Design, Synthesis, and Antibacterial Activity of Demethylvancomycin Analogues against Drug-Resistant Bacteria

Article abstract of DOI:10.1002/cmdc.201300011

Five novel N-substituted demethylvancomycin derivatives were rationally designed and synthesized by using a structure-based approach. The invitro antibacterial activities against methicillin-resistant Staphylococcus aureus (MRSA), gentamicin-resistant Enterococcus faecalis (GRE), methicillin-resistant Streptococcus pneumoniae (MRS), and vancomycin-resistant Enterococcus faecalis (VRE) were evaluated. One of the compounds, N-(6-phenylheptyl)demethylvancomycin (12a), was found to exhibit more potent antibacterial activity than vancomycin and demethylvancomycin. Compound 12a was also found to be ～18-fold more efficacious than vancomycin against MRSA; however, the two compounds were found to have similar efficacy against MRS. Furthermore, compound 12a exhibited a favorable pharmacokinetic profile with a half-life of 5.11±0.52h, which is longer than that of vancomycin (4.3±1.9h). These results suggest that 12a is a promising antibacterial drug candidate for further preclinical evaluation.

Full text of DOI:10.1002/cmdc.201300011

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DOI: 10.1002/cmdc.201300011
Design, Synthesis, and Antibacterial Activity of
Demethylvancomycin Analogues against Drug-Resistant
Bacteria
Jun Chang,[a] Si-Ji Zhang,[a] Yong-Wei Jiang,[a] Liang Xu,[b] Jian-Ming Yu,[a] Wen-Jiang Zhou,*[c]
and Xun Sun*[a]
Five novel N-substituted demethylvancomycin derivatives were
rationally designed and synthesized by using a structure-based
approach. The in vitro antibacterial activities against methicil-
lin-resistant Staphylococcus aureus (MRSA), gentamicin-resistant
Enterococcus faecalis (GRE), methicillin-resistant Streptococcus
pneumoniae (MRS), and vancomycin-resistant Enterococcus fae-
calis (VRE) were evaluated. One of the compounds, N-(6-phe-
nylheptyl)demethylvancomycin (12a), was found to exhibit
more potent antibacterial activity than vancomycin and deme-
thylvancomycin. Compound 12a was also found to be ~18-
fold more efficacious than vancomycin against MRSA; however,
the two compounds were found to have similar efficacy
against MRS. Furthermore, compound 12a exhibited a favora-
ble pharmacokinetic profile with a half-life of 5.11ꢀ0.52 h,
which is longer than that of vancomycin (4.3ꢀ1.9 h). These re-
sults suggest that 12a is a promising antibacterial drug candi-
date for further preclinical evaluation.
Introduction
Antimicrobial resistance is an emerging threat to global health.
Infections caused by drug-resistant Gram-positive bacteria,
such as methicillin-resistant Staphylococcus aureus (MRSA),
penicillin-resistant Streptococcus pneumoniae (PRSP), and van-
comycin-resistant Enterococcus faecalis (VRE), are the most seri-
ous.[1,2] There is therefore an urgent demand for new antibiot-
ics against drug-resistant bacteria.
and the resulting antibacterial activity of vancomycin is also
much weaker toward VRE than toward more sensitive bacte-
ria.[2,6] According to the proposed resistance mechanisms, strat-
egies for targeting drug-resistant bacteria could include:
1) screening for compounds with new chemical structures and
with novel mechanism(s) of action, 2) modifying the structures
of existing antibiotics and antibacterial drugs, which could be
aided by studying mechanism(s) of action and structure–activi-
ty relationship (SAR) data for the existing drugs, or 3) finding
a substance that can boost the host defense mechanism and/
or attenuate the microbial pathogenicity.
The glycopeptide antibiotic vancomycin (Figure 1) has
a unique history.[3] Regarded by some as the agent of last
resort for refractory Gram-positive bacterial infections; this first
and only commercially available glycopeptide antibiotic has
become the drug of choice for empiric therapy in much of the
world and is more widely used as a generic medication.[4] Its
antibacterial activity appears to result from the inhibition of
enzymatic peptidoglycan transglycosylation and/or transpepti-
dation reactions by binding to d-Ala-d-Ala, the terminus of
bacterial cell wall precursor peptides.[5] However, in the case of
VRE, the terminus of the precursor peptides is d-Ala-d-Lac in-
stead; the affinity of vancomycin for this terminus is only
about 1/1000 of that for the normal terminus (d-Ala-d-Ala),
Recent studies have shown that introducing a hydrophobic
side chain on the nitrogen of the amino sugar moiety in glyco-
peptide antibiotics was able to confer potent activity against
VRE while maintaining potency against MRSA. Telavancin, orita-
vancin, and dalbavancin are successful examples of this.[7,8]
Among these, telavancin was approved by FDA in 2009 for the
treatment of complicated skin and skin structure infections
(cSSSIs) caused by Gram-positive bacteria, including MRSA.
Both dalbavancin and oritavancin are in the late stages of
clinical trials for the treatment of cSSSIs. Telavancin, with
a long decylaminoethyl group as the side chain, has a strong
tendency to form dimers and become anchored to the mem-
brane. As a result, telavancin not only binds tightly to the
d-Ala-d-Ala terminus of the peptidoglycan in vancomycin-sen-
sitive bacterial strains but is also able to bind to the d-Ala-d-
Lac terminus of the peptidoglycan in vancomycin-resistant
bacterial strains.[9–11] On the other hand, oritavancin, with an N-
chlorobiphenyl group as the side chain, has been proposed to
have two cell wall binding sites: the well-known peptidoglycan
d-Ala-d-Ala terminus and the pentaglycyl bridging seg-
ment.[12,13]
[a] Dr. J. Chang, S.-J. Zhang, Y.-W. Jiang, Dr. J.-M. Yu, Prof. Dr. X. Sun
Department of Natural Products Chemistry
School of Pharmacy, Fudan University
826 Zhangheng Road, Shanghai 201203 (China)
E-mail: sunxunf@shmu.edu.cn
[b] Dr. L. Xu
Department of Chemistry of Medicinal Natural Products
West China School of Pharmacy, Sichuan University
Chengdu, Sichuan 610041 (China)
[c] Prof. W.-J. Zhou
Animal Center, School of Pharmacy, Fudan University
826 Zhangheng Road, Shanghai 201203 (China)
E-mail: wjzhou@shmu.edu.cn
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dues and bestow structural fea-
tures for activity against the
drug-resistant bacteria. Thus, we
designed
and
synthesized
a series of N-arylalkyl demethyl-
vancomycin derivatives. These
new compounds (12a–e), along
with our previously reported an-
alogues (12 f–v), were evaluated
in the current study for biologi-
cal activity against the drug-re-
sistant bacteria.
Results and Discussion
Design
The X-ray crystal structure of
vancomycin bound to the pepti-
doglycan of VRE provided the
starting point for the rational
design of new analogues. Molec-
ular docking showed
a large
Figure 1. Chemical structures of the antibacterial glycopeptides.
space between the amino sugar
moiety and the peptidoglycan in
the original vancomycin–pepti-
We recently reported the synthesis of seventeen novel N-
substituted (N-arylmethylene or -aliphatic substituents) deme-
thylvancomycin derivatives and their antibacterial activities
against Clostridium difficile.[14] It was found that several com-
pounds with N-arylmethylene substituents, structurally similar
to oritavancin, showed more potent antibacterial activity
against C. difficile than vancomycin (1) or demethylvancomycin
(2). More interestingly, preliminary data showed that one ana-
logue (i.e., N-undecyl-demethylvancomycin) exhibited potent
antibacterial activity against vancomycin-resistant Enterococcus
faecium (ATCC 700802). This observation suggested that fur-
ther modifications to this analogue, with a long N-alkyl group,
could potentially provide promising drug candidates against
drug-resistant bacteria.
doglycan complex. By retaining multiple hydrogen bonds with
d-Lac and l-Lys,[2] for a seven-carbon side chain, the distance
between the center of the phenyl group (such as in 12a) and
the peptidoglycan Gly4 would be 3.2 ꢁ, potentially creating
a NH–p interaction (Figure 2). Although this NH–p interaction
is not as strong as a hydrogen bonding or ionic interaction, it
may still make a binding energy contribution and could poten-
tially increase the binding affinity.[16] Therefore, we designed
a series of demethylvancomycin derivatives by connecting an
aromatic group to the amino sugar moiety of demethylvanco-
mycin through a seven-carbon linker. We hoped that the NH–p
As a part of our ongoing work to extend the SAR study of
vancomycin and demethylvancomycin, and to elucidate the
corresponding mechanisms of action, we decided to prepare
a set of analogues with long alkyl side chain substitutions at
the nitrogen of the amino sugar moiety of demethylvancomy-
cin. Thermodynamically, orienting the long flexible alkyl chains
might lead to a significant entropic penalty, due to the loss of
both rotational and translational degrees of freedom. However,
if optimal hydrophobic or aromatic fragments are introduced
to gain favorable binding enthalpy, the overall binding
strength can be improved by enthalpy–entropy compensation.
Considering the biphenyl substituted structure of oritavancin,
the reported binding mode of glycopeptide with peptidogly-
can,[12] and the crystal structure of vancomycin,[15] it was rea-
soned that introducing an aromatic ring at the end of an ali-
phatic side chain may enhance the binding affinity of the re-
sulting glycopeptide with the peptidoglycan amino acid resi-
Figure 2. Proposed binding mode of 12a with the peptidoglycan of VRE.
Vancomycin derivative 12a (green) and peptidoglycan (cyan) are shown as
stick models.
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interaction could enhance the binding affinity of demethylvan-
comycin derivatives for the peptidoglycan on the cell wall of
VRE.
Synthesis
The synthesis of aldehydes 9a–e started from commercially
available 1,6-hexanediol (3), as illustrated in Scheme 1. One of
the hydroxy groups of 3 was converted into a bromide by re-
acting with aqueous hydrobromide in toluene to afford 6-
bromo-hexanol (4) in 98% yield. The remaining hydroxy group
of 6-bromo-hexanol (4) was then protected with tetrahydro-
pyranyl (THP) in the presence of pyridinium para-toluenesulfo-
nate (PPTS) in dichloromethane to furnish 5 in 98% yield. Inter-
mediate 5 was heated with tri-
Scheme 1. Synthesis of compounds 9a–e. Reagents and conditions: a) HBr
(1.2 equiv), toluene, reflux, 72 h; b) 3,4-2H-dihydropyran (1.2 equiv), PPTs
(0.1 equiv), CH2Cl2, RT, 4 h; c) 1. PPh3 (1 equiv), neat, 1208C, 1 h, 2. LiHMDS,
RCHO, 08C–RT, THF; d) H2, Pd/C, MeOH, RT, 2 h; e) p-TsOH (1 equiv), MeOH,
RT, 1 h; f) PCC (1 equiv), SiO2 (1 weight equiv), CH2Cl2, RT, 3 h.
phenylphosphine
at 1208C
under neat conditions to form
the corresponding phosphoni-
um salt, which was then reacted
with the respective aldehydes
via a Wittig reaction (lithium
hexamethyldisilazide as a base)
to form 6a–e in 20–30% yield.
It should be noted that there
was no desired phosphonium
salt formation if toluene or
ethyl acetate was used as the
solvent, even at reflux. The re-
sulting compounds, 6a–e, were
subsequently reduced to 7a–
e in 80–98% yield using Pd/C
catalytic hydrogenation at room
temperature. After removing
the THP group of 7a–e with
para-toluenesulfonic acid (p-
TsOH), 8a–e were obtained in
85–98% yield. Intermediates
8a–e were then oxidized with
pyridinium chlorochromate and
silicon dioxide to afford the cor-
responding aldehydes 9a–e in
good yields.
Final analogues 12a–e were
synthesized from demethylvan-
comycin (2) with the methods
reported previously for the syn-
thesis of derivatives 12 f–v
(Scheme 2).[14] First, the N-termi-
nal free amino group of 2 was
protected with a 9-fluorenylme-
thoxycarbonyl (Fmoc) group in
dioxane/H2O (1:1) to afford 10
in 85% yield.[17] Then, com-
pound 10 was reacted with al-
dehydes 9a–e by reductive ami-
nation, using sodium cyanobor-
Scheme 2. Synthesis of compounds 12a–v. Reagents and conditions: a) DIEA (2 equiv), Fmoc-Cl (1.1 equiv) in diox-
ane/H2O (1:1 v/v), RT, 2.5 h; b) 1. aldehyde (5 equiv), DIEA (2 equiv)/DMF, RT, 1 h, 2. NaCNBH3 (3 equiv), TFA
ohydride as the reducing agent, (3 equiv) in MeOH, RT, 8–48 h; c) piperidine (15 equiv) in DMF, RT, 15 min.
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Table 1. In vitro antibacterial activity of compounds 12a–v against methicillin-resistant Staphylococcus aureus (MRSA) and gentamicin-resistant Enterococ-
cus (GRE) strains.
[a]
MIC [mgmLꢁ1
]
Compd
11001[b]
11002[b]
11004[b]
11005[b]
11006[b]
11031[c]
11032[c]
11033[c]
11034[c]
11035[c]
1
2
3.13
0.78
1.56
1.56
0.78
1.56
1.56
3.13
3.13
1.56
3.13
6.25
3.13
3.13
3.13
1.56
6.25
1.56
3.13
1.56
3.13
3.13
3.13
12.5
1.56
1.56
0.78
3.13
0.78
1.56
3.13
3.13
3.13
3.13
6.25
6.25
3.13
3.13
6.25
3.13
6.25
0.78
3.13
1.56
3.13
3.13
3.13
12.5
3.13
3.13
0.78
1.56
1.56
1.56
3.13
3.13
3.13
1.56
6.25
12.5
3.13
3.13
6.25
1.56
6.25
3.13
3.13
6.25
3.13
6.25
3.13
12.5
3.13
3.13
0.78
1.56
1.56
1.56
3.13
3.13
3.13
1.56
3.13
12.5
6.25
6.25
6.25
3.13
25
1.56
3.13
6.25
3.13
6.25
3.13
25
1.56
1.56
0.78
1.56
0.78
0.78
1.56
3.13
3.13
1.56
3.13
6.25
3.13
3.13
3.13
0.78
6.25
1.56
3.13
1.56
3.13
3.13
3.13
>50
>50
12.5
50
25
50
>50
>50
12.5
50
25
50
>50
>50
12.5
>50
50
3.13
3.13
0.78
1.56
1.56
3.13
0.78
3.13
3.13
1.56
3.13
6.25
3.13
3.13
3.13
3.13
6.25
0.78
3.13
3.13
3.13
6.25
6.25
12.5
3.13
1.56
0.78
1.56
1.56
0.78
0.78
3.13
3.13
1.56
6.25
6.25
3.13
3.13
1.56
3.13
6.25
0.78
3.13
3.13
3.13
3.13
3.13
12.5
12a
12b
12c
12d
12e
12 f
12g
12h
12i
12j
12k
12l
12m
12n
12o
12p
12q
12r
12s
12t
12u
12v
50
50
25
25
>50
>50
>50
>50
>50
>50
>50
50
>50
50
12.5
25
>50
>50
>50
>50
>50
>50
>50
50
>50
>50
>50
>50
>50
>50
>50
50
>50
>50
12.5
25
50
>50
12.5
25
50
50
50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
12.5
[a] Minimum inhibitory concentration; data are the mean of results from at least three independent experiments. [b] MRSA strain. [c] GRE strain.
to afford the corresponding N-alkylated Fmoc-demethylvanco-
mycin derivatives 11 a–e in 80–90% yield.[18] Finally, after re-
moving the Fmoc group on 11 a–e with piperidine in DMF at
room temperature, compounds 12a–e were obtained in good
yields after purification by preparative high pressure liquid
chromatography (HPLC). Their chemical structures were unam-
biguously confirmed by spectroscopic analyses, including
1H NMR and MS (ESI-MS and HRESI-MS).
design rationale based on molecular docking (vide supra). It
should also be noted that, among all the analogues tested,
compound 12a exhibited the most promising results in terms
of antibacterial activity against all seventeen Gram-positive
bacterial strains, and was two- to fourfold more potent than
1 and 2. Interestingly, for analogues 12b–e, the antibacterial
activity decreased with aryl group substitutions. Thus, 12a was
chosen for further in vivo evaluation.
In vitro antibacterial activity
In vivo antibacterial activity
Along with vancomycin (1) and demethylvancomycin (2), new
analogues 12a–e and those we reported previously (i.e., 12 f–
v) were evaluated in the current study for in vitro antibacterial
activity against Gram-positive bacteria, including MRSA, GRE,
MRS, and VRE. Minimum inhibitory concentration (MIC) values
were determined using the agar twofold dilution method ac-
cording to CLSI (Tables 1 and 2).
Compound 12a was evaluated for in vivo antibacterial efficacy
in a scalded rat model by smearing the burn wound once
a day (Table 3). The results showed that 12a significantly inhib-
ited MRSA (26003) growth when compared with the scald
MRSA control group at three different doses. Even at 20% of
the dose, compound 12a (80 mgmLꢁ1) still had a similar anti-
bacterial efficacy to vancomycin (400 mgmLꢁ1).
The assay results showed that 12a–e exhibited very good
antibacterial activities against both MRSA and GRE (Table 1). In
fact, they were found to be much more potent than all other
analogues tested in the current study except 12p, suggesting
that a substituted or unsubstituted phenyl group at the end of
a seven-carbon linker is able to confer a stronger antibacterial
effect than N-arylmethylene and N-aliphatic substitutions. This
same activity profile was also observed when the analogues
were tested against both MRS and VRE (Table 2), indicating
that the linker length of a N-substituent is a pivotal determi-
nant for antibacterial potency and is also consistent with our
The in vivo antibacterial activities of 12a against MRSA
(11001) and MRS (11061) in the mouse systemic infection
model were shown in Table 4. Compound 12a was found to
be ~18-fold more efficacious than 1 (ED50: 0.58 mgkgꢁ1 versus
10.49 mgkgꢁ1) against MRSA; however, 12a exhibited a similar
efficacy (ED50: 1.36 mgkgꢁ1) to 1 against MRS.
Pharmacokinetic profile of 12a
The highly potent compound 12a was subjected to pharmaco-
kinetic (PK) performance assessment in SD rats (Table 5). Com-
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and 1211.46ꢀ27.16 mghLꢁ1, re-
spectively. The clearance was
0.07 Lhꢁ1 kgꢁ1, and the Vz was
0.49ꢀ0.05 Lkgꢁ1, which is also
indicative of good PK perfor-
mance.
Table 2. In vitro antibacterial activity of compounds 12a–v against methicillin-resistant Streptococcus pneumo-
niae (MRS) and vancomycin-resistant Enterococcus faecalis (VRE) strains.
[a]
Compd
MIC [mgmLꢁ1
]
11061[b]
11062[b]
12031[c]
12032[c]
12033[c]
12034[c]
12035[c]
1
2
1.56
1.56
0.78
1.56
0.78
0.78
0.195
0.78
1.56
0.78
1.56
3.13
1.56
1.56
1.56
0.78
6.25
0.195
3.13
1.56
3.13
1.56
6.25
6.25
1.56
1.56
0.78
0.78
0.78
0.78
0.78
3.13
3.13
1.56
6.25
12.5
6.25
3.13
6.25
3.13
12.5
3.13
3.13
1.56
6.25
6.25
6.25
25
>50
50
12.5
50
25
25
>50
50
>50
>50
12.5
>50
50
>50
>50
50
50
25
>50
50
12.5
50
25
50
12a
12b
12c
12d
12e
12 f
12g
12h
12i
12j
12k
12l
12m
12n
12o
12p
12q
12r
12s
12t
12u
12v
12.5
>50
>50
>50
50
>50
>50
>50
>50
>50
>50
>50
50
>50
>50
6.25
25
50
50
Acute toxicity studies
>50
50
50
50
The acute toxicity investigation
of 12a showed a regular dose-
dependent increase in mortality
in both sexes of mice after i.v.
administration. The LD50 value
for this compound was calc-
ulated to be 43.3 mgkgꢁ1
(95% confidence limit: 41.139–
45.859 mgkgꢁ1).
25
50
>50
>50
>50
>50
>50
>50
>50
50
>50
50
6.25
25
50
50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
25
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
25
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
50
>50
>50
12.5
25
Pre-test study of long-term
toxicity in rats
50
>50
>50
>50
>50
>50
>50
>50
A two week preliminary toxicity
study for 12a (i.v. 10 mgkgꢁ1
,
once per day) showed no appar-
ent toxicity due to drug-induced
death. There were no abnormal
changes in weight and food
intake (p>0.05; Table 6). The
[a] Minimum inhibitory concentration; data are the mean of results from at least three independent experi-
ments. [b] MRS strain. [c] VRE strain.
Table 3. Staphylococcus aureus counts under scab tissue.
percentage
of
neutrophils
among leukocytes in the 12a-
treated male rats was decreased,
and the percentage of mono-
cytes and eosinophils was in-
creased, especially the percent-
age of reticulocytes (p<0.05),
whereas for the female rats, only
reticulocytes were significantly
increased (p<0.01; Table 7).
Compd
Dose
n
LogꢀSD[a]
[mgmLꢁ1
]
Day 3
Day 7
Day 10
Day 14
12a
12a
12a
1
80
40
20
12
12
12
12
12
12
5.31ꢀ0.76
5.291ꢀ1.20
6.05ꢀ0.45*
5.72ꢀ0.46*
4.19ꢀ0.80
4.87ꢀ0.37
6.13ꢀ0.71*
5.87ꢀ0.38**
6.77ꢀ0.29*
5.62ꢀ0.96*
6.21ꢀ0.10**
8.59ꢀ0.95
5.95ꢀ0.46**
5.94ꢀ0.96*
7.63ꢀ1.22
5.29ꢀ2.11*
8.46ꢀ1.74
9.53ꢀ1.25
6.10ꢀ0.97***
6.60ꢀ0.50***
7.90ꢀ0.39**
6.43ꢀ0.91**
8.66ꢀ1.12
400
Scald group
Scald MRSA group
10.08ꢀ0.61
[a] Significance compared with scald MRSA group: *p<0.05, **p<0.01, ***p<0.001 for n=number of animals
in the group.
There
were
no abnormal
changes found in the liver,
kidney function indicators, or
urine.
Table 4. In vivo antibacterial activity of compound 12a in systemic infec-
tion mouse models.
Conclusions
[a]
Compd
ED50 [mgkgꢁ1
]
In summary, a structure-based approach was taken in the cur-
rent study to design five new N-substituted demethylvancomy-
cin derivatives (12a–e) with an aromatic ring appended to
a seven-carbon linker on the nitrogen of the amino sugar
moiety. When evaluated for in vitro antibacterial activities
against MRSA, GRE, MRS, and VRE, compound 12a was found
to show the most potent antibacterial activity among all com-
pounds tested. When evaluated for in vivo antibacterial activi-
ty, compound 12a was found to be ~18-fold more efficacious
than vancomycin (1) against MRSA; however, the two com-
pounds were found to exhibit similar efficacy against MRS.
MRSA (11001)
MRS (11061)
12a
1
0.58
10.49
1.36
1.56
[a] Systemic infection with methicillin-resistant S. aureus (MRSA) strain
11001 and methicillin-resistant S. pneumoniae (MRS) strain 11061 (see Ex-
perimental Section for details).
pound 12a exhibited a favorable PK profile with a half-life of
5.11ꢀ0.52 h, longer than that of vancomycin (4.3ꢀ1.9 h).[19]
The AUC(0–t) and AUC(0–1) of 12a were 971.24ꢀ41.63 mghLꢁ1
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Table 5. Pharmacokinetic parameters of compound 12a determined in SD rats following i.v. compound administration.[a]
AUC(0–t) [mgLꢁ1 hꢁ1
]
AUC(0–1) [mgLꢁ1 hꢁ1
]
MRT(0–1) [h]
t1/2 [h]
tmax [h]
CL [Lhꢁ1 kgꢁ1
]
Vz [Lkgꢁ1
]
Cmax [mgLꢁ1
]
Rat 1
Rat 2
Rat 3
Mean
SD
923.96
1002.42
987.33
971.24
41.63
1190.86
1201.28
1242.23
1211.46
27.16
6.42
5.05
5.96
5.81
0.70
5.39
4.51
5.43
5.11
0.52
0.01
0.01
0.01
0.01
0.00
0.07
0.07
0.06
0.07
0.00
0.52
0.43
0.50
0.49
0.05
1283.68
1462.38
1029.15
1258.40
217.72
[a] AUC=area under concentration–time curve; MRT=mean residence time.
an IonSpec 4.7 Tesla FTMS (MALDI)
or Bruker Daltonics APEXIII7.0
TESLA FMS (ESI) for high-resolution
mass spectra.
Table 6. Changes in body weight of rats in a pre-test study of long-term toxicity.
Compd
Sex
n
Weight [g][a]
Day 0
Day 4
Day 8
Day 10
Day 12
Day 14
12a
F
M
F
10
10
10
10
163.0ꢀ4.9
165.0ꢀ4.4
164.5ꢀ4.9
166.5ꢀ4.0
179.1ꢀ4.4
191.6ꢀ5.3
180.1ꢀ4.7
194.4ꢀ6.2
190.1ꢀ10.3
214.7ꢀ5.5
193.3ꢀ7.8
221.8ꢀ11.2
195.7ꢀ12.3
234.6ꢀ7.6
200.7ꢀ9.1
238.8ꢀ14.3
204.3ꢀ10.6
249.1ꢀ8.9
209.1ꢀ12.2
253.4ꢀ14.1
209.0ꢀ13.7
247.1ꢀ8.5
212.0ꢀ12.7
261.7ꢀ16.5
6-Bromohexanol (4): 1,6-Hexane-
diol (3, 12.00 g, 66.27 mmol) was
dissolved in toluene (100 mL), and
the solution was heated at 1208C
before the dropwise addition of
48% hydrobromide (15 mL). After
the addition was complete, the re-
action mixture was stirred under
reflux for 72 h. After removing the
solvent under reduced pressure,
the residue was purified by silica
Control
M
[a] Data represent the mean ꢀSD; n=number of animals per group.
Table 7. Blood test data for rats in a pre-test study of long-term toxicity.
Sex
n
Cell type [%][a]
Lymphocyte Monocyte
Reticulocyte Neutrophil
Eosinophil
Basophils
gel
column
chromatography
(EtOAc/petroleum ether (PE), 1:4)
to afford 4 as a light-yellow oil
(11.76 g, 98%): 1H NMR (400 MHz,
CDCl3): d=3.64 (t, J=6.9 Hz, 2H),
3.41 (t, J=6.9 Hz, 2H), 1.87 (m,
2H), 1.58 (m, 2H), 1.48 (m, 2H),
1.38 ppm (m, 2H); MS-ESI: m/z 181
[M+H]+.
12a
F
M
F
10 20.7ꢀ6.2**
10 23.8ꢀ6.7*
19.67ꢀ4.12
74.39ꢀ4.46 4.99ꢀ0.92
0.93ꢀ0.39
0.06ꢀ0.06
14.56ꢀ2.73** 78.72ꢀ2.27 6.01ꢀ1.40** 0.64ꢀ0.30** 0.03ꢀ0.03
Control
10
9.0ꢀ3.7
22.79ꢀ5.61
18.61ꢀ2.86
71.80ꢀ4.90 4.58ꢀ1.12
77.28ꢀ2.76 3.71ꢀ0.86
0.78ꢀ0.36
0.36ꢀ0.19
0.06ꢀ0.06
0.03ꢀ0.03
M
10 17.3ꢀ6.8
[a] Data represent the mean ꢀSD; n=number of animals per group; significance compared with control
group: *p<0.05, **p<0.01.
Compound 12a also exhibited a favorable pharmacokinetic
profile with a half-life of 5.11ꢀ0.52 h, longer than that of van-
comycin (4.3ꢀ1.9 h). These findings strongly suggest that 12a
would be a promising antibacterial drug candidate for further
preclinical evaluation.
2-(6-Bromohexyloxy)tetrahydropyran (5): Compound 4 (13.5 g,
74.60 mmol), pyridinium para-toluenesulfonate (PPTs, 1.87 g,
7.46 mmol), and 3,4-dihydro-2H-pyran (10 mL, 111.84 mmol) were
dissolved in CH2Cl2 (100 mL). After the reaction mixture was stirred
at room temperature for 4 h, it was concentrated in vacuo. The ob-
tained residue was purified by silica gel column chromatography
(EtOAc/PE, 1:100) to give 5 as a light-yellow oil (18.36 g, 98%):
1H NMR (400 MHz, CDCl3): d=4.56 (d, J=4.1 Hz, 1H), 3.85 (t, J=
9.5 Hz, 1H), 3.72 (dd, J=9.5, 6.8 Hz, 1H), 3.49 (m, 1H), 3.38 (m, 3H),
1.84 (m, 3H), 1.70 (t, J=10.6 Hz, 1H), 1.57 (m, 4H), 1.42 ppm (m,
4H); MS-ESI: m/z 265 [M+H]+.
Experimental Section
Chemistry
General: Reagents were purchased from Sigma–Aldrich and TCI
Chemical companies. All solvents were purified and dried in ac-
cordance with standard procedures, unless otherwise indicated.
Oxygen- and water-free operations were carried out under argon
atmosphere in dried glassware unless otherwise noted. Reactions
were monitored by TLC using Yantai (China) GF254 silica gel plates
(5ꢂ10 cm). Silica gel column chromatography was performed on
General procedure for the preparation of 9a–e: Compound 5
(1.0 g, 4.0 mmol) and triphenylphosphine (1.0 g, 4.0 mmol) were
added to a dry three-necked flask (50 mL). After the mixture was
melted at 1208C and stirred for 1 h at this temperature, it was
washed with hot toluene twice and dried to give a pale yellow
phosphonium salt, to which was then added anhydrous THF
(10 mL). The resulting cloudy solution was then stirred at 08C, then
a solution of LiHMDS in THF (4.81 mL, 1 m) was added dropwise.
After the addition was complete, the reaction mixture was further
stirred at room temperature for 1 h, at which time the reaction
mixture became clear. At this point, the corresponding aldehyde
(2.67 mmol) was added at 08C, and after the addition was com-
plete, the reaction mixture was allowed to warm to room tempera-
ture and was stirred for 1 h before quenching with H2O. The reac-
tion mixture was extracted with EtOAc twice, and the combined
1
silica gel (300–400 mesh) from Yantai (China). H NMR spectra were
recorded on Bruker DRX-400 (400 MHz) and Bruker DRX-600
(600 MHz), and chemical shifts (d) were recorded in ppm with cou-
pling constants (J) in hertz (Hz). Splitting patterns describe appar-
ent multiplicities and are designated as s (singlet), d (doublet), t
(triplet), q (quartet), m (multiplet), or br (broad). 13C NMR spectra
data were recorded on Bruker DRX-400 (100 MHz) and Bruker DRX-
600 (150 MHz) spectrometer at room temperature. A Shimadzu
LCMS-2010EV was used for low-resolution mass spectra (ESI) and
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organic layers were washed with brine, dried over anhydrous
Na2SO4, and evaporated in vacuo. The desired product (i.e., 6a–e)
was isolated from the resulting residue using silica gel column
chromatography (CH2Cl2/PE, 1:5 to 1:4) to give colorless oil in 20–
30% yields.
e
(0.05 mmol) was dissolved in DMF (2 mL), and piperidine
(0.079 mL, 0.81 mmol) was added. After the reaction mixture was
stirred for 15 min at room temperature, anhydrous ether (5 mL)
was added. The resulting precipitate was filtered, washed with
EtOAc, and dried in vacuo to give crude product. Further HPLC pu-
rification (gradient eluent: CH3CN/H2O, 5–70% in 0.1% TFA) provid-
ed the desired fractions. The combined fractions were concentrat-
ed to a volume of 20 mL, neutralized with saturated sodium bicar-
bonate, and extracted with nBuOH (3ꢂ20 mL). The organic layers
were separated, washed with H2O, and evaporated in vacuo to dry-
ness to give pure compounds 12a–e as off white solids.
A solution of 6a–e in MeOH (10 mL) and 10% Pd/C (100 mg) were
added to a dried round-bottomed flask (25 mL). The reaction mix-
ture was stirred for 2 h at room temperature under hydrogen.
After filtering with a small amount of silica gel and washing with
CH2Cl2, the solvent in the filtrate was removed in vacuo, and 7a–
e were obtained as colorless oils in 80–98% yields.
N-(6-Phenylheptyl)demethylvancomycin (12a): 23.6%; 1H NMR
(600 MHz, [D6]DMSO): d=7.82 (s, 1H), 7.56 (m, 1H), 7.49 (s, 1H),
7.42 (m, 1H), 7.28 (d, J=8.4 Hz, 1H), 7.17 (d, J=8.4 Hz, 1H), 7.12 (s,
1H), 6.74 (d, J=8.4 Hz, 1H), 6.68 (d, J=8.4 Hz, 1H), 6.37 (d, J=
2.2 Hz, 1H), 6.25 (d, J=2.2 Hz, 1H), 5.73 (s, 1H), 5.63 (s, 1H), 5.32
(d, J=7.8 Hz, 1H), 5.27 (m, 1H), 5.11 (s, 1H), 5.08 (s, 2H), 4.79 (m,
1H), 4.57 (d, J=6.4 Hz, 1H), 4.41 (m, 2H), 4.15 (s, 1H), 4.09 (m, 1H),
4.02 (m, 1H), 3.65 (d, J=9.8 Hz, 1H), 3.55 (d, J=9.8 Hz, 1H), 3.00–
3.50 (5H), 2.65 (m, 2H), 2.58 (m, 1H), 2.51 (m, 1H), 1.80 (d, J=
13.0 Hz, 1H), 1.66 (d, J=13.0, 1H), 1.58 (m, 1H), 1.53 (m, 1H), 1.45
(m, 1H), 1.34 (m, 4H), 1.29 (m, 3H), 1.06 (d, J=6.4 Hz, 3H), 0.87 (m,
6H), 0.80 ppm (m, 6H); 13C NMR (150 MHz, [D6]DMSO) d=172.9,
170.7, 169.5, 168.2, 158.7, 158.5, 157.6, 156.9, 155.4, 152.9, 151.6,
150.3, 148.7, 142.9, 142.6, 136.5, 136.1, 135.4, 132.2, 129.0, 128.7,
128.6, 127.9, 127.5, 126.9, 126.6, 126.1, 125.9, 124.7, 123.8, 121.9,
118.5, 116.6, 108.2, 106.1, 105.2, 102.8, 101.3, 96.9, 78.3, 77.4, 77.3,
72.1, 71.9, 70.6, 69.0, 63.6, 62.3, 61.6, 59.4, 59.1, 57.1, 55.5, 54.1,
51.8, 51.2, 49.0, 39.0, 35.5, 33.1, 31.2, 28.8, 28.8, 26.5, 23.9, 23.2,
22.3, 19.4, 17.3 ppm; MS-ESI: m/z 804.8 [M+2H]2+; HRMS-ESI: m/z
[M+2H]2+ calcd for C78H91Cl2N9O24: 804.7850, found: 804.7862.
Compounds 7a–e were dissolved in 10 mL MeOH (10 mL), and p-
TsOH·H2O (1 equiv) was added. After the reaction mixture was
stirred at room temperature for 1 h, the solvent was removed in
vacuo. Compounds 8a–e were isolated from the resulting residues
using silica gel column chromatography (CH2Cl2/MeOH, 100: 1) as
colorless oils in 85–98% yields.
Pyridinium chlorochromate (1 equiv) and silica gel were added to
a dry two-necked flask (25 mL). After the addition of a solution of
8a–e in CH2Cl2 (2 mL), the reaction mixture was stirred at room
temperature for 4 h before it was filtered through a small amount
of silica gel and washed with CH2Cl2. The filtrate was evaporated in
vacuo to give 9a–e as colorless liquids (yields 85–98%), which
were used directly in the next reaction.
7-Phenylheptanal (9a): 1H NMR (400 MHz, CDCl3): d=9.68 (t, J=
2.2 Hz, 1H), 7.14 (m, 5H), 2.52 (t, J=7.9 Hz, 2H), 2.33 (dt, J=8.0,
2.2 Hz, 2H), 1.54 (m, 4H), 1.28 ppm (m, 4H); MS-ESI: m/z 191
[M+H]+.
1
7-(p-Methoxyphenyl)heptanal (9b): H NMR (400 MHz, CDCl3): d=
9.67 (t, J=2.2 Hz, 1H), 7.08 (d, J=8.6 Hz, 2H), 6.82 (d, J=8.6 Hz,
2H), 3.78 (s, 3H), 2.54 (m, 2H), 2.32 (m, 2H), 1.54 (m, 4H), 1.26 ppm
(m, 4H); MS-ESI: m/z 221 [M+H]+.
N-(6-(p-Methoxyphenyl)heptyl)demethylvancomycin
(12b):
1
19.3%; H NMR (600 MHz, [D6]DMSO): d=7.85 (s, 1H), 7.61 (m, 1H),
7.49 (m, 1H), 7.45 (m, 1H), 7.31 (m, 1H), 7.20 (m, 1H), 7.16 (m, 1H),
7.08 (d, J=8.6 Hz, 2H), 6.83 (m, 2H), 6.77 (m, 1H), 6.71 (m, 1H),
6.40 (d, J=2.1 Hz, 1H), 6.26 (d, J=2.1 Hz, 1H), 5.76 (m, 1H), 5.64
(m, 1H), 5.34 (s, 1H), 5.28 (m, 1H), 5.15 (m, 1H), 5.11 (m, 2H), 4.80
(m, 1H), 4.61 (m, 1H), 4.45 (m, 1H), 4.42 (m, 1H), 4.18 (m, 1H), 4.10
(m, 2H), 3.71 (s, 3H), 3.68 (m, 1H), 3.55 (m, 1H), 3.51 (m, 1H), 3.45
(m, 1H), 3.00–3.50 (4H), 2.77 (m, 1H), 2.68 (m, 1H), 2.40–2.60 (2H),
1.98 (m, 1H), 1.80 (m, 1H), 1.70 (m, 1H), 1.62 (m, 1H), 1.52 (m, 4H),
1.35 (s, 3H), 1.27 (m, 3H), 1.25 (m, 3H), 1.08 (d, J=6.3 Hz, 3H),
0.91 ppm (m, 6H); 13C NMR (150 MHz, [D6]DMSO) d=172.9, 170.7,
169.5, 168.2, 158.7, 158.5, 157.6, 156.9, 155.4, 152.9, 151.6, 150.3,
148.7, 142.9, 136.5, 136.1, 135.4, 134.5, 132.2, 129.6, 129.0, 127.9,
127.5, 126.9, 126.6, 125.9, 124.7, 123.8, 121.9, 118.5, 116.6, 114.1,
108.2, 106.1, 105.2, 102.8, 101.3, 96.9, 78.3, 77.4, 77.3, 72.1, 71.9,
70.6, 69.0, 63.6, 62.3, 61.6, 59.4, 59.1, 57.1, 55.5, 54.1, 51.8, 51.2,
49.0, 39.0, 35.5, 33.1, 31.2, 28.8, 28.8, 26.5, 23.9, 23.2, 22.3, 19.4,
17.3 ppm; MS-ESI: m/z 819.8 [M+2H]2+; HRMS-ESI: m/z [M+2H]2+
calcd for C79H93Cl2N9O25: 819.7903, found: 819.7866.
7-(p-Methylphenyl)heptanal (9c): 1H NMR (400 MHz, CDCl3): d=
9.67 (t, J=2.2 Hz, 1H), 7.08 (d, J=6.6 Hz, 2H), 6.64 (d, J=6.6 Hz,
2H), 2.57 (m, 2H), 2.32 (m, 5H), 1.52 (m, 4H), 1.28 ppm (m, 4H);
MS-ESI: m/z 205 [M+H]+.
7-(p-Fluorophenyl)heptanal (9d): 1H NMR (400 MHz, CDCl3): d=
9.67 (t, J=2.2 Hz, 1H), 7.11 (d, J=8.7 Hz, 2H), 6.95 (d, J=8.7 Hz,
2H), 2.52 (t, J=6.6 Hz, 2H), 2.33 (m, 2H), 1.49 (m, 4H), 1.28 ppm
(m, 4H); MS-ESI: m/z 209 [M+H]+.
1
7-(2-Naphthyl)heptanal (9e): H NMR (400 MHz, CDCl3): d=9.67 (t,
J=2.2 Hz, 1H), 7.78 (m, 3H), 7.61 (s, 1H), 7.43 (m, 2H), 7.33 (m,
1H), 2.78 (m, 2H), 2.33 (m, 2H), 1.71 (m, 2H), 1.57 (m, 2H),
1.38 ppm (m, 4H); MS-ESI: m/z 241 [M+H]+.
General procedure for the preparation of 12a–e: Compound 2
(2.0 g, 1.35 mmol) and 9-fluorenylmethoxycarbonyl chloride (Fmoc-
Cl; 384 mg, 1.49 mmol) were dissolved in dioxane/H2O (1:1, 20 mL),
then DIEA (0.45 mL, 2.70 mmol) was added. The resulting mixture
was stirred at room temperature for 2 h before the addition of
EtOAc. The resulting precipitate was filtered, washed with EtOAc,
and dried in vacuo to give compound 10 in 85% yield. After dis-
solving compound 10 (99 mg, 0.06 mmol) in DMF (2 mL), DIEA
(0.019 mL, 0.114 mmol) and aldehyde 9a–e (4 equiv) were added.
The mixture was stirred at room temperature for 1 h before the ad-
dition of NaBH3CN (10 mg, 0.17 mmol) and TFA (0.012 mL,
0.17 mmol). The reaction mixture was further stirred for 4 days
before the addition of anhydrous ether (5 mL). The resulting pre-
cipitate was filtered, washed with EtOAc, and dried in vacuo to
give compounds 11 a–e in 80–90% yields. Compound 11 a–
N-(6-(p-Methylphenyl)heptyl)demethylvancomycin (12c): 22.3%;
1H NMR (600 MHz, [D6]DMSO): d=7.85 (s, 1H), 7.60 (m, 1H), 7.51 (s,
1H), 7.46 (d, J=8.4 Hz, 1H), 7.32 (d, J=8.4 Hz, 1H), 7.19 (d, J=
8.4 Hz, 1H), 7.16 (s, 1H), 7.06 (q, J=8.2 Hz, 3H), 6.77 (m, 1H), 6.71
(d, J=8.5 Hz, 1H), 6.68 (d, J=8.5 Hz, 1H), 6.40 (d, J=1.9 Hz, 1H),
6.26 (d, J=1.9 Hz, 1H), 5.77 (m, 1H), 5.66 (s, 1H), 5.32 (m, 1H), 5.29
(s, 1H), 5.15 (s, 1H), 5.11 (s, 2H), 4.82 (m, 1H), 4.62 (d, J=6.5 Hz,
1H), 4.46 (m, 1H), 4.43 (m, 1H), 4.19 (m, 1H), 4.13 (m, 1H), 4.06 (s,
1H), 3.69 (d, J=10.2 Hz, 1H), 3.00–3.50 (6H), 2.76 (s, 1H), 2.69 (m,
1H), 2.40–2.60 (2H), 2.25 (s, 3H), 2.13 (m, 1H), 1.98 (m, 1H), 1.79
(d, J=12.8 Hz, 1H), 1.69 (d, J=12.8 Hz, 1H), 1.62 (m, 1H), 1.52 (m,
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4H), 1.35 (s, 3H), 1.25 (m, 6H), 1.09 (m, 3H), 0.90 ppm (m, 6H);
13C NMR (150 MHz, [D6]DMSO) d=173.8, 171.5, 170.4, 169.1, 158.4,
157.8, 156.3, 153.7, 152.4, 151.0, 149.5, 143.8, 140.3, 137.3, 136.1,
135.7, 133.0, 130.0, 129.4, 128.6, 128.4, 127.7, 127.4, 126.7, 125.5,
124.6, 122.8, 119.3, 117.4, 109.0, 106.9, 106.0, 102.1, 97.7, 79.2, 78.3,
78.1, 72.8, 71.4, 69.9, 64.4, 63.1, 62.4, 60.3, 59.9, 57.9, 56.1, 54.9,
52.6, 52.1, 39.0, 35.9, 32.1, 29.6, 27.3, 24.7, 24.1, 23.1, 21.9, 20.3,
18.1 ppm; MS-ESI: m/z 811.8 [M+2H]2+; HRMS-ESI: m/z [M+2H]2+
calcd for C79H93Cl2N9O24: 811.7928, found: 811.7905.
structed and optimized using the AMBER force field. Next, the au-
tomated molecular docking program AutoDock 4.2 (Morris et al.,
1998) was used to dock 12a into the peptidoglycan. By integrating
all available experimental data, the docking pose of the peptido-
glycan–12a complex was obtained, in which the appended phenyl
group could extend to the surface of the peptidoglycan and form
a favorable NH–p interaction with a Gly residue. This binding
mode was consistent with the previous binding model. A series of
the docking parameters were set: the number of generation,
energy evaluation, and docking runs were set to 370000,
1500000, and 10, respectively. The atom types, generations, and
the number of runs for the LGA algorithm were edited and as-
signed according to the requirement of the AMBER force field. Fi-
nally, the docked 12a–peptidoglycan complex was selected by
considering the geometrical complementarity and the lower bind-
ing energy. Specifically, the docked candidate complexes with the
five lowest binding energies were firstly selected, then the confor-
mations with rational geometrical matchings were chosen as the
final complex. These geometrical matchings were consistent with
those in the conserved interaction mode between vancomycin de-
rivatives and peptidoglycan. These complexes were used as the ini-
tial conformation for further geometric optimization.
N-(6-(p-Fluorophenyl)heptyl)demethylvancomycin (12d): 45%;
1H NMR (600 MHz, [D6]DMSO): d=7.85 (s, 1H), 7.59 (s, 1H), 7.51 (m,
1H), 7.46 (d, J=8.4 Hz, 1H), 7.32 (d, J=8.4 Hz, 1H), 7.20 (m, 2H),
7.16 (m, 1H), 7.09 (m, 2H), 6.77 (d, J=10.1 Hz, 1H), 6.70 (d, J=
10.1 Hz, 2H), 6.40 (d, J=2.1 Hz, 1H), 6.26 (d, J=2.1 Hz, 1H), 5.76
(m, 1H), 5.65 (s, 1H), 5.34 (m, 2H), 5.29 (s, 1H), 5.12 (m, 3H), 4.82
(m, 1H), 4.62 (d, J=6.4 Hz, 1H), 4.44 (m, 2H), 4.19 (m, 1H), 4.01 (m,
1H), 3.68 (s, 1H), 3.54 (m, 2H), 3.46 (m, 1H), 3.00–3.40 (6H), 2.76 (s,
1H), 2.69 (s, 1H), 2.40–2.60 (2H), 1.98 (d, J=13.0 Hz, 1H), 1.79 (d,
J=13.0 Hz, 1H), 1.70 (m, 1H), 1.62 (m, 1H), 1.55 (m, 4H), 1.35 (s,
3H), 1.26 (m, 6H), 1.08 (m, 3H), 0.89 ppm (m, 9H); 13C NMR
(150 MHz, [D6]DMSO) d=173.0, 170.7, 169.6, 168.3, 161.7, 158.4,
158.2, 157.6, 157.0, 155.5, 152.9, 151.6, 150.2, 148.7, 143.0, 138.8,
136.5, 136.1, 132.2, 130.4, 130.3, 129.0, 127.8, 127.6, 126.9, 126.6,
125.9, 124.7, 123.8, 122.0, 118.5, 116.6, 115.4, 115.2, 108.2, 106.1,
105.1, 102.8, 101.3, 96.9, 78.4, 77.5, 77.3, 72.0, 70.6, 69.1, 63.6, 62.3,
61.6, 59.5, 59.1, 57.1, 55.3, 54.1, 51.7, 51.4, 46.1, 39.0, 34.6, 33.2,
31.3, 28.8, 28.8, 26.5, 23.9, 23.3, 22.3, 19.4, 17.3 ppm; MS-ESI: m/z
Biological evaluation
Minimum inhibitory concentration (MIC) measurement: The mini-
mum inhibitory concentration (MIC) values were measured to de-
termine the antibacterial activities of the test compounds against
MRSA 11001–11006, GRE 11031–11035, MRS11061–11062, and
VRE12031–12035. Vancomycin and demethylvancomycin were
used as positive controls. MIC values were determined using an
agar dilution method according to the methods of CLSI. Com-
pound stock solutions (320 mgmLꢁ1) were prepared in DMSO/H2O
(50%). Serial twofold dilutions prepared from the stock solutions
with sterile H2O were further diluted tenfold with Mueller–Hinton
(MH) agar medium to obtain a concentration range of 0.024–
50 mgmLꢁ1. The test organisms were grown in MH broth medium
at 358C for 8 h and were adjusted to a turbidity of 0.5 using the
McFarland standard. The bacterial suspensions were inoculated
onto the drug-supplemented MH agar plates with a multipoint in-
oculator and incubated at 358C for 16 h.
813.8 [M+2H]2+
;
HRMS-ESI: m/z [M+2H]2+ calcd for
C78H90Cl2FN9O24: 813.7814, found: 813.7781.
N-(6-(2-Naphthyl)heptyl)demethylvancomycin
(12e): 20.4%;
1H NMR (600 MHz, [D6]DMSO): d=7.85 (m, 4H), 7.67 (s, 1H), 7.63
(m, 1H), 7.50 (d, J=8.4 Hz, 1H), 7.46 (m, 2H), 7.38 (d, J=8.4, 1H),
7.31 (d, J=8.3 Hz, 1H), 7.20 (d, J=8.3 Hz, 1H), 7.16 (s, 1H), 6.76 (d,
J=8.5 Hz, 1H), 6.71 (d, J=8.5 Hz, 1H), 6.40 (d, J=2.0 Hz, 1H), 6.26
(d, J=2.0 Hz, 1H), 5.77 (m, 1H), 5.65 (m, 1H), 5.33 (m, 2H), 5.29 (s,
1H), 5.12 (m, 3H), 4.82 (m, 1H), 4.61 (d, J=6.6 Hz, 1H), 4.44 (m,
2H), 4.19 (m, 1H), 4.11 (m, 1H), 4.04 (m, 1H), 3.69 (d, J=10.4 Hz,
1H), 3.56 (d, J=10.4 Hz, 1H), 3.52 (m, 1H), 3.46 (m, 1H), 3.00–3.30
(5H), 2.75 (m, 1H), 2.70 (s, 1H), 2.40–2.60 (2H), 1.98 (d, J=12.6 Hz,
1H), 1.80 (d, J=12.6 Hz, 1H), 1.70 (m, 1H), 1.62 (m, 1H), 1.55 (m,
4H), 1.35 (s, 3H), 1.26 (m, 6H), 1.08 (m, 3H), 0.89 ppm (m, 9H);
13C NMR (150 MHz, [D6]DMSO) d=173.0, 170.7, 169.6, 168.2, 158.3,
158.1, 157.6, 157.0, 155.5, 152.9, 151.6, 150.2, 148.7, 142.9, 140.3,
136.5, 136.1, 133.6, 132.2, 132.0, 128.9, 128.1, 127.9, 127.8, 127.8,
127.6, 127.5, 126.9, 126.5, 126.4, 126.4, 125.9, 125.6, 124.7, 123.8,
122.0, 118.5, 116.6, 108.1, 106.1, 105.1, 102.8, 101.2, 96.9, 78.3, 77.5,
77.3, 72.0, 70.6, 69.0, 63.6, 62.2, 61.6, 59.5, 59.1, 57.1, 55.3, 54.1,
51.7, 51.3, 46.0, 39.0, 35.7, 31.1, 28.9, 28.9, 26.5, 23.9, 23.2, 22.4,
19.4, 17.4 ppm; MS-ESI: m/z 829.8 [M+2H]2+; HRMS-ESI: m/z
[M+2H]2+ calcd for C82H93Cl2N9O24: 829.7928, found: 829.7923.
MRSA infection following scalding model: Sprague–Dawley (SD) rats
whose back hairs were removed using a shaver and 10% sodium
sulfide were housed in single cages before the experiment. Using
a YLS-5Q super temperature control scalding device, the #3 verte-
bral bit hot head was applied to the skin (1008C, 15 s, 500 g con-
tact pressure) to cause a deep (II) to superficial (III) scald with an
area of 4 cm2. After 20 min, the scald was scribbled with 0.2 mL of
an inoculum containing 109 CFUmLꢁ1 of MRSA (26003). Four hours
after the infection, SD rats were scribbled with test compound 12a
at doses of 20, 40, or 80 mgmLꢁ1; and vancomycin was used as
a positive control. The scalding group and the scalding MRSA
group were subsequently scribbled with saline (0.1 mL per day).
After 3, 7, 10, or 14 days, three SD rats in each group were sacri-
ficed, and the scald scab tissues were removed to prepare HE-
stained specimens (0.3ꢂ0.2 cm), fixed with 10% neutral buffered
formalin. The specimens were then homogenized with saline
(100 mgmLꢁ1) and diluted tenfold with the same saline. A portion
(0.5 mL) of the diluted liquid was dropped into MH medium and
cultured for 20 h in a 378C incubator.
Molecular docking and modeling
Sketching tools in the Discovery Studio 3.0 package were used to
build 3D structures of the demethylvancomycin derivatives using
the crystal structure of (1) as a template. Energy minimizations of
demethylvancomycin derivatives were performed using the Powell
method with Tripos force field and MMFF94 charge in SYBYL 6.9
for 1000 step iterations. The 3D structures of demethylvancomycin
derivatives were characterized by an l-shape (Figure 2). Starting
with a 1.8 ꢁ resolution X-ray co-crystal structure of 1 and the pep-
tidoglycan from VRSA, the 3D structure of peptidoglycan was con-
MRSA and MRS infection model: Mice (Kunming) weighing between
18 and 20 g, were used in the experiment, with 20 mice in each
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group. A lethal systemic infection was given to the mice as 0.5 mL
Keywords: antibiotics
resistance · structure–activity relationships · synthesis design
·
demethylvancomycin
·
drug
of an inoculum of MRSA (11001) or MRS (11061) (107–108 CFUmLꢁ1
)
via intraperitoneal injection. Compound 12a and vancomycin were
administered i.v. 1 h after infection. The ED50 values were calculated
7 days after treatment by the Bliss method.
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Acknowledgements
Financial support from the National Natural Science Foundation
of China (No. 81172920), the National Science and Technology
Major Project of China (No. 2009ZXJ09004-090, 2012ZX09J12108-
05C), and the National Basic Research Program of China (973
Program, No. 2010CB912603) are acknowledged. The authors
thank Prof. Q.-B. Mei and Prof. L. Liu (Shanghai Institute of Phar-
maceutical Industry) for assistance with the antibacterial assays.
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Received: January 7, 2013
Revised: March 12, 2013
Published online on April 10, 2013
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2013, 8, 976 – 984 984