Extra Sugar on Vancomycin: New Analogues for Combating Multidrug-Resistant Staphylococcus aureus and Vancomycin-Resistant Enterococci

Article abstract of DOI:10.1021/acs.jmedchem.7b01345

Lipophilic substitution on vancomycin is an effective strategy for the development of novel vancomycin analogues against drug-resistant bacteria by enhancing bacterial cell wall interactions. However, hydrophobic structures usually lead to long elimination half-life and accumulative toxicity; therefore, hydrophilic fragments were also introduced to the lipo-vancomycin to regulate their pharmacokinetic/pharmacodynamic properties. Here, we synthesized a series of new vancomycin analogues carrying various sugar moieties on the seventh-amino acid phenyl ring and lipophilic substitutions on vancosamine with extensive structure-activity relationship analysis. The optimal analogues indicated 128-1024-fold higher activity against methicillin-susceptible S. aureus, vancomycin-intermediate resistant S. aureus (VISA), and vancomycin-resistant Enterococci (VRE) compared with that of vancomycin. In vivo pharmacokinetics studies demonstrated the effective regulation of extra sugar motifs, which shortened the half-life and addressed concerns of accumulative toxicity of lipo-vancomycin. This work presents an effective strategy for lipo-vancomycin derivative design by introducing extra sugars, which leads to better antibiotic-like properties of enhanced efficacy, optimal pharmacokinetics, and lower toxicity.

Full text of DOI:10.1021/acs.jmedchem.7b01345

Subscriber access provided by University of Florida | Smathers Libraries
Article
Extra sugar on vancomycin: new analogues for combating multidrug-
resistant Staphylococcus aureus and vancomycin-resistant Enterococci
Dongliang Guan, Feifei Chen, Lun Xiong, Feng Tang, Faridoon Faridoon,
Yunguang Qiu, Naixia Zhang, Likun Gong, Jian Li, Lefu Lan, and Wei Huang
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01345 • Publication Date (Web): 15 Dec 2017
Downloaded from http://pubs.acs.org on December 16, 2017
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 92
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Extra sugar on vancomycin: new analogues for combating
multidrug-resistant Staphylococcus aureus and vancomycin-resistant
Enterococci
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1,2,†
1,†
1,3
1,2
Dongliang Guan , Feifei Chen , Lun Xiong , Feng Tang ,
1
Faridoon , Yunguang Qiu , Naixia Zhang , Likun Gong , Jian Li ,
1,2
1,2
1,2
3
1
,2,4*
1,2,5*
Lefu Lan
, Wei Huang
1
CAS Key Laboratory of Receptor Research, CAS Center for Excellence
in Molecular Cell Science, Shanghai Institute of Materia Medica, Chinese
Academy of Sciences, 555 Zuchongzhi Road, Pudong, Shanghai 201203,
China
2
University of Chinese Academy of Sciences, No.19A Yuquan Road,
Beijing 100049, China
3
Shanghai Key Laboratory of New Drug Design, School of Pharmacy,
East China University of Science and Technology, Shanghai 200237,
China
4
State Key Laboratory of Drug Research, Shanghai Institute of Materia
Medica, Chinese Academy of Sciences , Shanghai 201203, China.
5
Center for Bio-drug Development, Shanghai Institute of Materia Medica,
Chinese Academy of Sciences , Shanghai 201203, China.
†
These authors contribute to this work equally.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 92
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
*
Corresponding authors:
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Prof. Wei Huang
Tel: +86-21-20231000 ext. 2517
Fax: +86-21-50807088
Email: huangwei@simm.ac.cn
Prof. Lefu Lan
Tel: +86-21-50806600
Fax: +86-21-50807088
Email: llan@simm.ac.cn
Abstract
Lipophilic substitution on vancomycin is an effective strategy for
development of novel vancomycin analogues against drug-resistant
bacteria by enhancing bacterial cell wall interaction. However,
hydrophobic structures usually led to long elimination half-life and
accumulative toxicity, therefore hydrophilic fragments were also
introduced to the lipo-vancomycin to regulate their PK/PD properties.
Here, we synthesized a series of new vancomycin analogues carrying
ACS Paragon Plus Environment

Page 3 of 92
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
th
various sugar moieties on the 7 -amino acid phenyl ring and lipophilic
substitutions on vancosamine with extensive SAR analysis. The optimal
analogues indicated 128-1024 fold higher activity against MSSA, VISA,
and VRE compared with vancomycin. In vivo pharmacokinetics studies
demonstrated the effective regulation of extra sugar motifs, which
shortened the half-life and addressed the concerns of accumulative
toxicity of lipo-vancomycin. This work presented an effective strategy in
lipo-vancomycin derivative design by introducing extra sugars, which led
to better antibiotic-like properties in enhanced efficacy, optimal PK, and
lower toxicity.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Keywords: vancomycin analogues, drug-resistant bacteria, VISA, VRE,
sugar moiety
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 92
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Introduction
Vancomycin as the first member of glycopeptide antibiotics was approved
1
for clinical use in the 1950s . Its structure was not fully characterized
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
until 1983 . In antibacterial history, vancomycin opened a new era to fight
against
drug-resistant
Gram-positive
bacteria
including
multidrug-resistant Staphylococcus aureus (MRSA), and was rewarded as
“the last resort to fight against untreatable bacterial pathogens” and “ace
3
antibiotics” . Recent years, due to the abuse of antimicrobial agents,
drug-resistant bacterial infection has become a life-threatening problem in
public health. In an estimated survey, drug-resistant bacterial infection
4, 5
may cause 10 million deaths annually in worldwide by 2050 . The
emergence of vancomycin-resistant bacteria deteriorates the situation. In
6
1988, vancomycin-resistant enterococci (VRE) was firstly reported ,
thereafter vancomycin-intermediate resistant Staphylococcus aureus
(VISA) and vancomycin-resistant Staphylococcus aureus (VRSA) were
7-12
discovered in 1997 and 2002 . The spread of MRSA and
vancomycin-resistant relevant pathogens brought serious concerns in
outbreak of drug-resistant bacterial infection. Desire of new antibiotics
13
has become an urgent need to combat these pathogens .
The new-generation glycopeptide antibiotics derived from vancomycin
demonstrated improved antibacterial activities against various
ACS Paragon Plus Environment

Page 5 of 92
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
vancomycin-resistant strains, therefore providing a strategy to tackle the
14-18
crisis of drug-resistant infections . Three semi-synthetic vancomycin
analogues, Telavancin, Dalbavancin and Oritavancin, were recently
launched on the market in 2009 and 2014 for treatment of MRSA
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
19-21
infection . These analogues are also called lipoglycopeptides since
their structures bear a linear lipid or a biphenyl hydrophobic appendix
(
Figure 1). These lipo-structures anchor the antibiotics to bacterial cell
22
wall and dramatically enhance the antibacterial effect . The rigid
structure of chlorobiphenyl substitution could directly inhibit the
2
transglycosylase and contribute to the binding with pentaglycyl bridge
3
2
segments of cell wall peptidoglycan . However, hydrophobic groups
4
caused longer half-life and lower clearance rate in vivo that may lead to
25, 26
accumulative toxicity such as nephrotoxicity
. Hence, hydrophilic
groups such as extra sugar moieties (Figure 1) were usually introduced to
2
the scaffold to optimize their pharmacokinetics and toxicity .
7
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 92
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Cl
H
OHHN
N
OH
NH
2
H
3
C
O
H
3
C
O
HO
HO
OH
HN
CH
3O
OH
CH
3
O
O
O
O
HO
H
O
Cl OH
3
C
O
Cl OH
HO
O
O
CH3O
OH
O
O
O
NH
2
O
Cl OH
HO
OH
NH
HO
OH
NH
HO
H
Cl
H
N
O
Cl
H
N
O
3
C
O
O
O
O
H
N
O
H
N
O
O
CH3O
O
N
H
N
H
O
N
H
N
H
OH
NH
H
N
H
Cl
H
N
O
NH
O
O
O
NH
O
O
O
N
O
H
N
HO
HO
O
HO
HO
O
O
O
N
H
N
H
H
2
N
H
2
N
H
N
NH
O
O
O
HO
HO
O
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
OH
OH
Vancomycin
OH
OH
2
H N
N
H
3 2
PO H
Telavancin
OH
OH
Oritavancin
HO
OH OH
HO
OH OH
O
R:
O
R
O
O
N
H
N
H
HO
OH OH
O
O
O
O
Cl
Cl
2
Teicoplanin A -1
Ac
O
O
N
H
O
O
O
O
HO
OH
NH
OH
Cl
H
N
O
Cl
H
N
O
O
H
N
O
H
N
O
O
O
O
N
H
N
H
O
N
H
N
H
N
H
H
N
Teicoplanin A
2
-2
NH
O
O
O
NH
O
O
N
N
H
O
HO
HO
O
NH
2
2
H N
HO
O
Teicoplanin A -3
2
HO
O
OH
OH
O
OH
OH
OH
O
OH
O
OH
Dalbavancin
OH OH
OH OH
Figure 1. Structures of Vancomycin and its lipo-analogues. Extra sugar moieties or
hydrophilic groups are displayed in red color.
In structures of Dalbavancin, Oritavancin, and Teicoplanin (Figure 1),
extra sugar moieties such as mannose, N-acetylglucosamine, and
epi-vancosamine were substituted onto different hydroxyls of the
glycopeptide core and play important roles in structural optimization.
These extra sugar substitutions were all derived from corresponding
28-31
fermentation products
by biosynthesis that hampers the diversification
on various sugar substitutions for new analogue design. Telavancin
carries a phosphonomethylaminomethyl group as the hydrophilic tail onto
th 32
the phenyl of the 7 -amino acid via a selective Mannich reaction . With
2
6
the same reaction, direct assembly of an extra sugar glucosamine was
previously reported, but the antibacterial activities of these analogues
were significantly reduced even with a linear lipid tail on vancosamine.
ACS Paragon Plus Environment

Page 7 of 92
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
These results implicated the systematic structure-activity relationship
(SAR) investigation with various sugar moieties and hydrophobic
16,
fragments are required for this strategy. Recently, Haldar group reported
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
7
a
series of novel vancomycin analogues employing
“lipophilic-vancomycin-carbohydrate” conjugation by coupling of
different sugars onto the C-terminal carboxylic acid and introducing
linear lipid groups onto vancosamine. The successful improvement of
antibacterial activity against VISA and VRE demonstrated the assembly
of extra sugar moieties also modulated the drug efficacy. These examples
indicated that introducing the extra sugar to vancomycin by chemical
modification presented a new strategy for structural optimization on
vancomycin analogues. However, there are still many questions to
address, such as what kind of sugar to assemble? which position on
vancomycin to introduce the sugar especially when a rigid lipophilic tail
like chlorobiphenyl was linked on vancosamine? how the extra sugar
moieties influence the activity and pharmacokinetics? We sought to
answer these questions by investigation on precise SAR of
extra-sugar-bearing vancomycin analogues.
Results
Synthesis of new vancomycin analogues carrying extra saccharide
motif
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 92
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
In order to investigate the role of extra sugar moieties on
lipo-vancomycin for PK/PD optimization, we introduced various
th
monosaccharides and a disaccharide to the 7 -amino acid phenyl group of
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
lipo-vancomycin via a two-step synthesis (Schemes 1 and 2). Firstly,
decylaminoethyl (derived from Telavancin) and chlorobiphenylmethyl
(derived from Oritavancin) were assembled onto the amino group of
vancomycin by reductive amidation. Then, sugar motifs including
glucose (Glc), mannose (Man), galactose (Gal), glucosamine (GlcN),
mannosamine (ManN), galactosamine (GalN), and open-ring glucose
th
orGlc) and lactose (orLac) were attached on the 7 -amino acid resorcinol
(
via
Mannich
reaction
(compounds
compounds
5-14
from
from
decylaminoethyl-vancomycin
and
15-21
chlorobiphenylmethyl-vancomycin). In vitro anti-MRSA and anti-VISA
assays suggested GalN, Gal, and orGlc were the optimal sugar structures,
therefore in the second-round structural modification we chose these
three saccharides to investigate SAR with various lipophilic fragments on
vancosamine including linear alkyls and acyls, and rigid hydrophobic
structures containing benzene rings (compound 22-46). Some of the
lipo-fragments were selected from previous reports on the basis of the
2
5, 31
SAR studies
Table 1.
. The full structures of compounds 5-46 were listed in
ACS Paragon Plus Environment

Page 9 of 92
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
R2
R2
HO
C
HO
HN
OH
HN
NH
2
H
3
O
H
3
C
O
H
3
C
O
HO
HO
HO
CH
3O
O
OH
CH
3O
O
OH
Cl
CH
3O
OH
Cl
O
O
O
Cl OH
OH
O
OH
O
O
O
O
O
O
HO
OH
NH
HO
OH
NH
HO
OH
NH
i
Cl
H
N
O
Cl
H
N
O
Cl
H
N
O
O
H
N
ii
O
H
N
O
H
N
O
O
R2
-CHO
O
O
N
H
N
H
O
N
H
N
H
O
N
H
N
H
H
R
1
-NH2
H
H
(2a-2n)
or
R2
-COCl
NH
O
O
O
N
NH
O
O
O
O
N
NH
O
O
O
N
HO
HO
O
(4a-4k)
HO
HO
HO
HO
O
H
2
N
2
H N
H
2
N
(
2o,2p)
OH
OH
OH
R1
OH
OH
OH
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
3a-3p
5-46
O
CHO
CHO
HO
HO
HO
OH
O
HO
HO
HO
NOH
2
2
2
2
a
N
CHO
F
3
C
CHO
4a
4g
4h
4i
2g
2m
2n
Fmoc
NH
2
OH
O
Cl
O
CHO
OH
HO
Cl
CHO
2h
Si
4b
4c
4d
O
O
HO
HO
HO
O
b
c
HO
OH
NH
NH
2
OH
OH
2
HO OH
CHO
O
HO
HO
HO
O
O
CHO
CHO
2o
2p
O
HO
OH OH
2
2
i
Cl
OH
NH2
NH
2
CHO
O
2d
Cl
H HO O
HO
O
HO
HO
O
j
O
O
4j
N
H
NH
2
CHO
NHAc
NH
2
OH OH
OH OH O
2
2
e
f
CHO
2k
2l
4
e
f
NH
2
4k
N
H
CHO
HO
NH
2
N
CHO
O
OH
OH
4
OH
O
HO
P
NH
2
HOOH OH
O
Scheme 1. Synthesis of new vancomycin derivatives 5-46. Reagents and conditions: i) for
o
synthesis of 3a-3n: (a) 2a-n, DIPEA, DMF, room temperature or 55 C, 2-4h; (b) NaCNBH ,
3
TFA, MeOH, room temperature, 1h (for 3a, additional treatment of 20% piperidine in DMF
o
for 20 min); for synthesis of 3o-3p: (c) 2o-p, DIPEA, DMF, 0 C, 2h; ii) HCHO, 4a-k,
o
DIPEA, H O:MeCN=1:1, -10 C, 12h.
2
To understand how the C-terminus modification influences antibacterial
activity of lipo-vancomycin-carbohydrate structure, we introduced
dimethylaminopropylamine (derived from Dalbavancin) to the
C-terminus carboxylic acid by coupling reaction and synthesized
compound 54-56 (Scheme 3 and Table 2). We also utilized
aminoglycoside antibiotics (kanamycin and amikacin), which consist of
trisaccharide-like motifs, to assemble them onto vancomycin (compound
5
8-63) and test their antibacterial activities (see details in supporting
information).
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 92
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
AcO
AcO
AcO
OAc
HO
HO
HO
OH
O
AcO
AcO
AcO
OAc
O
O
NHCbz
i
ii iii
+
HO
NH
NH
2
O
Cbz
O
SPh
48
4
9
4a
4
7
R
AcO
1
R
1
R
1
HO
AcO
48
+
R
AcO
O
iv
R
AcO
O
ii iii
R
2
O
2
2
OAc
O
O
HO
R
3
R
NHCbz
R
3
NH
2
3
5
0a-c
51a-c
4b-d
50a,51a
R
1
=R
=R
=H R
3
=OAc
R
2
=H
=H
=NHAc
4b
R
1
=R
=R
=H R
3
=OH
R
2
=H
=H
=OH R =NHAc
4
c
R
R
2
3
=OH
R
1
5
0b,51b
0c,51c
R
2
3
=OAc
R
1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
d
1
2
3
5
R
1
2
=OAc R
3
2
H N
O
O
HO
HO
OH OH
O
v,vi
+
HO
N
H
NH
2
OH
BocHN
53a
OH
4j
OH
HO
5
2a
2
H N
O
HO HO
OH OH HO
O
O
O
v,iii
HO
N
H
NH
2
+
O
OH
HO
O
OH
OH
OH
HO
CbzHN
O
52b
53b
4k
HOOH OH
Scheme 2. Synthesis of saccharide intermediates 4a-k. Reagents and conditions: i), NIS,
o
TfOH, MS4A, dry DCM, 0 C to room temperature, 12h; ii), MeOH, NaOMe, room
temperature, 4h; iii), H
2
, Pd-C, MeOH, 2h. iv), BF
3
Et
2 4
O (for 51a, 51b) or SnCl (for 51c), dry
o
MeCN, 0 C to room temperature, 12h; v) CH
3
OH, reflux, 4h; vi）2N HCl, MeOH, room
temperature, 2h.
OH
HN
Cl
Cl
O HH N
Cl
OH
HN
H
3
C
O
HO
H
3
C
O
H
3
C
O
HO
CH
3O
OH
Cl
HO
O
CH
3O
OH
CH
3
O
OH
Cl
O
OH
O
O
O
O
OH
Cl OH
O
O
N
NH
2
O
O
O
O
4
a, 4j
HO
OH
NH
4
m
Cl
H
N
O
HO
OH
NH
HO
OH
NH
O
H
N
Cl
H
N
O
Cl
H
N
O
O
O
H
N
i
O
H
N
ii
O
N
H
N
H
O
O
H
N
O
N
H
N
H
O
N
H
N
H
NH
O
O
O
O
H
N
H
N
R
3
NH
O
O
O
NH
O
O
O
HO
HO
O
R
3
O
2
H N
2
H N
2
H N
HO
OH
OH
OH
OH
HO
OH
OH
R
1
3m
54
55, 56
Scheme 3. Synthesis of vancomycin analogues 54-56. Reagents and conditions: i) DIPEA,
o
O:MeCN = 1:1, -10 C,
HBTU, DMF, room temperature, 24h; ii) HCHO, 4a or 4j, DIPEA, H
2
12h.
NMR characterization of vancomycin analogues
th
Although chemical modification on vancomycin 7 -amino acid resorcinol
via Mannich reaction has been previously reported and NMR chemical
shifts of the aryl protons (Ar-Hs) of the modified position were described
ACS Paragon Plus Environment

Page 11 of 92
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
2
6, 32-34
in the literature
, the detailed assignment of these Ar-Hs with clear
2D spectrum data was lacked and not yet reported. Herein, we presented
the precise characterization with 2D NMR to confirm the assemble
position via Mannich reaction (Figure 2). Ha (6.41 ppm) and Hb (6.25
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
1
1
ppm) of intermediate 3m were assigned based on H NMR, H- H COSY,
1
13
H- C HSQC, H- C HMBC. In the HMBC spectrum (Figure 2, panel
1
13
B), Hb-Cα but no Ha-Cα correlation was observed that clearly
distinguished these two protons. After Mannich reaction, although
chemical shift of Hb (product 46) changed to downfield (6.56 ppm),
Hb-Cα correlation was still clearly identified and the disappearance of Ha
suggested it was substituted during the reaction. This is the first-time
report on the precise identification of vancomycin derivatives via
selective Mannich reaction with solid spectral data and is helpful for
future characterization on compounds of this type.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 92
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
A
2
HO C
α
Ha Hb
H
b
β 7
HO
γ
OH
δHa
3
m
2
HO C
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Hb
α
H
b
β 7
γ
HO
OH
OH
δ
O
OH
N
H
O
HO
OH
4
6
B
3
m：
46：
Hb
Ha
Hb
5
1
1
0
6
8
1
0
H /C
Hb/C
b
α
γ
α
0
00
50
00
Hb
/
/
C
C
H /C
γ
H /C
a
b
ß
120
H
b
δ
H /C
H /C
a
?
b
δ
1
40
60
1
6
.6
6.5
6.4
6.3
6.2
6.1
6
.4
6.3
6.2
6.1
f2 (ppm)
f2 (ppm)
1
Figure 2. NMR characterization and signal assignment of vancomycin analogues. Panel A: H
1 13
NMR spectra of compound 3m (upper) and 46 (bottom); Panel B: H- C HMBC spectra of
compound 3m (left) and 46 (right).
In vitro anti-MSSA and anti-VISA assay of vancomycin analogues
and SAR analysis
With these newly synthesized vancomycin analogues in hands, we sought
to investigate the SAR by in vitro antibacterial assays. Firstly, we chose a
3
5-37
MSSA (Methicillin-susceptible S. aureus) strain (Newman)
and a
37,38
VISA strain (Mu50)
to perform the assay and measure the MIC90
values of our compounds. As shown in Table 3, for
decylaminoethyl-vancomycins (compound 5-14 and Telavancin), extra
ACS Paragon Plus Environment

Page 13 of 92
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
sugar moieties of GalN, Gal, and orGlc indicated 4-16 fold higher
antibacterial activity than other sugars like Glc, GlcN, Man, ManN, orLac,
etc. at a similar MIC level of Telavancin in both Newman and Mu50
9
0
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
strains. For chlorobiphenylmethyl-vancomycins (compound 15-21), GalN,
Gal, and orGlc substitution also led to 8-64 fold higher activity than other
sugars and the phosphonomethylaminomethyl group (derived from
Telavancin) in Newman strain and 4-32 fold higher in Mu50 strain. For
lipophilic structures on vancosamine (compound 22-46), decyl and
biphenyl groups exhibited 8-128 fold better efficacy against MSSA and
VISA than other linear or rigid hydrophobic tails (18/36/40 vs 11; 32 vs
1
3; 46 vs 6). For C-terminus modified compound (54-56), the MIC90
increased to 1-2 ꢀg/mL compared with corresponding unmodified
compound 16 (MIC , 0.06 ꢀg/mL) and 19 (MIC , 0.06 ꢀg/mL), which
90
90
implicated the limited tolerance on C-terminus modification for
biphenyl-vancomycin and suggested the assembling position of extra
th
sugar moieties on 7 -amino acid resorcinol would be better than that on
C-terminus for MSSA and VISA. In summary, the extra sugar attachment
combined with suitable lipophilic modifications is an effective strategy to
develop new vancomycin analogues against MSSA and VISA. The
optimal compounds (18/32/36/40/46) bearing GlcN/Gal/orGlc and
decyl/biphenyl groups achieved the best activities as 64-128 fold higher
than vancomycin, 8-16 fold higher than Telavancin, and comparable
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 92
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
activity with Oritavancin in MSSA and VISA in vitro models.
*
Table 3. In vitro activities of vancomycin derivatives 5-56 against MSSA and VISA
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
MIC90 (ꢀg/ml)
Newman Mu 50
MIC90 (ꢀg/ml)
Newman Mu 50
MIC90 (ꢀg/ml)
Compd.
Compd.
Compd.
Newman
0.25
0.5
Mu 50
0.5
2
5
6
7
8
9
1
2
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
0.5
0.5
0.12
4
0.5
2
37
38
0.5
4
2
16
4
0.5
8
39
2
8
1
40
0.015
0.12
2
0.12
0.5
4
1
4
0.12
1
0.5
2
41
1
0
8
32
8
42
11
4
1
2
43
0.5
2
1
2
0.12
0.5
0.25
2
0.5
1
4
8
44
0.015
0.12
0.03
1
0.12
0.5
0.25
1
13
14
4
8
45
1
1
4
46
1
1
1
1
1
2
5
6
7
8
9
0
4
1
4
54
0.06
0.03
0.03
0.06
0.25
0.25
0.12
0.12
0.12
0.5
0.06
0.5
32
16
0.03
0.25
1
55
2
4
56
1
2
64
32
0.25
Vanco.
Tela.
Orita.
2
8
0.25
0.06
1
0.06
*Newman strain, vancomycin-susceptible Methicillin-susceptible S. aureus(MSSA), ATCC
35-37
5
904 . Mu 50 strain, a Healthcare associated Methicillin-resistant S. aureus(HA-MRSA)
3
7, 38
and vancomycin intermediate resistant S. aureus (VISA) strain isolated in Japan
.
In vitro anti-VRE assay of vancomycin analogues and SAR analysis
Next, we investigated the anti-VRE activities of selected compounds
(
Table 4). Five strains of three different subtypes of VRE (vanA, vanM,
3
9
and vanB) were used for the examination. Based on the results,
compounds containing the rigid hydrophobic tails demonstrated higher
activities than the linear lipophilic structures in all three VRE subtypes,
but extra sugar moieties and C-terminus modification indicated different
influence in three subtypes. For vanA and vanM subtypes, C-terminus
ACS Paragon Plus Environment

Page 15 of 92
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
modification enhanced the anti-VRE activity in 4-8 fold (55 vs 16; 56 vs
1
9); but for vanB, C-terminus modification decreased 8-32 fold. The extra
sugar motifs also implicated complicated influence against different VRE
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
subtypes. For vanA, the optimal sugar of GalN/Gal (compounds
1
6/18/40/46) indicated similar MIC compared with no extra sugar
90
compounds (17/44) and the phosphonomethylaminomethyl compound 21.
For vanM, the extra sugar caused 2-4 fold decrease in activity compared
with no extra sugar compounds (16/19 vs 17; 40/46 vs 44) and 8-64 fold
decrease compared with phosphonomethylaminomethyl compound
(
16/19 vs 21). However, for vanB, the optimal sugar modification
exhibited excellent activities with MIC at 0.06 ꢀg/mL (compound 40
9
0
and 46) which was 1024 fold better than vancomycin, 16 fold of
Telavancin, and 4-fold of Oritavancin. These data suggested VRE
subtypes may have diverse drug-resistant mechanisms that caused the
different response to different vancomycin analogues via various
modification strategies. The systematic SAR studies would help to
understand their individual mechanisms and to develop personalized
treatments. After all, the optimal compounds (15/40/46/55) bearing extra
sugars exhibited dramatically enhanced activities against VREs especially
for vanB subtype.
*
Table 4. In vitro activities of vancomycin derivatives against resistant Enterococci
MIC(ꢀg/ml)
Compd.
vanA
vanM
vanB
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 92
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Efm-HS-0649 Efm-HS-06188 Efm-HS-0847 Efm-HS-08257 (Efm-HS-
vb01)
9
16
64
64
16
2
16
64
64
8
8
32
32
8
1
8
4
10
16
11
8
16
12
13
15
16
8
1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
2
0.12
0.12
4
2
2
0.5
4
1
2
2
16
4
0.12
≤0.06
0.25
1
17
2
1
2
18
19
21
2
2
8
4
4
1
16
2
8
2
4
0.12
4
0.25
0.5
32
2
1
8
36
37
40
44
8
8
32
32
4
16
16
2
0.5
8
8
1
1
1
≤0.06
≤0.06
1
1
1
4
2
45
1
0.5
1
2
1
46
54
55
2
8
2
≤0.06
2
0.5
0.5
1
1
1
0.12
0.5
1
1
2
4
56
2
2
8
Vanco.
Tela.
>128
4
>128
8
>128
4
>128
0.12
0.5
64
1
Orita.
0.25
0.25
1
0.25
*
Efm-HS-0649 and Efm-HS-06188: Glycopeptide-resistant E.faecium, vanA phenotype,
39
isolated in China ; Efm-HS-0847 and Efm-HS-08257: Glycopeptide-resistant E.faecium,
39
vanM phenotype, isolated in China ; Efm-HS-vb01: Glycopeptide-resistant E.faecium, vanB
phenotype, isolated in China.
In vivo antibacterial assay of vancomycin analogues
To test the in vivo efficacy, we established two animal models, lethal
challenge assay and abscess formation assay respectively. Compounds 18,
3
2, 40, 44, and 46 were selected for lethal challenge assay on a mice
40
model infected by a MRSA strain USA300 LAC . Each group contains
1
5 infected mice and was treated with five selected compounds and
ACS Paragon Plus Environment

Page 17 of 92
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
control compounds of vancomycin and Telavancin via i.p. injection at a
single dose of 7 mg/kg. During 10-days monitoring, the survival numbers
of each group were recorded as shown in Figure 3. The survival rate of
vancomycin group after ten days was 6.67% (1/15), while that of
compound 18 group was 93.3% (14/15) as same as Telavancin. Treatment
of compound 46 also led to a survival rate of 86.7% (13/15) and the other
three compounds all indicated much better efficacy than vancomycin.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
For in vivo abscess formation assay, each group contains 12 mice infected
by a VISA strain (Mu50). Compounds 18, 46, vancomycin, and
Telavancin were administrated to the infected mice via twice i.p. injection
at 7 mg/kg dosage in 24 h interval. Five days post infection, the mice
were sacrificed and the liver CFUs (colony-forming units) were counted
for each mouse as shown in Figure 4. Vancomycin group did not shown
significant different in liver CFU level compared with the control group.
While, compound 18, 46, and Telavancin groups significantly reduced the
liver CFU to a similar lower level. These results demonstrated the in vivo
efficacy of our new compounds exceeding vancomycin and achieved
similar activity as the launched drug Telavancin in MRSA and VISA.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 92
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
00
8
0
Negative control
Vancomycin
Telavancin
6
0
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
0
18
32
40
2
0
44
46
0
0
1
2
3
4
5
6
7
8
9
10
Time(day)
Figure 3. Survival chart of USA300 LAC (MRSA) infected mice after treatment with
vancomycin derivatives in an in vivo lethal challenge assay. All compounds and positive
controls were administrated with a single dose of 7 mg/kg via i.p. injection at the first day of
infection.
Figure 4. Liver CFU (colony-forming unit) counts of Mu50 (VISA and MRSA) infected mice
after treatment with vancomycin derivatives in an in vivo abscess formation assay. All
compounds and positive controls were administrated via twice i.p. injection at 7 mg/kg
dosage. Statistical significance determined by the Mann−Whitney test (two-tailed): *p < 0.05,
*
*p < 0.01, n.s. indicates no significant difference. Each symbol represents the value for an
individual mouse. Horizontal bars indicate the observation means, and dashed lines mark the
limits of detection.
ACS Paragon Plus Environment

Page 19 of 92
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
In vivo pharmacokinetic assay of vancomycin analogues
Furthermore, we selected compounds 18, 32, 40, and 46 to investigate
how extra sugar moieties regulate the in vivo pharmacokinetics (PK)
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
properties
in
normal
CD-1
mice
compared
with
trifluoromethylbiphenyl-vancomycin (3m) and control groups of
vancomycin and Telavancin (Table 5). Compound 3m indicated dramatic
increase in T1/2 (~6 h) and AUC (64,194 h*ng/mL) compared with
vancomycin (T1/2 0.6 h, AUC 1242 h*ng/mL) that will cause high
accumulation in organs. While, our four compounds containing extra
sugars reduced half-life in ~2 fold and AUC in 3-6 fold that implicated
the extra sugar motifs are good fragments to enhance the in vivo clearance
and address the concerns of accumulative toxicity. Moreover, comparison
on their PK properties with Telavancin, these compounds exhibited
comparable T1/2 and AUC values that suggested the extra sugar
attachment is a good strategy to improve the drug-like features.
*
Table 5. Pharmacokinetic properties of selected compounds
T
1/2
AUClast
AUCINF_obs
(h*ng/ml)
CL_obs
(mL/min/kg)
1.24±0.12
7.0±1.38
6.4±1.39
3.45±0.40
4.42±1.42
68.7±18
MRTINF_obs
(h)
V
SS_obs
Compd.
(h)
(h*ng/ml)
(mL/kg)
509±39
3
m
6.04±0.30 64194±6049
3.81±0.31 12112±2483
3.72±0.70 10666±2063
3.45±1.26 20105±1714
2.94±1.39 16631±4960
67452±6633
12238±2486
13450±2725
24345±2804
20047±5489
1271±347
6.83±0.24
4.18±0.34
4.79±0.24
4.41±1.23
4.21±2.08
0.79±0.26
1.71±0.13
18
1754±380
1826±334
900±180
1077±449
3076±175
520±121
32
40
46
Vanco. 0.60±0.21
1242±335
Tela.
1.13±0.13 17143±5611
17304±5745
5.1±1.5
*T1/2: half-life; AUClast: area under the concentration-time curve up to last sampling time;
AUCINF_obs:area under the concentration-time curve up to infinity time; CL_obs: clearance;
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 92
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
MRTINF_obs: mean retention time up to infinity time;
state
VSS_obs:volume of distribution at steady
Cytotoxicity assay of vancomycin analogues
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
To evaluate the toxicity of our compounds, we performed cytotoxicity
assays of compound 16 and 46 on human renal proximal tubule epithelial
cells (HK-2) and human liver cell line (HL-7702). As shown in Figure 5,
compound 16 and 46 indicated significant lower toxicity in HL-7702
compared with vancomycin and Telavancin at three tested concentrations.
On HK-2 cells, these two compounds exhibited similar effect in cell
viability as Telavancin and slight reduced toxicity compared with
vancomycin. These data demonstrated the advantage in safety of our
selected compounds.
A
B
cell viability %
1
cell viability %
120
20
16
16
4
6
46
1
00
100
80
Telavancin
Telavancin
Vancomycin
Vancomycin
8
6
4
0
0
0
60
40
(ꢀM)
(ꢀM)
Figure 5. Cytotoxicity assays of selected compounds. Panel A: cell viability test on HL-7702
cells (human liver cells); Panel B: cell viability test on HK-2 cells (human renal proximal
tubule epithelial cells).
Interaction of vancomycin analogue 46 with bacterial peptidoglycan
precursor peptide
Vancomycin binds to the peptidoglycan precursor peptide Lys-dAla-dAla
ACS Paragon Plus Environment

Page 21 of 92
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
on bacterial cell wall therefore breaks the cell wall biosynthesis and kills
3
4, 41-43
the bacteria
. To test whether the attached extra sugar is involved in
this peptide ligand recognition, we investigated the interaction of
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
compound 46 with the peptidoglycan precursor tripeptide
1
13
Ac Lys-dAla-dAla by NMR measurement. Constant-time H- C HSQC
2
NMR analysis was performed for the solo compound 46 and after adding
the precursor tripeptide. The changes on chemical shifts represented the
corresponding signals were involved in the interaction. Figure 6
1
13
summarized all the changed H- C correlation signals observed in this
assay. The signals of x1, x2, x3, x5, V1, and V5 significantly changed or
disappeared which means these positions on original vancomycin
structure interacted with the target tripeptide ligand. Interestingly, we also
observed a signal change of Gal C /H or C /H (these two signals are
3
3
5
5
difficult to distinguish from each other) that suggested the Gal structure
of 46 might also contributed the interaction with the tripeptide. This
results explained a possible mechanism of extra sugar moiety with
enhanced interaction with the bacterial cell wall.
Then, we used the molecular modeling program Maestro (Schrȍdinger) to
investigate the binding mode of 46 and the tripeptide. Molecular
modeling was performed starting with X-ray crystal structure of
vancomycin cocrystallized with the precursor tripeptide ligand (PDB
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 92
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
4
3
code 1FVM ). Vancomycin was replaced with 46 then the complex was
optimized via energy minimalization to give a rational binding model
(
Figure S1 in Supporting Information). In this model, an H-bond
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
interaction of Gal motif with tripeptide was observed. Combined the
modeling result and NMR data, it is very likely the extra sugar moiety
participated the bacterial cell wall binding therefore enhanced the
antibacterial activity. This mechanism is important and helpful for the
future designs on novel vancomycin derivatives against drug-resistant
bacteria.
x1
x2
62.0
62.5
63.0
x3
3
4
4
9
0
1
5
6
9
0
6
3.5
61
.
20
4.15
4.10
4.05
f2 (ppm)
4.00
3.95
4.25
4.20
4.15
4.10
4.5
4.4
4.3
4.2
4.1
f2 (ppm)
f2 (ppm)
5
5
5
5
5
6
7.5
9
6
6
3
x5
V1
V5
8.0
8.5
9.0
9.5
0.0
97
6
4
9
9
8
9
65
5.35
5.30
5.25
5.20
5.15
5.10
4.90
4.85
4.80
4.75
4.70
4.65
4.60
.
65
4.60
4.55
4.50
4.45
f2 (ppm)
f2 (ppm)
f2 (ppm)
CF
3
OH
HN
4
6:
H C
3
O
CH
V1
HO
3O
O
Gal
Gal C /H3
3
V5
OH
Cl
C
4
/H
4
5
Gal C /H5
O
67
OH
6
6
8
9
O
O
Gal
C
2
/H
2
HO
O
OH
Cl
H
N
x5
O
7
7
7
7
0
1
2
3
O
H
N
x3
x2
O
N
N
NH
H
N
H
H
NH
O
O
HO
HO
O O
x1
H
2
N
3
.9
3.8
3.7
3.6
3.5
3.4
f2 (ppm)
OH
OH OH
OHGal
O
N
H
O
OH
HO
Figure 6. Interaction of compound 46 with bacterial peptidoglycan precursor peptide ligand
1
13
2
Ac Lys-dAla-dAla via H- C HSQC NMR measurement. The spectral signals in red
represented the solo compound 46, and the signals in green represented the mixture of 46 and
ACS Paragon Plus Environment

Page 23 of 92
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
peptide ligand. The significantly changed signals of 46 after adding the peptide ligand were
displayed in the selected spectral regions and assigned with their position information (x1, x2,
x3, x5, V1, V5, Gal C2-5/H2-5).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Discussion and Conclusions
Carbohydrate structure of vancomycin plays important roles in the in vivo
4
activity, PK properties, and drug distribution . Although vancomycin
4
45
aglycon also indicated good in vitro antibacterial activity , glycosylation
is still indispensable for vancomycin-based drug development. Previous
studies reported various strategies to replace the vancosamine/glucose
44, 46
with diverse sugar moities by enzymatic glycosylation
or chemical
4
7, 48
synthesis
C-terminus
vancomycin analogue design.
. Recently, assembly of extra sugars on vancomycin
16, 17
th
32
or 7 -amino acid
presented alternative chemistry in
Lipidation on vancosamine is an efficient approach to prepare
19-24
lipo-vancomycin derivatives and has been extensively reported
.
Introducing lipid structures to other hydroxyl groups of vancomycin by
4
9
various catalysts broadened the diversity of lipo-vancomycins . The
advantage of these lipo-analogues comes with dramatically enhanced
antibacterial activities against drug resistant bacteria. Meanwhile, these
compounds usually led to long half lives and brought concerns in
2
5, 26
accumulative toxicities
. Recently launched glycopeptide antibiotics
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 92
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(
Figure 1) feature with lipophilic fragments and extra sugar motifs on
vancomycin core that implicates the balance of lipophilic/hydrophilic
groups is important in the structural design.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Here, we synthesized a vancomycin analogue library with various sugar
motifs and lipophilic structures (Schemes 1 and 3) to understand the SAR
and optimal combination of lipo-fragments and extra sugars. The in vitro
antibacterial assay revealed that the GalN, Gal, and orGlc were the
optimal sugar moieties than other tested sugars for better antibacterial
activity. And decyl or biphenyl substitution indicated better activity than
other lipo-structures when combined with the sugar attachment in
vancomycin modification. The rigid lipo-vancomycin prefers the
th
assembly of extra sugar moieties on 7 -amino acid resorcinol rather than
on C-terminus for anti-MSSA and VISA activities. In vitro anti-VRE
assays on three VRE subtypes indicated different response to C-terminus
modified and extra sugar attached lipo-vancomycin derivatives and
suggested diverse drug-resistant mechanisms of these subtypes. The
optimal vancomycin derivatives exhibited excellent antibacterial activity
against MSSA, VISA, and VRE with 64-1024 fold enhanced activity
compared with vancomycin.
We evaluated in vivo activity of the optimal compounds on two infected
ACS Paragon Plus Environment

Page 25 of 92
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
mice models for lethal challenge assay and abscess formation assay. The
mice were infected by retro-orbital injection with MRSA or VISA strains
37, 50-52
following published literature
. In our previous work, treatment of
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
high-dose vancomycin (225 mg/kg within 108 h) showed an effective
37
therapeutic effect to Mu50 infection . In the current work, since our
compounds exceeded vancomycin in in vitro assays, we lowered the
dosage to 7 mg/kg for comparison. In lethal challenge experiment (Figure
3
) the survival rates of compounds 18 and 46 are significantly higher than
vancomycin. The abscess model is to mimic the S. aureus sepsis, a severe
and life-threatening disease caused by S. aureus infection. The abscess
was gradually developed, and could be most obvious between ~ 96-120
53
hours after infection with S.aurues . We euthanized mice for 5 day to
detect the accumulation of abscess in kidney, heart, and liver. The abscess
in liver is the most sensitive to antibiotics treatment therefore provides a
good model to evaluate the antibacterial efficacy. Compounds 18 and 46
indicated excellent therapeutic effect to relieve liver abscess but
vancomycin did not show significant activity (Figure 4).
Pharmacokinetic data also showed that the extra sugar significantly
regulates half-life and clearance rate of these analogues that proved our
design strategy (Table 5). In the time-course profiles of blood drug
concentration (Figure S3), all the tested compounds maintain > 1 ꢀg/mL
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 92
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
concentrations in blood after 4 h which is significantly higher than their
MIC values. These optimal PK properties explained their remarkable in
vivo efficacy.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
The NMR binding experiment elucidated that the extra sugar motif of
compound 46 is involved in D-Ala-D-Ala ligand interaction. This result
suggested an interesting mechanism of 46 in enhancing bacterial cell wall
attachment. One of the vancomycin-resistant mechanisms is the mutation
of D-Ala-D-Ala to D-Ala-D-Lac on bacterial cell wall that reduced its
14, 15
binding affinity with vancomycin. Bogar group
reported a series of
4
4
novel [Ψ[CH NH]Tpg ] and [Ψ[C(=NH)NH]Tpg ] vancomycin
2
analogues with enhanced binding with D-Ala-D-Lac which demonstrated
fantastic antibacterial activities against VREs. These works implicate the
enhancement of D-Ala-D-Ala/D-Ala-D-Lac binding is a good strategy to
fight against vancomycin-resistant bacteria. The extra sugar in our
analogues could interact with the D-Ala-D-Ala ligand that provides a new
modification strategy in vancomycin derivative design. Another possible
mechanism of these vancomycin analogues with enhanced antibacterial
activities may be due to the conformational change of vancomycin after
5
4
modification. It is reported that subtle alteration on vancomycin
hydroxyl groups may lead to significant change in conformation. On the
other hand, the detailed structural mechanism still remains to elucidate in
ACS Paragon Plus Environment

Page 27 of 92
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
the future.
In summary, we reported here an effective strategy to introduce extra
sugar moieties onto lipo-vancomycin for enhanced drug efficacy, optimal
PK properties, and lower toxicity against drug-resistant bacterial
including MRSA, VISA, and VRE.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Experimental Section
Material and Instrumentation. All reagents and solvents were
commercially purchased from Sinopharm, Bide Pharmatech Ltd. and
Shanghai Titan Ltd. (Shanghai, China) and used without further
purification. Analytical thin layer chromatography (TLC) was performed
on TLC plates precoated with silica gel HSGF254 (200ꢀm±30ꢀm
thickness). Analytic RP-HPLC analysis was performed on a Beijing
Chuang Xin Tong Heng LC-3000 (analytic model) instrument with a
o
C-18 column (5 ꢀm, 4.6 x 150 mm) at 40 C. The column was eluted with
a gradient of 2-90% acetonitrile containing 0.1% TFA in 30 min at a flow
rate of 1 mL/min. Preparative RP-HPLC preparation was performed on a
Beijing Chuang Xin Tong Heng LC-3000 (preparative model) instrument
with a C-18 column (10 ꢀm, 19 x 250 mm) at room temperature. The
column was eluted with a gradient of 2-70% acetonitrile containing 0.1%
TFA in 30 min at a flow rate of 10 mL/min. HPLC analysis showed that
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 92
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
the purity of all the final products were more than 95%. H-NMR,
1
3
C-NMR, HSQC and HMBC spectra were recorded on a BRUKER
TM
Ascend 400 MHz or 600 MHz instrument. Chemical shifts were
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
assigned in ppm and coupling constants were assigned in Hz.
1
Constant-time H-NMR and HSQC were recorded on AVANCE III HD
TM
BRUKER Ascend 600 MHz instrument. All the final products were
added 20ꢀl D O to exchange the active hydrogen of the target products
2
when the solvent was DMSO-d . ESI-MS spectra were measured with an
6
36
Agilent 6230 LC-TOF MS spectrometer. Bacterial strains Newman ,
3
Mu50 and USA300 LAC were from laboratory stock. Efm-HS-0649,
8
40
39
Efm-HS-06188, Efm-HS-0847, Efm-HS-08257 and VanB(R) were
obtained from Institute of antibiotics, Huashan hospital, Fudan University.
Female BALB/c mice were purchased from SIPPR-BK Lab Animal Ltd.
Male CD-1 mice were purchased from Shanghai Lingchang biological
technology co., LTD.
Synthesis and characterization
Aminoethyl glycoside (4a-4d).
Compounds 49 and 51a-51c were dissolved in methanol, and treated with
55
NaOMe to remove acetyls, followed by hydrogenation to generate
4a-4d in 70-90 % yield. 4a and 4d were used directly in next step.
ACS Paragon Plus Environment

Page 29 of 92
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Aminoethyl β-D-galactopyranoside 4b.
1
H NMR (400 MHz, D O) δ 4.31 (d, J = 7.8 Hz, 1H), 3.94 - 3.87 (m, 1H),
2
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
3
.81 (dd, J = 3.4, 1.0 Hz, 1H), 3.72 - 3.67 (m, 1H), 3.67 - 3.64 (m, 1H),
.60 (ddd, J = 6.9, 5.0, 3.9 Hz, 1H), 3.55 (dd, J = 9.9, 3.5 Hz, 1H), 3.43
(
dd, J = 9.9, 7.8 Hz, 1H), 2.94 - 2.87 (m, 2H).
2-Aminoethyl β-D-glucopyranoside 4c.
1
H NMR (400 MHz, D O) δ 4.38 (d, J = 7.8 Hz, 1H), 3.90 (ddd, J = 10.6,
2
5.7, 4.5 Hz, 1H), 3.81 (dd, J = 12.3, 2.3 Hz, 1H), 3.69 (ddd, J = 10.9, 6.1,
4
.6 Hz, 1H), 3.61 (dd, J = 12.3, 5.9 Hz, 1H), 3.44 - 3.33 (m, 2H), 3.29 (d,
J = 9.1 Hz, 1H), 3.19 (dd, J = 9.5, 7.9 Hz, 1H), 2.89 (ddd, J = 6.3, 4.4, 1.7
Hz, 2H).
Glycolactone derivatives (4j-4k).
D-gluconic acid lactone (52a, 2 g, 11.2 mmol) or lactobionolactone (52b,
2
g, 5.9 mmol) were refluxed with N-Boc-1,3-propanediamine (1.2 equiv)
or N-Cbz-1,3-propanediamine (1.2 equiv) in methanol for 4 hours
respectively. The solvent was removed and the residue was washed with
enough ethyl acetate to get rid of excess N-Boc-1,3-propanediamine or
N-Cbz-1,3-propanediamine. The corresponding products were obtained
1
after dried under vacuum (see supporting information for H-NMR
ACS Paragon Plus Environment