Synthesis and evaluation of a netropsin-proximicin-hybrid library for DNA binding and cytotoxicity

Article abstract of DOI:10.1016/j.bmcl.2009.04.042

The proximicins A-C (1-3) are novel naturally occurring γ-peptides with a hitherto unknown 2,4-disubstituted furan amino acid as a core structure. They show a moderate cytotoxic activity and induce upregulation of cell cycle regulating proteins (p53 and p21) and lead to cell cycle arrest in G0/G1-phase. Hybrid molecules combining structural motifs of the proximicins and of netropsin (4), a structurally related natural product, seem to have similar effects. Herein we describe the synthesis of a netropsin-proximicin-hybrid library and its evaluation regarding cytotoxicity and minor groove binding activity.

Full text of DOI:10.1016/j.bmcl.2009.04.042

Bioorganic & Medicinal Chemistry Letters 19 (2009) 3811–3815
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and evaluation of a netropsin–proximicin-hybrid library for DNA
binding and cytotoxicity
Falko E. Wolter a, Lise Molinari a, Elke R. Socher b, Kathrin Schneider a, Graeme Nicholson c, Winfried Beil d,
Oliver Seitz b, Roderich D. Süssmuth a,
*
a FG Organische Chemie, Institut für Chemie, Technische Universität Berlin, Straße des 17. Juni 124, 10623 Berlin, Germany
b Institut für Chemie, Humboldt Universität Berlin, Brook-Taylor-Str. 2, 12489 Berlin, Germany
c Institut für Chemie, Eberhard Karls Universität Tübingen, Auf der Morgenstelle 18, 72076 Tübingen, Germany
d Institut für Pharmakologie, Medizinische Hochschule Hannover, Carl-Neuberg-Str. 1, 30625 Hannover, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 15 February 2009
Revised 7 April 2009
Accepted 8 April 2009
Available online 18 April 2009
The proximicins A–C (1–3) are novel naturally occurring c-peptides with a hitherto unknown 2,4-disub-
stituted furan amino acid as a core structure. They show a moderate cytotoxic activity and induce upreg-
ulation of cell cycle regulating proteins (p53 and p21) and lead to cell cycle arrest in G0/G1-phase. Hybrid
molecules combining structural motifs of the proximicins and of netropsin (4), a structurally related nat-
ural product, seem to have similar effects. Herein we describe the synthesis of a netropsin–proximicin-
hybrid library and its evaluation regarding cytotoxicity and minor groove binding activity.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Netropsin–proximicin-hybrid
DNA binding
Cytotoxicity
Proximicin A, B, and C (1–3) are natural occurring
c
-peptides
the different N- and C-terminal residues attached to the central
dipeptide.
which were isolated from a marine Actinomycete strain of the
genus Verrucosispora (Fig. 1).1,2 As a main structural feature
these compounds bear a hitherto unknown 2,4-disubstituted
furan amino acid as a dipeptide core. This heterocyclic core is
structurally closely related to the prominent natural products
netropsin (4) and distamycin (5) (Fig. 1).3–5 These were argu-
ably the first compounds for which AT-selective DNA minor
groove binding was demonstrated.6 Furthermore, these com-
pounds served as scaffolds for a large variety of synthetic minor
groove binding polyamides capable to specifically recognize
DNA sequences.6–14 In contrast, no DNA binding activity could
be shown for the proximicins.2 Furthermore, cell cycle analysis
and analysis of the level of cell cycle regulating proteins (p53,
p21, and cyclinE) in comparison with distamycin revealed that
Toward this end, we synthesized a 19-membered library of
netropsin–proximicin hybrid molecules incorporating various N-
and C-terminal modifications attached to the dipeptide core of
netropsin and examined the cytotoxicity as well as potential minor
groove binding of these derivatives. We reported previously, that
netropsin–proximicin-hybrid C (8) is the most active hybrid.2 As
a consequence, its N-terminal methyl carbamate and its C-terminal
tryptamine served as template for the synthesis of a second gener-
ation of hybrids. The herein presented hybrid molecules can be di-
vided into two subsets: One group bears the methyl carbamate as a
constant element but differs in C-terminal modifications (13a–i).
Several biogenic amines, amino acids, and an ester function were
introduced at this position (Table 1). The other subset of hybrids
consists of members possessing the tryptamine moiety at the C-
terminus but different N-terminal residues (16a–j), like alkyl or
aryl carbamates, urea derivatives, and acylations (Table 2).
With these modifications at the N- and C-termini of the N-
methylpyrrol dipeptide the influence of different electronic and
steric effects on the biological activity should be determined.
The synthesis of all netropsin–proximicin-hybrids (13, 16)
starts off with commercially available compound 9 (Schemes 1
and 2). The various modifications in the respective subset of
the hybrid molecules were introduced in the final reaction step
in order to simplify the overall synthesis. The amide bonds of
all hybrids were mainly synthesized by peptide couplings
the proximicins might have
a different cellular target than
netropsin and distamycin despite their close structural relation-
ship.2 Similar results were obtained with a first set of synthetic
netropsin–proximicin-hybrids (6–8) which combine structural
features of both natural products (Fig. 1).2 This led to the
assumption that the difference in the biological activity be-
tween the proximicins (1–3) and netropsin (4) is caused by
* Corresponding author.
E-mail address: suessmuth@chem.tu-berlin.de (R.D. Süssmuth).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.04.042

3812
F. E. Wolter et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3811–3815
Figure 1. Structures of proximicin A–C (1–3), netropsin (4), distamycin (5), and of the three netropsin–proximicin-hybrids A–C (6–8).
applying an optimized protocol using EDCI and DMAP as acti-
vating reagents.15 This allows an easy purification by simple
acid/base liquid–liquid extracting procedures in order to avoid
column chromatography. Thus obtained products were pure en-
ough for the subsequent reaction steps.16 Only after the last
reaction step all compounds were purified by RP-HPLC for bio-
logical evaluation.17
(NH-function of an amide) to a hydrogen bond acceptor (O-atom
of an ester). Alternatively, the ester moiety lowers the overall
polarity of the molecule and might simplify penetration of the
cell membrane.
With regard to antitumor activity a great variety of N-termi-
nal modifications is tolerated. The comparison of active with
inactive members of this subset reveals subtle differences in
the effects caused by the different residues. Among the carba-
The antitumor activity of 13a–i and 16a–j was evaluated
against three carcinoma cell lines up to a maximum concentra-
mates it seems that a methylene group at the
a-position next
tion of 20
l
g/mL.18 The growth inhibition (GI50) values of the
to the oxygen is mandatory for biological activity. This is con-
firmed by the inactive tert-butyl carbamate 16b. But steric ef-
fects in the carbamate part appear negligible with regard to
the bulky residues of 16d–f.
compounds which were cytotoxic below this concentration are
shown (Table 3). The corresponding data of the proximicins
(1–3) and of netropsin–proximicin-hybrid C (8) are also given
in comparison.2 Whereas the antitumor activity of the hybrids
(13i, 16c–f, h, j) listed in Table 3 is higher or at least of the same
order as hybrid C (8), all other new synthesized derivatives
(13a–h, 16a+b, 16g, 16i) did not show a noteworthy antitumor
activity.
Remarkably, ester 13i is the only active compound from the
subset of C-terminally modified hybrids. Its cytoxicity is signifi-
cantly higher than that of hybrid C (8) and comparable to that of
the proximicins (1–3). The subset of N-terminal modifications con-
sists beside the derivative with the free amino group (16a) out of
carbamates (16b–f), ureas (16i+j), and acylated derivatives
(16g+h). Active derivatives can be found in all three chemical clas-
ses, which are 16c–f, 16h, and 16j showing a moderate cytotoxicity
(Table 3).
The results give an estimate of the influence of N- and C-ter-
minal modifications on the cytotoxicity of the netropsin–proxim-
icin-hybrids. Compound 13i, the only active hybrid out of the
subset of C-terminal modifications, reveals that an ester function
at the C-terminus raises the activity in comparison to an amide
function. This structural modification might have two distinctive
influences. One possibility is a shift from a hydrogen bond donor
Whereas 16e, a benzyl carbamate, shows a growth inhibiting
activity of tumor cells the corresponding urea derivative 16i is
inactive. Again, this corresponds to a shift from a hydrogen bond
acceptor (O-atom, 16e) to a hydrogen bond donor (NH, 16i). Inter-
estingly derivative 16j, which lacks a methylene group compared
with derivative 16i, displays a moderate cytotoxicity. In case of
an acetylation of the N-terminus (16g) no antitumor activity was
observed and only a moderate cytotoxicity for the corresponding
pivaloyl derivative (16h). These data support the view that an
overall non-polar structure of the molecule appears to be beneficial
for cytotoxicity.
The stem compounds 13a and 16a reveal that hybrids with a
polar free N-terminus or C-terminus lack cytotoxic activity. Nei-
ther the derivative with free amino group (16a) nor with the
carboxylic acid (13a) displays cytotoxic effects. Other polar
functional groups like phenols (13c, h) or carboxylic acids
(13e, f) in the C-terminal residues seem to prevent an activity,
too.
Based on the data obtained from cytotoxicity measurements,
we were interested in investigating the potential for minor
groove binding activity of our synthetic derivatives. The library

F. E. Wolter et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3811–3815
3813
Table 1
Table 2
Structures, reaction conditions and yields of the C-terminally modified library
members (Scheme 1)
Structures, reaction conditions and yields of the N-terminally modified library
members (Scheme 2)
Compd
Structure, R=a
OH
Reagents, conditions
H2/Pd
Yieldb
—
Compd
Structure, R=a
H
Reagents, conditions
—
Yieldb
—
13a
16a
H
N
O
O
13b
Phenylethylaminec
8 mg, 12%
16b
16c
Boc2Oc
7 mg, 12%
O
O
Alloc-Cld
13 mg, 23%
H
N
OH
OH
13c
13d
Dopaminec
5 mg, 8%
O
H
N
16d
16e
Troc-Cld
Cbz-Cld
8 mg, 13%
8 mg, 13%
Cl
Cl Cl
O
Isoamylaminec
18 mg, 29%
O
HOOC
O
N
H
13e
H-
L
-Trp-OMeÁHCld
4 mg. 5%
N
H
O
O
16f
Fmoc-OSud
9 mg, 13%
OH
N
H
13f
13g
13h
H-GABA-OMeÁHCld
6 mg, 10%
6 mg, 10%
24 mg, 32%
17 mg, 26%
O
O
O
16g
AcCld
11 mg, 22%
N
Me
Histaminee
N
H
N
H
16h
16i
Piv-Cld
BICd
6 mg, 12%
9 mg, 15%
N
H
OH
Serotoninec
O
N
H
N
H
O
13i
Phenylethanolc
O
16j
PICd
6 mg, 10%
N
a
H
Referring to compound 13 in Scheme 1.
After purification by preparative RP-HPLC; conditions (cf. Scheme 1).
EDCI/DMAP, DMF, 16 h, rt.
(1) EDCI/DMAP, DMF, 16 h, rt; (2) 0.1 N LiOH, rt.
PyBOP, DIPEA, DCM/DMF, 0 °C?rt.
b
c
a
Referring to compound 16 in Scheme 2.
b
c
After purification by preparative RP-HPLC; conditions (cf. Scheme 2).
1.3 equiv reagent, NaHCO3, dioxane/H2O, rt.
1.0 equiv reagent, TEA, THF, rt.
d
e
d
members of both subsets were incubated in various concentra-
tions with an AT-rich (cgcaaatttgcg) and with a GC-rich (catggc-
catg) oligonucleotide (Table 4, only compounds are shown which
display DNA interactions).19 Within our test panel we found that
not all compounds displaying cytotoxicity also showed DNA
binding capabilities. Incubation with 13i and 16j result in a shift
of Tm by 25 °C and 6 °C, which is selective for AT-rich DNA. In
contrast 16d and e lead to a stabilizing effect of both AT-rich
and GC-rich DNA sequences. Their stabilizing effect is even
stronger for GC-rich DNA sequence represented by a shift in
Tm of 21 °C (16d) and 23 °C (16e), respectively. It seems that
both compounds unspecifically bind into the minor groove of
DNA.
In summary, we presented the synthesis and biological evalua-
tion of new netropsin–proximicin-hybrids (Fig. 2). Compared to
the first generation of hybrids2 the antitumor activity of some
new hybrids could be increased to the same order of magnitude
of proximicin A, B, and C. In addition we were able to generate a
set of new minor groove binders based on the core structure of
netropsin. These DNA binding agents partially show selectivity
for an AT-rich oligonucleotide and lead to a very strong stabiliza-
tion of the double helix as determined by DNA melting point
analyses.
The netropsin–proximicin-hybrids are accessible by a simple
synthetic methodology. Further work is directed to the synthesis
of other hybrids based on 13i as a lead structure in order to exam-

3814
F. E. Wolter et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3811–3815
BocHN
1. Cs2CO3, EtOH/H2O
2. BnBr, DMF
1. 4 N HCl/dioxane
2. 9, EDCI, DMAP
BocHN
OH
OBn
N
(83 %)
N
(90 %)
O
O
Me
Me
9
10
MeO
BocHN
NH
1. 4 N HCl/dioxane
2. MeOCOCl, DIPEA,
THF
OBn
O
OBn
O
H
N
H
O
N
N
N
N
N
(93 %)
O
O
Me
Me
Me
Me
11
12
MeO
NH
1. H2/Pd
2. conditions
R
H
N
O
O
N
Me
N
O
Me
13
Scheme 1. Synthesis of the subset with C-terminal modifications. Conditions and yields of the ultimate reaction step are shown in Table 1.
BocHN
1. 4 N HCl/dioxane
2. 9, EDCI, DMAP
BocHN
Trpa,
EDCI, DMAP
H
N
OH
N
(88 %)
N
(79 %)
NH
O
O
Me
Me
9
14
R
1. 4 N HCl/
dioxane
BocHN
NH
2. conditions;
DIPEA
H
H
N
N
N
Me
N
Me
NH
NH
HN
HN
O
O
O
N
N
O
Me
Me
15
16
Scheme 2. Synthesis of the subset with N-terminal modifications. Conditions and yields of the ultimate reaction step are shown in Table 2.
Table 3
Antitumor activities of selected compounds against three carcinoma cell lines
AGSa GI50
(
lM)
HepG2a GI50
(l
M)
MCF7a GI50
(lM)
b
b
b
Compounds
Table 4
Proximicin A (1)
Proximicin B (2)
Proximicin C (3)
Hybrid C (8)
13i
16c
16d
16e
16f
2.0c
3.6c
0.6c
17.7c
2.8
2.8c
23.0c
1.8c
21.6c
2.0
14.3
4.0
5.6
24.6c
12.1c
20.6c
24.9c
13.0
15.4
10.3
10.8
18.8
17.4
19.7
DNA binding of netropsin (4), distamycin (5) and of 13i and 16d, e, j (values given in
°C)
Compounds
oligonucleotide 1 cgcaaatttgcg
oligonucleotide 2 catggccatg
a
a
b
a
a
b
Tm
51c
0
l
M
Tm
6
lM
D
Tm
Tm
0
lM
Tm
6
lM
DTm
1.7
10.0
9.3
Netropsin (4)
69c
65c
71
18
16
25
11
17
6
46c
46c
47
47
47
48c
45c
47
68
70
ꢀ0
ꢀ0
0
21
23
ꢀ0
Distamycin (5) 49c
>31.9d
23.5
16.8
4.6
13i
16d
16e
16j
47
48
48
47
>40.9e
14.3
59
65
53
16h
16j
47
45
Inactive derivatives (GI50 > 20
l
g/mL) are not shown.
a
Other synthetic derivatives showed no DNA binding.
Cell lines: AGS: gastric adenocarcinoma; HepG2: hepatocellularcarcinoma,
MCF7: breast adenocarcinoma.
a
Tm: melting point, given Tm-value is the mean of at least three denaturation
b
curves.
GI50: 50% growth inhibition.
Data obtained from literature (Ref. 2).
47% inhibition at 20
37% inhibition at 20
b
c
Differences in Tm of pure oligonucleotide and after adding 6
M oligonucleotide.
Data obtained from the literature (Ref. 2).
lM of ligand to
d
2
l
l
l
g/mL.
g/mL.
c
e

F. E. Wolter et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3811–3815
3815
Figure 2. Overview of synthesized netropsin–proximicin-hybrids. Structures of compounds with a cytotoxicity <20
with a cytotoxicity >20
l
g/mL are shown in solid black; structures of compounds
l
g/mL are given in light grey; structures of compounds capable to interact with double-stranded DNA are marked with an asterisk.
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Acknowledgment
The work was supported by the Deutsche Forschungsgemeins-
chaft (DFG project SU 239-5/1) and by the Cluster of Excellence
‘Unifying Concepts in Catalysis’ coordinated by the Technische Uni-
versität Berlin and funded by the DFG.
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References and notes
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17. Substantial mass-losses were observed after purification by RP-HPLC.
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