A unique class of duocarmycin and CC-1065 analogues subject to reductive activation

Article abstract of DOI:10.1021/ja075398e

N-Acyl O-amino phenol derivatives of CBI-TMI and CBI-indole₂ are reported as prototypical members of a new class of reductively activated prodrugs of the duocarmycin and CC-1065 class of antitumor agents. The expectation being that hypoxic tumor environments, with their higher reducing capacity, carry an intrinsic higher concentration of "reducing" nucleophiles (e.g., thiols) capable of activating such derivatives (tunable N-O bond cleavage) and increasing their sensitivity to the prodrug treatment. Preliminary studies indicate the prodrugs effectively release the free drug in functional cellular assays for cytotoxic activity approaching or matching the activity of the free drug, yet remain essentially stable and unreactive to in vitro DNA alkylation conditions (<0.1-0.01% free drug release) and pH 7.0 phosphate buffer, and exhibit a robust half-life in human plasma (t _1/2 = 3 h). Characterization of a representative O-(acylamino) prodrug in vivo indicates that they approach the potency and exceed the efficacy of the free drug itself (CBI-indole₂), indicating that not only is the free drug effectively released from the inactive prodrug but also that they offer additional advantages related to a controlled or targeted release in vivo.

Full text of DOI:10.1021/ja075398e

Published on Web 11/17/2007
A Unique Class of Duocarmycin and CC-1065 Analogues
Subject to Reductive Activation
†
†
†
‡
Wei Jin, John D. Trzupek, Thomas J. Rayl, Melinda A. Broward,
‡
‡
†
,†
George A. Vielhauer, Scott J. Weir, Inkyu Hwang, 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
Office of Therapeutics, DiscoVery and DeVelopment, UniVersity of Kansas Cancer Center,
3
901 Rainbow BouleVard, Kansas City, Kansas 66160
Received July 19, 2007; E-mail: boger@scripps.edu
2
Abstract: N-Acyl O-amino phenol derivatives of CBI-TMI and CBI-indole are reported as prototypical
members of a new class of reductively activated prodrugs of the duocarmycin and CC-1065 class of antitumor
agents. The expectation being that hypoxic tumor environments, with their higher reducing capacity, carry
an intrinsic higher concentration of “reducing” nucleophiles (e.g., thiols) capable of activating such derivatives
(tunable N-O bond cleavage) and increasing their sensitivity to the prodrug treatment. Preliminary studies
indicate the prodrugs effectively release the free drug in functional cellular assays for cytotoxic activity
approaching or matching the activity of the free drug, yet remain essentially stable and unreactive to in
vitro DNA alkylation conditions (<0.1-0.01% free drug release) and pH 7.0 phosphate buffer, and exhibit
a robust half-life in human plasma (t1/2 ) 3 h). Characterization of a representative O-(acylamino) prodrug
in vivo indicates that they approach the potency and exceed the efficacy of the free drug itself (CBI-indole ),
2
indicating that not only is the free drug effectively released from the inactive prodrug but also that they
offer additional advantages related to a controlled or targeted release in vivo.
Introduction
CC-1065, the duocarmycins, and yatakemycin constitute
exceptionally potent naturally occurring antitumor agents that
derive their biological properties through a characteristic
1-10
sequence-selective DNA alkylation reaction (Figure 1).
The
examination of the natural products, their synthetic unnatural
†
The Scripps Research Institute.
University of Kansas Cancer Center.
‡
(
(
(
(
(
1) Chidester, C. G.; Krueger, W. C.; Mizsak, S. A.; Duchamp, D. J.; Martin,
D. G. J. Am. Chem. Soc. 1981, 103, 7629.
2) Takahashi, I.; Takahashi, K.; Ichimura, M.; Morimoto, M.; Asano, K.;
Kawamoto, I.; Tomita, F.; Nakano, H. J. Antibiot. 1988, 41, 1915.
3) Ichimura, M.; Ogawa, T.; Takahashi, K.; Kobayashi, E.; Kawamoto, I.;
Yasuzawa, T.; Takahashi, I.; Nakano, H. J. Antibiot. 1990, 43, 1037.
4) Yasuzawa, T.; Muroi, K.; Ichimura, M.; Takahashi, I.; Ogawa, T.;
Takahashi, K.; Sano, H.; Saito, Y. Chem. Pharm. Bull. 1995, 43, 378.
5) (a) Igarashi, Y.; Futamata, K.; Fujita, T.; Sekine, A.; Senda, H.; Naoki,
H.; Furumai, T. J. Antibiot. 2003, 56, 107. (b) Structure, revision: Tichenor,
M. S.; Kastrinsky, D. B.; Boger, D. L. J. Am. Chem. Soc. 2004, 126, 8396.
6) CC-1065: (a) Warpehoski, M. A.; Hurley, L. H. Chem. Res. Toxicol. 1988,
(
(
1
1
, 315. (b) Hurley, L. H.; Needham-VanDevanter, D. R. Acc. Chem. Res.
986, 19, 230.
7) CC-1065: (a) Boger, D. L.; Johnson, D. S.; Yun, W.; Tarby, C. M. Bioorg.
Med. Chem. 1994, 2, 115. (b) 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.
(
8) 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. (b)
Boger, D. L.; Ishizaki, T.; Zarrinmayeh, H. J. Am. Chem. Soc. 1991, 113,
6
645. (c) Boger, D. L.; Yun, W.; Terashima, S.; Fukuda, Y.; Nakatani, K.;
Kitos, P. A.; Jin, Q. Bioorg. Med. Chem. Lett. 1992, 2, 759.
9) Duocarmycin SA: Boger, D. L.; Johnson, D. S.; Yun, W. J. Am. Chem.
Soc. 1994, 116, 1635.
10) Yatakemycin: (a) Parrish, J. P.; Kastrinsky, D. B.; Wolkenberg, S. E.;
Igarashi, Y.; Boger, D. L. J. Am. Chem. Soc. 2003, 125, 10971. (b) Tichenor,
M. S.; Trzupek, J. D.; Kastrinsky, D. B.; Shiga, F.; Hwang, I.; Boger, D.
L. J. Am. Chem. Soc. 2006, 128, 15683. (c) Trzupek, J. D.; Gottesfeld, J.
M.; Boger, D. L. Nat. Chem. Biol. 2006, 2, 79.
(
(
Figure 1. Natural products.
enantiomers, their derivatives, and synthetic analogues have
defined fundamental features that control the alkylation selectiv-
10.1021/ja075398e CCC: $37.00 © 2007 American Chemical Society
J. AM. CHEM. SOC. 2007, 129, 15391-15397
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15391

A R T I C L E S
Jin et al.
which the development of useful, stable, or safe prodrugs has
3
3-35
been conducted.
One feature limiting the attractiveness of this class of
cytotoxic agents is their remarkable potencies (IC50 5-20 pM)
creating special requirements for their preparation and handling.
In many instances, this has been addressed by the introduction
of chemically stable phenol protecting groups that are readily
cleaved at the final stage of their preparation or upon in vivo
administration. Such protected phenol precursors are intrinsically
much less potent yet readily release an active precursor to the
drug upon deprotection. Extensions of this protection and release
strategy have been pursued in which the free phenol release in
vivo is coupled to features that might facilitate tumor selective
3
6
Figure 2. Prodrug design.
delivery or cleavage. Such inactive prodrugs serve the dual
role of providing safer handling intermediates or final products
as well as potentially enhancing the therapeutic index of the
drug. As attractive and amenable as this approach is for this
class of drugs, a surprisingly small series of such studies have
ity, impact the alkylation efficiency, and are responsible for
DNA alkylation catalysis, providing a detailed understanding
of the relationships between structure, reactivity, and biological
activity.6
,11-13
25-32
been disclosed.
One of the most important and widely explored class of
Herein, we disclose a novel set of reductively activated phenol
prodrugs for the CC-1065 and duocarmycin class of compounds
that do not require enzymatic release and should prove general
for other phenolic drugs that may benefit from such a designed
1
4
analogues is CBI (1,2,9,9a-tetrahydrocyclopropa[c]benz[e]-
indol-4-one), being synthetically1
4,15
more accessible than the
natural products, yet indistinguishable in its DNA alkylation
selectivity (Figure 2).16 Moreover, the CBI derivatives proved
to be 4 times more stable and, correspondingly, 4 times more
potent than derivatives bearing the CC-1065 alkylation subunit
(
19) (a) Boger, D. L.; Ishizaki, T. Tetrahedron Lett. 1990, 31, 793. (b) Boger,
D. L.; Ishizaki, T.; Zarrinmayeh, H.; Kitos, P. A.; Suntornwat, O. Bioorg.
Med. Chem. Lett. 1991, 1, 55. (c) Boger, D. L.; Munk, S. A.; Ishizaki, T.
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H.; Han, N. Bioorg. Med. Chem. 1995, 3, 761. (g) Boger, D. L.; Han, N.
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(7-MeCPI), approaching the stability and potency of duocar-
mycin SA and yatakemycin derivatives, and they exhibit
efficacious in vivo antitumor activity in animal models at doses
that reflect this potency.17 Consequently, CBI and its derivatives
have served as the prototype analogues on which new design
1
4-32
concepts have been explored, developed, or introduced.
A unique feature of this class of molecules including the
natural products themselves is the observation that synthetic
phenol precursors (e.g., 1) to the final products, entailing a
Winstein Ar-3′ spirocyclization with displacement of an ap-
propriate leaving group, exhibit biological properties typically
indistinguishable from those of the cyclopropane-containing final
products (DNA alkylation rate or efficiency, in vitro cytotoxic
activity, and in vivo antitumor activity). This dependable
behavior of the precursor phenols has provided the basis on
6654. (o) Boger, D. L.; Stauffer, F.; Hedrick, M. P. Bioorg. Med. Chem.
Lett. 2001, 11, 2021. (p) Parrish, J. P.; Kastrinsky, D. B.; Stauffer, F.;
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3
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(
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(
11) Reviews: (a) Boger, D. L.; Garbaccio, R. M. Acc. Chem. Res. 1999, 32,
1
2
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12) Review of synthetic studies: Boger, D. L.; Boyce, C. W.; Garbaccio, R.
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(
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14) (a) Boger, D. L.; Ishizaki, T.; Wysocki, R. J., Jr.; Munk, S. A.; Kitos, P.
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(
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(
(
16) Boger, D. L.; Munk, S. A. J. Am. Chem. Soc. 1992, 114, 5487.
2
17) CBI-indole : (a) Boger, D. L.; Ishizaki, T.; Sakya, S. M.; Munk, S. A.;
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1
1
15. (b) Boger, D. L.; Yun, W.; Han, N. Bioorg. Med. Chem. 1995, 3,
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(
18) CBI-TMI: Boger, D. L.; Yun, W. J. Am. Chem. Soc. 1994, 116, 7996.
15392 J. AM. CHEM. SOC.
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VOL. 129, NO. 49, 2007

Reductively Activated Phenol Prodrugs
A R T I C L E S
activation. Alternative and prior efforts at incorporating a
reductive activation into the CC-1065 and duocarmycin class
include the Denny disclosures of nitro precursors to aryl amine
variants of the phenol precursors,30 Lee’s use of an ester that is
Scheme 1
3
1
subject to cleavage upon a tethered quinone reduction, and
our own report of mitomycin-like quinone precursors to a
reductively activated o-spirocyclization (versus p-spirocycliza-
tion) analogous to those observed with the duocarmycins or its
analogues.32 Although the approaches have provided some
increase in aerobic selectivity that results from the reductive
activation, none effectively or clearly utilizes an intrinsic enzyme
activity that differentiates normal versus tumor cells, and in
many instances it may be the ease of reoxidation in normal cells
(which occurs less readily in hypoxic tumors) that protects them
from the effects of the drug. The features contributing to and
limiting reductively activated hypoxia-selective cytotoxic agents
have been reviewed, and such agents include mitomycin C
(
MMC) and other aziridoquinones, nitro-aromatics, N-oxides,
37
various metal complexes, azides, and di- and trisulfides. Those
most related to the design disclosed herein are FR900482 and
its related congeners (FR66979 and FK-317) as well as a
dehydromonocrotaline progenitor39 bearing hydroxylamine hemi-
acetals which are irreversibly activated by reductive cleavage
of a N-O bond.
38
The approach detailed herein was not designed for reversible
or enzymatic reductive activation, but rather for irreversible
activation by cleavage of a weak N-O bond by reducing
nucleophiles (Figure 2). The expectation is that hypoxic tumor
cells, with their higher reducing capacity, may carry an intrinsic
high concentration of “reducing” nucleophiles (i.e., thiols)
capable of activating such derivatives, thus making them more
sensitive to the prodrug treatment.36 Moreover, as detailed
below, the design lends itself to a rational tuning of the ease of
reduction of the derivative, allowing empirical experience with
the series to guide future design.
Chemistry
(
24) (a) Wang, Y.; Yuan, H.; Wright, S. C.; Wang, H.; Larrick, J. W. Bioorg.
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Synthesis. A range of methods for direct conversion of a
precursor phenol to the corresponding O-amino phenol were
examined (O-amidation), and several routes to the final com-
pounds were explored. It was anticipated that this might best
be conducted on a seco-N-Boc-CBI derivative lacking the
capabilities of spirocyclization (e.g., 11). However, the lability
of the resulting N-acyl O-amino phenol derivatives to subsequent
chemical transformations proved significant, and this approach
proved less viable than a surprisingly effective direct O-
amidation reaction of seco-CBI-TMI or seco-CBI-indole2
(
c) Wang, Y.; Yuan, H.; Ye, W.; Wright, S. C.; Wang, H.; Larrick, J. W.
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(
(
(
25) Chari, R. V. J.; Jackel, K. A.; Bourret, L. A.; Derr, S. J.; Tadayoni, B. M.;
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2
002, 10, 1634. (b) Tietze, L. F.; Feuerstein, T.; Fecher, A.; Haunert, F.;
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2
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(Scheme 1). Thus, low-temperature phenol deprotonation of 2
(3 equiv of LiHMDS, 0 °C, ether-dioxane) followed by
1
569.
40
treatment with the amidating reagents, TsONHBoc or TsON-
(
28) Jeffrey, S. C.; Torgov, M. Y.; Andreyka, J. B.; Boddington, L.; Cerveny,
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41
Phth, provided 4 and 8 directly in good conversions. Competi-
tive spirocyclization of 2 to CBI-TMI itself was observed if
(
29) Kline, T.; Torgov, M. Y.; Mendelsohn, B. A.; Cerveny, C. G.; Senter, P.
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1
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J. AM. CHEM. SOC.
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A R T I C L E S
Scheme 2
Jin et al.
Scheme 3
the deprotonation was carried out at higher reaction temperatures
or in more polar solvents. It diminished as the solvent polarity
was reduced (glyme > THF > dioxane-ether > ether,
insoluble) and was less prominent with LiHMDS versus
NaHMDS. In most instances, recovered starting phenol was
present in the crude reaction product and was chromatographi-
cally close enough to the N-acyl O-amino phenols that special
precautions were taken to ensure its removal. This entailed
exposure of the product mixture to conditions that promote
deliberate spirocyclization of the seco-phenol derivatives (satu-
rated aqueous NaHCO3-THF (1:1), 23 °C, 2 h)15 and subse-
quent chromatographic separation of the much more polar CBI-
TMI or CBI-indole2. N-Acetylation of 4 (Ac2O, cat. DMAP,
CH2Cl2, 23 °C, 12 h, 81%) provided 6, and subsequent Boc
deprotection (TFA-CH2Cl2 (1:1), 23 °C, 3 h, 88%) afforded
such a contaminate phenol. Thus, following a route analogous
to that used for CBI itself, 20 was prepared from 16 and
15
converted to 21 enlisting a key 5-exo-trig-aryl radical-alkene
15h
cyclization, Scheme 3. The product 21, like 14 (R ) 1.19),
was chromatographically resolved on a semipreparative Chiral-
Cel OD column (R ) 1.42), providing each enantiomer that
was coupled with 5,6,7-trimethoxyindole-2-carboxylic acid (15)
upon N-Boc deprotection to provide 9.
5
. In an analogous fashion, seco-CBI-indole2 (3) was directly
converted to 8 (45%) upon LiHMDS deprotonation (3 equiv of
LiHMDS, ether-dioxane, 0 °C, 30 min) and subsequent
O-amidation with TsONHBoc.40
Stability and Reactivity of the N-Acyl O-Amino Phenol
Derivatives. Clear from efforts directed at their preparation,
the N-acyl amino phenol prodrugs displayed a useful range of
stability, yet were susceptible to cleavage of the critical N-O
bond. As might be anticipated, their relative stability followed
the order of 4 > 5 > 6 > 7 with 4 and 5 withstanding even
long-term storage effectively, but with 7 noticeably deteriorating
over time. Derivatives 4 and 6, as well as 7, proved surprisingly
robust to acidic conditions (TFA-CH2Cl2, 4 N HCl-EtOAc)
and stable to mild base treatment in nonpolar, aprotic solvents
For comparison purposes, two analogues of seco-CBI-TMI
were prepared that are incapable of spirocyclization to CBI-
TMI itself. The first incorporates the C4 phenol protected as
its methyl ether (10), and the second contains no C4 substituent
(
9). The former was prepared from 1115i by phenol O-
methylation, primary alcohol OTBS deprotection, and subse-
quent conversion to the primary chloride 14, followed by N-Boc
deprotection and coupling with 5,6,7-trimethoxyindole-2-car-
boxylic acid (15) to provide 10, Scheme 2. Throughout this
sequence and as a result of the multiple purifications, the chances
of residual, contaminant phenol (2) being present in the final
product 10 are remote. Nonetheless, since even trace quantities
of 2 can be misleadingly detected in the subsequent biological
evaluations (e.g., 0.01%), the analogue 9 was also prepared for
comparison and by an approach that precludes the presence of
(Et3N or DMAP, CH2Cl2), but exhibited a diminished stability
as the solvent polarity increases: stable to NaHCO3 in THF or
THF-H2O, but cleaved in NaHCO3/DMF-H2O or H2O and
DBU/CH2CN. Similarly, 4 proved stable in MeOH, but 2 was
released slowly upon treatment with NaHCO3 or Na2CO3 in
MeOH (2 h, 23 °C). Most pertinent to the potential source of
cleavage under physiological conditions, 4 was stable to
treatment with BnSH in THF (2-72 h, 23 °C) or MeOH (2-
72 h, 23 °C) and stable to treatment with BnSH in THF even
in the presence of insoluble NaHCO3 (2 h, 23 °C). However, 4
is cleaved to release 2 upon treatment with BnSH in MeOH in
the presence of NaHCO3 (2 h, 23 °C). Significantly, the stability
of 4 was assessed in pH 7.0 phosphate buffer, and within the
limits of detection (HPLC, UV) no significant cleavage of the
prodrug was observed over the time monitored (72 h). Finally,
the stability of 4 was monitored in human plasma (50 µg/100
µL, 10% DMSO) in which it displayed a half-life of 3 h with
release of the free drug 2.
(
36) (a) Wolkenberg, S. E.; Boger, D. L. Chem. ReV. 2002, 102, 2477. Reviews
on reductive activation: (b) Papadopoulou, M. V.; Bloomer, W. D. Drugs
Future 2004, 29, 807. (c) Jaffar, M.; Stratford, I. J. Exp. Opin. Ther. Pat.
1
1
999, 9, 1371. (d) Patterson, L. H.; Raleigh, S. M. Biomed. Health Res.
998, 25, 72.
(37) Denny, W. A. Hypoxia-Selective Cytotoxins. In Cancer Chemotherapeutic
Agents; Foye, W. O., Ed.; American Chemical Society: Washington DC,
1
995; pp 483-500.
(
(
(
(
38) Paz, M. M.; Hopkins, P. B. J. Am. Chem. Soc. 1997, 119, 5999. Williams,
R. M.; Rajski, S. R.; Rollins, S. B. Chem. Biol. 1997, 4, 127.
39) Tepe, J. J.; Kosogof, C.; Williams, R. M. Tetrahedron 2002, 58, 3553.
Tepe, J. J.; Williams, R. M. Angew. Chem., Int. Ed. 1999, 38, 3501.
40) Greck, C.; Bischoff, L.; Girard, A.; Hajicek, J.; Gen eˆ t, J. P. Bull. Soc. Chim.
Fr. 1994, 131, 429.
41) Neumann, U.; G u¨ tschow, M. J. Biol. Chem. 1994, 269, 21561.
15394 J. AM. CHEM. SOC.
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Reductively Activated Phenol Prodrugs
A R T I C L E S
Figure 4. In vitro cytotoxic activity.
equipotent against H460/H596 cells. More significantly and
unlike MMC, this reductive activation is not linked to the
expression levels of DT-diaphorase since 2 and 4-7 remain
equipotent in the H460 or H596 cell lines, although H596 is
10-fold less sensitive than H460 is to seco-CBI-TMI itself. This
illustrates that DT-diaphorase is not mediating the reductive
release of the drug from the O-amino phenol prodrugs, indicating
that their utility is orthogonal to that of MMC. While this does
not rule out enzymatic activation, the behavior is consistent with
our suggestion that the activation is nonenzymatic and likely is
mediated in situ by appropriate nucleophiles. Analogous trends
are also observed with the CBI-TMI unnatural enantiomers,
albeit at potencies that are approximately 100 to 1000-fold
higher than those of the natural enantiomers (Figure 3).
Especially interesting was the behavior of the CBI-indole2
prodrug. For this CBI analogue, only the NHBoc derivative was
examined since it was the most stable of the N-acyl O-amino
phenol prodrugs examined (Figure 4). In each cell line
examined, the prodrug 8 was essentially equipotent with CBI-
indole2 itself, indicating effective release of the free drug under
the conditions of the assay. In addition it proved to be
exceptionally potent being 100-1000 times more active than
MMC (IC50 ) 30-200 pM vs 20-40 nM), and it remained
remarkably active against the MMC-resistant H596 cell line
Figure 3. In vitro cytotoxic activity.
Biological Properties
Cytotoxic Activity. The O-amino phenol derivatives bearing
the N-O prodrug linkage and the various N-acyl substituents
were assayed for cytotoxic activity alongside the parent drugs
CBI-TMI (2)18 and CBI-indole2 (3)17 as well as the two control
standards, 9 and 10, incapable of free phenol release. Three cell
lines were examined including a standard L1210 cell line (mouse
leukemia) as well as the MMC-sensitive (H460, expresses high
levels of DT-diaphorase) and MMC-resistant (H596, lacks DT-
3
2,17
diaphorase) non-small cell lung cancer (NSCLC) cell lines.
Several important trends emerged from these studies. First, the
natural enantiomer control standards 9 and 10, incapable of free
phenol release, were inactive against all three cell lines (IC50
>
100 nM) being g10000-fold less active than the free drug 2
(IC50 ) 4 nM vs 5 µM). Even the unnatural enantiomer of 8,
(seco-CBI-TMI), Figure 3. In sharp contrast, the natural
which was found to be 10-100 fold less active than the natural
enantiomer, proved to be more active than MMC. Given the
efficacy of (+)-CBI-indole2 in animal tumor models,17 it was
especially interesting to compare 8 with 3 in vivo.
enantiomers of the O-amino phenol prodrugs exhibited potent
cytotoxic activity approaching that of the free drug itself (1-
0.1 times the activity of 2), indicating its successful release under
the assay conditions. Even more significantly, the relative
potency of the prodrugs, when distinguishable, mirrors the
expected ease of N-O bond cleavage (e.g., L1210: 7 > 6 > 5
DNA Alkylation Selectivity and Efficiency. The DNA
alkylation properties of 4 were examined alongside the parent
drug CBI-TMI (2) and the two control standards 9 and 10
>
4) suggesting that fundamental chemical principles may be
(
incapable of spirocyclization) within w794 duplex DNA42 for
used to “tune” the reductive free drug release. Provocatively,
the potency differences between the free drug 2 and the prodrugs
may vary with the character of the cell line; 4 is 10-fold less
potent than 2 against L1210, but 2 and 4 are essentially
which results for an extensive series of duocarmycin analogues
(
42) Boger, D. L.; Munk, S. A.; Zarrinmayeh, H.; Ishizaki, T.; Haught, J.; Bina,
M. Tetrahedron 1991, 47, 2661.
J. AM. CHEM. SOC.
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A R T I C L E S
Jin et al.
Figure 6. In vivo antitumor activity (L1210, ip).
stable to the DNA alkylation conditions examined. These
observations are consistent with the stability of 4 observed in
pH 7.0 phosphate buffer. Significantly, the results then suggest
that the in vitro cytotoxic activity of 4, and by analogy the
cytotoxicities of the related O-amino phenol prodrugs which
all approach that of the parent drug CBI-TMI (2), is derived
from in situ intracellular cleavage of the N-O bond and
productive release of the active drug under the cell culture
conditions.
In Vivo Antitumor Activity. The prodrug 8 was examined
for in vivo efficacy alongside the parent drug 3 in a standard
antitumor model consisting of L1210 murine leukemia cells
implanted ip into DBA/2J mice. This model has been reported
to respond well to the parent drugs of related compounds43 and
is a system that collaborators through the years have used to
assess an extensive series of (+)-CBI-indole2 analogues.
Although not published, these latter studies provided the
foundation on which we based our examination of 8. We used
the dose range (10-100 µg/kg) and the dosing schedule
Figure 5. Thermally induced strand cleavage of w794 DNA; DNA-agent
incubation at 4 °C for 18 h, removal of unbound agent by EtOH
precipitation, and 30 min of thermolysis (100 °C) followed by 8% denaturing
PAGE and autoradiography. Lane 1, control DNA; lanes 2-5, Sanger G,
-
4
-6
C, A, and T sequencing reactions; lanes 6-8, 2 (1 × 10 to 1 × 10 );
-
1
-3
-1
lanes 9-11, 10 (1 × 10 to 1 × 10 ); lanes 12-14, 4 (1 × 10 to 1 ×
-
3
-1
-3
1
0
), lanes 15-17, 9 (1 × 10 to 1 × 10 ). All compounds possess the
natural 1S-configuration.
have been reported. The sites of DNA alkylation and its
efficiency were directly assessed by thermally induced single-
strand cleavage of 5′-end-labeled duplex DNA following incu-
bation with the agents (Figure 5, natural enantiomers examined).
The reductively activated agent 4 was found to alkylate w794
DNA with a sequence selectivity identical to that of the parent
agent CBI-TMI (2), albeit with a substantially reduced efficiency
(
administered three times ip on days 1, 5, and 9) found suitable
17
for related parent drugs including (+)-CBI-indole2 (3) (Figure
). The dose at which a maximal response was observed for 8
6
corresponded closely to that of (+)-CBI-indole2 (3) while its
efficacy was significantly improved. This indicates that the
prodrug 8 (1) efficiently and effectively releases the free drug
3
in the in vivo model (reductive activation) and (2) that either
(1000-10000-fold). Similarly, the O-methyl ether 10 as well
the rate of release or the site of release enhances the efficacy
of the drug. Moreover, the efficacy of 8 is extraordinary,
providing 5/6 long-term survivors at 52 weeks (365 days, T/C
as 9 lacking a C4 substituent failed to exhibit significant
observable DNA alkylation. In fact, 9 showed no appreciable
DNA alkylation even under forcing conditions (37 °C, 18 h,
data not shown), whereas the potentially more reactive O-methyl
ether 10 (via assisted phenonium ion formation) displayed
perhaps a trace amount of DNA alkylation (<0.01% that of 2)
that could be attributed to either its direct, but much less facile,
DNA alkylation or the contaminant-free phenol present in the
synthetic sample of 10. With detection of DNA alkylation by
the prodrug 4 at the level observed (0.1-0.01% of 2), we cannot
distinguish whether this is due to direct alkylation by 4 itself,
trace release of 2 from 4 under the DNA incubation conditions
>
1550) at the optimal dosing examined (100 µg/kg). Notably,
little distinction between 3 and 8 was observed at days 30-
00 except that the prodrug-treated animals appeared healthier
1
displaying little or no weight loss which was evident with 3 at
the highest dosing. With the prolonged management of the
treated animals herein that exceeded the time frame typically
allotted for such an in vivo antitumor assessment, we observed
that the surviving mice at day 90 treated with the free drug 3,
but not the prodrug 8, eventually expired due to drug admin-
4
4
istration-related complications. While these would likely be
(in situ N-O cleavage), or attributable to trace contaminate 2
in the synthetic samples of 4. What the results do indicate is
that 4 is incapable of significant DNA alkylation in its own
right (requires N-O bond cleavage) and that 4 is essentially
(43) Li, L. H.; Kelly, R. C.; Warpehoski, M. A.; McGovern, J. P.; Gebhard, I.;
DeKoning, T. F. InVest. New Drugs 1991, 9, 137.
(
44) This appears to arise from damage to the intraperitioneal cavity or its organs
that originates with the bolus drug administration.
15396 J. AM. CHEM. SOC.
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Reductively Activated Phenol Prodrugs
A R T I C L E S
Conclusions
A unique class of N-acyl O-amino phenol prodrugs of CBI-
TMI and CBI-indole2 was explored as representative members
of the duocarmycin and CC-1065 class of antitumor agents. The
prodrugs, subject to reductive activation by nucleophilic cleav-
age of a weak N-O bond, effectively release the free drug in
functional cellular assays for cytotoxic activity approaching or
matching the activity of the free drug yet remain essentially
stable to in vitro DNA alkylation conditions (<0.1-0.01% free
drug release), pH 7.0 phosphate buffer, and exhibiting a robust
half-life in human plasma (t1/2 ) 3 h for 4). Most impressively,
assessment of the in vivo antitumor activity of a representative
O-(acylamino) prodrug, 8, indicates that they approach the
potency and exceed the efficacy of the free drug itself (CBI-
indole2), indicating that the inactive prodrugs not only effectively
release the free drug in vivo but that they offer additional
advantages related to a controlled or targeted release in vivo.
With cleavage release of the free drug being easily tunable using
fundamental chemical principles, the potential of developing
derivatives selectively or most effectively released in a reducing
hypoxic environment characteristic of solid tumors may further
improve on these impressive observations. An additional unique
feature of the O-amino phenol prodrugs is their capability to
serve as the linkage site for conjugation to targeting molecules
(i.e., antibodies). Thus, reductive cleavage of the drug may be
capable of a tunable release from a tethered, covalent delivery
vehicle offering a unique alternative to linkers (e.g., disulfide
or hydrazone) typically enlisted in studies to date.
Figure 7. In vivo antitumor activity (L1210, ip).
capable of being managed with an optimized dosing schedule,
this distinction between 3 and 8 in the long-term cures (>90
days) suggests the prodrug 8 offers significant advantages over
the free drug administration. Finally, it is worth noting that these
compounds are extraordinarily potent, requiring less than 1 mg
of sample to conduct the entire in vivo antitumor testing,
suggesting that clinical supplies of such agents could easily be
supplied by chemical synthesis.
Confirming these observations, an analogous antitumor as-
sessment was carried out independently at a second site utilizing
a slightly different and harsher protocol for drug administration
Acknowledgment. We gratefully acknowledge the financial
support of the National Institutes of Health (CA41986) and the
Skaggs Institute for Chemical Biology. We thank the American
Society for Engineering Education (NDSEG) for a predoctoral
fellowship (J.D.T). J.D.T. is a Skaggs Fellow.
(neat DMSO vs 30% DMSO in 0.1% glucose). Although this
assessment was terminated after 120 days, it similarly indicates
that administration of the prodrug 8 is significantly less toxic
than free drug 3 and that it is comparable or superior in terms
of reducing deaths due to the disease and tumor burden (Figure
Supporting Information Available: Full experimental details
and compound characterizations are provided.
7
). Again, 7/10 long-term survivors were observed with 8 at
day 120 at the optimal dosing (60 µg/kg).
JA075398E
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