One-pot synthesis and biological evaluation of aspergillamides and analogues

Article abstract of DOI:10.1016/S0960-894X(00)00305-X

A one-pot total synthesis of aspergillamide and analogues by a solution phase Ugi multi component reaction (MCR) is described. The reaction is easily performed in 96-well plates and offers a facile access to diverse aspergillamide analogue compound libraries. The antibiotic and cytotoxic activity of these compounds is measured. Several of them are more potent than the natural product. (C) 2000 Elsevier Science Ltd. All rights reserved.

Full text of DOI:10.1016/S0960-894X(00)00305-X

Bioorganic & Medicinal Chemistry Letters 10 (2000) 1701±1705
One-Pot Synthesis and Biological Evaluation of Aspergillamides
and Analogues
Barbara Beck,a,b Sibylle Hessa and Alexander Domlinga,b,
*
aMorphochem AG, Gmunderstr. 37-37a, 81379 Munchen, Germany
È
bInstitut fur Organische Chemie und Biochemie der Technischen Universitat Munchen,
Lichtenbergstr. 4, 85747 Garching, Germany
È
È
È
Received 13 March 2000; accepted 26 May 2000
AbstractÐA one-pot total synthesis of aspergillamide and analogues by a solution phase Ugi multi component reaction (MCR) is
described. The reaction is easily performed in 96-well plates and o€ers a facile access to diverse aspergillamide analogue compound
libraries. The antibiotic and cytotoxic activity of these compounds is measured. Several of them are more potent than the natural
product. # 2000 Elsevier Science Ltd. All rights reserved.
The indole-3-ethenamide substructure is a common ele-
ment in many bioactive natural products. Several examples
are shown in Figure 1. Recently described natural pro-
ducts of that class are the aspergillamides isolated by
Fenical et al. from Aspergillus spec. from a salt lake loca-
with an assembly of more than two starting materials are
called multi component reactions (MCRs).6,7 Often,
MCRs are advantageous over two component reactions in
terms of time, yield, complexity and diversity of products.
Therefore MCRs are convergent in contrast to the diver-
gent or linear two component reactions.
ted on the Bahamas. These compounds show moderate
1 1
cytotoxicity with an IC50 (HCT 116)=16 mg ml
.
The most famous and versatile MCR is the Ugi four
component (U-4CR) and related reactions.8 Inspection
of the backbone of aspergillamide reveals that it is ideally
suited for MCR chemistry of the Ugi-type (Scheme 1).
As part of our program to discover new anticancer drugs
and antibiotics with novel mechanisms of action we
recognised the presence of the indole-3-ethenamide sub-
structure in many biological active natural products as a
possible pharmacophore. Herein we describe the total
synthesis of one of these molecules, aspergillamide ``via'' a
one-pot multi component reaction. This method of synth-
esis allows us to generate libraries of aspergillamide ana-
logues with ®ve points of diversity. The compounds were
screened against two cancer cell lines and their antibiotic
activities were evaluated. Some initial results are reported.
Thus aspergillamide can be synthesised from N-acetyl-
leucin, methylamine, phenylacetaldehyde and E/Z-3-(2-
isocyanoethen)-indole. E/Z-3-(2-isocyanoethen)-indole
can be readily synthesised from commercially available
3-formylindole and isocyanomethylphosphonic acid
diethylester.9 In order to study the in¯uence of the
substituents on the cytotoxic and antiproliferative
activity we synthesised a library of aspergillamide and
its analogues in 96-well plates (Scheme 2).10
Complex natural products are normally synthesised by a
sequence of steps that can be distinguished by convergent
and divergent synthesis routes.2
In order to introduce more variability at the indole residue
we synthesised seven di€erent isocyanides based on simi-
lar heterocycles. Isocyanides in Table 1, 23±26, were syn-
thesised with a modi®ed Horner±Emmons reaction and
could be obtained as mixtures of E/Z-isomers.11 Isocya-
nide 27 was prepared by condensation of TOSMIC with
3-formylindole according to the van Leusen procedure.12
Convergent synthesis routes show advantages over diver-
gent or linear approaches with respect to speed, time, yields
and reproducibility. On the level of reactions, usually one
or two component reactions are applied to assemble more
complex products from their simpler precursor. Reactions
All the 224 possible combinations of Schi€-bases, car-
boxylic acids, and isocyanides have been carried out as
*Corresponding author. Fax: +1-4989-7800-5555; e-mail: alexander.
doemling@morphochem.de
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00305-X

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B. Beck et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1701±1705
tion, the activity of acidic phosphatase was determined.15
More than 50% of the compounds inhibited cell pro-
liferation (<50 mM). To determine an approximate IC50
selected compounds were screened at ®ve di€erent con-
centrations. The observed IC50 of the compounds
screened with the cell line MCF-7 range between 100 mM
and 100 nM (Table 2). Generally, they show a good ®t
with the activities recently described for Aspergillamides.1
Microscopic inspection of the cell cultures showed the
cytotoxic activity of the inspected compound class
(Scheme 3).
General cytotoxicity is often associated with lysis of the
cell membranes. Therefore, we quanti®ed the cytosolic
enzyme lactate dehydrogenase released in the supernatant
after incubation of MCF-7 with the compounds.16
Interestingly, most of the compounds showed no general
cytotoxic e€ects. Only several compounds that derive
from the heterocyclic indole analogue isocyanide 23
show general cytotoxicity (Scheme 4).
Some of the compounds also exhibited strong antifungal
and antibacterial activity. They were screened for their
antibiotic activity against Staphylococcus aureus, Bacillus
subtilis, Pseudomonas aeruginosa and Candida albicans.
Some of the compounds showed good activity (100%
growth inhibition at a concentration lower than 100 mM)
against the Gram positive S. aureus and B. subtilis
(Scheme 5). Also some compounds were identi®ed being
C. albicans inhibitors (100% growth inhibition at a
concentration lower than 100 mM) (Scheme 5).
Figure 1. Some naturally occuring bioactive indole-3-ethenamides.
Antifungal and cytotoxic chondriamide 1,3 fragilamide 2,4 cell cycle
inhibitor terpeptin 3,5 and cytotoxic aspergillamide A 4.1 The struc-
tural similarity between the aspergillamides and terpeptin suggest they
also act as cell cycle inhibitors.
A detailed discussion of the compounds antibiotic activity
is beyond the scope of this communication and will be
published later.
one-pot reactions. The formation of the corresponding
products were studied by HPLC-MS. Representative
compounds have been also synthesised on a larger scale,
puri®ed and fully characterised.13
In summary, we performed the ®rst total synthesis of
aspergillamides and analogues by a one-pot U-MCR, that
is amenable to combinatorial chemistry. Their proliferat-
ing activity and the cell toxicity is in the same range of
cytotoxicity as of the natural product aspergillamide.
The expected products were formed in each well according
to HPLC-MS, with the exception of 27, which did not
react under the conditions used.14 With these compounds
the biological screens were performed. In order to check
for the ability of the compounds to reduce cell prolifera-
A major draw back of the compounds synthesised is
their diasteromeric nature. Therefore the measured IC50
values are only approximate.
Scheme 1. Retrosynthetic analysis of the aspergillamide A total synthesis by U-MCR.

B. Beck et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1701±1705
1703
Scheme 2. The aspergillamide skeleton synthesised by a U-MCR can be divided in ®ve parts: the a,b-unsaturated amide derived from the a,b-
unsaturated isocyanide, the adjacent methylene unit which derived from the oxo component, the substituted secondary amide from the primary
amine and the adjacent amino acid from the carboxylic acid and ®nally the acyl substituent.
Table 1. Sketch of the starting materials in order to obtain aspergillamide and their analogues. Eight di€erent precondensed Schi€-bases 10±17,
four di€erent carboxylic acids 18±21, and seven di€erent isocyanides 8, 22±27 were used to prepare the library. Each of the wells were analysed by
HPLC-MS and occasionally NMRs were taken13
Scheme 3. Left photography (Axiovert 25 Zeiss, Jena, 1:1000); growth conditions: RPMI-1640-medium plus 5% FCS, 48 h, 37 ꢀC, 5% CO2: MCF-7
cells under normal growth conditions. The cells form a complete cell layer. Right photo: MCF-7 cell culture with compound 10±18±8. It shows the
lack of integrity of the cell colony and change of morphology and thus attachment of cell from the substrate.

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B. Beck et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1701±1705
Scheme 4. Compounds showing general cytotoxicity by cell membrane lysis. This is measured by the liberation of lactate dehydrogenase. All have in
common the pyridinoindole structure derived from isocyanide 23.
Scheme 5. Compounds showing activity against Gram positive bacteria (®rst row) and against candida (second row).
5. Kagamizono, T.; Sakai, N.; Arai, K.; Kobinata, K.; Osada,
H. Tetrahedron Lett. 1997, 38, 1223.
6. Domling, A.; Ugi, I. Angew. Chem., Int. Ed. Engl., 2000,
39, in press.
Table 2. IC50 of some selected compounds against MCF-7. The
compounds IDs are the numbers of the Schi€-base, the carboxylic acid
and the isocyanide according to Table 1. At a concentration of 50 mM
none of the compounds showed general cytotoxicity14
7. Domling, A. Comb. Chem. High Throughput Screening
1998, 1, 1.
8. Ugi, I.; Domling, A.; Horl, W. Endeavour., 1994, 18, 115;
Ugi, I., J. Prakt. Chem., 1997, 339, 499.
ID (SB±CA±IN)
10±18±8
10±20±23
12±20±8
14±18±8
IC50 (mM)
0.3
1.9
0.4
49.9
4.1
9. Hoppe, I.; Schollkopf, U. Liebigs Ann. Chem., 1984, 600.
10. Procedure (200 mM per well): 200 mL of a 100 mM solu-
tion of the aldehyde component in DCM and 200 mL of a 100
mM solution of the amine are dispensed in a 96-well plate. The
solvent is evaporated over night and the Schi€-base is formed.
200 mL of a 100 mM solution of isocyanide in methanol and
200 mL of a 100 mM solution of the carboxylic acid in metha-
nol are dispensed in the plate. The plate is shaken for 24 h and
the solvent is evaporated in vacuum (Gene Vac). From the
synthesis plates dilution plates are made for the HPLC-MS
analytics and the biological screening.
14±19±8
Studies are ongoing in our laboratory to determine the
cell cycle inhibitory character of selected compounds
out of this class of compounds related to the natural
products aspergillamides.
11. General procedure for the synthesis of a,b-unsaturated
isocyanides: All operations have to be done under nitrogen,
free of moisture, and in the dark, to avoid photoisomerization.
3.3 mmol isocyanomethylphosphonic acid diethylester in 3 mL
THF is dropped to a solution of 3 mmol sodium bis-
trimethylsilylamide in 5 mL THF at 78 ꢀC. The solution is
stirred for 15 min 3 mmol of a solution of the corresponding
aldehyde or ketone in 30 mL THF is dropped to the above
solution and the combined solutions are stirred for 20 h at
0 ꢀC. The reaction is quenched with 3.3 mmol of acetic acid in
References and Notes
1. Toske, S. G.; Jensen, P. R.; Kau€man, C. A.; Fenical, W.
Tetrahedron 1998, 54, 13459.
2. Nicolaou, K. C.; Sorensen, E. J. Classics in Total Synthesis,
Wiley-VCH, New York, 1996.
3. Palermo, J. A.; Blanch Flower, P.; Seldes, A. M. Tetra-
hedron Lett. 1992, 33, 3097.
4. Kirkup, M. P.; Moore, R. E. Tetrahedron Lett. 1983, 24, 2087.

B. Beck et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1701±1705
1705
1.5 mL THF. The solvent is carefully evaporated in vacuum.
The residue is taken up in 30 mL of ethyl acetate and washed
with 15 mL phosphate bu€er (pH 7) and 15 mL water. The
organic phase is dried with magnesium sulfate and the solvent
evaporated. The residual oil is puri®ed by chromatography on
silica gel with ether as eluent. Yields of the isocyanides: 8: 41%,
22: 35%, 23: 57%, 24: 65%, 25: 20%, 26: 14%. 1,5,6,7-Tetra-
hydro-4H-indol-4-vinyl-isocyanide 26: Mw (C10H10N2)=
158.20 g mol 1. Yield: 65 mg (14%). The isomers could not be
162.3 mg (42%) (total of E- and Z-derivative, which are formed
in a 1:1 ratio). E-derivative: 1H NMR (400 MHz:CD3OD):
d=6.69 (d, 1H, pyr); 6.62 (s, 1H, vinyl), 6.11 (d, 1H, pyr); 4.08
(s, 2H, CH2); 3.73 (s, 3H, N-CH3); 3.09 (s, 3H, CO±CH3); 2.66±
1.54 (m, 16H; CH2, cyclohexyl). 13C NMR (100 MHz:CD3OD):
d=173.66 (CˆO); 173.24 (CˆO); 171.70 (CˆO); 131.17 (C);
121.28 (C); 118.47 (CH); 111.87 (CH); 104.86 (ˆCHNH-);
66.87 (CˆCHNH); 44.65 (CH2); 33.25 (CO±CH2±NH); 31.84
(N-CH3), 26.59±23.97 (CH2); 22.76 (CO-CH3). Z-derivative:
1H NMR (400 MHz:CD3OD): d=6.52 (d, 1H, pyr); 6.21 (s, 1H,
vinyl); 6.09 (d, 1H, pyr); 4.06 (s, 2H, CH2); 3.34 (s, 3H,N±CH3);
3,05 (s, 3H, CO±CH3); 2.60±1.51 (m, 16H; CH2, cyclohexyl).
1
assigned. H NMR (400 MHz:CDCl3): d=8.00 (br, 1H, NH);
8.70 (d, 1H, pyrrol); 6.26 (d, 1H, pyrrol); 5.65 (s, 1H, vinyl-H);
2.59±1.03 (m, 6H, 3ÂCH2). 13C NMR (100 MHz:CDCl3):
d=140.19±114.43 (Pyrrol); 106.87 and 102.75 (CˆCHNC, E-
and Z-); 99.57 (CˆCHNC); 29,72 (CH2); 26.04 (CH2); 22.97
(CH2).
12. van Leusen, A. M.; Wildeman, J.; Oldenziel, O. H. J. Org.
Chem. 1977, 42, 1153.
13. Typical procedure (1 mmol): A solution of each of pre-
condensed Schi€-base, carboxylic acid and isocyanide in 1 mL
methanol are stirred for 24 h. The solvent is evaporated and the
product is extracted from the residue with ethyl acetate. The sol-
vent is evaporated and the residue is puri®ed by chromatography
with ethyl acetate as eluent. 1-[(Acetylamino)acetyl](methyl)
amino]-N-(1,5,6,7-tetrahydro-4H-indole-4-ylidenemethyl)-cyclo-
hexanecarboxamide.Mw (C21H30N4O3)=386.50 gmol 1. Yield:
14. Products of Ugi-reaction are generally obtained as mix-
tures of diastereomers.
15. Yang, T.; Sinai, P.; Kain, S. R. Analytic. Biochem. 1996,
241, 103.
16. Promega-Kit, CytoTox 961 Non-Radioactive Cytotoxi-
city Assay, Cat.Nr.: G1780.