Synthesis and Evaluation of Duocarmycin and CC-1065 Analogues Containing Modifications in the Subunit Linking Amide

Article abstract of DOI:10.1021/jo990452y

The preparation and evaluation of 6 and 7, analogues of the duocarmycins and CC-1065 in which the subunit linking amide has been replaced with an amidine and thioamide, are described. Consistent with the increased electron-withdrawing properties and conjugation of thioamides relative to amides, 7 showed increased solvolysis reactivity (t_1/2, 160 h versus 230 h) at pH 3, attributable to a diminished vinylogous amide stabilization of the reacting alkylation subunit. Amidine 6 proved to be even more unstable (t_1/2, 12 h) despite the diminished electron-withdrawing properties, but underwent preferential N² amidine linkage hydrolysis rather than solvolysis of the alkylation subunit, attributable to preferential N² vinylogous amide versus amidine conjugation. The natural isomers (+)-6 and (+)-7 exhibited an identical DNA alkylation selectivity as (+)-CBI-TMI and (+)-duocarmycin SA but were less efficient (10-100x). Biological studies of (+)-6 and (+)-7 (0.75 and 1.1 nM, respectively) indicated the analogues retained good cytotoxic activities (L1210), but were less potent than (+)-duocarmycin SA (0.01 nM, 100x) and (+)-CBI-TMI (0.02 nM, 50x). The enhanced properties of the linking amide versus amidine or thioamide established the N² amide as the optimal linking unit examined to date and revealed that it provides a beautiful balance between competing amide (reactivity) and vinylogous amide (stability) conjugation.

Full text of DOI:10.1021/jo990452y

J . Org. Chem. 1999, 64, 5241-5244
5241
Syn th esis a n d Eva lu a tion of Du oca r m ycin a n d CC-1065 An a logu es
Con ta in in g Mod ifica tion s in th e Su bu n it Lin k in g Am id e
Dale L. Boger,* Alejandro Santilla´n, J r., Mark Searcey, 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 March 12, 1999
The preparation and evaluation of 6 and 7, analogues of the duocarmycins and CC-1065 in which
the subunit linking amide has been replaced with an amidine and thioamide, are described.
Consistent with the increased electron-withdrawing properties and conjugation of thioamides
relative to amides, 7 showed increased solvolysis reactivity (t1/2, 160 h versus 230 h) at pH 3,
attributable to a diminished vinylogous amide stabilization of the reacting alkylation subunit.
Amidine 6 proved to be even more unstable (t1/2, 12 h) despite the diminished electron-withdrawing
properties, but underwent preferential N2 amidine linkage hydrolysis rather than solvolysis of the
alkylation subunit, attributable to preferential N2 vinylogous amide versus amidine conjugation.
The natural isomers (+)-6 and (+)-7 exhibited an identical DNA alkylation selectivity as (+)-CBI-
TMI and (+)-duocarmycin SA but were less efficient (10-100×). Biological studies of (+)-6 and
(+)-7 (0.75 and 1.1 nM, respectively) indicated the analogues retained good cytotoxic activities
(L1210), but were less potent than (+)-duocarmycin SA (0.01 nM, 100×) and (+)-CBI-TMI (0.02
nM, 50×). The enhanced properties of the linking amide versus amidine or thioamide established
the N2 amide as the optimal linking unit examined to date and revealed that it provides a beautiful
balance between competing amide (reactivity) and vinylogous amide (stability) conjugation.
The duocarmycins and CC-1065 (1-3) are the parent
members of an exceptionally potent class of antitumor
antibiotics which derive their biological activity through
the sequence selective alkylation of duplex DNA.1-6 A key
feature shared by 1-3 is the amide linking the DNA
binding and alkylation subunits. The important contribu-
tion of the N2 atom to the unusual stability of the
alkylation subunit via vinylogous amide conjugation has
been described, and studies have revealed an apparent
appropriate balance of reactivity versus stability with the
presence of the linking N2 amide.7-9 Replacement of the
linking amide with a methylene in CBI-TMI (4) provided
an analogue 5 possessing a fully engaged vinylogous
amide which exhibited extraordinary stability (t1/2 ) 3.5
years, pH 3), a loss of virtually all biological activity, and
the inability to alkylate DNA even under extreme reac-
tion conditions (37 °C, 0.1 M, 2 weeks).10 Alternatively,
removal of the nitrogen atom provided an exceptionally
reactive carbocycle alkylation subunit (t1/2 ) 4.0 h, pH
7).8 These studies establish the N2 site as the source of
stabilization and that disruption of the vinylogous amide
conjugation via a binding-induced conformational change
within minor groove AT-rich sites of DNA activates the
agents for nucleophilic attack (DNA alkylation cataly-
sis).11 To further probe the critical role of the N2 amide,
herein we report the synthesis and evaluation of the CBI-
(1) Boger, D. L.; Boyce, C. W.; Garbaccio, R. M.; Goldberg, J . Chem.
Rev. 1997, 97, 787. 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. Boger, D. L.; Coleman, R. S. In Studies in Natural Products
Chemistry; u-Rahman, A., Ed.; Elsevier: Amsterdam, 1989; Vol. 3, p
301.
(2) 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. H. 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;
Maryanoff, B. E., Maryanoff, C. E., Eds; 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: Dordrecht, The Netherlands,
1990; 531.
(3) Boger, D. L.; Ishizaki, T.; Zarrinmayeh, H.; Kitos, P. A.; Sun-
tornwat, O. J . Org. Chem. 1990, 55, 4499. Boger, D. L.; Ishizaki, T.;
Zarrinmayeh, H.; Munk, S. A.; Kitos, P. A.; Suntornwat, O. J . Am.
Chem. Soc. 1990, 112, 8961. Boger, D. L.; Munk, S. A.; Zarrinmayeh,
H.; Ishizaki, T.; Haught, J .; Bina, M. Tetrahedron 1991, 47, 2661.
Boger, D. L.; Ishizaki, T.; Zarrinmayeh, H. J . Am. Chem. Soc. 1991,
113, 6645. 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. Boger, D. L.;
J ohnson, D. S.; Yun, W. J . Am. Chem. Soc. 1994, 116, 1635.
(4) Boger, D. L.; J ohnson, D. S. J . Am. Chem. Soc. 1995, 117, 1443.
Boger, D. L.; J ohnson, D. S.; Yun, W.; Tarby, C. M. Bioorg. Med. Chem.
1994, 2, 115. Boger, D. L.; Zarrinmayeh, H.; Munk, S. A.; Kitos, P. A.;
Suntornwat, O. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 1431. Boger,
D. L.; Munk, S. A.; Zarrinmayeh, H. J . Am. Chem. Soc. 1991, 113,
3980. Boger, D. L.; Coleman, R. S.; Invergo, B. J .; Sakya, S. M.;
Ishizaki, T.; Munk, S. A.; Zarrinmayeh, H.; Kitos, P. A.; Thompson, S.
C. J . Am. Chem. Soc. 1990, 112, 4623.
(5) Sugiyama, H.; Hosoda, M.; Saito, I.; Asai, A.; Saito, H. Tetrahe-
dron Lett. 1990, 31, 7197. 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.
(6) Reynolds, V. L.; Molineux, I. J .; Kaplan, D. J .; Swenson, D. H.;
Hurley, L. H. Biochemistry 1985, 24, 6228. Hurley, L. H.; Lee, C.-S.;
McGovren, J . P.; Warpehoski, M. A.; Mitchell, M. A.; Kelly, R. C.;
Aristoff, P. A. Biochemistry 1988, 27, 3886. Hurley, L. H.; Warpehoski,
M. A.; Lee, C.-S.; McGovren, J . P.; Scahill, T. A.; Kelly, R. C.; Mitchell,
M. A.; Wicnienski, N. A.; Gebhard, I.; J ohnson, P. D.; Bradford, V. S.
J . Am. Chem. Soc. 1990, 112, 4633.
(7) Boger, D. L.; Turnbull, P. J . Org. Chem. 1998, 63, 8004.
(8) Boger, D. L.; Turnbull, P. J . Org. Chem. 1997, 62, 5849.
(9) Boger, D. L.; Nishi, T.; Teegarden, B. R. J . Org. Chem. 1994,
59, 4943.
(10) Boger, D. L.; Santilla´n, A., J r.; Searcey, M.; J in, Q. J . Am. Chem.
Soc. 1998, 120, 11554.
10.1021/jo990452y CCC: $18.00 © 1999 American Chemical Society
Published on Web 06/24/1999

5242 J . Org. Chem., Vol. 64, No. 14, 1999
Boger et al.
Sch em e 1
Ta ble 1. Solvolysis Stu d ies
agent
t1/2 (h)a
k (h-1
)
λmax
F igu r e 1.
CBI-TMI (4)
230
12
160
3.05 × 10-3
5.78 × 10-2
4.44 × 10-3
359
306b
366
6
7
TMI analogues 6 and 7 in which the linking amide has
been replaced with an amidine and thioamide.
a
b
Values determined at pH 3. CH3OH.
Syn th esis. The initial synthesis of thioamide 7 em-
ployed the seco precursor 8 first enlisted for the prepara-
tion of 5.10 Thus, treatment of 810 with base (DBU, 3
equiv, 25 °C, 30 min) yielded the thioamide linked
analogue 7 (30%), Scheme 1. A modification of a proce-
dure for the preparation of dithioesters from thioamides12
was enlisted to generate amidine 6 by substituting the
use of ammonia for hydrogen sulfide. Thus, activation of
thioamide 8 (CH3I, 15 equiv, CH3CN, 25 °C, 24 h)
followed by treatment with excess ammonia (THF, -78
°C) yielded the desired amidine linked precursor 9 in
excellent yield (91%). Suppression of spirocyclization to
6 was achieved by slow warming of the reaction mixture
over an extended period of time (20 h). Base initiated
cyclization of 9 (DBU, 3 equiv, 25 °C, 30 min) provided
the amidine linked analogue 6 (34%).
Alternatively, the synthesis of 9 was achieved by first
coupling13 the benzyl protected alkylation subunit pre-
cursor 11 with 5,6,7-trimethoxyindole-2-carboxylic acid
yielding the benzyl protected seco-CBI-TMI 12 (53%).
Thionation using Lawesson’s reagent ((pMeOPhPS2)2,
C6H6, 80 °C, 1.5 h) proceeded in improved yield (88%)
versus the corresponding free phenol (57%)10 for which
the more efficient conversion is attributed to the in-
creased solubility of 12 in benzene. Thioamide to amidine
conversion using the protocol detailed above proceeded
in excellent yield (95%). Final removal of the benzyl group
was achieved with TFA14 (70 °C, 80%) or upon hydroge-
nation (Pd-C, CH3OH, 100%).
carmycins and their analogues have provided insights
into their chemical and biological properties including the
demonstration of the source and extent of the alkylation
subunit stabilization. Replacement of the linking amide
with an amidine potentially could lead to enhanced
stabilization due to its decreased electron-withdrawing
effect, or the increased basicity of the amidine itself may
facilitate hydrolysis of the linkage site competitive with
solvolysis of the alkylation subunit. Conversely, the
slightly more powerful electron-withdrawing properties
of thioamides versus amides (σp ) 0.00 and 0.12, respec-
tively, for -NHC(X)CH3, X ) S, O)15 would be expected
to diminish the stabilizing vinylogous amide conjugation
resulting in enhanced reactivity. Analogous predictions
may be reached by considering the relative extent of
nitrogen conjugation in amides, amidines, and thioamides
established by measuring rotational barriers about the
C-N bond of N,N-dimethylacetamide derivatives (CH3C-
()X)-N(CH3)2, X ) O, NH, S).16 These studies estab-
lished the thioamide as having the highest barrier of
rotation followed by the amide and amidine (20.7, 18.1,
and 12.8 kcal/mol, respectively), indicative of enhanced
thioamide conjugation relative to amide and amidines
which would compete with and diminish the stabilizing
vinylogous amide conjugation.
The solvolysis of 4, 6, and 7 were conducted at pH 3
and monitored by UV (Table 1). Amide 4 (CBI-TMI) and
thioamide 7 behaved analogous to agents previously
studied in which a hypochromic shift was followed by UV.
Consistent with expectations, the thioamide 7 (t1/2, 160
h) exhibited an increased reactivity relative to the amide
(t1/2, 230 h) which proved to be the most stable agent in
Solvolysis of 6 a n d 7. Acid-catalyzed solvolysis stud-
ies of the alkylation subunits of CC-1065 and the duo-
(11) 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.
(12) J urayj, J .; Cushman, M. Tetrahedron 1992, 48, 8601.
(13) 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.
(14) Murphy, B. P.; Banks, H. D. Synth. Commun. 1985, 15, 321.
(15) Hansch, C.; Leo, A.; Enger, S. H.; Kim, K. H.; Nikaitani, D.;
Lien, E. J . J . Med. Chem. 1973, 16, 1207.
(16) Gilli, G.; Bertolasi, V.; Bellucci, F.; Ferretti, V. J . Am. Chem.
Soc. 1986, 108, 2420.

Duocarmycin and CC-1065 Analogues
J . Org. Chem., Vol. 64, No. 14, 1999 5243
Ta ble 2. In Vitr o Cytotoxic Activity
Sch em e 2
agent
IC50, nM (L1210)
natural enantiomer
(+)-duocarmycin SA
(+)-CBI-TMI (4)
0.01
0.02
(+)-6 (amidine)
0.75 (0.75)a
1.10 (1.00)a
(+)-7 (thioamide)
unnatural enantiomer
(-)-duocarmycin SA
(-)-CBI-TMI (4)
(-)-6 (amidine)
(-)-7 (thioamide)
0.10
0.90
70 (180)a
100 (210)a
a
The value in parentheses is the IC50 value established for the
corresponding seco precursors 8 or 9.
and amidine linked compounds alkylated DNA in a
manner analogous to CBI-TMI and retained the same
DNA alkylation sequence selectivity. Only adenine-N3
alkylation was detected under the conditions of limiting
agent and excess DNA. Therefore, changes in the linking
amide structure had no effect on the DNA alkylation
sequence selectivity of the agents.
In previous studies of the CBI-based analogues, the
natural enantiomer consistently proved to be 50-100×
more potent and effective at alkylating DNA than the
unnatural isomer.18 The thioamide and amidine-linked
compounds exhibited this same trend, with the unnatural
isomers being 100-fold less effective at alkylating DNA
than the natural enantiomers.
the series. In contrast to simple expectations, the amidine
6 (t1/2, 12 h) was the least stable analogue in the series.
Moreover, and unlike the behavior of 4 and 7, the initial
UV traces of amidine 6 indicated a large hypochromic
shift (decrease in intensity) indicative of protonation
followed by the slower appearance of two peaks (λmax
)
312 and 335 nm) consistent with the formation of CBI.
Under acidic conditions, protonation would be expected
to occur at the imino nitrogen (pKa 10.7),17 thus activating
the agent to hydrolysis rather than solvolysis of the
alkylation subunit.
The only significant difference in the agents rested
with the relative efficiency of DNA alkylation. Both the
thioamide 7 and the amidine 6 alkylated DNA 10-100×
less effectively than CBI-TMI. In part, this difference
presumably reflects the relative stability of the agents
and is similarly reflected in the cytotoxic activity of the
agents described below. Interestingly, both the DNA
alkylation efficiency and the cytotoxic potency of the
amidine 6 indicates that its DNA alkylation reaction
effectively competes with amidine hydrolysis under the
assay conditions.
Cytotoxic Activity. The cytotoxic activities of the
thioamide and amidine analogues and their seco deriva-
tives were established and are summarized in Table 2.
Previous studies have shown that the seco and ring-
closed agents possess identical activities, and a similar
finding was observed for the modified derivatives 6 and
7.1 Further consistent with previous studies, the natural
enantiomers exhibited more potent cytotoxic activity
(100×) than their unnatural counterparts. Consistent
with their relative DNA alkylation effectiveness and
relative stabilities, the modified analogues 6 and 7 were
less potent than (+)-DSA (100×) and (+)-CBI-TMI (50×),
establishing the amide as the optimum linkage unit.
Discu ssion a n d Con clu sion s. The role of the linking
amide shared by 1-3 has been further probed with the
preparation and evaluation of 6 and 7. The amide to
amidine and thioamide conversion led to an agent more
Preparative scale solvolysis reactions were conducted
to determine and confirm the product profile. Treatment
of 6 in MeOH with CF3SO3H (0.24 equiv, 72 h) provided
CBI (15, 87%) as the only product (Scheme 2). Even
conducting the reaction in the absence of an acid catalyst
(60 h) yielded CBI (15, 95%) and the O-methylimidate
16 (71%). This latter observation suggests that the
hydrolytic instability of amidine 6 may be due to more
than imino nitrogen protonation under acidic conditions,
but additionally derived from preferential vinylogous
amide conjugation within the alkylation subunit rather
than amidine conjugation itself. In contrast to 6, both 4
and 7 underwent typical acid-catalyzed solvolysis with
no evidence of hydrolysis of the linking amide or thioa-
mide.
DNA Alk yla tion P r op er ties of 6 a n d 7. The DNA
alkylation properties of both enantiomers of 6 and 7 were
examined within w794 duplex DNA, a 144 base pair
segment of duplex DNA for which comparative results
are available for related agents.1,2 Following treatment
of 5′-32P-labeled duplex DNA with a range of agent
concentrations at 25 °C for 24 h, the unbound agents were
removed by EtOH precipitation of the DNA. Subsequent
redissolution of the DNA in buffer, thermolysis (100 °C,
30 min) to induce strand cleavage at sites of alkylation,
followed by denaturing, high-resolution polyacryamide
gel electrophoresis (PAGE) next to Sanger sequencing
standards revealed the DNA cleavage and alkylation
sites.
susceptible to solvolysis in the case of the thioamide (t1/2
,
230 and 160 h, respectively), and one far more susceptible
to hydrolysis of the linking site in the case of the amidine
(t1/2, 12 h). The former is consistent with the greater
A representative comparison of 6 and 7 alongside (+)-
duocarmycin SA (1), (+)-CBI-TMI (4) and (-)-CBI-TMI
is illustrated in the figure in Supporting Information.
There are a number of important conclusions that can
be made from the comparisons. First, both the thioamide
(18) Boger, D. L.; Ishizaki, T.; Kitos, P. A.; Suntornwat, O. J . Org.
Chem. 1990, 55, 5823. 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.; Boyce, C. W.; Cai, H., J in, Q.; Kitos, P. A. J . Org. Chem.
1996, 61, 4894. Boger, D. L.; Mesini, P. J . Am. Chem. Soc. 1994, 116,
11335; 1995, 117, 11647.
(17) Oszczapowicz, J .; Raczyn´ska, E.; Pawlick, T. Polish J . Chem.
1984, 58, 117.

5244 J . Org. Chem., Vol. 64, No. 14, 1999
Boger et al.
believed to be obscured by the solvent peak at 30 ppm; IR (film)
νmax 3284, 1614, 1562, 1410, 1317, 1121 cm-1; UV (CH3OH)
λmax 306 nm (ꢀ 15700); FABHRMS (NBA/NaI) m/z 452.1574
(M+ + Na, C25H23N3O4 requires 452.1586).
thioamide conjugation reducing the effective vinylogous
amide stabilization of the alkylation subunit. The ease
in which the latter hydrolysis of the subunit linking
amidine was observed even at neutral pH indicates
preferential vinylogous amide conjugation and stabiliza-
tion at the expense of the stability of the linking amidine.
Both observations are consistent with the predicted
relative conjugation derived from estimates of barriers
to rotation:16 thioamide (20.7 kcal/mol) > amide (18.1
(+)-(8a R,9bS)-6. Pale yellow solid: [R]2D3 +30 (c 0.0002,
CH3OH).
(-)-(8a S,9bR)-6. Pale yellow solid: [R]2D3 -30 (c 0.0002,
CH3OH).
N2-[(5,6,7-Tr im eth oxyin d ol-2-yl)th ioca r bon yl]-1,2,9,9a -
tetr a h yd r ocyclop r op a [c]ben z[e]in d ol-4-on e (7). Yellow
semisolid: 1H NMR (acetone-d6, 500 MHz) δ 10.30 (br s, 1H),
8.04 (d, J ) 7.8 Hz, 1H), 7.60 (t, J ) 7.8 Hz, 1H), 7.42 (t, J )
7.8 Hz, 1H), 7.22 (d, J ) 7.8 Hz, 1H), 6.99 (d, J ) 2.2 Hz, 1H),
6.87 (s, 1H), 5.83 (s, 1H), 4.64 (dd, J ) 4.9, 12.0 Hz, 1H), 4.33
(d, J ) 12.0 Hz, 1H), 3.96 (s, 3H), 3.86 (s, 3H), 3.84 (s, 3H),
3.12 (dt, J ) 4.9, 5.2, 7.8 Hz, 1H), 2.13 (t, J ) 4.9 Hz, 1H),
2.00 (dd, J ) 4.9, 7.8 Hz, 1H); 13C NMR (acetone-d6, 125 MHz)
δ 192.2, 184.5, 161.7, 151.6, 142.2, 141.7, 140.1, 138.1, 133.4,
133.0, 128.4, 127.3, 127.0, 124.5, 123.1, 112.4, 108.1, 99.0, 61.5,
60.5, 56.5, 34.1, 32.4, 28.9, 25.9; IR (film) νmax 3298, 1732, 1615,
1372, 1299, 1236, 1112, 1047 cm-1; UV (pH 3 buffer) λmax 366
nm (ꢀ 15300), UV (CH3OH) λmax 365 nm (ꢀ 14700); FABHRMS
(NBA/NaI) m/z 447.1391 (M+ + H, C25H22N2O4S requires
447.1379).
kcal/mol) > vinylogous amide (12.2-14.5 kcal/mol)19
>
amidine (12.8 kcal/mol). The analogues showed identical
sequence selectivity patterns as (+)-duocarmycin SA and
(+)-CBI-TMI, but both were less efficient (10-100×) at
alkylating DNA. Consistent with these observations, the
cytotoxic activity of 6 and 7 was 50-100× less potent
than (+)-CBI-TMI and (+)-duocarmycin SA. The en-
hanced properties of the amide versus amidine or thio-
amide establish it as the optimal linking unit examined
to date, representing a beautiful balance between reac-
tivity and stability resulting from competing amide and
vinylogous amide conjugation.
(+)-(8a R,9bS)-7. Yellow semisolid: [R]2D3 +580 (c 0.0005,
EtOAc).
Exp er im en ta l Section
(-)-(8a S,9bR)-7. Yellow semisolid: [R]2D3 -576 (c 0.0005,
EtOAc).
N2-[(5,6,7-Tr im eth oxyin dol-2-yl)im in ocar bon yl]-1,2,9,9a-
tetr a h yd r ocyclop r op a [c]ben z[e]in d ol-4-on e (6). Pale yel-
low solid: mp 140-142 °C dec; 1H NMR (acetone-d6, 500 MHz)
δ 10.51 (br s, 1H), 8.99 (br s, 1H), 8.00 (d, J ) 7.6 Hz, 1H),
7.51 (t, J ) 7.6 Hz, 1H), 7.34 (t, J ) 7.6 Hz, 1H), 7.12 (d, J )
7.6 Hz, 1H), 6.88 (s, 1H), 6.74 (d, J ) 1.5 Hz, 1H), 5.37 (s,
1H), 4.43 (dd, J ) 5.1, 11.2 Hz, 1H), 3.95 (d, J ) 11.2 Hz, 1H),
3.93 (s, 3H), 3.84 (s, 3H), 3.80 (s, 3H), 3.04 (dt, J ) 4.9, 5.1,
7.8 Hz, 1H), 1.93 (t, J ) 4.9 Hz, 1H), 1.80 (dd, J ) 4.9, 7.8 Hz,
1H); 13C NMR (acetone-d6, 125 MHz) δ 184.0, 163.8, 151.1,
141.7, 140.9, 140.2, 133.8, 132.4, 132.3, 132.2, 126.9, 126.8,
126.7, 124.5, 122.8, 106.7, 106.1, 98.9, 61.5, 56.6, 55.4, 34.3,
24.9, the two remaining peaks were not observed and are
Ack n ow led gm en t. We gratefully acknowledge the
financial support of the National Institutes of Health
(CA41986) and the Skaggs Institute for Chemical Biol-
ogy.
Su p p or tin g In for m a tion Ava ila ble: Experimental de-
tails for the preparation of 6, 7, 9, and 12-14, solvolysis
studies of 4, 6, and 7 and the characterization of 16, experi-
mental details for the DNA alkylation studies, a gel figure
1
comparing the DNA alkylation of 1, 4, 6, and 7, and H NMR
spectra of 6, 7, 8, 9, 12, 13, 14, and 16 are provided. This
material is available free of charge via the Internet at
http://pubs.acs.org.
(19) Gottlieb, H. E.; Braverman, S.; Levinger, S. J . Org. Chem. 1990,
55, 3655; Filleux-Blanchard, M. L.; Mabon, F.; Martin, G. J . Tetrahe-
dron Lett. 1974, 3907.
J O990452Y