Systematic exploration of the structural features of yatakemycin impacting DNA alkylation and biological activity

Article abstract of DOI:10.1021/ja072777z

A systematic examination of the impact of the yatakemycin left and right subunits and their substituents is detailed along with a study of its unique three subunit arrangement (sandwiched vs extended and reversed analogues). The examination of the ca. 50 analogues prepared illustrate that within the yatakemycin three subunit structure, the subunit substituents are relatively unimportant and that it is the unique sandwiched arrangement that substantially increases the rate and optimizes the efficiency of its DNA alkylation reaction. This potentiates the cytotoxic activity of yatakemycin and its analogues overcoming limitations typically observed with more traditional compounds in the series (CC-1065, duocarmycins). Moreover, a study of the placement of the alkylation subunit within the three subunit arrangement (sandwiched vs extended and reversed analogues) indicates that it not only has a profound impact on the rate and efficiency of DNA alkylation but also controls and establishes the DNA alkylation selectivity as well, where both enantiomers of such sandwiched agents alkylate the same adenine sites exhibiting the same DNA alkylation selectivity independent of their absolute configuration.

Full text of DOI:10.1021/ja072777z

Published on Web 08/11/2007
Systematic Exploration of the Structural Features of
Yatakemycin Impacting DNA Alkylation and Biological Activity
Mark S. Tichenor, Karen S. MacMillan, John D. Trzupek, Thomas J. Rayl,
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
Received April 20, 2007; E-mail: boger@scripps.edu
Abstract: A systematic examination of the impact of the yatakemycin left and right subunits and their
substituents is detailed along with a study of its unique three subunit arrangement (sandwiched vs extended
and reversed analogues). The examination of the ca. 50 analogues prepared illustrate that within the
yatakemycin three subunit structure, the subunit substituents are relatively unimportant and that it is the
unique sandwiched arrangement that substantially increases the rate and optimizes the efficiency of its
DNA alkylation reaction. This potentiates the cytotoxic activity of yatakemycin and its analogues overcoming
limitations typically observed with more traditional compounds in the series (CC-1065, duocarmycins).
Moreover, a study of the placement of the alkylation subunit within the three subunit arrangement
(sandwiched vs extended and reversed analogues) indicates that it not only has a profound impact on the
rate and efficiency of DNA alkylation but also controls and establishes the DNA alkylation selectivity as
well, where both enantiomers of such sandwiched agents alkylate the same adenine sites exhibiting the
same DNA alkylation selectivity independent of their absolute configuration.
Introduction
Yatakemycin (1)1 is the newest and most potent (IC50, L1210
) 3-5 pM) member of a family of antitumor antibiotics that
includes CC-1065 (2, IC50 ) 20 pM),2 duocarmycin A (3, IC50
) 200 pM),3 and duocarmycin SA (4, IC50 ) 10 pM, Figure
1).4 Each derives its properties through a characteristic sequence-
selective alkylation of duplex DNA.5-9 The yatakemycin
structure is a remarkable hybrid of the preceding natural
products, incorporating a central DNA alkylation subunit
(1) Igarashi, Y.; Futamata, K.; Fujita, T.; Sekine, A.; Senda, H.; Naoki, H.;
Furumai, T. J. Antibiot. 2003, 56, 107-113.
(2) 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.
(3) Takahashi, I.; Takahashi, K.; Ichimura, M.; Morimoto, M.; Asano, K.;
Kawamoto, I.; Tomita, F.; Nakano, H. J. Antibiot. 1988, 41, 1915-1917.
(4) Ichimura, M.; Ogawa, T.; Takahashi, K.; Kobayashi, E.; Kawamoto, I.;
Yasuzawa, T.; Takahashi, I.; Nakano, H. J. Antibiot. 1990, 43, 1037-1038.
(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.
(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.
Figure 1. Natural products.
identical to that of duocarmycin SA, a left subunit identical to
the DNA-binding subunits found in CC-1065, and a right subunit
similar to that found in the duocarmycins. Distinct from the
(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.
(8) Duocarmycin SA: Boger, D. L.; Johnson, D. S.; Yun, W. J. Am. Chem.
Soc. 1994, 116, 1635-1656.
9
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J. AM. CHEM. SOC. 2007, 129, 10858-10869
10.1021/ja072777z CCC: $37.00 © 2007 American Chemical Society

Structural Features of Yatakemycin
A R T I C L E S
Scheme 1
Figure 2. DNA alkylation catalysis and substituent effects on DNA
alkylation efficiency, rate, and biological potency.
preceding agents, yatakemycin is the first naturally occurring
member possessing a DNA binding subunit flanking each side
of the alkylation subunit in a characteristic “sandwiched”
arrangement. Recently, we disclosed first and second generation
total syntheses10-13 of yatakemycin that served to reassign its
structure as 110 and reported preliminary evaluations of (+)-
and ent-(-)-yatakemycin and key partial structures.11
vinylogous amide conjugation and activates the cyclopropane
for nucleophilic attack. Through use of this DNA binding-
induced activation (shape-dependent catalysis), the compound
avoids reaction with competitive nucleophiles until being
selectively activated by and at its biological target.16
Prior to the discovery of yatakemycin, we had utilized the
C-terminus methyl ester of duocarmycin SA as a site for
attachment of a second DNA binding subunit providing the first,
albeit limited, series of such sandwiched analogues that were
found to exhibit potent cytotoxic activity, an enhanced DNA
alkylation rate, and a uniquely altered alkylation selectivity.14
The subsequent isolation of a naturally occurring member of
this class has focused greater interest in such sandwiched
compounds and now provides the incentive to more thoroughly
evaluate this unusual structural arrangement. Herein, we report
efforts to systematically define the relationships among structure,
reactivity, and biological activity of such sandwiched agents.
Extensive efforts have defined the structural features of the
duocarmycins that contribute to their remarkable rate, selectivity,
and efficiency of DNA alkylation.9 In these studies, the right-
hand DNA binding subunit was identified as one that not only
provides noncovalent DNA binding affinity and selectivity but
also is responsible for catalysis of the DNA alkylation reaction
(Figure 2).14,15 The alkylation subunit cyclopropane is stabilized
by the cross-conjugated delocalization of the N2 lone pair via a
vinylogous amide. Upon binding in the minor groove, the
compound is forced to adopt a twisted conformation to
accommodate the helical pitch of DNA, which disrupts the
Results and Discussion
Synthesis of Analogues with Modifications in the Right
Subunit. Studies conducted on duocarmycin SA have indicated
that only the C5-methoxy substituent of the DNA binding
subunit has a pronounced effect on the rate and efficiency of
DNA alkylation and the biological potency of the compounds.9,14
These effects are independent of the substituent electronic
properties and could be attributed simply to its presence and
the resulting increase in the rigid length of the agent.17 This
rigid extension increases the extent of the DNA binding-induced
conformational change, increasing the degree of vinylogous
amide disruption and enhancing the rate of DNA alkylation.
Consequently, a series of key yatakemycin analogues were
synthesized to establish the impact of its right subunit substit-
uents in such sandwiched structures.
In route to these key analogues and for comparison purposes,
the partial structures 9a-9f were first prepared and lack the
left subunit of yatakemycin (Scheme 1). As such, they represent
not only key partial structures of yatakemycin but also additional
key analogues of duocarmycin SA itself. Boc deprotection of
each enantiomer of 611 (only natural enantiomer shown, 4 N
HCl-EtOAc, 70 °C, 1 h), followed by coupling with the
corresponding indole-2-carboxylic acid (5a-f, 4 equiv of EDCI,
3 equiv of NaHCO3, DMF, 25 °C, 14 h, 62-78%) gave 7.
Benzyl ether deprotection of 7 (1 atm of H2, 10% Pd/C, 9:1
THF-MeOH, 2 h, 80-95%) provided 8, and subsequent
(10) Tichenor, M. S.; Kastrinsky, D. B.; Boger, D. L. J. Am. Chem. Soc. 2004,
126, 8396-8398.
(11) Tichenor, M. S.; Trzupek, J. D.; Kastrinsky, D. B.; Futoshi, S.; Hwang, I.;
Boger, D. L. J. Am. Chem. Soc. 2006, 128, 15683-15696.
(12) An independent route to the natural product has been reported: Okano,
K.; Tokuyama, H.; Fukuyama, T. J. Am. Chem. Soc. 2006, 128, 7136-
7137.
(13) Review of related efforts: Boger, D. L.; Boyce, C. W.; Garbaccio, R. M.;
Goldberg, J. A. Chem. ReV. 1997, 97, 787-828.
(14) (a) 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. (b) 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.
(16) 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.
(17) Parrish, J. P.; Kastrinsky, D. B.; Stauffer, F.; Hedrick, M. P.; Hwang, I.;
Boger, D. L. Bioorg. Med. Chem. 2003, 11, 3815-3838.
(15) Boger, D. L.; Garbaccio, R. M. Bioorg. Med. Chem. 1997, 5, 263-276.
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A R T I C L E S
Scheme 2
Tichenor et al.
Scheme 3
in which the left-hand subunit substituents or key structural
features are removed.
The synthesis of 16a-b incorporating a pyrrolidine in place
of the left subunit was accomplished as shown in Scheme 3.
Coupling of carboxylic acids 12a and 12c with pyrrolidine
hydrochloride (1.0 equiv, 4 equiv of EDCI, DMF, 25 °C, 16 h,
49-65%) gave 15a-b, and subsequent spirocyclization (sat.
NaHCO3 in 2:1 DMF-H2O, 25 °C, 1 h, 58-87%) provided
(+)- and ent-(-)-16a-b.
spirocyclization (sat. aq. NaHCO3 in 2:1 DMF-H2O, 25 °C, 1
h, 64-89%) provided (+)- and ent-(-)-9a-f.
The synthesis of sandwiched analogues 14b-f was ac-
complished as shown in Scheme 2. Methyl ester hydrolysis of
7 (only natural enantiomer shown, 4 equiv of LiOH, 3:2:1 THF-
MeOH-H2O, 25 °C, 14 h, 78-94%) followed by benzyl ether
deprotection of 11 (1 atm of H2, 10% Pd/C, 9:1 THF-MeOH,
2 h, 84-100%) gave 12. Coupling of 12 with the hydrochloride
salt of 1011 (4 equiv of EDCI, DMF, 25 °C, 16 h, 42-77%)
furnished 13, and subsequent spirocyclization (sat. aq. NaHCO3
in 2:1 DMF-H2O, 25 °C, 1 h, 63-98%) provided (+)- and
ent-(-)-14b-f.
Synthesis of Analogues with Modifications in the Left
Subunit. Yatakemycin is structurally unique among members
of its class, possessing the additional left-hand DNA binding
subunit. A series of left subunit analogues were prepared to
establish its contribution to yatakemycin’s properties. In previous
studies, the CDPI18 and indole subunits (Scheme 3) were found
to constitute unsubstituted and simplified replacements for the
PDE subunits found in CC-1065 that are readily accessible by
chemical synthesis, maintain the full cytotoxic potency of the
natural products, and impart more efficacious in vivo activity.19
Thus, yatakemycin analogues incorporating these and related
subunits were prepared for evaluation and constitute analogues
The synthesis of yatakemycin analogues incorporating ad-
ditional modifications in the left subunit was accomplished as
shown in Schemes 4 and 5. Coupling of carboxylic acid 12a
with the CDPI-based amine hydrochlorides derived from 1718
or 18 (1.0 equiv, 4 equiv of EDCI, DMF, 25 °C, 16 h, 37-
41%) gave 19a-b, and subsequent spirocyclization (sat. NaH-
CO3 in 2:1 DMF-H2O, 25 °C, 0.7 h, 66-91%) provided (+)-
and ent-(-)-20a-b. Similarly, coupling of carboxylic acid 12f
with 21 (1.0 equiv, 4 equiv of EDCI, DMF, 25 °C, 24 h, 53%)
gave 22, and subsequent spirocyclization (sat. NaHCO3 in 2:1
DMF-H2O, 25 °C, 1 h, 93%) provided (+)- and ent-(-)-23.
Design and Synthesis of Extended, Sandwiched, and
Reversed Analogues. Extended analogue 24, sandwiched
analogue 25, and reversed analogue 26 were prepared to
establish the importance of the agent structure and the location
of the DNA binding subunits on the DNA alkylation selectivity
(Figure 3). Each analogue in this series is composed of the
alkylation subunit of duocarmycin SA and two PDE subunits
attached in each of three possible arrangements. Extended ana-
logue 24 contains the same DNA binding subunits organized
in the same arrangement as that found in CC-1065 but incor-
porates the more stable (4-5-fold) and more potent (4-5-
fold) duocarmycin SA alkylation subunit.20 As such, it would
be expected to alkylate DNA with the same selectivity as that
(18) (a) Boger, D. L.; Coleman, R. S. J. Org. Chem. 1984, 49, 2240-2245. (b)
Boger, D. L.; Coleman, R. S.; Invergo, B. J. J. Org. Chem. 1987, 52, 1521-
1530. (c) Boger, D. L.; Coleman, R. S. J. Org. Chem. 1988, 53, 695-698.
(19) (a) Warpehoski, M. A.; Gebhard, I.; Kelly, R. C.; Krueger, W. C.; Li, L.
H.; McGovern, J. P.; Praire, M. D.; Wienienski, N.; Wierenga, W. J. Med.
Chem. 1988, 31, 590-603. (b) 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. (c) Boger, D. L.; Yun, W.; Han, N. Bioorg. Med.
Chem. 1995, 3, 1429-1453.
(20) (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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Structural Features of Yatakemycin
A R T I C L E S
Scheme 4
Figure 3. Extended, reversed, and sandwiched analogues.
both enantiomers of which exhibit identical alkylation selectivi-
ties (i.e., 5′-AAAAA)11 and indistinguishable cytotoxic poten-
cies. Reversed analogue 26 has two PDE subunits bound to the
left of the alkylation subunit and is capped on the right as its
Boc derivative. Unlike the extended and sandwiched agents,
the reversed agent lacks an extended heteroaromatic N2 acyl
substituent on the alkylation subunit to participate in a binding-
induced activation of the alkylation reaction. Consequently, both
its rate and efficiency of DNA alkylation would be expected to
be diminished relative to 24 and 25, resulting in a diminished
cytotoxic potency. Just as significantly and because the DNA
binding subunits extend in opposite directions relative to those
found in the extended analogue 24, they should exhibit a
reversed enantiomeric alkylation selectivity if it is controlled
by the noncovalent binding selectivity of the compounds. As
such, the natural enantiomer of 26 would be expected to alkylate
the same sites as the unnatural enantiomer of 24, whereas its
unnatural enantiomer should alkylate DNA with a selectivity
indistinguishable from the natural enantiomer of 24.14 Signifi-
cantly, all three agents have comparable DNA-binding surfaces
and the same inherent reactivity, thereby allowing a direct
comparison of the impact of the subunit arrangement on their
DNA alkylation profile and biological properties.
Scheme 5
The synthesis of extended analogue 24 was accomplished as
shown in Scheme 6. Acid-catalyzed Boc deprotection of 2711
(4 N HCl-EtOAc, 25 °C, 3 h) followed by coupling with 2822
(1.0 equiv, 4 equiv of EDCI, DMF, 25 °C, 22 h, 36%) and
subsequent spirocyclization (sat. aq. NaHCO3 in 2:1 DMF-
H2O, 25 °C, 0.5 h, 78%) provided (+)- and ent-(-)-24.
Sandwiched analogue 25 was prepared by the strategy outlined
in Scheme 7. Boc deprotection of 611 (4 N HCl-EtOAc, 25
°C, 3 h) followed by coupling with 3011,22 (1.0 equiv, 4 equiv
of EDCI, DMF, 25 °C, 14 h, 81%) provided 32. Methyl ester
hydrolysis (4 equiv of LiOH, 3:2:1 THF-MeOH-H2O, 25 °C,
5 h, 96%) followed by benzyl ether deprotection of (1 atm H2,
10% Pd/C, 9:1 THF-MeOH, 1 h, 93%) gave 34. Coupling of
34 with the hydrochloride salt of 3111 (4 equiv of EDCI, DMF,
25 °C, 16 h, 69%) furnished 35, and subsequent spirocyclization
(sat. aq. NaHCO3 in 2:1 DMF-H2O, 25 °C, 1 h, 89%) provided
(+)- and ent-(-)-25. The reversed analogue 26 (Scheme 8) was
prepared by Boc deprotection of 3622 (4 N HCl-EtOAc, 25
of CC-1065 but to be a more efficient DNA alkylating agent
and a more potent cytotoxic compound.21 Moreover, the two
enantiomers of 24, like CC-1065, would be expected to exhibit
characteristic and distinguishable alkylation selectivities con-
stituting an adenine N3 alkylation at the terminus of a five base-
pair AT-rich sequence albeit with opposite bound orientations
and with that of the unnatural enantiomer offset by one base
pair (i.e., nat. 5′-AAAAA vs unnat. 5′-AAAAA).6d Sandwiched
analogue 25 constitutes yatakemycin modified with incorpora-
tion of a PDE right-hand subunit. As such, the properties of 25
would be expected to be comparable to those of yatakemycin,
(21) (a) Parrish, J. P.; Hughes, T. V.; Hwang, I.; Boger, D. L. J. Am. Chem.
Soc. 2004, 126, 80-81. (b) Boger, D. L.; Munk, S. A.; Ishizaki, T. J. Am.
Chem. Soc. 1991, 113, 2779-2780.
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A R T I C L E S
Scheme 6
Tichenor et al.
Scheme 8
Scheme 7
Scheme 9
NaHCO3 in 2:1 DMF-H2O, 25 °C, 1 h, 96%) to provide (+)-
and ent-(-)-40 (Scheme 9).
Cytotoxic Activity. Summarized in Figure 4 is the cytotoxic
activity of the series of right subunit analogues against a cell
line (L1210 mouse leukemia) for which we have extensive
comparisons for this class. Compound 14b differs from yatake-
mycin only by switching the right subunit substituent locations
and exhibited cytotoxic potency that was indistinguishable from
that of the natural product or its unnatural enantiomer. Analogue
14c is a hybrid structure consisting of yatakemycin’s three
subunit sandwiched arrangement incorporating the 5,6,7-tri-
methoxyindole subunit found in the duocarmycins. Compound
14c (IC50 ) 5 pM) and ent-(-)-14c (IC50 ) 5 pM) were found
to be potent cytotoxic agents, indistinguishable in the cellular
assay and equipotent with (+)- and ent-(-)-yatakemycin.11 Both
compounds, like yatakemycin, are 2-fold more potent than
duocarmycin SA (4, IC50 ) 10 pM) and 20-fold more potent
than ent-(-)-duocarmycin SA. The substantial increase in
potency for the unnatural enantiomer relative to duocarmycin
SA is a characteristic feature observed with the sandwiched
agents. Presumably, the added left subunit contributes sufficient
additional binding affinity23 to compensate for the less effective
°C, 0.5 h) followed by coupling with 3714b (1.0 equiv, 4 equiv
of EDCI, DMF, 25 °C, 16 h, 67%) and subsequent spirocy-
clization (sat. aq. NaHCO3 in 2:1 DMF-H2O, 25 °C, 40 min,
68%) to provide (+)- and ent-(-)-26. Partial structure 40 having
the same two subunit arrangement as that of duocarmycin SA
incorporating a single PDE subunit was also synthesized by
benzyl ether deprotection of 32 (1 atm of H2, 10% Pd/C, 9:1
THF-MeOH, 2 h, 93%) followed by spirocyclization (sat. aq.
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Structural Features of Yatakemycin
A R T I C L E S
preceding sandwiched analogues, the unnatural enantiomer of
14f (IC50 ) 35 pM) was 6-fold less potent than the natural
enantiomer and 7-fold less potent than the ent-(-)-yatakemycin.
The corresponding partial structure 9f14b was found to be 10-
fold less potent (IC50 ) 60 pM) than 14f and 6-fold less potent
than 9a, whereas the unnatural enantiomer ent-(-)-9f was nearly
50-fold less potent (IC50 ) 1700 pM) than ent-(-)-14f and 4-5-
fold less potent than ent-(-)-9a. Clear from these comparisons
is the observation that the incorporation of a left subunit, which
increases the DNA binding affinity of the sandwiched analogues,
compensates for less effective right subunits and substantially
increases the cytotoxic potency of the unnatural enantiomers
relative to the corresponding partial structures incorporating only
the central and right-hand subunits. Only with the combination
of a less effective right-hand subunit and the unnatural enan-
tiomeric configuration in ent-(-)-14f was a detectable difference
in cytotoxic activity observed between enantiomers. Even here,
the difference in potency between the two enantiomers remains
remarkably small (5-fold) relative to the typical partial structures
incorporating only the central and right-hand subunits (10-40-
fold). An immediate conclusion to these studies is that the right-
hand subunit substituents significantly and predictably impact
the cytotoxic activity of duocarmycin-like compounds (series
9) especially improving the potency of the unnatural enantio-
mers,14 but they have a minor impact on the activity of
yatakemycin.
Compound 40 is the most potent compound in the partial
structure series (IC50 ) 5 pM), being approximately 2-fold more
potent than duocarmycin SA and equipotent with yatakemycin
and 25 (IC50 ) 6 pM). However, the unnatural enantiomer of
40 (IC50 ) 360 pM) is still 60-fold less potent than the unnatural
enantiomer of the corresponding sandwiched agent (ent-(-)-
25, IC50 ) 6 pM).
Figure 4. L1210 Cytotoxic activity of right subunit analogues (1, 4,
9a,b,d-f, 14b-f, 25, and 40).
DNA alkylation characteristic of the unnatural enantiomer of
the duocarmycins.
As shown in Figure 5, a separate series of compounds were
evaluated to establish the impact of the left subunit. Its removal
altogether providing 9a afforded an analogue that was not
distinguishable from duocarmycin SA and that was 2-fold
(natural enantiomer) or 70-fold (unnatural enantiomer) less
potent than yatakemycin and its unnatural enantiomer. The CDPI
subunit lacking the methoxy and hydroxyl substituents was
incorporated into analogue 20a (IC50 ) 5 pM, ent-(-)-20a IC50
) 6 pM) which was found to be equipotent with yatakemycin.
The thiomethyl ester derivative of CDPI was similarly incor-
The contribution of each right subunit substituent was
determined by evaluation of a series of key simplified analogues.
Yatakemycin partial structure 9a (IC50 ) 10 pM) served as a
valuable baseline comparison for identifying the contribution
of the left subunit for each analogue. Both enantiomers of 14d
lacking only the C6-methoxy group were equipotent (IC50 ) 5
pM) and indistinguishable from yatakemycin. In contrast, the
corresponding partial structure 9d (IC50 ) 30 pM) was 6-fold
less potent than yatakemycin and 3-fold less potent than 9a,
and its unnatural enantiomer (IC50 ) 600 pM) was considerably
less active. Similarly, 14e lacking the C5-hydroxy group (IC50
) 4 pM; ent-(-)-14e, IC50 ) 10 pM) was essentially indistin-
guishable from yatakemycin, whereas the corresponding partial
structure 9e (IC50 ) 25 pM) was 5-fold less potent than
yatakemycin, 2-3 times less active than 9a, and its unnatural
enantiomer (IC50 ) 1300 pm) was 260-fold less potent than
the sandwiched structures.
porated into analogue 20b (IC50 ) 7 pM, ent-(-)-20b IC50
)
6 pM) with no change in potency. Thus, like observations made
with yatakemycin itself,11 no unique effect was detected for the
presence of the methyl thioester. To probe the impact of the
pyrrolidine ring alone, 16a was prepared which significantly
reduces the size and length of the left subunit. The cytotoxic
potency of the natural enantiomer (IC50 ) 7 pM) was compa-
rable to 1 and perhaps a bit more potent than duocarmycin SA
and 9a. The unnatural enantiomer was 10-fold less potent (IC50
) 70 pM) but notably 5-fold more potent than the corresponding
methyl ester ent-(-)-9a (IC50 ) 400 pM). Pyrrolidine analogue
16b (IC50 ) 7 pM; ent-(-)-16b IC50 ) 80 pM) possessing a
5,6,7-trimethoxyindole right subunit was equipotent with 16a.
The small increase in agent length and rigidity provided by the
pyrrolidine amide results in an increase in the potency of the
unnatural enantiomer although a significant difference between
each enantiomer’s potency remains. The sandwiched analogue
A particularly interesting analogue was 14f, incorporating a
right subunit indole lacking additional substituents. The natural
enantiomer was essentially equipotent with yatakemycin (IC50
) 6 pM) despite lacking a C5 substituent. Distinct from the
(22) (a) Boger, D. L.; Coleman, R. S. J. Am. Chem. Soc. 1987, 109, 2717-
2727. (b) Boger, D. L.; Coleman, R. S. J. Am. Chem. Soc. 1988, 110, 1321-
1323; 1988, 110, 4796-4807.
(23) (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.
9
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Figure 6. Cytotoxic activity (L1210) and relative rates (krel) and efficiencies
of DNA alkylation (w836).
DNA binding subunits appears to compensate for the reduced
potency that is characteristic of the unnatural enantiomer of
duocarmycin SA. The extended agent 24 (IC50 ) 4 pM) was
essentially equivalent in potency with its unnatural enantiomer
(IC50 ) 6 pM), indistinguishable in potency with yatakemycin,
but 4-5-fold more potent than CC-1065. The greater potency
of 24 relative to CC-1065 can be attributed to the incorporation
of the more stable and more potent duocarmycin SA alkylation
subunit.21 The sandwiched analogue (+)-25 (IC50 ) 6 pM) was
equipotent with its unnatural enantiomer ent-(-)-25 (IC50 ) 6
pM) and both enantiomers of yatakemycin (IC50 ) 5 pM). In
contrast, the reversed analogues were approximately 10-fold less
potent than the preceding analogues ((+)-26, IC50 ) 40 pM;
ent-(-)-26, IC50 ) 60 pM) but displayed similar potencies. The
reversed agents lack an extended N2-acyl substituent that has
been shown to be largely responsible for catalysis of the DNA
alkylation reaction15 such that its removal results in less effective
DNA alkylating agents as shown below.
Relative Rate and Efficiency of DNA Alkylation. To date,
the biological properties of members of this class of natural
products have typically mirrored their relative efficiencies of
DNA alkylation. For the sandwiched analogues explored herein
which contain three subunits, little or no differences in the final
efficiencies of DNA alkylation were observed even though they
often incorporated suboptimal DNA binding subunits or even
possess the unnatural absolute configuration. These observations
mirror the near equivalent cytotoxic activities observed within
the 14 and 20 series and even between enantiomeric pairs of
such analogues. The exceptions to these generalizations include
only the unnatural enantiomers of the compounds bearing the
simplest, unsubstituted indole right-hand subunit which proved
Figure 5. L1210 Cytotoxic activity of left subunit analogues (1, 9a, 16a,b,
20a,b, 23).
with two indole subunits 23 (IC50 ) 15 pM) exhibited 3-fold
reduced cytotoxic activity relative to yatakemycin or its indole
analogue 14f, but it was still nearly as potent as duocarmycin
SA (IC50 ) 10 pM). Nonetheless, the indole left subunit
contributes sufficiently to exceed the potency of 9f (IC50 ) 60
pM). The unnatural enantiomer of 23 was approximately 20-
fold less potent (IC50 ) 275 pM) than the natural enantiomer,
50-fold less potent than yatakemycin and its enantiomer, and
8-fold less potent than the unnatural enantiomer of 14f. Clearly,
an optimal right subunit can compensate for substantial sim-
plifications in the left subunit, and this is especially true in the
natural enantiomer series.
The cytotoxic activity of the extended, sandwiched, and
reversed agents is summarized in Figure 6. The unnatural
enantiomers of CC-106522 and yatakemycin11 exhibit a cytotoxic
potency that is indistinguishable from that of the natural
enantiomers, which is distinct from the behavior of duocarmycin
SA where the unnatural enantiomer is approximately 10-fold
less active than the natural enantiomer.20 The presence of two
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Structural Features of Yatakemycin
A R T I C L E S
catalysis) or structural features (unnatural enantiomer config-
uration) that would limit the behavior of typical two subunit
members of this class including duocarmycin SA, so much so
that even the loss of 1000-fold in the rate of DNA alkylation
with the unnatural enantiomer of 14f relative to yatakemycin
itself provides a very effective DNA alkylating agent whose
DNA alkylation efficiency and resulting cytotoxic potency
approach that of the natural product.
A second set of comparisons is summarized in Figure 6
entailing the PDE-based sandwiched, extended, and reversed
analogues of yatakemycin which further illustrate the importance
and role of the right-hand subunit to the catalysis of the DNA
alkylation reaction. Both enantiomers of the sandwiched ana-
logue 25 alkylated DNA at extraordinary rates comparable to
yatakemycin itself, and the natural enantiomer of the extended
analogue 24 also alkylated DNA at a rate that was intermediate
of these two enantiomers. By contrast, the unnatural enantiomer
of the extended analogue 24 was considerably slower (100-fold)
but ultimately achieved the same efficiency of DNA alkylation.
Although the reversed natural enantiomer was faster than its
unnatural enantiomer, neither comes close to the rates exhibited
by the sandwiched or extended analogues 24 and 25 which more
closely approximate the rates of DNA alkylation observed with
N-Boc-DSA which lacks the DNA binding subunits altogether.8
Several important generalizations are illustrated with these
comparisons including the following: (1) the observation of
natural enantiomer > unnatural enantiomer rates within each
series which was especially pronounced for the extended
analogue (100-fold) versus the sandwiched (2-4-fold) and
reversed (3-fold) analogues, and (2) the relative DNA alkylation
rates follow the order sandwiched > extended . reversed
analogues for each enantiomeric series. By far, the most striking
observation is the extraordinarily slow rates (ca. 1000-fold) and
the reduced efficiency (10-fold) of DNA alkylation for the
reversed analogues that may be attributed to their lack of an
alkylation subunit N2-substituent that can participate in the
binding-induced catalysis derived from disruption of the alky-
lation subunit vinylogous amide.14
Figure 7. Relative rates (krel) and efficiencies of DNA alkylation (w836
DNA).
to alkylate DNA with a marginally reduced efficiency, Figure
7. However, these differences are magnified if one examines
the relative rates versus final efficiencies of DNA alkylation.
Here, readily detectable differences in the rates of DNA
alkylation are observed with each enantiomeric pair (natural >
unnatural enantiomer) as well as between pairs of analogues.
Illustrative of these differences and in a comparison that
highlights the importance and role of the right-hand subunit,
the relative rates of DNA alkylation in w836 DNA conducted
at 5 °C for each enantiomer of yatakemycin were compared
with those of 14f bearing the unsubstituted indole as its right-
hand subunit, Figure 7.
DNA Alkylation Selectivity. The DNA alkylation selectivity
of the yatakemycin analogues was examined within five 150
base-pair segments of DNA described previously.24 The alky-
lation site identification and the assessment of the relative
selectivity among the available sites were obtained by therm-
ally 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
including a description of the unaltered alkylation selectivity
of (+)- and ent-(-)-yatakemycin within nucleosome packaged
DNA5b as well as a detailed comparison of the DNA alkylation
properties of (+)- and ent-(-)-yatakemycin,11 we focused our
analysis on a select set of the new yatakemycin analogues. Most
representative of this set are the PDE-based sandwiched,
extended, and reversed analogues of yatakemycin (24-26).
Here, the placement of the alkylation subunit within the trimer
structure has a profound and controlling impact on the DNA
alkylation selectivity, as well as exerts an influence on the
As observed with yatakemycin, the natural enantiomer of 14f
alkylates DNA much faster than its unnatural enantiomer (700-
fold), and for each enantiomer, yatakemycin alkylates DNA
faster than 14f (1.5-fold for natural enantiomer and ca. 200-
fold for unnatural enantiomer). Consistent with its role in
catalysis, the right-hand subunit substituents (C5 substituent)14,17
increase the rate of DNA alkylation, and this effect is most
pronounced with the unnatural enantiomers.17
Notably, the natural enantiomer of 14f incorporating a
suboptimal right-hand subunit and even the unnatural enantiomer
of yatakemycin alkylate DNA faster than the natural enantiomer
of duocarmycin SA. In addition to highlighting the special
characteristics of such sandwiched compounds, these studies
illustrate that the incorporation of the yatakemycin left-hand
subunit can compensate for suboptimal right-hand subunits (for
(24) 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
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Figure 8. Thermally induced strand cleavage of w836 DNA (146 bp,
nucleotide no. 5189-91) after DNA-agent incubation with DSA-PDE-PDE
(24), PDE-DSA-PDE (25), and PDE-PDE-DSA (26, 23 h, 25 °C), removal
of unbound agent by EtOH precipitation and 30 min thermolysis (100 °C),
followed by denaturing 8% PAGE and autoradiography. Lanes 1 and 2,
(+)-yatakemycin and ent-(-)-yatakemycin (1 × 10-5 M); lanes 3 and 4,
(+)-PDE-DSA-PDE (25) and ent-(-)-PDE-DSA-PDE (25, 1 × 10-5 M);
lane 5, control DNA; lanes 6-9, Sanger G, C, A, and T sequencing
standards; lanes 10 and 11, (+)-DSA-PDE-PDE (24) and ent-(-)-DSA-
PDE-PDE (24, 1 × 10-5 M); lanes 12 and 13, (+)-PDE-PDE-DSA (26)
and ent-(-)-PDE-PDE-DSA (26, 1 × 10-4 M).
Figure 9. Alkylation selectivities.
the 3′-end of a five base-pair AT-rich site.6 In contrast, the
unnatural enantiomer alkylated the three adenines at the opposite
5′-end of the site (5′-AAAAAA-3′). These nonoverlapping
alkylated adenines for the two enantiomers of 24 represent
opposite bound orientations (3′ f 5′ for nat. enantiomer and 5′
f 3′ for unnat. enantiomer) of the compounds across a five
base-pair AT-rich site starting with (nat. enantiomer)6 or
preceding (unnat. enantiomer)6c the alkylated adenine.
efficiency of DNA alkylation, and exhibits a unique impact on
the rate of DNA alkylation that was detailed in the preceding
section.
Illustrated in Figure 8 is a representative comparison of the
DNA alkylation selectivity of the PDE-based sandwiched,
extended, and reversed analogues of yatakemycin within a six
base-pair A-rich site found in w836 DNA which beautifully
highlights the distinctions in the compounds. As might be
anticipated, the PDE-based sandwiched analogue 25 exhibited
a DNA alkylation selectivity essentially identical to the that of
natural product yatakemycin itself. Most significant in this
comparison is the observation that both enantiomers of PDE-
DSA-PDE (25), like both enantiomers of yatakemycin, alkylate
the same site(s) (adenines central to the six base-pair A-rich
site, 5′-AAAAAA-3′) with essentially the same relative ef-
ficiency.11
In contrast, the two enantiomers of the extended analogue
24, a close analogue of CC-1065 incorporating the yatakemycin
alkylation subunit, exhibited very distinct alkylation selectivities
which were both markedly different from those of yatakemycin
and its enantiomer. The natural enantiomer (+)-DSA-PDE-PDE
(24) alkylated the two adenines at the 3′ end of six base-pair
A-rich site (5′-AAAAAA-3′) consistent with alkylation only at
Most remarkable in this set of comparisons is the behavior
of the two enantiomers of the reversed analogues PDE-PDE-
DSA (26). Although much less efficient than preceding ana-
logues even with extended reaction times (g10-fold, requiring
10-4 vs 10-5 M concentrations), the two enantiomers of the
reversed analogues exhibited distinct DNA alkylation selectivi-
ties that were not only different from that of the sandwiched
compounds and their enantiomers but also opposite that of
extended analogues 24. That is, the natural enantiomer of the
reversed analogue exhibited the identical selectivity as that of
the unnatural enantiomer of the extended analogue 24 alkylating
the adenines at the 5′-end of the site, whereas its unnatural
enantiomer alkylated the same 3′ adenines as the extended
natural enantiomer 24, Figures 8 and 9. This complete reversal
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Structural Features of Yatakemycin
A R T I C L E S
of the enantiomeric selectivity14,25 between the extended versus
reversed analogues indicates that it is not the alkylation subunit
and its absolute configuration that dominates the alkylation
selectivity and rather that it is the compound’s AT-rich
noncovalent binding selectivity23 that delivers the modestly
reactive electrophile to accessible nucleophiles within a non-
covalent binding site.14
This distinct reversed enantiomer behavior of the extended
versus reversed analogues is also and even more clearly
illustrated in Figure 10. Within w794 DNA, the natural and
unnatural enantiomers of the extended analogues 24 each
alkylate a single distinct site within the 150 base-pair segment
of DNA consistent with observations first made while examining
CC-1065 and its unnatural enantiomer.25 Examination of the
unnatural enantiomer of the reversed analogue 26 alongside both
enantiomers of the extended analogue 24 illustrate that it
alkylates the natural enantiomer site (ent-(-)-PDE-PDE-DSA
) (+)-DSA-PDE-PDE) beautifully highlighting this reversal of
the enantiomer selectivity between the extended and reversed
analogues.
Models of the Alkylation Products. Presented in Figure 11
are models of the sandwiched, extended, and reversed PDE-
based analogue alkylations of their predominant w836 sites (see
Figure 8) that visually illustrate the selectivity features discussed
earlier. Most notable in the comparisons is the fact that all six
compounds (two enantiomers of three compounds) bind across
the identical five base-pair A-rich site with each enantiomer
pair binding with opposite orientations and undergoing alkyla-
tion at the resulting adenine proximal to the reactive cyclopro-
pane. The two enantiomers of the sandwiched analogues 25
alkylate the same adenine central to the site, the natural enan-
tiomer of the extended analogue 24 and the unnatural enantiomer
of the reversed analogue 26 alkylate the same adenine at the 3′
end of the site, whereas the unnatural enantiomer of the extended
analogue 24 and the natural enantiomer of the reversed analogue
26 alkylate an adenine at the opposite 5′ end of the site (Figure
12).
Conclusions
A systematic examination of the yatakemycin left- and right-
hand subunits and their substituents is detailed establishing their
impact on the cytotoxic potency and DNA alkylation properties
of such sandwiched compounds. Within the context of this three
subunit structural arrangement, the studies illustrate that the
subunit substituents are now relatively unimportant and that the
yatakemycin left-hand subunit, as well as those incorporating
significant simplifications, can compensate for suboptimal right-
hand subunits and substituents or structural features (i.e.,
unnatural enantiomer configuration) that limit the more tradi-
tional compounds in the series. Significantly, this sandwiched
subunit arrangement substantially increases the rate of DNA
alkylation, maximizes the efficiency of DNA alkylation, and
potentiates the cytotoxic potency of the compounds. Moreover,
the placement of the alkylation subunit within the three subunit
structure not only has a profound impact on the rate (PDE-
DSA-PDE > DSA-PDE2 . PDE2-DSA) and efficiency (PDE-
DSA-PDE g DSA-PDE2 > PDE2-DSA) of DNA alkylation but
also controls the DNA alkylation selectivity as well. Most
notable in this regard is the remarkable observation that both
Figure 10. Thermally induced strand cleavage of w794 DNA (144 bp,
nucleotide no. 5238-138) after DNA-agent incubation with DSA-PDE-
PDE (24) (25 °C, 5 days) and PDE-PDE-DSA (26) (37 °C, 5 days), 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; lane 6,
(+)-DSA-PDE-PDE (24, 25 °C, 1 × 10-5 M); lane 7, ent-(-)-DSA-PDE-
PDE (24, 25 °C, 1 × 10-5 M); lane 8, ent-(-)-PDE-PDE-DSA (26, 37 °C,
1 × 10-4 M).
(25) Boger, D. L.; Johnson, D. S. J. Am. Chem. Soc. 1995, 117, 1443-1444.
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A R T I C L E S
Tichenor et al.
Figure 11. (a) Models of (+)-DSA-PDE2 (left) and ent-(-)-DSA-PDE2
(right). (b) Models of (+)-PDE-DSA-PDE (left) and ent-(-)-PDE-DSA-
PDE (right). (c) Models of (+)-PDE2-DSA (left) and ent-(-)-PDE2-DSA
(right). See Supporting Information for larger images of the models.
Figure 12. Graphic representation of bound orientations and alkylation
sites for the extended, sandwiched, and reversed analogues.
extended or reversed analogues which not only are different
from that of the sandwiched compounds but also exhibit reversed
enantiomeric selectivities (e.g., (+)-DSA-PDE2 ) ent-(-)-
PDE2-DSA). These remarkable observations, which are easily
rationalized and visualized based on prevailing models of the
DNA alkylation reaction,26 are consistent with preferential
enantiomers of such sandwiched compounds alkylate the same
sites exhibiting the same DNA alkylation selectivity independent
of the enantiomeric configuration ((+)-PDE-DSA-PDE ) ent-
(-)-PDE-DSA-PDE). Contrasting this behavior is the distinct
DNA alkylation selectivities of the enantiomeric pairs of
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Structural Features of Yatakemycin
A R T I C L E S
noncovalent binding of the compounds across a five base-pair
AT-rich site delivering the natural enantiomer of a modestly
reactive electrophile to adenine positioned central (sandwiched)
or at the 3′ terminus (extended) and 5′ terminus (reversed) of
such sites, whereas the unnatural enantiomers bind within the
same sites but with reversed orientations delivering the elec-
trophile to adenine central (sandwiched) or at the opposite 5′
terminus (extended) and 3′ terminus (reversed) of the sites.
Superimposed on this binding-derived alkylation selectivity is
the impact of catalysis attributable to the alkylation subunit N2
substituent. The subunit arrangements incorporating an extended
heteroaromatic N2 acyl substituent on the alkylation subunit
(sandwiched, extended analogues) alkylate DNA with remark-
able rates and efficiencies for such a modest electrophile,
whereas those that lack this structural feature (reversed ana-
logues) do so only at markedly reduced rates (g1000-fold). We
have attributed this pronounced impact on the rate to a unique
source of catalysis for the DNA alkylation reaction that is
derived from a DNA binding-induced conformation change in
the molecule that activates them for nucleophilic attack. Thus,
the adoption of a DNA bound helical conformation disrupts the
N2 vinylogous amide conjugation stabilizing the alkylation
subunit (sandwiched and extended, but not reversed) activating
the cross-conjugated cyclopropane for nucleophilic attack.
Tucked into these studies, we have also prepared and
evaluated a key analogue of CC-1065. The extended analogue
24, (+)- and ent-(-)-DSA-PDE2, constitutes the substitution
of the duocarmycin SA/yatakemycin alkylation subunit into the
CC-1065 structure replacing its CPI alkylation subunit. This
substitution provided a hybrid analogue of CC-1065 and its
unnatural enantiomer (L1210 IC50 ) 20 pM, both enantiomers)
that exhibited an unaltered DNA alkylation selectivity relative
to CC-1065 but which proved to be 5-fold more potent (IC50
)
4 pM, Figure 6) than the natural product, precisely in line with
expectations21 based on its 5-fold enhanced stability.27
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) and American Society for Engineering Education (2003-
2005 J.D.T., NDSEG). J.D.T., K.S.M., and M.S.T. are Skaggs
Fellows.
Supporting Information Available: Full experimental details
are provided. This material is available free of charge via the
Internet at http://pubs.acs.org.
JA072777Z
(27) This stability was measured by assessing solvolysis reactivity of the
respective N-Boc derivatives at pH 3: N-Boc-CPI (t1/2 ) 37 h, k ) 5.26
× 10-6 s-1) versus N-Boc-DSA (t1/2 ) 177 h, k ) 1.08 × 10-6 s-1). See:
Boger, D. L.; Yun, W. J. Am. Chem. Soc. 1994, 116, 5523-5524. Boger,
D. L.; Yun, W. J. Am. Chem. Soc. 1994, 116, 7996-8006.
(26) Boger, D. L.; Johnson, D. S.; Yun, W. J. Am. Chem. Soc. 1994, 116, 1635-
1656.
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