H, m), 4.42 (1H, s), 4.43 (1H, d, Jϭ7.5 Hz),
4.92 (1H, d, Jϭ5 Hz), 5.11 (1H, dd, Jϭ11, 2 Hz), 7.14—7.20 (3H, m), 7.24—7.30 (2H, m)
(CϭO)

September 2001
1125
Table 4. (continued)
Compd.
No.
IR (KBr)
1H-NMR (CDCl3) d (ppm)
MS m/z
(cmϪ1
)
12r
12s
12t
0.84 (3H, t, Jϭ7.5 Hz), 0.95—1.81 (40H, m), 1.87—2.04 (2H, m), 2.21 (1H, d, Jϭ10 Hz), 2.29 (6H, s),
2.36 (1H, d, Jϭ15.5 Hz), 2.40—2.48 (1H, m), 2.59—2.68 (3H, m), 2.86—2.94 (1H, m), 3.02 (1H, t,
Jϭ10 Hz), 3.10 (1H, s), 3.22 (1H, dd, Jϭ10, 7.5 Hz), 3.32 (3H, s), 3.41 (1H, br s), 3.45—3.53 (1H, m),
3.59 (1H, d, Jϭ7.5 Hz), 3.64—3.72 (2H, m), 3.97—4.07 (4H, m), 4.43 (1H, d, Jϭ7.5 Hz), 4.44 (1H, s),
4.91 (1H, d, Jϭ5 Hz), 5.11 (1H, dd, Jϭ11, 2.5 Hz), 7.13—7.20 (3H, m), 7.23—7.30 (2H, m)
1738
(CϭO)
720
(MϪC8H16NO3)ϩ
0.84 (3H, t, Jϭ7.5 Hz), 0.97—1.75 (33H, m), 1.86—2.03 (3H, m), 2.22 (1H, d, Jϭ10.5 Hz), 2.28 (6H, s), 1738
694
2.34 (1H, d, Jϭ14.5 Hz), 2.38—2.47 (1H, m), 2.64—2.74 (1H, m), 2.83—2.93 (1H, m), 3.00 (1H, t,
Jϭ10 Hz), 3.09 (1H, s), 3.21 (1H, dd, Jϭ10, 7.5 Hz), 3.30 (3H, s), 3.37 (1H, br s), 3.42—3.57 (2H, m),
3.66—3.80 (2H, m), 3.87—4.04 (2H, m), 4.08—4.24 (2H, m), 4.33—4.44 (4H, m), 4.77 (1H, d, Jϭ
4.5 Hz), 5.10 (1H, dd, Jϭ11, 2.5 Hz), 6.84—6.99 (3H, m), 7.22—7.33 (2H, m)
0.85 (3H, t, Jϭ7.5 Hz), 1.02 (3H, d, Jϭ6.5 Hz), 1.08—1.80 (31H, m), 1.87—2.05 (2H, m), 2.08—2.18
(2H, m), 2.21 (1H, d, Jϭ10.5 Hz), 2.29 (6H, s), 2.33—2.48 (1H, m), 2.36 (1H, d, Jϭ15.5 Hz), 2.63—
2.72 (1H, m), 2.85—2.96 (1H, m), 3.02 (1H, t, Jϭ10 Hz), 3.10 (1H, s), 3.22 (1H, dd, Jϭ10, 7.5 Hz), 3.32
(3H, s), 3.41 (1H, s), 3.44—3.53 (1H, m), 3.57 (1H, d, Jϭ7.5 Hz), 3.64—3.76 (2H, m), 3.96—4.10 (4H,
m), 4.16—4.27 (2H, m), 4.38 (1H, s), 4.42 (1H, d, Jϭ7.5 Hz), 4.92 (1H, d, Jϭ5 Hz), 5.12 (1H, dd, Jϭ
11, 2 Hz), 6.84—6.96 (3H, m), 7.21—7.32 (2H, m)
(CϭO)
(MϪC8H16NO3)ϩ
1738
(CϭO)
708
(MϪC8H16NO3)ϩ
12u
12v
12w
12x
13t
0.85 (3H, t, Jϭ7.5 Hz), 0.96—2.05 (40H, m), 2.21 (1H, d, Jϭ10.5 Hz), 2.25—2.40 (1H, m), 2.32 (6H, s), 1738
896 (Mϩ)
2.40—2.55 (1H, m), 2.60—2.70 (1H, m), 2.85—2.95 (1H, m), 3.02 (1H, t, Jϭ9.5 Hz), 3.10 (1H, s),
3.20—3.27 (1H, m), 3.32 (3H, s), 3.40 (1H, s), 3.45—3.55 (1H, m), 3.59 (1H, d, Jϭ7.5 Hz), 3.65—3.75
(2H, m), 3.95—4.15 (6H, m), 4.41 (1H, s), 4.43 (1H, d, Jϭ7.5 Hz), 4.90 (1H, d, Jϭ5 Hz), 5.11 (1H, dd,
Jϭ11, 2 Hz), 6.90—7.00 (3H, m), 7.20—7.30 (2H, m)
0.85 (3H, t, Jϭ7.5 Hz), 0.92—1.70 (33H, m), 1.80—2.01 (3H, m), 2.21 (1H, d, Jϭ10 Hz), 2.28 (6H, s),
2.36 (1H, d, Jϭ14.5 Hz), 2.37—2.45 (1H, m), 2.60—2.70 (1H, m), 2.85—2.98 (1H, m), 2.95 (3H, s),
3.02 (1H, t, Jϭ10 Hz), 3.09 (1H, s), 3.21 (1H, dd, Jϭ10, 7.5 Hz), 3.32 (3H, s), 3.39 (1H, br s), 3.43—
3.58 (4H, m), 3.60—3.75 (2H, m), 3.95—4.05 (2H, m), 4.17—4.23 (2H, m), 4.35 (1H, s), 4.41 (1H, d, Jϭ
6.5 Hz), 4.90 (1H, d, Jϭ4.5 Hz), 5.11 (1H, dd, Jϭ11, 2 Hz), 6.68—6.77 (3H, m), 7.20—7.30 (2H, m)
(CϭO)
1738
(CϭO)
723
(MϪC8H16NO2)ϩ
0.85 (3H, t, Jϭ7.5 Hz), 0.97—1.70 (34H, m), 1.87—2.01 (2H, m), 2.20 (1H, d, Jϭ10.5 Hz), 2.28 (6H, s), 1738
858 (Mϩ)
884 (Mϩ)
2.37 (1H, d, Jϭ15.5 Hz), 2.37—2.46 (1H, m), 2.61—2.70 (1H, m), 2.86—2.96 (1H, m), 3.03 (1H, t, Jϭ
10 Hz), 3.08—3.25 (4H, m), 3.32 (3H, s), 3.40 (1H, s), 3.43—3.54 (2H, m), 3.61—3.70 (1H, m), 3.72
(1H, s), 3.96—4.06 (2H, m), 4.28 (2H, t, Jϭ6.5 Hz), 4.36—4.42 (2H, m), 4.93 (1H, d, Jϭ5 Hz), 5.13
(1H, dd, Jϭ11, 2 Hz), 6.85 (1H, d, Jϭ2.5 Hz), 6.94 (1H, dd, Jϭ5, 3.5 Hz), 7.15 (1H, dd, Jϭ5, 1 Hz)
(CϭO)
0.85 (3H, t, Jϭ7.5 Hz), 0.98—1.70 (33H, m), 1.85—2.05 (3H, m), 2.20 (1H, d, Jϭ10.5 Hz), 2.29 (6H, s), 1738
2.35 (1H, d, Jϭ15.5 Hz), 2.40—2.48 (1H, m), 2.65—2.72 (1H, m), 2.85—2.93 (1H, m), 3.01 (1H, t, Jϭ
10 Hz), 3.08 (1H, s), 3.09—3.18 (2H, m), 3.22 (1H, dd, Jϭ10.5, 7.5 Hz), 3.32 (3H, s), 3.39 (1H, brs),
3.45—3.53 (1H, m), 3.59 (1H, d, Jϭ3.5 Hz), 3.65—3.75 (2H, m), 3.98—4.07 (2H, m), 4.14—4.25 (3H,
m), 4.42 (1H, d, Jϭ6.5 Hz), 4.89 (1H, d, Jϭ5 Hz), 5.11 (1H, dd, Jϭ11, 2.5 Hz), 7.18—7.42 (5H, m)
0.84 (3H, t, Jϭ7.5 Hz), 0.97 (3H, d, Jϭ7.5 Hz), 1.02—1.75 (30H, m), 1.87—2.00 (2H, m), 2.07—2.25
(3H, m), 2.31 (6H, s), 2.36 (1H, d, Jϭ15.5 Hz), 2.39—2.49 (1H, m), 2.51—2.59 (1H, m), 2.82—2.92
(1H, m), 3.02 (1H, t, Jϭ9.5 Hz), 3.05 (3H, s), 3.15—3.25 (2H, m), 3.32 (3H, s), 3.37 (1H, br s), 3.43—
3.53 (1H, m), 3.59—3.80 (4H, m), 3.95—4.24 (5H, m), 4.43 (1H, d, Jϭ7.5 Hz), 4.59 (1H, s), 4.93 (1H,
d, Jϭ4.5 Hz), 5.11 (1H, dd, Jϭ11.5, 2 Hz), 6.86—6.94 (3H, m), 7.22—7.29 (2H, m)
(CϭO)
1736
(CϭO)
897 (Mϩ1)ϩ
A showed no activity against the resistant strains. On the thromycin A 9-[O-(3-phenoxypropyl)oxime] (12t) exhibited
other hand, some compounds (12e—g, m, p—u, x, 13t) in potent antibacterial activities against both the resistant and
type B showed good activity against both the resistant and susceptible strains. We also found that a couple of factors in-
susceptible strains. Our modeling study suggested that the fluenced the antibacterial activity against macrolide-resistant
difference in the spatial orientation of the 9-substituents strains. Our results suggest that the orientation of the sub-
might be an important factor for the antibacterial activity stituent of the 9-oxime ether group to the aglycon could be
against resistant strains. Six compounds (12i—l, n, o) in type an important factor for improved anti-MAC activity as well
B, however, showed no activity against the resistant strains. as the length of the 9-substituents. Further evaluations of the
The insufficient activity of these compounds would stem potent macrolides (12q, 12t), bearing antibacterial activity
from the inappropriate length at the 9-position, as mentioned against macrolide-resistant strains, are underway.
above. To demonstrate the spatial difference of the orienta-
Experimental
tion of the 9-substituents, nuclear Overhauser effect (NOE)
technique was considered to be useful, so we measured the
NOE in the representative type A (12d) and type B (12t)
compounds. In the actual experiments, the NOE between the
11-hydroxy group H atom and aromatic H atoms on the 9-
phenyl group was observed in 12t (Fig. 2), while it was not
observed in 12d.
In conclusion, our SAR studies of a series of erythromycin
A 9-oxime ether derivatives allowed us to find that ery-
thromycin A 9-[O-(4-phenylbutyl)oxime] (12q) and ery-
Melting points were measured with a Yanagimoto melting point apparatus
and are uncorrected. Infrared (IR) spectra were recorded on a Hitachi 270-
30 spectrometer. Proton nuclear magnetic resonance (1H-NMR) spectra were
recorded on a JEOL JNM-GX500 spectrometer. Chemical shifts are reported
1
in d downfield from internal tetramethylsilane. The difference H-NOE ex-
periments were carried out on the same spectrometer. Mass spectra (MS)
and high-resolution mass spectra (HR-MS) were taken on a Waters ZMD
2690 mass spectrometer and JEOL JMS-DX 300 mass spectrometer. HPLC
analyses were performed with Shimadzu LC-3A pumping system and Shi-
madzu ultraviolet–visible spectrophotomeric detector (210 nm). Column
chromatography was carried out with silica gel [Kieselgel 60 (Merck)]. TLC

1126
Vol. 49, No. 9
was run on 0.25 mm pre-coated silica gel plates (60 F254, Merck). All ex-
Nucleotide Sequencing Test Mycobacterium avium strains were sus-
tracted solvents were dried over anhydrous Na2SO4 and removed on a rotary pended in tris(hydromethyl)aminomethane–ethylenediaminetetraacetic acid
evaporator under reduced pressure. The known compounds (12a—c, 12n) (TE) buffer (12.0 ml) and mixed with phenol/CHCl3/isopentyl alcohol
were prepared according to the literature.11)
(12.0 ml), (Life Technologies, Inc., Grand Island, NY, U.S.A.) and 0.1 mm
4-Phenoxylbutyl Methanesulfonate (10u) To the mixture of 4-phe- diameter glass beads (10.0 ml). The mixture was sonicated (Sonifier 250,
noxybutyl alcohol (10.3 g, 62.0 mmol) and NEt3 (10.3 ml, 73.9 mmol) in Branson, Danbury, CT, U.S.A.) on ice for two 3 min periods, with a cooling
Et2O (100 ml), methanesulfonyl chloride (5.26 ml, 68.0 mmol) was added period in between. After centrifugation at 8000ϫg at room temperature for
dropwise under ice-cooling and the mixture was stirred at the same tempera- 15 min, the aliquot phase was removed to a new tube, and the DNA was pre-
ture for 30 min. To the reaction mixture was added saturated NaHCO3, and it
cipitated with 1/10 volume of NaOAc (pH 5.2) and 2 volumes of EtOH. The
was washed with water and brine successively, dried and concentrated to DNA was collected by centrifugation at 15000ϫg at 4 °C for 20 min, and the
give 10u (14.5 g, 96%) as a colorless oil. IR (liq.): 1355 (SO2), 1175 (SO2) pellet was dissolved in 100 ml of TE buffer. The mycobacterial DNA was
cmϪ1. MS m/z: 245 (Mϩ1)ϩ. 1H-NMR (CDCl3) d: 1.87—2.00 (4H, m), 3.01
amplified by a PCR designed in domain V of 23S rRNA gene using primers
(3H, s), 3.97—4.03 (2H, m), 4.27—4.37 (2H, m), 6.84—7.00 (3H, m), MA1 (5Ј-AACGGCGGTGGTAACTATAAC-3Ј) and 23.3 (5Ј-CCGACTTT-
7.22—7.35 (2H, m).
CGTCCCTGCTTGA-3Ј). These primers were designed with reference to
2-(N-Methylanilino)ethyl Methanesulfonate (10v) In a similar manner the 23S rRNA gene sequence of Mycobacterium avium.14b,c) DNA fragments
to that described above 10v (87 %) was obtained as a colorless oil. IR (liq.): were amplified with TakaRa Ex Taq polymerase (Takara, Osaka, Japan) by
1350 (SO2), 1175 (SO2) cmϪ1. MS m/z: 230 (Mϩ1)ϩ. H-NMR (CDCl3) d: using the following temperature profile in Thermal cycler 480 (Perkin-Elmer
2.92 (3H, s), 3.01 (3H, s), 3.67—3.73 (2H, m), 4.35—4.40 (2H, m), 6.67—
6.79 (3H, m), 7.21—7.28 (2H, m).
1
Cetus, Norwalk, CN, U.S.A.): One cycle of 4 °C for 10 min; 30 cycles of
94 °C for 1 min and 65 °C for 2 min and 72 °C for 1.5 min. The PCR prod-
Erythromycin A 9-[O-(3-Phenoxypropyl)oxime] (12t) Method A: A ucts were purified with Microcon-100 column (Millipore, Bedford, MA,
suspension of 8 (4.00 g, 5.34 mmol), 3-phenoxypropyl bromide (1.30 ml, U.S.A.). The DNA sequencing of the region was determined directly using
8.25 mmol), tetrabutylammonium iodide (0.10 g, 0.271 mmol), NaI (0.12 g,
primer MA1 with Sequenase 7-deaza-dGTP sequencing kit (Amersham,
0.801 mmol) and KOH (0.43 g, 6.51 mmol) in THF (40 ml) was stirred at Cleveland, OH, U.S.A.) in accordance with standard protocol,14d) or with
room temperature for 16.5 h. The reaction mixture was concentrated under Thermo sequence DNA polymerase (Amersham Pharmacia Biotech, Inc.
reduced pressure and the residue was made alkaline with saturated NaHCO3, Piscataway, NJ, U.S.A.) on auto sequencer (DSQ-2000L, Shimadzu, Kyoto,
and extracted with AcOEt. The organic layer was washed with brine, dried Japan) in according with the manufacturer’s instructions.
and concentrated. The residue was crystallized from Et2O and the precipitate
was filtered and washed with iso-Pr2O to give 12t as a colorless solid
Acknowledgements We are grateful to Dr. Osamu Nagata for encour-
(2.80 g, 59%). Recrystallization from iso-Pr2O gave 12t as colorless needles. agement and valuable comments.
Geometrical ratio: E : Zϭ99.7 : 0.3; HPLC using a TSK gel CN [4.6 mmϫ
100 mm, solvent; KH2PO4 : CH3CN (7 : 3), flow rate 1.0 ml/min]. The References
physicochemical and spectral data are listed in Tables 1 and 4, respectively.
A NOE was observed between the 11-hydroxy group H atom (d 4.38) and
the aromatic H atoms on the 9-phenyl group (d 6.84—6.96, 7.21—7.32).
Compounds 12d—k, o, p, s, x, 13t were prepared in a similar manner,
and their physicochemical and spectral data are listed in Tables 1 and 4, re-
spectively.
Method B: To a solution of 8 (0.50 g, 0.668 mmol) in THF (5.0 ml), NaH
(0.032 g, 0.800 mmol, 60% mineral oil dispersion) was added portionwise
under ice-cooling and the mixture was stirred at the same temperature for
5 min. Tetrabutylammonium iodide (0.012 g, 0.025 mmol) and 3-phe-
noxypropyl bromide (0.16 ml, 1.02 mmol) were successively added to the re-
action mixture and the mixture was stirred at room temperature for 27.5 h.
To the reaction mixture was added water, it was made alkaline with saturated
NaHCO3, and extracted with AcOEt. The organic layer was washed with
brine, dried and concentrated. The residue was recrystallized from iso-Pr2O
to give 12t as colorless needles (0.28 g, 47%), which were identical with the
compound (12t) synthesized by method A.
Erythromycin A 9-[O-(3-(Cyclohexyl)propyl)oxime] (12m) To a solu-
tion of 8 (15.0 g, 20.0 mmol) in THF (150 ml), NaH (0.96 g, 24.0 mmol, 60%
mineral oil dispersion) was added portionwise under ice-cooling and the
mixture was stirred at the same temperature for 5 min. Tetrabutylammonium
iodide (0.37 g, 1.00 mmol) and 3-(cyclohexyl)propyl methanesulfonate
(6.60 g, 30.0 mmol) were successively added to the reaction mixture and the
mixture was stirred at room temperature for 24.5 h. The reaction mixture
was concentrated under reduced pressure and to the residue was added
water, and the mixture was crystallized from iso-Pr2O. The precipitate was
dissolved in AcOEt to remove impurities and filtered. The filtrate was con-
centrated to give a pale brown amorphous solid (7.50 g, 43%). Recrystalliza-
tion from iso-Pr2O gave 12m as colorless crystals. Geometrical ratio:
E : Zϭ99.3 : 0.7; HPLC using a TSK gel CN [4.6 mmϫ100 mm, solvent;
KH2PO4 : CH3CN (7 : 3), flow rate 1.0 ml/min]. The physicochemical and
spectral data are listed in Tables 1 and 4, respectively.
Compounds 12l, q, r, u—w were prepared in a similar manner, and the
physicochemical and spectral data are listed in Tables 1 and 4, respectively.
In Vitro Antibacterial Test The MICs of the test drugs were deter-
mined by the agar dilution method. Mycobacterium avium and Mycobac-
terium intracellulare were tested in Middlebrook 7H11 agar (Becton Dickin-
son and Company, Sparks, MD, U.S.A.). Staphylococcus aureus was tested
in Mueller Hinton Agar (Becton Dickinson and Company). One loopful
(5 ml) of an inoculum corresponding to 106 CFU/ml was inoculated on drug-
containing agar plates, and the plates were incubated for 7 d (Mycobacteria)
or 18 h (Staphylococcus aureus) at 37 °C. The MIC was defined as the low-
est drug concentration which prevented visible growth of bacteria.
1) McGuire J. M., Bunch R. L., Anderson R. C., Boaz H. E., Flynn E. H.,
Powell H. M., Smith J. W., Antibiot. Chemother., 2, 281—283 (1952).
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