Proximicins A, B, and C - Antitumor furan analogues of netropsin from the marine actinomycete Verrucosispora induce upregulation of p53 and the cyclin kinase inhibitor p21

Article abstract of DOI:10.1002/anie.200705295

Drugs from the sea: Three new netropsin-type antibiotics (1-3) with a hitherto unknown furan core structure have been isolated from marine actinomycete strains and their structures elucidated by means of mass spectrometry and 2D NMR spectroscopy. The compounds show antitumor activity and, in contrast to netropsin, they induce upregulation of p53 and the cyclin kinase inhibitor p21. (Chemical Equation Presented)

Full text of DOI:10.1002/anie.200705295

Communications
DOI: 10.1002/anie.200705295
Marine Natural Products
Proximicins A, B, and C—Antitumor Furan Analogues of Netropsin
from the Marine Actinomycete Verrucosispora Induce Upregulation of
p53and the Cyclin Kinase Inhibitor p21**
Kathrin Schneider, Simone Keller, Falko E. Wolter, Lars Röglin, Winfried Beil, Oliver Seitz,
Graeme Nicholson, Christina Bruntner, Julia Riedlinger, Hans-Peter Fiedler,* and
Roderich D. Süssmuth*
Dedicated to Professor François Diederich
Netropsin (1) and distamycin (2) are naturally occurring g-
peptides with antiviral and antibacterial activity (Figure 1).[1]
Netropsin, formerly named congocidine, was isolated from
S. netropsis in 1951,[2] while distamycin was isolated from
S. distallicus in 1964.[3,4] Both compounds bind in the minor
groove of DNA, and they were arguably the first compounds
for which AT-selective DNA binding was demonstrated.[5–8]
The design of synthetic derivatives capable of addressing a
specific sequence of DNAwould allow the selective inhibition
of gene expression and thus result in compounds that act as
antitumor agents.[5] As a consequence, netropsin and dista-
mycin were used as the basic structures for the synthesis of
numerous analogues to enable the investigation and modu-
lation of their minor-groove binding. This was also achieved
by using combinatorial approaches.[5–11] A particular chal-
lenge was to generate selective binders for GC base pairs.[5–8]
One approach was based on the assumption that the
introduction of a hydrogen-bond acceptor in the pyrrole
Figure 1. Structures of netropsin (1), distamycin (2), and of the
compounds from the marine Verrucosispora: proximicin A (3), proximi-
[*] K. Schneider, S. Keller, F. E. Wolter, Prof. Dr. R. D. Süssmuth
Institut für Chemie, Technische Universität Berlin
Strasse des 17. Juni 124, 10623 Berlin (Germany)
Fax: (+49)30-314-24783
cin B (4), and proximicin C (5). The double helix illustrates the binding
of netropsin (1) in the minor groove of DNA to form a 1:1 complex.[13]
E-mail: suessmuth@chem.tu-berlin.de
Homepage: http://www2.tu-berlin.de/fb5/Suessmuth
C. Bruntner, J. Riedlinger, Prof. Dr. H.-P. Fiedler
Mikrobiologisches Institut, Universität Tübingen
rings of netropsin might allow the drug to bind to GC-rich
sequences.[5–8] By following this strategy, a large number of
Auf der Morgenstelle 28, 72076 Tübingen (Germany)
molecules were synthesized in which the N-methylpyrrole
Fax: (+49)7071-29-5999
ring of netropsin was substituted by other heterocycles, for
example, imidazole, thiazole, triazole, pyrazole, and furan.[5,8]
Since netropsin and distamycin lack selectivity and toxicity[8,9]
their use as drugs was prevented, except as an antiviral agent
in a topical application.[12] Recent efforts have aimed to
remedy these disadvantages.[9] A further important bioactivity
of netropsin and distamycin is their inhibitory activities
against the malaria-causing parasite Plasmodium falcipa-
rum.[7]
E-mail: hans-peter.fiedler@uni-tuebingen.de
G. Nicholson
Institut für Organische Chemie, Universität Tübingen
Auf der Morgenstelle 18, 72076 Tübingen (Germany)
Prof. Dr. W. Beil
Institut für Pharmakologie, Medizinische Hochschule Hannover
Carl-Neuberg-Strasse 1, 30625 Hannover (Germany)
L. Röglin, Prof. Dr. O. Seitz
Institut für Chemie, Humboldt-Universität zu Berlin
Brook-Taylor-Strasse 2, 12489 Berlin (Germany)
Herein we report the structure elucidation of new
netropsin analogues, named proximicin A (3), B (4), and C
(5), from marine actinomycetes of the genus Verrucosispora
(Figure 1). The proximicins bear the hitherto unknown g-
amino acid 4-aminofuran-2-carboxylic acid, which adds a new
element of structural diversity to the previously described
heterocyclic antibiotics. Furthermore, the antitumor activities
[**] The work was supported by the DFG (project SU 239-5/1) and from
the European Commission (project ACTINOGEN, 6th framework,
LSHM-CT-2004-005224). We thank G. Grewe (Universität Tübingen)
for technical assistance in fermentations, and Dr. P. Schmieder
(FMP, Berlin-Buch) for measurement of 15N NMR spectra.
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
3258
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2008, 47, 3258 –3261

Angewandte
Chemie
of netropsin and distamycin were compared to those of the
proximicins and synthetic netropsin–proximicin hybrids (6, 7,
8; Figure 2), with the latter found to be considerably more
cytotoxic in the investigated cell lines.
provided crucial evidence for a dipeptide of 4-aminofuran-2-
carboxylic acid, which represents the core structure of the
proximicins. The 1H-15N HSQC spectrum of 4 revealed three
signals: two corresponding to secondary amides and one
corresponding to a carbamate functionality. The N-terminal
methyl carbamate is present in all three proximicins, and with
the structural difference between the compounds lying in the
C-terminal modifications. Proximicin A (3) contains a C-
terminal amide, whereas proximicin B (4) and proximicin C
(5) have tyramine and tryptamine modifications, respectively.
Interestingly, the proximicins show a high structural analogy
to netropsin (1) and distamycin (2). The characteristic
fragment of these latter two compounds, the 2,4-disubstituted
N-methylpyrrole, is replaced by a 2,4-disubstituted furan ring,
which constitutes a hitherto unknown g-amino acid. Although
a considerable number of netropsin analogues have been
synthesized by exchanging the N-methylpyrrole ring with
other heterocycles, this structural motif was until now
unknown. Furthermore, the g-amino acid 4-aminofuran-2-
carboxylic acid has some structural analogy to GABA
mimetics.[18]
To evaluate the structural and functional relationships
between netropsin and the proximicins we synthesized the
netropsin-proximicin-hybrids 6–8 (Figure 2).[19] In these
hybrids the N-methylpyrrole core of netropsin is combined
with the corresponding N- and C-terminal modifications of
proximicins A, B, and C. Testing the proximicins 3–8 for
antitumor activity against three carcinoma cell lines showed
proximicins 3–5 had a significantly higher antitumor activity
than 1 and 2 (Table 1), while 7 and 8 had a moderately higher
activity. The DNA binding activity of the proximicins was
Figure 2. Structures of the synthetic netropsin–proximicin hybrids A
(6), B (7), and C (8).
Recently, we reported on the fermentation, isolation, and
structure elucidation of the polyketide antibiotics abyssomi-
cins B–D from the marine actinomycete Verrucosispora sp.
AB-18-032, which was isolated from sediment collected from
the Sea of Japan.[14,15] Careful evaluation of the HPLC
chromatograms of extracts of this strain revealed significant
amounts of another compound (3) that was not assignable to
any other known compound of our HPLC-DAD (HPLC
analysis with a diode array detector) database.[16] Remark-
ably, the extract from another marine member of the rare
genus Verrucosispora, strain MG-37, which was isolated from
sediment collected in the Raune Fjord (Norway) at a depth of
250 m, showed an HPLC-DAD signal identical to that of
compound 3. Furthermore, two further signals with similar
UV spectra as that of 3 were also seen, thus indicating the
presence of a family of structure-related peptide metabolites,
which were named proximicin A (3), B (4), and C (5).[17] The
compounds were purified by size-exclusion and adsorption
chromatography as well as by preparative reversed-phase
HPLC, and structure elucidation was carried out by means of
mass spectrometry (HPLC-ESI-MS, FT-ICR-MS, GC-MS)
and two-dimensional NMR spectroscopy.
Table 1: Antitumor activity of compounds 1–8 against three carcinoma
cell lines.[b]
Compound
AGS[a]
HepG2[a]
MCF7[a]
GI50[b] [mm]
GI50[b] [mm]
GI50[b] [mm]
netropsin (1)
distamycin (2)
proximicin A (3)
proximicin B (4)
>116.2
>103.8
2.0
60.4
54.0
2.8
23.0
1.8
119.0
34.1
21.6
74.3
14.3
24.6
12.1
20.6
131.5
41.0
24.9
3.6
0.6
93.9
28.4
17.7
The exact masses determined by high-resolution ESI-FT-
ICR-MS for proximicin A, B, and C were 294.0722 Da
[(M+H)+], 436.1116 Da [(M+Na)+], and 437.14566 Da
[(M+H)+], respectively. These values correspond to the
proximicin C (5)
6
7
8
molecular formulas C12H11N3O6 (3) [(M+H)+
=
theor
[a] Cell lines: AGS: gastric adenocarcinoma; HepG2: hepatocellular
carcinoma, MCF7: breast adenocarcinoma. [b] GI50: 50% growth
inhibition.
294.07206;
Dm = 0.477 ppm],
C20H19N3O7
(4)
and
[(M+Na)+theor = 436.11152;
Dm = 0.165 ppm],
C22H20N4O6 (5) [(M+H)+theor = 437.14556; Dm = 0.215 ppm].
After hydrolysis and derivatization of the compounds,
analysis by GC-MS revealed the presence of tyramine in the
case of proximicin B (4) and of tryptamine in the case of
proximicin C (5).
The subsequent 1D and 2D NMR analyses provided the
missing structural fragments. The chemical shifts and the
coupling constants in combination with the HMBC correla-
tions suggested that all the proximicins contained a furan ring
system with a 2,4-disubstitution pattern, and led to the
assignment of the 2-position as the carbonyl function and the
4-position as the amino function. The HMBC experiment
assessed by analyzing the DNA melting curves. In contrast to
netropsin and distamycin, no shift in the DNA melting
temperature Tm was detected for proximicin A, B, and C (3–5)
from Verrucosispora or for the synthetic hybrid molecules (6–
8).
A cell-cycle analysis in AGS cells revealed that 1
(116.2 mm) and 2 (103.8 mm) produce an accumulation of
cells in G2/M after incubation for 24 h (1: + 6.9%; 2: + 5.4%)
and 40 h (1: + 11.3%; 2: + 28.9%), and reduce the ratio of
cells in the G0/G1 phase (1: À9.2% and À9.5%; 2: À7% and
À20.5%). Proximicin C (5; 1.1 mm) and compound 8 (21.6 mm)
Angew. Chem. Int. Ed. 2008, 47, 3258 –3261ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
3259

Communications
produced cell arrest in the G0/G1 phase after incubation for
and furthermore increase the overall polarity of the molecule.
Although hybrid C (8) possesses a different molecular
structure and shows a lower antitumor activity, it seems to
address the same cellular target as the corresponding
proximicin C (5), thereby suggesting that the C-terminal
tryptamine residue can exert a significant influence on the
upregulation of p53 and p21. However, it seems as if the
methyl carbamate is the main contributor to the antitumor
activity. Boger et al. reported on a similar effect for distamy-
cins in which the formyl function was exchanged for a tert-
butyl carbamate moiety, while keeping a positive charge at
the C terminus.[11]
24 h (5: + 5.6%; 8: + 21.4%). After 40 h, there was an
increase in the number of cells in the sub-G1 phase, that is,
apoptotic cells (5: + 2.9%; 8: + 9.8%).
On the basis of these data we evaluated whether 5 and 8
activate cell-cycle regulatory proteins involved in the tran-
sition of cells from the G1 to the S phase (p53, p21, cyclin E).
It was found that proximicin C (5; data not shown) and
hybrid C (8) induce upregulation of p53 and of the cyclin
kinase inhibitor p21 in AGS cells (Figure 3). However,
Overall, in regard to the effect of the herein-described
proximicins on the cell cycle, we found an arrest of AGS cells
in G0/G1 and an increase in the levels of p53 and p21, whereas
distamycin, as reported by Poot et al.,[20] arrested the cells in
G2/M. Distamycin, in contrast to the proximicins, inhibited
cell growth in p53-mutated cells (Huh7). These data strongly
confirm that the proximicins act on a different cellular target,
that is, the transition of cells from the G1 to the S phase.
In summary, we have presented the structures of three
new netropsin-type antibiotics from marine actinomycete
strains that have interesting antitumor activity. Current work
is directed towards the total synthesis of proximicins as well as
to the generation of furan-based proximicin–netropsin
hybrids, to evaluate the antitumor activity of these com-
pounds in more detail.
Figure 3. Western blot analysis showing the effect of netropsin–prox-
imicin hybrid C (8) on the upregulated expression of p21, p53, and
cyclin E in AGS cells after 6 hand 13 hincubation.
distamycin (2; 103.8 mm) did not induce the expression of
p53 and p21 (see the Supporting Information). To further
compare the effects of 1, 2, 5, and 8, growth inhibition by the
compounds was tested in hepatoma cells with mutated p53
(Huh7).[20] The data obtained showed that 1 and 2 had similar
cytotoxicity as in the HepG2 cells (GI50: 1: 57.7 mm; 2:
62.3 mm), while 5 and 8 did not inhibit cell growth.
Netropsin (1; 58.1 mm) arrested Huh7 cells in G2/M (+
4.4%), while distamycin (2; 51.9 mm) arrested Huh7 cells in
G2/M (+ 8.7%) and in the S phase (+ 10.6%). Proximicin C
(5; 4.4 mm) and compound 8 (21.6 mm) did not alter the cell-
cycle distribution in the Huh7 cells.
Received: November 18, 2007
Revised: January 10, 2008
Published online: March 17, 2008
Keywords: antitumor activity · natural products · netropsin ·
.
proximcin · structure elucidation
[1] J. W. Lown, J. Mol. Recognit. 1994, 7, 79– 88.
[2] A. C. Finlay, F. A. Hochstein, B. A. Sobin, F. X. Murphy, J. Am.
Chem. Soc. 1951, 73, 341 – 343.
Remarkably, the experiments show that the antitumor
activities of the proximicins bearing an N-terminal methyl
carbamate are significantly higher than those of netropsin (1)
and distamycin (2), which have guanidinylglycine and formyl
functions, respectively. Analyses of the DNA melting curves
show that the antitumor activity of proximicins 3–5 as well as
of the hybrids 6–8 cannot be based on DNA binding. The
structural moieties which constitute the structural differences
of the proximicins compared to netropsin are modifications at
1) the N terminus, 2) the C terminus, and 3) the furan–N-
methylpyrrole exchange. The different C-terminal amide
modifications of proximicin A (3) and C (5) does not have a
great influence on the antitumor activity. This effect is more
pronounced in the synthetic hybrids 6–8; however, these N-
methylpyrrole derivatives display a generally lowered anti-
tumor activity compared to the furan analogues. The
exchange of the furan oxygen atom by an N-methyl group
leads to a 1.2- to ca. 30-fold decrease in antitumor activity.
This decrease may be based on a significant change in the
electronic properties of the proximicins, since the two furan
oxygen atoms may act as additional hydrogen-bond acceptors
[3] F. Arcamone, S. Penco, P. Orezzi, V. Nicolella, A. Pirelli, Nature
1964, 203, 1064 – 1065.
[4] P. G. Baraldi, D. Preti, F. Fruttarolo, M. A. Tabrizi, R. Romag-
noli, Bioorg. Med. Chem. 2007, 15, 17 – 35.
[5] C. Bailly, B. J. Chaires, Bioconjugate Chem. 1998, 9, 513 – 538.
[6] P. B. Dervan, B. S. Edelson, Curr. Opin. Struct. Biol. 2003, 13,
284 – 299.
[7] B. S. P. Reddy, S. K. Sharma, J. W. Lown, Curr. Med. Chem. 2001,
8, 475 – 508.
[8] D. E. Wemmer, Biopolymers 1999, 52, 197 – 211.
[9] C. J. Suckling, Expert Opin. Ther. Pat. 2004, 14, 1693 – 1724.
[10] D. L. Boger, M. A. Dechantsreiter, T. Ishii, B. E. Fink, M. P.
Hedrick, Bioorg. Med. Chem. 2000, 8, 2049– 2057.
[11] D. L. Boger, B. E. Fink, M. P. Hedrick, J. Am. Chem. Soc. 2000,
122, 6382 – 6394.
[12] L. De Angelis, Drugs Today 1977, 13, 319– 326.
[13] M. L. Kopka, C. Yoon, D. Goodsell, P. Pjura, R. E. Dickerson, J.
Mol. Biol. 1985, 183, 553 – 563.
[14] J. Riedlinger, A. Reicke, H. Zꢀhner, B. Krismer, A. T. Bull, L. A.
Maldonado, A. C. Ward, M. Goodfellow, B. Bister, D. Bischoff,
R. D. Sꢁssmuth, H.-P. Fiedler, J. Antibiot. 2004, 57, 271 – 279.
[15] B. Bister, D. Bischoff, M. Strꢂbele, J. Riedlinger, A. Reicke, F.
Wolter, A. T. Bull, H. Zꢀhner, H.-P. Fiedler, R. D. Sꢁssmuth,
3260 www.angewandte.org
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2008, 47, 3258 –3261

Angewandte
Chemie
Angew. Chem. 2004, 116, 2628 – 2630; Angew. Chem. Int. Ed.
2004, 43, 2574 – 2576.
[16] H.-P. Fiedler, Nat. Prod. Lett. 1993, 2, 119– 128.
[17] H.-P. Fiedler, C. Bruntner, J. Riedlinger, A. T. Bull, G. Knutsen,
A. L. Jones, M. Goodfellow, W. Beil, K. Schneider, S. Keller,
R. D. Sꢁssmuth, J. Antibiot. 2008, in press.
Chem. Lett. 2004, 14, 203 – 206; c) Z. Wang, H. Yuan, D. Nikolic,
R. B. Van Breemen, R. B. Silverman, Biochemistry 2006, 45,
14513 – 14522.
[19] For the synthesis of the netropsin–proximicin hybrids 6–8 see the
Supporting Information.
[20] M. Poot, K. H. Hiller, S. Heimpel, H. Hoehn, Exp. Cell Res. 1995,
218, 326 – 330.
[18] a) J. P. Burkhart, G. W. Holbert, B. W. Metcalf, Tetrahedron Lett.
1984, 25, 5267 – 5270; b) M. Fu, R. B. Silverman, Bioorg. Med.
Angew. Chem. Int. Ed. 2008, 47, 3258 –3261ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
3261