New synthetic siderophores and their β-lactam conjugates based on diamino acids and dipeptides

Article abstract of DOI:10.1016/S0968-0896(02)00044-5

Linking of siderophores to antibiotics improves the penetration and therefore increases the antibacterial activity of the antibiotics. We synthesized the acylated catecholates and hydroxamates as siderophore components for antibiotic conjugates to reduce

Full text of DOI:10.1016/S0968-0896(02)00044-5

Bioorganic & Medicinal Chemistry 10 (2002) 1659–1670
New Synthetic Siderophores and Their ꢀ-Lactam Conjugates
Based on Diamino Acids and Dipeptides
S. Wittmann,a,y M. Schnabelrauch,a,{ I. Scherlitz-Hofmann,y,a U. Mollmann,a
D. Ankel-Fuchsb and L. Heinischa,*
aHans Knoll Institute for Natural Product Research, Jena, Beutenbergstraße 11, D-07745 Jena, Germany
¨
bGrunenthal GmbH, Post box 50 04 44, D-52088 Aachen, Germany
¨
Received 7 December 2001; accepted 24 January 2002
Abstract—Linking of siderophores to antibiotics improves the penetration and therefore increases the antibacterial activity of the
antibiotics. We synthesized the acylated catecholates and hydroxamates as siderophore components for antibiotic conjugates to
reduce side effects of unprotected catecholate and hydroxamate moieties. In this paper, we report on bis- and tris-catecholates and
mixed catecholate hydroxamates based on diamino acids or dipeptides. These compounds were active as siderophores in a growth
promotion assay under iron limitation. Most of the conjugates with b-lactams showed high in vitro activity against Gram-negative
bacteria especially Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, Serratia marcescens and Stenotrophomonas
maltophilia. The compounds with enhanced antibacterial activity use active iron uptake routes to penetrate the bacterial outer
membrane barrier, demonstrated by assays with mutants deficient in components of the iron transport system. Correlation between
chemical structure and biological activity was studied. # 2002 Published by Elsevier Science Ltd.
0
0
Introduction
dihydroxybenzoyl)-l-threonine10 and N2-[N2 ,N6 -bis-(2,3-
dihydroxybenzoyl)-l-lysyl]-N6-(2,3-dihydroxybenzoyl)-l-
lysine.11 We synthesized a series of mono-, bis-, and tris-
catecholates of amino acids, among which the trisca-
techolates demonstrated the highest siderophore activity
due to their optimal iron chelation properties.12 b-
lactam conjugates with monocatecholates as siderophore
components were active as antibacterials.13 So far these
siderophore antibiotic conjugates were not used as
therapeutics probably due to side effects of the free
catechole moiety. Carbacephalosporins with bis-
catecholates based on l-lysine and with mixed catecho-
lates and hydroxamate ligands based on spermidine and
l-lysine were synthesized, but their antibacterial activity
was not significantly increased.8,14,15 In these com-
pounds the catecholate or hydroxamate moieties are
unprotected. We used the acylated catecholates or
hydroxamates as protected siderophore components to
facilitate the synthesis especially for sulfur containing
b-lactams. For iron chelating these protected side-
rophores obviously have to be cleaved enzymatically to
free catecholates or hydroxamates. By this process free
catecholates may become available only slowly which
possibly reduces their pharmacological side effects. The
feasibility of using protected siderophores was con-
firmed by recently prepared 8-acyloxy-1,3-benzoxazine-
Siderophores linked to b-lactam antibiotics can improve
their antibacterial activity significantly. Siderophore
antibiotic conjugates were used as a Trojan Horse
strategy to overcome penetration mediated bacterial
antibiotic resistance.1,2 Siderophores are bacterial iron
chelators mostly containing catecholate or hydroxamate
groups as chelating ligands. They are expressed under
iron starvation conditions and sequester extracellular
ferric ions.3 Specific bacterial outer membrane receptors
recognize the iron complexed siderophores and initiate
the active transport into the cell. Some siderophores
contain amino acids or dipeptides as scaffolds.4 Exam-
ples are enterobactin, a trimer of N-(2,3-dihydroxy-
benzoyl)-serine,5,6 found in Escherichia coli, or N2,N6-
bis-(2,3-dihydroxybenzoyl)-l-lysine7,8 and N-(2,3-dihy-
droxybenzoyl)-glycine,9 siderophores from Acotobacter
vinelandii and Bacillus subtilis, respectively. Published
synthetic analogues of these siderophores are N-(2,3-
*Corresponding author. Tel.: +49-3641-656714; fax: +49-3641-
656705; e-mail: heinisch@pmail.hki-jena.de
yPresent address: Jenapharm GmbH & Co. KG, Jena, Germany.
{Present address: INNOVENT Technologieentwicklung Jena, Ger-
many.
0968-0896/02/$ - see front matter # 2002 Published by Elsevier Science Ltd.
PII: S0968-0896(02)00044-5

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S. Wittmann et al. / Bioorg. Med. Chem. 10 (2002) 1659–1670
2,4-diones or 2,3-diacetoxybenzoyl derivatives and their
antibiotic conjugates.16ꢀ18 In this paper we report on the
synthesis and biological activity of new acylated 4- and
6-ligand catecholates or mixed catecholate hydro-
xamates as siderophores based on amino acids and on
dipeptides as well as of their conjugates with antibiotics.
lysine 15, respectively, by reacting with acid chloride 2a.
Reaction of N2-l-lysyl-l-lysine benzylester tritosylate
with acid chloride 2c gave after hydrogenolysis the tris-
(8-methoxycarbonyloxy-2,4-dioxo-benzoxazine) deriva-
tive 18 (Scheme 2). As a further tris-(8-methoxy-
carbonyloxy-2,4-dioxo-benzoxazine)
synthesized compound 21 with a more extensive and
derivative
we
possibly more flexible backbone. Synthesis started from
0
0
N6-[N2 ,N6 -di-Z-l-lysyl]-l-lysine 20, that was reacted in
succession with e-Z-aminocaproic acid, hydrogen and
finally with acid chloride 2c to form compound 21
(Scheme 3).
Results
To overcome penetration mediated antibiotic resistance
by linking of synthetic siderophores to b-lactams we
synthesized new acylated bis- and triscatecholates and
mixed biscatecholate hydroxamates based on diamino
acids (l- and d-ornithine, l-lysine) or corresponding
dipeptides. Catecholic moieties were attached in form of
2,3-diacetoxybenzoyl or heterocyclic 8-acyloxy-2,4-
dioxo-1,3-benzoxazine units. As acylated hydroxamates
we used O-benzoyl derivatives of dicarboxylic acid
monohydroxamates. In these hydroxamates the NOH
and CO groups are exchanged (reversed hydro-
xamates),19 compared with natural hydroxamates4
based on N-hydroxyamino acids such as N5-hydroxy-
ornithine.20 These synthetic acylated chelators were
active as siderophores for Gram-negative bacteria in a
growth promotion assay under iron deficient conditions.
The new siderophores were coupled with the b-lactam
antibiotics ampicillin (Ap), amoxicillin (Ax), cephalexin
(Ce) and cefaclor (Cf). Most of the conjugates were
highly active against Gram-negative bacteria especially
against strains of Pseudomonas aeruginosa and Streno-
trophomonas maltophilia.
Biscatecholate hydroxamates (Schemes 3 and 4)
We synthesized two types of biscatecholate hydrox-
amates derived from compounds 3a or 3b by linking the
hydroxamate unit via a l-glutamic or glutaric acid
spacer. Subsequent reaction of 3a or 3b with 1-benzyl
l-glutamate, N-benzoyloxy-N-methyl-amine and cata-
lytic hydrogenation with Pd/C led to the biscatecholate
hydroxamates 24a and b, respectively (Scheme 3). The
corresponding N-cyclohexyl derivatives 27a and b,
respectively, were synthesized from 1-benzyl Z-l-gluta-
mate, which was reacted in succession with N-benzoyl-
oxy-N-cyclohexyl-amine, hydrogen and finally 3a or 3b.
The biscatecholate hydroxamates 31a and b were syn-
thesized as follows. N-substituted N-benzoyloxy-glu-
taric amides 29a and b, prepared before from glutaric
anhydride and the corresponding N-substituted N-ben-
zoyloxyamines, were reacted with N6-Z-l-lysine. Fol-
lowing hydrogenolysis gave derivatives 30a and 30b,
which were acylated with 3a to the biscatecholate
hydroxamates 31a and b, respectively (Scheme 4).
Chemistry
ꢀ-Lactame conjugates (Schemes 1–5)
In this work we describe the synthesis of acylated bis-
and triscatecholates as well as biscatecholate hydro-
xamates with a diamino acid or dipeptide scaffold. As
acylated catecholate moieties, we used 2,3-diacetoxy-
benzoyl and 8-methoxycarbonyloxy-2,4-dioxo-benzox-
azine units. As acylated hydroxamates we used their
benzoyl derivatives.
The siderophore analogues 3a–d, 8a–c, 10a,b, 13a,b, 16,
18, 21, 23a,b, 27a,b and 31a,b were linked to the free
amino group of the antibiotics via mixed anhydride
method forming the ampicillin conjugates (X=Ap) 4a–
d, 9a–c, 11a,b, 14a,b, 17a, 19a, 22, 25a,b, 28a,b, 32a,b,
the amoxicillin conjugates (X=Ax) 5a,b, 17b and 19b,
the cephalexin conjugates (X=Ce) 6a,b and the cefaclor
conjugates (X=Cf) 7a,b and 19c. To obtain water solu-
ble compounds sodium salts were formed from the
conjugates by reacting the acids with sodium 2-ethyl-
hexanoate.
Biscatecholates (Scheme 1)
The acetylated biscatecholates 3a–d were synthesized
from the diamino acids 1a–c and 2,3-diacetoxybenzoyl
chloride 2a or its 5-bromo derivative 2b. The benzox-
azine compounds 8a–c were prepared from 1a–c and
2,3-di-methoxycarbonyloxybenzoyl chloride 2c. More-
over, the modification of the scaffold was accomplished
by attachment of the amino acids l-phenylalanine and
l-tryptophan which resulted in biscatecholates 10a, b.
Biology
Siderophore activity
Most of the compounds were active in a growth pro-
motion assay to study the siderophore activity (Table
1). The biscatecholates 3b, 3c based on d- and l-orni-
thine were comparable active in growth promotion of
all strains used, but the 5-bromocatecholate 3d was
inactive in Salmonella typhimurium enb7. The bisca-
techolate 3a, based on l-lysine, was active using all test
strains comparable to 3b and 3c. All other compounds
promoted growth of all test strains. The bis- and tris-
Triscatecholates (Schemes 2 and 3)
Triscatecholates and biscatecholate hydroxamates offer
optimal conditions for hexagonal iron chelating. In the
case of biscatecholates two molecules should be neces-
sary for iron complexing. The acetylated triscatecho-
lates 13a,b and 16 were prepared from N5-l-ornithyl-l-
ornithine 12a, N6-l-lysyl-l-lysine 12b and N2-l-lysyl-l-

S. Wittmann et al. / Bioorg. Med. Chem. 10 (2002) 1659–1670
1661
benzoxazine derivatives 8a–c and 18 were active as
siderophores in contrast to results obtained with mono-
benzoxazine derivatives, which can only act as side-
rophore components in b-lactam antibiotic con-
jugates.16 Especially high was the activity of the tris-
benzoxazine derivative 16 and the triscatecholate 13a.
The triscatecholate 13b was inactive in S. typhimurium
enb7.
which was resistant against the tested reference b-lac-
tams, was inhibited by the conjugates with MIC values
below 0.05 mg/L. Activity against Gram-negative
pathogens exceeded that of ampicillin, azlocillin, cefa-
clor and partly that of meropenem.
Studies of the mechanism of action of the conjugates
using E. coli strains deficient in siderophore receptors
showed the following results. Compared with the wild
type E. coli strain H 1443 there was no decrease in
activity of the conjugates against the fepA mutant H873.
Thus, activity does not depend on the enterobactin
receptor fepA. Against the mutants H 1875 and H 1877
missing one of the receptors cir and fiu (for uptake of
the breakdown products of enterobactin28) there were
only minor changes in activity. In contrast there was a
strong decrease in activity against the cir and fiu double
mutant H 1876 for most of the compounds, indicating
that entry of the compounds into the bacterial cells was
hindered. All tested compounds exhibited high activity
against E. coli AB2847 (tonB +) and reduced or no
activity against the tonB negative mutant BR158, indi-
cating a dependence of activity on active iron transport
mechanisms. On the other hand azlocillin, ampicillin or
cefaclor were not affected by the presence or the absence
of siderophore receptors or active iron transport sys-
tems. This demonstrates that the new conjugates, but
not ampicillin, cefaclor or azlocillin, reached their tar-
gets via iron transport uptake routes.
Antibacterialactivity
In a first approach all siderophores and siderophore
antibiotic conjugates were screened for antibacterial
activity in an agar diffusion assay. Siderophores alone
were generally inactive in a concentration of 100 mg/L
and an application volume of 50 mLper agar well. Con-
jugates were all active in different levels and minimal
inhibitory concentrations (MIC) were determined con-
sequently (Table 2). Most of the conjugates were highly
active against Gram-negative bacteria, but exhibited
only low activity against the Gram-positive strain S.
aureus SG 511. Generally ampicillin conjugates were
superior in activity compared to amoxicillin, cephalexin
and cefaclor conjugates. Amongst the biscatecholates
the acetylated derivatives, especially the bromo deriva-
tive 4d, showed the highest activity. The phenylalanine
moiety in 11a increased the activity against E. coli
ATCC 25922 compared to the basic compound 4c,
while the more polar tryptophan derivative 11b showed
no difference to 4c. Highly active were also the trisca-
techolate 17a and the biscatecholate hydroxamates
25a,b and 28b. The compounds 28a,b and 32b were
highly active against Klebsiella pneumoniae. The com-
pounds 4d, 11a, 25a,b and 28b showed the strongest
activity of all tested compounds. Beside the high activity
against P. aeruginosa and K. pneumoniae, S. maltophilia,
Conclusions
Acylated bis- or triscatecholates and mixed biscatecho-
late hydroxamates were synthesized. The compounds
showed high siderophore activity in a growth promotion
Scheme 1. Synthesis of biscatecholates. Ac=COCH3, Moc=COOCH3. Reagents and conditions: (I) (i) iBu-OCOCl, N-methylmorpholine; (ii) l-H-
Phe–OH, Et3N; II (i) iBu-OCOCl, N-methylmorpholine; (ii) l-H-Trp–OH, Et3N.

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S. Wittmann et al. / Bioorg. Med. Chem. 10 (2002) 1659–1670
assay under iron limitation. Their conjugates with
ampicillin, amoxicillin and cefaclor, exhibited strong in
vitro activity against Gram-negative bacteria, especially
P. aeruginosa and S. maltophilia, due to their MIC.
Whereas the cephalexin derivatives were less active, the
ampicillin derivatives were the most active ones. Com-
pounds with 8-acyloxy-2,4-dioxobenzoxazines as side-
rophore components were less active than conjugates
with acetylated siderophores. This improved anti-
bacterial activity was due to active uptake via iron
Scheme 2. Synthesis of triscatecholates. Ac=COCH3, Moc=COOCH3. Reagents and conditions: (I) (i) TsOH, C6H5CH2OH, 110 ꢁC; (ii) 2c,
NaHCO3; (iii) H2, Pd/C, rt.
Scheme 3. Synthesis of triscatecholates and biscatecholate hydroxamates. Bn=benzyl, Z=benzyloxycarbonyl, Bzl=benzoyl. Reagents: (I) (i) iBu-
OCOCl, N-methylmorpholine; (ii) NH2(CH2)5COOH, Et3N; (iii) H2, Pd/C 10%, rt; (iv) 2c, NaHCO3; (II) (i) iBu-OCOCl, N-methylmorpholine; (ii)
H-Glu-OBn, Et3N; (III) (i) iBu-OCOCl, N-methylmorpholine; (ii) CH3–NHOBzl, Et3N; (iii) H2, Pd/C 10%, rt; (IV) (i) iBu-OCOCl, N-methylmor-
pholine; (ii) c-C6H11–NHOBzl, Et3N; (iii) H2, Pd/C 10%, rt; (iv) 3a or 3b, iBu-OCOCl, N-methylmorpholine.

S. Wittmann et al. / Bioorg. Med. Chem. 10 (2002) 1659–1670
1663
Scheme 4. Synthesis of biscatecholate hydroxamates. Reagents and conditions: (I) R-NHOBzl, Et3N;. (II) (i) iBu-OCOCl, N-methylmorpholine; (ii)
l-H-Lys(Z)-OH, Et3N; (iii) H2, Pd/C 10%, rt; (III) 3a, iBu-OCOCl, N-methylmorpholine.
Scheme 5. Antibiotic components of the conjugates: R=siderophore components of Schemes 1–4; R1=H: Ap=ampicillino; R1=OH: Ax=amox-
icillino; R2=CH3: Ce=cephalexino; R2=Cl: Cf=cefacloro.
transport pathways. The triscatecholate 17a, the bisca-
techolates 4d, and 11a and the biscatecholate hydro-
xamates 25a,b and 28b were the most active compounds
from this series. For compound 25a in vivo therapeutic
activity in a P. aeruginosa septicaemic mouse model
(ED50: 10.4 mg/kg) could be demonstrated together with
moderate toxicity in mice (LD50: 125–250 mg/kg) (more
data will be presented in a following paper). This
compound is a promising candidate for further pre-
clinical and clinical investigation.
Experimental
Chemistry
General. The chemicals were purchased from Bachem
(amino acids and dipeptides), Aldrich-Chemie (Ger-
many) and Fluka (Switzerland). Tetrahydrofuran
(THF) was distilled over sodium/benzophenone prior to
use. CCl4 was dried over CaCl2, distilled and stored
over molecular sieves (4A). All other solvents were pur-
chased as pa (pro analysis) quality. Melting points
(corrected) were determined by a Boetius hot stage
Table 1. Growth promotion of Gram-negative bacteria by catecho-
late and catecholate hydroxamate derivatives under iron limitation
Compd
P. aeruginosa
E. coli
S.
typhimurium
enb7
ATCC 27853 SG137 NCTC ATCC ATCC
10662 9027 25922
1
microscope (Rapido, Germany). H NMR spectra were
3a
3b
3c
3d
8a
8b
8c
10a
10b
13a
13b
16
24
20
23
25
20
20
17
20
20
23
24
25
18
15
23
20
26
23
25
19
20
22
35
20
25
23
28
20
27
22
20
25
28
29
30
19
15
25
23
31
30
27
23
22
24
30
25
27
22
32
18
27
28
28
25
30
27
30
20
18
30
20
28
30
25
20
21
25
30
20
25
20
27
15
17
20
22
24
35
26
25
30
20
20
24
22
20
24
20
17
27
35
32
28
25
28
20
25
28
30
30
30
31
34
23
40
30
32
35
25
33
29
30
34
36
37
33
32
0
24
28
28
32
31
30
0
25
25
25
26
30
29
27
34
27
32
25
38
recorded on a 300 MHz Bruker NMR spectrometer.
The chemical shifts d are given in ppm related to tetra-
methyl silane as internal standard. The coupling con-
stants J are reported in Hz. High resolution mass
spectra were obtained by a Finnigan MAT 95 XV high
resolution mass spectrometer with fast-atom bombard-
ment (FAB) as well as positive (PI) and negative ion
(NI) electron-spray ionization (ESI). Preparative HPLC
was performed on a Gilson Abimed apparatus (Abimed
Analysentechnik, Germany) equipped with a 115 UV
detector (254 nm) and a Vertex reversed-phase column
(250ꢂ32 mm I.D., Knauer, Germany), packed with
Eurospher 100-C18 (7 mm). Eluents used were acetoni-
trile (HPLC quality, Merck, Germany) and double dis-
tilled water, beginning with a ratio of 20:80 (v/v) and
achieving 80:20 (v/v) after a period of 20 min (flux rate
20 mL/min).
18
21
23a
23b
24a
24b
27a
27b
31a
31b
Desferal
The following compounds were synthesized according
to published procedures: 2,3-diacetoxybenzoyl chlo-
ride 2a,21 5-bromo-2,3-diacetoxybenzoyl chloride 2b,21
5 mLof a 2 mM solution was applied on a 6 mm filter disc. Diameters
of growth zones (mm).

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S. Wittmann et al. / Bioorg. Med. Chem. 10 (2002) 1659–1670
Table 2. Antibacterial activities of siderophore b-lactam conjugates in vitro MIC (mg/L)
Compound
P. aeruginosa
E. coli
ATCC 25922
K. pneumoniae
ATCC 10031
S. maltophilia
GN 12873
S. marcescens
SG 621
S. aureus
SG 511
SG 137
ATCC 27853
4a
4b
4c
4d
5a
5b
6a
6b
7a
7b
9a
9b
0.05
0.1
0.2
<0.05
3.12
0.78
12.5
<0.05
0.1
<0.05
0.4
1.56
0.1
<0.05
0.2
1.56
0.78
0.78
0.78
0.2
0.78
50
6.25
0.01
<0.05
0.4
0.05
<0.05
0.78
6.25
>100
0.2
100
1.56
0.78
6.25
0.4
6.25
6.25
25
3.12
6.25
3.12
6.25
12.5
6.25
0.4
12.5
1.56
6.25
0.78
6.25
6.25
100
>100
6.25
3.12
50
0.2
0.4
0.2
<0.05
6.25
0.4
0.2
3.12
6.25
0.1
0.1
<0.05
50
50
0.2
1.56
0.1
n.d.
0.1
12.5
n.d.
12.5
25
6.25
n.d.
50
12.5
12.5
12.5
25
0.78
12.5
12.5
6.25
12.5
25
1.56
<0.05
6.25
6.25
100
n.d.
n.d.
n.d.
12.5
0.78
n.d.
<0.05
<0.05
1.56
0.1
<0.05
1.56
n.d.
0.78
25
0.78
0.05
<0.05
0.1
<0.05
0.05
1.56
0.2
3.125
1.56
0.1
<0.05
0.78
0.1
6.25
6.25
0.4
1.56
12.5
3.12
0.1
1.56
6.25
3.12
1.56
3.12
0.1
0.4
3.12
0.2
0.78
3.12
6.25
6.25
0.04
12.5
0.4
9c
<0.05
<0.05
<0.05
3.12
11a
11b
14a
14b
17a
17b
19a
19b
19c
6.25
25
0.78
3.12
3.12
0.78
6.25
50
12.5
0.2
0.4
0.4
0.2
<0.05
0.1
0.4
<0.05
>0.05
0.4
<0.05
0.05
0.4
0.1
100
1.56
0.04
<0.05
0.05
0.01
0.2
0.2
25
>100
>100
100
12.5
25
3.12
12.5
6.25
3.12
12.5
12.5
12.5
12.5
6.25
6.25
0.4
22
25a
25b
28a
28b
32a
32b
Azlocillin
Ampicillin
Meropenem
Cefaclor
0.01
<0.05
<0.005
<0.005
0.1
<0.005
6.25
6.25
1.56
0.78
1.56
3.12
6.25
>100
0.4
0.005
0.78
1.56
50
25
0.06
100
0.4
0.1
6.25
0.04
3.12
100
n.d., not determined.
2,3-dimethoxycarbonyloxybenzoyl chloride 2c,16 N-
benzoyloxy-N-methyl-amine hydrochloride,22 and N-
benzoyloxy-N-cyclohexyl-amine hydrochloride.23
component (1 mmol) and triethylamine (1 mmol per
amino and per carboxyl group) in THF (8 mL) and
water (2 mL) was added. The mixture was stirred for 1 h
at ꢀ10 ꢁC and for 1 h at ambient temperature and then
evaporated. The residue was dissolved in ethyl acetate
and water and carefully acidified with 2 M HCl. After
shaking the organic layer was separated, washed with
brine, dried over Na2SO4 and the solvent was eva-
porated. The residue was purified by preparative
HPLC.
Generalmethods
Method A: Attachment of catecholate moieties. In an
ultrasonic bath, a solution of substituted benzoyl chlo-
ride 2 (1 mmol per amino group) in absolute THF
(10 mL) was added to a solution of the amino acid or
dipeptide (1 mmol) in freshly prepared 0.5 M aqueous
sodium hydrogen carbonate solution (1 mmol per amino
and carboxyl group) at 0–5 ꢁC with stirring. The mix-
ture was stirred for 1 h at 0–5 ꢁC. Then the THF was
evaporated. The residual aqueous solution was acidified
to pH 2 at 0 ꢁC with 2 M aqueous HCl, and then
extracted with ethyl acetate. The organic layer was
washed three times with brine, dried over Na2SO4 and
the solvent was evaporated. The residue was purified by
preparative HPLC.
Preparation of bis- and triscatecholates
N2,N6-Bis-(2,3-diacetoxybenzoyl)-L-lysine (3a). Prepara-
tion according to method A from 1a and 2a gave com-
pound 3a as a white solid, yield 45%. 1H NMR (CDCl3)
d 1.28 (2H, m), 1.60 (2H, m,), 1.85 (1H, m), 1.99 (1H,
m), 2.29 (3H s), 2.30 (6H, s), 2.34 (3H, s), 3.38 (2H, m),
4.75 (1H, m), 6.59 (1H, t, J=5.7), 7.22–7.32 (5H, m),
7.47 (1H, dd, J=2.3, 7.2), 7,65 (1H, dd, J=1.9, 7.4).
Method B: Mixed anhydrides. Isobutyl chloroformate
(0.130 mL, 1 mmol) was added to a solution of
carboxylic acid (1 mmol) and N-methylmorpholine
(0.112 mL, 1 mmol) in absolute THF (10 mL) at ꢀ20 ꢁC
with stirring. The mixture was stirred for 45 min at
ꢀ10 ꢁC, and then a solution or suspension of the amino
N2,N5-Bis-(2,3-diacetoxybenzoyl)-D-ornithine (3b). Pre-
paration according to method A from 1b and 2a gave
compound 3b as a white solid, yield 60%. H NMR
(DMSO-d6) d 1.50–1.90 (4H, m), 2.22 (6H, s), 2.27 (6H,
s), 3.19 (2H,m), 4.33 (1H, m), 7.32–7.49 (6H, m), 8.34
(1H, m), 8.52 (1H, d).
1

S. Wittmann et al. / Bioorg. Med. Chem. 10 (2002) 1659–1670
1665
N2,N5-Bis-(2,3-diacetoxybenzoyl)-L-ornithine (3c). Pre-
paration according to method A from 1c and 2a gave
compound 3c as a white solid, yield 85%. H NMR
(DMSO-d6) d 1.50–1.90 (4H, m), 2.20 (6H, s), 2.27 (6H,s),
3.18 (2H, m), 4.35 (1H, m), 7.30–7.50 (6H, m), 8.35 (1H,
t), 8.51 (1H, d). MS (ESI-PI) m/z 595.2 [M+Na]+.
12a and 2a gave compound 13a as a white solid, yield
1
50%. H NMR (DMSO-d6) d 1.40–1.85 (8H, m), 2.19
1
(3H, s), 2.20 (3H, s), 2.22 (3H, s), 2.27 (9H, s), 3.10 (4H,
m), 4.35 (2H, m), 7.30–7.55 (9H, m), 7.93 (1H, d), 8.24
(1H, d), 8.32 (1H, m), 8.50 (1H, d). MS (ESI-NI) m/z
905.3 [MꢀH]ꢀ.
0
0
N2,N5 -Bis-(5-brom-2,3-diacetoxybenzoyl)-L -ornithine
(3d). Preparation according to method A from 1a and
N6-[N2 ,N6 -(2,3-diacetoxybenzoyl)-L-lysyl]-N2-(2,3-diace-
toxybenzoyl)-L-lysine (13b). Preparation according to
method A from N6-l-lysyl-l-lysine 12b and 2a gave
1
2b gave compound 3d as a white solid, yield 85%. H
1
NMR (DMSO-d6) d 1.50–1.90 (4H, m), 2.19 (3H, s),
2.21 (3H, s), 2.27 (6H, s), 3.19 (2H, m), 4.31 (1H, m),
7.61 (2H, dd), 7.71 (2H, dd), 8.46 (1H, t), 8.69 (1H, d).
MS (ESI-PI) m/z 753.3 [M+Na]+.
compound 13b as a white solid, yield 45%. H NMR
(DMSO-d6) d 1.35–1.82 (12H, m), 2.22 (9H, s), 2.30
(9H,s), 3.20 (4H, m), 4.21 (1H, m), 4.45 (1H, m), 7.20–
7.60 (9H, m), 8.15–8.40 (m, 4H).
0
0
2L,6-Bis-(8-methoxycarbonyloxy-3,4-dihydro-2,4-dioxo-
1,3-benzoxazine-3-yl)-n-hexanoic acid (8a). Preparation
according to method A from 1a and 2c gave compound
8a as a white solid, yield 80%. Mp 111–115 ꢁC. 1H
NMR (DMSO-d6) d 1.37–1.79 (4H, m), 2.20–2.35 (2H,
m), 3.95 (3H, s), 3.96 (3H, s), 3.97 (2H, m), 5.47 (1H,
m), 7.31 (1H, t, J=8.0), 7.36 (1H, t, J=8.0), 7.51 (1H,
dd, J1=8.0, J2=1.4), 7.57 (1H, dd, J1=8.0, J2=1.4),
7.86 (1H, dd, J1=8.0, J2=1.4), 7.94 (1H, dd, J1=8.0,
J2=1.4). MS (ESI-NI) m/z 585.2 [MꢀH]ꢀ.
N2-[N2 ,N6 -(2,3-diacetoxybenzoyl)-L-lysyl]-N6-(2,3-diace-
toxybenzoyl)-L-lysine (16). Preparation according to
method A from N2-l-lysyl-l-lysine 15 and 2a gave
1
compound 16 as a white solid, yield 40%. H NMR
(DMSO-d6) d 1.30–1.80 (12H, m), 2.21 (9H, s), 2.29 (9H,
s), 3.17 (4H, m), 4.20 (1H, m), 4.46 (1H, m), 7.30–7.55 (9H,
m), 8.10–8.35 (m, 4H). MS (ESI-NI) m/z 933.3 [MꢀH]ꢀ.
2L-[20L,60 -Bis-(8-methoxycarbonyloxy-3,4-dihydro-2,4-di-
oxo-1,3-benzoxazin-3-yl)-n-hexanoylamino]-6-(8-methoxy-
carbonyloxy-3,4-dihydro-2,4-dioxo-1,3-benzoxazin-3-yl)-
n-hexanoic acid (18). Benzylalcohol (4.32 g, 40 mmol)
and benzene (60 mL) were added to a mixture of N2-l-
lysyl-l-lysine 15 (dihydrochloride, 750 mg, 2.73 mmol)
and p-toluensulfonic acid monohydrate (1.87 g,
9.84 mmol). The mixture was boiled with water separa-
tor for 4 h and then the solvent was evaporated. Die-
thylether was added to the residue to give N2-l,l-
lysyllysine benzylester tritosylate as a white solid (1.58 g,
yield 66%). Triethylamine (1.48 mL, 10.6 mmol) was
added under argon to a solution of this benzylester
(1.55 g, 1.76 mmol) in absolute dimethylformamide
(20 mL), followed by a solution of 2c (1.53 g, 5.3 mmol)
in absolute dimethylformamide. The mixture was stirred
for 5 h at 0 ꢁC, stored over night at ambient temperature
and then the solvent was evaporated. Ethyl acetate and
water were added to the residue. The solution was cooled
down to 0 ꢁC and carefully acidified with dilute hydro-
chloric acid. After shaking, the organic layer was sepa-
rated, washed with brine and evaporated. The residue was
purified by preparative HPLC (eluent acetonitrile/water
1/1 v/v with 0.5% trifluoracetic acid) to give a yellow oil of
the benzylester of target compound 18 (X=OCH2C6H5)
(250mg, yield 14%, MS (FAB) m/z 1024.6 [M+H]+.
2D,5-Bis-(8-methoxycarbonyloxy-3,4-dihydro-2,4-dioxo-
1,3-benzoxazin-3-yl)-n-pentanoic acid (8b). Preparation
according to method A from 1b and 2c gave compound
1
8b as a white solid, yield 50%. H NMR (DMSO-d6) d
1.70 (2H, m), 1.95–2.25 (2H, m), 3.22 (2H, m), 3.90 (6H,
s), 5.35 (1H, m), 7.45–7.95 (6H, m). MS (ESI-NI) m/z
571.2 [MꢀH]ꢀ.
2L,5-Bis-(8-methoxycarbonyloxy-3,4-dihydro-2,4-dioxo-
1,3-benzoxazin-3-yl)-n-pentanoic acid (8c). Preparation
according to method A from 1c and 2c gave compound
1
8c as a white solid, yield 50%. H NMR (DMSO-d6) d
1.69 (2H, m), 1.90–2.25 (2H, m), 3.20 (2H, m), 3.90 (6H,
s), 5.34 (1H, m), 7.40–7.95 (6H, m). MS (ESI-NI) m/z
571.1 [MꢀH]ꢀ.
0
0
N2-[N2 ,N5 -Bis-(2,3-diacetoxybenzoyl)-L-ornithyl]-L-phe-
nylalanine (10a). Reacting 3c and l-phenylalanine
according to method B gave compound 10a as a white
1
solid, yield 30%. H NMR (DMSO-d6) d 1.90–1.50 (m,
4H), 2.22 (s, 6H), 2.27 (s, 6H), 2.99 (m, 2H), 3.17 (m, 2H),
4.45 (m, 2H), 7.50–7.15 (m, 11H), 8.15 (d, 1H), 8.20 (d,
1H), 8.30 (t, 1H). MS (FAB) m/z 720.3 [M+1]+.
0
0
N2-[N2 ,N5 -Bis-(2,3-diacetoxybenzoyl)-L-ornithyl]-L-tryp-
tophan (10b). Reacting 3c and l-tryptophan according
to method B gave compound 10b as a white solid, yield
60%. In contrast to the general procedure, citric acid
This benzylester was dissolved in ethanol (30 mL) and
then hydrogenolysed with Pd/C (10%, 60 mg) at ambi-
ent temperature and atmospheric pressure to give 18
(X=OH) as a white solid (220 mg, yield 98%). 1H
NMR (DMSO-d6) d 1.20–1.70 (12H, m); 3.80 (4H, m),
3.83 (3H, s), 3.89 (6H, s), 4.27 (1H, m), 5.03 (1H,m),
7.43 (3H, t, J=8.0), 7.73- 7.90 (6H, m), 8.27 (1H, d,
J=8.1). MS (ES-NI) m/z 933.8 [MꢀH]ꢀ.
N2-["-(8-Methoxycarbonyloxy-3,4-dihydro-2,4-dioxo-1,3
-benzoxazin-3-yl)-n-hexanoyl]-N6-[2,6-bis(8-methoxycar-
bonyloxy-3,4-dihydro-2,4-dioxo-1,3-benzoxazin-3-yl)-n-
hexanoyl]-L-lysine (21). According to method B, a tri-
1
was used instead of HCl. H NMR (DMSO-d6) d 1.90–
1.50 (m, 4H), 2.20 (s, 6H), 2.27 (s, 6H), 3.18 (m, 2H),
3.60 (m, 2H), 4.51 (m, 2H), 7.55–7.27 (m, 11H), 8.17 (d,
1H), 8.24 (d, 1H), 8.32 (t, 1H), 10.81 (s, 1H). MS (ESI-
NI) m/z 757.4 [MꢀH]ꢀ.
N5-[N2 ,N5 -(2,3-diacetoxybenzoyl)-L-ornithyl]-N2-(2,3-
diacetoxybenzoyl)-L-ornithine (13a). Preparation
according to method A from N5-l-ornithyl-l-ornithine
0
0

1666
S. Wittmann et al. / Bioorg. Med. Chem. 10 (2002) 1659–1670
0
0
Z-derivative was prepared from e-Z-aminohexanoic
N-(N2 ,N6 -Bis-(2,3-diacetoxybenzoyl)-L-lysyl)-L-glutamic
5-(N-benzoyloxy-N-cyclohexyl)-amide (27a). Z-l-Glu-
tamic 1-benzylester 5-(N-benzoyloxy-N-cyclohexyl)-amide
was prepared from 1-benzyl Z-l-glutamate 26 and N-
benzoyloxy-N-cyclohexyl-amine via mixed anhydride
method using isobutyl chloroformate (method B).
l-Glutamic 5-(N-benzoyloxy-N-cyclohexyl)-amide was
formed by catalytic hydrogenolysis with Pd/C (10%) at
ambient temperature and atmospheric pressure. MS
(FAB) m/z 349.3 [M+H]+.
0
0
acid and N6-[N2 ,N6 -di-Z-l-lysyl]-l-lysine 20 as a white
solid (yield 64%). MS (ES-PI) m/z 812.4 [M+Na]+.
The tri-Z-derivative was hydrogenolysed at ambient
temperature and atmospheric hydrogen pressure over
10% Pd/C to give N6-l-lysyl-N2-(e-aminocaproyl)-l-
lysine as a white solid (yield 50%). MS (ES-NI) m/z
388.3 [M+H]+.
This compound was reacted with acid chloride 2c
according to method A obtaining compound 21 as a
white solid in 50% yield. H NMR (DMSO-d6) d 1.00–
2.30 (20H, m), 2.97 (2H, m), 3.80 (4H, m), 3.90 (9H, s),
4.10 (1H, m), 5.07 (1H, m), 7.39- 7.99 (11H, m). MS
(ES-PI) m/z 1070.6 [M+Na]+.
Reacting the resulting amide with 3a according to
method B gave compound 27a as a white solid (yield
50%). H NMR (DMSO-d6) d 1.00–2.30 (16H, m), 2.19
(3H, s), 2.22 (3H, s), 2.27 (6H, s), 3.14 (2H, m), 4.26
(2H, m), 4.40 (1H, m), 7.31–8.05 (m, 11H), 8.18 (1H, d),
8.22 (1H, d), 8.30 (1H, m). MS (ESI-PI) m/z 939.6
[M+Na]+.
1
1
0
0
1-Benzyl N-[N2 ,N6 -bis-(2,3-diacetoxybenzoyl)-L-lysyl]
L-glutamate (23a). Preparation according to method B
from 3a and 1-benzyl l-glutamate gave compound 23a
0
0
N-(N2 ,N5 -Bis-(2,3-diacetoxybenzoyl)-D-ornithyl)-L-glu-
tamic 5-(N-benzoyloxy-N-cyclohexyl)-amide (27b). This
was prepared analogously to compound 27a using
biscatecholate 3b as a white solid (Yield 50%). 1H
NMR (DMSO-d6) d 1.00–2.30 (20H, m), 2.18 (3H, s),
2.20 (3H, s), 2.27 (6H, s), 3.15 (2H, m), 4.25 (1H, m),
4.35 (1H, m), 4.45 (1H, m), 7.32–8.05 (11H, m), 8.22
(2H, m), 8.30 (1H, d). MS (ESI-NI) m/z 901.7
[MꢀH]ꢀ.
1
as a white solid (Yield 30%). H NMR (DMSO-d6) d
1.30–2.18 (8H, m), 2.19 (3H, s), 2.21 (3H, s), 2.27 (6H,
s), 2.30 (2H, m), 3.15 (2H, m), 4.37 (2H, m), 5.11 (2H,
s), 7.30–7.50 (11H, m), 8.30 (3H, m).
0
0
1-Benzyl N-[N2 ,N5 -bis-(2,3-diacetoxy-benzoyl- D-orni-
thyl] L-glutamate (23b). Preparation according to
method B from 3b and 1-benzyl l-glutamate gave com-
pound 23b as a white solid (yield 40%). 1H NMR
(DMSO-d6) d 1.50–2.06 (6H, m), 2.19 (6H, s), 2.27 (6H,
s), 2.34 (2H, m), 3.18 (2H, m), 4.35 (1H, m), 4.50 (1H,
m), 5.11 (2H, s), 7.25–7.45 (10H, m), 7.53 (1H, dd), 8.24
(1H, d), 8.33 (1H, t), 8.42 (1H, d). MS (ESI-PI) m/z
814.2 [M+Na]+.
N2-[4-(N-Benzoyloxy-N-methyl-carbamoyl)-n-butanoyl]-
0
0
N6-(N2 ,N6 -bis-2,3-diacetoxybenzoyl)-L-lysyl]-L-lysine
(31a). Reacting l-glutaric 5-N-benzoyloxy-N-methyl-
monoamide 29a (prepared from glutaric anhydride and
N-benzoyloxy-N-methylamine hydrochloride) and N2-Z-
l-lysine according to method B gave N2-[4-(N-benzoyloxy-
N-methyl-carbamoyl)-n-butanoyl]-N6-Z-l-lysine as
a
Preparation of biscatecholate hydroxamates
white solid (yield 70%, MS (ESI-PI) m/z 528.3
[M+H]+). This compound was dissolved in methanol
(10 mL) and hydrogenolysed with Pd/C (0.1 g, 10%) at
ambient temperature and atmospheric pressure to give
N2-[4-(N-benzoyloxy-N-methyl-carbamoyl)-n-butanoyl]-
L-lysine 30a as a white solid (Yield 60%, MS (ESI-NI)
m/z 394.1 [MꢀH]ꢀ). Reacting 3a and 30a according to
0
0
N-[N2 ,N6 -Bis-(2,3-diacetoxybenzoyl)-L-lysyl]-L-glutamic
5-(N-benzoyloxy-N-methyl)-amide (24a). Reacting 23a
and N-benzoyloxy-N-methyl-amine according to
method B gave the benzylester of compound 24a
(X=OCH2C6H5) as a white solid (yield 40%). MS (ESI-
NI) m/z 923.5 [MꢀH]+. This benzylester was
hydrogenolysed at ambient temperature and atmo-
spheric hydrogen pressure over Pd/C (10%) to give
24a as a white solid, in 36% overall yield as a
white solid. 1H NMR (DMSO-d6) d 1.45–2.06 (8H,
m), 2.19 (3H, s), 2.22 (3H, s), 2.27 (6H, s), 2.39
(2H, m), 3.14 (2H, m), 3.30 (3H, m), 4.19 (1H, m),
4.37 (1H, m), 7.30–7.60 (8H, m), 7.75 (1H, t), 8.02 (2H,
d); 8.12 (H, d), 8.29 (2H, m). MS (ESI-NI) m/z 847.1
[MꢀH]ꢀ.
1
method B gave 31a as a white solid (Yield 30%). H
NMR (DMSO-d6) d 1.20–2.40 (18H, m), 2.19 (3H, s),
2.22 (3H, s), 2.27 (6H, s), 3.10 (4H, m), 4.03 (1H, m),
4.33 (1H, m), 7.30–8.05 (13H, m), 8.20 (1H, d), 8.29
(1H, m). MS (ESI-NI) m/z 959.9 [MꢀH]ꢀ.
N2-[4-(N-Benzoy0 loxy-N-cyclohexyl-carbamoyl)-n-buta-
0
noyl]-N6-[N2 ,N6 -bis-(2,3-diacetoxybenzoyl)-L-lysyl]-L-ly-
sine (31b). Reacting l-glutaric 5-N-benzoyloxy-N-
cyclohexyl-monoamide 29b (prepared from glutaric
anhydride and N-benzoyloxy-N-cyclohexylamine) and
N2-Z-l-lysine according to method B gave N2-[4-(N-
benzoyloxy-N-cyclohexyl-carbamoyl)-n-butanoyl)-N6-Z-
l-lysine as a white solid (yield 70%), MS (ESI-PI) m/z
618.4 [M+H]+.
0
0
N-[N2 ,N5 -Bis-(2,3-diacetoxybenzoyl)-D-ornithyl]-L-glu-
tamic 5-(N-benzoyloxy-N-methyl)-amide (24b). This was
prepared from 23a and N-benzoyloxy-N-methyl-amine
analogously to compound 24a via the corresponding
1
benzylester in 40% overall yield as a white solid, H
NMR (DMSO-d6) d 1.45–2.06 (6H, m), 2.18 (3H, s),
2.20 (3H, s), 2.27 (6H, s), 2.39 (2H, m), 3.16 (2H, m),
3.30 (3H, m), 4.22 (1H, m), 4.47 (1H, m), 7.30–7.65 (8H,
m), 7.75 (1H, t), 8.02 (2H, d), 8.27 (2H, m), 8.30 (1H).
MS (ESI-NI) m/z 833.1 [MꢀH]ꢀ.
This compound was dissolved in methanol (10 mL) and
hydrogenolysed with Pd/C (0.1 g, 10%) at ambient
temperature and atmospheric pressure to give N2-[4-(N-
benzoyloxy-N-cyclohexyl-carbamoyl)-n-butanoyl]-l -

S. Wittmann et al. / Bioorg. Med. Chem. 10 (2002) 1659–1670
1667
lysine 30b as a white solid (yield 60%). Reacting 3a and
30b according to method B gave compound 31b as a
white solid (yield 50%). H NMR (DMSO-d6) d 1.00–
2.30 (18H, m), 2.19 (3H, s), 2.22 (3H, s), 2.27 (6H, s),
3.03 (2H, m), 3.15 (2H, m), 4.08 (1H, m), 4.32 (2H, m)
7.30–8.06 (13H, m), 8.23 (1H, d), 8.31 (1H, m).
(4H, m), 2.16 (3H, s), 2.21 (3H, s), 2.27 (6H, s), 3.17
(2H, m), 4.19 (1H,s), 4.62 (1H, m), 5.38 (1H, d), 5.52
(1H, q), 5.75 (1H, d), 7.20–7.75 (9H, m), 8.45 (1H, t),
8.52 (1H, d), 8.67 (1H, d), 9.16 (1H, d), MS (ESI-NI) m/
z 1060.5 [MꢀH]ꢀ.
1
0
0
N-[N2 ,N6 -Bis-(2,3-diacetoxybenzoyl)-L-lysyl]-amoxicillin
sodium salt (5a). This was produced from 3a and
Antibiotic conjugates
1
amoxicillin hydrate as a white solid (yield 46%). H
The ampicillin conjugates (X=Ap) 4a–d, 9a–c, 11a,b,
14a,b, 17a, 19a, 22, 25a,b, 28a,b, 32a,b, the amoxicillin
conjugates (X=Ax) 5a,b, 17b and 19b, the cephalexin
conjugates (X=Ce) 6a,b and the cefaclor conjugates
(X=Cf) 7a,b and 19c were prepared from the correspond-
ing siderophore analogue and the antibiotic according to
method B. The obtained residue was purified by pre-
parative HPLC (Knauer Vertex B 31 Y 536, 250ꢂ32 mm,
Eurospher 100 D18 7 mm, flux 20mL/min, acetonitrile/
water 37.5:62.5). The fraction containing the desired pro-
duct was evaporated and the residual aqueous solution
dried by lyophilization to give the conjugate.
NMR (DMSO-d6) d 1.32 (m, 2H), 1.39 (3H, s), 1.45 (2H,
m), 1.54 (3H, s), 1.70 (2H, m), 2.19 (3H, s), 2.21 (3H, s),
2.27 (3H, s), 2.28 (3H, s), 3.13 (2H, m), 4.18 (1H, s), 4.56
(1H, m), 5.38 (1H, d), 5.51 (1H, q), 5.58 (1H, d), 6.68
(2H, d), 7.19 (2H, d), 7.38 (5H, m), 7.50 (1H, dd), 8.29
(2H, m), 8.44 (1H, d), 9.01 (1H, d), 9.35 (1H, s). MS
(ESI-PI) m/z 955.0 [M+H]+.
0
0
N-[N2 ,N5 -Bis-(2,3-diacetoxybenzoyl)-D-ornithyl]-amoxi-
cillin (5b). This was produced from 3b and amoxicillin
hydrate as a white solid (yield 60%). 1H NMR (DMSO-
d6) d 1.39 (3H, s), 1.53 (3H, s), 1.45–1.90 (4H, m), 2.17
(6H, s), 2.21 (6H, s), 3.18 (2H, m), 4.18 (1H, s), 4.55
(1H, m), 5.38 (1H, d), 5.52 (2H, m), 6.66–7.50 (10H, m),
8.34 (2H, m), 8.40 (1H, d), 8.95 (1H, d), 9.35 (1H, s).
MS (FAB) m/z 920.1 [M+H]+.
The sodium salts were prepared from the solution of the
corresponding conjugate (1 mmol) in ethyl acetate
(5 mL) by addition of a solution of sodium 2-ethylhex-
anoate (1.5 mmol) in ethyl acetate (3 mL). Precipitation
was completed by addition of petroleum ether. The
precipitate was filtered, washed with petroleum ether
and dried in vacuo.
0
0
N-[N2 ,N6 -Bis-(2,3-diacetoxybenzoyl)-L-lysyl]-cephalexin
(6a). This was produced from 3a and cephalexin as a
1
white solid (yield 45%). H NMR (DMSO-d6): 1.20–
1.80 (6H, m), 1.96 (3H, s), 2.19 (6H, s), 2.27 (6H, s), 3.12
(2H, m), 3.41 (2H, dd), 4.56 (1H, m), 5.11 (1H, d), 5.65
(1H, d), 5.70 (1H, q), 7.24–7.60 (11H, m), 8.25 (1H, m),
8.29 (1H, d), 8.61 (1H, d), 9.30 (1H, d).
0
0
N-[N2 ,N6 -Bis-(2,3-diacetoxybenzoyl)-L-lysyl]-ampicillin
(4a). This was produced from 3a and ampicillin
hydrate as a white solid (yield 65%). 1H NMR (DMSO-
d6) d 1.32 (2H, m), 1.39 (1H, s), 1.45 (2H, m), 1.54
(1H, s), 1.70 (2H, m), 2.19 (3H, s), 2.21 (3H, s), 2.27
(3H, s), 2.28 (3H, s), 3.13 (2H, m), 3.83 (1H, s), 4.57
(1H, m), 5.24 (1H, d), 5.35 (1H, q), 5.76 (1H, d),
7.22–7.45 (9H, m), 7.51 (1H, dd), 8.29 (1H, t), 8.35
(1H, d), 8.57 and 9.00 (1H, d), MS (ESI-NI) m/z
916.3 [MꢀH]ꢀ.
0
0
N-[N2 ,N5 -Bis-(2,3-diacetoxybenzoyl)-D-ornithyl]-cepha-
lexin (6b). This was prepared from 3b and cephalexin
as a white solid (yield 45%). 1H NMR (DMSO-d6):
1.50–1.90 (4H, m), 1.98 (3H,s), 2.22 (6H, s), 2.27 (6H, s),
3.22 (2H, m), 3.30 (2H, dd), 4.58 (1H, m), 4.96 (1H, d),
5.63 (1H, q), 5.67 (1H, d), 7.25–7.52 (11H, m), 8.35 (1H,
m), 8.40 (1H, d), 8.55 (1H, d), 9.28 (1H, d). MS (ES-)
900.3 [MꢀH]ꢀ.
0
0
N-[N2 ,N5 -Bis-(2,3-diacetoxybenzoyl)-D-ornithyl]-ampi-
cillin (4b). This was produced from 3b and ampicillin
hydrate as a white solid (yield 60%). 1H NMR (DMSO-
d6) d 1.39 (3H, s), 1.53 (3H, s), 1.45–1.90 (4H, m), 2.17
(3H, s), 2.18 (3H, s), 2.27 (6H, s), 3.18 (2H, m), 4.19 (1H,
s), 4.58 (1H, m), 5.38 (1H, d), 5.52 (1H, q), 5.69 (1H, d),
7.25–7.50 (11H, m), 8.33 (1H, m), 8.38 (1H, d), 8.55 (1H,
d), 9.12 (1H, d). MS (FAB) m/z 904.1 [M+H]+.
0
0
N-[N2 ,N6 -Bis-(2,3-diacetoxybenzoyl)-L-lysyl]-cefaclor
(7a). This was prepared from 3a and cefaclor as a
1
white solid (yield 40%). H NMR (DMSO-d6) d 1.20–
1.80 (6H, m), 2.19 (6H, s), 2.21 (6H, s), 2.27 (6H, s), 3.12
(2H, m), 3.71 (2H, dd), 4.56 (1H, m), 5.10 (1H, d), 5.67
(1H, d), 5.73 (1H, q), 7.25–7.58 (11H, m), 8.31 (1H, m),
8.32 (1H, d), 8.65 (1H, d), 9.44 (1H, d).
0
0
N-[N2 ,N5 -Bis-(2,3-diacetoxybenzoyl-L-ornithyl]-ampicil-
lin (4c). This was produced from 3c and ampicillin
hydrate as a white solid (yield 60%). 1H NMR (DMSO-
d6) d 1.40 (3H, s), 1.55 (3H, s), 1.45–1.90 (m, 4H), 2.22
(3H, s), 2.23 (3H, s), 2.27 (6H, s), 3.18 (2H, m), 4.20
(1H, s), 4.65 (1H, m), 5.39 (1H, d), 5.52 (1H, q), 5.76
(1H, d), 7.20–7.60 (11H, m), 8.32 (2H, m), 8.64 (1H, d),
9.22 (1H, d). MS (ESI-NI) m/z 902.7 [MꢀH]ꢀ.
0
0
N-[N2 ,N5 -Bis-(2,3-diacetoxybenzoyl)-D-ornithyl]-cefa-
clor (7b). This was produced from 3b and cefaclor as a
1
white solid (yield 40%). H NMR (DMSO-d6) d 1.50–
1.90 (m, 4H), 2.22 (6H, s), 2.27 (6H, s), 3.22 (2H, m),
3.65 (2H, dd), 4.56 (1H, m), 5.09 (1H, d), 5.62 (1H, d),
5.73 (1H, q), 7.25–7.52 (11H, m), 8.33 (1H, m), 8.37
(1H, d), 8.55 (1H, d), 9.38 (1H, d). MS (ESI-NI) m/z
919.9 [MꢀH]ꢀ.
0
0
N-[N2 ,N5 -Bis-(5-bromo-2,3-diacetoxybenzoyl)-L-orni-
thyl]-ampicillin (4d). This was produced from 3d and
ampicillin hydrate as a white solid (yield 40%). 1H
NMR (DMSO-d6) d 1.39 (3H, s), 1.53 (3H, s), 1.45–1.90
N-[2L,6-Bis-(8-methoxycarbonyloxy-3,4-dihydro-2,4-dioxo-
1,3-benzoxazin-3-yl)-n-hexanoyl]-ampicillin (9a). This was
produced from 8a and ampicillin hydrate as a white

1668
S. Wittmann et al. / Bioorg. Med. Chem. 10 (2002) 1659–1670
1
solid, yield 34%. H NMR (DMSO-d6) d 1.40 (3H, s),
1.53 (3H, s), 1.58 (2H, m), 2.01 (1H, m), 2.28 (1H, m),
3.79 (2H, m), 3.89 (3H, s), 3.91 (3H, s), 4.19 (1H, s), 5.20
(1H, m), 5.39 (1H, d), 5.50 (1H, q), 5.76 (1H, d), 7.20–
7.50 (7H, m), 7.76 (2H, m), 7.84 (2H, m), 8.79 (1H, d),
8.93 (1H, d). MS (ESI-NI) m/z 916.8 [MꢀH]ꢀ.
hydrate as a white solid (yield 70%). 1H NMR (DMSO-
d6) d 1.37 (3H, s), 1.50 (3H, s), 1.30–1.85 (12H, m),
2.19 (3H, s), 2.20 (3H, s), 2.22 (3H, s), 2.27 (9H, s),
3.15 (4H, m), 3.81 (1H, s), 4.32 (1H, m), 4.54 (1H,
m), 5.23 (1H, m), 5.34 (1H, m), 5.76 (1H, m), 7.20–7.55
(14H, m), 7.87 (1H, d), 8.22 (1H, d), 8.32 (2H, m), 8.57
(1H, d), 8.89 (1H, d). MS (ESI-PI) m/z 1288.5
[M+H]+.
N-[2D,5-Bis-(8-methoxycarbonyloxy-3,4-dihydro-2,4-di-
oxo-1,3-benzoxazin-3-yl)-n-pentanoyl]-ampicillin (9b). This
was produced from 8b and ampicillin hydrate as a white
0
00
00
0
N-{N2 -[N2 ,N6 -Bis-(2,3-diacetoxybenzoyl)-L-lysyl]-N6 -
(2,3-diacetoxybenzoyl)-L-lysyl}-ampicillin sodium salt
(17a). This was produced from 16 and ampicillin
hydrate as a white solid (yield 70%). 1H NMR (DMSO-
d6) d 1.38 (3H, s), 1.51 (3H, s), 1.20–1.80 (12H, m), 2.21
(9H, s), 2.29 (9H, s), 3.13 (4H, m), 3.80 (1H, s), 4.43
(2H, m), 5.23 (1H, m), 5.34 (1H, m), 5.75 (1H, m), 7.20–
7.55 (14H, m), 8.00–9.00 (6H, m). MS (ESI-PI) m/z
1310.5 [M+2Na]+.
1
solid (yield 60%). H NMR (DMSO-d6) d 1.41 (3H, s),
1.54 (3H, s), 1.64 (2H, m), 1.98–2.30 (2H, m), 3.84 (2H,
m), 3.90 (3H, s), 3.91 (3H, s), 4.20 (1H, s), 5.25 (1H, m),
5.41 (1H, d), 5.52 (1H, q), 5.77 (1H, d), 7.20–7.90 (11H,
m), 8.83 (1H, d), 9.07 (1H, d). MS (ESI-NI) m/z 902.5
[MꢀH]ꢀ.
N-[2L,5-Bis-(8-methoxycarbonyloxy-3,4-dihydro-2,4-dioxo-
1,3-benzoxazin-3-yl)-n-pentanoyl]-ampicillin (9c). This was
produced from 8c and ampicillin hydrate as a white
0
00
00
0
N-{N2 -[N2 ,N6 -Bis-(2,3-diacetoxybenzoyl)-L-lysyl]-N6 -
(2,3-diacetoxybenzoyl)-L-lysyl}-amoxicillin sodium salt
(17b). This was produced from 16 and amoxicillin
hydrate as a white solid (yield 50%). 1H NMR (DMSO-
d6) d 1.38 (3H, s), 1.50 (3H, s), 1.20–1.80 (12H, m), 2.21
(9H, s), 2.29 (9H, s), 3.13 (4H, m), 3.83 (1H, s); 4.44
(2H, m), 5.25 (1H, m), 5.34 (1H, m), 5.75 (1H, m), 6.65–
7.55 (13H, m), 8.00–9.00 (6H, m). MS (ESI-PI) m/z
1304.6 [M+Na]+.
1
solid (yield 60%). H NMR (DMSO-d6) d 1.40 (3H, s),
1.52 (3H, s), 1.64 (2H, m), 1.98–2.30 (2H, m), 3.84 (2H,
m), 3.89 (3H, s), 3.91 (3H, s), 4.19 (1H, s), 5.27 (1H, m),
5.37 (1H,d), 5.48 (1H, q), 5.74 (1H, d), 7.25–7.95 (11H,
m), 8.72 (1H, d), 8.90 (1H, d). MS (ESI-NI) m/z 901.9
[MꢀH]ꢀ.
N-{N2-[Bis-N2‘,N5‘-(2,3-diacetoxybenzoyl)-L-ornithyl]-L-
phenylalanyl}-ampicillin 11a. This was prepared from
10a and ampicillin hydrate as a white solid (Yield 30%).
1H NMR (DMSO-d6) d 1.40 (s, 3H), 1.55 (s, 3H), 1.90–
1.50 (m, 4H), 2.18 (s, 3H), 2.19 (s, 3H), 2.27 (s, 6H), 2.99
(m, 2H), 3.17 (m, 2H), 4.18 (s, 1H), 4.45 (m, 1H), 4.82
(m, 1H), 5.37 (d, 1H), 5.52 (q, 1H), 5.78 (d, 1H), 7.50–
7.10 (m, 16H), 8.10 (d, 1H), 8.21 (t, 1H), 8.30 (d, 1H),
8.85 (d, 1H), 9.22 (d, 1H). MS (ESI-NI) m/z 1049.5
[MꢀH]ꢀ.
N-{N2-[N2 ,N5 -(Bis-(2,3-diacetoxybenzoyl)-L-ornithyl]-
L-tryptophanyl}-ampicillin 11b. This was produced from
10b and ampicillin hydrate as a white solid (yield 60%).
1H NMR (DMSO-d6) d 1.39 (s, 3H), 1.54 (s, 3H), 1.90–
1.50 (m, 4H), 2.16 (s, 3H), 2.17 (s, 3H), 2.27 (s, 6H), 3.18
(m, 2H), 3.35 (m, 2H), 4.18 (s, 1H), 4.44 (m, 1H), 4.81
(m, 1H), 5.36 (d, 1H), 5.49 (q, 1H), 5.72 (d, 1H), 7.60–
6.85 (m, 16H), 8.07 (d, 1H), 8.32 (m, 2H), 8.71 (d, 1H),
9.20 (d, 1H), 10.78 (s, 1H). MS (ESI-NI) m/z 1088.4
[MꢀH]ꢀ.
N-{2L-[20 L,60 -Bis-(8-methoxycarbonyloxy-3,4-dihydro-
2,4-dioxo-1,3-benzoxazin-3-yl)-n-hexanoylamino]-6-(8-
methoxycarbonyloxy-3,4-dihydro-2,4-dioxo-1,3-benzoxa-
zin-3-yl)-n-hexanoyl}-ampicillin sodium salt (19a). This
was produced from 18 and ampicillin hydrate as a white
1
solid (yield 60%). H NMR (DMSO-d6) d 1.40 (3H, s),
1.52 (3H, s), 1.20–1.70 (12H, m), 3.80 (4H, m), 3.83 (3H,
s), 3.89 (6H, s), 4.18 (1H, s), 4.37 (1H, m), 5.09 (1H, m),
5.37 (1H,d), 5.47 (1H, q), 5.70 (1H, d), 7.20–7.90 (14H,
m), 8.25 (1H, d), 8.93 (1H, d), 9.13 (1H, d). MS (ESI-
NI) m/z 1264.5 [M–H]ꢀ.
00
00
N-{2L-[20L,60-Bis-[8-methoxycarbonyloxy-3,4-dihydro-2,4
-dioxo-1,3-benzoxazin-3-yl)-n-hexanoylamino]-6-(8-meth-
oxycarbonyloxy-3,4-dihydro-2,4-dioxo-1,3-benzoxazin-3-
yl)-n-hexanoyl}-amoxicillin (19b). This was produced
from 18 and amoxicillin hydrate as a white solid (Yield
50%). 1H NMR (DMSO-d6) d 1.50 (3H, s), 1.40 (3H, s),
1.25–1.80 (12H, m), 3.75 (4H, m), 3.80 (3H, s), 3.85 (6H,
s), 4.15 (1H, s), 4.35 (1H, m), 5.00 (1H, m), 5.35 (1H, d),
5.40 (1H, q), 5.45 (1H, d), 7.15–7.85 (13H, m), 8.25 (1H,
d), 8.50 (1H, d), 9.00 (1H, d), 9.35 (1H, s). MS (ESI-NI)
m/z 1279.7 [MꢀH]ꢀ.
0
00
00
N-0{N5 -[N2 ,N5 -Bis-(2,3-diacetoxybenzoyl)-L-ornithyl]-
N2 -(2,3-diacetoxybenzoyl)-L-ornithyl}-ampicillin (14a).
This was produced from 13a and ampicillin hydrate as
1
a white solid (yield 50%). H NMR (DMSO-d6) d 1.39
0
(3H, s), 1.54 (3H, s), 1.85–1.40 (8H, m), 2.17 (3H, s), 2.21
(6H, s), 2.27 (9H, s), 3.10 (4H, m), 4.,19 (1H, s), 4.37 (1H,
m), 4.62 (1H, m), 5.37 (1H, d), 5.50 (1H, q), 5.74 (1H, d),
7.20–7.50 (14H, m), 8.22 (1H, d), 8.32 (2H, m), 8.63
(1H, d), 8.93 (1H, m), 9.19 (1H, d). MS (ESI-NI) m/z
1236.0 [MꢀH]ꢀ.
N-{N6 -[N2 ,N6 -Bis-(2,3-diacetoxybenzoyl)-L-lysyl]-N2 -
(2,3-diacetoxybenzoyl)-L-lysyl}-ampicillin sodium salt
(14b). This was produced from 13b and ampicillin
N-{2L-[2L ,60-Bis-[8-methoxycarbonyloxy-3,4-dihydro-2,4
-dioxo-1,3-benzoxazin-3-yl)-n-hexanoylamino]-6-(8-meth-
oxycarbonyloxy-3,4-dihydro-2,4-dioxo-1,3-benzoxazin-3-
yl)-n-hexanoyl}-cefaclor (19c). This was produced from
1
18 and cefaclor as a white solid (yield 66%). H NMR
(DMSO-d6) d 1.20–1.90 (14H, m), 3.70 (4H, m), 3.80
(9H, s), 4.35 (1H, m), 5.00 (1H, m), 5.10 (1H, d), 5.61
(1H, d), 5.70 (1H, d), 7.27–7.86 (14H, m), 8.25 (1H, d),
8.64 (1H, d), 9.36 (1H,d). MS (ESI-NI) m/z 1282.1
[MꢀH]ꢀ.
0
00
00
0

S. Wittmann et al. / Bioorg. Med. Chem. 10 (2002) 1659–1670
1669
N-{N2-["-(8-Methoxycarbonyloxy-3,4-dihydro-2,4-dioxo
-1,3-benzoxazin-3-yl)-n-hexanoyl]-N6-[2L,6-bis-(8-meth-
oxycarbonyloxy-3,4-dihydro-2,4-dioxo-1,3-benzoxazin-3-
yl)-n-hexanoyl]-L-lysyl}-ampicillin (22). This was pro-
duced from 21 and ampicillin hydrate as a white solid
(18H, m), 2.19 (3H, s), 2.22 (3H, s), 2.27 (6H, s), 3.10
(4H, m), 4.19 (1H, s), 4.33 (2H, m), 5.37 (1H, d), 5.50
(1H, q), 5.68 (1H, d), 7.30–8.05 (18H, m), 8.20 (1H, d),
8.29 (1H, m), 8.40 (1H, m), 9.10 (1H, m). MS (ESI-NI)
m/z 1290.9 [MꢀH]ꢀ.
1
(yield 30%). H NMR (DMSO-d6) d 1.39 (3H, s), 1.53
(3H, s), 1.00–2.30 (20H, m), 2.97 (2H, m), 3.79 (4H, m),
3.90 (9H, s), 4.19 (1H, s), 4.25 (1H, m), 5.07 (1H, m),
5.37 (1H, d), 5.50 (1H, q), 5.65 (1H, m), 7.20–8.02 (16H,
m), 8.45 (1H, m), 9.10 (1H, m). MS (ESI-NI) m/z 1377.4
[MꢀH]ꢀ.
N-{2-{[N2,N6-Bis-(2,3-diacetoxybenzoyl)-L-lysyl]-amino}-
4-[(N-benzoyloxy-N-methyl)-carbamoyl]-L-n-butanoyl}-
ampicillin sodium salt (25a). This was produced from
24a and ampicillin hydrate as a white solid (yield 20%).
1H NMR (DMSO-d6) d 1.37 (3H, s), 1.50 (3H, s), 1.45–
2.06 (8H, m), 2.19 (3H, s), 2.21 (3H, s), 2.27 (6H, s), 2.39
(2H, m), 3.15 (2H, m), 3.33 (3H, m), 3.81 (1H, s), 4.38
(1H, m), 4.47 (1H, m), 5.23 (1H, d), 5.33 (1H, q), 5.75
(1H, d), 7.25–7.60 (13H, m), 7.72 (1H, t), 7.98 (2H, d),
8.12 (1H, m), 8.35 (2H, m), 8.56 (1H, d), 9.02 (1H, d).
MS (FAB) m/z 1224.5 [M+H]+.
N-{N2-[4-(N-Benzoyloxy-N-cyclohexyl)-carbamoyl)-n-
0
0
butanoyl]-N6-(N2 ,N6 -bis-2,3-diacetoxybenzoyl)-L-lysyl]-
L-lysyl}-ampicillin (32b). This was produced from 31b
1
and ampicillin hydrate as a white solid (yield 50%). H
NMR (DMSO-d6) d 1.32 (3H, s), 1.54 (3H, s), 1.00–2.30
(28H, m), 2.19 (3H, s), 2.22 (3H, s), 2.27 (6H, s), 3.03
(2H, m), 3.15 (2H, m), 4.33 (3H, m), 5.37 (1H, d), 5.51
(1H, q), 5.68 (1H, m), 7.20–7.95 (16H, m), 8.04 (2H, d),
8.29 (1H, m), 8.45 (1H, m), 9.10 (1H, m). MS (ESI-PI)
m/z 1383.7 [M+Na]+.
Biology
Siderophore activity. The siderophore activity of the
compounds 3a–d, 8a–c, 10a,b, 13a,b, 16, 18, 21, 23a,b,
24a,b, 27a,b, and 31a,b was tested under iron limitation
in growth promotion assays24,25 on agar diffusion plates
using the following Gram-negative bacterial strains: P.
aeruginosa ATCC 27853, SG 137, NCTC 10662 and
ATCC 9027, E. coli ATCC 25922, and S. typhimurium
N-{2-{[N2,N5 -Bis-(2,3-diacetoxybenzoyl)-D -ornithyl]-
amino}-4-[(N-benzoyloxy-N-methyl)-carbamoyl]-L-n-bu-
tanoyl}-ampicillin (25b). This was produced from 24b
1
and ampicillin hydrate as a white solid (yield 40%). H
Table 3. Susceptibility of E. coli—mutants depending on outer
membrane siderophore receptors and on tonB
NMR (DMSO-d6) d 1.39 (3H, s), 1.53 (3H, s), 1.45–2.06
(6H, m), 2.18 (6H, s), 2.27 (6H, s), 2.39 (2H, m), 3.15
(2H, m), 3.26 (3H, m), 4.18 (1H, s), 4.49 (2H, m), 5.35
(1H, d), 5.49 (1H, q), 5.73 (1H, d), 7.20–7.45 (11H, m),
7.54 (2H, t), 7.75 (1H, t), 8.00 (2H, d), 8.21 (2H, d), 8.32
(1H, m), 8.65 (1H, d), 9.18 (1H, d), MS (ESI-NI) m/z
1163.7 [MꢀH]ꢀ.
N-{2-[N2,N6-Bis-(2,3-diacetoxybenzoyl)-L-lysyl]-amino}-
4-[(N-benzoyloxy-N-cyclohexyl)-carbamoyl]-L-n-buta-
noyl}-ampicillin (28a). This was produced from 27a and
ampicillin hydrate as a white solid (yield 50%). 1H
NMR (DMSO-d6) d 1.39 (3H, s), 1.53 (3H, s), 1.00–2.30
(18H, m), 2.19 (3H, s), 2.22 (3H, s), 2.27 (6H, s), 3.14
(2H, m), 4.18 (1H, s), 4.20 (1H, m), 4.26 (1H, m), 4.40
(1H, m), 5.36 (1H, d), 5.48 (1H, q), 5.72 (1H, d), 7.20–
8.03 (16H, m), 8.08 (1H, d), 8.30 (2H, m), 8.53 (1H, d),
9.18 (1H, d). MS (ESI-PI) m/z 1270.5 [M+Na]+.
H1443 H1876 H873 H1877 H1875 AB2847 BR158
TonB
FepA
Cir
+
ꢀ
+
+
+
ꢀ
ꢀ
ꢀ
ꢀ
+
+
ꢀ
+
ꢀ
ꢀ
ꢀ
Fiu
+
Azlocillin 16
Ampicillin 23
16
17
23
22
21.5
28
23
22.5
20.5
22
14
21
16
22
22
17
16
21
22
21
20
24
23.5
20
20
24
21
19
20
21
21
0
24
22
13
14
14
13
10
14
15
16
11
0
14
13
13.5
14.5
13
14.5
13
14
10
10
12
10
12
14.5
14
12
14
13
Cefaclor
4a
4b
4c
4d
22
23
24
24
0
19
23
14
14
0
12
0
14.5
0
0
13.5
12.5
12
15
11.5
12.5
10.5
12.5
0
10
12
18
12
14
0
20.5
19.5
15
18
13
20
17.5
13
20.5
17.5
25
19.5
18.5
21
23
22
17
17
22.5
23.5
19.5
20
14
19
17.5
19
22
20
28
23.5
20
23.5
26.5
25
18
21
18.5
21
24
25
21
23
22.5
21
24
5a
5b
23.5
21
18
6b
7a
7b
9a
9b
9c
11a
11b
14a
14b
17a
17b
19a
19b
19c
22
13
13.5
19.5
20.5
18.5
23
20.5
27
26
20
23
23
22
18.5
19
19
0
25
27
22
24
21
14
21.5
21.5
17.5
23.5
20.5
29.5
26
21
24.5
28
21
19.5
22.5
20.5
29
25
21.5
24
25
23.5
19
20
20
22.5
26
N-{2-{[N2,N5 -Bis-(2,3-diacetoxybenzoyl)-D -ornithyl]-
amino}-4-[(N-benzoyloxy-N-cyclohexyl)-carbamoyl]-L-n-
butanoyl}-ampicillin (28b). This was produced from 27b
1
and ampicillin hydrate as a white solid (yield 50%). H
NMR (DMSO-d6) d 1.38 (3H, s, 1.53 (3H, s), 1.00–2.30
(18H, m), 2.18 (6H, s), 2.27 (6H, s), 3.15 (2H, m), 4.18
(1H, s), 4.20 (1H, m), 4.48 (2H, m), 5.35 (1H, d), 5.48
(1H, q), 5.74 (1H, d), 7.22–8.05 (16H, m), 8.22 (2H, d),
8.32 (1H, m), NH); 8.65 (1H, d), 9.19 (1H, d). MS (ESI-
PI) m/z 1256.4 [M+Na]+.
27
20.5
21.5
20.5
20
18.5
19
20
22.5
17
21
16
25a
25b
28b
28a
32a
32b
27
26.5
22.5
25
23.5
22.5
26
22
25
21.5
21
N-{N2-[4-(N0-Benzoyloxy-N-methyl)-carbamoyl)-n-buta-
0
12.5
0
0
noyl]-N6-(N2 ,N6 -bis-2,3-diacetoxybenzoyl)-L-lysyl]-L-ly-
12
syl}-ampicillin (32a). This was produced from 31a and
ampicillin hydrate as a white solid (yield 30%). 1H
NMR (DMSO-d6) d 1.39 (3H, s), 1.53 (3H, s), 1.20–2.40
Application of 5 mg/9 mm agar well.
Diameter of inhibition zones (mm).

1670
S. Wittmann et al. / Bioorg. Med. Chem. 10 (2002) 1659–1670
enb7 (blocked in enterobactin biosynthesis). Side-
rophore solutions were applied on filter discs (O 6 mm)
on the surface of the inocu•ated test agar plates. After
incubation for 18–20 h at 37 ꢁC, the zones of growth
surrounding the discs were determined. Desferal was
used as a control.
3. Winkelmann, G., van Helm, D., Neilands, J.B., Eds.; Iron
Transport in Microbes, Plants and Animals; Chemie-Verlags-
gesellschaft: Weinheim, 1987.
4. Drechsel, H.; Jung, G. J. Peptide Sci. 1998, 4, 147.
5. O’Brien, I. G.; Gibson, F. Biochim. Biophys. Acta 1970,
215, 393.
6. Pollack, J. R.; Neilands, J. B. Biochim. Biophys. Res. Com-
mun. 1970, 38, 989.
Antibacterialactivity. Minimal inhibititory concentra-
tions (MIC) of the ampicillin conjugates (X=Ap) 4a–d,
9a–c, 11a,b, 14a,b, 17a, 19a, 22, 25a,b, 28a,b, 32a,b, the
amoxicillin conjugates (X=Ax) 5a,b, 17b and 19b, the
cephalexin conjugates (X=Ce) 6a,b and the cefaclor
conjugates (X=Cf) 7a,b and 19c were determined by
the micro broth dilution method in Mueller-Hinton
broth (Difco) according to the NCCLS guidelines26
(Table 2). Test organisms were from Culture Collections
(ATCC, NCTC) or from the stock of the Hans-Knoll-
Institute (SG). S. maltophilia GN 12873 was kindly
provided by Prof. S. Mitsuhashi, Episome Institute,
Gunma (Japan).27
7. Corbin, J. L.; Bulen, W. A. Biochemistry 1969, 8, 757.
8. McKee, J. A.; Sharma, S. K.; Miller, M. J. Bioconjugate
Chem. 1991, 2, 281.
9. Ito, T.; Neilands, J. B. Amer. Chem. Soc. 1958, 80,
4645.
10. Kanai, F.; Kaneko, T.; Morishima, H.; Isshiki, K.;
Takita, T.; Takeuchi, T.; Umezawa, H. J. Antibiot. 1985, 38,
39.
11. Chimiak, A.; Neilands, J. B. Structure Bonding 1984, 58,
89.
12. Schnabelrauch, M.; Heinisch, L.; Wittmann, S.; Reiss-
brodt, R.; Mollmann, U. BioMetals 2000, 13, 333.
13. Arisawa, M.; Sekine, Y.; Shimizu, S.; Takano, H.;
Angehrn, P.; Then, R. L. Antimicrob. Agents Chemother. 1991,
35, 653.
14. Ghosh, A.; Ghosh, M.; Niu, C.; Malouin, F.; Mollmann,
U.; Miller, M. J. Chem. Biol. 1996, 3, 1011.
15. Mollmann, U.; Ghosh, A.; Dolence, E. K.; Dolence, J. A.;
Ghosh, M.; Miller, M. J.; Reissbrodt, R. BioMetals 1998, 11,
1.
16. Wittmann, S.; Scherlitz-Hofmann, I.; Mollmann, U.;
Ankel-Fuchs, D.; Heinisch, L. Arzneim. Forsch/Drug Res.
2000, 50, 752.
To investigate the mode of action of the conjugates the
antibacterial activity was tested in an agar diffusion
assay against E. coli strains deficient in siderophore
receptors28 or in the periplasmic TonB protein, which is
essential for energy transfer in active iron transport
systems. Results were read as inhibition zone diameters
(Table 3).
17. Heinisch, L.; Wittmann, S.; Mollmann, U.; Reiss-
brodt, R. EP 0,863,139-B1, 2001; Chem. Abstr. 1998, 129,
230578.
Acknowledgements
18. Heinisch, L.; Mollmann, U.; Schnabelrauch, M.; Reiss-
brodt, R. WO 9749670, 1997; Chem. Abstr. 1998, 128, 101912.
19. Tsafack, A.; Libman, J.; Shanzer, A.; Cabantchik, Z. J.
Antimicrob. Agents Chemother. 1996, 40, 2160.
20. Miller, J. M. Chem. Rev. 1989, 89, 1563.
21. Bergeron, R. J.; McGovern, K. A.; Channing, M. A.;
Burton, P. S. J. Org. Chem. 1980, 45, 1589.
22. Geffken, D. Chem. Ber. 1986, 119, 744.
23. Psiorz, M.; Zinner, G. Synthesis 1984, 3, 217.
24. Lin, Y.-M.; Miller, M. J.; Mollmann, U. BioMetals 2001,
14, 153.
The financial support of Bundesministerium fur Bil-
dung, Wissenschaft, Forschung und Technologie (Ber-
lin, Germany, FZ 0311232) is gratefully acknowledged.
We thank Antje Fritzsche and Silke Leonhardt for
technical assistance on the synthesis, Andrea Perner and
Renate Koch for mass spectra, Heike Heinecke for
NMR spectra, Irmgard Heinemann and Monika
Golembiewski for biological testing.
25. Schumann, G.; Mollmann, U. Antimicrob. Agents Che-
mother. 2001, 45, 1317.
26. National Committee for Clinical Laboratory Standards.
Methods for Dilution Antimicrobial Susceptibility Tests for
Bacteria that Grow Aerobically. Approved standard M7-A5.
NCCLS: Villanova, PA, 2000.
27. Sumita, Y.; Inoue, M.; Mitsuhashi, S. Eur. J. Clin.
Microb. Infect. Dis. 1998, 8, 908.
28. Hantke, K. FEMS Microbiol. Lett. 1990, 67, 5.
References and Notes
1. Miller, M.; Malouin, F. In The Development of Iron Chela-
tors for Clinical Use; Bergeron, B.J., Ed.; CRC: Boca Raton,
FL, 1994; p 275.
2. Munoz-Bellido, J.-L.; Garcia Rodriguez, J. A. Infect. Dis.
Clin. Practise 1996, 5, 232.