Selective metal cation activation of a DNA alkylating agent: Synthesis and evaluation of methyl 1,2,9,9a-tetrahydrocyclopropa[c]pyrido[3,2-e]indol-4-one-7-carboxylate (CPyI)

Article abstract of DOI:10.1021/jo000177b

The synthesis of methyl 1,2,9,9a-tetrahydrocyclopropa[c]pyrido[3,2-e]indol-4-one-7-carboxylate (CPyI) containing a one carbon expansion of the C ring pyrrole found in the duocarmycin SA alkylation subunit and its incorporation into analogues of the natural product are detailed. The unique 8-ketoquinoline structure of CPyI was expected to provide a tunable means to effect activation via selective metal cation complexation. The synthesis of CPyI was based on a modified Skraup quinoline synthesis followed by a 5-exo-trig aryl radical cyclization onto an unactivated alkene with subsequent TEMPO trap or 5-exo-trig aryl radical cyclization onto a vinyl chloride for synthesis of the immediate precursor. Closure of the activated cyclopropane, accomplished by an Ar-3′ spirocyclization, provided the CPyI nucleus in 10 steps and excellent overall conversion (29%). The evaluation of the CPyI-based agents revealed an intrinsic stability comparable to that of CC-1065 and duocarmycin A but that it is more reactive than duocarmycin SA and the CBI-based agents (3-4x). A pH-rate profile of the addition of nucleophiles to CPyI demonstrated that an acid-catalyzed reaction is observed below pH 4 and that an uncatalyzed reaction predominates above pH 4. The expected predictable activation of CPyI by metal cations toward nucleophilic addition was found to directly correspond to established stabilities of the metal complexes with the addition product (Cu²⁺ > Ni²⁺ > Zn²⁺ > Mn²⁺ > Mg²⁺) and provides the opportunity to selectively activate the agents upon addition of the appropriate Lewis acid. This tunable metal cation activation of CPyI constitutes the first example of a new approach to in situ activation of a DNA binding agent complementary to the well-recognized methods of reductive, oxidative, or photochemical activation. Resolution and synthesis of a full set of natural product analogues and subsequent evaluation of their DNA alkylation properties revealed that the CPyI analogues retain identical DNA alkylation sequence selectivity and near-identical DNA alkylation efficiencies compared to the natural products. Consistent with past studies and even with the deep-seated structural change in the alkylation subunit, the agents were found to exhibit potent cytotoxic activity that directly correlates with their inherent reactivity.

Full text of DOI:10.1021/jo000177b

4088
J . Org. Chem. 2000, 65, 4088-4100
Selective Meta l Ca tion Activa tion of a DNA Alk yla tin g Agen t:
Syn th esis a n d Eva lu a tion of Meth yl
1,2,9,9a -Tetr a h yd r ocyclop r op a [c]p yr id o[3,2-e]in d ol-4-on e-7-ca r boxyla te
(CP yI)
Dale L. Boger* and Christopher W. Boyce
Department of Chemistry and The Skaggs Institute for Chemical Biology, The Scripps Research Institute,
10550 North Torrey Pines Road, La J olla, California 92037
Received February 8, 2000
The synthesis of methyl 1,2,9,9a-tetrahydrocyclopropa[c]pyrido[3,2-e]indol-4-one-7-carboxylate
(CPyI) containing a one carbon expansion of the C ring pyrrole found in the duocarmycin SA
alkylation subunit and its incorporation into analogues of the natural product are detailed. The
unique 8-ketoquinoline structure of CPyI was expected to provide a tunable means to effect
activation via selective metal cation complexation. The synthesis of CPyI was based on a modified
Skraup quinoline synthesis followed by a 5-exo-trig aryl radical cyclization onto an unactivated
alkene with subsequent TEMPO trap or 5-exo-trig aryl radical cyclization onto a vinyl chloride for
synthesis of the immediate precursor. Closure of the activated cyclopropane, accomplished by an
Ar-3′ spirocyclization, provided the CPyI nucleus in 10 steps and excellent overall conversion (29%).
The evaluation of the CPyI-based agents revealed an intrinsic stability comparable to that of CC-
1065 and duocarmycin A but that it is more reactive than duocarmycin SA and the CBI-based
agents (3-4×). A pH-rate profile of the addition of nucleophiles to CPyI demonstrated that an
acid-catalyzed reaction is observed below pH 4 and that an uncatalyzed reaction predominates
above pH 4. The expected predictable activation of CPyI by metal cations toward nucleophilic
addition was found to directly correspond to established stabilities of the metal complexes with
the addition product (Cu2+ > Ni2+ > Zn2+ > Mn2+ > Mg2+) and provides the opportunity to selectively
activate the agents upon addition of the appropriate Lewis acid. This tunable metal cation activation
of CPyI constitutes the first example of a new approach to in situ activation of a DNA binding
agent complementary to the well-recognized methods of reductive, oxidative, or photochemical
activation. Resolution and synthesis of a full set of natural product analogues and subsequent
evaluation of their DNA alkylation properties revealed that the CPyI analogues retain identical
DNA alkylation sequence selectivity and near-identical DNA alkylation efficiencies compared to
the natural products. Consistent with past studies and even with the deep-seated structural change
in the alkylation subunit, the agents were found to exhibit potent cytotoxic activity that directly
correlates with their inherent reactivity.
(+)-Duocarmycin SA (DSA, 1) is a remarkably potent
antitumor antibiotic (IC50 ) 10 pM, L1210) disclosed in
19901 that has been shown to selectively bind and
alkylate DNA (Figure 1).2 (+)-Duocarmycin SA exhibits
enhanced stability and corresponding biological potency
compared to its predecessors, (+)-duocarmycin A (DA, 2)3
and (+)-CC-1065 (3),4 and therefore has been the subject
of extensive investigation. Studies have shown that CC-
(1) Ichimura, M.; Ogawa, T.; Takahashi, K.; Kobayashi, E.; Kawa-
moto, I.; Yasuzawa, T.; Takahashi, I.; Nakano, H. J . Antibiot. 1990,
43, 1037. Ichimura, M.; Ogawa, T.; Katsumata, S.; Takahashi, K.;
Takahashi, I.; Nakano, H. J . Antibiot. 1991, 44, 1045.
(2) Boger, D. L.; Machiya, K.; Hertzog, D. L.; Kitos, P. A.; Holmes,
D. J . Am. Chem. Soc. 1993, 115, 9025.
(3) Ohba, K.; Watabe, H.; Sasaki, T.; Takeuchi, Y.; Kodama, Y.;
Nakazawa, T.; Yamamoto, H.; Shomura, T.; Sezaki, M.; Kondo, S. J .
Antibiot. 1988, 41, 1515. Takahashi, I.; Takahashi, K.; Ichimura, M.;
Morimoto, M.; Asano, K.; Kawamoto, I.; Tomita, F.; Nakano, H. J .
Antibiot. 1988, 41, 1915. Yasuzawa, T.; Iida, T.; Muroi, K.; Ichimura,
M.; Takahashi, K.; Sano, H. Chem. Pharm. Bull. 1988, 36, 3728.
Ichimura, M.; Muroi, K.; Asano, K.; Kawamoto, I.; Tomita, F.;
Morimoto, M.; Nakano, H. J . Antibiot. 1988, 41, 1285. Ishii, S.;
Nagasawa, M.; Kariya, Y.; Yamamoto, H.; Inouye, S.; Kondo, S. J .
Antibiot. 1989, 42, 1713.
(4) Hanka, L. J .; Dietz, A.; Gerpheide, S. A.; Kuentzel, S. L.; Martin,
D. G. J . Antibiot. 1978, 31, 1211. Chidester, C. G.; Krueger, W. C.;
Mizsak, S. A.; Duchamp, D. J .; Martin, D. G. J . Am. Chem. Soc. 1981,
103, 7629.
F igu r e 1.
1065 and the duocarmycins tolerate and benefit from
structural modifications to the alkylation subunit and
10.1021/jo000177b CCC: $19.00 © 2000 American Chemical Society
Published on Web 05/10/2000

Metal Cation Activation of a DNA Alkylating Agent
J . Org. Chem., Vol. 65, No. 13, 2000 4089
activation (aflatoxin), disulfide or trisulfide cleavage
(calicheamicin), photochemical activation (psoralen), and
oxidant activation of metal complexes (bleomycin), we are
not aware of examples of tunable metal cation Lewis acid
activation of a DNA alkylating agent. Thus, the studies
represent the preparation of the first members of such a
class of DNA alkylating agents.
More intriguing, comparative trace metal analyses of
cancerous and noncancerous human tissue have revealed
significant distinctions.11 Although no generalizations
were possible across all tumor types, within a given
tumor type there were significant and potentially exploit-
able differences. For example, Zn was found in breast
carcinoma at levels 700% higher than normal breast cells
of the same type while lung carcinoma exhibited a
reversed and even larger 10-fold difference. Thus, che-
motherapeutic agents subject to Zn activation might
exhibit an enhanced activity against breast carcinoma
attributable to this difference in Zn levels.11
Syn th esis of N-BOC-CP yI. The CPyI synthesis was
based on a modified Skraup quinoline synthesis to
provide the core structure, followed by the TEMPO trap
of an aryl radical-alkene 5-exo-trig cyclization for intro-
duction of the A-ring and final Ar-3′ spirocyclization
(Scheme 1). Thus, 2-bromoacrolein was treated with 4
in the presence of bromine (1.0 equiv, AcOH, 100 °C, 1
h, 92%) to provide 5.12 Protection of phenol 5 (1.2 equiv
of BnBr, 1.1 equiv of NaH, DMF, 4-25 °C, 24 h, 85%)
was followed by nitro reduction with SnCl2 (5.0 equiv,
EtOAc, 0.5 h, 70 °C) and amine protection (4.0 equiv of
(BOC)2O, 2 equiv of Et3N, dioxane, 70 °C, 1 h, 74% for
two steps). The resultant 3-bromoquinoline 7 was sub-
jected to Pd(0)-catalyzed carboxybutylation13 (0.1 equiv
of (PPh3)4Pd, CO (g), 1.2 equiv of n-Bu3N, n-BuOH, 100
°C, 12 h, 78%) and treatment with LiOMe (1.1 equiv,
MeOH, 0-25 °C, 1.5 h, 91%) to afford 9. As detailed by
Heck,13 the use of MeOH as a reaction solvent for direct
Pd-mediated carboxymethylation of 7 is limited by the
achievable reaction temperatures (65 °C with MeOH
versus 115 °C with n-BuOH) and thus requires extended
reaction times (g3 d) and generally proceeds in lower
yields (e45%). Following selective, acid-catalyzed C5
iodination14 of 9 with N-iodosuccinimide (1.2 equiv,
catalytic TsOH, THF-CH3OH, 0-25 °C, 1-2 d, 88%),
N-alkylation of the sodium salt of 10 (1.1 equiv of NaH,
DMF, 4 °C, 30 min) with allyl bromide (3 equiv, DMF,
25 °C, 2.5 h, 94%) proceeded smoothly.
F igu r e 2.
that the resulting agents retain their ability to partici-
pate in the characteristic sequence selective DNA alky-
lation reaction.5,6 Such structural modifications and the
definition of their effects have served to advance the
understanding of the origin of sequence selectivity5,7 and
catalysis8,9 of the DNA alkylation reaction by 1-3.
The substitution of the fused pyrrole C ring of CPI,
the alkylating subunit of CC-1065 (3), with the six-
membered benzene ring in CBI was shown to increase
relative stability (4×) and biological potency (4×) without
affecting DNA alkylation selectivity.10 Herein, we report
the preparation of methyl 1,2,9,9a-tetrahydrocyclopropa-
[c]pyrido[3,2-e]indol-4-one-7-carboxylate (CPyI), which
contains a similar one carbon expansion of the C ring
pyrrole found in the DSA alkylation subunit but with
incorporation of a pyridine (Figure 2). We anticipated
that chelation of metal cations with the 8-ketoquinoline
functionality in CPyI would provide a tunable means to
effect Lewis acid-catalyzed functional reactivity, thereby
providing a means to selectively activate the agents.
Although there are several well-established methods
available for selective activation of DNA binding agents
including reductive activation (mitomycins), oxidative
In contrast to related substrates,6,14 cyclization of 11
under previously described conditions (6.0 equiv of Bu3-
SnH, 6.0 equiv of TEMPO, 70 °C, benzene, 3.5 h) did not
proceed in high yield (41-54%). Although tin hydride-
mediated reduction has been observed with similar
compounds,15 it had not been observed in other CC-1065/
duocarmycin systems. Consequently, it was surprising
to isolate significant amounts (g30%) of the halogen-
(5) For mechanistic aspects see: Boger, D. L.; J ohnson, D. S. Angew.
Chem., Int. Ed. Engl. 1996, 35, 1439.
(6) For synthetic aspects see: Boger, D. L.; Boyce, C. W.; Garbaccio,
R. M.; Goldberg, J . A. Chem. Rev. 1997, 97, 787.
(7) Sun, D.; Lin, C. H.; Hurley, L. H. Biochemistry 1993, 32, 4487
and references therein.
(8) Boger, D. L.; Garbaccio, R. M. Acc. Chem. Res. 1999, 32, 2, 1043.
Boger, D. L.; Garbaccio, R. M. Bioorg. Med. Chem. 1997, 5, 263. Boger,
D. L.; Hertzog, D. L.; Bollinger, B.; J ohnson, D. S.; Cai, H.; Goldberg,
J .; Turnbull, P. J . Am. Chem. Soc. 1997, 119, 4977. Boger, D. L.;
Bollinger, B.; Hertzog, D. L.; J ohnson, D. S.; Cai, H.; Goldberg, J .;
Me´sini, P.; Garbaccio, R. M.; J in, Q.; Kitos, P. A. J . Am. Chem. Soc.
1997, 119, 4987.
(11) Mulay, I. L.; Roy, R.; Knox, B. E.; Suhr, N. H.; Delaney, W. E.
J . Natl. Cancer Inst. 1971, 47, 1.
(12) Baker, R. H.; Tinsley, S. W., J r.; Butler, D.; Riegel, B. J . Am.
Chem. Soc. 1950, 72, 393. Tinsley, S. W. J . Am. Chem. Soc. 1955, 77,
4175.
(13) Schoenberg, A.; Bartoletti, I.; Heck, R. F. J . Org. Chem. 1974,
39, 3318. Heck, R. F. Palladium Reagents in Organic Synthesis;
Academic Press: London, Orlando. Fl, 1985.
(9) Warpehoski, M. A.; Hurley, L. H. Chem. Res. Toxicol. 1988, 1,
315.
(10) Boger, D. L.; Ishizaki, T.; Kitos, P. A.; Suntornwat, O. J . Org.
Chem. 1990, 55, 5823. Boger, D. L.; Ishizaki, T.; Wysocki, R. J ., J r.;
Munk, S. A.; Kitos, P. A.; Suntornwat, O. J . Am. Chem. Soc. 1989,
111, 6461.
(14) Boger, D. L.; McKie, J . A. J . Org. Chem. 1995, 60, 1271.
(15) Ueno, Y.; Chino, K.; Okawara, M. Tetrahedron Lett. 1982, 23,
2575. Shankaran, K.; Sloan, C. P.; Snieckus, V. Tetrahedron Lett. 1985,
26, 6001.

4090 J . Org. Chem., Vol. 65, No. 13, 2000
Boger and Boyce
Sch em e 1
Sch em e 2
3 h, 99%) and subsequent spirocyclization21 by treatment
of 15 with DBU (4 equiv, CH3CN, 25 °C, 3 h, 99%)
provided N-BOC-CPyI (16). Acid-catalyzed deprotection
of 16 (3 M HCl-EtOAc, 25 °C, 30 min), which is
accompanied by the addition of chloride to the cyclopro-
pane, followed by treatment of the crude hydrochloride
salt with K2CO3 (10 equiv, acetone, 25 °C, 24 h, 96%)
cleanly provided CPyI (17).
In an improved preparation of the advanced (chloro-
methyl)indoline precursor 14, adoption of a direct free
radical cyclization22 of a substrate bearing a vinyl chloride
acceptor alkene provided 14 in good conversion (Scheme
2). Thus, alkylation of the sodium salt of 10 (1.1 equiv of
NaH, DMF, 4 °C, 30 min) with E-1,3-dichloropropene (3
equiv, 25 °C, 12 h, 94%) was followed by free radical
cyclization of the vinyl chloride (1.5 equiv of Bu3SnH,
catalytic AIBN, benzene, 70 °C, 6.5 h, 87%) to provide
14. The incorporation of this improvement provided
N-BOC-CPyI (16) in 10 steps and superb overall conver-
sion (29%).
Resolu tion . To assess the properties of both enanti-
omers of the CPyI-based agents, a direct chromatographic
resolution on a ChiralCel-OD semipreparative HPLC
column (2 × 25 cm, 50% i-PrOH-hexanes eluant, 7 mL/
min, R ) 1.43) provided both enantiomers of 16 (>99%
ee). N-BOC-CPyI proved to be the only late-stage inter-
mediate that could be effectively resolved on a ChiralCel-
OD column, and similar efforts to separate 13-15 were
not successful. The slower eluting (+)-enantiomer of 16
(tR ) 38 min) was assigned the natural (3S)-configura-
tion, and agents derived from this (+)-enantiomer ex-
hibited the more potent biological activity, the more
effective DNA alkylation properties, and a DNA alkyla-
tion selectivity identical with the natural products. The
faster eluting (-)-enantiomer of 16 (tR ) 27 min) was
assigned the unnatural (3R)-configuration and agents
derived from this (-)-enantiomer exhibited the corre-
sponding less potent biological activity, the less effective
DNA alkylation properties, and a DNA alkylation selec-
tivity identical with the unnatural enantiomers of the
natural products.
reduced product. Characterization of this byproduct16 was
confirmed by direct comparison with an authentic sample
prepared by allylation of 9 (3 equiv of allyl bromide, 1.1
equiv of NaH, DMF, 4-25 °C, 89%). The radical cycliza-
tion was improved by utilizing tris(trimethylsilyl)silane
((CH3Si)3SiH, 5 equiv, 8.0 equiv of TEMPO, toluene, 80
°C, 16 h, 85%), which has a stronger metal-hydride bond
than Bu3SnH (79 vs 74 kcal/mol)17 and therefore a slower
rate of aryl radical reduction permitting clean intramo-
lecular 5-exo-trig cyclization. The TEMPO-trapped prod-
uct 12 was reduced with activated Zn14,18 (50 equiv, 3:1
HOAc-THF, 60 °C, 11 h, 60%) and the resulting alcohol
13 converted to the primary chloride19 (3.0 equiv of Ph3P,
9.0 equiv of CCl4, CH2Cl2, 25 °C, 3 h, 73%). Two-phase
transfer catalytic hydrogenolysis20 of the benzyl ether 14
(10% Pd-C, 10 equiv of 25% aqueous HCO2NH4, 25 °C,
Syn th esis of Du oca r m ycin a n d CC-1065 An a -
logu es. The CPyI alkylation subunit was incorporated
into duocarmycin and CC-1065 analogues as detailed in
Scheme 3. Deprotection and concurrent ring opening of
16 (3 M HCl-EtOAc) followed by immediate coupling (5
equiv of EDCI, DMF, 25 °C) of the resulting amine
hydrochloride salt with 5,6,7-trimethoxyindole-2-carboxy-
(16) Methyl 8-(benzyloxy)-6-[N-(tert-butyloxycarbonyl)-N-(2-prope-
nyl)amino]quinoline-3-carboxylate: 1H NMR (CDCl3, 500 MHz) δ 9.39
(d, J ) 2.0 Hz, 1H), 8.70 (d, J ) 2.0 Hz, 1H), 7.48 (m, 2H), 7.30 (m,
4H), 7.08 (br s, 1H), 5.85 (m, 1H), 5.40 (s, 2H), 5.05 (m, 2H), 4.20 (m,
2H), 3.98 (s, 3H), 1.39 (s, 9H); FABHRMS (NBA/CsI) m/z 581.1065 (M
+ Cs+, C26H28N2O5 requires m/z 581.1053).
(17) Giese, B.; Kopping, B. Tetrahedron Lett. 1989, 30, 681. Kana-
bus-Kaminska, J . M.; Hawari, J . A.; Griller, D. J . Am. Chem. Soc. 1987,
109, 5267.
(18) Newman, M. S.; Evans, F. J ., J r. J . Am. Chem. Soc. 1955, 77,
946.
(19) Hooz, J .; Gilani, S. S. H. Can. J . Chem. 1968, 46, 86.
(20) Ram, S.; Ehrenkaufer, R. E. Synthesis 1988, 91. Bieg, T.; Szeja,
W. Synthesis 1985, 76.
(21) Baird, R.; Winstein, S. J . Am. Chem. Soc. 1963, 85, 567. Baird,
R.; Winstein, S. J . Am. Chem. Soc. 1962, 84, 788. Winstein, S.; Baird,
R. J . Am. Chem. Soc. 1957, 79, 756.
(22) Patel, V. F.; Andis, S. L.; Enkema, J . K.; J ohnson, D. A.;
Kennedy, J . H.; Mohamadi, F.; Schultz, R. M.; Soose, D. J .; Spees, M.
M. J . Org. Chem. 1997, 62, 8868. Boger, D. L.; Boyce, C. W.; Garbaccio,
R. M.; Searcey, M. Tetrahedron Lett. 1998, 39, 2227.

Metal Cation Activation of a DNA Alkylating Agent
J . Org. Chem., Vol. 65, No. 13, 2000 4091
Sch em e 3
lic acid23 (TMI, 19, 10 h, 52%), 5-methoxyindole-2-
carboxylic acid (20, 10 h, 59%), indole-2-carboxylic acid
24
25
(21, 10 h, 71%), indole2 (22, 16 h, 64%), and CDPI1
F igu r e 3. Solvolysis study (UV spectra) of N-BOC-CPyI (16,
top) and CPyI (17, bottom) in 50% CH3OH-aqueous buffer
(pH 2, 4:1:20 (v:v:v) 1.0 M citric acid, 0.2 M Na2HPO4, and
H2O, respectively). The spectra were recorded at regular
intervals, and only a few are shown for clarity. Top: 0, 1, 2, 3,
4, 5, 7, 9, 12, 16, 41 h. Bottom: 0, 5, 10, 20, 26, 35, 47, 66, 93,
144, 283 h.
(23, 16 h, 41%) provided 24, 26, 28, 30, and 32, respec-
tively. DBU (3 equiv, 3 h, 25 °C) promoted spirocycliza-
tion of 24 (DMF, 86%), 26 (CH3CN, 90%), 28 (CH3CN,
93%), 30 (DMF, 97%), and 32 (DMF, 67%) provided 25,
27, 29, 31, and 33, respectively.
Solvolysis: Rea ctivity a n d Regioselectivity. Two
fundamental characteristics of the alkylation subunits
have proven important in past studies.5 The first is the
stereoelectronically controlled acid-catalyzed ring opening
of the activated cyclopropane which dictates preferential
addition of a nucleophile to the least substituted cyclo-
propane carbon. The second is the relative reactivity of
the agents as established by their rate of acid-catalyzed
solvolysis which has been found to accurately reflect a
direct relationship between intrinsic stability and in vitro
cytotoxicity.5
Ta ble 1. Solvolysis Ra tes w ith P h osp h a te Bu ffer a
compd
kobs (s-1
)
t1/2 (h)
16, N-BOC-CPyI
pH 2
pH 3
pH 2
pH 3
3.72 × 10-5
3.81 × 10-6
4.63 × 10-6
6.27 × 10-7
5
51
42
17, CPyI
310
a
pH 2: 50% CH3OH-aqueous buffer (4:1:20 (v:v:v) 1.0 M citric
acid, 0.2 M Na2HPO4, and H2O, respectively. pH 3: 50% CH3OH-
aqueous buffer (4:1:20 (v:v:v) 0.1 M citric acid, 0.2 M Na2HPO4,
and H2O, respectively).
Solvolysis was conducted in 50% CH3OH-buffer mix-
tures (pH 3 buffer ) 4:1:20 (v:v:v) 0.1 M citric acid:0.2 M
Na2HPO4:H2O; pH 2 buffer ) 4:1:20 (v:v:v) 1.0 M citric
acid:0.2 M Na2HPO4:H2O) and followed spectrophoto-
metrically by UV with the disappearance of the long-
wavelength absorption of the CPyI chromophore and with
the appearance of a short-wavelength absorption at-
tributable to the solvolysis product (Figure 3 and Table
1). N-BOC-CPyI (16) proved to be reasonably stable to
solvolysis even at pH 2.0 (k ) 3.72 × 10-5 s-1, t1/2 ) 5.2
h) and pH 3.0 (k ) 3.81 × 10-6 s-1, t1/2 ) 50.5 h) as
compared to N-BOC-CPI (34, pH 3, t1/2 ) 37 h) and
N-BOC-DA (35, pH 3, t1/2 ) 11 h) (Figure 4). However,
16 was less stable than N-BOC-DSA (36, pH 3, t1/2 ) 177
h) and N-BOC-CBI (37, pH 3, t1/2 ) 133 h). The rate of
solvolysis was found to be independent of the phosphate
buffer countercation (Na+, NH4+, or K+) within experim-
mental error.
The acid-catalyzed nucleophilic addition of CH3OH to
16 was conducted on a preparative scale to establish the
regioselectivity of addition and was confirmed by syn-
thesis of the expected product 41 derived from nucleo-
philic addition to the least substituted cyclopropane
carbon. Treatment of N-BOC-CPyI (16) with catalytic
CF3SO3H (0.3 equiv, CH3OH, 25 °C, 20 h, 91%) resulted
in the clean addition to provide a single product 41
(Scheme 4). Similarly, treatment of 16 with HCl-EtOAc
(2 equiv, THF, -78 °C, 2 min, 96%) provided 15 as a
single product. Clean cleavage of the C8b-C9 bond with
SN2 addition of CH3OH or HCl to the least substituted
C9 cyclopropane carbon was observed, and no cleavage
(23) Boger, D. L.; Ishizaki, T.; Zarrinmayeh, H.; Munk, S. A.; Kitos,
P. A.; Suntornwat, O. J . Am. Chem. Soc. 1990, 112, 8961.
(24) Boger, D. L.; Yun, W.; Han, N. Bioorg. Med. Chem. 1995, 3,
1429.
(25) Boger, D. L.; Coleman, R. S.; Invergo, B. J . J . Org. Chem. 1987,
52, 1521. Boger, D. L.; Coleman, R. S. J . Org. Chem. 1984, 49, 2240.

4092 J . Org. Chem., Vol. 65, No. 13, 2000
Boger and Boyce
Ta ble 2. Solvolysis Ra tes of N-BOC-CP yI w ith Un iver sa l
Bu ffer a
pH
kobs (s-1
)
t1/2 (h)
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
10.0
3.45 × 10-5
4.79 × 10-6
1.15 × 10-6
8.41 × 10-7
7.88 × 10-7
8.29 × 10-7
7.27 × 10-7
6.73 × 10-7
6.97 × 10-7
5.5
40
165
230
245
230
265
290
275
50% CH3OH-H2O
CH3OH
9.80 × 10-7
195
stable
stable
a
50% CH3OH-aqueous buffer (B(OH)3-citric acid-Na3PO4).
F igu r e 5. Plot of pH versus log kobs for solvolysis of N-BOC-
CPyI in universal buffer (pH 2-10).
dependenceon acid at pH 2-4 (Table 2 and Figure 5).
Above pH 4, the dependence on acid concentration
disappeared indicating a change in mechanism from acid-
catalyzed solvolysis to one which is uncatalyzed. A loss
of isobestic behavior in the UV trace at pH 11 indicated
a second reaction event at this pH, presumably arising
from hydrolysis of either the BOC or methyl ester. From
a regression analysis best fit plot of the kobs versus pH,
rate constants of 3.37 × 10-3 M-1s-1 and 8.36 × 10-7 s-1
for the acid-catalyzed and uncatalyzed reactions, respec-
tively, were established. This, plus the demonstration
that related reactions above pH 4 are not only not specific
acid-catalyzed, but also not general acid-catalyzed,29
suggests this is simply an uncatalyzed SN2 nucleophilic
addition. The demonstration of an uncatalyzed solvolysis
above pH 4 is consistent with the observation that the
DNA alkylation reaction at the physiological pH of 7.6
is not acid-catalyzed and that catalysis must come from
an alternative source.8,29
Solvolysis: Meta l Ca ta lysis. Central to the projected
use of the CPyI agents and their potential of metal cation
activation was their reactivity in the presence of metal
cations.30 Treatment of N-BOC-CPyI (16) with Zn(OTf)2
(1.1 equiv) in CH3OH (25 °C, 4 h, 92%) on a preparative
scale resulted in clean addition to provide a single
product 41 (Scheme 5). In contrast, N-BOC-CBI (37) did
not exhibit this metal-catalyzed reactivity in the presence
of Zn(OTf)2 (1.5 equiv, CH3OH, 27 h, 25 °C, 100%
recovery), confirming that the CPyI 8-ketoquinoline
structure is key to metal chelation, activation, and Lewis
acid-catalyzed reaction.
F igu r e 4.
Sch em e 4
of the C8b-C9a bond with ring expansion was detected
(>20:1). This is in sharp contrast to the natural products
where substantial amounts of the alternative ring expan-
sion addition products have been observed (6-1.5:1).26
Nonetheless, the observations are consistent with prior
studies with N-BOC-CBI (37), N-BOC-MCBI (38), and
N-BOC-CCBI (39), where no (>20:1) ring expansion
solvolysis product was detected (Figure 4).23,27
Solvolysis p H Dep en d en ce. Duocarmycin SA (1)
is known to be exceptionally stable at neutral condi-
tions, and it was interesting to observe that N-BOC-CPyI
(16) possessed measurable solvolytic reactivity in 50%
aqueous CH3OH (pH 7). A full pH-rate profile in 50%
CH3-OH-universal buffer (pH 2-12, B(OH)3-citric
acid-Na3PO4)28 demonstrated a near-first-order rate
(26) Warpehoski, M. A.; Harper, D. E. J . Am. Chem. Soc. 1994, 116,
7573. Warpehoski, M. A.; Harper, D. E. J . Am. Chem. Soc. 1995, 117,
2951. Boger, D. L.; Goldberg, J .; McKie, J . A. Bioorg. Med. Chem. Lett.
1996, 6, 1955. Boger, D. L.; McKie, J . A.; Nishi, T.; Ogiku, T. J . Am.
Chem. Soc. 1997, 119, 311.
(27) Boger, D. L.; McKie, J . A.; Cai, H.; Cacciari, B.; Baraldi, P. G.
J . Org. Chem. 1996, 61, 1710. Boger, D. L.; Han, N.; Tarby, C. M.;
Boyce, C. W.; Cai, H.; J in, Q.; Kitos, P. J . Org. Chem. 1996, 61, 4894.
(28) Perrin, D. D.; Dempsey, B. Buffers for pH and Metal Ion
Control; Chapman and Hall: London, 1979; p 156.
(29) Boger, D. L.; Garbaccio, R. M. J . Org. Chem. 1999, 64, 5666.
Boger, D. L.; Turnbull, P. J . Org. Chem. 1998, 63, 8004. Boger, D. L.;
Turnbull, P. J . Org. Chem. 1997, 62, 5849. Boger, D. L.; Boyce, C. W.;
J ohnson, D. S. Bioorg. Med. Chem. Lett. 1997, 7, 233.
(30) Boger, D. L.; Cassidy, K. C.; Nakahara, S. J . Am. Chem. Soc.
1993, 115, 10733. Yoshida, K.; Ishiguro, M.; Honda, H.; Yamamoto,
M.; Kubo, Y. Bull. Chem. Soc. J pn. 1988, 61, 4335. Yoshida, K.;
Yamamoto, M.; Ishiguro, M. Chem. Lett. 1986, 1059. Pratt, Y. T. J .
Org. Chem. 1962, 27, 3905.

Metal Cation Activation of a DNA Alkylating Agent
J . Org. Chem., Vol. 65, No. 13, 2000 4093
Sch em e 5
Ta ble 3. Meta l-Ca ta lyzed Ad d ition to N-BOC-CP yIa
are available for related agents.5 The alkylation site
identification and the assessment of the relative selectiv-
ity among the available sites were obtained by thermally
induced strand cleavage of the singly 5′ end-labeled
duplex DNA after exposure to the agents. Following
treatment of the end-labeled duplex DNA with a range
of agent concentrations and temperatures in the dark,
the unbound agent was removed by EtOH precipitation
of the DNA. Redissolution of the DNA in aqueous buffer,
thermolysis (100 °C, 30 min) to induce strand cleavage
at the sites of DNA alkylation, denaturing high-resolution
polyacrylamide gel electrophoresis (PAGE) adjacent to
Sanger dideoxynucleotide sequencing standards, and
autoradiography led to identification of the DNA cleavage
and alkylation sites. The full details of this procedure
have been disclosed elsewhere.32 A representative com-
parison of the DNA alkylation properties of both enan-
tiomers of N-BOC-CPyI (16) alongside both enantiomers
of N-BOC-DSA (36) is presented in Figure 6. Both
natural enantiomers exhibited approximately the same
efficiencies of DNA alkylation detectable at 10-3 M (37
°C, 48 h) and prominent at 10-2 M. (+)-N-BOC-CPyI was
slightly less efficient than the unnatural enantiomer, (-)-
N-BOC-CPyI, at both 24 and 48 h incubations which is
also reflected in the relative cytotoxic activities of the two
enantiomers. Like the preceding BOC derivatives exam-
ined,5 16 alkylated DNA much less efficiently than 24-
33 (104×), providing detectable alkylation at 10-2-10-3
M only under vigorous conditions (37 °C, 24-72 h) and
much less selectively than 24-33, exhibiting a two-base
pair AT-rich alkylation selectivity (5′-AA > 5′-TA). This
unusual behavior of the two enantiomers alkylating the
same sites is analogous to past observations.5 It is a
natural consequence of the reversed binding orientation
of the two enantiomers and the diastereomeric relation-
ship of the two adducts that result in the two enanti-
omers covering the exact same binding site surrounding
the alkylated adenine. This has been discussed in detail
illustrated elsewhere, and N-BOC-CPyI (16) conforms
nicely to these past observations and models.5
metal
equiv
kobs (s-1
)
t1/2 (h)
Cu(acac)2
Ni(acac)2
Zn(acac)2
Mn(acac)2
Mg(acac)2
Fe(acac)2
Cr(acac)2
Zn(OTf)2
Zn(OTf)2
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
0.2
1.0
1.0
1.0
5.57 × 10-5
3.51 × 10-5
1.67 × 10-5
9.79 × 10-7
<7 × 10-7
3.5
5.5
11.5
200
>250
47
4.09 × 10-6
9.73 × 10-6
2.48 × 10-4
9.19 × 10-6
5.86 × 10-6
1.23 × 10-6
5.53 × 10-5
20
0.8
21
33
16
3.5
b
Zn(OTf)2
Ti(OiPr)4
Cu(OMe)2
a
b
Solvolysis run in CH3OH except where noted. Solvolysis run
in 50% CH3OH-H2O.
The rates of metal-catalyzed (Cu2+, Ni2+, Zn2+, Mn2+
,
Mg2+, Fe3+, Cr3+, and Ti4+) addition to N-BOC-CPyI (16)
in CH3OH were measured spectrophotometrically by UV
with the disappearance of the long-wavelength absorption
of the CPyI chromophore and with the appearance of a
short-wavelength absorption (Table 3). Beautifully, within
a series of divalent metals with acetylacetonate (acac)
ligands, the relative rates of solvolysis (rate: Cu2+ > Ni2+
> Zn2+ > Mn2+ > Mg2+) corresponded directly with
established stability constants of the metal complexes
with 8-hydroxyquinoline (stability: Cu2+ > Ni2+ > Zn2+
> Mn2+ > Mg2+).31 Higher valence metals including Fe3+
,
Cr3+, and Ti4+ also demonstrated an analogous activation
of CPyI for nucleophilic addition. Notably, no apparent
catalysis was observed for Mg(acac)2 over and beyond the
background rate indicating that this endogenous metal
cation, like Na+, does not activate CPyI effectively.
Stronger Lewis acids were found to provide a faster
reaction rate (Zn(OTf)2, t1/2 ) 0.8 h, versus Zn(acac)2, t1/2
) 11.5 h) and incorporation of H2O in the solvent slowed
the reaction (Zn(OTf)2, 50% aqueous CH3OH, t1/2 ) 33
h). N-BOC-CBI (37) demonstrated no detectable reaction
in the Zn(OTf)2-CH3OH system after 7 d, further con-
firming the role of metal cation catalysis for CPyI. Thus,
the well-behaved reactivity of CPyI predictably tunable
by choice of the metal cation provides the opportunities
to selectively activate the agents.
A representative comparison of the DNA alkylation by
(+)-CPyI-TMI (25), (+)-CPyI-indole2 (31), and (+)-
CPyI-CDPI1 (33) alongside that of (+)-duocarmycin SA
(1) and (+)-CC-1065 (3) within w794 DNA is illustrated
in Figure 7. (+)-CPyI-TMI and (+)-duocarmycin SA
alkylate DNA with identical selectivity and near identical
efficiency with the latter agent being slightly more
effective. This is nicely illustrated in Figure 7 where the
two agents detectably alkylate the same high affinity site
DNA Alk yla tion Selectivity a n d Efficien cy. The
DNA alkylation properties of the agents were examined
within w794 duplex DNA32 for which comparative results
(31) Phillips, J . P. Chem. Rev. 1956, 56, 271. Irving, H.; Williams,
R. J . P. J . Chem. Soc. 1953, 3192.
(32) Boger, D. L.; Munk, S. A.; Zarrinmayeh, H.; Ishizaki, T.;
Haught, J .; Bina, M. Tetrahedron 1991, 47, 2661.

4094 J . Org. Chem., Vol. 65, No. 13, 2000
Boger and Boyce
F igu r e 7. Thermally induced strand cleavage of w794 DNA
(SV40 DNA segment, 144 bp, nucleotide nos. 138-5238), with
DNA-agent incubation for 24 h at 25 °C, removal of unbound
agent, and 30 min of thermolysis (100 °C), followed by
denaturing 8% PAGE and autoradiography: lane 1, control
DNA; lanes 2 and 3, (+)-duocarmycin SA (1, 1 × 10-5 and 1 ×
10-6); lanes 4-6, (+)-CPyI-TMI (25, 1 × 10-5-1 × 10-7); lanes
7-10, Sanger G, C, A, and T sequencing reactions; lanes 11
and 12, (+)-CC-1065 (3, 1 × 10-5 and 1 × 10-6); lanes 13-15,
(+)-CPyI-indole2 (31, 1 × 10-5-1 × 10-7); lanes 16-18, (+)-
CPyI-CDPI1 (33, 1 × 10-5-1 × 10-7).
F igu r e 6. Thermally induced strand cleavage of w794 DNA
(SV40 DNA segment, 144 bp, nucleotide nos. 138-5238), with
DNA-agent incubation for 24 or 48 h, as indicated, at 37 °C,
removal of unbound agent, and 30 min of thermolysis (100 °C),
followed by denaturing 8% PAGE and autoradiography: lanes
1 and 2, (+)-N-BOC-DSA (1 × 10-1 and 1 × 10-2); lanes 3 and
4, (-)-N-BOC-DSA (1 × 10-1 and 1 × 10-2); lane 5, control
DNA; lanes 6-9, Sanger G, C, A, and T sequencing reactions;
lanes 10 and 11, (+)-N-BOC-CPyI (1 × 10-2 and 1 × 10-3);
lanes 12 and 13, (-)-N-BOC-CPyI (1 × 10-2 and 1 × 10-3);
lanes 14 and 15, (+)-N-BOC-CPyI (1 × 10-2 and 1 × 10-3);
lanes 16 and 17, (-)-N-BOC-CPyI (1 × 10-2 and 1 × 10-3).
(1) were indistinguishable with the latter agent being
slightly more effective. The alkylation sites for the
unnatural enantiomers proved consistent with adenine
N3 alkylation with agent binding in the minor groove in
the reverse 5′ to 3′ direction across a 3.5 or 5 base-pair
AT-rich site surrounding the alkylation site (data not
shown). This is analogous to the natural enantiomer
alkylation selectivity except that it extends in the reverse
5′ to 3′ direction in the minor groove and, because of the
diastereomeric nature of the adducts, is offset by one base
pair relative to the natural enantiomers.
In Vitr o Cytotoxic Activity. Past studies with agents
in this class have defined a direct correlation between
inherent stability and cytotoxic potency.5 Consistent with
their relative reactivity, the CPyI-based agents exhibited
cytotoxic activity that closely followed this relationship
(Table 4 and Figure 9).5 The results, which also follow
trends established in the DNA alkylation studies, dem-
onstrate that the (+)-enantiomer of the analogues pos-
sessing the configuration of the natural products is the
more potent enantiomer by 3-30×. The exceptions to this
trend are the simple alkylation subunits 16 and 17
themselves which, like others in the series, typically
exhibit comparable activities. The seco precursors, which
lack the preformed cyclopropane but possess the capabili-
ties of ring closure, were found to possess cytotoxic
activity that was indistinguishable from the final ring-
closed agents. Consistent with the unique importance of
the C5 methoxy group in the binding subunit of the
duocarmycins,8 CPyI-TMI (25) and 27 were found to be
of 5′-AATTA at 10-6-10-7 M (25 °C, 24 h). This is
analogous to the observations made in comparisons of
duocarmycin SA and CBI-TMI.33 Like the preceding
agents, the CPyI-based agents exhibit AT-rich adenine
N3 alkylation selectivities that start at the 3′ adenine
N3 alkylation site with agent binding in the minor groove
in the 3′ to 5′ direction covering 3.5 or 5 base pairs (data
not shown).
A representative comparison of the DNA alkylation by
(-)-CPyI-TMI (25), (-)-CPyI-indole2 (31), and (-)-
CPyI-CDPI1 (33) alongside the unnatural enantiomer
of duocarmycin SA (1) and the natural enantiomer of CC-
1065 (3) within w794 DNA is illustrated in Figure 8. The
unnatural enantiomer DNA alkylation is considerably
slower, and the results shown in Figure 8 were obtained
only with their incubation for 72 h (25 °C) versus
incubation for 24 h (25 °C, Figure 7) for the natural
enantiomers. Despite the longer reaction times, the
extent of alkylation by the unnatural enantiomers is
lower, requiring higher agent concentrations to detect.
The DNA alkylation selectivity and efficiency observed
with ent-(-)-CPyI-TMI (25) and ent-(-)-duocarmycin SA
(33) Boger, D. L.; Yun, W. J . Am. Chem. Soc. 1994, 116, 7996.

Metal Cation Activation of a DNA Alkylating Agent
J . Org. Chem., Vol. 65, No. 13, 2000 4095
F igu r e 8. Thermally induced strand cleavage of w794 DNA
(SV40 DNA segment, 144 bp, nucleotide nos. 138-5238), with
DNA-agent incubation for 72 h at 25 °C, removal of unbound
agent, and 30 min of thermolysis (100 °C), followed by
denaturing 8% PAGE and autoradiography: lane 1, control
DNA; lanes 2 and 3, (-)-duocarmycin SA (1, 1 × 10-5 and 1 ×
10-6); lanes 4 and 5, (-)-CPyI-TMI (25, 1 × 10-5 and 1 ×
10-6); lanes 6-9, Sanger G, C, A, and T sequencing reactions;
lane 10, (+)-CC-1065 (3, 1 × 10-6); lanes 11 and 12, (-)-CPyI-
indole2 (31, 1 × 10-5 and 1 × 10-6); lanes 13 and 14, (-)-CPyI-
CDPI1 (33, 1 × 10-5 and 1 × 10-6).
F igu r e 9.
Ta ble 4. in Vitr o Cytotoxic Activity
displayed the most potent cytotoxic activity reflecting
their longer length and greater adduct stability.
agent
configuration
IC50 (L1210)a
Con clu sion s. A short and efficient 10 step (29%
overall) synthesis of CPyI featuring a modified Skraup
quinoline synthesis followed by a 5-exo-trig aryl radical
cyclization onto a vinyl chloride is detailed and consti-
tutes a one carbon expansion of the C ring pyrrole found
in the duocarmycin SA alkylation subunit. CPyI was
found to be 3-4× less stable than CBI and duocarmycin
SA but possesses a superior stability to CC-1065 and
duocarmycin A. Nucleophilic addition occurred at the
least substituted cyclopropane carbon with a regioselec-
tivity (>20:1) comparable to that of CBI but which
exceeds that of the natural products themselves (6-1.5:
1). A pH-rate profile of the addition of nucleophiles to
CPyI demonstrated that it is an acid-catalyzed reaction
below pH 4, but an uncatalyzed reaction above pH 4
consistent with the observation that the DNA alkylation
reaction at physiological pH is not acid-catalyzed.8,29 The
tunable activation of CPyI by metal cations toward
nucleophilic addition, which directly follows established
stabilities of the resulting metal complexes with the
addition product (Cu2+ > Ni2+ > Zn2+ > Mn2+ > Mg2+),
provides the opportunity to selectively and predictably
initiate reactions of the agent simply by addition of the
appropriate Lewis acid. This novel activation arises from
16, (+)-N-BOC-CPyI
16, (-)-N-BOC-CPyI
17, (+)-CPyI
natural
unnatural
natural
unnatural
natural
unnatural
natural
unnatural
natural
unnatural
natural
unnatural
natural
unnatural
110 nM
70 nM
1 µM
2 µM
27 pM (21 pM)
850 pM (350 pM)
30 pM (40 pM)
500 pM (500 pM)
400 pM (800 pM)
1000 pM (800 pM)
8 pM (7 pM)
45 pM (30 pM)
15 pM (10 pM)
40 pM (30 pM)
17, (-)-CPyI
25, (+)-CPyI-TMI
25, (-)-CPyI-TMI
27, (+)-27
27, (-)-27
29, (+)-CPyI-indole
29, (-)-CPyI-indole
31, (+)-CPyI-indole2
31, (-)-CPyI-indole2
33, (+)-CPyI-CDPI1
33, (-)-CPyI-CDPI1
a
The value in parentheses corresponds to the IC50 value
determined for the corresponding seco precursor. The assays were
conducted as detailed in ref 24.
equipotent illustrating that the C6 and C7 methoxy
groups of 25 are not contributing to its cytotoxic potency.
The indole derivative 29 was found to be less potent (10×)
than both 25 and 27, further demonstrating the impor-
tance of the C5 methoxy, which we have suggested is
derived from extending the rigid length of the agents and
contributing to the alkylation catalysis.8 Finally, the
longer agents, CPyI-indole2 (31) and CPyI-CDPI1 (33),

4096 J . Org. Chem., Vol. 65, No. 13, 2000
Boger and Boyce
J ) 2.2 Hz, 1H), 7.52 (apparent d, J ) 7.4 Hz, 2H), 7.38 (m,
2H), 7.32 (m, 1H), 5.47 (s, 2H); 13C NMR (CDCl3, 62.5 MHz) δ
155.4, 153.3, 146.4, 140.3, 138.8, 135.0, 128.7 (2C), 128.6, 128.4
(2C), 127.5, 119.9, 115.1, 103.3, 71.4; IR (film) νmax 3082, 3055,
2933, 2871, 1519 cm-1; FABHRMS (NBA/NaI) m/z 359.0040
(M + H+, C16H11BrN2O3 requires m/z 359.0031). Anal. Calcd
for C16H11BrN2O3: C, 53.50; H, 3.09; N, 7.80. Found: C, 53.81;
H, 3.23; N, 7.48.
chelation to the CPyI 8-ketoquinoline core, a unique
structural feature that is not found in the natural
products or alkylation subunit analogues disclosed to
date. Resolution and incorporation of CPyI into a full set
of duocarmycin and CC-1065 analogues allowed for
examination of their cytotoxic and DNA alkylation prop-
erties. The CPyI analogues were potent cytotoxic agents
exhibiting picomolar IC50’s which correlated with their
relative stability. In addition to smoothly following this
correlation, the analogues displayed a smooth trend of
increasing cytotoxic potency with the increasing length
in the DNA binding subunit. Analogous to the natural
products, the (+)-enantiomers possessing the absolute
configuration of 1-3 proved to be more potent (3-30×)
than the unnatural (-)-enantiomers. DNA alkylation
studies revealed that the CPyI analogues exhibited
identical DNA alkylation sequence selectivity and near-
identical DNA alkylation efficiencies compared to the
natural products. Thus, this set of analogues, which
contain a unique structural modification in the alkylation
subunit, retain full DNA alkylation and cytotoxic proper-
ties of the natural products, while possessing a novel
capability for predictable and tunable activation by
chelation of Lewis acids. Although this Lewis acid activa-
tion is of limited use for agents which already display
effective DNA alkylation properties, its use is especially
effective when applied to CPyI members which are poor
at alkylating DNA. This includes N-BOC-CPyI (16) for
which the alkylation efficiency is increased 103× and
reversed analogues of CPyI in which the DNA binding
subunits are attached through the C-terminus methyl
ester rather than N-terminus secondary amine.34 These
studies will be disclosed in due course.
8-(Ben zyloxy)-3-b r om o-6-(N-(ter t-b u t yloxyca r b on yl)-
a m in o)qu in olin e (7). A solution of 6 (0.20 g, 0.56 mmol, 1.0
equiv) in EtOAc (1.1 mL) at 25 °C was treated with SnCl2‚2H2O
(0.63 g, 2.8 mmol, 5.0 equiv). The reaction mixture was heated
to 70 °C under N2 until an orange slurry formed (ca. 0.5 h).
After being cooled to 25 °C, the reaction mixture was poured
on ice and neutralized with 1 N aqueous NaOH. The aqueous
layer was extracted with EtOAc, and the combined organic
layers were filtered, washed with saturated aqueous NaCl,
dried (Na2SO4), and concentrated. The yellow solid was placed
under vacuum for 0.5 h and then dissolved in anhydrous
dioxane (5.0 mL) and treated with di-tert-butyl dicarbonate
(0.49 g, 2.3 mmol, 4.0 equiv) and Et3N (0.16 mL, 1.1 mmol,
2.0 equiv). The reaction mixture was warmed to 70 °C under
Ar for 1 d. After cooling of the mixture to 25 °C, the solvent
was removed in vacuo. Chromatography (SiO2, 3 × 13 cm, 25%
EtOAc-hexane) afforded 7 (0.18 g, 74%) as a light yellow
1
solid: mp 162 °C; H NMR (CDCl3, 500 MHz) δ 8.77 (d, J )
2.0 Hz, 1H), 8.13 (d, J ) 2.5 Hz, 1H), 7.47 (apparent d, J )
7.5 Hz, 2H), 7.42 (d, J ) 2.0 Hz, 1H), 7.35 (m, 2H), 7.28 (m,
1H), 7.01 (d, J ) 2.0 Hz, 1H), 6.61 (s, 1H), 5.37 (s, 2H), 1.51
(s, 9H); 13C NMR (CDCl3, 125 MHz) δ 154.9, 152.4, 148.3,
137.9, 136.3, 136.2, 135.4, 131.0, 128.6 (2C), 128.0, 127.3 (2C),
118.6, 104.7, 103.6, 81.1, 70.9, 28.3 (3C); IR (film) νmax 3354,
2971, 2919, 1807, 1766, 1724 cm-1; FABHRMS (NBA/CsI) m/z
429.0825 (M + H+, C21H21BrN2O3 requires m/z 429.0814).
n -Bu t yl 8-(Ben zyloxy)-6-(N-(ter t-b u t yloxyca r b on yl)-
a m in o)qu in olin e-3-ca r boxyla te (8). A solution of 7 (4.4 g,
10 mmol, 1.0 equiv) in n-BuOH (85 mL) was degassed with
N2. Pd(PPh3)4 (1.2 g, 1.0 mmol, 0.1 equiv) and n-Bu3N (2.9 mL,
12 mmol, 1.2 equiv) were added, and the solution was again
purged with N2. The reaction mixture was flushed with CO
and then slowly heated to 100 °C under a CO atmosphere.
Upon complete reaction (ca. 12 h), H2O and saturated aqueous
NH4Cl were added. The organic layer was separated, and the
aqueous layer was extracted with EtOAc. The combined
organic layers were washed with saturated aqueous NaCl,
dried (Na2SO4), and concentrated. Chromatography (SiO2, 5.5
× 20 cm, 25% EtOAc-hexane) afforded 8 (3.6 g, 78%) as a
Exp er im en ta l Section
3-Br om o-8-h yd r oxy-6-n itr oqu in olin e (5). A solution of
2-bromoacrolein (5.0 g, 37 mmol, 1.0 equiv) in glacial acetic
acid (110 mL) at 25 °C was titrated to the appearance of a
faint reddish color with bromine (ca. 5.9 g, 37 mmol, 1.0 equiv).
2-Hydroxy-4-nitroaniline (4, 5.7 g, 37 mmol, 1.0 equiv) was
added, and the solution was gradually heated to 100 °C. The
solution was cooled to 25 °C after 1 h. Filtering and neutral-
ization of the precipitate with sodium phosphate buffer (1 M,
pH 7, Na2HPO4-NaH2PO4) afforded 9.2 g (92%) of 5 as a light
1
yellow solid: mp 135-136 °C; H NMR (CDCl3, 400 MHz) δ
9.31 (d, J ) 2.0 Hz, 1H), 8.66 (d, J ) 2.1 Hz, 1H), 7.61 (d, J )
1.8 Hz, 1H), 7.49 (apparent d, J ) 7.4 Hz, 2H), 7.35 (apparent
t, J ) 7.2 Hz, 2H), 7.29 (m, 1H), 7.11 (d, J ) 2.1 Hz, 1H), 6.64
(br s, 1H), 5.39 (s, 2H), 4.38 (t, J ) 6.6 Hz, 2H), 1.78 (m, 2H),
1.52 (s, 9H), 1.49 (m, 2H, buried under 1.52 ppm), 0.98 (t, J )
7.4 Hz, 3H); 13C NMR (acetone-d6, 100 MHz) δ 165.7, 155.7,
153.5, 146.8, 139.8, 139.5, 137.8, 137.7, 129.5, 129.1 (2C),
128.6, 128.4 (2C), 124.8, 107.1, 106.9, 80.4, 71.2, 65.5, 31.3,
28.3 (3C), 19.8, 13.9. IR (film) νmax 3222, 3049, 2958, 2930,
2876, 1717, 1617 cm-1; FABHRMS (NBA/CsI) m/z 451.2249
(M + H+, C26H30N2O5 requires m/z 451.2233).
1
yellow solid: mp 240-241 °C; H NMR (CDCl3, 400 MHz) δ
8.93 (d, J ) 2.0 Hz, 1H), 8.50 (d, J ) 2.0 Hz, 1H), 8.23 (d, J )
2.2 Hz, 1H), 8.18 (s, 1H), 7.92 (s, J ) 2.3 Hz, 1H); 13C NMR
(DMSO-d6, 62.5 MHz) δ 155.1, 152.1, 146.3, 139.5, 139.1, 128.7,
119.1, 113.5, 105.1; IR (film) νmax 3408 (br), 3089, 1587 cm-1
.
Anal. Calcd for C9H5BrN2O3: C, 40.18; H, 1.87; N, 10.41.
Found: C, 40.21; H, 1.91; N, 9.98.
8-(Ben zyloxy)-3-br om o-6-n itr oqu in olin e (6). A solution
of 5 (13.7 g, 51.0 mmol, 1.0 equiv) in anhydrous DMF (150
mL) was cooled to 4 °C under N2 and treated with KI (1.70 g,
10.0 mmol, 0.2 equiv) and NaH (60% dispersion in oil, 2.24 g,
56.0 mmol, 1.1 equiv). Benzyl bromide (7.30 mL, 6.10 mmol,
1.2 equiv) was added after 30 min, and the reaction was
allowed to warm to 25 °C. After 24 h, the reaction volume was
reduced by two-thirds in vacuo and EtOAc was added. The
reaction mixture was poured onto H2O and extracted with
EtOAc. The combined organic extracts were washed with
saturated aqueous NaCl, dried (Na2SO4), and concentrated.
Flash chromatography (SiO2, 5.5 × 20 cm, 50-100% CH2Cl2-
hexane gradient) afforded 6 (15.6 g, 85%) as a yellow solid:
Met h yl 8-(Ben zyloxy)-6-(N-(ter t-b u t yloxyca r b on yl)-
a m in o)qu in olin e-3-ca r boxyla te (9). A solution of 8 (2.9 g,
6.4 mmol, 1.0 equiv) in CH3OH (70 mL) was cooled to 4 °C
under N2 and treated with LiOMe (0.28 g, 7.1 mmol, 1.1 equiv).
The reaction mixture was allowed to warm to 25 °C after 20
min. Upon complete reaction (ca. 1.5 h), H2O was added. The
organic layer was separated, and the aqueous layer was
extracted with EtOAc. The organic layers were combined,
washed with saturated aqueous NaCl, dried (Na2SO4), and
concentrated. Chromatography (SiO2, 5 × 19 cm, 25-30%
EtOAc-hexane gradient) afforded 9 (2.4 g, 91%) as a yellow
1
1
mp 170 °C; H NMR (CDCl3, 400 MHz) δ 9.06 (d, J ) 2.2 Hz,
solid: mp 173-174 °C; H NMR (CDCl3, 400 MHz) δ 9.29 (d,
1H), 8.44 (d, J ) 2.2 Hz, 1H), 8.25 (d, J ) 2.2 Hz, 1H), 7.83 (d,
J ) 2.0 Hz, 1H), 8.66 (d, J ) 2.1 Hz, 1H), 7.56 (d, J ) 1.8 Hz,
1H), 7.45 (m, 2H), 7.30 (m, 2H), 7.25 (m, 1H), 7.18 (d, J ) 2.0
Hz, 1H), 6.85 (s, 1H), 5.36 (s, 2H), 3.98 (s, 3H), 1.50 (s, 9H);
13C NMR (CDCl3, 125 MHz) δ 165.9, 154.6, 152.6, 146.9, 138.6,
(34) Boger, D. L.; Wolkenberg, S. E.; Boyce, C. W. J . Am. Chem.
Soc., in press.

Metal Cation Activation of a DNA Alkylating Agent
J . Org. Chem., Vol. 65, No. 13, 2000 4097
137.9, 137.8, 136.0, 128.5, 128.4 (2C), 127.9, 127.3 (2C), 123.8,
106.5, 105.5, 80.8, 70.8, 52.4, 28.2 (3C); IR (film) νmax 3333,
3241, 2974, 1723, 1621 cm-1; FABHRMS (NBA/CsI) m/z
409.1773 (M + H+, C23H24N2O5 requires m/z 409.1763). Anal.
Calcd for C23H24N2O5: C, 67.63; H, 5.92; N, 6.86. Found: C,
68.00; H, 5.98; N, 6.75.
33.0, 32.8, 28.4 (3C), 20.1 (4C), 17.0; IR (film) νmax 2969, 2927,
2876, 1729, 1703, cm-1; FABHRMS (NBA/CsI) m/z 726.2383
(M + Cs+, C35H45N3O6 requires m/z 736.2363). Anal. Calcd for
C
35H45N3O6‚H2O: C, 67.61; H, 7.62; N, 6.76. Found: C, 67.70;
H, 7.11; N, 6.27.
Meth yl 5-(Ben zyloxy)-3-(ter t-bu tyloxyca r bon yl)-1-(h y-
d r oxym eth yl)-1,2-d ih yd r o-3H-p yr id o[3,2-e]in d ole-8-ca r -
boxyla te (13). A solution of 12 (20 mg, 33 µmol, 1.0 equiv) in
a 3:1 mixture of THF-H2O (0.90 mL) was treated with
activated zinc powder (54 mg, 0.80 mmol, 25 equiv) and HOAc
(0.20 mL), and the resulting suspension was warmed to 60 °C
with vigorous stirring. After 7 h, additional Zn (54 mg) was
added and the reaction was stirred for 4 h. The Zn powder
was removed by filtration through Celite with a CH2Cl2 wash,
and the mixture was concentrated in vacuo. The resulting
residue was dissolved in EtOAc and filtered through Celite,
and the solution was concentrated in vacuo. Chromatography
(SiO2, 1 × 12 cm, 20-50% EtOAc-hexane) provided 13 (9.2
Met h yl 8-(Ben zyloxy)-6-(N-(ter t-b u t yloxyca r b on yl)-
a m in o)-5-iod oqu in olin e-3-ca r boxyla te (10). A solution of
9 (2.1 g, 5.2 mmol, 1.0 equiv) in a 1:1 mixture of THF-
CH3OH (85 mL) was cooled to 4 °C and treated with catalytic
TsOH (40 mg) in THF (0.5 mL). N-Iodosuccinimide (1.4 g, 6.2
mmol, 1.2 equiv) in THF (10 mL) was slowly added over 10
min. After 1.5 h, the reaction mixture was warmed to 25 °C
and stirred 45 h. Upon complete reaction, saturated aqueous
NaHCO3, Et2O, and H2O were added. The organic layer was
separated, and the aqueous layer was extracted with Et2O and
EtOAc. The organic layers were combined, washed with
saturated aqueous NaHCO3 and saturated aqueous NaCl,
dried (Na2SO4), and concentrated. Chromatography (SiO2, 5
× 19 cm, hexanes and then 30% EtOAc-hexane) provided 10
(2.3 g, 84%, typically 80-88%) as a yellow solid: mp 182-183
°C; 1H NMR (CDCl3, 400 MHz) δ 9.27 (d, J ) 1.9 Hz, 1H),
8.96 (d, J ) 1.9 Hz, 1H), 8.40 (s, 1H), 7.58 (m, 2H), 7.36 (m,
2H), 7.27 (m, 1H), 7.26 (s, 1H), 5.43 (s, 2H), 4.01 (s, 3H), 1.55
(s, 9H); 13C NMR (CDCl3, 62.5 MHz) δ 165.4, 155.0, 152.3,
147.6, 141.9, 139.8, 139.7, 135.8, 129.6, 128.5 (2C), 128.1, 128.0
(2C), 125.1, 105.6, 81.7, 78.4, 71.1, 52.6, 28.2 (3C); IR (film)
1
mg, 60%): mp 186 °C; H NMR (CDCl3, 400 MHz) δ 9.05 (s,
1H), 8.65 (s, 1H), 8.06 (br s, 1H), 7.55 (br s, 2H), 7.36 (apparent
t, J ) 7.1 Hz, 2H), 7.30 (apparent t, J ) 7.2 Hz, 1H), 5.28 (s,
2H), 4.16 (m, 1H), 4.09 (m, 1H), 4.00 (s, 3H), 3.76 (m, 3H),
1.55 (s, 9H); 13C NMR (CDCl3, 125 MHz) δ 165.9, 154.8, 152.3,
146.0, 138.1, 136.2, 133.6, 128.5 (2C), 128.0 (2C), 127.9, 124.8,
123.5, 115.0, 102.4, 82.0, 70.9, 64.9, 52.5, 52.3, 41.0, 28.4 (3C);
IR (film) νmax 3214, 2961, 2924, 2850, 1717, 1701 cm-1
;
FABHRMS (NBA/CsI) m/z 465.2032 (M + H+, C26H28N2O6
requires m/z 465.2026).
ν
max 3384, 2974, 1723 cm-1; FABHRMS (NBA/CsI) m/z 535.0743
(M + H+, C23H23IN2O5 requires m/z 535.0730).
Meth yl 5-(Ben zyloxy)-3-(ter t-bu tyloxycar bon yl)-1-(ch lo-
r om eth yl)-1,2-d ih yd r o-3H-p yr id o[3,2-e]in d ole-8-ca r boxy-
la te (14). Meth od A. A solution of 13 (6.7 mg, 14 µmol, 1.0
equiv) in anhydrous CH2Cl2 (0.14 mL) under Ar was treated
sequentially with Ph3P (13 mg, 48 µmol, 3.0 equiv) and CCl4
(14 µL, 0.15 mmol, 9.0 equiv). The reaction mixture was stirred
at 25 °C for 3 h. The solvent was then evaporated under a
stream of N2. Radial chromatography (SiO2, 1.0 mm, 20%
EtOAc-hexane) afforded 14 (5.1 mg, 73%) as a gold solid: mp
Meth yl 8-(Ben zyloxy)-6-[(N-(ter t-bu tyloxyca r bon yl)-N-
(2-p r op en yl))a m in o]-5-iod oqu in olin e-3-ca r boxyla te (11).
A solution of 10 (0.40 g, 0.75 mmol, 1.0 equiv) in anhydrous
DMF (6.2 mL) at 4 °C in a flamed-dried round-bottom flask
was treated with NaH (60% dispersion in oil, 33 mg, 0.82
mmol, 1.1 equiv) and stirred under Ar. After 30 min, allyl
bromide (94 µL, 2.3 mmol, 3.0 equiv) was added, and the
reaction mixture was warmed to 25 °C and stirred 2.5 h.
Saturated aqueous NaHCO3 and EtOAc were added, and the
reaction was then poured on H2O. The aqueous layer was
extracted with EtOAc, and the combined organic extract
was washed with H2O and saturated aqueous NaCl, dried
(Na2SO4), and concentrated in vacuo. Chromatography (SiO2,
2.5 × 16 cm, 20-25% EtOAc-hexane gradient) afforded 11
(0.40 g, 94%) as a gold solid: mp 128-129 °C; major rotamer
1H NMR (CDCl3, 500 MHz) δ 9.40 (s, 1H), 9.12 (s, 1H), 7.43
(apparent d, J ) 7.5 Hz, 2H), 7.34 (apparent t, J ) 7.5 Hz,
2H), 7.26 (m, 1H), 6.89 (s, 1H), 5.74 (m, 1H), 5.43 (m, 2H),
4.86 (m, 2H), 4.41 (dd, J ) 5.5, 14.5 Hz, 1H), 4.02 (s, 3H), 3.76
(dd, J ) 7.0, 14.8 Hz, 1H), 1.23 (s, 9H); 13C NMR (CDCl3, 62.5
MHz) δ 165.2, 154.4, 153.3, 149.6, 144.3, 143.7, 141.6, 135.8,
132.8, 130.2, 128.8 (2C), 128.1 (2C), 126.9, 125.2, 118.5, 114.7,
94.7, 80.8, 71.0, 52.7, 51.7, 28.1 (3C); IR (film) νmax 3060, 2972,
2922, 1729, 1700 cm-1; FABHRMS (NBA/CsI) m/z 575.1036
(M + H+, C26H27IN2O5 requires m/z 575.1043).
1
194 °C (EtOAc-hexane); H NMR (CDCl3, 500 MHz) δ 9.29
(d, J ) 2.0 Hz, 1H), 8.62 (d, J ) 1.9 Hz, 1H), 8.16 (br s, 1H),
7.53 (br s, 2H), 7.37 (m, 2H), 7.30 (m, 1H), 5.41 (m, 2H), 4.24
(d, J ) 11.1 Hz, 1H), 4.13 (m, 1H), 4.00 (s, 3H), 3.98 (m, 1H,
buried under 4.00 ppm), 3.82 (dd, J ) 3.3, 11.1 Hz, 1H), 3.47
(t, J ) 10.5 Hz, 1H), 1.56 (s, 9H); 13C NMR (CDCl3, 100 MHz)
δ 165.7, 155.6, 152.2, 146.3, 143.0, 138.7, 136.0, 132.6, 128.5
(2C), 128.0 (3C), 124.4, 123.8, 114.5, 102.3, 81.6, 70.9, 53.0,
52.6, 46.8, 40.8, 28.3 (3C); IR (film) νmax 2976, 2954, 2942, 1723,
1702 cm-1; FABHRMS (NBA/NaI) m/z 483.1675 (M + H+,
C
26H27ClN2O5 requires m/z 483.1687). Anal. Calcd for C26H27-
ClN2O5: C, 64.66; H, 5.63; N, 5.80. Found: C, 64.78; H, 5.73;
N, 5.66.
Meth yl 3-(ter t-Bu tyloxyca r bon yl)-1-(ch lor om eth yl)-5-
h yd r oxy-1,2-d ih yd r o-3H-p yr id o[3,2-e]in d ole-8-ca r b oxy-
la te (15). A slurry of 14 (0.9 g, 1.9 mmol, 1.0 equiv) and 10%
Pd-C (0.36 g) in THF (6.2 mL) under N2 was cooled to -78
°C and degassed under vacuum. The reaction was warmed to
25 °C and treated with 25% aqueous HCO2NH4 (1.2 g in 4.7
mL H2O, 19 mmol, 10 equiv). After 3 h, the catalyst was
removed by filtration through Celite and the solvent was
evaporated under reduced pressure to afford 15 (0.72 g, 99%)
as a bright yellow solid: mp 162-163 °C; 1H NMR (CDCl3,
400 MHz) δ 9.09 (d, J ) 1.8 Hz, 1H), 8.62 (d, J ) 1.7 Hz, 1H),
8.28 (br s, 1H), 8.00 (br s, 1H), 4.22 (m, 1H), 4.13 (dd, J ) 8.9,
11.6 Hz, 1H), 4.00 (s, 3H), 3.94 (m, 1H), 3.78 (dd, J ) 3.5, 11.1
Hz, 1H), 3.47 (dd, J ) 9.9, 11.0 Hz, 1H), 1.53 (s, 9H); 13C NMR
(CDCl3, 100 MHz) δ 165.6, 153.5, 152.1, 144.9, 143.7, 136.2,
133.1, 124.4, 123.6, 113.6, 103.0, 81.7, 52.9 52.7, 46.6, 40.7,
28.3 (3C); IR (film) νmax 3385, 2978, 2919, 1701 cm-1; FAB-
HRMS (NBA/NaI) m/z 393.1228 (M + H+, C19H21ClN2O5
requires m/z 393.1217).
Met h yl
5-(Ben zyloxy)-3-(ter t-b u t yloxyca r b on yl)-1-
[[(2′,2′,6′,6′-tetr a m eth ylp ip er id in o)oxy]m eth yl]-1,2-d ih y-
d r o-3H-p yr id o[3,2-e]in d ole-8-ca r boxyla te (12). A solution
of 11 (20 mg, 35 µmol, 1.0 equiv) in anhydrous toluene (1.2
mL) was treated with a solution of TEMPO (16 mg, 0.11 mmol,
3.0 equiv) in toluene (0.11 mL) and (TMS)3SiH (11 µL, 37 µmol,
1.05 equiv). The solution was warmed to 80 °C, and 5 equiv of
TEMPO (2 × 14 mg in 0.29 mL toluene) and 4 equiv
(TMS)3SiH (4 × 11 µL) were added in portions over the next
4 h. After 16 h, the reaction mixture was cooled to 25 °C and
the volatiles were removed in vacuo. Chromatography (SiO2,
1.5 × 12 cm, 20% EtOAc-hexane) provided 12 (18 mg, 85%)
as a gold solid: mp 157-158 °C; 1H NMR (CDCl3, 400 MHz) δ
9.27 (d, J ) 2.0 Hz, 1H), 8.84 (d, J ) 2.0 Hz, 1H), 8.15 (br s,
1H), 7.55 (br s, 2H), 7.35 (apparent t, J ) 7.2 Hz, 2H), 7.28
(m, 1H), 5.42 (d, J ) 12.4 Hz, 1H), 5.38 (d, J ) 12.3 Hz, 1H),
4.09 (m, 2H), 3.95 (s, 3H), 3.92 (m, 2H), 3.80 (m, 1H), 1.54 (s,
9H), 1.30 (m, 6H), 1.06 (s, 3H), 1.01 (s, 3H), 0.95 (s, 3H), 0.85
(s, 3H); 13C NMR (CDCl3, 100 MHz) δ 166.0, 154.9, 152.4,
146.4, 142.2, 138.5, 136.3, 134.6, 128.5 (2C), 128.0 (3C), 125.0,
123.3, 117.3, 102.4, 79.1, 70.9, 59.8, 52.8, 52.5, 39.6, 39.5, 37.7,
Meth yl 2-(ter t-Bu tyloxyca r bon yl)-1,2,9,9a -tetr a h yd r o-
cyclop r op a [c]p yr id o[3,2-e]in d ol-4-on e-7-ca r boxyla te (16,
N-BOC-CP yI). A solution of 15 (81 mg, 0.21 mmol, 1.0 equiv)
in CH3CN (6.9 mL) at 25 °C under Ar was treated with DBU
(0.12 mL, 0.83 mmol, 4.0 equiv) and stirred 3 h. Flash
chromatography was applied directly to the reaction mixture

4098 J . Org. Chem., Vol. 65, No. 13, 2000
Boger and Boyce
(SiO2, 2.5 × 8 cm, 2% MeOH-CH2Cl2) and furnished 16 as a
light yellow-white solid (68 mg, 93%, typically 90-99%): mp
255 °C (dec); 1H NMR (CDCl3, 400 MHz) δ 9.28 (d, J ) 1.9
Hz, 1H), 7.84 (d, J ) 1.9 Hz, 1H), 6.98 (br s, 1H), 4.04 (m,
2H), 3.97 (s, 3H), 2.87 (dt, J ) 4.0, 8.0 Hz, 1H), 1.68 (dd, J )
4.8, 7.9 Hz, 1H), 1.55 (m, 1H, buried under 1.53), 1.53 (s, 9H);
13C NMR (CDCl3, 125 MHz) δ 182.9, 165.0, 159.3, 151.3, 150.9,
149.1, 135.8, 131.4, 127.0, 110.0, 83.9, 53.0, 52.8, 33.1, 28.7,
28.1 (3C), 23.4; IR (film) νmax 2974, 1728 cm-1; UV (CH3OH)
λmax 318 (ꢀ ) 9000), 240 (shoulder, ꢀ ) 11 000), 218 (ꢀ )
15 500); FABHRMS (NBA/NaI) m/z 379.1282 (M + Na+,
C19H20N2O5 requires m/z 379.1270).
Resolu tion of N-BOC-CP yI. Samples of racemic 16 were
resolved by semipreparative HPLC chromatography on a
Daicel ChiralCel OD column (10 µm, 2 × 25 cm) using 50%
i-PrOH-hexane eluant (7 mL/min). The enantiomers eluted
with retention times of 27.3 and 37.8 min (R ) 1.43). (+)-(8bR,
9aS)-16: [R]22D +120 (c 0.46, THF). (-)-(8bS,9aR)-16: -118 (c
0.47, THF).
Meth yl 1-(Ch lor om eth yl)-5-h ydr oxy-3-[(5,6,7-tr im eth ox-
yin dol-2-yl)car bon yl]-1,2-dih ydr o-3H-pyr ido[3,2-e]in dole-
8-ca r boxyla te (seco-CP yI-TMI, 24). A solution of 16 (10.0
mg, 28.1 µmol, 1.0 equiv) in 3 M HCl-EtOAc (0.935 mL) was
stirred for 30 min at 25 °C. The solvent was removed by a
stream of N2, and the residual salt was dried under vacuum.
The residue was dissolved in anhydrous DMF (0.300 mL) and
treated with 5,6,7-trimethoxyindole-2-carboxylic acid (19, 10.6
mg, 42.1 µmol, 1.5 equiv) and EDCI (27.0 mg, 140 µmol, 5.0
equiv). After being stirred for 10 h at 25 °C under Ar, the
reaction mixture was concentrated in vacuo and suspended
in H2O. The precipitate was collected by centrifugation and
washed with H2O (4 mL). Flash chromatography (SiO2, 0.7 ×
7 cm, 1-5% MeOH-CHCl3 gradient) afforded 24 (7.7 mg, 52%)
as a light yellow solid: 1H NMR (CDCl3, 400 MHz) δ 9.39 (br
s, 1H), 9.20 (d, J ) 1.7 Hz, 1H), 8.74 (d, J ) 1.7 Hz, 1H), 8.39
(s, 1H), 8.33 (br s, 1H), 7.00 (d, J ) 2.0 Hz, 1H), 6.86 (s, 1H),
4.78 (dd, J ) 2.0, 10.5 Hz, 1H), 4.70 (t, J ) 9.0 Hz, 1H), 4.16
(m, 1H), 4.08 (s, 3H), 4.04 (s, 3H), 3.93 (s, 3H), 3.90 (s, 3H),
3.88 (m, 1H, buried under 3.90 ppm), 3.52 (t, J ) 10.0 Hz,
1H); 13C NMR (DMSO-d6, 100 MHz) δ 165.3, 160.2, 154.3,
149.3, 145.0, 143.9, 140.1, 139.1, 137.4, 133.8, 130.6, 125.6,
123.7, 123.6, 123.2, 116.2, 106.6, 105.4, 98.0, 61.6, 61.2, 61.0,
56.0, 55.3, 52.6, 48.0; IR (film) νmax 3332, 2926, 2838, 1725,
1621 cm-1; FABHRMS (NBA/CsI) m/z 526.1397 (M + H+,
Meth yl 1,2,9,9a -Tetr a h yd r ocyclop r op a [c]p yr id o[3,2-e]-
in d ol-4-on e-7-ca r boxyla te (17, CP yI). A solution of 16 (4.0
mg, 11 µmol, 1.0 equiv) in 3 M HCl-EtOAc (0.37 mL) was
stirred for 30 min at 25 °C. The solvent was removed by a
stream of N2, and the residual salt was dried under vacuum.
The residue was taken up in acetone (0.28 mL) and treated
with K2CO3 (16 mg, 0.11 mmol, 10 equiv). After being stirred
for 24 h at 25 °C, the reaction mixture was filtered through
Celite to provide 17 as a yellow solid film (2.7 mg, 96%): 1H
NMR (acetone-d6, 400 MHz) δ 9.07 (d, J ) 2.0 Hz, 1H), 7.94
(d, J ) 2.0 Hz, 1H), 7.06 (br s, 1H), 5.72 (s, 1H), 3.93 (s, 3H),
3.91 (m, 1H), 3.72 (apparent d, J ) 10.8 Hz, 1H), 3.28 (m, 1H),
1.78 (dd, J ) 4.0, 8.0 Hz, 1H), 1.43 (t, J ) 4.6 Hz, 1H); 13C
NMR (CDCl3, 100 MHz) δ 180.9, 168.9, 165.4, 152.6, 148.7,
135.2, 130.9, 126.3, 98.8, 52.7, 50.7, 32.5, 29.4, 26.1; IR (film)
νmax 3163, 2923, 2853, 1727 cm-1; UV (CH3OH) λmax 360 (ꢀ )
7000), 298 (shoulder, ꢀ ) 5000), 226 (ꢀ ) 15 000); FABHRMS
(NBA/NaI) m/z 257.0927 (M + H+, C14H12N2O3 requires m/z
C
26H24ClN3O7Cl requires m/z 526.1381). (+)-(1S)-24: [R]25D +6
(c 0.33, CHCl3). (-)-(1R)-24: [R]25 -6 (c 0.33, CHCl3).
D
Meth yl 2-[(5,6,7-Tr im eth oxyin d ol-2-yl)ca r bon yl]-1,2,9,-
9a -t et r a h yd r ocyclop r op a [c]p yr id o[3,2-e]in d ol-4-on e-7-
ca r boxyla te (25, CP yI-TMI). A solution of 24 (1.5 mg, 2.8
µmol, 1.0 equiv) in anhydrous DMF (0.10 mL) at 25 °C was
treated with DBU (1.3 µL, 8.6 µmol, 3.0 equiv) and stirred 3
h under Ar. Direct chromatography of the reaction mixture
(0.7 × 3 cm, 5% MeOH-CH2Cl2) furnished 25 as a light yellow
solid (1.2 mg, 86%): 1H NMR (acetone-d6, 400 MHz) δ 10.50
(br s, 1H), 9.15 (d, J ) 1.7 Hz, 1H), 8.12 (d, J ) 1.7 Hz, 1H),
7.18 (d, J ) 2.6 Hz, 1H), 7.14 (s, 1H), 6.94 (s, 1H), 4.69 (dd, J
) 4.7, 9.9 Hz, 1H), 4.58 (d, J ) 10.3 Hz, 1H), 4.00 (s, 3H), 3.96
(s, 3H), 3.86 (s, 3H), 3.85 (s, 3H), 3.43 (m, 1H), 1.98 (dd, J )
5.8, 10.0 Hz, 1H), 1.82 (t, J ) 5.7 Hz, 1H); IR (film) νmax 3342,
2923, 2846, 1727, 1632 cm-1; FABHRMS (NBA/NaI) m/z
490.1629 (M + H+, C26H23N3O7 requires m/z 490.1614). (+)-
257.0926). (+)-(8bR,9aS)-17: [R]22 +41 (c 0.14, CH2Cl2). (-)-
D
(8bS,9aR)-17: -45 (c 0.09, CH2Cl2).
Meth yl 8-(Ben zyloxy)-6-[N-(ter t-bu tyloxyca r bon yl)-
N-(E-3-ch lor o-2-p r op en yl)a m in o]-5-iod oqu in olin e-3-ca r -
boxyla te (18). A solution of 10 (1.2 g, 2.2 mmol, 1.0 equiv) in
anhydrous DMF (20 mL) was cooled to 4 °C in a flamed-dried
round-bottom flask under Ar and was treated with NaH (60%
dispersion in oil, 98 mg, 2.5 mmol, 1.1 equiv). After 30 min,
E-1,3-dichloropropene (0.61 mL, 6.7 mmol, 3.0 equiv) was
added and the reaction mixture was gradually warmed to 25
°C and stirred 12 h. The reaction was poured on H2O and
saturated aqueous NaHCO3. The aqueous layer was extracted
with EtOAc, and the combined organic extract was washed
with saturated aqueous NaHCO3, H2O, and saturated aqueous
NaCl, dried (Na2SO4), and concentrated in vacuo. Chromatog-
raphy (SiO2, 5.5 × 14 cm, 20-30% EtOAc-hexane gradient)
afforded 18 (1.2 g, 85%, typically 84-94%) as a yellow foam
(mixture of amide rotamers in CDCl3). Major rotamer: 1H
NMR (CDCl3, 400 MHz) δ 9.43 (s, 1H), 9.12 (s, 1H), 7.47 (d, J
) 7.4 Hz, 2H), 7.35 (t, J ) 7.2 Hz, 2H), 7.30 (t, J ) 7.3 Hz,
1H), 6.87 (s, 1H), 5.92 (m, 1H), 5.85 (m, 1H), 5.45 (s, 2H), 4.34
(dd, J ) 6.8, 14.9 Hz, 1H), 4.04 (s, 3H), 3.79 (dd, J ) 7.6, 15.0
Hz, 1H), 1.22 (s, 9H); 13C NMR (CDCl3, 125 MHz) δ 165.2,
154.7, 153.2, 149.8, 144.0, 143.7, 141.6, 135.7, 130.2, 128.9
(2C), 128.3 (2C), 128.1, 127.0, 125.4, 122.3, 114.3, 94.8, 81.2,
71.2, 52.8, 48.8, 28.1 (3C); IR (film) νmax 3062, 2971, 1729, 1704
(8bR,9aS)-25: [R]25 +42 (c 0.085, CH2Cl2). (-)-(8bS,9aR)-25:
D
[R]25 -44 (c 0.045, CH2Cl2).
D
Meth yl 1-(Ch lor om eth yl)-5-h yd r oxy-3-[(5-m eth oxyin -
d ol-2-yl)ca r bon yl]-1,2-d ih yd r o-3H-p yr id o[3,2-e]in d ole-8-
ca r boxyla te (26). Flash chromatography (SiO2, 0.7 × 6 cm,
1-5% MeOH-CHCl3 gradient) afforded 26 (59%) as a yellow
solid: 1H NMR (CDCl3, 400 MHz) δ 9.30 (br s, 1H), 9.21 (d, J
) 1.7 Hz, 1H), 8.74 (d, J ) 1.8 Hz, 1H), 8.39 (s, 1H), 8.27 (br
s, 1H), 7.36 (d, J ) 8.8 Hz, 1H), 7.12 (d, J ) 2.2 Hz, 1H), 7.03
(m, 2H), 4.81 (dd, J ) 2.0, 10.8 Hz, 1H), 4.72 (t, J ) 8.8 Hz,
1H), 4.17 (m, 1H), 4.04 (s, 3H), 3.89 (m, 1H, buried under 3.86
ppm), 3.86 (s, 3H), 3.53 (t, J ) 10.3 Hz, 1H); IR (film) νmax
3340, 2924, 2857, 1718, 1603 cm-1; FABHRMS (NBA/NaI) m/z
466.1186 (M + H+, C24H20ClN3O5 requires m/z 466.1170). (+)-
(1S)-26: [R]25D +13 (c 0.16, CHCl3). (-)-(1R)-26: [R]25D -12 (c
0.27, CHCl3).
Meth yl 2-[(5-Meth oxyin d ol-2-yl)ca r bon yl]-1,2,9,9a -tet-
r a h yd r ocyclop r op a [c]p yr id o[3,2-e]in d ol-4-on e-7-ca r box-
yla te (27). Flash chromatography (0.7 × 3 cm, 5% MeOH-
CH2Cl2) furnished 27 as a light yellow solid (90%): 1H NMR
(acetone-d6, 400 MHz) δ 10.88 (br s, 1H), 9.15 (d, J ) 1.8 Hz,
1H), 8.13 (d, J ) 1.8 Hz, 1H), 7.48 (d, J ) 9.2 Hz, 1H), 7.24 (s,
1H), 7.21 (s, 1H), 7.14 (d, J ) 2.2 Hz, 1H), 6.97 (dd, J ) 2.4,
9.2 Hz, 1H), 4.74 (dd, J ) 4.9, 10.0 Hz, 1H), 4.65 (d, J ) 10.1
Hz, 1H), 3.95 (s, 3H), 3.81 (s, 3H), 3.46 (m, 1H), 1.98 (dd, J )
4.6, 7.9 Hz, 1H), 1.85 (t, J ) 4.6 Hz, 1H); IR (film) νmax 3205,
2951, 1719, 1624 cm-1; FABHRMS (NBA/NaI) m/z 452.1209
(M + Na+, C24H19N3O5 requires m/z 452.1222). (+)-(8bR,9aS)-
27: [R]25 +49 (c 0.07, CH2Cl2). (-)-(8bS,9aR)-27: [R]25 -44
cm-1; FABHRMS (NBA/CsI) m/z 740.9646 (M + Cs+, C26H26
ClN2O5I requires m/z 740.9629).
-
Meth yl 5-(Ben zyloxy)-3-(ter t-bu tyloxycar bon yl)-1-(ch lo-
r om eth yl)-1,2-d ih yd r o-3H-p yr id o[3,2-e]in d ole-8-ca r boxy-
la te (14). Meth od B. A solution of 18 (0.65 g, 1.1 mmol, 1.0
equiv) in benzene (20 mL) under Ar was treated with Bu3SnH
(0.15 mL, 0.50 mmol, 0.5 equiv) and catalytic AIBN (18 mg)
and stirred at 70 °C. Additional Bu3SnH (0.29 mL, 1.1 mmol,
1.0 equiv in 2 portions) was added over the next 1 h. After 3
h, the reaction mixture was concentrated in vacuo. Chroma-
tography (SiO2, 4 × 20 cm, 20-30% EtOAc-hexane gradient)
provided 14 (0.46 g, 87%).
D
D
(c 0.055, CH2Cl2).
Meth yl 1-(Ch lor om eth yl)-5-h yd r oxy-3-[(in d ol-2-yl)ca r -
b on yl]-1,2-d ih yd r o-3H -p yr id o[3,2-e]in d ole -8-ca r b oxy-
la te (seco-CP yI-in d ole, 28). Flash chromatography (SiO2,

Metal Cation Activation of a DNA Alkylating Agent
J . Org. Chem., Vol. 65, No. 13, 2000 4099
0.7 × 7 cm, 1-5% MeOH-CHCl3 gradient) afforded 28 (71%)
as a yellow solid: 1H NMR (CDCl3, 400 MHz) δ 9.38 (br s,
1H), 9.21 (d, J ) 1.6 Hz, 1H), 8.75 (d, J ) 1.7 Hz, 1H), 8.41 (s,
1H), 8.30 (br s, 1H), 7.73 (d, J ) 8.2 Hz, 1H), 7.47 (d, J ) 8.4
Hz, 1H), 7.35 (t, J ) 7.5 Hz, 1H), 7.18 (t, J ) 7.2 Hz, 1H), 7.13
(s, 1H), 4.83 (dd, J ) 2.0, 11.0 Hz, 1H), 4.75 (t, J ) 8.7 Hz,
1H), 4.18 (m, 1H), 4.04 (s, 3H), 3.89 (dd, J ) 3.5, 11.1 Hz, 1H),
3.55 (t, J ) 10.3 Hz, 1H); IR (film) νmax 3331, 2923, 2853, 1723,
1614 cm-1; FABHRMS (NBA/NaI) m/z 436.1052 (M + H+,
C
23H18ClN3O4I requires m/z 436.1064). (+)-(1S)-28: [R]25 +3
D
(c 0.35, CHCl3). (-)-(1R)-28: [R]25 -3 (c 0.29, CHCl3).
D
Meth yl 2-[(In d ol-2-yl)ca r bon yl]-1,2,9,9a -tetr a h yd r ocy-
clopr opa[c]pyr ido[3,2-e]in dol-4-on e-7-car boxylate (CP yI-
In d ole, 29). Flash chromatography (0.7 × 3 cm, 5% MeOH-
CH2Cl2) furnished 29 as a light yellow solid (93%): 1H NMR
(acetone-d6, 400 MHz) δ 10.98 (br s, 1H), 9.15 (d, J ) 1.6 Hz,
1H), 8.13 (d, J ) 2.0 Hz, 1H), 7.71 (d, J ) 8.0 Hz, 1H), 7.58 (d,
J ) 8.4 Hz, 1H), 7.31 (m, 2H), 7.26 (s, 1H), 7.12 (t, J ) 8.0 Hz,
1H), 4.77 (dd, J ) 4.8, 10.0 Hz, 1H), 4.68 (d, J ) 10.0 Hz, 1H),
3.96 (s, 3H), 3.47 (m, 1H), 1.99 (dd, J ) 4.8, 8.0 Hz, 1H), 1.85
(t, J ) 4.8 Hz, 1H); IR (film) νmax 3217, 2922, 2847, 1728, 1637
cm-1; FABHRMS (NBA/NaI) m/z 400.1310 (M + H+, C23H17N3O4
requires m/z 400.1297). (+)-(8bR, 9aS)-29: [R]25D +48 (c 0.065,
CH2Cl2). (-)-(8bS, 9aR)-29: [R]25 -43 (c 0.065, CH2Cl2).
D
Meth yl 1-(Ch lor om eth yl)-5-h yd r oxy-3-{[5-[N-(in d ol-2-
yl)ca r bon yl]a m in oin d ol-2-yl]ca r bon yl}-1,2-d ih yd r o-3H-
pyr ido[3,2-e]in dole-8-car boxylate (seco-CP yI-In dole2, 30).
Flash chromatography (SiO2, 0.7 × 6 cm, 10% DMF-CHCl3)
afforded 30 (64%) as a yellow solid: 1H NMR (DMSO-d6, 500
MHz) δ 11.82 (s, 1H), 11.73 (s, 1H), 10.45 (s, 1H), 10.20 (s,
1H), 9.14 (s, 1H), 8.89 (s, 1H), 8.23 (m, 2H), 7.67 (d, J ) 7.8
Hz, 1H), 7.59 (d, J ) 8.9 Hz, 1H), 7.47 (m, 2H), 7.42 (s, 1H),
7.28 (s, 1H), 7.21 (t, J ) 7.2 Hz, 1H), 7.06 (t, J ) 7.2 Hz, 1H),
4.87 (t, J ) 10.0 Hz, 1H), 4.62 (m, 1H), 4.46 (m, 1H), 4.02 (dd,
J ) 2.8, 11.1 Hz, 1H), 3.97 (s, 3H), 3.91 (m, 1H); IR (film) νmax
3282, 2921, 2840, 1702, 1620 cm-1; FABHRMS (NBA/CsI) m/z
726.0548 (M + Cs+, C32H24ClN5O5 requires m/z 726.0520). (+)-
(1S)-30: [R]25 +26 (c 0.105, DMF). (-)-(1R)-30: [R]25 -28 (c
D
D
0.06, DMF).
Meth yl2-{[5-[N-(In d ol-2-yl)ca r bon yl]a m in oin d ol-2-yl]-
ca r bon yl}-1,2,9,9a -tetr a h yd r ocyclop r op a [c]p yr id o[3,2-e]-
in d ol-4-on e-7-ca r boxyla te (CP yI-In d ole2, 31). Flash chro-
matography (SiO2, 0.7 × 2 cm, 10% DMF-CH2Cl2) afforded
31 (97%) as a yellow solid: 1H NMR (DMF-d7, 600 MHz) δ
11.85 (s, 1H), 11.75 (s, 1H), 10.29 (s, 1H), 9.19 (d, J ) 2.0 Hz,
1H), 8.41 (s, 1H), 8.29 (d, J ) 2.0 Hz, 1H), 7.75 (dd, J ) 2.0,
8.7 Hz, 1H), 7.70 (d, J ) 8.2 Hz, 1H), 7.60 (m, 2H), 7.53 (s,
1H), 7.38 (s, 1H), 7.26 (t, J ) 7.7 Hz, 1H), 7.22 (s, 1H), 7.10 (t,
J ) 7.7 Hz, 1H), 4.81 (dd, J ) 4.6, 10.2 Hz, 1H), 4.68 (d, J )
10.2 Hz, 1H), 3.99 (s, 3H), 3.60 (m, 1H), 2.08 (dd, J ) 4.6, 7.7
Hz, 1H), 1.93 (t, J ) 5.1 Hz, 1H); IR (film) νmax 3275, 2921,
2851, 1718, 1636 cm-1; MALDIHRMS (DHB) m/z 558.1757 (M
F igu r e 10.
J ) 8.5 Hz, 1H), 7.24 (s, 1H), 7.20 (s, 1H), 6.15 (br s, 2H), 4.79
(dd, J ) 4.7, 9.8 Hz, 1H), 4.68 (d, J ) 10.3 Hz, 1H), 4.15 (t, J
) 8.6 Hz, 2H), 3.98 (s, 3H), 3.59 (m, 1H), 3.39 (m, 2H, obscured
by H2O), 2.08 (dd, J ) 4.7, 8.2 Hz, 1H), 1.92 (t, J ) 5.1 Hz,
1H); IR (film) νmax 3338, 2917, 2848, 1718, 1653 cm-1
;
MALDIHRMS (DHB) m/z 484.1613 (M + H+, C26H21N5O5
requires m/z 484.1621). (+)-(1S)-33: [R]25D +50 (c 0.03, DMF).
+ H+, C32H23ClN5O5 requires m/z 558.1778). (+)-(1S)-31: [R]25
D
+50 (c 0.04, DMF). (-)-(1R)-31: [R]25 -46 (c 0.05, DMF).
(-)-(1R)-33: [R]25 -53 (c 0.03, DMF).
D
D
Meth yl 3-[(3-Ca r ba m oyl-1,2-d ih yd r o-3H-p yr r olo[3,2-e]-
in d ol-7-yl)ca r bon yl]-1-(ch lor om eth yl)-5-h yd r oxy-1,2-d i-
h yd r o-3H-p yr id o[3,2-e]in d ole-8-ca r boxyla te (seco-CP yI-
CDP I1, 32). Flash chromatography (SiO2, 0.7 × 7 cm, 5%
MeOH-10% DMF-CHCl3) afforded 32 (41%) as a yellow
solid: 1H NMR (DMSO-d6, 500 MHz) δ 11.70 (s, 1H), 10.45 (s,
1H), 9.13 (d, J ) 2.0 Hz, 1H), 8.88 (d, J ) 2.0 Hz, 1H), 8.20 (s,
1H), 8.01 (d, J ) 8.8 Hz, 1H), 7.23 (d, J ) 8.1 Hz, 1H), 7.07 (s,
1H), 6.13 (s, 2H), 4.84 (t, J ) 11.0, 1H), 4.58 (m, 1H), 4.45 (m,
1H), 4.00-3.91 (m, 4H, obscured by 3.97 ppm), 3.97 (s, 3H),
3.26 (m, 2H, obscured by H2O); IR (film) νmax 3344, 2923, 2841,
1715, 1660 cm-1; MALDIHRMS (DHB) m/z 520.1383 (M + H+,
Aqu eou s Solvolysis of N-BOC-CP yI a n d CP yI (p H 2
a n d p H 3, P h osp h a te Bu ffer ). Samples of 16 (0.15 mg) and
17 (0.05 mg) were dissolved in CH3OH (1.5 mL) and mixed
with pH 3.0 buffer (1.5 mL, 4:1:20 (v:v:v) 0.1 M citric acid, 0.2
M Na2HPO4, and H2O, respectively). Similarly, samples of 16
(0.1 mg) and 17 (0.05 mg) were dissolved in CH3OH (1.5 mL)
and mixed with pH 2.0 buffer (1.5 mL, 4:1:20 (v:v:v) 1.0 M
citric acid, 0.2 M Na2HPO4, and H2O, respectively). After
mixing of the reagents, the UV spectra of the solution were
measured against a reference solution containing CH3OH (1.5
mL), and the appropriate aqueous buffer (1.5 mL) and these
readings were used for the initial absorbance values (Ai). The
UV spectrum was measured at regular intervals for 30 d (16
at pH 3), 14 d (16 at pH 2), 40 d (17 at pH 3), and 8 d (17 at
pH 2). For 16, the decrease in the long-wavelength absorption
at 315 nm and increase in the short-wavelength absorption
at 278 nm were monitored. The solvolysis rate constant and
half-life (pH 3, k ) 3.81 × 10-6 s-1, t1/2 ) 51 h, r ) 0.99; pH 2,
k ) 3.72 × 10-5 s-1, t1/2 ) 5.2 h, r ) 0.99) were calculated from
the least-squares treatment of the slope of the plot of time
versus ln[(Af - Ai)/(Af - A)] (Figure 10). For 17, the decrease
C
26H22ClN5O5 requires m/z 520.1388). (+)-(1S)-32: [R]25D +23
(c 0.04, DMF). (-)-(1R)-32: [R]25 -20 (c 0.06, DMF).
D
Meth yl 2-[(3-Ca r ba m oyl-1,2-d ih yd r o-3H-p yr r olo[3,2-e]-
in d ol-7-yl)ca r bon yl]-1,2,9,9a -tetr a h yd r ocyclop r op a [c]p y-
r id o[3,2-e]in d ol-4-on e-7-ca r boxyla te (CP yI-CDP I1, 33).
Flash chromatography (SiO2, 0.7 × 4 cm, 5% MeOH-10%
DMF-CH2Cl2) afforded 33 (67%) as a yellow solid: 1H NMR
(DMF-d7, 500 MHz) δ 11.73 (br s, 1H), 9.19 (d, J ) 1.8 Hz,
1H), 8.28 (d, J ) 1.7 Hz, 1H), 8.20 (d, J ) 9.0 Hz, 1H), 7.37 (d,

4100 J . Org. Chem., Vol. 65, No. 13, 2000
Boger and Boyce
in the long-wavelength absorption at 362 nm and increase in
the short-wavelength absorption at 290 nm were monitored.
The solvolysis rate constant and half-life were calculated by
the same treatment providing k ) 6.27 × 10-7 s-1 (t1/2 ) 310
h, r ) 0.99) for pH 3 and k ) 4.63 × 10-6 s-1 (t1/2 ) 42 h, r )
0.99) for pH 2.
calculated from the least-squares treatment of the slope of the
plot of time versus ln[(Af - Ai)/(Af - A)].
Lew is Acid -Ca ta lyzed Ad d ition of CH3OH to N-BOC-
CP yI. A solution of 16 (1.2 mg, 3.4 µmol, 1.0 equiv) in
CH3OH (0.14 mL) was treated with Zn(OTf)2 (1.4 mg, 3.9 µmol,
1.1 equiv) at 25 °C. After 4 h, the solvent was removed with a
stream of N2. Flash chromatography (SiO2, 0.7 × 7 cm, 20-
50% EtOAc-CHCl3 gradient) afforded 41 (1.2 mg, 92%).
Meta l-Ca ta lyzed Solvolysis of N-BOC-CP yI. Samples of
16 (0.025 mg) were dissolved in CH3OH (1.9 mL), and the
resulting solutions were treated with 125 µL (1.0 equiv) or
25 µL (0.2 equiv) of a 0.56 mM solution (CH3OH) of the
desired metal (Cu(acac)2, Mg(acac)2, Ni(acac)2, Zn(acac)2,
Mn(acac)2, Mg(acac)2, Fe(acac)3, Cr(acac)3, Zn(OTf)2, Ti(Oi-Pr)4,
or Cu(OMe)2). The solvolysis solution was sealed and kept at
25 °C protected from light. After mixing of the reagents, the
UV spectra of the solution were measured against reference
solutions, and these readings were used for the initial absor-
bance values (Ai). The UV spectrum was measured at regular
intervals until no further change in absorbance was observed
(Af). The decrease in the long-wavelength absorption at 330
nm and increase in the short-wavelength absorption at 270
nm were monitored. The solvolysis rate constants and half-
lives were calculated from the least-squares treatment of the
slope of the plot of time versus ln[(Af - Ai)/(Af - A)].
Acid -Ca ta lyzed Ad d ition of CH3OH to N-BOC-CP yI:
Met h yl 3-(ter t-Bu t yloxyca r b on yl)-5-h yd r oxy-1-(m et h -
oxym eth yl)-1,2-d ih yd r o-3H-p yr id o[3,2-e]in d ole-8-ca r box-
yla te (41). A solution of 16 (3.0 mg, 8.4 µmol, 1.0 equiv) in
CH3OH (0.43 mL) was treated with CF3SO3H (0.13 mL, 2.5
µmol, 0.3 equiv) at 25 °C. After 20 h, the reaction was quenched
by the addition of NaHCO3 (9 mg), filtered through Celite, and
concentrated in vacuo. Chromatography (SiO2, 0.7 × 7 cm,
5-50% EtOAc-CHCl3 gradient) afforded 41 (3.0 mg, 91%) as
a yellow solid: 1H NMR (CDCl3, 400 MHz) δ 9.10 (d, J ) 1.9
Hz, 1H), 8.80 (s, 1H), 8.28 (br s, 1H), 8.07 (br s, 1H), 4.09 (m,
2H), 4.00 (s, 3H), 3.86 (m, 1H), 3.61 (dd, J ) 5.3, 9.0 Hz, 1H),
3.37 (m, 1H), 3.36 (s, 3H), 1.58 (s, 9H); IR (film) νmax 3386,
2979, 2926, 1721, 1708, 1624 cm-1; FABHRMS (NBA/NaI) m/z
389.1706 (M + H+, C20H24N2O6 requires m/z 389.1713).
Ad d ition of HCl to N-BOC-CP yI. A solution of 16 (2.3
mg, 6.5 µmol, 1.0 equiv) in THF (0.15 mL) was cooled to -78
°C and treated with 4 M HCl-EtOAc (3.0 µL, 11 µmol, 1.7
equiv). The mixture was stirred for 2 min before the solvent
was removed in vacuo. Chromatography (SiO2, 0.7 × 7 cm,
10% EtOAc-CHCl3) afforded 15 (2.4 mg, 96%).
Aqu eou s Solvolysis of N-BOC-CP yI (p H 2-11, Un iver -
sa l Bu ffer ). Samples of 16 (0.025 mg) were dissolved in CH3-
OH (1.0 mL), and the resulting solutions were mixed with a
universal aqueous buffer28 (pH 2-11, 1.0 mL, B(OH)3-citric
acid-Na3PO4). After mixing of the reagents, the UV spectra
of the solution were measured against reference solutions and
these readings were used for the initial absorbance values (Ai).
The UV spectrum was measured at regular intervals (pH 2-4,
every 1 h for 1 d and then every 24 h; pH 4-10, every 24 h)
until no further change in absorbance was observed (Af). The
decrease in the long-wavelength absorption at 320 nm and
increase in the short-wavelength absorption at 278 nm were
monitored. The solvolysis rate constants and half-lives were
Ack n ow led gm en t. We gratefully acknowledge the
financial support of the National Institutes of Health
(Grant CA41986), the Skaggs Institute for Chemical
Biology, and the award of an ACS Medicinal Division
fellowship sponsored by Bristol-Myers Squibb (1998-
1999, C.W.B.). We thank Michael Hedrick and Qing J in
for conducting the cytotoxicity studies. Cover art pre-
pared by Scott Wolkenberg and J iyong Hong.
Su p p or tin g In for m a tion Ava ila ble: 1H NMR spectra for
5-18, 24-33, and 41. This material is available free of charge
via the Internet at http://pubs.acs.org.
J O000177B