Synthesis and evaluation of CC-1065 and duocarmycin analogues incorporating the Iso-CI and Iso-CBI alkylation subunits: Impact of relocation of the C-4 carbonyl

J . Org. Chem. 1997, 62, 8875-8891

8875

Syn th esis a n d Eva lu a tion of CC-1065 a n d Du oca r m ycin An a logu es

In cor p or a tin g th e Iso-CI a n d Iso-CBI Alk yla tion Su bu n its: Im p a ct

of Reloca tion of th e C-4 Ca r bon yl

Dale L. Boger,* Robert M. Garbaccio, and Qing J in

Department of Chemistry and The Skaggs Institute for Chemical Biology, The Scripps Research Institute,

10550 North Torrey Pines Road, La J olla, California 92037

Received September 8, 1997^X

The synthesis of 2-(tert-Butyloxycarbonyl)-1,2,9,9a-tetrahydrocyclopropa[c]benzo[f]indol-8-one (31,

N-BOC-iso-CBI) and 1-(tert-Butyloxycarbonyl)-4-hydroxy-3-[[(methanesulfonyl)oxy]methyl]-2,3-

dihydroindole (19, seco-N-BOC-iso-CI) containing an isomeric structural modification in the CC-

1065 and duocarmycin alkylation subunits and their incorporation into analogues of the natural

products are detailed. The approach was based on a directed ortho metalation of an appropriately

functionalized benzene (13) or naphthalene (24) precursor to regiospecifically install iodine at the

C-2 position. Conversion of these respective intermediates to the dihydroindole skeleton utilized

an established 5-exo-trig aryl radical cyclization onto an unactivated alkene with subsequent TEMPO

trap or the more recent 5-exo-trig aryl radical cyclization onto a vinyl chloride for direct synthesis

of the immediate precursors. Closure of the activated cyclopropane to complete the iso-CBI nucleus

was accomplished by a selective ortho spirocyclization. The evaluation of the iso-CBI-based agents

revealed a significant stability comparable to that of CC-1065 and duocarmycin A, but that it is

more reactive than duocarmycin SA (6-7×) or the direct comparison CBI-based agents (5×) for

which X-ray structure comparisons served to establish the basis for their inherent reaction

regioselectivity and reactivity. Resolution and synthesis of a full set of natural product analogues

and subsequent evaluation of their DNA alkylation properties revealed that the iso-CBI analogues,

even with the relocation of the C-4 carbonyl and the most substantial structural modifications to

the alkylation subunit to date, reacted at comparable rates and retain the identical and characteristic

sequence selectivity of CC-1065 and the duocarmycins. This observation is inconsistent with the

proposal that a sequence-dependent C-4 carbonyl protonation by strategically located DNA backbone

phosphates controls the DNA alkylation selectivity but is consistent with the proposal that it is

determined by the AT-rich noncovalent binding selectivity of the agents and the steric accessibility

of the N3 alkylation site. Confirmation that the DNA alkylation reaction is derived from adenine

N3 addition to the least substituted carbon of the activated cyclopropane, and its quantitation

(95%) was established by isolation and characterization of the depurination adenine N3 adduct.

Consistent with past studies and despite the deep-seated structural change in the alkylation subunit,

the agents were found to exhibit potent cytotoxic activity that correlates with their inherent

reactivity.

(+)-CC-1065 (1)¹and the duocarmycins 2 and 3^2,3are

the initial members of a potent class of antitumor

antibiotics that derive their biological properties through

alkylation of DNA (Figure 1).⁴Prior studies have shown

^XAbstract published in Advance ACS Abstracts, November 1, 1997.

(1) Hanka, L. J .; Dietz, A.; Gerpheide, S. A.; Kuentzel, S. L.; Martin,

D. G. J . Antibiot. 1978, 31, 1211. Chidester, C. G.; Krueger, W. C.;

Mizsak, S. A.; Duchamp, D. J .; Martin, D. G. J . Am. Chem. Soc. 1981,

103, 7629.

(2) Yasuzawa, T.; Muroi, K.; Ichimura, M.; Takahashi, I.; Ogawa,

T.; Takahashi, K.; Sano, H.; Saitoh, Y. Chem. Pharm. Bull. 1995, 43,

378. Takahashi, I.; Takahashi, K.; Ichimura, M.; Morimoto, M.; Asano,

K.; Kawamoto, I.; Tomita, F.; Nakano, H. J . Antibiot. 1988, 41, 1915.

Yasuzawa, T.; Iida, T.; Muroi, K.; Ichimura, M.; Takahashi, K.; Sano,

H. Chem. Pharm. Bull. 1988, 36, 3728. Ichimura, M.; Muroi, K.; Asano,

K.; Kawamoto, I.; Tomita, F.; Morimoto, M.; Nakano, H. J . Antibiot.

1988, 41, 1285. Ohba, K.; Watabe, H.; Sasaki, T.; Takeuchi, Y.;

Kodama, Y.; Nakazawa, T.; Yamamoto, H.; Shomura, T.; Sezaki, M.;

Kondo, S. J . Antibiot. 1988, 41, 1515. Ishii, S.; Nagasawa, M.; Kariya,

Y.; Yamamoto, H.; Inouye, S.; Kondo, S. J . Antibiot. 1989, 42, 1713.

(3) Ichimura, M.; Ogawa, T.; Takahashi, K.; Kobayashi, E.; Kawa-

moto, I.; Yasuzawa, T.; Takahashi, I.; Nakano, H. J . Antibiot. 1990,

43, 1037. Ichimura, M.; Ogawa, T.; Katsumata, S.; Takahashi, K.;

Takahashi, I.; Nakano, H. J . Antibiot. 1991, 44, 1045.

(4) Boger, D. L.; J ohnson, D. S. Angew. Chem., Int. Ed. Engl. 1996,

35, 1439. Boger, D. L.; J ohnson, D. S. Proc. Natl. Acad. Sci. U.S.A.

1995, 92, 3642. Boger, D. L. Acc. Chem. Res. 1995, 28, 20. Boger, D. L.

Chemtracts: Org. Chem. 1991, 4, 329. Boger, D. L. Proceed. Robert A.

Welch Found. Conf. Chem. Res., XXXV. Chem. Front. Med. 1991, 35,

137. Boger, D. L. In Advances in Heterocyclic Natural Products

Synthesis; Pearson, W. H., Ed.; J AI: Greenwich, CT, 1992; Vol. 2, p 1.

F igu r e 1.

that the natural products can withstand and may benefit

from significant structural modifications to the alkylation

subunit and that the resulting agents retain their ability

8876 J . Org. Chem., Vol. 62, No. 25, 1997

Boger et al.

to participate in the characteristic sequence-selective

DNA alkylation reaction.^4,5These structural modifica-

tions and the definition of their effects have served to

advance the understanding of the origin of the sequence

selectivity^4,6and the catalysis^7,8of the DNA alkylation

reaction by 1-3.

Throughout our investigations,^4,7we have advanced the

proposal that the DNA alkylation sequence selectivity is

determined by the AT-rich noncovalent binding selectiv-

ity of the agents and their steric accessibility to the

adenine N3 alkylation site. This noncovalent binding

model accommodates and explains the reverse and offset

5 or 3.5 base-pair AT-rich adenine N3 alkylation selec-

tivities of the natural and unnatural enantiomers of 1⁹

and 2-3,^10,11respectively, and requires that simple

derivatives of the alkylation subunits exhibit alkylation

selectivities distinct from the natural products. It offers

a beautiful explanation for the identical alkylation

selectivities of both enantiomers of such simple deriva-

tives (5′-AA > 5′-TA), and the more extended AT-rich

selectivity of the advanced analogues of 1-3 corresponds

nicely to the length of the agent and the size of the

required binding region surrounding the alkylation site.

Further support of this model includes the demonstrated

AT-rich noncovalent binding of the agents,¹²their pref-

erential noncovalent binding coincidental with DNA

alkylation,¹³and a demonstration that the characteristic

DNA alkylation selectivity does not require the presence

of the C-4 carbonyl or even the activated cyclopropane.^14,15

A more recent demonstration of the complete switch in

the inherent enantiomeric DNA alkylation selectivity

that accompanied the reversal of the orientation of the

DNA binding subunits with reversed versus extended

analogues of the duocarmycins further established the

accuracy of this interpretation.¹⁶This model contrasts

an alternative proposal in which a sequence-dependent

backbone phosphate protonation of the C-4 carbonyl

activates the agent for DNA alkylation and controls the

F igu r e 2.

sequence selectivity.^6,8,17In efforts to further distinguish

the accuracy of these two interpretations, herein we

report the synthesis and evaluation of a series of ana-

logues related to the CI and CBI alkylation subunits 4

and 5 that isomerically relocate the key C-4 carbonyl to

the C-6 or C-8 position, now ortho to the cyclopropane

(Figure 2). From structural studies of DNA-agent

adducts,^17-19the C-4 carbonyl of the natural products

projects out of the minor groove lying on the outer face

of the complexes potentially accessible to the phosphate

backbone. In contrast, the relocated carbonyls of iso-CI

and iso-CBI (6 and 7) would be required to project into

the minor groove inaccessible to the phosphate backbone

if participating in an analogous adenine N3 alkylation

reaction. Thus, the examination of 6 and 7 and their

related analogues of 1-3, which incorporate the most

substantial structural modification to the alkylation

(5) For a review of synthetic efforts see: Boger, D. L.; Boyce, C. W.;

Garbaccio, R. M.; Goldberg, J . A. Chem. Rev. (Washington, D.C.) 1997,

97, 787.

(6) Warpehoski, M. A.; Hurley, L. H. Chem. Res. Toxicol. 1988, 1,

315. Hurley, L. H.; Needham-VanDevanter, D. R. Acc. Chem. Res 1986,

19, 230. Warpehoski, M. A. In Advances in DNA Sequence Specific

Agents; Hurley, L. H., Ed.; J AI: Greenwich, CT, 1992; Vol. 1, p 217.

Hurley, L. H.; Draves, P. In Molecular Aspects of Anticancer Drug-

DNA Interactions; Neidle, S., Waring, M., Eds.; CRC: Ann Arbor, 1993;

Vol. 1, p 89. Aristoff, P. A. In Advances in Medicinal Chemistry; J AI:

Greenwich, CT, 1993; Vol. 2, p 67. Warpehoski, M. A.; McGovren, P.;

Mitchell, M. A. In Molecular Basis of Specificity in Nucleic Acid-Drug

Interactions; Pullman, B., J ortner, J ., Eds.; Kluwer: Netherlands, 1990;

531.

(7) Boger, D. L.; Garbaccio, R. M. Biorg. Med. Chem. 1997, 5, 263.

(8) Warpehoski, M. A.; Harper, D. E. J . Am. Chem. Soc. 1994, 116,

7573. Warpehoski, M. A.; Harper, D. E. J . Am. Chem. Soc. 1995, 117,

2951.

(9) Boger, D. L.; Coleman, R. S.; Invergo, B. J .; Sakaya, S. M.;

Ishizaki, T.; Munk, S. A.; Zarrinmayeh, H.; Kitos, P. A.; Thompson, S.

C. J . Am. Chem. Soc. 1990, 112, 4623. Boger, D. L.; J ohnson, D. S.;

Yun, W.; Tarby, C. M. Bioorg. Med. Chem. 1994, 2, 115.

(10) 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.; Yun, W.; Terashima, S.; Fukuda, Y.; Nakatani, K.; Kitos, P. A.;

J in, Q. Bioorg. Med. Chem. Lett. 1992, 2, 759. Boger, D. L.; Yun, W. J .

Am. Chem. Soc. 1993, 115, 9872.

(11) Boger, D. L.; J ohnson, D. S.; Yun, W. J . Am. Chem. Soc. 1994,

116, 1635.

(12) Boger, D. L.; Colelman, R. S.; Invergo, B. J .; Zarrinmayeh, H.;

Kitos, P. A.; Thompson, S. C.; Leong, T.; McLaughlin, L. W. Chem.-

Biol. Interact. 1990, 73, 29. Boger, D. L.; Sakya, S. M. J . Org. Chem.

1992, 57, 1277.

(16) 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.; Mesini, P.;

Garbaccio, R. M.; J in, Q.; Kitos, P. A. J . Am. Chem. Soc. 1997, 119,

4987. Boger, D. L.; J ohnson, D. S. J . Am. Chem. Soc. 1995, 117, 1443.

(17) 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.

Lin, C. H.; Sun, D.; Hurley, L. H. Chem. Res. Toxicol. 1991, 4, 21. Lee,

C.-S.; Sun, D.; Kizu, R.; Hurley, L. H. Chem. Res. Toxicol. 1991, 4,

203. Lin, C. H.; Beale, J . M.; Hurley, L. H. Biochemistry 1991, 30, 3597.

Lin, C. H.; Hill, G. C.; Hurley, L. H. Chem. Res. Toxicol. 1992, 5, 167.

Ding, Z.-M.; Harshey, R. M.; Hurley, L. H. Nucl. Acids Res. 1993, 21,

4281. Sun, D.; Lin, C. H.; Hurley, L. H. Biochemistry 1993, 32, 4487.

Thompson, A. S.; Sun, D.; Hurley, L. H. J . Am. Chem. Soc. 1995, 117,

2371.

(13) Boger, D. L.; Zhou, J .; Cai, H. Bioorg. Med. Chem. 1996, 4, 859.

(14) Boger, D. L.; Munk, S. A.; Zarrinmayeh, H. J . Am. Chem. Soc.

1991, 113, 3980.

(18) Lin, C. H.; Patel, D. J . J . Mol. Biol. 1995, 248, 162. Lin, C. H.;

Patel, D. J . J . Am. Chem. Soc. 1992, 114, 10658.

(15) Boger, D. L.; Zarrinmayeh, H.; Munk, S. A.; Kitos, P. A.;

Suntornwat, O. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 1431.

(19) Eis, P. S.; Smith, J . A.; Rydewski, J . M.; Case, D. A.; Boger, D.

L.; Chazin, W. J . J . Mol. Biol. 1997, 272, 237.

Synthesis and Evaluation of CC-1065 and Duocarmycin Analogs

J . Org. Chem., Vol. 62, No. 25, 1997 8877

Sch em e 1

subunits to date, would permit an assessment of the

importance of the C-4 carbonyl positioning to the proper-

ties of the agents.

Syn th esis. Only one report of the synthesis of such

agents isomeric to the natural products has been detailed.

Sundberg and co-workers²⁰prepared an agent isomeric

with the alkylation subunit of CC-1065 employing an

intramolecular carbene insertion of an o-quinonediazide

onto a tethered alkene (Figure 2). Presumably because

of the perceived unique character of the authentic alkyl-

ation subunit at the time of the work, its chemical

behavior, biological characteristics, and DNA alkylation

properties were not examined. An alternative route was

devised for the synthesis of the isomeric CI²¹and CBI²²

analogues (Scheme 1). The strategy is complementary

to our most recent synthesis of CBI^5,22in which the

dihydroindole skeleton was constructed by a 5-exo-trig

radical cyclization of an aryl radical onto a tethered

alkene, and its extension to the isomeric analogues

required C2 versus C4 regiocontrol in the key aromatic

halogenation step. In the synthesis of CI and CBI, a

regioselective electrophilic halogenation served to install

iodine or bromine para to the phenol ether. For the

isomeric agents, an ortho halogenation protocol was

required, and a cooperative directed ortho metalation²³

was implemented to install the C2 halide. Its adoption

required two easily removed cooperative directing meta-

lation groups: OMOM²⁴and NHBOC.²⁵In addition, the

Winstein para Ar-3′ spirocyclization,²⁶utilized to close

the cyclopropane in the CI and CBI synthesis, is now

replaced by an ortho spirocyclization requiring C-alkyl-

ation with cyclopropane formation²⁷rather than competi-

tive O-alkylation and dihydrofuran formation.

Sch em e 2

This approach was first examined with iso-CI employ-

ing the commercially available 3-nitrophenol (10), Scheme

2. Protection of the phenol as the MOM ether 11 (NaH,

MOMCl, Bu₄NI, 89%), reduction of the nitro group with

(20) Sundberg, R. J .; Nishiguchi, T. Tetrahedron Lett. 1983, 24, 4773.

Sundberg, R. J .; Baxter, E. W.; Pitts, W. J .; Ahmed-Schofield, R.;

Nishiguchi, T. J . Org. Chem., 1991, 56, 3048.

(21) Boger, D. L.; Wysocki, R. J ., J r.; Ishizaki, T. J . Am. Chem. Soc.

1990, 112, 5230. For related efforts, see: Sundberg, R. J .; Pitts, W. J .

J . Org. Chem. 1991, 56, 3048. Drost, K. J .; J ones, R. J .; Cava, M. P. J .

Org. Chem. 1989, 54, 5985. Tidwell, J . H.; Buchwald, S. L. J . Am.

Chem. Soc. 1994, 116, 11797. Tietze, L. F.; Grote, T. Chem. Ber. 1993,

126, 2733. Sakamoto, T.; Kondo, Y.; Uchiyama, M.; Yamanaka, H. J .

Chem. Soc., Perkin Trans. 1 1993, 1941. Wang, Y.; Lown, J . W.

Heterocycles 1993, 36, 1399.

(22) Boger, D. L.; McKie, J . A. J . Org. Chem. 1995, 60, 1271. Boger,

D. L.; Yun, W.; Teegarden, B. R. J . Org. Chem. 1992, 57, 2873. Boger,

D. L.; Ishizaki, T.; Wysocki, R. J .; 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.; Ishizaki, T.;

Zarrinmayeh, H.; Kitos, P. A.; Suntornwat, O. Bioorg. Med. Chem. Lett.

1991, 1, 55. Boger, D. L.; Ishizaki, T.; Sakya, S.; Munk, S. A.; Kitos,

P. A.; J in, Q.; Besterman, J . M. Bioorg. Med. Chem. Lett. 1991, 1, 115.

Boger, D. L.; Munk, S. A. J . Am. Chem. Soc. 1992, 114, 5487. Boger,

D. L.; Yun, W.; Han, N.; J ohnson, D. S. Bioorg. Med. Chem. 1995, 3,

611. See also: Boger, D. L.; McKie, J . A.; Cai, H.; Cacciari, B.; Baraldi,

P. G. J . Org. Chem. 1996, 61, 1710. Boger, D. L.; Han, N.; Tarby, C.

M.; Boyce, C. W.; Cai, H.; J in, Q.; Kitos, P. A. J . Org. Chem. 1996, 61,

4894. Drost, K. J .; Cava, M. P. J . Org. Chem. 1991, 56, 2240. Aristoff,

P. A.; J ohnson, P. D. J . Org. Chem. 1992, 57, 6234.

(23) Snieckus, V. Chem. Rev. (Washington, D.C.) 1990, 90, 879.

(24) Winkle, M. R.; Ronal, R. C. J . Org. Chem. 1982, 47, 2101.

(25) Muchkowski, J . M.; Venuti, M. C. J . Org. Chem. 1980, 45, 4798.

(26) Baird, R.; Winstein, S. J . Am. Chem. Soc. 1963, 85, 567; 1962,

84, 788; 1957, 79, 756.

(27) Brown, R. F. C.; Matthews, B. R.; Rae, I. D. Tetrahedron Lett.

1981, 22, 2915. Smith III, A. B.; Yokoyama, Y.; Huryn, D. M.; Dunlap,

N. K. Tetrahedron Lett. 1987, 28, 3659. Kigoshi, H.; Kitamura, Y.;

Fujita, T.; Ohashi, H.; Atsumi, T.; Takagi, J .; Mutou, T.; Yamada, K.;

Kusakabe, T.; Sasaki, D.; Sugiura, Y. Tetrahedron Lett. 1997, 38, 3235.

(28) Meyers, A. I.; Durandetta, J . L. J . Org. Chem. 1975, 40, 2021.

Al-Hg amalgam²⁸(Et₂O-H₂O, 89%), and BOC protection

of the free amine 12 (BOC₂O, >95%) provided 13, a key

intermediate with which to examine the directed ortho

metalation. Treatment of 13 with 3.5 equiv of n-BuLi

and TMEDA at -20 °C (2 h) in THF, and reaction of the

8878 J . Org. Chem., Vol. 62, No. 25, 1997

Boger et al.

Sch em e 3

aryllithium intermediate with 1-chloro-2-iodoethane pro-

vided 14 (46%) along with recovered starting material

(41%). Although not extensively examined, this conver-

sion was not improved through use of longer reaction

times, different reaction temperatures or solvents, or

additional amounts of n-BuLi. However, the conversion

did allow the synthesis to proceed, and, as detailed, this

reaction proved much more effective in the iso-CBI series

where it was more carefully optimized. N-Alkylation

with allyl bromide (NaH, >95%) was followed by Bu₃-

SnH-promoted 5-exo-trig free radical cyclization of 15

with in situ TEMPO trap²²of the resulting primary

radical to provide 16 (91%). Subsequent reduction of the

N-O bond (Zn, HOAc, 87%) without the competitive

removal of either the MOM ether or the N-BOC protect-

ing groups afforded the free alcohol 17 in excellent overall

yield. Activation of the primary alcohol (MsCl, Et₃N,

94%) afforded the key intermediate 18.²⁹

To prepare N-BOC-iso-CI for direct comparison with

prior agents, selective removal of the MOM group in the

presence of the BOC group was required. Mild acid-

catalyzed deprotection (HCl, i-PrOH/THF, 48%) provided

seco-N-BOC-iso-CI (19) accompanied by 41% recovery of

starting material. Although not optimized, this selective

deprotection found greater success in the synthesis of

N-BOC-iso-CBI where it was more closely examined.

Exhaustive deprotection of the MOM ether and the BOC

group (3.6 M HCl/EtOAc) followed by EDCI-promoted³⁰

coupling with 5,6,7-trimethoxyindole-2-carboxylic acid

(TMI-COOH)^10,31provided 20 (55% overall). Consistent

with expectations, spirocyclization of 19 to N-BOC-iso-

CI (21) could be effected by treatment with DBU, but its

exceptional reactivity precluded attempts to isolate and

characterize the agent (eq 1).³²Sufficient for our con-

siderations, the studies revealed that N-BOC-iso-CI is

more reactive than its counterpart N-BOC-CI which,

although exceptionally reactive, can be isolated by quick

column chromatography. Since the seco agents can be

expected to behave analogous to their ring-closed cyclo-

propane counterparts in biological assays and DNA

alkylation studies,⁴and given that the careful compari-

sons could be made with the more stable iso-CBI based

agents, their isolation and characterization were not

further pursued.

phenol (MOMCl, NaH, Bu₄NI, 78%) as the MOM ether

provided the directed ortho metalation substrate 23.

Treatment of 23 with 3.5 equiv of n-BuLi and TMEDA

at -25 °C in THF for 2 h and reaction of the aryllithium

intermediate with 1-chloro-2-iodoethane gave the C2

iodide 24 in 80% yield, a substantial improvement over

the iso-CI directed metalation. N-Alkylation (NaH, allyl

bromide, 94%), 5-exo-trig free radical cyclization of 25

with in situ TEMPO trap (Bu₃SnH, TEMPO, 94%), and

N-O bond reduction of 26 (Zn, HOAc, 87%) provided the

primary alcohol 27 in excellent overall yield. Conversion

to the chloride 28 upon activation of the primary alcohol

under Mitsunobu conditions (Ph₃P, CCl₄, 90%) provided

a resolvable intermediate that served as a penultimate

precursor to all analogues. After the initial completion

of this route, a report detailing the synthesis of duocar-

mycin SA analogues described a further improvement in

this general approach.³³A 5-exo-trig radical cyclization

onto a tethered vinyl chloride was shown to provide the

With the additional stability provided by the fused

benzene ring, the CBI analogues are substantially more

stable⁴and biologically more potent than the CI series.

The extension of this approach to the preparation of the

iso-CBI alkylation subunit is detailed in Scheme 3 and

provided the opportunity to accurately document the

effects of this deep-seated structural change. Starting

with 22, available in two steps (70%) from commercially

available 1,3-dihydroxynaphthalene,²²protection of the

(29) Full experimental details and characterization for 11-18 is

provided in the Supporting Information.

(30) EDCI ) 1-(3-(dimethylamino)propyl)-3-ethylcarbodiimide hy-

drochloride.

(31) Boger, D. L.; Ishizaki, T.; Zarrinmayeh, H.; Kitos, P. A.;

Suntornwat, O. J . Org. Chem. 1990, 55, 4499.

(32) Diagnostic ¹H NMR (CD₃CN, 400 MHz) signals for 21 generated

in situ: δ 2.79 (dt, J ) 5.4, 7.8 Hz, 1H), 1.75 (dd, J ) 3.1, 7.8 Hz, 1H).

Synthesis and Evaluation of CC-1065 and Duocarmycin Analogs

J . Org. Chem., Vol. 62, No. 25, 1997 8879

Sch em e 4

remained unchanged after 48 h. Thus, the handling and

storage of the iso-CBI based agents were conducted,

minimizing their exposure to light typically at subzero

temperatures. Examination of the compounds over time

showed no appreciable decomposition or rearrangement

under these storage conditions.

Resolu tion . To assess the properties of both enanti-

omers of the iso-CBI based agents, a direct chromato-

graphic resolution of 28 on a semipreparative ChiralCel

OD column (2 × 25 cm, 3% i-PrOH/hexane, R ) 1.24)

was utilized.³⁶This procedure provided both enantiomers

(>99% ee) of an advanced intermediate and avoided

diastereomeric derivatization, separation, and dederiva-

tization. The assignment of absolute configuration was

based on the conversion of the slower eluting enantiomer

of 28 (t_R) 35.8 min) to 32 and subsequent single-crystal

X-ray structure determination which revealed the un-

natural (3R)-configuration (Scheme 3).³⁷Consistent with

this assignment, the agents derived from the (3S)-

enantiomer analogous to the absolute stereochemistry

found in the natural products 1-3 exhibited the more

potent biological activity, the more effective DNA alkyl-

ation properties, and a DNA alkylation selectivity identi-

cal with the natural products.

five-membered ring with a suitable leaving group already

in place. This concise strategy was adopted for the

synthesis of iso-CBI (eq 2). Thus, N-alkylation of 25 with

1,3-dichloropropene proceeded in 96% yield, providing the

key radical cyclization precursor (29). Treatment with

catalytic AIBN (0.1 equiv) and Bu₃SnH (1.1 equiv) at 80

°C (C₆H₆) yielded the tricyclic core of iso-CBI (28) in 96%

yield. This improved approach shortens the original

synthesis by two steps, avoiding reductive removal of the

TEMPO group and conversion to 28. With this improve-

ment, the preparation of 28 requires four steps and

proceeds in 58% yield overall. Removal of the MOM

ether (HCl, i-PrOH/THF, 90%) without competitive N-

BOC deprotection afforded seco-N-BOC-iso-CBI (30) in

superb yield. Ortho spirocyclization (DBU, CH₃CN, 96%)

provided N-BOC-iso-CBI (31) which could be purified by

standard chromatography. Similarly, exhaustive depro-

tection of 28 (3.6 N HCl/EtOAc) followed by N-acylation

with methyl chloroformate provided 32 (65%), and spi-

rocyclization (DBU, CH₃CN, 25 °C, 91%) afforded 33.

Syn th esis of Du oca r m ycin a n d CC-1065 An a -

logu es. The iso-CBI alkylation subunit was incorporated

into CC-1065 and duocarmycin analogues as detailed in

Scheme 5. Exhaustive deprotection of 28 (3.6 M HCl/

EtOAc, 30 min) followed by immediate coupling (3 equiv

of EDCI, DMF, 25 °C) of the amine hydrochloride salt

with 5,6,7-trimethoxyindole-2-carboxylic acid^10,31(37, 3

h, 91%), 38¹⁶(3 h, 95%), 39 (3 h, 87%), indole₂(40, 3 h,

38

74%), CDPI₁³⁹(41, 9 h, 80%), and CDPI₂³⁹(42, 12 h, 32%)

provided 43, 45, 47, 49, 51, and 53, respectively. The

poor solubility of CDPI₂precluded efficient coupling and

isolation, resulting in a lower yield. DBU (1.5 equiv, 25

°C) spirocyclization of 43 (CH₃CN, 30 min, 94%), 45 (CH₃-

CN, 30 min, 85%), 47 (CH₃CN, 30 min, 83%), 49 (DMF,

30 min, 88%), 51 (DMF, 60 min, 81%), and 53 (DMF, 60

min, 59%) afforded 44, 46, 48, 50, 52, and 54, respec-

tively, in excellent conversions.

Solvolysis Rea ctivity a n d Regioselectivity. Two

fundamental characteristics of the alkylation subunits

have proven important in past studies.⁴The first is the

stereoelectronically controlled acid-catalyzed ring opening

of the activated cyclopropane which dictates preferential

addition of a nucleophile to the least substituted cyclo-

propane carbon. The second is the relative rate of acid-

catalyzed solvolysis which has been found to accurately

reflect the functional reactivity of the agents and to follow

a direct relationship between solvolysis stability and in

vitro cytotoxicity.⁴While N-BOC-iso-CBI retains many

of the structural characteristics of N-BOC-CBI, the

isomeric modifications which may disrupt the vinylogous

amide conjugation were anticipated to reduce the sol-

Initial attempts to synthesize iso-CBI (34) by exhaus-

tive deprotection (3.6 M HCl/EtOAc) and subsequent ring

closure (5% aqueous NaHCO₃/THF) conducted in the

presence of air resulted in the isolation of the quinone

35 (Scheme 4). Presumably, adventious oxidation of the

intermediate iso-CBI after spirocyclization provided 35

and an interesting further modification of the iso-CBI

alkylation subunit.³⁴Consistent with this, spirocycliza-

tion conducted in an aprotic solvent under an inert

atmosphere (2.2 equiv of DBU, CH₃CN, 25 °C, 20 min,

Ar) provided the unstable iso-CBI (34, 55%), and subse-

quent exposure of 34 to air resulted in conversion to 35.

Exclusive spirocyclization with cyclopropane formation

was observed, and no competitive O-alkylation leading

to 36 was detected. However, prolonged exposure of neat

31 to ambient light at 25 °C (48 h) did lead to carbonyl-

cyclopropane rearrangement³⁵to form 36 (eq 3). An

identical neat sample of 31 protected from light by foil

(33) Patel, V. F.; Andis, S. L.; Enkema, J . K.; J ohnson, D. A.;

Kennedy, J . H.; Mohamadi, F.; Schultz, R. M.; Soose, D. J .; Spees, M.

M. J . Org. Chem. 1997, 62, 8868.

(34) Such agents may be subject to reductive activation.

(35) Wong, H. N. C.; Hon, M.-Y.; Tse, C.-W.; Yip, Y.-C.; Tanko, J .;

Hudlicky, T. Chem. Rev. (Washington, D.C.) 1989, 89, 165. See also:

Schweizer, E. E.; Kopay, C. M. J . Org. Chem. 1971, 36, 1489.

Danishefsky, S.; Dynak, J . Tetrahedron Lett. 1975, 79. Saalfrank, R.

W.; Gu¨ndel, J .; Rossman, G.; Hanek, M.; Rost, W.; Peters, K.; von

Schnering, H. G. Chem. Ber. 1990, 123, 1175.

(36) Boger, D. L.; Yun, W. J . Am. Chem. Soc. 1994, 116, 7996.

(37) The atomic coordinates for this structure have been deposited

with the Cambridge Crystallographic Data Centre and may be obtained

upon request from the Director, Cambridge Crystallographic Data

Centre, 12 Union Road, Cambridge, CB2 1EZ, UK.

8880 J . Org. Chem., Vol. 62, No. 25, 1997

Boger et al.

Ta ble 1. Solvolysis Rea ctivity a n d Regioselectivity

Sch em e 5

Sch em e 6

volytic stability of the agents. However, it was not clear

what the magnitude of this effect might be nor whether

solvolysis would still occur with the same high regiose-

lectivity.

N-BOC-iso-CBI (31, t_1/2) 27.6 h, k ) 6.97 × 10^-6s^-1

)

and N-CO₂Me-iso-CBI (33, t_1/2) 30.1 h, k ) 6.40 × 10^-6

s^-1) proved to be reasonably stable toward chemical

solvolysis at pH 3, exhibiting a reactivity comparable to

the CC-1065 alkylation subunit (N-BOC-CPI, 55, t_1/2

)

36.7 h), but more stable than the duocarmycin A alkyl-

ation subunit (N-BOC-DA, 56, t_1/2) 11 h), Table 1.

However, N-BOC-iso-CBI was significantly less stable

than N-BOC-DSA (57, t_1/2) 177 h) and N-BOC-CBI (58,

t_1/2) 133 h). Thus, N-BOC-iso-CBI (31) proved to be 5×

more reactive than its direct comparison analogue N-

BOC-CBI. The solvolysis was followed spectrophoto-

metrically by UV with the disappearance of the long-

wavelength absorption band of the iso-CBI chromophore

(397 nm), Figure 3. The reactivity of the quinone 35

was also examined at pH 3 (t_1/2) 5.1 h, k ) 3.80 × 10^-5

s^-1), and it proved to be 5-6× more reactive than 31 or

33.

more selective than the natural alkylation subunits

themselves (6-1.5:1).^16,40,41

X-r a y Str u ctu r e of N-CO₂Me-iso-CBI (33): Str u c-

tu r a l Cor r ela tion w ith Solvolysis Regioselectivity

a n d Rea ctivity. The single-crystal X-ray structure

determination of N-CO₂Me-iso-CBI (33)³⁷was conducted

and by direct comparison with that of N-CO₂Me-CBI⁴²

established a structural basis for the observed properties

(Figure 4). The first striking similarity between the two

systems is the perpendicular orientation of the bent

orbital of the cyclopropane bond extending to the least

substituted C9 cyclopropane carbon. This idealized ste-

reoelectronic alignment with the developing π-system of

The acid-catalyzed nucleophilic addition of CH₃OH to

31 was conducted on a preparative scale to establish the

regioselectivity of addition and confirmed by synthesis

of the expected product 62 derived from nucleophilic

addition to the least substituted cyclopropane carbon.

Treatment of N-BOC-iso-CBI with 0.1 equiv of CF₃SO₃H

in CH₃OH (25 °C, 17 h) resulted in the clean solvolysis

(94%) to provide a 40:1 mixture of 62 and 63 (Scheme

6). Consequently, the acid-catalyzed CH₃OH addition to

31 occurs with near exclusive regioselectivity (40:1)

analogous to N-BOC-CBI (58, >20:1)²²which is much

(38) Boger, D. L.; Yun, W.; Han, N. Bioorg. Med. Chem. 1995, 3,

1429.

(39) Boger, D. L.; Coleman, R. S.; Invergo, B. J . J . Org. Chem. 1987,

52, 1521. Boger, D. L.; Coleman, R. S. J . Org. Chem. 1984, 49, 2240.

(40) Boger, D. L.; Goldberg, J .; McKie, J . A. Bioorg. Med. Chem. Lett.

1996, 6, 1955.

(41) Boger, D. L.; McKie, J . A.; Nishi, T.; Ogiku, T. J . Am. Chem.

Soc. 1997, 119, 311.

(42) Boger, D. L.; Turnbull, P. J . Org. Chem. 1997, 62, 5849.

Synthesis and Evaluation of CC-1065 and Duocarmycin Analogs

J . Org. Chem., Vol. 62, No. 25, 1997 8881

F igu r e 4. Stick models of the side view and 90° rotation view

of the activated cyclopropane of N-CO₂Me-iso-CBI and N-CO₂-

Me-CBI illustrating data taken from the X-ray crystal struc-

tures and highlighting the stereoelectronic and geometric

alignment of the cyclopropane with the cyclohexadienone

π-system.

Å) extending to the more substituted carbon. Although

the ring expansion solvolysis would place a developing

positive charge on a preferred secondary versus primary

carbon, this inherent preference is overridden by the

stereoelectronic control of the reaction regioselectivity as

well as the characteristics of a S_N2 reaction which prefer

attack at the less substituted center.

In addition, the X-ray structures have provided in-

sights into the origin of the difference in stability between

CBI and iso-CBI. The stability of CC-1065, the duocar-

mycins, and their analogues is a result of at least three

structural features: the conjugative stability provided by

the fused aromatic system, the nonideal alignment of the

cyclopropane, and the strong cross-conjugative stability

provided by the vinylogous amide. The first of these

features, the fused aromatic ring, is present in both iso-

CBI and CBI. Like the CI/CBI comparison, the iso-CI/

iso-CBI reactivity comparison reveals that the diminished

aromatization driving force for iso-CBI relative to iso-CI

contributes significantly to the stability. In addition, the

cyclopropane alignments with the π-systems in N-CO₂-

Me-iso-CBI and N-CO₂Me-CBI are similar. Both are

bisected by the plane of the cyclohexadienone nearly

equally (41°/15° for iso-CBI and 41°/16° for CBI). In both,

the cyclopropane is not only pulled down but also to the

side by the constraints of the fused five-membered ring

(9° for iso-CBI and 12° for CBI). Thus, both benefit in

F igu r e 3. Solvolysis study (UV spectra) of N-BOC-iso-CBI

(31, top), N-CO₂Me-iso-CBI (33, middle), and 35 (bottom) in

50% CH₃OH-aqueous buffer (pH 3.0, 4:1:20 (v/v/v) 0.1 M citric

acid, 0.2 M NaH₂PO₄, and H₂O, respectively). The spectra were

recorded at regular intervals, and only a few are shown for

clarity. Top: (hours) 0, 10, 20, 28, 56, 84. Middle: (hours) 0,

10, 21, 29, 57, 86. Bottom: (hours) 0, 1, 3, 5, 10, 16.

the solvolysis product phenol imposes a preference for

nucleophilic addition to the less substituted C9 cyclopro-

pane carbon. In contrast, the cyclopropane bond extend-

ing to the tertiary C9a carbon is nearly orthogonal to the

π-system of the cyclohexadienone, and S_N2 addition to

this carbon is disfavored stereoelectronically as well as

sterically. The relative cyclopropane bond lengths of iso-

CBI reflect this orientation and π conjugation in which

the breaking C9-C8a cyclopropane bond (1.531 Å) is

longer, and thus weaker, than the C9a-C8a bond (1.513

8882 J . Org. Chem., Vol. 62, No. 25, 1997

Boger et al.

Ta ble 2

agent

N²-C^2abond length (Å)

t_1/2(h, pH 3)

N-CO₂Me-CBI⁴²

N-CO₂Me-iso-CBI

N-BOC-CBQ⁴³

1.390

1.400

1.415

1.428

133

28

2.1

N-CO₂Me-CNA⁴²

0.03

stability from the nonideal alignment and conjugation

of the cyclopropane which is imposed by the fused five-

membered ring. The important distinction between the

two systems is the direct cross-conjugated stability

afforded the activated cyclopropane by the vinylogous

amide. Diagnostic of this vinylogous amide conjugation

is the shortened length of the N²-C^2abond, reflecting this

resonance stabilization. As this cross-conjugated viny-

logous amide stabilization decreases, the conjugation and

inherent reactivity of the cyclopropane correspondingly

increases.^7,16,42Consistent with this, N-CO₂Me-iso-CBI

exhibits a N²-C^2abond length (1.400 Å versus 1.426 Å

for 32) indicative of a diminished but not eliminated

vinylogous amide conjugation relative to N-CO₂Me-CBI

(1.390 Å versus 1.416 Å for seco N-CO₂Me-CBI)⁴²and

that follows trends established in recent studies (Table

2).⁴²

DNA Alk yla tion Selectivity a n d Efficien cy. The

DNA alkylation properties of the agents were examined

within w794 and w836 duplex DNA⁴⁴for which compara-

tive results are available for related agents. The alky-

lation site identification and the assessment of the

relative selectivity among the available sites were ob-

tained by thermally induced strand cleavage of the singly

5′ end-labeled duplex DNA after exposure to the agents.

Following treatment of the end-labeled duplex DNA with

a range of agent concentrations and temperatures in the

dark, the unbound agent was removed by EtOH precipi-

tation of the DNA. Redissolution of the DNA in aqueous

buffer, thermolysis (100 °C, 30 min) to induce strand

cleavage at the sites of DNA alkylation, denaturing high-

resolution polyacrylamide gel electrophoresis (PAGE)

adjacent to Sanger dideoxynucleotide sequencing stan-

dards, and autoradiography led to identification of the

DNA cleavage and alkylation sites. The full details of

this procedure have been disclosed elsewhere.⁴⁴

A representative comparison of the DNA alkylation by

(-)-iso-CBI-TMI (44) alongside that of (+)-duocarmycin

SA and (+)-CBI-TMI is illustrated in Figure 5. There

are three important conclusions that can be drawn from

these comparisons. First, (-)-iso-CBI-TMI alkylates

DNA in a manner identical with (+)-duocarmycin SA and

(+)-CBI-TMI, exhibiting the same sequence selectivity.

No new sites of alkylation were detected, and only

adenine N3 alkylation was detected under the conditions

of limiting agent and excess DNA. Notably, such se-

quencing studies only detect the higher affinity alkylation

sites and minor sites of comparable affinities (1-0.01×).

Under these conditions, the studies illustrate that iso-

CBI-TMI, like duocarmycin SA and CBI-TMI, exhibits

an exclusive preference for adenine versus guanine N3

alkylation. However, given the reactivity of iso-CBI-TMI,

it is likely that a minor guanine alkylation could be

expected at incubations carried out at higher agent-base

pair ratios analogous to that observed with the more

F igu r e 5. Thermally induced strand cleavage of w794 DNA

(SV40 DNA segment, 144 bp, nucleotide nos. 138-5238); DNA-

agent incubation for 24 h at 25 °C, removal of unbound agent

and 30 min of thermolysis (100 °C), followed by denaturing

8% PAGE and autoradiography; lane 1, control DNA; lanes

2-5, Sanger G, C, A, and T sequencing reactions; lanes 6-7

(-)-iso-CBI-TMI (1 × 10^-3and 1 × 10^-4M); lanes 8-9, (+)-

CBI-TMI (1 × 10^-5and 1 × 10^-6M); lanes 10-11, (+)-

duocarmycin SA (1 × 10^-5and 1 × 10^-6).

reactive agents including CC-1065⁴⁵and duocarmycin

A.⁴⁶Importantly, the identical behavior of (-)-iso-CBI-

TMI and (+)-CBI-TMI illustrate that the position of the

C-4 carbonyl does not influence the sequence selectivity

of the DNA alkylation. This is inconsistent with the

proposal of a sequence-dependent phosphate backbone

protonation of the C-4 carbonyl for activation of the agent

for DNA alkylation that controls the sequence selectiv-

ity.^6,8It is, however, fully consistent with the model in

which it is controlled by the AT-rich noncovalent binding

selectivity of the agents and their steric accessibility to

the adenine N3 alkylation sites.⁴

Second, although there are no distinctions in the

sequence selectivity, there is a significant difference in

the relative efficiencies of DNA alkylation. Consistent

with its relative reactivity and cytotoxic potency, (-)-iso-

CBI-TMI alkylated DNA 50-100× less efficiently than

(+)-CBI-TMI and (+)-duocarmycin SA. Thus, (-)-iso-

CBI-TMI was found to alkylate DNA with an efficiency

comparable to duocarmycin A which has been shown to

be ca. 10× less efficient than duocarmycin SA.^10,11In

(43) Boger, D. L.; Mesini, P. J . Am. Chem. Soc. 1994, 116, 11335.

Boger, D. L.; Mesini, P.; Tarby, C. M. J . Am. Chem. Soc. 1994, 116,

6461. Boger, D. L.; Mesini, P. J . Am. Chem. Soc. 1995, 117, 11647.

(44) Boger, D. L.; Munk, S. A.; Zarrinmayeh, H.; Ishizaki, T.;

Haught, J .; Bina, M. Tetrahedron 1991, 47, 2661.

(45) Park, H.-J .; Hurley, L. H. J . Am. Chem. Soc. 1997, 119, 629.

(46) Sugiyama, H.; Ohmori, K.; Chan, K. L.; Hosoda, M.; Asai, A.;

Saito, H.; Saito, I. Tetrahedron Lett. 1993, 34, 2179. Yamamoto, K.;

Sugiyama, H.; Kawanishi, S. Biochemistry 1993, 32, 1059. Asai, A.;

Nagamura, S.; Saito, H. J . Am. Chem. Soc. 1994, 116, 4171.

Synthesis and Evaluation of CC-1065 and Duocarmycin Analogs

J . Org. Chem., Vol. 62, No. 25, 1997 8883

F igu r e 6. Thermally induced strand cleavage of w836 DNA

(146 bp, nucleotide nos. 5189-91): DNA-agent incubation for

24 h ((-)-iso-CBI-TMI) at 25 °C, removal of unbound agent

and 30 min of thermolysis (100 °C), followed by denaturing

8% PAGE and autoradiography; lane 1, control DNA; lanes

2-5, Sanger G, C, A, and T sequencing reactions; lanes 6-8

(-)-iso-CBI-TMI (1 × 10^-3to 1 × 10^-5M); lanes 9-10, (+)-

duocarmycin SA (1 × 10^-5to 1 × 10^-6M); lanes 11-12, (+)-

iso-CBI-TMI (1 × 10^-2).

addition and analogous to the observations made with

the agents containing the more reactive alkylation sub-

units including duocarmycin A^10,21,43,46but unlike the

more stable agents,⁴the alkylation efficiency of (-)-iso-

CBI-TMI was found to increase as the incubation tem-

perature was decreased from 25 °C to 4 °C. This suggests

that the differences may be attributed in part to the

nonproductive solvolysis of iso-CBI-TMI which competes

with alkylation and lowers the overall efficiency of DNA

alkylation.

Third, although the rate of DNA alkylation by 44 was

not accurately quantitated, it is qualitatively similar to

those of CBI-TMI and duocarmycin SA and much faster

than agents we have examined which exhibited substan-

tially diminished rates (e.g. reversed analogues of duo-

carmycin SA).¹⁶Thus, the relocation of the C-4 carbonyl

did not impact the rate of DNA alkylation in a manner

that would be consistent with a phosphate backbone

protonation (or cation Lewis acid complexation) required

of catalysis in the alkylation site model.

F igu r e 7. Thermally induced strand cleavage of w794 DNA

(SV40 DNA segment, 144 bp, nucleotide nos. 138-5238); DNA-

agent incubation for 24 h at 25 °C, removal of unbound agent

and 30 min of thermolysis (100 °C), followed by denaturing

8% PAGE and autoradiography; lane 1, control DNA; lanes

2-5, Sanger G, C, A, and T sequencing reactions; lanes 6-8

(+)-duocarmycin SA (1 × 10^-4to 1 × 10^-6M); lanes 9-11, seco-

iso-CI-TMI (1 × 10^-3to 1 × 10^-5M).

illustrated elsewhere⁴and both enantiomers of iso-CBI-

TMI conform nicely to the models. Thus, the relocation

of the C-4 carbonyl from the outer face of a bound

complex potentially proximal to the phosphate backbone

to a position deep in the minor groove inaccessible to the

phosphates had no impact on the DNA alkylation selec-

tivity of either enantiomer.

Similar results were obtained within w836 DNA (Fig-

ure 6). The natural enantiomer (-)-iso-CBI-TMI alky-

lated the same sites as (+)-duocarmycin SA but did so

with a 50-100 fold lower efficiency. Within the segment

illustrated, the two natural enantiomers alkylated the

first three 3′ adenines in the sequence 5′-AAAAAA and

less effectively the fourth 3′ adenine corresponding to

alkylation and 3′f5′ binding across a 3-4 base-pair AT-

rich site (i.e., 5′-AAAA > 5′-CAAA). Like the unnatural

enantiomer of CBI-TMI, the unnatural enantiomer, (+)-

iso-CBI-TMI, alkylated DNA 50-100× less effectively

than the natural enantiomer and did so with a selectivity

identical with that of ent-(-)-duocarmycin SA. Within

w836, this constitutes the central adenines within the

sequence 5′-AAAAAA and corresponds to alkylation of

3-4 base-pair AT-rich sites with binding in the reverse

5′f3′ direction. Because of the diastereomeric nature of

the adducts, the unnatural enantiomer alkylates the

second 5′ base (adenine) within the sequence (i.e., 5-AAAA

> 5′-CAAAA). This has been discussed in detail and

In addition, the DNA alkylation properties of 20, seco

iso-CI-TMI, were examined alongside (+)-duocarmycin

SA, and a representative comparison is illustrated in

Figure 7 with w794 DNA. In past studies, such seco

agents have behaved in a manner indistinguishable from

their cyclopropane ring-closed counterparts exhibiting

identical DNA alkylation selectivities, efficiencies, and

cytotoxic activity, indicating that ring closure is not

limiting under the assay conditions.⁴Since we were not

able to isolate the ring closed iso-CI agents because of

their exceptional reactivity, the examination was con-

ducted with the seco precursor 20. Analogous to the

observations made with iso-CBI-TMI, 44 alkylated DNA

in a manner identical with duocarmycin SA alkylating

only adenine and exhibiting the same sequence selectiv-

ity. Although no new sites of alkylation were detected,

the relative selectivity among the available sites was

slightly lower with 20. This is illustrated nicely in Figure

7 where 20 alkylates the minor sites more prominently

than does (+)-duocarmycin SA. Interestingly and im-

8884 J . Org. Chem., Vol. 62, No. 25, 1997

Boger et al.

Ta ble 3. Ca lf Th ym u s DNA Alk yla tion

authentic materials. The preparative DNA alkylation

reaction and subsequent isolation of (+)-66 was carried

out under conditions determined to provide complete

consumption of the agent in the presence of a large excess

of DNA. Thus, extraction (EtOAc) of the aqueous buffer

solution containing calf thymus DNA following alkylation

(4 °C, 72 h, 150 bp) afforded no recovered (-)-44, and

only a small amount (<5%) of the rearranged product

65. Conducting the reaction at 4 °C in the presence of a

large excess of DNA precluded competitive solvolysis.

Thermal treatment of the alkylated DNA in aqueous 10

mM sodium phosphate buffer (100 °C, 30 min, pH 7.0)

followed by EtOAc extraction provided 66 in 90-95%

conversion, G95% purity (by HPLC). Repeating this

thermal treatment provided little or no additional adduct.

No trace of a competing guanine adduct could be detected.

This high conversion to a single adduct established that

44 participates exclusively in the adenine N3 alkylation

reaction under the conditions examined.

base-pair

conditions

equiv

66

65

44

solvolysis

4 °C, 72 h

4 °C, 120 h

75^a

150^a

150^b

94%

95%

92%

95%

<5%

nd

a

Analytical scale, HPLC separation and UV quantitation.

Preparative scale, isolation and weight quantitation. ^cnd ) not

b

detected.

portantly, 20 alkylated DNA only 10× less efficiently

than (+)-duocarmycin SA being far more effective than

the ring closed iso-CI-TMI might be projected to be. To

date, this observation is unique to the iso-CI series and,

as yet, has not been observed in the iso-CBI series or with

other analogues incorporating modified alkylation sub-

units. Whether this stems from the use of the mesylate

versus chloro seco precursor for 20 or is unique to the

iso-CI series remains to be established. However, it does

suggest that in selected instances the seco precursors

may exhibit productive properties that exceed those of

the corresponding cyclopropane ring-closed materials

especially with the more reactive agents. Unlike the

more stable agents which readily undergo cyclopropane

ring closure, the ring closure of 20 to iso-CI-TMI under

the conditions of the assay is unlikely. Rather, the DNA

alkylation most likely occurs directly with 20 without the

intermediate generation of the free cyclopropane agent.

Thus, not only did the relocation of the C-4 carbonyl not

alter the DNA alkylation selectivity, but its removal

altogether may be possible,¹⁴providing a class of agents

which, depending on the nature of the electrophile, also

exhibit comparable DNA alkylation selectivities, efficien-

cies, and rates. This is consistent with early observations

that related electrophiles that lack the capabilities for

ring closure to an activated cyclopropane exhibit DNA

alkylation selectivities identical with the corresponding

natural products.¹⁴

Full characterization of 66 unambiguously established

its structure, and the spectral characteristics showed

strong homology to the duocarmycin SA¹¹and A⁴⁷adenine

1

N3 adducts and N3 methyl adenine (Table 4). In the H

NMR, the C3-H of adduct 66 was observed as a single

proton (1H) at a characteristic chemical shift of 4.36-

4.37. The C3-H NMR signal for the benzylic center

resulting from alternative adenine N3 addition to the

more substituted cyclopropane carbon would be readily

distinguishable appearing as two protons (3.5-3.6 ppm,

2H) with a large geminal coupling constant (19.5 Hz).²

Additionally diagnostic of the structure were the chemical

shifts and coupling constants for C2-H₂and C4-H₂.

Characteristic of an adenine N3 alkylation product, the

adenine C2-H and C8-H were readily distinguishable.

The ¹H NMR of the protonated base was taken in DMSO/

1% TFA, and again a strong correlation to the spectral

data obtained for the duocarmycin SA adduct was

observed. The two adenine C6-NH₂protons were seen

as separate signals indicating restricted rotation about

6

the CdNH₂⁺bond as is present in protonated N-methyl

Qu a n tita tion , Isola tion , a n d Ch a r a cter iza tion of

th e (-)-iso-CBI-TMI Ad en in e Ad d u ct. The initial

alkylation studies established that (-)-iso-CBI-TMI al-

kylated adenine within the minor groove in a manner

identical with (+)-duocarmycin SA. The thermal cleav-

age of DNA used to identify the alkylation sites in these

studies only detects adducts susceptible to thermal

glycosidic bond cleavage (adenine N3, guanine N3, or

guanine N7 alkylation), and potential alkylation events

involving other nucleophilic centers in DNA may not be

detected in this assay. To confirm that (-)-iso-CBI-TMI

alkylates DNA in a manner identical with (+)-duocar-

mycin SA, and in efforts that established the relative

extent of adenine N3 versus alternative alkylation events,

the quantitation of the adenine N3 alkylation reaction

and confirmation of the structure of the product of the

reaction were established through isolation and charac-

terization of the thermally released adenine adduct.

This was addressed through a study of the alkylation

of calf thymus DNA. Optimized conditions for the alkyl-

ation were established for (-)-44 on an analytical scale

(100 µg of agent). For this purpose, the long-wavelength

UV absorption of the agent and the adduct provided a

useful quantitative measure of the adenine adduct, unre-

acted starting material, and any side products (solvolysis

or rearrangement), Table 3. Analytical HPLC analysis

was used to confirm the identity of the products through

correlation of retention times and UV spectral data with

adenine. In addition, a downfield shift as a result of

protonation of both the adenine C8-H and adenine C2-H

was also observed. The ¹³C NMR of 66 was also found

to be in excellent agreement with that of the duocarmycin

SA¹¹and A⁴⁷adducts. The key distinguishing signals are

found within or proximal to the fused five- versus six-

membered ring with 66 exhibiting chemical shifts con-

sistent only with the former, i.e. C3 at δ 39.7 consistent

with duocarmycin SA (δ 41.1) and inconsistent with the

six-membered ring in duocarmycin B₁/C₁(δ 33-34).

Thus, the adenine N3 addition to the least substituted

cyclopropane carbon of (-)-iso-CBI-TMI, as with (+)-

(47) Boger, D. L.; Ishizaki, T.; Zarrinmayeh, H. J . Am. Chem. Soc.

1991, 113, 6645.

Synthesis and Evaluation of CC-1065 and Duocarmycin Analogs

J . Org. Chem., Vol. 62, No. 25, 1997 8885

Ta ble 4. ¹H (400 MHz) a n d ¹³C NMR (150 MHz)

66, (-)-iso-CBI-TMI-adenine

(+)-duocarmycin SA-adenine

N3 adduct

N3-Me adenine

DMSO-d₆+

DMSO-d₆

+

DMSO-d₆

+

assignment

acetone-d₆

8.22

1% d-TFA

acetone-d₆

8.12

1% d-TFA

acetone-d₆

1% d-TFA

Ade-C8-H (1H, s)

Ade-C2-H (1H, s)

N3-CH₃(3H, s)

C6-NH₂(2H)

8.45

8.55

8.22

7.79

3.99

8.72

8.65

3.97

8.06

n/a

8.39

n/a

7.98

n/a

8.46

n/a

9.27 (br s)

8.78 (br s)

9.25 (br s)

8.94 (br s)

4.71 (dd, J )

13.6, 5.4 Hz)

4.60 (dd, J )

13.6, 8.0 Hz)

4.24

4.52 (br d, J )

11.0 Hz)

4.47 (dd, J )

11, 10 Hz)

9.20 (br s)

9.17 (br s)

C4-H (1H)

4.92 (dd, J )

10.8, 1.9 Hz)

4.73 (dd, J )

10.8, 8.5 Hz)

4.37

4.89 (dd, J )

14.5, 7.0 Hz)

4.73 (dd, J )

14.5, 7.6 Hz)

4.72 (dd, J )

10.8, 5.6 Hz)

4.68 (dd, J )

10.8, 5.6 Hz)

4.36

4.55 (dd, J )

9, 9 Hz)

4.54 (br d, J )

9 Hz)

4.89 (dd, J )

13.6, 4.9 Hz)

4.67 (dd, J )

13.6, 7.4 Hz)

4.55

4.82 (br d, J )

11.0 Hz)

4.61 (dd, J )

11.0, 8.6 Hz)

C3-H (1H, m)

C2-H (1H)

(CD₃OD)

152.9

151.1

120.8

156.8

146.0

36.9

Ade-C2

Ade-C4

Ade-C5

Ade-C6

Ade-C8

N3-CH₃

C2

152.2

151.0

123.0

156.9

143.9

n/a

153.0

149.9

122.7

155.6

146.4

n/a

56.4

53.9

C3

39.7

41.4

C4

57.5

58.9

Ta ble 5. In Vitr o Cytotoxic Activity

duocarmycin SA, was found to account for 90-100% of

the consumption of the agent in the presence of duplex

DNA and confirmed that it binds and alkylates DNA in

a manner identical with the natural products despite the

relocation of the C-4 carbonyl.

agent

configuration

IC₅₀(L1210)^a

31, (-)-N-BOC-iso-CBI

natural

unnatural

racemic

natural

unnatural

natural

unnatural

natural

unnatural

natural

unnatural

natural

5 µM

31, (+)-N-BOC-iso-CBI

34, (()-34

3 µM

12 µM

35, (()-35

15 µM

In Vitr o Cytotoxic Activity. Past studies with

agents in this class have defined a direct correlation

between solvolysis stability and cytotoxic potency.⁴Con-

sistent with their relative reactivity, the iso-CBI based

agents exhibited cytotoxic activity that closely followed

this relationship despite the deep-seated structural modi-

fication (Table 5, Figure 8).⁴The results, which also

follow trends established in the DNA alkylation studies,

demonstrate that the (-)-enantiomer of the analogues

possessing the (S)-configuration analogous to the natural

products is the more potent enantiomer by 10-50×. The

exception to this generalization is N-BOC-iso-CBI where

the two enantiomers were not readily distinguishable and

the unnatural enantiomer was consistently slightly more

potent (1-2×). The seco precursors, which lack the

preformed cyclopropane but possess the capabilities of

ring closure, were found to possess cytotoxic activity that

was indistinguishable from the final ring-closed agents.

Consistent with the unique importance of the C5 methoxy

group of the duocarmycins, iso-CBI-TMI (44) and 46 were

found to be equipotent, illustrating that the C6 and C7

methoxy groups of 44 are not contributing to its cytotoxic

potency. The cinnamate derivative 48 was found to be

substantially less potent (40-50×), suggestive of the

requirement for a rigid N²DNA binding subunit.⁷Fi-

44, (-)-iso-CBI-TMI

44, (+)-iso-CBI-TMI

46, (-)-46

2 nM

50 nM

2 nM (2 nM)

110 nM (60 nM)

90 nM (40 nM)

350 nM

3 nM (6 nM)

20 nM (60 nM)

1 nM (0.2 nM)

70 nM (6 nM)

0.05 nM (0.03 nM)

0.5 nM (0.5 nM)

46, (+)-46

48, (-)-48

48, (+)-48

50, (-)-iso-CBI-indole₂

50, (+)-iso-CBI-indole₂

52, (-)-iso-CBI-CDPI₁

52, (+)-iso-CBI-CDPI₁

54, (-)-iso-CBI-CDPI₂

54, (+)-iso-CBI-CDPI₂

unnatural

natural

unnatural

a

The value in parentheses corresponds to the IC₅₀value

determined for the corresponding seco precursor.

nally, the agents exhibited a smooth trend of increasing

cytotoxic potency as the size and length of the DNA

binding subunits increased with iso-CBI-CDPI₁and iso-

CBI-CDPI₂displaying the most potent cytotoxic activity

in the series exhibiting IC₅₀values of 200 and 50 pM,

respectively.⁴⁸

(48) While we were unable to isolate N-BOC-iso-CI (21) and iso-CI-

TMI for direct comparison, their seco precursors (()-19 and (()-20

exhibited surprisingly potent cytotoxic activity (IC₅₀, L1210 ) 10 µM

and 6 nM, respectively) that likely exceeds that of the ring closed

materials themselves.

8886 J . Org. Chem., Vol. 62, No. 25, 1997

Boger et al.

for the origin of the DNA alkylation selectivity with the

duocarmycins and CC-1065. These agents were prepared

by application of a directed ortho metalation and ortho

spirocyclization in an improved synthetic scheme comple-

mentary to that reported for CI and CBI. Iso-CBI was

found to be 5× less stable than CBI and possess a

reactivity comparable with CC-1065 and duocarmycin A.

In addition, nucleophilic addition occurred at the least

substituted cyclopropane carbon with a regioselectivity

(40:1) comparable to that of CBI but which exceeds that

of the natural products themselves (6-1.5:1). Compari-

son of the X-ray structures of iso-CBI and CBI revealed

the near identical nonideal conjugation of the cyclopro-

panes and the exclusive stereoelectronic alignment of the

cleaved cyclopropane bond. Consistent with recent ob-

servations, the lower stability of iso-CBI relative to CBI

itself can be attributed to a diminished cross-conjugated

vinylogous amide stabilization for which the N²-C^2abond

length is diagnostic.^7,42Resolution and incorporation of

iso-CBI into a full set of duocarmycin and CC-1065

analogues allowed for comparison of their properties and

a further distinguishing test for the origin of the DNA

alkylation sequence selectivity. The iso-CBI analogues

were highly potent cytotoxic agents exhibiting picomolar

IC₅₀’s which correlated with their relative stability. In

addition to smoothly following this correlation, the

analogues displayed a smooth trend of increasing cyto-

toxic potency with the increasing length in the DNA

binding subunit. Analogous to the natural products, the

(S)-enantiomer possessing the absolute configuration of

1-3 proved to be more potent (10-50×) than the (R)-

enantiomer. DNA alkylation studies revealed a strong

correlation between DNA alkylation efficiency and cyto-

toxic potency as (-)-iso-CBI-TMI was approximately 50-

100× less efficient than (+)-CBI-TMI and (+)-duocarmy-

cin SA. In addition, both the iso-CI and iso-CBI analogues,

with the repositioned C-4 carbonyl, exhibited the identi-

cal sequence selectivity and alkylated the same sites as

CBI-TMI and duocarmycin SA derived from adenine N3

addition to the least substituted cyclopropane carbon at

comparable reaction rates. This is inconsistent with a

proposal that the DNA alkylation selectivity is controlled

by a sequence-dependent DNA backbone phosphate pro-

tonation of the C-4 carbonyl for activation of alkylation

but is consistent with the noncovalent binding and steric

accessibility model. Finally, this set of isomeric ana-

logues contains the most significant structural modifica-

tion to the alkylation subunit to date, yet remain effective

DNA alkylating agents with properties comparable to the

natural products themselves.

Exp er im en ta l Section ²⁹

1-(ter t-Bu tyloxycar bon yl)-4-h ydr oxy-3[[(m eth an esu lfo-

n yl)oxy]m eth yl]-2,3-d ih yd r oin d ole (19). A solution of 18²⁹

(22 mg, 0.057 mmol) in 2 mL of 1:1 i-PrOH/THF was treated

with 12 N HCl (33 µL, 0.4 mmol) and stirred for 48 h at 25 °C.

The reaction solution was concentrated under reduced pres-

sure. Flash chromatography (SiO₂, 1.5 × 10 cm, 40% EtOAc/

hexane) afforded recovered 18 (8 mg, 41%) and 19 (9.5 mg,

48%) as a white film: ¹H NMR (CDCl₃, 400 MHz) δ 7.43 (m,

1H), 7.07 (m, 1H), 6.39 (d, J ) 8.6 Hz, 1H), 5.44 (br s, 1H),

4.55 (dd, J ) 4.5, 9.8 Hz, 1H), 4.26 (dd, J ) 8.1, 9.8 Hz, 1H),

4.00 (m, 2H), 3.79 (m, 1H); IR (film) ν_max3347, 2977, 1682,

1622, 1602, 1467, 1394, 1172, 1145, 948 cm^-1; FABHRMS

(NBA/CsI) m/z 476.0156 (C₁₅H₂₁NO₆S + Cs⁺requires 476.0144).

4-Hyd r oxy-3[[(m et h a n esu lfon yl)oxy]m et h yl]-1-[5,6,7-

tr im eth oxyin d ol-2-yl)ca r bon yl]-2,3-d ih yd r oin d ole (20). A

sample of 18²⁹(9.0 mg, 0.023 mmol) was dissolved in 3.6 N

F igu r e 8.

Con clu sion s. In summary, iso-CI and iso-CBI were

designed to test a key element of the different proposals

Synthesis and Evaluation of CC-1065 and Duocarmycin Analogs

J . Org. Chem., Vol. 62, No. 25, 1997 8887

HCl/EtOAc (2.4 mL), and the solution was stirred for 30 min

at 25 °C. The solvents were removed by a stream of N₂, and

the residual salt was thoroughly dried under high vacuum.

The salt was dissolved in anhydrous DMF (1.2 mL) and treated

with 37¹⁰(7 mg, 0.028 mmol) and EDCI (13 mg, 0.07 mmol).

The resulting solution was stirred at 25 °C for 3 h under Ar.

The reaction mixture was then diluted with H₂O (5 mL) and

extracted with EtOAc (3 × 5 mL). The combined organic layer

was dried (Na₂SO₄) and concentrated under reduced pressure.

Flash chromatography (SiO₂, 1.5 × 10 cm, 50% EtOAc/hexane)

yielded pure 20 (6.0 mg, 55%) as a white film: ¹H NMR (CDCl₃,

400 MHz) δ 9.33 (br s, 1H), 7.87 (d, J ) 8.0 Hz, 1H), 7.19 (m,

1H), 6.92 (d, J ) 2.4 Hz, 1H), 6.84 (s, 1H), 6.52 (dd, J ) 0.5,

8.0 Hz, 1H), 4.65 (dd, J ) 4.3, 10.1 Hz, 1H), 4.58 (dd, J ) 9.2,

10.8 Hz, 1H), 4.51 (dd, J ) 3.9, 10.8 Hz, 1H), 4.31 (dd, J )

8.4, 10.1 Hz, 1H), 4.05 (s, 3H), 4.01 (m, 1H), 3.92 (s, 3H), 3.89

(s, 3H), 2.95 (s, 3H); IR (film) ν_max3261, 2938, 1659, 1611, 1462,

1352, 1306, 1282, 1174, 1107 cm^-1; FABHRMS (NBA/CsI) m/z

609.0319 (C₂₂H₂₄N₂O₈S + Cs⁺requires 609.0308).

N-(ter t-Bu tyloxycar bon yl)-4-(m eth oxym eth oxy)-2-n aph -

th yla m in e (23). A solution of 22²²(6.67 g, 25.0 mmol) in 125

mL of anhydrous DMF at 0 °C was treated with NaH (1.13 g,

28.0 mmol) in several portions over 5 min. After 10 min, Bu₄-

NI (0.925 g, 2.5 mmol) was added followed by the dropwise

addition of ClCH₂OCH₃(2.9 mL, 38 mmol). The reaction

mixture was stirred at 25 °C for 5 h before the reaction was

quenched by the slow addition of 100 mL of H₂O. The aqueous

layer was extracted with EtOAc (3 × 100 mL). The organic

layers were combined, washed with 10% aqueous NaHCO₃

(100 mL) and H₂O (4 × 50 mL), dried (Na₂SO₄), and concen-

trated under reduced pressure. Flash chromatography (SiO₂,

4 × 15 cm, 0-15% EtOAc/hexane gradient) provided 23 (6.0

g, 78%) as a peach colored solid: mp 64-66 °C; ¹H NMR

(CDCl₃, 250 MHz) δ 8.15 (d, J ) 7.8 Hz, 1H), 7.67 (m, 2H),

7.38 (m, 2H), 7.05 (d, J ) 1.9 Hz, 1H), 6.87 (br s, 1H), 5.34 (s,

2H), 3.51 (s, 3H), 1.54 (s, 9H); ¹³C NMR (CDCl₃, 100 MHz) δ

153.3, 152.8, 136.0, 134.8, 127.0, 126.9, 123.7, 122.5, 121.6,

108.0, 101.8, 94.6, 80.5, 56.1, 28.2; IR (film) ν_max3334, 2977,

1713, 1634, 1538, 1392, 1367, 1248, 1160, 1057 cm^-1; FAB-

HRMS (NBA) m/z 303.1463 (C₁₇H₂₁NO₄requires 303.1471).

Anal. Calcd for C₁₇H₂₁NO₄: C, 67.31; H, 6.98; N, 4.62.

Found: C, 67.13; H, 7.18; N, 4.89.

N-(ter t-Bu tyloxycar bon yl)-3-iodo-4-(m eth oxym eth oxy)-

2-n a p h th yla m in e (24). A solution of 23 (0.435 g, 1.43 mmol)

in 5.7 mL of anhydrous THF was cooled to -25 °C and treated

with TMEDA (0.758 mL, 5.0 mmol) followed by n-BuLi (2.29

mL of a 2.5 M solution in hexane, 5.0 mmol) in a slow dropwise

manner. The resulting gold solution was stirred for 2 h at

-25 °C. The reaction mixture was treated with 1-chloro-2-

iodoethane (0.37 mL, 5.0 mmol) and stirred for 15 min at 25

°C. The reaction was diluted with H₂O (40 mL) and extracted

with Et₂O (3 × 20 mL), and the combined organic extracts were

washed with saturated aqueous NaCl, dried (Na₂SO₄), and

concentrated under reduced pressure. Flash chromatography

(SiO₂, 2.5 × 15 cm, 0-7% EtOAc/hexane gradient) yielded 24

(490 mg, 80%) as a yellow oil: ¹H NMR (CDCl₃, 400 MHz) δ

8.35 (s, 1H), 8.03 (d, J ) 12.8 Hz, 1H), 7.78 (d, J ) 12.5 Hz,

1H), 7.42 (m, 2H), 7.14 (br s, 1H), 5.20 (s, 2H), 3.74 (s, 3H),

1.56 (s, 9H);¹³C NMR (CDCl₃, 100 MHz) δ 154.7, 152.6, 135.1,

134.8, 127.6, 127.4, 125.1, 125.0, 122.2, 113.0, 100.5, 87.7, 81.1,

58.5, 28.4; IR (film) ν_max3391, 2977, 2933, 1732, 1524, 1367,

1340, 1276, 1228, 1157 cm^-1; FABHRMS (NBA/NaI) m/z

430.0507 (C₁₇H₂₀INO₄+ H⁺requires 430.0515).

Flash chromatography (SiO₂, 2.5 × 15 cm, 10% EtOAc/hexane)

yielded 25 (503 mg, 94%) as a colorless oil: ¹H NMR (CDCl₃,

250 MHz) δ 8.16 (m, 1H), 7.77 (m, 1H), 7.50 (m, 3H), 5.97 (m,

1H), 5.23 (m, 2H), 5.07 (m, 2H), 4.56 (m, 1H), 3.80 (m, 1H),

3.72 (s, 3H), 1.55 and 1.33 (s, 9H); ¹³C NMR (CDCl₃, 100 MHz)

δ 155.6 and 155.4, 154.1 and 153.9, 141.3 and 141.1, 133.8

and 133.5, 127.7 and 127.6, 127.1, 126.8, 125.2, 124.5, 122.4,

117.8 and 117.3, 100.6, 95.7, 80.6 and 80.3, 58.3, 53.6, 52.4,

28.3 and 28.2; IR (film) ν_max2975, 2930, 1703, 1581, 1566, 1385,

1366, 1159, 1047 cm^-1; FABHRMS (NBA/CsI) m/z 601.9825

(C₂₀H₂₄INO₄+ Cs⁺requires 601.9804).

1-(t er t -Bu t yloxyc a r b on yl)-4-(m e t h oxym e t h oxy)-3-

[[(2′,2′,6′,6′-tetr a m eth yl-p ip er id in o)oxy]m eth yl]-2,3-d ih y-

d r o-1H-ben zo[f]in d ole (26). A solution of 25 (470 mg, 1.00

mmol) and TEMPO (468 mg, 3.0 mmol) in 43 mL of anhydrous

benzene was treated with Bu₃SnH (0.283 mL, 1.05 mmol). The

solution was warmed at 50 °C, and an additional 1.05 equiv

of Bu₃SnH (0.283 mL, 1.05 mmol) was added twice during the

next 30 min. Another 3.0 equiv of TEMPO (468 mg, 3.0 mmol)

was added in 10 mL of anhydrous benzene, along with an

additional 1.05 equiv of Bu₃SnH added twice during the next

45 min. After 1.5 h total, the solution was cooled to 25 °C,

and the volatiles were removed under reduced pressure. Flash

chromatography (SiO₂, 2.5 × 15 cm, 0-12% EtOAc/hexane

gradient) provided 26 (470 mg, 94%) as a yellow oil: ¹H NMR

(CDCl₃, 250 MHz) δ 8.05 (br s, 1H), 7.97 (d, J ) 8.0 Hz, 1H),

7.74 (d, J ) 7.7 Hz, 1H), 7.36 (m, 2H), 5.24 (d, J ) 5.9 Hz,

1H), 5.16 (d, J ) 5.9 Hz, 1H), 4.26 (m, 1H), 4.15 (m, 1H), 4.01

(m, 1H), 3.81 (m, 2H), 3.63 (s, 3H), 1.61 (s, 9H), 1.23 (s, 3H),

1.17 (s, 3H), 1.04 (s, 3H), 1.04 (s, 3H), 0.99 (s, 3H), 1.48-0.89

(m, 6H); ¹³C NMR (CDCl₃, 62.5 MHz) δ 152.0, 149.3, 141.1,

135.3, 127.2, 125.7, 124.4, 123.4, 122.6, 121.0, 107.0, 99.1, 80.2,

76.0, 59.3, 57.1, 51.2, 39.1, 32.6, 27.9, 19.6, 16.6; IR (film) ν_max

2974, 2931, 1709, 1634, 1446, 1374, 1352, 1332, 1147 cm^-1

;

FABHRMS (NBA/CsI) m/z 631.2168 (C₂₉H₄₂N₂O₅+ Cs⁺re-

quires 631.2148).

1-(ter t-Bu tyloxyca r bon yl)-3-(h yd r oxym eth yl)-4-(m eth -

oxym eth oxy)-2,3-d ih yd r o-1H-ben zo[f]in d ole (27). A solu-

tion of 26 (220 mg, 0.44 mmol) in 15 mL of 3:1:1 HOAc/H₂O/

THF was treated with Zn powder (1.15 g, 17.6 mmol), and the

resulting suspension was warmed at 70 °C under a reflux

condenser and with vigorous stirring for 1 h. The reaction

mixture was cooled to 25 °C, and the Zn was removed by

filtration through Celite with a 25 mL CH₂Cl₂wash. The

volatiles were removed under reduced pressure, and the

resulting residue was dissolved in 25 mL of EtOAc and filtered.

The solution was concentrated under reduced pressure. Flash

chromatography (SiO₂, 2.5 × 10 cm, 30-40% EtOAc/hexane

gradient) provided 27 (138 mg, 87%) as a colorless oil: ¹H NMR

(CDCl₃, 250 MHz) δ 8.05 (br s, 1H), 7.91 (d, J ) 8.0 Hz, 1H),

7.72 (d, J ) 7.5 Hz, 1H), 7.36 (m, 2H), 5.23 (d, J ) 6.0 Hz,

1H), 5.16 (d, J ) 6.0 Hz, 1H), 4.10 (m, 1H), 3.94-3.78 (m, 4H),

3.63 (s, 3H), 2.04 (d, J ) 8.4 Hz, 1H), 1.58 (s, 9H); ¹³C NMR

(CDCl₃, 125 MHz) δ 152.5, 149.7, 142.0, 136.0, 127.9, 126.4,

124.6, 124.1, 123.1, 121.3, 107.8, 100.0, 81.1, 65.1, 57.8, 51.3,

40.7, 28.3; IR (film) ν_max3447, 2975, 1704, 1634, 1447, 1353,

1336, 1149 cm^-1; FABHRMS (NBA) m/z 360.1821 (C₂₀H₂₅NO₅

+ H⁺requires 360.1811).

1-(ter t-Bu t yloxyca r b on yl)-3-(ch lor om et h yl)-4-(m et h -

oxym eth oxy)-2,3-d ih yd r o-1H-ben zo[f]in d ole (28). F r om

27: A solution of 27 (55 mg, 0.16 mmol) in 3 mL of anhydrous

CH₂Cl₂was treated with CCl₄(155 µL, 1.6 mmol) and Ph₃P

(212 mg, 0.81 mmol), and the mixture was stirred at 25 °C for

2 h. The solution was concentrated in vacuo. Flash chroma-

tography (SiO₂, 1.5 × 15 cm, 0-12% EtOAc/hexane gradient)

provided 28 (52 mg, 90%) as a white solid: mp 99-101 °C; ¹H

NMR (CDCl₃, 250 MHz) δ 8.03 (br s, 1H), 7.89 (d, J ) 8.1 Hz,

1H), 7.72 (d, J ) 7.6 Hz, 1H), 7.37 (m, 2H), 5.24 (d, J ) 6.1

Hz, 1H), 5.15 (d, J ) 6.1 Hz, 1H), 4.06 (m, 4H), 3.65 (s, 3H),

3.51 (app t, J ) 10.1 Hz, 1H), 1.59 (s, 9H); ¹³C NMR (CDCl₃,

100 MHz) δ 152.3, 150.5, 141.1, 136.2, 127.9, 126.6, 124.6,

124.1, 122.4, 121.4, 107.7, 100.0, 81.2, 57.5, 52.1, 45.9, 40.8,

28.4; IR (film) ν_max2976, 1704, 1634, 1449, 1336, 1147, 1055,

2-[[N-(ter t-Bu tyloxycar bon yl)-N-(2-pr open -1-yl)]am in o]-

3-iod o-4-(m eth oxym eth oxy)n a p h th a len e (25). A solution

of 24 (0.490 g, 1.1 mmol) in 36 mL of anhydrous DMF was

cooled to -10 °C and treated with NaH (69 mg, 1.7 mmol) in

small portions. The resulting suspension was stirred 15 min

and treated with neat allyl bromide (0.49 mL, 5.7 mmol) in a

slow dropwise manner. The reaction mixture was warmed to

25 °C and stirred for 1 h. The reaction mixture was quenched

with the addition of 5% aqueous NaHCO₃(50 mL), and the

aqueous layer was extracted with EtOAc (3 × 20 mL). The

combined organic extracts were washed with H₂O (5 × 10 mL),

983 cm^-1; FABHRMS (NBA) m/z 378.1463 (C₂₀H₂₄Cl NO₄

⁺requires 378.1472). Anal. Calcd for C₂₀H₂₄INO₄: C, 63.75;

H, 6.40; N, 3.71. Found: C, 63.42; H, 6.11; N, 3.41.

+

dried (Na₂SO₄), and concentrated under reduced pressure to

yield 25 as a 2:1 mixture of amide rotamers as a yellow oil.

H

8888 J . Org. Chem., Vol. 62, No. 25, 1997

Boger et al.

F r om 29: A solution of 29 (500 mg, 1.0 mmol) in 10 mL of

anhydrous benzene was treated with AIBN (15.7 mg, 0.1

mmol) and Bu₃SnH (295 µL, 1.1 mmol) and warmed at 80 °C

for 2 h. The reaction mixture was concentrated under reduced

pressure, and flash chromatography (SiO₂, 2.5 × 15 cm, 0-20%

EtOAc/hexanes gradient) yielded 28 (360 mg, 96%) as a white

solid identical with that described above.

1635, 1472, 1410, 1324, 1143, 1009 cm^-1; UV (THF) λ_max(ꢀ)

387 (2700), 302 (6000 sh), 293 (6900), 250 (20400) nm;

FABHRMS (NBA/NaI) m/z 298.1450 (C₁₈H₁₉NO₃+ H⁺requires

298.1443). (-)-(8aS,9aS)-31: [R]²⁵-172 (c 0.08, CH₃OH);

D

(+)-(8aR,9aR)-31: [R]²⁵+179 (c 0.12, CH₃OH).

D

3-Ch lor o-4-h yd r oxy-1-(m eth oxyca r bon yl)-2,3-d ih yd r o-

1H-ben zo[f]in d ole (32). A solution of 28 (11 mg, 29.1 µmol)

was treated with 1.0 mL of 3.6 N HCl/EtOAc, and the resulting

solution was stirred for 30 min at 25 °C. The solvent was

removed by a stream of N₂, and the residual salt was dried

under vacuum. This salt was suspended in 0.6 mL of

anhydrous THF and treated with NaHCO₃(5.4 mg, 64.1 µmol,

2.2 equiv) and ClCO₂CH₃(4.5 µL, 58.2 µmol, 2.0 equiv) and

the mixture was stirred for 3 h at 25 °C. Upon completion,

the reaction mixture was diluted with 10 mL of H₂O, extracted

with EtOAc (3 × 10 mL), dried (Na₂SO₄), and concentrated in

vacuo. Flash chromatography (SiO₂, 1.5 × 15 cm, 0-40%

EtOAc/hexane gradient) provided pure 32 (5.5 mg, 65%) as a

white solid: ¹H NMR (CDCl₃, 400 MHz) δ 7.86 (d, J ) 8.2 Hz,

1H), 7.86 (br s, 1H), 7.73 (d, J ) 8.0 Hz, 1H), 7.42 (m, 1H),

7.35 (m, 1H), 5.80 (s, 1H), 4.11 (m, 2H), 4.01-3.87 (m, 5H),

3.63 (app t, J ) 9.4 Hz, 1H); IR (film) ν_max3379, 2956, 1673,

1435, 1372, 1283, 1218 cm^-1; FABHRMS (NBA/NaI) m/z

291.0656 (M⁺, C₁₅H₁₄ClNO₃requires 291.0662).

Resolu tion of 28. A sample of 28 (4.0 mg) in 0.9 mL of

5% i-PrOH/hexane was resolved on a semipreparative Daicel

Chiralcel OD column (10 µm, 2 × 25 cm, 7.0 mL/min flow rate,

3% i-PrOH/hexane). The effluent was monitored at 254 nm,

and the enantiomers were eluted with retention times of 29.0

and 35.8 min (R ) 1.25). The first enantiomer (S) was found

to be >99% enantiomerically pure. The second fraction (R)

was reinjected in order to obtain a sample of >99% ee. The

fractions containing the separated enantiomers were collected

and concentrated to afford (+) and (-)-28. (+)-(3S)-28: [R]²⁵

D

+29 (c 0.50, CH₂Cl₂); (-)-(3R)-28: [R]²⁵_D-30 (c 0.50, CH₂Cl₂).

2-[N-(ter t-Bu tyloxyca r bon yl)-N-(3-ch lor o-2-p r op en -1-

yl)a m in o]-3-iod o-4-(m eth oxym eth oxy)n a p h th a len e (29).

A solution of 24 (0.480 g, 1.1 mmol) in 11 mL anhydrous DMF

was cooled to 0 °C, and treated with NaH (67 mg, 2.2 mmol)

in small portions. The resulting suspension was stirred 15

min and treated with neat 1,3-dichloropropene (0.52 mL, 5.5

mmol) in a slow dropwise manner, followed by catalytic Bu₄-

NI (40 mg, 0.1 mmol). The reaction mixture was warmed to

25 °C and stirred for 12 h. The reaction mixture was quenched

with the addition of 5% aqueous NaHCO₃(50 mL), and the

aqueous layer was extracted with EtOAc (3 × 20 mL). The

combined organic extracts were washed with H₂O (5 × 10 mL),

dried (Na₂SO₄), and concentrated under reduced pressure to

yield 29 as a mixture of rotamers and E and Z alkenes as a

yellow oil. Flash chromatography (SiO₂, 2.5 × 15 cm, 0-20%

EtOAc/hexanes gradient) yielded 29 (540 mg, 96%) as a yellow

oil: ¹H NMR (CDCl₃, 250 MHz) δ 8.16 (m, 1H), 7.80 (m, 1H),

7.55 (m, 2H), 7.44 (s, 1H), 6.11 (m, 2H), 5.25 (d, J ) 5.6 Hz,

1H), 5.20 (d, J ) 5.6 Hz, 1H), 4.51 (m, 1H), 3.75 (m, 1H), 3.73

(s, 3H), 1.55 and 1.31 (s, 9H); ¹³C NMR (CDCl₃, 62.5 MHz) δ

156.2, 153.9, 140.7, 134.0, 128.7, 127.9, 127.7, 127.4, 127.1,

124.7, 122.5, 121.7, 100.7, 80.8, 77.2, 58.5, 49.5, 28.2; IR (film)

The structure and absolute configuration of (3R)-32 derived

from the slower eluting enantiomer of 28 (t_R) 35.8 min, [R]²⁵

D

-9 (c 0.10, CH₃OH)) was obtained from a single-crystal X-ray

structure determination conducted on prisms grown from

EtOAc/hexane.³⁷

2-(Meth oxycar bon yl)-1,2,9,9a-tetr ah ydr ocyclopr opa[c]-

ben zo[f]in d ol-8-on e (N-CO₂Me-iso-CBI, 33). A solution of

32 (5.0 mg, 0.017 mmol) in 0.5 mL of CH₃CN was treated with

DBU (3.9 µL, 0.025 mmol), and the mixture was stirred at 25

°C for 15 min. Flash chromatography (SiO₂, 1.5 × 10 cm,

0-40% EtOAc/hexane gradient) afforded 33 (4.0 mg, 91%) as

a pale yellow solid:¹H NMR (C₆D₆, 400 MHz) δ 8.33 (d, J )

6.0 Hz, 1H), 7.40 (br s, 1H), 7.10 (app t, J ) 6.0 Hz, 1H), 7.03

(d, J ) 6.0 Hz, 1H), 6.91 (m, 1H), 3.36 (s, 3H), 3.17 (m, 1H),

2.93 (m, 1H), 2.30 (dt, J ) 4.0, 6.0 Hz, 1H), 1.49 (dd, J ) 2.4,

6.4 Hz, 1H), 0.64 (dd, J ) 2.8, 4.4 Hz, 1H); IR (film) ν_max2954,

1731, 1633, 1445, 1323, 1196, 1018 cm^-1; UV (THF) λ_max(ꢀ)

386 (2300), 302 (4500 sh), 293 (5100), 250 (18000) nm;

FABHRMS (NBA/NaI) m/z 255.0901 (M⁺, C₁₅H₁₃NO₃requires

ν

_max2975, 1699, 1565, 1387, 1366, 1328, 1294, 1254, 1162 cm^-1

;

FABHRMS (NBA/NaI) m/z 504.0424 (C₂₀H₂₃CIINO₄+ H⁺

requires 504.0439).

255.0895). (+)-(8aR,9aR)-33: [R]²⁵+31 (c 0.07, CH₃OH).

1-(ter t-Bu tyloxyca r bon yl)-3-(ch lor om eth yl)-4-h yd r oxy-

2,3-d ih yd r o-1H-ben zo[f]in d ole (30). A solution of 28 (18

mg, 47.5 µmol) in 1.5 mL of 1:1 i-PrOH/THF was treated with

12 N HCl (0.20 mL, 0.38 mmol), and the mixture was stirred

at 25 °C for 6 h before the volatiles were removed in vacuo.

Flash chromatography (SiO₂, 1.5 × 15 cm, 0-20% EtOAc/

hexane gradient) provided 30 (14.5 mg, 90%) as a pale yellow

oil: ¹H NMR (CDCl₃, 250 MHz) δ 7.85 (d, J ) 8.0 Hz, 1H),

7.71 (d, J ) 7.8 Hz, 1H), 7.43-7.30 (m, 2H), 5.67 (s, 1H), 4.09-

D

The structure of 33 was confirmed with a single-crystal

X-ray structure determination conducted on plates grown from

EtOAc/hexane.³⁷

1,2,9,9a -Tetr a h yd r ocyclop r op a [c]ben zo[f]in d ol-8-on e

(iso-CBI, 34). A solution of 28 (10.6 mg, 28.1 µmol) was

treated with 3.6 M HCl/EtOAc and the mixture was stirred

at 25 °C for 30 min. The volatiles were removed by a stream

of N₂and the residual salt was suspended in 1.0 mL of

degassed CH₃CN. The suspension was treated with DBU (9.3

µL, 61.8 µmol, 2.2 equiv) and stirred under Ar for 20 min at

25 °C in the dark. Flash chromatography (SiO₂, 1.5 × 15 cm,

40% EtOAc/hexane) provided pure 34 (3.0 mg, 55%) as an

unstable bright yellow oil: ¹H NMR (C₆D₆, 400 MHz) δ 8.38

(d, J ) 8.0 Hz, 1H), 7.18 (m, 1H), 6.99 (d, J ) 8.0 Hz, 1H),

6.87 (m, 1H), 5.27 (s, 1H), 2.60 (m, 1H), 2.52 (m, 1H), 2.45 (m,

1H), 1.70 (m, 1H), 1.00 (m, 1H); IR (film) ν_max3358, 1682, 1594,

1470, 1284, 1262, 1223 cm^-1; FABHRMS (NBA/NaI) m/z

198.0926 (C₁₃H₁₁NO + H⁺requires 198.0919).

9,9a -1H -Dih yd r ocyclop r op a [c]b e n zo[f]in d ol-3,8-d i-

on e (35). A solution of 28 (10.6 mg, 28.1 µmol) was treated

with 4 M HCl/EtOAc, and the mixture was stirred at 25 °C

for 30 min. The volatiles were removed by a stream of N₂,

and the residual salt was suspended in 1.0 mL of THF. The

suspension was treated with 1.0 mL of 5% aqueous NaHCO₃

and stirred exposed to the air for 2 h at 25 °C. The bright

orange solution was extracted with EtOAc (3 × 5 mL), washed

with H₂O (2 × 5 mL), dried (Na₂SO₄), and concentrated in

vacuo. Flash chromatography (SiO₂, 1.5 × 15 cm, 60% EtOAc/

hexane) provided pure 35 (3.5 mg, 63%) as an orange film:

¹H NMR (C₆D₆, 400 MHz) δ 8.14 (m, 1H), 8.05 (m, 1H), 7.00

(m, 2H), 3.62 (dd, J ) 6.2, 19.4 Hz, 1H), 3.52 (ddt, J ) 0.6,

2.2, 19.4 Hz, 1H), 2.55 (ddt, J ) 2.2, 6.0, 8.3 Hz, 1H), 1.54

3.86 (m, 4H), 3.65 (dd, J ) 8.0, 10.0 Hz, 1H), 1.59 (s, 9H); ¹³

C

NMR (CDCl₃, 62.5 MHz) δ 147.9, 135.8, 132.7, 128.8, 128.0,

126.6, 123.7, 121.2, 119.5, 119.5, 104.4, 77.2, 52.6, 46.4, 34.7,

28.5; IR (film) ν_max3368, 2976, 1668, 1477, 1450, 1369, 1145,

978, 746 cm^-1; FABHRMS (NBA/CsI) m/z 466.0197 (C₁₈H₂₀

-

ClNO₃+ Cs⁺requires 466.0186). (-)-(3S)-30: [R]²⁵-54 (c

D

0.25, CH₃OH); (+)-(3R)-30: [R]²⁵+51 (c 0.30, CH₃OH).

D

2-(ter t-Bu tyloxyca r bon yl)-1,2,9,9a -tetr a h yd r ocyclopr o-

p a [c]ben zo[f]in d ol-8-on e (N-BOC-iso-CBI, 31). A solution

of 30 (5.0 mg, 0.015 mmol) in 0.6 mL of CH₃CN was treated

with DBU (2.7 µL, 0.018 mmol), and the mixture was stirred

at 25 °C for 15 min. Flash chromatography (SiO₂, 1.5 × 10

cm, 10% EtOAc/hexane) afforded 31 (4.3 mg, 96%) as a light

golden oil: ¹H NMR (CDCl₃, 400 MHz) δ 7.98 (d, J ) 7.8 Hz,

1H), 7.50 (dt, J ) 1.4, 7.8 Hz, 1H), 7.28 (d, J ) 7.8 Hz, 1H),

7.21 (dt, J ) 1.1, 7.8 Hz, 1H), 6.97 (br s, 1H), 3.88 (br d, J )

11.2 Hz, 1H), 3.81 (dd, J ) 4.9, 11.1 Hz, 1H), 2.88 (dt, J ) 5.5,

7.8 Hz), 1.87 (dd, J ) 3.2, 7.8 Hz, 1H), 1.53 (s, 9H), 1.41 (dd,

1

J ) 3.2, 5.5 Hz, 1H); H NMR (C₆D₆, 400 MHz) δ 8.34 (dd, J

) 0.6, 7.8 Hz, 1H), 7.10 (m, 2H), 6.92 (m, 1H), 3.18 (br m, 1H),

2.32 (m, 1H), 1.49 (dd, J ) 3.1, 7.8 Hz, 1H), 1.39 (s, 9H), 0.66

(dd, J ) 3.1, 5.4 Hz, 1H); ¹³C NMR (CDCl₃, 62.5 MHz) δ 194.7,

154.3, 142.0, 134.3, 127.7, 127.3, 125.9, 125.2, 103.2, 77.2, 51.5,

46.1, 32.0, 28.2, 25.7; IR (film) ν_max2973, 2927, 1716, 1670,

Synthesis and Evaluation of CC-1065 and Duocarmycin Analogs

J . Org. Chem., Vol. 62, No. 25, 1997 8889

(ddd, J ) 0.6, 3.3, 8.4 Hz, 1H), 0.30 (dd, J ) 3.3, 5.8 Hz, 1H);

¹³C NMR (CDCl₃, 100 MHz) δ 191.6, 182.0, 168.9, 135.8, 135.6,

135.0, 134.6, 128.4, 127.0, 63.8, 49.7, 35.0, 30.4; IR (film) ν_max

2923, 1692, 1680, 1585, 1361, 1262, 1223, 1082 cm^-1; UV (CH₃-

OH) λ_max(ꢀ) 271 (5200), 239 (13300) nm; FABHRMS (NBA/

NaI) m/z 212.0715 (C₁₃H₉NO₂+ H⁺requires 212.0712).

4-(ter t-Bu t yloxyca r b on yl)-1,2,2a ,3-t et r a h yd r ofu r a n o-

[4,3,2-c,d ]ben zo[f]in d ole (36). A neat sample of 31 (6.0 mg,

16 µmol) was allowed to sit under room light at 25 °C for 48 h

to yield 36 (6.0 mg, 100%): ¹H NMR (CD₃CN, 500 MHz) δ 7.80

(d, J ) 8.0 Hz, 1H), 7.67 (d, J ) 7.7 Hz, 1H), 7.35 (m, 1H),

7.29 (m, 1H), 7.05 (br s, 1H), 5.22 (dd, J ) 8.0, 8.5 Hz, 1H),

4.64 (dd, J ) 9.0, 11.0 Hz, 1H), 4.40 (dd, J ) 8.5, 10.5 Hz,

1H), 4.22 (m, 1H), 3.88 (m, 1H), 1.55 (br s, 9H); IR (film) ν_max

2-[(5-Meth oxyin d ol-2-yl)ca r bon yl]-1,2,9,9a -tetr a h yd r o-

cyclop r op a [c]ben zo[f]in d ol-8-on e (46). Flash chromatog-

raphy (SiO₂, 1.5 × 5 cm, 40% EtOAc/hexane) afforded 46 (85%)

as a light golden oil: ¹H NMR (C₆D₆, 400 MHz) δ 9.15 (s, 1H),

8.38 (m, 1H), 7.80 (s, 1H), 7.12-7.05 (m, 4H), 6.94 (ddd, J )

1.6, 6.8, 8.3 Hz, 1H), 6.85 (app dt, J ) 0.8, 8.6 Hz, 1H), 6.39

(d, J ) 1.6 Hz, 1H), 3.51 (s, 3H), 3.48 (d, J ) 10.0 Hz, 1H),

3.21 (dd, J ) 5.2, 10.0 Hz, 1H), 2.42 (dt, J ) 5.3, 7.8 Hz, 1H),

1.56 (dd, J ) 3.3, 7.8 Hz, 1H), 0.68 (dd, J ) 3.3, 5.3 Hz, 1H);

IR (film) ν_max3303, 2927, 1667, 1643, 1597, 1518, 1408, 1277,

1030, 1013 cm^-1; FABHRMS (NBA/NaI) m/z 371.1388 (C₂₃H₁₈

-

N₂O₃+ H⁺requires 371.1396). (-)-(8aS,9aS)-46: [R]²⁵-66

D

(c 0.04, CH₃OH); (+)-(8aR,9aR)-46: [R]²⁵+71 (c 0.03, CH₃-

D

OH).

2975, 2932, 1699, 1475, 1456, 1418, 1353, 1166, 1132 cm^-1

;

3-(C h lor o m e t h y l)-4-h y d r oxy-1-[((E )-3-(2-m e t h o x y -

p h en yl)p r op en yl)ca r b on yl]-2,3-d ih yd r o-1H -b en zo[f]in -

d ole (47). Flash chromatography (SiO₂, 1.5 × 10 cm, 0-40%

EtOAc/hexane gradient) afforded 47 (87%) as a pale yellow

solid: ¹H NMR (CDCl₃, 400 MHz) δ 8.40 (br s, 1H), 7.87 (m,

3H), 7.40 (m, 2H), 7.31 (m, 1H), 7.22 (m, 1H), 7.20 (s, 1H),

6.93 (m, 2H), 5.81 (m, 1H), 4.36 (m, 2H), 4.09 (m, 2H), 3.85 (s,

3H), 3.65 (m, 1H); IR (film) ν_max3266, 2963, 1659, 1598, 1445,

1377, 1269, 1158, 1048 cm^-1; FABHRMS (NBA/NaI) m/z

394.1217 (C₂₅H₂₀ClNO₃+ H⁺requires 394.1210). (-)-(3S)-

47: [R]²⁵_D-18 (c 0.15, CH₃OH); (+)-(3R)-47: [R]²⁵_D+22 (c 0.04,

CH₃OH).

FABHRMS (NBA/NaI) m/z 297.1451 (M⁺, C₁₈H₁₉NO₃requires

297.1365).

3-(Ch lor om eth yl)-4-h yd r oxy-1-[(5,6,7-tr im eth oxyin d ol-

2-yl)ca r b on yl]-2,3-d ih yd r o-1H -b en zo[f]in d ole (43).

A

sample of 28 (8.4 mg, 0.022 mmol) was treated with 1.0 mL of

3.6 N HCl/EtOAc, and the resulting solution was stirred for

30 min at 25 °C. The solvent was removed by a stream of N₂,

and the residual salt was dried under vacuum. This salt was

dissolved in 0.9 mL of anhydrous DMF and treated with 5,6,7-

trimethoxyindole-2-carboxylic acid¹⁰(6.8 mg, 0.026 mmol) and

EDCI (12.7 mg, 0.067 mmol), and the mixture was stirred for

3 h at 25 °C. Upon completion, the reaction mixture was

diluted with 20 mL of EtOAc, washed with H₂O (5 × 3 mL),

dried (Na₂SO₄), and concentrated in vacuo. Flash chromatog-

raphy (SiO₂, 1.5 × 15 cm, 0-40% EtOAc/hexane gradient)

provided 43 (11.2 mg, 91%) as a white solid: ¹H NMR (CDCl₃,

400 MHz) δ 9.41 (s, 1H), 8.35 (s, 1H), 7.88 (d, J ) 8.4 Hz, 1H),

7.82 (d, J ) 7.8 Hz, 1H), 7.45 (m, 1H), 7.41 (m, 1H), 6.96 (d, J

) 2.3 Hz, 1H), 6.85 (s, 1H), 5.95 (br s, 1H), 4.58 (m, 2H), 4.07

(m, 2H), 4.07 (s, 3H), 3.94 (s, 3H), 3.90 (s, 3H), 3.64 (dd, J )

10.3, 12.0 Hz, 1H); ¹³C NMR (acetone-d₆, 400 MHz) δ 193.6,

161.1, 151.0, 149.7, 143.4, 141.5, 136.7, 131.6, 128.8, 127.3,

126.5, 124.7, 124.5, 124.0, 121.9, 115.3, 107.6, 107.2, 99.0,

61.44, 61.37, 56.4, 54.9, 46.6, 41.7; IR (film) ν_max3405, 3301,

2937, 1594, 1446, 1312, 1228, 1106 cm^-1; FABHRMS (NBA/

CsI) m/z 467.1358 (C₂₅H₂₃ClN₂O₅+ H⁺requires 467.1374).

(-)-(3S)-43: [R]²⁵-4 (c 0.22, CHCl₃); (+)-(3R)-43: [R]²⁵+4

2-[((E)-3-(2-Meth oxyph en yl)pr open yl)car bon yl]-1,2,9,9a-

tetr a h yd r ocyclop r op a [c]ben zo[f]in d ol-8-on e (48). Flash

chromatography (SiO₂, 1.5 × 5 cm, 40% EtOAc/hexane) af-

forded 48 (83%) as a light golden oil: ¹H NMR (C₆D₆, 400 MHz)

rotamers δ 8.39 (d, J ) 7.5 Hz, 1H), 8.09 (d, J ) 15.4 Hz, 1H),

7.15-6.95 (m, 8H), 6.76 (ddd, J ) 1.0, 2.5, 8.1 Hz, 1H), 3.70-

3.50 (m, 1H), 3.31 (s, 3H), 3.00-2.80 (m, 1H), 2.35 (m, 1H),

1.63 (m, 1H), 0.73 (m, 1H); IR (film) ν_max2926, 1673, 1626,

1471, 1408, 1383, 1265, 1156, 1092, 1044, 1009 cm^-1; FAB-

HRMS (NBA/NaI) m/z 357.1360 (M⁺, C₂₅H₁₉NO₃requires

357.1365). (-)-(8aS,9aS)-48: [R]²⁵-21 (c 0.08, CH₃OH);

D

(+)-(8aR,9aR)-48: [R]²⁵+23 (c 0.03, CH₃OH).

D

3-(Ch lor om eth yl)-4-h yd r oxy-1-{[5-[N-(in d ol-2-yl)ca r bo-

n yl]a m in oin d ol-2-yl]ca r bon yl}-2,3-d ih yd r o-1H-ben zo[f]-

in d ole (49). Flash chromatography (SiO₂, 1.5 × 10 cm, 40-

80% EtOAc/hexane gradient) afforded 49 (74%) as a light

brown solid: ¹H NMR (DMF-d₇, 400 MHz) δ 11.76 (s, 2H),

10.30 (s, 1H), 10.28 (s, 1H), 8.41 (s, 1H), 8.29 (s, 1H), 8.25 (d,

J ) 8.0 Hz, 1H), 7.81 (d, J ) 8.4 Hz, 1H), 7.71 (dd, J ) 2.8,

9.2 Hz, 1H), 7.69 (d, J ) 9.6 Hz, 1H), 7.59 (d, J ) 8.6 Hz, 2H),

7.52 (s, 1H), 7.46 (m, 1H), 7.40 (m, 1H), 7.30 (d, J ) 1.6 Hz,

1H), 7.25 (m, 1H), 7.09 (m, 1H), 4.83 (m, 1H), 4.70 (dd, J )

2.4, 10.4 Hz, 1H), 4.30 (m, 1H), 4.16 (dd, J ) 2.8, 11.2 Hz,

1H), 3.98 (dd, J ) 8.8, 11.2 Hz, 1H); IR (film) ν_max3280, 2929,

1660, 1650, 1594, 1519, 1443, 1389, 1314, 1250, 1098, 749

D

(c 0.35, CHCl₃).

2-[(5,6,7-Tr im et h oxyin d ol-2-yl)ca r b on yl]-1,2,9,9a -t et -

r a h yd r ocyclop r op a [c]ben zo[f]in d ol-8-on e (iso-CBI-TMI,

44). A solution of 43 (5.7 mg, 0.012 mmol) in 0.5 mL of CH₃-

CN was treated with DBU (2.2 µL, 0.015 mmol) and stirred

at 25 °C for 15 min. Flash chromatography (SiO₂, 1.5 × 10

cm, 50% EtOAc/hexane) provided pure 44 (4.2 mg, 94%) as a

light golden oil: ¹H NMR (C₆D₆, 400 MHz) δ 9.65 (s, 1H), 8.36

(m, 1H), 7.76 (s, 1H), 7.10 (m, 1H), 7.03 (m, 1H), 6.94 (m, 1H),

6.77 (s, 1H), 6.45 (d, J ) 2.2 Hz, 1H), 3.78 (s, 3H), 3.70 (s,

3H), 3.54 (s, 3H), 3.52 (d, J ) 9.9 Hz, 1H), 3.26 (dd, J ) 5.2,

9.9 Hz, 1H), 2.43 (m, 1H), 1.57 (dd, J ) 3.3, 7.8 Hz, 1H), 0.70

(dd, J ) 3.3, 5.4 Hz, 1H); ¹³C NMR (acetone-d₆, 400 MHz) δ

194.5, 191.8, 161.7, 151.4, 147.1, 143.1, 142.0, 140.4, 135.6,

131.3, 129.4, 128.9, 127.3, 126.9, 126.7, 125.0, 112.7, 108.1,

107.6, 99.3, 63.7, 63.6, 57.0, 54.3, 40.6; IR (film) ν_max3295,

2934, 1667, 1651, 1409, 1306, 1279, 1109 cm^-1; UV (CH₃OH)

cm^-1; FABHRMS (NBA/CsI) m/z 667.0529 (C₃₁H₂₃ClN₄O₃

+

Cs⁺requires 667.0513). (-)-(3S)-49: [R]²⁵-18 (c 0.10, CH₃-

D

OH); (+)-(3R)-49: [R]²⁵+21 (c 0.06, CH₃OH).

D

2-{[5-[[N-(in d ol-2-yl)ca r bon yl]a m in o]in d ol-2-yl]ca r bo-

n yl}-1,2,9,9a -t et r a h yd r ocyclop r op a [c]b en zo[f]in d ol-8-

on e (iso-CBI-in d ole₂, 50). Flash chromatography (SiO₂, 1.5

× 10 cm, 10% DMF/toluene) afforded 50 (88%) as a light golden

solid: ¹H NMR (DMF-d₇, 400 MHz) δ 11.75 (s, 1H), 11.72 (s,

1H), 10.28 (s, 1H), 8.37 (s, 1H), 7.96 (d, J ) 7.9 Hz, 1H), 7.73-

7.64 (m, 3H), 7.58 (t, J ) 8.1 Hz, 2H), 7.50 (m, 2H), 7.35 (m,

2H), 7.23 (m, 2H), 7.08 (t, J ) 7.3 Hz, 1H), 4.57 (m, 2H), 3.09

(dt, J ) 5.5, 7.7 Hz, 1H), 1.86 (dd, J ) 3.1, 7.7 Hz, 1H), 1.77

(dd, J ) 3.2, 5.5 Hz, 1H); IR (film) ν_max3289, 2927, 1668, 1652,

1557, 1520, 1409, 1313 cm^-1; FABHRMS (NBA/CsI) m/z

499.1752 (C₃₁H₂₂N₄O₃+ H⁺requires 499.1770). (-)-(8aS,9aS)-

50: [R]²⁵-21 (c 0.10, DMF); (+)-(8aR,9aR)-50: [R]²⁵+21 (c

λ

_max390 (ꢀ 7100), 332 (ꢀ 14100), 245 nm (ꢀ 19100); FABHRMS

(NBA/NaI) m/z 431.1615 (C₂₅H₂₂N₂O₅+ H⁺requires 431.1607).

(-)-(8aS,9aS)-44: [R]²⁵-41 (c 0.13, CH₃OH); (+)-(8aR,9aR)-

D

44: [R]²⁵+38 (c 0.08, CH₃OH).

D

3-(Ch lor om eth yl)-4-h yd r oxy-1-[(5-m eth oxyin d ol-2-yl)-

ca r bon yl]-2,3-d ih yd r o-1H-ben zo[f]in d ole (45). Flash chro-

matography (SiO₂, 1.5 × 10 cm, 0-40% EtOAc/hexane gradi-

ent) afforded pure 45 (95%) as a clear oil: ¹H NMR (CDCl₃,

400 MHz) δ 9.38 (br s, 1H), 8.35 (s, 1H), 7.93 (d, J ) 7.6 Hz,

1H), 7.82 (d, J ) 7.9 Hz, 1H), 7.41 (m, 2H), 7.36 (d, J ) 9.0

Hz, 1H), 7.11 (d, J ) 2.2 Hz, 1H), 7.01 (m, 2H), 6.43 (s, 1H),

4.63 (d, J ) 5.4 Hz, 2H), 4.11 (m, 2H), 3.86 (s, 3H), 3.64 (m,

1H); IR (film) ν_max3287, 2927, 1665, 1596, 1518, 1445, 1402,

1389, 1290, 1231, 1167 cm^-1; FABHRMS (NBA/NaI) m/z

D

0.06, DMF).

1-[(3-Ca r ba m oyl-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]-3-(ch lor om et h yl)-4-h yd r oxy-2,3-d ih yd r o-

1H-ben zo[f]in d ole (51). Flash chromatography (SiO₂, 1.5 ×

10 cm, 10-50% DMF/toluene gradient) afforded pure 51 (80%)

as a yellow solid: ¹H NMR (DMF-d₇, 400 MHz) δ 11.64 (s, 1H),

10.32 (s, 1H), 8.28 (s, 1H), 8.25 (d, J ) 8.4 Hz, 1H), 8.16 (d, J

) 8.9 Hz, 1H), 7.81 (d, J ) 8.1 Hz, 1H), 7.46 (m, 1H), 7.39 (m,

2H), 7.15 (d, J ) 1.7 Hz, 1H), 6.13 (s, 2H), 4.82 (m, 1H), 4.68

406.1071 (C₂₅H₁₉ClN₂O₃requires 406.1084). (-)-(3S)-45: [R]²⁵

D

-26 (c 0.07, CH₃OH); (+)-(3R)-45: [R]²⁵+25 (c 0.04, CH₃-

D

OH).

8890 J . Org. Chem., Vol. 62, No. 25, 1997

Boger et al.

(dd, J ) 2.7, 10.8 Hz, 1H), 4.30 (m, 1H), 4.15 (m, 3H), 3.97

(dd, J ) 8.3, 10.8 Hz, 1H), 3.39 (m, 2H); IR (film) ν_max3337,

2924, 1662, 1652, 1513, 1456, 1441, 1400, 1344, 1272 cm^-1

;

ESIMS m/z 461 (M + H⁺, C₂₅H₂₁ClN₄O₃requires 461).

(-)-(3S)-51: [R]²⁵_D-21 (c 0.18, DMF); (+)-(3R)-51: [R]²⁵_D+22

(c 0.13, DMF).

2-[(3-Ca r ba m oyl-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]-1,2,9,9a -t et r a h yd r ocyclop r op a [c]b en zo[f]-

in d ol-8-on e (iso-CBI-CDP I₁, 52). Flash chromatography

(SiO₂, 1.5 × 10 cm, 30-50% DMF/toluene gradient) afforded

52 (81%) as a brown solid: ¹H NMR (DMF-d₇, 400 MHz) δ

11.60 (s, 1H), 8.15 (d, J ) 8.8 Hz, 1H), 7.95 (d, J ) 7.6 Hz,

1H), 7.66 (t, J ) 7.4 Hz, 1H), 7.49 (d, J ) 7.8 Hz, 1H), 7.35

(m, 3H), 7.10 (s, 1H), 6.13 (s, 2H), 4.55 (m, 2H), 4.13 (t, J )

8.8 Hz, 2H), 3.36 (t, J ) 9.1 Hz, 2H), 3.09 (m, 1H), 1.85 (dd, J

) 2.9, 7.6 Hz, 1H), 1.76 (dd, J ) 3.3, 5.4 Hz, 1H); IR (film)

ν

_max3346, 2927, 1667, 1651, 1505, 1435, 1408, 1343, 1279, 1098

cm^-1; ESIMS m/z 425 (M + H⁺, C₂₅H₂₀N₄O₃requires 425).

(-)-(8aS,9aS)-52: [R]²⁵-13 (c 0.06, DMF); (+)-(8aR,9aR)-

D

52: [R]²⁵+12 (c 0.07, DMF).

D

1-{[3-[N-(3-Ca r ba m oyl-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 bon yl}-3-ch lor om eth yl-4-h yd r oxy-2,3-d ih yd r o-1H-

ben zo[f]in d ole (53). Flash chromatography (SiO₂, 1.5 × 15

cm, 10-50% DMF/toluene gradient) afforded pure 53 (32%)

as a red solid: ¹H NMR (DMF-d₇, 400 MHz) δ 11.85 (s, 1H),

11.54 (s, 1H), 10.33 (s, 1H), 8.39 (m, 1H), 8.30 (s, 1H), 8.26 (d,

J ) 8.4 Hz, 1H), 8.13 (d, J ) 8.9 Hz, 1H), 7.82 (d, J ) 8.0 Hz,

1H), 7.48 (m, 2H), 7.38 (m, 2H), 7.31 (s, 1H), 7.08 (s, 1H), 6.10

(s, 2H), 4.86 (m, 1H), 4.73 (m, 3H), 4.31 (m, 1H), 4.15 (m, 3H),

3.99 (dd, J ) 8.2, 10.7 Hz, 1H), 3.55 (m, 2H), 3.39 (m, 2H); IR

(film) ν_max3338, 2956, 2927, 1727, 1659, 1650, 1604, 1510,

1402, 1365, 1286 cm^-1; ESIMS m/z 645/647 (M + H⁺, C₃₆H₂₉

-

ClN₆O₄requires 645/647). (-)-(3S)-53: [R]²⁵-19 (c 0.10,

D

DMF); (+)-(3R)-53: [R]²⁵+21 (c 0.08, DMF).

D

2-{[3-[N-(3-Ca r ba m oyl-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 bon yl}-1,2,9,9a -t et r a h yd r ocyclop r op a [c]b en zo[f]-

in d ol-8-on e (iso-CBI-CDP I₂, 54). Flash chromatography

(SiO₂, 1.5 × 10 cm, 50% DMF/toluene) afforded 53 (59%) as a

brown solid: ¹H NMR (DMF-d₇, 400 MHz) δ 11.80 (s, 1H),

11.53 (s, 1H), 8.37 (m, 1H), 8.13 (d, J ) 8.9 Hz, 2H), 7.65 (m,

1H), 7.49 (d, J ) 7.8 Hz, 1H), 7.47 (d, J ) 9.1 Hz, 1H), 7.36

(m, 3H), 7.24 (d, J ) 1.5 Hz, 1H), 7.07 (s, 1H), 6.12 (s, 2H),

4.74 (t, J ) 8.4 Hz, 2H), 4.58 (m, 2H), 4.14 (t, J ) 8.9 Hz, 2H),

3.52 (m, 2H), 3.38 (t, J ) 8.8 Hz, 2H), 3.11 (m, 1H), 1.87 (dd,

J ) 3.1, 7.8 Hz, 1H), 1.78 (dd, J ) 3.1, 5.5 Hz, 1H); IR (film)

ν_max3293, 2926, 1665, 1612, 1581, 1503, 1431, 1409, 1363,

1344, 1278, 1185 cm^-1; ESIMS m/z 607 (M - H⁺, C₃₆H₂₈N₆O₄

requires 607). (-)-(8aS,9aS)-54: [R]²⁵-34 (c 0.05, DMF);

D

(+)-(8aR,9aR)-54: [R]²⁵+33 (c 0.03, DMF).

D

Solvolysis Regioselectivity: 1-(ter t-Bu tyloxycar bon yl)-

4-h yd r oxy-3-(m eth oxym eth yl)-2,3-d ih yd r o-1H-ben zo[f]-

in d ole (62). A solution of 31 (7.7 mg, 0.026 mmol) in 2.5 mL

of CH₃OH was cooled to 0 °C, and CF₃SO₃H in CH₃OH (311

µL, 0.01 N, 0.12 equiv) was added. After slowly warming to

25 °C over 17 h, the reaction was quenched by the addition of

NaHCO₃(2.1 mg), filtered through Celite, and concentrated

under reduced pressure. Flash chromatography (SiO₂, 1.5 ×

15 cm, 0-40% EtOAc/hexane gradient) yielded the major

isomer 62 (8.0 mg, 94%) as a white film: ¹H NMR (CDCl₃,

400 MHz) δ 9.05 (s, 1H), 8.12 (d, J ) 6.4 Hz, 1H), 7.80 (m,

1H), 7.65 (s, 1H), 7.37 (m, 1H), 7.29 (m, 1H), 4.17 (m, 1H),

3.83 (m, 1H), 3.77 (dd, J ) 2.8, 6.4 Hz, 1H), 3.60 (dd, J ) 6.8,

8.8 Hz, 1H), 3.54 (s, 3H), 3.49 (m, 1H), 1.56 (s, 9H); IR (film)

ν

_max3233, 2975, 1704, 1644, 1435, 1348, 1148, 1026, 956 cm^-1

;

FABHRMS (NBA/NaI) m/z 352.1535 (C₁₉H₂₃NO₄+ Na⁺re-

quires 352.1525).

F igu r e 9.

For the minor isomer 63 (<2% by ¹H NMR of the crude

reaction mixture): ¹H NMR (CDCl₂, 500 MHz) δ 8.10 (m, 1H),

7.95 (d, J ) 6.5 Hz, 1H), 7.73 (m, 1H), 7.38 (m, 1H), 7.33 (m,

1H), 7.24 (m, 1H), 4.13 (m, 1H), 3.98 (s, 3H), 3.92 (m, 1H),

3.81 (m, 2H), 3.50 (m, 1H), 1.57 (s, 9H); FABHRMS (NBA/

NaI) m/z 352.1534 (C₁₉H₂₃NO₄+ Na⁺requires 352.1525).

P r ep a r a tion of Au th en tic 62. A solution of 27 (17.3 mg,

0.05 mmol) in 1.6 mL of anhydrous DMF was treated with

NaH (4.3 mg, 0.14 mmol) in small portions, and the resulting

suspension was stirred for 15 min at 0 °C. Methyl iodide (18

µL, 0.29 mmol) was added neat and the resulting mixture

warmed to 25 °C over 2 h. The reaction mixture was quenched

with the addition of 5% aqueous NaHCO₃(10 mL), and the

aqueous layer was extracted EtOAc (3 × 5 mL). The combined

organic extracts were washed with H₂O (5 × 5 mL), dried (Na₂-

Synthesis and Evaluation of CC-1065 and Duocarmycin Analogs

J . Org. Chem., Vol. 62, No. 25, 1997 8891

SO₄), and concentrated under reduced pressure. The crude

mixture was subjected to flash chromatography (SiO₂, 1.5 ×

15 cm, 10% EtOAc-hexanes) to yield the intermediate methyl

ether 64 (14.0 mg, 78%) as a colorless oil. 64 was subjected

to the selective MOM deprotection reaction and purification

as detailed for 30 to yield the major solvolysis product 62,

identical in all aspects.

Solvolysis Rea ctivity. N-BOC-iso-CBI (31, 0.2 mg) was

dissolved in CH₃OH (1.5 mL) and mixed with pH 3 aqueous

buffer (1.5 mL). The buffer contained 4:1:20 (v/v/v) 0.1 M citric

acid, 0.2 M Na₂HPO₄, and H₂O, respectively. The solvolysis

solution was sealed and kept at 25 °C protected from light.

The UV spectrum was measured at regular intervals every 1

h during the first 24 h, every 4 during the next 72 h, and every

12 h for an additional week. The decrease in the long

wavelength absorption at 397 nm was monitored, Figure 3.

The solvolysis rate constant (k ) 6.98 × 10^-6s^-1) and the half-

life (t_1/2) 28 h) were calculated from data recorded at the long

wavelength from the least-squares treatment (r ) 0.99) of the

slope of the plot of time versus ln[(A_f- A_i)/(A_f- A)] (Figure

9).

raphy (SiO₂, 0.5 × 3 cm, 0-7% CH₃OH/CHCl₃gradient elution)

afforded (+)-66 as a pale yellow solid (1.25 mg, 1.31 mg

theoretical, 95%, 90-95% in four runs) contaminated with a

small impurity (<5% by HPLC). For 66: [R]²³+28 (c 0.06,

D

CH₃OH); R_f) 0.3 (SiO₂, 10% CH₃OH/CHCl₃); ¹H NMR

(acetone-d₆, 400 MHz) δ 10.37 (s, 1H, N1′-H), 8.58 (s, 1H,

ArOH), 8.31 (d, J ) 7.9 Hz, 1H, ArH), 8.22 (s, 1H, Ade-C8-

H), 8.07 (s, 1H, Ade-C2-H), 7.78 (d, J ) 7.9 Hz, 1H, ArH),

7.49 (br s, 1H, ArH), 7.45 (ddd, J ) 1.3, 6.8, 7.9 Hz, 1H, ArH),

7.38 (ddd, J ) 1.3, 6.8, 7.9 Hz, 1H, ArH), 7.10 (d, J ) 2.3 Hz,

1H, C3′-H), 6.93 (s, 1H, C5′-H), 4.92 (dd, J ) 10.6, 1.9 Hz, 1H,

CHH-Ade), 4.89 (dd, J ) 14.5, 7.0 Hz, 1H, CHHNCO), 4.74

(dd, J ) 14.5, 7.6 Hz, 1H, CHHNCO), 4.73 (dd, J ) 10.8, 2.2

Hz, 1H, CHH-Ade), 4.37 (m, 1H, CH₂CHCH₂), 4.04 (s, 3H,

OCH₃), 3.87 (s, 3H, OCH₃), 3.86 (s, 3H, OCH₃); ¹H NMR

(DMSO-d₆+ 1% d-TFA, 400 MHz) δ 11.40 (s, 1H, N1′-H), 9.28

(br s, 1H, NHH), 8.78 (br s, 1H, NHH), 8.45 (s, 1H, Ade-C8-

H), 8.40 (s, 1H, Ade-C2-H), 8.07 (d, J ) 7.2 Hz, 1H, Ar-H),

8.05 (br s, 1H, ArOH), 7.76 (d, J ) 7.2 Hz, 1H, ArH), 7.43 (dd,

J ) 6.4, 6.4 Hz, 1H, ArH), 7.35 (dd, J ) 6.4, 6.4 Hz, 1H, ArH),

6.95 (s, 1H, C3′-H), 6.90 (s, 1H, C5′-H), 4.72 (dd, J ) 10.8, 5.6

Hz, 1H, CHH-Ade), 4.68 (dd, J ) 10.8, 5.6 Hz, 1H, CHH-Ade),

4.55 (dd, J ) 9.0, 9.0 Hz, 1H, CHHNCO), 4.54 (br d, J ) 9.0

Hz, 1H, CHHNCO), 4.36 (m, 1H), 3.93 (s, 3H, OCH₃), 3.80 (s,

3H, OCH₃), 3.79 (s, 3H, OCH₃); ¹³C NMR (150 MHz, acetone-

d₆) δ 170.5, 161.0, 156.9, 152.2, 151.2, 151.0, 150.0, 143.9,

142.9, 141.4, 140.0, 136.7, 131.8, 128.4, 127.2, 126.4, 124.7,

124.6, 124.1, 123.0, 114.2, 107.3, 106.1, 98.9, 61.5, 61.4, 57.5,

56.5, 56.4, 39.8; IR (film) ν_max3251, 2911, 2850, 1684, 1647,

1458, 1312, 1200, 1024 cm^-1; UV (CH₃OH) λ_max331 (ꢀ 12400),

300 (ꢀ 12600), 240 nm (ꢀ 17700); FABHRMS (NBA) m/z

566.2074 (M + H⁺, C₃₀H₂₇N₇O₅requires 566.2083).

Similarly, N-CO₂Me-iso-CBI (33, 0.2 mg) was dissolved in

CH₃OH (1.5 mL) and mixed with pH 3 aqueous buffer (1.5 mL).

The solvolysis solution was sealed and kept at 25 °C protected

from light. The UV spectrum was measured at regular

intervals every 1 h during the first 24 h, every 4 h during the

next 72 h, and every 12 h for an additional week. The decrease

in the long wavelength absorption at 395 nm was monitored,

Figure 3. The solvolysis rate constant (k ) 6.40 × 10^-6s^-1

)

and the half-life (t_1/2) 30 h) were determined as detailed above

(r ) 0.99).

Similarly, 35 (0.2 mg) was dissolved in CH₃OH (1.5 mL) and

mixed with pH 3 aqueous buffer (1.5 mL). The solvolysis

solution was sealed and kept at 25 °C protected from light.

The UV spectrum was measured at regular intervals every 1

h during the first 12 h, every 2 h for the next 24 h, and every

4 h for an additional day. The decrease in the short wave-

length absorption at 239 nm was monitored, Figure 9. The

solvolysis rate constant (k ) 3.80 × 10^-5s^-1) and the half-life

(t_1/2) 5 h) were determined as detailed above (r ) 0.99).

Isola tion , Ch a r a cter iza tion , a n d Qu a n tita tion of th e

Th er m a lly Relea sed (-)-iso-CBI-TMI Ad en in e Ad d u ct 66.

(-)-iso-CBI-TMI (44, 1.0 mg, 2.32 µmol) in 500 µL of DMSO

was added to a solution of calf thymus DNA (Sigma, 220 mg,

ca. 150 bp) in 10 mM sodium phosphate buffer (13 mL, pH

7.0) in a 50 mL centrifuge tube. The mixture was cooled at 4

°C for 72 h and then extracted with EtOAc (3 × 10 mL) to

remove hydrolyzed, rearranged (65), or unreacted 44. UV and

HPLC assay of the EtOAc extracts revealed no unreacted 44

(0%) and <5% rearranged 65. The centrifuge tube containing

the aqueous DNA layer was sealed with Teflon tape and

warmed at 100 °C for 30 min. The resulting solution was

cooled to 25 °C and extracted with EtOAc (3 × 10 mL). The

combined organic layer was dried (Na₂SO₄) and concentrated

under reduced pressure to afford a yellow solid. Chromatog-

HPLC t_R(4 × 250 mm column, 1.0 mL/min, 35% CH₃CN/

0.05 N aqueous HCO₂NH₄) were 44 (21.6 min), 65 (39.0 min),

solvolysis product (24.0 min), and 66 (17.5 min).

Ack n ow led gm en t. We gratefully acknowledge the

financial support of the National Institutes of Health

(CA55276) and the Skaggs Institute for Chemical Biol-

ogy, and the award of an ACS Organic Division fellow-

ship sponsored by Zeneca Pharmaceutical Co. (1997-

1998, R.M.G.).

Su p p or tin g In for m a tion Ava ila ble: Experimental pro-

cedures and characterization for 11-18, experimental for the

DNA alkylation studies, tables of data employed to prepare

Figure 8, copies of ¹H NMR spectra for 12, 13, 15-20, 24-27,

29-36, 43-54, 62, 63, and 66, and details of the X-ray

structure determinations of 32 and 33 are provided (70 pages).

This material is contained in libraries on microfiche, im-

mediately follows this article in the microfilm version of the

journal, and can be ordered from the ACS; see any current

masthead page for ordering information.

J O971686P

Article Doi

Synthesis and evaluation of CC-1065 and duocarmycin analogues incorporating the Iso-CI and Iso-CBI alkylation subunits: Impact of relocation of the C-4 carbonyl

DOI: 10.1021/jo971686p

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