A Novel Class of CC-1065 and Duocarmycin Analogues Subject to Mitomycin-Related Reductive Activation

Article abstract of DOI:10.1021/jo991301y

A new class of DNA alkylating agents is described that incorporate the quinone of the mitomycins, which is thought to impart tumor cell selectivity as a result of preferential reduction and activation in hypoxic tumors, into the AT-selective binding framework of the duocarmycins capable of mitomycin-like reductive activation and duocarmycin-like spirocyclization and subsequent DNA alkylation. Consistent with this design, the quinone prodrugs fail to alkylate DNA unless reductively activated and then do so with an adenine N3 alkylation sequence selectivity identical to that of the duocarmycins. Additionally, the agents exhibit a selectivity toward DT-Diaphorase (NQO1)-containing versus DT-Diaphorase-deficient (resistant) tumor cell lines, and they were shown to be effective substrates for reduction by recombinant human DT-Diaphorase. As such, the agents constitute effective duocarmycin and CC-1065 analogues subject to reductive activation. In addition, the solvolysis pH rate dependence of a series of reactive spirocyclopropanes revealed a unique and inverted order of reactivity at pH 7 versus pH 3. This behavior and the structural features responsible for it are consistent with an acid-catalyzed reaction at pH 3, but a direct uncatalyzed S_N2 reaction at pH 7 that is not subject to acid catalysis.

Full text of DOI:10.1021/jo991301y

8350
J . Org. Chem. 1999, 64, 8350-8362
A Novel Cla ss of CC-1065 a n d Du oca r m ycin An a logu es Su bject to
Mitom ycin -Rela ted Red u ctive Activa tion
Dale L. Boger* and Robert M. Garbaccio
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 August 17, 1999
A new class of DNA alkylating agents is described that incorporate the quinone of the mitomycins,
which is thought to impart tumor cell selectivity as a result of preferential reduction and activation
in hypoxic tumors, into the AT-selective binding framework of the duocarmycins capable of
mitomycin-like reductive activation and duocarmycin-like spirocyclization and subsequent DNA
alkylation. Consistent with this design, the quinone prodrugs fail to alkylate DNA unless reductively
activated and then do so with an adenine N3 alkylation sequence selectivity identical to that of
the duocarmycins. Additionally, the agents exhibit a selectivity toward DT-Diaphorase (NQO1)-
containing versus DT-Diaphorase-deficient (resistant) tumor cell lines, and they were shown to be
effective substrates for reduction by recombinant human DT-Diaphorase. As such, the agents
constitute effective duocarmycin and CC-1065 analogues subject to reductive activation. In addition,
the solvolysis pH rate dependence of a series of reactive spirocyclopropanes revealed a unique and
inverted order of reactivity at pH 7 versus pH 3. This behavior and the structural features
responsible for it are consistent with an acid-catalyzed reaction at pH 3, but a direct uncatalyzed
SN2 reaction at pH 7 that is not subject to acid catalysis.
The duocarmycins and CC-1065 (1-3)1,2 and the mi-
tomycins (4 and 5)3 are two families of potent antitumor
antibiotics that derive their biological activity through
alkylation of DNA4 (Figure 1). Prior studies have shown
that CC-1065 and the duocarmycins tolerate and benefit
from structural modifications to the alkylation subunit
and that the resulting agents retain their ability to
participate in the characteristic sequence selective DNA
alkylation reaction.4,5 Such structural modifications and
the definition of their effects have served to advance the
understanding of the origin of sequence selectivity4,6 and
the catalysis7,8 of the DNA alkylation reaction by 1-3.
The recent observation that isomeric analogues of the
duocarmycins, iso-CBI-based agents 6,9 retained not only
(1) 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.
(2) 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.
(3) Tomasz, M. In Molecular Aspects of Anticancer Drug-DNA
Interactions; Neidle, S., Waring, M., Eds.; MacMillan: London, 1994;
Vol. 2, pp 312-349.
F igu r e 1.
the characteristic DNA alkylation selectivity of the
natural products but also comparable in vitro biological
activity, provided the basis for the preparation of syn-
thetic hybrids combining the DNA alkylation features of
the duocarmycins and the reductive activation of the
mitomycins described herein.10
One compelling feature of mitomycin C is its effective
use in the clinic, either as a single agent or in combina-
tion with other agents,11 and it is especially useful in the
(4) For mechanistic aspects see: Boger, D. L.; J ohnson, D. S. Angew.
Chem., Int. Ed. Engl. 1996, 35, 1439.
(5) For synthetic aspects see: Boger, D. L.; Boyce, C. W.; Garbaccio,
R. M.; Goldberg, J . A., Chem Rev. 1997, 97, 787.
(6) Sun, D.; Lin, C. H.; Hurley, L. H. Biochemistry 1993, 32, 4487
and references therein.
(7) Boger, D. L.; Garbaccio, R. M. Acc. Chem. Res., submitted. Boger,
D. L.; Garbaccio, R. M. Bioorg. Med. Chem. 1997, 5, 263.
(8) Warpehoski, M. A.; Hurley, L. H. Chem. Res. Toxicol. 1988, 1,
315.
(9) Boger, D. L.: Garbaccio, R. M.; J in, Q. J . Org. Chem. 1997, 62,
8875.
(10) Quyand, A., Skibo, E. B. J . Org. Chem. 1998, 63, 1893.
(11) Carter, S. K.; Crooke, S. T. Mitomycin C: Current Status and
New Developments; Academic Press: New York, 1979. Mitomycin C
in Cancer Chemotherapy Today; Taguchi, T., Aigner, K. R., Eds.
Selected Proceedings of the 15th International Cancer Congress,
Hamburg, Germany, Aug 16-22, 1990, Excerpta Med. 1991.
10.1021/jo991301y CCC: $18.00 © 1999 American Chemical Society
Published on Web 10/29/1999

A Novel Class of CC-1065 and Duocarmycin Analogues
J . Org. Chem., Vol. 64, No. 22, 1999 8351
Sch em e 1
F igu r e 2.
Sch em e 2
treatment of solid tumors. Following reductive activation,
mitomycin C is known to cross-link DNA through bifunc-
tional alkylation (Scheme 1),12 and it is believed that this
interstrand mitomycin/DNA adduct inhibits DNA repli-
cation, resulting in inhibition of cell division.13
The synthesis of analogues of both these families has
proven fruitful, yielding the current clinical mitomycin-
like candidates KW-2149,14 BMY-25067,15 and the duo-
carmycin-like candidate KW-2189.16 Herein, we report
the incorporation of the quinone of the mitomycins, which
is thought to impart tumor cell selectivity as a result of
preferential reduction in the hypoxic environment of
tumors, into the AT-selective binding framework of the
duocarmycins to form a new hybrid agent 7 (Figure 2)
capable of mitomycin-like reductive activation and duo-
carmycin-like DNA alkylation.
An activation cascade for such hybrid agents was
envisioned to involve bioreduction, followed by in-situ
spirocyclization unleashing a highly reactive DNA alky-
lating agent isomeric to the duocarmycins (Scheme 2).
Importantly, unlike many simple mitomycin analogues,
this agent is a mitosane which allows for spirocyclization
and prevents the mitomycin-like release of a basic
nitrogen lone pair promoting competitive elimination of
chlorine. In addition, the quinone not only imparts
potential tumor cell selectivity but also acts as a prodrug
increasing the stability of what is predicted to be a highly
reactive cyclopropylcyclohexadienone.
Syn th esis. The synthetic route to the hybrid analogue
7 relied upon a cooperative directed ortho-metalation to
install iodine at the more-hindered position on a substi-
tuted aryl precursor9 and a subsequent 5-exo-trig radical
cyclization onto a tethered vinyl chloride to complete
construction of the functionalized dihydroindole.17 For the
synthesis of the cyclopropane-containing hybrids, the
ortho-variant18 of the Winstein Ar-3′ spirocyclization19
could be expected to effect cyclopropane spirocyclization.
In turn, the quinone-based analogues were anticipated
to be accessible from a late stage intermediate using a
selective p-quinone oxidation protocol of a substituted 5,7-
dimethoxy-4-hydroxy-2,3-dihydroindole.
(12) Iyer, V. N.; Szybalski, W. Proc. Natl. Acad. Sci. U.S.A. 1963,
50, 355.
(13) Iyer, V. N.; Szybalski, W. Science 1964, 145, 55. Zwelling, L.
A.; Anderson, T.; Kohn, K. W. Cancer Res. 1979, 39, 365.
(14) Vyas, D. M.; Chiang, Y.; Benigni, D.; Rose, W. C.; Bradner, W.
T. In Recent Advances in Chemotherapy. Anticancer Section; Tshigami,
J ., Ed.; University of Tokyo Press: Tokyo, 1985; pp 4385-486.
(15) Kono, M.; Saitoh, Y.; Kasai, M.; Sato, A.; Shirahata, K.;
Morimoto, M.; Ashizawa, T. Chem. Pharm. Bull. 1989, 37, 1128-1130.
(16) Yasuzawa, T.; Muroi, K.; Ichimura, M.; Takahashi, I.; Ogawa,
T.; Takahashi, K.; Sano, H. Saitoh, Y. Chem. Pharm. Bull. 1995, 43,
378. Nagamura, S.; Asai, A.; Kanda, Y.; Kobayashi, E.; Gomi, K.; Saito,
H. Chem. Pharm. Bull. 1996, 44, 1723. Nagamura, S.; Kobayashi, E.;
Gomi, K.; Saito, H. Bioorg. Med. Chem. 1996, 4, 1379. Asai, A.;
Nagamura, S.; Kobayashi, E.; Gomi, K.; Saito, H. Bioorg. Med. Chem.
Lett. 1996, 6, 1215. Nagamura, S.; Kobayashi, E.; Gomi, K.; Saito, H.
Bioorg. Med. Chem. Lett. 1996, 6, 2147.
The approach began with 2,4-dimethoxy-3-methylphe-
nol (10), readily available from 2,6-dimethoxytoluene
through the protocol described by Kishi20 (Scheme 3).
Protection of the phenol as the MOM ether 11 (NaH,
MOMCl, Bu4NI, 88%), regiospecific nitration (Cu(NO3)2‚
(17) Boger, D. L.; Boyce, C. W.; Garbaccio, R. M.; Searcey, M.
Tetrahedron Lett. 1998, 39, 2227.
(18) Brown, R. F. C.; Matthews, B. R.; Rae, I. D. Tetrahedron Lett.
1981, 22, 2915. Smith, A. B., III; Yokoyama, Y.; Huryn, D. M.; Dunlap,
N. K. Tetrahedron Lett. 1987, 28, 3659.
(19) Baird, R.; Winstein, S. J . Am. Chem. Soc. 1963, 85, 567.

8352 J . Org. Chem., Vol. 64, No. 22, 1999
Boger and Garbaccio
Sch em e 3
Sch em e 4
without competitive N-BOC deprotection afforded the
seco agent in excellent yield. Ortho spirocyclization (DBU,
CH3CN, 88%) provided the first target N-BOC-19 which,
although reactive, could be purified by rapid chromatog-
raphy.22
For purposes related to establishing the stability of the
novel ortho spirocyclopropyl-cyclohexadienones and the
regioselectivity of nucleophilic addition, the correspond-
ing N-pivaloyl and N-methyl derivatives (N-PIV-19 and
N-Me-19) were also prepared (Scheme 3). The N-pivaloyl
derivative was prepared by a route analogous to that
outlined above23 or by BOC deprotection of 20 and
N-acylation with pivaloyl chloride. Similarly, N-methy-
lation was effected following BOC deprotection and
reductive amination (9 equiv of CH2O, 3 equiv of AcOH,
3 equiv of NaCNBH3) to provide 22.23 In each case,
spirocyclization effected by treatment with DBU afforded
the corresponding cyclopropylcyclohexadienones in su-
perb yield.
Resolu tion a n d Assign m en t of Absolu te Ster eo-
ch em istr y. To establish the properties of both enanti-
omers of the agents, a direct chromatographic resolution
of 17 on a semipreparative ChiralCel OD column (2 ×
25 cm, 1% i-PrOH/hexane, R ) 1.63) was utilized.24 This
procedure provided both enantiomers (>99% ee) of an
advanced intermediate and avoided diastereomeric de-
rivatization, separation, and dederivatization. The superb
separation of racemic 17 allowed 200 mg injections
suitable for our preparative efforts. In addition, the
carbomethoxy derivatives of these and related alkylation
subunits prove to be crystalline.25 Thus, the slower
eluting enantiomer (+)-17 was deprotected and acylated
with methyl chloroformate (90%) in a one-pot procedure
to yield 23, which was recrystallized from EtOAc/hexanes
(Scheme 4). Its absolute configuration was established
by a single-crystal X-ray structure determination which
revealed the natural (3S) configuration.26
2.5H2O, 88%), reduction of the nitro group with Al-Hg
amalgam21 (Et2O-H2O, 85%), and BOC protection of the
free amine 13 (BOC2O, 73%) provided 14, a key inter-
mediate on which to examine the directed ortho-meta-
lation for completion of the preparation of the hexasub-
stituted benzene. Treatment of 14 with 3.7 equiv of
n-BuLi and TMEDA at -25 °C (2 h) in THF and reaction
of the aryllithium intermediate with 1-chloro-2-iodo-
ethane provided 15 in 74% yield. N-Alkylation of 15 with
1,3-dichloropropene proceeded in 96% yield, providing the
key radical cyclization precursor 16. Treatment of 16 with
catalytic AIBN (0.1 equiv) and Bu3SnH (1.1 equiv) at 74
°C effected 5-exo-trig cyclization and provided the dihy-
droindole core of the hybrid analogue in 96% yield.17
Removal of the MOM ether (HCl, i-PrOH/THF, 88%)
Syn th esis of th e Mitom ycin -Rela ted A-Rin g. A key
challenge in the preparation of this set of agents was to
(22) Even neat at subzero temperatures, 19 was subject to slow
decomposition to a complex mixture.
(23) See Supporting Information.
(24) Boger, D. L.; Yun, W. J . Am. Chem. Soc. 1994, 116, 7996.
(25) Boger, D. L.; Turnbull, P. J . Org. Chem. 1997, 62, 5849.
(26) The atomic coordinates for this structure have been deposited
with the Cambridge Crystallographic Data Centre and may be obtained
upon request of the Director, Cambridge Crystallographic Data Centre,
12 Union Road, Cambridge, CB2 1EZ, UK.
(20) Kishi, Y. J . Nat. Prod. 1979, 42, 549. Nakatsubo, F.; Fukuyama,
T.; Cocuzza, A. J .; Kishi, Y. J . Am. Chem. Soc. 1977, 99, 8115.
Fukuyama, T.; Nakatsubo, F.; Cocuzza, A. J .; Kishi, Y. Tetrahedron
Lett., 1977, 49, 4295.
(21) Meyers, A. I.; Durandetta, J . L. J . Org. Chem. 1975, 40, 2021.

A Novel Class of CC-1065 and Duocarmycin Analogues
J . Org. Chem., Vol. 64, No. 22, 1999 8353
Ta ble 1
oxidant
solvent (25 °C)
DMF
1:1 CH3CN:H2O
1:1 CH3CN:H2O (5 °C)
20:1 acetone:H2O
CH2Cl2
24, %
25, %
F igu r e 3.
salcomine/O2
CAN
CAN
40
21
24
<5
55
60
58
86
85
<5
12
even with annelated quinones. In addition, the o-quinone
displayed a pronounced tendency, when compared to the
p-quinone, to form the [M + 2]+ peak during analysis by
DDQ
25
PhSeO2H
PhSeO2H
•ON(KSO3)2
•ON(KSO3)2
<10
mass spectroscopy. Generally, o-quinones display key 13
C
Ac2O
0
1:1 acetone/KH2PO4
1:1 CH2Cl2/KH2PO4
<10
25
carbonyl resonances at higher fields than p-quinones, as
well as a characteristic IR carbonyl stretch at a higher
wavenumber, but with the case of 24 (13C NMR 180.3
and 179.3 ppm, IR 1715 and 1651 cm-1) and 25 (13C NMR
181.6 and 175.4 ppm, IR 1728 and 1646 cm-1), the key
peaks were too close to be considered diagnostic. Con-
clusive evidence was established by 1D GOESY34 which
for 25 clearly indicated an NOE between the C-7 methoxy
and the BOC group at N-1 (Figure 3) which was not
observed for the p-quinone 24.
selectively access the p-quinone present in the mitomy-
cins. Recognizing the susceptibility of 18 to both base-
catalyzed cyclization and ensuing degradation and over-
oxidation to the indole, mild oxidants were examined
(Table 1). Bis(salicylidene)-ethyenediiminocobalt (II) (sal-
comine)27 provided the most selective oxidation to the
p-quinone 24 (DMF, 40%) of the reagents examined and
only trace amounts of the o-quinone 25 could be observed
in the NMR of the crude reaction products. To our
knowledge such an oxidative demethylation has not been
observed previously with the salcomine/O2 system. In
preliminary studies, CAN28 and DDQ29 were found to be
less successful even though they appeared to be the
reagents of choice for selective oxidative demethylation
in the synthesis of a number of similar mitosane ana-
logues.30 With 18, both oxidants provided the correspond-
ing o-quinone 25 as the major product in a 2.4:1 ratio to
the desired p-quinone. Fremy’s salt,31 well recognized for
its mild p-selective oxidations, provided the desired
quinone 24 as the major product in a 2:1 ratio to the
undesired o-quinone 25, but in lower yield (20%) and was
unsuccessful under the single phase (acetone-water)
conditions typically employed. The mild oxidant
PhSeO2H32 yielded a highly selective (9:1) and compli-
mentary oxidation to the o-quinone (86%). When this
oxidation was run in Ac2O, thus forming the mixed
selenic-anhydride (a known ortho-oxidant), none of the
p-quinone was formed.
Syn th esis of th e Du oca r m ycin -Mitom ycin Hybr id
An a logu es. The hybrid subunit 24 was incorporated in
to duocarmycin-like analogues. Exhaustive deprotection
of 17 (3.6 M HCl/EtOAc, 30 min) followed by immediate
coupling (3 equiv of EDCI,35 DMF, 25 °C) of the amine
hydrochloride salt with 5,6,7-trimethoxyindole-2-carboxy-
lic acid (26)36 produced the seco agent 27 (Scheme 5).37
DBU (1.3 equiv, 25 °C, 30 min, 82%) spirocyclization of
27 afforded the labile 28, containing the reactive cyclo-
propane representing a key comparison analogue.
The seco agent 27 was treated with PhSeO2H, and the
labile o-quinone analogue 29 was isolated in 80% yield
(Scheme 5). Treatment of 27 with salcomine/O2 in DMF
led to a complex mixture of products, probably arising
from competitive oxidation of the trimethoxyindole sub-
unit. Thus, an alternative pathway to the p-quinone was
developed utilizing 24. Reduction of 24 (H2, 10 wt % Pd/
C, CH3OH, 5 min, 100%, Scheme 6) provided the air-
stable hydroquinone 30 which was subsequently depro-
tected (3.6 M HCl/EtOAc, 30 min) and immediately
coupled with 26 (1.2 equiv, 3 equiv of EDCI, DMF, 25
°C) to give the suprisingly air-stable hydroquinone 31 in
44% yield. The hydroquinone was then easily oxidized
(air, 1.5 wt equiv Pd/C, EtOAc, 5 h, 100%)38 without
competitive oxidation of the indole binding subunit to
provide 7, the duocarmycin-mitomycin hybrid (Scheme
6).
Preliminary indications differentiating between the o-
and p-quinones were provided by the compound’s color
and their UV and mass spectrum. The o-quinone 25 was
deep red and the p-quinone 24 was bright orange
consistent with related observations.33 Also consistent
with this assignment, the o-quinone 25 absorbs at a
higher wavelength and with a lower extinction coefficient
(UV λmax (CH3OH) 332 nm ꢀ ) 6900) than the p-quinone
(UV λmax (CH3OH) 314 nm ꢀ ) 13600) as is generally seen
Solvolysis Rea ctivity a n d Regioselectivity. Two
fundamental characteristics of the alkylation subunits
have proven important in past studies.4 The first is the
stereoelectronically-controlled ring opening of the acti-
vated cyclopropane which dictates preferential addition
of a nucleophile to the least-substituted cyclopropane
carbon. The second is the relative rate of acid-catalyzed
(27) Van Dort, H. M.; Geursen, H. J . Recl. Trav. Chim. Pays-Bas
1967, 86, 520. Hibino, S.; Weinreb, S. M. J . Org. Chem. 1977, 42, 332.
Yoshida, K.; Nakajima, S.; Ohnuma, T.; Ban, Y.; Shibasaki, M. J . Org.
Chem. 1988, 53, 5355.
(28) CAN ) Ceric ammonium nitrate Ce(NH4)2(NO3)6.
(29) DDQ ) 2,3-Dichloro-5,6-dicyano-1,4-benzoquinone.
(30) Remers, W. A.; Iyengar, B. S. Synthesis of Mitomycins In Recent
Progress in the Chemical Synthesis of Antibiotics; Springer-Verlag:
Berlin, 1990; 415-445.
(34) Stonehouse, J .; Adell, P.; Keeler, J .; Shaka, A. J . J . Am. Chem.
Soc. 1994, 116, 6037.
(35) EDCI ) 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hy-
drochloride.
(36) Boger, D. L.; Ishizaki, T.; Zarrinmayeh, H.; Kitos, P. A.;
Suntornwat, O. J . Org. Chem. 1990, 55, 4499.
(31) Zimmer, H.; Lankin, D. C.; Horgan, S. W. Chem. Rev. 1971,
71, 229.
(32) Barton, D. H. R.; Finet, J .-P.; Thomas, M. Tetrahedron 1988,
44, 6397.
(33) Berger, S.; Rieker, A. In The Chemistry of the Quinonoid
Compounds; Patai, S., Ed.; Wiley: London, 1974; pp 163-229.
(37) The low yield is believed to arise from the propensity for the
seco agent to cyclize under the reaction conditions and then quickly
decompose during reaction and slow chromatography.
(38) Luly, J . R.; Rapoport, H. J . Org. Chem. 1984, 49, 1671.

8354 J . Org. Chem., Vol. 64, No. 22, 1999
Boger and Garbaccio
Sch em e 5
F igu r e 4.
Ta ble 2. Solvolysis Rea ctivity a n d Regioselectivity
Sch em e 6
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
biological activity.4 Although N-BOC-19 is highly reac-
tive, it was sufficiently stable to purify and characterize
and its solvolysis behavior may be considered representa-
tive of that expected of 9, 28, and related intermediates.
Its solvolysis at pH 3 and pH 7, as well as that for N-PIV-
19 and N-Me-19, were followed spectrophotometrically
by UV with the disappearance of the long-wavelength
absorption band (Figure 4).
The agents N-BOC-19 (t1/2 ) 95 s, k ) 7.28 × 10-3 s-1),
N-PIV-19 (t1/2 ) 108 s, k ) 6.41 × 10-3 s-1), and N-Me-
19 (t1/2 ) 77 s, k ) 9.00 × 10-3 s-1) proved to be slightly
more stable than N-BOC-CI (35, t1/2 ) 35 s, k ) 1.98 ×
10-2 s-1) and much more reactive than a related isomeric
system N-BOC-iso-CBI (6, t1/2 ) 28 h, k ) 6.98 × 10-6
s-1) (Table 2). From past studies,4 it is known that the
lack of a fused aromatic ring and the corresponding
increased aromatization energy is the source of the
substantial increase in reactivity relative to iso-CBI (6)
and the increased stability of 19 relative to the analogous
CI (3×) was a welcomed property. Interestingly, the order
of stability for N-BOC-19 (t1/2 ) 17 min, k ) 6.79 × 10-4

A Novel Class of CC-1065 and Duocarmycin Analogues
J . Org. Chem., Vol. 64, No. 22, 1999 8355
Sch em e 7
to the least-substituted carbon of the cyclopropane under
acidic conditions (Scheme 7). Consequently, the nucleo-
philic additions to N-BOC-19 and N-Me-19 occur with
exclusive regioselectivity (>20:1) analogous to N-BOC-
iso-CBI,9 which are much more selective than the natural
alkylation subunits themselves 32-34 (6-1.5:1).4
DNA Alk yla tion Selectivity a n d Efficien cy. The
DNA alkylation properties of the agents were examined
within w794 duplex DNA40 for which comparative results
are available for related duocarmycin-based agents. The
alkylation 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, the
unbound agent was removed by EtOH precipitation of
the DNA. Redissolution of the DNA in aqueous buffer,
thermolysis (100 °C, 30 min) to induce strand cleavage
at the sites of DNA alkylation, denaturing high-resolution
polyacrylamide gel electrophoesis (PAGE) adjacent to
Sanger dideoxynucleotide sequencing standards, and
autoradiography led to identification of the DNA cleavage
and alkylation sites. The full details of this procedure
have been disclosed elsewhere.40
s-1), N-PIV-19 (t1/2 ) 13 min, k ) 8.88 × 10-4 s-1), and
N-Me-19 (t1/2 ) 275 min, k ) 4.20 × 10-5 s-1) was
reversed at pH ) 7. Recent studies39 enlisting pH rate
profiles have shown that closely related nucleophilic
addition reactions of modified alkylation subunits are
acid-catalyzed at pH ) 3, and it is the uncatalyzed
reactions that dominate at pH ) 7. In agreement with
these studies, N-PIV-19 (108 s) was the most stable
derivative at pH ) 3 (acid-catalyzed) followed in order
by N-BOC-19 (95 s) and N-Me-19 (77 s). Thus, the most
easily protonated compound exhibits the greater reactiv-
ity at pH 3. At pH ) 7, where the uncatalyzed reaction
dominates the reaction rate, the order is reversed and
N-Me-19 (275 min) is the most stable agent with the
greatest degree of vinylogous amide stabilization of the
reactive spirocyclopropylcyclohexadienone, followed in
order by N-BOC-19 (17 min) and N-PIV-19 (13 min) in
which the nitrogen lone pair conjugation is partially and
progressively diminished by carbamate and amide con-
jugation, respectively. This simple observation has very
significant ramifications. The preceding studies of the
solvolysis pH rate profiles of reactive alkylation subunits
did not show a rate dependence on the buffer concentra-
tion above pH 6. This indicated that the solvolysis
reactions at pH > 6 were not only not specific acid-
catalyzed, but that they also were not general acid-
catalyzed. Though surprising, these counterintuitive
conclusions are confirmed with this reverse order of
reactivity at pH 7 which is only consistent with the switch
from an acid-catalyzed reaction to one which is now
uncatalyzed.
A representative comparison of DNA alkylation by the
hybrid agents 7, and 31 alongside that of (+)-duocarmy-
cin SA (2) is illustrated below (Figure 5). There are three
important conclusions that can be drawn from these
comparisons. First, the quinone-based analogue 7, under
nonreductive conditions, did not alkylate DNA even at
10-2 M concentration. This is consistent with expectations
that this agent, in its oxidized form, cannot undergo in
situ spirocyclization to form the reactive cyclopropane
and thus alkylate DNA efficiently. Second, the reduced
form of 7, the hydroquinone 31, alkylates DNA in a
sequence-specific manner identical to the alkylation
pattern of the natural product (+)-duocarmycin SA and
does so with approximately only 100× less efficiency.
Duocarmycin SA is the most efficient DNA alkylating
agent of the natural products in this class, and despite
this 100-fold difference in efficiency, 31 represents a very
effective agent. For example, duocarmycin A (1) is 20×
less efficient than duocarmycin SA (2) and only 5× more
efficient than 31. No new sites of DNA alkylation were
detected with 31, and only adenine N3 alkylation was
observed under the conditions of limiting agent and
excess DNA. Notably, such sequencing studies only detect
the high affinity alkylation sites and minor sites of
comparable affinity (1-0.01×). Under these conditions,
the studies illustrate that despite the similarity of the
alkylation subunit to that of the guanine-selective mito-
mycin A, the binding selectivity of the full structure of
31 including the trimethoxyindole subunit of the hybrid
dominates the observed sequence selectivity retaining the
adenine alkylation. Last, it was demonstrated that
Na2S2O4 mediated reduction of the quinone 7 to presum-
ably produce the hydroquinone which alkylated DNA
with the same selectivity and near identical efficiency
as the synthetic hydroquinone, supporting a reaction
cascade in which DNA alkylation is only observed fol-
lowing reduction (see Scheme 2).
The acid-catalyzed nucleophilic addition of HCl and
CH3OH to the N-BOC derivative 19 was conducted on a
preparative scale to establish the regioselectivity of
addition. Treatment of 19 with HCl (1.2 equiv, -78 °C)
resulted in clean addition to provide the seco-agent 18
(Scheme 7) as did CH3OH (cat. TfOH) to give 37.
Additionally, similar observations were made with the
N-Me-19 in which chloride (38, HCl, 89%), methanol (39,
cat. TfOH, 71%), and water (40, cat. TfOH, 88%) all added
Although the data are not shown herein, the seco-agent
(40) Boger, D. L.; Munk, S. A.; Zarrinmayeh, H.; Ishizaki, T.;
Haught, J .; Bina, M. Tetrahedron 1991, 47, 2661.
(39) Boger, D. L.; Garbaccio, R. M. J . Org. Chem. 1999, 64, 5666.

8356 J . Org. Chem., Vol. 64, No. 22, 1999
Boger and Garbaccio
Sch em e 7
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
2-4, (+)-duocarmycin SA (1 × 10-5 to 1 × 10-7); lanes 5-8,
Sanger G, C, A, and T sequencing reactions; lanes 9-11, 7 (1
× 10-2 to 1 × 10-4); lanes 12-14, 7 + 1 µL 100 mM Na2S2O4
(1 × 10-2 to 1 × 10-4), lanes 15-18, 31 (1 × 10-2 to 1 × 10-5).
In addition to the trimethoxyindole derivative 44, a
naphthalene-linked naphthoquinone 46 was also synthe-
sized. N-BOC deprotection of 41 (3.6 M HCl/EtOAc)
followed by immediate coupling with 2-naphthalenecar-
boxylic acid (3 equiv of EDCI, DMF, 25 °C) produced the
seco agent 45 (47%). Oxidation of 45 (cat. salcomine, O2,
CH3CN) provided the final naphthoquinone 46 in excel-
lent yield (82%).
27 and the corresponding analogue 28 containing the
preformed activated cyclopropane also alkylated DNA in
a manner exactly analogous to 31 and duocarmycin SA
(2). Thus, only adenine N3 alkylation was observed, and
it occurred with a selectivity that was identical to that
of duocarmycin SA. This indicates that a likely interme-
diate in the alkylation of DNA by the reduced hydro-
quinone 31 is the cyclopropane-containing intermediate
9. In addition and although not an objective of the present
work, neither 27 nor its cyclopropane-containing ana-
logue 28 can be physically subject to a DNA phosphate
backbone protonation of the C6 carbonyl for activation
of the DNA alkylation reaction. Thus, such an activation
event cannot be contributing to or controlling the sites
of DNA alkylation. Finally, both 27 and 28 alkylated
DNA with an efficiency that was 10× lower than the
hydroquinone 31 and 100-1000× less efficient than
duocarmycin SA.
Secon d Gen er a tion An a logu es. The remarkable
stability of the iso-CBI based analogues relative to the
iso-CI based structures9 and the corresponding low
stability of 19 suggested a benzannulated set of ana-
logues to be examined in parallel. These agents, following
bioreduction and cyclization, would be more stable and
possibly more potent than 7 or 24.
Our prior efforts entailing the preparation of iso-CBI-
TMI had provided the key precursors 41 and 42.9
Naphthols 41 and 42 were oxidized to the bright yellow
naphthoquinones 43 (78%) and 44 (70%), respectively,
by the action of salcomine/O2 (cat., CH3CN, Scheme 8).
Red u ction by DT-Dia p h or a se. To confirm that the
agents were substrates for enzymatic reduction, recom-
binant human DT-Diaphorase41 (NQO1) was acquired
and an established HPLC assay42 was enlisted to mea-
sure the rate of substrate reduction and cofactor oxida-
tion. Mitomycin C (4, MMC) and mitomycin A (5, MMA)
were used as control agents as the former is a poor
substrate for DT-diaphorase, and the latter is a good
substrate at pH 7.4. The simple BOC-protected quinone
24 was an excellent substrate for DT-diaphorase and was
reduced at a greater rate than mitomycin A (Table 3).
Nitrogen lone pair conjugation with the BOC carbamate
makes 24 easier to reduce than mitomycin A, and this is
consistent with the observed rates of metabolism. Naph-
thoquinone analogues 43 and 46 were also good sub-
strates for DT-diaphorase. Again, a correlation between
the expected reduction potential and DT-diaphorase
metabolism was observed with 43 and 46 where the
amide in 46 is more strongly conjugated with the nitrogen
lone pair than the carbamate in 43. Thus 24, 43, and 46
(41) Beall, H. D.; Mulcahy, R. T.; Siegel, D.; Traver, R. D.; Gibson,
N. W.; Ross, D. Cancer Res. 1994, 54, 3196.
(42) Winski, S. L.; Hargreaves, R. H. J .; Butler, J .; Ross, D. Clin.
Canc. Res. 1998, 4, 3083.

A Novel Class of CC-1065 and Duocarmycin Analogues
J . Org. Chem., Vol. 64, No. 22, 1999 8357
NADH or NADPH as a cofactor44 to catalyze the two-
electron reduction of quinones and can protect cells
against their toxic effects. Paradoxically, DT-Diaphorase
is also involved in the reductive activation of mitomycin
C (4) and related analogues.45 Central to the design of
the mitomycin-duocarmycin hybrid would be a dif-
ferential activity against H460 and H596 nonsmall cell lung
cancer (NSCLC) cell lines which display high DT-Dia-
phorase activity (1360 nmol min-1 mg-1) and no DT-
Diaphorase activity, respectively.46 Increased potency in
the H460 cell line versus the H596 cell line would be
consistent with their preferential activation by DT-
Diaphorase. The results indicate a modest (3-6×) in-
crease in cytotoxic potency for the quinones 7 and 24
against the H460 cell line, which has high DT-diaphorase
activity, compared to the H596 cell line which has no
measurable DT-diaphorase activity. In addition, the
benzannelated naphthoquinone analogues (43, 44, and
46) displayed even greater levels of selectivity with 46
exhibiting a 24× increased potency against H460 consis-
tent with reductive activation by DT-Diaphorase.
Con clu sion s. A new class of bioreductively activated
DNA alkylating agents were designed and synthesized,
incorporating the mitomycin-like quinone structure into
the framework of the duocarmycins. The first generation
analogues were studied and found to be highly reactive
in their cyclopropane form, and a second generation set
of analogues were synthesized in parallel to achieve
increased potency. Both these sets of analogues were
found to alkylate DNA only upon quinone reduction and
to do so with the same characteristic sequence selectivity
as the duocarmycins and with only 100× less efficiency.
Biological activity assays revealed potency of a similar
magnitude. A cell-based assay established that these
analogues, like the mitomycins, were more active in a
DT-Diaphorase-enriched cell line versus a DT-Diapho-
rase-deficient (resistant) cell line. Further studies con-
firmed that these analogues are good substrates for
human recombinant DT-Diaphorase, establishing the
viable potential for these and related agents to be tumor-
selective DNA-alkylating agents subject to bioreductive
alkylation.
Ta ble 3. Red u ction Ra tes of Qu in on es by Recom bin a n t
NQO1a
5, mitomycin A
4, mitomycin C
24
43
46
1.05 ( 0.03
not detectedb
1.14 ( 0.08
0.53 ( 0.04
0.85 ( 0.05
a
Metabolism of quinones (50 µM) at 23 °C in pH 7.4 buffer by
human recombinant NQO1 (0.1-1.0 µg) was measured as ir-
reversible NADH oxidation (200 µM) by HPLC,43 rates of reduction
b
are expressed as µmol/min/mg NQO1. MMC does reduce at pH
5.8 (0.15 mmol/min/mg).40
Ta ble 4. In Vitr o Cytotoxic Activity
IC50 (µM)
agent
configuration L1210
H460
H596
selectivity
(-)-18
(+)-18
(-)-30
(+)-30
(-)-25
(+)-25
(-)-24
(+)-24
(-)-27
(+)-27
(+)-31
(-)-31
(+)-7
(-)-7
43
natural
unnatural
natural
unnatural
natural
unnatural
natural
unnatural
natural
unnatural
natural
unnatural
natural
unnatural
racemic
85
85
9
9
9
9
9
9
0.9
>20
0.6
1
1
8
0.3
0.1
0.01
nd
70
70
5
50
70
10
10
15
14
12
6
6
12
10
4
2
3
>20
0.07
5
3
3
9
>20
0.08
9
0.8
6
0.9
0.2
0.08
0.01
3
3.8
1.7
16
5
25
10
14
1
2
2
44
46
racemic
racemic
5, MMA natural
200
were all found to be good substrates for DT-diaphorase
with comparable rates of reaction to mitomycin A,
confirming their ability to undergo bioreduction in re-
ductase enriched cell lines and consistent with results
obtained in the cytotoxic assays below.
In Vitr o Cytotoxic Activity. Past studies with agents
in this class have defined a direct correlation between
solvolysis stability and cytotoxic potency. Consistent with
their reactivity, the hybrid agents exhibited cytotoxic
activity against L1210 that closely mirrored this relation-
ship even with the deep-seated structural modifications
(Table 4). These results, which also parallel trends
established in the DNA alkylation studies, demonstrated
that the enantiomer with the natural (3S) configuration
corresponding to that of the natural product, is the more
potent enantiomer by about 1-10× for the extended
agents 7, 27, 31, and little to no difference in the
enantiomers was observed for the simple BOC-substi-
tuted agents 18, 24, 25, 30. The p-quinones bearing the
mitomycin A substitution (7 and 24) exhibited cytotoxic
potencies comparable to the corresponding air-stable
hydroquinones (30 and 31), both of which were typically
more potent than the methyl ether precursors (18 and
27). Consistent with expectations, the intrinsically more
stable naphthoquinone analogues (43, 44, and 46) were
found to be more potent.
Exp er im en ta l Section
2,4-Dim eth oxy-3-m eth yl-1-(m eth oxym eth oxy)ben -
zen e (11). A solution of 2,4-(dimethoxy)-3-methylphenol
(1.0 g, 5.95 mmol) in 60 mL of anhydrous DMF at 0 °C
was treated with NaH (357 mg, 9.91 mmol) in several
portions over 5 min. After 10 min, Bu4NI (219 mg, 0.60
mmol) was added followed by the dropwise addition of
ClCH2OCH3 (0.68 mL, 8.91 mmol). The reaction mixture
was stirred at 25 °C for 36 h before the reaction was
quenched by the slow addition of 30 mL of H2O. The
aqueous layer was extracted with EtOAc. The organic
layers were combined, washed with 10% aqueous NaH-
CO3 and H2O, dried (Na2SO4), and concentrated under
(44) Ernster, L. Methods Enzymol. 1967, 10, 309. Ernster, L. Chim.
Scripta 1987, 27A, 1.
A second set of assays were conducted in order to
evaluate the efforts to impart tumor cell selectivity on a
duocarmycin-mitomycin hybrid. It has been reported
that a variety of tumor types have elevated DT-diapho-
rase (NQO1) activities relative to normal tissue.43 DT-
Diaphorase is a two-electron reductase that uses either
(45) Beall, H. D.; Hudnott, A. R.; Winski, S.; Siegel, D.; Swann, E.;
Ross, D.; Moody, C. J . Bioorg. Med. Chem. Lett. 1998, 8, 545. Beall, H.
D.; Mulcahy, T.; Siegel, D.; Traver, R. D.; Gibson, N. W.; Ross, D.
Cancer Res. 1994, 54, 3196. Ross, D.; Siegel, D.; Beall, H.; Prakash,
A. S.; Mulcahy, R. T.; Gibson, N. W. Cancer Metastasis Rev. 1993, 12,
83. Ross, D.; Beall, H.; Traver, R. D.; Siegel, D.; Phillips, R. M.; Bibson,
N. W. Oncol. Res. 1994, 6, 493.
(46) Traver, R. D.; Siegel, D.; Beall, H. D.; Phillips, R. M.; Gibson,
N. W.; Franklin, W. A.; Ross, D. Br. J . Cancer 1997, 75, 69.
(43) Schlager, J . J .; Powis, G. Int. J . Cancer 1990, 45, 403.

8358 J . Org. Chem., Vol. 64, No. 22, 1999
Boger and Garbaccio
reduced pressure. Flash chromatography (SiO2, 3 × 10
cm, 10% EtOAc/hexane) provided 11 (1.11 g, 88%) as a
light yellow oil: 1H NMR (CDCl3, 250 MHz) δ 6.92 (d, J
) 8.8 Hz, 1H), 6.51 (d, J ) 8.8 Hz, 1H), 5.13 (s, 2H), 3.79
(s, 3H), 3.76 (s, 3H), 3.50 (s, 3H), 2.13 (s, 3H); 13C NMR
(CDCl3, 62.5 MHz) δ 153.5, 149.2, 144.3, 121.0, 114.4,
105.3, 96.0, 60.4, 56.0, 55.7, 8.9; IR (film) νmax 2937, 2833,
1595, 1487, 1440, 1420 cm-1; FABHRMS (NBA) m/z
212.1040 (C11H16O4 requires 212.1049).
2,4-Dim eth oxy-3-m eth yl-5-n itr o-1-(m eth oxym eth -
oxy)ben zen e (12). A solution of 11 (1.11 g, 5.21 mmol)
in 18 mL of freshly distilled Ac2O at 0 °C was treated
with Cu(NO3)2‚2.5H2O (2.41 g, 10.4 mmol) in several
portions over 5 min. The reaction mixture was stirred
for 2 h at 0 °C, and 1 h at 25 °C before the reaction was
poured over H2O and extracted with EtOAc. The com-
bined organic layers were washed with saturated aque-
ous NaCl, dried (Na2SO4), and concentrated under re-
duced pressure. The crude light yellow oil (1.18 g, 88%)
was carried on to the next transformation: 1H NMR
(CDCl3, 250 MHz) δ 7.54 (s, 1H), 5.18 (s, 2H), 3.87 (s,
3H), 3.81 (s, 3H), 3.48 (s, 3H), 2.21 (s, 3H); 13C NMR
(CDCl3, 62.5 MHz) δ 153.0, 147.8, 145.8, 138.9, 128.2,
110.5, 95.3, 61.8, 60.5, 56.2, 9.5; IR (film) νmax 2942, 2829,
1522, 1481 cm-1; FABHRMS (NBA) m/z 258.0977 (C11H15-
NO6 + H+ requires 258.0978).
5-Am in o-2,4-dim eth oxy-3-m eth yl-1-(m eth oxym eth -
oxy)ben zen e (13). A solution of 12 (1.18 g, 4.57 mmol)
in 90 mL of moist Et2O (8:2:1 Et2O:EtOH:H2O) was cooled
to 0 °C, and treated with freshly prepared Al-Hg19 (1.23
g Al, 45.7 mmol) which had been prepared from small 1
× 1 cm pieces of Al. The reaction mixture was stirred
vigorously for 0.5 h at 0 °C and 1 h at 25 °C. The reaction
mixture was filtered through Celite, and the Celite was
washed thoroughly with Et2O. The solution was washed
with saturated aqueous NaCl, dried (Na2SO4), and con-
centrated under reduced pressure to afford 13 (0.88 g,
85%) as a crude brown oil, which was immediately carried
on to the next step: 1H NMR (CDCl3, 250 MHz) δ 6.42
(s, 1H), 5.11 (s, 2H), 3.70 (s, 3H), 3.66 (s, 3H), 3.56 (m,
2H), 3.47 (s, 3H), 2.16 (s, 3H); 13C NMR (CDCl3, 250 MHz)
δ 147.0, 140.4, 140.3, 135.8, 125.4, 102.0, 95.4, 60.6, 59.4,
56.0, 9.34; IR (film) νmax 3446, 3359, 2935, 2826, 1617,
1492 cm-1; ESIMS m/z 228 (C11H17NO4 + H+ requires
228).
mL of a 2.5 M solution in hexane, 6.18 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 (0.45 mL, 6.18 mmol) and stirred
for 15 min at 25 °C. The reaction was diluted with H2O
and extracted with Et2O, and the combined organic
extracts were washed with saturated aqueous NaCl,
dried (Na2SO4), and concentrated under reduced pres-
sure. Flash chromatography (SiO2, 2.5 × 10 cm, 20%
EtOAc/hexane) yielded 15 (560 mg, 74%) as a colorless
oil: 1H NMR (CDCl3, 400 MHz) δ 5.99 (br s, 1H), 5.10 (s,
2H), 3.75 (s, 3H), 3.69 (s, 3H), 3.65 (s, 3H), 2.17 (s, 3H),
1.49 (s, 9H); 13C NMR (CDCl3, 100 MHz) δ 153.6, 151.8,
150.5, 146.9, 129.3, 126.7, 99.1, 95.8, 80.6, 60.5, 60.3,
58.5, 28.3, 9.8; IR (film) νmax 3321, 2975, 2936, 1722, 1485,
1455 cm-1; FABHRMS (NBA/CsI) m/z 585.9688 (C16H24
INO6 + Cs+ requires 585.9703).
-
[N-(ter t-Bu tyloxyca r bon yl)-N-(3-ch lor o-2-p r op en -
1-yl)a m in o]-2,4-d im eth oxy-6-iod o-5-(m eth oxym eth -
oxy)-3-m eth ylben zen e (16). A solution of 15 (0.610 g,
1.34 mmol) in 13.4 mL of anhydrous DMF was cooled to
0 °C and treated with NaH (60% dispersion in oil, 121
mg, 4.03 mmol) in small portions. The resulting suspen-
sion was stirred for 15 min and treated with neat 1,3-
dichloropropene (0.52 mL, 5.5 mmol) dropwise, followed
by catalytic n-Bu4NI (50.0 mg, 0.13 mmol). The reaction
mixture was warmed to 25 °C and stirred for 3 h. The
reaction mixture was quenched with the addition of
saturated aqueous NaHCO3, and the aqueous layer was
extracted with EtOAc. The combined organic extracts
were washed with H2O, dried (Na2SO4), and concentrated
under reduced pressure. Flash chromatography (SiO2, 3
× 10 cm, 0-20% EtOAc/hexane gradient) yielded 16
(0.681 g, 96%) as a colorless oil as a mixture of rotam-
ers: 1H NMR (CDCl3, 400 MHz) 2:1 rotamers δ 6.15-
6.03 (m, 1H), 6.00-5.90 (m, 1H), 5.11-5.03 (m, 2H),
4.17-3.87 (m, 2H), 3.77 and 3.74 (s, 3H), 3.65 and 3.63
(s, 3H), 3.627 and 3.622 (s, 3H), 2.14 and 2.13 (s, 3H),
1.50 and 1.34 (s, 9H); 13C NMR (CDCl3, 100 MHz)
rotamers δ 153.65 and 153.62, 152.8 and 152.1, 151.0 and
150.7, 147.0 and 146.7, 134.5 and 134.0, 129.5 and 129.0,
127.0 and 126.7, 121.0 and 120.6, 99.0, 97.7 and 97.3,
80.8 and 80.6, 60.5 and 60.4, 60.3 and 60.2, 58.5 and 58.4,
50.4, 48.8, 28.3, and 28.2, 9.9; IR (film) νmax 2973, 2936,
1704, 1456 cm-1; FABHRMS (NBA/CsI) m/z 659.9655
(C19H27ClINO6 + Cs+ requires 659.9626).
[N-(ter t-Bu tyloxyca r bon yl)a m in o]-2,4-d im eth oxy-
5-(m eth oxym eth oxy)-3-m eth ylben zen e (14). A solu-
tion of crude 13 (0.88 g, 3.85 mmol) in 40 mL of
anhydrous THF was treated with BOC2O (1.73 g, 7.72
mmol), and the reaction mixture was warmed at reflux
(65 °C) for 18 h. The solvents were removed under
reduced pressure, and flash chromatography (SiO2, 3 ×
10 cm, 10% EtOAc/hexane) provided pure 14 as a yellow
oil (0.96 g, 76%): 1H NMR (CDCl3, 250 MHz) δ 7.72 (br
s, 1H), 6.86 (br s, 1H), 5.18 (s, 2H), 3.75 (s, 3H), 3.67 (s,
3H), 3.51 (s, 3H), 2.19 (s, 3H), 1.50 (s, 9H); 13C NMR
(CDCl3, 100 MHz) δ 152.7, 146.4, 143.6, 141.6, 127.8,
124.8, 105.3, 95.5, 80.4, 60.5, 60.4, 56.4, 28.3, 9.5; IR
1-(ter t-Bu t yloxyca r b on yl)-3-(ch lor om et h yl)-5,7-
d im eth oxy-4-(m eth oxym eth oxy)-6-m eth yl-2,3-d ih y-
d r oin d ole (17). A solution of 16 (580 mg, 1.09 mmol) in
10.9 mL of anhydrous benzene was treated with AIBN
(17.0 mg, 0.11 mmol) and Bu3SnH (311 µL, 1.15 mmol)
and warmed at 75 °C for 2 h. The reaction mixture was
concentrated under reduced pressure. Flash chromatog-
raphy (SiO2, 2.5 × 15 cm, 0-20% EtOAc/hexane gradient)
yielded 17 (430 mg, 96%) as a colorless oil: 1H NMR
(CDCl3, 400 MHz) δ 5.10 (d, J ) 5.9 Hz, 1H), 5.04 (d, J
) 5.9 Hz, 1H), 4.22 (dd, J ) 2.6, 11.8 Hz, 1H), 4.02-3.89
(m, 2H), 3.68 (s, 3H), 3.62 (s, 3H), 3.53 (s, 3H), 3.51 (m,
1H), 3.42 (dd, J ) 9.8, 18.8 Hz, 1H), 2.12 (s, 3H), 1.49 (s,
9H); 13C NMR (CDCl3, 100 MHz) δ 153.6, 147.8, 144.9,
142.3, 130.1, 126.3, 125.0, 99.1, 81.0, 60.2, 58.3, 57.2,
54.4, 44.9, 42.7, 28.1, 9.8; IR (film) νmax 2935, 1703, 1471,
(film) νmax 3437, 3341, 2977, 2935, 1731, 1519, 1454 cm-1
;
FABHRMS (NBA/CsI) m/z 460.0723 (C16H25NO6 + Cs+
requires 460.0736).
[N-(ter t-Bu tyloxyca r bon yl)a m in o]-2,4-d im eth oxy-
6-iod o-5-(m eth oxym eth oxy)-3-m eth ylben zen e (15).
A solution of 14 (0.55 g, 1.67 mmol) in 6.6 mL of
anhydrous THF was cooled to -25 °C and treated with
TMEDA (0.94 mL, 6.18 mmol) followed by n-BuLi (2.5
1417 cm-1; FABHRMS (NBA/NaI) m/z 424.1490 (C19H28
ClNO6 + Na+ requires 424.1503).
-
Resolu tion of 17. A sample of 17 (200 mg) in 1.0 mL
of 1% i-PrOH/hexane was resolved on a semipreparative

A Novel Class of CC-1065 and Duocarmycin Analogues
J . Org. Chem., Vol. 64, No. 22, 1999 8359
Daicel Chiralcel OD column (10 mm, 2 × 25 cm, 7.0 mL/
min flow rate, 1% i-PrOH/hexane). The effluent was mon-
itored at 254 nm, and the enantiomers were eluted with
retention times of 18.0 (R) and 31.0 (S) min (R ) 1.63).
Both enantiomers were found to be >99% enantiomeri-
cally pure by analytical HPLC. The fractions containing
the separated enantiomers were collected and concen-
trated to afford (+) and (-)-17. (-)-(3R)-17: [R]25D -13.9
2-yl)ca r bon yl]-1,2,7,7a -tetr a h yd r ocyclop r op [1,2-c]-
in d ol-6-on e (28). A solution of 27 (1.0 mg, 2.0 µmol) in
0.5 mL of CH3CN was treated with DBU (0.5 µL, 4.0
µmol) and stirred for 20 min at 25 °C. The reaction
solution was then loaded onto a 1% Et3N/EtOAc-slurried
silica gel column, and quick flash chromatography (SiO2,
1.5 × 3 cm, EtOAc) afforded 28 (0.76 mg, 82%) as a
golden oil: 1H NMR (C6D6, 400 MHz) δ 9.20 (s, 1H), 6.81
(s, 1H), 6.64 (s, 1H), 3.81 (s, 3H), 3.80 (m, 1H), 3.75 (s,
3H), 3.66 (s, 3H), 3.44 (s, 3H), 3.33 (m, 1H), 3.18 (s, 3H),
2.13 (m, 1H), 1.81 (dd, J ) 3.5, 7.7 Hz, 1H), 1.63 (s, 3H),
0.97 (dd, J ) 3.5, 5.8 Hz, 1H); IR (film) νmax 3291, 2931,
1623, 1464 cm-1; FABHRMS (NBA/NaI) m/z 455.1711
(C24H26N2O7 + H+ requires 455.1740).
(c 1.0, CH2Cl2); (+)-(3S)-17: [R]25 +13.1 (c 1.0, CH2Cl2).
D
1-(ter t-Bu t yloxyca r b on yl)-3-(ch lor om et h yl)-5,7-
d im et h oxy-4-h yd r oxy-6-m et h yl-2,3-d ih yd r oin d ole
(18). A solution of 17 (88.0 mg, 0.22 mmol) in 11.0 mL
1:1 i-PrOH/THF was treated with 12 N HCl (0.13 mL,
1.53 mmol), and the mixture was stirred for 44 h at 25
°C before the volatiles were removed in vacuo. Flash
chromatography (SiO2, 1.5 × 15 cm, 0-20% EtOAc/
hexane gradient) provided 18 (62 mg, 82%) as a white
1-(ter t-Bu tyloxycar bon yl)-3-(ch lor om eth yl)-5-m eth -
oxy-6-m eth yl-2,3-d ih yd r oin d ole-4,7-d ion e (24). A so-
lution of 17 (48.0 mg, 0.13 mmol) in 6.7 mL of anhydrous
DMF was treated with salcomine (22.0 mg, 67.0 µmol),
and O2 was bubbled through the dark brown solution for
20 h at 25 °C. The reaction mixture was filtered through
Celite, and the Celite was washed thoroughly with
EtOAc. The filtrate was washed with H2O and saturated
aqueous NaCl, dried (Na2SO4), and concentrated under
reduced pressure. Flash chromatography (SiO2, 1.5 × 10
cm, 10% EtOAc/hexane) provided 24 (18.0 mg, 40%) as a
bright orange oil: 1H NMR (C6D6, 400 MHz) δ 3.75 (dd,
J ) 6.0, 11.9 Hz, 1H, CHHCl), 3.62 (s, 3H, OCH3), 3.56
(dd, J ) 10.6, 11.9 Hz, 1H, CHHCl), 3.36 (dd, J ) 6.0,
11.1 Hz, 1H, CHHN), 3.16 (dd, J ) 3.1, 11.1 Hz, 1H,
CHHN), 2.81 (m, 1H, C3-H), 1.84 (s, 3H, C6-CH3), 1.44
(s, 9H, C(CH3)3); 13C NMR (CDCl3, 100 MHz) δ 180.3 (C-
4), 179.3 (C-7), 155.0 (C-5), 151.9 (CO2t-Bu), 148.7 (C-
3b), 127.5 (C-6), 125.1 (C-3a), 82.2 (C(CH3)3), 60.5 (OCH3),
53.8 (CH2Cl), 45.2 (C2), 40.5 (C3), 27.8 (C(CH3)3), 9.0
1
solid: mp 95-97 °C (white needles, EtOAc); H NMR
(CDCl3, 250 MHz) δ 5.49 (s, 1H), 4.15 (dd, J ) 3.9, 11.8
Hz, 1H), 4.05 (dd, J ) 7.7, 11.8 Hz, 1H), 3.93 (dd, J )
3.3, 10.7 Hz, 1H), 3.71 (s, 3H), 3.61 (s, 3H), 3.59 (m, 1H),
3.48 (dd, J ) 9.4, 10.7 Hz, 1H), 2.19 (s, 3H), 1.50 (s, 9H);
13C NMR (CDCl3, 100 MHz) δ 153.5, 142.4, 141.6, 141.0,
131.0, 124.8, 117.0, 80.1, 61.0, 58.6, 54.7, 44.8, 42.0, 28.1,
10.0; IR (film) νmax 3386, 2978, 2937, 1699, 1476 cm-1
;
FABHRMS (NBA/NaI) m/z 357.1334 (C17H24ClNO5 re-
quires 357.1343); (+)-(3R)-18: [R]25D +7.1 (c 1.0, CH2Cl2);
(-)-(3S)-18: [R]25 -7.2 (c 1.0, CH2Cl2).
D
N-(ter t-Bu tyloxyca r bon yl)-3,5-d im eth oxy-4-m eth -
yl-1,2,7,7a -t et r a h yd r ocyclop r op [1,2-c]in d ol-6-on e
(19). A solution of 18 (3.1 mg, 8.30 µmol) in 0.5 mL of
CH3CN was treated with DBU (1.6 µL, 10.9 µmol) and
stirred for 20 min at 25 °C. The reaction solution was
loaded onto a 1% Et3N/hexane-slurried silica gel column,
and quick flash chromatography (SiO2, 1.5 × 10 cm, 50%
EtOAc/hexane) afforded 19 (2.5 mg, 93%) as a golden
oil: 1H NMR (C6D6, 400 MHz) δ 3.73 (s, 3H), 3.71 (d, J )
11.4 Hz, 1H), 3.26 (s, 3H), 3.06 (dd, J ) 4.5, 11.4 Hz,
1H), 2.07 (s, 3H), 2.05 (m, 1H), 1.69 (dd, J ) 3.2, 7.5 Hz,
1H), 1.42 (s, 9H), 0.81 (dd, J ) 3.2, 5.8 Hz, 1H); IR (film)
(C6-CH3); IR (film) νmax 2931, 1716, 1651, 1590 cm-1
;
UV (MeOH) λmax (ꢀ) 313 (13600), 215 (37400) nm; FAB-
HRMS (NBA/NaI) m/z 342.1121 (C16H20NO5Cl + H+
requires 342.1108); (+)-(3R)-24: [R]25D +29 (c 0.45, CH2-
Cl2); (-)-(3S)-24: [R]25 -30 (c 0.42, CH2Cl2).
D
1-(ter t-Bu tyloxycar bon yl)-3-(ch lor om eth yl)-7-m eth -
oxy-6-m eth yl-2,3-d ih yd r oin d ole-4,5-d ion e (25). A so-
lution of 17 (6.5 mg, 18.2 µmol) in 1.1 mL of CH2Cl2 was
treated with PhSeO2H (4.1 mg, 21.8 µmol) in 0.9 mL of
CH2Cl2, and the resulting deep red solution was stirred
for 20 min at 25 °C. The reaction mixture was quenched
with the addition of saturated aqueous NaHCO3, and the
aqueous layer was extracted with CH2Cl2. The combined
organic extracts were dried (Na2SO4), and concentrated
under reduced pressure. Flash chromatography (SiO2, 1.5
× 5 cm, 40% EtOAc/hexane) yielded 25 (5.4 mg, 87%) as
a red/purple oil: 1H NMR (C6D6, 400 MHz) δ 3.69 (dd, J
) 5.0, 11.8 Hz, 1H, CHHCl), 3.48 (dd, J ) 10.0, 11.8 Hz,
1H, CHHCl), 3.44 (dd, J ) 5.8, 11.1 Hz, 1H, CHHN), 3.23
(s, 3H, OCH3), 3.22 (dd, J ) 3.1, 11.1 Hz, 1H, CHHN),
2.84 (m, 1H, C3-H), 1.70 (s, 3H, C6-CH3), 1.33 (s, 9H,
C(CH3)3); 13C NMR (CDCl3, 100 MHz) δ 181.6 (C-5), 175.4
(C-4), 157.7 (C-7), 153.0 (C-3b), 152.3 (CO2t-Bu), 123.6
(C-6), 120.6 (C-3a), 82.4 (C(CH3)3), 60.3 (OCH3), 54.5
(CH2Cl), 45.2 (C2), 40.5 (C3), 27.7 (C(CH3)3), 9.8 (C6-
CH3); IR (film) νmax 2979, 2930, 1728, 1646, 1575, 1445
cm-1; UV (MeOH) λmax (ꢀ) 332 (6900), 223 (10200) nm;
ESIMS (NBA/NaI) m/z 342 (C16H20NO5Cl + H+ requires
342).
ν
max 2978, 2933, 1699, 1633 cm-1; FABHRMS (NBA/CsI)
m/z 454.0645 (C17H23NO5 + Cs+ requires 454.0631).
3-(Ch lor om eth yl)-5,7-dim eth oxy-4-h ydr oxy-6-m eth -
yl-1-[(5,6,7-tr im eth oxyin d ol-2-yl)ca r bon yl]-2,3-d ih y-
d r oin d ole (27). A sample of 17 (17.4 mg, 0.038 mmol)
was dissolved in 3.6 N HCl/EtOAc (3.8 mL) and stirred
for 30 min at 25 °C. The solvents were removed by a
stream of N2, and the residual salt was thoroughly dried
on the pump. The salt was then dissolved in DMF (1.5
mL) and treated with 2629 (11.5 mg, 0.045 mmol) and
EDCI (21.5 mg, 0.11 mmol). The resulting mixture was
stirred at 25 °C for 3 h under Ar. The reaction was then
diluted with H2O and extracted with EtOAc. The collected
organic layer was dried (Na2SO4) and condensed under
reduced pressure to yield crude 27. Flash chromatogra-
phy (40-60% EtOAc/hexanes gradient) yielded pure 27
(9.8 mg, 47%) as a white film: 1H NMR (CDCl3, 400 MHz)
δ 9.15 (s, 1H), 6.90 (d, J ) 2.4 Hz, 1H), 6.82 (s, 1H), 5.52
(s, 1H), 4.53 (m, 2H), 4.06 (s, 3H), 3.97 (dd, J ) 3.3, 11.0
Hz, 1H), 3.92 (s, 3H), 3.89 (s, 3H), 3.76 (s, 3H), 3.73 (m,
1H), 3.58 (s, 3H), 3.50 (dd, J ) 9.8, 11.0 Hz, 1H), 2.24 (s,
3H); IR (film) νmax 3296, 2937, 1634, 1462 cm-1; FAB-
HRMS (NBA/CsI) m/z 491.1601 (C24H27N2O7Cl + H+
requires 491.1585). (+)-(3R)-27: [R]25D +10.0 (c 0.12, CH2-
1-(ter t-Bu tyloxyca r bon yl)-3-(ch lor om eth yl)-4,7-d i-
h ydr oxy-5-m eth oxy-6-m eth yl-2,3-dih ydr oin dole (30).
A solution of 24 (10.0 mg, 32.2 µmol) in 1.5 mL anhydrous
Cl2); (-)-(3S)-27: [R]25 -10.7 (c 0.075, CH2Cl2).
D
3,5-Dim eth oxy-4-m eth yl-1-[(5,6,7-tr im eth oxyin dol-

8360 J . Org. Chem., Vol. 64, No. 22, 1999
Boger and Garbaccio
CH3OH was treated with Pd/C (1.0 mg, 10 wt %) and
stirred under H2 for 10 min at 25 °C. The reaction
mixture was filtered through Celite, and the Celite was
washed thoroughly with CH3OH. The combined organic
layers were then washed with H2O and saturated aque-
ous NaCl, dried (Na2SO4), and concentrated under re-
duced pressure to yield pure 30 as a clear oil: 1H NMR
(C6D6, 400 MHz) δ 10.63 (s, 1H), 5.23 (s, 1H), 3.98 (m,
3H), 3.71 (s, 3H), 3.68 (m, 1H), 3.49 (dd, J ) 9.5, 10.7
Hz, 1H), 2.16 (s, 3H), 1.52 (s, 9H); 13C NMR (CDCl3, 100
MHz) δ 154.8, 142.9, 137.9, 136.6, 124.5, 120.3, 114.8,
83.0, 61.1, 53.4, 45.6, 40.7, 28.1, 10.0; IR (film) νmax 3422,
2980, 1667, 1471 cm-1; FABHRMS (NBA/NaI) m/z
366.1078 (C16H22NO5Cl + Na+ requires 366.1084); (+)-
(3R)-30: [R]25 +6.5 (c 0.40, CH2Cl2); (-)-(3S)-30: [R]25
was treated with salcomine (catalytic), and O2 was
bubbled through the solution for 1 h at 25 °C. The
reaction mixture was filtered through Celite, and the
Celite was washed thoroughly with EtOAc. The solution
was then concentrated under reduced pressure and
subjected directly to flash chromatography (SiO2, 1.5 ×
5 cm, 20% EtOAc/hexane) to provide 43 (7.2 mg, 78%) as
a bright yellow oil: 1H NMR (CDCl3, 400 MHz) δ 7.99
(m, 2H), 7.67 (m, 2H), 4.15 (dd, J ) 10.5, 11.9 Hz, 1H),
4.06 (dd, J ) 5.7, 11.9 Hz, 1H), 3.87 (m, 2H), 3.81 (m,
1H), 1.51 (s, 9H); 13C NMR (CDCl3, 100 MHz) δ 182.2,
177.2, 151.5, 150.1, 133.6, 133.4, 133.3, 132.2, 130.0,
126.7, 125.7, 83.0, 53.3, 45.3, 41.1, 27.9; IR (film) νmax
2978, 2929, 1721, 1644, 1597, 1403 cm-1; FABHRMS
(NBA/NaI) m/z 370.0820 (C18H18NO4Cl + Na+ requires
370.0822).
D
D
-6.1 (c 0.38, CH2Cl2).
3-(Ch lor om eth yl)-4,7-dih ydr oxy-5-m eth oxy-6-m eth -
yl-1-[(5,6,7-tr im eth oxyin d ol-2-yl)ca r bon yl]-2,3-d ih y-
d r oin d ole (31). A sample of 30 (6.9 mg, 20.0 µmol) was
dissolved in 3.6 N HCl/EtOAc (1.5 mL), and the solution
was stirred for 30 min at 25 °C. The solvents were
removed under reduced pressure, and the residual salt
was thoroughly dried under high vacuum. The salt was
dissolved in anhydrous DMF (1.0 mL) and treated with
2629 (5.6 mg, 22.0 µmol) and EDCI (11.4 mg, 60.0 µmol).
The resulting solution was stirred at 25 °C for 3 h under
Ar. The reaction mixture was then diluted with H2O, and
extracted with EtOAc. The combined organic layer was
dried (Na2SO4) and concentrated under reduced pressure.
Flash chromatography (SiO2, 1.5 × 10 cm, 0-50% EtOAc/
hexane gradient) yielded 31 (4.2 mg, 44%) as a yellow
oil: 1H NMR (C6D6, 400 MHz) δ 10.87 (s, 1H), 9.32 (br s,
1H), 6.63 (d, J ) 2.4 Hz, 1H), 6.62 (s, 1H), 5.06 (s, 1H),
4.23 (m, 1H), 3.86 (m, 2H), 3.79 (s, 3H), 3.69 (s, 3H), 3.53
(m, 1H), 3.51 (s, 3H), 3.21 (s, 3H), 3.18 (m, 1H), 2.41 (s,
3H); 13C NMR (C6D6, 125 MHz) δ 160.5, 151.2, 145.1,
139.7, 139.5, 138.3, 129.0, 126.6, 126.4, 123.9, 121.6,
115.6, 108.2, 98.2, 61.1, 60.7, 60.5, 56.1, 56.0, 45.3, 42.2,
10.7; IR (film) νmax 3425, 2939, 1574, 1432 cm-1; FAB-
HRMS (NBA/NaI) m/z 476.1338 (C23H25N2O7Cl requires
3-(Ch lor om eth yl)-1-[(5,6,7-tr im eth oxyin d ol-2-yl)-
ca r bon yl]-2,3-d ih yd r o-1H-ben zo[f]in d ole-4,9-d ion e
(44). A solution of 42 (2.5 mg, 5.34 µmol) in 1.0 mL of
anhydrous CH3CN was treated with salcomine (catalytic),
and O2 was bubbled through the solution for 1 h at 25
°C. The reaction mixture was filtered through Celite, and
the Celite was washed thoroughly with EtOAc. The
solution was then concentrated under reduced pressure
and subjected HPLC purification. Semipreparative reverse-
phase HPLC (Waters Bondapak C-18 column, 300 Å, 25
× 100 mm, gradient 10-90% CH3CN/H2O over 30 min,
10 mL/min) afforded 44 (tR ) 25 min, 1.8 mg, 70%) as a
bright yellow oil: 1H NMR (C6D6, 400 MHz) δ 9.12 (s,
1H), 8.01 (d, J ) 7.3 Hz, 1H), 7.78 (d, J ) 7.3 Hz, 1H),
6.97 (m, 1H), 6.88 (m, 1H), 6.71 (d, J ) 2.1 Hz, 1H), 6.55
(s, 1H), 3.93 (dd, J ) 5.4, 11.0 Hz, 1H), 3.78 (m, 1H),
3.76 (s, 3H), 3.63 (s, 3H), 3.57 (dd, J ) 6.2, 11.0 Hz, 1H),
3.44 (s, 3H), 3.38 (dd, J ) 3.2, 11.4 Hz, 1H), 3.15 (m, 1H);
IR (film) νmax 3320, 2926, 1682, 1644, 1596, 1463 cm-1
;
FABHRMS (NBA/NaI) m/z 481.1153 (C25H21N2O6Cl + H+
requires 481.1166).
3-(Ch lor om eth yl)-4-h yd r oxy-1-[(n a p h th -2-yl)ca r -
bon yl]-2,3-d ih yr d o-1H-ben zo[f] in d ole (45). A sample
of 41 (11.8 mg, 31.2 µmol) was dissolved in 3.6 N HCl/
EtOAc (2.0 mL), and the solution was stirred for 30 min
at 25 °C. The solvents were removed under reduced
pressure, and the residual salt was thoroughly dried
under high vacuum. The salt was dissolved in anhydrous
DMF (1.0 mL) and treated with naphthalene-2-carboxylic
acid (6.4 mg, 37.0 µmol) and EDCI (17.8 mg, 93.0 µmol).
The resulting solution was stirred at 25 °C for 3 h under
Ar. The reaction mixture was then diluted with H2O and
extracted with EtOAc. The combined organic layer was
dried (Na2SO4) and concentrated under reduced pressure.
Flash chromatography (SiO2, 1.5 × 10 cm, 0-30% EtOAc/
hexane gradient) yielded 45 (5.7 mg, 47%) as a colorless
film: 1H NMR (acetone-d6, 400 MHz) δ 8.88 (s, 1H), 8.20
(m, 2H), 8.03 (m, 4H), 7.74 (dd, J ) 1.6, 8.4 Hz, 1H), 7.70
(br s, 1H), 7.63 (m, 2H), 7.43 (m, 1H), 7.37 (m, 1H), 4.39
(dd, J ) 9.2, 11.4 Hz, 1H), 4.19 (m, 1H), 4.08-4.01 (m,
2H), 3.85 (dd, J ) 8.9, 10.6 Hz, 1H); 13C NMR (CDCl3,
100 MHz) δ 169.9, 150.3, 143.3, 137.0, 136.3, 135.4, 132.2,
130.6, 130.0, 129.7, 129.2, 129.1, 128.7, 128.3, 127.8,
126.8, 125.7, 125.0, 122.6, 116.2, 56.0, 46.9, 41.6; IR (film)
476.1350); (-)-(3R)-31: [R]25 -30 (c 0.21, CH2Cl2); (+)-
D
(3S)-31: [R]25 +31 (c 0.13, CH2Cl2).
D
3-(Ch lor om et h yl)-5-m et h oxy-6-m et h yl-1-[(5,6,7-
t r im et h oxyin d ol-2-yl)ca r b on yl]-2,3-d ih yd r oin d ol-
4,7-d ion e (7). A solution of 31 (3.0 mg, 6.2 µmol) in 1.5
mL of anhydrous EtOAc was treated with Pd/C (4.5 mg,
150 wt %) and stirred under air for 6 h at 25 °C. The
reaction mixture was filtered through Celite, and the
Celite was washed thoroughly with EtOAc. The solution
was concentrated under reduced pressure to yield pure
7 (2.9 mg, 97%) as an orange oil: 1H NMR (C6D6, 400
MHz) δ 9.10 (s, 1H), 6.69 (d, J ) 2.3 Hz, 1H), 6.61 (s,
1H), 3.91 (dd, J ) 5.4, 11.2 Hz, 1H), 3.76 (m, 1H), 3.76
(s, 3H), 3.66 (s, 3H), 3.63 (s, 3H), 3.49 (dd, J ) 6.2, 11.2
Hz, 1H), 3.47 (s, 3H), 3.27 (dd, J ) 3.2, 11.2 Hz, 1H),
3.01 (m, 1H), 1.80 (s, 3H); 13C NMR (C6D6, 100 MHz) δ
180.4, 179.4, 162.3, 155.5, 151.3, 150.1, 141.7, 139.6,
130.0, 127.9, 127.2, 126.3, 123.5, 108.7, 98.1, 61.1, 60.65,
60.64, 56.3, 55.9, 45.1, 42.0, 9.0; IR (film) νmax 3298, 2938,
1651, 1586 cm-1; FABHRMS (NBA/NaI) m/z 607.0227
(C23H23N2O7Cl + Cs+ requires 607.0248); (-)-(3R)-7:
[R]25D -81 (c 0.15, CH2Cl2); (+)-(3S)-7: [R]25D +80 (c 0.10,
CH2Cl2).
ν
max 2923, 1688, 1598, 1468 cm-1; FABHRMS (NBA/NaI)
m/z 388.1097 (C24H18NO2Cl + H+ requires 388.1104).
3-(Ch lor om et h yl)-1-[(n a p h t h -2-yl)ca r b on yl]-2,3-
d ih yd r o-1H-ben zo[f]in d ole-4,9-d ion e (46). A solution
of 45 (2.0 mg, 5.17 µmol) in 1.0 mL of anhydrous CH3CN
was treated with salcomine (catalytic), and O2 was
1-(ter t-Bu tyloxyca r bon yl)-3-(ch lor om eth yl)-2,3-d i-
h yd r o-1H-ben zo[f]in d ole-4,9-d ion e (43). A solution of
41 (8.0 mg, 26.6 µmol) in 1.5 mL of anhydrous CH3CN

A Novel Class of CC-1065 and Duocarmycin Analogues
J . Org. Chem., Vol. 64, No. 22, 1999 8361
19 (2.5 mg, 7.8 µmol) in CH3OH (1.0 mL) under Ar at 0
°C was treated with 0.01 N TfOH (93 µL, 0.12 equiv) and
allowed to warm to 25 °C for 2 h. The reaction was
directly subjected to flash chromatography (SiO2, 1.0 ×
3 cm, 30% EtOAc/hexane) to provide pure 37 (2.2 mg,
82%) as a colorless oil as the only detectable reaction
product: 1H NMR (CDCl3, 400 MHz) δ 7.62 (s, 1H), 4.10
(dd, J ) 8.1, 11.6 Hz, 1H), 3.76 (s, 3H), 3.66 (dd, J ) 5.4,
11.6 Hz, 1H), 3.60 (m, 1H), 3.59 (s, 3H), 3.54 (m, 2H),
3.44 (s, 3H), 2.16 (s, 3H), 1.49 (s, 9H); IR (film) νmax 3332,
2933, 1682, 1470; MALDIFTMS (NBA) m/z 376.1733
(C18H27NO6 + Na+ requires 376.1736).
Solvolysis Regioselectivity: 5,7-Dim eth oxy-1,6-
d im et h yl-4-h yd r oxy-3-(m et h oxy m et h yl)-2,3-d ih y-
d r oin d ole (39). A solution of N-Me-19 (3.5 mg, 0.015
mmol) in CH3OH (1.5 mL) under Ar at 0 °C was treated
with 0.01 N TfOH (180 µL, 0.12 equiv) and allowed to
warm to 25 °C for 5 h. The reaction was quenched with
NaHCO3 (2.5 mg, 0.03 mmol), diluted with EtOAc,
filtered through Celite, and concentrated. Flash chroma-
tography (SiO2, 1.0 × 3 cm, 30% EtOAc/hexane) provided
pure 39 (2.8 mg, 71%) as a colorless oil as the only
detectable reaction product: 1H NMR (C6D6, 250 MHz)
δ 7.70 (s, 1H), 3.78 (s, 3H), 3.45 (s, 3H), 3.45 (s, 3H), 3.38
(m, 1H), 3.15 (dd, J ) 8.4, 9.6 Hz, 1H), 2.95 (m, 2H), 2.82
(s, 3H), 2.80 (s, 3H), 2.52 (dd, J ) 7.1, 9.6 Hz, 1H), 2.35
F igu r e 6.
bubbled through the solution for 1 h at 25 °C. The
reaction mixture was then filtered through Celite, and
the Celite was washed thoroughly with EtOAc. The
solution was then concentrated under reduced pressure
and subjected to HPLC purification. Semipreparative
reverse-phase HPLC (Waters Bondapak C-18 column,
300 Å, 25 × 100 mm, gradient 10-100% CH3CN/H2O
over 20 min, 10 mL/min afforded 46 (tR ) 17.0 min, 1.7
mg, 82%) as a bright yellow oil: 1H NMR (acetone-d6,
400 MHz) δ 8.31 (s, 1H), 8.05 (dd, J ) 1.1, 7.8 Hz, 1H),
7.96 (m, 3H), 7.85-7.78 (m, 2H), 7.72 (m, 1H), 7.65-7.60
(m, 2H), 7.55 (m, 1H), 4.55 (dd, J ) 10.0, 11.6 Hz, 1H),
4.28 (dd, J ) 5.2, 11.6 Hz, 1H), 4.18 (dd, J ) 6.5, 11.3
1H), 4.12-4.08 (m, 2H); IR (film) νmax 2925, 2854, 1687,
1650, 1596 cm-1; FABHRMS (NBA/NaI) m/z 402.0813
(C24H16NO3Cl + H+ requires 402.0819).
Rep r esen ta tive Solvolysis Rea ctivity. A sample of
N-BOC-19 (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 30 s until
completion. The decrease in the long wavelength absorp-
tion at 397 nm was monitored, as was the absorption at
285 nm, Figure 6. The solvolysis rate constant (k ) 7.28
× 10-3 s-1) and the half-life (t1/2 ) 95 s) were calculated
from data recorded at the long wavelength from the least-
squares treatment (r2 ) 0.99) of the slope of the plot of
time versus ln [(Af - Ai)/(Af - A)].
(s, 3H); IR (film) νmax 3254, 2930, 2827, 1474, 1452 cm-1
;
FABHRMS (NBA) m/z 267.1480 (M+, C14H21NO4 requires
267.1471).
Solvolysis Regioselectivity: 3-(Ch lor om eth yl)-5,7-
d im et h oxy-1,6-d im et h yl-4-h yd r oxy-2,3-d ih yd r oin -
d ole (38). A solution of N-Me-19 (3.6 mg, 0.015 mmol)
in THF (1.5 mL) under Ar at -78 °C was treated with
3.6 N HCl/EtOAc (85 µL, 0.03 mmol) and stirred 10 min.
The solvents were removed under reduced pressure, and
the residue was dissolved in H2O (3 mL), treated with
NaHCO3 (2.5 mg, 0.03 mmol), and quickly extracted with
Et2O (10 mL). The organic layer was dried (Na2SO4), and
concentrated under reduced pressure to yield pure 38 (3.7
mg, 89%) as a colorless oil as the only detectable reaction
product: 1H NMR (C6D6, 250 MHz) δ 5.08 (s, 1H), 3.96
(dd, J ) 3.4, 10.1, 1H), 3.66 (m, 1H), 3.49 (t, J ) 10.1
Hz, 1H), 3.35 (s, 3H), 3.29 (dd, J ) 4.4, 9.8 Hz, 1H), 3.15
(s, 3H), 3.02 (t, J ) 9.5 Hz, 1H), 2.72 (s, 3H), 2.14 (s,
3H); IR (film) νmax 3383, 2939, 2850, 1476, 1456 cm-1
;
FABHRMS (NBA) m/z 271.0985 (M+, C13H18ClNO3 re-
quires 267.1471).
Solvolysis Regioselectivity: 5,7-Dim eth oxy-1,6-
dim eth yl-4-h ydr oxy-3-(h ydr oxym eth yl)-2,3-dih ydr o-
in d ole (40). A solution of N-Me-19 (2.1 mg, 9 µmol) in
1:1 THF/H2O (1.8 mL) under Ar at 0 °C was treated with
0.01 N TfOH (107 µL, 0.12 equiv) and stirred 3 days at
25 °C. The reaction mixture was then extracted with
Et2O, dried (Na2SO4), and subjected to flash chromatog-
raphy (SiO2, 1.0 × 5 cm, 70% EtOAc/hexane) which
yielded pure 40 (2.0 mg, 88%) as a colorless oil as the
only detectable reaction product: 1H NMR (C6D6, 400
MHz) δ 7.11 (s, 1H), 3.60 (s, 3H), 3.42 (t, J )10.5 Hz,
1H), 3.25 (m, 2H), 2.92 (t, J ) 9.2 Hz, 1H), 2.77 (s, 3H),
2.50 (dd, J ) 6.6, 9.2 Hz, 1H), 2.29 (s, 3H); IR (film) νmax
3359, 3251, 2933, 2862, 2830, 1462, 1453 cm-1; FAB-
HRMS (NBA) m/z 253.1321 (M+, C13H19NO4 requires
253.1314).
The same procedure was also carried out at pH 7, and
for N-PIV-19 (390 nm) and N-Me-19 (445 nm) at both
pH 3 and pH 7.
Solvolysis Regioselectivity: 1-(ter t-Bu tyloxyca r -
bon yl)-3-(ch lor om eth yl)-5,7-d im eth oxy-4-h yd r oxy-
6-m eth yl-2,3-d ih yd r oin d ole (18). A solution of N-BOC-
19 (3.6 mg, 15.0 µmol) in THF (1.5 mL) under Ar at -78
°C was treated with 3.6 N HCl/EtOAc (85 µL, 30.0 µmol)
and stirred 10 min. The solvents were removed under
reduced pressure to yield pure 18 (3.7 mg, 89%).
Solvolysis Regioselectivity: 1-(ter t-Bu tyloxyca r -
bon yl)-5,7-dim eth oxy-4-h ydr oxy-3-(m eth oxym eth yl)-
6-m eth yl-2,3-d ih yd r oin d ole (37). A solution of N-BOC-
Su bstr a te Red u ction by DT Dia p h or a se (NQO1):
HP LC Assa y. To measure the ability of NQO1 to reduce
the synthetic quinones, metabolism was followed by

8362 J . Org. Chem., Vol. 64, No. 22, 1999
Boger and Garbaccio
HPLC measuring NADH consumption, loss of the parent
quinone peak, and appearance of the hydroquinone
peak.37 NADH and the quinones were separated on a
Waters HPLC system (W-600 Pumps and Controller, 996
PDA detector (340 nm)) with a Waters Bondapak C-18
column (125 Å, 3.9 × 300 mm). Separation was ac-
complished using a linear gradient of 5-95% CH3OH-
buffer over 12 min (with 10 mM potassium phosphate
(pH 6.0) as the aqueous buffer) followed by 95% CH3OH
for 8 min. Retention times were as follows: mitomycin
A (5) (14.0 min), mitomycin C (4) (12.3 min), 24 (17.1
min), 43 (17.4 min), and 46 (17.3 min). Relative rates of
metabolism were calculated through autointegration as
loss of NADH, as the quinones were reversibly reduced
and were determined from the integration of the corre-
sponding peaks at t ) 0 min and t ) 35 min. A
representative trace depicting the oxidation of NADH and
the reduction of 24 in the presence of mitomycin C (4) is
shown below (Figure 7).
Ack n ow led gm en t. We gratefully acknowledge the
financial support of the National Institutes of Health
(CA41986), The Skaggs Institute for Chemical Biology,
the award of an ACS Organic Division graduate fellow-
ship sponsored by Zeneca Pharmaceuticals Group and
an Eli Lilly graduate fellowship (R.M.G.). We thank
Shannon Winski and Professor David Ross of the
University of Colorado for the generous sample of
recombinant human DT-Diaphorase (NQO1) and their
helpful discussions. In addition, we thank Dr. Dolatrai
Vyas from Bristol Myers Squibb for providing a sample
F igu r e 7.
of mitomycin A and Qing J in for conducting the cyto-
toxicity studies.
Su p p or tin g In for m a tion Ava ila ble: Full details of an
alternative synthesis of N-BOC-19 and the preparation and
cyclization of N-PIV-19 and N-Me-19 and copies of H NMR
spectra of the new compounds disclosed herein. This material
is available free of charge via the Internet at http://pubs.acs.org.
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