Design, synthesis, and biological evaluation of achiral analogs of duocarmycin SA

Article abstract of DOI:10.1016/j.bmcl.2004.10.021

The design, synthesis, as well as biochemical and biological evaluation of two novel achiral analogs of duocarmycin SA (DUMSA), 1 and 2, are described. Like CC-1065 and adozelesin, compounds 1 and 2 covalently reacted with adenine-N3 in AT-rich sequences

Full text of DOI:10.1016/j.bmcl.2004.10.021

Bioorganic & Medicinal Chemistry Letters 15 (2005) 177–180
Design, synthesis, and biological evaluation of achiral analogs
of duocarmycin SA
Kristen Daniell,a Michelle Stewart,a Erik Madsen,a Minh Le,a Heather Handl,a
Natalie Brooks,b Konstantinos Kiakos,b John A. Hartleyb and Moses Leea,*
aDepartment of Chemistry, Furman University, Greenville, SC 29613, USA
bCancer Research UK Drug-DNA Interactions Research Group, Department of Oncology, University College London, 91 Riding
House Street, London, W1W 7BS UK
Received 31 August 2004; revised 4 October 2004; accepted 5 October 2004
Available online 28 October 2004
Abstract—The design, synthesis, as well as biochemical and biological evaluation of two novel achiral analogs of duocarmycin SA
(DUMSA), 1 and 2, are described. Like CC-1065 and adozelesin, compounds 1 and 2 covalently reacted with adenine-N3 in AT-rich
sequences and led to the formation of DNA strand breaks upon heating. The cytotoxicity of compounds 1 and 2 against human
cancer cells (K562, LS174T) was determined using a MTT assay giving IC50 values in the low nanomolar. Further cytotoxicity
screening of compound 2 conducted by the NCI against a panel of 60 different human cancer cell lines indicated that it was partic-
ularly active against several solid tumor cells lines derived from the lung, colon, CNS, skin, and breast.
Ó 2004 Elsevier Ltd. All rights reserved.
CC-10651 and duocarmycin SA2 (Fig. 1) are cyclopro-
panepyrroloindolone- or CPI-containing natural prod-
ucts isolated from Streptomyces. Both compounds
exhibit potent anticancer activity, with IC50 values in
the picomolar range against the growth of mouse
L1210 leukemia cells in culture.3 They derive their cyto-
toxic property through covalent reaction with adenine-
N3 in the minor groove of AT-rich sequences.3 Due to
their potent cytotoxic properties, the CC-1065 and duo-
carmycin class of compounds have received significant
attention, and four analogs were selected for clinical
evaluation, including adozelesin (Fig. 1).3b Presently,
N
NH2
OCH3
H
O
N
N
H3C
OH
OCH3
OCH3
N
H
N
O
N
O
N
O
OCH3
CH3
N
H
MeO2C
O
OH
OCH3
N
O
H
N
N
N
H
H
O
H
O
O
N
H
Adozelesin
O
(+)-DUMSA
(+)-CC-1065
Cl
OCH3
OCH3
OCH3
OCH3
OCH3
H
N
OCH3
OCH3
OCH3
Cl
MeO2C
MeO2C
Cl
H
N
O
MeO2C
MeO2C
H
N
N
O
MeO2C
H
N
N
N
N
O
H
N
OCH3
N
H
MeO2C
H
O
H
O
H
O
N
N
OH
H
H
2
1
OH
3
4
OH
O
Figure 1. Structures of (+)-CC-1065, doucarmycin SA (DUMSA), adozelesin, and compounds 1–4.
Keywords: Achiral duocarmycins; Cytotoxicity; Anticancer; DNA; Sequence specificity.
*
Corresponding author. Tel.: +1 864 294 3368; fax: +1 864 294 3559; e-mail: moses.lee@furman.edu
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.10.021

178
K. Daniell et al. / Bioorg. Med. Chem. Lett. 15 (2005) 177–180
only one of the four compounds, bizelesin, remains in
clinical trial.4 One of the severe dose limiting toxicities
of these compounds is bone marrow suppression.3b,4,5
Consequently, there is a strong interest in the design,
synthesis, and testing of novel analogs that have compa-
rable antitumor activity, but with reduced systemic tox-
icity. A wide range of analogs of CC-1065 and the
duocarmycins, including modifications of the alkylating
subunit, such as analog 3,6 seco-iso-cyclopropanefuran-
oindoline (seco-iso-CFI) analogs, and other heterocycles
have been investigated.7 Studies were also conducted in
which the non-covalent binding subunit of the molecules
was altered, which included the use of water soluble pyr-
idyl systems.8
The achiral seco-duocarmycin analog 2 should lose
HCl to generate the ultimate cyclopropane-containing
drug, which should react with adenine-N3 (Fig. 2).
Compound 2 is analogous to the previously reported
DUMSA analog 3, which has an IC50 value of
1.38nM against P388 murine leukemia cancer cells in vi-
tro5 Along with the synthesis of compounds 1 and 2,
their biochemical and cytotoxic properties are described
herein (Scheme 1).
Synthesis of compounds 1 and 2 began with the reaction
of 2-amino-4-chloro-5-nitrophenol with benzyl bromide
to give aniline 5 in 56% yield. Reaction of 5 with benzoyl
chloride provided benzamide 6 in 85% yield, which was
reacted with sodium dimethyl malonate to afford malo-
nate 7 in 36% yield. Hydrolysis of the ester groups of
compound 7, followed by decarboxylation afforded acid
8 in 93% yield. The carboxylic acid group was selectively
reduced with borane in THF to produce alcohol 9 in
53% yield. Reaction of compound 9 with dimethyl
acetylenedicarboxylate in methanol gave compound 10
in 96% yield. The alcohol group in compound 10 was
converted to a chloride 11 in 85% yield with carbon tet-
rachloride and triphenylphosphine. Reaction of com-
pound 11 with palladium(II) acetate in DMA at 70°C
gave indole 12 in 23% yield.6 Hydrogenation of com-
pound 12 with 10% palladium-on-carbon in THF gave
an amine intermediate, which was directly coupled with
5,6,7-trimethoxyindole-2-carboxylic acid11 and 5-(ben-
zofuran-2-carboxamido)-indole-2-carboxylic acid12 in
the presence of EDCI in DMF at room temperature
for three days. The target achiral seco-duocarmycin ana-
logs 1 and 2 were isolated in 16% and 20% yield, respec-
tively. All compounds reported in this paper were
characterized by NMR, IR, high resolution MS. Com-
pounds 1 and 2 were further characterized by elemental
analysis.
One aspect of the CPI structure that has not been inves-
tigated with respect to DNA interaction and anticancer
properties is the chiral center present in CC-1065 and
the duocarmycins. Studies have revealed that the opti-
cally active (+)-(S) enantiomers are generally more cyto-
toxic than their mirror images. For example, natural
(+)-(S)-DUMSA has an IC50 of 10pM, compared to
100pM for the unnatural isomer. This is consistent with
(+)-(S)-DUMSA being ten times more effective in react-
ing with DNA.3b,3c,9 Further, the binding orientation of
(+)-DUMSA is 30–50 over an AT-rich 3–5 base pair site,
but the (ꢀ)-enantiomer orients in the 50–30 direction.
Evidently, chirality can influence the biological proper-
ties of the duocarmycin compounds. This has led our
laboratories to initiate a program to investigate whether
the chiral effects could be eliminated while retaining
DNA interaction and cytotoxicity. In an earlier study,
we have shown that the hydroxyphenethyl chloride in
compound 4, an achiral seco-cyclopropaneindoline and
the simplest analog of the duocarmycins was able to
interact with DNA and inhibited the growth of cancer
cells in vitro10 Based on the structure–activity relation-
ship that a DUMSA alkylating subunit, which is solvo-
lytically one of the most stable analogs and the most
cytotoxic,3 compounds 1 and 2 (Fig. 1) were designed.
The cytotoxic and DNA binding properties of com-
pounds 1 and 2 were assessed. The cytotoxicity studies
were conducted with 3-day continuous exposure on
two human cancer cell lines using a MTT based colori-
metric assay.13 The IC50 values given in Table 1 indicate
that both compounds have activity in the nanomolar
range and they are active against leukemia and solid
tumors. The results showed that achiral duocarmycins
1 and 2 were 58–3450 times more cytotoxic than the
achiral-CI compound 4, and that was likely to be a re-
sult of enhanced stability of the duocarmycin alkylating
subunit.3b,3c More significantly, compound 2 has com-
parable cytotoxic potency to its chiral counterpart 3, al-
beit the latter was against P388 cells.6 These results
suggest that the chiral center present in the duocarmyc-
ins is not critical for cytotoxicity. Compound 2 was fur-
ther tested by the NCI against their panel of 60 different
human cancer cells. The agent has potent activity with
50% net growth inhibition conferred by 5.6–330nM
(95nM mean). Compound 2 was found to exhibit selec-
tivity against several solid tumor cells of the lung (NCI-
H522, NCI-H226, EKVX), colon (HT29, KM12), CNS
(SF-268, SF-539, SNB-75), melanoma (M14, SK-MEL-
2, UACC-62), ovarian (OVCAR-8), and breast (HS-
578T).
OMe
Cl
MeO2C
OMe
H
N
N
H
MeO2C
OMe
O
HN
2
OH
DNA - A(N3)
OMe
OMe
OMe
H
MeO2C
MeO2C
N
N
H
O
N
H
O
+
H
Figure 2. Proposed mechanism of activation and DNA alkylation by
achiral duocarmycin analog 2.

K. Daniell et al. / Bioorg. Med. Chem. Lett. 15 (2005) 177–180
179
MeO2C
CO2Me
NO2
Cl
Cl
CO2H
NO2
Cl
NO2
NO2
BnBr
NO2
NaH, dimethyl
malonate, DMSO,
100˚C, (36%)
K2CO3,
nBu4NI
(56%)
benzoyl chloride
triethylamine
(85%)
H2N
1. NaOH
H2N
BzNH
BzNH
H2N
2. HCl-THF-H2O
reflux
OH
OBn
OBn
OBn
OBn
(93%)
5
6
8
7
BH3, THF
(53%)
OH
Cl
OH
Cl
MeO2C
NO2
NO2
MeO2C
MeO2C
dimethyl acetylene-
MeO2C
MeO2C
NO2
NO2
dicarboxylate, MeOH
N
MeO2C
H2N
CCl4
Ph3P
(85%)
H
N
Pd(OAc)2
N
H
OBn
H
OBn
reflux overnight
(96%)
DMA, 70˚C
(23%)
OBn
OBn
11
9
12
10
1, H2, Pd/C
2, RCO2H, EDCI
DMF
Cl
OCH3
OCH3
OCH3
Cl
H
N
MeO2C
MeO2C
H
MeO2C
H
O
N
N
O
N
H
N
MeO2C
O
H
N
O
N
H
H
OH
2 (20%)
1 (16%)
Scheme 1. Synthesis of achiral duocarmycin 1 and 2.
OH
seco-DUMSA 2 generally showed similar covalent se-
quences selectivity to CC-1065 and adozelesin, at 50-
TTAA-30 and AAAAAA sequences. However, the gel
showed additional sites of alkylation at some adenine-
N3 sites that were absent for adozelesin and CC-1065.
Table 1. Cytotoxicity of compounds 1 and 2 determined using the
MTT assay
Compound
IC50 (nM)
K562
LS174T
1
2
4
15
25
29
4.0
1470
13800
K562 = chronic human myeloid leukemia cells; LS174T = human
colon carcinoma.
The ability of compounds 1 and 2 to covalently interact
with DNA was studied using a thermal induced DNA
cleavage assay.7,10 Treatment of supercoiled pBR322
DNA with 6.6lM of the compounds for 40h at 40°C
produced 30 3% and 23 3% of Form I DNA, respec-
tively. Control pBR322 DNA gave 5% spontaneous
DNA cleavage under similar conditions. These results
are consistent with the alkylation of purine-N3 positions
in the minor groove of the DNA. DNA sequence specif-
icity was ascertained using a Taq DNA polymerase stop
assay.14 The pBR322 DNA was linearized to provide a
stop for the Taq downstream from the primer. The olig-
odeoxynucleotide primers were 50-end labeled prior to
amplification using T4 polynucleotide kinase and
[c32P]-ATP
(5000Ci/mmol).
The
primer
50-
GCAGCAGATTACGCGCAGAA-30 binds to the
complementary strand at position 3090–3109 and was
used to examine the alkylation on the bottom strand.
The primer 50-GCATTGGTAACTG-TCAGACC-30
binds in the sequence 3303–3284 and was used to exam-
ine the top strand. From the gels shown in Fig. 3, achiral
Figure 3. Taq DNA polymerase stop studies on compounds 2.

180
K. Daniell et al. / Bioorg. Med. Chem. Lett. 15 (2005) 177–180
3642; (d) Boger, D. L. Acc. Chem. Res. 1995, 28, 20;
(e) Boger, D. L. Chemtracts: Org. Chem. 1991, 4,
329.
The sequence preference of compound 1 was identical to
that for adozelesin and CC-1065 (data not shown).
These results further support our contention that the
chiral center is not needed for this class of compounds
to display potent biological activities. They also provide
indication that, like the chiral compounds, improvement
of the chemical stability of the achiral alkylating subunit
can increase the cytotoxic potency of the compounds. In
summary, achiral analogs of CC-1065 and the duocar-
mycins represent a novel class of agents with potential
as anticancer drugs.
4. Pitot, H. C., IV; Erlichman, C.; Reid, J. M.; Sloan, J. A.;
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Acknowledgements
The authors thank ACS-PRF, NSF (REU), Enzacta
Ltd, UK, Taiho Pharmaceutical Co., and the NCI for
their support. The authors also thank Dr. Robert Kelley
of UpJohn Pharmacia for a generous gift of CC-1065
and adozelesin.
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