Rational design, synthesis, and evaluation of key analogues of CC-1065 and the duocarmycins

Article abstract of DOI:10.1021/ja073989z

The design, synthesis, and evaluation of a predictably more potent analogue of CC-1065 entailing the substitution replacement of a single skeleton atom in the alkylation subunit are disclosed and were conducted on the basis of design principles that emerg

Full text of DOI:10.1021/ja073989z

Published on Web 10/19/2007
Rational Design, Synthesis, and Evaluation of Key Analogues
of CC-1065 and the Duocarmycins
Mark S. Tichenor,† Karen S. MacMillan,† James S. Stover,† Scott E. Wolkenberg,†
Maria G. Pavani,‡ Lorenzo Zanella,‡ Abdel N. Zaid,‡ Gianpiero Spalluto,‡
Thomas J. Rayl,† Inkyu Hwang,† Pier Giovanni Baraldi,*,‡ and Dale L. Boger*,†
Contribution from the Department of Chemistry and the Skaggs Institute for Chemical Biology,
The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, and
Dipartimento di Scienze Farmaceutiche, UniVersita` degli Studi di Ferrara,
Via Fossato di Mortara 17/19, 44100 Ferrara, Italy
Received June 18, 2007; E-mail: boger@scripps.edu
Abstract: The design, synthesis, and evaluation of a predictably more potent analogue of CC-1065 entailing
the substitution replacement of a single skeleton atom in the alkylation subunit are disclosed and were
conducted on the basis of design principles that emerged from a fundamental parabolic relationship between
chemical reactivity and cytotoxic potency. Consistent with projections, the 7-methyl-1,2,8,8a-tetrahydro-
cyclopropa[c]thieno[3,2-e]indol-4-one (MeCTI) alkylation subunit and its isomer 6-methyl-1,2,8,8a-tetrahy-
drocyclopropa[c]thieno[2,3-e]indol-4-one (iso-MeCTI) were found to be 5-6 times more stable than the
MeCPI alkylation subunit found in CC-1065 and slightly more stable than even the DSA alkylation subunit
found in duocarmycin SA, placing it at the point of optimally balanced stability and reactivity for this class
of antitumor agents. Their incorporation into the key analogues of the natural products provided derivatives
that surpassed the potency of MeCPI derivatives (3-10-fold), matching or slightly exceeding the potency
of the corresponding DSA derivatives, consistent with projections made on the basis of the parabolic
relationship. Notable of these, MeCTI-TMI proved to be as potent as or slightly more potent than the natural
product duocarmycin SA (DSA-TMI, IC50 ) 5 vs 8 pM), and MeCTI-PDE2 proved to be 3-fold more potent
than the natural product CC-1065 (MeCPI-PDE2, IC50 ) 7 vs 20 pM). Both exhibited efficiencies of DNA
alkylation that correlate with this enhanced potency without impacting the intrinsic selectivity characteristic
of this class of antitumor agents.
Introduction
CC-10651 (1) and duocarmycin SA (2)2 represent the key
members of a small class of exceptionally potent naturally
occurring antitumor agents1-4 that derive their biological
properties through a now characteristic sequence-selective
DNA alkylation reaction (Figure 1).5-9 Extensive investiga-
tions on the naturally occurring members of this class as
well as their synthetic analogues have defined key and subtle
† The Scripps Research Institute.
‡ Universita di Ferrara.
(1) CC-1065: Martin, D. G.; Biles, C.; Gerpheide, S. A.; Hanka, L. J.; Krueger,
W. C.; McGovren, J. P.; Mizsak, S. A.; Neil, G. L.; Stewart, J. C.; Visser,
J. J. Antibiot. 1981, 34, 1119-1125.
(2) Duocarmycin SA: Ichimura, M.; Ogawa, T.; Takahashi, K.; Kobayashi,
E.; Kawamoto, I.; Yasuzawa, T.; Takahashi, I.; Nakano, H. J. Antibiot.
1990, 43, 1037-1038.
(3) Duocarmycin A: Takahashi, I.; Takahashi, K.; Ichimura, M.; Morimoto,
M.; Asano, K.; Kawamoto, I.; Tomita, F.; Nakano, H. J. Antibiot. 1988,
41, 1915-1917.
Figure 1. Natural products.
features that contribute to their properties.9,10 Most notable of
these are the structural features that contribute to the AT-rich
noncovalent binding selectivity dominating the alkylation
selectivity,11 those that define the source of catalysis for the
DNA alkylation reaction,12,13 and those that subtly impact
the unusual and intrinsic stability of their alkylation sub-
units.9,13,14
(4) Yatakemycin: Igarashi, Y.; Futamata, K.; Fujita, T.; Sekine, A.; Senda,
H.; Naoki, H.; Furumai, T. J. Antibiot. 2003, 56, 107-113.
(5) Yatakemycin: (a) Parrish, J. P.; Kastrinsky, D. B.; Wolkenberg, S. E.;
Igarashi, Y.; Boger, D. L. J. Am. Chem. Soc. 2003, 125, 10971-
10976. (b) Trzupek, J. D.; Gottesfeld, J. M.; Boger, D. L. Nat. Chem.
Biol. 2006, 2, 79-82. (c) Tichenor, M. S.; Trzupek, J. D.; Kastrinsky,
D. B.; Shiga, F.; Hwang, I.; Boger, D. L. J. Am. Chem. Soc. 2006, 128,
15683-15696. (d) Tichenor, M. S.; MacMillan, K. S.; Trzupek, J. D;
Rayl, T. J.; Hwang, I.; Boger, D. L. J. Am. Chem. Soc. 2007, 129, 10858-
10869.
9
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10.1021/ja073989z CCC: $37.00 © 2007 American Chemical Society

Key Analogues of CC-1065 and the Duocarmycins
A R T I C L E S
Figure 2. Relationship between reactvity (solvolysis -log k, pH 3) and
cytotoxic potency (-log IC50, L1210), natural enantiomers. See Supporting
Information for abbreviations and key to letter notations.
Results and Discussion
Figure 3. Alkylation subunits and AM1 calculated heats of reaction for
hydride addition to the activated cyclopropane.
Design. Throughout the course of our investigations, we have
chronicled a direct relationship between the intrinsic chemical
stability of the alkylation subunit and the cytotoxic potency of
the resulting derivatives.15 Recently, a compilation of the data
derived from more than 30 deep-seated modifications resulted
in the establishment of a well-defined parabolic relationship
between the alkylation subunit reactivity and the resulting
cytotoxic potency that spanned a 104-106 range of reactivities
and activities (Figure 2).16 Presumably, this fundamental
relationship simply reflects the fact that the compound must be
sufficiently stable to reach its biological target yet remain
sufficiently reactive to alkylate DNA once it does. Significantly,
the work defined this optimal balance between reactivity and
stability, providing a fundamental design feature that is subject
to investigational interrogation.
Herein, we report our first such efforts, culminating in the
synthesis and evaluation of 7-methyl-1,2,8,8a-tetrahydrocyclo-
propa[c]thieno[3,2-e]indol-4-one (MeCTI, 3) as well as 6-meth-
yl-1,2,8,8a-tetrahydrocyclopropa[c]thieno[2,3-e]indol-4-one (iso-
MeCTI, 4) and their incorporation into analogues of both CC-
1065 and the duocarmycins (Figure 3).17 The design of MeCTI
and the decision to invest in its exploration rested with
expectations that it would be substantially more stable than the
alkylation subunit found in CC-1065 (MeCPI, 5), leading to a
substantially more potent CC-1065 analogue approaching the
stability and activity of duocarmycin SA. Intuitively, this might
be anticipated to arise from the strain release provided by a
fused thiophene versus pyrrole, which in turn may further benefit
from the intrinsic electron-withdrawing character of a thiophene.
More quantitatively, this increased stability could be ap-
proximated using semiempirical calculations (AM1, MNDO)
for heats of reaction for hydride addition to the activated
cyclopropane (Figure 3). Using this approximation, MeCTI was
selected among several candidate alkylation subunits as being
more stable than CBI (6, 1,2,9,9a-tetrahydrocyclopropa[c]benzo-
[e]indol-4-one) and approaching or exceeding that of DSA (7),
potentially approaching the optimal stability defined by the
parabolic relationship. Significantly, MeCTI represents a single
atom change in the backbone structure of the CC-1065 alkylation
subunit that in turn was projected to provide a nearly optimal
increase in stability and potency (Figure 4).
(6) (a) CC-1065: Hurley, L. H.; Lee, C.-S.; McGovren, J. P.; Warpehoski, M.
A.; Mitchell, M. A.; Kelly, R. C.; Aristoff, P. A. Biochemistry 1988, 27,
3886-3892. (b) 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.; Johnson, P. D.; Bradford, V. S. J. Am. Chem. Soc. 1990, 112,
4633-4649. (c) Boger, D. L.; Johnson, D. S.; Yun, W.; Tarby, C. M.
Bioorg. Med. Chem. 1994, 2, 115-135. (d) 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, 4623-4632.
(7) Duocarmycin A: (a) Boger, D. L.; Ishizaki, T.; Zarrinmayeh, H.; Munk,
S. A.; Kitos, P. A.; Suntornwat, O. J. Am. Chem. Soc. 1990, 112, 8961-
8971. (b) Boger, D. L.; Ishizaki, T.; Zarrimayeh, H. J. Am. Chem. Soc.
1991, 113, 6645-6649. (c) Boger, D. L.; Yun, W.; Terashima, S.; Fukuda,
Y.; Nakatani, K.; Kitos, P. A.; Jin, Q. Bioorg. Med. Chem. Lett. 1992, 2,
759-765.
(8) Duocarmycin SA: Boger, D. L.; Johnson, D. S.; Yun, W. J. Am. Chem.
Soc. 1994, 116, 1635-1656.
(9) Reviews: (a) Boger, D. L.; Johnson, D. S. Angew. Chem., Int. Ed. Engl.
1996, 35, 1438-1474. (b) Boger, D. L. Acc. Chem. Res. 1995, 28, 20-29.
(c) Boger, D. L.; Johnson, D. S. Proc. Natl. Acad. Sci. U.S.A. 1995, 92,
3642-3649. (d) Boger, D. L.; Garbaccio, R. M. Acc. Chem. Res. 1999,
32, 1043-1052.
(10) Warpehoski, M. A.; Gebhard, I.; Kelly, R. C.; Krueger, W. C.; Li, L.;
McGovern, J. P.; Praire, M. D.; Wienienski, N.; Wierenga, W. J. Med.
Chem. 1988, 31, 590-603.
(11) (a) Boger, D. L.; Coleman, R. S.; Invergo, B. J.; Zarrinmayeh, H.; Kitos,
P. A.; Thompson, S. C.; Leong, T.; McLaughlin, L. W. Chem.-Biol. Interact.
1990, 73, 29-52. (b) Boger, D. L.; Zarrinmayeh, H.; Munk, S. A.; Kitos,
P. A.; Suntornwat, O. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 1431-1435.
(c) Boger, D. L.; Munk, S. A.; Zarrinmayeh, H. J. Am. Chem. Soc. 1991,
113, 3980-3983. (d) Boger, D. L.; Johnson, D. S. J. Am. Chem. Soc. 1995,
117, 1443-1444.
(12) Boger, D. L.; Bollinger, B.; Hertzog, D. L.; Johnson, D. S.; Cai, H.; Mesini,
P.; Garbaccio, R. M.; Jin, Q.; Kitos, P. A. J. Am. Chem. Soc. 1997, 119,
4987-4998.
(13) Boger, D. L.; Garbaccio, R. M. Bioorg. Med. Chem. 1997, 5, 263-276.
(14) Reviews: (a) Wolkenberg, S. E.; Boger, D. L. Chem. ReV. 2002, 102, 2477-
2496. (b) Tse, W. C.; Boger, D. L. Chem. Biol. 2004, 11, 1607-1617.
(15) (a) Boger, D. L.; Ishizaki, T. Tetrahedron Lett. 1990, 31, 793-796. (b)
Boger, D. L.; Munk, S. A.; Ishizaki, T. J. Am. Chem. Soc. 1991, 113, 2779-
2780. (c) Boger, D. L.; Yun, W. J. Am. Chem. Soc. 1994, 116, 5523-
5524.
(16) (a) Parrish, J. P.; Hughes, T. V.; Hwang, I.; Boger, D. L. J. Am. Chem.
Soc. 2004, 126, 80-81. (b) Parrish, J. P.; Trzupek, J. D.; Hughes, T. V.;
Hwang, I.; Boger, D. L. Bioorg. Med. Chem. 2004, 12, 5845-5856.
Synthesis of N-Boc-MeCTI. Stobbe condensation of 4-me-
thylthiophene-3-carboxaldehyde (9, 4 equiv of t-BuOK, 6 equiv
(17) (a) Muratake, H.; Hayakawa, A.; Natsume, M. Chem. Pharm. Bull. 2000,
48, 1558-1566. (b) Muratake, H.; Okabe, K.; Takahashi, M.; Tonegawa,
M.; Natsume, M. Chem. Pharm. Bull. 1997, 45, 799-806. (c) Muratake,
H.; Hayakawa, A.; Natsume, M. Tetrahedron Lett. 1997, 38, 7577-7580.
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A R T I C L E S
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Scheme 2
Figure 4. Structure of (+)-MeCTI-PDE2.
Scheme 1
6 equiv of CCl4, 25 °C, 20 h, 95%) provided 20. Intermediate
20 was separated into its two enantiomers by resolution on a
semipreparative Chiralcel OD column (20% i-PrOH/hexane, R
) 1.30).21 Hydrogenolysis of the benzyl ether (25% aqueous
HCO2NH4, 10% Pd/C, 60 °C, 2 h) followed by immediate
spirocyclization of the crude product by treatment with DBU
(10 equiv, DMF-CH3CN, 25 °C, 10 min, 70%) provided each
enantiomer of 3.
Synthesis of MeCTI-TMI, MeCTI-Indole2, and MeCTI-
PDE2. Acid-catalyzed deprotection of 20 (4 N HCl-EtOAc,
25 °C, 1 h) followed by coupling of the resulting hydrochloride
salt with 5,6,7-trimethoxyindole-2-carboxylic acid (22,22 4 equiv
of EDCI, 2 equiv of NaHCO3, 25 °C, 15 h, 92%) provided 23
(Scheme 2). Hydrogenolysis of the benzyl ether (25% aqueous
HCO2NH4, 10% Pd/C, 25 °C, 2 h, 83%) afforded 24, and its
spirocyclization was effected by treatment with DBU (4 equiv,
DMF-CH3CN, 25 °C, 10 min, 70%), providing 25.
A subtle modification of this sequence was used to access
MeCTI-indole2 and MeCTI-PDE2. Thus, acid-catalyzed depro-
tection of 21 (4 N HCl-EtOAc, 25 °C, 1 h) followed by
coupling of the resulting hydrochloride salt with PDE2-CO2H
(26)23 and indole2-CO2H (27) (4 equiv of EDCI, 1.5 equiv of
RCO2H, 25 °C, 0.5 h, 40-72%) provided 28 and 29 (Scheme
3). This coupling with an alkylation subunit precursor in which
the C5-benzyl ether was already removed permitted direct
spirocyclization, effected by treatment with NaHCO3 (4 equiv,
2:1 DMF-H2O, 25 °C, 10 min, 46-81%), providing 8 and 30.
Synthesis of N-Boc-iso-MeCTI. N-Boc-iso-MeCTI was
prepared using a synthetic strategy analogous to the route
described for 3, beginning with the Stobbe condensation of the
isomeric aldehyde 31 (Scheme 4). Compound 42 was resolved
(Chiralcel OD, 5% i-PrOH/hexane, R ) 1.44) and deprotected
(25% aqueous HCO2NH4, 10% Pd/C, 60 °C, 2 h), and the crude
product was spirocyclized using DBU (10 equiv, DMF-CH3-
CN, 25 °C, 4 h, 39%) to give each enantiomer of 4. Notably,
the Ar-3′ spirocyclization of this isomer was much slower and
less facile than that of 3.
of diethyl succinate, 83 °C, 1.5 h) gave a mixture of half-esters
10 which were subjected to Friedel-Crafts acylation (excess
Ac2O/NaOAc, 140 °C, 5 h, 40%) to provide 11 (Scheme 1).18
Compound 11 was hydrolyzed to phenol 12 (1.1 equiv of K2-
CO3, 78 °C, 14 h, 83%), which was protected as the benzyl
ether 13 (1.2 equiv of BnBr, 1.2 equiv of K2CO3, 25 °C, 5 h,
98%). Subsequent hydrolysis of the ethyl ester (3 equiv of LiOH,
4:1:1 THF-MeOH-H2O, 25 °C, 18 h, 98%) followed by
Curtius rearrangement of the resulting carboxylic acid 14 (1.2
equiv of DPPA, 1.2 equiv of Et3N, 83 °C, 18 h, 76%) provided
carbamate 15. Regioselective acid-catalyzed C4-iodination (0.1
equiv of H2SO4, 1.1 equiv of NIS, 25 °C, 2 h, 89%) followed
by carbamate alkylation of 16 with allyl bromide (1.2 equiv of
NaH, 3 equiv of allyl bromide, 25 °C, 3 h, 95%) provided 17.
5-Exo-trig radical cyclization conducted in the presence of
TEMPO19 (2.7 equiv, 3 equiv of (Me3Si)3SiH, 100 °C, 5 h, 78%)
gave 18 in good yield, whereas the more conventional use of
Bu3SnH was unsuccessful.20 Zinc-mediated reductive cleavage
of 18 (16 equiv of Zn, 70 °C, 3 h, 83%) followed by chlorination
displacement of the released primary alcohol (2 equiv of Ph3P,
Synthesis of iso-MeCTI-TMI. Deprotection of 42 (4 N
HCl-EtOAc, 25 °C, 1 h) followed by coupling of the resulting
(18) (a) Boger, D. L.; McKie, J. A.; Cai, H.; Cacciari, B.; Baraldi, P. G. J. Org.
Chem. 1996, 61, 1710-1729. (b) Boger, D. L.; Han, N.; Tarby, C. M.;
Boyce, C. W.; Cai, H.; Jin, Q.; Kitos, P. A. J. Org. Chem. 1996, 61, 4894-
4912. (c) Review of related efforts: Boger, D. L.; Boyce, C. W.; Garbaccio,
R. M.; Goldberg, J. A. Chem. ReV. 1997, 97, 787-828.
(19) Boger, D. L.; McKie, J. A. J. Org. Chem. 1995, 60, 1271-1275.
(20) (a) Boger, D. L.; Boyce, C. W.; Garbaccio, R. M.; Searcey, M. Tetrahedron
Lett. 1998, 39, 2227-2230. (b) Patel, V. F.; Andis, S. L.; Enkema, J. K.;
Johnson, D. A.; Kennedy, J. H.; Mohamadi, F.; Schultz, R. M.; Soose, D.
J.; Spees, M. M. J. Org. Chem. 1997, 62, 8868-8874.
(21) Boger, D. L.; Yun, W. J. Am. Chem. Soc. 1994, 116, 7996-8006.
(22) Boger, D. L.; Ishizaki, T.; Zarrinmayeh, H.; Kitos, P. A.; Suntornwat, O.
J. Org. Chem. 1990, 55, 4499-4502.
(23) (a) Boger, D. L.; Coleman, R. S. J. Am. Chem. Soc. 1987, 109, 2717-
2727. (b) D. L.; Coleman, R. S. J. Am. Chem. Soc. 1988, 110, 1321-
1323; 1988, 110, 4796-4807.
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Key Analogues of CC-1065 and the Duocarmycins
A R T I C L E S
Scheme 3
Scheme 5
Scheme 4
Figure 5. Solvolysis reactivity.
N-Boc-MeCTI (3) and N-Boc-iso-MeCTI (4) underwent mea-
surable solvolysis, monitored spectrophotometrically by UV with
the disappearance of the long-wavelength absorption of the CTI
chromophore and the appearance of a short-wavelength absorp-
tion attributable to the solvolysis product. The Boc derivatives
of the two CTI isomers exhibited nearly indistinguishable
reactivities (t1/2 ) 206 and 209 h), being ca. 5-6-fold more
stable than the alkylation subunit of CC-1065 (N-Boc-MeCPI,
t1/2 ) 37 h),23 significantly more stable than N-Boc-CBI (t1/2
)
133 h),24 and measurably more stable than even N-Boc-DSA
(t1/2 ) 177 h)25 (Figure 5). This places N-Boc-MeCTI (3) and
N-Boc-iso-MeCTI (4) among the most stable alkylation subunits
explored to date that still exhibit a measurable reactivity at
pH 3.
Cytotoxic Activity. The compounds displayed cytotoxic
activity (L1210) consistent with their relative stabilities. Sum-
marized in Figure 6 is the L1210 cytotoxic activity of the MeCTI
and iso-MeCTI derivatives examined along with that of the key
comparison compounds,26 and a full table of comparison
derivatives is provided in the Supporting Information (Tables
S1-S5). The natural enantiomers of N-Boc-MeCTI (IC50 ) 30
nM) and N-Boc-iso-MeCTI (IC50 ) 25 nM) proved to be ca.
hydrochloride salt with 5,6,7-trimethoxyindole-2-carboxylic acid
(22,22 1.4 equiv, 4 equiv of EDCI, 3 equiv of NaHCO3, 25 °C,
15 h, 78%) provided 44 (Scheme 5). Hydrogenolysis of the
benzyl ether (25% aqueous HCO2NH4, 10% Pd/C, 60 °C, 2 h,
83%) afforded 45. Spirocyclization of 45 was particularly slow
(DBU/DMF) with this isomer, such that hydrolysis of the labile
amide was competitive. The use of an alternative solvent system
(2:1 CH3CN/DMF) under strictly anhydrous conditions (6 equiv
of DBU, 25 °C, 1 h, 71%) gave 46 in improved conversions.
Chemical Reactivity. The relative reactivity of the alkylation
subunits was established by measuring their rates of acid-
catalyzed solvolysis. At pH 3 (50% CH3OH-buffer, buffer )
4:1:20 v/v/v 0.1 M citric acid, 0.2 M Na2HPO4, H2O), both
(24) (a) Boger, D. L.; Ishizaki, T.; Wysocki, R. J., Jr.; Munk, S. A.; Kitos, P.
A.; Suntornwat, O. J. Am. Chem. Soc. 1989, 111, 6461-6463. (b) Boger,
D. L.; Ishizaki, T.; Kitos, P. A.; Suntornwat, O. J. Org. Chem. 1990, 55,
5823-5832.
(25) (a) Boger, D. L.; Machiya, K. J. Am. Chem. Soc. 1992, 114, 10056-10058.
(b) Boger, D. L.; Machiya, K.; Hertzog, D. L; Kitos, P. A; Holmes, D. J.
Am. Chem. Soc. 1993, 115, 9025-9036.
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A R T I C L E S
Tichenor et al.
Figure 6. Cytotoxic activity.26
Figure 8. Cytotoxic activity of indole2 derivatives.26
Figure 7. Cytotoxic activity of PDE2 derivatives.26
10-fold more potent than the Boc derivative of the CC-1065
alkylation subunit (N-Boc-MeCPI, IC50 ) 330 nM)23 and 2-3-
fold more potent than (+)-N-Boc-CBI (IC50 ) 80 nM),24 but
4-5-fold less potent than (+)-N-Boc-DSA (IC50 ) 6 nM).25
With the exception of the latter comparison with (+)-N-Boc-
DSA, which exhibits an anomalously potent activity, the
cytotoxic activity of 3 and 4 proved to be in line with
Figure 9. Relationship between reactivity (solvolysis k, pH 3) and cytotoxic
potency (L1210), natural enantiomers.
expectations. The unnatural enantiomers of 3 and 4 (IC50
)
600 nM) exhibited potencies 20-fold less active than the natural
enantiomers and in line with expectations based on the preceding
observations with 5-7.
exhibiting a distinction between enantiomeric activities that is
greater than that observed with duocarmycin SA (10-fold)25 or
CBI-TMI (70-fold).21
Similarly, the natural enantiomers of TMI derivatives 25 and
46 were found to be exceptionally potent cytotoxic agents (IC50
) 5 and 7 pM, respectively), perhaps slightly more active than
even (+)-duocarmycin SA (IC50 ) 8-10 pM)25 and notably
more potent than (+)-CBI-TMI (IC50 ) 30 pM)21 (Figure 6).26
Their unnatural enantiomers, ent-(-)-25 and ent-(-)-46, proved
to be 100-300-fold less active than the natural enantiomers,
Especially interesting is the comparison of MeCTI-PDE2 (8)
with CC-1065 (1) and the synthetic hybrid natural product DSA-
PDE2 (48)5d (Figure 7).26 Consistent with expectations, the
natural enantiomer of MeCTI-PDE2 (IC50 ) 7 pM) was found
to be 3-fold more potent than CC-1065 (IC50 ) 20 pM) and
nearly equipotent with DSA-PDE2 (48, IC50 ) 4 pM). Thus,
the deep-seated change of a single skeleton atom in CC-1065
(NHfS) served to increase its potency 3-fold, in line with
expectations based on its 5-6-fold increased stability.
In a series of indole2 analogues that are more manageable to
work with because of their improved physical properties,
including their solubility in conventional organic solvents, and
which have exhibited efficacious in vivo antitumor activity,10,27
(26) The L1210 cytotoxic activities for (+)-duocarmycin SA (8-10 pM) and
(+)-CC-1065 (20 pM; 23-18 pM) have been tested as standards (>100
times) through the years and side-by-side with the samples disclosed herein.
The small 2-3-fold differences are always observed, and the absolute
potencies always fall in this narrow range indicated ((20%). We have
developed highly refined, reproducible conditions for conducting such
cytotoxic assays ((20%) that avoid the more variable results typically
experienced with such assays. The IC50’s reported in Figures 6-8 are the
average values ((20%) obtained typically from multiple rounds of testing,
and the number of times the samples disclosed herein were tested in
triplicate alongside the standards are as follow: (+)-3, 4×; ent-(-)-3, 2×;
(+)-4, 3×; ent-(-)-4, 3×; (+)-25, 7×; ent-(-)-25, 3×; (+)-46, 3×; ent-
(-)-46, 1×; (+)-8, 4×; (+)-30, 4×; and ent-(-)-30, 3×.
(27) (a) Boger, D. L.; Ishizaki, T.; Sakya, S. M.; Munk, S. A.; Kitos, P. A.; Jin,
Q.; Besterman, J. M. Bioorg. Med. Chem. Lett. 1991, 1, 115-120. (b)
Boger, D. L.; Yun, W.; Han. N. Bioorg. Med. Chem. 1995, 3, 1429-1453.
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Key Analogues of CC-1065 and the Duocarmycins
A R T I C L E S
the MeCTI derivative 30 exhibited a cytotoxic potency precisely
in line with expectations based on its enhanced stability (Figure
8).26 Thus, the natural enantiomer of MeCTI-indole2 (30) was
found to be ca. 8-fold more potent than MeCPI-indole2 (49),10,28
in line with its 5-6-fold increased stability, 2-fold more potent
than CBI-indole2,27 consistent with its 1.5-fold increased stabil-
ity, and ca. 2-fold less potent than DSA-indole2 (1.2-fold
difference in reactivity). Similarly, the unnatural enantiomer of
MeCTI-indole2 (IC50 ) 250 pM) proved to be 50-fold less active
than the natural enantiomer, but similarly 4-fold more potent
than MeCPI-indole2 (IC50 ) 1000 pM) and essentially equipo-
tent with DSA-indole2 (IC50 ) 150 pM).
The Parabolic Relationship. Among the most important of
the features established to date with this class of compounds is
the relationship between chemical stability and biological
potency (cytotoxic activity). This parabolic relationship extends
over a 106-fold range in reactivities (-log k, pH 3) and activities
(-log IC50, L1210), covering an extensive range of modified
alkylation subunits. Consistent with the design features, N-Boc-
MeCTI (pH 3 solvolysis t1/2 ) 206 h) and N-Boc-iso-MeCTI
(pH 3 solvolysis t1/2 ) 209 h) both exhibit a reactivity
comparable to that of N-Boc-DSA (t1/2 ) 177 h) that lies at the
pinnacle of the parabolic relationship at a point that is 5-6 times
more stable than N-Boc-MeCPI. In line with this enhanced
stability, the Boc derivatives of both CTI isomers exhibit a
cytotoxic activity roughly 10-fold more potent than that of
N-Boc-MeCPI (IC50 ) 330 nM) and 4-5-fold less potent than
that of N-Boc-DSA (IC50 ) 6 nM), placing them near expecta-
tions based on the parabolic relationship (Figure 9).26 Impor-
tantly, and aside from the anomalously potent activity of N-Boc-
DSA, this places both MeCTI derivatives among the most
potent analogues examined to date, on par with the activity of
N-Boc-CCBI, further defining the pinnacle of the parabolic
relationship.
DNA Alkylation Selectivity and Efficiency. The DNA
alkylation selectivity of the new analogues was examined within
a 150 base-pair segment of DNA described previously (w794).30
The alkylation site identification and the assessment of the
relative selectivity among the available sites were obtained by
thermally induced strand cleavage of the singly 5′-end-labeled
duplex DNA after exposure to the compounds as detailed.5-8
Since the DNA alkylation properties of members of each class
of these agents have been established in preceding studies, we
focused our analysis on a select set of the new analogues to
simply confirm their DNA alkylation selectivity and relative
efficiency. Most representative of this set are the TMI- and PDE-
based analogues, constituting key analogues of duocarmycin SA
and CC-1065, respectively.
Figure 10. Thermally induced strand cleavage of w794 DNA (144 bp,
nucleotide no. 5238-138) after DNA-agent incubation with duocarmycin
SA, MeCTI-TMI, and CBI-TMI (20 °C, 24 h), removal of unbound agent
by EtOH precipitation, and 30 min thermolysis (100 °C), followed by
denaturing 8% PAGE and autoradiography. Lane 1, control DNA; lanes
2-5, Sanger G, C, A, and T sequencing standards; lanes 6 and 7, (+)-
duocarmycin SA and ent-(-)-duocarmycin SA (1 × 10-5 M); lanes 8 and
9, (+)-MeCTI-TMI and ent-(-)-MeCTI-TMI (1 × 10-5 M); lane 10, (+)-
CBI-TMI (1 × 10-5 M).
Illustrated in Figure 10 is a representative comparison of the
DNA alkylation selectivity of the TMI-based analogues which
highlights the similarity as well as subtle distinctions in the
compounds. As anticipated, the TMI-based analogues 25 and
46 exhibited a DNA alkylation selectivity identical to all such
TMI derivatives, including duocarmycin SA itself. Within this
segment of DNA, the natural and unnatural enantiomers alkylate
a single major site, and the only significant distinction detected
was their relative efficiencies of DNA alkylation. In each case,
the natural enantiomer alkylates DNA with a greater efficiency
than the corresponding unnatural enantiomer. Throughout both
enantiomeric series, duocarmycin SA, MeCTI-TMI, and iso-
MeCTI-TMI (not shown) exhibited no distinction in their
relative efficiencies of DNA alkylation, whereas CBI-TMI (CBI-
TMI > MeCPI-TMI)21 was less effective.
(28) Aristoff, P. A.; Johnson, P. D.; Sun, D. J. Med. Chem. 1993, 36, 1956-
1963.
In a subtle contrast to this behavior, the PDE2 derivatives
illustrated in Figure 11 alkylated the same major sites in both
enantiomeric series, with DSA-PDE2 and MeCTI-PDE2 each
(29) Boger, D. L.; Hertzog, D. L.; Bollinger, B.; Johnson, D. S.; Cai, H.;
Goldberg, J.; Turnbull, P. J. Am. Chem. Soc. 1997, 119, 4977-4986.
(30) Boger, D. L.; Munk, S. A.; Zarrinmayeh, H.; Ishizaki, T.; Haught, J.; Bina,
M. Tetrahedron 1991, 47, 2661-2682.
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A R T I C L E S
Tichenor et al.
Figure 12. In vivo antitumor activity (L1210, i.p.).
In Vivo Antitumor Activity. The natural enantiomer of
MeCTI-indole2 (30) was examined for in vivo efficacy in a
standard antitumor model enlisting L1210 murine leukemia
implanted i.p. into DBA/2J mice. This model has been shown
to respond well to related compounds31 and is a system that
collaborators through the years have used to assess an extensive
series of (+)-CBI-indole2 analogues.27 Although not published,
these latter studies provided the foundation on which we based
our examination of 30. Thus, (+)-30 was examined with the
dose range (10-100 µg/kg) and the dosing schedule (admin-
istered three times i.p. on days 1, 5, and 9) found suitable for
related agents including (+)-CBI-indole227 (Figure 12). Although
the higher doses (100 and 60 µg/kg) were found to be toxic to
the treated animals, efficacious antitumor activity was observed
at the lower doses, producing 4/6 (at 10 µg/kg) and 3/6 (at 30
µg/kg) long-term survivors (>80 days) in the experiment. This
efficacy is at least as good as and may exceed that observed
with related drugs in this class in this tumor model and occurs
at an optimal dose that is lower than that observed with (+)-
CBI-indole2 (30-60 µg/kg) or related MeCPI-based agents.
Thus, the enhanced chemical stability of the alkylation subunit
leads to a greater in vivo potency and a maintained or further
enhanced efficacy.
Conclusions
Figure 11. Thermally induced strand cleavage of w794 DNA (144 bp,
nucleotide no. 5238-138) after DNA-agent incubation with DSA-PDE-
PDE (23 °C, 24 h), MeCTI-PDE-PDE (23 °C, 24 h), and CC-1065 (23 °C,
24 h), removal of unbound agent by EtOH precipitation, and 30 min
thermolysis (100 °C), followed by denaturing 8% PAGE and autoradiog-
raphy. Lane 1, control DNA; lanes 2-5, Sanger G, C, A, and T sequencing
standards; lane 6, (+)-DSA-PDE-PDE (1 × 10-6 M); lane 7, ent-(-)-DSA-
PDE-PDE (1 × 10-6 M); lane 8, (+)-MeCTI-PDE-PDE (1 × 10-6 M);
lane 9, ent-(-)-MeCTI-PDE-PDE (1 × 10-6 M); lane 10, (+)-CC-1065 (1
× 10-6 M).
The MeCTI alkylation subunit was designed on the basis of
the fundamental relationship between reactivity and biological
potency observed in this class of DNA alkylating agents. The
pH 3 solvolysis reactivity of MeCPI (t1/2 ) 37 h) found in the
natural product CC-1065 is greater than that of the optimal
naturally occurring alkylation subunit DSA (t1/2 ) 177 h).
Computational studies (AM1, MNDO) predicted that a single
atom change in the MeCPI alkylation subunit (S, MeCTI vs
NH, MeCPI) would impart an increased stability that was
expected to increase the biological potency of the alkylation
subunit, approaching that of DSA.
The alkylation subunit MeCTI and its isomer iso-MeCTI were
prepared and found to be slightly more stable than DSA (pH 3
solvolysis: MeCTI, t1/2 ) 206 h; iso-MeCTI, t1/2 ) 209 h),
placing it at the point representing an optimal balance between
chemical reactivity and stability on the basis of the established
parabolic relationship. Consistent with their increased relative
exhibiting an efficiency that subtly exceeds that of (+)-CC-
1065. Most significant in this series, and consistent with their
relative cytotoxic potencies, the unnatural enantiomers now
approach or match the DNA alkylation efficiencies of the natural
enantiomers.
To date, the biological properties of members of this class
of natural products have typically mirrored their relative
efficiencies of DNA alkylation. As illustrated in the preceding
section, the observations illustrated in Figure 10 and 11 mirror
the cytotoxic activities observed within the series and even
between enantiomeric pairs of such analogues.
(31) Li, L. H.; Kelly, R. C.; Warpehoski, M. A.; McGovren, J. P.; Gebhard, I.;
DeKoning, T. F. InVest. New Drugs 1991, 9, 137-150.
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Key Analogues of CC-1065 and the Duocarmycins
A R T I C L E S
stabilities, the natural enantiomers of N-Boc-MeCTI (IC50
)
alkylation subunit derivatives further verified the direct impact
of the relative reactivities on the cytotoxic potency. These
observations and a subsequent demonstration of its in vivo
antitumor potency and efficacy places (+)-MeCTI-indole2
among the most interesting such derivatives disclosed to date.
Most importantly, these observations are derived from and
in line with expectations based on the extensive examination
of a series of alkylation subunit analogues that defined a direct
relationship between chemical stability and biological potency
characteristic of this class of antitumor agents. Presumably, the
underlying parabolic relationship reflects the fact that the
compounds must be sufficiently stable to reach their biological
target yet reactive enough to effectively alkylate DNA once they
do.26
20 nM) and N-Boc-iso-MeCTI (IC50 ) 25 nM) proved to be
10-fold more potent than the CC-1065 alkylation subunit (+)-
N-Boc-MeCPI (IC50 ) 330 nM) and 2-3-fold more potent than
(+)-N-Boc-MeCBI (IC50 ) 80 nM), but 4-5-fold less potent
than the anomalously potent (+)-N-Boc-DSA. The unnatural
enantiomers of N-Boc-MeCTI and N-Boc-iso-MeCTI (IC50
)
600 nM) each exhibited potencies 20-fold less active than the
natural enantiomers, consistent with expectations for this class
of agents.
Key natural product analogues evaluated included (+)-
MeCTI-TMI (5 pM) and its isomer (+)-iso-MeCTI-TMI (7 pM),
which matched or slightly exceeded the potency of duocarmycin
SA (DSA-TMI, 8-10 pM), a natural product incorporating the
optimal naturally occurring alkylation subunit. Also prepared
and evaluated was a key MeCTI analogue of CC-1065. The
analogue 8 (MeCTI-PDE2) constitutes the substitution of a single
atom in the alkylation subunit of CC-1065, providing a
compound that exhibited a DNA alkylation profile identical to
that of CC-1065 (IC50 ) 20 pM) but is 3-fold more potent (IC50
) 7 pM, natural enantiomer) than the natural product. Ad-
ditionally, consistent with expectations based on their similar
inherent reactivities, MeCTI-PDE2 was also comparable in
potency with DSA-PDE2 (IC50 ) 4 pM). Extending these
observations, the incorporation of MeCTI into an indole2
derivative and its comparison with an important series of
Acknowledgment. We gratefully acknowledge the financial
support of the National Institutes of Health (CA41986 and
CA42056), the Skaggs Institute for Chemical Biology, and
predoctoral fellowship support from the American Chemical
Society (2005-2006 M.S.T., sponsored by Roche Pharmaceu-
ticals). K.S.M. and M.S.T. are Skaggs Fellows.
Supporting Information Available: Full experimental details.
This material is available free of charge via the Internet at
http://pubs.acs.org.
JA073989Z
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