Synthesis and antibacterial evaluation of a series of 11,12-cyclic carbonate azithromycin-3-O-descladinosyl-3-O-carbamoyl glycosyl derivatives

Article abstract of DOI:10.3390/molecules22122146

A novel series of 11,12-cyclic carbonate azithromycin-3-O-descladinosyl-3-O-carbamoyl glycosyl derivatives were designed, synthesized, and evaluated for their antibacterial activities in vitro. Most of these compounds had significant antibacterial activity against seven kinds of susceptible strains. In particular, compound G1 exhibited the most potent activity against methicillin-resistant Streptococcus pneumoniae 943 (MIC: 1 μg/mL), Staphylococcus pneumoniae 746 (MIC: 2 μg/mL), Streptococcus pyogenes 447 (MIC: 8 μg/mL), and Escherichia coli 236 (MIC: 32 μg/mL), which were two-, four-, four-, four-, and eight-fold stronger activity than azithromycin, respectively. Additionally, compound G2 exhibited improved activity against methicillin-resistant Staphylococcus aureus MRSA-1 (MIC: 8 μg/mL), Streptococcus pneumoniae 943 (MIC: 2 μg/mL), Staphylococcus pneumoniae 746 (MIC: 2 μg/mL), and Escherichia coli 236 (MIC: 32 μg/mL), which were two-, two-, four-, and eight-fold better activity than azithromycin, respectively. As for methicillin-resistant Staphylococcus aureus MRSA-1, compound G6 presented the most excellent activity (MIC: 4 μg/mL), showing four-fold higher activity than azithromycin (MIC: 16 μg/mL) and erythromycin (MIC: 16 μg/mL). However, compared with other compounds, compounds G7 and G8 with the disaccharide side chain were observed the lower activity against seven strains.

Full text of DOI:10.3390/molecules22122146

molecules
Article
Synthesis and Antibacterial Evaluation of a Series of
1,12-Cyclic Carbonate Azithromycin-3-O-
descladinosyl-3-O-carbamoyl Glycosyl Derivatives
1
1
1
1,2
2,
1,
Chao-Ming Wang , Feng-Lan Zhao , Lei Zhang , Xiao-Yun Chai * and Qing-Guo Meng *
1
School of Pharmacy, Key Laboratory of Molecular Pharmacology and Drug Evaluation, Ministry of Education,
Collaborative Innovation Center of Advanced Drug Delivery System and Biotech Drugs in Universities of
Shandong, Yantai University, 30 Qingquan Road, Yantai 264005, China;
wangchaoming315@163.com (C.-M.W.); zfl@ytu.edu.cn (F.-L.Z.); fuben19911221@163.com (L.Z.)
Department of Organic Chemistry, College of Pharmacy, Second Military Medical University,
2
3
25 Guohe Road, Shanghai 200433, China
*
Correspondence: chaixy1207@163.com (X.-Y.C.); qinggmeng@163.com (Q.-G.M.);
Tel./Fax: +86-535-670-6022 (Q.-G.M.)
Received: 2 November 2017; Accepted: 27 November 2017; Published: 4 December 2017
Abstract: A novel series of 11,12-cyclic carbonate azithromycin-3-O-descladinosyl-3-O-carbamoyl
glycosyl derivatives were designed, synthesized, and evaluated for their antibacterial activities in vitro
Most of these compounds had significant antibacterial activity against seven kinds of susceptible
strains. In particular, compound G1 exhibited the most potent activity against methicillin-resistant
.
Streptococcus pneumoniae 943 (MIC: 1
µg/mL), Staphylococcus pneumoniae 746 (MIC: 2
µg/mL),
Streptococcus pyogenes 447 (MIC: 8 g/mL), and Escherichia coli 236 (MIC: 32
µ
µg/mL), which were
two-, four-, four-, four-, and eight-fold stronger activity than azithromycin, respectively. Additionally,
compound G2 exhibited improved activity against methicillin-resistant Staphylococcus aureus MRSA-1
(
(
MIC: 8
MIC: 2 µg/mL), and Escherichia coli 236 (MIC: 32 µg/mL), which were two-, two-, four-, and eight-fold
better activity than azithromycin, respectively. As for methicillin-resistant Staphylococcus aureus MRSA-1,
compound G6 presented the most excellent activity (MIC: 4 g/mL), showing four-fold higher activity
than azithromycin (MIC: 16 g/mL) and erythromycin (MIC: 16 g/mL). However, compared with
µg/mL), Streptococcus pneumoniae 943 (MIC: 2 µg/mL), Staphylococcus pneumoniae 746
µ
µ
µ
other compounds, compounds G7 and G8 with the disaccharide side chain were observed the lower
activity against seven strains.
Keywords: azithromycin; glycosyl derivatives; synthesis; antibacterial activity
1
. Introduction
Macrolide antibiotics are polyketides composed of a 14-, 15-, or 16-membered macrocyclic lactone
ring to which several sugars and/or side chains have been attached. They exhibit potent activity against
human pathogens in clinical treatment of respiratory tract, genital, and skin infections, inhibiting
bacterial protein synthesis by blocking the formation of peptide bond [
the broad use of macrolide antibiotics has led to bacterial resistance that can even increase the likelihood
of death from infection by common pathogens in developed and developing countries alike [ ].
1,2]. In the past five decades,
3–5
Therefore, it is still urgently necessary to seek and extend efficacious and safe chemotherapeutic agents
to improve present situation. As is known to all, the most widespread approach with combating the
resistance is to continue modifying existing classes of antibacterial agents to design and synthesize
novel analogues.
Commonly-used clinical macrolide antibiotics include erythromycin, clarithromycin,
and azithromycin (Figure 1). The 14-membered macrolide erythromycin has been widely used for
Molecules 2017, 22, 2146; doi:10.3390/molecules22122146
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over five decades, but the extensive emergence of erythromycin resistance is extraordinarily high [6].
The 15-membered macrolide azithromycin, which is prepared from erythromycin by expanding
semi-synthetically, is characterized by good oral bioavailability, high concentrations in tissues and
fluids, wide spectrum of activity, and superior pharmacokinetic and safety properties in comparison
to erythromycin [
7
–
10]. Efforts to find macrolides that were active against macrolide-resistant
13].
strains led to the modifications of C-3, C-5, C-6 and C11/C12 positions of the azithromycin [11
–
Additionally, 9a-N substituted 15-membered azalides with amide and amine functionalities
also had marked advantages over azithromycin in terms of high selectivity for the bacteria and
moderate oral bioavailability in vivo [9, –16]. Besides, with intensive research of azithromycin,
14
a novel modified measure of the 4 -position of the cladinose was potent against emergence of
drug resistance [17 21]. To resolve these therapeutic problems of tolerance, we investigated and
0
0
–
found that the 3-O-acylerythromycin derivatives, which were named ‘acylides’, not only recovered
the abolished antibacterial activity but also enhanced pharmacological activity against resistant
pathogens [22]. There have been few reports about the other glycosylation modification of the
3-O-position. Our previous studies have found that the hydroxy group of the glycosyl moiety was
significant for hydrogen bond formation, greatly improving soluble in water [19]. Compound F1
,
0
0
the 4 -position of the cladinose modified with glucosyl group, was the best active glycosylated
compound we reported (Figure 2). It showed improved activity against all the tested bacterial strains
relative to the positive control erythromycin, even showed the same or higher antibacterial activities
as azithromycin. Besides, we also found that compounds with disaccharide side chains expressed
the lowest activity. We deduced that the monosaccharide side chain may help the target compounds
fit into the binding pocket and interact with the bacterial ribosome, while the bulky disaccharide
side chain might hinder the interactions of the derivative with the bacterial ribosome. Therefore,
the group which connected to 3-O-position was very important for the activity. Considering these
aspects, we hydrolyzed the cladinose of azithromycin and introduced different glycosyl moieties to
the 3-O-position to examine the antibacterial activities of the derivatives in vitro and discussed the
structure–activity relationship.
Figure 1. Chemical structure of macrolide antibiotics.
Figure 2. Chemical structure of compound F1.

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2
. Results and Discussions
2
.1. Chemistry
The eight glycosylated intermediate compounds 1f
–
8f were prepared from inexpensive and
available carbohydrates with a series of reported synthetic methods (Figure 3) [19]. The general
synthetic route of the target compounds G1 G8 was shown in Scheme 1. First, to remove
–
the L-cladinose, azithromycin was stirred with aqueous HCl and compound
B was obtained.
0
Then, the 2 -hydroxyl group of compound
B
was protected with acetic anhydride to obtain compound
was prepared by treatment of with
Et N and CDI in toluene [23]. NaH and DMF were abandoned in this reaction, greatly improving yield
C. 11,12-Cyclic carbonate azithromycin 3-O-acylimidazolide
D
C
3
in contrast to previous studies [19]. The compounds E1
saccharide group in the presence of DBU. The intermediates, E1
F1 F8 by Pd/CaCO under H . Finally, the target compounds G1
of the 2 -O-acetyl group. Structures of all the objective compounds and intermediates were determined
–
E8 were obtained by benzyl protection of the
E8, were converted to compounds
G8 were prepared by deprotection
–
–
–
3
2
0
1
13
by H-NMR, C-NMR, and ESI-MS.
Figure 3. Structure of the intermediates 1f–8f.
Scheme 1. The synthesis of the target compounds G1
r.t., 5 h, in 91% yield; ( ) Ac O, DCM, Et N, r.t., 20 h, in 88% yield; (
in 93% yield; ( 1f 8f, DBU, DMF, r.t., 24 h, in 17–25% yield; (
f) CH OH, 55 C, 20 h, 62–75% yield.
–
G8. Reactions and conditions: (
a) HCl/H O,
2
b
c
) CDI, Et N, toluene, reflux, 48 h,
2
3
3
d
)
–
e) Pd/C, H , MeOH, r.t., in 70–80% yield;
2
◦
(
3

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2
.2. Antibacterial Activity
The in vitro antibacterial activities were reported as minimum inhibitory concentrations (MICs),
which were determined using a standard dilution assay as recommended by the CLSI [24].
2
.3. Biological Evaluation
The results of the assays are summarized in Table 1. The data points express the mean of replicate
experiments. All of our susceptibility tests were performed three times using each antibacterial agent.
The antibacterial activities indicated that 11,12-cyclic carbonate azithromycin-3-O-
descladinosyl-3-O-carbamoyl glycosyl derivatives exhibited apparent activity against seven
strains compared with azithromycin and erythromycin. Noticeably, all the title compounds exhibited
more potent against S. aureus and S. pneumoniae than the other strains. The MIC value indicated
that the compounds G1 and G2 showed excellent activity against the other compounds, even more
potent than the positive control erythromycin and azithromycin. The MIC value of compound
G1 and G2 (2
µ
g/mL) was eight times lower than that of the positive control erythromycin
g/mL) against S. aureus MSSA-1 in vitro and even the same as the positive control azithromycin
g/mL). In particular, compound G1 exhibited the most potent activity against methicillin-resistant
g/mL), S. pneumoniae 746 (2 g/mL), S. pyogenes 447 (8 g/mL), and E. coli 236
g/mL), which were two-, four-, four-, four-, and eight-fold better activity than azithromycin,
respectively. Additionally, compound G2 exhibited improved activity against methicillin-resistant
S. aureus MRSA-1 (8 g/mL), S. pneumoniae 943 (2 g/mL), S. pneumoniae 746 (2 g/mL), and E. coli
36 (32 g/mL), which were two-, two-, four-, and eight-fold better activity than azithromycin,
respectively. The compounds G3 G6 showed slightly higher activities than the positive control
(
(
16 µ
2
µ
S. pneumoniae 943 (1
32
µ
µ
µ
(
µ
µ
µ
µ
2
µ
–
erythromycin and azithromycin, while compounds G7 and G8 presented the slightly less activities
according to the MIC values. As for methicillin-resistant S. aureus MRSA-1, compound G6 presented
the most excellent activity (4
µg/mL), showing four- and four-fold higher activity than azithromycin
(16 µg/mL) and erythromycin (16 µg/mL). However, compared with other compounds, compounds
G7 and G8 with the disaccharide side chain were observed the lower activity against seven strains.
Activity of the tested compounds was either maintained or slightly improved against E. coli 236 and
E. coli ATCC 25922. Unfortunately, all compounds exhibited similar or only slightly better activity than
erythromycin and azithromycin against S. pyogenes 447, E. coli 236, and E. coli ATCC 25922.
Table 1. Antibacterial activities of the title compounds in vitro (MIC, µg/mL).
S. aureus
MSSA-1
S. aureus
MRSA-1
S. pneumoniae
S. pneumoniae
S. pyogenes
E. coli
236
E. coli ATCC
Compd.
943
746
447
25922
G1
G2
G3
G4
G5
G6
G7
G8
Azi
Ery
2
2
4
8
4
2
8
16
2
16
8
8
8
8
8
1
2
8
4
4
2
2
4
8
32
32
32
32
32
32
32
64
64
128
128
128
256
128
128
256
256
256
256
8
16
16
32
16
8
4
8
64
64
16
16
16
16
32
16
4
128
128
32
128
256
256
256
8
16
32
Abbreviations: Azi, azithromycin; Ery, erythromycin.
0
0
Compared with the 11,12-cyclic carbonate azithromycin-4 -O-carbamoyl glycosyl derivatives we
reported in the past [19], the title compounds displayed more potent activity against S. aureus MSSA-1
and S. aureus MRSA-1. Most of them exhibited 2–4 fold higher activity than that before. As for the
other five strains, this series did not show notably improved antibacterial activity.

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3
. Materials and Methods
3
.1. Chemistry
All reactions were monitored by thin-layer chromatography (Huanghai, Yantai, China). A part of
each compound was visualized by UV light (Yukang, Shanghai, China). NMR spectra were recorded
1
13
on a Bruker Avance II 600 spectrometer at 600 MHz for H-NMR and 150 MHz for C-NMR with TMS
as the internal standard. Chemical shifts are expressed in (ppm). The HPLC-MS were recorded on
δ
Agilent 1100 series LC/MS. Column chromatography was carried out using silica gel (300–400 mesh,
Huanghai, Yantai, China). Silica gel chromatography solvents were of analytical grade (Huanghai,
Yantai, China). All reaction solvents were dried prior to use according to standard procedures (Taitan,
Shanghai, China).
3
.1.1. 3-O-descladinosyl Azithromycin (B)
We added 36% aqueous HCl dropwise to a solution of azithromycin (10 g, 13 mmol) in water
(
60 mL) at room temperature until the reaction mixture was adjusted to pH = 1.0–2.0. The resulting
solution was allowed to stir for 5 h at the same temperature, and then it was adjusted to pH = 9
with 25% aqueous NH . The precipitate was collected by filtration, and washed with cold water,
3
crystallized from acetone to afford 7.17 g (91%) of 3-O-descladinosyazithromycin (
M.p. 139–141 C.
B) as white solid,
◦
0
3
.1.2. 2 -O-Acetyl-3-O-descladinosyl Azithromycin (C)
Ac O (2.3 mL, 49 mmol) and Et N (6.7 mL, 49 mmol) were added to a solution of
2
3
3
-O-descladinosylazithromycin (7.17 g, 12 mmol) prepared above in CH Cl (80 mL) at room
2
2
temperature. The resulting solution was allowed to stir for 24 h at the same temperature. The reaction
was quenched with saturated NaHCO (60 mL) and the aqueous layer was extracted with CH Cl
3
2
2
(
2
×
10 mL). The combined organic layers were washed with water and brine, then dried over
anhydrous Na SO . The organic layer was filtered and concentrated in vacuum to afford 6.77 g (88%)
of 2 -O-acetyl-3-O-descladinosy azithromycin (C) as white solid, M.p. 143–145 C.
2
4
0
◦
0
3
.1.3. 2 -O-Acetyl-3-O-acylimidazolyl-3-O-descladinosyl Azithromycin-11,12-Cyclic Carbonate (D)
Et N (2.2 mL, 14 mmol) and CDI (2.27 g, 14 mmol) were added to a solution of
3
0
2
-O-acetyl-3-descladinosy azithromycin (
C, 3.59 g, 5.6 mmol) in toluene (30 mL) . The resulting
◦
solution was stirred for 48 h at 80 C.The reaction was quenched with saturated NaHCO (30 mL) and
3
the aqueous layer was extracted with toluene (2
×
15 mL). The combined organic layers were dried
over anhydrous Na SO , filtered, and concentrated in a vacuum to afford 4.21 g (93%) of compound
D
2
4
◦
as white solid, M.p. 149–151 C.
0
3
.1.4. 2 -O-Acetyl-3-O-amido-[ethyl-2-(2,3,4,6-tetra-O-benzyl-β-D-glucopyranoside)]-3-O-
descladinosyl Azithromycin-11,12-Cyclic Carbonate (E1)
DBU (0.15 mL, 1.03 mmol) and 1f (0.47 g, 0.79 mmol) were added to a solution of
D
(0.4 g,
0
.53 mmol) in DMF (15 mL). The resulting solution was stirred for 24 h at the room temperature.
The reaction was quenched with saturated NaHCO (30 mL) and the aqueous layer was extracted with
3
ethyl acetate (2
×
15 mL). The combined organic layers were washed with water and brine, and dried
over NaSO . After filtering and concentrating the reaction solution in vacuum to afford the crude
4
compound E1 (0.21 g) as white solid. The syntheses of other intermediates (E2–E8) are carried out
according to the above conditions.

Molecules 2017, 22, 2146
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0
3
.1.5. 2 -O-Acetyl-3-O-amido-(ethyl-2-β-D-glucopyranoside)-3-O-descladinosyl
Azithromycin-11,12-Cyclic Carbonate (F1)
A solution of E1 (140 mg, 0.11 mmol) Pd/C (50 mg) in methanol (15 mL) was adjusted to pH = 4–5
by acetic acid and stirred for 24 h under an atmosphere of hydrogen at the room temperature. After,
filtering and concentrating the reaction solution in vacuum afforded the crude compound F1 as white
solid. The syntheses of other intermediates (F2–F8) are carried out according to the above conditions.
3
.1.6. General Procedure for the Preparation of Azithromycin Derivatives G1–G8
A solution of the above crude intermediate product in MeOH (15 mL) was heated to 55 C
◦
and stirred for 36 h at the same temperature. After concentrating the reaction solution in vacuum,
the residue was purified by flash chromatography (DCM/MeOH, 4:1, v/v) to afford the title compound.
3
-O-Amido-(ethyl-2-
β-d-glucopyranoside)-3-O-descladinosyl azithromycin-11,12-cyclic carbonate (G1): White
◦
1
solid; Yield: 75%, M.p. 108–110 C; H-NMR (600 MHz, CDCl )
δ
5.17 (1H, s), 5.05–5.04 (1H, m), 4.54
3
(
(
(
2H, m), 4.38–4.36 (1H, m), 4.26–4.19 (2H, m), 3.99–3.97 (1H, m), 3.87 (1H, t), 3.82 H, m), 3.72–3.70
1H, m), 3.68 (1H, d, J = 1.8 Hz), 3.66 (1H, d, J = 5.4 Hz) 3.64–3.61 (1H, m), 3.60 (1H, m), 3.57–3.55
2H, m), 3.36 (2H, m), 3.27–3.23 (1H, m), 2.96 (1H, m), 2.93 (2H, m), 2.88–2.82 (2H, m), 2.67–2.62 (1H, m),
2
(
7
3
.34 (3H, s), 2.52–2.49 (2H, m), 2.25 (9H, m), 1.98 (3H, m), 1.92–1.86 (6H, m), 1.31–1.26 (5H, m), 1.25–1.23
7H, m), 0.94–0.90 (9H, m); 13C-NMR (150 MHz, CDCl3)
176.7, 170.1, 153.7, l03.6, 100.2, 95.6, 85.9,
9.7, 76.8, 76.7, 76.2, 74.7, 73.4, 73.5, 70.8, 70.3, 68.8, 67.4, 65.3, 63.2, 61.4, 61.1, 50.4, 45.5, 44.2, 41.6, 40.4,
5.7, 34.2, 29.4, 26.7, 22.9, 22.1, 21.2, 15.1, l4.3, 12.2, 10.4, 5.7; MS (ESI) calcd. for C H N O 907.49;
δ
42
73
3
18
+
found [M + H] 908.51.
3
-O-Amido-(ethyl-2-galactosyl)-3-O-descladinosyl azithromycin-11,12-cyclic carbonate (G2): White solid;
◦
1
Yield: 68%, M.p. 113–115 C; H-NMR (600 MHz, CDCl )
δ
5.40 (1H, d, J = 3.6 Hz), 5.11 (1H, m), 5.02
3
(
1H, d, J = 10.5 Hz), 4.50–4.39 (2H, m), 4.34–4.21 (2H, m), 4.33–4.21 (2H, m), 4.18 (1H, d, J = 11.4 Hz),
4
2
.12 (1H, m), 4.03 (1H, m), 3.91 (2H, m), 3.70 (2H, m), 3.68–3.54 (2H, m), 3.30 (2H, m), 3.27–3.22 (1H, m),
.88–2.82 (1H, m), 2.69–2.60 (2H, m), 2.53–2.43 (1H, m), 2.37 (2H, m), 2.25 (9H, m), 2.21 (3H, s), 2.19
(
1H, m), 1.91–1.85 (2H, m), 1.68–1.63 (4H, m), 1.45 (3H, m), 1.31–1.23 (4H, m), 1.06–1.01 (9H, m),
0
7
3
.94–0.90 (9H, m); 13C-NMR (150 MHz, CDCl3)
6.8, 74.7, 74.2, 73.9, 73.7, 73.2, 71.1, 70.3, 70.1, 68.1, 65.5, 62.4, 61.7, 51.2, 45.3, 42.4, 42.1, 40.2, 36.1,
5.2, 29.1, 27.5, 26.8, 22.1, 21.1, 16.4, 14.7, 11.3, 9.2, 5.4; MS (ESI) calcd. for C H N O 907.49; found
δ 178.5, 171.1, 153.7, l03.2, 100.7, 94.9, 83.3, 78.4, 77.3,
42
73
3
18
+
[
M + H] 908.51.
3
-O-Amido-(ethyl-2-mannosyl)-3-O-descladinosyl azithromycin-11,12-cyclic carbonate (G3): White solid;
◦
1
Yield: 64%, M.p. 114–116 C; H-NMR (600 MHz, CDCl )
δ
5.35 (1H, m), 5.04 (1H, m), 4.88 (1H, d,
3
J = 3.0 Hz), 4.60 (1H, s), 4.55 (1H, m), 4.40 (1H, d, J = 4.2 Hz), 4.30 (1H, d, J = 12 Hz), 4.13 (1H, m), 4.04
(
(
(
(
7
2
1H, m), 3.95 (1H, m), 3.88 (2H, m), 3.82–3.80 (1H, m), 3.68 (2H, m), 3.66 (2H, m), 3.56 (1H, m), 3.47
2H, m), 3.36–3.35 (2H, m), 3.00–2.95 (3H, m), 2.83 (4H, m), 2.68–2.60 (1H, m), 2.50–2.47 (1H, m), 2.34
3H, s), 2.25 (6H, m), 2.19 (2H, m), 1.89 (2H, m), 1.66 (2H, m), 1.45–1.42 (5H, m), 1.23 (1H, m), 1.07–1.00
12H, m), 0.94–0.89 (9H, m); 13C-NMR (150 MHz, CDCl3)
δ
177.6, 171.4, 157.6, 102.3, 101.6, 95.1, 83.7,
8.4, 76.5, 74.4, 74.1, 73.9, 73.2, 70.8, 70.1, 67.8, 65.5, 62.6, 62.1, 50.3, 45.2, 42.1, 40.3, 36.1, 35.1, 29.7, 27.3,
6.6, 21.9, 21.4, 21.2, 20.8, l6.2, l4.4, 11.5, 9.4, 5.3. MS (ESI) calcd. for C H N O 907.49; found [M +
42
73
3
18
+
H] 908.51.
3
7
-O-Amido-(ethyl-2-ribosyl)-3-O-descladinosyl azithromycin-11,12-cyclic carbonate (G4): White solid; Yield:
◦
1%, M.p. 105–107 C; H-NMR (600 MHz, CDCl ) δ 5.16 (1H, d, J = 4.8 Hz), 5.05–5.04 (1H, m), 4.54
3
1
(2H, m), 4.38–4.36 (1H, m), 4.26–4.19 (2H, m), 3.99–3.97 (1H, m), 3.72–3.70 (1H, m), 3.64–3.61 (1H, m),
3.59 (2H, m), 3.57–3.55 (2H, m), 3.53 (1H, d, J = 1.8 Hz), 3.51 (1H, d, J = 5.4 Hz), 3.47 (1H, m), 3.36
(
(
(
2H, m), 3.27–3.23 (1H, m), 2.96 (1H, m), 2.91 (2H, m), 2.88–2.82 (2H, m), 2.67–2.62 (1H, m), 2.52–2.49
2H, m), 2.25 (9H, m), 1.98 (3H, m), 1.92–1.86 (6H, m), 1.31–1.26 (5H, m), 1.25–1.23 (7H, m), 0.94–0.90
9H, m); 13C-NMR (150 MHz, CDCl ) δ 177.1, 153.4, 153.2, 103.5, 100.4, 95.6, 85.7, 79.6, 76.7, 76.4, 76.1,
3

Molecules 2017, 22, 2146
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7
5.1, 74.2, 73.6, 70.6, 68.2, 67.4, 64.8, 62.4, 61.1, 51.4, 45.3, 43.4, 40.0, 35.3, 34.3, 30.l, 26.7, 23.2, 22.0, 21.2,
+
1
5.1, 14.4, 11.3, 10.4, 5.8. MS (ESI) calcd.for C H N O 835.47; found [M + H] 836.54.
39 69 3 16
3
6
-O-Amido-(ethyl-2-xylosyl)-3-O-descladinosyl azithromycin-11,12-cyclic carbonate (G5): White solid; Yield:
◦
5%, M.p. 107–109 C; H-NMR (600 MHz, CDCl ) δ 5.21(1H, d, J = 4.8 Hz), 5.11 (1H, m), 4.50–4.39
3
1
(
2H, m), 4.34–4.21 (2H, m), 4.33–4.21 (2H, m), 3.83 (1H, d, J = 12 Hz), 3.77 (2H, m), 3.74 (1H, d,
J = 1.8 Hz), 3.71 (1H, d, J = 5.4 Hz), 3.68–3.54 (2H, m), 3.47 (2H, m), 3.27–3.22 (1H, m), 3.15(2H, m),
.88–2.82 (1H, m), 2.69–2.60 (2H, m), 2.53–2.43 (1H, m), 2.37 (2H, m), 2.25 (9H, m), 2.19 (1H, m),
.91–1.85 (2H, m), 1.68–1.63 (4H, m), 1.45 (3H, m), 1.31–1.23 (4H, m), 1.06–1.01 (9H, m), 0.94–0.90
9H, m); 13C-NMR (150 MHz, CDCl ) 177.7, 153.4, 103.2, 101.2, 95.1, 83.6, 78.5, 75.7, 75.4, 74.4, 74.1, 73.5,
2
1
(
3
7
9
1.2, 70.3, 69.4, 68.3, 65.7, 62.3, 51.1, 45.1, 42.6, 40.4, 36.1, 35.4, 29.2, 27.5, 26.8, 21.9, 21.2, 16.2, 14.7, 11.3,
+
.1, 5.4; MS (ESI) calcd. for C H N O 835.47; found [M + H] 836.54.
39
69
3
16
3
-O-Amido-(ethyl-2-rhamnosyl)-3-O-descladinosyl azithromycin-11,12-cyclic carbonate (G6): White solid;
◦
1
Yield: 70%, M.p. 110–112 C; H-NMR (600 MHz, CDCl )
δ
5.55 (1H, s), 5.03 (1H, m), 4.62–4.60 (2H, m),
.44 (1H, m), 3.79 (1H, m), 3.65–3.55 (2H, m), 3.57 (2H, m), 3.53 (1H, d, J = 1.8 Hz), 3.52–3.44 (2H, m),
.42 (1H, d, J = 5.4 Hz), 3.33 (1H, m), 3.28–3.25 (3H, m), 2.49 (1H, m), 2.39–2.37 (1H, m), 2.32–2.20
3
4
3
(
1
1
3
8
10H, m), 2.18 (3H, m),1.91–1.86 (1H, m), 1.77–1.67 (4H, m), 1.48–1.43 (4H, m), 1.30–1.24 (4H, m),
13
.04–1.00 (9H, m), 0.96–0.89 (9H, m); C-NMR (150 MHz, CDCl3)
03.1, 101.3, 95.6, 85.9, 79.6, 76.6, 76.1, 74.7, 73.5, 73.3, 71.2, 68.4, 67.5, 64.8, 61.2, 45.1, 43.4, 42.6, 40.2,
5.3, 34.2, 30.1, 26.8, 22.9, 22.0, 21.2, 16.7, l 5.1, l 4.2, l1.4, 10.3, 5.7; MS (ESI) calcd. for C H N O
δ 177.2, 171.3, 153.4, 145.5, 133.6,
42
73
3
17
+
91.49; found [M + H] 892.46.
3
-O-Amido-(ethyl-2-maltosyl)-3-O-descladinosyl azithromycin-11,12-cyclic carbonate (G7): White solid;
◦
1
Yield: 62%, M.p. 117–119 C; H-NMR (600 MHz, CDCl )
δ
5.41 (1H, d, J = 4.2 Hz), 5.03 (1H, m), 4.85
3
(2H, m), 4.62–4.60 (2H, m), 4.62 (3H, m), 4.53 (2H, m), 4.44 (1H, m), 3.96 (5H, m), 3.65–3.55 (2H, m),
3
.33 (1H, m), 3.30 (2H, m), 3.28–3.25 (3H, m), 2.49 (1H, m), 2.39–2.37 (1H, m), 2.32–2.20 (10H, m), 2.14
(
3H, m), 1.91–1.86 (1H, m), 1.77–1.67 (4H, m), 1.48–1.43 (4H, m), 1.30–1.24 (4H, m), 1.04–1.00 (9H, m),
0
7
2
.96–0.89 (9H, m); 13C-NMR (150 MHz, CDCl3)
6.6, 76.3, 74.5, 74.2, 73.8, 73.9, 71.2, 70.1, 68.1, 65.5, 63.2, 62.4, 62.4, 62.3, 51.5, 45.2, 42.5, 40.3, 36.2, 35.2,
9.1, 27.5, 26.8, 22.1, 21.3, l6.3, 14.8, 11.1, 9.4, 5.2; MS (ESI) calcd. for C H N O 1069.54; found
δ 177.6, 172.3, 153.4, 103.6, 103.1, 101.7, 95.2, 84.1, 78.4,
48
83
3
23
+
[
M + H] 1070.51.
3
6
4
-O-Amido-(ethyl-2-lactosyl)-3-O-descladinosyl azithromycin-11,12-cyclic carbonate (G8): White solid; Yield:
◦
1
3%, M.p. 119–121 C; H-NMR (600 MHz, CDCl )
δ
5.24 (1H, d, J = 4.8 Hz), 5.13 (1H, m), 4.53 (1H, m),
3
.47–4.45 (1H, m), 4.38 (1H, m), 4.34–4.28 (2H, m), 3.92 (2H, d, J = 1.8 Hz), 3.83 (2H, m), 3.71 (2H, d,
J = 10.4 Hz), 3.66 (4H, m), 3.63 (4H, m), 3.55 (2H, m), 3.51–3.47 (2H, m), 3.27–3.22 (2H, m), 3.18 (2H, m),
2
.84–2.82 (1H, m), 2.67–2.63 (1H, m), 2.50–2.45 (1H, m), 2.28–2.20 (7H, m), 2.25–2.23 (6H, m), 2.18 (3H, s),
.01 (1H, m), 1.89 (1H, m), 1.75 (m, 1H), 1.67–1.63 (2H, m), 1.46 (2H, m), 1.25–1.23 (4H, m), 1.05–1.01
2
(
9H, m), 0.94–0.88 (9H, m); 13C-NMR (150 MHz, CDCl3)
δ 177.3, 170.4, l56.7, l53.2, l03.1, 111.2, 101.5,
95.7, 86.1, 85.2, 80.4, 76.5, 76.3, 76.1, 73.5, 73.1, 70.9, 68.5, 67.6, 65.4, 63.1, 62.2, 61.8, 61.2, 53.5, 45.3, 43.5,
4
1.8, 40.3, 35.5, 34.3, 29.6, 26.8, 26.2, 22.1, 21.9, 21.2, 20.7, 20.7, 15.1, l4.1, l1.4, l0.2, 5.7; MS (ESI) calcd. for
+
C H N O 1069.54; found [M + H] 1070.51.
48
83
3
23
3
.2. Antibacterial Activity
The in vitro antibacterial activities were reported as minimum inhibitory concentrations (MICs),
which were determined using a standard dilution assay as recommended by the CLSI [24].
For the assays, the title compounds to be tested were dissolved in dimethyl sulfoxide (DMSO),
◦
serially diluted in growth medium, inoculated and incubated at 35 C. The selected strains evaluated
were methicillin-susceptible Staphylococcus aureus MSSA-1 (S. aureus MSSA-1), methicillin-resistant
Staphylococcus aureus MRSA-1 (S. aureus MRSA-1), Streptococcus pneumoniae 943 (S. pneumoniae 943),
Staphylococcus pneumoniae 746 (S. pneumoniae 746), Streptococcus pyogenes 447 (S. pyogenes 447),

Molecules 2017, 22, 2146
8 of 9
Escherichia coli 236 (E. coli 236), and Escherichia coli ATCC 25922 (E. coli ATCC 25922). Erythromycin and
azithromycin served as the positive control and were obtained from their respective manufacturers.
4
. Conclusions
In summary, in our endeavor to develop promising antibacterial molecules against the strains
based on the naturally occurring azithromycin, we have designed and synthesized a series of related
glycosyl derivatives possessing potent in vitro antibacterial activity. The analysis of the in vitro results
demonstrated that 11,12-cyclic carbonate azithromycin-3-O-descladinosyl-3-O-carbamoyl glycosyl
derivatives expressed improved antibacterial activities compared with the parent compound. Above all,
the compounds G1–G6 with monosaccharide side chain could be suitable to inhibit bacteria protein
synthesis in the bacterial ribosome, while compounds G7 and G8 with disaccharide side chain exhibited
the least activity, which could be due to the long side chain not binding well to the ribosomal RNA
of bacteria. Besides, all the compounds showed significantly potent activity (2–16 µg/mL) against
0
0
S. aureus than 11,12-cyclic carbonate azithromycin-4 -O-carbamoyl glycosyl derivatives reported in our
published paper [19]. Maybe the hydrolyzation of L-cladinose is beneficial for activity improvement.
This discovery, as well as further studies on azithromycin glycosyl derivatives, could develop a potent
method to overcome the bacterial strain.
Acknowledgments: We thank Qiuye Wu for valuable scientific discussion. This work was supported by the
National Natural Science Foundation of China (No. 81473104) and Shandong graduate education innovation
program (SDYY16132).
Author Contributions: Chao-Ming Wang and Lei Zhang designed and carried out the experiments.
Chao-Ming Wang and Feng-Lan Zhao wrote the paper. Xiao-Yun Chai and Qing-Guo Meng provided study
design and guidance. All authors have read and approved the final manuscript.
Conflicts of Interest: The authors have declared that no competing interests exist.
References
1
.
Fyfe, C.; Grossman, T.H.; Kerstein, K.; Sutcliffe, J. Resistance to Macrolide Antibiotics in Public Health
Pathogens. Cold Spring Harb. Perspect. Med. 2016, 6, 1–37. [CrossRef] [PubMed]
2
3
.
.
Deane, C. Antibiotic discovery: Macrolides en masse. Nat. Chem. Biol. 2016, 12, 467. [CrossRef]
Doern, G.V.; Heilmann, K.P.; Huynh, H.K.; Rhomberg, P.R.; Coffman, S.L.; Brueggemann, A.B. Antimicrobial
resistance among clinical isolates of Streptococcus pneumoniae in the United States during 1999–2000,
including a comparison of resistance rates since 1994–1995. Antimicrob. Agents Chemother. 2001, 45, 1721–1729.
[CrossRef] [PubMed]
4.
5.
6.
Kamruzzaman, M.; Shoma, S.; Thomas, C.M.; Partridge, S.R.; Iredell, J.R. Plasmid interference for curing
antibiotic resistance plasmids in vivo. PLoS ONE 2017, 12, e0172913. [CrossRef] [PubMed]
Fernandes, P.; Martens, E.; Pereira, D. Nature nurtures the design of new semi-synthetic macrolide antibiotics.
J. Antibiot. 2017, 70, 527–533. [CrossRef] [PubMed]
Song, J.H.; Jung, S.I.; Ko, K.S.; Kim, N.Y.; Son, J.S.; Chang, H.H.; Ki, H.K.; Oh, W.S.; Suh, J.Y.; Peck, K.R.; et al.
High prevalence of antimicrobial resistance among clinical Streptococcus pneumoniae isolates in Asia
(an ANSORP study). Antimicrob. Agents Chemother. 2004, 48, 2101–2107. [CrossRef] [PubMed]
7.
8.
9.
Zhanel, G.G.; Dueck, M.; Hoban, D.J.; Vercaigne, L.M.; Embil, J.M.; Gin, A.S.; Karlowsky, J.A. Review
of macrolides and ketolides: Focus on respiratory tract infections. Drugs 2001, 61, 443–498. [CrossRef]
[PubMed]
Seiple, I.B.; Zhang, Z.Y.; Jakubec, P.; Mercier, A.L.; Wright, P.M.; Hog, D.T.; Yabu, K.; Allu, S.R.; Fukuzaki, T.;
Carlsen, P.N.; et al. A platform for the discovery of new macrolide antibiotics. Nature 2016, 533, 338–345.
[CrossRef] [PubMed]
Kraja cˇ i c´ , M.B.; Peri c´ , M.; Smith, K.S.; Schönfeld, Z.I.; Žiher, D.; Fajdeti c´ , A.; Kujundži c´ , N.; Schönfeld, W.;
Landek, G.; Padovan, J.; et al. Synthesis, structure-activity relationship, and antimalarial activity of ureas
and thioureas of 15-membered azalides. J. Med. Chem. 2011, 54, 3595–3605. [CrossRef] [PubMed]

Molecules 2017, 22, 2146
9 of 9
1
0. Kobayashi, Y.; Wada, H.; Rossios, C.; Takagi, D.; Higaki, M.; Mikura, S.; Goto, H.; Barnes, P.J.; Ito, K. A novel
macrolide solithromycin exerts superior anti-inflammatory effect via NF- B inhibition. J. Pharmacol. Exp. Ther.
013, 345, 76–84. [CrossRef] [PubMed]
1. Champney, W.S.; Tober, C.L. Structure-activity relationships for six ketolide antibiotics. Curr. Microbiol. 2001
2, 203–210. [PubMed]
2. Heggelund, A.; Rømming, C.; Undheim, K. Preparation and antibacterial activity of cyclic 2 ,3 -carbamate
derivatives of azithromycin. Eur. J. Med. Chem. 2008, 43, 1657–1664. [CrossRef] [PubMed]
3. Zhang, L.; Song, L.; Liu, Z.; Li, H.; Lu, Y.; Li, Z.; Ma, S. Synthesis and antibacterial activity of novel
κ
2
1
1
1
,
4
0
0
3-O-carbamoyl derivatives of clarithromycin and 11,12-cyclic carbonate azithromycin. Eur. J. Med. Chem.
2
010, 45, 915–922. [CrossRef] [PubMed]
1
1
4. Bukvi c´ , K.M.; Novak, P.; Dumi c´ , M.; Cindri c´ , M.; Paljetak, H.C.; Kujundzi c´ , N. Novel ureas and thioureas of
5-membered azalides with antibacterial activity against key respiratory pathogens. Eur. J. Med. Chem. 2009
4, 3459–3470. [CrossRef] [PubMed]
1
4
,
5. Peri c´ , M.; Fajdeti c´ , A.; Rup cˇ i c´ , R.; Alihodži c´ , S.; Žiher, D.; Kraja cˇ i c´ , M.B.; Smith, K.S.; Schönfeld, Z.I.;
Padovan, J.; Landek, G.; et al. Antimalarial activity of 9a-N substituted 15-membered azalides with improved
in vitro and in vivo activity over azithromycin. J. Med. Chem. 2012, 55, 1389–1401. [CrossRef] [PubMed]
6. Lee, Y.; Choi, J.Y.; Fu, H.; Harvey, C.; Ravindran, S.; Roush, W.R.; Boothroyd, J.C.; Khosla, C. Chemistry and
biology of macrolide antiparasitic agents. J. Med. Chem. 2011, 54, 2792–2804. [CrossRef] [PubMed]
7. Pavlovi c´ , D.; Mutak, S. Discovery of 4”-ether linked azithromycin-quinolone hybrid series: Influence of the
central linker on the antibacterial activity. ACS Med. Chem. Lett. 2011, 2, 331–336. [CrossRef] [PubMed]
8. Stimac, V.; Škugor, M.M.; Jakopovi c´ , I.P.; Vinter, A.; Ilijaš, M.; Alihodži c´ , S.; Mutak, S. Initial Scale-Up and
Process Improvements for the preparation of a lead antibacterial macrolone compound. Org. Process Res. Dev.
1
1
1
2
010, 14, 1401–1409. [CrossRef]
1
9. Zhang, L.; Chai, X.; Wang, B.; Yu, S.; Hu, H.; Zou, Y.; Zhao, Q.; Meng, Q.; Wu, Q. Design, synthesis and
biological evaluation of azithromycin glycosyl derivatives as potential antibacterial agents. Bioorg. Med.
Chem. Lett. 2013, 23, 5057–5060. [CrossRef] [PubMed]
2
0. Li, X.; Ma, S.T.; Yan, M.; Wang, Y.Z.; Ma, S.T. Synthesis and antibacterial evaluation of novel
11,4”-disubstituted azithromycin analogs with greatly improved activity against erythromycin-resistant
bacteria. Eur. J. Med. Chem. 2013, 59, 209–217. [CrossRef] [PubMed]
2
2
1. Ma, S.T.; Ma, R.X.; Xian, R.Q.; Hao, B. Synthesis of novel 15-membered macrolide dimers. Chin. Chem. Lett.
2
009, 20, 931–934. [CrossRef]
2. Tanikawa, T.; Asaka, T.; Kashimura, M.; Suzuki, K.; Sugiyama, H.; Sato, M.; Kameo, K.;
Morimoto, S.; Nishida, A. Synthesis and antibacterial activity of a novel series of acylides:
3
-O-(3-pyridyl)acetylerythromycin A derivatives. J. Med. Chem. 2003, 46, 2706–2715. [CrossRef] [PubMed]
3. Yan, M.; Ma, R.X.; Jia, L.; Venter, H.; Ma, S.T. Synthesis and antibacterial activity of novel
-O-descladinosylazithromycin derivatives. Eur. J. Med. Chem. 2017, 127, 874–884. [CrossRef] [PubMed]
4. Clinical and Laboratory Standards Institute. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria
that Grow Aerobically; Approved Standard-Eight Edition: CLSI Document M07-A8; Clinical and Laboratory
Standards Institute: Wayne, PA, USA, 2009.
2
2
3
Sample Availability: Samples of the compounds G1–G8 are available from the authors.
©
2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).