DNA interstrand crosslinking agents: Synthesis, DNA interactions, and cytotoxicity of dimeric achiral seco-amino-CBI and conjugates of achiral seco-amino-CBI with pyrrolobenzodiazepine (PBD)

Article abstract of DOI:10.1016/j.bmcl.2006.08.005

The design and synthesis of three novel bisalkylating agents derived from the achiral seco-duocarmycin or CC-1065 analogs and pyrrolobenzodiazepines (PBDs) are described: achiral seco-CBI (cyclopropanebenz[e]indoline)-PBD 11, achiral seco-CI-PBD 12, and a

Full text of DOI:10.1016/j.bmcl.2006.08.005

Bioorganic & Medicinal Chemistry Letters 16 (2006) 5677–5681
DNA interstrand crosslinking agents: Synthesis, DNA interactions,
and cytotoxicity of dimeric achiral seco-amino-CBI and conjugates
of achiral seco-amino-CBI with pyrrolobenzodiazepine (PBD)
Bethany Purnell,a Atsushi Sato,a,b Amanda O’Kelley,a Carly Price,a
Kaitlin Summerville,a Stephen Hudson,a Caroline O’Hare,c Konstantinos Kiakos,c
c
Tetsuji Asao,b Moses Leea,d, and John A. Hartley
*
aDepartment of Chemistry, Furman University, 3300 Pointsett Highway, Greenville, SC 29613, USA
bTaiho Pharmaceutical Co., Ltd., 1-27, Misugidai Hanno-City, Saitama, 357-8527, Japan
cCancer Research UK Drug-DNA Interactions Research Group, Department of Oncology, University College London,
91 Riding House Street, London W1W 7BS, UK
dDivision of Natural Sciences and Department of Chemistry, Hope College, Holland, MI 49423, USA
Received 23 June 2006; revised 27 July 2006; accepted 1 August 2006
Available online 21 August 2006
Abstract—The design and synthesis of three novel bisalkylating agents derived from the achiral seco-duocarmycin or CC-1065 ana-
logs and pyrrolobenzodiazepines (PBDs) are described: achiral seco-CBI (cyclopropanebenz[e]indoline)-PBD 11, achiral seco-CI-
PBD 12, and achiral seco-CBI dimer 13. Compounds 11 and 12 demonstrated enhanced cytotoxicity over the monomer counterparts
against the growth of P815 murine mastocytoma cells in culture. Conjugate 11 was found to covalently react with adenine-N3 posi-
tions within the minor groove at AT-rich sequences and to produce DNA interstrand crosslinks. Both compounds were found to
induce apoptosis in P815 cells. Due to its poor water solubility, dimer 13 did not give any appreciable DNA binding or cytotoxicity.
Ó 2006 Elsevier Ltd. All rights reserved.
Compounds that exhibit selectivity for binding to
DNA sequences and covalently react with specific
nucleobases continue to be important targets for the
design and synthesis of novel medicinal agents.1 Figure
1 gives examples of DNA minor groove binding and
AT sequence-specific compounds that exhibit potent
anticancer activities. It includes analogs of (+)-CC-
1065 (1),2 duocarmycin SA (2),3 seco-CBI-TMI (3),4
and the recently described achiral congeners from our
laboratories, 45 and 5.6 The latter class of compounds
have been found to alkylate adenine-N3 and promote
cell death through apoptosis. The achiral seco-amino-
CBI-TMI 4 has low toxicity and strong in vivo anti-
cancer activity against mouse B16 melanoma.5
Another class of DNA minor groove binding agents that
are being extensively examined contain a pyrrolo-
benzodiazepine (PBD) moiety, and they are analogous
to the anthramycins, for example, compound 6.7 PBD
analogs display specificity of binding to 50-PuGPu-30
sequences, and they covalently react to form stable ad-
ducts between the imine group on the PBD with G-2-
NH2.8
In an effort aimed at enhancing the biological properties
of CC-1065 (1) and PBD analogs, a number of dimers
and conjugates between these two classes of compounds
(Fig. 1) were designed and biologically tested. Bizelesin
(7) was found to effectively produce interstrand DNA
crosslinks at AT-rich sequences and it was endowed
with potent cytotoxicity and anticancer activity. It has
undergone clinical evaluation for cancer treatment.9 Di-
mer compounds of PBD analogs were also developed,
such as, DSB-120 (8)10 and SJG-136 (9).11 These com-
pounds span six base pairs in the minor groove and pro-
duce DNA interstrand crosslinks between guanine-N2
positions at 50-GATC-30 sequences.11 The latter
Keywords: CC-1065; Duocarmycins; Pyrrolobenzodiazepines; Achiral;
Seco-amino-CBI; Dimer.
*
Corresponding author. Tel.: +1 616 395 7190; fax: +1 616 395
7923; e-mail: lee@hope.edu
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.08.005

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B. Purnell et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5677–5681
N
NH2
OMe
OMe
OMe
O
OMe
OMe
OMe
N
OH
OMe
Cl
N
O
H
N
H3C
N
H
OH
OMe
N
N
N
CH3O2C
O
N
O
H
N
H
O
O
N
H
O
H
OH
O
(+)-CC-1065, 1
3
(+)-DUMSA, 2
OMe
OMe
OMe
OMe
Cl
Cl
OMe
OMe
H
H
N
N
H
N
MeO2C
N
N
H
H
O
O
MeO
N
O
NH2
OH
5
4
6
CH3
H
H
N
Cl
Cl
H
N
N
O
N
O
MeO
HN
O
H
O
O
H3C
CH3
N
O
N
N
OMe
N
N
O
N
R
H
N
N
H
N
H
R
O
H
O
O
O
N
N
N
MeO
N
H
DSB-120, 8, R = H2
SJG-136, 9, R = CH2
10
H
H
bizelesin, 7
OH
OH
O
H
N
H
N
Cl
H
H
Cl
O
N
N
N
O
N
Cl
Cl
H
H
H
N
H
N
H
N
O
H
O
O
MeO
MeO
N
N
N
O
O
O
O
O
O
O
O
O
O
12
11
13
NH2
NH2
NH2
OH
Figure 1. Structures of (+)-CC-1065 1, (+)-duocarmycin SA 2, seco-CBI-TMI 3, achral seco-amino-CBI-TMI 4, achiral seco-CI-TMI 5, PBD
monomer 6, bizelesin 7, DSB-128 8, SJG-136 9, CPI-PBD 10, achiral seco-amino-CBI-PBD 11, achiral seco-CI-PBD 12, Dimer of achiral seco-amino
CBI 13.
compound 9 is presently undergoing phase I clinical trial
for cancer treatment in Europe and the USA.12
The target compounds were prepared following a syn-
thetic strategy given in Scheme 1. Reaction of the
aromatic amine functional group of compound 14
with 5-nitrobenzofuran-2-carboxylic acid proceeded
readily using an EDCI and HOBt coupling method.
The desired compound was isolated in 54% yield.
The acetyl moiety was removed by treatment with a
methanolic solution of potassium carbonate to yield
compound 15 in 36% yield. Compound 15 was trans-
formed into chloride 16 in three steps (mesylation of
the primary alcohol, displacement of the mesylate
group with LiCl, followed by catalytic hydrogenation
to reduce the nitro group to an amine). The overall
yield for converting alcohol 15 to chloride 16 was
achieved at 5.4%.
In addition to dimeric compounds, conjugates of these
two classes of compounds have also been reported.13
For example, the CPI (cyclopropylpyrolo[e]indolone)-
PBD conjugate 10 was found to exhibit potent cytotox-
icity against human cancer cells (such as breast MCF-7,
colon SW480, and lung A549) grown in culture, and it
also showed selectivity for producing interstrand cross-
links at a 50-T[A]AATTG-30 site (alkylation at the
underlined and bold faced A and G residues).13a
With this background and the recent discovery that
seco-achiral analogs of CC-1065 (1) are potent DNA
interactive anticancer agents, we began to focus atten-
tion on preparing and testing the biological properties
of hybrid PBD and achiral seco-amino-CBI conjugates.
Accordingly, compounds 11 and 12, which are conju-
gates of the PBD analog 6 and an achiral seco-amino-
CBI analog of 4, as well as a conjugate of compound
6 and an achiral seco-CI analog of 5, respectively, were
designed and prepared. These compounds offer a unique
advantage over the published conjugates because they
contain a single stereocenter that can be readily intro-
duced early in the synthetic scheme through the use of
2(S)-(+)-pyrrolidinyl methanol as a synthon.7 A dimeric
compound of the achiral seco-CBI compound 13 was
also prepared, and it is analogous to the bizelesin design.
This communication describes the synthesis and the
cytotoxicity of compounds 11–13, along with an investi-
gation of the sequence specificity, DNA alkylation, and
interstrand crosslinking abilities of the compounds.
The amino group of compound 16 was reacted with a
PBD-carboxylic acid 177 in the presence of EDCI, giving
the desired product 18 in 62% yield. Final removal of the
BOC-protecting groups with concd HCl, followed by
elimination of the Troc protecting group using a Pb–
Cd reaction, produced compound 19, and target 11, in
97% and 84% yields, respectively. A similar strategy
was employed in the synthesis of target molecule 12,
except amine 20 was used.
Synthesis of the symmetrical dimer 13 was achieved in
30% yield by reaction of amine 16 with triphosgene.
Removal of the BOC group of the intermediate yielded
the desired compound 13 (68%). All compounds gave
acceptable spectroscopic data (500 MHz 1H NMR,
FTIR, high resolution mass spectrometry, and melting
points for solid samples).

B. Purnell et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5677–5681
5679
NH2
NO2
OAc
NH2
Cl
OH
b
H
N
a
H
f,g
c,d,e
N
13
O
O
O2N
O
O
CO2H
NHBOC
O
NHBOC
NHBOC
14
15
16
NH2
Cl
k-m
H
N
HO
H
Troc
12
O
HO2C
N
O
N
O
OBn
O
20
h
O
MeO
17
HO
HO
H
Troc
H
O
Troc
HN
N
HN
Cl
N
Cl
N
N
O
H
N
O
i
H
N
HCl
O
j
O
O
MeO
19
11
O
MeO
O
18
O
NH2
NHBOC
Scheme 1. Reagents and conditions: (a) EDCI, HOBt, pyridine, 54%; (b) K2CO3, MeOH, 36%; (c) mesyl chloride, Et3N, THF; (d) LiCl, DMF; (e)
H2, 10% Pd–C, THF, 5.4% from 16; (f) triphosgene, DIPEA, CH2Cl2, THF, 30%; (g) concd HCl, THF, 68%; (h) EDCI, HOBt, DMF, 62%; (i) 3M
HCl, EtOAc, 97%; (j) ammonium acetate, 10% Pb–Cd, THF, 84%; (k) 17, EDCI, HOBt, DMF, 92%; (l) 10% Pd–C, THF, 50%; (m) ammonium
acetate, 10% Pb–Cd, THF, 31%.
An MTT colorimetric study was used to determine the
cytotoxic properties of compounds 11–13.4,5 P815 mur-
ine mastocytoma cells were continuously exposed to the
compounds for 3 days. Viability was determined by the
ability of the cells to convert MTT to a purple formazan
dye. The results are shown in Table 1, and they are listed
according to their ability to inhibit cell growth by 50%
(IC50) relative to an untreated control. It is worthy to
note that for this class of compounds, the racemic com-
pound 3, which contained a stereocenter, was most cyto-
toxic, despite compounds 11–13 being theoretically
capable of producing cytotoxic DNA interstrand cross-
links. However, the non-symmetrical conjugate 11 was
more cytotoxic than its monomeric analogs: achiral-
seco-amino-CBI 4 and PBD 6, indicating that inter-
strand crosslink formation could increase the cytotoxic
potency of these DNA reactive agents. The data, howev-
er, showed that dimer 13 was least active among the new
compounds, and that could be due to the poor solubility
of 13 in water and cell culture media.
dose for 18 h were fixed and treated with propidium
iodine according to an earlier published procedure.5
The cells were sorted on a flow cytometer, and the pop-
ulation of sub-G0 cells was determined: 21% for treated
versus 3% for an untreated control (data not shown).
These results are consistent with earlier studies indicat-
ing that achiral CBI compounds are capable of inducing
cancer cells to undergo apoptosis.5,14
An agarose DNA thermally induced cleavage study was
conducted to determine the ability of compound 11 to
induce DNA strand breaks. Supercoiled pBR322
DNA was treated with compound 11 at 6.6 lM for
40 h at 37 °C. DNA strand breaks were observed
through the percent conversion of supercoiled DNA
(Form I) to open circular DNA (Form II). In this case,
the experiment gave 16 2% Form II DNA, compared
to the control DNA (0%), and free PBD monomer 6
(4%).
Since compound 11 was found to be more cytotoxic
than its parent monomeric units (4 and 6), the inter-
strand crosslinking ability of compound 11 with a
50-32P-labeled linear pBR322 DNA was examined. As
depicted in Figure 2, compound 9 effectively produced
interstrand crosslinks, evidenced by formation of the
DS or double stranded band in a sample of denatured
DNA (SS).15 This result is consistent with earlier re-
ports, and the covalent adducts were thermally stable.
For compound 11, a crosslink DNA band appeared at
10 lM. It is worth noting that this gel crosslink study
was performed using alkali denaturation because when
the treated DNA sample was denatured by heating to
95 °C for 2 min no crosslink band was observed consis-
tent with the presence of thermally labile adenine-N3
covalent adducts (data not shown).
Further cell-based studies were conducted to ascertain
the mechanism by which compound 11 derived its cyto-
toxic activity. Cultured P815 cells treated at the IC50
Table 1. In vitro cytotoxicity studies in P815 cells for compounds 3–6
and 11–13, following a continuous exposure for 3 days
Compound
Cytotoxicity IC50 (lM)
3
4
0.0086
0.068
5.6
5
6
55
11
12
13
0.026
0.56
42

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B. Purnell et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5677–5681
The results demonstrated that the DNA alone did not
have any strand breaks. Consistent with literature re-
ports,2 at 0.1 lM, compound 1 (CC1065) demonstrated
selectivity for 50-TAA sequences, with alkylation at the
underlined adenine-N3 position and consistent with
the known sequence preference of compound 4.5 PBD
ester 6 did not show evidence of DNA alkylation of this
fragment, even at 100 lM, consistent with adducts at
guanine-N2 not resulting in thermally induced strand
breaks. In contrast, at a low concentration of 1 lM,
molecule 11 exhibited a clear and selective alkylation
band at A(3233). This is an interesting site because it
lines up with the 50-AAA(3233)TTTTC[G]C-30, which
is potentially an interstrand crosslink site due to the
proximity between the alkylated adenine residue and a
50-PuGPu site in the complementary strand.13 However,
when the concentration of compound 11 was increased
to 10 lM, additional alkylation sites common to CC-
1065 1 became evident.
Figure 2. Agarose gel DNA crosslink studies. SS is single stranded
DNAs, DS double stranded DNA. The 50-32P labeled DNA was
treated with compounds for 24 h, and the sample denatured with alkali
before it was analyzed by neutral agarose gel electrophoresis.
The covalent sequence specificity of duocarmycin ana-
logs can be measured using a thermally induced cleav-
age-DNA sequencing method.5,6
A
singly 50-32P-
The results given in this communication demonstrate
that conjugates of achiral analogs of CC-1065 or duo-
carmycins with PBDs are biologically active and are
capable of producing interstrand crosslinks. Agent 11
has the potential of being developed as an anticancer
agent. Further biological studies are underway and the
results will be reported in due course.
radiolabeled linear fragment of pBR322 plasmid DNA
was used as a template for these studies. A gel showing
the sites of thermally induced cleavage for compounds 1,
6, and 11 is shown in Figure 3.
Acknowledgments
Support from Taiho Pharmaceutical Company of
Japan, the National Cancer Institute, GlaxoSmithKline
USA, and Furman University is acknowledged. The
authors also thank Ms. Lillia Holmes at the Greenville
Oncology Center for assistance in the flow cytometry
experiments. Ipsen has acquired an exclusive worldwide
license to develop SJG-136, a clinical antitumor agent,
from Spirogen.
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