Synthesis of new N-analogous corollosporine derivatives with antibacterial activity by laccase-catalyzed amination

Article abstract of DOI:10.1248/cpb.56.781

Corollosporine isolated from the marine fungus Corollospora maritima and N-analogous corollosporines are antimicrobial substances. Owing to the basic structure of the N-analogous corollosporines, they have become an attractive target for laccase-catalyzed

Full text of DOI:10.1248/cpb.56.781

June 2008
Chem. Pharm. Bull. 56(6) 781—786 (2008)
781
Synthesis of New N-Analogous Corollosporine Derivatives with
Antibacterial Activity by Laccase-Catalyzed Amination
Annett MIKOLASCH, ,a Susanne HESSEL,a Manuela GESELL SALAZAR,b Helfried NEUMANN,c
*
Katrin MANDA,a Dirk GÖRDES,d Enrico SCHMIDT,d Kerstin THUROW,d Elke HAMMER,b
a
Ulrike LINDEQUIST,e Matthias BELLER,c and Frieder SCHAUER
a Institute of Microbiology, Ernst-Moritz-Arndt-University Greifswald; b Interfacultary Institute for Genetics and
Functional Genomics, Ernst-Moritz-Arndt-University Greifswald; e Institute of Pharmacy, Ernst-Moritz-Arndt-University
Greifswald; 17489 Greifswald, F.-L.-Jahn-Str. 15, Germany: c Leibniz-Institute of Catalysis E.V., University Rostock;
Germany: and d Institute of Automation University Rostock; 18119, Germany.
Received November 13, 2007; accepted March 11, 2008; published online March 19, 2008
Corollosporine isolated from the marine fungus Corollospora maritima and N-analogous corollosporines are
antimicrobial substances. Owing to the basic structure of the N-analogous corollosporines, they have become an
attractive target for laccase-catalyzed derivatisation. In this regard we report on the straightforward laccase-cat-
alyzed amination of dihydroxylated arenes with N-analogous corollosporines. In biological assays the obtained
amination products are more active than the parent compounds.
Key words corollosporine; laccase; Corollospora maritima; biotransformation; resistance
Laccase-catalyzed aminations represent an efficient phenyl-acetamido)cephalosporinic acid,13) the dimerization
method for the construction of biologically important C–N of penicillin X14) and the oxidative coupling of hydroquinone
bonds and allows for the use of mild reaction conditions, and mithramicine.15) Unfortunately, in these examples real-
aqueous solvent systems, normal pressure, and room temper- ized so far, the goal of enhancement of the bioactive effect
ature. Recently, this type of reaction has been applied to syn- has not been achieved.13—15) Recently we reported on novel
thesize novel antibiotics.1,2) The synthesis and biological penicillins and cephalosporins synthesized by biotransforma-
evaluation of potentially new antibiotic agents is undoubtedly tion using laccase.1,2) These results showed that derivatization
an important topic in current chemical and medicinal re- of antibiotics by laccase-catalyzed reaction can be achieved
search. Beside the design of more effective antibiotics with a without any loss of antibacterial activity.
lower number of unwanted side effects, this demand is espe-
In the present study we have employed laccase from Tram-
cially forced by the ongoing multi-resistance of several bac- etes sp. to derivatize N-analogous corollosporines and to cou-
teria, e.g. Streptococcus pneumoniae strains3—5) and Staphy- ple them both with 4-methylcatechol and with derivatives of
lococcus strains, against currently available antibiotics.6—9)
2,5-dihydroxybenzoic acid. The 2,5-dihydroxybenzoic acid
The discovery of suitable new antibiotics is generally gov- derivatives are structurally related to the ganomycins, a new
erned by the isolation of active compounds from biological chemical class of antibacterial compounds16) and to other an-
resources. In a recent example, Lindequist and co-workers tibacterial active substances,17,18) and therefore they are inter-
isolated the novel antibacterial agent corollosporine (Fig. 1) esting as coupling partner for N-analogous corollosporines.
from the marine fungus Corollospora maritima.10) Corol- To our delight the resulting products inhibit the growth of
losporine [(ꢀ)-3-hexyl-3,7-dihydroxy-1(3H)-isobenzofuran- several Gram positive bacterial strains in the agar diffusion
1-one] is a typical member of antimicrobial compounds with assay, among them methicillin-resistant Staphylococcus au-
phthalide structure and is active against Staphylococcus au- reus (MRSA).
reus and Bacillus subtilis. Beside the total syntheses of the
corollosporine,11) a synthesis protocol of N-analogous com- Results and Discussion
pounds was developed to study their antibiotic behaviour.12)
Biotransformation of N-Analogous Corollosporines
Some of the obtained products revealed antibiotic activity, Laccase-catalyzed reaction between N-analogous corol-
which was comparable to that of the natural product corol- losporines (2a—e) on the one hand and several derivates of
losporine.
2,5-dihydroxybenzoic acid and 4-methylcatechol (1a—d) on
As a consequence of the promising activity of the corol- the other hand leads to a small library of cross coupling
losporines in antibacterial assays, we became interested in products (Table 1). Altogether 34 transformation products
laccase-catalyzed amination of dihydroxylated aromatics have been performed and analyzed by high performance
with N-analogous corollosporines. Until to date the use of liquid chromatography (HPLC). In the course of incubation,
laccase for the derivatization of antibiotics is limited to a few a color change of the initially clear or yellow mixture was
examples including the phenolic oxidation of 7-(4-hydroxy- noted. Within the first hour, the reaction mixture turned from
yellow to dark red.
Among the different model transformations four reactions
were selected for scaling up to prove our general approach
(from 4 to 200 ml reaction volume), because of the high
yields, the easy to apply separation from the reaction mix-
ture, and the stability of the products. The reaction between
Fig. 1. Structure of Corollosporine
∗ To whom correspondence should be addressed. e-mail: annett.mikolasch@gmx.de
© 2008 Pharmaceutical Society of Japan

782
Vol. 56, No. 6
Table 1. Products Obtained in Laccase-Catalyzed Biotransformation
Substances
1a
1b
1c
1d
2a
2b
2c
2d
2e
1a) [3a]b)
1 [3b]
2 [3c]
2
3
1
4
c)
1
3
3
3
3
—
1 [3d]
2
0
0
1
1
2
a) Number of products. b) Description of products analyzed in more detail. c) Not tested.
2a and 1a led to the selective formation of cross coupling ylamino)-3,6-dioxocyclohexa-1,4-dienecarboxylic acid de-
product 3a. HPLC analysis of the reaction mixture revealed rivatives are shown in Table 2.
full conversion of both reacting partners to give 3a. A suffi-
Mass spectroscopic analyses of the compounds 3a to 3d
cient amount of 3a (approximately 95% yield) for detailed showed a molecular mass which corresponds to the coupling
structural characterization was formed within an incubation of one 2,5-dihydroxybenzoic acid derivative (1a or 1b) with
period of 1 h.
one N-analogous corollosporine derivative (2a or 2b). Electro
The reactions between 2b or 2c and derivatives of 2,5-di- spray ionization (ESI) (negative and positive ion mode) and
hydroxybenzoic acid led to one or two different cross cou- atmospheric pressure chemical ionization (APCI) (negative
pling products. After incubation times of 1 h the substrates ion mode) measurements directly after dissolution showed
1a or 1b and 2b or 2c were no longer detectable in the reac- the corresponding quinonoid products. However, during
tion mixture and the corresponding coupling products were HPLC-MS analysis performed 20 min after dissolution the
observed (80—90% yield). Yields of more than 80% showed occurrence of the hydroquinone form of the products is al-
the high efficiency of the reaction. Some years ago we found ready detectable in solvents like methanol as we reported
comparable straightforward biotransformation providing cou- previously for other amination products of 2,5-dihydroxyben-
pling products between 2,5-dihydroxybenzoic acid deriva- zoic acid derivatives.23)
1
tives and primary aromatic amines19,20) or b-lactam antibi-
More specifically H-NMR spectral data of 3a contained
otics,1,2) as well as between 3-(3,4-dihydroxy-phenyl)-propi- the characteristic signals for both substrate 1a and compound
onic acid and 1-hexylamine.21) In all these reactions transfor- 2a. The number of CH proton signals of the dihydroxylated
mation rates and product yields are rather high and byprod- phenyl rings changed from three—in the substrate–to two
ucts could be neglected. In contrast to these findings are re- signals—in the product. The multiplicity of H4 and H5 of the
action kinetics which were described for the coupling reac- product indicated a further substituent at the C2. The chemi-
tion between 3,4-dichloroaniline and syringic acid22) and be- cal shift to lower field of H4 and H5 demonstrated the pres-
tween 3-(3,4-dihydroxy-phenyl)-propionic acid and 4- ence of an electron-withdrawing group. 13C-NMR measured
aminobenzoic acid.21) In these experiments the formation of in CH2Cl2 showed two typical signals for quinones in the
byproducts diminished the yield of the coupling product up range of 180 ppm, an important indication for the quinonoid
to 40%. The dihydroxylated compounds used in this study character of 3a, confirming the oxidation of the p-hydro-
showed fast reactions in the control experiments without a quinone to a quinone. This observation is in accordance with
second coupling partner, too. However, these undesired reac- our previous findings of coupling products with a benzo-
tions were suppressed almost completely in the presence of quinone structure motif which were formed between 2,5-
the N-analogous corollosporine derivatives.
dihydroxybenzoic acid derivatives and primary aromatic
The products 3a to 3d were isolated by separation from amines19,20) or b-lactam antibiotics.1,2) A related coupling
buffer and laccase. Their structures were established unam- product was synthesized between 3,4-dichloraniline and pro-
1
biguously by H-, 13C-NMR and mass spectroscopic investi- tocatechuic acid or syringic acid.22,24)
gations. The resulting 2-(3-oxo-2,3-dihydro-1H-isoindol-4-
1H–1H-COSY measurements did not include correlations

June 2008
783
Table 2. Coupling Reaction with Laccase
2,5-Dihydroxybenzoic
acid derivative
Aniline
Coupling product
Yield [%]
2a
2b
3a
3b
94
1a
82
2c
3c
80
1b
2c
3d
85
Conditions: Aniline (1 mM), 2,5-dihydroxybenzoic acid derivative (1 mM) dissolved in 200 ml 0.02 M sodium acetate buffer pH 5.0 10% methanol, laccase C of Trametes spec
(final activity 0.15 unit mlꢁ1).
between the aromatic amine proton of the N-analogous corol-
losporine and any other proton, proving the coupling between Escherichia coli, whereas all other tested substances did not
1a and 2a to be a C–N bond at C2. exhibit any growth inhibition of E. coli. Products 3a to 3d
3c showed low activity against the Gram negative strain
Biological Activity of the Biotransformation Products were not active against the Gram negative strain
According to Table 2 all products (3a to 3d) obtained by bio- Pseudomonas aeruginosa and against the yeast Candida
transformation showed a moderate growth inhibition of sev- maltosa. Investigations regarding the stability of the synthe-
eral Gram positive strains, among them multidrug resistant sised compounds showed limited lifetime in aqueous solu-
Staphylococcus strains (Table 3), in the agar diffusion test. tion. Incubation of compounds 3a to 3d (in solution at 30 °C)
Notably, the antibacterial activity of the reaction products (3a showed decomposition after 2 h. Therefore for further studies
to 3d) was increased in comparison to both N-analogous on the biological activity non-aqueous application systems
corollosporines (2a to 2c) and originally isolated natural have to be implemented.
compound corollosporine. In comparison to the 2,5-dihy-
In conclusion a series of novel biologically active com-
droxybenzoic acid derivatives their cross coupling products pounds has been prepared by laccase-catalyzed amination.
were significantly more active. The increase of the activity of This technology for derivatization of potentially active leads
coupling products was synergistic, compared with the com- has many advantages over other classical synthetic technolo-
ponent compounds, 2,5-dihydroxybenzoic acid derivatives gies. Within short time a variety of new substances can be
and amino-corollosporines, and showed the advantage of the synthesized smoothly. The enzyme laccase can be isolated
effect of laccase-catalyzed coupling of one laccase substrate easily and is highly stable. Hence, it is possible to functional-
with bioactive amino-corollosporines.
ize also sensitive natural substances. Noteworthy, active com-

784
Vol. 56, No. 6
Table 3. Antimicrobial Activity of Products 3a to 3d and 2a to 2c
Amount
[mmol]
E. coli
11229
B. subtilis
6051
S. aureus
ATCC 6538
S. aureus
S. epidermidis
S. haemolyticus
Substances
NESe,f)
847f)
535f)
a)
Corollosporine
1
0.4
0.2
—
—
—
14b) (2.0)c)
11 (3.5)
9 (2.0)
10 (0.6)
8 (1.0)
7 (0.0)
12 (0.6)
10 (2.1)
8 (0.6)
14 (1.5)
12 (0.6)
10 (1.0)
8 (0.6)
rd)
r
1a
1b
2a
2b
2c
3a
3b
3c
3d
1
0.4
0.2
r
r
r
r
r
r
r
r
r
r
r
r
r
r
r
r
r
r
1
0.4
0.2
r
r
r
r
r
r
r
r
r
r
r
r
r
r
r
r
r
r
1
0.4
0.2
r
r
r
r
r
r
r
r
r
r
r
r
r
r
r
r
r
r
1
0.4
0.2
r
r
r
10 (1.2)
r
r
r
r
r
r
r
r
r
r
r
r
r
r
1
0.4
0.2
r
r
r
r
r
r
12 (2.3)
10 (1.0)
r
r
r
r
r
r
r
r
r
r
1
0.4
0.2
r
r
r
7 (1.5)
7 (0.6)
r
10 (0.6)
8 (0.6)
r
16 (1.7)
12 (0.6)
10 (0.0)
16 (1.2)
12 (0.6)
r
r
r
r
1
0.4
0.2
r
r
r
8 (0.6)
r
r
11 (0.6)
18 (0.6)
14 (1.5)
10 (0.6)
16 (0.0)
12 (0.0)
r
8 (0.6)
r
r
r
r
1
0.4
0.2
8 (0.6)
r
r
14 (2.1)
13 (2.3)
10 (1.2)
15 (2.1)
11 (0.6)
8 (1.5)
18 (1.5)
12 (0.6)
8 (0.6)
12 (0.6)
10 (2.1)
8 (0.6)
12 (0.6)
10 (0.6)
8 (0.6)
1
0.4
0.2
r
r
r
12 (0.0)
11 (0.6)
10 (0.6)
14 (2.0)
13 (1.5)
10 (0.6)
16 (1.7)
10 (1.2)
8 (0.6)
14 (0.6)
10 (1.2)
8 (0.6)
12 (0.6)
10 (1.5)
8 (0.6)
a) Not tested. b) Zones of inhibition (mm) calculated from 3 replicates. c) Standard deviation calculated from 3 replicates. d) r resistant (no zone of inhibition). e)
North German Epidemic Strain. f) Multi-resistant strains.
pounds, which do not belong to substrates of laccase, can be transformation was started by addition the enzyme laccase C of Trametes
spec (final activity 0.15 unit mlꢁ1). Reaction was performed at room temper-
ature with agitation at 400 rpm.
linked with the appropriate substrates of the enzyme.
Experimental
Four adapted reactions were selected to scale up the reaction approach.
One of the N-analogous corollosporines 2a to 2c was dissolved in 20 ml
methanol thereafter 100 ml 0.02 M sodium acetate buffer pH 5.0 was added.
1a or 1b was diluted in 80 ml of 0.02 M sodium acetate buffer pH 5.0 by ul-
trasonics. The concentration of the reacting partners was 1 mM in the reac-
tion mixture. The accomplishment of the reactions was proceeded as de-
scribed above.
High Performance Liquid Chromatography In order to follow the
process of reaction, the mixture was analyzed by a HPLC system (Kontron,
Neufahrn, Germany), consisting of a Kontron series pump model 522, a
Kroma Automatic Sample Injector 565 and a Diode Array Detector system
540. For the separation of metabolites an endcapped, 5-mm, LiChroCart 125-
4 RP 18 column (Merck, Darmstadt, Germany) was used at a flow rate of
1 ml·minꢁ1. The mobile phase consisted of methanol (eluent A) and phos-
phoric acid (0.1%, eluent B), starting from an initial ratio of 10% A and
90% B and reaching then 100% A within 14 min. After a reaction time of 50
to 100 min one or two coupling products could be detected.
Enzymes Extra cellular laccase C of Trametes sp. (EC 1.10.3.2) was ob-
tained from ASA Spezialenzyme (Wolfenbüttel, Germany) and used as re-
ceived (activity 1000 U/g; substrate: syringaldazine).
Syntheses of N-Analogous Corollosporines N-Analogous corol-
losporines [7-amino-3-hydroxy-2-methyl-3-hexyl-2,3-dihydro-isoindol-1-on
(2a), 4-amino-2,6-dimethyl-isoindol-1,3-dion (2b), 7-amino-3-hydroxy-
2,4,6-trimethyl-3-pentyl-2,3-dihydro-isoindol-1-on (2c), 7-amino-3-hy-
droxy-2,4,6-trimethyl-3-heptyl-2,3-dihydro-isoindol-1-on (2d), 7-amino-3-
hydroxy-2,4,6-trimethyl-3-hexyl-2,3-dihydro-iso-indol-1-on (2e)] were syn-
thesized as described before.12)
Synthesis of the 2,5-Dihydroxybenzoic Acid Derivate 1d The 2,5-di-
hydroxy-N-(2-methyl-1,3-dioxo-2,3,3a,4,7,7a-hexahydro-1H-isoindol-4-
yl)benzamide was synthesized as described before.25)
Amination of Different Substituted Hydroquinones with N-Analogous
Corollosporines by Laccase C After dissolving of an N-analogous corol-
losporine in 1 ml methanol 4 ml 0.02 M sodium acetate buffer pH 5.0 was
added. For complete dissolving of N-analogous corollosporines ultrasonics
was used. One of the 2,5-dihydroxybenzoic acid derivatives or 4-methylcate-
chol was diluted in 5 ml of 0.02 M sodium acetate buffer pH 5.0 by ultrason-
ics as well. The two solutions were transferred to one reaction tube. The
concentration of the reacting partners was 1 mM in the reaction mixture. The
Isolation of Transformation Products with Solid Phase Extraction
Solid phase extraction was utilized for isolation and the transfer of the cou-
pling products into an organic matrix, using a Strata C18-E column (55 mm,
10 g capacity, 60 ml, Phenomenex, Germany). After activation and equilibra-
tion the RP-18 column was charged with 100 ml of the incubation mixture.

June 2008
785
3
The column was washed twice with 60 ml methanol (10%) and aqua dest.
(90%). More than 80% of the coupling products were eluted by acetonitrile
100%. The eluted metabolites were evaporated to dryness using a vacuum
rotator at 30 °C.
d2) dH 13.28 (s, 1H, NH aromatic), 9.77 (t, 1H, Jꢃ5.4 Hz, NH, H2ꢅ), 7.35
(dd, 3Jꢃ7.7 Hz, 1H, H6ꢄ), 7.33 (d, 3Jꢃ7.5 Hz, 1H, H5ꢄ or H7ꢄ), 7.14 (d,
3Jꢃ7.9 Hz, 1H, H5ꢄ or H7ꢄ), 6.52 (d, 3Jꢃ10.2 Hz, 1H, H4), 6.47 (d,
3Jꢃ10.2 Hz, 1H, H5), 3.91 (s, 1H, OH heterocyclic), 3.65 (m, Jꢃ5.1 Hz,
3
2H, H4ꢅ), 3.46 (m, 3Jꢃ5.5 Hz, Jꢃ3.9 Hz, 1H, H3ꢅ), 3.41 (m, 3Jꢃ5.5 Hz,
Jꢃ3.9 Hz, 1H, H3ꢅ), 3.18 (t, 3Jꢃ5.1 Hz, 1H, H5ꢅ OH aliphatic), 3.08 (s, 3H,
H8ꢄ), 2.02 (m, 2H, H9ꢄ), 1.17 (m, 6H, H11ꢄ, H12ꢄ, H13ꢄ), 0.82 (m, 3H,
H14ꢄ), 0.76 (m, 1H, H10ꢄ), 0.65 (m, 1H, H10ꢄ). 13C-NMR (100 MHz;
CH2Cl2-d2; determination of proton assignment by hetcor) d 184.9, 181.2
(C6/C3); 170.0 (C1ꢅ); 166.1 (C3ꢄ); 154.8 (C1); 149.1, 136.8, and 121.8
(C7aꢄ/C3aꢄ/C4ꢄ); 139.0 (C4); 134.3 (C5); 133.5 (C6ꢄ); 124.9, 119.4
(C7ꢄ/C5ꢄ); 101.1 (C1); 90.3 (C1ꢄ); 62.7 (C4ꢅ); 42.6 (C3ꢅ); 35.5 (C9ꢄ); 31.8,
29.2, and 22.8 (C13ꢄ/C12ꢄ/C11ꢄ); 23.41 (C10ꢄ); 23.42 (C8ꢄ); 14.1 (C14ꢄ).
LC/MS m/z: APCI, neg. ion mode, 454.2 [MꢁH]ꢁ; API-ES, pos. ion mode,
478.1 [MꢂNa]ꢂ, 933.3 [2MꢂNa]ꢂ; API-ES, neg. ion mode 454.2 [MꢁH]ꢁ.
After 20 min: APCI, neg. ion mode, 454.2 [MꢁH]ꢁ and 456.2 [MꢁH]ꢁ of
the hydroquinone form of 3a; ESI, pos. ion mode, 478.1 [MꢂNa]ꢂ and
480.1 [MꢂNa]ꢂ of the hydroquinone form of 3a; ESI, neg. ion mode 454.2
[MꢁH]ꢁ and 456.2 [MꢁH]ꢁ of the hydroquinone form of 3a. HR-MS
Calcd for C24H29N3O6Na [MꢂNa]ꢂ: 478.1949; Found: 478.1944. HR-MS
Calcd for C24H31N3O6Na [MꢂNa]ꢂ, the hydroquinone form of 3a:
480.2106; Found: 480.2108 (all confirmed by FT-ICR MS).
LC/MS, NMR The products were characterized by liquid chromatogra-
phy/mass spectrometry (LC/MS). Atmospheric pressure ionization (API)
mass spectrometry experiments were performed using an Agilent Series
1100 HPLC system and an Agilent 1946C quadrupole mass spectrometer
(Waldbronn, Germany). The mass spectrometer was used with both, APCI
and ESI sources.
HPLC-MS separation was performed on
a
LiChroCART® 125-4,
LiChrosphere® 100 RP-18e column (Merck, Darmstadt, Germany) with the
following binary standard gradient system at a flow rate of 1 ml/min: 14-min
gradient elution from 10 to 100% eluent B (MeOH) where eluent A was
0.1% formic acid in water; 100% eluent B (MeOH) for further 2 min, 10%
eluent B and 90% A for 2 min to equilibrate the column for next run. Chro-
matography was performed at 25 °C and a UV signal recorded at 220 nm
with a variable wavelength detector (VWD). APCI conditions (positive and
negative ion mode) were as follows: nebulizer and drying gas, nitrogen; neb-
ulizer pressure, 30 psig; drying gas flow, 10 l/min; vaporizer temperature,
350 °C; drying gas temperature, 250 °C; capillary voltage, 4 kV; corona cur-
rent, 4 mA.
ESI conditions (positive and negative ion mode): nebulizer and drying
gas, nitrogen; nebulizer pressure, 30 psig; drying gas flow, 10 l/min; drying
gas temperature, 350 °C; capillary voltage, 4 kV.
2-(2,6-Dimethyl-1,3-dioxo-2,3-dihydro-1H-isoindol-4-ylamino)-3,6-
dioxo-cyclohexa-1,4-dienecarboxylic acid (2-hydroxy-ethyl)amide (3b)
Synthesis and isolation as described above. Red solid. Yield 63 mg (82%).
LC/MS m/z: ESI, neg. ion mode 382.1 [MꢁH]ꢁ and 787.1 [2(MꢁH)ꢂ
Na]ꢁ; ESI, pos. ion mode, 406.0 [MꢂNa]ꢂ. HR-MS Calcd for C19H18N3O6
[MꢂH]ꢂ: 384.1190; Found: 384.1195 (confirmed by FT-ICR MS).
2-(1ꢀ-Heptyl-1ꢀ-hydroxy-2ꢀ,5ꢀ,7ꢀ-trimethyl-3-oxo-2,3-dihydro-1H-
isoindol-4-ylamino)-3,6-dioxocyclohexa-1,4-dienecarboxylic acid (2ꢁ-hy-
droxyethyl)amide (3c) Synthesis and isolation as described above. Red
solid. Yield 75 mg (80%). LC/MS m/z: ESI, pos. ion mode, 492.2 [MꢂNa]ꢂ,
452.2 [MꢁOH]ꢂ, and 961.4 [2MꢂNa]ꢂ; ESI, neg. ion mode 468.2
[MꢁH]ꢁ and 959.5 [2(MꢁH)ꢂNa]ꢁ. HR-MS Calcd for C25H31N3O6Na
[MꢂNa]ꢂ: 492.2110; Found: 492.2091 (confirmed by FT-ICR MS).
2-(1ꢀ-Heptyl-1ꢀ-hydroxy-2ꢀ,5ꢀ,7ꢀ-trimethyl-3-oxo-2,3-dihydro-1H-
isoindol-4-ylamino)-3,6-dioxocyclohexa-1,4-dienecarboxylic acid methyl
ester (3d) Synthesis and isolation as described above. Red solid. Yield
75 mg (85%). LC/MS m/z: ESI, pos. ion mode, 441.2 [MꢂH]ꢂ, 463.2
[MꢂNa]ꢂ, and 903.4 [2MꢂNa]ꢂ; ESI, neg. ion mode 439.1 [MꢁH]ꢁ and
901.2 [2(MꢁH)ꢂNa]ꢁ. HR-MS Calcd for C24H29N2O6 [MꢂH]ꢂ: 441.2020;
Found: 441.2019 (confirmed by FT-ICR MS).
All FT-ICR MS high-resolution mass spectrometry (HR-MS) experiments
were performed on a Bruker Daltonics APEX III FT-ICR mass spectrometer
(Bremen, Germany) equipped with a 7.0 T shielded superconducting mag-
net. The flow rate for the eluent (H2O/ACN/HCOOH 49/49/2, all HPLC-
grade) was 2 ml/min, using a syringe pump (Cole-Palmer 74900 series). The
ions were generated from an external electrospray ionization source (Apollo
ESI-Source) with nebulizing gas pressure at 20 psi, heated drying gas at 10
psi (back-pressure) and 150 °C, and a capillary entrance voltage of ꢁ4500 V
in negative ion mode and ꢂ4500 V in positive ion mode.26—28)
Mass spectra were acquired with both, positive and negative, ion modes
with broadband detection (32 scans each experiment) from 100 to 2.000 Da
using 1024 K data points. All experimental sequences, including scan accu-
mulation and data processing, were performed with XMASS 6.1.2 on Win-
dows 2000.
The nuclear magnetic resonance (NMR) spectra were recorded on a
Bruker (Karlsruhe, Germany) Avance 600 instrument (1H, 600 MHz) or on a
Bruker ARX 400 with QNP probe head (1H, 400.13 MHz; 13C, 100.61 MHz)
at 25 °C.
Antimicrobial Activity To determine the antimicrobial activity an agar
diffusion assay according to Burkhardt29) was used. Sterile Mueller-Hinton
II-Agar in Stacker Petri discs (Becton Dickinson Microbiology systems,
Cockeysvill, U.S.A.) was inoculated with cells (200 ml of a yeast or bacterial
cell suspension in 20 ml medium) of the yeast Candida maltosa SBUG 700,
the bacterial strains Escherichia coli 11229, Pseudomonas aeruginosa
27853, Bacillus subtilis 6051 and Staphylococcus aureus ATCC 6538 and
the multiresistant bacteria strains Staphylococcus aureus North German Epi-
demic Strain, Staphylococcus epidermidis 847 and Staphylococcus
haemolyticus 535. Samples were applied in different concentration on sterile
paper discs (Sensi-Disc, 6 mm diameter, Becton Dickinson Microbiology
systems, Cockeysvill, U.S.A.). Plates were kept for 3 h in a refrigerator to
enable prediffusion of the substances into the agar and then incubated for
24 h at 37 °C. Inhibition zone diameters around each of the disc were meas-
ured and recorded at the end of the incubation time. An average zone of in-
hibition was calculated from 3 replicates.
Chemicals Chemicals were purchased from commercial suppliers: 2,5-
dihydroxy-N-(2-hydroxyethyl)-benzamide (97%, 1a) was received from Mi-
dori Kagaku Co., Ltd. (Japan). Methyl 2,5-dihydroxybenzoic acid (99%, 1b)
and 4-methylcatechol (95%, 1c) are commercially available from Aldrich
(Steinheim, Germany).
2-(1ꢀ-Hexyl-1ꢀ-hydroxy-2ꢀ-methyl-3-oxo-2,3-dihydro-1H-isoindol-4-
ylamino)-3,6-dioxocyclohexa-1,4-dienecarboxylic acid (2ꢁ-hydroxy-
ethyl)amide (3a) Synthesis and isolation as described above. Red solid.
Yield 86 mg (94%). 1H-NMR (400 MHz; methanol-d4) dH 7.35 (m,
3Jꢃ8.3 Hz, 3Jꢃ7.5 Hz, 1H, H6ꢄ), 6.99 (d, 3Jꢃ8.7 Hz, 1H, H4), 6.95 (d,
3Jꢃ7.5 Hz, 1H, H5ꢄ or H7ꢄ), 6.74 (d, 3Jꢃ8.7 Hz, 1H, H5), 6.51 (d,
3Jꢃ8.3 Hz, 1H, H5ꢄ or H7ꢄ), 3.71 (m, 3Jꢃ5.6 Hz, 2H, H4ꢅ), 3.49 (m,
3Jꢃ5.7 Hz, 2H, H3ꢅ), 3.39 (s, 3H, H8ꢄ), 2.11 (m, 2H, H9ꢄ), 1.25 (m, 8H,
H10ꢄ, H11ꢄ, H12ꢄ, H13ꢄ), 0.89 (m, 3H, H14ꢄ). 1H-NMR (600 MHz; CH2Cl2-
Acknowledgments Financial support from the government of Mecklen-
burg-Vorpommern, Germany, and from the European Union (Landes-
forschungsschwerpunkt “Neue Wirkstoffe und Biomaterialien–Innovative
Screeningverfahren and Produktentwicklung”) is gratefully acknowledged.
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