Synthesis and characterization of a cyclobutane duocarmycin derivative incorporating the 1,2,10,11-tetrahydro-9 H-cyclobuta[ c ]benzo[ e ]indol-4-one (CbBI) alkylation subunit

Article abstract of DOI:10.1021/ja106986f

The synthesis of 1,2,10,11-tetrahydro-9H-cyclobuta[c]benzo[e]indol-4-one (17, CbBI), which contains a deep-seated fundamental structural modification in the CC-1065 and duocarmycin alkylation subunit consisting of the incorporation of a ring-expanded fused cyclobutane (vs cyclopropane), its chemical and structural characterization, and its incorporation into a key analogue of the natural products are detailed. The approach to the preparation of CbBI was based on a precedented (Ar-3′ and Ar-5′) but previously unknown Ar-4′ spirocyclization of a phenol onto a tethered alkyl halide to form the desired cyclobutane. The conditions required for the implementation of the Ar-4′ spirocyclization indicate that the entropy of activation substantially impacts the rate of reaction relative to that for the much more facile Ar-3′ spirocyclization, while the higher enthalpy of activation slows the reaction relative to an Ar-5′ spirocyclization. The characterization of the CbBI-based agents revealed their exceptional stability and exquisite reaction regioselectivity, and a single-crystal X-ray structure analysis of N-Boc-CbBI (13) revealed their structural origins. The reaction regioselectivity may be attributed to the stereoelectronic alignment of the two available cyclobutane bonds with the cyclohexadienone π-system, which resides in the bond that extends to the less substituted cyclobutane carbon for 13. The remarkable stability of N-Boc-CbBI (which is stable even at pH 1) relative to N-Boc-CBI containing a cyclopropane (t_1/2 = 133 h at pH 3) may be attributed to a combination of the increased extent of vinylogous amide conjugation, the nonoptimal geometric alignment of the cyclobutane with the activating cyclohexadienone, and the intrinsic but modestly lower strain energy (1.8 kcal/mol) of a cyclobutane versus a cyclopropane.

Full text of DOI:10.1021/ja106986f

Published on Web 09/14/2010
Synthesis and Characterization of a Cyclobutane
Duocarmycin Derivative Incorporating the
1,2,10,11-Tetrahydro-9H-cyclobuta[c]benzo[e]indol-4-one
(CbBI) Alkylation Subunit
James P. Lajiness and Dale L. Boger*
Department of Chemistry and The Skaggs Institute for Chemical Biology, The Scripps Research
Institute, 10550 North Torrey Pines Road, La Jolla, California 92037
Received August 4, 2010; E-mail: boger@scripps.edu
Abstract: The synthesis of 1,2,10,11-tetrahydro-9H-cyclobuta[c]benzo[e]indol-4-one (17, CbBI), which
contains a deep-seated fundamental structural modification in the CC-1065 and duocarmycin alkylation
subunit consisting of the incorporation of a ring-expanded fused cyclobutane (vs cyclopropane), its chemical
and structural characterization, and its incorporation into a key analogue of the natural products are detailed.
The approach to the preparation of CbBI was based on a precedented (Ar-3′ and Ar-5′) but previously
unknown Ar-4′ spirocyclization of a phenol onto a tethered alkyl halide to form the desired cyclobutane.
The conditions required for the implementation of the Ar-4′ spirocyclization indicate that the entropy of
activation substantially impacts the rate of reaction relative to that for the much more facile Ar-3′
spirocyclization, while the higher enthalpy of activation slows the reaction relative to an Ar-5′ spirocyclization.
The characterization of the CbBI-based agents revealed their exceptional stability and exquisite reaction
regioselectivity, and a single-crystal X-ray structure analysis of N-Boc-CbBI (13) revealed their structural
origins. The reaction regioselectivity may be attributed to the stereoelectronic alignment of the two available
cyclobutane bonds with the cyclohexadienone π-system, which resides in the bond that extends to the
less substituted cyclobutane carbon for 13. The remarkable stability of N-Boc-CbBI (which is stable even
at pH 1) relative to N-Boc-CBI containing a cyclopropane (t1/2 ) 133 h at pH 3) may be attributed to a
combination of the increased extent of vinylogous amide conjugation, the nonoptimal geometric alignment
of the cyclobutane with the activating cyclohexadienone, and the intrinsic but modestly lower strain energy
(1.8 kcal/mol) of a cyclobutane versus a cyclopropane.
Introduction
CC-1065 (1) and duocarmycin SA (2) represent key members
of a class of antitumor agents that derive their biological activity
from their ability to selectively alkylate duplex DNA (Figure
1).1-3 The study of the natural products and their synthetic
unnatural enantiomers,4 derivatives, and key analogues has
defined the fundamental features that control the DNA alkylation
selectivity, efficiency, and catalysis, providing a detailed
understanding of fundamental relationships between structure,
reactivity, and biological activity.3
Among the most studied duocarmycin analogues is CBI5
(Figure 2), which is synthetically more accessible and also has
been found to enhance both the chemical stability (4×) and the
biological potency (4×) of the corresponding derivatives relative
to the alkylation subunit found in CC-1065. Thus, it is on this
Figure 1. Natural products.
scaffold that new design concepts are often explored, developed,
and evaluated.3 Despite the extensive synthetic studies on the
duocarmycins, there have been very few examples of modifica-
tion of the alkylation subunit’s cyclopropane, which is intimately
involved in the DNA alkylation reaction responsible for their
biological activity.6 For some time, we have been interested in
probing the deep-seated change in this cyclopropane involving
expansion to a cyclobutane, which provides the corresponding
(1) For CC-1065, see: (a) 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. For
duocarmycin SA, see: (b) Ichimura, M.; Ogawa, T.; Takahashi, K.;
Kobayashi, E.; Kawamoto, I.; Yasuzawa, T.; Takahashi, I.; Nakano,
H. J. Antibiot. 1990, 43, 1037. For duocarmycin A, see: (c) Takahashi,
I.; Takahashi, K.; Ichimura, M.; Morimoto, M.; Asano, K.; Kawamoto,
I.; Tomita, F.; Nakano, H. J. Antibiot. 1988, 41, 1915. For yatakemycin,
see: (d) Igarashi, Y.; Futamata, K.; Fujita, T.; Sekine, A.; Senda, H.;
Naoki, H.; Furumai, T. J. Antibiot. 2003, 56, 107.
9
13936 J. AM. CHEM. SOC. 2010, 132, 13936–13940
10.1021/ja106986f  2010 American Chemical Society

Synthesis and Characterization of a Duocarmycin Derivative
A R T I C L E S
established methods for forming four-membered rings (e.g.,
[2 + 2] cycloaddition) cannot be easily adapted to its prepara-
tion. The approach pursued and ultimately implemented entailed
a previously unreported Ar-4′ alkylative spirocyclization of the
corresponding phenol that is analogous to the Ar-3′ spirocy-
clization used for the synthesis of CBI (Figure 2). Pioneering
efforts by Winstein and Baird first demonstrated the spirocy-
clization of such phenol precursors to form reactive cyclopro-
panes (Ar-3′ alkylation) and the more stable cyclopentanes (Ar-
5′ alkylation), but no reports of its extension to an Ar-4′
spirocyclization for the preparation of cyclobutanes have been
disclosed.8
Results and Discussion
Figure 2. CBI vs CbBI and retrosynthetic analysis.
Synthesis. The synthesis of CbBI began with the N-alkylation
of 59,10 with 6, which proceeded in 83% yield (Scheme 1). Key
5-exo-trig free-radical cyclization11 of 7 upon treatment with
tributyltin hydride gave the desired ethyl ester 8, and reduction
of 8 with lithium borohydride provided 9 in quantitative yield.
Treatment of the resulting alcohol 9 with methanesulfonyl
chloride in pyridine followed by the addition of LiCl cleanly
provided the desired chloride 10. Removal of the benzyl
protecting group was achieved by hydrogenolysis with 10%
Pd/C and ammonium formate, providing the first key spirocy-
clization substrate, 11. However, all attempts to spirocyclize
11 to give 13 were unsuccessful, providing only recovered
starting material in most cases and chloride elimination and/or
Boc-deprotected products upon heating at high reaction tem-
peratures (150-160 °C). An extensive range of bases, solvents,
and reaction temperatures were examined without success. Thus,
in spite of the relative release of ring strain, the Ar-4′
1,2,10,11-tetrahydro-9H-cyclobuta[c]benzo[e]indol-4-one (CbBI)
alkylation subunit (Figure 2). We recognized that the modestly
smaller strain energy of the cyclobutane in CbBI (1.8 kcal/mol;
27.6 kcal/mol vs 25.8 kcal/mol)7 should make these agents
chemically more stable, but the extent of the changes in
reactivity and reaction regioselectivity and their impact on the
DNA alkylation properties and biological activity of the resulting
analogues was not clear. Herein, we report the synthesis of
N-Boc-CbBI (13), its incorporation into a key analogue of the
duocarmycins (16, CbBI-TMI), the X-ray structure characteriza-
tion of 13, and its correlation with the reactivity and reaction
regioselectivity that impact the resulting DNA alkylation and
biological properties of this class of compounds.
In addition, the parent spirocyclobutylcyclohexadienone
ring system embedded in CbBI is unknown, and many well-
(2) For duocarmycin SA, see: (a) Boger, D. L.; Johnson, D. S.; Yun, W.
J. Am. Chem. Soc. 1994, 116, 1635. For yatakemycin, see: (b) Parrish,
J. P.; Kastrinsky, D. B.; Wolkenberg, S. E.; Igarashi, Y.; Boger, D. L.
J. Am. Chem. Soc. 2003, 125, 10971. (c) Trzupek, J. D.; Gottesfeld,
J. M.; Boger, D. L. Nat. Chem. Biol. 2006, 2, 79. (d) Tichenor, M. S.;
MacMillan, K. S.; Trzupek, J. D.; Rayl, T. J.; Hwang, I.; Boger, D. L.
J. Am. Chem. Soc. 2007, 129, 10858. For CC-1065, see: (e) 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. (f)
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.
(g) Boger, D. L.; Johnson, D. S.; Yun, W.; Tarby, C. M. Bioorg. Med.
Chem. 1994, 2, 115. (h) 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. For duocarmycin
A, see: (i) Boger, D. L.; Ishizaki, T.; Zarrinmayeh, H.; Munk, S. A.;
Kitos, P. A.; Suntornwat, O. J. Am. Chem. Soc. 1990, 112, 8961. (j)
Boger, D. L.; Ishizaki, T.; Zarrinmayeh, H. J. Am. Chem. Soc. 1991,
113, 6645. (k) Boger, D. L.; Yun, W.; Terashima, S.; Fukuda, Y.;
Nakatani, K.; Kitos, P. A.; Jin, Q. Bioorg. Med. Chem. Lett. 1992, 2,
759. (l) Boger, D. L.; Yun, W. J. Am. Chem. Soc. 1993, 115, 9872.
(m) Boger, D. L.; Munk, S. A.; Zarrinmayeh, H.; Ishizaki, T.; Haught,
J.; Bina, M. Tetrahedron 1991, 47, 2661.
(5) (a) Boger, D. L.; Ishizaki, T.; Kitos, P. A.; Suntornwat, O. J. Org.
Chem. 1990, 55, 5823. (b) Boger, D. L.; Ishizaki, T.; Wysocki, R. J.,
Jr.; Munk, S. A.; Kitos, P. A.; Suntornwat, O. J. Am. Chem. Soc. 1989,
111, 6461. (c) Boger, D. L.; Ishizaki, T. Tetrahedron Lett. 1990, 31,
793. (d) Boger, D. L.; Ishizaki, T.; Zarrinmayeh, H.; Kitos, P. A.;
Suntornwat, O. Bioorg. Med. Chem. Lett. 1991, 1, 55. (e) Boger, D. L.;
Ishizaki, T.; Sakya, S. M.; Munk, S. A.; Kitos, P. A.; Jin, Q.;
Besterman, J. M. Bioorg. Med. Chem. Lett. 1991, 1, 115. (f) Boger,
D. L.; Munk, S. A.; Ishizaki, T. J. Am. Chem. Soc. 1991, 113, 2779.
(g) Boger, D. L.; Munk, S. A. J. Am. Chem. Soc. 1992, 114, 5487.
(h) Boger, D. L.; Yun, W. J. Am. Chem. Soc. 1994, 116, 5523. (i)
Boger, D. L.; Yun, W.; Han, N.; Johnson, D. S. Bioorg. Med. Chem.
1995, 3, 611. (j) Boger, D. L.; Yun, W.; Cai, H.; Han, N. Bioorg.
Med. Chem. 1995, 3, 761. (k) Boger, D. L.; Yun, W.; Han, N. Bioorg.
Med. Chem. 1995, 3, 1429. (l) Drost, K. J.; Cava, M. P. J. Org. Chem.
1991, 56, 2240. (m) Aristoff, P. A.; Johnson, P. D. J. Org. Chem.
1992, 57, 6234. (n) Boger, D. L.; Wysocki, R. J.; Ishizaki, T. J. Am.
Chem. Soc. 1990, 112, 5230. (o) Boger, D. L.; Zarrinmayeh, H.; Munk,
S. A.; Kitos, P. A.; Suntornwat, O. Proc. Natl. Acad. Sci. U.S.A. 1991,
88, 1431. (p) Boger, D. L.; Munk, S. A.; Zarrinmayeh, H. J. Am. Chem.
Soc. 1991, 113, 3980.
(6) (a) Boger, D. L.; Jenkins, T. J. J. Am. Chem. Soc. 1996, 118, 8860.
(b) Boger, D. L.; Palanki, M. S. S. J. Am. Chem. Soc. 1992, 114,
9318. (c) Boger, D. L.; Johnson, D. S.; Palanki, M. S.; Kitos, P. A.;
Chang, J.; Dowell, P. Bioorg. Med. Chem. 1993, 1, 27. (d) Tietze,
L. F.; Herzig, T.; Fecher, A.; Haunert, F.; Schuberth, I. ChemBioChem
2001, 2, 758.
(3) For reviews, see: (a) Boger, D. L.; Johnson, D. S. Angew. Chem., Int.
Ed. Engl. 1996, 35, 1438. (b) Boger, D. L. Acc. Chem. Res. 1995, 28,
20. (c) Boger, D. L.; Johnson, D. S. Proc. Natl. Acad. Sci. U.S.A.
1995, 92, 3642. (d) Boger, D. L.; Garbaccio, R. M. Acc. Chem. Res.
1999, 32, 1043. (e) Tichenor, M. S.; Boger, D. L. Nat. Prod. Rep.
2008, 25, 220. (f) MacMillan, K. S.; Boger, D. L. J. Med. Chem. 2009,
52, 5771. (g) Searcey, M. Curr. Pharm. Des. 2002, 8, 1375.
(4) (a) Boger, D. L.; Coleman, R. S. J. Am. Chem. Soc. 1988, 110,
1321. (b) Boger, D. L.; Coleman, R. S. J. Am. Chem. Soc. 1988, 110,
4796. (c) Boger, D. L.; Machiya, K. J. Am. Chem. Soc. 1992, 114, 10056.
(d) Boger, D. L.; Machiya, K.; Hertzog, D. L.; Kitos, P. A.; Holmes,
D. J. Am. Chem. Soc. 1993, 115, 9025. (e) Boger, D. L.; McKie, J. A.;
Nishi, T.; Ogiku, T. J. Am. Chem. Soc. 1996, 118, 2301. (f) Boger,
D. L.; McKie, J. A.; Nishi, T.; Ogiku, T. J. Am. Chem. Soc. 1997,
119, 311. (g) Tichenor, M. S.; Kastrinsky, D. B.; Boger, D. L. J. Am.
Chem. Soc. 2004, 126, 8346. (h) Tichenor, M. S.; Trzupek, J. D.;
Kastrinsky, D. B.; Shiga, F.; Hwang, I.; Boger, D. L. J. Am. Chem.
Soc. 2006, 128, 15683.
(7) Dudev, T.; Lim, C. J. Am. Chem. Soc. 1998, 120, 4450.
(8) (a) Winstein, S.; Baird, R. J. Am. Chem. Soc. 1957, 79, 756. (b) Baird,
R.; Winstein, S. J. Am. Chem. Soc. 1957, 79, 4238. (c) Baird, R.;
Winstein, S. J. Am. Chem. Soc. 1962, 84, 788. (d) Baird, R.; Winstein,
S. J. Am. Chem. Soc. 1963, 85, 567.
(9) (a) Boger, D. L.; Yun, W.; Teegarden, B. R. J. Org. Chem. 1992, 57,
2873. (b) Boger, D. L.; McKie, J. A. J. Org. Chem. 1995, 60, 1271.
(c) Boger, D. L.; McKie, J. A.; Boyce, C. W. Synlett 1997, 515. (d)
Kastrinsky, D. B.; Boger, D. L. J. Org. Chem. 2004, 69, 2284.
(10) Boger, D. L.; Boyce, C. W.; Garbaccio, R. M.; Goldberg, J. A. Chem.
ReV. 1997, 97, 787.
(11) Boger, D. L.; Boyce, C. W.; Garbaccio, R. M.; Searcey, M.
Tetrahedron Lett. 1998, 39, 2227.
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A R T I C L E S
Lajiness and Boger
Scheme 1
Scheme 2
In order to accurately assess the biological properties of such
cyclobutane derivatives, the corresponding analogue 16 (CbBI-
TMI) of duocarmycin SA (2) was prepared. Thus, Boc depro-
tection of 11 (4 N HCl in EtOAc, 23 °C, 30 min) followed by
direct coupling of the resulting indoline hydrochloride salt with
5,6,7-trimethoxyindole-2-carboxylic acid13 [1.1 equiv, 3 equiv
of 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDCI),
N,N-dimethylformamide (DMF), 23 °C, 16 h, 65%] afforded
14 (Scheme 2). Conversion of the chloride to the iodide 15 (5.0
equiv of NaI, 2-butanone, 65 °C, 3 h, 82%) provided the
cyclization substrate that was subjected to the optimized
spirocyclization conditions. However, the hydrolytic lability of
the linking amide bond proved to be significant, resulting in
the generation of only small amounts of 16 (<5%) and the
production of predominantly the free amine of CbBI, which was
formed via spirocyclization and subsequent hydrolysis of the
resulting labile amide. When the spirocyclization reaction was
run at a lower temperature for a longer reaction time (110 °C
for 5 h), amide bond hydrolysis was minimized, and the yield
of 16 was improved. As discussed below, the linking amide
bond in 16 is weak and diagnostic of the preferential nitrogen
lone-pair vinylogous amide (vs amide) conjugation with the
cyclohexadienone system of CbBI. Since this vinylogous amide
is cross-conjugated with the cyclobutylcyclohexadienone, its
dominance serves to stabilize the otherwise reactive cyclobutane.
Reactivity and Reaction Regioselectivity. The study of both
the rate of acid-catalyzed solvolysis and the regioselectivity of
addition to the activated cyclopropane has proven to be key to
understanding the structural features that contribute to the
biological properties of the CC-1065 and duocarmycin alkylation
subunits. The rates of solvolysis have provided insights into
the structural features that stabilize or activate the cyclopropane
for nucleophilic attack as well as into the source of catalysis
for the DNA alkylation reaction,14 and their study has defined
a fundamental parabolic relationship between the intrinsic
reactivity of the alkylation subunit and the biological activity.15
spirocyclization of 11 is kinetically much slower than the
analogous Ar-3′ spirocyclization used to provide N-Boc-CBI,
which occurs at room temperature upon exposure to even
aqueous NaHCO3 (2 h). The reactivity of the cyclization
substrate was increased by conversion of 11 to the corresponding
iodide in nearly quantitative yield using NaI in 2-butanone.
Although spirocyclization of 12 was not observed at or near
room temperature under a wide range of reaction conditions,
warming a solution of this substrate in 1:1 saturated aqueous
NaHCO3/tetrahydrofuran (THF) at 110 °C for 3 h provided the
desired spirocycle 13, as confirmed by X-ray diffraction
analysis12 (Figure 3). Further optimization of the spirocyclization
to minimize competitive Boc deprotection and iodide solvolysis
(130 °C for 1 h) provided N-Boc-CbBI in 55% yield. It is
remarkable, but not unprecedented, that the four-membered ring
closure to provide 13 is kinetically so much slower than the
corresponding three-membered ring spirocyclization used for
the preparation of N-Boc-CBI. This may be attributed largely
to the increased entropy of activation required for the Ar-4′
spirocyclization and does not reflect the relative stabilities of
the resulting products. In contrast, the slow rate of ring closure
relative to an Ar-5′ spirocyclization8 may be related to the
corresponding enthalpy of activation and likely reflects the
relative stabilities of the resulting products.
(12) The X-ray structure of 13 has been deposited with the Cambridge
Crystallographic Data Centre (CCDC 787358).
(13) Boger, D. L.; Ishizaki, T.; Zarrinmayeh, H.; Kitos, P. A.; Suntornwat,
O. J. Org. Chem. 1990, 55, 4499.
(14) (a) Boger, D. L.; Hertzog, D. L.; Bollinger, B.; Johnson, D. S.; Cai,
H.; Goldberg, J.; Turnbull, P. J. Am. Chem. Soc. 1997, 119, 4977. (b)
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. (c) Boger, D. L.; Garbaccio, R. M. Bioorg. Med.
Chem. 1997, 5, 263.
Figure 3. X-ray structure of N-Boc-CbBI (13).
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13938 J. AM. CHEM. SOC. VOL. 132, NO. 39, 2010

Synthesis and Characterization of a Duocarmycin Derivative
A R T I C L E S
The regioselectivity of the ring-opening reaction has helped
define the mechanism of DNA alkylation, where the preferred
site of nucleophilic addition was found to depend on the
stereoelectronic alignment of the cyclopropane (or, in this case,
cyclobutane) bonds with the cyclohexadienone π-system.
The solvolysis reactivity of N-Boc-CbBI (13) was followed
by UV spectrophotometry in pH 3, pH 2, and even pH 1
phosphate buffer, but no measurable solvolysis was observed.
Treatment of 13 with 10% trifluoroacetic acid or 10% F3CSO3H
in MeOH led only to Boc cleavage over time (72 h) with no
evidence of cyclobutane ring opening. Treatment of 13 with 4
N HCl in EtOAc (-78 to 25 °C) afforded only 17 resulting
from Boc deprotection, with no observed chloride addition to
the cyclobutane. In order to avoid the competitive acid-catalyzed
Boc deprotection of 13 while assessing the relative reactivity
of CbBI, the corresponding methyl carbamate 18 was prepared
(eq 1). This was most easily accomplished by heating a solution
of 12 at 160 °C in saturated aqueous NaHCO3/THF (1:1) for
30 min in a microwave reactor to provide 17 in 74% yield via
spirocyclization followed by in situ Boc deprotection. Depro-
tonation of 17 with sodium hydride (2.5 equiv, DMF, 30 min,
0 °C) followed by treatment with methyl chloroformate (5.0
equiv) added slowly afforded 18 in superb yield (96%).
Analogous to observations made with 13, no measurable
solvolysis of 18 was observed even in pH 1 phosphate buffer.
Whereas treatment of 18 with 4 N HCl in EtOAc at -78 °C
provided only recovered starting material, warming the solution
to room temperature for 2 h provided a single chloride addition
product, 19, in quantitative yield (eq 2). Exclusive chloride
addition to the less substituted cyclobutane carbon was observed,
and no seven-membered ring addition product resulting from
attack on the more hindered carbon was observed. Thus, the
CbBI alkylation subunit exhibits a remarkable stability, being
unreactive to solvolysis even at pH 1. This contrasts with N-Boc-
CBI, which is stable at pH 7 but exhibits readily measurable
solvolysis reactivity at pH 3 (k ) 1.45 × 10-6 s-1, t1/2 ) 133 h)
and pH 2 (t1/2 ) 12.5 h).16 A lower limit on the relative reactivity
of CbBI versus CBI indicates that it is at least 100 times more
stable than CBI toward acid-catalyzed solvolysis.
Figure 4. Comparison of X-ray crystal structures of CBI analogues (data
taken from refs 17-19).
across the series of modified alkylation subunits illustrated in
Figure 4.17-19 That is, the shorter length of the c bond
corresponds to increased vinylogous amide conjugation, which
in turn is correlated with a remarkable progressive increase in
compound stability. Similarly observed is the smaller ꢀ1 dihedral
angle and the longer amide bond length (bond d, 1.379 Å;
indicative of less amide vs vinylogous amide conjugation) across
the series. Further contributing to the stability of CbBI relative
to CBI is the modestly diminished ring strain intrinsic in a
cyclobutane versus a cyclopropane (1.8 kcal/mol),7 which makes
its cleavage less facile. The net effect is that CbBI exhibits a
remarkable stability in which its intrinsic reactivity is masked
in part by the strong stabilizing vinylogous amide conjugation.
Additionally, the X-ray crystal structure indicates that the
geometric alignment of the cyclobutane deviates slightly with
respect to the plane bisecting the cyclohexadienone (Figure 5A).
This results from the fusion of the five-membered ring, which
precludes an ideal alignment and overlap between the cyclobu-
tane orbitals and the cyclohexadienone.18
The reaction regioselectivity originates in the stereoelectronic
alignment of the two available cyclobutane bonds with the
cyclohexadienone π-system. As shown in Figure 5B, the orbital
of the cyclobutane bond extending to the less substituted carbon
overlaps best with the developing π-system of the phenol
reaction product. In contrast, the bond extending to the more
substituted carbon nearly lies in the plane of this developing
π-system and is not stereoelectronically aligned to undergo
cleavage. Notably, this stereoelectronic control overrides any
intrinsic preference to place a developing positive charge on a
preferred secondary versus primary center (ring expansion).
Furthermore, diagnostic of the regioselectivity of nucleophilic
addition, the cleaved bond is longer (1.590 vs 1.551 Å) and
(15) Parrish, J. P.; Hughes, T. V.; Hwang, I.; Boger, D. L. J. Am. Chem.
Soc. 2004, 126, 80.
(16) Boger, D. L.; Garbaccio, R. M. J. Org. Chem. 1999, 64, 5666.
(17) For CBI, see: Boger, D. L.; Ishizaki, T.; Kitos, P. A.; Suntornwat, O.
J. Org. Chem. 1990, 55, 5823.
(18) For CBQ, see: (a) Boger, D. L.; Mesini, P.; Tarby, C. M. J. Am. Chem.
Soc. 1994, 116, 6461. (b) Boger, D. L.; Mesini, P. J. Am. Chem. Soc.
1994, 116, 11335.
The remarkable stability of CbBI is due in large part to the
stabilizing vinylogous amide conjugation. Most diagnostic of
this conjugation is the shortened N2-C2a bond length (bond c,
1.382 Å), which shows a progressive shortening as one moves
(19) For CNA, see: Boger, D. L.; Turnbull, P. J. Org. Chem. 1997, 62,
5849.
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A R T I C L E S
Lajiness and Boger
effective alkylation of duplex DNA and follows trends depicted
in the parabolic relationship between reactivity and cytotoxic
activity.15
Conclusions
The subtle change in the structure of CbBI versus CBI that
results in such a dramatic alteration of the chemical and
biological properties of the compound highlight the remarkable
constellation of properties that are incorporated into the compact
natural product structures 1 and 2 and are used to mask or tame
an otherwise reactive electrophile.21 Even though CbBI could
be expected to be more stable than CBI, the remarkable stability
exhibited by N-acyl-CbBI (stable at pH 1) is not intuitively
obvious from a cursory inspection of its structure. Contributing
to this unusual stability is the dominant vinylogous amide
conjugation,22 the nonideal alignment of the cyclobutane with
the activating cyclohexadienone imposed by the fused five-
membered ring,18 and the modestly reduced strain energy (1.8
kcal/mol)7 intrinsic to a four-membered cyclobutane ring versus
a three-membered cyclopropane ring.
Acknowledgment. We gratefully acknowledge the financial
support of the National Institutes of Health (CA 41986) and the
Skaggs Institute for Chemical Biology, and we thank William M.
Robertson for conducting the cytotoxic activity assays. J.P.L. is a
Skaggs Fellow.
Note Added in Proof. In the interim while the manuscript was
under review, we successfully secured the X-ray crystal structure
of 17 (CDCC 792209), and details of its comparison with CBI and
related compounds (CBQ and CNA) are provided in Figure S1 in
the Supporting Information. Most notable in these comparisons is
the shorter c bond length (1.330 vs 1.337 Å for CBI and 1.382 Å
for N-Boc-CbBI), indicative of the greater vinylogous amide
conjugation and further reduced reactivity. Accompanying this
change is a shorter b bond length (1.577 vs 1.590 Å for N-Boc-
CbBI), indicative of a further diminished cyclobutane conjugation
with the cyclohexadienone.
Figure 5. (A) Back view and (B) side view of 13.
weaker than the bond extending to the more substituted carbon,
reflecting its conjugation with the π-system. Steric factors
resulting from preferential SN2 attack on the less hindered site
may also play a role.
Cytotoxic Activity. Compounds 15 and 16 were assayed for
cytotoxic activity against the L1210 tumor cell line (mouse
leukemia cell line). These compounds were found to display
mean inhibitory concentration (IC50) values of 1.2 and 6.3 µM,
respectively. This represents a 106-fold decrease in potency in
comparison with the natural products themselves (IC50 ) 6-10
pM for 2) and a 105-106-fold loss in activity in comparison
with CBI-TMI (IC50 ) 30 pM),20 a testament to the remarkable
stability of these CbBI derivatives. Although relatively inactive,
this level of cytotoxic activity is in line with expectations based
on the exceptional stability of compounds that preclude their
Supporting Information Available: Full experimental details
and compound characterizations along with crystallographic data
(CIF). This material is available free of charge via the Internet
at http://pubs.acs.org.
JA106986F
(21) (a) Wolkenberg, S. E.; Boger, D. L. Chem. ReV. 2002, 102, 2477. (b)
Tse, W. C.; Boger, D. L. Chem. Biol. 2004, 11, 1607. (c) Tse, W. C.;
Boger, D. L. Acc. Chem. Res. 2004, 37, 61.
(20) Boger, D. L.; Yun, W. J. Am. Chem. Soc. 1994, 116, 7996.
(22) Boger, D. L.; Turnbull, P. J. Org. Chem. 1998, 63, 8004.
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