Synthesis and antibacterial activity of 4″-O-heteroarylcarbamoyl derivatives of macrolide

Article abstract of DOI:10.1016/j.bmcl.2008.09.022

A series of novel 4″-position modified macrolide derivatives has been synthesized via a facile procedure. Their in vitro antibacterial activities against constitutively erythromycin-resistant strains were evaluated. Among the derivatives tested, compound 8a which has 11,12-carbamate and 4″-O-heteroarylcarbamoyl groups was found to have potent activity against most resistant bacteria.

Full text of DOI:10.1016/j.bmcl.2008.09.022

Bioorganic & Medicinal Chemistry Letters 18 (2008) 5507–5511
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and antibacterial activity of 400-O-heteroarylcarbamoyl derivatives
of macrolide
Peng Xu a, Lu Liu a, Zhi-ping Jin b, Guang-qiang Wang b, Jian Liu c, Yun Li c, Ping-sheng Lei a,
*
a Institute of Materia Medica, Chinese Academy of Medical Sciences, Beijing 100050, China
b Jingxin Pharmaceutical Co., Xinchang, Zhejiang 312400, China
c Institute of Clinical Pharmacology, Peking University, Beijing 100083, China
a r t i c l e i n f o
a b s t r a c t
A series of novel 400-position modified macrolide derivatives has been synthesized via a facile procedure.
Their in vitro antibacterial activities against constitutively erythromycin-resistant strains were evalu-
ated. Among the derivatives tested, compound 8a which has 11,12-carbamate and 400-O-heteroarylcarba-
moyl groups was found to have potent activity against most resistant bacteria.
Ó 2008 Elsevier Ltd. All rights reserved.
Article history:
Received 2 June 2008
Revised 29 August 2008
Accepted 4 September 2008
Available online 9 September 2008
Keywords:
400-Modified macrolide
Synthesis
Macrolide resistance
The mechanism of macrolides binding to the ribosome RNA of
bacteria has been getting clearer since the last few years of 20th
century. The high-resolution X-ray cocrystal structures of the bac-
terial ribosome with macrolides have revealed the detailed interac-
tion at atomic level.1a–d It demonstrated that the macrolides inhibit
bacterial protein synthesis by sterically blocking the passage of
nascent polypeptides through the exit tunnel of the ribosome.2
The second-generation macrolide antibiotics, such as clarithro-
mycin and azithromycin, have been widely prescribed for upper
and lower respiratory tract infection because of their superior anti-
bacterial activity. However, the therapeutic utility of these macro-
lides has been severely compromised by the emergence of resistant
pathogens.3 Two of the most important mechanisms of macrolide
The study of high-resolution X-ray cocrystal structures shows
that cladinose group of erythromycin is located at and fits with
the cavity formed by G2505, C2610 and C2611 in domain V of
the erythromycin binding site.1a It has been reported10 that the
induction of methylase in macrolide-resistant bacteria could be
dissociated from inhibition of the bacteria by using erythromycin
analogs with modification at the 400-position of the cladinose sugar.
Some papers reported11,12 that introduction of certain groups in
400-position of the cladinose sugar resulted in decreased inducer
activity but had a negligible effect on antibacterial activity.
400-Modification can alter the relative potency an antibiotic has
for induction and inhibition. In these researches, the derivatives
modified at 400-position were obtained, for example, A-6056510
and CP-54437211 (Fig. 1). The compound CP-544372, introduc-
tion of [2-N-substituted aminoethyl] amino carbonyl chain to
400-hydroxyl group of cladinose, was identified effective against
macrolide-lincosamide-streptogramin B (MLSB) resistant strains.
According to the result of X-ray cocrystal structure study, Taka-
shima, H. suggested1a that the prolonged 400-aryl alkyl group of CP-
544372 reached the chloramphenicol binding site (the peptidyl
transferase region) and inhibited peptide formation of bacterial
ribosomes. This perspective is very informative for structure-based
drug design.13 We focused on the 400-position of the cladinosyl moi-
ety as the target. In the case of CP-544372, the length of phenyl
side chain was six atoms distance from 400-O. In our work, four aryl
and heteroaryl substituted primary amines were chosen (Fig. 2).
The length of the 400-side chains is just six atoms distance from
400-O to aromatic ring. Since VDW interaction and aromatic stack-
ing may increase binding affinity, the aromatic moiety chosen here
resistance were ribosomal mutation (erm) and efflux (mef).4
A
great effort has been making to discover novel macrolides to ad-
dress these status.5 For example, Telithromycin6 and Cethromy-
cin,7 known as ketolide, have been investigated. Telithromycin
possesses a 3-keto group and a 11,12-carbamate functionality. In
addition, it has a proper aromatic side chain which can interact
with nucleotide A752 in domain II of the 23S rRNA. This interaction
makes telithromycin show strong activity against major macro-
lide-resistant strains.8 It is worthy of notice that ketolide is not
the only class of new macrolides for the effective management of
macrolide resistance. Many derivatives of nonketolide families
have been synthesized by different research groups.9
* Corresponding author. Tel.: +86 10 63162006; fax: +86 10 63017757.
E-mail address: Lei@imm.ac.cn (P. Lei).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.09.022

5508
P. Xu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5507–5511
N
O
N
O
HO
O
HO
O
H2N
OH
OH
O
O
O
O
HO
O
HO
O
O
O
O
O
O
O
H
N
N
O
O
O
O
N
H
O
O
O
O
CP-544372
Figure 1. Structures of A-60565 and CP-544372.
A-60565
are phenyl, diaryl or fused bicyclic aryl group with mono, di and tri
nitrogen atoms in the heteroaryl systems. These structures have
different bulk of molecular and different distribution of hetero
atoms. Some of these long anchor groups were proved having sig-
nificant effect for the antibacterial activity in ketolides.14
By substituting of 400-position with various aryl alkyl side
chains, a series of novel 14-membered macrolide derivatives was
obtained. The further modification at 11-N,12-O-carbamate
sub-structure was carried out. We hoped that a combination of
400-O-heteroarylcarbamoyl group and 11-N,12-O-cyclic carbamate
anchor group might enhance the antibacterial activity against
resistant strains.
a
b
N
H2N
H2N
N
N
N
N
c
H2N
d
H2N
N
Figure 2. Structures of aryl alkyl side chains.
N
N
O
HO
OCH3
O
AcO
OCH3
HO
HO
O
O
HO
HO
O
O
b
a
O
O
O
O
O
O
O
OH
OCH3
O
OH
OCH3
1
2
N
O
N
O
AcO
OCH3
O
AcO
OCH3
O
c
O
O
O
O
HO
O
O
O
O
O
O
O
H
N
O
N
O
OCH3
N
O
O
OCH3
R
O
O
4
3
N
N
O
O
HO
4a,5a R=
N
OCH3
N
O
d
HO
4b,5b R=
4c,5c R=
N
O
O
O
H
O
O
OCH3
N
R
O
5
Scheme 1. Reagentsandconditions:(a)Et3N, aceticanhydride, CH2Cl2, 5 h,0 °C,64%;(b)CDI,NaH, DMF, 0 °C,1 h,89%;(c)DBU,RNH2, DMF,10 h,30–36%;(d)MeOH, reflux, 78–85%.

P. Xu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5507–5511
5509
Our approach to get 400-substituted derivatives is to regioselec-
tively protect 20-OH of clarithromycin 1 and to modify the 400-OH.
20-O-Acetyl clarithromycin 2 provided a convenient starting mate-
rial for the chemical modification leading to the 400-substituted
derivatives.
treatment with various primary arylamines in presence of DBU.
13C NMR study suggested that 7(a–d) were got because two carbon
peaks of carbamates exist (at about d 157 and d 156). Deprotection
of the 20-O-acetate compounds 7(a–d) in refluxing methanol pro-
vided the target compounds 8(a–d) in yields ranging from 70% to
80% (Scheme 2). The structures of 8(a–d) were confirmed by 13C
NMR, 1H NMR and HRMS spectra.
By using the method of Baker et al.,6a 2 was treated with excess
1,10-carbonyldiimidazole (CDI) and sodium hydride in DMF at 0 °C
for 1 h. 11,12-Carbonate 400-O-acylimidazolide 3 was obtained in a
yield of 89%. The structure of 3 was confirmed by 13C NMR spec-
trum in which two carbon peaks of carbonate and carbamate could
be found at d 154.0 and d 148.5. Compound 4(a–c), a series of de-
sired compounds, was prepared by reacting compound 3 with pri-
mary arylamines and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU).
Deprotection of the 20-O-acetates 4(a–c) in refluxing methanol pro-
vided compounds 5(a–c) in yields ranging from 78% to 85%. The
structures of 5(a–c) were determined by 13C NMR and HRMS spec-
tra (Scheme 1).
The newly prepared macrolide derivatives, 5a, 5b, 8a, 8b, and
8d, together with clarithromycin and azithromycin as references,
were tested against a panel of representative respiratory tract
pathogens ( Table 1). The strains are Staphylococcus aureus
ATCC29213, 01-430, 01-431, and 01-481 (methicillin-sensitivity
staphylococcus aureus (MSSA)), S. aureus 01-433, 01-429, 01-483
(methicillin-resistant staphylococcus aureus (MRSA)), Streptococ-
cus pneumoniae ATCC49619, 04-050 (erythromycin-susceptible
strain (Ery-S)), S. pneumoniae 03-436, 03-437, 03-554 (erythromy-
cin-resistance strain (Ery-R)), Streptococcus pyogenes 01-469, 03-
804, 03-740 (Ery-S), S. pyogenes 03-474, 03-475, 03-476, 01-781
(Ery-R), S. pyogenes 03-480 (Ery-R encoded by ermB gene), Entero-
coccus faecalis 03H065, 03I076 (Ery-S), E. faecalis 03A056, 03A080,
03A133 (Ery-R). All the MRSA and Ery-R strains chosen in this test
are constitutively resistant strains15 supplied by the Ministry of
Health National Antimicrobial Resistance Investigation Net
(MOHNARIN, China). The in vitro antibacterial activity was re-
ported as minimum inhibitory concentrations (MICs), which was
determined by the broth microdilution method as recommended
by the NCCLS.16
Compound 2 could be further elaborated to a series of com-
pounds modified at both 400-position and 11-N,12-O-cyclic carba-
mate sub-structure. Agouridas et al. synthesized ketolides with
introduction of acylimidazoles by using CDI and sodium hydride.6b
The desired 11,12-cyclic carbamate ketolides were synthesized by
reacted 12-O-acylimidazolyl ketolide in acetonitrile with the
appropriate amines.6c In our case, 2 was treated with excess CDI
and sodium hydride in DMF at 25 °C for 10 h. 12-O,400-O-Diacylim-
idazolide 6 was obtained in a yield of 92%. Compound 6 was con-
verted without purification to designed compounds 7(a–d) by
N
N
O
AcO
OCH3
O
AcO
OCH3
O
HO
HO
O
O
O
O
a
O
N
N
O
O
O
O
O
O
O
O
c
OH
OCH3
O
OCH3
N
N
6
2
O
N
O
O
AcO
OCH3
R
N
O
b
O
O
O
O
O
H
N
O
O
OCH3
R
O
7
N
N
O
7a,8a R=
N
N
O
HO
OCH3
R
N
O
7b,8b R=
7c,8c R=
7d,8d R=
O
N
O
O
O
O
H
N
O
O
OCH3
R
N
O
8
N
Scheme 2. Reagents and conditions: (a) CDI, NaH, DMF, 0 °C, 10 h, 92%; (b) DBU, RNH2, DMF, 10 h, 20–62%; (c) MeOH, reflux, 70–80%.

5510
P. Xu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5507–5511
Table 1
Antibacterial activity of 5(a and b), 8(a,b, and d)
Pathogens
5a
5b
8a
8b
8d
Clar
Azi
ATCC 29213
01-430 MSSA
01-431 MSSA
01-481 MSSA
01-433 MRSA
01-429 MRSA
01-483 MRSA
16
8
16
8
1
1
1
16
16
16
16
16
16
16
4
4
4
0.25
0.25
0.25
0.25
>256
>256
>256
1
0.5
1
16
16
16
16
16
16
16
16
16
16
1
4
1
16
16
16
16
16
16
>256
>256
>256
S. pneumoniae
ATCC 49619
2
0.5
0.125
16
0.25
0.031
0.025
S. pneumoniae
04-050 (Ery-S)
03-436 (Ery-R)
03-437 (Ery-R)
03-554 (Ery-R)
0.062
0.031
0.062
0.25
0.125
0.25
16
16
16
16
0.25
0.25
0.125
0.25
0.031
128
64
0.25
>256
256
2
2
8
8
8
8
128
256
S. pyogenes
01-469 (Ery-S)
03-804 (Ery-S)
03-740 (Ery-S)
03-474 (Ery-R)
03-475 (Ery-R)
03-476 (Ery-R)
03-480 (Ery-R)
01-781 (Ery-R)
0.5
0.5
0.5
0.5
1
1
0.125
0.125
0.125
0.5
0.5
0.5
2
2
2
8
8
8
8
8
0.5
0.5
0.5
2
2
2
0.016
0.031
0.031
256
256
256
0.062
0.125
0.125
>256
>256
>256
>256
>256
16
16
16
16
16
16
16
16
16
16
0.5
0.5
2
2
256
256
E. faecalis
03H065 (Ery-S)
03I076 (Ery-S)
03A056 (Ery-R)
03A080 (Ery-R)
03A133 (Ery-R)
2
2
16
16
16
4
4
16
16
16
0.5
0.5
16
16
16
4
4
16
16
16
1
1
16
16
16
0.125
0.125
>256
>256
>256
0.25
0.25
>256
>256
>256
The tabulated results exhibited that clarithromycin and azithro-
mycin were potent against erythromycin-susceptible strains and
inactive against all the resistant strains.
Typical of 400-substituted macrolides, the compounds 5a, 5b,
8a, 8b, and 8d showed improved activity against MRSA 01-433,
01-429, and 01-483. The active against MSSA of the 400-substituted
macrolides were, except 8a, markedly decrease. Compared to
azithromycin, 8a still remained the activity against MSSA 01-430,
01-431, and 01-481. It is worthy of note that all the compounds
in this paper showed mild activity against MRSA tested. This seems
a result of modification on 400-position. With increase in MRSA
infections worldwide, and expanding an existing antibacterial
activity is noteworthy. This work may provide significant opportu-
nities for the design of new macrolide antibiotics having potent
activity against MRSA.
Compounds 5a and 5b showed improved activity against all the
erythromycin-resistant strains, for example S. pneumoniae 03-436,
03-437, and 03-554; S. pyogenes 03-474, 03-475, 03-476, and 01-
781; as well as S. pyogenes 03-480 strain encoded by ermB gene.
The active against erythromycin-susceptible strains of 5a and 5b
decreased in a certain extent.
With the presence of anchor groups at both 400-position and 11-
N,12-O-carbamate of macrolide lactone ring, 8a and 8d were active
against erythromycin-resistant strains. 8d showed
a greater
improvement against erythromycin-resistant strains than 5a and
5b. But the activity against erythromycin-susceptible strains of
8d was less than that of the references.
For E. faecalis strains, the activity of the tested compounds ap-
peared weaker than that of the references against Ery-S strains. 400-
Modified macrolides showed modest activity against Ery-R strains.
As a highlight, all the target compounds could be obtained
through few steps procedure from clarithromycin. The improved
antibacterial activity against resistant bacteria achieved by these
derivatives is the result of the possible interaction of the aryl alkyl
group of 400-position with ribosome RNA bases in the exit tunnel by
aromatic stacking or VDW interaction. These studies present a con-
siderable opportunity for the development of new macrolide anti-
biotics to combat the growing problems of MLS-resistance.
The most active compound 8a was obtained when the fused
bicyclic aryl alkyl side chain (4-(3H-imidazo[4,5-b]pyridin-3-yl)-
butyl)amine was appended to the macrolide core. The compound
8a demonstrated more than 500-fold better active than clarithro-
mycin and azithromycin against resistant S. pneumoniae strains
and resistant S. pyogenes strains. To the resistant S. pyogenes 03-
480 strain encoded by ermB gene, 8a showed also potent activity.
Meanwhile, 8a retained almost the same activity against erythro-
mycin-susceptible strains comparable to one of the references,
azithromycin. We believe that the potent activity achieved by 8a
against resistant strains was a result of the secondary interaction
between the 11,12-anchor group and the nucleotide A752 in do-
main II of 23S rRNA.
Compound 8b was less active against both erythromycin-sus-
ceptible strains and erythromycin-resistant strains compared to
8a and 8d. The contrast of the antibacterial activity between 8a
and 8b showed that the difference was caused not only by the po-
sition of the aryl alkyl side chains on the macrolide core, but also
by the structure of the side chain.
Acknowledgements
This work is finance supported by the National Natural Science
Foundation of China (30572275) and Natural Science Foundation of
Beijing (7062047).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2008.09.022.

P. Xu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5507–5511
5511
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