Asymmetric total synthesis of (+)- and ent-(-)-yatakemycin and duocarmycin SA: Evaluation of yatakemycin key partial structures and its unnatural enantiomer

Article abstract of DOI:10.1021/ja064228j

Complementary to studies that provided the first yatakemycin total synthesis resulting in its structure revision and absolute stereochemistry assignment, a second-generation asymmetric total synthesis is disclosed herein. Since the individual yatakemycin subunits are identical to those of duocarmycin SA (alkylation subunit) or CC-1065 (central and right-hand subunits), the studies also provide an improvement in our earlier total synthesis of CC-1065 and, as detailed herein, have been extended to an asymmetric total synthesis of (+)-duocarmycin SA. Further extensions of the studies provided key yatakemycin partial structures and analogues for comparative assessments. This included the definition of the DNA selectivity (adenine central to a five-base-pair AT sequence, e.g., 5′-AAAAA), efficiency, relative rate, and reversibility of ent-(-)-yatakemycin and its comparison with the natural enantiomer (identical selectivity and efficiency), structural characterization of the adenine N3 adduct confirming the nature of the DNA reaction, and comparisons of the cytotoxic activity of the natural product (L1210, IC₅₀ = 5 pM) with those of its unnatural enantiomer (IC₅₀ = 5 pM) and a series of key partial structures including those that probe the role of the C-terminus thiomethyl ester. The only distinguishing features between the enantiomers is that ent-(-)-yatakemycin alkylates DNA at a slower rate (k_rel = 0.13) and is reversible, whereas (+)-yatakemycin is not. Nonetheless, even ent-(-)-yatakemycin alkylates DNA at a faster rate and with a greater thermodynamic stability than (+)-duocarmycin SA, illustrating the unique characteristics of such "sandwiched" agents.

Full text of DOI:10.1021/ja064228j

Published on Web 11/18/2006
Asymmetric Total Synthesis of (+)- and ent-(-)-Yatakemycin
and Duocarmycin SA: Evaluation of Yatakemycin Key Partial
Structures and Its Unnatural Enantiomer
Mark S. Tichenor, John D. Trzupek, David B. Kastrinsky, Futoshi Shiga,
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 June 15, 2006; E-mail: boger@scripps.edu
Abstract: Complementary to studies that provided the first yatakemycin total synthesis resulting in its
structure revision and absolute stereochemistry assignment, a second-generation asymmetric total synthesis
is disclosed herein. Since the individual yatakemycin subunits are identical to those of duocarmycin SA
(alkylation subunit) or CC-1065 (central and right-hand subunits), the studies also provide an improvement
in our earlier total synthesis of CC-1065 and, as detailed herein, have been extended to an asymmetric
total synthesis of (+)-duocarmycin SA. Further extensions of the studies provided key yatakemycin partial
structures and analogues for comparative assessments. This included the definition of the DNA selectivity
(adenine central to a five-base-pair AT sequence, e.g., 5′-AAAAA), efficiency, relative rate, and reversibility
of ent-(-)-yatakemycin and its comparison with the natural enantiomer (identical selectivity and efficiency),
structural characterization of the adenine N3 adduct confirming the nature of the DNA reaction, and
comparisons of the cytotoxic activity of the natural product (L1210, IC50 ) 5 pM) with those of its unnatural
enantiomer (IC50 ) 5 pM) and a series of key partial structures including those that probe the role of the
C-terminus thiomethyl ester. The only distinguishing features between the enantiomers is that ent-(-)-
yatakemycin alkylates DNA at a slower rate (krel ) 0.13) and is reversible, whereas (+)-yatakemycin is
not. Nonetheless, even ent-(-)-yatakemycin alkylates DNA at a faster rate and with a greater thermodynamic
stability than (+)-duocarmycin SA, illustrating the unique characteristics of such “sandwiched” agents.
Introduction
sented a remarkable hybrid of the preceding natural products
containing a central alkylation subunit identical to that found
Yatakemycin (1)1 was isolated from the culture broth of
Streptomyces sp. TP-AO356 and represents the newest and most
potent member of a class of antitumor compounds that includes
CC-1065,2 duocarmycin A,3 and duocarmycin SA4 (Figure 1).
Each derives its biological properties through a characteristic
and distinguishable DNA alkylation reaction.5-9 The structure
of (+)-yatakemycin was disclosed in 2003 as 5 on the basis of
extensive spectroscopic studies (Figure 2).1 As such, it repre-
in duocarmycin SA, a right-hand subunit similar to that of the
duocarmycins, and a left-hand subunit similar to the central and
right-hand subunits of CC-1065. Distinct from the preceding
natural products, it represented the first naturally occurring
member of this class that contains DNA binding subunits
flanking each side of the alkylation subunit. Since our earlier
studies established that this “sandwiched” arrangement conveyed
remarkable properties to a series of duocarmycin SA analogues
(enhanced DNA alkylation rate, uniquely altered alkylation
selectivity, more potent cytotoxic activity),10 we became
especially interested in yatakemycin. With a sample of natural
material provided by Igarashi, we defined natural yatakemycin’s
(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.
(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. (e) Boger, D. L.; Garbaccio, R. M. Bioorg. Med. Chem.
1997, 5, 263-276.
9
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J. AM. CHEM. SOC. 2006, 128, 15683-15696
15683

A R T I C L E S
Tichenor et al.
Figure 3. Model comparison.
shift in the indole C8-H, and this reformulated structure (6)
was more consistent with other members of the natural product
family. Most notably, the left-hand subunit was now identical
to the central and right-hand subunits of CC-1065, albeit capped
as a thiomethyl ester. Diagnostically, aryl thiomethyl esters12a
exhibit a methyl 13C NMR chemical shift of roughly δ 11,
matching the chemical shift reported for yatakemycin (δ 11.14),
whereas aryl thioacetates12b occur at δ 30. Thus, yatakemycin
was reformulated as 6 and targeted for synthesis.
Figure 1. Natural products.
Total synthesis of this alternative structure provided a
compound nearly identical to, but still subtly distinct from, the
1
natural product. However, the H NMR chemical shifts in the
left-hand and central subunits of 6 matched those of authentic
yatakemycin, suggesting this portion of the structure incorporat-
ing the reformulated thiomethyl ester was now correct. The
remaining discrepancies rested exclusively with subtle perturba-
1
tions in the H NMR chemical shifts in the right-hand subunit
of 6. After we convinced ourselves that these 1H NMR
differences were not due to concentration or solvent effects (e.g.,
water in pyridine-d5 or partial phenol deprotonation) and that 6
and yatakemycin were chromatographically distinguishable
(coelute in most solvents), a reexamination of the 1H NMR and
HMBC data disclosed in the structure identification established
that the substituent locations, but not their identity, had been
defined. Model indole 7 was synthesized14 and found to correlate
well with the right-hand subunit of yatakemycin in the same
NMR solvent (pyridine-d5), particularly with respect to the most
deshielded aromatic proton δ 7.66 (vs δ 7.62 for yatakemycin),
which was absent in both previous candidate structures (Figure
3). Further characterization of 7 by NMR (pyridine-d5, ROESY,
HMBC, HMQC, 13C-APT) and correlation with yatakemycin
Figure 2. Original yatakemycin structure and first structure reformulation.
DNA alkylation properties which proved characteristic of such
a sandwiched compound.5
In 2004, we reported the total synthesis of 5 and its lack of
correlation with the natural product (Figure 2).11 On the basis
of spectroscopic distinctions between 5 and yatakemycin, the
natural product structure was reformulated as 6. Notably, the
1H NMR of 5 did not match that of natural yatakemycin, and
the greatest discrepancies occurred in the left-hand subunit. The
1H NMR chemical shift of the yatakemycin left subunit indole
C8-H is found at δ 7.52, significantly downfield of the
corresponding proton in 5 at δ 6.85, and the thioacetate methyl
group was found 0.16 ppm upfield of the corresponding protons
in the natural material.1 Model substrates and computer 1H NMR
predictions supported a reassignment of the C7-substituent as
a thiomethyl ester versus thioacetate. The electron-withdrawing
character of a C7-thioester would account for the downfield
1
indicated that the reported H NMR chemical shifts of C4-H
and C7-H of the right-hand subunit most likely had been
(12) (a) Barbero, M.; Cadamuro, S.; Degani, I.; Fochi, R.; Gatti, A.; Regondi,
V. Synthesis 1988, 300-302. (b) Jordan, F.; Kudzin, Z.; Witczak, Z.; Hoops,
P. J. Org. Chem. 1986, 51, 571-573.
(13) Okano, K.; Tokuyama, H.; Fukuyama, T. J. Am. Chem. Soc. 2006, 128,
7136-7137.
(14) Synthesis of 5-hydroxy-6-methoxyindole-2-carboxylic acid (45): 3-(Ben-
zyloxy)-4-methoxybenzaldehyde was condensed with N3CH2CO2Me (4
equiv, 4 equiv of NaOMe, MeOH, -15 °C, 3 h, then 0 °C, 24 h, 87%) and
then subjected to cyclization in xylenes (130 °C, 12 h, 72%). Methyl ester
hydrolysis (4 equiv of LiOH, 25 °C, 14 h, 93%) and benzyl ether
deprotection (1 atm of H2, 10% Pd/C, 25 °C, 1 h, 98%) provided 45. The
conversion of 45 to 7 was accomplished by coupling with pyrrolidine (3
equiv, 3 equiv of EDCI, 25 °C, 4 h, 69%). See: (a) MacKenzie, A. R;
Moody, C. J.; Rees, C. W. J. Chem. Soc., Chem. Commun. 1983, 22, 1372-
1373. (b) Bolton, R. E.; Moody, C. J.; Pass, M.; Rees, C. W.; Tojo, G. J.
Chem. Soc., Perkin Trans. 1 1988, 2491-2499.
(10) (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.
(11) Tichenor, M. S.; Kastrinsky, D. B.; Boger, D. L. J. Am. Chem. Soc. 2004,
126, 8396-8398.
9
15684 J. AM. CHEM. SOC. VOL. 128, NO. 49, 2006

Synthesis of Yatakemycin and Duocarmycin SA
A R T I C L E S
Scheme 1
Scheme 2
discussed in the following sections, whereas the right-hand
subunit (5-hydroxy-6-methoxyindole-2-carboxylic acid) was
readily available in four steps from commercially available
material.14
switched, which could have caused the substituent locations to
be misassigned. As a result, 1 was targeted for synthesis and
bears this right-hand subunit substituent reassignment as well
as the left-hand subunit thiomethyl ester. This further reformula-
tion of the yatakemycin structure was confirmed by total
synthesis of (+)- and ent-(-)-1 in studies that additionally
established the absolute configuration of the natural product.11
Herein we report full details of studies providing a second-
generation, asymmetric total synthesis of (+)- and ent-(-)-
yatakemycin,11,13 the extension of our preceding studies to the
preparation of key partial structures and analogues, and details
of the assessments of their properties. This includes the
definition of the DNA alkylation properties of synthetic ent-
(-)-yatakemycin and its comparison with those of the natural
enantiomer, the isolation and structure determination of the
thermally released DNA-adenine adduct confirming the nature
of the DNA alkylation reaction, and a comparison of the
cytotoxic activity of the natural product (L1210, IC50 ) 5 pM)
with those of its unnatural enantiomer (IC50 ) 5 pM) and a
series of key partial structures and analogues. Since the
individual subunits of yatakemycin are identical to those found
in duocarmycin SA (alkylation subunit) or CC-1065 (central
and right-hand subunits), the studies also provide an improve-
ment in our reported total synthesis of CC-1065 and, as also
detailed herein, have provided a second-generation, asymmetric
total synthesis of (+)-duocarmycin SA.15
Synthesis of the Alkylation Subunit. Complementary to our
original synthesis of the alkylation subunit first developed for
the total synthesis of (+)- and (-)-duocarmycin SA,16-21 which
relied on a late-stage resolution of an advanced intermediate
for accessing optically active material,16 an asymmetric synthesis
was developed and is disclosed herein. Key to its implementation
is a surprisingly effective and regioselective intramolecular
epoxide opening inspired by the studies of Sakamoto,22
a
uniquely concise synthesis of the requisite iodoindole precursor
16, and a final-stage transannular Ar-3′ spirocyclization for
introduction of the activated cyclopropane (Scheme 2).17,21,23,24
Importantly, the late-stage introduction of the chiral center
derived from (R)- or (S)-glycidol permits ready access to either
enantiomer.
The asymmetric synthesis of the alkylation subunit began with
aldehyde 8,25 which is prepared in a single step from com-
mercially available 3,5-dinitrobenzyl alcohol (Scheme 3). Nu-
cleophilic displacement of one nitro group in 8 using benzal-
dehyde oxime26 (1.5 equiv, 3 equiv of K2CO3, DMF, 90 °C,
1.25 h) occurred with in situ elimination to the phenol, which
was trapped as the benzyl ether (1.6 equiv of BnBr, DMF, 25
°C, 2 h, 86%) to provide 10 in superb overall yield. This one-
pot substitution of a benzyloxy group (hydroxy group) for a
nitro substituent serves as a superb alternative to a low-yielding
Chemistry
Like our original synthesis of yatakemycin, the natural
product was accessed from three subunits that compose its
structure, using a defined order for their coupling (Scheme 1).
Unlike our prior approach, a final transannular Ar-3′ spirocy-
clization was used to close the activated cyclopropane, enlisting
a Mitsunobu activation of a precursor secondary alcohol,
permitting the free phenol 19 to be utilized directly in the
coupling sequence without protection and shortening the number
of late-stage steps. Additionally, an asymmetric synthesis of the
central alkylation subunit was developed and incorporates a late-
stage introduction of the chiral center, permitting ready access
to either enantiomer. Key elements of the strategic design of
the routes used to access the central and left-hand subunits are
(17) (a) Muratake, H.; Matsumura, N.; Natsume, M. Chem. Pharm. Bull. 1995,
43, 1064-1066. (b) Muratake, H.; Abe, I.; Natsume, M. Chem. Pharm.
Bull. 1996, 44, 67-79. (c) Muratake, H.; Tonegawa, M.; Natsume, M.
Chem. Pharm. Bull. 1998, 46, 400-412.
(18) Fukuda, Y.; Terashima, S. Tetrahedron Lett. 1997, 38, 7207-7208.
(19) Yamada, K.; Kurokawa, T.; Tokuyama, H.; Fukuyama, T. J. Am. Chem.
Soc. 2003, 125, 6630-6631.
(20) Tietze, L. F.; Haunert, F.; Feuerstein, T.; Herzig, T. Eur. J. Org. Chem.
2003, 3, 562-566.
(21) Hiroya, K.; Matsumoto, S.; Sakamoto, T. Org. Lett. 2004, 6, 2953-2956.
(22) (a) Uchiyama, M.; Kameda, M.; Mishima, O.; Yokoyama, N.; Koike, M.;
Kondo, Y.; Sakamoto, T. J. Am. Chem. Soc. 1998, 120, 4934-4946. (b)
Kondo, Y.; Matudaire, T.; Sato, J.; Murata, N.; Sakamoto, T. Angew. Chem.,
Int. Ed. Engl. 1996, 35, 736-738. (c) Uchiyama, M.; Koike, M.; Kameda,
M.; Kondo, Y.; Sakamoto, T. J. Am. Chem. Soc. 1996, 118, 8733-8734.
(23) Boger, D. L.; McKie, J. A.; Nishi, T.; Ogiku, T. J. Am. Chem. Soc. 1996,
118, 2301-2302; 1997, 119, 311-325.
(24) Boger, D. L.; McKie, J. A.; Boyce, C. E. Synlett 1997, 515-517.
(25) Barrett, A. G.; Braddock, D. C.; McKinnell, R. M.; Waller, F. J. Synlett
1999, 1489-1490. The Swern oxidation is a more effective alternative to
synthesize 8 (1.5 equiv of (ClCO)2, 3 equiv of DMSO, CH2Cl2, -78 °C,
1 h, then 5 equiv of Et3N, -30 °C, 1 h, 95%).
(26) (a) Shevelev, S. A.; Vatsadze, I. A.; Dutov, M. D. MendeleeV Commun.
2002, 5, 196-198. (b) Knudsen, R. D.; Snyder, H. R. J. Org. Chem. 1974,
39, 3343-3346.
(15) Review of synthetic studies: Boger, D. L.; Boyce, C. W.; Garbaccio, R.
M.; Goldberg, J. A. Chem. ReV. 1997, 97, 787-828.
(16) (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.
9
J. AM. CHEM. SOC. VOL. 128, NO. 49, 2006 15685

A R T I C L E S
Scheme 3
Tichenor et al.
Scheme 4
to the generation of an appropriate cuprate reagent. Metal-
halogen exchange to the Grignard reagent proceeded without
such competitive nucleophilic additions, and the intermediate
aryl Grignard was unreactive (at -42 °C) toward the epoxide
until treated with a catalytic amount of the copper reagent. In
contrast to the present example, analogous intramolecular
epoxide additions with both organolithium and organocuprate
reagents typically give mixtures of 5-endo and 6-exo products
in modest yields.30 Transfer hydrogenolysis of the benzyl ether
(excess 25% aqueous HCO2NH4, 10% Pd/C, 9:1 THF-MeOH,
25 °C, 3 h, 82%) gave the alkylation subunit precursor 19.
In addition, N-alkylation of 16 with 1,3-dichloropropene
followed by 5-exo-trig free radical ring closure following a
protocol introduced in 199731,32 provides an expedient route to
the racemic alkylation subunit precursor 21 (Scheme 4) analo-
gous to the approach enlisted by Tietze and co-workers20 in
their total synthesis of (()-duocarmycin SA. Resolution of 21
by chromatographic separation33 (Chiralcel OD, 2 × 25 cm, 7
mL/min, R ) 1.28) provides each enantiomer of the alkylation
subunit (g99.9% ee), suitable for elaboration to optically active
yatakemycin or duocarmycin SA. Notably, the route to 16
detailed herein (seven steps) reduces this approach to a simple
nine-step synthesis of an alkylation subunit precursor, resolvable
at step nine.
Total Synthesis of (+)-Duocarmycin SA. The asymmetric
alkylation subunit synthesis described herein was applied to a
short and efficient preparation of duocarmycin SA. Boc depro-
tection of 19 (4 N HCl-EtOAc, 25 °C, 3 h) followed by
coupling with 5,6,7-trimethoxyindole-2-carboxylic acid7,34 (22;
1.5 equiv, 4 equiv of EDCI, DMF, 0 °C, 3 h, 64%) provided
seco-duocarmycin SA (23) (Scheme 5). The final Ar-3′ spiro-
cyclization was achieved using a Mitsunobu activation and
displacement of the secondary alcohol (6 equiv of ADDP, 6
equiv of Bu3P, THF, 0.5 h, 25 °C, 62%) to give (+)-4. This
asymmetric synthesis of (+)-duocarmycin SA was accomplished
in 12 steps from 3,5-dinitrobenzaldehyde and constitutes one
of the most efficient reported to date.16-21
monoreduction/diazotization procedure commonly described in
the literature27 and merits more widespread recognition and
adoption than is presently the case. Condensation of aldehyde
10 with methyl azidoacetate (4 equiv, 5 equiv of NaOMe,
MeOH, 4 °C, 48 h, 78%) provided 11. Thermolysis of the
resulting styryl azide (xylenes, 140 °C, 7 h, 68%) provided the
desired Hemetsberger-Rees product 12,14,28 and protection of
the indole, nitro reduction, and Boc protection of the resulting
amine provided 15 (91% overall).20
Regioselective iodination of 15 (2 equiv of NIS, toluene-
HOAc, 25 °C, 8 h, 91%) set the stage for the key intramolecular
epoxide opening and the final stages of the synthesis. Alkylation
of 16 by treatment with (S)-glycidyl-3-nosylate29 (1.2 equiv,
1.2 equiv of NaH, DMF, 0 °C, 3 h, 96%) provided 17 (for the
natural enantiomer) with clean SN2 displacement of the nosylate
versus SN2′ epoxide opening and subsequent displacement of
the nosylate. Competitive SN2′ displacement would give the
enantiomeric epoxide, reducing the enantiomeric purity of the
product epoxide,29 but 17 was isolated in high, nearly perfect
optical purity, confirming the superb alkylation regioselectivity.
Formation of the Grignard reagent by metal-halogen exchange
(1.1 equiv of i-PrMgCl, -42 °C, 1 h) followed by transmeta-
lation to the cuprate (0.2 equiv of CuI-Bu3P, -78 °C, 2 h,
69%) induced rapid intramolecular ring opening to give
exclusively 18 (99% ee, Chiralcel OD HPLC), the result of
6-endo versus 5-exo addition to the epoxide. Notably, the
corresponding aryllithium reagent failed to give any of the
desired product due to preferential or competitive reactions of
the metalating reagent (n-BuLi, 1.0 equiv, -78 °C) with the
methyl ester or Boc group, precluding the use of this approach
Synthesis of the Left-Hand Subunit. In our initial studies,11
the left-hand subunit of yatakemycin was established to possess
a structure identical to that of the subunits found in CC-1065,
albeit capped as a thiomethyl ester. Thus, the originally disclosed
C7-thioacetate was reassigned as the C7-thiomethyl ester
following the noncorrelation of 5 with yatakemycin and an
assessment of the spectroscopic discrepancies. In this initial
work, the total synthesis of 1 and its successful correlation with
(30) Erdik, E. Tetrahedron 1984, 40, 641-657.
(31) Boger, D. L.; Boyce, C. W.; Garbaccio, R. M.; Searcey, M. Tetrahedron
Lett. 1998, 39, 2227-2230.
(32) 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.
(27) Abraham, D. J.; Gazze, D. M.; Kennedy, P. E.; Mokotoff, M. J. Med. Chem.
1984, 27, 1594-1559.
(28) The undesired regioisomer, methyl 5-(benzyloxy)-7-nitroindole-2-carboxy-
late, was also produced in 18% yield (3.8:1 selectivity favoring 12).
(29) Hanson, R. M. Chem. ReV. 1991, 91, 437-475.
(33) Boger, D. L.; Yun, W. J. Am. Chem. Soc. 1994, 116, 7996-8006.
(34) Boger, D. L.; Ishizaki, T.; Zarrinmayeh, H.; Kitos, P. A.; Suntornwat, O.
J. Org. Chem. 1990, 55, 4499-4502.
9
15686 J. AM. CHEM. SOC. VOL. 128, NO. 49, 2006

Synthesis of Yatakemycin and Duocarmycin SA
A R T I C L E S
Scheme 5
Scheme 7
Scheme 6
7). Subsequent Curtius rearrangement (1.05 equiv of DPPA, 3
equiv of Et3N, THF, 25 °C, 3 h) with water addition to the
intermediate isocyanate provided amine 27 (78% overall), which
was N-tosylated (1.5 equiv of p-TsCl, pyridine, 25 °C, 2 h, 89%)
to give 28. Nitro reduction (5 equiv of SnCl2‚2H2O, 1:1
dioxane-EtOH, 85 °C, 0.5 h, 92%), Boc protection of the
resulting amine 29 (1.2 equiv of Boc2O, THF, 65 °C, 14 h,
94%), and subsequent oxidation of 30 (1.05 equiv of Pb(OAc)4,
CHCl3, 25 °C, 2 h, 96%) provided the selectively activated
p-quinodiimide 31. Throughout this preparation of 31, the easily
purified crystalline intermediates permitted the multigram
synthesis of advanced intermediates. The key Diels-Alder
reaction of 31 with 2-methoxy-1,3-butadiene (20 equiv, 40 °C,
48 h) provided a single cycloadduct regioisomer that was treated
with Et3N to effect in situ aromatization to 32 (57% overall).36
As expected, the cycloaddition regioselectivity was dominated
by the stronger electron-withdrawing character of the N-
tosylimine and set the stage for cleavage of the enol ether with
release of two differentially oxidized side chains suitable for
closure to an appropriately functionalized dihydropyrroloin-
dole.37 Thus, ozonolysis of 32 (O3, 1:1 CH2Cl2-MeOH, -78
°C) followed by reductive workup (10 equiv of Me2S, 71%)
provided the labile 2-hydroxyindoline 33, which in turn was
treated with 4 N HCl-EtOAc (25 °C, 0.6 h, 96%) to induce
aromatization and N-Boc deprotection. Further cyclization of
34 to lactam 35 was achieved upon treatment with HOAc (25
°C, 24 h, 91%). The indole N-tosyl group was reductively
cleaved using Mg in MeOH (20 equiv of Mg, sonication, 25
°C, 1.5 h, 96%). Subsequent indole reduction (2.0 equiv of
NaCNBH3, AcOH, 25 °C, 2 h, 96%) afforded 37, which was
protected (2.0 equiv of Boc2O, THF, 65 °C, 1.5 h, 93%) to
provide 38.
the natural product relied on a preparation of this subunit
enlisting a late-stage intermediate used in the synthesis of the
CC-1065 central and right-hand subunits. Herein, we report an
improved synthesis of this intermediate and its incorporation
into yatakemycin. The approach represents an extension of our
earlier yatakemycin studies that provided the misassigned left-
hand subunit in which the amide that was used to introduce the
thioacetate (via the thioamide) is now enlisted to introduce a
methyl ester via its conversion to the corresponding bromoindole
and subsequent Pd(0)-catalyzed carbonylation (eq 1).
Key to conception of this concise synthesis of the left-hand
subunit of first 5 and now 1 was a regioselective Diels-Alder
reaction of a selectively activated p-quinodiimide in which it is
the toluenesulfonyl (versus Boc) group of 31 that expectantly
controls the regioselectivity35 of the [4 + 2] cycloaddition
reaction (Scheme 6). Subsequent oxidative cleavage of the enol
ether in the cycloadduct 32 was anticipated to provide the
appropriately substituted dihydropyrroloindole skeleton of the
left-hand subunit.
This synthesis of the left-hand subunit began with 24, which
is readily available from o-vanillin.35 Phenol protection as the
benzyl ether (1.5 equiv of BnBr, 2.0 equiv of K2CO3, DMF, 25
°C, 14 h, 88%) followed by aldehyde oxidation of 25 (1.2 equiv
of NaClO2, t-BuOH, 25 °C, 2 h, 97%) provided 26 (Scheme
Direct conversion of lactam 38 to the bromoindole 39 was
effected by treatment with POBr3 (6 equiv, 8 equiv of imidazole,
(35) (a) Review: Adams, R.; Reifschneider, W. Bull. Soc. Chim. Fr. 1958, 1,
23-65. (b) Regioselectivity: Boger, D. L.; Zarrinmayeh, H. J. Org. Chem.
1990, 55, 1379-1390.
(36) (a) Kraus, G. A.; Yue, S.; Sy, J. J. Org. Chem. 1985, 50, 283-284. (b)
Zawada, P. V.; Banfield, S. C.; Kerr, M. A. Synlett 2003, 971-974.
(37) Kato, S.; Morie, T. J. Heterocycl. Chem. 1996, 33, 1171-1178.
9
J. AM. CHEM. SOC. VOL. 128, NO. 49, 2006 15687

A R T I C L E S
Scheme 8
Tichenor et al.
Scheme 9
4 equiv of anisole, dioxane, 25 °C, 24 h, 72%) in the presence
of imidazole (Scheme 8). The anisole additive to the reaction
mixture avoided additional C8 bromination, and conducting the
reaction in the presence of imidazole38 precluded competitive
O-debenzylation and N-Boc deprotection. Palladium-catalyzed
carbonylation (0.1 equiv of PdCl2(PPh3)2, 3 equiv of Et3N, CO
(1 atm), 20% MeOH-toluene, 90 °C, 36 h, 97%) installed the
C7-methyl ester, providing 40 in superb yield. Benzyl ether
hydrogenolysis (1 atm of H2, 10% Pd/C, THF, 2 h, 92%),
followed by ester hydrolysis of 41 (4 equiv of LiOH, THF-
MeOH-H2O, 25 °C, 14 h, 95%), provided carboxylic acid 42,
which was coupled with CH3SH (excess, 4 equiv of EDCI,
DMF, 0 °C, 2 h, 82%)11 to provide the key thiomethyl ester
43. Deprotection of 43 (4 N HCl-EtOAc, 25 °C, 0.5 h, 100%)
provided the indoline hydrochloride salt, which was used
immediately and directly in subsequent coupling reactions.
Scheme 10
hydrochloride 44 (2 equiv, 4 equiv of EDCI, DMF, 25 °C, 5 h,
45%). The final transannular Ar-3′ spirocyclization was achieved
using Mitsunobu activation and intramolecular displacement of
the secondary alcohol (3 equiv of DEAD, 9 equiv of Ph3P, 10
min, THF, 25 °C, 70%) and proceeded extraordinarily rapidly
to give (+)- and ent-(-)-1.
Several variations on the coupling protocol used to link the
central and right-hand subunits were examined. The direct
coupling of 19 and 45 was much less successful if conducted
in the presence of base to liberate the free amine from its
hydrochloride salt (4 equiv of EDCI, 4 equiv of NaHCO3, DMF,
3 h, 25 °C, 9%) or when conducted at room temperature (4
equiv of EDCI, DMF, 3 h, 25 °C, 29%) due principally to
competitive or subsequent secondary alcohol O- versus N-
acylation. In addition, the coupling of the O-benzyl ethers 18
and 49 (Scheme 10) in the presence of added base proceeded
cleanly and rapidly (4 equiv of EDCI, 4 equiv of NaHCO3,
DMF, 3 h, 25 °C, 50%) to provide 50, and subsequent
O-debenzylations (10% Pd/C, 9:1 THF-MeOH, 25 °C, 3 h,
83%) also provided 46.
Key Partial Structures and Analogues. The alkylation
subunit precursor 21 was deprotected by transfer hydrogenolysis
(excess 25% aqueous HCO2NH4, Pd/C, 25 °C, 3 h, 72%) to
provide 51. Concurrent with our original studies,11 the right-
hand partial structures 55 and 56 were prepared by acid-
catalyzed deprotection of 51 (natural or unnatural enantiomer,
4 N HCl-EtOAc, 70 °C, 1 h) followed by coupling with
5-hydroxy-6-methoxyindole-2-carboxylic acid (45)14 or 6-hy-
droxy-5-methoxyindole-2-carboxylic acid (52;37 1.5 equiv, 4
equiv of EDCI, DMF, 25 °C, 12 h, 60-78%) to provide 53
and 54 (Scheme 11). Base-induced spirocyclization (NaHCO3,
2:1 DMF-H2O, 1 h, 25 °C, 64-89%) gave the key partial
structures 55 and 56, the former of which bears the authentic
yatakemycin right-hand subunit and the latter of which incor-
porates the original misassigned indole.
This route to 43, by virtue of proceeding through 41, also
represents an improved, alternative synthesis of the central and
right-hand subunits of CC-1065 (PDE-I and -II).39 As such, it
also constitutes an improvement in our total synthesis of (+)-
and ent-(-)-CC-106540 that was disclosed at the start of our
interest in this class of natural products.
Completion of the Total Synthesis of (+)- and ent-(-)-
Yatakemycin. The asymmetric alkylation subunit synthesis and
improved left-hand subunit synthesis were combined to provide
an efficient synthesis of (+)- and ent-(-)-yatakemycin (Scheme
9). Boc deprotection of 19 (4 N HCl-EtOAc, 25 °C, 3 h)
followed by coupling of the resulting amine hydrochloride with
5-hydroxy-6-methoxyindole-2-carboxylic acid14 (45; 1.5 equiv,
4 equiv of EDCI, DMF, 0 °C, 3 h, 58%) provided 46. Notably,
the successful coupling in the presence of the free secondary
alcohol and two free phenols reduces the number of late-stage
protecting group manipulations. Saponification of the methyl
ester (excess LiOH, 3:2:1 THF-MeOH-H2O, 25 °C, 94%)
gave carboxylic acid 47, which was coupled with amine
(38) Chen, J. J.; Wei, Y.; Drach, J. C.; Townsend, L. B. J. Med. Chem. 2000,
43, 2449-2456.
(39) (a) Boger, D. L.; Coleman, R. S. J. Am. Chem. Soc. 1987, 109, 2717-
2727. (b) Boger, D. L.; Coleman, R. S. J. Org. Chem. 1986, 51, 3250-
3252. (c) Carter, P.; Fitzjohn, S.; Halazy, S.; Magnus, P. J. Am. Chem.
Soc. 1987, 109, 2711-2717. (d) Rawal, V. H.; Cava, M. P. J. Am. Chem.
Soc. 1986, 108, 2110-2112. (e) Bolton, R. E.; Moody, C. J.; Rees, C. W.;
Tojo, G. J. Chem. Soc., Perkin Trans. 1 1987, 931-935. (f) Komoto, N.;
Enomoto, Y.; Tanaka, Y.; Nitanai, K.; Umezawa, H. Agric. Biol. Chem.
1979, 43, 559-561. (g) see also: Boger, D. L.; Coleman, R. S. J. Org.
Chem. 1984, 49, 2240-2245.
(40) Boger, D. L.; Coleman, R. S. J. Am. Chem. Soc. 1988, 110, 1321-1323;
1988, 110, 4796-4807.
9
15688 J. AM. CHEM. SOC. VOL. 128, NO. 49, 2006

Synthesis of Yatakemycin and Duocarmycin SA
A R T I C L E S
Scheme 11
Scheme 13
Scheme 12
Scheme 14
The left-hand partial structures 61 and 62 were prepared by
acid-catalyzed deprotection of 43 and 41 to give 44 and 57,
followed by coupling with the alkylation subunit precursor 5810b
(1.0 equiv, 4 equiv of EDCI, DMF, 25 °C, 24 h, 41-45%).
Spirocyclization was accomplished under mild, basic conditions
(NaHCO3, 2:1 DMF-H2O, 25 °C, 1 h, 68-76%) to provide
61 or 62 (Scheme 12).
Analogues That Address the Impact of the Thiomethyl
Ester. A unique feature of yatakemycin that is not found in the
preceding natural products is the C-terminus thiomethyl ester.
As a consequence, four key analogues or partial structures were
prepared that might shed insight into its role or impact on the
properties of 1. The first (65) represents the C-terminus methyl
versus thiomethyl ester of yatakemycin (Scheme 13), the second
and third (70 and 72) represent the thiomethyl versus methyl
ester of duocarmycin SA (Scheme 14) and N-Boc-DSA (Scheme
15), and the fourth (62, presented in Scheme 12) represents the
methyl versus thiomethyl ester of the key partial structure 61
that also lacks the right-hand subunit.
give 64, which was spirocyclized under basic conditions
(NaHCO3, 2:1 DMF-H2O, 25 °C, 1 h, 100%) to provide 65
(Scheme 13). The thiomethyl ester of duocarmycin SA (70) was
prepared from 21 by acid-catalyzed deprotection (4 N HCl-
EtOAc, 70 °C, 1 h) followed by coupling with 5,6,7-trimethoxy-
indole-2-carboxylic acid7,34 (22; 1.5 equiv, 4 equiv of EDCI, 2
equiv of NaHCO3, DMF, 25 °C, 24 h, 79%) to provide 66.
Saponification of the methyl ester (4 equiv of LiOH, THF-
MeOH-H2O, 25 °C, 3 h, 89%) followed by hydrogenation of
the benzyl ether (1 atm of H2, 10% Pd/C, THF, 2 h, 98%)
provided 68. Coupling with methyl thiol (excess, 4 equiv of
EDCI, DMF, 0 °C, 2 h, 71%) installed the thiomethyl ester,
and subsequent spirocyclization (10 equiv of DBU, CH3CN, 1
h, 25 °C, 82%) gave 70 (Scheme 14). The thiomethyl ester of
the alkylation subunit (72) was prepared by an analogous
sequence of coupling 5810b with methyl thiol (excess, 4 equiv
of EDCI, DMF, 0 °C, 2 h, 67%) followed by spirocyclization
(10 equiv of DBU, 1 h, 25 °C, 70%) to give 72 (Scheme 15).
The yatakemycin methyl ester analogue 65 was prepared by
coupling of a late-stage precursor of yatakemycin (63)11 with
57 (1.5 equiv, 4 equiv of EDCI, DMF, 25 °C, 24 h, 60%) to
9
J. AM. CHEM. SOC. VOL. 128, NO. 49, 2006 15689

A R T I C L E S
Scheme 15
Tichenor et al.
DNA Alkylation Studies and Cytotoxic Activity
Figure 4. Adenine adduct.
Isolation, Quantitation, and Characterization of the (+)-
Yatakemycin-Adenine Adduct. With limited amounts of
naturally occurring yatakemycin available (1-2 mg), our initial
studies carried out in the collaboration with Igarashi focused
on defining its DNA alkylation selectivity, efficiency, revers-
ibility, and relative rate.5 With larger quantities of the natural
product acquired through its total synthesis now being available
to us, we had the opportunity to address additional features of
the reaction not yet examined. The first of these was the isolation
and characterization of the thermally released adenine adduct
in efforts that confirm the site of nucleophilic attack (adenine
N3) and the structure of the DNA alkylation product (addition
to the least substituted cyclopropane carbon). The structural
similarity of yatakemycin to duocarmycin SA, for which this
has been established,8 suggested that the two compounds would
behave in an analogous manner, and this expectation was
confirmed with the studies detailed below.
to the duocarmycin SA-adenine adduct, most importantly the
1H NMR C10-H chemical shift at δ 4.52 (vs δ 4.48, duocar-
mycin SA)8 and the 13C NMR C10 chemical shift at δ 41.1 (vs
δ 41.1, duocarmycin SA).8 The assignment was corroborated
by analysis of the alkyl carbon 13C chemical shifts, in which
the C10 methine (δ 41.1) is located upfield of the adjacent C11
and C13 methylene peaks (δ 54.8 and 54.3). This indicates that
both methylenes are bound to heteroatoms and is consistent only
with the five-membered (vs ring expansion six-membered) ring
structure. The carbon shifts corresponding to the adenine portion
were assigned on the basis of the high degree of correlation
1
with the carbon shifts of 3-methyladenine.8 An ROESY H-
1H cross-peak between the C2-H of adenine (δ 8.28), identified
by the associated C2 carbon shift at δ 144.6 (vs δ 144.0,
3-methyladenine), and the C13-H methylene of the adduct
established the adenine N3 as the alkylation site.
Analogous to the studies conducted with CC-1065,6 duocar-
mycin A,7b and duocarmycin SA,8 a solution of calf thymus
DNA (100 bp equiv) in pH 7.4 phosphate buffer was treated
with (+)-yatakemycin under conditions (25 °C, 6 h) that achieve
complete DNA alkylation. The alkylated DNA was isolated by
precipitation (EtOH), and no residual unreacted yatakemycin
was observed in the supernatant. Thermal depurination of the
alkylated calf thymus DNA (pH 7.4 phosphate buffer, 100 °C,
30 min, 2×) followed by 1-butanol extraction and subsequent
purification by precipitation (DMF-CH2Cl2) afforded the
adenine adduct 73 in 72% yield (Figure 4). Because of the
lability of the thiomethyl ester, we were not able to establish
whether the remaining balance of material represents alternative
non thermally labile DNA alkylation events that are not detected
including potential intra- or interstrand cross-linking reactions
or simply an instability of the thiomethyl ester of the adduct 73
to the depurination conditions, leading to its diminished
recovery. Complementary studies with duocarmycin SA (ad-
enine adduct recovery 90-95%)8 suggests that alternative, non
thermally labile DNA alkylation events are unlikely, and
additional studies detailed herein failed to detect interstrand
DNA cross-links via the thiomethyl ester. At present, it appears
that the nonquantitative recovery of 73, which is also extraor-
dinarily insoluble making its isolation and purification chal-
lenging, may simply reflect its stability to the thermal depuri-
nation reaction conditions or our ability to quantitatively recover
it from the reaction. Nonetheless, the 72% recovery of 73
accounts for the majority of the compound and indicates that it
constitutes the predominant alkylation event.
Exploratory studies were also performed with the methyl ester
analogue 65 of yatakemycin, which lacks the reactive thiomethyl
ester and was expected to be more stable to the thermal
depurination conditions. Alkylation of calf thymus DNA and
isolation as described above yielded the adenine adduct in 69%
yield. The spectroscopic characteristics of its adenine adduct
were analogous to those of the yatakemycin adenine adduct. It,
like 73, was remarkably insoluble and challenging to work with.
Unlike 73, it would not be expected to be unstable to the thermal
depurination conditions, and yet it was isolated with comparable
recovery, suggesting the quantitation of the recovered adduct
73 (72%) reflects the challenges of its isolation rather than
reactivity intrinsic to the thiomethyl ester.
DNA Alkylation Selectivity of ent-(-)-Yatakemycin. With
ample supplies of the synthetic samples of both enantiomers of
yatakemycin available, the DNA alkylation properties of the
unnatural enantiomer were established for comparison with those
of the natural product.5 Thus, the DNA alkylation of ent-(-)-
yatakemycin was examined in five 150-base-pair segments of
DNA42 for which the alkylation sites of the natural enantiomer5
as well as an extensive range of related compounds (e.g., 2-4)
have been established. After exposure to the compounds and
thermally induced depurination of the adenine N3 adducts in
singly 5′-32P-end-labeled duplex DNA, denaturing PAGE
alongside Sanger dideoxynucleotide sequencing standards per-
mitted alkylation site identification used in the subsequent
assessment of the alkylation selectivity. This DNA alkylation
selectivity was assessed and established under a range of reaction
conditions (25 °C, 1-8 days, typically 18 h) and agent
The full NMR spectroscopic characterization (1H, 13C-APT,
HMQC, ROESY) of the adenine adduct led to the unambiguous
assignment of structure 73 in which adenine N3 addition to the
unsubstituted cyclopropane carbon of yatakemycin was estab-
lished. This was first evident from the spectroscopic similarity
(41) 6-Hydroxy-5-methoxyindole-2-carboxylic acid (52) was prepared by benzyl
deprotection (H2, 10% Pd/C, MeOH, 1 h, 99%) of 6-(benzyloxy)-5-
methoxyindole-2-carboxylic acid (Spectrum Chemical).
(42) (a) Boger, D. L.; Munk, S. A.; Zarrinmayeh, H.; Ishizaki, T.; Haught, J.;
Bina, M. Tetrahedron 1991, 47, 2661-2682. (b) Boger, D. L.; Munk, S.
A. J. Am. Chem. Soc. 1992, 114, 5487-5496.
9
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Synthesis of Yatakemycin and Duocarmycin SA
A R T I C L E S
alkylation sites observed were at adenine exclusively, and no
minor guanine N3 or N7 alkylation was detected. This lack of
guanine alkylation is characteristic of the more stable com-
pounds (also not observed with duocarmycin SA or (+)-
yatakemycin), but is increasingly more prevalent with the more
reactive natural products (duocarmycin A > CC-1065).8,43 All
the observed adenine N3 alkylation sites were flanked on both
sides by an A or T base. As shown in Table 2, the preference
for this three-base-pair sequence was 5′-AAA > 5′-AAT = 5′-
TAA > 5′-TAT.44 This A/T sequence preference extended
outward an additional base, revealing a strong preference for
the second 5′- and 3′-bases to be A or T. In general, it was
observed that typically one, but not both, of these positions could
be G or C in a given alkylation sequence, highlighting the
preference for ent-(-)-yatakemycin to alkylate sites central to
an extended AT-rich tract. Thus, alkylation was observed at
adenine N3 central to a five-base-pair A/T tract (e.g., 5′-
AAAAA) as summarized in Table 1. The consensus alkylation
sequence and sequence preference data for (+)- and ent-(-)-1
were found to be essentially indistinguishable.
Figure 5. Thermally induced strand cleavage of w836 DNA (146 bp’s,
nucleotide 5189-91) after DNA-agent incubation at 25 °C (24 h), removal
of unbound agent by EtOH precipitation, thermal depurination (100 °C, 30
min), denaturing 8% PAGE, and autoradiography: lane 1, control DNA;
lanes 2-4, Sanger G, C, A, and T sequencing standards; lane 5, (+)-CC-
1065 (1 × 10-6 M); lane 6, (+)-duocarmycin SA (1 × 10-6 M); lane 7,
(+)-yatakemycin (1 × 10-6 M); lane 8, ent-(-)-yatakemycin (1 × 10-6
M); lane 9, ent-(-)-CC-1065 (1 × 10-6 M).
Illustration of these alkylation properties in w836 DNA is
provided in Figure 5. This six-base-pair polyA sequence in w836
provides a unique setting in which all compounds can be
compared and for which the relative alkylation selectivity can
be nicely contrasted. (-)-Yatakemycin preferentially alkylates
adenine central to this polyA tract as was found for (+)-
yatakemycin, but contrasts the 3′-terminal adenine alkylation
observed for the natural enantiomers (+)-CC-1065 and (+)-
duocarmycin SA as well as the 5′-terminal adenine alkylation
observed for the unnatural enantiomers (represented by ent-
(-)-CC-1065).6d Although a slight erosion in selective alkylation
of the central adenine by ent-(-)-1 relative to (+)-1 was
observed, a clear and smooth trend from 3′- to 5′-selective
alkylation can be observed simply by changing the DNA
alkylation subunit position and cyclopropane stereochemistry
in a controlled fashion. As was observed with (+)-1, the
differences in DNA alkylation selectivity among ent-(-)-1, 2,
and 4 are more pronounced than depicted in Figure 5. As a
result of alkylation central to AT sequences, many alkylation
sites observed for 2 and 4 do not overlap with those for ent-
(-)-1, whereas this unnatural enantiomer exhibits an alkylation
site selectivity essentially identical to that of its natural
enantiomer. In summary, ent-(-)-1 was found to alkylate
adenine central to a five-base-pair A/T sequence (preference
5′-AAA > 5′-AAT = 5′-TAA > 5′-TAT)44 in agreement with
the selectivity established for the natural enantiomer (+)-1, but
differing from that of the 3′-terminal adenine alkylation of DNA
by (+)-CC-1065 (five-base-pair sequence, e.g., 5′-AAAAA) and
(+)-duocarmycin SA (three- to four-base-pair sequence, e.g.,
5′-AAAA) and the offset 5′-terminal adenine alkylation observed
for their unnatural enantiomers including ent-(-)-CC-1065 (five-
base-pair sequence, e.g., 5′-AAAAA). Models of the natural
and unnatural enantiomer alkylation of a w794 high-affinity site
are shown in Figure 6 and illustrate nicely these features. Both
Figure 6. Models of the yatakemycin natural (left, anti-thioester shown)
and unnatural (right, syn-thioester shown) enantiomer DNA alkylation of a
w794 high-affinity site. Both enantiomers alkylate and bind across the site
5′-TAATT, but with reversed binding orientations.
concentrations (1 × 10-4 to 1 × 10-7 M). A statistical treatment
of the alkylation sites also considering those sites not alkylated
was performed in establishing a consensus sequence of DNA
alkylation for ent-(-)-1 and highlighted features that would
otherwise be missed upon examination of only the alkylated
sites.
One of the exciting features of yatakemycin was that it
represented the first naturally occurring sandwiched agent
containing an alkylation subunit flanked with DNA binding
subunits on each side. As previously disclosed, not only does
this provide compounds that alkylate DNA with extraordinary
rates and efficiencies that exceed those of the more classical
compounds including duocarmycin SA and CC-1065, but it
occurs remarkably and predictably with both enantiomers
alkylating the same sites exhibiting essentially the same
selectivity.10 Consistent with this, ent-(-)-yatakemycin alkylated
DNA in a manner essentially identical to the selectivity of (+)-
yatakemycin, and distinct from that of 2-4 (Figure 5). The
consensus alkylation sequence for ent-(-)-1 is detailed in Table
1, the sequence preference for alkylation is outlined in Table
2, and illustration of the DNA alkylation sites within the DNA
sequences is provided in the Supporting Information. All
(43) Guanine N3 alkylation even for duocarmycin A and CC-1065 is detected
only upon exhaustion of the available adenine N3 alkylation sites and
generally requires excess compound to detect. See ref 8.
(44) Unlike 5′-AAA, the mixed sequences can contain competitive alkylation
sites on the complementary unlabeled strand that diminishes the apparent
alkylation efficiency on the labeled strand. The majority of the observed
three-base A/T selectivity is simply a statistical preference exaggerated by
competitive unlabeled strand alkylation rather than unique inherent
characteristics present in individual sequences.
9
J. AM. CHEM. SOC. VOL. 128, NO. 49, 2006 15691

A R T I C L E S
Tichenor et al.
3
Table 1. (-)-Yatakemycin Consensus DNA Alkylation Sequence (5′ f 3′)
basea
−3
−2
−1
0
1
2
A (30)b
T (26)
G (21)
C (23)
A/T (56)
composite
56
12
19
12
69
59
19
12
9
88
12
0
0
100
A/T
100
0
0
0
100
A/T
91
9
0
0
100
A/T
59
19
19
3
44
19
22
15
78
78
63
A/T g G/C
A/T > G/C
A/T > G/C
A/T g G/C
a Percentage of the indicated base located at the designated position relative to the adenine N3 alkylation site. b Percentage composition within the DNA
examined.
Table 2. (-)-Yatakemycin Sequence Preferences
toxic potency typically exhibited by such analogues. Since
ent-(-)-1 was found to be identical to its natural enantiomer in
nearly all of these respects, we elected to examine its DNA
alkylation reversibility.
sequence
no. of ASa
no. of TSb
percentagec
5′-NAAAN-3′
5′-NTAAN-3′
5′-NAATN-3′
5′-NTATN-3′
25
4
3
39
18
15
15
64
22
20
0
The reversibility of DNA alkylation by ent-(-)-1 was
examined under a variety of conditions (pH 6.0-8.4, 25-47
°C, 1-8 days) alongside duocarmycin SA8 as a positive control
and (+)-yatakemycin5 as a negative control. The compounds
were incubated with unlabeled w836 DNA at 25 °C for 24 h
(∼90% DNA alkylation under identical conditions with labeled
DNA) to ensure complete alkylation before any remaining
unbound compound was extracted using an exhaustive protocol,
which was followed by EtOH precipitation of the DNA to ensure
complete compound removal. Covalently modified unlabeled
DNA was mixed with singly 5′-end-labeled w836 DNA (1:1
ratio), and they were incubated together under a range of
conditions. The reversibility of DNA alkylation was established
by observation of compound transfer from the unlabeled to
labeled DNA as detected using denaturing PAGE following
thermal depurination, with the results visualized by autorad-
iography.
0
a Number of alkylated sites in the DNA examined (AS ) alkylated sites).
b Total number of sites in the DNA examined (TS ) total sites). c (Number
of AS/number of TS) × 100, percentage of sites alkylated in the DNA
examined.
enantiomers alkylate and bind across the same 5′-TAATT site
with adenine alkylation central to this five base-pair AT site,
but with reversed binding orientations and as diastereomeric
complexes. In these models, it is easy to recognize that the
thiomethyl ester lies on the outer face of the complexes
sandwiched in the minor groove. Its positioning in either syn
or anti orientation appears distal from any nucleophilic site in
DNA that might provide inter- or intrastrand DNA cross-linking,
but it is ideally located to capture external nucleophiles (e.g.,
Lys or Cys in proteins) when adopting its anti conformation.
Relative Efficiency and Rate of DNA Alkylation. The
efficiency of DNA alkylation was indistinguishable for (+)- and
ent-(-)-1 under the conditions examined (25 °C, 1-8 days).
The lowest concentration at which alkylation was observed was
10-6-10-7 M in our studies, the reactions were extraordinarily
fast even at 4 °C, and even subtle differences in the DNA
alkylation efficiencies were not detected when the reactions were
taken to completion. The relative rates of alkylation (krel at 1 ×
10-6 M) were established by observation of alkylation in w836
DNA (25 °C). Impressively, (+)-1 consumed >50% of the DNA
in less than 30 s at room temperature. Under identical conditions,
ent-(-)-1 was found to alkylate DNA with minimal loss of rate
(∼8 fold), an observation in accord with previous work with
sandwiched agents where (+)-CDPI-DSA-CDPI exhibited a
∼1.6-fold faster rate of alkylation than its enantiomer (Figure
7).10
No reversibility of the ent-(-)-yatakemycin DNA alkylation
was observed when the transfer incubation was conducted at
the lower pH and temperature conditions (pH 6.0, 25-47 °C,
and pH 7.4, 25 °C) even when conducted for up to 8 days. Under
intermediate incubation conditions including those close to
physiological conditions (pH 7.4, 37 °C, and pH 8.4, 25 °C),
minimal (∼10%) alkylation reversibility was observed but only
after extended reaction times (8 days). However, under more
forcing conditions enlisting higher incubation temperatures and
pH (pH 7.4, 47 °C, and pH 8.4, 37 or 47 °C), more extensive
reversibility of alkylation was observed, with the transfer being
complete at pH 8.4 and 47 °C at the extended times (4-8 days).
The quantitation of transfer of ent-(-)-1 from unlabeled to
labeled DNA is shown in Figure 8 for these more extreme
incubation conditions.
Reversibility of the DNA Alkylation Reaction. Previous
studies established that (+)-CC-1065 and (+)-yatakemycin
constituted irreversible DNA-alkylating agents,5,45 whereas
smaller (e.g., duocarmycin SA) compounds were found to be
slowly reversible under select conditions.8,46 Moreover, no study
on the reversibility of an unnatural enantiomer in this class has
been reported, and this is likely the result of the decreased rates
and efficiency of DNA alkylation and the decreased cyto-
This alkylation reversibility was not observed with the natural
enantiomer and was slower than that of (+)-duocarmycin SA,
which were examined alongside ent-(-)-1. Consequently, the
diastereomeric relationships of the DNA adducts for ent-(-)-1
versus (+)-1 are sufficient to manifest itself in a detectibly
reversible versus irreversible DNA alkylation reaction. Although
this diastereomeric nature of the resulting adducts has often been
correlated with relative rates or efficiencies of DNA alkylation
(natural g unnatural enantiomers) and inferences have been
made on the relative stabilities of the resulting adducts,8,9 it has
not been previously exemplified. As such, the comparison of
(+)- and ent-(-)-yatakemycin illustrates an important and third
structural feature that affects the reversibility of the DNA
alkylation reaction. From previous studies, we established that
(45) Li, L. H.; Swenson, D. H.; Schpok, S. L. F.; Kuentzel, S. L.; Dayton, B.
D.; Krueger, W. C. Cancer Res. 1982, 42, 999-1004. Swenson, D. H.; Li,
L. H.; Hurley, L. H.; Rokem, J. S.; Petzold, G. L.; Dayton, B. D.; Wallace,
T. L.; Lin, A. H.; Krueger, W. C. Cancer Res. 1982, 42, 2821-2828.
(46) (a) Boger, D. L.; Yun, W. J. Am. Chem. Soc. 1993, 115, 9872-9873. (b)
Warpehoski, M. A.; Harper, D. E.; Mitchell, M. A.; Monroe, T. J.
Biochemistry 1992, 31, 2502-2508. (c) Lee, C.-S.; Gibson, N. W.
Biochemistry 1993, 32, 9108-9114.
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Synthesis of Yatakemycin and Duocarmycin SA
A R T I C L E S
Figure 7. Relative rates of DNA alkylation.
the compound relative reactivity (duocarmycin A > SA)
correlates with a slower adenine N3 adduct retroalkylation,8 and
the extent of noncovalent binding stabilization (size of the
compound) also results in less effective adduct reversibility
(1 vs 4).5 Herein, we additionally demonstrate that the diaster-
eomeric interaction of enantiomeric compounds with duplex
DNA affects the reversibility (stability) of such adducts,
illustrating that the unnatural enantiomers not only alkylate DNA
at slower rates (kinetic effect), but also provide thermodynami-
cally less stable adducts (thermodynamic effect). Moreover, even
the unnatural enantiomer of yatakemycin alkylates DNA at a
faster rate and with a greater thermodynamic stability than the
natural enantiomer of duocarmycin SA, illustrating the unique
characteristics of such sandwiched agents.
Interstrand DNA Cross-Link. (+)-Yatakemycin represents
the first member of this class of antitumor agents to contain a
thioester. Although an analogous methyl ester has been en-
countered in many structures of this class (e.g., duocarmycin
SA), the corresponding thiomethyl ester in 1 represents a unique
second, modestly reactive electrophile susceptible to nucleophilic
attack.47 With this in mind, a series of conditions were examined
(25-37 °C, 1-9 days) to probe formation of DNA interstrand
cross-links. The DNA cross-linking studies were performed on
both singly 5′-end-labeled w836 DNA48 and a 146-base-pair
segment (R-satellite) of DNA.49 Following incubation with (+)-1
under standard conditions (1 × 10-5 M) where typically one
alkylation site per DNA stand is occupied or saturating
Figure 8. Time (a), pH (b), and temperature (c) dependence of the
reversible ent-(-)-yatakemycin DNA alkylation reaction. No reversibility
was detected at pH 6.0 (25-47 °C) or pH 7.4 (25 °C), and minimal
reversibility was observed at pH 7.4 (37 °C) and pH 8.4 (25 °C).
conditions (1 × 10-4 M) where complete consumption of DNA
occurs and multiple alkylation sites per DNA strand are
observed, removal of unbound agent by EtOH precipitation, no
thermal depurination, and denaturation, denaturing PAGE (w836
DNA) or nondenaturing PAGE (R-satellite DNA) failed to reveal
evidence of detectable quantities of a DNA-DNA cross-link
(Supporting Information Figure S1).
Cytotoxic Activity. A key element of the studies detailed
herein was the opportunity the synthetic studies provide for
assessing the properties of not only the natural product, which
has been available in only limited quantities, but also the
unnatural enantiomer, key partial structures and analogues, and
the misassigned structures 5 and 6. Most central to these is
synthetic ent-(-)-1, which was anticipated and found to be
essentially indistinguishable from the natural enantiomer itself
(Figure 9). Summarized in Figure 9 is the cytotoxic activity of
the key series of compounds against a cell line (L1210 mouse
leukemia cell line) that has been used historically to compare
compounds in this class. Included in this study are several key
partial structures (55, 56, 61) that were prepared (Schemes 11
and 12) independent of the yatakemycin total synthesis in efforts
to define the role of each subunit.
(47) (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.
(48) Boger, D. L.; Johnson, D. S.; Palanki, M. S. S. Bioorg. Med. Chem. 1993,
1, 27-38.
(49) Luger, K.; Maeder, A. W.; Richmond, R. K.; Sargent, D. F.; Richmond,
T. J. Nature 1997, 389, 251-260.
Both natural (+)- and synthetic ent-(-)-yatakemycin proved
to be exceptionally potent cytotoxic compounds (IC50 ) 5 pM)
indistinguishable from one another in this functional cellular
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A R T I C L E S
Tichenor et al.
Like CC-1065, the two enantiomers of yatakemycin exhibit
indistinguishable cytotoxic potency, and this serves as a contrast
to the behavior of duocarmycin SA where the unnatural
enantiomer is substantially less active than the natural enanti-
omer. On the surface, these observations may appear unusual,
but they fit a pattern that has emerged in the studies to date.
The unnatural enantiomers of compounds in this class that
contain a single DNA binding subunit exhibit significantly less
potent cytotoxic activity than the natural enantiomers, whereas
the activity of those that contain two or more DNA binding
subunits approaches or matches the activity of the corresponding
natural enantiomers. These observations mirror the relative
differences in their efficiencies of DNA alkylation.
The original structure 5 disclosed for yatakemycin was found
to be much less active than the natural product. Although
perhaps surprising on the surface, we found that this compound
is not very stable and undergoes ready hydrolysis of the
thioacetate. Most likely its reduced activity is related to this
reduced stability, highlighting the fact that the reformulation
of the yatakemycin structure as 1 also entailed the removal of
a structural liability in the molecule (thioacetate) and its
replacement with a structural asset (thiomethyl ester). In contrast
and consistent with this, both enantiomers of the first alternate
structure 6 prepared11 that differs from yatakemycin only in the
right-hand subunit substituent locations (substitution switched)
exhibited cytotoxic activity indistinguishable from that of the
natural product and its unnatural enantiomer.
Significantly, removal of the left-hand subunit with 55 (or
its isomer 56) provided a key partial structure that was slightly
less potent than yatakemycin (2-fold for the natural enantiomer)
and whose unnatural enantiomer was now 75-fold less potent
than ent-(-)-yatakemycin. Nonetheless, the activity of the
natural and unnatural enantiomers mirrors that of the two
enantiomers of duocarmycin SA, albeit with the unnatural
enantiomer being even less potent (4-fold). Most significantly,
removal of the right-hand subunit with 61 resulted in a >10-
fold loss in activity for the natural enantiomer and a >100-fold
loss in activity for the unnatural enantiomer. The alkylation
subunit N2-acyl substituent composed of an extended hetero-
cyclic chromophore has been shown to contribute to and be
largely responsible for catalysis of the DNA alkylation reac-
tion.9,10 Consequently, it is not surprising that its removal with
61 provided a much less effective compound. Typically, the
loss in activity with such “reversed” analogues of duocarmycin
SA has been greater than 20-fold (ca 100-fold),10 suggesting
that either the unique left-hand subunit of 61 (PDE) or the
terminal thiomethyl ester is now productively enhancing the
activity of 61. Finally, the comparison of yatakemycin and the
preceding partial structures 55 and 61 with N-Boc-DSA (74)
illustrates that removal of both DNA binding domains reduces
the activity 1000-fold (natural enantiomer) and 10000-fold
(unnatural enantiomer) relative to that of yatakemycin, 600- and
200-fold relative to that of 55, and 50-100-fold relative to that
of 61.
Figure 9. Cytotoxic activity.
assay.50,51 Both are roughly 2-fold more active than (+)-
duocarmycin SA (IC50 ) 8-10 pM),16 20-fold more potent than
ent-(-)-duocarmycin SA (IC50 ) 100 pM),16 and 4-5 times
more potent than (+)- or ent-(-)-CC-1065 (IC50 ) 20 pM).40
(51) The IC50 (L1210) for (+)-yatakemycin ranges from 3 to 5 pM and has
been tested g20 times over several years, side-by-side with (+)-duocar-
mycin (IC50 ) 8-10 pM). The 2-3-fold difference in potency is always
observed, and the absolute potencies always fall in this narrow range
indicated. We have developed highly refined, reproducible conditions for
conducting such cytotoxic assays and are confident in other similarly close
comparisons made herein. We are happy to share our insights with others
who might experience typically more variable results with such assays.
(50) Parrish, J. P.; Hughes, T. V.; Hwang, I.; Boger, D. L. J. Am. Chem. Soc.
2004, 126, 80-81.
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Synthesis of Yatakemycin and Duocarmycin SA
A R T I C L E S
the thiomethyl ester 61, both being >10-fold less active than
yatakemycin. Similarly, the unnatural enantiomers exhibited
indistinguishable cytotoxic activities (IC50 ) 760-790 pM),
both being >100-fold less active than either enantiomer of
yatakemycin and 10-fold less active than their respective natural
enantiomers. As such, the surprising potency of 61 relative to
typical reversed analogues of duocarmycin SA10 (ca. 100-fold
less potent) is not attributable to the thiomethyl ester, but appears
to be unique to the incorporation of the yatakemycin left-hand
subunit.
The final comparison was that of 72 with 74 (N-Boc-DSA),
and it proved to be the most interesting. Here both enantiomers
of the thiomethyl ester of the simple alkylation subunit (72)
were found to be 10-fold more potent than the respective
enantiomer of N-Boc-DSA (74) which bears the C-terminus
methyl ester. Moreover, this level of activity (IC50 ) 600 pM
or 0.6 nM) for the natural enantiomer is remarkable for such a
simple derivative, and it is only 100-fold less potent than
duocarmycin SA or yatakemycin. To put this into perspective,
N-Boc-DSA (74; IC50 ) 6 nM) itself is the most potent of such
simplified N-Boc derivatives (IC50 ) 300 nM for N-Boc-CPI
and 80 nM for N-Boc-CBI),50 a correlation that mirrors the
relative stability of the alkylation subunits,50 and its thiomethyl
ester is now even 10-fold more potent. This effect is larger than
one could expect of a simple substituent effect and suggests a
special role for the thioester. Of those that can be envisioned,
protein conjugation is most attractive. Whether this entails the
intervention of other or additional biological targets or whether
this might represent protein conjugation before or following
DNA alkylation cannot be distinguished from the studies to date,
but the latter would provide DNA-protein cross-links, and
investigations to probe such possibilities are in progress.
Figure 10. Cytotoxic activity.
Additionally, four important comparisons were conducted in
efforts to establish a functional role for the C-terminus thio-
methyl ester (Figure 10).
Conclusions
The first, the comparison of both enantiomers of yatakemycin
with their corresponding methyl esters 65, revealed that the
cytotoxic activity was indistinguishable (IC50 ) 5 pM). Thus,
both enantiomers of the yatakemycin methyl ester (65) exhibited
identical cytotoxic activities independent of their absolute
configuration and at a potency that was indistinguishable from
that of the natural product and its unnatural enantiomer.
Complementary to our earlier studies that provided the
structural reassignment of yatakemycin and its first total
synthesis,11 herein we detailed a second-generation, asymmetric
total synthesis of (+)- and ent-(-)-yatakemycin enlisting a final
transannular Ar-3′ spirocyclization of the free alcohol 48 to close
the activated cyclopropane within the full trimer structure. In
turn, the left-hand subunit was assembled using a key, regiose-
lective Diels-Alder reaction of a selectively activated p-
quinodiimide followed by oxidative cleavage of a resulting enol
ether with release of two differentially oxidized side chains
suitable for closure to an appropriately functionalized dihydro-
pyrroloindole. Extending our preceding studies, the lactam 38
used to prepare the misassigned thioacetate of the original
structure 5 now served as a key precursor to the C-terminus
thiomethyl ester via its conversion to 2-bromoindole 39,
followed by an effective Pd(0)-catalyzed carbonylation. The
central alkylation subunit was prepared enlisting a surprisingly
effective regioselective intramolecular 6-endo-epoxide addition
in which the late-stage introduction of the chiral center derived
from (R)- or (S)-glycidol permits ready access to either
enantiomer.
The second entailed the examination of the duocarmycin SA
thiomethyl ester (70) and its comparison with duocarmycin SA
itself, which possesses a C-terminus methyl ester. Here, interest-
ingly, the natural enantiomer of the modified C-terminus
thiomethyl ester was found to be reproducibly 2-fold more
potent (IC50 ) 5 pM) than the natural product (IC50 ) 10 pM)
and essentially equipotent with yatakemycin. In this case, the
C-terminus thiomethyl ester enhanced the cytotoxic potency of
the compound in this functional assay 2-fold. Provocatively,
given the results below and this behavior of 70 and its structural
relationship with 1, we would suggest that it may be simply a
matter of time before 70 is identified as a natural product in its
own right. In contrast, the unnatural enantiomer of 70 (IC50
)
200 pM) was 2-fold less potent than the unnatural enantiomer
of duocarmycin SA (IC50 ) 100 pM) and 40-fold less active
than the natural enantiomer of 70.
With the larger quantities of the natural product now being
available through its total synthesis and completing our char-
acterization of the (+)-yatakemycin DNA alkylation reaction,5
the isolation, characterization, and quantitation of the thermally
released adenine adduct was accomplished, confirming the
In contrast to naive expectations, the comparison of 61 with
62 similarly revealed little or no apparent role for the thiomethyl
ester. Thus, the natural enantiomer of the methyl ester 62
exhibited a cytotoxic potency equivalent to that displayed by
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A R T I C L E S
Tichenor et al.
exclusive adenine N3 addition (no guanine N3 or N7 addition)
to only the least substituted cyclopropane carbon of yatakemycin
and establishing that this represents the predominant (g72%),
perhaps exclusive, alkylation event.
subunit being more pronounced than that of the left-hand subunit
especially with regard to the activity of the unnatural enanti-
omers. Significantly and surprisingly on the surface, the
thiomethyl ester in yatakemycin and a series of key partial
structures was shown not to have an apparent impact on their
cytotoxic properties. In contrast, the enhanced properties of the
thiomethyl ester of duocarmycin SA (2-fold enhancement vs
the properties of duocarmycin SA methyl ester for the natural
enantiomer) suggests that it may well constitute a natural product
itself, and the remarkable potentiation of the cytotoxic activity
of the simple N-Boc derivative 72 of the alkylation subunit (IC50
) 600 pM, 10-fold enhancement vs that of N-Boc-DSA) implies
there may be a special role that the thiomethyl ester can play in
enhancing their properties. Additional studies of these and
related structural features of yatakemycin continue to be
explored, and the results of such studies will be disclosed in
due time.
Just as significantly and with the synthetic unnatural enan-
tiomer now available, the DNA alkylation properties of ent-
(-)-yatakemycin were established. Although unusual on the
surface, the unnatural enantiomer was found to alkylate DNA
with essentially the same selectivity (adenine central to a five-
base AT-rich sequence, e.g., 5′-AAAAA) and efficiency as the
natural enantiomer, consistent with prevailing models.8-10
Differentiating the two enantiomers and consistent with the
models, the unnatural enantiomer alkylates DNA at a slower
rate (8-fold) and in a reaction that is reversible under forcing
conditions whereas that of the natural enantiomer is not,
illustrating that the unnatural enantiomer diastereomeric adducts
are both kinetically and thermodynamically less favorable than
those of the natural enantiomer. Nonetheless, even the unnatural
enantiomer of yatakemycin alkylates DNA at a faster rate and
with a thermodynamic stability that exceeds that of the natural
enantiomer of duocarmycin SA, illustrating the special charac-
teristics such sandwiched structures possess.
Finally, the synthetic studies provided the opportunity to
assess the cytotoxic properties of not only the natural product,
but also its unnatural enantiomer, key partial structures and
analogues, and misassigned structures 5 and 6. Both synthetic
(+)- and ent-(-)-yatakemycin were found to be exceptionally
potent and indistinguishable in the functional cellular assay
(L1210, IC50 ) 5 pM),51 being 2-fold more potent than (+)-
duocarmycin SA, 20-fold more potent than ent-(-)-duocarmycin
SA, and 4-5-fold more potent than the two enantiomers of CC-
1065. Each of the flanking subunits contributes to the cytotoxic
potency of the natural product, with the effect of the right-hand
Acknowledgment. We gratefully acknowledge the financial
support of the National Institutes of Health (Grants CA41986
and CA42056) and 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). We thank Professor Igarashi of the
Toyama Perfectural University for authentic samples of yatake-
mycin. J.D.T, D.B.K., and M.S.T. are Skaggs Fellows.
Supporting Information Available: Full experimental details,
comparison of 1H NMR spectra of natural and synthetic 1, and
figures illustrating the DNA alkylation sites. This material is
available free of charge via the Internet at http://pubs.acs.org.
JA064228J
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