Synthesis and antibacterial activity of novel ketolides with 11,12-sulfur contained aryl alkyl side chains

Article abstract of DOI:10.1016/j.ejmech.2010.11.004

A novel series of ketolides with 11,12-sulfur contained aryl alkyl side chains were synthesized and evaluated for their antibacterial activity. These ketolides exhibited potent activity against key macrolide sensitive and resistant respiratory pathogens.

Full text of DOI:10.1016/j.ejmech.2010.11.004

European Journal of Medicinal Chemistry 46 (2011) 208e217
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and antibacterial activity of novel ketolides with 11,12-sulfur contained
aryl alkyl side chains
*
Xiao-zhuo Chen, Peng Xu, Lu Liu, Dan Zheng, Ping-sheng Lei
Key Laboratory of Bioactivity Substance and Resources Utilization of Chinese Herbal Medicine, Ministry of Education, Institute of Materia Medica, Chinese Academy of Medical
Sciences & Peking Union Medical College, Beijing 100050, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of ketolides with 11,12-sulfur contained aryl alkyl side chains were synthesized and
evaluated for their antibacterial activity. These ketolides exhibited potent activity against key macrolide
sensitive and resistant respiratory pathogens. The newly synthesized 9a, 9e, 9k and 9n showed a similar
antimicrobial spectrum and comparable activity to telithromycin, the commercial ketolide antibacterial.
Ó 2010 Elsevier Masson SAS. All rights reserved.
Received 13 July 2010
Received in revised form
25 October 2010
Accepted 2 November 2010
Available online 10 November 2010
Keywords:
Ketolide
Sulfur contained alkyl side chain
Antibacterial activity
Macrolide-resistant strain
Synthesis
1. Introduction
can interact with nucleotide A752 in domain II of the 23S rRNA [8].
In recent years, many ketolides have been synthesized in order to
Macrolide antibiotics have been used safely and effectively for
treating community-acquired respiratory tract infections since 1950s.
In recent years, with intensive emergence of antibacterial resistance
against antibiotics [1], great efforts have been made to find new
structures which are potent against resistant pathogens. The typical
third generation macrolide [2], telithromycin for example, has been
launched to the market [3]. Another ketolide, cethromycin, has been
approved by FDA and now is in late-stage development for the
treatment of community acquired pneumonia and infections with
biodefensepathogens[4]. Theadvantageofketolidesisthattheycover
macrolide sensitive and resistant strains and are poor inducers of the
erm resistance mechanism as well as mef efflux-mediated resistance
[5,6]. However, with the use of telithromycin, some safety concerns
emerged [7]. So new drug developments are crucially needed (Fig. 1).
Both telithromycin and cethromycin comprise the same key
structure features: a 3-keto group and an 11,12-carbamate func-
tionality. In addition, they have a proper aryl alkyl side chain which
find macrolides with improved activity and low toxicity. For
instance, the fluoroketolide antibiotic CEM-101 (OPT-1068; Cempra
Pharmaceuticals) is to enter into phase II investigation in 2010 [9].
The azetidinyl ketolides Thomas V-6s synthesized by Pfizer, Inc.
with low hepatotoxicity has come into phase Iclinical trials [10].
The molecule of Thomas V-6s showed that a tiny change in the
11,12-side chain made notable decrease of hepatotoxicity.
In our previous work [11], the introduction of a sulfur atom to
the 11,12-cyclic carbamate side chains retained the antibacterial
activity against erythromycin sensitive and resistant test strains.
Compared to telithromycin, the 14-membered ketolide with imi-
dazo[4,5-b]pyridinyl sulfur contained alkyl side chain Xu-15a
demonstrated improved in vitro activity against erythromycin
susceptible and resistant strains including Streptococcus pneumo-
niae (Ery-S) and Streptococcus pyogenes (Ery-R).
For sulfur atom is slightly larger than carbon atom and the
electron density is also higher, inserting a sulfur atom to carbon
chains could slightly adjust the length and nucleophilicity of the
side chain in macrolides. Hunziker et al. [12] developed a series of
novel ketolides with a fused five-membered lactone ring and S-
alkyl or aryl substituted
a-thio acetic acid side chain. These sulfur
contained ketolide products exhibited high activity against
a representative set of erythromycin sensitive and resistant strains.
* Corresponding author. Institute of Materia Medica, Peking Union Medical
College & Chinese Academy of Medical Sciences, Room 408, East building, No. 1 Xia
Xuan Wu district, Beijing 100050, PR China. Tel.: þ86 10 63162006; fax: þ86 10
63017757.
E-mail address: lei@imm.ac.cn (P.-s. Lei).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.11.004

X.-z. Chen et al. / European Journal of Medicinal Chemistry 46 (2011) 208e217
209
N
N
N
N
N
O
R
O
HO
O
N
O
N
O
O
HO
HO
HO
O
O
HO
O
O
O
H
N
O
O
O
O
N
O
O
O
O
O
O
OH
O
O
O
O
O
O
erythromycinA:R=H
clarithromycin:R=CH3
telithromycin
cethromycin
O
N
N
S
HN
OH
N
N
N
HO
O
O
N
O
HO
O
O
N
O
N
HO
N
N
N
O
O
O
N
N
O
O
O
N
O
O
O
O
O
O
H2N
O
O
O
O
O
O
F
O
O
O
CEM-101
Xu-15a
Thomas V-6s
Fig. 1. Structures of representative macrolides.
The best compound showed a similar antimicrobial spectrum and
comparable in vitro activity to telithromycin.
work, simple isolation and purification procedures were used in the
last two steps. For instance, the product of 5 was recrystallized from
ethyl acetate as a white solid without chromatography [14]. In the
preparation of 6, the great quantity of solvent (DMF) was not easy to
remove under vacuum. The reaction mixture was poured into ice
water after the reaction was completed. The crude product of 6 was
obtained as white solid, which was separated easily by filtration.
The synthesis of sulfur contained aryl alkyl side chains was
depicted in Scheme 2 using the method published previously [15]. A
series of heterocyclic with a mercaptan substituent were treated
with one equivalent of N-(3-bromopropyl) phthalimide in DMF at
90 ꢀC toaffordproducts 7aer with ayield of 70e99%. After removing
the phthalimide group of 7aer in a solution of 85% aqueous hydra-
zine in ethanol, a series of free amines 8aer were obtained.
It is reported that, the hepatotoxicity can be decreased by
increasing protonation or diminishing the hydrophobicity of the
erythromycin structure [13]. It is presumed that by varying the side
chain with different protonation contained heterocyclics, novel
ketolides with improved antibacterial activity or lower hepato-
toxicity could be found.
We sought to further optimize the 11,12-cyclic carbamate sulfur
contained aryl alkyl side chains and to introduce several new
heterocyclic groups in the side chain. In this report, a novel series of
ketolides were described with the aim of finding new macrolide
antibiotics with higher antibacterial activity and/or improved
pharmacokinetics.
The 11,12-cyclic carbamate ketolides with sulfur contained aryl
alkyl side chains 9aeo were obtained from the reaction of 6 with
excessive 8aeo in 10% aqueous acetonitrile at 30e40 ꢀC [16]. The
acetyl group was removed to afford 9aeo by refluxing in CH3OH
without any additional base (Scheme 3).
2. Results and discussion
2.1. Chemistry
The synthesis of 6, an important intermediate of ketolides, star-
ted from commercially available clarithromycin (Scheme 1). Decla-
dinose on the 3-position to form the 3-hydroxy group 1 in a yield of
96%, followed byacetylation of the desosamine to form 2 in a yield of
86%. Formation of 11,12-carbonate 3 was carried out with tri-
chloromethyl chloroformate in a mixture of CH2Cl2 and pyridine at
0 ꢀC in 75% yield. The Swern Oxidation was carried to convert the 3-
hydroxy group to the carbonyl group 4 in a yield of 76%. The double
bond on the 10,11-positions was formed underwent smoothly
elimination by treating 4 with 1,8-Diazabicyclo [5.4.0] undec-7-ene
(DBU) in acetone to afford 5 in a yield of 81%. 1,10-carbon-
yldiimidazole (CDI) was used to transform of the 11-hydroxyl to the
acyl imidazole 6 in a yield of 82%. The structure of this key inter-
mediate was characterized by its 1H NMR,13C NMR and HRMS. In our
Some uncyclized O-carbamate ketolides with sulfur contained
aryl alkyl side chains 10aec were also obtained under this
procedure. The HRMS showed the same molecular weight as the
11,12-cyclic carbamate products. But the 1H NMR signals for the 11-
H of 10aec appeared at around 6.76 ppm, while it was shifted
upfield to 3.54 ppm in cyclized compounds. The low field signal
indicated the existence of 10,11-ene. A broad single signal for the
NeH was found at about 5.50 ppm. And the 13C NMR signals for C-9
were about 10 ppm higher than that in cyclized compounds,
which were in accord with the shift of
(Scheme 4).
a, b-unsaturated ketones
Considering the base-catalyzed mechanism of Michael reaction,
the uncyclized O-carbamates 10aec were treated with sodium
hydride in THF. Unfortunately, no cyclic carbamate was formed.

210
X.-z. Chen et al. / European Journal of Medicinal Chemistry 46 (2011) 208e217
N
O
HO
O
N
O
N
O
O
N
O
AcO
O
O
HO
O
AcO
O
O
O
O
O
O
O
HO
HO
a
c
HO
HO
HO
HO
b
O
O
O
O
O
O
OH
OH
OH
O
O
O
O
OH
O
O
O
O
3
2
clarithromycin
1
d
N
O
N
O
N
AcO
O
AcO
O
AcO
O
O
O
O
O
O
O
e
O
f
O
O
O
N
HO
N
O
O
O
O
O
O
O
O
O
O
O
6
5
4
Scheme 1. Preparation of compound 6.
2.2. Pharmacology
worth notice that strain 07A006 was resistant to clarithromycin
(MIC 8 mg/L), but sensitive to telithromycin and all the synthesized
ketolides. 9a, 9e, 9f, 9k and 9n showed excellent activity which
were on equality with telithromycin against 07A006.
Against erythromycin-sensitivity S. pyogenes 07U084 and
07U086, the synthesized ketolides also showed excellent antibac-
terial activity. The MIC of 9a, 9e, 9f, 9n were 4 folds lower compared
to telithromycin, 9k was 2 times more potent than telithromycin.
Compared to telithromycin, compound 9k demonstrated improved
(2 folds lower) activity against erythromycin-resistant S. pyogenes
(ERSPy) of 03-233 and 01-533. All the tested compounds showed
potent activity (MIC<4 mg/L) against strain 01-533 which was
encoded by mef-gene.
All the tested compounds showed excellent activities against
erythromycin-sensitivity S. pneumoniae ATCC49619, but against
07H252 which was also sensitive to clarithromycin, the ketolides
we prepared showed weak activities except compound 9a
(ꢁ0.016 mg/L). Against erythromycin-resistant S. pneumoniae
07D201 and 07P390, the tested compounds showed improved
activity compared to clarithromycin, 9k has nearly equal activity
with telithromycin.
All the compounds were tested against four H. influenzae strains.
9a, 9c, 9f, 9k, 9n showed equal or improved activity compared to
telithromycin.
The antibacterial activity of uncyclized O-carbamate ketolides
10aec was weak. This phenomena confirmed that the 11,12-cyclic
carbamate was a very important factor for the improvement of
the macrolide antibacterial activity. This 5-member ring could
increase the rigidity of the ketolide conformation. The carbamate
group could form a network of hydrophobic interactions with
domain V of 23S rRNA; And N2 of the carbamate group could
The obtained compounds 9aeo and 10aec were tested against
a panel of representative respiratory tract pathogens together with
telithromycin and clarithromycin as references (Table 1 and 2).
Various macrolide sensitive and resistant strains were included in
order to evaluate the activity of this novel series of macrolide
derivatives.
ATCC29213 is a methicillin-sensitivity Staphylococcus aurous
(MSSA). ANS46 (SCCmecIII) is methicillin-resistant Staphylococcus
aurous (MRSA). 07C134 and 07T202 are methicillin-sensitivity
Staphylococcus epidermidis (MSSE). 07R066 and 07A006 are methi-
cillin-resistant Staphylococcus epidermidis (MRSE). ATCC49619 and
07H252 are erythromycin-sensitivity Streptococcus pneumoniae
(ESSP). 07D201 and 07P390 are erythromycin-resistant Streptococcus
pneumoniae(ERSP), 07D201 wasencoded bybothermB andmefgenes
while 07P390 was encoded by only ermB gene. 07U084 and 07U086
are erythromycin-sensitivity Streptococcus pyogenes (ESSPy); 03-233
and 01-533 are erythromycin-resistant Streptococcus pyogenes
(ERSPy), which are encoded by the ermB and mef genes, respectively.
ATCC49247, 07O114, 07O339 and 07O394 are Haemophilus influenzae
strains which were sensitive to erythromycin.
All the strains chosen in this test were supplied by the Ministry
of Health National Antimicrobial Resistance Investigation Net
(MOHNARIN, China). The in vitro antibacterial activity was reported
as minimum inhibitory concentrations (MICs), which was deter-
mined by the broth microdilution method as recommended by the
NCCLS [17].
All the tested ketolides with 11,12-cyclic carbamate sulfur con-
tained side chains showed potent antibacterial activity against
MSSA strain ATCC29213.
It is interesting that, against the methicillin-resistant strains of
S. epidermidis 07R066 and 07A006, all the tested compounds
showed potent activity, while against the methicillin-sensitivity
strain of S. epidermidis 07T202, all the tested compound showed
weak antibacterial activity. This could explain by the different
resistant mechanisms of bacteria to methicillin and macrolides. It is
form a hydrogen bond with O4 of U2588DR (U2609EC) in
domain V of 23S rRNA. These interactions seem to be the main
contributors for the enhancement of the binding of ketolides to the
ribosome [18].
From Tables 1 and 2, some structureeactivity relationships could
be summarized. First at all, among the various ketolide derivatives,

X.-z. Chen et al. / European Journal of Medicinal Chemistry 46 (2011) 208e217
211
O
O
R
a
S
NH2
b
R
SH
+
Br
N
S
N
R
O
O
CH3
N
N
N
N
N
N
N
N
N
H3C
N
N
S
S
H3C
CH3
CH3
Ph
N
N
N
H3C
N
N
Ph
CH3
O
S
O
N
Ph
N
N
N
H3CO
N
N
N
S
NH
N
N
H
N
S
N
Ph
O2N
N
N
N
N
N
H
O
N
H
N
Scheme 2. Preparation of compounds 8aer.
9k and 9n exhibited the most potent activity against both suscep-
tible and resistant bacteria. It was revealed that introduction of
sulfur atom to the 11,12-cyclic carbamate side chains did make sense
for further research in macrolide antibiotics. Then, too many
nitrogen atoms resulted in no improvement of activity. For instance,
there are four nitrogen atoms contained in the aryl alkyl side chains
of 9d and 9m, both of which showed weaker activity against
susceptible and resistant bacteria. The last, compared 9j with 9g,
R
N
AcO
O
S
N
O
O
HO
O
O
O
O
O
N
S
NH2
N
O
a,b
N
O
+
O
O
R
O
O
O
O
O
O
6
8a-o
9a-o
Scheme 3. Preparation of compounds 9aeo.

212
X.-z. Chen et al. / European Journal of Medicinal Chemistry 46 (2011) 208e217
R'
N
O
S
AcO
O
O
N
O
O
HO
O
O
O
O
N
NH
O
S
NH2
a,b
N
O
+
O
O
R'
O
O
O
O
O
10a-c
8p-r
6
10a
10b
10c
O2N
N
N
N
R' =
N
O
N
H
N
H
N
Scheme 4. Preparation of compounds 10aec.
Table 1
In vitro antibacterial activity [MICs (mg/L)] of synthesized ketolides against selected respiratory pathogens(1).
S. aureus
S. epidermidis
S. pyogenes
ATCC29213
ANS46
07C134
07T202
07R066
07A006
07U084
07U086
03-233
01-533
9a
9b
9c
9d
9e
9f
9g
9h
9i
9j
9k
9l
9m
9n
9o
10a
10b
10c
Cla
Teli
0.25
0.5
>32
>32
>16
>32
>16
>32
>16
>16
>32
>16
>32
>16
>32
>16
32
0.125
0.5
>32
>32
>16
>32
>16
>32
>16
>16
>32
>16
32
2
0.5
0.5
0.125
0.5
0.25
1
0.125
0.125
0.25
1
0.5
2
0.125
0.5
0.5
ꢁ0.016
0.125
0.062
0.062
0.016
ꢁ0.016
0.125
0.125
0.125
0.5
0.031
0.062
0.125
0.016
0.25
0.031
0.125
0.062
0.062
0.016
ꢁ0.016
0.125
0.125
0.125
0.5
0.031
0.062
0.125
0.016
0.25
>32
>32
>16
>32
nt
>32
>16
nt
2
nt
1
nt
>32
nt
32
>16
>16
nt
>32
2
1
2
1
1
0.25
0.25
0.125
0.125
0.25
0.5
0.25
0.25
0.125
0.125
0.25
0.5
0.25
4
0.125
0.25
0.5
0.062
0.5
4
0.5
1
>32
0.125
0.062
0.25
0.5
0.25
4
0.125
0.25
0.25
0.062
0.5
4
0.5
1
0.125
0.125
nt
0.5
0.5
nt
2
nt
0.125
nt
2
nt
1
>16
8
0.5
4
0.125
0.5
0.25
0.125
0.5
4
0.5
1
16
>32
>16
32
>16
>16
>16
>32
>16
0.125
0.5
4
0.5
1
>16
>16
>16
>32
>16
2
0.25
0.125
0.031
0.062
2
0.25
0.125
0.031
0.062
nt
2
0.25
0.25
0.125
0.25
0.125
8
0.125
adding two phenyls to the heterocyclic made activity against both
susceptible and resistant bacteria significantly descend. The similar
trend could be seen from the comparison between 9o and 9f.
laboratory emerged as a valuable lead series that might be useful in
thefightagainstmacrolide resistance pathogens. Theseketolides are
promising candidates for further efficacy evaluation.
3. Conclusion
4. Experimental section
In summary, a series of ketolides with 11,12-sulfur contained aryl
alkyl side chains were synthesized. Some of the newly prepared
ketolides displayed excellent in vitro antimicrobial activity against
macrolide sensitive and resistant pathogens. Against erythromycin
sensitivity and resistant S. pyogenes, erythromycin-sensitivity S.
pneumoniaes, erythromycin-sensitivity H. influenzaes, 9n and 9k
showed better antibacterial activity than telithromycin. 9a, 9e, 9k
and 9n have in vitro antibacterial spectrum similar to telithromycin
and are potent against erm and mef-gene containing resistant
strains. In summary, the novel class of ketolides presented in our
4.1. General chemistry methods
All NMR spectra were recorded on Mercury-300 spectrometers
in CDCl3 or DMSO. The chemical shifts are reported impart per
million (ppm) with tetramethylsilane (TMS) as an internal stan-
dard. HRMS experiments were performed using an Aglient 1100
series LC/MSD TOF. Analytical thin layer chromatography (TLC) was
carried out on TLC plates silica gel HSGF254 percolated by Branch of
Qingdao Haiyang Chemical Plant. Chromatography was performed
with silica gel H (HG/T2354-92). The major chemicals were

X.-z. Chen et al. / European Journal of Medicinal Chemistry 46 (2011) 208e217
213
Table 2
In vitro antibacterial activity [MICs (mg/L)] of synthesized ketolides against selected respiratory pathogens (2).a
S. pneumoniae
H. influenzae
ATCC49619
07H252
07D201
07P390
ATCC49247
07O114
07O339
07O394
9a
9b
9c
9d
9e
9f
9g
9h
9i
9j
9k
9l
9m
9n
9o
10a
10b
10c
Cla
Teli
ꢁ0.016
0.062
ꢁ0.016
0.031
0.016
0.031
0.031
0.125
0.062
1
0.062
0.125
0.125
0.016
0.25
ꢁ0.016
2
2
8
nt
0.5
1
nt
8
nt
16
0.125
1
0.5
0.5
nt
0.25
0.25
nt
0.25
nt
0.031
nt
0.5
nt
0.25
>16
16
2
4
2
8
8
2
8
2
8
8
4
8
16
8
>16
2
16
16
2
32
>16
>16
16
4
2
4
2
8
4
2
8
2
8
8
>32
>16
>32
nt
>32
>16
nt
>32
nt
4
4
4
4
16
16
8
>16
2
16
16
4
16
>16
>16
16
4
16
16
8
>16
2
16
32
2
32
>16
>16
>16
8
16
16
8
>16
2
16
32
2
32
>16
>16
>16
8
0.25
nt
1
nt
>32
nt
nt
0.5
16
8
nt
0.062
0.062
8
2
0.5
0.125
0.031
0.031
>16
>16
nt
>32
1
nt
2
0.031
4
4
4
4
a
‘nt’ ¼ not test.
purchased from Alfa Chemical Corporation. All other chemicals
were analytical grade. All reactions were performed under argon
unless otherwise noted.
Clarithromycin forantibacterialtesting wasobtainedfromHUAYI
Pharmaceutical Co., China. Bacterial strains were from the American
Type Culture Collection (ATCC) as indicated inTables 1 and 2, or were
clinical isolates from the Ministry of Health national antimicrobial
resistance investigation net, China (MOHNARIN, China).
2.234 (m, 2H). 13C NMR (75 MHz, CDCl3):
d 168.32, 151.30, 134.04,
131.98, 123.33, 36.63, 31.32, 28.28. HRMS (ESI) m/z found 306.0351,
calcd for C13H12N3O2S2 (M þ H)þ 306.0365.
4.2.4. 2-(3-(1-Methyltetrazole-5-thio) propyl) 1H-isoindole-1,3
(2H)-dione (7d)
Yield: 88%. 1H NMR (300 MHz, CDCl3):
d
7.848 (m, 2H), 7.732
(m, 2H), 3.920 (s, 3H), 3.862 (t, J ¼ 6.9 Hz, 2H), 3.380 (t, J ¼ 6.9 Hz,
2H), 2.215 (m, 2H). 13C NMR (75 MHz, CDCl3):
168.35,
d
4.2. General procedure for compounds 7aer
153.91, 134.12, 131.92, 123.34, 36.26, 33.36, 30.65, 28.46. HRMS
(ESI) m/z found 304.0852, calcd for C13H14N5O2S (M þ H)þ
304.0863.
To a suspension of mercaptan substituent heterocyclic in DMF,
one equivalent of N-(3-bromopropyl) phthalimide and K2CO3 (1
equivalent) were added. The reaction was stirred for 6 h at 90 ꢀC
before cooled to room temperature. After evaporation of the DMF
under vacuum, the residue was diluted with methylene chloride,
washed with water and brine, and dried over MgSO4. After evap-
oration of the solution, the crude products were purified by column
chromatography eluting with 2.5:1 petroleum ether/acetone to
afford the desired products as yellow or white solid.
4.2.5. 2-(3-(Pyridine-2-thio) propyl) 1H-isoindole-1,3(2H)-dione
(7e)
Yield: 99%. 1H NMR (300 MHz, CDCl3):
d
8.276 (dt, J ¼ 5.1, 0.9 Hz,
1H), 7.822 (dd, J ¼ 5.4, 3.0 Hz, 2H), 7.691 (dd, J ¼ 5.4, 3.0 Hz, 2H),
7.416 (t, J ¼ 7.8 Hz, 1H), 7.120 (dd, J ¼ 7.8, 0.9 Hz, 1H), 6.907 (dd,
J ¼ 7.8, 5.1 Hz, 1H), 3.821 (t, J ¼ 6.6 Hz, 2H), 3.189 (t, J ¼ 6.6 Hz, 2H),
2.084 (m, 2H). 13C NMR (75 MHz, CDCl3):
d 168.28, 158.40, 149.30,
135.73, 133.84, 132.06, 123.13, 122.30, 119.27, 37.04, 28.60, 26.98.
HRMS (ESI) m/z found 299.0959, calcd for C16H15N2O2S (M þ H)þ
299.0916.
4.2.1. 2-(3-(5-Methyl-1,3,4-thiadiazole-2-thio) propyl) 1H-
isoindole-1,3(2H)-dione (7a)
Yield: 99%. 1H NMR (300 MHz, CDCl3):
d
7.384 (m, 2H), 7.714 (m,
2H), 3.846 (t, J ¼ 6.9 Hz, 2H), 3.334 (t, J ¼ 7.2 Hz, 2H), 2.702 (s, 3H),
2.195 (m, 2H). 13C NMR (75 MHz, CDCl3):
168.31, 164.95, 134.03,
4.2.6. 2-(3-(Thiazole-2-thio) propyl) 1H-isoindole-1,3(2H)-dione
d
(7f)
132.00, 123.31, 36.66, 31.16, 28.34, 15.60. HRMS (ESI) m/z found
Yield: 93%. 1H NMR (300 MHz, CDCl3):
d 7.842 (m, 2H), 7.715 (m,
320.0537, calcd for C14H14N3O2S2 (M þ H)þ 320.0527.
2H), 7.595 (d, J ¼ 3.3 Hz, 1H), 7.192 (d, J ¼ 3.3 Hz, 1H), 3.838 (t,
J ¼ 6.9 Hz, 2H), 3.262 (t, J ¼ 6.9 Hz, 2H), 2.154 (m, 2H). 13C NMR
4.2.2. 2-(3-(4,6-Dimethylpyrimidine-2-thio) propyl) 1H-isoindole-
(75 MHz, CDCl3): d 168.31, 142.42, 133.99, 132.03, 123.28, 118.94,
1,3(2H)-dione (7b)
36.77, 31.65, 28.46. HRMS (ESI) m/z found 305.0411, calcd for
Yield: 98%. 1H NMR (300 MHz, CDCl3):
d
7.832 (m, 2H), 7.702 (m,
2H), 6.634 (s, 1H), 3.828 (t, J ¼ 6.9 Hz, 2H), 3.147 (t, J ¼ 7.8 Hz, 2H),
2.314 (s, 6H), 2.124 (m, 2H). 13C NMR (75 MHz, CDCl3):
168.31,
C14H13N2O2S2 (M þ H)þ 305.0418.
d
4.2.7. 2-(3-(2-Methylfurane-3-thio) propyl) 1H-isoindole-1,3(2H)-
166.92,133.87,132.10,123.17,115.56, 37.24, 28.75, 27.87, 23.73. HRMS
dione (7g)
(ESI) m/z found 328.1107, calcd for C17H18N3O2S (M þ H)þ 328.1120.
Yield: 80%. 1H NMR (300 MHz, CDCl3):
d 7.828 (m, 2H), 7.701 (m,
2H), 7.243 (d, J ¼ 1.5 Hz, 1H), 6.352 (d, J ¼ 1.5 Hz, 1H), 3.779 (t,
4.2.3. 2-(3-(1,3,4-Thiadiazole-2-thio) propyl) 1H-isoindole-1,3
J ¼ 6.9 Hz, 2H), 2.636 (t, J ¼ 7.2 Hz, 2H), 2.335 (s, 3H), 1.885 (m, 2H).
(2H)-dione (7c)
13C NMR (75 MHz, CDCl3):
d 168.26, 155.18, 140.57, 133.92, 132.03,
Yield: 80%. 1H NMR (300 MHz, CDCl3):
d
8.988 (s, 1H), 7.847 (m,
123.19, 114.99, 36.84, 33.21, 28.36, 11.75. HRMS (ESI) m/z found
2H), 7.721 (m, 2H), 3.867 (t, J ¼ 6.9 Hz, 2H), 3.415 (t, J ¼ 6.9 Hz, 2H),
302.0797, calcd for C16H16N3S (M þ H)þ 302.0845.

214
X.-z. Chen et al. / European Journal of Medicinal Chemistry 46 (2011) 208e217
4.2.8. 2-(3-(5-n-Propylpyrimidine-2-thio) propyl) 1H-isoindole-1,3
2H), 7.167 (dd, J ¼ 6.0, 3.0 Hz, 2H), 3.862 (t, J ¼ 6.6 Hz, 2H), 3.323 (t,
(2H)-dione (7h)
J ¼ 6.6 Hz, 2H), 2.135 (m, 2H). 13C NMR (75 MHz, CDCl3):
d 168.44,
Yield: 99%. 1H NMR (300 MHz, CDCl3):
d
8.262(s, 2H), 7.844 (dd,
149.33, 133.99, 131.90, 123.25, 122.30, 114.12, 36.45, 30.23, 28.55.
HRMS (ESI) m/z found 338.0961, calcd for C18H16N3O2S (M þ H)þ
338.0963.
J ¼ 5.4, 3.0 Hz, 2H), 7.712 (dd, J ¼ 5.4, 3.0 Hz, 2H), 3.839 (t, J ¼ 6.9 Hz,
2H), 3.157 (t, J ¼ 6.9 Hz, 2H), 2.462 (t, J ¼ 7.2 Hz, 2H), 2.132 (m, 2H),
1.596 (m, 2H), 0.935 (t, 3H). 13C NMR (75 MHz, CDCl3):
d 168.37,
157.16, 133.92, 132.12, 130.07, 123.20, 37.18, 31.74, 28.62, 28.02,
23.90, 13.47. HRMS (ESI) m/z found 342.1265, calcd for C18H20N3O2S
(M þ H)þ 342.1271.
4.2.15. 2-(3-(4-Phenylthiazole-2-thio) propyl) 1H-isoindole-1,3
(2H)-dione (7o)
Yield: 97%. 1H NMR (300 MHz, CDCl3):
d 7.696 (m, 4H), 7.562 (m,
2H), 7.224 (d, J ¼ 7.5 Hz, 1H), 7.170 (d, J ¼ 5.1 Hz, 2H), 7.113 (m, 1H),
4.2.9. 2-(3-(4-Methylpyrimidine-2-thio) propyl) 1H-isoindole-1,3
3.733 (t, J ¼ 6.9 Hz, 2H), 3.185 (t, J ¼ 6.9 Hz, 2H), 2.098 (m, 2H). 13C
(2H)-dione (7i)
NMR (75 MHz, CDCl3): d 168.29, 163.99, 155.26, 133.96, 132.00,
Yield: 96%. 1H NMR (300 MHz, CDCl3):
d
8.251 (d, J ¼ 4.8 Hz, 1H),
128.64, 128.07, 126.23, 123.26, 112.35, 36.86, 31.61, 28.57. HRMS
(ESI) m/z found 381.0717, calcd for C20H17N2O2S2 (M þ H)þ
381.0726.
7.835 (dd, J ¼ 5.4, 3.3 Hz, 2H), 7.704 (dd, J ¼ 5.4, 3.3 Hz, 2H), 6.773
(d, J ¼ 4.8 Hz, 1H), 3.832 (t, J ¼ 6.6 Hz, 2H), 3.153 (t, J ¼ 6.6 Hz, 2H),
2.379 (s, 3H), 2.128 (m, 2H). 13C NMR (75 MHz, CDCl3):
d 171.25,
168.31, 167.50, 156.62, 133.89, 132.09, 123.17, 116.08, 37.18, 28.62,
27.93, 23.97. HRMS (ESI) m/z found 314.0958, calcd for C16H16N3O2S
(M þ H)þ 314.0958.
4.2.16. 2-(3-(Benzoxazole-2-thio) propyl) 1H-isoindole-1,3(2H)-
dione (7p)
Yield: 82%. 1H NMR (300 MHz, CDCl3):
d 7.863 (m, 2H), 7.736 (m,
2H), 7.488 (dd, J ¼ 6.9, 1.8 Hz, 1H), 7.408 (dd, J ¼ 6.9, 1.8 Hz, 1H),
4.2.10. 2-(3-(4,5-Diphenyloxazole-2-thio) propyl) 1H-isoindole-1,3
7.229 (m, 2H), 3.895 (t, J ¼ 6.6 Hz, 2H), 3.342 (t, J ¼ 7.2 Hz, 2H), 2.266
(2H)-dione (7j)
(m, 2H). 13C NMR (75 MHz, CDCl3):
d 168.37, 164.54, 151.87, 141.87,
Yield: 98%. 1H NMR (300 MHz, CDCl3):
d
7.836 (dd, J ¼ 5.4,
134.04, 132.04, 124.21, 123.81, 123.34, 118.35, 109.77, 36.67, 29.47,
28.68. HRMS (ESI) m/z found 339.0815, calcd for C18H15N2O3S
(M þ H)þ 339.0803.
3.0 Hz, 2H), 7.704 (dd, J ¼ 5.4, 3.0 Hz, 2H), 7.632 (d, J ¼ 2.1 Hz, 1H),
7.607 (d, J ¼ 2.1 Hz, 1H), 7.553 (d, J ¼ 2.1 Hz, 1H), 7.527 (d, J ¼ 2.1 Hz,
1H), 7.330 (m, 6H), 3.891 (t, J ¼ 6.9 Hz, 2H), 3.292 (t, J ¼ 6.9 Hz, 2H),
2.266 (m, 2H). 13C NMR (75 MHz, CDCl3):
d
168.31, 158.80, 147.12,
4.2.17. 2-(3-(Pyrazolo [3,4d] pyrimidine-4-thio) propyl) 1H-
136.27, 133.96, 132.00, 128.61, 128.50, 128.16, 127.86, 126.30, 123.26,
36.74, 29.81, 28.83. HRMS (ESI) m/z found 441.1265, calcd for
C26H21N2O3S (M þ H)þ 441.1267.
isoindole-1,3(2H)-dione (7q)
Yield: 70%. 1H NMR (300 MHz, CDCl3):
d 8.649 (s, 1H), 8.123 (s,
1H), 7.866 (dd, J ¼ 5.4, 3.0 Hz, 2H), 7.735 (dd, J ¼ 5.4, 3.0 Hz, 2H),
3.891 (t, J ¼ 6.9 Hz, 2H) 3.442 (t, J ¼ 6.9 Hz, 2H), 2.212 (m, 2H). 13C
4.2.11. 2-(3-(4-(3-Pyridine)-1H-imidazole-2-thio) propyl) 1H-
NMR (75 MHz, CDCl3): d 168.43, 165.91, 153.64, 151.67, 134.07,
isoindole-1,3(2H)-dione (7k)
132.97, 132.03, 123.31, 36.83, 28.55, 26.28. HRMS (ESI) m/z found
Yield: 84%. 1H NMR (300 MHz, CDCl3):
d
8.955 (d, J ¼ 1.5 Hz, 1H),
340.0865, calcd for C16H14N5O2S (M þ H)þ 340.0862.
8.470 (dd, J ¼ 4.8, 1.5 Hz, 1H), 8.087 (d, J ¼ 7.8 Hz, 1H), 7.839 (dd,
J ¼ 5.4, 3.3 Hz, 2H), 7.718 (dd, J ¼ 5.4, 3.3 Hz, 2H), 7.461 (s, 1H), 7.299
(dd, J ¼ 5.1, 7.8 Hz, 1H), 3.946 (t, J ¼ 7.2 Hz, 2H), 3.038 (t, J ¼ 7.2 Hz,
4.2.18. 2-(3-(5-Nitrobenzimidazole-2-thio) propyl) 1H-isoindole-1,
3(2H)-dione (7r)
2H), 1.977 (m, 2H). 13C NMR (75 MHz, CDCl3):
d
168.64, 147.69,
Yield: 80%. 1H NMR (300 MHz, DMSO):
d 13.236 (s, 1H), 8.013 (d,
146.08, 134.19, 132.29, 131.90, 123.65, 123.39, 36.03, 32.71, 28.19.
HRMS (ESI) m/z found 365.1074, calcd for C19H17N4O2S (M þ H)þ
365.1072.
J ¼ 8.7 Hz, 1H), 7.881 (m, 4H), 7.475 (d, J ¼ 8.7 Hz, 1H), 3.722 (t,
J ¼ 6.6 Hz, 2H), 3.354 (t, J ¼ 6.6 Hz, 2H), 2.029 (m, 2H). 13C NMR
(75 MHz, DMSO):
d 168.11, 134.36, 131.77, 123.02, 117.37, 54.90,
36.33, 30.69, 28.56. HRMS (ESI) m/z found 383.0814, calcd for
4.2.12. 2-(3-(5-Methoxybenzothiazole-2-thio) propyl) 1H-
isoindole-1,3(2H)-dione (7l)
C18H15N4O4S (M þ H)þ 383.0814.
Yield: 99%. 1H NMR (300 MHz, CDCl3):
d
7.840 (dd, J ¼ 5.4,
4.3. General procedure for compounds 8aer
3.0 Hz, 2H), 7.710 (dd, J ¼ 5.4, 3.0 Hz, 2H), 7.553 (d, J ¼ 9.0 Hz, 1H),
7.275 (d, J ¼ 2.4 Hz, 1H), 6.910 (dd, J ¼ 9.0, 2.4 Hz, 1H), 3.872 (t,
J ¼ 6.9 Hz, 2H), 3.843 (s, 3H), 3.371 (t, J ¼ 6.9 Hz, 2H), 2.232 (m, 2H).
To a stirred suspension of compounds 7aer in dry ethanol,
2 equiv. of 85% aqueous hydrazine was added. After refluxing for
4 h, the solvent was concentrated. The mixture was diluted with
ethyl acetate, washed with 2 mol/L NaOH aqueous, water and brine,
dried over MgSO4. After evaporation of the solvent, the crude
products were used for the next step without isolation.
13C NMR (75 MHz, CDCl3):
d 168.28, 167.42, 158.83, 154.26, 133.96,
131.98, 126.76, 123.25, 121.07, 113.89, 104.46, 55.56, 36.77, 30.61,
28.51. HRMS (ESI) m/z found 385.0668, calcd for C19H17N2O3S2
(M þ H)þ 385.0675.
4.2.13. 2-(3-(1-Phenyl-tetrazole-5-thio) propyl) 1H-isoindole-1,3
4.4. General procedure for compounds 9aeo and 10aec
(2H)-dione (7m)
Yield: 88%. 1H NMR (300 MHz, CDCl3):
d
7.838 (m, 2H), 7.720 (m,
2H), 7.552 (m, 5H), 3.856 (t, J ¼ 6.6 Hz, 2H), 3.425 (t, J ¼ 7.2 Hz, 2H),
2.249 (m, 2H). 13C NMR (75 MHz, CDCl3):
168.34, 134.07, 131.92,
Macrolide intermediate 6 and 5 equiv. of amino side chains 8aer
were dissolved in 10% aqueous acetonitrile, the reaction was stirred
at 30 ꢀC until 6 disappeared by TLC. The mixture was diluted with
CH2Cl2, washed with 5% KH2PO4 aqueous, water and brine, and
dried over MgSO4. After evaporation of the solvent, the products
were purified by column chromatography eluting with 3:1 petro-
leum ether/acetone to afford products with 20-OAc as white foam.
After refluxing with CH3OH, purified by column chromatography
eluting with 1:1 petroleum ether/acetone, the desired products
were obtained as white foam.
d
130.09, 129.77, 123.81, 123.31, 36.38, 30.60, 28.31. HRMS (ESI) m/z
found 366.1020, calcd for C18H16N5O2S (M þ H)þ 366.1025.
4.2.14. 2-(3-(Benzoxazole-2-thio) propyl) 1H-isoindole-1,3(2H)-
dione (7n)
Yield: 70%. 1H NMR (300 MHz, CDCl3):
d
7.798 (dd, J ¼ 5.4,
3.0 Hz, 2H), 7.686 (dd, J ¼ 5.4, 3.0 Hz, 2H), 7.478 (dd, J ¼ 6.0, 3.0 Hz,

X.-z. Chen et al. / European Journal of Medicinal Chemistry 46 (2011) 208e217
215
4.4.1. 11,12-Didehydro-3-O-descladinosyl-6-O-methyl-3-oxo-12,11-
(oxycarbonyl (3-(5-methyl-1,3,4-thiadiazole-2-mercapto) propyl)
imino) erythromycin A (9a)
J ¼ 4.5, 1.8 Hz, 1H), 4.930 (dd, J ¼ 10.2, 1.8 Hz, 1H), 4.304 (d,
J ¼ 6.9 Hz, 1H), 4.211 (d, J ¼ 8.4 Hz, 1H), 3.813 (q, J ¼ 6.9 Hz, 1H),
3.727 (t, J ¼ 8.4 Hz, 2H), 3.581 (s, 1H), 3.548 (m, 1H), 3.281 (q, 1H),
3.175 (t, 2H), 3.083 (m, 2H), 2.600 (s, 3H), 2.429 (s, 6H),1.450 (s, 3H),
1.329 (d, J ¼ 6.6 Hz, 3H), 1.145 (d, J ¼ 6.9 Hz, 3H), 0.995 (d, J ¼ 6.6 Hz,
Yield: 30%. 1H NMR (300 MHz, CDCl3):
d
4.917 (d, J ¼ 9.3 Hz, 1H),
4.274 (d, J ¼ 7.2 Hz, 1H), 4.228 (d, J ¼ 8.4 Hz, 1H), 3.831 (q, J ¼ 6.6 Hz,
1H), 3.754 (m, 2H), 3.566 (s, 1H), 3.534 (d, J ¼ 3.9 Hz, 1H), 3.483 (s,
1H), 3.331 (m, 2H), 3.115 (m, 3H), 2.708 (s, 3H), 2.623 (s, 3H), 2.450
(m, 1H), 2.555 (s, 6H), 2.165 (s, 1H), 1.457 (s, 3H), 1.148 (d, J ¼ 6.6 Hz,
3H), 1.000 (d, J ¼ 6.6 Hz, 3H), 0.851 (t, J ¼ 7.2 Hz, 3H). 13C NMR
3H), 0.826 (t, J ¼ 7.2 Hz, 3H). 13C NMR (75 MHz, CDCl3):
d 215.84,
203.87, 169.36, 157.06, 149.35, 135.66, 122.06, 119.08, 103.39, 82.08,
79.38, 77.99, 77.23, 70.01, 68.97, 66.10, 60.61, 55.60, 51.14, 49.76,
47.39, 44.81, 42.96, 40.22, 39.38, 39.01,29.07, 27.30, 26.98, 22.23,
21.01, 19.66, 18.27, 15.67, 14.63, 14.34, 13.81, 10.33. HRMS (ESI) m/z
found 764.4120, calcd for C39H62N3O10S (M þ H)þ 764.4150.
(75 MHz, CDCl3):
d 215.97, 203.70, 169.52, 165.46, 164.69, 157.15,
103.96, 82.22, 79.58, 78.14, 70.35, 69.62, 65.88, 60.60, 51.18, 49.89,
47.54, 44.87, 42.66, 40.21, 39.54, 39.01, 31.64, 28.15, 26.73, 22.26,
21.16, 19.74, 18.33, 15.71, 15.57, 14.61, 14.37, 13.85, 10.39. HRMS (ESI):
m/z found 785.3879, calcd for C37H61N4O10S2 (M þ H)þ 785.3824.
4.4.6. 11,12-Didehydro-3-O-descladinosyl-6-O-methyl-3-oxo-12,11-
(oxycarbonyl (3-(thiazole-2-mercapto) propyl) imino) erythromycin
A (9f)
4.4.2. 11,12-Didehydro-3-O-descladinosyl-6-O-methyl-3-oxo-12,11-
(oxycarbonyl (3-(4, 6-dimethylpyrimidine-2-mercapto) propyl)
imino) erythromycin A (9b)
Yield: 48%. 1H NMR (300 MHz, CDCl3):
d
7.616 (d, J ¼ 3.3 Hz, 1H),
7.163 (d, J ¼ 3.3 Hz, 1H), 4.911 (dd, J ¼ 10.5, 2.1 Hz, 1H), 4.269 (d,
J ¼ 7.2 Hz, 1H), 4.211 (d, J ¼ 8.7 Hz, 1H), 3.817 (q, J ¼ 6.6 Hz, 1H),
3.732 (m, 2H), 3.561 (s, 1H), 3.535 (m, 2H), 2.602 (s, 3H), 2.283 (s,
6H), 1.513 (s, 3H), 1.137 (d, J ¼ 7.2 Hz, 3H), 0.988 (d, J ¼ 6.6 Hz, 3H),
Yield: 34%. 1H NMR (300 MHz, CDCl3):
d 6.635 (s, 1H), 4.930 (d,
J ¼ 10.5 Hz, 1H), 4.271(d, J ¼ 7.2 Hz, 1H), 4.212 (d, J ¼ 8.4 Hz, 1H),
3.817 (q, J ¼ 6.6 Hz, 1H), 3.713 (m, 2H), 3.582 (s, 1H), 3.519 (m, 2H),
3.169 (m, 3H), 2.603 (s, 3H), 2.370 (s, 6H), 2.265 (s, 6H),1.483 (s, 3H),
1.139 (d, J ¼ 6.9 Hz, 3H), 0.999 (d, J ¼ 6.6 Hz, 3H), 0.832 (t, J ¼ 7.5 Hz,
0.831 (t, J ¼ 7.5 Hz, 3H). 13C NMR (75 MHz, CDCl3):
d 215.98, 203.73,
169.42, 164.83, 157.09, 142.62, 118.52, 103.77, 82.16, 79.52, 78.05,
77.17, 70.21, 69.43, 65.89, 60.56, 51.14, 49.80, 47.51, 44.82, 42.73,
40.20, 39.47, 38.96, 31.74, 29.21, 28.31, 26.81, 22.20, 21.11, 19.69,
18.28, 15.75, 14.60, 14.33, 13.82, 10.34. HRMS (ESI) m/z found
770.3732, calcd for C37H60N3O10S2 (M þ H)þ 770.3720.
3H). 13C NMR (75 MHz, CDCl3):
d 215.78, 203.84, 171.07, 169.36,
166.81, 157.06, 115.38, 103.83, 82.10, 79.47, 78.03, 70.28, 69.52,
65.88, 60.64, 51.15, 49.82, 47.51, 44.85, 43.18, 40.21, 39.45, 39.00,
28.17, 28.02, 26.99, 23.79, 22.22, 21.13, 19.69, 18.31, 15.74, 14.59,
14.33, 13.85, 10.37. HRMS (ESI): m/z found 793.4402, calcd for
C40H65N4O10S (M þ H)þ 793.4416.
4.4.7. 11, 12-Didehydro-3-O-descladinosyl-6-O-methyl-3-oxo-
12,11-(oxycarbonyl (3-(2-methylfurane-3-mercapto) propyl) imino)
erythromycin A (9g)
4.4.3. 11,12-Didehydro-3-O-descladinosyl-6-O-methyl-3-oxo-12,11-
(oxycarbonyl (3-(1, 3,4-thiadiazole-2-mercapto) propyl) imino)
erythromycin A (9c)
Yield: 52%. 1H NMR (300 MHz, CDCl3):
d
7.230 (d, J ¼ 1.5 Hz, 1H),
6.362 (d, J ¼ 1.5 Hz, 1H), 4.881 (d, J ¼ 9.0 Hz, 1H), 4.275 (d, J ¼ 7.2 Hz,
1H), 4.224 (d, J ¼ 8.4 Hz,1H), 3.826 (q, J ¼ 6.6 Hz, 1H), 3.681 (m, 2H),
3.540 (s, 1H), 3.506 (m, 1H), 2.584 (s, 3H), 2.320 (s, 3H), 2.272 (s,
6H), 1.444 (s, 3H), 1.142 (d, J ¼ 6.9 Hz, 3H), 0.983 (d, J ¼ 6.6 Hz, 3H),
Yield: 30%. 1H NMR (300 MHz, CDCl3):
d 8.969 (s, 1H), 4.915 (dd,
J ¼ 10.8, 1.8 Hz, 1H), 4.262 (d, J ¼ 6.9 Hz, 1H), 4.216 (d, J ¼ 8.7 Hz,
1H), 3.788 (m, 3H), 3.561 (s, 1H), 3.521 (m, 1H), 3.406 (t, J ¼ 7.2 Hz,
2H), 2.618 (s, 3H), 2.434 (m, 1H), 2.247 (s, 6H), 2.155 (d, J ¼ 2.4 Hz,
1H), 2.103 (t, J ¼ 7.5 Hz, 1H), 1.449 (s, 3H), 1.138 (d, J ¼ 6.9 Hz, 3H),
0.990 (d, J ¼ 6.6 Hz, 3H), 0.839 (t, J ¼ 7.2 Hz, 3H). 13C NMR (75 MHz,
0.829 (t, J ¼ 7.2 Hz, 3H). 13C NMR (75 MHz, CDCl3):
d 216.05, 203.78,
169.39, 157.06, 155.00, 140.37, 115.07, 103.82, 82.07, 79.49, 78.04,
77.23, 70.25, 69.50, 65.88, 60.44, 51.14, 49.76, 47.46, 44.88, 42.55,
40.19, 39.47, 38.97, 33.40, 28.22, 27.32, 22.22, 21.14, 19.69, 18.34,
15.68, 14.63, 14.34, 13.85, 11.73, 10.38. HRMS (ESI) m/z found
767.4152, calcd for C39H63N2O11S (M þ H)þ 767.4147.
CDCl3): d 216.05, 203.67, 169.54, 157.15, 151.07, 103.88, 82.22, 79.56,
78.10, 77.17, 70.27, 69.55, 65.80, 60.50, 51.13, 49.85, 47.53, 44.82,
42.57, 40.17, 39.47, 38.95, 31.67, 28.10, 26.55, 22.17, 21.12, 19.70,
18.28, 15.73, 14.56, 14.31, 13.81, 10.33. HRMS (ESI): m/z found
771.3668, calcd for C36H59N4O10S2 (M þ H)þ 771.3673.
4.4.8. 11,12-Didehydro-3-O-descladinosyl-6-O-methyl-3-oxo-12,11-
(oxycarbonyl (3-(5-n-propylpyrimidine-2-mercapto) propyl) imino)
erythromycin A (9h)
4.4.4. 11,12-Didehydro-3-O-descladinosyl-6-O-methyl-3-oxo-12,11-
(oxycarbonyl (3-(1-methyltetrazole-5-mercapto) propyl) imino)
erythromycin A (9d)
Yield: 35%. 1H NMR (300 MHz, CDCl3):
d 8.315 (s, 2H), 4.946 (d,
J ¼ 9.0 Hz, 1H), 4.276 (d, J ¼ 7.5 Hz, 1H), 4.219 (d, J ¼ 8.4 Hz, 1H),
4.107 (q, J ¼ 7.5 Hz, 1H), 3.819 (q, J ¼ 6.9 Hz, 1H), 3.720 (t, J ¼ 6.9 Hz,
2H), 3.593 (s, 1H), 3.522 (m, 1H), 2.636 (s, 3H), 2.477 (t, J ¼ 7.5 Hz,
3H), 2.266 (s, 6H), 1.450 (s, 3H), 1.142 (d, J ¼ 6.9 Hz, 3H), 1.001 (d,
J ¼ 7.2 Hz, 3H), 0.944 (t, J ¼ 7.2 Hz, 3H), 0.829 (t, J ¼ 7.2 Hz, 3H). 13C
Yield: 40%. 1H NMR (300 MHz, CDCl3):
d
4.905(d, J ¼ 9.0 Hz, 1H),
4.280 (d, J ¼ 7.5 Hz, 1H), 4.218 (d, J ¼ 8.4 Hz, 1H), 3.909 (s, 3H), 3.824
(m, 1H), 3.755 (m, 2H), 3.543 (s, 1H), 3.394 (m, 2H), 3.215 (m, 1H),
3.080 (m, 2H), 2.562 (s, 3H), 2.333 (s, 6H), 2.073 (q, 1H), 1.453 (s,
3H), 1.143 (d, J ¼ 7.2 Hz, 3H), 0.972 (d, J ¼ 6.6 Hz, 3H), 0.846 (t,
NMR (75 MHz, CDCl3):
d 215.84, 203.83, 169.33, 157.10, 129.75,
103.85, 82.11, 79.49, 78.04, 70.28, 69.52, 65.86, 60.67, 53.75, 51.16,
49.82, 47.54, 44.86, 43.10, 40.20, 39.47, 39.01, 31.81, 29.65, 29.23,
28.17, 26.94, 23.94, 22.25, 21.14, 19.70, 18.30, 15.75, 14.60, 14.31,
13.84, 13.49, 10.34. HRMS (ESI) m/z found 807.4570, calcd for
C41H67N4O10S (M þ H)þ 807.4578.
J ¼ 7.2 Hz, 3H). 13C NMR (75 MHz, CDCl3):
d 216.08, 203.67, 169.64,
157.21, 103.70, 82.28, 79.55, 78.11, 77.23, 70.16, 69.34, 66.03, 60.45,
51.14, 49.83, 47.50, 44.84, 42.17, 40.23, 39.45, 38.95, 33.36, 30.94,
28.54, 26.83, 22.19, 21.09, 19.70, 18.31, 15.73, 14.59, 14.34, 13.81,
10.33. HRMS (ESI): m/z found 769.4150, calcd for C36H61N6O10
S
(M þ H)þ 769.4170.
4.4.9. 11,12-Didehydro-3-O-descladinosyl-6-O-methyl-3-oxo-12,11-
(oxycarbonyl (3-(4-methylpyrimidine-2-mercapto) propyl)imino)
erythromycin A (9i)
4.4.5. 11,12-Didehydro-3-O-descladinosyl-6-O-methyl-3-oxo-12,11-
(oxycarbonyl (3-(pyridine-2-mercapto) propyl) imino)
erythromycin A (9e)
Yield: 36%. 1H NMR (300 MHz, CDCl3):
d
8.300 (d, J ¼ 5.1 Hz, 1H),
6.760 (d, J ¼ 5.1 Hz, 1H), 4.917 (dd, J ¼ 9.0, 1.8 Hz, 1H), 4.266 (d,
J ¼ 7.2 Hz, 1H), 4.194 (d, J ¼ 9.0 Hz, 1H), 3.803 (q, J ¼ 6.9 Hz, 1H),
3.700 (m, 2H), 3.565 (s, 1H), 3.518 (m, 1H), 3.349 (q, J ¼ 6.9 Hz, 1H),
Yield: 50%. 1H NMR (300 MHz, CDCl3):
7.423 (td, J ¼ 8.1, 1.8 Hz, 1H), 7.135 (d, J ¼ 8.1 Hz, 1H), 6.920 (td,
d
8.378 (d, J ¼ 4.5 Hz, 1H),

216
X.-z. Chen et al. / European Journal of Medicinal Chemistry 46 (2011) 208e217
2.598 (s, 3H), 2.406 (s, 3H), 2.312 (s, 6H), 1.970 (s, 3H), 1.433 (s, 3H),
J ¼ 9.9, 7.5 Hz, 1H), 3.074 (m, 2H), 2.593 (s, 3H), 2.316 (s, 6H), 1.448
(s, 3H), 1.077 (d, J ¼ 6.3 Hz, 3H), 0.981 (d, J ¼ 6.9 Hz, 3H), 0.839 (t,
1.127 (d, J ¼ 6.9 Hz, 3H), 0.981 (d, J ¼ 6.9 Hz, 3H), 0.812 (t, J ¼ 7.2 Hz,
3H). 13C NMR (75 MHz, CDCl3):
d
215.88, 203.84, 169.33, 167.31,
J ¼ 7.2 Hz, 3H). 13C NMR (75 MHz, CDCl3):
d 216.02, 203.69, 169.60,
156.63, 115.91, 103.64, 82.11, 79.40, 77.98, 70.13, 69.28, 65.89, 60.59,
51.10, 49.74, 47.48, 44.81, 43.04, 40.18, 39.38, 29.27, 28.49, 28.02,
27.75, 26.84, 23.99, 22.17, 21.06, 19.66, 18.24, 15.78, 14.56, 14.30,
13.81, 10.31. HRMS (ESI): m/z found 779.4274, calcd for
C39H63N4O10S (M þ H)þ 779.4259.
157.20, 154.28, 133.69, 129.95, 129.69, 123.94, 103.73, 82.27, 79.53,
78.08, 77.18, 70.18, 69.37, 65.95, 60.52, 51.13, 49.80, 47.50, 44.81,
42.32, 40.22, 39.44, 38.93, 30.74, 28.43, 26.62, 22.16, 21.09, 19.69,
18.27, 15.75, 14.57, 14.31, 13.79, 10.30. HRMS (ESI) m/z found
831.4322, calcd for C41H63N6O10S (M þ H)þ 831.4326.
4.4.10. 11,12-Didehydro-3-O-descladinosyl-6-O-methyl-3-oxo-
12,11-(oxycarbonyl (3-(4,5-diphenyloxazole-2-mercapto) propyl)
imino) erythromycin A (9j)
4.4.14. 11,12-Didehydro-3-O-descladinosyl-6-O-methyl-3-oxo-
12,11-(oxycarbonyl (3-(benzoxazole-2-mercapto) propyl) imino)
erythromycin A (9n)
Yield: 17%. 1H NMR (300 MHz, CDCl3):
d
7.655 (d, J ¼ 6.6 Hz, 2H),
Yield: 15%. 1H NMR (300 MHz, CDCl3):
d 10.550 (br s, 1H), 7.169
7.556 (d, J ¼ 6.6 Hz, 2H), 7.359 (m, 6H), 4.949 (dd, J ¼ 8.7, 1.2 Hz, 1H),
4.267 (d, J ¼ 7.2 Hz, 1H), 4.209 (d, J ¼ 8.4 Hz, 1H), 3.810 (m, 2H),
3.594 (s, 1H), 3.525 (m, 1H), 3.300 (t, J ¼ 7.2 Hz, 2H), 2.624 (s, 3H),
2.257 (s, 6H), 1.460 (s, 3H), 1.336 (d, J ¼ 6.9 Hz, 3H), 1.146 (d,
J ¼ 6.9 Hz, 3H), 1.013 (d, J ¼ 7.2 Hz, 3H), 0.823 (t, J ¼ 7.2 Hz, 3H). 13C
(dd, J ¼ 6.0, 3.0 Hz, 2H), 7.094 (d, J ¼ 3.0 Hz, 1H), 4.933 (d, J ¼ 9.0 Hz,
1H), 4.262 (d, J ¼ 7.2 Hz, 1H), 4.197 (d, J ¼ 9.0 Hz, 1H), 3.832 (m, 3H),
3.580 (s,1H), 3.518 (m, H), 2.566 (s, 3H), 2.444 (m,1H), 2.255 (s, 6H),
2.052 (q, J ¼ 6.9 Hz, 2H), 1.461 (s, 3H), 1.360 (d, J ¼ 6.9 Hz, 3H), 1.136
(d, J ¼ 6.9 Hz, 3H), 1.007 (d, J ¼ 6.9 Hz, 3H), 0.809 (t, J ¼ 7.5 Hz, 3H).
NMR (75 MHz, CDCl3):
d
215.92, 203.71, 169.51, 159.08, 157.17,
13C NMR (75 MHz, CDCl3):
d 216.37, 203.54, 169.86, 157.47, 149.06,
146.93, 136.44, 132.28, 129.55, 128.81, 128.54, 128.44, 128.24,
128.02, 126.34, 125.75, 103.88, 82.23, 79.54, 78.08, 77.19, 70.31,
69.56, 65.85, 60.66, 53.78, 51.17, 49.89, 47.49, 45.81, 44.86, 42.83,
40.21, 39.51, 39.00, 31.71, 29.90, 29.67, 29.25, 28.15, 27.16, 22.23,
21.15, 19.70, 18.34, 15.67, 14.60, 14.34, 13.87, 10.34. HRMS (ESI) m/z
found 906.4543, calcd for C49H68N3O11S (M þ H)þ 906.4569.
129.51, 125.71, 122.07, 118.53, 103.85, 82.46, 79.46, 78.10, 70.27,
69.55, 65.77, 60.97, 53.74, 51.16, 49.76, 47.59, 44.87, 42.76, 40.15,
39.47, 38.93, 30.86, 29.20, 28.13, 27.24, 22.17, 21.09, 19.64, 18.22,
15.87, 14.53, 14.30, 13.84, 10.33. HRMS (ESI) m/z found 803.4263,
calcd for C41H63N4O10S (M þ H)þ 803.4259.
4.4.15. 11,12-Didehydro-3-O-descladinosyl-6-O-methyl-3-oxo-
12,11-(oxycarbonyl (3-(4-phenylthiazole-2-mercapto) propyl)
imino) erythromycin A (9o)
4.4.11. 11, 12-Didehydro-3-O-descladinosyl-6-O-methyl-3-oxo-
12,11-(oxycarbonyl(3-(4-(3-pyridine)-1H-imidazole-2-mercapto)
propyl) imino)erythromycin A (9k)
Yield: 74%. 1H NMR (300 MHz, CDCl3):
d
7.880 (d, J ¼ 7.2 Hz, 2H),
Yield: 42%. 1H NMR (300 MHz, CDCl3):
d
11.201 (br s, 1H), 8.942
7.392 (t, J ¼ 7.8 Hz, 2H), 7.314 (s, 1H), 7.304 (d, J ¼ 6.3 Hz, 1H), 4.930
(d, J ¼ 8.7 Hz, 1H), 4.268 (d, J ¼ 7.2 Hz, 1H), 4.207 (d, J ¼ 8.4 Hz, 1H),
3.803 (m, 3H), 3.583 (s, 1H), 3.530 (m, 2H), 3.323 (t, J ¼ 7.2 Hz, 2H),
3.180 (dd, J ¼ 7.2, 9.9 Hz, 1H), 3.079 (m, 2H), 2.611 (s, 3H), 2.274 (s,
6H), 1.457 (s, 3H), 1.146 (d, J ¼ 6.9 Hz, 3H), 1.006 (d, J ¼ 7.2 Hz, 3H),
(s, 1H), 8.441 (d, J ¼ 4.5 Hz, 1H), 8.111 (s, 1H), 7.439 (s, 1H), 7.284 (dd,
J ¼ 7.5, 3.3 Hz, 1H), 4.901 (d, J ¼ 9.6 Hz, 1H), 4.264 (d, J ¼ 7.2 Hz, 1H),
4.203 (d, J ¼ 9.0 Hz, 1H), 3.848 (m, 3H), 3.583 (s, 1H), 3.519 (m, 1H),
2.856 (s, 3H), 2.278 (s, 6H), 1.468 (s, 3H), 1.111 (d, J ¼ 6.9 Hz, 3H),
1.029 (d, J ¼ 6.6 Hz, 3H), 0.833 (t, J ¼ 7.2 Hz, 3H). 13C NMR (75 MHz,
0.829 (t, J ¼ 7.2 Hz, 3H). 13C NMR (75 MHz, CDCl3):
d 215.94, 203.72,
CDCl3):
d
216.72, 203.54, 169.91, 157.78, 147.64, 146.25, 123.55,
169.47, 164.66, 157.13, 134.15, 128.58, 127.95, 126.33, 112.11, 103.80,
82.16, 79.53, 78.05, 77.18, 70.24, 69.48, 65.87, 60.56, 51.14, 49.85,
47.51, 44.82, 42.83, 40.20, 39.48, 38.98, 31.77, 29.23, 28.22, 26.94,
22.20, 21.12, 19.66,18.31, 15.72, 14.60, 14.31, 13.84, 10.34. HRMS (ESI)
m/z found 846.3976, calcd for C43H64N3O10S2 (M þ H)þ 846.4027.
103.85, 82.85, 79.46, 78.16, 77.18, 70.24, 69.49, 65.84, 61.20, 51.14,
49.69, 47.63, 45.88, 44.96, 42.46, 40.15, 39.53, 38.90, 33.35, 29.64,
29.23, 28.28, 27.03, 22.19, 21.12, 19.67, 18.21, 15.90, 14.30, 13.93,
10.38. HRMS (ESI) m/z found 830.4305, calcd for C42H64N5O10
S
(M þ H)þ 830.4368.
4.4.16. 11,12-Didehydro-3-O-descladinosyl-6-O-methyl-3-oxo-
10,11-dehydrate-12-((3-(benzoxazole-2-mercapto)propyl)
oxycarbonylimino) erythromycin A (10a)
4.4.12. 11,12-Didehydro-3-O-descladinosyl-6-O-methyl-3-oxo-
12,11-(oxycarbonyl(3-(5-methoxybenzothiazole-2-mercapto)
propyl) imino) erythromycin A (9l)
Yield: 25%. 1H NMR (300 MHz, CDCl3):
d
7.333 (d, J ¼ 7.5 Hz, 1H),
Yield: 41%. 1H NMR (300 MHz, CDCl3):
d
7.557 (d, J ¼ 9.0 Hz, 1H),
7.222 (d, J ¼ 8.1 Hz, 1H), 7.138 (t, J ¼ 7.5 Hz, 1H), 7.006 (t, J ¼ 7.5 Hz,
1H), 6.763 (s, 1H), 5.648 (dd, J ¼ 9.3, 2.7 Hz, 1H), 5.501 (s, 1H), 4.234
(d, J ¼ 7.5 Hz, 1H), 4.156 (d, J ¼ 9.3 Hz, 1H), 3.730 (q, J ¼ 6.6 Hz, 1H),
3.497 (s, 3H), 2.895 (t, J ¼ 6.6 Hz, 2H), 2.844 (s, 3H), 2.269 (s, 6H),
1.967 (t, J ¼ 6.3 Hz, 2H), 1.868 (s, 3H), 1.668 (s, 3H), 1.170 (d,
J ¼ 6.6 Hz, 3H), 0.901 (t, J ¼ 7.2 Hz, 3H). 13C NMR (75 MHz, CDCl3):
7.376 (d, J ¼ 2.1 Hz, 1H), 6.898 (dd, J ¼ 9.0, 2.1 Hz, 1H), 4.928 (d,
J ¼ 9.3 Hz, 1H), 4.262 (d, J ¼ 7.2 Hz, 1H), 4.202 (d, J ¼ 9.0 Hz, 1H),
3.857 (s, 3H), 3.807 (m, 2H), 3.581 (s, 1H), 3.521 (m, 1H), 2.601 (s,
3H), 2.257 (s, 6H), 2.161 (s, 3H), 1.456 (s, 3H), 1.330 (d, J ¼ 6.9 Hz,
3H), 1.145 (d, J ¼ 6.9 Hz, 3H), 1.003 (d, J ¼ 6.9 Hz, 3H), 0.836 (t,
J ¼ 7.2 Hz, 3H). 13C NMR (75 MHz, CDCl3):
d
215.96, 203.69, 169.48,
d 205.18, 203.38, 168.96, 161.82, 148.46, 142.89, 140.13, 137.41,
158.78, 157.13, 154.52, 126.87, 120.97, 113.74, 104.52, 82.17, 79.55,
78.07, 77.20, 70.27, 69.52, 65.83, 60.55, 55.54, 53.75, 51.14, 49.83,
47.54, 44.82, 42.78, 40.18, 39.48, 38.98, 37.50, 30.86, 29.21, 28.14,
26.87, 22.22, 21.11, 19.66, 18.30, 15.75, 14.60, 14.30, 13.81, 10.34.
HRMS (ESI) m/z found 850.3962, calcd for C42H64N3O11S2 (M þ H)þ
850.3982.
123.83, 120.79, 116.26, 108.69, 104.08, 83.41, 78.16, 70.22, 69.40,
65.72, 50.98, 50.50, 47.16, 40.98, 40.20, 38.06, 29.71, 28.36, 27.93,
22.31, 21.04, 20.94, 19.88, 18.45, 14.57, 12.89, 10.41. HRMS (ESI) m/z
found 804.3999, calcd for C41H62N3O11S (M þ H)þ 804.4099.
4.4.17. 11, 12-Didehydro-3-O-descladinosyl-6-O-methyl-3-oxo-
10,11-dehydrate-12-((3-(pyrazolo[3,4d]pyrimidine-4-mercapto)
propyl) oxycarbonylimino) erythromycin A (10b)
4.4.13. 11,12-Didehydro-3-O-descladinosyl-6-O-methyl-3-oxo-
12,11-(oxycarbonyl (3-(1-phenyl-tetrazole-5-mercapto) propyl)
imino) erythromycin A (9m)
Yield: 31%. 1H NMR (300 MHz, CDCl3):
d 8.381 (s, 1H), 8.075 (s,
1H), 6.771 (s, 1H), 5.650 (dd, J ¼ 9.9, 3.3 Hz, 1H), 5.590 (s, 1H), 4.224
(d, J ¼ 7.2 Hz, 1H), 4.151 (d, J ¼ 9.3 Hz, 1H), 3.733 (m, 1H), 3.671 (m,
1H), 3.477 (m, 2H), 3.047 (t, J ¼ 8.1 Hz, 1H), 2.923 (t, J ¼ 6.6 Hz, 2H),
2.266 (s, 6H), 1.869 (s, 3H), 1.436 (s, 3H), 0.898 (t, J ¼ 7.5 Hz, 3H). 13C
Yield: 58%. 1H NMR (300 MHz, CDCl3):
d 7.546 (m, 5H), 4.923 (d,
J ¼ 9.0 Hz, 1H), 4.276 (d, J ¼ 7.2 Hz, 1H), 4.209 (d, J ¼ 8.7 Hz, 1H),
3.822 (q, J ¼ 6.9 Hz, 1H), 3.720 (m, 2H), 3.548 (s, 1H), 3.206 (dd,

X.-z. Chen et al. / European Journal of Medicinal Chemistry 46 (2011) 208e217
217
NMR (75 MHz, CDCl3):
d
205.90, 169.24, 155.87, 140.22, 137.69,
References
135.14, 132.38, 124.96, 103.10, 83.38, 78.34, 69.63, 68.47, 65.92,
63.28, 50.90, 50.32, 47.16, 34.41, 32.14, 31.85, 30.80, 29.29, 28.41,
26.33, 23.35, 22.62, 22.17, 21.09, 20.72, 19.75, 18.73, 14.50, 13.00,
[1] D. Hoban, F. Baquero, V. Reed, D. Felmingham, Int. J. Infect. Dis. 9 (2005)
262e273.
[2] G.G. Zhanel, M. Walters, A. Noreddin, L.M. Vercaigne, A. Wierzbowski,
J.M. Embil, A.S. Gin, S. Douthwaite, D.J. Hoban, Drugs 62 (2002) 1771e1804.
[3] T.M. File Jr., J. Allergy Clin. Immunol. 115 (2005) S361e370.
[4] DailyDrugNews.com, May 28, 2010.
10.41. HRMS (ESI) m/z found 805.4135, calcd for C39H61N6O10
S
(M þ H)þ 805.4164.
[5] A.M. Nilius, Z. Ma, Curr. Opin. Pharmacol. 2 (2002) 493e500.
[6] S. Douthwaite, L.H. Hansen, P. Mauvais, Mol. Microbiol. 36 (2000) 183e193.
[7] S. Bolesta, B.P. Roslund, Am. J. Health-Syst. Ph 65 (2008) 37e41.
[8] P. Pfister, S. Jenni, J. Poehlsgaard, A. Thomas, S. Douthwaite, N. Ban,
E.C. Bottger, J. Mol. Biol. 342 (2004) 1569e1581.
4.4.18. 11,12-Didehydro-3-O-descladinosyl-6-O-methyl-3-oxo-
10,11-dehydrate-12-((3-(5-nitrobenzimidazole-2-mercapto)propyl)
oxycarbonylimino) erythromycin A (10c)
Yield: 20%. 1H NMR (300 MHz, CDCl3):
d 8.121 (s, 1H), 8.016 (d,
[9] DailyDrugNews.com, April 23, 2010.
[10] T.V. Magee, J. Med. Chem. 52 (2009) 7446e7457.
J ¼ 8.7 Hz, 1H), 7.276 (d, J ¼ 8.7 Hz, 1H), 6.727 (s, 1H), 5.861 (br s,
1H), 5.665 (dd, J ¼ 9.9, 2.1 Hz, 1H), 4.280 (d, J ¼ 7.2 Hz, 1H), 4.150 (d,
J ¼ 9.3 Hz, 1H), 3.891 (m, 1H), 3.184 (t, J ¼ 7.5 Hz, 2H), 3.041 (t,
J ¼ 6.9 Hz, 1H), 2.629 (s, 3H), 2.265 (s, 6H), 1.152 (d, J ¼ 6.9 Hz, 3H),
0.902 (t, J ¼ 6.0 Hz, 3H). HRMS (ESI): m/z found 848.4071, calcd for
C41H62N5O12S (M þ H)þ 848.4110.
[11] P. Xu, L. Liu, X.Z. Chen, Y. Li, J. Liu, Z.P. Jin, G.Q. Wang, P.S. Lei, Bioorg. Med.
Chem. Lett. 19 (2009) 4079e4083.
[12] D. Hunziker, P.C. Wyss, P. Angehrn, A. Mueller, H.P. Marty, R. Halm,
L. Kellenberger, V. Bitsch, G. Biringer, W. Arnold, A. StVampfli, A.S. Hoffmann,
D. Cousot, Bioorgan. Med. Chem. 12 (2004) 3503e3519.
[13] M. Delaforge, P. Ladam, G. Bouillé, J.G. Benarous, M. Jaouen, J.P. Girault, Chem.
Biol. Interact. 85 (1992) 215e227.
[14] X. Wei, Q.D. You, Org. Process Res. Dev. 10 (2006) 446e449.
[15] L. Liu, P. Xu, L. Zhou, P.S. Lei, Chin. Chem. Lett. 19 (2008) 1e4.
[16] W.R. Baker, J.D. Clark, R.L. Stephens, K.H. Kim, J. Org. Chem. 53 (1988)
2340e2345.
Acknowledgment
[17] National Committee for Clinical Laboratory Standards, Methods for Dilution
Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically, fifth ed.,
vol. 20, (2000) Approved Standard: NCCLS Document M7-A5.
[18] Frank Schlünzen, Jörg M. Harms, Francois Franceschi, Harly A.S. Hansen,
Heike Bartels, Raz Zarivach, Ada Yonath, Structure 11 (2003) 329e338.
This work was supported by the National S&T Major Special
Project on Major New Drug Innovation (Item Number:
2008ZX09401-004 and 2009ZX09301-003) and National Natural
Science Foundation of China (81072524).