Parallel synthesis and evaluation of 132 (+)-1,2,9,9a-tetrahydrocyclopropa[c]benz[e]indol-4-one (CBI) analogues of CC-1065 and the duocarmycins defining the contribution of the DNA-binding domain

Article abstract of DOI:10.1021/jo010454u

The solution-phase, parallel synthesis and evaluation of a library of 132 (+)-1,2,9,9a-tetrahydrocyclopropa[c]benz[e]indol-4-one (CBI) analogues of CC-1065 and the duocarmycins containing dimeric monocyclic, bicyclic, and tricyclic heteroaromatic replacements for the DNA-binding domain are described. This systematic study revealed clear trends in the structural requirements for observation of potent cytotoxic activity and DNA alkylation efficiency, the range of which spans a magnitude of ≥10 000-fold. Combined with related studies, these results highlight that the role of the DNA-binding domain goes beyond simply providing DNA-binding selectivity and affinity (10-100-fold enhancement in properties), consistent with the proposal that it contributes significantly to catalysis of the DNA alkylation reaction accounting for as much as an additional 1000-fold enhancement in properties.

Full text of DOI:10.1021/jo010454u

6654
J . Org. Chem. 2001, 66, 6654-6661
P a r a llel Syn th esis a n d Eva lu a tion of 132
(+)-1,2,9,9a -Tetr a h yd r ocyclop r op a [c]ben z[e]in d ol-4-on e (CBI)
An a logu es of CC-1065 a n d th e Du oca r m ycin s Defin in g th e
Con tr ibu tion of th e DNA-Bin d in g Dom a in
Dale L. Boger,* Harald W. Schmitt, Brian E. Fink, and Michael P. Hedrick
Department of Chemistry and The Skaggs Institute for Chemical Biology, The Scripps Research Institute,
10550 North Torrey Pines Road, La J olla, California 92037
boger@scripps.edu
Received May 3, 2001
The solution-phase, parallel synthesis and evaluation of a library of 132 (+)-1,2,9,9a-tetrahydro-
cyclopropa[c]benz[e]indol-4-one (CBI) analogues of CC-1065 and the duocarmycins containing
dimeric monocyclic, bicyclic, and tricyclic heteroaromatic replacements for the DNA-binding domain
are described. This systematic study revealed clear trends in the structural requirements for
observation of potent cytotoxic activity and DNA alkylation efficiency, the range of which spans a
magnitude of g10 000-fold. Combined with related studies, these results highlight that the role of
the DNA-binding domain goes beyond simply providing DNA-binding selectivity and affinity (10-
100-fold enhancement in properties), consistent with the proposal that it contributes significantly
to catalysis of the DNA alkylation reaction accounting for as much as an additional 1000-fold
enhancement in properties.
In tr od u ction
CC-1065 (1)1 and the duocarmycins (2 and 3)2 are
among the most potent antitumor antibiotics discovered
to date, and they have been shown to derive their
biological activity through the sequence selective alky-
lation of duplex DNA (Figure 1).2-4 An extensive series
of studies has defined the nature of the alkylation
reaction, which proceeds by adenine N3 addition to the
least-substituted cyclopropane carbon of the left-hand
alkylation subunit, and the alkylation sequence selectiv-
ity.5-8 For the natural enantiomers, this entails 3′
adenine N3 alkylation with binding across a 3.5-4
(duocarmycins) or 5 (CC-1065) base-pair AT-rich site
(e.g., 5′-AAAAA), whereas the unnatural enantiomers
bind in the reverse 5′f3′ direction (e.g., 5′-AAAAA) across
analogous 3.5-5 base-pair AT-rich sites.4 An alternative
(1) Hanka, L. J .; Dietz, S. A.; Gerpheide, S. A.; Ku¨ntzel, S. L.;
Martin, D. G. J . Antibiot. 1978, 31, 1211.
F igu r e 1.
(2) Boger, D. L. Chemtracts: Org. Chem. 1991, 4, 329.
(3) Warpehoski, M. A. In Advances in DNA Sequence Specific Agents;
Hurley, L. H., Ed.; J AI Press: Greenwich, CT, 1992; Vol. 1, p 217.
Hurley, L. H.; Warpehoski, M. A. Chem. Res. Toxicol. 1988, 1, 315.
(4) Boger, D. L.; J ohnson, D. S. Angew. Chem., Int. Ed. Engl. 1996,
35, 1438. Boger, D. L.; J ohnson, D. S. Proc. Natl. Acad. Sci. U.S.A.
1995, 92, 3642.
(5) Hurley, L. H.; Reynolds, V. L.; Swenson, D. H.; Petzold, G. L.;
Scahill, T. A. Science 1984, 226, 843. Hurley, L. H.; Lee, C.-S.;
McGovren, J . P.; Warpehoski, M. A.; Mitchell, M. A.; Kelly, R. C.;
Aristoff, P. A. Biochemistry 1988, 27, 3886. Hurley, L. H.; Warpehoski,
M. A.; Lee, C.-S.; McGovren, J . P.; Scahill, T. A.; Kelly, R. C.; Mitchell,
M. A.; Wicnienski, N. A.; Gebhard, I.; J ohnson, P. D.; Bradford, V. S.
J . Am. Chem. Soc. 1990, 112, 4633.
way of visualizing this behavior of the two enantiomers
is that, from a common bound orientation and within a
common binding site, they alkylate adenine on comple-
mentary strands of duplex DNA at sites offset by one base
5′-AATTA(natural)
pair (e.g., 3′-TTAAT(unnatural)).9 Early studies demonstra-
ted that the right-hand segment(s) of the natural prod-
ucts effectively delivers the alkylation subunit to AT-rich
sequences of duplex DNA increasing the selectivity and
efficiency of DNA alkylation.10 Because the preferential
AT-rich noncovalent binding affinity, like that of dista-
mycin and netropsin,11 is related to the deeper and
narrower shape of the AT-rich minor groove, it is often
(6) Boger, D. L.; J ohnson, D. S.; Yun, W.; Tarby, C. M. Bioorg. Med.
Chem. 1994, 2, 115. Boger, D. L.; Coleman, R. S.; Invergo, B. J .; Sakya,
S. M.; Ishizaki, T.; Munk, S. A.; Zarrinmayeh, H.; Kitos, P. A.;
Thompson, S. C. J . Am. Chem. Soc. 1990, 112, 4623.
(7) Boger, D. L.; Ishizaki, T.; Zarrinmayeh, H.; Kitos, P. A.; Sun-
tornwat, O. J . Org. Chem. 1990, 55, 4499. Boger, D. L.; Ishizaki, T.;
Zarrinmayeh, H.; Munk, S. A.; Kitos, P. A.; Suntornwat, O. J . Am.
Chem. Soc. 1990, 112, 8961. Boger, D. L.; Ishizaki, T.; Zarrinmayeh,
H. J . Am. Chem. Soc. 1991, 113, 6645. Boger, D. L.; Yun, W. J . Am.
Chem. Soc. 1993, 115, 9872.
(8) Boger, D. L.; J ohnson, D. S.; Yun, W. J . Am. Chem. Soc. 1994,
116, 1635. Asai, A.; Nagamura, S.; Saito, H. J . Am. Chem. Soc. 1994,
116, 4171.
(9) Smith, J . A.; Bifulco, G.; Case, D. A.; Boger, D. L.; Gomez-Paloma,
L.; Chazin, W. J . J . Mol. Biol. 2000, 300, 1195. Eis, P. S.; Smith, J . A.;
Rydzewski, J . M.; Case, D. A.; Boger, D. L.; Chazin, W. J . J . Mol. Biol.
1997, 272, 237. Schnell, J . R.; Ketchem, R. R.; Boger, D. L.; Chazin,
W. J . J . Am. Chem. Soc. 1999, 121, 5645.
(10) Boger, D. L.; Coleman, R. S.; Invergo, B. J .; Zarrinmayeh, H.;
Kitos, P. A.; Thompson, S. C.; Leong, T.; McLaughlin, L. W. Chem.-
Biol. Interact. 1990, 73, 29.
10.1021/jo010454u CCC: $20.00 © 2001 American Chemical Society
Published on Web 09/13/2001

Synthesis and Evaluation of Duocarmycin Analogues
J . Org. Chem., Vol. 66, No. 20, 2001 6655
referred to as shape-selective recognition. However, it is
only in more recent studies that it has become apparent
that the DNA-binding domain also plays an important
role in catalysis of the DNA alkylation reaction.12 Because
this is also related to the shape characteristics of the
minor groove and results in preferential activation in the
narrower, deeper AT-rich minor groove, we have come
to refer to this as shape-dependent catalysis.12 We have
suggested that this catalysis is derived from a DNA-
binding-induced conformational change in the agents,
which adopt a helical DNA-bound conformation requiring
a twist in the amide linking of the alkylation subunit and
the first DNA-binding subunit. This conformational
change serves to partially deconjugate the stabilizing
vinylogous amide, activating the cyclopropane for nu-
cleophilic attack. For activation, this requires a rigid,
extended heteroaromatic N2-amide substituent,12-14 and
the shape, length, and strategically positioned substitu-
ents on the first DNA-binding subunit can have a
pronounced effect on the DNA alkylation rate and ef-
ficiency and the resulting biological properties of the
agents.
F igu r e 2.
Because of its synthetic accessibility, its potency and
efficacy, which matches or exceeds that of the CC-1065
MeCPI alkylation subunit, and the extensive documenta-
tion of the biological properties of its derivatives, the
library was assembled using the seco precursor 4 to the
(+)-1,2,9,9a-tetrahydrocyclopropa[c]benz[e]indol-4-one
(CBI) alkylation subunit (Figure 2).21-24 To date, no
distinctions between the seco-CBI and CBI derivatives
have been detected in a range of in vitro and in vivo
assays in accordance with past studies of all such
alkylation subunits,25 indicating that in situ spirocycliza-
tion is not rate-determining or property-limiting.
The combination of the effects is substantial. The DNA
alkylation rate and efficiency increase approximately
10 000-fold, and the resulting biological potency also
increases proportionally 10 000-fold when simple N-
acetyl or N-Boc derivatives of the alkylation subunits,
which lack the DNA-binding domain, are compared with
1-3. In three independent studies, the DNA-binding
subunit contribution to the DNA alkylation rate could
be partitioned into that derived from an increased
binding selectivity/affinity and that derived from a
contribution to catalysis of the DNA alkylation reaction.
The former was found to increase the rate approximately
10-100-fold, whereas the latter increases the rate ap-
proximately 1000-fold indicating a primary impor-
tance.13,15,16
Syn th esis of th e 132-Mem ber ed Libr a r y
In a recent study,26 we detailed the parallel synthesis
of a 132-membered library of heteroaromatic dimers
Throughout these investigations, the complementary
roles of the DNA-binding subunits have been examined
with relatively limited numbers of compounds, and no
systematic study has been disclosed. Moreover, there is
some confusion in the disclosures as to the relative
effectiveness of the distamycin/lexitropsin substitutions
for the DNA-binding subunits, with regard to both DNA
alkylation selectivity and alkylation efficiency.14,17-20
Consequently, we have examined and describe herein the
effects of the DNA-binding subunits with a complete
series of dimeric monocyclic, bicyclic, and tricyclic het-
eroaromatics. The results of these studies underscore the
observation that the contribution of the DNA-binding
domain goes beyond simply providing AT-rich noncova-
lent binding affinity supporting an additional primary
role in catalysis.
(17) Wang, Y.; Lown, J . W. Heterocycles 1993, 36, 1399. Fregeau,
N. L.; Wang, Y.; Pon, R. T.; Wylie, W. A.; Lown, J . W. J . Am. Chem.
Soc. 1995, 117, 8917. Wang, Y.; Gupta, R.; Huang, L.; Luo, W.; Lown,
J . W. Anti-Cancer Drug Des. 1996, 11, 15. Iida, H.; Lown, J . W. Recent
Res. Dev. Synth. Org. Chem. 1998, 1, 17. J ia, G.; Iida, H.; Lown, J . W.
Heterocycl. Commun. 1998, 4, 557. J ia, G.; Iida, H.; Lown, J . W. Chem.
Commun. 1999, 119.
(18) Tao, Z.-F.; Fujiwara, T.; Saito, I.; Sugiyama, H. Angew. Chem.,
Int. Ed. 1999, 38, 650. Tao, Z.-F.; Fujiwara, T.; Saito, I.; Sugiyama, H.
J . Am. Chem. Soc. 1999, 121, 4961. Tao, Z.-F.; Fujiwara, T.; Saito, I.;
Sugiyama, H. J . Am. Chem. Soc. 1999, 121, 4961. Amishiro, N.;
Nagamura, S.; Kobayashi, E.; Okamoto, A.; Gomi, K.; Saito, H. Chem.
Pharm. Bull. 1999, 47, 1393. Tao, Z.-F.; Saito, I.; Sugiyama, H. J . Am.
Chem. Soc. 2000, 122, 1602.
(19) Chang, A. Y.; Dervan, P. B. J . Am. Chem. Soc. 2000, 122, 4856.
(20) Atwell, G. J .; Milbank, J . J .; Wilson, W. R.; Hogg, A.; Denny,
W. A. J . Med. Chem. 1999, 42, 3400. Baraldi, P. G.; Balboni, G.; Pavani,
M. G.; Spalluto, G.; Tabrizi, M. A.; DeClercq, E.; Balzarini, J .; Bando,
T.; Sugiyama, H.; Romagnoli, R. J . Med. Chem. 2001, 44, 2536.
(21) Boger, D. L.; Ishizaki, T.; Wysocki, R. J ., J r.; Munk, S. A.; Kitos,
P. A.; Suntornwat, O. J . Am. Chem. Soc. 1989, 111, 6461. Boger, D.
L.; Ishizaki, T.; Kitos, P. A.; Suntornwat, O. J . Org. Chem. 1990, 55,
5823. Boger, D. L.; Ishizaki, T. Tetrahedron Lett. 1990, 31, 793. Boger,
D. L.; Yun, W.; Teegarden, B. R. J . Org. Chem. 1992, 57, 2873. Boger,
D. L.; Yun, W. J . Am. Chem. Soc. 1994, 116, 7996. Boger, D. L.; McKie,
J . A. J . Org. Chem. 1995, 60, 1271. Boger, D. L.; McKie, J . A.; Boyce,
C. W. Synlett 1997, 515. Boger, D. L.; Boyce, C. W.; Garbaccio, R. M.;
Searcey, M. Tetrahedron Lett. 1998, 39, 2227. Boger, D. L.; Boyce, C.
W.; Garbaccio, R. M.; Searcey, M.; J in, Q. Synthesis 1999, 1505.
(22) Boger, D. L.; Yun, W.; Han, N. Bioorg. Med. Chem. 1995, 3,
1429.
(11) J ohnson, D. S.; Boger, D. L. In Supramolecular Chemistry;
Lehn, J .-M., Ed.; Pergamon Press: Oxford, 1996; Vol. 4, p 73.
(12) Boger, D. L.; Garbaccio, R. M. Bioorg. Med. Chem. 1997, 5, 263.
Boger, D. L.; Garbaccio, R. M. Acc. Chem. Res. 1999, 32, 1043.
(13) Boger, D. L.; Hertzog, D. L.; Bollinger, B.; J ohnson, D. S.; Cai,
H.; Goldberg, J .; Turnbull, P. J . Am. Chem. Soc. 1997, 119, 4977. Boger,
D. L.; Bollinger, B.; Hertzog, D. L.; J ohnson, D. S.; Cai, H.; Me´sini, P.;
Garbaccio, R. M.; J in, Q.; Kitos, P. A. J . Am. Chem. Soc. 1997, 119,
4987.
(14) Boger, D. L.; Han, N. Bioorg. Med. Chem. 1997, 5, 233.
(15) Boger, D. L.; Wolkenberg, S. E.; Boyce, C. W. J . Am. Chem.
Soc. 2000, 122, 6325. Boger, D. L.; Boyce, C. W. J . Org. Chem. 2000,
65, 4088.
(23) Boger, D. L.; Yun, W.; Cai, H.; Han, N. Bioorg. Med. Chem.
1995, 3, 761.
(24) Boger, D. L.; Munk, S. A. J . Am. Chem. Soc. 1992, 114, 5487.
(25) Boger, D. L.; Boyce, C. W.; Garbaccio, R. M.; Goldberg, J . A.
Chem. Rev. 1997, 97, 787.
(16) Boger, D. L.; Ellis, D. A.; Wolkenberg, S. E. J . Am. Chem. Soc.,
in press.
(26) Boger, D. L.; Fink, B. E.; Hedrick, M. P. J . Am. Chem. Soc.
2000, 122, 6382.

6656 J . Org. Chem., Vol. 66, No. 20, 2001
Boger et al.
sufficiently stable for use in the next conversion. After
deprotection of 4 (4 M HCl-EtOAc, 25 °C, 45 min), the
resulting hydrochloride 19 was coupled with the dimer
carboxylic acids using 1-[3-(dimethylamino)propyl]-3-
ethylcarbodiimide hydrochloride (EDCI) to provide 20.
Simple acid-base extraction and purification with aque-
ous 3 N HCl and saturated aqueous Na2CO3 yielded each
analogue sufficiently pure for direct assay.
Cytotoxic Activity
Evaluation of the CBI-based analogues in a cellular
functional assay for L1210 cytotoxic activity revealed a
clear relationship between the potency of the agents and
the structure of the DNA-binding domain (Figure 4). For
comparison, the L1210 IC50 for (+)-N-Boc-CBI, which
lacks an attached DNA-binding domain, is 80 nM (80 000
pM). With a few exceptions, all group 1 compounds
containing two monocyclic subunits (5-10 in positions
X and Y) exhibited IC50 values between 1 and 10 nM or
higher indicating an increase in potency of approximately
10-fold relative to N-Boc-CBI. The exception is the
thiophene subunit 8, which, when incorporated as the X
subunit adjacent to the DNA alkylation subunit, exhib-
ited slightly greater potency. The best in this series were
X8-Y8 (290 pM, 275-fold enhancement) and X8-Y10
(310 pM, 260-fold enhancement). Notably, the distamy-
cin/netropsin dipyrrole was also effective with X10-Y10
(440 pM) exhibiting a 180-fold enhancement. Nonethe-
less, even the best in this series exhibited a modest ca.
100-fold enhancement over (+)-N-Boc-CBI, and typically
the series exhibited a much more modest 10-100-fold
enhancement. Within the group 1 dimers, it is also
interesting that the 4-aminobenzoic acid subunit (5, X
group) compares favorably with the distamycin N-methyl-
4-aminopyrrole-2-carboxylic acid subunit (10) providing
IC50’s that are within 2-3-fold of one another, whereas
the 3-aminobenzoic acid subunit (6) and the imidazole
(9) are not effective.
F igu r e 3.
Sch em e 1
An analogous level of potency (10-100-fold enhance-
ment) was observed with the group 2 monocyclic het-
eroaromatics (X group) when they were coupled to a
terminal bicyclic heteroaromatic subunit (12-15), and a
slightly greater enhancement was observed when the Y
subunit was tricyclic (11). Notably, none of the com-
pounds in this group 1 or 2 series exhibit IC50’s lower
than 100 pM or approach the potency of the natural
products.
In contrast to these analogues, the group 3 dimers with
the bicyclic and tricyclic subunits 11-14 bound directly
to the DNA alkylation subunit constitute an array of
substances with much greater cytotoxic potency. The
potency enhancement observed with the analogues con-
taining a bicyclic or tricyclic X subunit linked directly to
the alkylation subunit (group 3, X11-14 subunits) typi-
cally ranges from 27 000-1 000 (IC50 ) 3-80 pM) relative
to N-Boc-CBI. This is also roughly a 100-1000-fold
enhancement over the monocyclic X subunits. All com-
pounds in the library with IC50’s below 10 pM can be
found in this collection, and two-thirds of them contain
the tricyclic CDPI subunit (11) in this key position, i.e.,
X11-Y7 (5 pM), X11-Y8 (3 pM), X11-Y9 (3 pM), X11-
Y10 (5 pM), X11-Y11 (5 pM), and X11-Y14 (7 pM). In
this regard, it seems advantageous to have a five-
membered heterocycle in the Y position with CDPI (11)
in the X position.
related to the structures of distamycin and CC-1065. This
included the monocyclic, bicyclic, and tricyclic heteroaro-
matic amino acids 5-16 (Figure 3), which have been
explored in the examination of these two natural prod-
ucts. The 132 dimers composed of these subunits were
assembled by parallel synthesis through formation of the
linking amide enlisting a simple acid-base liquid-liquid
extraction protocol for isolation and purification. Each
of the 132 dimers was fully characterized26 and used for
the formation of the library of CBI analogues. Each dimer
was saponified by treatment with LiOH (4 M aqueous
solution in dioxane-water 4:1 for 12 h at 25 °C) to afford
the lithium salts of the carboxylic acids (Scheme 1).
Hydrolysis of the compounds that possessed the 4-amino-
1-methylpyrrole-2-carboxylate (10) or 5-aminoindole-2-
carboxylate (14) subunits at the C-terminus was slower,
and the reactions were conducted at 40 °C. Acidifying
the aqueous Li-salt solutions gave the free carboxylic
acids 18 that were used for the subsequent couplings
without further purification. Notably, the dimers with
the 6-aminobenzoxazole-2-carboxylate (15) and 5-ami-
nobenzimidazole-2-carboxylate (16) subunits at the C-
terminus, which are prone to decarboxylation,26 were

Synthesis and Evaluation of Duocarmycin Analogues
J . Org. Chem., Vol. 66, No. 20, 2001 6657
F igu r e 4. Cytotoxic activity (IC50 values ( 10%, average of two or more assays run in triplicate) of the CBI analogues, L1210
IC50 (pM). The values in parentheses represent those previously reported (see ref. 22).
The proposal of binding-induced catalysis for DNA
alkylation by CC-1065 (1) and related compounds in
which the shape and size of the substituent directly
bound to the vinylogous amide make a major contribution
to the properties is supported by the trends within the
library. Compounds having the extended subunits 11-
14 in the X position and smaller subunits 7-10 in the Y
position show higher potency (typically 10-100-fold) than
the corresponding compounds with inverted sequences.
Because the bound agent is forced to follow the inherent
helical twist of the minor groove, the helical rise induced
in the molecule can only be adjusted by twisting the
linking amide that connects the noncovalent binding
subunit with the vinylogous amide of the alkylation
subunit. The more extended the subunit is, the greater
the twist is in the linking amide resulting in an increased
activation of the agent. The lower cytotoxicity exhibited
by analogues made from dimers consisting of the five-
membered heterocycles 7-10 is also consistent with this
explanation. Although these subunits are well-known as
minor groove-binding constituents of distamycin, netrop-
sin, and lexitropsins, they lack the rigid length that the
fused aromatic heterocycles possess.
Compared to the analogues possessing benzothiophene
(12), benzofuran (13), or indole (14) at the X position of
the dimer, agents containing benzoxazole (15) or benz-
imidazole (16) in this position (group 4) exhibit a con-
siderable decrease in potency, up to 130-fold for X15-
Y13. Similar observations have been made in a previous
study concerning deep-seated modifications of the DNA-
F igu r e 5.
binding subunit of CC-1065 (Figure 5).22,23 The introduc-
tion of an additional heteroatom in the carboxylate
destabilizing electrostatic interactions between the amide
carbonyl lone pair and the heteroatom lone pairs present
when the amide carbonyl adopts either of the in-plane
conjugated conformations (Figure 5). This interaction
results in a twist of the C-terminal bicyclic aromatic ring
out of the plane defined by the carboxamide precluding
bearing aromatic ring of (+)-CBI-CDPI (21) led to a 40-
fold decrease in cytotoxic activity and an analogous
decrease in the DNA alkylation efficiency observed with
(+)-CBI-CDPBO (22) and (+)-CBI-CDPBI (23) but no
alteration in the alkylation selectivity compared to that
of the parent compound. This was attributed to the

6658 J . Org. Chem., Vol. 66, No. 20, 2001
Boger et al.
strand cleavage at the sites of DNA alkylation, denatur-
ing high-resolution polyacrylamide gel electrophoresis
(PAGE) adjacent to Sanger dideoxynucleotide sequencing
standards, and autoradiography led to identification of
the DNA cleavage and alkylation sites.28
Representative of the comparisons made and the
trends observed, the analogues 25 and 26 were found to
detectably alkylate DNA at 10-5-10-6 and 10-3 M,
respectively, whereas alkylation by 24 (not shown) could
not be observed even at 10-3 M (Figure 7). Throughout
the comparisons, the relative DNA alkylation efficiencies
were found to parallel the cytotoxic potencies of the
compounds. Thus, the 100-fold lower cytotoxicity of 26
compared to 25 is also reflected in the 100-1000-fold
lower alkylation efficiency of 26. This behavior is dra-
matic with 26 being only 10-100-fold more effective than
N-Boc-CBI which alkylates DNA at 10-1-10-2 M under
comparable reaction conditions albeit with a reduced
selectivity. Thus, while the dipyrrole binding subunit
does enhance the DNA alkylation efficiency and selectiv-
ity relative to N-Boc-CBI, it is also substantially less
effective (100-1000-fold) than the compounds containing
bicyclic or tricyclic X groups. The significance of those
observations should not be underestimated, and the
observations suggest that hybrid agents composed of the
CC-1065/duocarmycin-related alkylation subunits and
distamycin/netropsin DNA-binding subunits are intrinsi-
cally poor DNA alkylating agents.
Notably, no alterations in the DNA alkylation selec-
tivities were observed despite the changes in the DNA-
binding domain except for the minor differences noted
before. Thus, although the efficiency of DNA alkylations
was altered greatly, the selectivity was not. Within the
w794 DNA, a major alkylation site (5′-AATTA-3′) and two
minor sites (5′-ACTAA-3′, 5′-GCAAA-3′) are observed
with the natural enantiomers. The relative extent to
which alkylation at the minor sites is observed is de-
pendent on the overall size (length) of the agent and the
extent of DNA alkylation. For example, neither 27 nor
28 alkylates the minor 5′-ACTAA-3′ site to a significant
extent, while the shorter agent 25, like 21,24 does. In
addition, the minor 5′-GCAAA-3′ site only appears on the
gel after nearly complete consumption of the end-labeled
DNA indicative of extensive, multiple DNA alkylations
resulting in cleavage to shorter fragments of DNA. Other
than these minor distinctions in the DNA alkylation
selectivity, which have been noted in prior studies of CBI
derivatives,24 no significant changes were observed with
variations in the DNA-binding subunits. Thus, while it
may appear reasonable to suggest that the alkylation of
the 5′-ACTAA-3′ site by 25 is a result of imidazole
H-bonding to the intervening GC base pair, the identical
behavior of (+)-CBI-CDPI (21), which lacks this subunit,
suggests it is simply a natural consequence of a shorter
agent binding and alkylating DNA within a shorter AT-
rich sequence.24,29
F igu r e 6.
preferential adoption of a near-planar conformation that
facilitates minor groove binding.
DNA Alk yla tion Efficien cy a n d Selectivity
The DNA alkylation properties of the compounds
including those of CBI-X9-Y9 (24), CBI-X11-Y9 (25), and
CBI-X10-Y10 (26) (Figure 6) were examined within a 150
base-pair segment of duplex DNA and compared with (+)-
duocarmycin SA (2), (+)-CBI-CDPI2 (27), and (+)-CBI-
indole2 (28). One clone of phage M13mp10 that contained
the SV40 nucleosomal DNA insert w794 (nucleotide no.
5238-138) was selected for the study.27 The alkylation site
identification and the assessment of the relative selectiv-
ity among the available sites were obtained by thermally
induced strand cleavage of the singly 5′-end-labeled
duplex DNA after exposure to the agents. After treatment
of the end-labeled duplex DNA with a range of agent
concentrations, the unbound agent was removed by EtOH
precipitation of the DNA. Redissolution of the DNA in
aqueous buffer, thermolysis (100 °C, 30 min) to induce
Con clu sion s
The parallel synthesis of 132 CBI analogues of CC-1065
and the duocarmycins was described utilizing the solu-
tion-phase technology of acid-base liquid-liquid extrac-
tion for their isolation and purification. The 132 ana-
(27) Ambrose, C.; Rajadhyaksha, A.; Lowman, H.; Bina, M. J . Mol.
Biol. 1989, 210, 255.
(28) Boger, D. L.; Munk, S. A.; Zarrinmayeh, H.; Ishizaki, T.;
Haught, J .; Bina, M. Tetrahedron 1991, 47, 2661.

Synthesis and Evaluation of Duocarmycin Analogues
J . Org. Chem., Vol. 66, No. 20, 2001 6659
matic subunits, is a contribution to catalysis of the DNA
alkylation reaction that provides additional enhance-
ments of 100-1000-fold for a total that exceeds a 25 000-
fold enhancement. Aside from the significance of these
observations in the design of future CC-1065/duocarmy-
cin analogues, their significance to the design of hybrid
structures containing the CC-1065/duocarmycin alkyla-
tion subunit should not be underestimated. Those that
lack an attached bicyclic or tricyclic X subunit, i.e.,
duocarmycin/distamycin or CC-1065/distamycin hybrids,
can be expected to be intrinsically poor or slow DNA
alkylating agents.
Exp er im en ta l Section
Gen er a l P r oced u r e for P r ep a r a tion of th e CBI An a -
logu es. A solution of the dimer ester 1726 (20 µmol) in
dioxane-water (4:1, 250-300 µL) was treated with aqueous
LiOH (4 M, 20 µL), and the mixture was stirred for 12 h at
20-25 °C. After lyophilization, the crude material was dis-
solved in water (500 µL) and treated with aqueous HCl (3 M,
100 µL), and the precipitate was collected by centrifugation.
Decantation and lyophilization of the residue from water (500
µL) yielded material (18) that was sufficiently pure for the
subsequent coupling. A sample of 421 (1 mg, 3 µmol) was
treated for 45 min with HCl-EtOAc (4 M, 300 µL). After
evaporation of the solvent under a steady stream of N2, the
residue was dried in vacuo. The crude material was dissolved
in DMF (40 µL) together with EDCI (9 µmol, 1.7 mg) and 18
(4.5 µmol) and allowed to stand at 20-25 °C. The reaction was
quenched after 12 h by adding saturated aqueous NaCl (400
µL). Isolation of the product was performed by extraction with
EtOAc (4 × 600 µL) and subsequent washing of the organic
layer with aqueous 3 M HCl (4 × 400 µL), saturated aqueous
Na2CO3 (4 × 400 µL), and saturated aqueous NaCl (1 × 400
µL). The combined organic layers were dried (Na2SO4) and
concentrated to afford the CBI analogue in yields between 30
and 97%.
F igu r e 7. Thermally induced strand cleavage of double-
stranded DNA (SV40 DNA fragment, 144 bp, nucleotide no.
5238-138, clone w794) after 24 h of incubation of agent-DNA
at 25 °C followed by removal of unbound agent and 30 min
incubation at 100 °C, denaturing PAGE, and autoradiogra-
phy: lane 1, control DNA; lanes 2-4, (+)-CBI-CDPI2 (27, 1
× 10-5 to 1 × 10-7 M); lanes 5-7, (+)-CBI-indole2 (28, 1 ×
10-5 to 1 × 10-7 M); lanes 8-11, Sanger G, C, A, and T
reactions; lanes 12 and 13, CBI-X10-Y10 (26, 1 × 10-3 and 1
× 10-4 M); lanes 14-17, CBI-X11-Y9 (25, 1 × 10-4 to 1 × 10-7
M).
The diagonal elements of the library and additional selected
1
members were characterized by H NMR and high-resolution
matrix-assisted laser desorption ionization (HRMALDI)-
FTMS.
1-(Ch lor om eth yl)-5-h yd r oxy-3-{4-[4-(ter t-bu toxyca r bo-
n ylam in o)ben zoylam in o]ben zoyl}-1,2-dih ydr o-3H-ben z[e]-
1
logues constitute a systematic study of the DNA-binding
domain with the incorporation of dimers composed of
monocyclic, bicyclic, and tricyclic heteroaromatic sub-
units. From examination of these analogues, clear trends
in cytotoxic potency and DNA alkylation efficiency emerge
highlighting the principle importance of the first attached
DNA-binding subunit (X subunit): tricyclic > bicyclic >
monocyclic heteroaromatic subunits. Notably, the trends
observed in the cytotoxic potencies parallel those ob-
served in the relative efficiencies of DNA alkylation. Our
interpretation of these results is that the trends represent
the partitioning of the role of the DNA-binding
subunit(s) into two distinct contributions. The first is
derived from an increase in DNA-binding selectivity and
affinity which leads to property enhancements of 10-
100-fold and is embodied in the monocyclic group 1 series.
The second, which we suggest is additionally and ef-
fectively embodied in the bicyclic and tricyclic heteroaro-
in d ole (seco-CBI-X5-Y5): 0.99 mg, 58%; H NMR (acetone-
d6, 600 MHz) δ 9.67 (s, 1H), 9.27 (s, 1H), 8.75 (s, 1H), 8.21 (d,
J ) 8.3 Hz, 1H), 8.03-7.94 (m, 4H), 7.84 (d, J ) 8.3 Hz, 1H),
7.70 (d, J ) 8.8 Hz, 2H), 7.67 (d, J ) 8.3 Hz, 2H), 7.53 (dd, J
) 7.7, 7.9 Hz, 1H), 7.36 (dd, J ) 7.4, 7.5 Hz, 1H), 4.44 (dd, J
) 9.6, 9.9 Hz, 1H), 4.30-4.24 (m, 1H), 4.10-4.05 (m, 1H), 3.99
(dd, J ) 3.1, 10.9 Hz, 1H), 3.82-3.75 (m, 1H), 1.50 (s, 9H);
HRMALDI-FTMS (DHB) m/z 572.1943 (C32H30ClN3O5 + H+
requires 572.1952).
1-(Ch lor om eth yl)-5-h yd r oxy-3-{3-[3-(ter t-bu toxyca r bo-
n ylam in o)ben zoylam in o]ben zoyl}-1,2-dih ydr o-3H-ben z[e]-
1
in d ole (seco-CBI-X6-Y6): 0.95 mg, 55%; H NMR (acetone-
d6, 600 MHz) δ 9.73 (s, 1H), 9.29 (s, 1H), 8.61 (s, 1H), 8.25-
8.20 (m, 1H), 8.17 (s, 1H), 8.12 (br s, 1H), 8.05 (br s, 1H), 7.84
(d, J ) 8.5 Hz, 1H), 7.73 (d, J ) 7.1 Hz, 1H), 7.60 (d, J ) 7.6
Hz, 1H), 7.55-7.48 (m, 2H), 7.43-7.35 (m, 3H), 4.47-4.40 (m,
1H), 4.22 (br s, 1H), 4.14-4.08 (m, 1H), 3.99 (d, J ) 9.5 Hz,
1H), 3.84-3.76 (m, 1H), 1.48 (s, 9H); HRMALDI-FTMS (DHB)
m/z 558.1995 (C32H30ClN3O5 - HCl + Na+ requires 558.2005).
1-(Ch lor om eth yl)-5-h yd r oxy-3-{[2-[[2-(ter t-bu toxyca r -
bon yla m in o)-1,3-th ia zol-4-yl]ca r bon yla m in o]-1,3-th ia zol-
4-yl]ca r bon yl}-1,2-d ih yd r o-3H-ben z[e]in d ole (seco-CBI-
X7-Y7): 1.12 mg, 64%; 1H NMR (acetone-d6, 300 MHz) δ 10.64
(br s, 1H), 9.29 (s, 1H), 8.23 (d, J ) 8.8 Hz, 1H), 8.09 (s, 2H),
7.94 (br s, 1H), 7.86 (d, J ) 8.3 Hz, 1H), 7.57-7.50 (m, 1H),
7.43-7.35 (m, 1H), 4.74-4.67 (m, 2H), 4.20-4.11 (m, 1H),
4.04-3.94 (m, 1H), 3.78-3.68 (m, 1H), 1.54 (s, 9H); HRMALDI-
FTMS (DHB) m/z 608.0814 (C26H24ClN5O5S2 + Na+ requires
608.0805).
(29) It is important to recognize that the X subunit C5 substituent
contributes significantly to the rate and efficiency of DNA alkylation
and cytotoxic activity presumably by extending the rigid length of the
X subunit. In studies of analogues which lack a third Y subunit, the
presence of a C5 substituent on the bicyclic X subunit substantially
(g10-1000-fold) enhances the properties providing analogues compa-
rable in cytotoxic potency and DNA alkylation efficiency to the best
analogues detailed herein. See the following: (a) ref 13. (b) Boger, D.
L.; Stauffer, F.; Hedrick, M. P. Bioorg. Med. Chem. Lett. 2001, 11, 2021.

6660 J . Org. Chem., Vol. 66, No. 20, 2001
Boger et al.
1-(Ch lor om eth yl)-5-h yd r oxy-3-{[2-[[4-(ter t-bu toxyca r -
bon yla m in o)-1-m eth ylim id a zol-2-yl]ca r bon yla m in o]-1,3-
th iazol-4-yl]car bon yl}-1,2-dih ydr o-3H-ben z[e]in dole (seco-
CBI-X7-Y9): 1.10 mg, 63%; 1H NMR (acetone-d6, 600 MHz) δ
10.73 (br s, 1H), 9.27 (s, 1H), 8.69 (br s, 1H), 8.23 (d, J ) 8.7
Hz, 1H), 8.09 (br s, 1H), 7.93 (br s, 1H), 7.85 (d, J ) 8.2 Hz,
1H), 7.53 (dd, J ) 7.4, 7.6 Hz, 1H), 7.42-7.35 (m, 2H), 4.76-
4.65 (m, 2H), 4.19-4.13 (m, 1H), 4.12 (s, 3H), 3.99 (dd, J )
3.1, 11.2 Hz, 1H), 3.73 (dd, J ) 8.7, 11.3 Hz, 1H), 1.48 (s, 9H);
HRMALDI-FTMS (DHB) m/z 583.1519 (C27H27ClN6O5S + H+
requires 583.1525).
(m, 2H), 7.20 (s, 1H), 7.00 (s, 1H), 6.56 (s, 1H), 4.88-4.83 (m,
1H), 4.82-4.78 (m, 1H), 4.41 (dd, J ) 8.2, 8.2 Hz, 2H), 4.31-
4.26 (m, 1H), 4.06 (dd, J ) 3.1, 11.2 Hz, 1H), 3.83 (dd, J )
8.7, 11.3 Hz, 1H), 3.78 (s, 3H), 3.42-3.31 (m, 2H), 1.46 (s, 9H);
HRMALDI-FTMS (DHB) m/z 626.2377 (C35H34ClN5O5 - HCl
+ Na+ requires 626.2374).
1-(Ch lor om eth yl)-5-h yd r oxy-3-{[3-[[3-(ter t-bu toxyca r -
bon yl)-1,2-d ih yd r o-3H-p yr r olo[3,2-e]in d ol-7-yl]ca r bon yl]-
1,2-d ih yd r o-3H -p yr r olo[3,2-e]in d ol-7-yl]ca r b on yl}-1,2-
d ih yd r o-3H-ben z[e]in d ole (seco-CBI-X11-Y11): 1.06 mg,
50%; 1H NMR (acetone-d6, 600 MHz) δ 10.93 (br s, 1H), 10.75
(br s, 1H), 9.26 (s, 1H), 8.47-8.38 (m, 1H), 8.25 (d, J ) 8.2
Hz, 1H), 8.09 (s, 1H), 7.88 (d, J ) 8.2 Hz, 1H), 7.55 (dd J )
7.1, 7.2 Hz, 1H), 7.47 (d, J ) 8.7 Hz, 1H), 7.43-7.37 (m, 2H),
7.24 (s, 1H), 7.06 (s, 1H), 4.89-4.85 (m, 1H), 4.82 (dd, J ) 1.5,
10.7 Hz, 1H), 4.79-4.73 (m, 2H), 4.32-4.27 (m, 1H), 4.11-
4.04 (m, 3H), 3.84 (dd, J ) 8.7, 11.3 Hz, 1H), 3.63-3.52 (m,
2H), 3.32 (t, J ) 9.2 Hz, 2H), 1.56 (s, 9H); HRMALDI-FTMS
(DHB) m/z 702.2478 (C40H36ClN5O5 + H+ requires 702.2478).
1-(Ch lor om eth yl)-5-h yd r oxy-3-{[3-[[5-(ter t-bu toxyca r -
bon ylam in o)in dole-2-yl]car bon yl]-1,2-dih ydr o-3H-pyr r olo-
[3,2-e]in d ol-7-yl]ca r b on yl}-1,2-d ih yd r o-3H -b e n z[e]in -
1-(Ch lor om eth yl)-5-h yd r oxy-3-{[2-[[5-(ter t-bu toxyca r -
bon ylam in o)ben zofu r an -2-yl]car bon ylam in o]-1,3-th iazol-
4-yl]ca r bon yl}-1,2-d ih yd r o-3H-ben z[e]in d ole (seco-CBI-
X7-Y13): 1.00 mg, 54%; 1H NMR (acetone-d6, 600 MHz) δ 9.27
(br s, 1H), 8.58 (br s, 1H), 8.23 (d, J ) 8.7 Hz, 1H), 8.13 (br s,
2H), 7.96 (br s, 1H), 7.87-7.83 (m, 2H), 7.63-7.58 (m, 1H),
7.57-7.51 (m, 2H), 7.38 (dd, J ) 7.4, 7.7 Hz, 1H), 4.70 (br s,
2H), 4.18-4.13 (m, 1H), 4.01-3.96 (m, 1H), 3.75-3.69 (m, 1H),
1.50 (s, 9H); HRMALDI-FTMS (DHB) m/z 641.1215 (C31H27
-
ClN4O6S + Na+ requires 641.1232).
1-(Ch lor om eth yl)-5-h yd r oxy-3-{[4-[[4-(ter t-bu toxyca r -
b on yla m in o)t h iop h en -2-yl]ca r b on yla m in o]t h iop h en -2-
yl]ca r bon yl}-1,2-d ih yd r o-3H-ben z[e]in d ole (seco-CBI-X8-
1
d ole (seco-CBI-X11-Y14): 0.91 mg, 45%; H NMR (acetone-
d6, 600 MHz) δ 10.94 (br s, 1H), 10.70 (br s, 1H), 9.27 (s, 1H),
8.44-8.40 (m, 1H), 8.29-8.21 (m, 2H), 8.09 (br s, 1H), 7.93-
8.01 (m, 2H), 7.89 (d, J ) 8.2 Hz, 1H), 7.55 (dd, J ) 7.7, 7.9
Hz, 1H), 7.48 (dd, J ) 9.0, 12.2 Hz, 2H), 7.41-7.37 (m, 2H),
7.25 (s, 1H), 7.12 (s, 1H), 4.90-4.86 (m, 1H), 4.80-4.83 (m,
1H), 4.77-4.73 (m, 1H), 4.32-4.27 (m, 1H), 4.07 (dd, J ) 3.1,
11.3 Hz, 1H), 3.84 (dd, J ) 8.4, 10.7 Hz, 1H), 3.62-3.52 (m,
2H), 1.50 (s, 9H); HRMALDI-FTMS (DHB) m/z 676.2309
(C38H34ClN5O5 + H+ requires 676.2321).
1
Y8): 1.51 mg, 86%; H NMR (acetone-d6, 500 MHz) δ 10.30
(br s, 1H), 9.26 (s, 1H), 8.82 (s, 1H), 8.23 (d, J ) 8.3 Hz, 1H),
8.00 (d, J ) 1.2 Hz, 1H), 7.97 (br s, 1H), 7.92 (s, 1H), 7.87 (d,
J ) 8.3 Hz, 2H), 7.54 (t, J ) 8.3 Hz, 1H), 7.41-7.36 (m, 2H),
4.68 (t, J ) 10.3 Hz, 1H), 4.61 (dd, J ) 1.9, 10.7 Hz, 1H), 4.25-
4.20 (m, 1H),4.05 (dd, J ) 3.1, 11.5 Hz, 1H), 3.81 (dd, J ) 8.5,
11.1 Hz, 1H), 1.48 (s, 9H); HRMALDI-FTMS (DHB) m/z
570.1118 (C28H26ClN3O5S2 - HCl + Na+ requires 570.1133).
1-(Ch lor om eth yl)-5-h yd r oxy-3-{[4-[[4-(ter t-bu toxyca r -
b on yla m in o)-1-m et h ylim id a zol-2-yl]ca r b on yla m in o]-1-
m eth ylim id a zol-2-yl]ca r bon yl}-1,2-d ih yd r o-3H-ben z[e]-
1-(Ch lor om eth yl)-5-h yd r oxy-3-{[5-[[4-(ter t-bu toxyca r -
b on yla m in o)-1-m et h ylp yr r ol-2-yl]ca r b on yla m in o]b en -
zot h iop h e n -2-yl]ca r b on yl}-1,2-d ih yd r o-3H -b e n z[e]in -
1
1
in d ole (seco-CBI-X9-Y9): 1.48 mg, 85%; H NMR (acetone-
d ole (seco-CBI-X12-Y10): 1.05 mg, 57%; H NMR (acetone-
d6, 600 MHz) δ 9.26 (br s, 1H), 9.21 (s, 1H), 8.56 (br s, 1H),
8.24 (d, J ) 8.4 Hz, 1H), 8.10 (br s, 1H), 7.87 (d, J ) 8.2 Hz,
1H), 7.57-7.52 (m, 3H), 7.39 (t, J ) 7.2 Hz, 1H), 7.24 (br s,
1H), 4.95-4.88 (m, 1H), 4.80-4.71 (m, 1H), 4.19-4.13 (m, 1H),
4.08 (s, 3H), 4.05-3.98 (m, 4H), 3.75-3.69 (m, 1H), 1.48 (s,
9H); HRMALDI-FTMS (DHB) m/z 580.2060 (C28H30ClN7O5 +
H+ requires 580.2075).
d6, 600 MHz) δ 9.39 (br s, 1H), 9.31 (br s, 1H), 8.61 (br s, 1H),
8.25 (d, J ) 8.2 Hz, 1H), 8.18 (br s, 1H), 8.06 (s, 1H), 7.93 (d,
J ) 8.7 Hz, 1H), 7.89 (d, J ) 8.7 Hz, 1H), 7.77 (d, J ) 8.2 Hz,
1H), 7.55 (dd, J ) 7.2, 7.4 Hz, 1H), 7.40 (dd, J ) 7.4, 7.7 Hz,
1H), 6.99 (br s, 1H), 6.94 (br s, 1H), 4.84-4.79 (m, 1H), 4.70-
4.67 (m, 1H), 4.26-4.21 (m, 1H), 4.05 (dd, J ) 3.1, 11.3 Hz,
1H), 3.93 (s, 3H), 3.83 (dd, J ) 8.4, 10.7 Hz, 1H), 1.46 (s, 9H);
HRMALDI-FTMS (DHB) m/z 495.1504 (C33H31ClN4O5S - Boc
- HCl + H+ requires 495.1491).
1-(Ch lor om eth yl)-5-h yd r oxy-3-{[4-[[4-(ter t-bu toxyca r -
bon yla m in o)-1-m eth ylp yr r ol-2-yl]ca r bon yla m in o]-1-m e-
t h ylp yr r ol-2-yl]ca r b on yl}-1,2-d ih yd r o-3H -b e n z[e]in -
1-(Ch lor om eth yl)-5-h yd r oxy-3-{[5-[[5-(ter t-bu toxyca r -
b on yla m in o)b en zot h iop h en e-2-yl]ca r b on yla m in o]b en -
zot h iop h en e-2-yl]ca r b on yl}-1,2-d ih yd r o-3H -b en z[e]in -
1
d ole (seco-CBI-X10-Y10): 1.18 mg, 68%; H NMR (acetone-
d6, 600 MHz) δ 9.30 (br s, 1H), 9.21 (s, 1H), 8.21 (d, J ) 8.7
Hz, 1H), 8.14 (br s, 1H), 7.82 (d, J ) 8.2 Hz, 1H), 7.66 (br s,
1H), 7.52 (t, J ) 6.7 Hz, 1H), 7.46 (d, J ) 1.5 Hz, 1H), 7.36 (t,
J ) 8.2 Hz, 1H), 6.93 (br s, 1H), 6.81 (br s, 1H), 6.72 (br s,
1H), 4.50 (d, J ) 5.1 Hz, 2H), 4.10-4.04 (m, 1H), 3.99 (dd, J
) 3.6, 11.0 Hz, 1H), 3.92 (s, 3H), 3.83 (s, 3H), 3.74-3.69 (m,
1H), 1.44 (s, 9H); HRMALDI-FTMS (DHB) m/z 564.2233
(C30H32ClN5O5 - HCl + Na+ requires 564.2223).
1-(Ch lor om eth yl)-5-h yd r oxy-3-{[3-[[2-(ter t-bu toxyca r -
bon yla m in o)-1,3-t h ia zol-4-yl]ca r b on yl]-1,2-d ih yd r o-3H-
pyr r olo[3,2-e]in dol-7-yl]car bon yl}-1,2-dih ydr o-3H-ben z[e]-
in d ole (seco-CBI-X11-Y7): 1.23 mg, 64%;1H NMR (acetone-
d6, 600 MHz) δ 10.92 (br s, 1H), 10.42 (br s, 1H), 9.28 (s, 1H),
8.40 (br s, 1H), 8.24 (d, J ) 8.2 Hz, 1H), 8.08 (br s, 1H), 7.88
(d, J ) 8.7 Hz, 1H), 7.73 (br s, 1H), 7.54 (dd, J ) 7.1, 7.2 Hz,
1H), 7.47-7.42 (m, 1H), 7.39 (dd, J ) 7.4, 7.7 Hz, 1H), 7.20 (s,
1H), 4.88-4.82 (m, 1H), 4.82-4.77 (m, 1H), 4.62-4.52 (m, 2H),
4.31-4.25 (m, 1H), 4.06 (dd, J ) 3.6, 11.8 Hz, 1H), 3.86-3.80
(m, 1H), 3.43-3.35 (m, 1H), 1.54 (s, 9H); HRMALDI-FTMS
(DHB) m/z 544.1195 (C33H30ClN5O5S - Boc + H+ requires
544.1205).
1
d ole (seco-CBI-X12-Y12): 1.81 mg, 88%; H NMR (acetone-
d6, 600 MHz) δ 9.91 (br s, 1H), 9.33 (br s, 1H), 8.61-8.59 (m,
2H), 8.28-8.23 (m, 2H), 8.18 (s, 1H), 8.10 (s, 1H), 8.01, (d, J
) 8.7 Hz, 1H), 7.95 (br s, 1H), 7.94-7.88 (m, 2H), 7.85-7.80
(m, 1H), 7.62 (dd, J ) 1.8, 8.4 Hz, 1H), 7.55 (t, J ) 7.0 Hz,
1H), 7.40 (t, J ) 8.0 Hz, 1H), 4.87-4.81 (m, 1H), 4.72-4.67
(m, 1H), 4.28-4.23 (m, 1H), 4.05 (dd, J ) 3.1, 11.3 Hz, 1H),
3.85 (dd, J ) 8.2, 11.2 Hz, 1H), 1.51 (s, 9H); HRMALDI-FTMS
(DHB) m/z 684.1366 (C36H29ClN3O5S2 + H+ requires 684.1388).
1-(Ch lor om eth yl)-5-h yd r oxy-3-{[5-[[4-(ter t-bu toxyca r -
b on yla m in o)b en zoyla m in o]b en zofu r a n -2-yl]ca r b on yl}-
1,2-d ih yd r o-3H-ben z[e]in d ole (seco-CBI-X13-Y5): 1.78 mg,
1
97%; H NMR (acetone-d6, 600 MHz) δ 9.58 (br s, 1H), 9.34
(br s, 1H), 8.73 (br s, 1H), 8.41 (d, J ) 2.0 Hz, 1H), 8.25 (d, J
) 8.7 Hz, 1H), 8.03-7.98 (m, 2H), 7.89 (d, J ) 8.2 Hz, 1H),
7.84 (dd, J ) 2.0, 8.7 Hz, 1H), 7.74-7.63 (m, 5H), 7.56 (dd, J
) 7.4, 7.7 Hz, 1H), 7.41 (dd, J ) 7.7, 8.2 Hz, 1H), 4.86-4.80
(m, 1H), 4.31-4.24 (m, 1H), 4.05 (dd, J ) 3.1, 10.7 Hz, 1H),
3.83 (dd, J ) 10.7, 11.3 Hz, 1H), 3.60-3.56 (m, 1H), 1.50 (s,
9H); HRMALDI-FTMS (DHB) m/z 598.1946 (C34H30ClN3O6 -
HCl + Na+ requires 598.1949).
1-(Ch lor om eth yl)-5-h yd r oxy-3-{[3-[[4-(ter t-bu toxyca r -
bon yla m in o)-1-m eth ylp yr r ol-2-yl]ca r bon yl]-1,2-d ih yd r o-
3H -p yr r olo[3,2-e]in d ol-7-yl]ca r b on yl}-1,2-d ih yd r o-3H -
ben z[e]in d ole (seco-CBI-X11-Y10): 1.19 mg, 62%; 1H NMR
(acetone-d6, 600 MHz) δ 10.90 (br s, 1H), 9.27 (br s, 1H), 8.24
(d, J ) 8.2 Hz, 1H), 8.13 (br s, 1H), 8.09-8.02 (m, 2H), 7.88
(d, J ) 8.7 Hz, 1H), 7.54 (dd, J ) 7.7, 8.2 Hz, 1H), 7.45-7.36
1-(Ch lor om eth yl)-5-h yd r oxy-3-{[5-[[4-(ter t-bu toxyca r -
bon yla m in o)th iop h en -2-yl]ca r bon yla m in o]ben zofu r a n -
2-yl]ca r bon yl}-1,2-d ih yd r o-3H-ben z[e]in d ole (seco-CBI-
X13-Y8): 0.91 mg, 48%; 1H NMR (acetone-d6, 600 MHz) δ 9.88
(br s, 1H), 9.33 (br s, 1H), 8.85 (br s, 1H), 8.36-8.33 (m, 1H),
8.24 (d, J ) 8.2 Hz, 1H), 8.18 (br s, 1H), 8.00 (br s, 1H), 7.89

Synthesis and Evaluation of Duocarmycin Analogues
J . Org. Chem., Vol. 66, No. 20, 2001 6661
(d, J ) 8.7 Hz, 1H), 7.82-7.88 (m, 1H), 7.67-7.63 (m, 2H),
7.55 (dd, J ) 7.1, 7.2 Hz, 1H), 7.42-7.38 (m, 2H), 4.82-4.79
(m, 2H), 4.28-4.24 (m, 1H), 4.04 (dd, J ) 3.1, 11.3 Hz, 1H),
3.84 (dd, J ) 8.7, 11.3 Hz, 1H), 1.49 (s, 9H); HRMALDI-FTMS
(DHB) m/z 517.0855 (C32H28ClN3O6S+ - Boc requires 517.0863).
1-(Ch lor om eth yl)-5-h yd r oxy-3-{[5-[[5-(ter t-bu toxyca r -
bon ylam in o)ben zofu r an -2-yl]car bon ylam in o]ben zofu r an -
2-yl]ca r bon yl}-1,2-d ih yd r o-3H-ben z[e]in d ole (seco-CBI-
X13-Y13): 1.32 mg, 67%; 1H NMR (acetone-d6, 600 MHz) δ
9.90 (br s, 1H), 9.34 (s, 1H), 8.55 (br s, 1H), 8.47-8.44 (m, 1H),
8.25 (d, J ) 8.7 Hz, 1H), 8.08 (br s, 1H), 7.93-7.88 (m, 2H),
7.71-7.68 (m, 2H), 7.64 (s, 1H), 7.60-7.54 (m, 2H), 7.51 (d, J
) 8.7 Hz, 1H), 7.41 (ddd, J ) 1.0, 7.7, 7.7 Hz, 1H), 4.84-4.80
(m, 2H), 4.30-4.25 (m, 1H), 4.05 (dd, J ) 3.1, 11.3 Hz, 1H),
3.83 (dd, J ) 8.7, 11.3 Hz, 1H), 1.50 (s, 9H); HRMALDI-FTMS
(DHB) m/z 638.1883 (C36H30ClN3O7 - HCl + Na+ requires
638.1903).
7.98 (m, 1H), 7.95-7.90 (m, 1H), 7.78 (d, J ) 8.9 Hz, 1H),
7.60-7.54 (m, 2H), 7.44-7.37 (m, 2H), 5.50 (dd, J ) 10.9, 11.1
Hz, 1H), 4.98-4.88 (m, 1H), 4.36-4.27 (m, 1H), 4.12-4.04 (m,
1H), 3.88-3.80 (m, 1H), 1.49 (s, 9H); HRMALDI-FTMS (DHB)
m/z 618.1584 (C31H28ClN5O5S + H+ requires 618.1572).
DNA Alk yla tion Stu d ies: Selectivity a n d Efficien cy.
The preparation of singly 32P-5′-end-labeled double-stranded
DNA, agent binding studies, gel electrophoresis, and autora-
diography were conducted according to procedures described
in full detail elsewhere.28 Eppendorf tubes containing the 5′-
end-labeled DNA (9 µL) in TE buffer (10 mM Tris, 1 mM
EDTA, pH 7.5) were treated with the agent in DMSO (1 µL at
the specified concentration). The solution was mixed by
vortexing and brief centrifugation and subsequently incubated
at 25 °C for 24 h. The covalently modified DNA was separated
from the unbound agent by EtOH precipitation and resus-
pended in TE buffer (10 µL). The solution of DNA in an
Eppendorf tube sealed with Parafilm was warmed at 100 °C
for 30 min to introduce cleavage at the alkylation sites, allowed
to cool to 25 °C, and centrifuged. Formamide dye (0.03% xylene
cyanol FF, 0.03% bromophenol blue, 8.7% Na2EDTA 250 mM)
was added (5 µL) to the supernatant. Prior to electrophoresis,
the sample was denatured by warming at 100 °C for 5 min,
placed in an ice bath, and centrifuged, and the supernatant
(3 µL) was loaded directly onto the gel. Sanger dideoxynucle-
otide sequencing reactions were run as standards adjacent to
the reaction samples. Polyacrylamide gel electrophoresis
(PAGE) was run on an 8% sequencing gel under denaturing
conditions (8 M urea) in TBE buffer (100 mM Tris, 100 mM
boric acid, 0.2 mM Na2EDTA) followed by autoradiography.
1-(Ch lor om eth yl)-5-h yd r oxy-3-{[5-[[5-(ter t-bu toxyca r -
bon yla m in o)in d ole-2-yl]ca r bon yla m in o]in d ole-2-yl]ca r -
bon yl}-1,2-d ih yd r o-3H-ben z[e]in d ole (seco-CBI-X14-Y14):
1
1.39 mg, 71%; H NMR (acetone-d6, 600 MHz) δ 10.86 (br s,
1H), 10.83 (d, J ) 1.0 Hz, 1H), 9.59 (br s, 1H), 9.23 (s, 1H),
8.38-8.34 (m, 1H), 8.24 (d, J ) 8.2 Hz, 1H), 8.08 (br s, 1H),
7.96 (br s, 1H), 7.89 (d, J ) 8.2 Hz, 1H), 7.63 (dd, J ) 2.0, 8.9
Hz, 1H), 7.59-7.52 (m, 2H), 7.49 (d, J ) 8.7 Hz, 1H), 7.41-
7.37 (m, 1H), 7.35 (d, J ) 8.2 Hz, 1H), 7.26 (d, J ) 5.6 Hz,
1H), 4.89-4.84 (m, 1H), 4.83-4.77 (m, 1H), 4.32-4.27 (m, 1H),
3.84 (dd, J ) 8.2, 11.2 Hz, 1H), 1.49 (s, 9H); HRMALDI-FTMS
(DHB) m/z 650.2149 (C36H32ClN5O5 + H+ requires 650.2165).
1-(Ch lor om eth yl)-5-h yd r oxy-3-{[6-[[6-(ter t-bu toxyca r -
b on yla m in o)b en zoxa zole-2-yl]ca r b on yla m in o]b en zox-
a zole-2-yl]ca r bon yl}-1,2-d ih yd r o-3H-ben z[e]in d ole (seco-
CBI-X15-Y15): 1.06 mg, 50%; 1H NMR (DMSO-d6, 600 MHz)
δ 11.51 (s, 1H), 10.57 (s, 1H), 9.83 (s, 1H), 8.51 (s, 1H), 8.14
(d, J ) 8.2 Hz, 1H), 8.15-8.00 (m, 4H), 7.88 (d, J ) 8.7 Hz,
1H), 7.84 (d, J ) 8.4 Hz, 1H), 7.60-7.51 (m, 2H), 7.45-7.39
(m, 1H), 4.98 (d, J ) 12.3 Hz, 1H), 4.86-4.79 (m, 1H), 4.29-
4.24 (m, 1H), 3.91-3.86 (m, 1H), 1.51 (s, 9H); HRMALDI-
FTMS (DHB) m/z 653.1692 (C34H28ClN5O7+ requires 653.1671).
1-(Ch lor om eth yl)-5-h yd r oxy-3-{[5-[[4-(ter t-bu toxyca r -
bon yla m in o)th iop h en e-2-yl]ca r bon yla m in o]ben zim id a -
zole-2-yl]ca r bon yl}-1,2-d ih yd r o-3H-ben z[e]in d ole (seco-
Ack n ow led gm en t. We gratefully acknowledge the
financial support of the National Institutes of Health
(Grant CA 41986), Corixa Pharmaceuticals, and The
Skaggs Institute for Chemical Biology.
Su p p or tin g In for m a tion Ava ila ble: 1H NMR spectra of
the diagonal elements of the library, seco-CBI-X5-Y5-seco-
CBI-X15-Y15, seco-CBI-X7-Y9, seco-CBI-X7-Y13, seco-CBI-
X11-Y7, seco-CBI-X11-Y10, seco-CBI-X11-Y14, seco-CBI-X12-
Y10, seco-CBI-X13-Y5, seco-CBI-X13-Y8, and seco-CBI-X16-
Y8. This material is available free of charge via the Internet
at http://pubs.acs.org.
1
CBI-X16-Y8): 1.40 mg, 75%; H NMR (acetone-d6, 500 MHz)
δ 9.89 (br s, 1H), 9.82 (br s, 1H), 9.30 (s, 1H), 8.84 (br s, 1H),
8.48-8.45 (m, 1H), 8.26 (d, J ) 8.5 Hz, 1H), 8.23 (s, 1H), 8.01-
J O010454U