Antiretroviral activity of semisynthetic derivatives of glycopeptide antibiotics

Article abstract of DOI:10.1021/jm0300882

A variety of semisynthetic derivatives of natural antibacterial glycopeptide antibiotics such as vancomycin, eremomycin, ristocetin A, teicoplanin A₂-2, DA-40926, their aglycons, and also the products of their partial degradation with a destroyed or modified peptide core show marked anti-retroviral activity in cell culture. In particular, aglycon antibiotic derivatives containing various substituents of a preferably hydrophobic nature displayed activity against human immunodeficiency virus type 1 (HIV-1), HIV-2, and Moloney murine sarcoma virus at a 50% inhibitory concentration in the lower micromolar (1-5 μM) concentration range while not being cytostatic against human lymphocytic cells at 250 μM or higher. The mode of anti-HIV action of the antibiotic aglycon derivatives could be ascribed to inhibition of the viral entry process.

Full text of DOI:10.1021/jm0300882

J . Med. Chem. 2003, 46, 2755-2764
2755
An tir etr ovir a l Activity of Sem isyn th etic Der iva tives of Glycop ep tid e An tibiotics
J an Balzarini,† Christophe Pannecouque,† Erik De Clercq,† Andrey Y. Pavlov,#,‡ Svetlana S. Printsevskaya,#
Olga V. Miroshnikova,# Marina I. Reznikova,# and Maria N. Preobrazhenskaya*,#
Rega Institute for Medical Research, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium, and
Gause Institute of New Antibiotics, Russian Academy of Medical Sciences, B. Pirogovskaya 11, Moscow 119021, Russia
Received February 20, 2003
A variety of semisynthetic derivatives of natural antibacterial glycopeptide antibiotics such as
vancomycin, eremomycin, ristocetin A, teicoplanin A2-2, DA-40926, their aglycons, and also
the products of their partial degradation with a destroyed or modified peptide core show marked
anti-retroviral activity in cell culture. In particular, aglycon antibiotic derivatives containing
various substituents of a preferably hydrophobic nature displayed activity against human
immunodeficiency virus type 1 (HIV-1), HIV-2, and Moloney murine sarcoma virus at a 50%
inhibitory concentration in the lower micromolar (1-5 µM) concentration range while not being
cytostatic against human lymphocytic cells at 250 µM or higher. The mode of anti-HIV action
of the antibiotic aglycon derivatives could be ascribed to inhibition of the viral entry process.
In tr od u ction
induced cytopathicity in CEM cell cultures and MSV-
induced transformation of murine fibroblast cells.
Glycopeptide antibiotics (i.e., vancomycin, teicoplanin)
are vital therapeutic agents used worldwide for the
treatment of serious life-threatening infections caused
by Gram-positive bacteria. Other antibiotics of this type
(eremomycin, chloroeremomycin, ristocetin, teicoplanin
aglycon, and some others) are also highly active against
Gram-positive microorganisms including methicillin-
resistant staphylococci.1 The antibacterial activity of
natural glycopeptide antibiotics is based on their ability
to inhibit bacterial cell wall biosynthesis by a reversible,
noncovalent binding of the drugs to the -D-Ala-D-Ala
fragments of the peptidoglycan precursor.2 Emerging
bacterial resistance to vancomycin, which has recently
become a major public health threat, was a stimulus
for the synthesis and investigation of various derivatives
of glycopeptide antibiotics. This research has resulted
in the discovery of the activity of hydrophobic deriva-
tives of these antibiotics against glycopeptide-resistant
enterococci (GRE).3-5 Some hydrophobic derivatives of
eremomycin or vancomycin also demonstrate antibacter-
ial activity despite decreased binding to D-Ala-D-Ala and
D-Ala-D-lactate.6,8 This antibacterial activity appears to
be due to inhibition of the transglycosylase reaction,
which is of crucial importance in the bacterial pepti-
doglycan synthesis.6-10 The nature of the hydrophobic
substituent on these molecules plays a major role in the
eventual antibacterial activity of the glycopeptide an-
tibiotic derivatives against GRE.8 In the present study,
a wide variety of lipophilic glycopeptide antibiotic
derivatives have been evaluated against the cytopath-
icity of HIV-1 and HIV-2 in human CEM cell cultures
and against the transforming effect of Moloney murine
sarcoma virus (MSV) on murine C3H/3T3 embryo
fibroblast cell cultures. Several glycopeptide antibiotics
proved to be highly efficacious in preventing HIV-
Resu lts
The goal of our studies was the search for low toxicity
anti-HIV compounds among various semisynthetic de-
rivatives of natural vancomycin, eremomycin, teicopla-
nin, DA-40926 and ristocetin glycopeptide antibiotics,
the aglycons thereof, and also glycopeptide antibiotics
with the peptide core partially destroyed or modified.
The methods for introducing chemical modifications in
the sugar moieties, at the amide part, at the resorcinol
fragment and at the N-end of the antibacterial glyco-
peptide antibiotics were elaborated earlier and used for
the preparation of a variety of semisynthetic glycopep-
tides.3,4 Here, we present for the first time the results
of the investigations of the antiretroviral properties of
these glycopeptide antibiotic derivatives. The structures
of the compounds investigated are presented in Tables
1a-7a and 8. The cytotoxic and antiviral data are given
in Tables 1b-7b and 8. They represent the inhibitory
effects (IC50 in µM) of the compounds investigated on
the proliferation of murine leukemia cells (L1210) and
human T-lymphocytic cells (Molt4/C8, CEM), HIV-1 and
HIV-2 infection (EC50 in µM) in human T-lymphocytic
(CEM) cells, MSV-induced transformation of C3H/3T3
embryo murine fibroblasts (EC50 in µM), and cytotoxic
activity against C3H/3T3 cell cultures (minimum inhibi-
tory concentration (MIC) in µM).
Vancomycin and eremomycin were neither toxic to the
human CEM and Molt4/C8 and murine embryo fibro-
blast cells nor inhibitory to HIV-1, HIV-2, and MSV. The
introduction of a hydrophobic substituent on the van-
comycin and eremomycin molecules resulted in the
appearance of moderate anti-HIV-1 activity for some of
the derivatives (e.g., 11); however, their cytotoxicity was
also increased in comparison with the unsubstituted
antibiotics (Table 1). Compound 11 preferentially in-
hibited HIV-1 above HIV-2.
* To whom correspondence should be addressed. E-mail: lcta@
space.ru. Phone: (7095) 245-37-53. Fax: (7095) 245-02-95.
† Katholieke Universiteit Leuven.
Interestingly, teicoplanin (14), but not ristocetin (12),
was active against HIV-1 (EC50 ) 17 µM). The deman-
#
Gause Institute of New Antibiotics.
‡ Deceased April 17, 2002.
10.1021/jm0300882 CCC: $25.00 © 2003 American Chemical Society
Published on Web 05/24/2003

2756 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13
Balzarini et al.
Ta ble 1. Structures of Vancomycin, Eremomycin, and Their Derivatives and Inhibitory Data
(a) Vancomycin, Eremomycin, and Their Derivatives
compd
ref
X
Y
R
Brutto formula
MW
Vancomycin (Van) and Its Derivatives
W ) CI, S1 ) Glc, S2 ) Vancosamine, S3 d H
1 (Van)
H
H
H
H
OH
H
C
66H75N9O24Cl2
1448
1587
1647
1648
2
3
4
11
11
15, 16
NHC10H21
NHBn(PhCl-p)-p
OH
H
H
C76H96N10O23Cl2
C
C
79H85N10O23Cl3
79H84N9O24Cl3
Bn(PhCl-p)-p
Eremomycin (Ere) and Its Derivatives
W ) H, S1 ) Glc, S2 ) S3 ) Eremosamine
5 (Ere)
6
7
8
9
10
11
H
H
OH
H
H
H
H
H
H
H
C
C
73H89N10O26Cl
74H93N11O25Cl
1556
1570
1725
1693
1756
1721
1786
17
18
19
8
NHMe
OH
NHC10H21
NHBn(PhCl-p)-p
NHBnPh-p
OH
CH2NHC10H21
H
H
H
C84H112N11O26Cl
C83H110N11O25Cl
C
C
86H100N11O25Cl2
86H100N11O25Cl
19
CH2NHBn(PhCl-p)-p
C87H102N11O26Cl2
(b) Inhibitory Effects (IC50, µM) of Vancomycin-Type Glycopeptides and Their Derivativesa
IC50,b µM
EC50,c µM
MIC,d µM
C3H/3T3
compd
L1210
MOLT-4/C8
CEM
HIV-1
HIV-2
MSV
Vancomycin Derivatives
1 (vancomycin)
>500
30 ( 22
>500
>10
>500
30 ( 2
>250
>10
>250
>10
>100
>100
20
20
g25
2
3
4
>4
>2
7.7 ( 4.3
Eremomycin Derivatives
5 (eremomycin)
6
7
8
9
10
11
>500
>500
>500
>250
>250
>250
>20
>10
>10
>10
>50
>100
>100
>500
>500
>500
>250
>20
19 ( 6
29 ( 11
15 ( 11
41 ( 5
136 ( 12
27 ( 8
21 ( 1
7.7 ( 0.5
34 ( 0.5
72 ( 40
24 ( 13
9.4 ( 4.7
8.6 ( 0.2
35 ( 1
>10
>10
>0.8
>0.8
>4
4
4
g20
>10
106 ( 65
22 ( 3
a
Compounds with IC50 e 30 µM and MIC e 30 µM are shown in bold. Compounds with EC50 e 20 µM are shown in bold and italic.
b 50% inhibitory concentration, or compound concentration required to inhibit tumor cell proliferation by 50%. c 50% effective concentration,
or compound concentration required to protect cells against the cytopathogenicity of HIV or MSV by 50%. Minimal inhibitory concentration,
d
or compound concentration required to cause a microscopic detectable alteration of cell morphology.
nosyl ristocetin (13) and the methylamide of teicoplanin
(15) were both active predominantly against HIV-1, and
the last two compounds also proved to be inhibitory
against MSV (Table 2).
were not cytotoxic (IC50 > 500 µM) but active against
HIV-1 at rather high concentrations (EC50 ) 50-65 µM)
(Table 4). However, the hydrophobic derivatives of
eremomycin aglycon 25-28 invariably inhibited both
HIV-1 and HIV-2 to a comparable extent at EC50 values
between 3.5 and 12 µM. These concentrations were at
least 15- to 20-fold lower than required for the unsub-
stituted eremomycin aglycon to inhibit HIV replication.
The aglycon antibiotic derivatives were invariably non-
toxic for CEM cells (IC50 > 100-500 µM). Splitting off
the first amino acid (N-methyl-D-leucine) from eremo-
mycin aglycon led to the eremomycin aglycon hexapep-
tide (EAH) 29, which had lost activity against HIV.
However, its 2-aminoadamantane derivatives 30 and 31
were active against both HIV-1 and HIV-2 at EC50
values of 13-40 µM and were not cytotoxic at 250 µM
(Table 4).
The antibiotic deacyl-A40926 (16) and its demanno-
sylated derivative DMDA 40926 (17) were not cytotoxic
and did not show marked antiviral properties, but the
removal of the D-mannose residue and the introduction
of hydrophobic substituents led to antibiotic derivatives
with marked anti-HIV-1 activities (i.e., EC50 ) 3.5 µM
for 20 and 21), though some of the compounds were toxic
for the cells at rather low drug concentrations (Table
3). Compounds 20, 21 also inhibited the replication of
HIV-2, albeit at markedly higher (2.5- to 7-fold) con-
centrations than required for inhibition of HIV-1. Com-
pounds 20 and 22 also proved to be inhibitory to MSV
at subtoxic concentrations.
Removing all carbohydrates from vancomycin and
eremomycin led to aglycon antibiotics (23 and 24), which
The aglycons of ristocetin, DA40926, and teicoplanin
(Table 5, compounds 32-34) were predominantly en-

Derivatives of Glycopeptide Antibiotics
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13 2757
Ta ble 2. Structures of Teicoplanin-Type Glycopeptides and Their Derivatives and Inhibitory Data
(a) Teicoplanin-Type Glycopeptides and Their Derivatives
compd
ref
Y
Brutto formula
MW
2068
1304
Ristocetin A
W1 ) W2 ) W3 ) H, W4 ) Me, S1 ) Tetrasaccharide, S2 ) Ristosamine, S3 ) Man, S4 ) OH
12 (risto)
20 OMe 95H110N8O44
C
Ristosaminylaglycon of Ristocetin
W1 ) W2 ) W3 ) S1 ) S3 ) H, W4 ) Me, S2 ) Ristosamine, S4 ) OH
20 OMe 66H64N8O21
13
C
Teicoplanin and Its Derivatives
W1 ) W2 ) CI, W3 ) W4 ) H, S1 ) GlcNAcyl, S2 ) GlcNAc, S3 ) Man, S4 ) OH
14 (teico)
15
OH
C
88H97N9O33Cl2
2006
1893
21
NHMe
C89H100N10O32Cl2
(b) Inhibitory Effects (IC50, µM) of Teicoplanin-Type Glycopeptides and Their Derivativesa
IC50,b µM
EC50,c µM
MIC,d µM
compd
L1210
MOLT-4/C8
CEM
HIV-1
HIV-2
MSV
>100
C3H/3T3
12 (risto)
13
>500
>500
>500
>100
>500
>500
>250
g50
17 ( 3.5
80 ( 28
>250
g250
100 ( 0
190 ( 84
>100
g100
>100
>100
52 ( 30
>100
14 (teico)
15
>500
>500
>500
>500
75 ( 3
a
b
Compounds with EC50 e 20 µM are shown in bold and italic. 50% inhibitory concentration, or compound concentration required to
inhibit tumor cell proliferation by 50%. c 50% effective concentration, or compound concentration required to protect cells against the
d
cytopathogenicity of HIV or MSV by 50%. Minimal inhibitory concentration, or compound concentration required to cause a microscopic
detectable alteration of cell morphology.
dowed with moderate anti-HIV-1 activity while not
being toxic for mammalian cells. A large variety of
teicoplanin aglycon derivatives with hydrophobic sub-
stituents were synthesized and explored for their anti-
HIV and MSV activity (Table 5). Interestingly, all of
them showed very pronounced anti-HIV-1 and anti-
HIV-2 activity in cell culture, often with a tendency of
being slightly more active against HIV-1 than HIV-2
(∼2- to 5-fold). The methyl ester of the teicoplanin
aglycon (42) was active against both HIV-1 and HIV-2
nin aglycon with the first peptide bond disrupted (Table
7, compounds 69, 70) were still clearly active against
HIV-1 and HIV-2, and also MSV. Even when the bond
between amino acids 6 and 7 in the teicoplanin aglycon
peptide core was disrupted (71, Table 8), this compound
maintained measurable activity against both HIV strains
and MSV (EC50 ) 22-32 and 14 µM, respectively).
Discu ssion
There are common structural features for glycopep-
tide antibiotics to be active against glycopeptide-
resistant bacterial strains and retroviruses. The intro-
duction of a hydrophobic substituent is beneficial for
both types of activity. However, the presence of carbo-
hydrate moieties in glycopeptides usually results in a
dramatic decrease of anti-HIV activity, whereas for
antibacterial activity the presence of sugars represents
a critical determinant, although some hydrophobic
derivatives of aglycons of eremomycin, teicoplanin, and
des(N-methyl-D-leucyl)eremomycin also demonstrate
rather good anti-GRE properties.11
It seems that the peptide framework formed by amino
acids 2 and 4-7 containing a hydrophobic substituent
is a prerequisite for the antiretroviral activity. Modifica-
tions of the aglycons resulting in the disruption of the
macrocycles or deleting amino acid 1 leads to compounds
with surprisingly high anti-HIV activity, though the
antiviral efficacy is lower than for the derivatives with
the intact peptide core.
(EC50 ) 9.5 and 15 µM, respectively) and MSV (EC50
)
12 µM). However, the most active congeners were
inhibitory against HIV-1 in the range 0.75-2.5 µM
(compounds 41, 44, 61, and 62). Compounds 41, 61, and
62 were not cytotoxic at 250 µM. This means that these
compounds reached a selectivity index (ratio IC50/EC50)
that was g100. The antiviral activity of the latter
compounds (0.75-4.5 µM) was also at least 10- to 20-
fold improved over that of the unsubstituted teicoplanin
aglycon 34 (EC50 ) 17-20 µM; IC50 > 500 µM). Many
of the compounds active against HIV-1 were also potent
inhibitors of MSV-induced transformation of murine
fibroblast cell cultures at comparable drug concentra-
tions (Table 5b).
The elimination of amino acids 1 and 3 from teico-
planin aglycon and the introduction of hydrophobic
substituents was not accompanied by the disappearance
of the antiviral properties, although the antiviral po-
tency was ∼5-fold reduced compared to that of the intact
teicoplanin aglycon derivatives (Table 6, compounds
65-68). Similarly, hydrophobic derivatives of teicopla-
A group of hexapeptide antibiotics with a peptide core
similar to that of the antibacterial glycopeptides have

2758 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13
Balzarini et al.
Ta ble 3. Structures of N-Deacyl-A40926 (DA40), Demannosyl-N-deacyl-A40926 (DMDA40), and Their Derivatives and
Inhibitory Data
(a) N-Deacyl-A40926 (DA40), Demannosyl-N-deacylA40926 (DMDA40), and Their Derivatives
compd
ref
X
Y1 ) Y2
Z1
Z2
Brutto formula
MW
DA40 and Its Derivatives
S ) Man
16
H
OH
H
H
H
C
C
71H66N8O28Cl2
65H56N8O23Cl2
1551
DMDA40 and Its Derivatives
S ) H
17
18
19
20
21
22
22
23
24
24
24
23
H
H
H
OH
H
1389
1881
1703
1799
1967
1633
NH(CH2)3NMe2
NH(CH2)3NMe2
OH
NH(CH2)3NMe2
OH
p-BuOBn
p-BuOBn
p-BuBn
p-BuBn
p-BuBn
H
C97H108N12O23Cl2
C86H94N12O21Cl2
C94H90N10O23Cl2
C104H114N14O21Cl2
C81H80N10O23Cl2
H
H
H
H
CH2N[CH2CH2]2NBnPh-p
CH2N[CH2CH2]2NBnPh-p
CH2N[CH2CH2]2NBnBu-p
(b) Inhibitory Effects of DA40 and DMDA40 and Their Derivativesa
IC50,b µM
EC50,c µM
MIC,d µM
compd
L1210
MOLT-4/C8
CEM
HIV-1
>250
115 ( 21.2
5.0 ( 0.7
12 ( 3.5
3.5 ( 0.7
3.5 ( 2.1
20 ( 7.1
HIV-2
MSV
C3H/3T3
16 (DA40926)
17 (DMDA40926)
>500
>500
>500
>500
18 ( 2.5
66 ( 20
>500
>500
80 ( 6
>250
81 ( 20
212 ( 34
106 ( 2
>250
g250
>10
>100
>100
>4
>100
>100
20
100 to >20
g20
18
19
20
21
22
20 ( 7.5
36 ( 14
>50
>20
22 ( 3.5
20 ( 0.0
g50
6.9 ( 4.1
>4
25 ( 0.7
20 ( 7.1
35 ( 6.1
g50
20
100
6.9 ( 4.1
a
Compounds with IC50 e 30 µM and MIC e 30 µM are shown in bold. Compounds with EC50 e 20 µM are shown in bold and italic.
b 50% inhibitory concentration, or compound concentration required to inhibit tumor cell proliferation by 50%. c 50% effective concentration,
d
or compound concentration required to protect cells against the cytopathogenicity of HIV or MSV by 50%. Minimal inhibitory concentration,
or compound concentration required to cause a microscopic detectable alteration of cell morphology.
been reported to also exhibit antiviral properties. Chlo-
ropeptins I and II (complestatin) and kistamicins A and
B (Figure 1) have been shown to inhibit in vitro binding
of HIV-1 gp120 to the CD4 receptor and, consequently,
HIV replication in human lymphocytes.12-14 These
antibiotics and the aglycons of antibacterial glycopep-
tides have a common structural motif, although the size
of their macrocycles, i.e., the framework of these anti-
biotics, is different. Indeed, the structures of these hexa-
or heptapeptide antibiotics and the structures of anti-
bacterial glycopeptide antibiotics and their aglycons
show profound differences in amino acid sequence,
composition, and stereochemistry. The cycle formed with
the participation of the C-terminal amino acid present
in glycopeptide antibiotics is absent in chloropeptin and
complestatin and is of a different type and size in
kistamicin. Kistamicins, complestatin, and chloropeptin
I contain a tryptophan moiety linked to the central
amino acid 4 (F and D cycles), whereas a substituted
phenylalanine moiety is present in the antibacterially
active glycopeptides (Figure 1).
study, the drug was added to the virus-infected cell
cultures at different time points after infection. The
results strongly suggest that inhibition of viral entry is
the most likely molecular event for the anti-HIV activity
of this type of compound. The compound (38) lost its
antiviral activity when added at 1-2 h postinfection.
The HIV adsorption inhibitor dextran sulfate (DS-5000)
already lost its activity when added at 1 h postinfection,
whereas addition of the reverse transcriptase inhibitor
AZT and the HIV protease inhibitor ritonavir could be
delayed for up to 3 and 18 h postinfection, respectively,
before their antiviral efficacy was lost (data not shown).
This is also in agreement with the report that related
compounds interfere with gp120-CD4 binding.12-14 Also,
the inhibitory activity of 38 and 40 against fusion
between uninfected Molt4/C8 and persistently HIV-1-
infected HUT-78 cells (resulting in syncytium formation
between both cell types) is in agreement with these
findings (EC50 ) 20 and 11 µM, respectively).
The viral entry process is the result of a specific
interplay between viral glycoproteins (gp120, gp41) and
cellular (co)receptors (CD4, CCR5, CXCR4). Given our
preliminary findings that viral entry is a target for the
anti-HIV activity of the glycopeptide antibiotics, it is not
so surprising that we did not find a close correlation
Preliminary mechanism of action studies on the anti-
HIV activity of the modified antibiotic derivatives have
been performed in “time-of-addition” experiments car-
ried out with the glycopeptide antibiotic (38). In this

Derivatives of Glycopeptide Antibiotics
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13 2759
Ta ble 4. Structures of Vancomycin-Type Aglycons and Their Derivatives and Inhibitory Data
(a) Vancomycin-Type Aglycons and Their Derivatives
compd
ref
25
26
X
Y
Z
Brutto formula
MW
Vancomycin Aglycon (VA)
W ) Cl
23 (VA)
H
OH
H
C
C
53H52N8O17Cl2
53H53N8O17Cl
1143
Eremomycin Aglycon (EA) and Its Derivatives
W ) H
24 (EA)
25
26
27
28
H
OH
OH
OH
NHMe
NHMe
H
H
Boc
Boc
H
1108
1374
1474
1487
1387
CH2N[CH2CH2]2 NBnPh-p
CH2N[CH2CH2]2NBnPh-p
CH2N[CH2CH2]2NBnPh-p
CH2N[CH2CH2]2NBnPh-p
C71H75N10O17Cl
C76H83N10O19Cl
C77H86N11O18Cl
C72H78N11O16Cl
Eremomycin Aglycon Hexapeptide (EAH) and Its Derivatives
W) H, First Amino Acid (N-Me-D-Leu) Replaced by H
29 (EAH)
30
31
27
H
OH
C
46H40N7O16Cl
981
1130
1143
CH2NHAdam-2a
CH2NHAdam-2a
OH
NHMe
C57H57N7O16Cl
C58H60N8O15Cl
(b) Inhibitory Effects of Vancomycin-Type Aglycons and Their Derivativesb
d
IC50,c µM
EC50
,
µM
MIC,e µM
C3H/3T3
compd
L1210
MOLT-4/C8
CEM
HIV-1
HIV-2
MSV
23 (aglycon Van)
24 (aglycon Ere)
>500
>500
>500
>500
>500
>500
65 ( 7.1
50 ( 28.3
g250
g250
>100
>100
>100
>100
Aglycon Eremomycin Derivatives
25
26
27
28
250 ( 39
>500
>500
>100
>100
>100
5.5 ( 0.7
4.5 ( 0.7
4.0 ( 0.0
4.0 ( 1.7
12 ( 3.5
4.5 ( 2.1
3.5 ( 0.7
5.5 ( 0.7
13 ( 2
8.4 ( 3.0
>4
100 to >20
100 to >20
20
84 ( 22
>100
>100
>100
12 ( 1
100 to >20
EAH Derivatives
29 (EAH)
30
31
>250
>250
>250
115 ( 21.2
22 ( 11
13 ( 9.9
>250
40 ( 14.1
20 ( 7.1
>100
47 ( 5
14 ( 5
g100
g100
g100
>250
>250
a
b
Adam-2 means admantyl-2. Compounds with IC50 e 30 µM and MIC e 30 µM are shown in bold. Compounds with EC50 e 20 µM
are shown in bold and italic. c 50% inhibitory concentration, or compound concentration required to inhibit tumor cell proliferation by
d
50%. 50% effective concentration, or compound concentration required to protect cells against the cytopathogenicity of HIV or MSV by
50%. e Minimal inhibitory concentration, or compound concentration required to cause a microscopic detectable alteration of cell morphology.
between the anti-HIV activity and anti-MSV activity of
the glycopeptide antibiotics in cell culture. In fact, the
correlation coefficients (r) between the EC50 for MSV
and the EC50 for HIV-1 and HIV-2 are 0.50 and 0.41,
respectively, and the r value between the EC50 for HIV-1
and HIV-2 is 0.64 (figures not shown). These observa-
tions may point to a rather specific interaction of the
compounds with a viral factor (i.e., gp120 or gp41 for
HIV-1 and HIV-2 and an envelope protein for MSV) that
has different structural requirements for interaction
with the modified antibiotics. The elucidation of the
antiretroviral target(s) of the glycopeptide aglycon
antibiotics and of the molecular mode of interaction with
their target is currently a subject of investigation in our
laboratories.
glycopeptide derivatives can be envisaged as potential
lead compounds for application as microbicides against
sexual HIV transmission.
Exp er im en ta l Section
1. Ch em istr y. Eremomycin sulfate and ristocetin A were
produced at the pilot plant of the Gause Institute of New
Antibiotics, Moscow. Vancomycin hydrochloride was obtained
from Sigma Corporation (St. Louis, MO). Teicoplanin, teico-
planin aglycon, and the antibiotic DA-40926 were kindly
provided by Dr. R. Ciabatti and Dr. A. Malabarba (Biosearch
S.p.A., Gerenzano (Varese), Italy). All reagents and solvents
were purchased from Aldrich (Milwaukee), Fluka (Deisen-
hofen, Germany), and Merck (Darmstadt, Germany). The
references for the methods of the preparation of the compounds
previously described are presented in the Tables 1a-6a and
8. The novel compounds were obtained by the methods (e.g.,
amidation, Mannich reaction, N-acylation, alkylation) previ-
ously described for the synthesis of analogous glycopeptide
derivatives.4,5,8,17-19,30 The homogeneity and identity of the
compounds obtained were assessed by HPLC and ESI mass
spectrometry (see the Supporting Information).
In conclusion, semisynthetic, lipophilic aglycon gly-
copeptide derivatives have been discovered to be selec-
tively active against retroviruses including HIV-1, HIV-
2, and MSV. In the case of HIV, the compounds seem
to impede viral entry into the cells, and thus, these

2760 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13
Balzarini et al.
Ta ble 5. Structures of Teicoplanin-Type Aglycons and Their Derivatives and Inhibitory Data
(a) Teicoplanin-Type Aglycons and Their Derivatives
compd
ref
X
Y
Z
Brutto formula
MW
Ristocetin Aglycon
W1) W2 ) W3 ) H, W4 ) Me, S4 ) OH
32
28
22
H
H
H
OMe
H
C
C
C
60H52N7O19
1174
1212
Aglycon DA40
W1 ) W3 ) Cl, W4 ) S4 ) H,
33
OH
Me
59H47N7O18Cl2
58H45N7O18Cl2
Teicoplanin Aglycon (TD) and Its Derivatives
W1 ) W2 ) Cl, W3 ) W4 ) S4 ) H
34 (TD)
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
29
30
30
11
30
21
OH
H
1199
1452
1366
1338
1446
1212
1527
1456
1213
1297
1552
1353
1474
1487
1387
1299
1421
1377
1333
1312
1390
1334
1332
1362
1375
1480
1493
1495
1318
1368
1366
CH2NHC10H21
CH2N(Me)BnCl-p
NH(CH2)3NMe2
OH
H
H
C74H80N10O17Cl2
C67H55N8O18Cl3
C68H66N8O17Cl2
C74H74N10O17Cl2
C59H48N8O17Cl2
C79H81N11O17Cl2
C75H72N10O17Cl2
H
NHC10H21
NH(CH2)3NMe2
H
H
CH2NHAdam-2a
H
NHMe
NH(CH2)3NMe2
H
H
CH2N[CH2CH2]2NBnBu-p
CH2N[CH2CH2]2NBnBu-p
NHMe
OMe
H
H
31
H
H
C59H47N7O18Cl2
NH(CH2)6NH2
NH(CH2)6NH2
H
H
C64H59N9O17Cl2
C80H94N11O17Cl2
C68H67N9O17Cl2
C75H70N8O20Cl2
C76H73N9O19Cl2
C71H65N9O17Cl2
C
C
C
C
C
C
C
C68H60N8O17Cl2
C69H62N8O18Cl2
C70H65N9O17Cl2
C77H84N8O18Cl2
C78H87N9O17Cl2
C79H77N9O17Cl2
C
C
C
CH2NH(CH2)3N+Me2C10H21
H
NH(CH2)10NH2
OH
NHMe
NHMe
H
CH2NHBnBu-p
CH2NHBnBu-p
CH2NHBnBu-p
H
H
H
H
H
Boc
Boc
H
32
OH
OH
OH
OH
NHMe
NHMe
Boc
Fmoc
Adoc
Cbz
Boc
Adoc
63H53N7O20Cl2
73H55N7O20Cl2
69H59N7O20Cl2
66H51N7O20Cl2
64H56N8O19Cl2
70H6 2N8O19Cl2
65H50N8O18Cl2S
32
21
H
H
H
33
OH
C(S)NHPh
H
NHAdam-2a
CH2NHAdam-2a
OH
NHMe
H
H
CH2NHAdam-2a
CH2NHC18H37
OH
H
H
H
CH2NHC18H37
NHMe
CH2NHAdam-2a
NHAdam-2a
H
H
H
perhydroisoquinolinyl-
OH
OH
H
67H60N8O17Cl2
68H50N8O20 Cl2
69H66N7O19Cl2
(glyoxalyl-indol-3-yl)-
1-adamantoyl-
(b) Inhibitory Effects of Teicoplanin-Type Aglycons and Their Derivativesb
d
IC50,c µM
EC50
,
µM
IC,e µM
C3H/3T3
compd
L1210
MOLT-4/C8
CEM
HIV-1
HIV-2
MSV
32 (aglycon risto)
33 (aglycon DA40)
34 (aglycon teico)
>100
25 ( 7.1
40 ( 14.1
17 ( 3.5
g50
>50
20 ( 0.0
19 ( 1
82 ( 26
25 ( 17
>100
>100
>100
>500
>500
g500
>500
>500
>500
Aglycon Teicoplanin Derivatives
35
36
37
38
48 ( 8
>100
>100
>100
>100
1.4 ( 0.6
17 ( 3.5
2.6 ( 2.0
2.5 ( 0.7/
4.0 ( 0.0
15 ( 7.1
3 ( 0/
6.1 ( 3.9
20 ( 7.1
5.8 ( 0.4
8.0 ( 2.8
22 ( 3.5
17 ( 3.5
5 ( 1.4/
13 ( 9.9
3.0 ( 1.4
6.3 ( 1.1
15 ( 7.1
17 ( 3.5
7 ( 0
12 ( 3.5
8.3 ( 5.86
12 ( 5.2
12 ( 6.81
20 ( 0.0
15 ( 0.0
6.0 ( 1.4
8.0 ( 2.8
17 ( 3.5
7.8 ( 3.2
8.0 ( 2.8
>100
11 ( 5
12 ( 5
100 to >20
21 ( 0.2
>500/
>500
>500
20
>250
39
40
>500
29 ( 7
>500
108 ( 79
>500
g20
2.4/
3.5 ( 0.7
3.6/6.9 ( 0.4
100 to >4
20
>500/
>250
4.0 ( 1.4
1.7 ( 0.42
3.5 ( 0.7
9.5 ( 7.8
15 ( 0
41
61 ( 10
>500
>500/
g20
>250
42
43
44
45
46
47
48
49
50
51
52
53
54
55
364 ( 192
>500
38 ( 1
500
>500
>500
72 ( 6.0
225 ( 8.0
248 ( 1
>500
12 ( 2
>4
g100
20
66 ( 2
402 ( 138
60 ( 11.9
>100
1.8 ( 0.49
6.5 ( 0.7
20 ( 0
g4
20
7.6 ( 6.1
>4
>4
12 ( 0.0
10 ( 0.0
9.8 ( 1.8
11 ( 2.0
11 ( 5.0
8.7 ( 4.6
7.5 ( 5.0
100 to >20
20
20
g100
100
g20
>100
>100
100
70 ( 23
>100
>100
>100
6 ( 1
>100
>250
114 ( 1
104 ( 3
>250
229 ( 30
88 ( 16
220 ( 43
9.7 ( 9.0
13 ( 9.9
17 ( 3.5
13 ( 9.9
12 ( 3.5
13 ( 9.2
17 ( 17.7
6.0 ( 1.4
22 ( 0.1
25 ( 0.99
100

Derivatives of Glycopeptide Antibiotics
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13 2761
Ta ble 5. (Continued)
(b) Inhibitory Effects of Teicoplanin-Type Aglycons and Their Derivativesb (Continued)
d
IC50,c µM
EC50, µM
IC,e µM
C3H/3T3
compd
L1210
MOLT-4/C8
CEM
HIV-1
HIV-2
MSV
56
57
58
59
60
61
62
63
64
30 ( 5.7
26 ( 6.0
123 ( 6.0
>250
7.0 ( 4.2
25 ( 0.0
5.0 ( 1.4
4.5 ( 0.7
4.5 ( 0.7
2.5 ( 07
0.75 ( 0.07
10 ( 0.0
10 ( 7.1
6.0 ( 1.4
37 ( 17.7
17 ( 3.5
7.8 ( 3.2
6.0 ( 1.4
3.5 ( 2.1
4.5 ( 0.7
15 ( 0.0
4.0 ( 1.4
2.1 ( 0.1
5.6 ( 3.9
9.5 ( 0.8
1.7 ( 0.3
4.7 ( 2.8
2.0 ( 1.2
>4
g100
100
g20
100
100
g20
20
212 ( 54
>250
>250
22 ( 1.0
60 ( 1.0
>250
202 ( 68
>250
192 ( 15
33 ( 1.0
>250
>250
>250
108 ( 5
39 ( 13
108 ( 30
83 ( 17
4.4 ( 2.1
11 ( 1.0
g100
100
a
b
Adam-2 means admantyl-2. Compounds with IC50 e 30 µM and MIC e 30 µM are shown in bold. Compounds with EC50 e 20 µM
are shown in bold and italic. c 50% inhibitory concentration, or compound concentration required to inhibit tumor cell proliferation by
d
50%. 50% effective concentration, or compound concentration required to protect cells against the cytopathogenicity of HIV or MSV by
50%. e Minimal inhibitory concentration, or compound concentration required to cause a microscopic detectable alteration of cell morphology.
Ta ble 6. Structures of Teicoplanin Aglycon Derivatives with the Eliminated Amino Acids 1 and 3 and Inhibitory Data
(a) Teicoplanin Aglycon Derivatives with
the Eliminated Amino Acids 1 and 3
compd
ref
X
Y
Brutto formula
MW
65
66
67
68
34
34
H
H
H
C
C
51H43N5O16Cl2
56H51N5O18Cl2
1053
1153
1315
1215
Boc
Boc
H
CH2NHAdam-2a
CH2NHAdam-2a
C67H68N6O18Cl2
C62H60N6O16Cl2
(b) Inhibitory Effects of Teicoplanin Aglycon Derivatives
with the Eliminated Amino Acids 1 and 3b
d
IC50,c µM
EC50
,
µM
MIC,e µM
C3H/3T3
compd
L1210
>500
MOLT-4/C8
CEM
HIV-1
HIV-2
MSV
65
66
67
68
>500
>500
25 ( 7.1
17 ( 3.5
17 ( 3.5
17 ( 3.5
85 ( 21.2
17 ( 3.5
11 ( 5.7
37 ( 17.7
>250
87 ( 19
26 ( 1
39 ( 15
>100
g100
g100
95 ( 10
181 ( 4.0
122 ( 13
>250
240 ( 13
>250
a
b
Adam-2 means admantyl-2. Compounds with EC50 e 20 µM are shown in bold and italic. c 50% inhibitory concentration, or compound
d
concentration required to inhibit tumor cell proliferation by 50%. 50% effective concentration, or compound concentration required to
protect cells against the cytopathogenicity of HIV or MSV by 50%. e Minimal inhibitory concentration, or compound concentration required
to cause a microscopic detectable alteration of cell morphology.
1.1. Meth od A. Am in om eth yla ted Der iva tives 11, 25,
26, 28, 30, 36, 57, 59, 67, 68, 70. To a stirred solution of 0.5
mmol of an antibiotic or its degradation product and 4 mmol
of an appropriate amine in 10 mL of an acetonitrile/water, 1:1,
mixture was added 3 mmol of 37% aquous formaldehyde. If a
salt of an amine was used, 1 N NaOH was added to attain pH
10. The reaction mixture was stirred at room temperature for
18 h, and then 100 mL of water was added. After adjustment
of the reaction mixture at pH 3 with 1 N HCl, the resulting
solution (or suspension) was extracted with n-BuOH (∼25 mL
× 2). The organic layer was washed with water (∼15 mL × 2)
and then concentrated at 45 °C in a vacuum to a small volume
(∼3 mL). On addition of ether (∼100 mL), a solid that
precipitated was collected and dried in a vacuum at room
temperature for 4 h. Then it was dissolved in a minimal
amount of MeOH and applied to a chromatographic column
with Sephadex LH-20 (2 × 100 cm) preequilibrated with
MeOH. The product was eluted with MeOH at a rate of 10
mL/h, while collecting 5 mL fractions. The suitable fractions
were combined and concentrated to a small volume (∼3 mL).
After addition of ether (∼100 mL), the formed
precipitate was collected, rinsed with ether, and dried in a
vacuum at room temperature.
The starting compound for 67, N2-Cbz-N4-Boc-TDTP-Me
(66), was obtained as previously described.34 Compound 68 was
obtained from 67 by the removal of the Boc group in TFA as
previously described for N2-Cbz-N4-Boc-TDTP-Me.34
The starting compound for 70, the N-terminal phenylthio-
hydantoin derivative of teicoplanin aglycon, was obtained by
Edman degradation of teicoplanin aglycon.
To a solution of teicoplanin aglycon (34) (100 mg, ∼0.08
mmol) in a mixture of Py/H2O (6:1, 4 mL), triethylamine (0.26
mL, 2 mmol) and PhNCS (0.02 mL, ∼0.16 mmol) were added
at room temperature under argon. The reaction mixture was
stirred for 16 h, an amount of 8 mL of H2O was added, and
the reaction mixture was evaporated with n-BuOH to dryness.
The precipitate was dissolved in a mixture of TFA/CH2Cl2, 1:1
(3 mL), at 0-5 °C and then was stirred at this temperature
for 1 h. Water (3 mL) was then added, and the mixture was
neutralized with 25% NH4OH and washed with EtOAc (3 mL
× 3), and the aqueous fraction was concentrated in a vacuum
with the addition of n-BuOH and applied to a column of
silanized silica gel (2 × 100 cm) previously equilibrated with

2762 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13
Balzarini et al.
Ta ble 7. Structures of Teicoplanin Aglycon with the Disrupted Bond between Amino Acids 1 and 2 and Inhibitory Data
(a) Teicoplanin Aglycon with the Disrupted Bond between Amino Acids 1 and 2
compd
X
Brutto formula
65H51N8O18Cl2S
MW
69
70
H
C
1335
1498
CH2NHAdam-2a
C76H68N9O18Cl2S
(b) Inhibitory Effects of Teicoplanin aglycon with the Disrupted Bond between Amino Acids 6 and 7 and Their Derivativesb
d
IC50,c µM
EC50
,
µM
MIC,e µM
compd
L1210
MOLT-4/C8
CEM
>250
242 ( 11
HIV-1
HIV-2
MSV
27 ( 0
2.9 ( 0.8
C3H/3T3
69
70
15 ( 7.1
13 ( 9.9
37 ( 17.7
17 ( 3.5
>100
g100
73 ( 24
g250
a
b
Adam-2 means admantyl-2. Compounds with EC50 e 20 µM are shown in bold and italic. c 50% inhibitory concentration, or compound
d
concentration required to inhibit tumor cell proliferation by 50%. 50% effective concentration, or compound concentration required to
protect cells against the cytopathogenicity of HIV or MSV by 50%. e Minimal inhibitory concentration, or compound concentration required
to cause a microscopic detectable alteration of cell morphology.
Ta ble 8. Teicoplanin Aglycon with the Disrupted Bond between Amino Acids 6 and 7 and Its Inhibitory Effectsa
IC50,b µM
EC50,c µM
MIC,d µM
C3H/3T3
compd
ref
35
L1210
MOLT-4/C8
CEM
HIV-1
HIV-2
MSV
71
>250
22 ( 3.5
32 ( 3.5
14 ( 2.0
>100
a
b
Compounds with EC50 e 20 µM are shown in bold and italic. 50% inhibitory concentration, or compound concentration required to
inhibit tumor cell proliferation by 50%. c 50% effective concentration, or compound concentration required to protect cells against the
cytopathogenicity of HIV or MSV by 50%. Minimal inhibitory concentration, or compound concentration required to cause a microscopic
d
detectable alteration of cell morphology.
0.01 M acetic acid. Compound 70 was eluted with acetic acid
(0.01M) at a flow rate of 30 mL/h. Fractions were pooled and
concentrated with the addition of n-BuOH in a vacuum, and
acetone (50 mL) was added to yield the precipitate, which was
filtered off, washed with acetone, and dried to yield 68 mg
(54%) of 70.
organic layer was washed with water (∼15 mL × 3) and then
concentrated at 45 °C in a vacuum to a small volume (∼3 mL).
On addition of ether (∼100 mL), a solid precipitate was
collected and dried in a vacuum at room temperature for 4 h.
Addition of 100 mL of acetone led to the formation of a
precipitate, which was collected to give a pure carboxamide.
1.3. Meth od C. Ca r boxa m id es of Am in om eth yla ted
Der iva tives 31, 40, 41, 44, 48, 58, 60, 61. The compounds
were obtained by method B, starting from the aminomethy-
lated derivatives obtained by the method A.
1.4. Meth od D. N-Ca r ba m oyla ted Der iva tive 51. To a
stirred solution of 0.5 mmol of teicoplanin aglycon (34) in 15
mL of THF/water, 1:1, mixture adjusted to pH 10 with 1 N
NaOH 0.55 mmol of adamantyloxycarbonyl chloride was
added. The reaction mixture was stirred at room temperature
for 4 h, and then it was diluted with 100 mL of water. After
adjustment of the reaction mixture at pH 3 with 1 N HCl, the
resulting solution (or suspension) was extracted with n-BuOH
(∼25 mL × 2). The organic layer was washed with water (∼15
mL × 2) and then concentrated at 45 °C in a vacuum to a small
volume (∼3 mL). The precipitate that formed on addition of
ether (∼100 mL) was collected and dried in a vacuum at room
temperature for 4 h.
1.2. Meth od B. Ca r boxa m id es 43, 45, 56, 62, 63, 64. To
a mixture of an antibiotic or its degradation product (0.5 mmol)
and 5 mmol of an amine hydrochloride dissolved in 5 mL of
DMSO were added portionwise Et3N to give pH 8.5-9 and
afterward during 1 h 1 mmol of PyBOP reagent [(benzotriazol-
1-yloxy)tris(pyrrolidino)phosphonium hexafluorophosphate] or
HBPyU reagent [O-(benzotriazol-1-yloxy)-N,N,N′,N′-bis(tet-
ramethylene)uronium hexafluorophosphate]. The reaction mix-
ture was stirred at room temperature for 3 h.
Addition of ether (∼100 mL) to the reaction mixture led to
an oily residue, which was shaken successively with ether (15
mL × 2) and acetone (∼15 mL). After addition of 100 mL of
acetone, a precipitate of crude amide was collected and
dissolved in 50 mL of water and the pH was adjusted to 9 using
a 1 N sodium hydroxide solution. The resulting solution (or
suspension) was extracted with n-BuOH (∼25 mL × 3). The

Derivatives of Glycopeptide Antibiotics
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13 2763
defined as the concentration of the compound that inhibited
cell proliferation by 50%.
2.4. An ti-Molon ey Mu r in e Sa r com a Vir u s (MSV) As-
sa ys. The inhibitory effect of the test compounds on MSV-
induced transformation of murine embryo fibroblast C3H/3T3
cell cultures was examined microscopically at day 6 postin-
fection. MSV was added at 75 focus-forming units to monolayer
cell cultures in 48-well microtiter plates. The detailed proce-
dures for the antiretroviral evaluations have been previously
described in detail.36,37
2.5. Tim e-of-Ad d ition Assa ys. MT-4 cells were infected
with HIV-1(IIIB) at a multiplicity of infection (moi) of 0.5. The
test compound 38 (final concentration of 100 µM) was added
at different times after infection as described before.39 Viral
p24 Ag production was determined at 31 h postinfection by
enzyme linked immunosorbent assay (NEN, Brussels, Bel-
gium). The reference compounds, dextran sulfate (20 µM), AZT
(1.87 µM), and ritonavir (2.84 µM), were added at 100 times
their 50% inhibitory concentration (IC50) as obtained in the
MT-4/MTT assay.
2.6. Cocu ltiva tion Assa ys betw een Un in fected Molt4/
C8 a n d P er sisten tly HIV-1-In fected HUT-78/HIV-1 Cells.
Persistently HIV-1-infected HUT-78 cells continuously releas-
ing HIV-1 particles without dying (designated HUT-78/HIV-
1) were washed to remove free virus from the culture medium,
and 5 × 104 cells (50 µL) were transferred to 96-well microtiter
plates (Sterilin). Then, 5 × 104 uninfected MOLT-4 (clone 8)
cells (50 µL) and an appropriate concentration of test com-
pound (100 µL) were added to each well. The mixed cell
cultures were cultured at 37 °C in a CO2-controlled atmo-
sphere. The first syncytia (giant cell formation as a result of
fusion between the HUT-78/HIV-1 and MOLT-4 cells) arose
after about 6 h of cocultivation. After 16-20 h, marked
syncytium formation was noted and the number of syncytia
was examined and quantified under a microscope.
F igu r e 1. Structural formulas of chloropeptin I, kistamicins
A and B, and complestatin.
Ack n ow led gm en t. The authors thank Dr. Evgenia
Olsufyeva (Gause Institute) for fruitful discussion, Mrs.
Ann Absillis, Lizette van Berckelaer, Cindy Heens (Rega
Institute), Tatiana Loim, and Natalia Maliutina (Gause
Institute) for excellent technical assistance, and Mrs.
Christiane Callebaut (Rega Institute) for dedicated
editorial assistance. Financial support from the Gecon-
certeerde Onderzoeksacties (Grant No. GOA-00/12), the
“Fonds voor Wetenschappelijk Onderzoek” (FWO) (Grant
No. G-0104.98), the European Union (EU) Rene´ Des-
cartes Prizes2001 (Grant No. HPAW-CT2002-90001),
and ISEP/FORTIS is gratefully acknowledged.
1.5. Meth od E. N-Ca r ba m oyla ted Der iva tives of Ca r -
boxa m id es of Am in om eth yla ted Der iva tives 27, 47. The
compounds were obtained by method D, using Boc2O reagent
starting from carboxamides of aminomethylated derivatives
obtained by method C.
2. An tivir a l a n d Cytosta tic Assa y Meth od s. 2.1. Vi-
r u ses. The origins of MSV, HIV-1 (strain IIIB) (kindly provided
by Dr. R. Gallo and Dr. M. Popovic, at that time at the National
Cancer Institute of the National Institutes of Health, Be-
thesda, MD), and HIV-2 (strain ROD) (kindly provided by Dr.
L. Montagnier, at that time at the Pasteur Institute, Paris,
France) have been described previously.36-38 HIV-1(IIIB) and
HIV-2(ROD) stocks were obtained from supernatants of virus-
infected MT-4 cell cultures. Moloney murine sarcoma virus
(MSV) was obtained as described.37
Su p p or tin g In for m a tion Ava ila ble: HPLC, molecular
formulas, and mass spectral data (ESI MS) for compounds 11,
25-28, 30, 31, 40, 41, 43-48, 50, 51, 54, 56-64, 67-70. This
material is available free of charge via the Internet at http://
pubs.acs.org.
2.2. An ti-HIV Activity Assa ys. Inhibition of HIV-1(IIIB)-
and HIV-2(ROD)-induced cytopathicity in CEM cell cultures
was measured in microtiter 96-well plates containing ∼3 ×
105 CEM cells/mL infected with 100 CCID50 of HIV per
milliliter and containing appropriate dilutions of the test
compounds. After 4-5 days of incubation at 37 °C in a CO2-
controlled humidified atmosphere, CEM giant (syncytium) cell
formation was examined microscopically. The EC50 (50%
effective concentration) was defined as the compound concen-
tration required to inhibit HIV-induced giant cell formation
by 50%.
Refer en ces
(1) Nagarajan, R. Glycopeptide Antibiotics; Marcel Dekker: New
York, 1994.
(2) Walsh, C. T. Vancomycin resistance: decoding the molecular
logic. Science 1993, 261, 308-309.
(3) Malabarba, A.; Nicas, T. I.; Thompson, R. S. Structural Modifica-
tions of Glycopeptide Antibiotics. Med. Res. Rev. 1997, 17, 69-
137.
(4) Pavlov, A. Y.; Preobrazhenskaya, M. N. Chemical Modification
of Glycopeptide Antibiotics. Russ. J . Bioorg. Chem. 1998, 24,
570-587.
(5) Cooper, R. D. G.; Snyder, N. J .; Zweifel, M. J .; Staszak, M. A.;
Rodrigues, M. J .; Huff, B. E.; Thompson, R. C. Reductive
Alkylation of Glycopeptide Antibiotics: Synthesis and Antibacter-
ial Activity. J . Antibiot. 1996, 49, 575-581.
(6) Ge, M.; Chen, Zh.; Onishi, H. R.; Kohler, J .; Silver, L. L.; Kerns,
R.; Fukuzawa, S.; Thompson, C.; Kahne, D. Vancomycin Deriva-
tives That Inhibit Peptidoglycan Synthesis without Binding
D-Ala-D-Ala. Science 1999, 284, 507-511.
2.3. Cytosta tic Activity Assa ys. All assays were per-
formed in 96-well microtiter plates. To each well were added
(5-7.5) × 104 cells and a given amount of the test compound.
The cells were allowed to proliferate for 48 h (murine leukemia
L1210 cells) or 72 h (human lymphocytic CEM and Molt4/clone
8 cells) at 37 °C in a humidified CO2-controlled atmosphere.
At the end of the incubation period, the cells were counted in
a Coulter counter. The IC50 (50% inhibitory concentration) was

2764 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13
Balzarini et al.
(7) Goldman, R. C.; Baizman, E. R.; Longley, C. B.; Branstrom, A.
A. Chlorobiphenyl-Desleucyl-Vancomycin Inhibits the Trans-
glycosylase Required for Peptidoglycan Synthesis in Bacteria.
FEMS Microbiol. Lett. 2000, 183, 209-214.
(8) Printsevskaya, S. S.; Pavlov, A. Y.; Olsufyeva, E. N.; Mirchink,
E. P.; Isakova, E. B.; Reznikova, M. I.; Goldman, R. C.; Bran-
strom, A. A.; Baizman, E. R.; Longley, C. B.; Sztaricskai, F.;
Batta, G.; Preobrazhenskaya, M. N. Synthesis and Mode of
Action of Hydrophobic Derivatives of the Glycopeptide Antibiotic
Eremomycin and Des-(N-methyl-D-leucyl)eremomycin against
Glycopeptide-Sensitive and -Resistant Bacteria. J . Med. Chem.
2002, 45, 1340-1347.
(9) Roy, R. S.; Yang, P.; Kodali, S.; Xiong, Y.; Kim, R. M.; Griffin,
P. R.; Onishi, R,; Kohler, J .; Silver, L. L.; Chapman, K. Direct
Interaction of a vancomycin derivative with bacterial enzymes
involved in cell wall biosynthesis. Chem. Biol. 2001, 8, 1095-
1106.
(10) Eggert, U. S.; Ruiz, N.; Falcone, B. V.; Branstrom, A. A.;
Goldman, R. C.; Silhavy, T. J .; Kahne, D. Genetic Basis for
Activity Differences between Vancomycin and Glycolipid Deriva-
tives of Vancomycin. Science 2001, 294, 361-364.
(11) Printsevskaya, S. S.; Pavlov, A. Y.; Olsufyeva, E. N.; Mirchink,
E. P.; Preobrazhenskaya, M. N. The Role of the Glycopeptide
Framework in the Antibacterial Activity of Hydrophobic Deriva-
tives of Glycopeptide Antibiotics. J . Med. Chem. 2003, 46, 1204-
1209.
(12) Matsuzaki, K.; Ikeda, H.; Ogino, T.; Matsumoto, A.; Woodruff,
H. B.; Tanaka, H.; Omura, S. Chloropeptins I and II. Novel
inhibitors against gp-120-CD4 binding from Streptomyces sp. J .
Antibiot. 1994, 47, 1173-1174.
(24) Maffiolli, S. I.; Romano, G.; Marzorati, E.; Pavlov, A. Y.;
Preobrazhenskaya, M. N.; Ciabatti, R. The Synthesis and
Antibacterial Activity of Monoalkylated Derivatives of the
Glycopeptide Antibiotic A40926 and Their Amides. 42nd ICAAC
Abstracts, San Diego, CA, September 27-30, 2002; ICAAC:
Washington, DC, 2002; p F-357.
(25) Kannan, R.; Harris, C. M.; Harris, T. M.; Waltho, J . P.; Skeiton,
N. J .; Williams, D. H. Function of the amino sugar and
N-terminal acid of the antibiotic vancomycin in its complexation
with cell wall peptides. J . Am. Chem. Soc. 1988, 110, 2946-
2953.
(26) Berdnikova, T. F.; Lomakina, N. N.; Olsufyeva, E. N.; Alexan-
drova, L. G.; Potapova, N. P.; Rozinov, B. V.; Malkova, I. V.;
Orlova, G. I. Structure and Antimicrobial Activity of Products
of Partial Degradation of Antibiotic Eremomycin. Antibiot.
Chemother. (Moscow) 1991, 36, 28-31.
(27) Miroshnikova, O. V.; Berdnikova, T. F.; Olsufyeva, E. N.; Pavlov,
A. Y.; Reznikova, M. I.; Preobrazhenskaya, M. N.; Ciabatti, R.;
Malabarba, A.; Colombo, L. A Modification of the N-Terminal
Amino Acid in the Eremomycin Aglycone. J . Antibiot. 1996, 49,
1157-1161.
(28) Bognar, R.; Sztaricskai, F.; Munk, M. E.; Tamas, J . Structure
and Stereochemistry of Ristosamine. J . Org. Chem. 1974, 39,
2971-2974.
(29) Malabarba, A.; Ferrari, P.; Gallo, G. G.; Kettenring, J .; Cavalleri,
B. Teicoplanin, Antibiotics from Actinoplanes teichomyceticus
nov. sp. VII. Preparation and NMR Characteristics of the
Aglycone of Teicoplanin. J . Antibiot. 1986, 39, 1430-1442.
(30) Pavlov, A. Y.; Preobrazhenskaya, M. N.; Malabarba, A.; Ciabatti,
R.; Colombo, L. Mono and Double Modified Teicoplanin Aglycon
Derivatives on the Amino Acid No. 7; Structure-Activity
Relationship. J . Antibiot. 1998, 51, 73-81.
(31) Malabarba, A.; Trani, A.; Ferrari, P.; Pallanza, R.; Cavalleri, B.
Synthesis and Biological Activity of Some Esters of the N-
Acetylglucosaminyl Aglycone and of the Aglycone of Teicoplanin.
J . Antibiot. 1987, 40, 1572-1587.
(13) Naruse, N.; Oka, M.; Konishi, M.; Oki, T. New antiviral
antibiotics, kistamycins A and B. II. Structural determination.
J . Antibiot. 1993, 46, 1812-1818.
(14) Nicolau, K. C.; Boddy, C. N. C.; Brase, S.; Winssinger, N.
Chemistry, Biology, and Medicine of the Glycopeptide Antibiot-
ics. Angew. Chem., Int. Ed.1999, 38, 2096-2152.
(15) Nagarajan, R.; Schabel, A. A.; Occolowitz, J . L.; Counter, F. T.;
Ott, J . L.; Felty-Duckworth, A. M. Syntesis and Antimicrobial
Activity of N-alkyl Vancomycins. J . Antibiot. 1989, 42, 63-72.
(16) Nagarajan, R. Structure-activity relationships of vancomycin-
type glycopeptides antibiotics. J . Antibiot. 1993, 46, 1181-1195.
(17) Pavlov, A. Y.; Berdnikova, T. F.; Olsufyeva, E. N.; Miroshnikova,
O. V.; Filiposyanz, S. T.; Preobrazhenskaya, M. N.; Scottani, C.;
Colombo, L.; Goldstein, B. P. Carboxamides and Hydrazide of
Glycopeptide Antibiotic Eremomycin. Synthesis and Antibacte-
rial Activity. J . Antibiot. 1996, 49, 194-198.
(18) Pavlov, A. Y.; Lazhko, E. I.; Preobrazhenskaya, M. N. A New
Type of Chemical Modification of Glycopeptides Antibiotics;
Aminomethylated Derivatives of Eremomycin and Their Anti-
bacterial Activity. J . Antibiot. 1997, 50, 509-513.
(19) Miroshnikova, O. V.; Printsevskaya, S. S.; Olsufyeva, E. N.;
Pavlov, A. Y.; Nilius, A.; Hensey-Rudloff, D.; Preobrazhenskaya,
M. N. Structure-Activity Relationships in the Series of Eremo-
mycin Carboxamides. J . Antibiot. 2000, 53, 286-293.
(20) Gerhard, U.; Mackay, J . P.; Maplestone, R. A.; Williams, D. H.
The Role of the Sugar and Chlorine Substituents in the Dimer-
ization of Vancomycin Antibiotics. J . Am. Chem. Soc. 1993, 115,
232-237.
(21) Malabarba, A.; Trani, A.; Strazzolini, P.; Cietto, G.; Ferrari, P.;
Tarzia, G.; Pallanza, R.; Berti, M. Synthesis and Biological
Properties of N63-Carboxamides of Teicoplanin Antibiotics.
Structure-Activity Relationships. J . Med. Chem. 1989, 32,
2450-2460.
(32) Malabarba, A.; Ciabatti, R.; Kettenring, J .; Scotti, R.; Candiani,
G.; Pallanza, R.; Berti, M.; Goldstein, B. P. Synthesis and
Antibacterial Activity of a Series of Basic Amides of Teicoplanin
and Deglucoteicoplanin with Polyamines. J . Med. Chem. 1992,
35, 4054-4060.
(33) Trani, A.; Ferrari, P.; Pallanza, R.; Ciabatti, R. Thiooureas and
Isothiouronium Salts of the Aglycone of Teicoplanin. 1. Synthesis
and Biological Activity. J . Antibiot. 1989, 42, 1268-1282.
(34) Malabarba, A.; Ciabatti, R.; Maggini, M.; Ferrari, P.; Vekey, K.;
Colombo, L.; Denaro, M. Structural Modifications of the Active
Site in Teicoplanin and Related Glycopeptides. 2. Deglucoteico-
planin-Derived Tetrapeptide. J . Org. Chem. 1996, 61, 2151-
2157.
(35) Cavalleri, B.; Ferrari, P.; Malabarba, A.; Magni, A.; Pallanza,
R.; Gallo, G. G. Teicoplanin, Antibiotics from Actinoplanes
teichomyceticus nov. sp. VIII. Opening of the Polypeptide Chain
of Teicoplanin Aglycone under Hydrolytic Conditions. J . Antibiot.
1987, 40, 49-59.
(36) Balzarini, J .; Naesens, L.; Slachmuylders, J .; Niphuis, H.;
Rosenberg, I.; Holy´, A.; Schellekens, H.; De Clercq, E. 9-(2-
Phosphonylmethoxyethyl)adenine (PMEA) effectively inhibits
retrovirus replication in vitro and simian immunodeficiency
virus infection in rhesus monkeys. AIDS 1991, 5, 21-28.
(37) De Clercq, E.; Merigan, T. C. Moloney sarcoma virus-induced
tumors in mice: inhibition of stimulation by (poly rI):(poly rC).
Proc. Soc. Exp. Biol. Med. 1971, 137, 590-594.
(38) Popovic, M.; Sarngadharan, M. G.; Read, E.; Gallo, R. C.
Detection, isolation, and continuous production of cytopathic
retroviruses (HTLV-III) from patients with AIDS and pre-AIDS.
Science 1984, 224, 497-500.
(39) Pauwels, R.; Andries, K.; Desmyter, J .; Schols, D.; Kukla, M. J .;
Breslin, H. J .; Raeymaeckers, A.; Van Gelder, J .; Woestenborghs,
R.; Heykants, J . Potent and selective inhibition of HIV-1
replication by a novel series of TIBO derivatives. Nature 1990,
343, 470-474.
(22) Hermann, R.; Ripamonti, F.; Romano, G.; Restelli, E.; Ferrari,
P.; Goldstein, B. P.; Berti, M.; Ciabatti, R. Synthesis and
Antibacterial Activity of Derivatives of the Glycopeptide Anti-
biotic A-40926 and Its Aglycone. J . Antibiot. 1996, 49, 1236-
1247.
(23) Preobrazhenskaya, M. N.; Pavlov, A. Y.; Maffioli, S. I.; Romano,
H.; Ciabatti, R. The Synthesis and Antibacterial Activity of Di-
Alkylated Derivatives of the Glycopeptide Antibiotic A-40926 and
Their Amides. 42nd ICAAC Abstracts, San Diego, CA September
27-30, 2002; ICAAC: Washington, DC, 2002; p F-358.
J M0300882