Highly antibacterial active aminoacyl penicillin conjugates with acylated bis-catecholate siderophores based on secondary diamino acids and related compounds

Article abstract of DOI:10.1021/jm010546b

New acylated bis-catecholates and 1,3-benzoxazine-2,4-dione derivatives based on secondary diamino acids (N-(aminoalkyl)glycines, N-aminopropyl-alanine, and N-aminopropyl-4-amino-valeric acid), on N-(aminoalkyl)aminomethyl benzoic acids, on N-(aminoalkyl)

Full text of DOI:10.1021/jm010546b

3032
J . Med. Chem. 2002, 45, 3032-3040
High ly An tiba cter ia l Active Am in oa cyl P en icillin Con ju ga tes w ith Acyla ted
Bis-Ca tech ola te Sid er op h or es Ba sed on Secon d a r y Dia m in o Acid s a n d Rela ted
Com p ou n d s
Lothar Heinisch,*,† Steffen Wittmann,†,‡ Thomas Stoiber,†,§ Albrecht Berg,† Dorothe Ankel-Fuchs,| and
Ute Mo¨llmann†
Hans Kno¨ll-Institute for Natural Products Research, J ena, Germany, and Gru¨nenthal GmbH,
Division of Anti-Infectives/ New IndicationsAachen, Germany
Received December 3, 2001
New acylated bis-catecholates and 1,3-benzoxazine-2,4-dione derivatives based on secondary
diamino acids (N-(aminoalkyl)glycines, N-aminopropyl-alanine, and N-aminopropyl-4-amino-
valeric acid), on N-(aminoalkyl)aminomethyl benzoic acids, on N-(aminoalkyl)aminomethyl
phenoxyacetic acids, or on 3,5-diaminobenzoic acid were synthesized as artificial siderophores.
The corresponding diamino acids were obtained from the diamines and oxocarboxylic acids by
catalytic hydrogenation. The acylated bis-catecholates and 1,3-benzoxazine-2,4-diones were
coupled with ampicillin or amoxicillin to new siderophore aminoacylpenicillin conjugates. These
conjugates exhibited very strong antibacterial activity in vitro against Gram-negative bacterial
pathogens including Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Escherichia coli,
Klebsiella pneumoniae, and Serratia marcescens. The ampicillin derivative 7b (HKI 9924154)
and the corresponding amoxicillin derivative 8 (HKI 9924155) represent the most active
compounds. The conjugates can use bacterial iron siderophore uptake routes to penetrate the
Gram-negative outer membrane permeability barrier. This was demonstrated by assays with
mutants deficient in components of the iron transport systems. New siderophore penicillin V
conjugates with the siderophore component attached to the phenyl ring of penicillin V are
inactive against these Gram-negative bacteria.
In tr od u ction
biotic conjugates with one catecholate moiety and
enhanced in vitro antibacterial activities have been pub-
lished.9,10 Ampicillin conjugates with natural pyoverdins
were specifically active only against that Pseudomonas
strain producing the corresponding pyoverdin.11 Pyover-
din-quinolone adducts were synthesized, too.12 â-Lac-
tam conjugates with a series of different noncatecholate
siderophore structures seem to act independently of iron
transport systems and can reach activities near that of
the cephalosporin moiety.13
In this paper, we report on the synthesis of N-(amino-
alkyl)amino acids from diamines and oxocarboxylic acids
by catalytic hydrogenation, on the acylation of these
compounds or of 3,5-diaminobenzoic acid to acylated bis-
catecholate and 1,3-benzoxazine-2,4-dione derivatives as
new artificial siderophores and on their condensation
with aminoacyl penicillins and on the investigation of
the biological activities of the siderophores and their
conjugates with ampicillin and amoxicillin. We used
acylated catecholates and the corresponding benz-
oxazine derivatives as siderophore components to fa-
cilitate the synthesis and to decrease pharmacological
side effects in comparison to free catecholates.
The outer membrane permeability barrier of bacteria
is one important reason for â-lactam antibiotic resist-
ance of Gram-negative bacterial pathogens. This applies
especially to Pseudomonas aeruginosa and Stenotropho-
monas maltophilia strains. To overcome this membrane-
mediated resistance, siderophore structures covalently
bound to the antibiotic can function as a shuttle for
active transport into the bacterial cell. Siderophores are
microbial iron chelators excreted to sequester extra-
cellular ferric ions under iron starvation conditions such
as found in infected tissues. Specific outer membrane
receptors recognize the siderophore iron complex and
initiate its active transport into the cell. Many natural
siderophores contain two or three catecholate groups as
chelating ligands based on di- or triamines.1 Examples
are bis-catecholate derivatives of spermidine2 or of
diamino acids such as N,N′-bis-(2,3-dihydroxybenzoyl)-
L-lysine.3 Artificial siderophores of these structures
based on spermidine or norspermidine were published.4-6
Recently, we synthesized artificial siderophores based
on amino acids or dipeptides7 and mono-1,3-benzox-
azine-2,4-dione derivatives as masked catecholates.
These compounds can act as siderophore components
in â-lactam conjugates.8 Examples of siderophore anti-
Resu lts a n d Discu ssion
Syn th esis of th e Sid er op h or es. As the backbone
for new artificial siderophores, we synthesized N-
(aminoalkyl)amino acids 3a -d ,o,p , N-(aminoalkyl)-
aminomethyl benzoic acids 3k ,l, and N-(aminoalkyl)-
aminomethyl phenoxyacetic acids 3m ,n from the di-
amines 1a -e and oxocarboxylic acids 2a -f by hydro-
* To whom correspondence should be addressed. Tel.: (+49)3641
656714. Fax: (+49)3641 656705. E-mail: Heinisch@pmail.hki-jena.de.
† Hans Kno¨ll-Institute for Natural Products Research.
‡ Present address: J enapharm GmbH & Co. KG, J ena, Germany.
§ Present address: Institute of Molecular Biotechnology, J ena,
Germany.
| Division of Anti-Infectives/New IndicationsAachen.
10.1021/jm010546b CCC: $22.00 © 2002 American Chemical Society
Published on Web 06/06/2002

Antibacterial Active Aminoacyl Penicillin Conjugates
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 14 3033
F igu r e 1. Syntheses of the siderophore components 5 and 6 and of the antibiotic conjugates 7-9; Ac ) COCH3; Moc ) COOCH3;
1a : n ) 0, R1 ) H; 1b: n ) 1, R1 ) H; 1c: n ) 1, R1 ) CH3; 1d : n ) 2, R1 ) H; 1e: n ) 3, R1 ) H; 2a : X ) CH; 2b: X ) C-CH3;
2c: X ) C-CH3-(CH2)2-; 2d : X ) CH-oC6H4; 2e: X ) CH-pC6H4-OCH2; 2f: X ) CH-oC6H4-OCH2; 4a : R2 ) 2,3-OAc, R3 ) H; 4b:
R2 ) 2,3-OMoc, R3 ) H; 4c: R2 ) 2,3-OMoc, R3 ) 5-Cl; 4d : R2 ) 2,3-OMoc, R3 ) 5-Br; 4e: R2 ) 2,3-OMoc, R3 ) 5,6-di-Cl; 4f: R2
) 3,4-OAc, R3 ) H; 6a , 9a : n ) 2, X ) CH2; 6b, 9b: n ) 1, X ) CHCH3; 6c, 9c: n ) 1, X ) CH(CH3)CH2CH2; other substituents,
see Table 1.
F igu r e 2. Structure of the aminoacyl penicillin components;
R ) siderophore components of Figures 1 and 4; R1 ) H:
ampicillin derivatives, Ap; R1 ) OH: amoxicillin derivative,
Ax.
genation catalyzed by palladium on active carbon. The
synthesis of N-(aminoethyl)glycine 3a by this procedure
was known.14 The intermediate in this reaction is
possibly an N-heterocyclic acid like hexahydro-pyrimi-
dine-2-carboxylic acid as a derivative of 1,3-diamino-n-
propane15 and not the corresponding azomethine, be-
F igu r e 3. Penicillin Vsderivatives 10a ,b, Ac ) COCH3.
1
cause no CHdN signal was found in H nuclear magnetic
resonance (NMR) spectra. Compounds 3 were purified
according to structure 5 were cyclized to structure 6 by
by codistillation with toluene to separate unreacted
reaction in acetonitrile. These compounds represent a
diamines or via the synthesis of CBZ derivatives with
heterocyclic acylated form of catecholates, which can be
benzyl chloroformate, separation from impurities by
transformed enzymatically into the free catecholates.
preparative high-performance liquid chromatography
(HPLC), and hydrogenolysis with Pd/C. The diamino
â-Lactam conjugates with one benzoxazindione moiety
showed high antibacterial activity.8 As a further di-
acids 3a -n were acylated with diacyloxybenzoyl chlo-
amino acid, we used 3,5-diaminobenzoic acid as the
backbone for siderophores, which were acylated with 4a
to compound 11 and with 4b , with [8-(methoxycar-
rides 4a -f in aqueous sodium hydrogen carbonate
solution to the bis-catecholate compounds 5a -n (Figure
1). The 4,5-dichloro-2,3-di-methoxycarbonyloxy-benzoyl
bonyloxy)-1,3-benzoxazine-2,4-dione-3-yl]acetyl chloride
chloride 4e was prepared analogously to 4b 13 from 4,5-
4g and the corresponding propionyl chloride 4h ,8 re-
dichloro-2,3-dihydroxybenzoic acid16 and methyl chlo-
spectively, to the compounds 13a -c (Figure 4).
roformate by reaction of the obtained 4,5-dichloro-2,3-
Syn th esis of th e â-La cta m Con ju ga tes. The com-
pounds 5a -m , 6a -c, 11, and 13a -c were coupled with
ampicillin or amoxicillin via their mixed anhydrides
with isobutyl chloroformate to the ampicillin conjugates
7a -m , 9a -c, 12, and 14a -c and to the amoxicillin
conjugates 8 and 15c (Figure 2). The bis-catecholates
5m ,n were also coupled with 6-aminopenicillanic acid
to give derivatives of penicillin V 10a (p-derivative) and
10b (o-derivative) (Figure 3, the structure of penicillin
V is marked). In these compounds, the siderophore
component is placed on the phenyl of the phenoxy-
di-methoxycarbonyloxy-benzoic acid with PCl5. Com-
pounds 5a -c,l-n and the corresponding â-lactam con-
jugates 7a -c,l,m , 8, and 10a ,b obviously exist in two
rotameric forms because two triplets for CONH were
found in the H NMR spectra, which change to singlets
at 360 K studied on compounds 5b,m and 5b and 10a ,
respectively.
The 1,3-benzoxazine-2,4-dione derivatives 6a -c were
synthesized from the diamino acids 3b,o, or p and 2,3-
dimethoxycarbonyloxybenzoyl chloride 4b (Figure 1).
Bis-2,3-di-(methoxycarbonyloxy)-benzoyl intermediates
1

3034 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 14
Heinisch et al.
F igu r e 4. Synthesis of 3,5-diaminobenzoic acid derivatives 11-15, Ac ) COCH3, Moc ) COOCH3; 13a and 14a : X ) direct
bond; 13b and 14b: X ) CH2CONH; 13c, 14c, and 15c: X ) CH2CH2CONH-.
Ta ble 1. Substituent Pattern of Compounds 3, 5, 7, 8, Ac )
COCH3, and Moc ) COOCH3
Ta ble 2. Growth Promotion of the Bis-catecholate Derivatives
5a -n , 6a -c, 11, and 13a -c of Gram-Negative Bacteria under
Iron Limitation (5 µL of a 2 mM Solution Was Applicated on a
6 mm Paper Disk) and the Results of CAS Assaya
compd
n
R1
R2
R3
X
3a
5a
7a
3b
5b
7b
8
3c
5c
7c
3d
5d
7d
5e
7e
5f
0
0
0
2
2
2
2
3
3
3
1
1
1
1
1
1
1
1
1
1
1
2
2
1
1
1
2
2
2
2
2
2
2
2
1
1
H
H
H
H
H
H
H
H
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
P. aeruginosa
E. coli
2,3-OAc
2,3-OAc
H
H
ATCC NCTC ATCC K799/ ATCC CAS
compd 27853 SG 137 10662 9027
WT
25922 assay
2,3-OAc
2,3-OAc
2,3-OAc
H
H
H
5b
5c
5d
5e
5f
5g
5h
5i
5k
5l
5m
5n
6a
6b
6c
11
20
30
19
15
25
22
25
25
20
22
22
18
18
15
13
0
26
28
15
9
26
25
30
10
19
21
19
nt
27
19
15
20
25
20
23
30
25
30
23
18
25
27
30
14
18
23
23
27
24
25
20
17
18
20
20
30
20
20
18
13
24
20
25
19
nt
25
25
22
25
25
27
25
20
17
15
12
25
23
22
14
nt
28
27
20
20
30
32
30
21
27
34
27
26
30
22
15
27
20
15
20
38
++
+++
+++
+
H
H
CH3
2,3-OAc
2,3-OAc
H
H
+
+
++
++
+
CH3 2,3-OAc
CH3 2,3-OAc
CH3 2,3-OMoc
CH3 2,3-OMoc
CH3 2,3-OMoc 5-Cl
CH3 2,3-OMoc 5-Cl
CH3 2,3-OMoc 5-Br
CH3 2,3-OMoc 5-Br
CH3 2,3-OMoc 5,6-di-Cl CH2
CH3 2,3-OMoc 5,6-di-Cl CH2
H
H
CH3
H
H
H
H
nt
+
25
26
nt
nt
nt
10
20
20
20
35
+
+
7f
+
5 g
7 g
5h
7h
5i
++
+
++
+
13a
13b
13c
desferal
15
15
18
35
nt
nt
nt
36
3,4-OAc
3,4-OAc
H
H
CH2
CH2
CH2-(o-C6H4)
CH2-(o-C6H4)
CH2-(o-C6H4)
CH2-(o-C6H4)
CH2-(o-C6H4)
CH2-(o-C6H4)
CH2-(p-C6H4)-OCH2
CH2-(p-C6H4)-OCH2
CH2-(p-C6H4)-OCH2
CH2-(o-C6H4)-OCH2
CH2-(o-C6H4)-OCH2
CH3CH
(+)
(+)
7i
3k
5k
7k
3l
5l
7l
3m
5m
7m
3n
5n
3o
3p
CH3 2,3-OAc
CH3 2,3-OAc
H
H
H
a
nt ) not tested.
H
H
H
H
H
H
H
H
H
2,3-OAc
2,3-OAc
H
H
activity for some of the strains. The acylated cate-
cholates active as siderophores obviously were trans-
formed to free catecholates in bacterial cultures.
Ir on -Com p lexin g Ca p a city. All bis-catecholates
showed a positive chromazurol-S (CAS) reaction (Table
2), which is associated with iron chelation, a basic
requirement for siderophore activity.
2,3-OAc
2,3-OAc
H
H
2,3-OAc
H
CH3CH-(CH2)2
An tiba cter ia l Activity. Compounds 7-10, 12, 14,
and 15c were tested for their antibacterial activity in
vitro against the Gram-negative bacteria P. aeruginosa
SG 137 and ATCC 27853, S. maltophilia GN 12873,
Escherichia coli ATCC 25922, Klebsiella pneumoniae
ATCC 10031, Serratia marcescens SG 621, and against
the Gram-positive strain Staphylococcus aureus SG 511
(Table 3). The compounds 7a -h , 8, and 9a exhibited
extremely high antibacterial activity against the Gram-
negative test strains, by far exceeding the activity of
the ampicillin moiety, azlocillin, and partly also the
activity of meropenem, especially against S. maltophilia.
acetylpenicillanic acid representing a new type of sidero-
phore â-lactam conjugate.
Sid er op h or e Activity. Siderophore activity of the
synthesized compounds was determined by the assay
of their growth-promoting activity in Gram-negative
bacteria under iron-limited conditions.
According to Table 2, the bis-catecholates of N-
(aminoalkyl)glycines 5b-d ,f-h and 6a ,b efficiently act
as siderophores and promote the growth of the bacterial
test strains. The growth promotion activities of com-
pounds 5e,i, 6c, 11, and 13b exhibited decreased

Antibacterial Active Aminoacyl Penicillin Conjugates
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 14 3035
Ta ble 3. Antibacterial Activities In Vitro of the Siderophore-â-Lactam Conjugates 7-15 (MIC in mg/L)
P. aeruginosa
E. coli
K. pneumoniae
S. maltophilia
S. marcescens
S. aureus
compd
SG 137
ATCC 27853
ATCC 25922
ATCC 10031
GN 12873
SG 621
SG 511
7a
7b
7c
7d
7e
7f
7g
7h
7i
7k
7l
7m
8
9a
9b
9c
10a
10b
12
14a
14b
14c
0.4
<0.005
0.04
0.005
0.04
<0.005
<0.005
0.05
0.2
0.1
0.04
0.2
0.01
0.04
0.1
0.78
0.05
0.16
0.08
0.31
0.1
0.05
0.1
0.78
0.4
0.78
<0.005
0.16
0.02
0.02
0.05
<0.005
0.02
0.78
1.56
0.4
0.78
0.01
0.01
0.4
1.56
nt
100
100
25
25
6.25
1.56
6.25
6.25
0.04
0.1
<0.005
<0.005
<0.005
0.005
<0.005
<0.005
<0.005
0.4
0.78
0.005
0.02
0.02
0.08
0.02
0.02
0.05
50
<0.05
<0.05
0.2
0.005
0.04
0.4
0.4
25
25
1.56
0.78
3.12
1.56
6.25
25
0.78
<0.005
0.04
0.05
0.02
0.02
0.02
0.05
3.12
0.4
0.24
6.25
<0.005
<0.005
0.2
0.1
nt
50
nt
12.5
12.5
3.12
0.4
50
25
3.12
5
2.5
3.12
5
12.5
6.25
3.12
0.78
0.78
6.25
1.56
5
0.2
0.2
<0.05
<0.05
<0.005
<0.005
<0.05
0.2
nt
100
3.12
6.25
1.56
0.2
0.2
6.25
6.25
0.04
6.25
0.16
0.156
3.12
3.12
50
2.5
1.56
1.56
1.56
3.12
25
25
25
6.25
6.25
0.4
0.1
50
25
0.4
50
3.12
1.56
1.56
0.2
0.2
0.05
0.05
0.2
15c
1.56
6.25
>100
0.4
azlocillin
ampicillin
meropenem
6.25
>100
>100
>100
0.4
0.1
0.2
0.06
Ta ble 4. Influence of Porins and TonB on Antibacterial
Activity in E. Coli (Diameter of Inhibition Zones in mm, 50 µL
of a 0.2 mM Solution Was Filled in 9 mm Agar Wells)a
As known from literature, the increase in activity
against Gram-negative bacteria is more or less at the
expense of activity against Gram-positive S. aureus.17,18
An exception is the derivative 7k based on 2-[N-
(aminobutyl)aminomethyl]benzoic acid, which inhibited
both Gram-positive and Gram-negative strains. The 3,5-
diaminobenzoic acid derivatives 12, 14a -c, and 15c
generally had a lower antibacterial activity than the
derivatives 7-9 based on N-(aminoalkyl)amino acids.
The most active antibacterial compounds from this
series were the 2,3-diacyloxybenzoyl derivatives based
on N-(aminobutyl)glycine, the ampicillin derivatives
7b,d ,f-h and the amoxicillin derivative 8. The corre-
sponding 3,4-diacyloxy derivative 7i was less active.
In contrast to the ampicillin and amoxicillin conju-
gates, derivatives of penicillin V 10a ,b were nearly
inactive against the Gram-negative strains. From these
results, we can deduce that the distance of the sidero-
phore component to the â-lactam ring affects the activity
of the conjugates. The siderophore moiety is closer to
the â-lactam ring if attached to the NH group of
aminoacyl penicillins than if attached to the phenyl ring
of penicillin V derivatives. Possibly, the â-lactam struc-
ture is involved in the iron complexation.
compd
OmpF
OmpC
TonB
azlocillin
ampicillin
7a
7b
7c
7d
7e
7f
7g
7h
7i
7k
7l
KB4
KB5
PLB268
AB2847
BR158
-
+
+
+++
+
+
+
+
-
+
+
+
+
+
-
16
15
17
20
31
25
29
28.5
30
31
29
19
19
20
29.5
29.5
20
19
11
13
18
22
22.5
32
25
25
34
30
32.5
33
33
33
32.5
25
27
27
33
33.5
29
25.5
18.5
19
20
20
21
12
12.5
11.5
13
14.5
13
12.5
14.5
16
14
12
11.5
15.5
13
10
nt
10.5
20
31.5
26
29.5
30.5
31
30.5
30.5
23
25
25
31.5
31
24
20
20.5
29.5
24
28
31.5
30
29.5
29
21
24
25
30
32
22.5
20.5
nt
8
9a
9b
9c
22
12
15.5
15.5
20
14a
14b
14c
15c
16
22.5
21
0
0
0
0
23.5
24
22
19.5
19.5
23.5
a
Mech a n ism of Action . The mechanism of action of
the highly active conjugates was studied by agar diffu-
sion assays using E. coli-porin and iron transport
mutants. The outer membrane porin proteins OmpF
and OmpC allow the diffusion of small hydrophilic
molecules such as â-lactam antibiotics. The TonB pro-
tein is essential for energy transfer in the active
siderophore iron transport. FepA, Cir, and Fiu are outer
membrane receptors for catecholate type siderophores.19
Table 4 demonstrates the inhibition of E. coli mutants
either deficient in OmpF or OmpC or overexpressing
OmpC as well as the inhibition of a TonB mutant by
ampicillin, azlocillin, and the siderophore conjugates.
Activity of ampicillin alone but not of the acylamino
nt ) not tested.
penicillin conjugates is strongly influenced by the pres-
ence of the outer membrane porins. In contrast, activity
of the siderophore acylamino penicillin conjugates, but
not of ampicillin or azlocillin, essentially depends on the
presence of TonB and thus on the function of active iron
transport mechanisms. This was investigated in more
detail using catecholate siderophore receptor mutants
of E. coli. As shown in Table 5, the activities of the new
conjugates are drastically decreased against strain
H1876, which is deficient in all three catecholate
siderophore receptors. In contrast, activity against
strain H873, deficient only in the enterobactin receptor

3036 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 14
Heinisch et al.
Ta ble 5. Influence of Outer Membrane Siderophore Receptors
on Antibacterial Activity in E. Coli (Diameter of Inhibition
Zones in mm, 50 µL of a 0.2 mM Solution Was Filled in 9 mm
Agar Wells)
results in strongly increased activity against Gram-
negative bacteria including strains of P. aeruginosa, S.
maltophilia, E. coli, K. pneumoniae, and S. marcescens.
The bis-catecholates of N-(aminoalkyl)aminomethyl phen-
oxyacetic acids were linked to 6-aminopenicillanic acid
to form a new type of siderophore-â-lactam conjugate
of penicillin V in which the siderophore moiety is
attached to the phenyl ring of aminoacyl penicillins.
This constellation, in which the siderophore part is more
distant to the â-lactam part than in conjugates with the
siderophore moiety linked to the NH group of aminoacyl
penicillins, resulted in inactive compounds. We can
assume that the â-lactam moiety is involved in the iron
complexation in highly active conjugates.
The ampicillin and amoxicillin derivatives of N,N′-
(2,3-diacetoxybenzoyl)-N-aminobutyl-glycine 7b (HKI
9924154) and 8 (HKI 9924155) were also highly active
in vivo in a murine septicaemic model and exhibited low
acute toxicity (data will be presented in a following
paper). Accompanied with low synthetic effort, these
results favor compounds 7b and 8 as promising candi-
dates for further preclinical and clinical investigations.
H1443
H1876
H873
H1877
H1875
FepA
Cir
Fiu
ampicillin
azlocillin
7a
7b
7c
7d
7e
+
-
-
-
-
+
-
+
+
-
+
20.5
16
22
34
29
32
30
30
34
31
24.5
25
-
+
19.5
17
22
33
28.5
31.5
30
29.5
32.5
32
-
20
16
21
30.5
25
29.5
27.5
28
32.5
31
22
+
20.5
16
20
27
23
27
26
26.5
29
20
16
11
12
0
11
12.5
12
12
16
13
0
7f
7g
7h
7i
28.5
23
21
24
25
7k
24
7l
8
9a
9b
9c
12
25
32
32
24.5
22.5
18
0
25.5
32
31
25
23
24
29
28
22.5
19.5
0
22
26
24
22
20.5
16
11
14
11.5
0
0
17
14a
14b
14c
15c
16.5
21
20.5
22.5
0
0
0
0
18.5
21
23
13
18.5
18
18
20
21.5
21.5
Exp er im en ta l Section
Gen er a l. 1H NMR spectra were recorded on a 300 MHz
Bruker NMR spectrometer. Chemical shifts (δ) are given in
ppm related to tetramethylsilane as internal standard. Cou-
pling constants (J ) are reported in Hz. Mass spectrometry (MS)
and high-resolution MS (HRMS) spectra were obtained by a
Finnigan MAT 95 XV high-resolution mass spectrometer with
electron-spray ionization (ESI) technique. Purification of the
compounds by preparative HPLC was performed on an
ABIMED GILSON apparatus equipped with an 115 UV
detector (254 nm) and a KNAUER VERTEX reversed phase
column (250 mm × 32 mm or 50 mm × 20 mm) packed with
Eurosper 100-C18 (7 µm). Eluents used were acetonitrile and
water, beginning with ratio 30:70 (v/v) and achieving 100:0
(v/v) after a period of 20 min (flux rate, 20 or 10 mL/min).
Column chromatography was performed using silica gel
(Merck 60, 0.040-0.063 mm). The purity of all compounds was
controlled by 1H NMR spectra and by thin-layer chromatog-
raphy (TLC) using precoated silica plates (Merck 60 F254) and
n-butyl acetate/acetic acid (4:1) or n-butyl acetate/methanol/
formic acid/water (32.5:15:2.5:5) as solvents.
22
19
FepA, is the same as against the parent strain H1443
where the complete set of catecholate receptors is
available. Thus, activity of the conjugates depends on
the receptors Cir or Fiu for the building blocks or
breakdown products of enterobactin, namely, mono-, di-,
and trimers of 2,3-dihydroxybenzoic acid, not on the
receptor FepA. There is evidence for a coordinating
function of Cir and Fiu as there is only a minor or no
decrease in activity of the conjugates against the
mutants H1875 and H1877, deficient only in one of the
receptors Cir or Fiu. Cir may play a more dominant role
for uptake of the conjugates 7a -l and 8 (acetylated
catecholates based on N-(aminoalkyl)amino acids and
Fiu for the conjugates 12, 14a -c, and 15c (diaminoben-
zoic acid derivatives). The activity of ampicillin and
azlocillin is independent of siderophore receptors. There
is no marked change in activity against the parent
strain H1443 and the different receptor mutants. These
results confirm the importance of iron transport for the
high antibacterial activities of the new conjugates 7-9,
12, 14, and 15.
The following compounds were prepared according to pub-
lished procedures: 2,3-Di-acetoxy-benzoyl chlorides 4a ,20 2,3-
di(methoxycarbonyloxy)benzoyl chloride 4b,8 5-chloro-2,3-di-
(methoxycarbonyloxy)benzoyl chloride 4c,8 5-bromo-2,3-di-
(methoxycarbonyloxy)benzoyl chloride 4d ,8 and 3,4-diacetoxy-
benzoyl chloride 4f.21 2,3-Dichloro-5,6-dimethoxycarbonyloxy-
benzoyl chloride 4e was synthesized analogously to compound
4b as follows: 2,3-dichloro-5,6-dihydroxybenzoic acid
16
and
methyl chloroformate in sodium hydroxide solution gave 2,3-
dichloro-5,6-di-(methoxycarbonyloxy)benzoic acid, yield 60%.
1H NMR (DMSO-d6): δ 8.06 (s, 1H), 3,85 (s, 6H). MS: ESI
337.0 [M - H]-. Subsequent reaction with PCl5 in tetrachloro
methane resulted in 4e as a yellowish oil (yield 89%).
Con clu sion
New N-(aminoalkyl)amino acids, N-(aminoalkyl)amino-
methyl benzoic acids or N-(aminoalkyl)aminomethyl
phenoxyacetic acids were prepared from oxocarboxylic
acids and diamines by catalytic hydrogenation. These
compounds and 3,5-diaminobenzoic acid are useful
scaffolds for new acylated bis-catecholates. The acylated
catecholates were synthesized as 2,3-di-acetoxybenzoyl
derivatives and in heterocyclic form as 2,4-dioxo-1,3-
benzoxazine derivatives. These acylated bis-catecholates
can function as artificial siderophores for Gram-negative
bacteria as demonstrated for P. aeruginosa and E. coli.
The acylated bis-catecholates were coupled with ampi-
cillin or amoxicillin to new siderophore-antibiotic con-
jugates, which were able to use siderophore iron uptake
routes to penetrate the bacterial outer membrane. This
Gen er a l P r oced u r e for th e P r ep a r a tion of Secon d a r y
Dia m in o Acid s 3b -d ,o,p , N-(Am in oa lk yl)a m in om et h yl
Ben zoic Acid s 3k ,l, a n d N-(Am in oa lk yl)a m in om eth yl
P h en oxya cetic Acid s 3m ,n Accor d in g to th e Syn th esis
of 3a .14 In an ice bath, a solution of 0.1 mol of the respective
oxocarboxylic acid 2a -f in methanol (20 mL) was dropped with
stirring into a solution of 0.12 mol of the respective diamine
1a -e in methanol (30 mL), and then, 1.0 g Pd/C (10%) was
added under nitrogen. The mixture was hydrogenated at
ambient temperature and atmospheric pressure. Then, the
reaction mixture was filtered by Celite and the solvent was
evaporated. Three times toluene (20 mL) was added to the
residue and distilled off at 120 °C. The residue was dried in a
vacuum to give a light yellow or colorless oil of 3. In most cases,

Antibacterial Active Aminoacyl Penicillin Conjugates
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 14 3037
the product was pure enough for the following reaction. The
product can be further purified by reaction with benzyl
chloroformate to obtain the di-Z-derivative, separating from
impurities by preparative HPLC and following hydrogenolysis
with Pd/C (10%). 3b: N-(Amino-n-butyl)glycine, from 1d and
(DMSO-d6): δ 8.04 (m, 2H), 4.00 (m, 2H), 3.81 (m, 12H), 2.96-
3.40 (m, 4H), 2.78 (s, 3H), 1.80 (m, 2H). 5i: N,N′-Bis-(3,4-
diacetoxy-benzoyl)-N-(aminobutyl)glycine, from 3b and 4f. The
crude product was purified by column chromatography on
silicagel using ethyl acetate/methanol (3:1) as eluent, yield
1
1
2a , yield 93%. H NMR (D2O): δ 3.21 (s, 2H), 2.58-2.93 (m,
17%. H NMR (DMSO-d6): δ 8.10, 8.20 (2× t, 1H), 6.69-7.80
4H), 1.53-1.62 (m,4H). 3c: N-(Amino-n-pentyl)glycine, from
1e and 2a , yield 90%. 1H NMR (DMSO-d6): δ 3.30 (s, 2H),
2.71-2.74 and 2.77-2.80 (2× t, 4H), 1.40-1.64 (t, 2H), 1.59-
1.64 (m, 4H). 3d : N-(Methylaminopropyl)glycine, from 1c and
2a , yield 80%. 1H NMR (DMSO-d6): δ 3.01 (s, 2H), 2.71 (t,
2H), 2.57 (t, 2H), 2.32 (s, 3H), 1.63 (t, 2H). MS: 146.9 [M +
H]+. 3k: 2-[N-(Methylamino-n-propyl)aminomethyl]benzoic acid,
(m, 6H), 3.52-3.68 (m, 2H), 3.13-3.30 (m, 4 H), 2.26-2.28
(m, 12H), 1.19-1.56 (m, 4H). MS: 609.5 [M + Na]+. 5k :
2-[N,N′-Bis-(2,3-diacetoxybenzoyl)-N-(methylamino-n-propyl)-
aminomethyl]benzoic acid, from 3k and 4a , yield 34%. 1H NMR
(DMSO-d6): δ 6.83-7.92 (m, 10H), 4.75-5.12 (m, 2H), 2.70-
3.29 (m, 4H), 2.53 (s, 3H), 2.10-2.28 (m, 12H), 1.73-1.91 (m,
2H). MS: 661.8 [M - H]-. 5l: 2-[N,N′-Bis-(2,3-diacetoxyben-
zoyl)-N-(aminobutyl)aminomethyl]benzoic acid, from 3l and 4a ,
yield 15%. 1H NMR (DMSO-d6): δ 8.20, 8.29 (2× t, 1H), 7.07-
7.90 (m, 10H), 4.71-5.04 (m, 2H), 2.99-3.29 (m, 4 H), 2.15-
2.27 (m, 12H), 1.22-1.56 (m, 4H). MS: 661.3 [M - H]-.
5m : 4-[N,N′-Bis-(2,3-diacetoxybenzoyl)-N-(aminobutyl)amino-
methyl]phenoxyacetic acid, from 3m and 4a , yield 47%. 1H
NMR (DMSO-d6): δ 13.01 (s, 1H), 8.20, 8.28 (2× t, at 360 K
changed to s, 1H), 6.88-7.36 (m, 10H), 4.65 (s, 2H), 2.90-
3.08 (m, 4H), 1.97-2.49 (m, 14H), 1.42-1.55 (m, 4H). MS:
715.2 [M + Na]+, 691.3 [M - H]+. 5n : 2-[N,N′-Bis-(2,3-di-
acetoxybenzoyl)-N-(aminobutyl)aminomethyl]phenoxyacetic acid,
1
from 1c and 2d , yield 60%. H NMR (D2O): δ 7.32-7.55 (m,
4H), 3.96 (d, J ) 8.2, 2H), 2.70-2.76 (m, 4H), 1.50-1.59 (m,
4H). 3l: 2-[N-(Aminobutyl)aminomethyl]benzoic acid, from 1d
1
and 2d , yield 95%. H NMR (D2O): δ 7.32-7.55 (m, 4H), 3.96
(d, J ) 8.2, 2H), 2.70-2.76 (m, 4H), 1.50-1.59 (m, 4H). 3m :
4-[N-(Aminobutyl)aminomethyl]phenoxyacetic acid, from 1d
and 2e, yield 90%. 1H NMR (DMSO-d6): δ 7.23-7.28 (m, 2H),
6.89-6.91 (m, 2H), 4.40-4.44 (m, 2H), 3.75 (s, 2H), 2.75-2.80
(m, 2H), 2.62-2.68 (m, 2H), 1.54-1.55 (m, 4H). 3n : 2-[N-
(Aminobutyl)aminomethyl]phenoxyacetic acid, from 1d and 2f,
yield 60%. MS: 275.0 [M + Na]+. 3o: N-(Amino-n-propyl)-
alanine), from 1b and 2b, yield 65%. MS: 147.0 [M + H]+.
3p : N-(Aminopropyl)-4-aminovaleric acid, from 1b and 2c,
yield 70%. MS: 175.2 [M + H]+.
1
from 3n and 4a , yield 50%. H NMR (DMSO-d6): δ 8.18, 8.30
(2× t, 1H); 6.85-7.39 (m, 12H); 4.65, 4.73 (s, 2 × 2H); 3.01-
3.29 (m, 4H); 2.05-2.26 (m, 12 H); 1.49-1.54 (m, 4H). MS:
715.3 [M + Na]+, 691.3 [M - H]-. 11: 3,5-Di-[(2,3-diacetoxy-
benzoyl)amino]benzoic acid, from 3,5-diaminobenzoic acid and
4a , mp 197-200 °C (ethyl acetate), yield 55%. 1H NMR
(DMSO-d6): δ 10.65 (s, 2H), 8.33 (m, 1H), 8.06 (d, J ) 1.9,
2H), 7.59 (dd, J 1)5.5, J 2)1.9, 2H), 7.56 (s, 2H), 7.44 (d, J )
1.9, 2H), 2.30 (s, 3H), 2.22 (s, 3H). MS: 591.3 [M - H]-. 13b:
3,5-[Bis-(8-methoxycarbonyloxy-1,3-benzoxazine-2,4-dione-3-
yl)acetylamino]benzoic acid, from 3,5-diaminobenzoic acid and
4g (8-methoxycarbonyloxy-1,3-benzoxazine-2,4-dione-3-yl)-
Gen er a l P r oced u r e for th e P r ep a r a tion of Acyla ted
Bis-ca tech ola tes 5a -n a n d 11 a n d of th e Ben zoxa zin e-
2,4-d ion e Der iva tives 13b,c. In an ultrasonic bath, a solution
of 20 mmol of the respective compounds 4a -h in absolute
tetrahydrofuran (20 mL) was added dropwise at 0-5 °C with
stirring to a solution of 10 mmol of compounds 3a -d ,k -n or
3,5-diaminobenzoic acid, respectively, in a 0.5 M aqueous
sodium hydrogen carbonate solution (60 mL, 30 mmol). The
mixture was stirred for 1 h at 0-5 °C, and then, the
tetrahydrofuran was removed in a vacuum. The obtained
aqueous solution was cooled to 0 °C, acidified to pH 2 using 6
N HCl, and extracted with ethyl acetate. The organic layer
was separated, washed with saturated NaCl solution, dried
over Na2SO4, filtered, and evaporated. The residue was dried
in a vacuum to give compounds 5a -n and 13b,c, respectively,
as colorless to yellow solid foams. The compounds could be
purified by preparative HPLC. 5a : N,N′-Bis-(2,3-diacetoxy-
benzoyl)-N-(aminoethyl)glycine, from 3a 14 and 4a , yield 35%.
1H NMR (DMSO-d6): δ 8.30, 8.40 (2× t, 1H), 7.11-7.78 (m,
6H), 3.28-3.41 (m, 4H), 2.14-2.29 (m, 12H). 5b: N,N′-Bis-
(2,3-diacetoxybenzoyl)-N-(aminobutyl)glycine, from 3b and 4a ,
1
acetylchloride, yield 53%, mp 190-195 °C. H NMR (DMSO-
d6): δ 10.48 (s, 2H), 8.14 (m, 1H), 7.96-7.86 (m, 6H), 7.49 (t,
J ) 7.9 Hz, 2H), 4.69 (s, 4H), 3.91 (s, 6H). MS: 705.1 [M -
H]-. 13c: 3,5-Bis-[3-(8-methoxycarbonyloxy-1,3-benzoxazine-
2,4-dione-3-yl)propionyl-amino]benzoic acid, from 3,5-diamino-
benzoic acid and 4h (8-methoxycarbonyloxy-1,3-benzoxazine-
2,4-dione-3-yl)propionyl chloride, yield 51%, mp 160-165 °C.
1H NMR (DMSO-d6): δ 10.23 (s, 2H), 8.08 (m, 1H), 7.94-7.80
(m, 6H), 7.46 (t, J ) 7.9, 2H), 4.16 (t, J ) 8,0, 4H), 3.90 (s,
6H), 2.70 (t, J ) 8,0, 4H). MS: 733.0 [M - H]-.
Gen er a l P r oced u r e for th e P r ep a r a tion of th e 1,3-
ben zoxa zin e-2,4-d ion e Der iva tives 6a -c or 13a . Analo-
gously to the procedure for compound 5, a solution of 2 mmol
of 2,3-di-(methoxycarbonyloxy)benzoyl chloride 4b in tetra-
hydrofuran was added to a solution of 1 mmol of compound
3b,o,p or 3,5- diaminobenzoic acid, respectively, in 0.5 M
aqueous sodium hydroxide solution at 0-5 °C in an ultrasonic
bath. After it was stirred for 1 h, the tetrahydrofuran was
removed and the residual aqueous solution was acidified using
6 N HCl and extracted with ethyl acetate. The organic phase
was washed with brine, dried over Na2SO4, filtered, and
evaporated. The residue was stirred in acetonitrile for 3 h at
ambient temperature. The solvent was then evaporated, and
the residue was purified by preparative HPLC. Acetonitrile
was evaporated in a vacuum from the fraction containing the
target product, and the residual aqueous solution was dried
by lyophilization to give 6a -c and 13a , respectively, as
colorless solids. 6a : 7-(8-Methoxycarbonyloxy-1,3-benzoxazine-
2,4-dione-3-yl)-3-(2,3-dimethoxy-carbonyl-oxybenzoyl)-3-aza-
1
yield 60%. H NMR (DMSO-d6): δ 8.25, 8.35 (2× t, at 360 K
changed to s, 1H), 6.94-7.42 (m, 6H), 3.95 (m, 2H), 3.22 (m,
2H), 2.77 (s, 3H), 2.27 (s, 6H), 2.20 (s, 6H), 1.40-1.60 (m, 4H).
MS: 609.2 [M + Na]+. 5c: N,N′-Bis-(2,3-diacetoxybenzoyl)-
1
N-(aminopentyl)glycine, from 3c and 4a , yield 70%. H NMR
(DMSO-d6): δ 8.22, 8.29 (2× t, 1H), 7.09-7.80 (m, 6H), 4.00-
4.03 (m, 4H), 3.16-3.28 (m, 4H), 2.16-2.49 (m, 12H), 1.14-
1.49 (m, 6H). MS: (ESI) 623.2, [M + Na]+, 599.4 [M - H]-.
5d : N,N′-Bis-(2,3-diacetoxybenzoyl)-N-(methylaminopropyl)-
glycine, from 3d and 4a , yield 40%. 1H NMR (DMSO-d6): δ
6.94-7.39 (m, 6H), 3.98 (m, 2H), 2.87-3.15 (m, 2H), 2.77 (s,
3H), 2.27 (s, 6H), 2.20 (s, 6H), 1.75 (m, 2H). MS: 587.2 [M +
Na]+. 5e: N,N′-Bis-(2,3-dimethoxycarbonyloxy-benzoyl)-N-
(methylamino-n-propyl)glycine, from 3d and 4b, yield 89%. 1H
NMR (DMSO-d6): δ 7.2-7.6 (m, 6H), 4.00 (s, 2H), 3.80 (m,
12H), 2.96-3.40 (m, 4H), 1.75 (m, 2H). MS: 673.0 [M + Na]+.
5f: N,N′-Bis-(2,3-dimethoxycarbonyloxy-5-chloro-benzoyl)-N-
(methylamino-n-propyl)glycine, from 3d and 4c, yield 30%. 1H
NMR (DMSO-d6): δ 7.80-7.20 (m, 4H), 4.00 (m, 2H), 3.81 (m,
12H), 2.96-3.40 (m, 4H), 2.78 (s, 3H), 1.75 (m, 2H). 5g:
N,N′-Bis-(2,3-dimethoxycarbonyloxy-5-bromo-benzoyl)-N-(methyl-
aminopropyl)glycine, from 3d and 4d , yield 40%. 1H NMR
(DMSO-d6): δ 7.35-7.90 (m, 4H), 4.00 (m, 2H), 3.81 (m, 12H),
2.96-3.40 (m, 4H), 2.78 (s, 3H), 1.75 (m, 2H). 5h : N,N′-Bis-
(2,3-dimethoxycarbonyloxy-5,6-dichloro-benzoyl)-N-(methyl-
aminopropyl)glycine, from 3d and 4e, yield 40%. 1H NMR
1
heptanoic acid, from 3b and 4b, yield 50%. H NMR (DMSO-
d6): δ 7.25-7.60 (m, 6H), 4.15 (s, 2H), 3.72-3.94 (m, 9H), 3.65
(t, 2H), 3.10 (t, 2H), 1.35-1.65 (m, 4H). MS: 641.0 [M + Na]+.
6b: 6-(8-Methoxycarbonyloxy-1,3-benzoxazine-dione-3-yl)-3-
(2,3-d im et h oxy-ca r bon yloxyben zoyl)-2-m et h yl-3-a za -
hexanoic acid, from 3o and 4b. 1H NMR (DMSO-d6): δ 3.75
(t, 2H), 7.28-7.89 (m, 6H), 4.15 (s, H), 3.78-3.91 (m, 9H), 3.20
(t, 2H), 1.80-1.95 (m, 2H), 1.32-1.40 (m, 3H). MS: 641.2 [M
+ Na]+. 6c: 8-(8-Methoxycarbonyloxy-1,3-benzoxazine-2,4-

3038 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 14
Heinisch et al.
dione-3-yl)-5-(2,3-dimethoxy-carbonyloxybenzoyl)-5-aza-4-methyl-
octanoic acid, from 3p a nd 4b, yield 60%. 1H NMR (DMSO-
d6): δ 7.20-7.90 (m, 6H), 3.85-4.05 (m, 9H), 3.40-3.85 (m,
4H), 3.05 (q, 1H), 1.9-2.2 (m, 4H), 1.75 (m, 2H), 1.15 (d, 3H).
13a : 3,5-Bis-(8-methoxycarbonyloxy-1,3-benzoxazin-2,4-dione-
3-yl)benzoic acid, yield 64%, mp 164-166 °C. 1H NMR (DMSO-
d6): δ 8.20 (d, J ) 1.9, 2H), 7.92 (dd, J 1)7.8, J 2)1.5, 2H), 7.86
(dd, J 1)7.8, J 2)1.5, 2H), 7.78 (t, J ) 1.8, 1H), 7.50 (t, J ) 8,0,
2H), 3.92 (s, 6H). MS: 591.4 [M - H]-.
Gen er a l P r oced u r e for th e P r ep a r a tion of th e Am in o-
a cylp en icillin Con ju ga tes 7a -m , 8, 9a -c, 12, 14a -c, a n d
15c. To a solution of 1 mmol of compounds 5a -m , 6a -c, 11,
or 13a -c, respectively, and N-methylmorpholine (112 µL) in
absolute tetrahydrofuran (10 mL), methylchloroformate (131
µL) was added at - 20 °C with stirring. The mixture was
stirred for 1 h at -10 °C, and then, a solution of ampicillin
trihydrate (445 mg, 1.1 mmol) or amoxicillin trihydrate (461
mg), respectively, and triethylamine (140 µL, 1 mmol) in
tetrahydrofuran (4 mL) and water (1 mL) was added at -20
°C. The mixture was stirred for 1 h at -10 °C and 1 h at
ambient temperature and then evaporated. Ethyl acetate and
water were added to the residue. The mixture was acidified
to pH 3 using diluted HCl with shaking. The organic phase
was separated, washed with brine, dried with Na2SO4, filtered,
and evaporated. The obtained residue was purified by pre-
parative HPLC. The fraction containing the respective conju-
gate was evaporated, and the residual aqueous solution was
dried by lyophilization to give 7a -m , 8 or 9a -c, 12, 14a -c,
or 15c, respectively, as a colorless solid.
1H), 5.52 (q, 1H), 4.18 (s, 1H), 3.95 (m,2H), 3.85 (m,6H), 3.80
(m, 6H), 2.96-3.40 (m, 4H), 2.78 (s, 3H), 1.75 (m, 2H), 1.53 (s,
3H), 1.39 (s, 3H). MS: 1038.5 [M - H]-. 7h : N-[N′,N′′-Bis-
(2,3-dimethoxycarbonyloxy-5,6-dichloro-benzoyl)-N-(methyl-
aminopropyl)glycyl]ampicillin, from 5h , yield 30%. 1H NMR
(DMSO-d6): δ 9.19 (d, 1H), 8.65 (m, 1H), 7.20-8.05 (m, 7H),
5.70 (m, 1H), 5.48 (m, 1H), 5.38 (m, 1H), 4.19 (s, 1H), 4.05 (m,
2H), 3.85 (m, 6H), 3.82 (m, 6H), 2.96-3.40 (m, 4H), 2.78 (s,
3H), 1.80 (m, 2H), 1.53 (s, 3H), 1.39 (s, 3H). MS: 1118.4 [M -
H]-. 7i: N-[N′,N′′-Bis-(3,4-diacetoxy-benzoyl)-N-(aminobutyl)-
glycyl]ampicillin, from 5i, yield 9%. 1H NMR (DMSO-d6): δ
8.91-9.12 (m, 2H), 8.44-8.74 (m, 1H), 7.25-7.87 (m, 11H),
5.69-5.75 (m, 1H), 5.46-5.52 (m, 1H), 5.37-5.41 (m, 1H), 4.16
(s, 1H), 3.12-3.36 (m, 6H), 2.28-2.29 (m, 12H), 1.53 (s, 3H),
1.40 (s, 3H), 1.24-1.45 (m, 4H). MS: 939.7 [M + Na]+, 917.7
[M + H]+. 7k: N-{2-[N,N′-Bis-(2,3-diacetoxybenzoyl)-N-(methyl-
aminopropyl)aminomethyl]benzoyl}ampicillin, from 5k , yield
20%. 1H NMR (DMSO-d6): δ 8.96-9.26 (m, 2H), 6.88-7.49
(m, 15H), 5.84-5.93 (m, 1H), 5.50-5.55 (m, 1H), 5.38-5.40
(m, 1H), 4.18 (s, 1H), 2.74-3.29 (m, 6H), 2.71 (s, 3H), 2.12-
2.27 (m, 12H), 1.52 (s, 3H), 1.40 (s, 3H), 1.45-1.82 (m, 2H).
MS (ESI + NH4OAc): 1012.2 [M + NH4]+. 7l: N-{2-[N,N′-Bis-
(2,3-diacetoxybenzoyl)-N-(aminobutyl)aminomethyl]benzoyl}-
ampicillin, from 5l, yield 36%. 1H NMR (DMSO-d6):δ 8.92-
9.12 (m, 2H), 8.20, 8.30 (2× t, 1H), 7.24-7.54 (m, 15H), 5.88-
5.91 (m, 1H), 5.48-5.59 (m, 1H), 5.39-5.40 (m, 1H), 4.19 (s,
1H), 2.95-3.23 (m, 6H), 2.15-2.26 (m, 12H), 1.52 (s, 3H), 1.40
(s, 3H), 1.16-1.45 (m, 4H). MS: 1016. [M + Na]+. 7m : N-{4-
[N′,N′′-Bis-(2,3-diacetoxybenzoyl)-N′-(aminobutyl)aminomethyl]-
phenoxyacetyl}ampicillin, from 5m , yield 65%. 1H NMR
(DMSO-d6): δ 9.18-9.20 (m, 1H), 8.54-8.57 (m, 1H), 8.20, 8.30
(2× t, 1H), 6.88-7.41 (m, 15H), 5.75-5.85 (m, 1H), 5.51-5.52
(m, 1H), 5.39 (d, J ) 4.0, 1H), 4.63 (s, 2H), 4.18 (s, 2H), 2.90-
3.15 (m, 4H), 2.16-2.27 (m, 12H), 1.39-1.53 (m, 10H). MS:
1047.6 [M + Na]+. 8: N-[N′,N′′-Bis-(2,3-diacetoxybenzoyl)-N′-
(aminobutyl)glycyl]amoxicillin, from 5b and amoxicillin, yield
The sodium salts were prepared from the solution of 1 mmol
of the compounds 7-9, 12, 14, or 15c, respectively, in ethyl
acetate (5 mL) by addition of sodium ethylhexanoate (1.5
mmol) in ethyl acetate (3 mL). Precipitation was completed
by adding petroleum ether. The obtained colorless solid was
filtered, washed with petroleum ether, and dried in a vacuum.
7a : N-[N′,N′′-Bis-(2,3-diacetoxybenzoyl)-N-(aminoethyl)gly-
cyl]ampicillin, from 5a , yield 23%. 1H NMR (DMSO-d6): δ
9.10-9.20 (m, 1H), 8.72-8.74 (m, 1H), 8.25, 8.35 (2× t, 1H),
7.24-7.49 (m, 11H), 5.72 (m, 1H), 5.49-5.51 (m, 1H), 5.35-
5.38 (m, 1H), 4.14 (s, 1H), 3.10-3.40 (m, 6 H), 2.05-2.27 (m,
12H), 1.40-1.52 (m, 6H). MS: 912.3 [M + Na]+. 7b: N-[N′,N′′-
Bis-(2,3-diacetoxybenzoyl)-N′-(aminobutyl)glycyl]ampicillin, from
5b, yield 50%. 1H NMR (DMSO-d6): δ 8.70 (m, 2H), 8.25, 8.34
(2× t, changed to s at 360 K, 1H), 6.94-7.52 (m, 11H), 5.72
(m, 1H), 5.51 (m, 1H), 5.38 (m, 1H), 4.19 (s, 1H), 3.95 (m, 2H),
3.30 (s, 3H), 3.22 (m, 2H), 2.27 (s, 6H), 2.21 (s, 3H), 2.17 (s,
3H), 1.54 (s, 3H), 1.40 (s, 3H), 1.40-1.60 (m, 4H). MS: 940.1
[M + Na]+. 7c: N-[N′,N′′-Bis-(2,3-diacetoxybenzoyl)-N-(amino-
pentyl)glycyl]ampicillin, from 5c, yield 40%. 1H NMR (DMSO-
d6): δ 9.11-9.15 (m, 1H), 8.68 (m, 1H), 8.29, 8.38 (2× t, 1H),
7.25-7.49 (m, 11H), 5.72 (m, 1H), 5.48 (m, 1H), 5.35 (m, 1H),
4.15 (m, 2H), 3.90 (m, 1H), 3.16-3.31 (m, 4H), 2.16-2.23 (m,
12H), 1.40-1.54 (m, 12H). MS: 954.6 [M + Na]+, 930.2 [M -
H]+. 7d : N-[N′,N′′-Bis-(2,3-diacetoxybenzoyl)-N-(methylamino-
propyl)glycyl]ampicillin from 5d , yield 40%. 1H NMR (DMSO-
d6): δ 9.15 (m, 1H), 8.73 (m, 1H), 6.94-7.48 (m, 11H), 5.85
(m, 1H), 5.52 (m, 1H), 5.39 (m, 1H), 3.98 (m, 2H), 2.87-3.15
(m, 2H), 2.75 (s, 3H), 2.27 (s, 6H), 2.20 (s, 6H), 1.75 (m, 2H),
1.53 (s, 3H), 1.40 (s, 3H). 7e: N-[N′,N′′-Bis-(2,3-dimethoxy-
carbonyloxy-benzoyl)-N-(methylaminopropyl)glycyl]ampi-
cillin, from 5e, yield 45%. 1H NMR (DMSO-d6): δ 8.71, 9.15
(m, 2 × 1H), 7.25-7.60 (11H), 5.72 (q, 1H), 5.50 (m, 1H), 5.38
(m, 1H), 4.17 (s, 1H), 3.77-4.20 (m, 14H), 2.96-3.40 (m, 4H),
2.76 (s, 3H), 1.75 (m, 2H), 1.53 (s, 3H), 1.40 (s, 3H). MS: 1004.0
[M + Na]+. 7f: N-[N′,N′′-Bis-(2,3-dimethoxycarbonyloxy-5-
chloro-benzoyl)-N-(methylaminopropyl)glycyl]ampicillin, from
5f, yield 40%. 1H NMR (DMSO-d6): δ 9.19 (d, 1H), 8.75 (m,
1H), 7.75-7.20 (m, 9H), 5.74 (d, 1H), 5.48 (q, 1H), 5.38 (d, 1H),
4.18 (s, 1H), 3.93 (m, 2H), 3.85 (m, 6H), 3.81 (m, 6H), 2.96-
3.40 (m, 4H), 2.78 (s 3H), 1.75 (m, 2H), 1.53 (s, 3H), 1.39 (s,
3H). MS: 1048.1 [M - H]-. 7g: N-[N′,N′′-Bis-(2,3-dimethoxy-
carbonyloxy-5-chloro-benzoyl)-N-(methylaminopropyl)glycyl]-
ampicillin, from 5g, yield 50%. 1H NMR (DMSO-d6): δ 9.20
(d, 1H), 8.75 (m, 1H), 7.85-7.25 (m, 9H), 5.74 (d, 1H), 5.38 (d,
1
40%. H NMR (DMSO-d6): δ 9.38 (s, 1H), 9.02 (m, 1H), 8.56
(m, 1H), 8.25, 8.35 (2× t, 1H), 6.60-7.45 (m, 10H), 5.55 (m,
2H), 5.38 (m, 1H), 4.18 (s, 1H), 3.95 (m, 2H), 3.03 (m, 2H),
3.13 (m, 3H), 2.27 (s, 6H), 2.21 (s, 3H), 2.17 (s, 3H), 1.54 (s,
3H), 1.41 (s, 3H), 1.40-1.60 (m, 4H). MS: 956.6 [M + Na]+.
9a : N-[7-(8-Methoxycarbonyloxy-1,3-benzoxazine-2,4-dione-3-
yl)-3-(2,3-dimethoxycarbonyloxy-benzoyl)-3-aza-heptanoyl]ampi-
1
cillin, from 6a , yield 55%. H NMR (DMSO-d6): δ 8.67-9.15,
2H), 7.25-7.90 (m, 11H), 5.67 (q, 1H), 5.51 (m, 1H), 5.38 (m,
1H), 4.19 (s, 1H), 4.15 (s, 2H), 3.90 (s, 3H), 3.83 (s, 3H), 3.77
(s, 3H), 3.71 (t, 2H), 3.08 (t, 2H), 1.53 (s, 3H), 1.40 (s, 3H),
1.35-1.65 (m, 4H). MS: 980.1 [M + Na]+. 9b: N-[6-(8-
Methoxycarbonyloxy-1,3-benzoxazine-2,4-dione-3yl)-3-(2,3-
dimethoxycarbonyloxy-benzoyl)-3-aza-2-methyl-hexanoyl]ampi-
cillin, from 6b, yield 40%. MS: 972.4 [M + Na]+, 948.6 [M -
H]-. 9c: N-[8-(8-Methoxycarbonyloxy-1,3-benzoxazine-2,4-di-
one-3-yl)-5-(2,3-dimethoxycarbonyloxy-benzoyl)-5-aza-4-meth-
1
yl-octanoyl]ampicillin, from 6c, yield 73%. H NMR (DMSO-
d6): δ 8.50-9.10 (m, 2H), 7.2-7.9 (m, 11H), 5.67 (q,1H), 5.51
(m, 1H), 5.37 (m, 1H), 4.19 (s, 1H), 3.8-4.0 (m, 9H), 3.05 (q,
1H), 1.8-2.2 (m, 4H), 1.75 (m, 2H), 1.55 (s, 3H), 1.40 (s, 3H),
1.15 (d, 3H). MS: 978.3 [M + H]+. 12: N-[3,5-Di-(2,3-di-
acetoxybenzoylamino)benzoyl]ampicillin, from 11, yield 25%.
1H NMR (DMSO-d6): δ 10.61 (s, 2H), 9.08 (d, 1H), 8.67 (d,
1H), 8.29 (m, 1H), 7.86 (d, 2H), 7.58 (m, 2H), 7.51 (d, 2H), 7.45
(s, 2H), 7.43 (d, 2H), 7.32 (m, 3H), 5.86 (d, 1H), 5.54 (q, 1H),
5.40 (d, 1H), 4.19 (s, 1H), 2.29 (s, 6H), 2.21 (s, 6H), 1.51 (s,
3H), 1.40 (s, 3H). MS: 922.3 [M - H]-. 14a : N-[3,5-N′,N′′-
Bis-(8-methoxycarbonyloxy-1,3-benzoxazine-2,4-dione-3-yl)ben-
1
zoyl]ampicillin, from 13a , yield 81%. H NMR (DMSO-d6): δ
9,13 (d, J ) 7.6, 1H), 8.94 (d, J ) 7.9, 1H), 8.15 (d, J ) 1.9,
2H), 7.92 (dd, J 1)7.8, J 2)1.5, 2H), 7.86 (dd, J 1)7.8, J 2)1.5,
2H), 7.72 (t, J ) 1.8, 1H), 7.49 (m, 4H), 7.32 (m, 3H), 5.89 (d,
J ) 8,0, 1H), 5.53 (q, 1H), 5.41 (d, J ) 3.8, 1H), 3.92 (s, 6H),
4.18 (s, 1H), 1.50 (s, 3H), 1.38 (s, 3H). MS: 922.2 [M - H]-.
14b: N-{[3,5-N,N′-Bis-(8-methoxycarbonyloxy-1,3-benzoxazine-
2,4-dione-3-yl)acetylamino]benzoyl}ampicillin, from 13b, yield
1
90%. H NMR (DMSO-d6): δ 10.48 (s, 2H), 9.06 (d, 1H), 8.86

Antibacterial Active Aminoacyl Penicillin Conjugates
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 14 3039
(d, 1H), 8.14 (m, 1H), 7.96-7.86 (m, 6H), 7.55-7.25 (m, 7H),
5.82 (d, 1H), 5.48 (q, 1H), 5.39 (d, 1H), 4.69 (s, 4H), 4.17 (s,
1H), 3.91 (s, 6H), 1.48 (s, 3H), 1.37 (s, 3H). MS: 1036.7 [M -
H]-. 14c: N-{[3,5-Bis-3-(8-methoxycarbonyloxy-1,3-benzox-
azine-2,4-dione-3-yl)propionylamino]benzoyl}ampicillin, from
the natural siderophore ferrioxamin B (Desferal mesylate,
Sigma, Germany) was used as control (Table 2).
In parallel to the growth promotion assays, the relative iron-
complexing capacity of the siderophore derivatives was checked
by the CAS assay according to Schwyn and Neilands (1987).23
A positive CAS reaction is associated with iron chelation (Table
2).
1
13c, yield 80%. H NMR (DMSO-d6): δ 10.23 (s, 2H), 9.11 (d,
1H), 9.01 (d, 1H), 8.07 (m, 1H), 7,89 (dd, J 1)8, J 2)1,5, 2H),
7,81 (dd, J 1)8.0, J 2)1.5, 2H), 7.66 (d, J ) 1.8,0, 2H), 7.46 (m,
4H), 7.30 (m, 3H), 5.83 (d, 1H), 5.48 (q, 1H), 5.38 (d, 1H), 4.17
(s, 1H), 4.16 (t, J ) 8.0, 4H), 3.91 (s, 6H), 2.69 (t, J ) 8, 4H),
1.51 (s, 3H), 1.39 (s, 3H). MS: 1064.5 [M - H]-. 15c: N-{3,5-
Bis-[3-(8-methoxycarbonyloxy-1,3-benzoxazin-2,4-dione-3-yl)-
The antibacterial activities of the conjugates 7-9, 10, 12,
14, and 15 were determined as minimal inhibitory concentra-
tions (MIC) by the microbroth dilution method in Mueller-
Hinton broth (Difco) according to the NCCLS guidelines24
(Table 3). Test organisms were from the American Type
Culture Collection (ATCC) and from the stock of the Hans
1
propionylamino]benzoyl}amoxicillin, from 13c, yield 40%. H
25
Kno¨ll institute (SG). S. maltophilia GN 12873 was kindly
NMR (DMSO-d6): δ 10.18 (s, 2H), 9.46 (br s, 1H), 8.70 (d, 1H),
8.43 (d, 1H), 8.11 (s, 1H), 7.89 (d, 2H), 7.79 (d, 2H), 7.63 (br s,
2H), 7.45 (t, 2H), 7.26 (d, 2H), 6.71 (d, 2H), 5.69 (d, 1H), 5.37
(q, 1H), 5.27 (d, 1H), 4.16 (t, 4H), 3.91 (s, 6H), 3.81 (s, 1H),
2.70 (t, 4H), 1.47 (s, 3H), 1.39 (s, 3H). MS: 1080.6 [M - H]-.
provided by the Episome Institute, Gunma (J apan).
The mechanism of action, e.g., the influence of FeIII uptake
routes and efflux systems on the activities of the conjugates,
was studied either in agar diffusion assays in Nutrient Agar
(Serva) or by determination of MIC by a microbroth dilution
technique (inoculum 105 CFU/mL) in Nutrient Broth (Serva).
The strains used were mutants of E. coli K-12 with alterations
in outer membrane porin proteins OmpC and OmpF (deleted
- or overexpressed +++), in the iron transport protein TonB
(Table 4), and in the catecholate Fe3+ siderophore receptors
Cir, Fiu, and FepA (strains were kindly provided by K. Hantke,
Tu¨bingen, Germany19) (Table 5). Compounds were tested with
addition of clavulanic acid in a concentration of 10 µg/mL to
neutralize the activity of the penicillinase integrated as a
selection marker in the mutants. In the agar diffusion assay,
50 µL of a 0.2 mM solution of the compounds was filled in agar
wells of 9 mm in diameter. Inhibition zones were read after
overnight incubation at 37 °C.
P r oced u r e for th e P r ep a r a tion of th e P en icillin V
Der iva tives 10a ,b. Isobutyl chloroformate (65 µL, 0.5 mmol)
was added with stirring at -15 °C to a solution of 0.5 mmol of
5m or n , respectively, and 56 µL of N-methyl morpholine in
absolute tetrahydrofuran (10 mL). The mixture was stirred
for 1 h at -10 °C, and then, 6-aminopenicillanic acid (108 mg,
0.5 mmol) and triethylamine (70 µL) in tetrahydrofuran/water
(5 mL, 4:1) were added. The mixture was stirred for 1 h at
-10 °C and for 1 h at ambient temperature and then
evaporated. Water was added to the residue, and the solution
was then carefully acidified using dilute aqueous HCl. The
mixture was extracted with ethyl acetate. The organic phase
was washed with brine, dried over Na2SO4, filtered, and
evaporated. The residue was purified by preparative HPLC.
From the obtained main fraction, the acetonitrile was removed
in a vacuum and the residual aqueous suspension was dried
by lyophilization to afford a colorless solid of 10a or b, respec-
tively. 10a : N-{4-[N,N′-Bis-(2,3-diacetoxybenzoyl)-N-(amino-
butyl)aminomethyl]phenoxyacetyl}-6-aminopenicillanic acid,
from 5m , yield 65%. 1H NMR (DMSO-d6): δ 8.59-8.65 (m,
1H), 8.24, 8.32 (2× t, changed to s at 360 K, 1H), 6.90-7.50
(m, 10H), 5.50-5.65 (m, 2H), 4.50 (m, 3H), 4.25 (m, 2H), 3.05-
3.25 (m, 4H), 2.15-2.25 (m, 12H), 1.40-1.60 (m, 10H). MS:
913.4 [M + Na]+, 889.2 [M - H]+. 10b: N-{2-[N,N′-Bis-(2,3-
diacetoxybenzoyl)-N-(aminobutyl)aminomethyl]phenoxyacetyl}-
6-aminopenicillanic acid, from 5n , yield 85%. 1H NMR (DMSO-
d6): δ 8.63-8.65 (2× d, 1H), 8.21, 8.31 (2× t, 1H), 6.95-7.40
(m, 10H), 5.47-5.57 (m, 2H), 4.70 (m, 3H), 4.24-4.35 (m, 2H),
3.03-3.19 (m, 4H), 2.12-2.27 (m, 12H), 1.46-1.57 (m, 10H).
MS: 913.6 [M + Na]+, 889.4 [M - H]-.
Ack n ow led gm en t. The financial support of Bundes-
ministerium fu¨r Bildung, Wissenschaft, Forschung und
Technologie (Berlin, Germany, FZ 0311232) and of
Gru¨nenthal GmbH, Aachen, Germany, is gratefully
acknowledged. We thank Andrea Perner, Renate Koch,
and Heike Heinecke for HRMS and NMR spectra and
Antje Fritzsche, Silke Leonhardt, Roswitha Peters,
Irmgard Heinemann, and Monika Golembiewski for
excellent technical assistance.
Su p p or tin g In for m a tion Ava ila ble: HRMS data of
â-lactam conjugates. This material is available free of charge
via Internet at http://pubs.acs.org.
Refer en ces
Det er m in a t ion of Biologica l Act ivit ies. Utilization of
siderophores was determined by growth promotion assays.
Bacterial strains were grown overnight on Nutrient Agar
(Serva, Germany), suspended in medium VA (glycerol, 50 mL/
L; NaCl, 8.5 g/L; pH 7.2), and diluted to the density of a 1
McFarland standard (approximately 1 × 109 cfu per mL). A
1.5 mL amount of this suspension was mixed with 99 mL of
test medium prepared according to Hall and Ratledge22
(glycerol, 20 mL/L; L-asparagin, 5 g/L; KH2PO4, 5 g/L in
distilled water, pH 7.5). After 20 g of Al2O3 was added and it
was sterilized at 121 °C for 15 min, the suspension was filtered
and the pH was adjusted to 6.8. A 12 g amount of agar No. 1
(Unipath, Germany) was added, and the medium was steril-
ized at 121 °C for 15 min. Prior to inoculation, trace elements
were added to the medium as a filter-sterilized solution (ZnSO4
× 7H2O, 2.03 mg; MnSO4 × 4H2O, 0.405 mg; MgSO4, 0.2 mg;
(1) Drechsel, H.; J ung, G. Peptide Siderophores. J . Pept. Sci. 1998,
4, 147-181.
(2) Bergeron, R. J .; Burton, P. S.; McGovern, K. A.; Onge, E. J . St.;
Streiff, R. R. Synthesis, Absorption, and Toxicity of N1,N8-Bis-
(2,3-dihydroxybenzoyl)spermidine, a Potent Iron Chelator. J .
Med. Chem. 1980, 23, 1130-1133.
(3) Corbin, J . L.; Bulen, W. A. The Isolation and Identification of
2,3-Dihydroxybenzoic Acid and 2-N,6-N-Di-(2,3-dihydroxyben-
zoyl)-L-lysine Formed by Iron-Deficient Azotobacter vinelandii.
Biochemistry 1969, 8, 757-762.
(4) Bergeron, R. J .; Kline, S. J .; Stollowich, N. J .; McGovern, K. A.;
Burton, P. S. Flexible Synthesis of Polyamine Catecholamides.
J . Org. Chem. 1981, 46, 4524-4529.
(5) Weitl, F.; Harris, W. R.; Raymond, K. N.; Sulfonated Catechol-
amide Analogues of Enterobactin as Iron Sequestering Agents.
J . Med. Chem. 1979, 22, 1281-1283.
(6) Buckley, G. M.; Pattenden, G.; Whiting, D. A. New Synthetic
Probes of the Iron Transport System of Paracoccus denitrificans.
Tetrahedron 1994, 50, 11781-11792.
(7) Schnabelrauch, M.; Wittmann, S.; Rahn, K.;. Reissbrodt, R.;
Mo¨llmann, U.; Heinisch, L. New synthetic catecholate-type
siderophores based on amino acids and dipeptides. BioMetals
2000, 13, 333-348.
CaCl2 × 2H2O, 1 mg; Na2MoO4 × 2H2O, 0.2 mg; CuSO4
×
5H2O, 0.2 mg; CoCl2 × 6H2O, 0.4 mg; and ethylenediamine-
di(o-hydroxy-phenylacetic acid) (EDDHA), 3.6 g). Siderophore
solutions were applied on assay disks (L 6 mm) on the surface
of the inoculated test agar plates, and the zones of growth
surrounding the disks were determined after incubation for
18-20 h at 37 °C.
Gram-negative bacterial strains were used as follows: P.
aeruginosa ATCC 27853, SG 137, NCTC 10662 and E. coli
ATCC 25922. For comparison with the synthetic siderophores,
(8) Wittmann, S.; Heinisch, L.; Scherlitz-Hofmann, I.; Mo¨llmann,
U.; Ankel-Fuchs, D. 8-Acyloxy-1,3-benzoxazine-2,4-diones as
siderophore components of antibiotics. Arzneim.-Forsch./ Drug
Res. 2000, 50, 752-757.
(9) Arisawa, M.; Sekine, Y.; Shimizu, S.; Takano, H.; Angehrn, P.;
Then, R. L. In vitro and in vivo evaluation of Ro 09-1428, a new
parenteral cephalosporin with high antipseudomonal activity.
Antimicrob. Agents Chemother. 1991, 35, 653-659.

3040 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 14
Heinisch et al.
(10) Mun˜oz-Bellido, J .-L.; Garcia Rodriguez, J . A. New trends in
cephalosporin developments: catechole and isoxazolidine cepha-
losporins. Infect. Dis. Clin. Practice 1996, 5, 232-243.
(11) Budzekiewicz, H. Siderophore-Antibiotic Conjugates Used as
Trojan Horses Against Pseudomonas aeruginosa. Curr. Top.
Med. Chem. 2001, 1, 73-82.
(12) Hennard, Ch.; Truong, Q. C.; Desnottes, J .-F.; Paris, J .-M.;
Moreau, N. J .; Abdallah, M. A. Synthesis and Activities of
Pyoverdin-Quinolone Adduct: A Prospective Approach to a
Specific Therapy against Pseudomonas aeruginosa. J . Med.
Chem. 2001, 44, 2139-2151.
(13) Kline, T.; Fromhold, M.; Mckennon, T. E.; Cai, S.; Treiberg, J .;
Ihle, N.; Sherman, D.; Schwan, W.; Hickey, M. J .; Warrener, P.;
Witte, P. R.; Brody, L. L.; Goltry, L.; Barker, L. M.; Anderson,
S. U.; Tanaka, S. K.; Shawar, R. M.; Nguyen, L. Y.; Langhorne,
M.; Bigelow, A.; Embuscado, L.; Naeemi, E. Antimicrobial Effects
of Novel Siderophores Linked to â-Lactam Antibiotics. Bioorg.
Med. Chem. 2000, 8, 73-93.
(14) Byk, G.; Gilon, Ch. Building Units for N-Backbone Cyclic
Peptides. 1. Synthesis of Protected N-(ω-Aminoalkylene)amino
Acids and Their Incorporation into Dipeptide Units. J . Org.
Chem. 1992, 57, 5687-5692.
(15) Craig, D. C.; Doughty, A.; Kassiou, M.; MacIntosh, M. G.; Read,
R. W. Reactions of Perhydro-2,2′-bipyrimidines with Carbonyl
Compounds Bearing R-Carbonyl Functionality. J . Org. Chem.
1993, 58, 5753-5758.
(16) Daukshas, W. K. Electrophilic substitution in 2,3-dihydroxy-
benzoic acid and its derivatives. J . Org. Chem. USSR 1979, 15,
1740-1743.
(17) Basker, M. J .; Frydrych, C. H.; Harrington, F. P.; Milner, P. H.
Antibacterial activity of catecholic piperacillin analogues. J .
Antibiot. 1989, 17, 1328-1330.
(18) Nakagawa, S.; Sanada, M.; Matsuda, K.; Hashizume, T.; Asahi,
Y.; Ushijima, R.; Ohtake, N.; Tanaka, N. In vitro and in vivo
antibacterial activities of BO-1341, a new antipseudomonal
cephalosporin. Antimicrob. Agents Chemother. 1989, 33, 1423-
1427.
(19) Hantke, K. Dihydroxybenzoylserinesa Siderophore for E. coli.
FEMS Microbiol. Lett. 1990, 67, 5-8.
(20) Bergeron, R. J .; McGovern, K. A.; Channing, M. A.; Burton, P.
S. Synthesis of N4-Acylated N1,N8-Bis(acyl)spermidines: An
Approach to the synthesis of Siderophores. J . Org. Chem. 1980,
45, 1589-1592.
(21) Heinisch, L.; Ulbricht, H.; Willitzer, H.; Hanschke, K. G.;
Tresselt, D.; Mo¨llmann, U.; Eckardt, K.; Haupt, I. Synthesis and
antibacterial activity of benzoylaminoacyl-penicillins and related
compounds. Arzneim.-Forsch./ Drug Res. 1992, 42, 6-673.
(22) Hall, M. J .; Ratledge, C. A simple method for the production of
mycobactin, the lipid-soluble siderophore, from mycobacteria.
FEMS Microbiol. Lett. 1982, 15, 133-136.
(23) Schwyn, B.; Neilands, J . B. Universal chemical assay for the
detection and determination of siderophores. Anal. Biochem.
1987, 160, 47-56.
(24) National Committee for Clinical Laboratory Standards. Methods
for Dilution Antimicrobial Susceptibility Tests for Bacteria that
Grow Aerobically, 4th ed.; Villanova, Ed.; Approved Standard
Document M7-A4. NCCLS, 1997.
(25) Sumita, Y.; Inoue, M.; Mitsuhashi, S. In vitro antibacterial
activity and beta-lactamase stability of new carbapenem SM-
7338. Eur. J . Clin. Microbial. Infect. Dis. 1998, 8, 908-916.
J M010546B