Novel β-lactam antibiotics synthesized by amination of catechols using fungal laccase

Article abstract of DOI:10.1248/cpb.56.902

Novel cephalosporins, penicillins, and carbacephems were synthesized by amination of catechols with amino-β-lactams like cefadroxil, amoxicillin, ampicillin and the structurally related carbacephem loracarbef using laccase from Trametes sp. All isolated monoaminated products inhibited the growth of several Gram positive bacterial strains in the agar diffusion assay, among them methicillin-resistant Staphylococcus aureus strains and vancomycin-resistant Enterococci. Observed differences in the cytotoxicity and in vivo activity in a Staphy-lococcus-infected, immune suppressed mouse model are discussed.

Full text of DOI:10.1248/cpb.56.902

902
Chem. Pharm. Bull. 56(7) 902—907 (2008)
Vol. 56, No. 7
b
Novel -Lactam Antibiotics Synthesized by Amination of Catechols Using
Fungal Laccase1)
Annett MIKOLASCH,*,a Martina WURSTER,b Michael LALK,b Sabine WITT,c Simone SEEFELDT,c
b
Elke HAMMER,d Frieder SCHAUER,a Wolf-Dieter JÜLICH,b and Ulrike LINDEQUIST
a Institute of Microbiology, Ernst-Moritz-Arndt-University Greifswald; b Institute of Pharmacy, Ernst-Moritz-Arndt-
University Greifswald; d Interfacultary Institute for Genetics and Functional Genomics Ernst-Moritz-Arndt-University
Greifswald; 17489 Greifswald, F.-L.-Jahn-Str. 15, Germany: and c Ganomycin Society of Biomedical Research Ltd.; 17489
Greifswald, Germany. Received January 11, 2008; accepted March 31, 2008; published online April 1, 2008
Novel cephalosporins, penicillins, and carbacephems were synthesized by amination of catechols with
amino-b-lactams like cefadroxil, amoxicillin, ampicillin and the structurally related carbacephem loracarbef
using laccase from Trametes sp. All isolated monoaminated products inhibited the growth of several Gram posi-
tive bacterial strains in the agar diffusion assay, among them methicillin-resistant Staphylococcus aureus strains
and vancomycin-resistant Enterococci. Observed differences in the cytotoxicity and in vivo activity in a “Staphy-
lococcus-infected, immune suppressed mouse” model are discussed.
Key words laccase; catechol; biotransformation; b-lactam antibiotic; resistance
In preceding papers, we discussed the laccase-catalyzed Laccase-catalyzed reaction between catechols (substrates 1a
amination of 2,5-dihydroxybenzoic acid derivatives with to 1c) on one hand and the amino-b-lactams cefadroxil 2a,
aminopenicillins and aminocephalosporins.2,3) The motive for amoxicillin 2b, ampicillin 2c, and the structurally related car-
our work was the global issue with resistant pathogenic bac- bacephem loracarbef 2d on the other hand led to cross cou-
teria, e.g. Streptococcus pneumoniae strains4—7) and Staphy- pled products (Table 1).
lococcus strains, against currently available b-lactam antibi-
otics.8—11) Aminated products like 2-(3,6-dioxocyclohexa- after an incubation time of 1.5 h, and monoaminated prod-
1,4-dienylamino)-2-phenyl-acetylamino-penicillanic acids ucts (3a to 3d) were detectable by high-performance liquid
3-Methylcatechol and amino-b-lactams were consumed
and 2-(3,6-dioxocyclohexa-1,4-dienylamino)-2-phenyl-acetyl- chromatography (HPLC). If other catechols or longer reac-
amino-cephalosporanic acids2,3) inhibited the growth of sev- tion times were used, monoaminated products were only ob-
eral Gram positive bacterial strains and protected mice tained in low yields and undesirable side reactions pro-
against an infection with Staphylococcus aureus. After ami- duced a number of by-products. Reaction kinetics similar to
nation the 2,5-dihydroxybenzoic acid derivatives were units these findings were described for hybrid dimer formation
of the C-7 substituent of the b-lactam antibiotics.
from 3,4-dichloroaniline and syringic acid24) or 3-(3,4-dihy-
The possibility of utilizing catechol units as an element of droxy-phenyl)-propionic acid and 4-aminobenzoic acid.25)
the C-7 substituent of penicillins and cephalosporins was in- In contrast, for hybrid dimer formation from 2,5-dihydroxy-
vestigated for a long time.12—17) Through the tonB dependent benzoic acid derivatives with aminopenicillins and amino-
iron transport mechanism,18) the use of catechol substituted cephalosporins,2,3) amino acids,26) and primary aromatic
b-lactams was known to increase the drug’s penetration into amines,27,28) we found straightforward biotransformations.
the bacterial cell walls. Thus, several catechol-substituted b- Therefore the amination of 2,5-dihydroxybenzoic acid deriv-
lactams have been synthesized and showed good antimicro- atives with laccase from Trametes sp. is an excellent method
bial activities.19—23)
for the synthesis of novel b-lactams whereas the amination of
As a consequence of the promising activity of the catechol catechols is limited to a few specific educts (Fig. 1).
substituted b-lactams on one hand and the novel laccase-syn-
Table 1. Products Obtained in Laccase-Catalyzed Biotransformation
thesized b-lactams on the other hand, we got interested in
laccase-catalyzed amination of catechols with aminopeni-
cillins and aminocephalosporins.
In this study, we have employed laccase from Trametes sp.
to derivatize amino-b-lactams and to couple them both with
catechol and with substituted catechols. The novel b-lactams
Substances
1a
1b
1c
were structurally characterized as new coupling products in-
hibiting the growth of several Gram positive bacterial strains
in the agar diffusion assay, among them methicillin-resistant
Staphylococcus aureus and vancomycin-resistant Entero-
cocci. The cytotoxicity of the new compounds and the effec-
tiveness in a “Staphylococcus-infected, immune suppressed
mouse” model are discussed.
2a
2b
2c
2d
1a) [3a]b)
(ꢀ5)c)
1 [3b]
(ꢀ5)
1 [3c]
(ꢀ5)
1 [3e]
(ꢀ5)
4
(ꢀ5)
4
(ꢀ5)
3
(ꢀ5)
ꢀ5
(ꢀ5)
ꢀ5
(ꢀ5)
ꢀ5
(ꢀ5)
ꢀ5
(ꢀ5)
1 [3d]
(ꢀ5)
Results and Discussion
a) Number of products after 1.5 h. b) Description of products analyzed in more
Biotransformation of Amino-b-lactams by Laccases detail. c) Number of products in parentheses after 5 h.
∗ To whom correspondence should be addressed. e-mail: annett.mikolasch@gmx.de © 2008 Pharmaceutical Society of Japan

July 2008
903
Fig. 1. Catechols (1a, 1b), b-Lactam Antibiotics (2a to 2d), and the Products 3a to 3e
After separation of the products 3a to 3e by solid phase logical activity because of the described activities of other
extraction, mass spectral analyses (LC/MS with API-ES pos- ortho-quinonoid30—32) or para-quinonoid33—35) substances.
itive modes) of the compounds showed a molecular mass at-
Biological Activity of the Biotransformation Products
tributed to the coupling of methylcatechol (1a or 1b) with Growth inhibition of the new b-lactam antibiotics 3a to 3e
one amino-b-lactam (2a to 2d) under loss of four hydrogen against Gram positive strains was determined by the agar dif-
atoms indicating a quinonoid structure of the products. Fur- fusion method according to Burkhardt,36) and the results for
1
thermore, H-NMR spectral data of 3a to 3e contained the selected strains—among them multidrug resistant Staphylo-
characteristic signals for both substrates (1a, 1b) and educts coccus and Enterococcus strains—are summarized in Table
(2a to 2d). The number of CH proton signals of the dihy- 2. The growth inhibition values of cefadroxil, amoxicillin,
droxylated phenyl rings changed from three—in the sub- ampicillin, and loracarbef (2a to 2d) and the catechol deriva-
strates—to two signals—in the products. The multiplicity of tives are also presented for comparison. 3b and 3c showed
the two signals of the product indicated a further substituent balanced antibacterial activity against all Gram positive
at the C1ꢁ. HMBC spectra measured in DMSO showed two strains except against S. epidermidis 535 which is strongly
typical correlations for quinones in the range of 180 ppm. resistant to penicillin and cephalosporin families. The new
This is an important indication for the quinonoid character of ortho-quinonoid substituted penicillins (3b, 3c) showed more
3a to 3e, confirming the oxidation of catechols to quinones potent activity against the tested strains than the new ortho-
similar to the hybrid dimer quinones formed from 2,5-di- quinonoid substituted cephalosporin and carbacephem deriv-
hydroxybenzoic acid derivatives with penicillins,2) amino atives (3a, 3d, 3e) comparable with the results of the para-
acids,26) and primary aromatic amines,27,28) and for the hybrid quinonoid substituted penicillins,2) cephalosporins, and car-
dimer quinones produced from 3,4-dichloraniline with proto- bacephems.3) Investigations towards the stability of the syn-
catechuic acid and syringic acid.24,29) Although 3a to 3e have thesized compounds showed limited lifetime in aqueous so-
quinone units as an element of the C-7 or C-6 substituent in- lution. Incubation at 30 °C of solutions of 3a to 3e showed
stead of catechol units these products should also have bio- decomposition after 2 h. Therefore the survey of the antimi-

904
Vol. 56, No. 7
Table 2. Antimicrobial Activity of Products 3a to 3e, and Educts 1a, 1b, 2a to 2d
S. aureus
Nord-
deutscher
Stamm
Entero-
coccus
faecalis 769 aureus 315
Staphylo-
coccus
S. aureus
ATCC
6538
S. epider- S. epider- S. epider-
Amount
n [mmol]
S. aureus
36881
S. aureus
38418
S. aureus
520
Compound
midis
midis
midis
125
535
847
3a
0.02
0.1
0.2
ra)
r
10
r
10
14
r
r
10
10b)
18
22
r
r
10
16
20
26
r
10
12
12
20
26
r
r
10
r
14
16
3b
3c
3d
3e
2a
2b
2c
2d
1a
1b
0.02
0.1
0.2
20
26
28
r
12
14
r
12
14
30
ꢀ30
ꢀ30
r
10
14
30
ꢀ30
ꢀ30
r
10
14
20
24
28
r
r
10
14
20
22
0.02
0.1
0.2
20
28
30
r
12
14
r
12
14
30
ꢀ30
ꢀ30
10
14
18
30
ꢀ30
ꢀ30
r
10
14
20
26
28
r
r
10
16
22
24
0.02
0.1
0.2
r
r
r
r
8
12
r
r
10
14
24
28
r
r
10
20
30
ꢀ30
r
8
14
14
22
26
r
r
10
r
14
18
0.02
0.1
0.2
r
r
r
r
r
10
r
r
12
12
22
26
r
r
r
18
30
ꢀ30
r
r
12
16
26
28
r
r
r
r
16
18
0.02
0.1
0.2
r
10
16
r
r
r
r
14
20
20
30
ꢀ30
r
r
12
30
ꢀ30
ꢀ30
r
r
r
22
30
ꢀ30
r
r
r
10
22
26
0.02
0.1
0.2
20
26
28
r
12
14
r
12
14
30
ꢀ30
ꢀ30
r
10
14
ꢀ30
ꢀ30
ꢀ30
r
8
10
20
24
28
r
r
r
18
24
26
0.02
0.1
0.2
24
28
30
r
12
14
r
12
16
30
ꢀ30
ꢀ30
10
14
18
30
ꢀ30
ꢀ30
r
10
14
24
28
30
r
r
10
18
24
28
0.02
0.1
0.2
r
r
12
16
20
22
r
20
28
24
30
ꢀ30
r
8
14
28
ꢀ30
ꢀ30
r
r
8
22
30
ꢀ30
r
r
r
10
20
24
0.02
0.1
0.2
r
r
r
r
r
r
r
r
r
r
r
r
r
r
r
r
r
r
r
r
10
r
r
8
r
r
r
r
r
10
0.02
0.1
0.2
r
r
r
r
r
r
r
r
r
r
r
r
r
r
r
r
r
r
r
r
10
r
r
r
r
r
r
r
r
r
a) Resistent (no zone of inhibition). b) Diameter of inhibition zone (mm).
Table 3. Cytotoxic Activity of Products 3a to 3e, and Educts 1a, 1b, 2a to
2d
crobial effects was concentrated on the initial screening
using the agar diffusion test.
Cytotoxicity of the new b-lactam antibiotics (3a to 3e) and
of cefadroxil, amoxicillin, ampicillin, and loracarbef (2a to
2d) was determined by the neutral red uptake assay37) using
FL-cells, a human amniotic epithelial cell line (Table 3).
Whereas 3a was not cytotoxic against FL cells in concentra-
tions tested, 3b to 3e showed slight cytotoxicity. Remarkable
is the fact, that the methylcatechol substrates were strong cy-
totoxic in all concentrations tested whereas cytotoxicity of
products (3b to 3e) were comparable low, but not negligible.
Aminopenicillins and aminocephalosporins synthesized by
amination of 2,5-dihydroxybenzoic acid derivatives showed
no or only weak cytotoxicity against FL cells in concentra-
tions up to 100 mg/ml.2,3)
Compound
IC50 (mM)
3a
3b
3c
3d
3e
2a
2b
2c
2d
1a
1b
ꢀ200
250
197
142
128
ꢀ275
ꢀ275
ꢀ285
ꢀ285
ꢀ100
ꢀ100
A “Staphylococcus-infected, immune suppressed mouse”

July 2008
905
Experimental
Table 4. Effectiveness of in Vitro Active Products in the “Staphylococcus-
Enzymes Extracellular laccase C of Trametes sp. (EC 1.10.3.2) was ob-
tained from ASA Spezialenzyme (Wolfenbüttel, Germany) and used in an
activity of 800 nmol·mlꢂ1·minꢂ1 (substrate: 2,2ꢃ-amino-bis-3-ethylbenzthi-
azoline-6-sulfonic acid).
Infected, Immune Suppressed Mouse” Model
Survived/treated Survived/control
Compound
Dose
mice n/n
mice n/n
Chemicals Chemicals were purchased from commercial suppliers: ce-
fadroxil, cefaclor and cefalexin from Sigma-Aldrich Germany, loracarbef
from Eli Lilly Germany, 2,5-dihydroxy-N-(2-hydroxyethyl)benzamide from
Midori Kagaku Co., Japan, 2,5-dihydroxybenzoic acid methyl ester and 2,5-
dihydroxybenzoic acid ethyl ester from Sigma-Aldrich. All chemicals were
used as received. 2,5-Dihydroxybenzamide was synthesized as described
previously.28)
Conditions of Biotransformation b-Lactam antibiotics [2a to 2d
(13 mg)] were dissolved in 15 ml sodium acetate buffer, 20 mM pH 5.6. After
addition of laccase C (activity 800 nmol·mlꢂ1·minꢂ1), the reaction mixtures
were completed by a solution of 5 mg catechols (1a to 1c) in 5 ml sodium
acetate buffer, pH 5.6. The reaction mixtures were incubated for 5 h at room
temperature with agitation at 400 rpm.
For preparative scale the same procedure was used as described above
apart from lager amount of educts [1a (31 mg in 25 ml for production of 3a,
9.9 mg in 10 ml for production of 3b, 37 mg in 25 ml for production of 3c,
44 mg in 25 ml for production of 3d), 1b (31 mg in 25 ml for production of
3e), 2a, 2d (58 mg in 75 ml), 2b (26 mg in 30 ml), 2c (49 mg in 75 ml)] and
shorter reaction time (1.5 h).
Analytical High-Performance Liquid Chromatography (HPLC) For
routine analysis, samples of the incubation mixture were analyzed by an
HPLC system (Dionex, Idstein, Germany) consisting of a Gynkotek High
Precision Pump Model 480, Dionex ASI-100 Automated Sample-Injector,
Staphylococcus aureus ATCC 6538
2a
2b
3a
2ꢆ0.5 mg
10/10
10/10
3/6
0/10
(25 mg/kg)
2ꢆ0.5 mg
(25 mg/kg)
2ꢆ0.5 mg
(25 mg/kg)
2ꢆ1.0 mg
(50 mg/kg)
2ꢆ0.5 mg
(25 mg/kg)
2ꢆ1.0 mg
(50 mg/kg)
2ꢆ1.5 mg
(75 mg/kg)
2ꢆ0.5 mg
(25 mg/kg)
2ꢆ1.0 mg
(50 mg/kg)
0/10
0/10
0/5
1/3
3b
3c
4/6
0/10
0/5
3/3
1/3
0/5
4/6
0/10
0/5
2/3
model was used for the examination of in vivo effectiveness Dionex UVD 340S Diode-Array-Detector, and Chromeleon Version 6.30.
An endcapped, 5-mm, LiChroCart 125-4 RP 18 column (Merck, Darmstadt,
Germany) was used. A solvent system consisting of methanol (eluent A) and
phosphoric acid, 0.1% pH 2 (eluent B), starting from an initial ratio of 10%
A and 90% B and reaching 100% A within 14 min, was used at a flow rate
of 1 ml/min.
Procedure for Isolation of Biotransformation Products After activa-
tion and equilibration, an RP18 silicagel column (20 cc, 5 g absorbent mate-
rial, Waters, Manchester, U.K.) was charged with the incubation mixture.
The column was washed twice with 20 ml of a mixture of methanol (10%)
and distilled water (90%). The products were eluted with 10 ml acetonitrile.
The acetonitrile eluates were dried using a vacuum rotator at 30 °C.
Characterization of Biotransformation Products Products were ana-
lyzed by mass spectrometry (LC/MS with API-ES in positive modes).
The nuclear magnetic resonance (NMR) spectra were obtained at
600 MHz (1H and HMBC) in dimethyl sulfoxide-d6 (DMSO-d6) or acetoni-
trile-d3.
against Gram positive strains of in vitro active products
(Table 4). 30 to 60% of the mice treated i.p. with biotransfor-
mation products survived the infection with Staphylococcus
aureus ATCC 6538, whereas all untreated mice died after in-
fection within 2 d. But all mice treated with cefadroxil and
amoxicillin survived the infection with Staphylococcus au-
reus ATCC 6538 without any noticeable problems, whereas
the mice treated with biotransformation products showed
symptoms of intoxication. In contrast to these results we re-
ported on para-quinonoid substituted penicillins,2) cephalo-
sporins and carbacephems,3) which protected mice against an
infection with Staphylococcus aureus without any hints of in-
toxication. Of the two series of cephalosporins and peni-
cillins, the ones bearing para-quinonoid structures2,3) showed
more potent activity in protecting mice against an infection
with Staphylococcus aureus than another with ortho-
quinonoid structures (3a to 3e), possibly due to the different
cytotoxicity.
7-[2-(5-Methyl-3,4-dioxocyclohexa-1,5-dienylamino)-2-(4-hydroxy-
phenyl)-acetylamino]-cephalosporanic Acid 3a Synthesis and isolation
as described above. Dark red solid. Yield 27% (21 mg). 1H-NMR d (DMSO-
d6) 1.86 (s, 3H, H7ꢁ), 1.99 (s, 3H, H10), 3.29 (d, Jꢄ18.4 Hz, 1H, H2), 3.49
(d, Jꢄ18.4 Hz, 1H, H2), 4.89 (d, Jꢄ4.4 Hz,1H, H6), 5.19 (d(s), Jꢄ1.5 Hz,
1H, H2ꢁ), 5.31 (d, Jꢄ7.0 Hz, 1H, H13), 5.65 (dd, Jꢄ4.7, 8.1 Hz, 1H, H7),
Concluding, the activity in vivo of the novel penicillins 6.76 (d, Jꢄ8.2 Hz, 2H, H3ꢃ, H5ꢃ), 7.14 (d(s), Jꢄ1.5 Hz, 1H, H6ꢁ), 7.27 (d,
Jꢄ8.2 Hz, 2H, H2ꢃ, H6ꢃ), 8.71 (d, Jꢄ7.1 Hz, 1H, H14), 9.29 (d, Jꢄ8.3 Hz,
and cephalosporins with ortho-quinonoid structure is unsatis-
factory because of the cytotoxicity based on the ortho-
quinonoid structure. But the laccase-catalyzed amination of
1H, H11). 13C-NMR d (DMSO-d6, determination of carbon assignment by
HMBC) 15.6 (C7ꢁ), 19.7 (C10), 29.1 (C2), 57.1 (C6), 58.8 (C7), 59.5 (C13),
95.1 (C2ꢁ), 115.7 (C3ꢃ, C5ꢃ), 122.9 (C4), 126.8 (C1ꢃ), 128.9 (C2ꢃ, C6ꢃ),
catechols with aminopenicillins and aminocephalosporins
is a new method to synthesize novel penicillins and
cephalosporins by enzymatic transformation. These reactions
represent new, low-cost processes, which allow the use of
mild reaction conditions, aqueous solvent systems, normal
pressure, and room temperature. Further advantage is the
specificity of the reaction, which can comprise the ami-
nation of para- and ortho-dihydroxylated aromatic com-
pounds2,3,25,27,28) on one hand and the combination of two an-
tibiotics containing a phenol moiety,38) the introduction of a
phenolic compound into an antibiotic containing a phenolic
moiety,39) and the oxidation of an antibiotic40) on the other
hand. In summary it can be concluded that the laccase has a
high potential for the synthesis of new antibiotic substances.
Further studies are in progress.
129.6 (C3), 133.7 (C6ꢁ), 140.0 (C5ꢁ), 155.2 (C1ꢁ), 157.8 (C4ꢃ), 163.8 (C9),
1
164.1 (C8), 170.0 (C12), 174.7 (C3ꢁ), 183.6 (C4ꢁ). HMBC H–13C correla-
tions H7ꢁ (C1ꢁ, C4ꢁ, C5ꢁ, C6ꢁ), H10 (C2, C3, C4, C9), H2 (C3, C4, C6, C9,
C10), H6 (C2, C8), H2ꢁ (C13, C3ꢁ, C4ꢁ, C5ꢁ, C6ꢁ), H13 (C12, C1ꢃ, C2ꢃ,
C6ꢃ, C1ꢁ), H7 (C6, C8, C12), H3ꢃ, H5ꢃ (C1ꢃ, C3ꢃ, C4ꢃ, C5ꢃ), H6ꢁ (C2ꢁ, C4ꢁ,
C5ꢁ, C7ꢁ), H2ꢃ,H6ꢃ (C13, C2ꢃ, C3ꢃ, C4ꢃ, C5ꢃ, C6ꢃ), H14 (C12, C13, C1ꢃ,
C1ꢁ, C2ꢁ, C3ꢁ, C6ꢁ), H11 (C7, C8, C12). LC/MS m/z 483.4 ([M]ꢅ, 504.8
[MꢅNa]ꢅ API-ES pos. mode).
6-[2-(5-Methyl-3,4-dioxocyclohexa-1,5-dienylamino)-2-(4-hydroxy-
phenyl)-acetylamino]-penicillanic Acid 3b Synthesis and isolation as de-
scribed above. Dark red solid. Yield 28% (10 mg). 1H-NMR (acetonitrile-d3)
d 1.46 (s, 3H, H9 or H10), 1.54 (s, 3H, H9 or H10), 4.33 (s, 1H, H3), 5.13
(d, Jꢄ6.7 Hz, 1H, H13), 5.14 (s, 1H, H2ꢁ), 5.41 (d, Jꢄ3.8 Hz,1H, H5), 5.55
(dd, Jꢄ3.9, 8.7 Hz, 1H, H6), 6.82 (s, 1H, H6ꢁ), 6.84 (d, Jꢄ8.4 Hz, 2H, H3ꢃ,
H5ꢃ), 7.29 (d, Jꢄ8.3 Hz, 2H, H2ꢃ, H6ꢃ). LC/MS m/z 485.3 ([M]ꢅ, 507.3
[MꢅNa]ꢅ API-ES pos. mode).
6-[2-(5-Methyl-3,4-dioxocyclohexa-1,5-dienylamino)-2-phenyl-acetyl-
amino]-penicillanic Acid 3c Synthesis and isolation as described above.

906
Vol. 56, No. 7
Dark red solid. Yield 83% (55 mg). 1H-NMR d (DMSO-d6) 1.41 (s, 3H, H9
or H10), 1.55 (s, 3H, H9 or H10), 1.87 (s, 3H, H7ꢁ), 4.18 (s, 1H, H3), 5.22
(s, 1H, H2ꢁ), 5.39 (d, Jꢄ3.7 Hz, 1H, H5), 5.52 (dd, Jꢄ3.7, 7.8 Hz, 1H, H6),
5.55 (br s, 1H, H13), 7.18 (s, 1H, H6ꢁ), 7.34 (dd, Jꢄ7.1 Hz, 1H, H4ꢃ), 7.40
(dd, Jꢄ7.4, 7.1 Hz, 2H, H3ꢃ, H5ꢃ), 7.47 (d, Jꢄ7.6 Hz, 2H, H2ꢃ, H6ꢃ), 8.84
(br s, 1H, H14), 9.20 (d, Jꢄ7.6 Hz, 1H, H11). 13C-NMR d (DMSO-d6, deter-
mination of carbon assignment by HMBC) 15.1 (C7ꢁ), 26.4 (C9 or C10),
30.1 (C9 or C10), 57.7 (C6), 58.8 (C13), 63.8 (C2), 66.8 (C5), 70.7 (C3),
95.3 (C2ꢁ), 127.0 (C2ꢃ, C6ꢃ), 128.3 (C4ꢃ), 128.7 (C3ꢃ, C5ꢃ), 136.3 (C1ꢃ),
133.0 (C6ꢁ), 139.8 (C5ꢁ), 154.7 (C1ꢁ), 168.4 (C12), 169.1 (C8), 172.6 (C7),
pleted by L-glutamin (1%, Sigma), penicillin G/streptomycin (1%, Sigma)
and FCS (10%, Biocrom). After 24 h 50 ml of the test solution (test sub-
stance dissolved in 20 ml DMSO under stirring in an ultrasonic bath for
5 min and then diluted with 1 ml medium) or medium with equal amounts of
DMSO (control) were added. After a further incubation for 72 h cells were
washed three times with phosphate buffered saline solution (PBS). One hun-
dred microliter neutral red solution (Sigma, 0.3% in DMEM) was added per
well. The cells were then incubated for 3 h at 37 °C, followed by another
three times washing with PBS. A solution (100 ml) of acetic acid (1%, v/v)
and ethanol (50%, v/v) in distilled water were added. After shaking for
15 min the optical density was measured at 492 nm with a Micro Screener
LB 9260 (EG&G Berthold, Bad Wildbad, Germany). The mean of three
measurements for each concentration was determined (nꢄ3).
1
182.8 (C4ꢁ). HMBC H–13C correlations H9, H10 (C3, C5, C9, C10), H7ꢁ
(C1ꢁ, C4ꢁ, C5ꢁ, C6ꢁ), H3 (C2, C5, C7, C8, C9, C10), H2ꢁ (C3ꢁ, C4ꢁ, C6ꢁ),
H5 (C6, C7), H6 (C5, C7, C12), H13 (C12, C1ꢃ, C2ꢃ, C6ꢃ, C1ꢁ), H6ꢁ (C2ꢁ,
C4ꢁ, C7ꢁ), H4ꢃ (C2ꢃ, C6ꢃ), H3ꢃ, H5ꢃ (C1ꢃ, C3ꢃ, C5ꢃ), H2ꢃ, H6ꢃ (C13, C2ꢃ,
C4ꢃ, C6ꢃ), H11 (C6, C7, C12). LC/MS m/z 469.4 ([M]ꢅ, 491.3 [MꢅNa]ꢅ
API-ES pos. mode).
Animal Assays
A “Staphylococcus-infected, immune suppressed
mouse” model was established for the examination of in vivo effectiveness
of in vitro selected drugs. In this model 8-weeks old female BALB/C mice
(3 mice/group/assay) were pre-treated with cyclophosphamide (250 mg/kg
intraperitoneal (i.p.) day ꢂ3 and 100 mg/kg i.p. day ꢂ1, Sigma) to suppress
the immune answer. Three days later they were infected with Staphylococcus
aureus ATCC 6538, i.p., in a lethal dose (1010—1012 colony forming units).
The test agents were injected 30 min and 6 h after the infection with Staphy-
lococcus aureus ATCC 6538. The concentration of the test agents was se-
lected according to therapeutically used doses of ampicillin. The antibiotic
effectiveness was recognised within the next 2—6 d.
3-Chloro-7-[2-(5-methyl-3,4-dioxocyclohexa-1,5-dienylamino)-2-
phenyl-acetylamino}-8-oxo-5-azabicyclo[4.2.0]oct-3-ene-4-carboxylic
Acid 3d Synthesis and isolation as described above. Dark red solid. Yield
1
81% (63 mg). H-NMR d (DMSO-d6) 1.35 (m, 2H, H1), 1.86 (s, 3H, H7ꢁ),
2.48 (m, 2H, H2), 3.72 (m, Jꢄ7.8 Hz, 1H, H6), 5.16 (s, 1H, H2ꢁ), 5.25 (dd,
Jꢄ7.9, 5.6 Hz, 1H, H7), 5.33 (s, 1H, H13), 7.19 (s, 1H, H6ꢁ), 7.35 (dd,
Jꢄ7.4 Hz, 1H, H4ꢃ), 7.40 (dd, Jꢄ7.4 Hz, 2H, H3ꢃ, H5ꢃ), 7.49 (d, Jꢄ7.6 Hz,
2H, H2ꢃ, H6ꢃ), 8.88 (br, 1H, H14), 9.39 (d, Jꢄ8.0 Hz, 1H, H11). 13C-NMR d
(DMSO-d6, determination of carbon assignment by HMBC) 21.5 (C1), 28.0
(C2), 51.4 (C6), 57.7 (C7), 60.1 (C13), 95.0 (C2ꢁ), 120.2 (C4), 127.2 (C4ꢃ),
127.6 (C2ꢃ, C6ꢃ), 128.5 (C3), 128.7 (C3ꢃ, C5ꢃ), 133.5 (C6ꢁ), 136.5 (C1ꢃ),
Acknowledgment Parts of this work were supported financially by the
Stiftung Industrieforschung (S574). We thank R. Jack (Institute of Immunol-
ogy, University of Greifswald) for help in preparing the manuscript.
1
163.9 (C8), 169.1 (C12), 183.0 (C4ꢁ). HMBC H–13C correlations H2 (C1,
C3, C4, C6), H6 (C2, C8), H2ꢁ (C4ꢁ, C6ꢁ), H7 (C6, C8, C12), H13 (C12,
C1ꢃ, C2ꢃ, C6ꢃ, C1ꢁ), H6ꢁ (C2ꢁ, C4ꢁ, C7ꢁ), H4ꢃ (C2ꢃ, C6ꢃ), H3ꢃ,H5ꢃ (C1ꢃ,
C3ꢃ, C5ꢃ), H2ꢃ, H6ꢃ (C13, C2ꢃ, C4ꢃ, C6ꢃ), H11 (C7, C8, C12). LC/MS m/z
469.5 ([M]ꢅ, 491.3 [MꢅNa]ꢅ API-ES pos. mode).
References and Notes
1) Part III of “Enzymatic Synthesis and Activity of Novel b-Lactam An-
tibiotics”; for Part I see ref. 2 and for Part II see ref. 3.
2) Mikolasch A., Niedermeyer T. H. J., Lalk M., Witt S., Seefeldt S.,
Hammer E., Schauer F., Gesell M., Hessel S., Jülich W. D., Lindequist
U., Chem. Pharm. Bull., 54, 632—638 (2006).
3) Mikolasch A., Niedermeyer T. H. J., Lalk M., Witt S., Seefeldt S.,
Hammer E., Schauer F., Gesell M., Hessel S., Jülich W. D., Lindequist
U., Chem. Pharm. Bull., 55, 412—416 (2007).
4) Appelbaum P. C., Eur. J. Clin. Microbiol., 6, 367—377 (1987).
5) Critchley I. A., Karlowsky J. A., Draghi D. C., Jones M. E., Thorns-
berry C., Murfitt K., Sahm D. F., Antimicrob. Agents Chemother., 46,
550—555 (2002).
6) Fluit A. C., Schmitz F. J., Jones M. E., Acar J., Gupta R., Verhoef J.,
Int. J. Infect. Dis., 3, 153—156 (1997).
7) Klugman K. P., Clin. Microbiol. Rev., 3, 171—196 (1990).
8) Shimizu M., Shiota S., Mizushima T., Ito H., Hatano T., Yoshida T.,
Tsuchiya T., Antimicrob. Agents Chemother., 45, 3198—3201 (2001).
9) Fisher J. F., Meroueh S. O., Mobashery S., Chem. Rev., 105, 395—424
(2005).
10) Waley S. G., “The Chemistry of b-Lactams,” ed. by Page M. I.,
Blackie A & P, London, 1992, pp. 198—228.
11) Helfand M. S., Bonomo R. A., Curr. Opin. Pharmacol., 5, 452—458
(2005).
12) Ohi N., Aoki B., Shinozaki T., Moro K., Noto T., Nehashi T., Okazaki
H., Matsunaga I., J. Antibiot., 39, 230—241 (1986).
13) Ohi N., Aoki B., Shinozaki T., Moro K., Kuroki T., Noto T., Nehashi
T., Matsumoto M., Okazaki H., Matsunaga I., Chem. Pharm. Bull., 35,
1903—1909 (1987).
14) Ohi N., Aoki B., Moro K., Kuroki T., Sugimura N., Noto T., Nehashi
T., Matsumoto M., Okazaki H., Matsunaga I., J. Antibiot., 39, 242—
250 (1986).
15) Ohi N., Aoki B., Kuroki T., Matsumoto M., Kojima K., Nehashi T., J.
Antibiot., 40, 22—28 (1987).
7-[2-(6-Methyl-3,4-dioxocyclohexa-1,5-dienylamino)-2-(4-hydroxy-
phenyl)-acetylamino]-cephalosporanic Acid 3e Synthesis and isolation
as described above. Dark red solid. Yield 78% (61 mg). 1H-NMR d (DMSO-
d6) 1.99 (s, 3H, H10), 2.32 (s, 3H, H7ꢁ), 3.29 (d, Jꢄ18.1 Hz, 1H, H2), 3.48
(d, Jꢄ18.4 Hz, 1H, H2), 4.99 (d, Jꢄ4.4 Hz, 1H, H6), 5.12 (s, 1H, H2ꢁ), 5.25
(d, Jꢄ6.1 Hz, 1H, H13), 5.69 (dd, Jꢄ4.9, 7.8 Hz, 1H, H7), 6.32 (s, 1H, H5ꢁ),
6.76 (d, Jꢄ8.2 Hz, 2H, H3ꢃ, H5ꢃ), 7.11 (d, Jꢄ6.1 Hz, 1H, H14), 7.34 (d,
Jꢄ8.2 Hz, 2H, H2ꢃ, H6ꢃ), 9.29 (d, Jꢄ8.4 Hz, 1H, H11). 13C-NMR d
(DMSO-d6, determination of carbon assignment by HMBC) 17.5 (C7ꢁ), 19.5
(C10), 28.6 (C2), 56.8 (C6), 58.8 (C7), 59.5 (C13), 97.7 (C2ꢁ), 115.9 (C3ꢃ,
C5ꢃ), 123.0 (C4), 126.7 (C1ꢃ), 128.7 (C2ꢃ, C6ꢃ), 129.4 (C5ꢁ), 129.6 (C3),
147.2 (C6ꢁ), 154.3 (C1ꢁ), 158.0 (C4ꢃ), 163.6 (C9), 163.9 (C8), 170.2 (C12),
1
175.6 (C3ꢁ), 182.8 (C4ꢁ). HMBC H–13C correlations H2 (C3, C4, C6, C9,
C10), H6 (C2, C8), H2ꢁ (C3ꢁ, C4ꢁ, C5ꢁ, C6ꢁ, C7ꢁ), H13 (C12, C1ꢃ, C2ꢃ, C6ꢃ,
C1ꢁ), H7 (C6, C8, C12), H5ꢁ (C1ꢁ, C3ꢁ, C7ꢁ), H3ꢃ, H5ꢃ (C1ꢃ, C3ꢃ, C4ꢃ, C5ꢃ),
H14 (C12, C2ꢁ, C6ꢁ), H2ꢃ, H6ꢃ (C13, C2ꢃ, C3ꢃ, C4ꢃ, C5ꢃ, C6ꢃ), H11 (C7,
C8, C12). LC/MS m/z 483.4 ([M]ꢅ, 504.9 [MꢅNa]ꢅ API-ES pos. mode).
Determination of Antibacterial Activity An agar diffusion method ac-
cording to Burkhardt36) was used to determine antibacterial activity. Sterile
Mueller-Hinton II-Agar in Stacker petri dishes (Becton Dickinson Microbi-
ology systems, Cockeysville, U.S.A.) was inoculated with bacterial cells
(200 ml of bacterial cell suspension—1.5ꢆ108 cells—on 20 ml medium).
The following bacterial strains were used: Staphylococcus aureus ATCC
6538 and S. aureus Norddeutscher Epidemiestamm. Besides these the agents
were tested against the following multidrug resistant strains isolated from
patients: S. aureus 315, S. aureus 36881, S. aureus 38418, S. aureus 520, S.
epidermidis 125, S. epidermidis 535, S. epidermidis 847, and Enterococcus
faecalis 769. The test samples were applied in different concentrations on
sterile paper discs (Sensi-Disc, 6 mm diameter, Becton Dickinson Microbi-
ology systems). Test concentrations were selected according to the concen-
tration of the standard antibiotics (cefadroxil, amoxicillin, ampicillin, and
loracarbef) on the Sensi discs. Plates were kept for 3 h in a refrigerator to
enable prediffusion of the substances into the agar and were then incubated
for 24 h at 37 °C. Average inhibition zone diameters were calculated from 3
replicates.
16) Choi K. I., Cha J. H., Pae A. N., Cho Y. S., Kang H.-Y., Koh H. Y.,
Chang M. H., J. Antibiot., 48, 1371—1374 (1995).
17) Choi K. I., Cha J. H., Pae A. N., Cho Y. S., Kang H.-Y., Koh H. Y.,
Chang M. H., J. Antibiot., 48, 1375—1377 (1995).
18) Silley P., Griffiths J. W., Monsey D., Harris A. M., Antimicrob. Agents
Chemother., 34, 1806—1808 (1990).
19) Erwin M. E., Jones R. N., Barrett M. S., Briggs B. M., Johnson D. M.,
Antimicrob. Agents Chemother., 35, 929—937 (1991).
20) Watanabe N.-A., Nagasu T., Katsu K., Kitoh K., Antimicrob. Agents
Chemother., 31, 497—504 (1987).
Cytotoxic Activity The cytotoxicity was determined by the neutral red
uptake assay37) using FL-cells, a human amniotic epithelial cell line. The test
allows the differentiation between dead and living cells on the fact that only
living cells are able to manage the active uptake of neutral red. FL-cells were
cultivated in a 96-well microtiter plate (105 cells/ml Hepes modified Dul-
becco, Sigma, 150 ml/well) at 37 °C in a humidified 5% carbon dioxide
atmosphere. The Dulbecco’s Modified Eagle’s Medium (DMEM) was com-
21) Jones R. N., Erwin M. E., Antimicrob. Agents Chemother., 36, 233—

July 2008
907
238 (1992).
22) Mochizuki H., Yamada H., Oikawa Y., Murakami K., Ishiguro J., Ko-
31) Oogose K., Hafuri Y., Takemori E., Nakata E., Inouye Y., Nakamura
S., Kubo A., J. Antibiot., 40, 1778—1781 (1987).
suzume H., Aizawa N., Mochida E., Antimicrob. Agents Chemother., 32) Hafuri Y., Takemori E., Oogose K., Inouye Y., Nakamura S., Kitahara
32, 1648—1654 (1988). Y., Nakahara S., Kubo A., J. Antibiot., 41, 1471—1478 (1988).
23) Nakagawa S., Sanada M., Matsuda K., Hashizume T., Asahi Y., Ushi- 33) Yoshimoto Y., Kawada M., Kumagai H., Someno T., Inoue H., Kawa-
jima R., Ohtake N., Tanaka N., Antimicrob. Agents Chemother., 33,
1423—1427 (1989).
24) Tatsumi K., Freyer A., Minard R. D., Bollag J. M., Environ. Sci. Tech-
nol., 28, 210—215 (1994).
25) Mikolasch A., Hammer E., Jonas U., Popowski K., Stielow A.,
Schauer F., Tetrahedron, 58, 7589—7593 (2002).
26) Manda K., Hammer E., Mikolasch A., Gördes D., Thurow K., Schauer
F., Amino Acids, 31, 409—419 (2006).
mura N., Isshiki K., Ikeda D., J. Antibiot., 58, 590—593 (2005).
34) Charan R. D., Schlingmann G., Bernan V. S., Feng X., Carter G. T., J.
Antibiot., 58, 271—274 (2005).
35) Chiung Y. M., Fujita T., Nakagawa M., Nozaki H., Chen G. Y., Chen
Z. C., Nakayama M., J. Antibiot., 46, 1819—1826 (1993).
36) Burkhardt F., “Mikrobiologische Diagnostik,” Georg Thieme Verlag,
Stuttgart, New York, 1992.
37) Lindl T., Bauer J., “Zell und Gewebekultur,” Gustav Fischer Verlag,
Jena, 1989.
27) Manda K., Hammer E., Mikolasch A., Niedermeyer T. H. J., Dec J.,
Jones A. D., Benesi A. J., Schauer F., Bollag J.-M., J. Mol. Catal. B, 38) Agematu H., Tsuchida T., Kominato K., Shibamoto N., Yoshioka T.,
35, 86—92 (2005).
Nishida H., Okamoto R., Shin T., Murao S., J. Antibiot., 46, 141—148
(1993).
39) Anyanwutaku I. O., Petroski R. J., Rosazza J. P. N., Bioorg. Med.
Chem., 2, 543—551 (1994).
40) Agematu H., Kominato K., Shibamoto N., Yoshioka T., Nishida H.,
Okamoto R., Shin T., Murao S., Biosci. Biotechnol. Biochem., 57,
1387—1388 (1993).
28) Niedermeyer T. H. J., Mikolasch A., Lalk M., J. Org. Chem., 70,
2002—2008 (2005).
29) Tatsumi K., Freyer A., Minard R. D., Bollag J. M., Soil Biol. Biochem.,
26, 735—742 (1994).
30) Shin D.-Y., Kim H.-S., Min K.-H., Hyun S.-S., Kim S.-A., Huh H.,
Choi E.-C., Choi Y. H., Kim J., Choi S.-H., Kim W.-B., Suh Y.-G.,
Chem. Pharm. Bull., 48, 1805—1806 (2000).