Synthesis and in vitro antibiotic activity of 16-membered 9-O-arylalkyloxime macrolides

Article abstract of DOI:10.1016/j.bmcl.2005.11.061

A series of novel 9-O-arylalkyloxime analogs based on three different 16-membered macrolide scaffolds-5-O-mycaminosyltylonolide (OMT), tilmicosin, and 20-deoxy-20-(3,5-dimethyl-1-piperidin-1-yl)-OMT-was synthesized. In vitro antibiotic activities were assayed against Gram-positive Streptococcus pneumoniae and Staphylococcus aureus and Gram-negative Haemophilus influenzae bacterial strains. Analogs derived from OMT (3-15) showed similar or better antibacterial activities against macrolide-susceptible strains and enhanced activities against macrolide-resistant strains compared with erythromycin A, tylosin, or OMT. Similar results were observed for tilmicosin 9-O-arylalkyloxime analogs (18-24). In contrast, most of the 20-deoxy-20-(3,5-dimethyl-1- piperidin-1-yl)-OMT analogs (25-33) showed reduced antibacterial activities compared with OMT. Ribosome-binding studies were performed on compounds 12 (OMT derivative), 20 (tilmicosin derivative), and 29 [20-deoxy-20-(3,5-dimethyl-1- piperidin-1-yl)-OMT derivative]. It was found that these compounds interacted with both domain V and domain II of the Escherichia coli 23S rRNA.

Full text of DOI:10.1016/j.bmcl.2005.11.061

Bioorganic & Medicinal Chemistry Letters 16 (2006) 1259–1266
Synthesis and in vitro antibiotic activity of 16-membered
9-O-arylalkyloxime macrolides
Hong Fu,*, Saul Marquez, Xiangrong Gu, Leonard Katz and David C. Myles
Department of Chemistry, Kosan Biosciences Inc., 3832 Bay Center Place, Hayward, CA 94545, USA
Received 28 October 2005; revised 17 November 2005; accepted 18 November 2005
Available online 15 December 2005
Abstract—A series of novel 9-O-arylalkyloxime analogs based on three different 16-membered macrolide scaffolds—5-O-mycamino-
syltylonolide (OMT), tilmicosin, and 20-deoxy-20-(3,5-dimethyl-1-piperidin-1-yl)-OMT—was synthesized. In vitro antibiotic activ-
ities were assayed against Gram-positive Streptococcus pneumoniae and Staphylococcus aureus and Gram-negative Haemophilus
influenzae bacterial strains. Analogs derived from OMT (3–15) showed similar or better antibacterial activities against macrolide-
susceptible strains and enhanced activities against macrolide-resistant strains compared with erythromycin A, tylosin, or OMT.
Similar results were observed for tilmicosin 9-O-arylalkyloxime analogs (18–24). In contrast, most of the 20-deoxy-20-(3,5-dimeth-
yl-1-piperidin-1-yl)-OMT analogs (25–33) showed reduced antibacterial activities compared with OMT. Ribosome-binding studies
were performed on compounds 12 (OMT derivative), 20 (tilmicosin derivative), and 29 [20-deoxy-20-(3,5-dimethyl-1-piperidin-1-yl)-
OMT derivative]. It was found that these compounds interacted with both domain V and domain II of the Escherichia coli 23S
rRNA.
Ó 2005 Elsevier Ltd. All rights reserved.
We describe the preparation and evaluation of a series
of novel functionalized 16-membered macrolides based
on the tylonolide scaffold. These compounds show a
promising spectrum of activity against a range of impor-
tant pathogenic bacteria. Both 14- and 16-membered
macrolide antibiotics have been used extensively in hu-
man and veterinary medicine. These compounds bind
to bacterial ribosomes and inhibit protein synthesis.1
The steady and continuous emergence of strains resis-
tant to macrolides has prompted a large effort in the de-
sign of semisynthetic analogs, mainly around
erythromycin A, the prototype 14-membered macrolide,
resulting in the creation of the recently approved keto-
lide telithromycin.2
macrolides from the cells before they reach their
target.4 It is believed that the ketolides overcome
macrolide resistance in Streptococcus pneumoniae
and Streptococcus pyogenes through enhanced binding
to the bacterial ribosome via their aromatic side
chains.5 These compounds also evade the efflux
pumps. In our search for novel macrolides to combat
resistance, we have designed and synthesized a series
of 16-membered macrolides functionalized with
9-O-arylalkyloximes, wherein the aryl group of the
oxime side chain may mimic the function of the aryl
groups of the ketolides.
The 9-O-ether oxime derivatives of tylosin6 and erythro-
mycin7 were prepared previously and showed antibacte-
rial activity comparable to that of their parent, tylosin
(Tyl) or Ery A. The 9-O-arylalkyloxime compounds in
this study were prepared from 5-O-mycaminosyltylono-
lide (OMT), tilmicosin, and 20-deoxy-20-(3,5-dimethyl-
1-piperidin-1-yl)-OMT scaffolds. The preparation of
OMT and tilmicosin from tylosin and desmycosin,
respectively, has been described previously.8,9 20-De-
oxy-20-(3,5-dimethyl-1-piperidin-1-yl)-OMT was pre-
pared from OMT using a similar protocol as that for
preparing tilmicosin from desmycosin.9 Synthesis of
9-O-arylalkyloxime OMTs (compounds 3–15) starting
from OMT is depicted in Scheme 1.
The two most important mechanisms by which bacte-
ria become resistant to macrolides are through N6-
mono or N6,6-dimethylation of the adenosine residue
at nucleotide 2058 of the 23S rRNA component of
the 50S subunit of the bacterial ribosome3 and
through the acquisition of efflux pumps that export
Keywords: 16-Membered macrolide antibiotics; Oximes; Ribosome
binding.
*
Corresponding author. Tel.: +1 510 731 5221; fax: +1 510 732
8401; e-mail: fu@kosan.com
Present address: 3832 Bay Center Place, Hayward, CA 94545, USA.
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.11.061

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H. Fu et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1259–1266
O
O
O
CHO
O
O
HO OH
NMe2
OH
NMe2
O OH
O
O
O
HO
HO
HO
HO
CSA/CH2Cl2
O
O
O
OH
OH
OMT
NOH
1
O
NOR
O
O
NH2OH.HCl
Py./MeOH
O
RBr/DMF
OK
NMe2
OH
O
O
HO
HO
NMe2
OH
O
O
O
HO
HO
O
OH
O
O
OH
2
RO
OR
N
N
CHO
O
CHO
CAS
acetone
NMe2
O
NMe2
OH
HO
HO
O
O
HO
HO
O
OH
O
O
OH
O
OH
3, 4, 6, 8, 9, 11, 13-15
5, 7, 10, 12
O
O
14 (Z) R =
3 (E/Z) R =
N
N
N
9 (Z) R =
N
O
N
N
4 (Z) & 5 (E) R =
10 (E) & 11 (Z) R =
12 (E) & 13 (Z) R =
O
15 (Z) R =
6 (Z) R =
N
N
N
7 (E) & 8 (Z) R =
Scheme 1. Synthesis of 9-O-arylalkyloxime OMTs.
The C20 aldehyde functional group of OMT was pro-
tected as the 1,3-dioxolane by treatment with ethylene
glycol in the presence of camphorsulfonic acid (CSA)
in dichloromethane. Conversion of the C-9 ketone to
the oxime was carried out using hydroxylamine hydro-
chloride in the presence of pyridine.10 The oxime could
then be alkylated selectively on oxygen using arylalkyl-
bromides and potassium t-butoxide in DMF to generate
the arylalkyloxime analogs. In many cases, the E and Z
oximes generated could be separated by reversed-phase
HPLC. The oxime configuration was determined by
improved potency, equal to that of telithromycin,
against a number of macrolide-resistant strains of
S. pneumoniae (ATCC700676, ATCC700677, and
ATCC700905). The basis of resistance to macrolides in
these strains has not been determined. It is worth noting
that the optimal chain length between the aromatic side
chain and the oxime oxygen is four atoms, with com-
pounds 3 and 15 (4-atom linker) showing potencies
superior to those of compound 6 (5-atom linker), and
compounds 12 and 13 (3-atom linker). The Z oximes
consistently show better potencies than those of their
E counterparts (i.e., 4 > 5, 8 > 7, and 11 > 10).
1
comparison of the H and 13C NMR spectra of both
E and Z isomers according to the literature.11,12 In gen-
eral, the chemical shift for H8 in E isomers is more
down-field (ca. 1 ppm) than that in the Z isomer. In con-
trast, the chemical shift for C8 in the E isomer is more
up-field (7–10 ppm) than that in the Z isomer. Finally,
de-protection of the 20-aldehyde was achieved by stir-
ring the alkylated oxime in acetone and CSA.13 The in vi-
tro activities of compounds 3–15 are summarized in
Table 1.
The improved potencies of 3 and 6 are also maintained
against the macrolide-susceptible S. aureus strains ATCC
6538p and ATCC29213, and S. epidermidis ATCC12228.
In general, staphylococci are inherently less susceptible to
macrolides than streptococci. The basis for erythromycin/
azithromycin/clarithromycin resistance in S. aureus
ATCC141541 is ribosome methylation induced by the
presence of the 14-membered macrolide. The 16-mem-
bered macrolides (tylosin and OMT) analogs are poor
inducers of macrolide resistance in this strain, hence the
modest activities (MIC = 1.56 lg/ml). The OMT analogs,
in particular 3 and 15, are very potent against this strain,
likely due to their enhanced activities as antibiotics and,
possibly, to their weakened ability to act as inducers of
resistance. The basis of resistance in the remaining S.
aureus strains in Table 1, ATCC33591, ATCCBAA-39,
Many of the arylalkyloxime OMT analogs have compa-
rable or better activity than that of Ery A or OMT
against the macrolide-susceptible strains S. pneumoniae
ATCC6301, ATCC700671, and ATCC49618, Staphylo-
coccus aureus ATCC 6538p and ATCC 29213, and
Staphylococcus epidermidis ATCC12228. In addition,
the analogs, in particular 3 and 15, show substantially

Table 1. Antibacterial activities of compounds 3–9 and 10–15
MIC (lg/ml)
Compound:
EryA
Tyl
OMT
3
4
5
6
7
8
9
10
11
12
13
14
15
S. pneumoniae
ATCC6301
0.025
0.098
0.025
0.025
0.025
0.025
0.20
0.025
0.025
0.025
0.39
0.098
0.39
0.20
1.56
0.78
0.025
0.025
0.025
0.39
0.20
0.39
0.20
3.12
0.39
0.025
0.025
0.025
0.39
0.025
0.049
0.049
0.025
0.025
0.025
0.025
0.049
0.049
0.025
0.025
0.049
0.025
0.049
0.049
0.025
0.025
0.025
0.025
0.025
ATCC700671
ATCC700676a
ATCC700677a
ATCC700905a
ATCC700906a
ATCC49619
0.049
6.25
6.25
3.12
0.20
0.20
0.049
0.78
6.25
0.78
0.20
0.025
1.56
0.20
>12.5
0.20
>12.5
6.25
0.20
>12.5
0.049
>12.5
0.049
>12.5
0.049
>12.5
0.025
>12.5
0.049
>12.5
0.025
>12.5
0.049
>12.5
0.025
>12.5
0.049
>12.5
0.025
0.025
>12.5
0.01
0.025
>12.5
0.01
0.025
>12.5
0.01
>12.5
>12.5
>12.5
0.20
>12.5
0.20
>12.5
>12.5
0.01
>12.5
0.049
0.049
0.098
0.098
0.20
S. aureus
ATCC6538p
ATCC33591a
ATCC141541a
0.098
0.20
0.39
0.025
>12.5
0.20
>12.5
>12.5
0.098
0.098
>12.5
0.39
>12.5
>12.5
0.20
1.56
>12.5
3.12
>12.5
>12.5
3.12
0.20
>12.5
0.78
>12.5
>12.5
0.78
0.78
>12.5
1.56
>12.5
>12.5
1.56
0.39
0.78
>12.5
1.56
>12.5
>12.5
1.56
0.098
>12.5
0.78
>12.5
>12.5
0.39
0.098
>12.5
0.39
>12.5
>12.5
0.39
0.098
>12.5
0.39
>12.5
>12.5
0.39
0.20
>12.5
0.78
>12.5
>12.5
0.78
0.20
>12.5
0.39
>12.5
>12.5
0.39
0.025
>12.5
0.20
>12.5
>12.5
0.20
>12.5
>12.5
>12.5
1.56
>12.5
>12.5
1.56
>12.5
1.56
>12.5
>12.5
0.78
>12.5
1.56
>12.5
>12.5
1.56
ATCCBAA-39a >12.5
ATCCBAA-44a >12.5
ATCC29213
0.20
S. epidermidis
ATCC12228
0.20
0.39
0.39
0.098
0.20
1.56
0.20
1.56
0.78
0.20
0.20
0.098
0.049
0.39
0.20
0.20
E. faecalis
ATCC51575
>12.5
>12.5
>12.5
>12.5
>12.5
>12.5
>12.5
>12.5
>12.5
>12.5
>12.5
>12.5
>12.5
>12.5
>12.5
>12.5
Abbreviations: Ery A, erythromycin A; Tyl, tylosin, S. pneumoniae, Streptococcus pneumoniae; S. aureus, Staphylococcus aureus; S. epidermidis, Staphylococcus epidermidis; E. faecalis, Enterococcus faecalis.
a Macrolide-resistant strains.

1262
H. Fu et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1259–1266
and ATCCBAA-44, is constitutive ribosomal methyla-
tion. These strains are resistant to 14- and 16-membered
macrolides, the ketolides, and the arylalkyloxime OMT
analogs, indicating that no macrolide or analog can bind
to dimethylated S. aureus ribosomes. The Enterococcus
faecalis ATCC51575 strain in Table 1, resistant to all mac-
rolides and OMT analogs, likely also contains methylated
ribosomes.
(31–33) restored antibacterial activity only slightly (Ta-
ble 3). Steitz et al. have suggested that the C-6 ethylalde-
hyde of 16-membered macrolides forms a covalent bond
with the N6 atom of the A2103 residue (corresponding
to A2062 in Escherichia coli) in the 23S rRNA compo-
nent of the ribosome of Haloarcula marismortui.1 They
also suggested that the mycinosyl moiety of tylosin
interacts with A841 (A748, E. coli numbering) in do-
main II of the 50S ribosome. It is possible that the bind-
ing of 16-membered macrolides to ribosomes requires
the mycinose residue if the 20-aldehyde is missing. Addi-
tion of extensions at C-9 would therefore not restore
binding substantially, resulting in the poor activity of
25. On the other hand, when the scaffold contains either
the 20-aldehyde (OMT) or the mycinose residue (tilmic-
osin), addition of arylalkyl side chains at C-9 via the
oxime appeared to enhance binding to the ribosome. It
is also possible that the poor activities of the 20-
deoxy-20-(3,5-dimethyl-1-piperidin-1-yl)-OMT 9-oxime
derivatives are due to reduced penetration into the bac-
terial cell.
The synthesis of 9-O-arylalkyloxime tilmicosin analogs
follows a straightforward path (Scheme 2). Tilmicosin
was prepared from desmycosin via reductive amination,
using 3,5-dimethylpiperidine (a mixture of cis and trans
isomers) in the presence of formic acid. Formation of
the oxime of tilmicosin was achieved by treatment of til-
micosin with hydroxylamine hydrochloride in MeOH/
THF/H2O.14 Alkylation of tilmicosin 9-oxime with
arylalkylbromides gave rise to the corresponding
9-O-arylalkyloxime tilmicosins 18–24. Similarly,
9-O-arylalkyloxime-20-deoxy-20-(3,5-dimethyl-1-piperi-
din-1-yl)-OMT analogs were prepared from OMT via
reductive amination of the 19-aldehyde followed by oxi-
mation and alkylation leading to compounds 27–33
(Scheme 3). The in vitro antibacterial activities of com-
pounds 18–24 and compounds 27–33 are summarized in
Table 2 and Table 3, respectively.
One compound from each scaffold was chosen for ribo-
some binding studies. Results are shown in Figure 1 and
Table 4. The studies were performed as described previ-
ously using reconstituted E. coli 70S ribosomes.15 Briefly,
the ribosomes are first methylated with dimethylsulfate in
the presence or absence of saturating amounts of a given
macrolide. The RNA is then extracted from the ribosomes
and subjected to transcription employing a 32P-labeled
primer, deoxyribonucleotides, and reverse transcriptase.
Transcription stops when it encounters a methylated
nucleotide; thus a band is observed on the autoradio-
grams of a polyacrylamide sequencing gel.16 Quantitation
of the bands is achieved by scanning phosphorimages
with a gel reader. Sites on the rRNA that are methylated
can be uncovered by use of primers corresponding to do-
main II, domain V, etc. Binding of macrolides protects
In general, the 9-arylalkyloxime tilmicosin analogs show
significantly increased antibacterial potencies over the
parent tilmicosin against both macrolide-susceptible
and macrolide-resistant S. pneumoniae strains (Table
2), but none of the compounds exhibited the overall
potencies of 3 and 15. Interestingly, 20-deoxy-20-(3,5-di-
methyl-1-piperidin-1-yl)-OMT (25) and 20-deoxy-20-
(3,5-dimethyl-1-piperidin-1-yl)-OMT
9-oxime
(26)
exhibited reduced antibiotic activity compared to
OMT, and addition of aromatic side chains to 20-
deoxy-20-(3,5-dimethyl-1-piperidin-1-yl)-OMT 9-oxime
O
O
N
HO
CHO
N
NH2OH.HCl
H
NMe2
O
MeOH/THF/H2O
OH
O
O
OMe
HO
NMe2
O
HO
O
O
HCOOH
OH
O
OMe
O
HO
O
OH
OMe
O
OH
OMe
16
Desmycosin
NOR
NOH
N
N
RBr
NMe2
O OH
HO
O
O
OMe
O
KOH/DMF
HO
NMe2
O OH
HO
O
O
OMe
O
O
HO
OH
OMe
O
OH
OMe
18-24
17
O
18 (Z) & 19 (E) R =
20 (Z) & 21 (E) R =
N
O
N
22 (E/Z) R =
O
23 (Z) & 24 (E) R =
F
Scheme 2. Synthesis of 9-O-arylalkyloxime tilmicosins.

H. Fu et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1259–1266
1263
O
O
N
CHO
HO
N
H
HO
NMe2
OH
O
O
HO
NMe2
OH
O
HCOOH
O
O
HO
O
O
OH
O
OH
25
NOH
NOR
N
N
NH2OH.HCl
MeOH/THF/H2O
RBr
HO
HO
KOH/DMF
NMe2
OH
NMe2
OH
O
O
O
O
HO
HO
O
O
O
O
OH
OH
27-33
26
O
O
27 (Z) & 28 (E) R =
29 (Z) & 30 (E) R =
31 (E/Z) R =
N
N
O
32 (E/Z) R =
33 (E/Z) R =
F
N
N
O
Scheme 3. Synthesis of 9-O-arylalkyloxime-20-deoxo(3,5-dimethyl-1-piperidine)OMTs.
Table 2. Antibacterial activities of compounds 18–24
MIC (lg/ml)
19 20
Compound:
EryA
Tyl
Til
18
21
22
23
24
S. pneumoniae
ATCC6301
0.025
0.098
0.39
0.78
0.78
6.25
1.56
0.01
0.01
0.01
0.01
0.01
1.56
6.25
1.56
0.025
0.049
1.56
0.39
0.39
1.56
6.25
0.78
0.39
0.78
3.12
6.25
3.12
0.20
ATCC700671
ATCC700676a
ATCC700677a
ATCC700905a
ATCC700906a
ATCC49619
0.049
6.25
6.25
3.12
0.20
0.20
0.025
0.049
0.049
0.20
0.39
0.78
6.25
0.78
0.049
0.025
0.098
>12.5
0.20
>12.5
6.25
0.78
>12.5
>12.5
1.56
>12.5
0.098
>12.5
>12.5
>12.5
0.098
12.5
12.5
12.5
0.049
0.098
0.025
0.01
0.78
0.78
0.39
S. aureus
ATCC6538p
ATCC33591a
ATCC141541a
ATCCBAA-39a
ATCCBAA-44a
ATCC29213
0.098
0.20
0.098
>12.5
0.39
>12.5
>12.5
0.20
0.20
>12.5
0.39
>12.5
>12.5
0.39
0.20
0.78
0.78
>12.5
1.56
>12.5
>12.5
3.12
0.20
>12.5
0.78
0.39
>12.5
6.25
0.20
12.5
0.78
>12.5
>12.5
1.56
>12.5
>12.5
1.56
>12.5
0.20
>12.5
>12.5
0.20
>12.5
1.56
>12.5
>12.5
1.56
>12.5
>12.5
>12.5
>12.5
>12.5
>12.5
>12.5
>12.5
>12.5
0.20
0.39
0.78
0.78
S. epidermidis
ATCC12228
0.20
0.39
0.098
0.39
0.20
1.56
3.12
0.39
0.78
0.78
E. faecalis
ATCC51575
>12.5
>12.5
>12.5
>12.5
>12.5
>12.5
>12.5
>12.5
>12.5
>12.5
Strains and abbreviations as in Table 1; Til, tilmicosin.
a Macrolide-resistant strains.
sites from methylation, resulting in loss of intensity of
bands in the gel. Both 14- and 16-membered macrolides
interact with nucleotides A2058 and A2059 in domain V
of 23S RNA via their desosamine or mycaminose
moieties, respectively.1
enhancement of methylation at A752 in domain II.
The ketolide cethromycin (ABT-773) shows protection
not only of A2058 and 2059 but also of A752 in domain
II. Our data indicate that tylosin, tilmicosin (16), and til-
micosin 9-oxime (17) also interact with A752, probably
via the mycinose moiety. The OMT analog 12, which
lacks the mycinose sugar at the 23-position, has a very
similar protection pattern to ABT-773. The interaction
As expected, erythromycin A protects both A2058 and
A2059 from methylation16 and also causes slight

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H. Fu et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1259–1266
Table 3. Antibacterial activity of compounds 25–33
MIC (lg/ml)
Compound:
EryA
Tyl
25
26
27
28
29
30
31
32
33
S. pneumoniae
ATCC6301
0.025
0.098
0.39
0.78
1.56
0.78
1.56
6.25
6.25
6.25
6.25
6.25
1.56
3.12
6.25
6.25
12.5
0.20
0.39
0.78
0.39
0.39
0.39
0.049
0.098
1.56
ATCC700671
ATCC700676a
ATCC700677a
ATCC700905a
ATCC700906a
ATCC49619
0.049
6.25
6.25
3.12
0.20
0.20
>12.5
>12.5
>12.5
>12.5
12.5
>12.5
12.5
>12.5
>12.5
12.5
>12.5
0.20
>12.5
>12.5
1.56
>12.5
0.78
>12.5
6.25
>12.5
1.56
>12.5
6.25
>12.5
6.25
>12.5
0.39
>12.5
0.39
>12.5
0.39
>12.5
0.39
>12.5
0.78
>12.5
0.049
>12.5
>12.5
6.25
>12.5
12.5
0.049
0.098
6.25
S. aureus
ATCC6538p
ATCC33591a
ATCC141541a
ATCCBAA-39a
ATCCBAA-44a
ATCC29213
0.098
0.20
0.39
>12.5
1.56
>12.5
>12.5
1.56
3.12
>12.5
12.5
>12.5
>12.5
>12.5
>12.5
>12.5
>12.5
>12.5
>12.5
>12.5
>12.5
>12.5
6.25
>12.5
>12.5
>12.5
>12.5
>12.5
>12.5
>12.5
0.78
>12.5
6.25
>12.5
>12.5
6.25
0.78
>12.5
3.12
>12.5
>12.5
3.12
0.39
>12.5
3.12
>12.5
>12.5
1.56
>12.5
>12.5
1.56
>12.5
>12.5
1.56
>12.5
>12.5
>12.5
>12.5
>12.5
>12.5
12.5
>12.5
>12.5
>12.5
12.5
0.20
S. epidermidis
ATCC12228
0.20
0.39
0.78
>12.5
>12.5
>12.5
>12.5
>12.5
>12.5
>12.5
>12.5
>12.5
>12.5
6.25
3.12
0.78
E. faecalis
ATCC51575
>12.5
>12.5
>12.5
>12.5
>12.5
>12.5
Strains and abbreviations as in Table 1.
a Macrolide-resistant strains.
A
B
C
D
1
2
3
1
2
3
1
2
3
1
2
3
--A752
--A752
A2058
A2059
--
--
--A2062
--A2058
--A2059
--A2062
Figure 1. Polyacrylamide gels showing antibiotic-induced alterations in the degree of methylation of nucleotides in domain V (A and C) and domain
II (B and D) of Escherichia coli 23S rRNA. Antibiotics were used at concentrations of 50 lM. (A and B) lane 1, no antibiotic; lane 2, erythromycin A;
lane 3, compound 12. (C and D) lane 1, no antibiotic, lane 2, compound 20; lane 3, compound 29.
Table 4. Ribosome (Escherichia coli MRE600 23S rRNA) accessibility studya of selected compounds
A752
A2058
A2059
A2062
No drug
EryA
ABT773
Tyl
1
1
1
1
1.28 0.02
0.105 0.002
0.069 0.027
0.10 0.03
0.44 0.16
0.35 0.11
0.23 0.12
0.44 0.12
0.08 0.03
0.033 0.020
0.082 0.020
0.031 0.011
0.070 0.032
0.035 0.025
0.12 0.020
0.093 0.022
0.22 0.07
0.11 0.03
0.16 0.03
0.054 0.037
0.14 0.02
0.073 0.046
0.30 0.05
0.28 0.17
1.46 0.32
1.83 0.30
0.15 0.07
2.17 0.19
1.30 0.20
1.90 0.10
1.10 0.06
1.8 0.7
12
Til
17
20
29
The intensities of A782 and A2082 were not affected by drug binding16 and were therefore used to normalize the intensities of A752 or A2058, A2059,
and A2062 within each lane.
a Quantification was obtained by scanning the phosphorimages of the sequencing gels.
of compound 12 with A752 is probably via the aromatic
side chain extending from the 9-oxime. Likewise, tilmic-
osin analog 20 and 20-deoxy-20-(3,5-dimethyl-1-piperi-
din-1-yl)-OMT analog 29 protect A752 to a lesser
degree than does 12.
The two most potent analogs (3 and 15) were tested
against two Haemophilus influenzae strains (ATCC9006
and ATCC49766) (Table 5). Each showed ca. 4-fold
decreased activity against H. influenzae compared to
OMT. It has been documented that replacement of the

H. Fu et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1259–1266
Table 5. In vitro activities of 3, 15, 38, and 39
1265
MIC (lg/ml)
Compound:
EryA
Teli
OMT
3
15
38
39
S. pneumoniae
ATCC6301
0.025
0.025
0.025
0.098
0.025
0.025
0.20
0.025
0.025
0.025
0.025
0.20
0.025
0.025
0.025
0.025
0.025
0.025
0.098
0.025
0.025
0.049
0.025
ATCC700671
ATCC700676a
ATCC700677a
ATCC700905a
ATCC700906a
ATCC49619
0.049
6.25
6.25
3.12
0.049
0.78
6.25
0.78
>12.5
0.025
>12.5
0.025
>12.5
0.025
>12.5
0.025
0.025
>12.5
0.01
>12.5
>12.5
>12.5
0.049
0.049
0.049
0.098
H. influenzae
ATCC9006
ATCC49766
EH001
1.56
6.25
3.12
6.25
3.12
1.56
3.12
1.56
6.25
1.56
1.56
1.56
3.12
6.25
1.56
6.25
6.25
6.25
6.25
1.56
3.12
3.12
6.25
3.12
1.56
3.12
3.12
3.12
1.56
N/D
N/D
N/D
N/D
EH002
EH003
N/D
N/D
Abbreviations as in Tables 1 and 2. H. influenzae, Haemophilus influenzae.
a Macrolide-resistant strains.
23-OH of OMT by a basic group such as dialkylamine
enhances potency against Gram-negative bacteria.17,18
Therefore, 3 was converted to its 23-deoxy-23-dimeth-
ylamino analogs (38 and 39). The activity of 38 and 39
against a panel of five H. influenzae strains was found
to have regained potencies similar to those of OMT
and telithromycin; their potencies against S. pneumoniae
essentially remained unchanged compared to 3 (except
against ATCC700677). The preparation of 38 and 39
is illustrated in Scheme 4. The 19-protected 3 was con-
verted to 34 by treating it with diphenylphosphoryl
azide and subsequently converted to the 23-azido com-
pound 35 by heating 34 in the presence of sodium azide
and a catalytic amount of sodium iodide in DMF.
Reduction of 35 with trimethylphosphine resulted in
36, followed by reductive amination with NaCNBH3/
HOAc/HCHO to give 37. Deprotection of 37 using
CSA/acetone yielded 38 and 39, which were separated
by HPLC.
The work presented here represents an initial attempt to
produce potent macrolide analogs that can be used
against macrolide-resistant bacterial pathogens. Three
scaffolds were used to produce a limited series of C9-aryl-
alkyloximes. The most potent compounds employed the
simplest scaffold, OMT, and contained a 4-atom linker
between the aryl group and the oxygen atom, and exhib-
ited properties similar to those of the recently developed
ketolides: clinically relevant activity against macrolide-re-
sistant S. pneumoniae and inducible-resistant S. aureus
strains, although their in vivo potencies, pharmacology,
and toxicities have yet to be examined. Footprinting data
NOR
O
NOR
O
O
O
NaN3/NaI
DMF, 65oC
(PhO)2P(O)N3 PhO
NMe2
HO
NMe2
OH
O
O
O
HO
O
O
HO
P
DBU/THF
O
OH
PhO
O
O
OH
O
O
OH
N
34
R =
O
NOR
NOR
O
O
O
O
Me3P
HCHO/NaCNBH3
AcOH/MeOH
NMe2
OH
THF/H2O
NMe2
HO
O
N3
O
H2N
O
O
HO
O
O
OH
O
O
OH
OH
35
36
NOR
NOR
O
O
CHO
O
CSA
NMe2
OH
Me2N
O
O
NMe2
OH
HO
Me2N
O
acetone
HO
O
O
O
OH
O
OH
37
Scheme 4. Synthesis of compounds 38 and 39.
38 (E) & 39 (Z)

1266
H. Fu et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1259–1266
4. Katz, L.; Ashley, G. W. Chem. Rev. 2005, 105, 449.
5. Douthwaite, S.; Champney, W. S. J Antimicrob. Chemo-
ther. 2001, 48, 1.
6. Ruggeri, C.; Laborde, M. A.; Dessinges, A.; Ming, L.;
Olesker, A.; Lukacs, G. J. Antibiot. 1989, 42, 1443.
7. Gasc, J.-C.; DÕAmbrieres, S. G.; Lutz, A.; Chantot, J.-F.
J. Antibiot. 1991, 44, 313.
suggest that the restoration of potency against the macro-
lide-resistant strains are through binding to the ribosome
in domain II to offset the disruption of macrolide–ribo-
some interaction in domain V that takes place through
methylation at nucleotide 2058. As in the case of the keto-
lides, the OMT analogs were not active against the consti-
tutive-resistant staphylococci and potencies against H.
influenzae, an important respiratory disease causing
agent, were somewhat lower than that of the ketolides,
but could be improved through modification of the scaf-
fold. Additional modifications, including optimization
of the aryl group, should also enhance the properties of
this exciting class of molecules.
8. Gorman, M.; Morin, R.B. U.S. Patent 3,459,853, 1969.
9. Debono, M.; Willard, K. E.; Kirst, H. A.; Wind, J. A.;
Crouse, G. D.; Tao, E. V.; Vicenzi, J. T.; Counter, F.
ꢀ
T.; Ott, J. L.; Ose, E. E.; Omura, S. J. Antibiot. 1989,
42, 1253.
10. Lukacs, G.; Duchatelle-Ruggeri, C. U.S. patent 4,918,058,
1990.
11. Hawkes, G. E.; Herwig, K.; Roberts, J. D. J. Org. Chem.
1974, 39, 1017.
12. Naranda, A.; Lopotar, N. J. Antibiot. 1999, 52, 68.
13. Fu, H.; Katz, L; Myles, C.D. U.S. patent application
11/154,435, 2005.
14. Ojima, J.; Yagi, Y.; Ejiri, E.; Ishizaka, S.; Kato, T. Bull.
Chem. Soc. Jpn. 1986, 59, 1791.
Acknowledgments
We thank Yiqing Zhou and Wei Ma for performing the
in vitro MIC assays.
15. Stern, S.; Moazed, D.; Noller, H.F. In Methods in
Enzymology; Abelson, J.N., Moldave, K., Simon, M.I.,
Noller, H.F., Eds.; Elsevier Science and Technology
Books, 1988; Vol. 164, pp 481–489.
References and notes
16. Hansen, L. H.; Mauvais, P.; Douthwaite, S. Mol. Micro-
biol. 1999, 31, 623.
17. Sakamoto, S.; Tsuchiya, T.; Tanaka, A.; Umezawa, S.;
Hamada, M.; Umezawa, H. J. Antibiot. 1984, 37, 1628.
18. Tanaka, A.; Tsuchiya, T.; Okada, Y.; Umezawa, S.
J. Antibiot. 1982, 35, 113.
1. Hansen, J. L.; Ippolito, J. A.; Ban, N.; Nissen, P.; Moore,
P. B.; Steitz, T. A. Mol. Cell 2002, 10, 117.
2. Yassin, H. M.; Dever, L. L. Expert. Opin. Investig. Drugs
2001, 10, 353.
3. Liu, M.; Douthwaite, S. Proc. Natl. Acad. Sci. U.S.A.
2002, 99, 14658.