A novel series of 11-O-carbamoyl-3-O-descladinosyl clarithromycin derivatives bearing 1,2,3-triazole group: Design, synthesis and antibacterial evaluation

Article abstract of DOI:10.1016/j.bmcl.2019.126850

A series of novel 11-O-carbamoyl-3-O-descladinosyl clarithromycin derivatives bearing the 1,2,3-triazole group were designed, synthesized, and evaluated for their in vitro antibacterial activity. The antibacterial results indicated that most of the target compounds not only increased their activity against resistant bacterial strains, but also partially retained the activity against sensitive bacterial strains compared with clarithromycin. Among them, 13d had the best antibacterial activity against resistant strains, including Streptococcus pneumoniae B1 expressing the ermB gene (16 μg/mL), Streptococcus pneumoniae AB11 expressing the mefA and ermB genes (16 μg/mL) and Streptococcus pyogenes R1 (16 μg/mL), showing >16, 8 and 16-fold higher activity than that of CAM, respectively. Moreover, 13d and 13g exhibited the best antibacterial activity against sensitive bacterial strains, including Staphylococcus aureus ATCC25923 (4 μg/mL) and Bacillus Subtilis ATCC9372 (1 μg/mL). The MBC results showed that the most promising compounds 13d and 13g exhibited antibacterial activity through bacteriostatic mechanism, while the time-kill kinetic experiment revealed bactericidal kinetics of 13g from microscopic point of view. In vitro antibacterial experiments and molecular docking results further confirmed that it was feasible to our initial design strategy by modifying the C-3 and C-11 positions of clarithromycin to increase the activity against resistant bacteria.

Full text of DOI:10.1016/j.bmcl.2019.126850

Journal Pre-proofs
A novel series of 11-O-carbamoyl-3-O-descladinosyl clarithromycin deriva-
tives bearing 1,2,3-triazole group: design, synthesis and antibacterial evaluation
Yuetai Teng, Yinhui Qin, Di Song, Xingbang Liu, Yingang Ma, Panpan Zhang,
Shutao Ma
PII:
S0960-894X(19)30824-8
DOI:
https://doi.org/10.1016/j.bmcl.2019.126850
BMCL 126850
Reference:
Bioorganic & Medicinal Chemistry Letters
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Accepted Date:
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30 October 2019
20 November 2019
Please cite this article as: Teng, Y., Qin, Y., Song, D., Liu, X., Ma, Y., Zhang, P., Ma, S., A novel series of 11-O-
carbamoyl-3-O-descladinosyl clarithromycin derivatives bearing 1,2,3-triazole group: design, synthesis and
antibacterial evaluation, Bioorganic & Medicinal Chemistry Letters (2019), doi: https://doi.org/10.1016/j.bmcl.
2019.126850
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A novel series of 11-O-carbamoyl-3-O-descladinosyl clarithromycin derivatives
bearing 1,2,3-triazole group: design, synthesis and antibacterial evaluation
Yuetai Teng, Yinhui Qin, Di Song, Xingbang Liu, Yingang Ma, Panpan Zhang, Shutao Ma*
Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (Ministry of Education),
School of Pharmaceutical Sciences, Shandong University, 44 West Culture Road, Jinan 250012, China
Running title: 11-O-Carbamoyl-3-O-descladinosyl derivatives
* Corresponding author.
E-mail addresses: mashutao@sdu.edu.cn (S. Ma)
1

Abstract
A series of novel 11-O-carbamoyl-3-O-descladinosyl clarithromycin derivatives bearing the
1,2,3-triazole group were designed, synthesized, and evaluated for their in vitro antibacterial activity.
The antibacterial results indicated that most of the target compounds not only increased their activity
against resistant bacterial strains, but also partially retained the activity against sensitive bacterial
strains compared with clarithromycin. Among them, 13d had the best antibacterial activity against
resistant strains, including Streptococcus pneumoniae B1 expressing the ermB gene (16 µg/mL),
Streptococcus pneumoniae AB11 expressing the mefA and ermB genes (16 µg/mL) and Streptococcus
pyogenes R1 (16 µg/mL), showing more than 16, 8 and 16-fold higher activity than that of CAM,
respectively. Moreover, 13d and 13g exhibited the best antibacterial activity against sensitive bacterial
strains, including Staphylococcus aureus ATCC25923 (4 µg/mL) and Bacillus Subtilis ATCC9372 (1
µg/mL). The MBC results showed that the most promising compounds 13d and 13g exhibited
antibacterial activity through bacteriostatic mechanism, while the time-kill kinetic experiment revealed
bactericidal kinetics of 13g from microscopic point of view. In vitro antibacterial experiments and
molecular docking results further confirmed that it was feasible to our initial design strategy by
modifying the C-3 and C-11 positions of clarithromycin to increase the activity against resistant
bacteria.
Keywords: 11-O-Carbamoyl-3-O-descladinosyl derivatives; Antibacterial activity; Bacterial
resistance; Design and synthesis.
2

The term "macrolide" was first proposed by R. B. Woodward in 1957.1 Commonly used macrolides
contain a 14-, 15- or 16-membered macrolide ring and glycosyl groups, which are linked by glycoside
bonds.2 They are widely used in clinic due to their strong antibacterial activity, broad antibacterial
spectrum and remarkable curative effect. The antibacterial mechanism of macrolide antibiotics is to
prevent the extension of nascent polypeptide chains and to further inhibit the synthesis of proteins by
binding with the peptide exit tunnel of peptidyl-transferase center (PTC) which is located at the large
subunit (50S) of bacterial ribosome.3 Erythromycin A (EMA) as a representative of first-generation
macrolides (Fig. 1) is clinically safe and effective in the treatment of upper and lower respiratory tract
infections, and is especially suitable for the patients who are resistant or allergic to β-lactam
antibiotics.4 In view of the degradation of EMA under acidic conditions leading to poor bioavailability
and gastrointestinal side effects, second-generation macrolides, represented by clarithromycin (CAM)
and azithromycin (AZM), with improved antibacterial activity, higher bioavailability and fewer
gastrointestinal side effects, have been obtained further development and widespread application.5
O
O
N
11
12
OH
O
OH
HO
HO
HO
OH
OH
OH
N
N
N
2'
5 HO
O
HO
HO
O
O
O
O
O
O
O
O
1
3
O
O
O
O
O
O
O
O
O
OH
OH
OH
O
O
O
4''
Azithromycin
Erythromycin A
Clarithromycin
Fig. 1. Structures of erythromycin A, clarithromycin and azithromycin
However, with the increased clinical application of macrolide antibiotics, the problem of bacterial
resistance to them has become quite serious.6 The two main resistance mechanisms of
macrolide-resistant pathogens are ribosomal methylation modification and efflux pump active efflux.
The former is a methyltransferase encoded by the erm gene that dimethylates the nucleotide A2058 of
the 23S rRNA in the bacterial ribosome, weakening the interaction between the macrolide antibiotic
and bacterial ribosome while the latter is an efflux transporter encoded by the mef gene that vents the
macrolide out of the bacterial cell.7, 8
The emergence and prevalence of resistant pathogens has led to severe limitations in the
effectiveness of macrolide antibiotics. An X-ray cocrystal structure research stated clearly that the
3-O-descladinosyl moiety at the C-3 position of macrolides is not essential for its antibacterial activity,
but it can induce bacterial mef gene expression and produce efflux resistance.9 Therefore,
third-generation macrolide antibiotics with anti-drug-resistant bacterial activity, represented by
ketolides, have been developed. Among them, telithromycin (Fig. 2) was approved by FDA in 2004, it
binds to wild bacterial ribosomes more tightly than CAM and AZM, and exhibits strong activity
against erythromycin-resistant Streptococcus pneumoniae and Haemophilus influenza.10, 11 The clinical
candidate drug cethromycin exhibits remarkable activity against multidrug-resistant respiratory
pathogens,
effectively
treating
community-acquired
pneumonia
(CAP)
caused
by
macrolide-lincosamide-streptogramin B (MLSB) resistant bacteria.12 Solithromycin, also in clinical
research, shows strong efficacy against a variety of resistant bacteria including methicillin-resistant
14
Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE).13,
Seiple et al.15
established a pathway for the total synthesis of macrolide antibiotics and ascertained that ketolides
bearing 1,2,3-triazole side chains were extremely effective against common macrolide-resistant strains.
In addition to ketolides, other chemical modifications can also improve the antibacterial activity of
3

macrolides. For instance, EP-1553, an 11-O-substituted clarithromycin derivative, exhibits fairly good
activity against sensitive and resistant Streptococcus pyogenes, S. aureus and S. pneumoniae.16, 17
H2N
N
N
N
N
N
N
N
N
O
N
N
O
N
O
O
HO
O
HO
OCH3
O
O
O
O
O
N
O
NH
O
O
O
O
N
O
O
O
O
N
N
HO
O
HO
HO
O
O
O
O
O
O
O
O
O
O
OH
OMe
O
O
O
F
O
O
EP-1553
Telithromycin
Solithromycin
Cethromycin
Fig. 2. Structures of telithromycin, cethromycin, solithromycin and EP-1553
Based on the above-mentioned causes of macrolide resistance and current research, we have
proposed a new modification strategy for the second-generation macrolide antibiotic CAM. Firstly, we
retained the 2'-OH which interacts with A2058 and A2059 in domain V of the bacterial ribosomal 23S
rRNA, thereby producing antibacterial activity against susceptible bacteria.18 Secondly, substitution of
a ketone group for the cladinose moiety at the C-3 position with the purpose of reducing the effect of
active efflux mediated by the mef gene.19 Thirdly, we noticed that there are few literature reports on the
chemical modification of the C-11 position of CAM. Moreover, our previous work20, 21 also have
confirmed that introduction of aralkyl side chain at the C-11 position of CAM can increase the
anti-resistant bacteria activity. thus, we designed 11-O-carbamoyl-3-O-descladinosyl clarithromycin
derivatives with potent activity against A2058 and A2059 methylated modified resistant strains through
introducing a carbamate side chain bearing 1,2,3-triazole with appropriate length, flexibility and spatial
extension to the C-11 position of CAM, which would generate new binding site with the nucleotide
residue U790 of bacterial ribosome by hydrogen bond interaction, π-π stacking interaction and
hydrophobic interaction.22-24
The synthetic methods for 11-O-carbamoyl-3-O-descladinosyl clarithromycin derivatives 9a-9t and
13a-h are shown in Scheme 1 and 2, respectively. CAM was treated with diluted hydrochloric acid
(HCl) to produce the 3-descladinosylclarithromycin 2.25 Using triethylamine as acid binding agent, the
2'-OH of 2 was selectively protected by acetic anhydride (Ac2O) in dichloromethane (DCM) to
generated the acetyl product 3 due to the reactivity of the 2'-OH is stronger than that of the 3-OH.26
Then, cyclization of the 11-OH and the 12-OH of
3
to 11,12-cyclic carbonate by
27
bis(triehloromethyl)carbonate (BTC) in the presence of pyridine gave intermediate 4. Subsequently,
the 3-OH was oxidized by pyridinium chlorochromate (PCC) to obtain ketolide 5.28 Catalyzed by
pyridine hydrochloride, 6 was afforded by treating 5 with ethanolamine. The terminal hydroxyl group
on the C-11 side chain of 6 was esterified with methanesulfonyl chloride (MsCl) to obtain mesylate
intermediate 7. Intermediate 7 was treated with sodium azide (NaN3) in the mixed solvent of water and
N,N-dimethylformamide (DMF) at 60°C to produce the key intermediate 8. After that, intermediate 8
clicked reaction with the corresponding phenylacetylene catalyzed by cuprous iodide (CuI) in toluene
solution29 and followed by deprotection in methanol (MeOH) to afford target compounds 9a-9t
(Scheme 1).30 The synthetic method for another series of target compounds 13a-h is shown in Scheme
2. Intermediate 5 reacted with 3-aminopropanol to afford 10 in the presence of pyridine hydrochloride
at 15°C. The subsequent synthetic method for target compounds was same as that of target compounds
9a-9t. The structures of all the target compounds were confirmed by 1H NMR and MS spectra.
4

O
O
O
OH
O
OH
O
O
OH
O
OH
OH
N
N
OH
N
a
b
c
HO
HO
AcO
O
O
O
O
O
O
O
O
O
O
O
OH
O
O
OH
OH
O
2
3
clarithromycin
O
O
O
O
O
O
HO
N
O
O
H
O
O
O
O
O
O
O
O
HO
N
N
N
d
e
AcO
O
AcO
AcO
O
O
O
O
O
O
O
O
O
OH
O
O
6
4
5
O
S
O
O
O
O
N3
O
H3C
N
H
N
O
H
O
O
O
O
O
O
O
HO
HO
N
N
g
h
f
AcO
AcO
O
O
O
O
O
O
O
8
7
9a R1 =phenyl
9k R1=2-methoxyphenyl
9l R1=4-ethoxyphenyl
9m R1=4-fluorophenyl
9n R1=3-fluorophenyl
9o R1=4-chlorophenyl
9p R1=3-chlorophenyl
9q R1=2-chlorophenyl
9r R1=3-bromophenyl
9s R1=3-nitrophenyl
9t R1=4-nitrophenyl
N
N
N
9b R1=4-methylphenyl
9c R1=3-methylphenyl
9d R1=4-ethylphenyl
9e R1=4-n-propylphenyl
9f R1=4-n-butylphenyl
9g R1=4-tert-butylphenyl
9h R1=4-biphenyl
R1
O
O
N
H
O
O
O
O
HO
N
HO
O
O
O
9i R1= 3-aminophenyl
9j R1=4-methoxyphenyl
9
Scheme 1. Synthesis of 11-O-carbamoyl-3-O-descladinosyl clarithromycin derivatives (9a–9t).
Reagents and conditions: (a) HCl, H2O, rt, 5 h, 83%; (b) Ac2O, Et3N, DCM, 0 °C~rt, 36 h, 94%; (c)
pyridine, BTC, DCM, 0 °C~rt, 20 h, 99%; (d) PCC, DCM, rt, 24 h, 88%; (e) ethanolamine, pyridine
hydrochloride, 15 °C, 2 h, 37%; (f) MsCl, Et3N, DCM, 0~25 °C, 2 h, 91%; (g) NaN3, DMF, H2O,
60 °C, 12 h, 76%; (h) i) phenylacetylene, CuI, toluene, 90 °C, 24 h; ii) CH3OH, 55 °C, 24 h, 56~68%.
5

O
O
O
O
HO
N
O
H
O
O
O
O
O
O
O
HO
N
N
b
a
AcO
AcO
O
O
O
O
O
O
O
O
O
5
10
O
O
O
O
N3
S
H3C
N
N
H
H
O
O
O
O
O
O
O
O
HO
HO
N
N
c
AcO
AcO
O
O
O
O
O
O
O
11
12
N
N
N
O
R2
O
13a R2=phenyl
N
H
13b R2=4-n-butylphenyl
13c R2= 4-tert-butylphenyl
13d R2=4-biphenyl
13e R2=3-aminophenyl
13f R2=2-methoxyphenyl
13g R2=4-chlorophenyl
13h R2=3-bromophenyl
O
O
HO
N
d
HO
O
O
O
O
O
13
Scheme 2. Synthesis of 11-O-carbamoyl-3-O-descladinosyl clarithromycin derivatives (13a–13h).
Reagents and conditions: (a) 3-aminopropanol, pyridine hydrochloride, 15 °C, 2 h, 42%; (f) MsCl,
Et3N, DCM, 0~25 °C, 2 h, 89%; (g) NaN3, DMF, H2O, 60 °C, 12 h, 78%; (h) i) phenylacetylene, CuI,
toluene, 90 °C, 24 h; ii) CH3OH, 55 °C, 24 h, 56~65%.
The in vitro antibacterial activity of target compounds were determined by the standard broth
microdilution procedures recommended by NCCLS. The tested strains included four susceptible strains
of S. aureus ATCC25923 (erythromycin-susceptible strain), Bacillus subtilis ATCC9372
(penicillin-susceptible strain), Escherichia coli ATCC25922 (penicillin-susceptible strain) and
Pseudomonas aeruginosa ATCC27853 (penicillin-susceptible strain), and four resistant strains of S.
aureus ATCC43300 (methicillin-resistant strain), S. pyogenes R1 (erythromycin-resistant strain
isolated clinically), S. pneumoniae B1 (erythromycin-resistant strain expressing the ermB gene), and S.
pneumoniae AB11 (erythromycin-resistant strain expressing the ermB and mefA genes). The minimal
inhibitory concentration (MIC) values for the above target compounds as well as CAM and AZM as
the references were tested against a panel of sensitive and resistant bacteria strains as shown in Table 1.
6

Table 1
In vitro antibacterial activity of 11-O-carbamoyl-3-O-descladinosyl clarithromycin derivatives
Minimum inhibitory concentration (MIC) (µg/mL)
S. aureus
E. coli
B. Subtilis
P. aeruginosa
S. pneumoniae
S. pneumoniae
S. pyogenes
S. aureus
Compound
ATCC25923a
ATCC25922b
ATCC9372c
ATCC27853d
AB11e
B1f
R1g
ATCC43300h
CLM
AZM
13a
13b
13c
13d
13e
13f
13g
13h
9a
0.25
0.25
8
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
0.25
0.25
8
32
128
256
64
>256
>256
128
32
256
256
64
>256
>256
256
4
128
128
128
128
128
128
128
128
128
128
128
128
128
128
128
16
16
4
2
32
16
64
16
1
64
64
32
128
16
16
16
64
16
8
4
32
128
256
64
128
64
>256
>256
128
2
32
4
1
16
32
8
2
32
64
32
128
32
32
32
32
32
16
32
8
256
256
256
256
128
64
256
>256
>256
>256
256
128
128
256
256
256
128
64
>256
>256
>256
>256
256
9b
8
9c
8
9d
4
9e
8
9f
4
32
256
9g
8
128
64
256
7

9h
9i
32
32
16
16
16
16
8
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
4
32
4
128
128
128
128
128
128
128
128
128
128
128
128
128
64
256
256
>256
256
128
128
128
128
128
128
128
256
128
>256
>256
>256
>256
256
32
256
256
256
128
64
128
>256
>256
>256
>256
>256
256
9j
9k
9l
4
16
4
9m
9n
9o
9p
9q
9r
9s
4
256
128
64
8
4
128
256
8
4
128
64
256
8
4
128
64
256
16
8
8
256
128
64
>256
>256
>256
4
256
9t
16
4
256
64
\aS. aureus ATCC25923: erythromycin-susceptible strain;
bE. coli ATCC25922: penicillin-susceptible strain;
cB. Subtilis ATCC9372: penicillin-susceptible strain;
dP.aeruginosa ATCC27853: penicillin-susceptible strain;
eS. pneumoniae AB11: erythromycin-resistant strain expressing the ermB and mefA genes;
fS. pneumonia B1: erythromycin-resistant strain expressing the ermB gene;
gS. pyogenes R1: erythromycin-resistant strain isolated clinically;
hS. aureus ATCC43300: methicillin-resistant strain.
8

Compared to the references of CAM and AZM, the 11-O-carbamoyl-3-O-descladinosyl
clarithromycin derivatives universally showed greatly improved activity against S. pneumoniae B1, S.
pneumoniae AB11, S. pyogenes R1 and S. aureus ATCC43300, but slightly weaker activity against S.
aureus ATCC25923 and B. subtilis ATCC9372 than the references. Unfortunately, none of them
displayed improved activity against E. coli ATCC25922 and P. aeruginosa ATCC27853. Among them,
13d showed significantly improved activity (16 µg/mL) against S. pneumoniae B1, which was >16-fold
more active than that of CAM and AZM (>256 and >256 µg/mL), while 13b, 13d, 13e, 13f, 13g and
13h exhibited the most effective activity against S. pneumoniae AB11 with MIC values of 16-32
µg/mL, showing 4 to 8-fold better than CAM (128 µg/mL) and 8 to 16-fold better than AZM (256
µg/mL), respectively. In addition, 9f, 9h, 13b, 13c, 13d, 13g and 13h displayed the best antibacterial
activity with MIC values of 16-32 µg/mL against S. pyogenes R1, 8 to 16-fold better than CAM and
AZM (256 and 256 µg/mL). In the inhibition of S. aureus ATCC43300, the most active 13b and
13d showed an MIC value of 64 mg/mL, >4-fold better than CAM and AZM (>256 µg/mL).
Furthermore, 13d and 13g with the best anti-sensitive bacteria activity, had an MIC value of 4 and 1
µg/mL against S. aureus ATCC25923 and B. subtilis ATCC9372, respectively. Besides, many
compounds demonstrated antibacterial activity with MIC values of 8-32 µg/mL and 2-8 µg/mL against
S. aureus ATCC25923 and B. subtilis ATCC9372, respectively.
In general, the target compounds exhibited greatly improved activity against resistant bacteria and
slightly weak activity against susceptible bacteria in comparison with CAM and AZM. Furthermore,
most of target compounds showed favorable activity against Gram-positive bacteria but weak activity
against Gram-negative bacteria, probably because the target compounds are difficult to pass through
the cell wall of Gram-negative bacteria. The results indicate that simultaneous modification of the C-3
and C-11 positions of CAM can greatly enhance the activity against resistant strains and partially retain
the activity against sensitive strains. We believe that the C-11 side chain can directly interact with the
nucleotide A752 and U790, and the alteration of the length of the C-11 side chain and the different
substituents on the terminal benzene ring of the C-11 side chain lead to the change of interaction force,
thereby result in the difference of antibacterial activity for the target compounds.
Preliminary structure-activity relationship (SAR) studies on the 11-O-carbamoyl-3-O-descladinosyl
clarithromycin derivatives indicated that when the length between the carbon atom at the C-11 position
and the 1,2,3-triazole moiety was extended from 5 atoms to 6 atoms (9a and 13a, 9f and 13b, 9g and
13c, 9h and 13d, 9i and 13e, 9k and 13f, 9o and 13g, 9r and 13h), the activity against sensitive bacteria
increased by 2 to 8-fold and the activity against resistant bacteria increased by 2 to 4-fold, which
suggests that the 1,2,3-triazole side chain with the appropriate length may be more susceptible to
interaction with the U790 of 23S rRNA. The electron-donating groups on the terminal benzene ring of
the 1,2,3-triazole side chain (9a-9d and 9i-9l) result in loss of activity against the drug-resistant strains,
which indicates that these compounds only retain partial interaction with sensitive bacteria, without
new binding force with the ribosomes of the drug-resistant bacteria. Aryl substituents (9h and 13d) and
electron-withdrawing groups (9m-9t, 13g and 13h) on the benzene ring are beneficial to the
antibacterial activity. In contrast, weak electron-donating groups on the benzene ring (9e-9g and 13b
and 13c) are detrimental to the antibacterial activity. However, new binding force is generated in space
due to the flexibility of their alkyl substituent, resulting in improved activity. In particular, linear
alkane groups (9f and 13b) are more active than branched alkane groups (9g and 13c). Position of the
substitutes on the benzene ring has no prominent effect on the activity (9j-9k, 9m-9q and 9s-9t). For
halogen-substituted compounds, the chlorine atom substitution is superior to the fluorine and bromine
9

substitution (9m-9r and 13g-13h) in the activity. Therefore, we speculated that the appropriate length
of the 1,2,3-triazole side chain can reach the chloramphenicol binding site in the bacterial ribosome,
and the different substituents on the benzene ring affect the binding ability of the target compounds to
the binding site, thereby inhibiting the formation of bacterial peptide.
To investigate whether the antibacterial activity of these target compounds was bactericidal or
bacteriostatic in nature, we determined the minimum bactericidal concentration (MBC) values of 13d
and 13g against susceptible bacteria (B. Subtilis ATCC9372) and resistant bacteria (S. pneumoniae
AB11) (Table 2). By comparing their MBC values with their corresponding MIC values, we discovered
that 13d and 13g exhibiting bacteriostatic action against B. Subtilis ATCC9372 with MBC/MIC ratio of
128 and 32, respectively. Similarly, 13d and 13g were observed as bacteriostatic agents against S.
pneumoniae AB11 with the MBC/MIC ratio of 16 and 8, respectively. The above results reveal that the
test compounds exert an antibacterial effect against sensitive and resistant bacterial strains by
bacteriostatic action.
Table 2
Comparison of MIC and MBC values for 13d and 13g in B. Subtilis ATCC9372 and S. pneumoniae AB11.
Compound
MBC(µg/mL)
MIC(µg/mL)
MBC/MIC
B. subtilis ATCC9372a
13d
13g
128
32
1
1
128
32
CAM
128
0.25
512
S. pneumoniae AB11b
13d
13g
256
128
128
16
16
16
8
CAM
128
1
aB. Subtilis ATCC9372: penicillin-susceptible strain;
bS. pneumoniae AB11: erythromycin-resistant strain expressing the ermB and mefA genes.
In order to further examine the antibacterial potency of the target compounds, time-kill kinetic
experiment of 13g against S. pneumoniae AB11 was tested. The growth of bacteria was observed when
it was incubated without 13g or with 0.5 MIC of 13g as shown in Fig. 3. On the contrary, 13g exhibited
certain bactericidal effect (0-9 h) and bacteriostatic effect (9-24 h) at the concentrations of 1, 2 and 4
MIC. Time-kill kinetic experiment further proved that 13g was a concentration-dependent
bacteriostatic agent from a microscopic point of view.
12
10
DMSO
0.5 MIC
1 MIC
2 MIC
4 MIC
8
6
4
2
0
0
3
6
9
12
24
TIME/H
Fig. 3. Time-kill kinetics of compound 13g against S. pneumoniae AB11.
10

By analyzing the activity data, we found that these target compounds had good antibacterial activity,
and especially their anti-resistant bacterial activity showed obvious improvement compared with that of
CAM and AZM, indicating the combined modifications of the C-3 and the C-11 positions of CAM are
instrumental in activity against multiple drug-resistant bacterial strains. It is worth noting that these
compounds have significantly improved activity against S. pneumoniae B1 and S. pneumoniae AB11,
which confirms the correctness of our design mentality. We believe that removal of the cladinose group
at the C-3 position of CAM and oxidation of the resulting hydroxyl group to ketone, and further
introduction of the C-11 carbamate side chains containing 1,2,3-triazole group not only weaken the
active efflux of the resistant bacteria but also produce additional affinity for the bacterial ribosome,
thereby effectively inhibiting the type of erm and mef resistant bacteria. Docking model (Fig. 4)
performed through Le-dock further indicated that 13g has similar binding mode to CAM when it binds
to E. coli ribosome (PDB code, 4v7s), preserving the interaction between 2'-OH and A2058, which
explains why the compound displays the activity against susceptible bacteria. What's more, the
1,2,3-triazole side chain at the C-11 position of 13g forms hydrogen bonds with the A752 and U790
nucleotide residues of 23S rRNA. Meanwhile, 13g also interacts with the pocket formed by G2505,
C2610 and C2611 in domain V, thereby increasing the activity against resistant bacteria.
a
b
Fig. 4. (a) The docking model of compound 13g (blue) and clarithromycin (green) in E. coli ribosome
(PDB code, 4v7s). Both are located in approximately the same spatial area in E. coli ribosome. (b)
Model of compound 13g docked into E. coli ribosome (PDB code, 4v7s). In the model, the predicted
hydrogen bonds between compound 13g and the nucleotide residues are indicated by dotted lines.
In summary, a novel series of 11-O-carbamoyl-3-O-descladinosyl clarithromycin derivatives bearing
the 1,2,3-triazole group were designed, synthesized and evaluated for their in vitro antimicrobial
activity against various drug-susceptible and -resistant bacterial strains. The antibacterial results
indicated that the target compounds greatly enhanced the activity against resistant bacterial strains and
partially retained the activity against sensitive bacterial strains. Among them, 13d had the best
antibacterial activity against resistant strains, including S. pneumoniae B1 (16 µg/mL), S. pneumoniae
AB11 (16 µg/mL) and S. pyogenes R1 (16 µg/mL), showing more than 16, 8 and 16-fold higher
activity than that of CAM, respectively. Moreover, 13d and 13g exhibited the best antibacterial activity
against sensitive bacterial strains, including S. aureus ATCC25923 (4 µg/mL) and B. Subtilis
ATCC9372 (1 µg/mL). On the other hand, measurement of MBC showed that the most promising
compounds 13d and 13g exhibited antibacterial activity through bacteriostatic mechanism, while the
time-kill kinetic experiment revealed bactericidal kinetics of 13g from microscopic point of view. In
vitro antibacterial experiments and molecular docking results further confirmed that it was feasible to
11

our initial design strategy by modifying the C-3 and C-11 positions of CAM to enhance the activity
against resistant bacteria.
Conflict of interest
The authors declare that this study was carried out only with public funding. There is no funding or
no agreement with commercial for profit firms.
Acknowledgements
This research was supported financially by the National Natural Science Foundation of China
(81973179 and 81673284) and the Key Research and development project of Shandong Province
(2017CXGC1401).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
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14

Figure captions
Fig. 1. Structures of erythromycin A, clarithromycin and azithromycin.
Fig. 2. Structures of telithromycin, cethromycin, solithromycin and EP-1553.
Scheme 1. Synthesis of 11-O-carbamoyl-3-O-descladinosyl clarithromycin derivatives (9a–9t).
Reagents and conditions: (a) HCl, H2O, rt, 5 h, 83%; (b) Ac2O, Et3N, DCM, 0 °C~rt, 36 h, 94%; (c)
pyridine, BTC, DCM, 0 °C~rt, 20 h, 99%; (d) PCC, DCM, rt, 24 h, 88%; (e) ethanolamine, pyridine
hydrochloride, 15 °C, 2 h, 37%; (f) MsCl, Et3N, DCM, 0~25 °C, 2 h, 91%; (g) NaN3, DMF, H2O,
60 °C, 12 h, 76%; (h) i) phenylacetylene, CuI, toluene, 90 °C, 24 h; ii) CH3OH, 55 °C, 24 h, 56~68%.
Scheme 2. Synthesis of 11-O-carbamoyl-3-O-descladinosyl clarithromycin derivatives (13a–13h).
Reagents and conditions: (a) 3-aminopropanol, pyridine hydrochloride, 15 °C, 2 h, 42%; (f) MsCl,
Et3N, DCM, 0~25 °C, 2 h, 89%; (g) NaN3, DMF, H2O, 60 °C, 12 h, 78%; (h) i) phenylacetylene, CuI,
toluene, 90 °C, 24 h; ii) CH3OH, 55 °C, 24 h, 56~65%.
Fig. 3. Time-kill kinetics of compound 13g against S. pneumoniae AB11.
Fig. 4. (a) The docking model of compound 13g (blue) and clarithromycin (green) in E. coli ribosome
(PDB code, 4v7s). Both are located in approximately the same spatial area in E. coli ribosome. (b)
Model of compound 13g docked into E. coli ribosome (PDB code, 4v7s). In the model, the predicted
hydrogen bonds between compound 13g and the nucleotide residues are indicated by dotted lines.
15

Fig. 1
O
O
N
11
12
OH
O
OH
HO
HO
HO
OH
OH
OH
N
N
N
2'
5 HO
O
HO
HO
O
O
O
O
O
O
O
O
1
3
O
O
O
O
O
O
O
O
O
OH
OH
OH
O
O
O
4''
Azithromycin
Erythromycin A
Clarithromycin
16

Fig. 2
H2N
N
N
N
N
N
N
N
N
N
N
O
N
O
O
HO
O
HO
OCH3
O
O
O
O
O
N
O
NH
O
O
O
O
N
O
O
O
O
N
N
O
HO
O
HO
HO
O
O
O
O
O
O
O
O
O
O
OH
OMe
O
O
O
F
O
O
EP-1553
Telithromycin
Solithromycin
Cethromycin
17

Scheme 1
O
O
O
OH
O
OH
OH
O
O
OH
O
OH
N
N
OH
N
a
b
c
HO
HO
AcO
O
O
O
O
O
O
O
O
O
O
O
O
OH
O
OH
OH
O
2
3
clarithromycin
O
O
O
O
O
O
HO
N
O
O
H
O
O
O
O
O
O
O
O
HO
N
N
N
d
e
AcO
O
AcO
O
AcO
O
O
O
O
O
O
O
O
OH
O
O
6
4
5
O
S
O
O
O
O
N3
O
H3C
N
H
N
O
H
O
O
O
O
O
O
O
HO
HO
N
N
g
h
f
AcO
AcO
O
O
O
O
O
O
O
8
7
9a R1 =phenyl
9k R1=2-methoxyphenyl
9l R1=4-ethoxyphenyl
9m R1=4-fluorophenyl
9n R1=3-fluorophenyl
9o R1=4-chlorophenyl
9p R1=3-chlorophenyl
9q R1=2-chlorophenyl
9r R1=3-bromophenyl
9s R1=3-nitrophenyl
9t R1=4-nitrophenyl
N
N
N
9b R1=4-methylphenyl
9c R1=3-methylphenyl
9d R1=4-ethylphenyl
9e R1=4-n-propylphenyl
9f R1=4-n-butylphenyl
9g R1=4-tert-butylphenyl
9h R1=4-biphenyl
R1
O
O
N
H
O
O
O
O
HO
N
HO
O
O
O
9i R1= 3-aminophenyl
9j R1=4-methoxyphenyl
9
18

Scheme 2
O
O
O
O
HO
N
O
H
O
O
O
O
O
O
O
HO
N
N
b
a
AcO
AcO
O
O
O
O
O
O
O
O
O
5
10
O
O
O
O
N3
S
H3C
N
N
H
H
O
O
O
O
O
O
O
O
HO
HO
N
N
c
AcO
AcO
O
O
O
O
O
O
O
11
12
N
N
N
O
R2
O
13a R2=phenyl
N
H
13b R2=4-n-butylphenyl
13c R2= 4-tert-butylphenyl
13d R2=4-biphenyl
13e R2=3-aminophenyl
13f R2=2-methoxyphenyl
13g R2=4-chlorophenyl
13h R2=3-bromophenyl
O
O
HO
N
d
HO
O
O
O
O
O
13
19

Fig. 3
12
10
8
DMSO
0.5 MIC
1 MIC
2 MIC
4 MIC
6
4
2
0
0
3
6
9
12
24
TIME/H
20

Fig. 4
a
b
21

Table 1
In vitro antibacterial activity of 11-O-carbamoyl-3-O-descladinosyl clarithromycin derivatives
Minimum inhibitory concentration (MIC) (µg/mL)
S. aureus
E. coli
B. Subtilis
P. aeruginosa
S. pneumoniae
S. pneumoniae
S. pyogenes
S. aureus
Compound
ATCC25923a
ATCC25922b
ATCC9372c
ATCC27853d
AB11e
B1f
R1g
ATCC43300h
CLM
AZM
13a
13b
13c
13d
13e
13f
13g
13h
9a
0.25
0.25
8
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
0.25
0.25
8
32
128
256
64
>256
>256
128
32
256
256
64
>256
>256
256
4
128
128
128
128
128
128
128
128
128
128
128
128
128
128
128
16
16
4
2
32
16
64
16
1
64
64
32
128
16
16
16
64
16
8
4
32
128
256
64
128
64
>256
>256
128
2
32
4
1
16
32
8
2
32
64
32
128
32
32
32
32
32
16
32
8
256
256
256
256
128
64
256
>256
>256
>256
256
128
128
256
256
256
128
64
>256
>256
>256
>256
256
9b
8
9c
8
9d
4
9e
8
9f
4
32
256
9g
8
128
64
256
22

9h
9i
32
32
16
16
16
16
8
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
4
32
4
128
128
128
128
128
128
128
128
128
128
128
128
128
64
256
256
>256
256
128
128
128
128
128
128
128
256
128
>256
>256
>256
>256
256
32
256
256
256
128
64
128
>256
>256
>256
>256
>256
256
9j
9k
9l
4
16
4
9m
9n
9o
9p
9q
9r
9s
4
256
128
64
8
4
128
256
8
4
128
64
256
8
4
128
64
256
16
8
8
256
128
64
>256
>256
>256
4
256
9t
16
4
256
64
\aS. aureus ATCC25923: erythromycin-susceptible strain;
bE. coli ATCC25922: penicillin-susceptible strain;
cB. Subtilis ATCC9372: penicillin-susceptible strain;
dP.aeruginosa ATCC27853: penicillin-susceptible strain;
eS. pneumoniae AB11: erythromycin-resistant strain expressing the ermB and mefA genes;
fS. pneumonia B1: erythromycin-resistant strain expressing the ermB gene;
gS. pyogenes R1: erythromycin-resistant strain isolated clinically;
hS. aureus ATCC43300: methicillin-resistant strain.
23

Table 2
Comparison of MIC and MBC values for 13d and 13g in B. Subtilis ATCC9372 and S. pneumoniae AB11.
Compound
MBC(µg/mL)
MIC(µg/mL)
MBC/MIC
B. subtilis ATCC9372a
13d
13g
128
32
1
1
128
32
CAM
128
0.25
512
S. pneumoniae AB11b
13d
13g
256
128
128
16
16
16
8
CAM
128
1
aB. Subtilis ATCC9372: penicillin-susceptible strain;
bS. pneumoniae AB11: erythromycin-resistant strain expressing the ermB and mefA genes.
24

Graphical abstract:
A novel series of 11-O-carbamoyl-3-O-descladinosyl clarithromycin derivatives bearing 1,2,3-triazole group: design, synthesis and
antibacterial evaluation
Yuetai Teng, Yinhui Qin, Di Song, Xingbang Liu, Yingang Ma, Panpan Zhang, Shutao Ma*
25

O
OH
O
O
OH
N
Necessary group
HO
O
O
O
O
O
Relevant with efflux resistance
encoded by the mef gene
OH
O
CAM
H2N
Conferring a secondary
interaction with A752
N
N
N
N
N
N
O
N
HO
O
HO
OCH3
O
N
O
O
O
O
O
O
O
HO
O
O
O
O
O
O
OH
F
OMe
O
Solithromycin
EP-1553
N
N
N
N
N
O
O
O
O
O
Cl
N
N
N
H
H
O
O
O
O
O
HO
HO
N
N
HO
HO
O
O
O
O
O
O
O
O
13d
13g
11-O-carbamoyl-3-O-descladinosyl clarithromycin derivatives
26

Research Highlights
> Novel 11-O-carbamoyl-3-O-descladinosyl clarithromycin derivatives were synthesized and evaluated. > Some of them showed effective activity against both
susceptible and resistant strains. > Compound 13d and 13g exhibited prominent antibacterial activity.
27