Vancomycin disulfide derivatives as antibacterial agents

Article abstract of DOI:10.1016/j.bmcl.2003.11.040

A series of lipidated vancomycin analogues 1 bearing disulfide bonds within their lipid chains was designed and synthesized to optimize their ADME profiles while retaining antibacterial potency. These compounds exhibited good activity against resistant organisms and low accumulation in tissues such as kidney and liver.

Full text of DOI:10.1016/j.bmcl.2003.11.040

Bioorganic & Medicinal Chemistry Letters 14 (2004) 735–738
Vancomycin disulfide derivatives as antibacterial agents
y
YongQi Mu,* Matthew Nodwell, John L. Pace, Jeng-Pyng Shaw and J. Kevin Judice
Theravance, Inc., 901 Gateway Blvd., South San Francisco, CA 94080, USA
Received 5 August 2003; accepted 13 November 2003
Abstract—A series of lipidated vancomycin analogues 1 bearing disulfide bonds within their lipid chains was designed and synthe-
sized to optimize their ADME profiles while retaining antibacterial potency. These compounds exhibited good activity against
resistant organisms and low accumulation in tissues such as kidney and liver.
#
2003 Elsevier Ltd. All rights reserved.
Vancomycin isa glycopeptide antibiotic that iswidely
used in the treatment of Gram-positive bacterial infec-
tions. Over the past decades, resistance to vancomycin
We designed a series of analogues (e.g., 1) bearing dis-
ulfide bondswithin their lipid appendage .s We antici-
pated that these disulfide analogues could be
metabolized to more water-soluble thiol derivatives in
vivo and be excreted in urine. The latent reactive func-
tionality could also allow self-dimerization to form
vancomycin disulfide dimers linked at vancosamine,
which also had the potential for the activity against
1
hasbecome an increa si ng problem. Thishascreated an
urgent need for new clinical agentswith activity against
2
resistant organisms. Over the past 20 years, extensive
research has been done on the chemical modification of
3ꢀ5
vancomycin.
Through thiswork, it hasbeen found
8
,9
that vancomycin derivativesbearing hydrophobic us b-
stituents on the disaccharide moiety have potent activity
against resistant strains of bacteria including methi-
cillin-resistant Staphylococcus aureus (MRSA) and van-
resistant organisms. Herein, we report the synthesis of
vancomycin di us lfide analoguesaswell astheir anti-
bacterial propertiesand rat ADME profiles( Chart 1).
3
,6,7
comycin-resistant enterococci (VRE).
However,
Reductive N-alkylation on the vancosamine was carried
out with aldehydesbearing an un ys mmetrical di us lfide
lipophilic modificationsthrough alkyl-linked groupson
vancosamine impart unfavorable absorption, distribu-
tion, metabolism and excretion (ADME) properties
such as high tissue accumulation and long elimination
1
0
linkage (see Scheme 1). The unsymmetric disulfide bond
wasformed between a thiol-e st er 2 and an alkane-thiol 3
using diethyl azodicarboxylate (DEAD) as the oxidizing
3
,10
11
half-life.
We hypothesized that these unfavorable
agent. Thisreaction proceeded under mild and neutral
ADME propertieswere correlated with the increa es d
hydrophobicity of these analogues, as the parent van-
comycin is not accumulated in tissues and is cleared
quite efficiently in the urine. Our program in thisarea
sought to retain potent antibacterial properties in com-
poundswhile improving ADME profilesrelative to the
parent molecules in this class. One approach to this
would be to introduce a metabolically labile linkage into
the lipid tail, which can be degraded in vivo to a more
hydrophilic product for subsequent excretion.
*
Corresponding author. Tel.: +1-650-808-6028; fax: +1-650-808-
120; e-mail: ymu@theravance.com
Current address: Genetech, Inc., 1 DNA Way, MS#18, South San
Francisco, CA 94080, USA.
6
y
Chart 1. Lipidated vancomycin derivativeswith di su fide bondswithin
lipid appendages.
0
960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.11.040

736
Y. Mu et al. / Bioorg. Med. Chem. Lett. 14 (2004) 735–738
conditionsgiving 4 in good yield. The esters 4 were then
ꢁ
duct 1 substituted at the amino group of vancosamine.12,13
The final productswere purified via rever se d-pha se HPLC
and characterized by ion-spray mass spectrometry.
reduced to aldehydes 5 using DIBAL-H at ꢀ78 C with-
out concomitant reduction of the disulfide bond. Reduc-
tive alkylation of vancomycin with aldehydes 5 could yield
two mono-N-alkylated products, on the vancosamine and
N-methyl leucine residue, as well as one bis-N-alkylated
product. Under our optimized conditions, the highly
selective reductive alkylation of vancomycin yields pro-
In order to study the metabolites of vancomycin dis-
ulfide-lipid analogues, we also prepared compound 8, a
vancomycin dimer linked at vancosamine via a disulfide
linker (Scheme 2). The symmetric disulfide bond was
Scheme 1. General synthesis of vancomycin disulfide analogues. Reagents and conditions: (a) diethyl azodicarboxylate, CH
ꢁ
2
Cl
2
, rt, 1.5 h;
, TFA,
(b) C
methanol, rt, 1 h.
2
H
5
(CH
2
)
m
CH
2
SH 3, rt, 12 h; (c) DIBAL-H, ether, ꢀ78 C, 2 h; (d) vancomycin, diisopropylethylamine, DMF, rt, 1 h; (e) NaCNBH
3
ꢁ
Scheme 2. Synthesis of vancomycin dimer 8. Reagentsand condition s: (a) I , methanol, rt, 5 h; (b) DIBAL-H, ether, ꢀ78 C, 2 h; (c) vancomycin,
2
diisopropylethylamine, DMF, rt, 1 h; (d) NaCNBH , TFA, methanol, rt, 1 h.
3

Y. Mu et al. / Bioorg. Med. Chem. Lett. 14 (2004) 735–738
737
formed by iodine oxidation of thiol-ester 2, followed by
DIBAL-H reduction to give bis-aldehyde 7. Reductive
alkylation of vancomycin with 7 gave disulfide dimer 8.
strains including staphylococci and enterococci were
determined by standard in vitro broth microdilution
susceptibility tests. The data is summarized in Table 1.
Hydrophobic vancomycin monomerswith a d ius lfide
1
4
The antibacterial activitiesof vancomycin di su lfide deri-
vatives against a selection of pathogenic Gram-positive
bond within the lipid tail (1a–e) were aspotent astheir
nitrogen (1f) and carbon (1g) analoguesagain st st aphyl-
ococci and against VRE strains of both the VanB and
VanA phenotypes. Within the disulfide series there was
a trend favoring shorter lipids for activity against
MRSA. Compound 1b, which hasa 12-atom lipid chain
length, was12-fold more active again st MRSA than 1e
Table 1. Antibacterial activity of lipidated vancomycin derivatives
1a–g and metabolite 8
(lipid length of 14 atoms). These compounds were
rapidly bactericidal, retaining a key property of hydro-
phobically substituted glycopeptides. Time–kill curves
against MRSA and VRE are shown in Figure 1. Again,
in comparison with their close analogues bearing an
amine-nitrogen 1f, or the saturated hydrocarbon 1g, the
Compd
X
n
m
MSSA MRSA VanB VanA Fm VanA Fs
1
1
1
1
1
1
1
8
a
b
c
d
e
f
S–S
S–S
S–S
S–S
S–S
N
2
1
1
2
1
1
1
2
5
5
6
6
7
7
7
3.1
0.2
0.3
0.6
1.2
0.4
2.8
0.4
0.2
0.6
1.2
2.4
0.4
1.5
18.8
1.8
2.4
1.2
1.8
0.6
1.8
1.6
4.7
4.7
6.3
6.3
3.1
3.1
4.1
4.1
6.3
3.8
4.7
3.1
3.1
3.1
3.6
3.8
12.5
g
CH
S–S
2
Dimer 6.3
0.7
Vanco na na na
2.0 >256
>256
>256
Figure 1. Bactericidal activity of vancomycin disulfides. Top: time–kill
curvesof MRSA 33591 in the pre se nce of vancomycin derivativesat 4
mg/mL. Bottom: time–kill curvesof VRE st rain MGH-01 in the pre-
sence of vancomcycin derivatives at 10 mg/mL.
Results are expressed as minimal inhibitory concentration (mg/mL).
na, not available.
Figure 2. Possible metabolic pathway for 1a.

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Y. Mu et al. / Bioorg. Med. Chem. Lett. 14 (2004) 735–738
Table 2. Tissue distribution and metabolites of lipidated vancomycin
derivatives 1a, 1f and 1g
References and notes
Compd
X
% dose % dose % dose % dimer 8 % thiol 9
in liver in kidney in urine in urine15 in urine
15
1. (a) Leclerq, R.; Derlot, E.; Duval, J.; Courvalin, P. N.
Engl. J. Med. 1988, 319, 157. (b) Hiramatsu, K. Drug
Resist. Updates 1998, 1, 135.
1
1
1
a
f
g
S–S
N
CH
4.9
37
70
0.5
22
3.4
1
16
0.4
14
na
na
2.3
na
na
2
. (a) Dougherty, T. J.; Barrett, J. F. Curr. Opin. Anti-Infect.
Invest. Drugs 1999, 1, 18. (b) Zhu, J. Expert Opin. Ther.
Pat. 1999, 8, 1005.
2
3
4
. Nagarajan, R. J. Antibiot. 1993, 46, 1181.
. Malabarba, A.; Nicas, T. I.; Thompson, R. C. Med. Res.
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introduction of disulfide linkage into the lipid tail retained
good bactericidal activity against MRSA and VRE.
5
. Niccolai, D.; Tarsi, L.; Thomas, R. J. J. Chem. Soc.,
Chem. Commun. 1997, 2333.
To test the hypothesis that lipidated vancomycin dis-
ulfide analoguescan be cleaved in vivo, we st udied the
rat ADME propertiesof compound 1a aswell asits
nitrogen and hydrocarbon analogues, 1f and 1g respec-
tively. The tissue distribution data was determined 24 h
after a 50 mg/kg dose was administered intravenously to
male Sprague–Dawley rats. The data is summarized in
Table 2.
6
. Cooper, R. D. G.; Snyder, N. J.; Zweifel, M. J.; Staszak,
M. A.; Wilkie, S. C.; Nicas, T. I.; Mullen, D. L.; Futler,
T. F.; Rodriguez, M. J. J. Antibiot. 1996, 49, 575.
7. Setti, E. L.; Quattrocchio, L.; Micetich, R. G. Drugs
Future 1997, 22, 271.
8. Sundram, U.; Griffin, J.; Nicas, T. J. Am. Chem. Soc.
1
996, 118, 13107.
9
. (a) Nicolaou, K. C.; Hughes, R.; Cho, S. Y.; Winssinger,
N.; Smethurst, C.; Labischinski, H.; Endermann, R.
Angew. Chem., Int. Ed. 2000, 39, 3823. (b) Nicolaou,
K. C.; Hughes, R.; Cho, S. Y.; Winssinger, N.; Labi-
schinski, H.; Endermann, R. Chem. Eur. J. 2001, 7, 3824.
As shown above, introducing a disulfide bond into the
lipid appendage had a significant effect on the tissue
distribution. The lipid chains of 1a, 1g and 1f are all 13
atomsin length. However, the accumulationsof 1a in
liver and kidney are much lower than that of 1g and 1f.
Intriguingly, there was15% of dimer 8 observed in
urine along with 1% of parent compound 1a and 2.3%
of free thiol metabolite 9. These findings support our
hypothesis that lipophilic vancomycin disulfides can be
metabolized into more hydrophilic compounds su ch as 8
and 9 for urinary excretion. The thiol 9 wasconcentrated
during urinary production, and allowed to self-dimerize
to form 8 linked via a disulfide bond (Fig. 2). Compound
1
0. Nagarajan, R.; Schabel, A. A.; Occolowitz, J. L.; Coun-
ter, F. T.; Ott, J. L.; Felty-Duckworth, A. M. J. Antibiot.
1989, 42, 63.
11. Mukaiyama, T.; Takahashi, K. Tetrahedron Lett. 1968, 9,
5907.
1
1
2. Judice, K. J.; Fatheree, P. R.; Lam, B. M. T.; Leadbetter,
M. R.; Linsell, M. S.; Mu, Y.; Trapp, S. G.; Yang, G.;
Zhu, Y. US Patent 6,392,012, 2002.
3. General procedure for the reductive alkylation on vanco-
samine: To the mixture of vancomycin (1 mmol, 1 equiv)
and aldehyde (1.3 mmol, 1.3 equiv) in DMF (10 mL) was
added diisopropylethyl amine (2 mmol, 2 equiv). The
8
1
still exhibited antibacterial activity against VRE (Table
). The concentration of 8 in urine iswell above itsMIC
reaction was stirred at rt for 1 h. A solution of NaCNBH
(1 mmol, 1 equiv) in methanol (5 mL) wasadded to the
3
level, which should be effective for the treatment of
VRE-associated urinary tract infections.
reaction, followed by 3 mmol TFA, and stirring was con-
tinued for an additional h. The reaction mixture wascon-
centrated and then precipitated in acetonitrile. Purification
on RP-HPLC gave the desired product as a TFA salt.
4. MIC was determined against clinical isolates of MRSA
In summary, we have successfully synthesized vanco-
mycin derivativesbearing di us lfide bondsin the lipid
tail. These compounds retained good potency and rapid
bactericidal activity against vancomycin-resistant
strains. In vivo ADME studies showed that lipophilic
vancomycin disulfide analogues exhibited an improved
ADME profile in that they could be converted into
more hydrophilic metabolitesand excreted in urine.
1
(
ATCC 33591), MSSA (ATCC 13709) and vancomycin
resistant Enterococcus faecium (KPB-01) and E. faecalis
MGH-01) of Van A and Van B (ATCC 51575) pheno-
(
type utilizing the NCCLS broth microdilution method.
Bactericidal activity was evaluated by time–kill analysis.
15. Calculation wasba se d on the peak area ratio of metabo-
lite peak versus parent peak.