Synthesis and antibacterial activity of alkyl derivatives of the glycopeptide antibiotic A40926 and their amides

Article abstract of DOI:10.1016/j.bmcl.2005.05.076

New derivatives of the glycopeptide antibiotic A40926 were synthesized and evaluated for antimicrobial activity against VRE. Deacylated A40926 was obtained by microbial transformation of the parent antibiotic with the use of Actinoplanes teichomyceticus ATCC 31121. Regioselective synthesis of alkylated derivatives of Deacyl A40926 was carried out using lipophilic aliphatic and aromatic halides or aldehydes. Further modification of the two carboxylic acids was performed to increase antibiotic activity. Poor antimicrobial activity was observed for the derivatives obtained by lipophilic mono- or dialkylation of the amino groups present on the molecule, while simultaneous condensation of both carboxylic groups, in hydrophobic derivatives, with dibasic amines led to a strong increase in antibiotic activity.

Full text of DOI:10.1016/j.bmcl.2005.05.076

Bioorganic & Medicinal Chemistry Letters 15 (2005) 3801–3805
Synthesis and antibacterial activity of alkyl derivatives
of the glycopeptide antibiotic A40926 and their amidesq
a
a
a
Sonia I. Maffioli,a, Romeo Ciabatti, Gabriella Romano, Ettore Marzorati,
*
`
Maria Preobrazhenskayab and Andrej Pavlovb
aVicuron Pharmaceuticals via R. Lepetit 34, 21040 Gerenzano (Varese), Italy
bGause Institute of New Antibiotics (RAMS), Moscow, Russian Federation
Received 20 January 2005; revised 10 May 2005; accepted 11 May 2005
Available online 29 June 2005
Abstract—New derivatives of the glycopeptide antibiotic A40926 were synthesized and evaluated for antimicrobial activity against
VRE. Deacylated A40926 was obtained by microbial transformation of the parent antibiotic with the use of Actinoplanes teichomy-
ceticus ATCC 31121. Regioselective synthesis of alkylated derivatives of Deacyl A40926 was carried out using lipophilic aliphatic
and aromatic halides or aldehydes. Further modification of the two carboxylic acids was performed to increase antibiotic activity.
Poor antimicrobial activity was observed for the derivatives obtained by lipophilic mono- or dialkylation of the amino groups pres-
ent on the molecule, while simultaneous condensation of both carboxylic groups, in hydrophobic derivatives, with dibasic amines led
to a strong increase in antibiotic activity.
Ó 2005 Elsevier Ltd. All rights reserved.
1. Introduction
The aim of this work was to take advantage of a previ-
ous description and to investigate if new mono- and
dialkylated derivatives of Deacyl A40926 (DA40, see
Fig. 1) could have an improved antibiotic activity
against Van-A enterococci. Encouraging preliminary
results were obtained in a previous study carried out
on this molecule in which a slightly improved antimicro-
bial activity against Van-A enterococci was observed for
an alkyl derivative of the DA40 glycopeptide.6
Recent chemical modification of glycopeptide antibiot-
ics has been directed toward the synthesis of derivatives
active against vancomycin-resistant enterococci (VRE),
while retaining activity against susceptible Gram-posi-
tive bacteria including methicillin-resistant and coagu-
lase-negative staphylococci. This effort resulted in the
discovery of compounds with enhanced activity against
VRE produced by reductive alkylation of vancomycin,1
eremomycin,2 and teicoplanin.3 Among these new com-
pounds, oritavancin4 (Ly333328), derived from chloro
eremomycin, is currently under clinical development.
Recent studies have shown that hydrophobic substitu-
ents play a major role in the antibacterial activity against
VRE. Specific hydrophobic derivatives of eremomycin
and vancomycin demonstrate that antibacterial activity
against resistant strains is not based on binding to
D-Ala-D-Lac. This activity appears due to inhibition of
the transglycosylation step of bacterial peptidoglycan
biosynthesis.5
Keywords: Glycopeptides; VRE; Antibiotic.
q Part of this work was presented as a poster at 42° ICAAC 2002, San
Diego CA, USA.
Figure 1. Structures of A40926, N-Boc-N0-deacylA40926, and Deacyl
A40926.
*
Corresponding author. Tel.: +39 0296474370; fax: +39 0296474358;
e-mail: SMaffioli@Vicuron.it
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.05.076

3802
S. I. Maffioli et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3801–3805
This program started with from N-Boc-N0-DA407 (1)
obtained from a recently described microbial deacyla-
tion of the Boc-protected natural scaffold.8
At the beginning of the project, selective N0-alkylation
of DA40 was easily obtained by treatment of 1 with
the desired aliphatic or aromatic aldehyde in the pres-
ence of NaCNBH3 as a reducing agent, in THF/H2O
1:1 as a solvent (see Chart 1).
To obtain N-alkylated isomers, we also studied the
reductive alkylation on the deprotected DA40 (2) ob-
tained from 1 by Boc removal with TFA at rt. We sub-
jected 2 to the same reductive alkylation conditions
described above. The sugar amine was again selectively
alkylated, showing it to be the most reactive in this type
of reaction. When a large excess (10–20 eqiuv) of alde-
hyde was used, second alkylation was observed on
the same sugar amine, obtaining N0,N0-dialkylated
derivatives, while the variation of the conditions (e.g.,
solvents, temperature or other reducing agents: NaBH4
or Na(AcO)3BH) did not lead to any desired alkylation
of the peptidic N-terminal amino group.
Chart 2. Reagents and conditions: (i) Me2(CH2)3NH2, PyBOP, in
DMSO, pH 7.5–8, with TEA, rt 2 h.
alkylated derivative, we preferred to use a different
approach to produce dialkylated derivatives. Preliminary
activation of the N-terminal peptide amino group of
DA40 (2) was achieved by nitrogen silylation with the
use of trimethylsilyl-acetamide.9 Following this proce-
dure, 2 was reacted for 15 min at 50 °C with the desired
aldehyde (5 mmol), of Me3SiNHCOMe (10 mmol), and
TEA (2 mmol) in DMF and then Na(OAc)3BH was
added and the reaction was stirred for 2 h at rt.
The peptidic amine was selectively alkylated by reaction
with 1–2 equivalents of primary alkyl bromides in DMF
or DMSO. Alkylation on the sugar amine was observed
only in the presence of a large excess of alkyl bromide.
Depending on the nature of the alkyl bromide, carbox-
ylic acids did not react when the reaction was carried
out with pH in the range 6–8.
Mono- and dialkylated derivatives described above were
transformed into the corresponding basic amides by
condensation with Me2N(CH2)3NH2 in the presence of
PyBOP as a condensing agent in DMF or DMSO at
pH 7.5–8 (see Chart 2). All the compounds were tested
for the antimicrobial activity against an appropriate
panel of microorganisms. Tests were performed in
microtiter plates following the NCCLS procedure.10
Even if the N,N0-dialkylated derivative could be
obtained by further reductive alkylation of the N-mono-
2. Results
A40 and DA40 (2) have no activity against VRE but
have a range of 0.25–16 mg/L of activity against the
other tested microorganisms (Table 1).
N- and N0-monoalkylated compounds (Table 1, 3–13)
maintained good or moderate activity against staphylo-
cocci, streptococci,11 and Van-S enterococci (VSE),
while only a few compounds showed marginal activity
(Table 1, 3–6) against VRE. All these activities were
negatively affected by the presence of serum.
Further derivatization of the two carboxylic moieties,
present on the DA40 alkyl derivatives, by amidation
with alkyl or aryl amines resulted in derivatives having
an antibacterial profile very much similar to that of
the acidic derivatives of Table 1, while the correspond-
ing basic diamides, produced by condensation with
diamine Me2N(CH2)3NH2, proved to have interesting
activity against VRE and showed only a moderate inac-
tivation in the presence of serum (Table 1, 15–20). In
this class of basic amides, compounds 18 and 19 gave
Chart 1. Reagents and conditions: (i) 1. RCHO, NaCNBH3, THF/
H2O (1:1), rt 8 h; 2. TFA/CH2Cl2 (1:1), rt 15 min; (ii) TFA/CH2Cl2
(1:1), rt 15 min; (iii) RCHO, NaCNBH3, THF/H2O (1:1), rt 8 h; (iv)
RCH2Br, DMF or DMSO, pH 6–8, with TEA, rt 16 h; (v) 1. RCHO,
Me3SiNHCOMe, TEA in DMF 50 °C 15 min; 2. Na(OAc)3BH, rt 2 h.

Table 1. Monoalkylated derivatives
ID
MIC or MIC range (mg/L)
S. aureus
S. epidermidis
S. haemolyticus
E. faecalis
E. faecium
R1
R2
R3
Met-S (1)
Met-R (2) Met-S (1) Met-R (1) (1)
Van-S (3)
Van-A (4)
Van-S (1) Van-A (4)
A40 Natural fatty acid
H
H
H
H
H
H
H
H
H
OH
OH
OH
OH
OH
OH
OH
OH
OH
0.25 [4]
16 [nt]
4 [16]
2 [4]
0.5
16
4
nt
nt
nt
nt
nt
4
16
nt
0.125 (1)
8–4 (2)
>64 [nt]
0.5
8
>64
2
H
nt
>128 [nt]
>128
3
4-n-Decyl-Ph-CH2–
4-OctyloxyPh-CH2–
4-HexylOPh-CH2–
4-n-BuPhCH2–
4-n-BuOPhCH2–
Naphthyl-2-CH2–
n-HexylCH(n-Bu)-CH2–
H
2
32
32
16
nt
60.125–0.25 (2) 4–1 (2) [>128]
60.125–0.25 (2) 8–64 (2) [>128]
60.125 (2)
60.125
0.5–1
0.5
0.5
0.25
2
2–64 (2)
32–>128 (2)
64–>128 (2)
64–>128
64–>128
128–>128
128–>128
64–>128
4–>128
64–>128
4–>128
>128
4
1
16
5
0.5 [16]
0.25 [8]
1 [8]
0.25
8
16–64 (2) [>128]
64–128 [>128]
128–>128
128–>128
>128
6
0.25
0.25
16
32
32
32
>128
32
128
32
0.5
2
7
1
1
16
16
32
128
16
32
8
1
8
2 [8]
1
2
0.5–1
2
9
4 [32]
4 [16]
1 [32]
2 [16]
1 [8]
2
2
60.125–0.25
1–2
1
10
11
12
13
14
15
16
17
18
19
20
4-n-BuOPhCH2– OH
4-Ph-PhCH2– OH
Naphthyl-2-CH2 OH
2
32
128–>128
128–>128
>128
2
H
1
2
0.25–0.5
0.5–1
0.5
1
H
2
16
H
4-n-BuPhCH2–
OH
1
1
0.25–0.5
1 (2)
128–>128
128–>128 (2)
4 (2) [>32]
0.5 [2]–8 (2)
0.5
1
H
H
H
H
H
H
H
H
NH(CH2)3NMe2 1 [1]
NH(CH2)3NMe2 2 [8]
NH(CH2)3NMe2 0.5 [0.5]
2
1
nt
nt
nt
nt
nt
nt
nt
4-n-Decyl-Ph-CH2–
4-OctyloxyPh-CH2–
4-HexylOPh-CH2–
CH2CH(Bu)C6H13
3-EtO-4n-C6H13O-Ph-CH2–
4-BuCH(Et)Oph-CH2–
2
1
0.25
0.25
0.13
4–8 (2)
8 (2)
0.25
0.125
60.125
60.125
60.125
60.125
4
0.25
NH(CH2)3NMe2 60.125 [60.125] 60.125
NH(CH2)3NMe2 0.5 [1]
16
0.25
2
60.125 (2)
60.125 (2)
60.125 (2)
60.125 (2)
60.125 [0.25]–16 (2) 60.125
0.25–8 (2) [nt]
60.125
60.125 [0.25]–16 (2) 60.125
60.125–16 (2) 60.125
8–16 (2)
16 (1)
1
NH(CH2)3NMe2 60.125 [0.25]
NH(CH2)3NMe2 60.125 [0.25]
60.125
60.125
16 (2)
4
16 (2)
( ), Number of tested microorganisms; [ ], MIC values for one strain in the presence of 30% bovine serum; nt, not tested.

Table 2. Dialkylated derivatives
ID
MIC or MIC range (mg/L)
S. aureus
S. epidermidis
S. haemolyticus
E. faecalis
E. faecium
R1
R2
R3
Met-S (1) Met-R (2) Met-S (1) Met-R (1) (1)
Van-S (3)
Van-A (4)
Van-S (1) Van-A (4)
21 p-Ph-PhCH2–
p-Ph-PhCH2–
OH
OH
OH
OH
OH
OH
0.5 [64]
2 [64]
2 [128]
2 [32]
2 [64]
4 [32]
4 [4]
0.5
1
0.5
2
4
16
8
64
16
8
0.25–0.5
60.125
0.25–1
1–16 [>128]
4–128 [>128]
2–4 [>128]
0.5
2
4–>128
2–>128
0.5–>128
2–>128
2–>128
32–>128
1–>128
0.5–>128
2–32
22 2-Naphthyl-CH2–
23 p-nBu-Ph-CH2–
24 p-nBuO-Ph-CH2–
25 p-PhOCO-Ph-CH2–
26 3-Indolyl-CH2–
2-Naphthyl-CH2–
p-nBu-Ph-CH2–
p-nBuO-Ph-CH2–
p-PhOCO-Ph-CH2–
3-Indolyl-CH2–
2
4
1
1
2
8
32
64
>128
64
8
60.125–0.5 1–8 [>128]
0.25–1
0.5–1
0.25
2
2
4
64
16
128
32
2
4 [>128]
32–>128 [>128]
1
2
2
27 N-nBu-3-Indolyl-CH2– N-nBu-3-Indolyl-CH2– OH
28 N-Bn-3-Indolyl-CH2– N-Bn-3-Indolyl-CH2– OH
2
4
16–>128 [>128] 60.125
2–>128 [>128] 60.125
60.125 [4] 60.125
60.125
0.25
60.125
0.25
29 p-Ph-PhCH2–
p-Ph-PhCH2–
NH(CH2)3NMe2 1 [16]
1
1
2
1–16 [4]
0.5
30 2-Naphthyl-CH2–
31 p-nBu-Ph-CH2–
32 p-nBuO-Ph-CH2–
33 p-PhOCO-Ph-CH2–
34 3-Indolyl-CH2–
2-Naphthyl-CH2–
p-nBu-Ph-CH2–
p-nBuO-Ph-CH2–
p-PhOCO-Ph-CH2–
3-Indolyl-CH2–
NH(CH2)3NMe2 1 [16]
NH(CH2)3NMe2 1 [8]
NH(CH2)3NMe2 0.25 [2]
NH(CH2)3NMe2 1 [16]
NH(CH2)3NMe2 2 [4]
2
60.125
0.25
2
1
60.125–0.25 0.5–64 [2]
60.125–0.25 0.25–8 [8]
0.25
0.5
0.25–32
0.5–16
2
2
0.5
2
1
60.125
0.25
1
60.125
0.25–8 [1
0.125
0.5
0.125–16
0.5–16
1
2
2
60.125–0.25 1–8 [4]
0.5–8 [8]
1
1
0.25
2
2
60.125
0.25
0.125
0.125
0.25–32
0.125–128
0.125–32
35 N-nBu-3-Indolyl-CH2– N-nBu-3-Indolyl-CH2– NH(CH2)3NMe2 0.5 [1]
36 N-Bn-3-Indolyl-CH2– N-Bn-3-Indolyl-CH2– NH(CH2)3NMe2 0.25 [1]
2
0.25
2
0.5
0.25
0.25
60.125–0.5 2–128 [2]
60.125–0.25 4–32 [16]
0.125
( ), Number of tested microorganisms; [ ], MIC values for one strain in the presence of 30% bovine serum; nt, not tested.

S. I. Maffioli et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3801–3805
3805
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the best results. The relevant role of hydrophobic chain
the activity against resistant strains was confirmed by a
lack of activity, against VRE, of the simple basic
diamide of DA40 (Table 1, 14).
4. (a) Nicas, T. I.; Mullen, D. L.; Flokowitsch, J. E.;
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L.; Kerns, R.; Fukuzawa, S.; Thompson, C.; Kahne, D.
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6. Pavlov, A. Y.; Preobrazhenskaya, M. N.; Malabarba, A.;
Ciabatti, R. J. Antibiot. 1998, 51, 525.
7. The nitrogen atom at the N-terminus of the peptide in
A40926 and its derivatives is designated as N, the
nitrogen atom in the 2-aminoglucuronyl residue is desig-
nated as N0.
8. (a) Borghi, A.; Spreafico, F.; Beretta, G.; Ferrari, P.;
Goldstein, B. P.; Berti, M.; Denaro, M.; Selva, E. J.
Antibiot. 1996, 49, 607; (b) Carelli, A.; Borghi, A.;
Carrano, L.; Gastaldo, L.; Marinelli, F. J. Ind. Microbiol.
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N,N0-disubstituted derivatives of DA40 demonstrated
high activity against susceptible enterococci and only
modest activity against VRE (Table 2, 21–28) but, as
observed in the case of monoalkylated compounds,
activity disappeared in the presence of serum. Again,
the corresponding basic diamides increased the activity
against VRE and revealed only moderate inactivation
in the presence of serum (Table 2, 29–36). In this class,
the best compound appears to be 32.
3. Conclusion
A40 and DA40 (2) are completely inactive against VRE.
Mono- and dialkylation of the two amines slightly
improved the antimicrobial activity. Basic diamides of
N-mono- and N,N0-dialkylated derivatives did show
emergence of any interesting activity against VRE.
Among these diamides only the monoalkylated had good
activity against the sensitive strain. In the presence of ser-
um only, the diamides demonstrated activity against
VRE. Compounds 18, 19, and 32 showed antimicrobial
activity of particular interest and deserve further study.
References and notes
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10. NCCLS Document M7-A6, Vol. 23, No. 2. Methods
for dilution antimicrobial susceptibility tests for bacte-
ria that grow aerobically. Approved standard January
2003.
11. Against streptococci all the compounds showed MIC
range <0.125–0.5 mg/L but DA40 that showed 8 mg/L.