Multivalent Antibiotics via Metal Complexes: Potent Divalent Vancomycins against Vancomycin-Resistant Enterococci

Article abstract of DOI:10.1021/jm030417q

Dimers of vancomycin (Van), linked by a rigid metal complex, [Pt(en)(H ₂O)₂]²⁺, exhibit potent activities (MIC ～0.8 μg/mL, ～720 times more potent than that of Van itself) against vancomycin-resistant enterococci (VRE). The result suggests that combining metal complexation and receptor/ligand interaction offers a useful method to construct multivalent inhibitors.

Full text of DOI:10.1021/jm030417q

4904
J . Med. Chem. 2003, 46, 4904-4909
Mu ltiva len t An tibiotics via Meta l Com p lexes: P oten t Diva len t Va n com ycin s
a ga in st Va n com ycin -Resista n t En ter ococci
Bengang Xing,† Chun-Wing Yu,† Pak-Leung Ho,‡ Kin-Hung Chow,‡ Terence Cheung,‡ Hongwei Gu,†
Zongwei Cai,§ and Bing Xu†,*
Department of Chemistry, The Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong, China,
Center of Infection and Department of Microbiology, Faculty of Medicine, The University of Hong Kong, Pokfulam Road,
Hong Kong, China, and Department of Chemistry, Hong Kong Baptist University, Kowloon Tong, Hong Kong, China
Received August 28, 2003
Dimers of vancomycin (Van), linked by a rigid metal complex, [Pt(en)(H2O)2]2+, exhibit potent
activities (MIC ∼0.8 µg/mL, ∼720 times more potent than that of Van itself) against vancomycin-
resistant enterococci (VRE). The result suggests that combining metal complexation and
receptor/ligand interaction offers a useful method to construct multivalent inhibitors.
In tr od u ction
coordination and receptor/ligand interactions39-42 offers
a useful method to construct multivalent receptors.
Drug resistance1 of bacteria poses a serious public
health threat and demands effective counter measures.
Among promising approaches,2-8 multi/polyvalencys
multiple simultaneous binding of two or more ligands
and receptorssis beginning to be explored system-
atically.9-18 In the research of multivalency, the ligand-
receptor pair of vancomycin (Van)-D-Ala-D-Ala has at-
tracted a great deal of research attention because it
relates to vancomycin19-22-resistant enterococci (VRE).23,24
Walsh and colleagues21,24-27 have deciphered the mech-
anism of vancomycin resistance: VRE mutates its
terminal peptides from D-Ala-D-Ala to D-Ala-D-Lac (i.e.,
D-alanine-D-lactate), which has substantially lowered
(∼103 times decrease) its affinity to Van.25,26,28 Though
Van self-associates to form homodimers upon binding
to D-Ala-D-Ala, as elucidated by Williams et al.,29-33 this
noncovalent dimerization of Van alone is insufficient to
act against VRE. Griffin et al.,17,34 Nicolaou et al.,15,16,19,20
and an Eli Lilly group35 have used organic linkers to
synthesize dimers of Van, and demonstrated that co-
valently linked dimeric Vans exhibit enhanced to potent
activity against VRE. It was, however, suggested that
the flexibility of the organic linker limited the avidity
of the multivalent binding due to the loss of conforma-
tional entropy upon binding.9,36,37 We believe that the
combination of receptor/ligand interaction and a metal
complex, which has special geometry, structural rigidity,
and stability, can serve as an alternative approach to
minimize the loss of conformational entropy.
Resu lts a n d Discu ssion s
We chose [Pt(en)]2+ as the rigid linker to form dimeric
Van via complexation due to its extensive developed
chemistry, well understood properties, and well pre-
served planar rigidity. Moreover, the ionic nature of the
[Pt(en)]2+ retainssif not increasessthe solubility of Van
in aqueous media, which is necessary for in vitro study.
As shown in Scheme 1, commercially available vanco-
mycin (1) reacted with 3-picolylamine, 4-picolylamine,
3-(2-aminoethyl)pyridine, or 1-(3-aminopropyl)imidazole
to give the vancomycin carboxamide derivatives (Van-
CONH-L) 2a -d in good yields (>65%), respectively.
Compounds 2a -d were purified using reversed-phase
HPLC according to modified literature procedure13 and
1
characterized by high field H NMR spectroscopy and
mass spectrometry (MS). The attachment of the ligands
to the C-terminal of Van hardly changes conformation
1
of Van, as indicated by H NMRsthe chemical shifts of
the protons belonging to Van on 2a -d remain es-
sentially the same as that in 1, suggesting that the
binding pockets of 2a -d are undisrupted and should
function similarly as that of 1. [Pt(en)(H2O)2]2+ (3a )
coordinates with 2a -d to give dimeric Vans 4a -d , and
[Pt(en)(H2O)(N-pyridin-3-ylmethylacetamide)]2+ (3b )
binds to 2a ,b to afford monomeric Vans 5a ,b.
As shown in Table 1, 2a -d are inactive against VRE,
and 3a or 3b alone is inactive against both the Van-
sensitive strain and VREs (MIC > 128 µg/mL). A simple
mixture of Van and [Pt(en)(H2O)2](NO3)2 (1+3a ) or the
monovalent complexes of Van and Pt2+(5a or 5b) behave
similarly to the corresponding monomeric Vans. These
results exclude the possibility that enhanced activities
of dimeric Vans against VRE originate from some
unrelated synergistic effects between monomeric Van
and [Pt(en)]2+. 4a -d exhibits enhanced activity against
VRE in comparison to Van. In fact, 4a is ∼103 times
more potent against VRE (genotype VanA) than Van
itself. In an attempt to form the tetravalent compound,
[Zn(2d )4]10+, the activity of the mixture of 2d and Zn-
(OAc)2 (4:1, at pH ) 7) against VRE is the same as that
To test this strategy, we used a derivative of cispl-
atin,38 [Pt(en)(H2O)2]2+(en: ethylenediamine)sa rigid,
square planar metal complexsto form dimeric Vans,
and evaluated their activities against VRE. These
rigidly linked dimeric Vans exhibit enhanced activities
against VRE and are up to ∼720 times more potent
against VRE than Van itself in the best case (MIC: 0.8
µg/mL). Our results suggest that combining metal
* To whom correspondence should be addressed. E-mail:
chbingxu@ust.hk.
† The Hong Kong University of Science and Technology.
‡ The University of Hong Kong.
§ Hong Kong Baptist University.
10.1021/jm030417q CCC: $25.00 © 2003 American Chemical Society
Published on Web 10/10/2003

Multivalent Antibiotics via Metal Complexes
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 23 4905
Sch em e 1. The Structures of Divalent and Monovalent Derivatives of Van
Ta ble 1. Antibacterial Activity (MIC: µg/mL) of Vancomycin
Derivatives and Dimeric Vans
ATCC29212
(sensitive)
E. gall
(VanC)
E. faecium E. faecalis
compound
(VanB)
(VanA)
1
2a
2b
2c
2
2
2
2
8
4
4
2
102
76.8
70.4
19
576
>128
>128
104
64
2d
2
2
22
3a
3b
1+3a
4a
>128
>128
>128
>128
64
>128
>128
160
0.8
>128
>128
F igu r e 1. The illustration of possible divalent interaction of
the dimeric Vans with the terminal peptides of VRE (D-Lac
for VanA and VanB strains; D-Ser for the VanC strains).
4
1
1
8
0.03
0.05
0.28
2
4b
14
4c
4d
5a
5b
1
1
1
1
1
0.5
4
1
1
2
2
2
2
2
1.2
4.8
14
32
22
3.3
38
84
88
60
4
5.5
Let the conformational entropies (∆Sconf) of Van plus the
CONHCH2 segment to be the same as in 4-7, we
compared the ∆Sconf of the different linkers (green
portions) upon dimerization (assuming the ∆Sconf of the
metal complex linker to be zero due to the rigidity of
[Pt(en)]2+). For example, the ∆Sconf of 6 would be the
sum of torsional entropies of two C-C bonds and an
S-S bond (-ΣStor ) 7.3 × 2 + 3.5 ) 18.1 J /mol‚K).36 If
we consider only the contribution of ∆Sconf to the ∆G
and assume that the binding occurs at room tempera-
ture, the ratios of binding constants K4a/Kn of 4a -d , 6,
and 7 can be calculated. In the case of VanA strains
(Table 2), the ratios of binding constants K4a/Kn agree
qualitatively with the MIC values (except 4b). For
example, 4a is ∼5 times more potent than 6,17 which
agrees with their ∆Sconf values. 4c resembles 4a struc-
turally except that it has an extra CH2 increasing the
flexibility its linker; therefore, 4c exhibits lower activity
against VRE. Similarly, additional flexibility in 4d or
2d +Zn 2+
6
7
1
1.6
of 2d , suggesting that there is no multivalent Van
formed by metal complexation when 2d and Zn(OAc)2
react at the condition of the in vitro experiment. We also
did not observe the formation of [Zn(2d )n](2n+2)+ (n )
1-4) by ESI-MS, which is consistent with the observed
activity.
Figure 1 illustrates the plausible divalent interaction
between 4a and the terminal peptides of peptidoglycan
precursors. According to this binding mode, both the
configuration and rigidity of the dimeric Vans determine
their activities.34 To further understand the structural-
activity relationship, we performed semiquantitative
entropy analysis according to the reported methods.36

4906 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 23
Xing et al.
Ta ble 2. Comparison between Conformational Entropies and Activity of Divalent Vans against VRE (VanA and VanB strains)
MIC (µg/mL)
n
∆Sconf(J /mol‚K) (-ΣStor
)
K4a /Kn
VanA
VanB
4a
4b
4c
4d
6
0
0
14.6
29.2
18.1
11.8
1
1
5.9
34
9
0.8
14
3.3
38
4
5.5
0.28
2
1.2
4.8
1
7
4
1.6
(TFA) in water (solution A)] and [0.1% TFA in acetonitrile
(solution B)]. The linear gradient started from 90% solution
A and 10% solution B, changed to 80% solution A and 20%
solution B in 50 min, and to 0% solution A and 100% solution
B in the following 5 min, and then to 90% solution A and 10%
solution B in the next 5 min. The ESI-MS spectra were
obtained on LCQ DECA XP/ESI&APCI (ThermoQuest/Finni-
gan), and the HR ToF-MS spectra of the key compounds, 4a
and 4b, were obtained on Agilent HP1100/Sciex Q-Star Pulsar
I (Applied Biosystem).
7 results in the loss of ∆Sconf upon divalent binding and
the decrease of its activity. Similar qualitative agree-
ment also exhibits between ∆Sconf and the MIC values
in the case of VanB strains. The discrepancy between
K4a/K4b and the MIC of 4b apparently originates from
the configuration of 4b, which contributes to the changes
of enthalpy. Though other mechanisms cannot be ruled
out at this moment, the above analysis supports the
hypothesis of the roles of rigidity in divalency for the
case of VanA and VanB strains.
Syn th esis. Synthesis of dimeric Van 4c: 2.5 mg of [Pt(en)-
(H2O)2](NO3)2 (0.006 mmol, 1.0 equiv) was added to a solution
of 2c (20 mg, 0.0129 mmol, 2.15 equiv) in 1 mL of DMSO,.
The mixture was stirred for 20 h at room temperature in dark.
During the whole procedure, RP-HPLC was used to monitor
the reaction. In the first 3 h, a vancomycin-3-ethylene pyridine-
carboxamide peak exists at the elution time of 24 min. With
increased time, a new peak at 18 min was found and the peak
intensity of the starting material at 24 min was decreased.
After 14 h, RP-HPLC indicated that almost all vancomycin-
3-ethylene pyridine-carboxamide was consumed completely.
Then by quenching the reaction with 10 mL of acetone, a white
solid was precipitated. This crude product was filtered and
washed three times with acetone and dried under vacuum
before it was redissolved in H2O and separated by RP-HPLC.
After purification by HPLC, 15.6 mg of pure product was
obtained (yield: 75.8%): 1H NMR (500 MHz, DMSO-d6) δ 9.18-
(v br s), 9.1(v br s), 8.77(v br s), 8.74(d, 5.5 Hz), 8.67(br s),
8.54(s), 8.30(s), 8.09(d, 7.8 Hz), 7.98 (s), 7.75(overlapped), 7.73-
(d, 7.0 Hz), 7.59(d, 7.8 Hz), 7.57(s), 7.45(d, 8.6 Hz), 7.33(s),
7.31(d, 8.6 Hz), 7.15(v br s), 7.08(d, 10.1 Hz), 6.89(d, 8.5 Hz),
6.82(d, 8.5 Hz), 6.80(v br s), 6.64(v br s), 6.47(s), 6.34(s), 6.09-
(br s), 5.87(d, 7.8 Hz), 5.69(s), 5.57(s), 5.38(s), 5.37(s), 5.35(s),
5.31(br s), 5.04(br m), 4.79(d, 6.2 Hz), 4.57(d, 3.1 Hz), 4.43(br
q, 5.5 Hz), 4.34(d, 10.1 Hz), 4.04(d, 11.0 Hz), 3.37(s), 3.29(s),
2.96(m), 2.75(s), 2.65(d, 4.7 Hz), 2.52(s), 2.27(dd, 16.4 Hz, 6.5
Hz), 2.01(br d, 9.4 Hz), 1.85(br d, 10.6 Hz), 1.79(non 7.0 Hz),
1.69(q, 7.0 Hz), 1.66(q, 7.0 Hz), 1.42(s), 1.18(d, 6.2 Hz), 1.01-
(d, 6.2 Hz), 0.96(d, 6.2 Hz). ESI-MS: The peaks at m/z 1121.3,
1680.4, 1158.6, and 1737.5 correspond to M3+, M2+, (M +
TFA)3+, and (M + TFA)2+, respectively.
∆Sconf correlates, however, to little enhancement of
the activity of dimeric Vans against vancomycin-sensi-
tive strains and the less resistant VRE (VanC), which
has been observed in other systems of divalent
Vans,16,17,34 suggesting the enhancement of the activity
of dimeric Vans may depend on the composition of
peptidoglycan precursors produced by the strains.43
Con clu sion
In summary, we have demonstrated that a metal
complex can be used as a new platform to construct
multivalent inhibitors, which are as effective as other
rigid linkers44-46 used for multivalency. One of the
concerns on platinum-based complexes is its cytotoxicity.
Our preliminary study has shown that these cis-platin-
based divalent Vans are not toxic toward mammalian
cells (the detailed work will be published elsewhere).
Our future work will examine other metal complex
linkers, which may help further elucidate the structural
basis of vancomycin resistance,43,47 as well as the
mechanism of multivalent Vans binding to vancomycin-
sensitive strains,48,49 which has yet to be established.
Exp er im en ta l Section
Gen er a l. Chemical reagents and solvents were used as
received from commercial sources. Dimethyl sulfoxide (DMSO)
was dried over 4 Å molecular sieves and dimethylformamide
(DMF) was dried over silica gel. 1H NMR spectra were obtained
at 500 MHz Varian XL-500 in Me2SO-d6 and D2O. Reversed-
phase HPLC was carried out with Waters 600 Controller and
996 photodiode Array Detector, using XTerra RP18 C18 7 µm
columns for both analytical and preparative purpose. HPLC
elution employed linear gradients of [0.1% trifluoroacetic acid
Synthesis of dimeric Van 4a : Similar to the synthesis of
4c, 2.5 mg of [Pt(en)(H2O)2](NO3)2 (0.0060 mmol, 1.0 equiv)
was added to a solution of 2a (20 mg, 0.0130 mmol, 2.16 equiv)
in 1 mL of DMSO. After being purified by HPLC, 14.0 mg of
pure product was obtained (yield: 69.6%): 1H NMR (500 MHz,
DMSO-d6) δ 9.18(v br s), 9.09(v br s), 8.81(d, 4.9 Hz), 8.76(s),
8.72(s), 8.69(br s), 8.54(v br s), 8.09(d, 8.6 Hz), 7.95 (s), 7.76-

Multivalent Antibiotics via Metal Complexes
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 23 4907
(d, 8.5 Hz), 7.68(d, 8.6 Hz), 7.63(s), 7.59(s), 7.57(s), 7.56(s), 7.43-
(d, 8.6 Hz), 7.39(br s), 7.30(d, 7.3 Hz), 7.11(br s), 6.89(d, 8.6
Hz), 6.82(d, 8.6 Hz), 6.80(v br s), 6.64(v br s), 6.51(s), 6.34(s),
6.00(v br s), 5.87(d, 7.3 Hz), 5.69(s), 5.58(v br s), 5.42(s), 5.37-
(d, 7.3 Hz), 5.35(s), 5.31(br s), 5.21(br s), 5.05(br m), 4.78(q,
7.3 Hz), 4.66(d, 5.6 Hz), 4.59(s), 4.47(br q, 5.6 Hz), 4.40(d, 11.0
Hz), 3.79(d, 10.4 Hz), 3.67(t, 7.8 Hz), 3.57(br s), 3.37(s), 3.27-
(br s), 2.97(s), 2.83(s), 2.26(dd, 15.5, 6.5 Hz), 2.01(br d, 10.4
Hz), 1.80(br d, 10.6 Hz), 1.77(non 7.1 Hz), 1.68(q, 7.1 Hz), 1.62-
(q, 7.1 Hz), 1.41(s), 1.17(d, 6.5 Hz), 1.01(d, 6.5 Hz), 0.96(d, 6.5
Hz). ESI-MS: The peak at m/z 667.3, 833.7, 1111.5, 689.6,
at m/z 972.9 and 1028.5 correspond to M2+ and (M + TFA)2+
,
respectively.
Synthesis of monomeric Van 5b: 6.0 mg of [Pt(en)(H2O)(N-
pyridin-3-ylmethylacetamide)](NO3)2 (0.0124 mmol, 1.0 equiv)
was added to a solution of 2b (20 mg, 0.0130 mmol, 1.05 equiv)
in 1 mL of DMSO. After being purified by HPLC, 15.5 mg of
pure product was obtained (yield: 62.0%): 1H NMR (500 MHz,
DMSO-d6) δ 9.43(v br s), 9.26(v br s), 9.13(v br s), 8.84(s), 8.8-
(d, 4.7 Hz), 8.75(triple), 8.67(d, 5.9 Hz), 8.64(s), 8.62(d, 4.2 Hz),
8.28(v br s), 8.07(d, 8.2 Hz), 7.93 (s), 7.91(d, 7.6 Hz), 7.80(s),
7.77(multiple), 7.75(v br s), 7.67(d, 8.8 Hz), 7.66(v br s), 7.63
(v br s), 7.58(d, 5.3 Hz), 7.57(s), 7.56(s), 7.43(d, 8.2 Hz), 7.34-
(s), 7.29(d, 8.2 Hz), 7.12(br s), 6.86(d, 8.8 Hz), 6.82(d, 8.8 Hz),
6.79(v br s), 6.64(overlapped), 6.50(d, 2.34 Hz), 6.35(d, 2.3 Hz),
6.00(v br s), 5.87(d, 8.1 Hz), 5.68(v br s), 5.36(s), 5.35(s), 5.33-
(d, 3.7 Hz), 5.29(br s), 5.22(br s), 5.00(br m), 4.78(q, 6.6 Hz),
4.72(d, 5.9 Hz), 4.55(d, 5.1 Hz), 4.47(br q, 5.6 Hz), 4.43(d, 5.9
Hz), 4.39(d, 11.0 Hz), 4.05(v br s), 3.79(d, 11.0 Hz), 3.66(t, 8.8
Hz), 3.55(br s), 3.39(s), 3.29(br s), 2.75(s), 2.26(dd, 15.1 Hz,
6.5 Hz), 2.01(overlapped), 1.82(br d, 10.6 Hz), 1.77(non 7.1 Hz),
1.68(q, 7.1 Hz), 1.62(q, 7.1 Hz), 1.41(s), 1.17(d, 6.4 Hz), 1.01-
(d, 6.5 Hz), 0.96(d, 6.5 Hz). ESI-MS: The peaks at m/z 648.9,
862.2, and 1149.6 correspond to M5+, M4+, M3+, (M + TFA)5+
,
(M + TFA)4+, and (M + TFA)3+, respectively.
Synthesis of dimeric Van 4b: 2.5 mg of [Pt(en)(H2O)2](NO3)2
(0.0060 mmol, 1.0 equiv) was added to a solution of 2b (20
mg, 0.0130 mmol, 2.16 equiv) in 1 mL of DMSO. After being
purified by HPLC, 15.3 mg of pure product was obtained
(yield: 76.1%): 1H NMR (500 MHz, DMSO-d6) δ 9.43(v br s),
9.26(v br s), 9.13(v br s), 8.84(s), 8.8(d, 4.7 Hz), 8.75(triple),
8.67(d, 5.9 Hz), 8.64(s), 8.62(d, 4.2 Hz), 8.28(v br s), 8.07(d,
8.2 Hz), 7.93 (s), 7.91(d, 7.6 Hz), 7.80(s), 7.77(multiple), 7.75-
(v br s), 7.67(d, 8.8 Hz), 7.66(v br s), 7.63 (v br s), 7.58(d, 5.3
Hz), 7.57(s), 7.56(s), 7.43(d, 8.2 Hz), 7.34(s), 7.29(d, 8.2 Hz),
7.12(br s), 6.86(d, 8.8 Hz), 6.82(d, 8.8 Hz), 6.79(v br s), 6.64-
(overlapped), 6.50(d, 2.34 Hz), 6.35(d, 2.3 Hz), 6.00(v br s), 5.87-
(d, 8.1 Hz), 5.68(v br s), 5.36(s), 5.35(s), 5.33(d, 3.7 Hz), 5.29(br
s), 5.22(br s), 5.00(br m), 4.78(q, 6.6 Hz), 4.72(d, 5.9 Hz), 4.55-
(d, 5.1 Hz), 4.47(br q, 5.6 Hz), 4.43(d, 5.9 Hz), 4.39(d, 11.0 Hz),
4.05(v br s), 3.79(d, 11.0 Hz), 3.66(t, 8.8 Hz), 3.55(br s), 3.39-
(s), 3.29(br s), 2.75(s), 2.26(dd, 15.1 Hz, 6.5 Hz), 2.01-
(overlapped), 1.82(br d, 10.6 Hz), 1.77(non 7.1 Hz), 1.68(q, 7.1
Hz), 1.62(q, 7.1 Hz), 1.41(s), 1.17(d, 6.4 Hz), 1.01(d, 6.5 Hz),
0.96(d, 6.5 Hz). ESI-MS: The peaks at m/z 834.16, 1111.15,
and 1666.25 correspond to M4+, M3+, and M2+, respectively.
971.9, and 1028.5 correspond to M3+, M2+, and (M + TFA)2+
respectively.
,
Synthesis of dimeric Van 6: Following to the same procedure
as for 2a , 6.8 mg of cystamide dihydrochloride (30 µmol, 1.0
equiv) was added to a solution of vancomycin hydrochloride
(100 mg, 67 µmol, 2.2 equiv) in 1 mL of dry DMSO. The
mixture was cooled to 0 °C and HBTU (90 µmol, 3 equiv) in 1
mL of DMF was added, followed by DIEA (0.057 mL, 328 µmol,
4.88 equiv). The reaction was allowed to rise to room temper-
ature and stirred for overnight. At this time, analytical RP-
HPLC showed that a vancomycin peak still existed. Further
addition of HBTU (10 mg, 26 µmol, 0.39 equiv) and DIEA
(0.024 mL, 164 µmol, 2.44 equiv) was made. After another 24
h, the reaction was monitored with HPLC again and almost
all vancomycin was found to have been consumed. To quench
the reaction, the reaction mixture was added dropwise into
15 mL of acetone by using syringe. A white solid was
precipitated out and filtered, and 5 mL of acetone was used to
wash the solid once. The white solid was purified by reversed-
phase HPLC (RP-HPLC). The percentage yield is 52%. 1H
NMR (500 MHz, DMSO-d6) δ 9.88(v br s), 9.44(v br s), 9.10(v
br s), 8.71(br s), 8.51(br s), 8.27(d, 5.1 Hz), 7.98 (s), 7.83-
(overlapped), 7.69(d, 7.8 Hz), 7.56(d, 8.6 Hz), 7.43(d, 8.6 Hz),
7.31(d, 8.6 Hz), 7.29 (s), 7.09(v br s), 6.86(d, 8.6 Hz), 6.82(d,
8.6 Hz), 6.80(v br s), 6.62(v br s), 6.49(s), 6.35(s), 6.07(v br s),
5.87(d, 7.8 Hz), 5.85(s), 5.69(s), 5.50(v br s), 5.41(s), 5.39(s),
5.37(s), 5.35(d, 2.8 Hz), 5.30(br s), 5.03(br m), 4.78(d, 6.2 Hz),
4.53(br q, 5.5 Hz), 4.32(d, 10.9 Hz), 4.03(s), 3.79(d, 10.9 Hz),
3.37(s), 3.27(s), 3.00(m), 2.72(s), 2.26(dd, 16.1 Hz, 6.3 Hz), 2.07-
(m), 2.01(br d, 9.4 Hz), 1.82(br d, 10.4 Hz), 1.79(non 7.0 Hz),
1.74(q, 7.0 Hz), 1.67(q, 7.0 Hz), 1.44(s), 1.18(d, 6.7 Hz), 1.02-
(d, 6.2 Hz), 0.97(d, 6.2 Hz). ESI-MS: The peak at m/z 1508.8
Synthesis of dimeric Van 4d : Again, similar to the synthesis
of 4c, 2.5 mg of [Pt(en)(H2O)2](NO3)2 (0.0060 mmol, 1.0 equiv)
was added to a solution of 2d (20 mg, 0.0128 mmol, 2.15 equiv)
in 1 mL of DMSO. After being purified by HPLC, 16.0 mg of
pure product was obtained (yield: 78.8%): 1H NMR (500 MHz,
DMSO-d6) δ 9.47(v br s), 9.19(s), 9.12(v br s), 8.79(br s), 8.63-
(s), 8.58(br s), 8.42(s), 8.25(d, 6.2 Hz), 7.94 (s), 7.78(s), 7.77-
(overlapped), 7.70(s), 7.67(d, 8.6 Hz), 7.59(s), 7.57(d, 7.8 Hz),
7.45(d, 8.6 Hz), 7.40(s), 7.31(d, 8.6 Hz), 7.15(v br s), 6.88(s),
6.87(d, 8.2 Hz), 6.82(d, 8.2 Hz), 6.80(v br s), 6.64(v br s), 6.48-
(s), 6.36(m), 6.31(s), 6.00(v br s), 5.87(d, 7.8 Hz), 5.68(s), 5.57-
(s), 5.43(s), 5.37(s), 5.36(s), 5.34(d, 2.9 Hz), 5.30(br s), 5.02(br
m), 4.79(d, 7.0 Hz), 4.58(d, 4.4 Hz), 4.42(br q, 5.4 Hz), 4.37(d,
10.2 Hz), 4.24(m), 4.16(m), 4.04(s), 3.37(s), 3.29(s), 3.15(m),
2.83(s), 2.76(s), 2.52(v br s), 2.27(dd, 16.3 Hz, 6.4 Hz), 2.07-
(m), 2.02(br d, 9.4 Hz), 1.84(br d, 10.6 Hz), 1.81(non 7.1 Hz),
1.72(q, 7.1 Hz), 1.65(q, 7.1 Hz), 1.40(s), 1.17(d, 6.2 Hz), 1.01-
(d, 6.2 Hz), 0.96(d, 6.2 Hz). ESI-MS:The peak at m/z 1161.3
corresponds to M3+
.
Synthesis of monomeric Van 5a : 6.0 mg of [Pt(en)(H2O)-
(N-Pyridin-3-ylmethyl-acetamide)](NO3)2 (0.0124 mmol, 1.0
equiv) was added to a solution of 2a (20 mg, 0.0130 mmol,
1.05 equiv) in 1 mL of DMSO. After being purified by HPLC,
14.7 mg of pure product was obtained (yield: 58.9%): 1H NMR
(500 MHz, DMSO-d6) δ 9.46(v br s), 9.26(v br s), 9.17(v br s),
9.09(v br s), 8.84(s), 8.8(d, 4.7 Hz), 8.69(d, 5.9 Hz), 8.67-
(overlapped), 8.64(br d, 4.4 Hz), 8.61(d, 4.2 Hz), 8.28(v br s),
8.07(d, 8.2 Hz), 7.93 (s), 7.77(multiple), 7.67(d, 8.8 Hz), 7.66(v
br s), 7.63 (v br s), 7.58(d, 5.3 Hz), 7.57(s), 7.55(multiple), 7.44-
(d, 8.2 Hz), 7.32(s), 7.29(d, 8.2 Hz), 7.14(s), 6.87(br s), 6.85(d,
8.8 Hz), 6.81(d, 8.8 Hz), 6.78(v br s), 6.64(overlapped), 6.50(d,
2.34 Hz), 6.35(d, 2.3 Hz), 6.00(v br s), 5.87(d, 8.1 Hz), 5.68(v
br s), 5.36(s), 5.35(s), 5.33(d, 3.7 Hz), 5.29(br s), 5.22(br s), 5.00-
(br m), 4.78(q, 6.6 Hz), 4.55(d, 5.1 Hz), 4.47(br q, 5.6 Hz), 4.44-
(d, 5.9 Hz), 4.39(d, 11.0 Hz), 4.05 (v br s), 3.79(d, 11.0 Hz),
3.66(t, 8.8 Hz), 3.55(br s), 3.39(s), 3.29(br s), 2.75(s), 2.26(dd,
15.1 Hz, 6.5 Hz), 2.01(overlapped), 1.82(br d, 10.6 Hz), 1.77-
(non 7.1 Hz), 1.68(q, 7.1 Hz), 1.62(q, 7.1 Hz), 1.41(s), 1.17(d,
6.4 Hz), 1.01(d, 6.5 Hz), 0.96(d, 6.5 Hz). ESI-MS: The peaks
corresponds to M2+
.
5,5′-Bis(br om om eth yl)-2,2′-bip yr id in e (7a ). A solution
of 5,5′-dimethyl-2,2′-bipyridine (1 g, 5.43 mmol, 1 equiv), NBS
(5.1 g, 28.7 mmol, 5.3 equiv), and VAZO (265 mg, 1.09 mmol,
0.2 equiv) in CCl4 (100 mL) was refluxed under nitrogen for 1
h, and the precipitated succinimide was removed immediately
from the hot mixture by filtration. The precipitate was washed
with CCl4, and the combined CCl4 phases were evaporated.
The remaining solid was dissolved in CH2Cl2 (100 mL) and
extracted with 1 M Na2S2O3 solution (2 × 150 mL). The
combined Na2S2O3 fractions were extracted with CH2Cl2 (50
mL), and the combined CH2Cl2 layers were dried by Na2SO4.
The crude product was purified by flash column chromatog-
raphy (silica gel, EtOAc/hexane, 1:4) and yielded 412 mg (22%)
1
to give white solid. H NMR (CDCl3, δ 7.26 ppm) δ 8.68 (d, J
) 2.2 Hz, 1H, CH), 8.40 (d, J ) 8.2 Hz, 1H, CH), 7.86 (dd, J
) 8.2 Hz, 2.2 Hz, 1H, CH), 4.54 (s, 2H, CH2). 13C NMR (CDCl3,
δ 77.7 ppm) δ 156.08, 150.05, 138.32, 134.59, 121.87, 30.22.
ESI-MS: The peak atm/z 343 corresponds to (M + 1)+.

4908 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 23
Xing et al.
5,5′-Bis(a zid om eth yl)-2,2′-bip yr id in e (7b). NaN3 (42 mg,
0.64 mmol, 2.2 equiv) was added to a solution of 5,5′-bis-
(bromomethyl)-2,2′-bipyridine (100 mg, 0.29 mmol, 1 equiv)
in dry DMF (3 mL). The solution was heated for 17 h at 90 °C
and then concentrated under vacuum to yield a mixture, which
was treated with CH2Cl2. The solid (NaBr) was filtered off,
and the extract was concentrated in a vacuum to yield the
product (white solid), which was purified by flash column
chromatography (silica gel, EtOAc/hexane, 40/60) and yielded
69 mg (90%) of pure white solid. 1H NMR (CDCl3, δ 7.26 ppm)
δ 8.64 (d, J ) 1.8 Hz, 1H, CH), 8.45 (d, J ) 8.2 Hz, 1H, CH),
7.81 (dd, J ) 8.2 Hz, 2.3 Hz, 1H, CH), 4.44 (s, 2H, CH2). 13C
NMR (CDCl3, δ 77.7 ppm) δ 156.35, 149.44, 137.41, 131.99,
121.81, 52.68. ESI-MS: The peak atm/z 267 corresponds to
(M + 1)+.
5,5′-Bis(a m in om eth yl)-2,2′-bip yr id in e (7c). 10% Pd on
activated carbon (15 mg) was dissolved in 1 mL of dry CH2Cl2
in a closed round-bottom flask with vigorous stirring. 6 M HCl
was added to another round-bottom flask that contained zinc
powder in order to generate hydrogen gas. These two round-
bottom flasks were connected by a rubber pipe. Make sure that
there is no leakage. An outlet was introduced in the round-
bottom flask containing 10% Pd on activated carbon. 5,5′-bis-
(azidomethyl)-2,2′-bipyridine (60 mg, 0.23 mmol, 1 equiv) in 2
mL of dry CH2Cl2 was added to the round-bottom flask
contained 10% Pd on activated carbon 3 min after the outlet
was introduced. The reaction was completed within 4 h. 10%
Pd on activated carbon was filtered by Celite and CH2Cl2 was
dried to obtain 39 mg of white solid (yield, 80%). 1H NMR
(CDCl3, δ 7.26 ppm) δ 8.62 (d, J ) 2.0 Hz, 1H, Ar-H), 8.35 (d,
J ) 8.1 Hz, 1H, Ar-H), 7.80 (dd, J ) 8.1 Hz, 2.0 Hz, 1H, Ar-
H), 3.97 (s, 2H, CH2). 1.54(s, 2H, NH2). 13C NMR (CDCl3, δ
77.7 ppm) δ 156.15, 150.12, 137.11, 131.08, 121.61, 43.8. ESI-
MS: The peak atm/z 215 corresponds to (M + 1)+.
Refer en ces
(1) Neu, H. C. The Crisis in Antibiotic-Resistance. Science 1992,
257, 1064-1073.
(2) Breukink, E.; Wiedemann, I.; van Kraaij, C.; Kuipers, O. P.; Sahl,
H. G. et al. Use of the cell wall precursor lipid II by a pore-
forming peptide antibiotic. Science 1999, 286, 2361-2364.
(3) Chiosis, G.; Boneca, I. G. Selective Celavage of D-Ala-D-Lac by
Small Molecules: Re-Sensitizing Resistant Bacteria to Vanco-
mycin. Science 2001, 293, 1484-1487.
(4) Ge, M.; Chen, Z.; Onishi, H. R.; Kohler, J .; Silver, L. L. et al.
Vancomycin derivatives that inhibit peptidoglycan biosynthesis
without binding D-Ala-D-Ala. Science 1999, 284, 507-511.
(5) Kohli, R. M.; Walsh, C. T.; Burkart, M. D. Biomimetic synthesis
and optimization of cyclic peptide antibiotics. Nature 2002, 418,
658-661.
(6) Trauger, J . W.; Kohli, R. M.; Mootz, H. D.; Marahiel, M. A.;
Walsh, C. T. Peptide cyclization catalysed by the thioesterase
domain of tyrocidine synthetase. Nature 2000, 407, 215-218.
(7) Koeller, K. M.; Wong, C. H. Emerging themes in medicinal
glycoscience. Nat. Biotech. 2000, 18, 835-841.
(8) Liu, H. T.; Sadamoto, R.; Sears, P. S.; Wong, C. H. An efficient
chemoenzymatic strategy for the synthesis of wild- type and
vancomycin-resistant bacterial cell-wall precursors: UDP-N-
acetylmuramyl-peptides. J . Am. Chem. Soc. 2001, 123, 9916-
9917.
(9) Mammen, M.; Choi, S. K.; Whitesides, G. M. Polyvalent interac-
tions in biological systems: Implications for design and use of
multivalent ligands and inhibitors. Angew. Chem., Int. Ed. 1998,
37, 2755-2794.
(10) Cooper, M. A.; Williams, D. H. Binding of glycopeptide antibiotics
to a model of a vancomycin- resistant bacterium. Chem. Biol.
1999, 6, 891-899.
(11) Rao, J . H.; Lahiri, J .; Isaacs, L.; Weis, R. M.; Whitesides, G. M.
A trivalent system from vancomycin center dot D-Ala-D-Ala with
higher affinity than avidin center dot biotin. Science 1998, 280,
708-711.
(12) Rao, J .; Yan, L.; Xu, B.; Whitesides, G. M. Using surface plasmon
resonance to study the binding of vancomycin and its dimer to
self-assembled monolayers presenting D-Ala-D-Ala. J . Am. Chem.
Soc. 1999, 121, 2629-2630.
5,5′-Bis(a m in om eth yl)-2,2′-bip yr id in e Va n com ycin (7).
The experiment procedure was same as for 2a . The crude white
solid was purified by reversed-phase HPLC (RP-HPLC). The
percentage yield is 43%. 1H NMR (500 MHz, DMSO-d6) δ 9.11-
(v br s), 8.76(v br s), 8.71(v br s), 8.68(br s), 8.62(br s), 8.40(d,
7.0 Hz), 8.04 (s), 7.95(s), 7.67(d, 8.5 Hz), 7.57(d, 8.6 Hz), 7.44-
(d, 8.6 Hz), 7.34(d, 8.6 Hz), 7.29 (s), 7.12(v br s), 6.88(d, 9.0
Hz), 6.82(d, 8.6 Hz), 6.80(v br s), 6.63(v br s), 6.48(s), 6.36(s),
6.10(br s), 5.86(d, 7.3 Hz), 5.68(s), 5.39(s), 5.36(s), 5.34(s), 5.33-
(d, 2.8 Hz), 5.29(br s), 5.04(br m), 4.78(d, 6.2 Hz), 4.65(d, 10.9
Hz), 4.54(br q, 4.9 Hz), 4.37(d, 10.9 Hz), 4.04(s), 3.79(d, 10.1
Hz), 3.37(s), 3.28(s), 3.02(m), 2.74(s), 2.26(dd, 16.6 Hz, 6.6 Hz),
2.00(br d, 9.4 Hz), 1.84(br d, 10.4 Hz), 1.78(non 7.1 Hz), 1.74-
(q, 7.1 Hz), 1.67(q, 7.1 Hz), 1.62(q, 7.1 Hz), 1.39(s), 1.16(d, 6.2
Hz), 1.00(d, 6.2 Hz), 0.95(d, 6.2 Hz). ESI-MS: The peaks atm/z
(13) Rao, J . H.; Lahiri, J .; Weis, R. M.; Whitesides, G. M. Design,
synthesis, and characterization of
system derived from vancomycin and L-Lys-D-Ala-D-Ala. J . Am.
Chem. Soc. 2000, 122, 2698-2710.
a high-affinity trivalent
(14) Rao, J . H.; Yan, L.; Lahiri, J .; Whitesides, G. M.; Weis, R. M. et
al. Binding of a dimeric derivative of vancomycin to L-Lys-D-
Ala-D-Lact in solution and at a surface. Chem. Biol. 1999, 6,
353-359.
(15) Nicolaou, K. C.; Hughes, R.; Cho, S. Y.; Winssinger, N.; La-
bischinski, H. et al. Synthesis and biological evaluation of
vancomycin dimers with potent activity against vancomycin-
resistant bacteria: target- accelerated combinatorial synthesis.
Chem. Eur. J . 2001, 7, 3824-3843.
(16) Nicolaou, K. C.; Hughes, R.; Cho, S. Y.; Winssinger, N.; Smethurst,
C. et al. Target-accelerated combinatorial synthesis and discov-
ery of highly potent antibiotics effective against vancomycin-
resistant bacteria. Angew. Chem., Int. Ed. 2000, 39, 3823-3828.
(17) Sundram, U. N.; Griffin, J . H.; Nicas, T. I. Novel vancomycin
dimers with activity against vancomycin- resistant enterococci.
J . Am. Chem. Soc. 1996, 118, 13107-13108.
770.3, 1026.8 and 1539.4 correspond to M4+, M3+ and M2+
respectively.
,
B. In Vitr o Stu d y. Minimum concentrations of the Van,
[Pt(en)(H2O)2](NO3)2+Van, and dimers of Van required to
inhibit the growth of bacterial cells were measured using
cation-adjusted Muller-Hinton broth as the growth media. One
vancomycin-susceptible strain, one strain exhibiting low-level
resistance to vancomycin, five strains exhibiting midlevel
resistance to vancomycin, and four strains exhibiting high-
level resistance to vancomycin were used to determine the MIC
values. The average MIC values for the last two cases were
shown in Table 1. The genotype of the strains was confirmed
by PCR.
(18) Thoma, G.; Katopodis, A. G.; Voelcker, N.; Duthaler, R. O.;
Streiff, M. B. Novel glycodendrimers self-assemble to nanopar-
ticles which function as polyvalent ligands in vitro and in vivo.
Angew. Chem., Int. Ed. 2002, 41, 3195-3198.
(19) Nicolaou, K. C.; Mitchell, H. J .; J ain, N. F.; Winssinger, N.;
Hughes, R. et al. Total synthesis of vancomycin. Angew. Chem.,
Int. Ed. 1999, 38, 240-244.
(20) Nicolaou, K. C.; Boddy, C. N. C.; Brase, S.; Winssinger, N.
Chemistry, biology, and medicine of the glycopeptide antibiotics.
Angew. Chem., Int. Ed. 1999, 38, 2097-2152.
(21) Hubbard, B. K.; Walsh, C. T. Vancomycin assembly: Nature’s
way. Angew. Chem., Int. Ed. 2003, 42, 730-765.
(22) Evans, D. A.; Wood, M. R.; Trotter, B. W.; Richardson, T. I.;
Barrow, J . C. et al. Total syntheses of vancomycin and eremo-
mycin aglycons. Angew. Chem., Int. Ed. 1998, 37, 2700.
(23) Novak, R.; Henriques, B.; Charpentier, E.; Normark, S.;
Tuomanen, E. Emergence of vancomycin tolerance in Strepto-
coccus pneumoniae. Nature 1999, 399, 590-593.
Ack n ow led gm en t. This work was partially sup-
ported by the Research Grant Council of Hong Kong,
DuPont Young Faculty Grant (for Xu), Direct Allocation
Grants (HKUST), and University Development Fund
(HKU). We thank Prof. Zhongwan Mao for helps on ESI-
MS.
(24) Walsh, C. Molecular mechanisms that confer antibacterial drug
resistance. Nature 2000, 406, 775-781.
(25) Walsh, C. T. Vancomycin Resistance - Decoding the Molecular
Logic (Vol 261, Pg 469, 1993). Science 1993, 262, 164-164.
(26) Walsh, C. Microbiology - Deconstructing vancomycin. Science
1999, 284, 442-443.
Su ppor tin g In for m ation Available: The synthetic scheme
of 7 and the high-resolution mass spectra (in tabular form) of
4a and 4b. This material is available free of charge via the
Internet at http://pubs.acs.org.
(27) Walsh, C. Antibiotics: Actions, Origins, Resistance, 1st ed.; ASM
press: Washington, D. C., 2003.
(28) Popieniek, P. H.; Pratt, R. F. Anal. Biochem. 1987, 165, 108-
113.

Multivalent Antibiotics via Metal Complexes
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 23 4909
(29) Waltho, J . P.; William, D. H. J . Am. Chem. Soc. 1989, 111, 2475-
2480.
(41) Cuenoud, B.; Schepartz, A. Altered Specificity of DNA-Binding
Proteins with Transition- Metal Dimerization Domains. Science
1993, 259, 510-513.
(42) Mack, D. P.; Dervan, P. B. Sequence-Specific Oxidative Cleavage
of DNA by a Designed Metalloprotein, Ni(Ii).Ggh(Hin139-190).
Biochemistry 1992, 31, 9399-9405.
(43) Dong, S. D.; Oberthur, M.; Losey, H. C.; Anderson, J . W.; Eggert,
U. S. et al. The structural basis for induction of VanB resistance.
J . Am. Chem. Soc. 2002, 124, 9064-9065.
(44) Xing, B.; Yu, C.-W.; Chow, K.-H.; Ho, P.-L.; Fu, D. et al.
Hydrophobic Interaction and Hydrogen Bonding Cooperatively
(30) Williams, D. H.; Maguire, A. J .; Tsuzuki, W.; Westwell, M. S.
An analysis of the origins of a cooperative binding energy of
dimerization. Science 1998, 280, 711-714.
(31) Shiozawa, H.; Chia, B. C. S.; Davies, N. L.; Zerella, R.; Williams,
D. H. Cooperative binding interactions of glycopeptide antibiot-
ics. J . Am. Chem. Soc. 2002, 124, 3914-3919.
(32) Williams, D. H.; Cox, J . P. L.; Doig, A. J .; Gardner, M.; Gerhard,
U. et al. Toward the Semiquantitative Estimation of Binding
Constants - Guides for Peptide Peptide Binding in Aqueous-
Solution. J . Am. Chem. Soc. 1991, 113, 7020-7030.
(33) Williams, D. H.; Bardsley, B. The vancomycin group of antibiotics
and the fight against resistant bacteria. Angew. Chem., Int. Ed.
1999, 38, 1173-1193.
(34) Griffin, J . H.; Linsell, M. S.; Nodwell, M. B.; Chen, Q.; Pace, J .
L. et al. Multivalen Drug Design. Synthesis and In Vitro
Analysis of an Array of Vancomycin Dimers. J . Am. Chem. Soc.
2003, 125, 6517-6531.
Confer
a Vancomycin Hydrogel: A Potential Candidate for
Biomaterials. J . Am. Chem. Soc. 2002, 124, 14846-14847.
(45) Gu, H.; Ho, P. L.; Tong, E.; Xu, C.; Xu, B. Presenting Vancomycin
on Nanoparticles to Enhance Antimicrobial Activities. Nano Lett.
2003, 3, 1261-1263.
(46) Xing, B.; Ho, P. L.; Yu, C.-W.; Chow, K.-H.; Gu, H. et al. Self-
Assembled Multivalent Vancomycin on Cell Surfaces Against
Vancomycin-Resistant Enterococci (VRE). Chem. Commun. 2003,
2224-2225.
(47) Chen, Z.; Eggert, U. S.; Dong, S. D.; Shaw, S. J .; Sun, B. Y. et
al. Structural requirements for VanA activity of vancomycin
analogues. Tetrahedron 2002, 58, 6585-6594.
(48) Kim, S. J .; Cegelski, L.; Studelska, D. R.; O’Connor, R. D.; Mehta,
A. K. et al. Rotational-echo double resonance characterization
of vancomycin binding sites in Staphylococcus aureus. Biochem-
istry 2002, 41, 6967-6977.
(35) Stack, D. R.; Thompson, R. G. EP 0801075 A1, 1997.
(36) Mammen, M.; Shakhnovich, E. I.; Whitesides, G. M. Using a
convenient, quantitative model for torsional entropy to establish
qualitative trends for molecular processes that restrict confor-
mational freedom. J . Org. Chem. 1998, 63, 3168-3175.
(37) Searle, M. S.; Williams, D. H. The cost of conformational order:
entropy changes in Molecular associations. J . Am. Chem. Soc.
1992, 114, 10690-10697.
(38) Rosenberg, B.; VanCamp, L.; Trosko, J . E.; Mansour, V. H.
Nature 1969, 222, 385-386.
(49) Cegelski, L.; Kim, S. J .; Hing, A. W.; Studelska, D. R.; O’Connor,
R. D. et al. Rotational-echo double resonance characterization
of the effects of vancomycin on cell wall synthesis in Staphylo-
coccus aureus. Biochemistry 2002, 41, 13053-13058.
(39) Sakai, S.; Sasaki, T. Multivalent Carbohydrate Ligands As-
sembled on a Metal Template. J . Am. Chem. Soc. 1994, 116,
1587-1588.
(40) Cuenoud, B.; Schepartz, A. Design of a Metallo Bzip-Protein That
Discriminates between Cre and Ap1 Target Sites - Selection
against Ap1. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 1154-1159.
J M030417Q