Diazo transfer-click reaction route to new, lipophilic teicoplanin and ristocetin aglycon derivatives with high antibacterial and anti-influenza virus activity: An aggregation and receptor binding study

Article abstract of DOI:10.1021/jm900950d

Semisynthetic, lipophilic ristocetin and teicoplanin derivatives were prepared starting from ristocetin aglycon and teicoplanin ψ-aglycon (N-acetyl-D-glucosaminyl aglycoteicoplanin). The terminal amino functions of the aglycons were converted into azido f

Full text of DOI:10.1021/jm900950d

J. Med. Chem. 2009, 52, 6053–6061 6053
DOI: 10.1021/jm900950d
Diazo Transfer-Click Reaction Route to New, Lipophilic Teicoplanin and Ristocetin Aglycon
Derivatives with High Antibacterial and Anti-influenza Virus Activity: An Aggregation and
Receptor Binding Study
†
Gabor Pinter, Gyula Batta,* Sandor Keki, Attila Mandi, Istvan Komaromi, Krisztina Takacs-Novak,
,‡
§
^
#
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
Ferenc Sztaricskai, Erzsebet Roth, Eszter Ostorhazi, Ferenc Rozgonyi, Lieve Naesens, and Pal Herczegh*
†
†
3
3
O
,†
ꢀ
ꢀ
ꢀ
†
‡
ꢀ
Department of Pharmaceutical Chemistry, University of Debrecen, Egyetem ter 1, H-4010 Debrecen, Hungary, Department of Organic
Chemistry, University of Debrecen, Egyetem teꢀr 1, H-4010 Debrecen, Hungary, §Department of Applied Chemistry, University of Debrecen,
Egyetem teꢀr 1, H-4010 Debrecen, Hungary, Research Group for Carbohydrates of the Hungarian Academy of Sciences, Egyetem teꢀr 1, H-4010
Debrecen, Hungary, ^Thrombosis, Hemostasis and Vascular Biology Research Group of the Hungarian Academy of Sciences at the University of
Debrecen, Nagyerdei krt. 98., H-4032 Debrecen, Hungary, #Department of Pharmaceutical Chemistry, Semmelweis University, Hoogyes E. u. 9,
H-1092 Budapest, Hungary, 3Microbiology Laboratory, Department of Dermatology, Venerology and Dermatooncology, Semmelweis
University, Maꢀria u. 41, H-1085 Budapest, Hungary, and ORega Institute for Medical Research, Katholieke Universiteit, B-3000-Leuven, Belgium
Received June 29, 2009
Semisynthetic, lipophilic ristocetin and teicoplanin derivatives were prepared starting from ristocetin
aglycon and teicoplanin ψ-aglycon (N-acetyl-D-glucosaminyl aglycoteicoplanin). The terminal amino
functions of the aglycons were converted into azido form by triflic azide. Copper catalyzed 1,3-dipolar
cycloaddition reaction with lipophilic alkynes resulted in the title compounds. Two of the teicoplanin
derivatives showed very good MIC and MBC values against various Gram-positive bacteria, including
vanA enterococci. The aggregation and interaction of a n-decyl derivative with bacterial cell wall
components was studied. One of the lipophilic ristocetin derivatives displayed favorable anti-influenza
virus activity.
Introduction
Through various chemical modifications of such glyco-
peptides, Nagarajan et al.4 obtained the first lipophilic
N-alkyl derivatives of vancomycin active against vanco-
mycin resistant bacteria. Since that work, the same prin-
ciple has been applied to prepare many vancomycin,
eremomycin, and teicoplanin derivatives, carrying lipo-
philic substituents. Three of the lipophilic glycopeptides,
oritavancin,5 telavancin,6 and dalbavancin,7 are before
introduction into clinical practice. However, of these three
antibiotics, only oritavancin has excellent (MIC < l μg/mL)
bacteriostatic effect against enterococci possessing the
vanA resistance gene, which is associated with resistance
to both glycopeptides vancomycin and teicoplanin. In
order to study the role of sugar components in the anti-
bacterial action of such derivatives, we decided to syn-
thesize lipophilic derivatives from the teicoplanin and
ristocetin derivatives possessing no or diminished sugar
content, i.e., using antibiotic aglycons or pseudoaglycons
as starting compounds. Recently, we have started a sys-
tematic study on semisynthetic derivatization of vanco-
mycin and ristocetin aglycons.8-10 The latter compounds
can be obtained from the parent antibiotics using anhy-
drous hydrogen fluoride treatment according to Boger
et al.11 We have recognized that attachment of lipophilic
side chains to the aglycons through a squaramide moiety
yields new vancomycin and ristocetin derivatives with
good antibacterial8,9 and, in some cases, anti-influenza
virus activities.10
Glycopeptide antibiotics vancomycin and teicoplanin are
used for treating serious Gram-positive bacterial infections
that are resistant to other antibiotics. From the late 1980s,
glycopeptide-resistant enterococci (GREa) and glycopeptide
intermediate resistant Staphylococcus aureus (GISA) as well
as teicoplanin resistant Staphylococcus hemolyticus have emer-
ged and are widespread. This problem induced an urgent need
for new antibiotics active against resistant bacteria and pro-
voked an intensive research in this field in the past 20 years.1-3
*To whom correspondence should be addressed. H-4010 Debrecen
POB 70, Hungary. (G.B.) Tel: þ36-52-512-900/22895. Fax: þ36-52-512-
914. E-mail: batta@tigris.unideb.hu. (P.H.) E-mail: herczeghp@gmail.
com.
a Abbreviations: MIC, minimum inhibition concentration; MBC,
minimum bactericidal concentration; ATCC, American typed culture
collection; MSSA, methicillin sensitive Staphylococcus aureus; MRSA,
methicillin resistant Staphylococcus aureus; Vanco/Teico R, vancomy-
cin/teicoplanin resistant; Vanco/Teico S, vancomycin/teicoplanin sensi-
tive; vanA þ, vanA gene positive; vanB þ, vanB gene positive; GRE,
glycopeptide-resistant enterococci; GISA, glycopeptide intermediate
resistant Staphylococcus aureus; VRE, vancomycin resistant enterococ-
cus; CC50, 50% cytotoxic concentration; EC50, 50% effective concen-
tration; CPE, cytopathic effect; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-
(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; D-Ala,
D-alanine; D-Lac, D-lactic acid; GlucNAc, N-acetylglucosamine; DOSY,
diffusion ordered spectroscopy; STD, saturation transfer difference;
NOESY, nuclear Overhauser effect spectroscopy; TMS, tetramethylsi-
lane; DSS, 2,2-dimethyl-2-silapentane-5-sulfonic acid; HSQC, hetero-
nuclear single quantum correlation; COSY, correlation spectroscopy;
TOCSY, total correlation spectroscopy; MALDI-TOF MS, matrix
assisted laser desorption ionization time-of-flight mass spectrometry;
DLS, dynamic light scattering; TLC, thin layer chromatography; Et3N,
triethylamine; DMF, N,N-dimethyl formamide.
In the present work, we report on the extension of those
studies to ristocetin and teicoplanin derivatives using a new
derivatization methodology.
r
2009 American Chemical Society
Published on Web 09/04/2009
pubs.acs.org/jmc

6054 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19
Pinteꢀr et al.
Figure 1. List of ristocetin derivatives.
Results and Discussion
was prepared by esterification of 4c, followed by the click
reaction of the intermediate 4f with 6. Similarly, starting from
teicoplanin aglycon methyl ester (4b), the diazo transfer
reaction led to 4h, which was coupled with 6 to give the 4i
methyl ester derivative. These chemical transformations are
summarized in Scheme 2. All of the new compounds were
tested for antibacterial and antimyxoviral activity.
There are several structural differences between the risto-
cetin and teicoplanin aglycons. Compared to the teicoplanin
aglycon, the ristocetin derivative bears one additional alipha-
tic hydroxyl, an aromatic C-methyl substituent, and the
carboxyl group in the form of a methyl ester, while the chloro
substituents are lacking. Moreover, we also used the teicopla-
nin pseudoaglycon 4a carrying the N-acetylglucosaminyl
substituent. All of the differences in biological activity can
be attributed to these factors.
The 2b and 4c azides showed intermediate bacteriostatic and
bactericidal activity on all Staphylococcus aureus and Staphy-
lococcus epidermidis strains, regardless of their methicillin-
resistance status (Table 1). They exerted similar activity against
the vancomycin- and teicoplanin-sensitive Enterococcus faeca-
lis strain. The lipophilic ristocetin derivatives 2c and 2d (log
P=3.36 and 2.71, respectively) proved to be completely inactive.
However, the teicoplanin derivatives 4d and 4e exhibited
significantly stronger bacteriostatic and bactericidal effect at
very low concentrations than did either vancomycin or teico-
planin. They inhibited the growth of all strains tested, regardless
During our previous studies for the preparation of the
starting vancomycin and ristocetin aglycons, we used Boger’s
method, i.e., hydrogen fluoride treatment of the parent anti-
biotics. Now, when we applied this cleaving procedure for
teicoplanin, we obtained the N-acetylglucosaminyl aglycon 4a
as an exclusive product, even after prolonged reaction time.
Compound 4a was obtained earlier12 by means of a laborious
hydrolysis and purification method.
The Cu(I) ion catalyzed version of the Huisgen 1,3-dipolar
cycloaddition reaction of organic azides and alkynes is one of
the most versatile ligation methods known as the click reac-
tion.13 In order to use this methodology for the derivatization
of 2a (Figure 1) and 4a (Figure 2) with lipophilic molecules,
the primary amino group of 2a and 4a was exchanged for an
azido function with the help of the diazo transfer reaction, i.e.,
reacting the amines with trifluoromethanesulfonyl azide in
pyridine solution, in the presence of a catalytic amount of
copper ion.14 In this way, azides 2b and 4c were prepared. For
the introduction of lipophilic groups, n-decyl-propargyl ether
(6) and 4-propargyloxybiphenyl (8) were prepared by propar-
gylation of the appropriate alcohols (Scheme 1).
The latter compounds were allowed to react with azides 2b
and 4c in the presence of a catalytic amount of cuprous iodide,
resulting in the 1,2,3-triazoles 2c, 2d, 4d, and 4e. In order to
study the contribution of methyl ester and N-acetyl-D-gluco-
samine residues on biological activity, 4g (methyl ester of 4d)

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19 6055
Figure 2. List of teicoplanin derivatives.
of staphylococcal methicillin resistance or enterococcal vanco-
mycin-teicoplanin resistance. Their minimal bactericidal con-
centrations were close to or the same as their MICs for all
strains examined, indicating a very strong bactericidal activity.
In this respect, these two derivatives are more active than
oritavancin, for which the concentration yielding bactericidal
activity is much higher compared to its MIC value. (In the case
of vanA-positive E. faecium, MIC=1-2 μg/mL, and MBC=
8-32 μg/mL).15 In spite of the inactivity of ristocetin deriva-
tives, interestingly, conversion of 4d to the 4g ester did not
reduce the very good bacteriostatic activity of the former, while
the bactericidal effect against enterococci was lost after such a
simple chemical transformation. The ester derivative of the
n-decyl aglycon (4i) proved to be inactive, demonstrating the
strong contribution of the N-acetylglucosaminyl residue to the
antibacterial activity of this group of compounds.

6056 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19
Pinteꢀr et al.
Recently, we have reported the synthesis of a series of
ristocetin aglycon derivatives containing lipophilic side chains
connected to the N-terminal group by a squaramide linker.10
Since some of these compounds showed excellent activity
against three (sub)types of influenza virus, as an extension
of these studies, we evaluated the present triazolyl compounds
for anti-influenza virus activity. The teicoplanin derivatives
4c, 4d, and 4e proved to be completely inactive (Table 2). The
ristocetin azido derivative 2b showed moderate activity
against influenza A/H1N1 and A/H3N2 viruses, but it was
inactive against type B. The more lipophilic n-decyl derivative
2c exerted good and reproducible activity against all
three (sub)types tested, with a mean antiviral EC50 value of
0.71 μM and a selectivity index (ratio of cytotoxic to antiviral
concentration) of 17. In fact, the EC50 values of 2c were
superior to that of the therapeutically used oseltamivir car-
boxylate. Compound 2d and the methyl ester derivatives
4f-4i were inactive.
Since the aim of this work was to study the influence of
lipophilicity of glycopeptide antibiotic derivatives on their
antibacterial activity, we performed some theoretical calcula-
tions on their log P values. These experimental value-adjusted
calculations were based on the experimental log P of com-
pound 2b (see Table 3). From a comparison of the log P values
of the compounds (see Table 3) to their antibacterial activities,
we established that the most active derivatives have medium
level of lipophilicity, i.e., log P of 0.3-1.3, while the very
lipophilic (2c, 2d, and 4i) and hydrophilic (4c and 4f) com-
pounds possessed minimal or no activity.
Scheme 1a
The probable mechanism of the antibacterial action of
glycopeptide antibiotics is based on the strong complex for-
mation with the N-acyl-D-Ala-D-Ala termini of a biosynthetic
intermediate of the bacterial cell wall peptidoglycan.3 In this
way, completion of the biosynthesis of the cell wall is inhib-
ited. According to their susceptibility to vancomycin and
teicoplanin-vancomycin, resistant enterococci (GRE) can
be divided into two phenotypes vanA and vanB.16,17 The
former phenotype shows 1000-fold increased resistance to
both antibiotics, while vanB GRE are resistant to vancomycin
but remain susceptible to teicoplanin.18 At the molecular
basis, this resistance results from a change in the peptidogly-
can termini from D-Ala-D-Ala to D-Ala-D-lactate,19 thus
weakening the complex formation with antibiotics. Lipophilic
derivatives, such as oritavancin can overcome both vanA and
vanB resistance. It has been postulated that lipophilic substit-
uents of glycopeptide antibiotics facilitate dimerization and
membrane localization, resulting in a better binding to the
target D-Ala-D-Lac peptides.20 It has indeed been demon-
strated that dimerization can enhance the binding potency
of such ligands presented in multiple copies on a bacterial cell
wall model. In general, applying multivalency to glycopeptide
antibacterials, including covalent dimers21,22 and trimers,23 is
a good strategy to improve the activity against resistant
strains. Using ring-opening metathesis polymerization of a
a (a) Propargyl bromide/NaH/dry dioxane; (b) propargyl bromide/
K2CO3/dry DMF.
Scheme 2a
a (a) HF/anisole; (b) 90% TFA; (c) TfN3/pyridine/CuSO4/Et3N; (d)
HCl/MeOH; (e) 6/CuI/Et3N/DMF; (f) 8/CuI/Et3N/DMF.
Table 1. Antibacterial Activities of Ristocetin and Teicoplanin Derivativesa
MIC/MBC (μg/mL)
test microorganism
ATCC 6633 B. subtilis
vancomycin teicoplanin
2b
4/1
2c
2d
4c
4d
4e
4f
4g
2/4
4h
2/4
4i
1/4
1/4
2/4
2/4
0.5/8
1/4
>256 >256 64/32 2/0.25 0.5/0.25 8/8
0.5/0.5
ATCC 33591 MSSA
ATCC 29213 MRSA
ATCC 35984 S. epidermidis biofilm-
positive
2 /1
2/2
>256 >256 4/2
>256 >256 8/8
>256 >256 16 /16 0.5/0.25 0.5
0.5/0.25 0.5/0.25 4/8
0.5/0.25 0.5/0.25 4/8
0.5/0.5 64/256 64/256
8/32
1/4
1/1
1/1
64/256 128/256
64/256 256/256
16/8
8/8
S. epidermidis
ATCC 29212 E. faecalis Vanco:
S Teico: S
2/4
4/16
32/32
4/16
16/16 >256 >256 4/4
16/8
0.5/0.5 0.5
4/4
1/1
>256 >256 32/32 0.5/0.25 0.5/0.25 16/256 2/256 128/256 32/128
64/256 16/64
ATCC 51299 E. faecalis Vanco:
R Teico: S VanB þ
16/16
>256
16/64
>256
128/64 >256 >256 64/32 0.5/0.25 0.5/0.25 16/256 2/128 128/256 64/256
>256 >256 >256 >256 0.5/0.25 0.5/0.25 16/256 2/256 128/256 128/256
15376b E. faecalis Vanco: R Teico:
R VanA þ
a MIC, minimum inhibition concentration; MBC, minimum bactericidal concentration; ATCC, American Type Culture Collection; MSSA,
methicillin sensitive Staphylococcus aureus; MRSA, methicillin resistant Staphylococcus aureus; Vanco/Teico R, vancomycin/teicoplanin resistant;
Vanco/Teico S, vancomycin/teicoplanin sensitive; vanA þ, vanA gene positive; vanB þ, vanB gene positive. b Ref 33.

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19 6057
Table 2. Antimyxoviral Activity of Ristocetin and Teicoplanin Derivatives
c
antiviral EC50
influenza A H1N1
subtype
influenza A H3N2
subtype
cytotoxicity
influenza B
concentration
unit
minimum cytotoxic
concentrationb
visual scored
CPE
visual scored
CPE
visual scored
CPE
a
compound
CC50
MTS
MTS
MTS
2b
2c
μM
μM
>100
53.2
11.4
8.5
100
100
20
10
8.9
8.8
20
9.2
N.A.
N.A.
0.5
N.A.
N.A.
0.5
9
0.8
N.A.d
2
N.A.
2.8
0.5
0.7
0.1
4
0.1
0.2
0.2
0.2
2d
4c
4d
4e
4f
μM
μM
μM
μM
μM
μM
μM
μM
17.4
59.7
3.7
g4
20
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
4
2.6
4
20
48.7
0.8
4g
4h
4i
0.8
0.2
g0.8
0.1
4.6
Oseltamivir carboxylate
Ribavirin
Amantadine
μM
μM
μM
μM
>100
>100
>500
241
>100
>100
>500
500
2.0
9.0
45
8.6
9.7
35
0.8
9
1.1
6.0
0.4
0.2
12
9
6.4
3.6
0.8
0.1
N.A.
N.A.
N.A.
N.A.
Rimantadine
10
13
a Fifty percent cytotoxic concentration, as determined by measuring the cell viability with the colorimetric formazan-based MTS assay. b Minimum
compound concentration that causes a microscopically detectable alteration of normal cell morphology. c Fifty percent effective concentration or the
concentration producing 50% inhibition of virus-induced cytopathic effect, as determined by visual scoring of the CPE or by measuring the cell viability
with the colorimetric formazan-based MTS assay. d N.A.: not active at the highest concentration tested or at subtoxic concentration.
aggregates. For comparison of the results of these physical
measurements, the molecular volume of 4d has been calcu-
lated with Gaussian 0326 and MOPAC 200727 from optimized
lowest energy geometries obtained from molecular dyna-
mics.25 The estimated computed volume was 1.0 dm3/mol.
Usingthis volumeandthe average diameter of 10nm obtained
bylight scatteringexperiment, wecouldcalculateanaggregate
containing about 240 molecules, having a molecular mass of
about 390 kDa. This is in good agreement with the result of
DOSY NMR.
Table 3. log P Values of Ristocetin and Teicoplanin Derivatives
derivative
log P (calculated)
2a
2b
2c
2d
4a
4c
4d
4e
4f
-0.91
0.29
3.36
2.71
-2.48
-1.83
1.24
0.59
-1.96
1.11
1.57
Elucidation of the nature of the interaction of one of our
most effective lipophilic teicoplanin derivatives 4d with the
model peptide derivatives of the bacterial cell wall of non-
resistant and resistant bacteria could help to explain the high
antibacterial potency of these compounds against VRE.
Saturation transfer difference (STD) NMR spectroscopy is
a powerful tool for studying receptor-ligand interactions on
the binding epitope level.28-32 Therefore, we attempted to use
it for obtaining information on the interaction of compound
4d with NR,Nε-diacetyl-L-lysyl-D-alanyl-D-alanine (A) (the
terminal tripeptide model of the vancomycin-sensitive
bacteria) and with NR,Nε-diacetyl-L-lysyl-D-alanyl-D-lactic
acid (B), which is the component of VRE strains. STD spectra
obtained with and without presaturation of the n-decyl region
of the spectra of a solution of 4d and compound A (Figure 3)
and of 4d and compound B provided evidence for a strong
apolar interaction between the lipophilic decyl chain of the
antibiotic and the methyl groups of N-acetyl substituents of A
or B, as well as with the two alanine methyl groups in A or
the alanine and lactate methyls in compound B (Figure 4).
These interactions are similarly defined for A and B, revealing
a binding force in addition to the hydrogen bonds of the
classical D-Ala-D-Ala binding pocket. In addition to the
strong STD responses observed at the tripeptide ligands
due to saturation of the decyl side chain in the micelles
of 4d, standard NOESY experiments revealed some more
intermolecular interactions. Using ligand A, negative NOEs
4g
4h
4i
4.64
a Experimentally measured value (see text).
vancomycin monomer, Arimoto et al.24 obtained multivalent
polymers which exhibited significant activity against VRE.
From an analysis of the structures of our lipophilic teico-
planin derivatives possessing the highest antibacterial activity
(for example4dor 4e), it canbe postulated thatthey must have
an amphiphilic character. As a result of their lipophilic side
chain and negatively charged carboxylate group, micelle
formation of these molecules can be expected. Therefore,
the high bacteriostatic and bactericidal activity of 4d could
be explained by self-assembly in aqueous medium into a
multivalent aggregate.
We examined the aggregation of 4d sodium salt in water
using dynamic light scattering measurements. This experi-
ment showed the presence of aggregates with an effective
diameter of 10 nm in monomodal distribution. We also
performed diffusion ordered NMR (DOSY) experiments to
measure the molecular mass of the aggregates, using DSS
(M=196) as the internal reference compound. Disregarding
uncertainties caused by the simplification that shapes and
densities of the studied molecules must be similar, we obtained
a 350 ( 60 kDa value for the molecular mass of the 4d

6058 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19
Pinteꢀr et al.
Figure 3. Saturation transfer difference (STD) NMR spectrum of 4d with ligand A.
Figure 4. Comparison of the STD spectra of 4d with ligand A (top) and of 4d with ligand B (bottom). In the bottom spectrum, the broad micelle
signals are less efficiently suppressed because of the shorter spin-lock relaxation filter time (20 ms vs 100 ms).
(slow motion regime) are clearly detected between the Me
group of R-NHAc (1Lys) and nearly all protons of the
GlucNAc in 4d. Furthermore, the anomeric proton of Gluc-
NAc interacts with the methyl group 2β of A. Consequently,
thereis an interaction at the GlucNAc moietyof4d in addition
to the apolar interactions at the decyl chain of the modified
glycopeptide. These two effects together may more than out-
weigh the impact of a less efficient binding pocket. Probably,
other molecules with apolar parts could also bind nonspeci-
fically to the glycopeptide micelles. However, in our case,
the apolar anchoring works as a useful hand for consecutive
antibiotic binding. It is suggested, therefore, that the tripep-
tides bind to antibiotic self-assemblies most likely close to the
decyl hydrophobic patches of the modified antibiotics that
could be further enhanced with interactions around the
GlucNAc part of the molecules. The hydrophobic surfaces
may support nonspecific interactions with the peptidoglycan
cell wall and provide a possible explanation for the strong
antibacterial activity of 4d and similar lipophilic derivatives
against resistant VRE.
Structural modification of teicoplanin pseudoaglycon by
introduction of lipophilic side chains with concomitant change

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19 6059
Figure 5. Interactions of 4d with ligands A and B based on STD
experiments.
Figure 6. Numbering of the atoms of the compounds for the NMR
assignment (above, teicoplanin derivatives; below, ristocetin de-
rivatives).
of the terminal amino group by a neutral triazole linker group
has a beneficial influence on antibacterial activity. This effect
can be explained by the aggregation of the amphiphilic
derivatives, as well as by secondary interactions of the lipo-
philic substituent with the target bacterial peptide. Esterifica-
tion of the carboxyl group (4fand 4g) resulted in nosignificant
change of the antibacterial activity; however, removal of the
N-acetyl-D-glucosamine residue (4h and 4i) rendered the
compounds inactive. Introduction of similar lipophilic side
chains into the ristocetin aglycon yielded inactive derivatives.
Therefore, it can be postulated that the N-acetyl-D-glucosa-
mine component of the lipophilic teicoplanin derivatives has a
crucial role in antibacterial activity.
Interestingly, the presence of the above-mentioned sugar in
the lipophilic derivatives caused a lack of anti-influenza virus
activity. Remarkably, the n-decyl-triazolyl ristocetin aglycon
derivative 2c displayed favorable anti-influenza action. Cur-
rently, we have no clear insight into the antiviral mode of
action of these lipopeptide ristocetin derivatives, but it can be
concluded that, besides obtaining potent antibacterials, intro-
duction of lipophilic side chains into the aglycon derivatives of
glycopeptide antibiotics also represents an attractive strategy
for the development of new anti-influenza virus compounds.
Mass spectra were recorded with a Bruker Biflex-III MALDI
TOF mass spectrometer. Purity of the new compounds was
>95% according to the combustion analysis (C,H,N). The
dynamic light scattering mesurements were performed on a
Brookhaven Light Scattering device equipped with a BI-9000
digital correlator. For purification of the products, flash chro-
matography on Merck silica gel (Kieselgel 60), 0.040-0.063 mm
(70-230 mesh) was used. Thin layer chromatography (TLC)
was performed on Kieselgel 60 F254 (Merck).
Spots were visualized by irradiation under UV lamp and/or
by spraying with an ammonium-molibdenate/sulfuric acid solu-
tion and heating, or using Pauly reagent. Evaporations were
carried out under diminished pressure at 35-40 ꢀC (bath
temperature).
Synthesis. Ristocetin Aglycon (2a). Compound 1 (5.0 g, 2.31
mmol) was dissolved in anisole (6.0 mL), and anhydrous HF (50 mL)
was passed into the reaction vessel at -70 to -80 ꢀC. The
reaction mixture was stirred at -18 ꢀC for 2 h. After evaporation
of the anisole and HF, the residue was diluted with dry ether.
The precipitate was filtered off and washed with dry ether. The
crude product was purified by flash chromatography (CH2Cl2:
MeOH =8: 2 f 6: 4) to give 2a (1.7 g, 61%) .
MALDI-TOF MS: [M þ Na]þ = 1196.4 m/z. Calcd. for
C60H51N7O19Na 1196.3. 1H NMR, 13C NMR: for the chemical
shift assignment, see ref 8.
Experimental Section
General Method for Preparation of the Azido Derivatives (2b,
4c, and 4h). The corresponding amino derivative (2a, 4a, and 4b)
was dissolved in pyridine. Two equivalents of Et3N and 0.1 equi-
valent of aqueous copper(II) sulfate solution (c = 10 mg/mL)
were added and cooled to 0 ꢀC. Then, 2.5 equivalent of freshly
prepared trifluoromethanesulphonyl azide in dry pyridine was
added, and the reaction mixture was stirred at room tempera-
ture overnight and diluted with toluene, and the solvents were
evaporated, and dry toluene was destilled from the residue. The
purification was carried out by flash chromatography to give the
corresponding azido derivative (2b, 4c, and 4h).
Materials and Methods. The starting materials and solvents
were purchased from commercial sources (Sigma-Aldrich or
Fluka and Hallochem Pharma - teicoplanin) and used as recei-
ved. 1H and 13C NMR spectra were recorded at 360 (500) and
90 (125) MHz, respectively, with a Bruker DRX-360 (DRX 500)
spectrometer at 300 K, using CDCl3, MeOH-d4, DMSO-d6, or
D2O as solvents and TMS (tetramethylsilane) or DSS (2,2-
dimethyl-2-silapentane-5-sulfonic acid) as internal standards.
Signal assignment was aided by 2D HSQC, COSY, and TOCSY
techniques. Multiplicities of the 1H NMR signals are reported as
follows: d, doublet; t, triplet; m, multiplet. For numbering of
atoms in ristocetin and teicoplanin derivatives see Figure 6. IR
spectra were recorded on a Perkin-Elmer 16 PC FTIR spectrometer.
Teicoplanin ψ-Aglycon (4a). Compound 3 (3.5 g, 1.87 mmol)
was dissolved in anisole (3.0 mL), and anhydrous HF (50 mL)

6060 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19
Pinteꢀr et al.
was passed into the reaction vessel at -70 to -80 ꢀC. The
reaction mixture was stirred at -18 ꢀC for 2 h. After evaporation
of the anisole and HF, the residue was diluted with a cold ethyl
acetate/methanol = 4:1 mixture (100 mL), and the precipitate
was filtered off and washed with cold ethyl acetate and ether to
result in 4a (2.1 g, 80%).
(4) Nagarajan, R.; Schabel, A. A.; Occolowitz, J. L.; Counter, F. T.;
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Wilkie, S. C.; Nicas, T. I.; Mullen, D. L.; Butler, T. F.; Rodriguez,
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Glycopeptide Antibiotics: Synthesis and Antibacterial Activity.
J. Antibiot. 1996, 49, 575–581.
MALDI-TOF MS: [M þ Na]þ=1423.3 m/z,[M- Hþ 2Na]þ=
1
11445.3 m/z. Calcd. for C66H58Cl2N8O23Na 1423.3. H NMR
(DMSO-d6, 500 MHz) δ: 2.81/3.33 (z2), 4.33 (G1), 5.07 (4f), 5.29
(z6), 5.53 (4b). 13C NMR (DMSO-d6, 500 MHz) δ: 37.2 (z2),
76.8 (z6), 99.9 (G1), 105.4 (4f), 108.6 (4b).
(6) Judice, J. K.; Pace, J. L. Semi-Synthetic Glycopeptide Antibacter-
ials. Bioorg. Med. Chem. Lett. 2003, 13, 4165–4168.
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Chem. 2001, 8, 1759–1773. Van Bambeke, F. Glycopeptides in Clinical
Development: Pharmacological Profile and Clinical Perspectives. Curr.
Opinion Pharmacol. 2004, 4, 471–478.
Methyl Ester of Teicoplanin Aglycon (4b). Compound 4a
(250 mg, 0.18 mmol) was dissolved in 90% TFA (5.0 mL) and
was stirred at 80 ꢀC for 5 h. The reaction mixture was poured on
cold dry ether, and the precipitate was filtered out. The crude
product, without further purification, was dissolved in metha-
nol (18.0 mL), and hydrochloric acid in methanol (2.0 mL,
0.5 M) was added and refluxed for 4 h. After evaporation of the
solvent, the residue was purified by flash chromatography
(CH2Cl2/MeOH=8:2) to give 4b (98 mg, 45%) as a pale yellow
substance.
ꢀ
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Rooth, E.; Szabo, P. T.; Kardos, S.; Rozgonyi, F.; Boda, Z. A
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Acid Moiety. Synthesis, Structural and Antibacterial Studies.
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(9) Sztaricskai, F.; Pinter, G.; Rooth, E.; Herczegh, P.; Kardos, S.;
Rozgonyi, F.; Boda, Z. N-Glycosylthioureido Aglyco-Ristocetins
Without Platelet Aggregation Activity. J. Antibiot. 2007, 60, 529–
533.
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Snoeck, R.; Panneconque, C.; Illyes, E.; Batta, G.; Herczegh, P.;
MALDI-TOF MS: [M þ Na]þ = 1234.4 m/z. Calcd. for
C59H47Cl2N7O18Na 1234.2. 1H NMR, 13C NMR: for the chemi-
cal shift assignment, see ref 8.
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ity Relationship of Aglycoristocetin Derivatives with Cyclobutene-
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Methyl Ester of Azido-Teicoplanin ψ-Aglycon (4f). Compound
4c (70 mg, 0.01 mmol) was dissolved in methanol (1.8 mL), and
hydrochloric acid in methanol (0.2 mL, 0.5 M) was added and
refluxed for 2 h. The reaction mixture was concentrated, and the
residue was purified by flash chromatography (toluene/MeOH =
6:4) to give 4f (30 mg, 41%).
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MALDI-TOF MS: [M þ Na]þ = 1463.4 m/z. Calcd. for
.
C67H58Cl2N10O23Na 1463.3. IR: 2110 cm-1 1H NMR
(MeOH-d4, 500 MHz) δ: 1.89 (NHAc), 2.97/3.38 (z2), 3.84
(COOCH3), 4.49 (G1), 5.19 (x1), 5.2 (4f), 5.39 (z6), 5.69 (4b).
13C NMR (MeOH-d4, 500 MHz) δ: 22.1 (NHAc), 37.1 (z2), 52
(COOCH3), 64.6 (x1), 78.7 (z6), 101.2 (G1), 104.6 (4f), 108 (4b).
General Method for Preparation of the Triazol Derivatives (2c,
2d, 4d, 4e, 4g, and 4i). In a solution of the corresponding azide
and 1.2 equivalents of propargyl ether 6 or 8 in dry DMF, argon
was bubbled for 10 min, then Et3N (1 equivalent) and CuI
(0.1 equivalent) were added. The reaction mixture was stirred at
room temperature overnight. The copper was precipitated as
copper sulfide passing hydrogen sulfide into the reaction mix-
ture. After removal of the solvent under reduced pressure, the
residue was purified by flash chromatography.
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Solid Phase: [1,2,3]-Triazoles by Regiospecific Copper(I)-Cata-
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and Terminal Alkynes. Angew. Chem., Int. Ed. 2002, 41, 2596–
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Acknowledgment. This work was supported by the Hun-
garian ResearchFund OTKAthroughgrants 79126, T-46186,
OTKA-NKTH CK 77515, and NK 68578. L.N. acknowl-
edges the technical assistance from Leentje Persoons and the
financial support from the Flemish Fonds voor Wetenschap-
pelijk Onderzoek (FWO No. 9.0188.07) and the International
Consortium for Anti-Virals (ICAV).
(16) Arthur, M.; Courvalin, P. Genetics and Mechanisms of Glycopep-
tide Resistance in Enterococci. Antimicrob. Agents Chemother.
1993, 37, 1563–1571.
(17) Evers, S.; Quintiliani, R., Jr; Courvalin, P. Genetics of Glycopep-
tide Resistance in Enterococci. Microb. Drug Resist. 1996, 2, 219–
223.
(18) Walsh, C. T. Antibiotics: Actions, Origins, Resistance; ASM Press:
New York, 2003; pp 148-155.
(19) Walsh, C. T.; Fisher, S. L.; Park, I. S.; Prahalad, M.; Wu, Z.
Bacterial Resistance to Vancomycin: Five Genes and one Missing
Hydrogen Bond Tell the Story. Chem. Biol. 1996, 3, 21–28.
(20) Sharman, G. J.; Try, A. C.; Dancer, R. J.; Cho, Y. R.; Staroske, T.;
Bardsley, B.; Maguire, A. J.; Cooper, M. A.; O’Brien, D. P.;
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choring in Activity of Glycopeptide Antibiotics Against Vanco-
mycin-Resistant Bacteria. J. Am. Chem. Soc. 1997, 119, 12041–
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Supporting Information Available: Synthetic procedures, MS,
NMR, and combustion analysis data, antibacterial and antiviral
assays, log P calculations, DLS experiments, computational
methods, DOSY, NOESY, and STD measurements This ma-
terial is available free of charge via the Internet at http://pubs.
acs.org.
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Dimers with Activity against Vancomycin-Resistant Enterococci.
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