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

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 (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.

Introduction

sented a remarkable hybrid of the preceding natural products

containing a central alkylation subunit identical to that found

Yatakemycin (1)¹was 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-1065,²duocarmycin A,³and duocarmycin SA⁴(Figure 1).

Each derives its biological properties through a characteristic

and distinguishable DNA alkylation reaction.^5-9The structure

of (+)-yatakemycin was disclosed in 2003 as 5 on the basis of

extensive spectroscopic studies (Figure 2).¹As 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),¹⁰we 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

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 esters^12a

exhibit a methyl ¹³C NMR chemical shift of roughly δ 11,

matching the chemical shift reported for yatakemycin (δ 11.14),

whereas aryl thioacetates^12boccur 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-d₅or 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 synthesized¹⁴and 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).¹¹On 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.¹Model 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,^11,13the 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,^16-21which

relied on a late-stage resolution of an advanced intermediate

for accessing optically active material,¹⁶an 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 8,²⁵which 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.³⁰Transfer 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 1997^31,32provides an expedient route to

the racemic alkylation subunit precursor 21 (Scheme 4) analo-

gous to the approach enlisted by Tietze and co-workers²⁰in

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 acid^7,34(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 literature²⁷and 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,^14,28and 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 S_N2 displacement of the nosylate

versus S_N2′ epoxide opening and subsequent displacement of

the nosylate. Competitive S_N2′ displacement would give the

enantiomeric epoxide, reducing the enantiomeric purity of the

product epoxide,²⁹but 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.³⁷Thus, 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 regioselectivity³⁵of 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.³⁵Phenol 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 imidazole³⁸precluded 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%)¹¹to 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,¹¹the 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)¹⁴or 6-hy-

droxy-5-methoxyindole-2-carboxylic acid (52;³⁷1.5 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).³⁹As such, it

also constitutes an improvement in our total synthesis of (+)-

and ent-(-)-CC-1065⁴⁰that 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 58^10b

(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 acid^7,34(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 58^10bwith 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)¹¹with

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.⁵With 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,⁸suggested 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)⁸and the ¹³C NMR C10 chemical shift at δ 41.1 (vs

δ 41.1, duocarmycin SA).⁸The 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.⁸An 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-1065,⁶duocar-

mycin A,^7band duocarmycin SA,⁸a 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%)⁸suggests 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.⁵Thus, the DNA alkylation of ent-(-)-

yatakemycin was examined in five 150-base-pair segments of

DNA⁴²for 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

15690 J. AM. CHEM. SOC. VOL. 128, NO. 49, 2006

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).^8,43All

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.⁴⁴This 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^-6M); lane 6, (+)-duocarmycin SA (1 × 10^-6M); lane 7,

(+)-yatakemycin (1 × 10^-6M); lane 8, ent-(-)-yatakemycin (1 × 10^-6

M); lane 9, ent-(-)-CC-1065 (1 × 10^-6M).

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).^6dAlthough 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)⁴⁴in 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^-4to 1 × 10^-7M). 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.¹⁰Consistent 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′)

base^a

−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

100

A/T

100

0

100

A/T

91

9

0

100

A/T

59

19

3

44

19

22

15

78

63

A/T g G/C

A/T > G/C

A/T g G/C

^aPercentage of the indicated base located at the designated position relative to the adenine N3 alkylation site. ^bPercentage 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 AS^a

no. of TS^b

percentage^c

5′-NAAAN-3′

5′-NTAAN-3′

5′-NAATN-3′

5′-NTATN-3′

25

4

3

39

18

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 SA⁸as a positive control

and (+)-yatakemycin⁵as 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

^aNumber of alkylated sites in the DNA examined (AS ) alkylated sites).

^bTotal 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^-7M 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 (k_relat 1 ×

10^-6M) 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,^5,45whereas

smaller (e.g., duocarmycin SA) compounds were found to be

slowly reversible under select conditions.^8,46Moreover, 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,^8,9it 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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15692 J. AM. CHEM. SOC. VOL. 128, NO. 49, 2006

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,⁸and

the extent of noncovalent binding stabilization (size of the

compound) also results in less effective adduct reversibility

(1 vs 4).⁵Herein, 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.⁴⁷With 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 DNA⁴⁸and a 146-base-pair

segment (R-satellite) of DNA.⁴⁹Following incubation with (+)-1

under standard conditions (1 × 10^-5M) 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^-4M) 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

9

J. AM. CHEM. SOC. VOL. 128, NO. 49, 2006 15693

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 prepared¹¹that 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.^9,10Consequently, 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),¹⁰suggesting

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.^50,51Both are roughly 2-fold more active than (+)-

duocarmycin SA (IC₅₀) 8-10 pM),¹⁶20-fold more potent than

ent-(-)-duocarmycin SA (IC₅₀) 100 pM),¹⁶and 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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15694 J. AM. CHEM. SOC. VOL. 128, NO. 49, 2006

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),⁵⁰a correlation that mirrors the

relative stability of the alkylation subunits,⁵⁰and 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,¹¹herein 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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J. AM. CHEM. SOC. VOL. 128, NO. 49, 2006 15695

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),⁵¹being 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

9

15696 J. AM. CHEM. SOC. VOL. 128, NO. 49, 2006

Article Doi

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

DOI: 10.1021/ja064228j

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Article abstract of DOI:10.1021/ja064228j

Full text of DOI:10.1021/ja064228j

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