Design and synthesis of new vancomycin derivatives

Article abstract of DOI:10.1016/j.bmcl.2014.03.093

A set of vancomycin derivatives with lipid chain attached via a glyceric acid linker was designed and synthesized. A concise synthesis towards these derivatives was developed and the IC₅₀s of these new lipoglycopeptides were tested. Some of them showed very potent activity against both vancomycin sensitive and resistant strains.

Full text of DOI:10.1016/j.bmcl.2014.03.093

Accepted Manuscript
Design and Synthesis of New Vancomycin Derivatives
Wen Gu, Bei Chen, Min Ge
PII:
S0960-894X(14)00329-1
DOI:
http://dx.doi.org/10.1016/j.bmcl.2014.03.093
BMCL 21486
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
4 December 2013
23 March 2014
25 March 2014
Please cite this article as: Gu, W., Chen, B., Ge, M., Design and Synthesis of New Vancomycin Derivatives,
Bioorganic & Medicinal Chemistry Letters (2014), doi: http://dx.doi.org/10.1016/j.bmcl.2014.03.093
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Design and Synthesis of New Vancomycin Derivatives
Wen Gua, Bei Chena, Min Gea,b,*
a College of Biotechnology and Pharmaceutical Engineering, Nanjing University of Technology, Nanjing, 210009, China
b. Acesys PharmaTech, Inc., Science and Technology Building 24B, 5 Xing Mo Fang Road, Nanjing, 210009, China
This is where the receipt/accepted dates will go; Received Month XX, 2000; Accepted Month XX, 2000 [BMCL RECEIPT]
Abstract—A set of vancomycin derivatives with lipid chain attached via a glyceric acid linker was designed and synthesized. A concise
synthesis towards these derivatives was developed and the IC50s of these new lipoglycopeptides were tested. Some of them showed very
potent activity against both vancomycin sensitive and resistant strains.
R1
HO
The glycopeptides antibiotics are the most
important drugs in current use for the treatment of
Gram-positive bacterial infections. They function
NH
OH
O
Me
OH
Me
O
OH
O
O
through binding to the bacterial cell wall substrate D-
Ala-D-Ala and thus inhibiting the transpeptidation step.
In 1990s, vancomycin resistant enterococci (VREs) and
vancomycin-resistant staphylococcus aureus (VRSA)
emerged which posed a very serious public health
problem. This led to new interests in the development
of antibiotics of different class as well as new
derivatives of the glycopeptides.
Cl
O
O
O
Cl
OH
R2
O
O
H
N
O
H
N
H
N
Me
O
N
H
N
H
N
H
H
O
O
HN
O
HO
NH2
C
O
OH
OH
HO
R1=H, R2=H
R1=
vancomycin:
Oritavancin:
NH2
HO
Me
Cl
R2=
O
Me
O
Many different types of vancomycin derivatives
were prepared and studied. The most successful finding
is the discovery by Nagarajan et al that attaching lipid
chain to the vancomycin carbohydrate portion led to
significant potency boost towards both vancomycin
sensitive and resistant strains.[1] These modifications
H2
N
HO
O
CH3
OH
O
O
O
O
NH2
HO
HO
H3C
O
HO
HO
O
OH
O
NH
CH2
OH
O
NHAc
O
O
O
O
O
HO
NHAc
HO
OH
O
P
O
O
OH
O
eventually led to the development of
a
new
HO2C
glycopeptide antibiotic called oritavancin which is now
close to FDA filing (Figure 1). Studies suggest that
these new vancomycin derivatives with lipid chain
attached (lipoglycopeptides) have dual mechanisms by
Moenomycin
Figure 1. Chemical structures of vancomycin, oritavancin and
moenomycin.
inhibiting
both
transpeptidation
and
the
During the development of the lipoglycopeptides,
it was found that these derivatives have poorer
solubility in water compared to vancomycin. In
addition, the greasy chain caused unwanted ion-channel
side effect. How to improve aqueous solubility while
improving the potency is a challenging problem in this
area.
transglycosylation steps of the cell wall biosynthesis.[2]
The alycone portion binds the cell wall substrate and the
lipid carbohydrate portion inhibits the transglycosylases
at the same time. Interestingly, we notice that another
transglycosylase inhibitor – moenomycin (Figure 1) -
also has a similar structure feature where a long lipid
chain is attached to its carbohydrate portion via a
glyceric acid linker. Kahne group has extensively
studied the functions of the C55 lipid chain as well as
the carbohydrate portion.[3] However, the function of
this glyceric acid phosphate linker remains unknown.
We have noticed the similarity between the
lipoglycopeptides and moenomycin. It was interesting
to understand the role of the glyceric acid-phosphate
portion of moenomycin is. Here we designed a set of
vancomycin derivatives with lipid chains attached to the
carbohydrate portion via a similar glyceric acid linker.
Our assumption is that an important structural feature
“borrowed” from a transglycosylase inhibitor should
-------------------------------
* Corresponding author. Tel.: +86 025 83421757; fax: +86 025
83172563; e-mail: minge88@gmail.com
1

O-K+
O
also be helpful to the transglycosylase inhibiting effect
of the vancomycin carbohydrate portion. In addition,
the added polarity of this acid linker should also help to
lower the LogP, improve the solubility and reduce the
unwanted ion channel side effect. On the other hand,
studies on these analogs may also give us some hints of
the role of this glyceric acid linker in moenomycin.
a,b
O
c
R-OH
RO
OMe
OMe
Product
OR
Entry
1
Yield[%]
R=
OMe
O
benzyl
63
O
+K-O
OMe
1a
A straightforward way for making these new
derivatives is through the reductive-amination between
ester 1 and vancomycin (Scheme 1). We tried a few
methods to make this unique ester. At first, we
O
Cl
2
3
chlorobiphenyl methyl
79
66
O
+K-O
1b
O
OMe
n-butyl
+K-O
converted
serine
into
methyl
2-bromo-3-
O
hydroxypropanoate 2 through diazonium reaction and
esterification. However, all attempts to displace the
alfa-bromide with alkoxide only led to the
decomposition of 2. We then tried the Kahne procedure
and prepared the ester 5.[4] However, the final oxidation
of the hydroxyl group failed completely with every
oxidation reagent we tried, presumably due to the
instability of the final product.
1c
O
OMe
n-hexyl
4
55
+K-O
O
1d
aReagents and conditions: (a) chloroacetic acid, NaH, THF; (b) SOCl2,
MeOH; (c) KOtBu, methyl formate, ether.
Next we studied the reductive-amination reaction
between compounds 1a-1d and vancomycin. Following
the known condition (DMF, NaBH3CN, 80℃)[1], we
only obtained the enamine intermediate. Attempts to
reduce this intermediate with various reducing agents all
failed (entry 1, 2, 3, table 2). During the isolation of
this intermediate, we noticed that it was less stable
under acidic conditions. With this in mind, we adjusted
the acidity of the reaction media. A 1:1 mixture of
DMF and acetic acid turned out to be the best reaction
solvent and high temperature (80 ℃) was also needed.
Complete reduction of the enamine intermediate was
achieved to give 6a (entry 4, table 2) [6]. Compounds
6b-6d were synthesized in similar fashion (entry 5-8,
table 2). Thus, a concise synthesis of these vancomycin
derivatives with lipid-glyceric linker was developed.
Further hydrolysis of the methyl esters was achieved
using LiOH to give the final products 7a-7d.[7]
Scheme 1a.
Attempted syntheses of compound 1.
O
O
OH
O
OH
O
a, b
OMe
OMe
OH
OR
Br
NH2
1
2
OH OH OR
OH OH OH
OH
O
c, d, e
f, g
OMe
OR OH OH
OH OH OH
OR
5
4
3
aReagents and conditions: (a) NaNO2, HBr; (b) MeOH, HCl; (c) PMB
dimethoxy ketal, TsOH; (d) RBr, NaH; (e) AcOH, H2O; (f) NaIO4, THF, H2O;
(g) NaClO2, NaH2PO4, H2O, 2-methyl-2-butene; then MeOH, HCl.
After
lots
of
literature
searching
and
experimentation, we found that the condensation of
methyl 2-benzoxy-acetate with methyl formate can
provided the sodium salt of desire product in one single
step (entry 1, table 1).[5] The product is quite stable as
potassium salt. This method can also be applied to
other 2-alkoxy-acetates to give a series of 3-oxy
Table 2a Synthesis of targeted vancomycin derivatives.
propionates.
Proton NMR indicated that these
compounds are in their enolate form which makes them
much more stable.
Table 1a Synthesis of various methyl 2-aloxy-3-oxo-
propanoates.
2

Reaction conditions
Yield[%]
0
Entriy
1
Substrates
Products
MICs
Newman
Entry
Compound
vancomycin
O
O
Mu50
NaBH(OAc)3/
DMF
1a
O
6a
8
2
1
2
2
NH
Vancomycin
chlorobiphenyl
vancomycin
<0.125
NaBH(OAc)3/
THF
0
0
1a
1a
6a
2
3
3
4
8
32
2
6a
6b
NaBH3CN/
DMF
6a
6a
5
6
16
2
64
4
6c
NaBH3CN/
DMF/AcOH
62
1a
4
6d
OH
O
O
O
O
Cl
7
16
64
O
NaBH3CN/
DMF/AcOH
7a
54
80
NH
1b
1c
5
6
Vancomycin
6b
6c
NH
Vancomycin
OH
O
O
O
Cl
O
NaBH3CN/
DMF/AcOH
O
NH
Vancomycin
8
9
<0.125
64
2
NH
Vancomycin
7b
7c
OH
O
O
O
NaBH3CN/
DMF/AcOH
68
O
1d
7
O
128
6d
NH
Vancomycin
NH
aReagents and conditions: (i) NaBH3CN, DMF/AcOH=1/1, 80℃; (b)
LiOH•H2O, THF/H2O.
Vancomycin
O
OH
O
The anti-bacterial activities of these derivatives
were tested against vancomycin sensitive and medium
resistant strains.[8] Their IC50s were listed in table 3.
From the IC50 data, it showed that the new analogs with
longer lipid chain were generally more potent (entry 8
vs. 9, table 3). The acid analogs 7a-7d have similar
activities as the esters 6a-6d. The most potent
compound, 7b, is as active as the known chlorobiphenyl
vancomycin.
NH
10
4
8
7d
Vancomycin
Thus, we have developed a concise synthetic route
towards a set of vancomycin lipid analogs with a
glyceric acid linker. Some of these analogs showed
good activities towards both vancomycin sensitive and
resistant strains. We are actively testing their solubility
as well as their ion-channel binding activities.
Table 3. Sixteen-hour MICs (µg/mL) of vancomycin,
chlorobiphenyl vancomycin and compounds 6a-6d, 7a-
7d for selected bacterial strains. Vancomycin sensitive
strains: Staphylococcus aureus Newman; vancomycin
intermediate resistance strain: Staphylococcus aureus
Mu 50.
Acknowlegement
Financial support for this work was provided by the
Chinese national Science Foundation (21072094/
B0206). We like to thank professor Lefu Lan’s group in
Shanghai Institute of Materia Medica, Chinese
Academy of Science for the biological testing.
References and Notes
1. Nagarajan, R.; Schabel, A. A.; Occolowitz, J. L.;
Counter, F. T.; Ott, J. L.; Felty-Duckworth, A. M. J.
Antibiotics 1989, 42, 63.
2. Ge, M.; Chen, Z.; Onishi, R.; Kohler, J.; Silver, L.;
Kerns, R.; Fukuzawa, S.; Thompson, C.; Kahne, D.
Science 1999, 284, 507-511.
3. (a) Fuse, S.; Tsukamoto, H.; Yuan, Y.; Wang, T.-S. A.;
Zhang, Y.; Bolla, M.; Walker, S.; Sliz, P.; Kahne, D. ACS
Chem. Biol. 2010, 5, 701; (b) Taylor, J. G.; Li, X.;
Oberthür, M.; Zhu, W.; Kahne, D. E. J. Am. Chem. Soc.
3

2006, 128, 15084; (c) Zhang, Y.; Fechter, E. J.; Wang, T.-
S. A.; Barrett, D.; Walker,S.; Kahne, D. E. J. Am. Chem.
Soc. 2007, 129, 3080. (d) Gampe, C. M.; Tsukamoto, H.;
Wang, T.-S. A.; Walker, S.; Kahne, D. Tetrahedron 2011,
67, 9771.
Hz, 1H), 5.55 (s, 1H), 5.43 (s, 1H), 5.24-5.38 m, 3H), 5.11-
5.19 (m, 2H), 4.88 (m, 1H), 4.61 (q, J=11.7 Hz, 1H), 4.51
(s, 2H), 4.43 (d, J=2.8 Hz, 1H), 4.42 (d, J=5.6 Hz, 1H),
4.34 (q, J=7.2 Hz, 1H), 4.19 (d, J=7.5 Hz, 1H), 4.09 (m,
1H), 3.71 (m, 2H), 3.67 (s, 3H), 3.59 (m, 1H), 3.50 (m,
1H), 3.31 (m, 3H), 3.27 (s, 1H), 3.17 (m, 2H), 2.96 (m,
1H),2.78 (m,1H), 2.35 (s, 3H), 2.14 (m, 2H), 1.49-1.59 (m,
4H), 1.10 (s, 3H), 1.04 (s, 3H), 0.89 (d, J=6.3 Hz, 3H),
0.84 (d, J=6.4 Hz, 3H) ppm. HRMS (ESI): calcd for
C83H90Cl3N9O27 1749.5012, found 1750.5076[M+H].
Synthesis of 7b: Compound 7b was synthesized
similar to compound 7a. 1H NMR (300 MHz, DMSO, δ) :
9.44 (s,1H), 9.12 (s, 1H), 8.64 (d, J=2.8 Hz, 1H), 8.48 (d,
J=5.6 Hz, 1H), 8.25 (s, 1H), 8.17 (m, 2H), 7.93 (s, 1H),
7.86 (s, 1H), 7.53-7.63 (m, 4H), 7.47-7.52 (m, 2H), 7.34-
7.42 (m, 4H), 7.18-7.26 (m, 2H), 6.92 (s, 2H), 6.77 (m,
1H), 6.72 (d, J=6.0 Hz, 1H), 6.67 (s, 1H), 6.62 (s, 1H),
6.42 (d, J=2.1 Hz, 1H), 6.26 (d, J=2.1 Hz, 1H), 5.74 (m,
1H), 5.69 (d, J=5.7 Hz, 1H), 5.55 (s, 1H), 5.43 (s, 1H),
5.24-5.38 m, 3H), 5.11-5.19 (m, 2H), 4.88 (m, 1H), 4.61 (q,
J=11.7 Hz, 1H), 4.51 (s, 2H), 4.43 (d, J=2.8 Hz, 1H), 4.42
(d, J=5.6 Hz, 1H), 4.34 (q, J=7.2 Hz, 1H), 4.19 (d, J=7.5
Hz, 1H), 4.09 (m, 1H), 3.71 (m, 2H), 3.59 (m, 1H), 3.50
(m, 1H), 3.31 (m, 3H), 3.27 (s, 1H), 3.17 (m, 2H), 2.96 (m,
1H),2.78 (m,1H), 2.35 (s, 3H), 2.14 (m, 2H), 1.49-1.59 (m,
4H), 1.10 (s, 3H), 1.04 (s, 3H), 0.89 (d, J=6.3 Hz, 3H),
0.84 (d, J=6.4 Hz, 3H) ppm. HRMS (ESI): calcd for
C82H88Cl3N9O27 1735.4855, found 1736.4874[M+H].
4. Adachi, M.; Zhang, Y.; Leimkuhler, C.; Sun, B.;
LaTour, J. V.; Kahne, D. E. J. Am. Chem. Soc. 2006, 128,
14012.
5. Synthesis of 1a: To a solution of methyl 2-
(benzyloxy)acetate (1.0 g, 5.2 mmol, 1 eq) in 6 mL of
anhydrous ether was added potassium tert-butoxide (0.63
g, 5.2 mmol, 1.1 eq) and methyl formate (0.53 g,. 8.8
mmol, 1.7 eq). The mixture was stirred at room
temperature for 15 hours. The precipitated solid was
filtered and washed with ether (15 mL) to give 1a (734mg,
63%) as a white solid. 1H NMR (300 MHz，DMSO, δ) :
8.55 (dd, J=12.4Hz, 1H), 7.30 (m, 5H), 4.52 (s, 2H), 3.40
(s, 3H) ppm. LRMS (ESI): calcd for C11H12KO4 246.0,
found 206.9[M-39].
6. Synthesis of 1b: The synthesis of compound 1b is
1
similar to the synthesis of compound 1a. H NMR (300
MHz，DMSO, δ): 8.57 (s, 1H), 7.48-7.71 (m, 8H), 4.61 (s,
2H), 3.40 (s, 3H) ppm. 13C NMR (75 MHz，DMSO, δ):
166.98, 166.07, 140.23, 139.09, 137.14, 132.08, 128.90,
128.36, 128.24, 126.03, 123.64, 72.65, 48.60 ppm. LRMS
(ESI): calcd for C17H14KClO4 356.0, found 317.0[M-39].
8. IC50 measurement: Media and bacterial strains:
Tryptic Soy Broth (TSB) medium was used to cultivate
Staphylococcus aureus Newman strain (vancomycin
sensitive) and Staphylococcus aureus Mu 50 strain
(vancomycin intermediate resistance).
7. Synthesis of 6a: To a solution of vancomycin (0.74 g,
0.5 mmol, 1 eq) in 40 mL of anhydrous DMF was added
1a (150 mg, 0.6 mmol, 1.2 eq). The mixture was stirred at
80℃ for 2 hours until HPLC indicated the disappearance
of vancomycin. To the mixture was added AcOH (8mL)
and NaBH3CN (63 mg, 1.0mmol, 2 eq). The reaction was
stirred for another 2 hours. The reaction mixture was
directly subjected to preparative reverse phase HPLC
purification to give product 6a (508 mg, 62% yield) as a
white solid. HRMS (ESI): calcd for C77H87Cl2N9O27
1639.5088, found 1640.5133[M+H].
Synthesis of 7a: To a solution of LiOH•H2O (7.2 mg,
0.17 mmol, 10 eq) in 0.57 mL of THF and 0.57 ml of
water was added compound 6a (30 mg, 0.018 mmol, 1 eq).
The mixture was stirred at room temperature for 25
minutes. The reaction was quenched with AcOH (0.1 mL)
and then concentrated under reduced pressure. The residue
was purified by preparative reverse phase HPLC to give
product 7a (19 mg, 64% yield). LRMS (ESI): calcd for
C76H85Cl2N9O27 1625.4932, found 1626.4990 [M+H] and
1648.4827 [M+Na].
MIC (Minimum Inhibitory Concentration) values were
tested for all compounds. Compounds were dissolved with
DMSO to 1.28 mg/mL as stock solution. All samples were
diluted with culture broth to 128 µg/mL as the initial
concentration. Further 1:2 serial dilutions were performed
by addition of culture broth to reach concentrations
ranging from 64 µg/mL to 0.125 µg/mL. 100 µL of each
dilution was distributed in 96-well plates, as well as sterile
controls, growth controls (containing culture broth plus
DMSO, without compounds) and positive controls
(containing culture broth plus vancomycin hydrochloride).
Each test and growth control well was inoculated with 5
µL of a bacterial suspension (about 105 CFU/well). The
96-well plates were incubated at 37 ºC for 16 h. MIC
values of these compounds against Staphylococcus aureus
Newman strain and Staphylococcus aureus Mu 50 strain,
was defined as the lowest concentration to inhibit the
bacterial growth completely.
Synthesis of 6b: Compound 6b was synthesized
similar to compound 6a. 1H NMR (300 MHz, DMSO, δ):
9.12 (s, 1H), 8.64 (d, J=2.8 Hz, 1H), 8.48 (d, J=5.6 Hz,
1H), 8.25 (s, 1H), 8.17 (m, 2H), 7.93 (s, 1H), 7.86 (s, 1H),
7.53-7.63 (m, 4H), 7.47-7.52 (m, 2H), 7.34-7.42 (m, 4H),
7.18-7.26 (m, 2H), 6.92 (s, 2H), 6.77 (m, 1H), 6.72 (d,
J=6.0 Hz, 1H), 6.67 (s, 1H), 6.62 (s, 1H), 6.42 (d, J=2.1 Hz,
1H), 6.26 (d, J=2.1 Hz, 1H), 5.74 (m, 1H), 5.69 (d, J=5.7
4

Graphical abstract
5