Lipophilic vancomycin aglycon dimer with high activity against vancomycin-resistant bacteria

Article abstract of DOI:10.1016/j.bmcl.2015.10.083

Antibiotic-resistant superbugs such as vancomycin-resistant Enterococci (VRE) and Staphylococci have become a major global health hazard. To address this issue, we synthesized vancomycin aglycon dimers to systematically probe the impact of a linker on biological activity. A dimer having a pendant lipophilic moiety in the linker showed ～300-fold more activity than vancomycin against VRE. The high activity of the compound is attributed to its enhanced binding affinity to target peptides which resulted in improved peptidoglycan (cell wall) biosynthesis inhibition. Therefore, our studies suggest that these compounds, prepared by using facile synthetic methodology, can be used to combat vancomycin-resistant bacterial infections.

Full text of DOI:10.1016/j.bmcl.2015.10.083

Bioorganic & Medicinal Chemistry Letters 25 (2015) 5477–5480
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Lipophilic vancomycin aglycon dimer with high activity against
vancomycin-resistant bacteria
⇑
Venkateswarlu Yarlagadda, Paramita Sarkar, Goutham B. Manjunath, Jayanta Haldar
Chemical Biology and Medicinal Chemistry Laboratory, New Chemistry Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur PO, Bengaluru 560064, Karnataka, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Antibiotic-resistant superbugs such as vancomycin-resistant Enterococci (VRE) and Staphylococci have
become a major global health hazard. To address this issue, we synthesized vancomycin aglycon dimers
to systematically probe the impact of a linker on biological activity. A dimer having a pendant lipophilic
moiety in the linker showed ꢀ300-fold more activity than vancomycin against VRE. The high activity of
the compound is attributed to its enhanced binding affinity to target peptides which resulted in improved
peptidoglycan (cell wall) biosynthesis inhibition. Therefore, our studies suggest that these compounds,
prepared by using facile synthetic methodology, can be used to combat vancomycin-resistant bacterial
infections.
Received 17 September 2015
Accepted 27 October 2015
Available online 27 October 2015
Keywords:
Antibiotic resistance
Vancomycin
Vancomycin-resistant bacteria
Cell wall biosynthesis inhibition
Antibacterial activity
Ó 2015 Elsevier Ltd. All rights reserved.
Vancomycin, a glycopeptide antibiotic, has long been consid-
ered as ‘antibiotic of last resort’ for the treatment of Gram-positive
bacterial infections especially those caused by multidrug-resistant
bacteria such as methicillin-resistant Staphylococcus aureus
(MRSA).1 The extensive use of vancomycin for MRSA infections
resulted in emergence of vancomycin resistance in MRSA (van-
comycin-intermediate-resistant S. aureus, VISA and vancomycin-
resistant S. aureus, VRSA).2 In addition to this, the emergence and
spread of vancomycin-resistant Enterococci (VRE) is a growing
concern worldwide.3,4 The perennial persistence of vancomycin
resistance calls for urgent measures to develop more potent ana-
logs to tackle vancomycin-resistant bacteria.
skin infections caused by MRSA.18 However, both dalbavancin and
telavancin are moderately active against more virulent VanA phe-
notypes of vancomycin-resistant bacteria.19 In our own efforts,
membrane active vancomycin analogs13,20 and vancomycin–sugar
conjugates12,21 have been developed which showed potent
antibacterial activity against more virulent drug-resistant bacteria.
Glycopeptide antibiotics such as vancomycin are known to self-
associate into homodimers via hydrogen bonding and hydrophobic
interactions in both solution and solid states.22 This noncovalent
dimerization is highly favorable and cooperative with the binding
of cell wall precursors which could lead to enhancement in
antibacterial activity.23 This observation prompted the scientific
community to study the effects of covalent dimerization on
antibacterial properties of glycopeptides.24–27 Also, it has been
shown that the overall binding affinity of a covalent dimer is more
than its corresponding monomer towards the bacterial ligands.
Here, we report the synthesis of a series of bis-(vancomycin agly-
con)carboxamides with variable linkers that differ in lipophilicity
and cationic charge. The dimer having a pendant hydrophobic moi-
ety in the linker showed high antibacterial activity against VRE
(VanA phenotype). Compared to vancomycin, this dimer caused
more accumulation of cell wall (peptidoglycan) precursor indicat-
ing the inhibition of cell wall biosynthesis in a greater extent.
Unlike vancomycin, the compound was found to be highly bacteri-
cidal with improved ex vivo antibacterial activity in whole blood.
In the synthetic strategy employed for preparing the van-
comycin aglycon dimers, bis(vancomycin aglycon)carboxamides,
polyamines were used as linkers with variable hydrophobicity
and positive charge. The linkers bearing a primary amine group
Vancomycin inhibits bacterial cell wall biosynthesis by binding
to the peptidoglycan peptide terminus D-Ala-D-Ala found in cell
wall precursors, sequesters the substrate from transpeptidase
and inhibits cell wall cross-linking.1 Conversely, bacteria discern-
ing this vancomycin stress, have altered their cell wall precursors
from D-Ala-D-Ala to D-Ala-D
-Lac (depsipeptide).5,6 This alteration
is induced by five van genes and is ample to lessen the binding effi-
ciency of vancomycin to its target by 1000-fold and as a result, its
antibacterial activity reduced by >1000-fold (VanA).7 Significant
approaches have been intended for the development of next-gen-
eration glycopeptide antibiotics that address the specter of wide-
spread vancomycin resistance.8–17 Among numerous semi-
synthetic glycopeptides, very few of them such as telavancin, dal-
bavancin and oritavancin were FDA approved for the treatment of
⇑
Corresponding author. Tel.: +91 8022082565.
E-mail address: jayanta@jncasr.ac.in (J. Haldar).
http://dx.doi.org/10.1016/j.bmcl.2015.10.083
0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.

5478
V. Yarlagadda et al. / Bioorg. Med. Chem. Lett. 25 (2015) 5477–5480
on either side were coupled to the carboxyl group of vancomycin
aglycon via amide bond formation by using N,N,N0,N0-tetram-
ethyl-O-(1H-benzotriazol-1-yl)uronium
hexafluorophosphate
(HBTU) as a coupling reagent. These vancomycin aglycon dimers
were purified by reverse-phase HPLC (High-performance liquid
chromatography) using 0.1% trifluoroacetic acid (TFA) in water/
acetonitrile solvent system to more than 95% purity in 40–45%
yield and characterized by 1H NMR spectroscopy and high resolu-
tion mass spectrometry (HR-MS). Also, the corresponding mono-
meric adducts were obtained in 25% yield as trifluoroacetamides
probably due to the possible reactivity of the second free amine
group of the monomers with TFA of solvents during purification.
Firstly, we synthesized vancomycin aglycon (2) from van-
comycin (1), which was deglycosylated by using trifluoroacetic
acid.28 Then, the carboxylic group of vancomycin aglycon (2) was
coupled to the amine group of 1,8-diaminooctane (which does
not have permanent positive charge) to give monomer 3 and its
dimer 4 (Scheme 1).
Next, we sought to incorporate permanent positively charged
centres (quaternary ammonium centres) and lipophilicity in the
linker due to the anticipated favorable electrostatic interactions
between the more negatively charged bacterial cell membrane
and the cationic centres of molecule, which might lead to favor-
able biological properties. Utilizing this concept, we synthesized
N1,N8-bis(3-aminopropyl)-N1,N1,N8,N8-tetramethyloctane-1,8-dia-
minium bromide (5c) and coupled to vancomycin aglycon
(Scheme 2). Here, to synthesize compound 5c, N,N-dimethyl-
1,3-propanediamine was protected using di-t-butylpyrocarbonate
to give t-butyl (3-(dimethylamino)propyl)carbamate (5a). Then,
the tertiary amine group of compound 5a was quaternized with
1,8-dibromooctane to give N-Boc protected quaternized com-
pound (5b). Now, compound 5b was subjected to deprotection
in presence of acid to give N-Boc free compound 5c. Then, the
compound 5c was coupled to the carboxylic group of vancomycin
aglycon (2) to yield monomer 5 and dimer 6 (Scheme 2).
Next, we synthesized dimer 8 using N0-(3-aminopropyl)-N0-
octylpropane-1,3-diamine (7b) as a linker comprising a pendant
lipophilic moiety and tertiary amine (which becomes cationic
under physiological conditions). It has been shown in the literature
that inclusion of a pendant hydrophobicity to the periphery of gly-
copeptides leads to enhanced dimerization and stronger associa-
tion with bacterial membrane. This further leads to enhanced
Scheme 2. Synthesis of monomer (5) and dimer (6).
inhibition of cell wall biosynthesis compared to parent
glycopeptide thereby accounting for high antibacterial activity.29
In order to have this beneficial effect along with increased binding
affinity, dimer 8 has been synthesized. Initially, octyl amine was
subjected to Michael addition with acrylnitrile to give an adduct
(7a). Then, the nitrile group of the adduct was reduced by lithium
aluminium hydride (LAH) to primary amine and then finally
coupled to vancomycin aglycon to yield monomer 7 and dimer 8
(Scheme 3).
The antibacterial activities (minimum inhibitory concentration,
MIC) of all the compounds were determined against vancomycin-
sensitive strains of Staphylococci (MSSA and MRSA) and
Enterococci (VSE), as well as against vancomycin-resistant strains
of Staphylococci (VISA) and Enterococci (VRE) and the results are
summarized in Table 1. The activity of vancomycin aglycon was
found to be similar to vancomycin against sensitive bacteria with
the MIC of ꢀ1
lM. Against VISA, vancomycin aglycon showed
ꢀ3-fold more activity than vancomycin whereas in case of VRE it
was found to be ineffective. Then, we evaluated the activities of
vancomycin aglycon derivatives having various linkers (monomers
3, 5 and 7) and their MICs were found to be ꢀ1
comycin-sensitive bacteria. In case of VISA, compounds 3, 5 and
7 showed potent activity with MICs ranging from 0.5 to 0.8 M.
When checked against VRE, monomers 3 and 5 showed moderate
activity with the MICs of 30 and 48 M, respectively, whereas
monomer 7 exhibited good activity with the MIC of 6.5 M which
lM against van-
l
l
l
is ꢀ115-fold more active than vancomycin. Next, we have evalu-
ated the antibacterial activity of vancomycin aglycon dimers (4, 6
and 8) and the results are compared with their corresponding
monomers (3, 5 and 7). The dimer 4 comprising 1,8-diaminoocty-
lene in the linker showed ꢀ65-fold increase in activity against VISA
(MIC = 0.2
l
M) and ꢀ30-fold increase in case of VRE (MIC = 25
lM)
compared to vancomycin. Further, dimer 4 exhibited ꢀ3-fold more
activity than its corresponding monomer 3 against both VISA and
Scheme 1. Synthesis of vancomycin aglycon (2), monomer (3) and dimer (4).
Reagents: CF3COOH, trifluoroacetic acid; DIPEA, N,N-diisopropylethylamine; HBTU,
N,N,N0,N0-tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate.
Scheme 3. Synthesis of monomer (7) and dimer (8).

V. Yarlagadda et al. / Bioorg. Med. Chem. Lett. 25 (2015) 5477–5480
5479
Table 1
In vitro antibacterial activity of the compounds
Compound
Minimum inhibitory concentration (lM)
MSSAa
MRSAb
VISAc
VSEd
VREme
Vancomycin
2 (VAG)
3 (monomer)
4 (dimer)
5 (monomer)
6 (dimer)
7 (monomer)
8 (dimer)
0.6
1.0
1.0
0.8
1.0
0.2
0.8
0.6
0.7
1.0
2.0
0.6
1.0
0.3
1.0
0.6
13.0
4.0
0.6
0.2
0.5
0.1
0.8
0.6
0.6
1.8
0.6
0.6
0.2
0.05
0.4
0.5
750
>100
30.5
25.8
>100
48.0
6.5
2.5
a
b
c
MSSA (methicillin-sensitive S. aureus).
MRSA (methicillin-resistant S. aureus).
VSE (vancomycin-sensitive E. faecium).
VISA (vancomycin-intermediate-resistant S. aureus).
VREm (vancomycin-resistant E. faecium, VanA).
d
e
MRSA whereas it showed slightly better activity against VRE.
Dimer (having two permanent positively charged centres
connected together by an octylene linker) was found to be ꢀ2-fold,
ꢀ12-fold and ꢀ130-fold more active than vancomycin against
MRSA, VSE and VISA, respectively, with the MICs ranging from
6
Figure 1. Intracellular accumulation of the cell wall precursor UDP-MurNAc-
pentadepsipeptide after treatment of VRE with vancomycin and dimer 8 at 5 lM (A
and B). (A) Identification of intracellular UDP-MurNAc-pentadepsipeptide by
monitoring absorbance at 260 nm wavelength (B) UDP-MurNAc-pentadepsipeptide
was identified by mass spectrometry as indicated by the peak at m/z 1150.35; (C)
Bactericidal properties of dimer 8 and vancomycin against MRSA in media. Single
stars correspond to reduction of 3log10 CFU/mL and double stars correspond to
0.05 to 0.3 lM. Against VRE, dimer 6 exhibited an MIC of 48 lM
which is ꢀ15-fold more active than vancomycin. Further, dimer 6
was ꢀ4-fold more active than its corresponding monomer 5
against MRSA, VISA and VSE whereas it showed slightly lower
activity against VRE. Dimer 8 comprising a pendant octyl chain
<50 CFU/mL. (D) Antibacterial activity of vancomycin and dimer
incubation in 90% human whole blood against MRSA.
8 after 3 h
in the linker exhibited an activity of 2.5 lM against VRE which is
observation was reported for semi-synthetic lipoglycopeptides
such as oritavancin and telavancin wherein they show significantly
better inhibition of cell wall biosynthesis compared to parent drug
due to the installed additional lipophilicity in the molecule.18,29
In order to study the bactericidal activity of the optimum com-
pound, we carried out in vitro time-kill assay with the dimer 8 and
vancomycin against MRSA (starting bacterial concentration of
ꢀ300-fold higher than vancomycin. Further, dimer 8 was ꢀ3-fold
more active than its corresponding monomer 7 against VRE
whereas against rest of the bacteria dimer 8 showed an MIC of
ꢀ0.6
lM which is similar to monomer 7. Among compounds 3–8,
dimer 8 showed the best activity against VRE and dimer 6 demon-
strated the best activity against VISA and VSE.
To substantiate these findings, the binding constants of the best
active dimer, compound 8 and vancomycin were evaluated using
UV-difference spectroscopy against both sensitive and resistant
ꢀ8log10 CFU/mL), at two different concentrations (2 and 4
lM).
Our results demonstrated a rapid bactericidal activity with dimer
8, which increased with increasing concentration. Further, dimer
8 caused ꢀ3log10 CFU/mL reduction in bacterial concentration
model ligands: N,N0-diacetyl-Lys-
Lys- -Ala-
D
-Ala-
D
-Ala and N,N0-diacetyl-
D
D-Lac, respectively (Supplementary Figs. S1 and S2).
within 3 h of incubation at 4 lM (Fig. 1C). Also, it displayed com-
The binding affinity of the dimer 8 was found to be similar to van-
plete bactericidal activity in 24 h at 4 lM. On the other hand, van-
comycin against N,N0-diacetyl-Lys-
When evaluated against N,N0-diacetyl-Lys-
D
-Ala-
D
-Ala (ꢀ1 ꢁ 105 Mꢂ1).
comycin showed bacteriostatic effect in the initial 6 h and did not
show any effect on bacterial growth irrespective of the concentra-
tion beyond that time (Fig. 1C). The potent bactericidal activity of
compound 8 can be attributed to its improved cell wall biosynthe-
sis inhibition.
Next, an ex vivo assay was developed to provide a relevant chal-
lenge to the antimicrobial agent in complex biomatrices such as
whole blood. The assay design includes simultaneous introduction
of antimicrobial agent and bacterial cells into biomatrices. Here,
we performed ex vivo whole blood assay with dimer 8 and
vancomycin against MRSA. In whole blood, an inoculum of
ꢀ5.0log10 CFU/mL yielded an end point in MRSA viability of
ꢀ7.0log10 CFU/mL (Fig. 1D) after a 3 h incubation at 37 °C in
growth control (no compound). In whole blood containing the
D
-Ala-D-Lac, this dimer
(Ka = 5.7 ꢁ 103 Mꢂ1) exhibited the binding affinity of 10-fold more
than vancomycin (Ka = 5 ꢁ 102 Mꢂ1). This observation indicates
that compound 8 binds more effectively with bacterial ligands
compared to vancomycin and leads to improved inhibition of cell
wall biosynthesis.
In order to investigate the effect of enhanced binding affinity
on peptidoglycan biosynthesis, we determined the accumulation
of UDP-linked peptidoglycan precursor, UDP-N-acetyl-muramyl-
pentadepsipeptide (UDP-MurNAc-pp) after treating bacteria
(VRE) with the compound 8 and vancomycin at 5 lM. In case of
compound 8, a more intense peak was observed at 260 nm com-
pared to vancomycin, which corresponds to more accumulation
of UDP-MurNAc-pp and confirmed by high resolution mass spec-
trometry (m/z = 1150.94 (calcd), 1150.35 (obsd) for [M+H]+)
(Fig. 1A and B). This suggests that dimer 8 causes more accumula-
tion of cell wall (peptidoglycan) precursor than vancomycin indi-
cating a higher inhibition of peptidoglycan biosynthesis. This
might be due to increased association of the dimer 8 with bacterial
ligands and bacterial membranes because of the presence of pen-
dant lipophilicity which presumably serves to anchor the drug
thereby allowing it to stay for a longer time at cell wall region
and inhibiting the cell wall biosynthesis in a greater extent, which
leads to improvement in antibacterial activity against VRE. Similar
dimer at a concentration of 2 lM, 4.5log10 CFU/mL viable bacterial
cells were detectable after a 3 h incubation, which indicates a
reduction of ꢀ2.5log10 CFU/mL, in comparison to vancomycin
which showed only 1.0log10 CFU/mL reduction at 4 lM. These
results indicate that the dimer
8 can potentially maintain
antibacterial activity in vivo with nominal loss due to non-specific
interactions with tissue components.
To summarize, bis(vancomycin aglycon)carboxamides with
variable linkers have been developed that differ in permanent pos-
itive charge and lipophilicity. The dimer comprising a pendant
lipophilic moiety in the linker exhibited 300-fold more activity

5480
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Acknowledgments
We thank Prof. C. N. R. Rao (JNCASR) for his constant support
and encouragement. J. H. acknowledges Department of Science
and Technology (DST), Govt. of India for Ramanujan Fellowship
[SR/S2/RJN-43/2009]. V. Y. thanks CSIR for research fellowship.
Supplementary data
20. Yarlagadda, V.; Konai, M. M.; Manjunath, G. B.; Prakash, R. G.; Mani, B.;
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Supplementary data (synthetic protocols, assay details, supple-
mentary figures and compound characterization data) associated
with this article can be found, in the online version, at http://
dx.doi.org/10.1016/j.bmcl.2015.10.083.
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