DNA cross-linking by a phototriggered pyrrolic progenitor developed from monocrotaline

Article abstract of DOI:10.1016/S0040-4039(01)01341-7

A wide variety of pyrrolizidine alkaloids, such as monocrotaline, exert their cytotoxicity through the formation of DNA-DNA interstrand cross-links and DNA-protein cross-links. These pyrrolizidine alkaloids are oxidatively activated in vivo forming a highly reactive pyrrolic-type intermediate, which is responsible for the DNA cross-linking reaction. The oxidative pathway of activation leads to undesired toxicity. Based on a previously reported photochemically triggered progenitor of monocrotaline, we describe here the semi-synthesis and DNA cross-linking of a dicarbamate analogue of the original phototriggered progenitor.

Full text of DOI:10.1016/S0040-4039(01)01341-7

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 42 (2001) 6641–6643
DNA cross-linking by a phototriggered pyrrolic progenitor
developed from monocrotaline
Christi Kosogof,b Jetze J. Tepea and Robert M. Williamsb,*
aDepartment of Chemistry, Michigan State University, East Lansing, MI 48824, USA
bDepartment of Chemistry, Colorado State University, Fort Collins, CO 80523, USA
Received 20 July 2001; accepted 23 July 2001
Abstract—A wide variety of pyrrolizidine alkaloids, such as monocrotaline, exert their cytotoxicity through the formation of
DNA–DNA interstrand cross-links and DNA–protein cross-links. These pyrrolizidine alkaloids are oxidatively activated in vivo
forming a highly reactive pyrrolic-type intermediate, which is responsible for the DNA cross-linking reaction. The oxidative
pathway of activation leads to undesired toxicity. Based on a previously reported photochemically triggered progenitor of
monocrotaline, we describe here the semi-synthesis and DNA cross-linking of a dicarbamate analogue of the original phototrig-
gered progenitor. © 2001 Elsevier Science Ltd. All rights reserved.
Pyrrolizidine alkaloids (PAs) are potent hepatotoxins
and carcinogens isolated from a wide variety of
plants.1,2 Natural members of this large family, such as
monocrotaline 1, exert their cytotoxicity through the
formation of DNA–DNA and DNA–protein cross-
links.3 Pyrrolizidine alkaloids are activated in vivo by
liver cytochrome P450 mixed-function oxidases.4,5 Oxi-
dation of the dihydropyrrole moiety results in the in
situ generation of electrophilic pyrrolic intermediates
that have been shown to cross-link DNA primarily at
the 5%CpG3% site in the minor groove of DNA.6 The
pyrrolic pyrrolizidine alkaloids are potent DNA–DNA
and DNA–protein cross-linking agents and are there-
fore of interest as potential antitumor and antibacterial
agents.3a,7–9 The clinical utility of such substances has,
unfortunately, been obviated by the acute hepatotoxic-
ity that these compounds display; a direct manifestation
hydromonocrotaline upon photochemical activation.8
In this system, photochemical cleavage of the NVOC
group on the tetrahydropyrrole ring nitrogen atom,
lead to the formation of dehydromonocrotaline, which
induced DNA–DNA cross-link formation in linear
pBR322 DNA. However, this original phototriggered
compound (2) displayed poor solubility in water,
requiring the DNA interstrand cross-linking experi-
ments to be conducted in 10% DMSO/water solutions.
In an attempt to improve the water solubility of such
substances, we have endeavored to remove the macro-
cyclic diester backbone and replace this substituent with
primary carbamates. The carbamate groups were envi-
sioned to impart good water-solubility as well as
providing for good leaving groups in the covalent DNA
interstrand cross-linking reactions, as illustrated by the
antitumor agent mitomycin C and the FR-class of
antitumor antibiotics.10,11
of the mechanism for their activation in vivo.
The synthesis of the photoactivated dicarbamate
derivative 8 was accomplished from commercially avail-
able monocrotaline (Scheme 2).12 Monocrotaline was
In 1999, Tepe and Williams reported the first photo-
chemically triggered progenitor of a pyrrolizidine alka-
loid (compound 2, Scheme 1), that generated de-
Me
OH
Me
OH
O
Me
Me
O
O
O
DNA
DNA
HO
Me
HO
Me
O
O
hv (365 nm)
DNA
O
O
H
H
H
N
N
N
CHO
NVOC
3, dehydromonocrotaline
4, interstrand DNA cross-link
2
Scheme 1.
* Corresponding author. E-mail: rmw@chem.colostate.edu
0040-4039/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(01)01341-7

6642
C. Kosogof et al. / Tetrahedron Letters 42 (2001) 6641–6643
365 nm light for 1 hour results in DNA cross-link
formation (lanes 10 and 12). As shown, only the reac-
tions depicted in lanes 10 and 12 produced the inter-
strand DNA–DNA cross-link product similar to that
observed from treatment with dehydromonocrotaline
(lane 4).
condensed with 9-fluorenylmethyl chloroformate in the
presence of KI in acetonitrile to afford the allylic iodide
5.8,9 The allylic iodide was oxidized to the correspond-
ing aldehyde under Kornblum conditions (DMSO and
AgBF4). The aldehyde was subsequently protected as
the ethylene glycol acetal. The 9-fluorenylmethyl pro-
tecting group was cleaved with piperidine in THF (1:1
mixture) and the free amine was acylated with 6-nitro-
veratryl chloroformate, to give the NVOC-derivative 6.
The diester backbone was cleaved with KCN in 95%
ethanol to give the diol 7. The carbamate moieties were
installed using trichloroacetylisocyanate, followed by
removal of the trichloroacetyl residues over neutral
alumina.13 Finally, the acetal was deprotected with 1%
aqueous HCl in acetone to give the masked
pyrrolizidine derivative 8.
These studies suggest that cleavage of the diester back-
bone does not adversely affect the cross-linking poten-
tial of the pyrrolic substrate. In addition, this work
further demonstrates the viability that more structurally
diverse masked DNA-reactive pyrrolizidine progenitors
The DNA–DNA cross-linking ability of dicarbamate
derivative 8 was investigated using linear pBR322 DNA
by denaturing alkaline agarose gel electrophoresis
according to the protocol reported by Cech.14 Com-
pound 8 (various concentrations of a 10 mM stock
solution) and 0.5 mg DNA (EcoR1 linearized pBR322)
in a DMSO–water solvent mixture (10 mL, 0.001%
DMSO/H2O) was exposed to 365 nm light at 23°C for
1 h, followed by incubation at 37°C for 9 h. The crude
reaction mixture was loaded onto a denaturing alkaline
agarose gel and provided the results shown in Fig. 1.
Figure 1. All dark (control) reactions were incubated at 37°C
for 10 hours. The reactions exposed to UV radiation (1 hour)
were incubated an additional 9 hours at 37°C. Lane (1) 0.5 mg
lambda DNA-BstE II digest (molecular weight standard);
lane (2) 0.5 mg pBR322 (control); lane (3) 0.5 mg pBR322+hw,
1 hour (light control); lane (4) 0.5 mg pBR322+10 mM dehy-
dromonocrotaline; lane (5) 0.5 mg pBR322+10 mM NVOC-
piperidine (dark control); lane (6) 0.5 mg pBR322+10 mM
NVOC-piperidine+hw, 1 hour (light control); lane (7) 0.5 mg
pBR322+1 mM NVOC-piperidine+hw, 1 hour (light control);
lane (8) 0.5 mg pBR322+100 nM NVOC-piperidine+hw, 1 hour
(light control); lane (9) 0.5 mg pBR322+10 mM compound 8
(dark control); lane (10) 0.5 mg pBR322+10 mM compound
8+hw, 1 hour; lane (11) 0.5 mg pBR322+1 mM compound 8
(dark control); lane (12) 0.5 mg pBR322 +1 mM compound
8+hw, 1 hour; lane (13) 0.5 mg pBR322+100 nM compound 8
(dark control); lane (14) 0.5 mg pBR322+100 nM compound
8+hw, 1 hour.
Lambda DNA-BstE II was employed as a molecular
weight standard (lane 1). Control reactions were per-
formed with NVOC-protected piperidine (10 mM, 1
mM, 100 nM) to assure that the photocleaved side
product, 6-nitroveratryl aldehyde was not responsible
for the observed cross-linked DNA. An authentic spec-
imen of dehydromonocrotaline, 3 (10 mM) was also
used as a DNA cross-link standard. As illustrated in
Fig. 1, incubation of compound 8 with the DNA
duplex in the dark leads to no detectable cross-linked
product (lanes 9, 11, 13). Incubation of compound 8
with linear pBR322 at 10 and 1 mM with exposure to
Me
Me
OH
Me
OH
OH
Me
Me
Me
1. AgBF4, DMSO,
O
O
O
O
TEA, rt
O
O
HO
Me
HO
HO
Me
Me
2. HOCH2CH2OH,
TMSCl, CH2Cl2
O
O
O
O
H
O
FMOC-Cl, KI
MeCN
O
H
H
H
H
H
3. Piperidine, THF
4. NVOC-Cl
N
N
N
O
30%
NVOC
Fmoc
Hunig's base, THF
I
O
51%
1, monocrotaline
5
6
O
O
H
NH2
H2N
O
OH
O
O
H
N
HO
H
H
1. Cl3CCONCO, Al2O3
2% KCN in 95% EtOH
68%
2. 1% HCl, acetone
50%
N
CHO
NVOC
NVOC
O
7
8
Scheme 2.

C. Kosogof et al. / Tetrahedron Letters 42 (2001) 6641–6643
6643
should be capable of being designed and synthesized for
more selective oligonucleotide modifications. Such
agents hold promise as useful tools to gain insight into
the mechanism of DNA–DNA and DNA–protein
cross-linking. It has been demonstrated that dehy-
dromonocrotaline undergoes rapid polymerization that
generates a structure capable of cross-linking several
fragments of DNA and that the cross-linked adducts
are structurally complex.16 As such, an attempt to
ascertain the sequence selectivity of interstrand DNA
cross-linking for compound 8 was not undertaken. It
might be further noted that Hincks et al. concluded
that pyrrolizidine alkaloids require both a macrocyclic
necic ester and an a,b-unsaturated ester function for
potent cross-linking.17,18 Our preliminary results with
compound 8 that lacks both such functionalities, sug-
gests that additional structure–activity data needs to be
acquired and considered. With the increasing demand
for new and less cytotoxic antitumor agents, and the
recent success of the clinically significant photopheresis
technologies, these agents can provide a conceptual
framework for the development of new pyrrolizidine-
type pro-drugs.15 Studies towards these ends are under
investigation in these laboratories and will be reported
on in due course.
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This work was supported by the National Institutes of
Health (grant cRO1CA51875). We are grateful to Ted
Judd for helpful discussions.
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