Synthesis and Evaluation of Duocarmycin and CC-1065 Analogues Incorporating the 1,2,9,9a-Tetrahydrocyclopropa[c]benz[e]-3-azaindol-4-one (CBA) Alkylation Subunit

Article abstract of DOI:10.1021/jo035119f

An efficient eight-step synthesis (53% overall) and the evaluation of 1,2,9,9a-tetrahydrocyclopropa-[c]benz[e]-3-azaindol-4-one (CBA) and its derivatives containing an aza variant of the CC-1065/ duocarmycin alkylation subunit are detailed. This unique de

Full text of DOI:10.1021/jo035119f

Syn th esis a n d Eva lu a tion of Du oca r m ycin a n d CC-1065 An a logu es
In cor p or a tin g th e
1,2,9,9a -Tetr a h yd r ocyclop r op a [c]ben z[e]-3-a za in d ol-4-on e (CBA)
Alk yla tion Su bu n it
J ay P. Parrish, David B. Kastrinsky, Inkyu Hwang, and Dale L. Boger*
Department of Chemistry and the Skaggs Institute for Chemical Biology, The Scripps Research Institute,
10550 North Torrey Pines Road, La J olla, California 92037
boger@scripps.edu
Received J uly 30, 2003
An efficient eight-step synthesis (53% overall) and the evaluation of 1,2,9,9a-tetrahydrocyclopropa-
[c]benz[e]-3-azaindol-4-one (CBA) and its derivatives containing an aza variant of the CC-1065/
duocarmycin alkylation subunit are detailed. This unique deep-seated aza modification provided
an unprecedented 2-aza-4,4-spirocyclopropacyclohexadienone that was characterized chemically
and structurally (X-ray). CBA proved structurally identical with CBI, the carbon analogue, including
the stereoelectronic alignment of the key cyclopropane, its bond lengths, and the bond length of
the diagnostic C3a-N2 bond, reflecting the extent of vinylogous amide (amidine) conjugation.
Despite these structural similarities, CBA and its derivatives were found to be much more reactive
toward solvolysis and hydrolysis, much less effective DNA alkylating agents (1000-fold), and
biologically much less potent (100- to 1000-fold) than the corresponding CBI derivatives.
In tr od u ction
CC-1065 (1)1 and the duocarmycins (2 and 3)2,3 are the
parent members of a class of potent antitumor antibiot-
ics4 that derive their properties through a sequence-
selective alkylation of duplex DNA (Figure 1).5,6 Exten-
sive studies have characterized their structural features
responsible for the DNA alkylation reaction and have
established fundamental relationships between their
structure and reactivity or activity.5-10 Aside from the
structural complexity inherent in the alkylation subunit,
they possess a stability that defies intuition. This is due
principally to the vinylogous amide conjugation with and
stabilization of the cyclohexadienone structure, which is
dominant over that activating the cross-conjugated
cyclopropane.11-13 Accordingly, disruption of this vinylo-
* Corresponding author.
(1) Chidester, C. G.; Krueger, W. C.; Mizsak, S. A.; Duchamp, D. J .;
Martin, D. G. J . Am. Chem. Soc. 1981, 103, 7629.
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K.; Kawamoto, I.; Tomita, F.; Nakano, H. J . Antibiot. 1988, 41, 1915.
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moto, I.; Yasuzawa, T.; Takahashi, I.; Nakano, H. J . Antibiot. 1990,
43, 1037.
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T.; Takahashi, K.; Sano, H.; Saitoh, Y. Chem. Pharm. Bull. 1995, 43,
378.
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35, 1438. For earlier reviews, see: Boger, D. L. Acc. Chem. Res. 1995,
28, 20. Boger, D. L.; J ohnson, D. S. Proc. Natl. Acad. Sci., U.S.A. 1995,
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F IGURE 1. Structures of CC-1065 and the duocarmycins.
gous amide conjugation leads to remarkable increases in
reactivity as large as 104-fold13 that we have suggested
is the source of catalysis for the DNA alkylation
reaction.7-11
(11) Boger, D. L.; Bollinger, B.; Hertzog, D. L.; J ohnson, D. S.; Cai,
H.; Mesini, P.; Garbaccio, R. M.; J in, Q.; Kitos, P. A. J . Am. Chem.
Soc. 1997, 119, 4987.
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(8) Boger, D. L.; Garbaccio, R. M. Acc. Chem. Res. 1999, 32, 1043.
(9) Wolkenberg, S. E.; Boger, D. L. Chem. Rev. 2002, 102, 2477.
(10) Ambroise, Y.; Boger, D. L. Bioorg. Med. Chem. Lett. 2002, 12,
303.
(12) 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.
(13) Boger, D. L.; Turnbull, P. J . Org. Chem. 1997, 62, 5849. Boger,
D. L.; Turnbull, P. J . Org. Chem. 1998, 63, 8004. CSD NILJ UN for
N-CO2Me-CBI.
10.1021/jo035119f CCC: $25.00 © 2003 American Chemical Society
Published on Web 10/24/2003
8984
J . Org. Chem. 2003, 68, 8984-8990

CBA and Its Derivatives
The synthesis of analogues containing deep-seated
structural changes have been central to these studies
providing insights not accessible through examination of
the natural products themselves.14 Among those intro-
duced, the 1,2,9,9a-tetrahydrocyclopropa[c]benz[e]indol-
4-one (CBI) alkylation subunit has emerged as the most
extensively examined series, Figure 2.15-59 Not only is it
(14) Review: Boger, D. L.; Boyce, C. W.; Garbaccio, R. M.; Goldberg,
J . Chem. Rev. 1997, 97, 787.
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(16) For an alternative source of catalysis, see: Ellis, D. A.;
Wolkenberg, S. E.; Boger, D. L. J . Am. Chem. Soc. 2001, 123, 9299.
Boger, D. L.; Wolkenberg, S. E.; Boyce, C. W. J . Am. Chem. Soc. 2000,
122, 6325. Boger, D. L. Boyce, C. W. J . Org. Chem. 2000, 65, 4088.
For an alternative source of activation, see: Boger, D. L.; Garbaccio,
R. M. J . Org. Chem. 1999, 64, 8350.
F IGURE 2. Structures of CBI and CBA.
the most synthetically accessible alkylation subunit in a
rich series, but its derivatives exhibit biological properties
that surpass those of 1 and 2 while approaching those of
3, and it exhibits a stability and inherent reaction
regioselectivity that are near optimal.15
Characteristic of this class of compounds, the cyclo-
propane precursors such as 5 not only stand up to
prolonged storage more effectively than 4 but readily
close in vitro and in vivo, displaying biological properties
that are not distinguishable from the final cyclopropane-
containing compounds. In a continued examination of
modified alkylation subunits, we targeted the pyridone
7 as an especially storage-stable precursor to the unique
1,2,9,9a-tetrahydrocyclopropa[c]benz[e]-3-azaindol-4-
one (CBA, 6) alkylation subunit. Although 7 was antici-
pated to be more stable than the CBI derivative 5, the
properties of 6, incorporating an unprecedented 2-aza-
4,4-spirocyclopropacyclohexadienone, were unknown.
Herein, we provide full details60 of the synthesis and
X-ray structural characterization of this system and an
examination of its chemical and biological properties.
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Garbaccio, R. M. J . Org. Chem. 2001, 66, 5163. Iso-CBI: Boger, D. L.;
Garbaccio, R. M.; J in, Q. J . Org. Chem. 1997, 62, 8875. C2BI: Boger,
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L.; J ohnson, D. L.; Palanki, M. S. S.; Kitos, P. A.; Chang, J .; Dowell,
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Parrish et al.
SCHEME 1
F IGURE 3. X-ray structure of 16.
Likewise, N-Boc deprotection of 15 and subsequent
coupling of the amine hydrochloride salt with 5,6,7-
trimethoxyindole-2-carboxylic acid65 (TMI) provided 18
(50%), which was spirocyclized to provide 19 using DBU
(71%).
Resu lts a n d Discu ssion
The assignment of the absolute configuration is con-
fidently, but not unambiguously, assigned on the basis
of the more potent biological activity of the assigned
natural enantiomers, the distinguishable and readily
assignable DNA alkylation selectivities of the natural and
unnatural enantiomers, and the characteristic strong
dextrorotatory [R]D of the natural enantiomers of the final
cyclopropane-containing agents.
X-r a y Str u ctu r e of N-Boc-CBA (16). With crystalline
16 in hand, its single-crystal X-ray structure determi-
nation65 was conducted on needles obtained by recrys-
tallization from 9:1 hexanes-CH2Cl2. Not only are its
overall structural characteristics identical to those of
N-CO2Me-CBI,13 including the stereoelectronic alignment
of the key cyclopropane (Figure 3), but the two structures
are nearly superimposable. Essentially all bond lengths
of N-Boc-CBA were within (0.02 Å of those of N-CO2-
Me-CBI, including not only all three cyclopropane bonds
of which the reacting C8b-C9 bonds are identical (1.544
vs 1.544 Å) but also the C3a-N2 bond (1.383 vs 1.390 Å,
respectively) whose length is diagnostic of the extent of
vinylogous amide conjugation.7,8 The only exceptions are
the slightly more perturbed C3a-N3 versus C3a-C3
Syn th esis. The synthesis of CBA began by treatment
of 8 under conditions disclosed to provide 1-bromo-2-
aminoisoquinoline (9, Scheme 1).61 Nucleophilic introduc-
tion of the C1 benzyloxy group and subsequent Boc
protection of the resulting amine 10 provided 11 (59%, 2
steps). Regioselective C4 iodination of 11 and subsequent
N-alkylation of 12 with 1,3-dichloropropene afforded 13
and set the stage for a key 5-exo-trig aryl radical-alkene
cyclization to form 14.62 Resolution of 14 by chromato-
graphic separation on a semiprep Chiralcel OD column
provided both enantiomers cleanly, which were then
subjected to hydrogenolysis to provide 15 (R enantiomer
not shown).63,64 Spirocyclization of 15 was effected by
treatment with DBU in anhydrous CH3CN to give N-Boc-
CBA (16) in superb yield (95%) in eight steps and 53%
overall yield. Additionally, treatment of 15 with HCl and
subsequent spirocyclization afforded CBA (17, 89%).
(61) J ohnson, F.; Nasutavicus, W. A. J . Org. Chem. 1962, 27, 3953.
(62) 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.
(63) Resolution was performed at this stage as a result of optimum
solubility. Separation of the enantiomers of 15 (R ) 1.17) and 16 (R )
1.26) is also possible.
(65) The atomic coordinates for 16 have been deposited with the
Cambridge Crystallographic Data Centre under deposition number
CDCC 209699.
(64) Boger, D. L.; Yun, W. J . Am. Chem. Soc. 1994, 116, 7996.
8986 J . Org. Chem., Vol. 68, No. 23, 2003

CBA and Its Derivatives
SCHEME 2
SCHEME 3
bond (1.30 vs 1.35 Å) and the adjacent N3-C4 vs C3-
C4 bond (1.40 vs 1.44 Å) reflecting the intrinsically
shorter CdN vs CdC bond lengths.
Notably, the cyclopropane alignment of 16, like that
of CBI, is such that the bent orbitals of the longer C8b-
C9 cyclopropane bond are aligned with the conjugated
π-system, whereas those of the alternative and shorter
C8b-C9a bond are nearly orthogonal. Additionally re-
markable is the near identical relative orientation of the
N2 carbamate. With 16, this places the carbamate
carbonyl proximal to N3, introducing what is likely a
destabilizing electrostatic repulsion between the N3 lone
pair and the carbamate carbonyl lone pair. Despite this
interaction, the ø1 dihedral angle (12°) exhibits no
significant distortion in efforts to relieve the destabilizing
electrostatic interaction, nor does it adopt the alternative
transoid versus cisoid conformation.
The stability of 16 and its unprecedented 2-aza-4,4-
spirocyclopropacyclohexadienone may be attributed to the
geometrical constraints of the fused five-membered ring
that prevents an ideal, bisected conjugation of the cyclo-
propane with the π-system13,40 and, most importantly, the
N2 amidine cross-conjugation.
Rea ction Regioselectivity. Consistent with the ster-
eoelectronic alignment of the cyclopropane, acid-catalyzed
nucleophilic addition proceeded exclusively (>20:1) at the
least substituted cyclopropane carbon with cleavage of
the reactive and aligned C8b-C9 bond (Scheme 2).
Notably, this stereoelectronic control overrides any in-
trinsic preference for ring expansion ring opening with
partial positive charge development on the more substi-
tuted cyclopropane carbon and may benefit from the
preference for SN2 nucleophilic attack to occur at the least
substituted carbon. Thus, treatment of 16 with anhy-
drous HCl resulted in exclusive formation of 15 in 5 min
at -78 °C (99%). Similarly, treatment of 16 with CF3-
SO3H-MeOH (0.02 M CF3SO3H, 0-23 °C, 30 min) led
to exclusive formation of 20 (90%) derived from MeOH
addition to the least substituted cyclopropane carbon.
Significantly, 16 was much more sensitive to base
treatment under protic conditions than CBI. Thus, rapid
hydrolysis (1 N LiOH, 3:1 THF-MeOH) of the carbamate
of 16 to provide CBA (17) is observed at room tempera-
ture (e5 min, 98%) or 0 °C (30 min, >95%). In contrast,
an identical treatment of N-Boc-CBI led to a much slower
hydrolysis at room temperature (6 h) and no reaction at
0 °C, indicating that it is roughly 100-fold less reactive
(Scheme 3). Similarly, CBA-TMI (19) proved very sensi-
tive to protic base, undergoing rapid and quantitative
hydrolysis to CBA (17) upon treatment with LiOH (23
°C, 15 min).
TABLE 1. Solvolysis Rea ctivitya
CBA (17)
CBI13
k, s-1 (pH 3)
t1/2, h (pH 3)
6.44 × 10-5
2.07 × 10-7
3.0
903
a
pH ) 3: 50% CH3OH-buffer, buffer is 4:1:20 (v/v/v) 0.1 M
citric acid, 0.2 M Na2HPO4, and deionized H2O.
ity of 16 and 19 indicates that there is little, if any,
stabilizing conjugation of the N2 lone pair with the Boc
carbonyl. Rather, the N2 lone pair is intimately tied into
the amidine conjugation. Thus, unlike CBI, which prefers
to maintain the linking amide conjugation and relinquish
its vinylogous amide conjugation if perturbed, CBA in
structures such as CBA-TMI (19) prefers to relinquish
the linking amide (an acyl amidine) rather than disrupt
the more intimate amidine conjugation. Notably, this is
analogous to the behavior observed with modifications
in the CBI-TMI linking amide.42
Solvolysis Rea ctivity. The chemical solvolysis of 16
at pH ) 3 (50% CH3OH-buffer, buffer is 4:1:20 (v/v/v)
0.1 M citric acid, 0.2 M Na2HPO4, and deionized H2O)
and pH ) 7 (50% H2O-CH3OH) was not observed and
led instead to the hydrolysis of the N-Boc group, Thus,
complete hydrolysis of the Boc was observed to provide
17 at both pH ) 3 and 7 in 0.25 and 1.3 h, respectively.
In contrast, the apparent solvolysis of CBA (17) was
measurable and was found to be >100-fold more reactive
than CBI (Table 1). This reactivity was assumed, but not
confirmed, to represent cyclopropane solvolysis. Regard-
less of the origin of this increased reactivity, this behavior
was found to correlate with a corresponding loss in
biological potency.
DNA Alk yla tion P r op er ties. A comparison of DNA
alkylation efficiency and selectivity of (+)-CBA-TMI (19)
and (+)-duocarmycin SA (3) was conducted following
protocols previously detailed66 (Figure 4). These compari-
sons revealed that the alkylation selectivity for (+)-19
was indistinguishable from that of (+)-3, and this is
illustrated nicely in Figure 4 where both compounds
exclusively alkylate the same 5′-AATTA-3′ high affinity
site. This selectivity confirmed the tentatively assigned
This latter reactivity proved to be key to understanding
the behavior of the CBA system. This hydrolytic reactiv-
(66) Boger, D. L.; Munk, S. A.; Zarrinmayeh, H.; Ishizaki, T.;
Haught, J .; Bina, M. Tetrahedron 1991, 47, 2661.
J . Org. Chem, Vol. 68, No. 23, 2003 8987

Parrish et al.
potent cytotoxic activity (1000-fold vs (+)-CBI-TMI and
1000- to 10 000-fold vs (+)-3).
Cytotoxic Activity. The in vitro cytotoxic activity of
the CBA-based analogues is summarized in Table 2. In
general, the CBA-based analogues were found to be 100-
to 1000-fold less active than the corresponding CBI-based
agents. For example, N-Boc-CBA (16) was 50-fold less
potent than N-Boc-CBI. Similarly, CBA-TMI (19) was
1000-fold less potent than CBI-TMI. Removing the N2-
acyl group with 17 (IC50 ) 5 µM) did not result in a large
decrease in potency relative to 16, but both 16 and 17
were roughly 100-fold less potent than 19, which is
consistent with past observations. As expected, the
unnatural enantiomers exhibited reduced cytotoxic activ-
ity compared to that of the natural enantiomers and the
seco precursors 15 and 18 exhibited cytotoxic activity
indistinguishable from that of the cyclopropane-contain-
ing final products.
Con clu sion
An efficient eight-step synthesis (53% overall) of CBA
and its derivatives is detailed constituting the prepara-
tion of an unprecedented CC-1065/duocarmycin alkyla-
tion subunit aza analogue. Although an X-ray comparison
of N-Boc-CBA (16) with N-Boc-CBI revealed nearly
identical structures, the solvolysis and hydrolysis reactiv-
ity of CBA was much greater than that of CBI. Presum-
ably this is a consequence of a CBA preference to
relinquish the linking acyl conjugation over that of the
imbedded amidine itself. Characteristic of this behavior,
the CBA derivatives displayed a DNA alkylation rate and
efficiency that was 1000- to 10 000-fold lower than the
corresponding CBI derivatives while exhibiting the same
DNA alkylation sequence selectivity. Similarly, the cy-
totoxic activity of the CBA derivatives was 100- to 1000-
fold less potent than the corresponding CBI-based agents.
F IGURE 4. Thermally induced strand cleavage of w794 DNA
(SV40 DNA segment, 144 bp, nucleotide nos. 138-5238). DNA-
agent incubation at 25 °C for (+)-duocarmycin SA (24 h) and
37 °C for (+)-CBA-TMI (120 h), removal of unbound agent and
30 min thermolysis (100 °C), followed by denaturing 8% PAGE
and autoradiography. Lane 1, control DNA; lane 2, (+)-
duocarmycin SA ((+)-3, 1 × 10-6 M); lanes 3-6, Sanger G, C,
A, and T reactions; lane 7, (+)-CBA-TMI ((+)-19, 1 × 10-3 M).
natural absolute configuration of (+)-19. In contrast to
(+)-3 and (+)-CBI-TMI, the rate and efficiency of DNA
alkylation by (+)-CBA-TMI (19) was low. The detectable
alkylation for 19 was observed only at high agent
concentrations (10-3 vs 10-6 M), at increased reaction
times (120 vs 2-24 h), and at elevated reaction temper-
atures (37 vs 5-23 °C). Potentially, this may be at-
tributed to either competitive hydrolysis of 19 under the
pH 8 incubation conditions or the lack of alkylation
catalysis upon DNA binding. The former may well be
expected on the basis of the enhanced sensitivity of 19
to aqueous base hydrolysis (Scheme 3) and the latter
anticipated to arise from a DNA binding induced confor-
mation change that would preferentially relinquish the
acyl amidine conjugation over that of the amidine itself.
Regardless of the origin of the effect, the less effective
DNA alkylation by (+)-19 correlates well with its less
Exp er im en ta l Section
3-Am in o-1-ben zyloxyisoqu in olin e (10). A solution of
benzyl alcohol (0.70 mL, 6.72 mmol) in DMF (8 mL) cooled to
0 °C was treated with NaH (60% mineral oil dispersion, 0.269
g, 6.72 mmol) in portions and stirred at 0 °C for an additional
0.5 h. A solution of 3-amino-1-bromoisoquinoline61 (0.500 g,
2.24 mmol) in DMF (4 mL) was treated dropwise with this
solution. The deep red mixture was warmed to 25 °C and
stirred for 1.5 h. The reaction was quenched with the addition
of saturated aqueous NaHCO3 (25 mL) and extracted with
Et2O (3 × 50 mL). The combined organic layers were washed
with H2O (3 × 20 mL), dried (Na2SO4), and concentrated in
vacuo. Flash chromatography (SiO2, 10% EtOAc-hexanes)
afforded 3-amino-1-benzyloxyisoquinoline (10, 0.372 g, 66%)
as a tan oil: Rf ) 0.61 (33% EtOAc-hexanes); 1H NMR (CDCl3,
400 MHz) δ 8.15 (d, J ) 8.2 Hz, 1H), 7.55 (d, J ) 7.0 Hz, 2H),
7.49 (m, 2H), 7.43 (t, J ) 7.0 Hz, 2H), 7.36 (t, J ) 7.3 Hz, 1H),
TABLE 2. In Vitr o Cytotoxic Activity
compound
IC50 (nM)a
80
0.03
4000 (4000)
5000
compound
IC50 (nM)a
900
(+)-N-Boc-CBI
(+)-CBI-TMI
(-)-N-Boc-CBI
(-)-CBI-TMI
2
(+)-N-Boc-CBA, 16 (15)
(+)-CBA, 17
(-)-N-Boc-CBA, 16 (15)
(-)-CBA, 17
9000 (10 000)
60000
3000 (5000)
(+)-CBA-TMI, 19 (18)
50 (40)
(-)-CBA-TMI, 19 (18)
a
L1210 cytotoxic activity, average of 2-7 determinations in triplicate.
8988 J . Org. Chem., Vol. 68, No. 23, 2003

CBA and Its Derivatives
7.19 (m, 1H), 6.33 (s, 1H), 5.55 (s, 2H), 4.26 (br s, 2H); 13C
NMR (CDCl3, 100 MHz) δ 160.3, 152.2, 141.3, 137.7, 130.8,
128.6 (2C), 128.0 (2C), 127.9, 124.6, 124.3, 122.4, 114.6, 92.6,
28.5 (3C); IR (film) νmax 3445, 1704, 1568, 1162 cm-1; MALDIFT-
HRMS (DHB) m/z 573.0429 (M + Na+, C24H24ClIN2O3 requires
573.0412).
67.9; IR (film) νmax 3473, 3382, 1633, 1336, 1156 cm-1
;
5-Be n zyloxy-3-N -(ter t -b u t yloxyca r b on yl)-1-(ch lor o-
m eth yl)-1,2-d ih yd r op yr r olo[2,3-c]isoqu in olin e (14). A so-
lution of 13 (0.0623 g, 0.113 mmol) in C6H6 (1.5 mL) was
degassed by three freeze-pump-thaw cycles and treated with
Bu3SnH (37 µL, 0.136 mmol) and AIBN (3.7 mg, 0.0227 mmol).
A stream of Ar was bubbled through the solution for 10 min
before the reaction vessel was closed and warmed at 80 °C for
2 h. The mixture was cooled to 25 °C and concentrated in
vacuo. Flash chromatography (SiO2, 0-10% EtOAc-hexanes
gradient) afforded 14 (0.0461 g, 96%) as a white solid: mp
116-117 °C; Rf ) 0.57 (25% EtOAc-hexanes); 1H NMR
(acetone-d6, 500 MHz) δ 8.25 (d, J ) 8.4 Hz, 1H), 7.58 (m, 4H),
7.43 (app t, J ) 6.6 Hz, 2H), 7.35 (m, 2H), 5.64 (d, J ) 12.5
Hz, 1H), 4.28 (d, J ) 2.2 Hz, 1H), 4.17 (m, 1H), 3.84 (m, 2H),
3.51 (t, J ) 10.2 Hz, 1H), 1.64 (s, 9H); 13C NMR (CD2Cl2, 150
MHz) δ 161.8, 152.1, 137.8, 136.4, 131.9, 129.0, 128.9, 128.8,
128.5 (2C), 128.4 (2C), 126.0, 124.3, 121.7, 116.2, 107.1, 81.3,
68.8, 52.3, 47.3, 39.6, 28.8 (3C), 28.2; IR (film) νmax 3446, 1723,
1682, 1564, 1349, 1154 cm-1; MALDIFT-HRMS (DHB) m/z
447.1449 (M + Na+, C24H25ClN2O3 requires 447.1446).
MALDIFT-HRMS (DHB) m/z 251.1182 (M + H+, C16H14N2O
requires 251.1179).
1-Ben zyloxy-3-[(ter t-bu tyloxyca r bon yl)a m in o]isoqu in -
olin e (11). A solution of 10 (0.372 g, 1.48 mmol) in t-BuOH
(4.2 mL) was treated with Boc2O (0.581 g, 2.66 mmol) and
stirred for 24 h at 25 °C during which time the yellow solution
turned light orange and a precipitate formed. The mixture was
filtered, and the solid was washed with hexanes and collected.
The filtrate was concentrated in vacuo and purified by flash
chromatography (SiO2, 10% EtOAc-hexanes). The precipitate
was determined to be pure product and was combined with
the product isolated by chromatography to afford 11 (0.462 g,
89%) as an orange solid: mp 109-110 °C; Rf ) 0.65 (33%
1
EtOAc-hexanes); H NMR (CDCl3, 400 MHz) δ 8.18 (d, J )
8.0 Hz, 1H), 7.77 (s, 1H), 7.68 (d, J ) 8.2 Hz, 1H), 7.57 (m,
1H), 7.51 (d, J ) 7.0 Hz, 2H), 7.41 (t, J ) 7.6 Hz, 2H), 7.34 (t,
J ) 7.0 Hz, 2H), 7.07 (br s, 1H) 5.51 (s, 2H), 1.57 (s, 9H); 13C
NMR (CDCl3, 100 MHz) δ 159.7, 152.5, 146.9, 143.9, 140.5,
137.4, 131.0, 128.7 (2C), 128.0 (2C), 127.9, 126.3, 124.2, 116.9,
99.1, 85.4, 81.0, 28.5 (3C); IR (film) νmax 2980, 1809, 1631, 1331,
1156 cm-1; MALDIFT-HRMS (DHB) m/z 351.1705 (M + H+,
Resolu tion of 14. The enantiomers of 14 were resolved on
a HPLC semipreparative Daicel Chiralcel OD column (10 µm,
2 × 25 cm) using 2% i-PrOH-hexane eluant (7 mL/min). The
enantiomers eluted with retention times of 14.6 min (natural
enantiomer) and 12.5 min (unnatural enantiomer, R ) 1.17).
(1S)-14: [R]23 -13 (c 0.047, CH2Cl2). (1R)-14: [R]23 +14 (c
C
21H22N2O3 requires 351.1703).
1-Ben zyloxy-3-(ter t-b u t yloxyca r b on yl)a m in o-4-iod o-
isoqu in olin e (12). A solution of 11 (50 mg, 0.14 mmol) in THF
(1.5 mL) at -20 °C was treated with N-iodosuccinimide (38
mg, 0.17 mmol) and p-TsOH (10 mg, 0.053 mmol). The mixture
was stirred at -20 °C for 0.5 h and then warmed to 25 °C for
24 h. The THF was removed by a stream of N2, and the
mixture was diluted with EtOAc (5 mL) and treated with
aqueous saturated Na2SO3 (5 mL). The aqueous layer was
extracted with EtOAc (3 × 25 mL), dried (Na2SO4), and
concentrated in vacuo. Flash chromatography (SiO2, 10%
EtOAc-hexanes) afforded 12 (67 mg, 100%) as a white solid:
mp 93-94 °C; Rf ) 0.48 (20% EtOAc-hexanes); 1H NMR
(CDCl3, 400 MHz) δ 8.19 (d, J ) 8.2 Hz, 1H), 7.93 (d, J ) 8.5
Hz, 1H), 7.68 (t, J ) 8.2 Hz, 1H), 7.58 (d, J ) 7.6 Hz, 2H),
7.46 (t, J ) 7.6 Hz), 7.42 (t, J ) 7.6 Hz, 2H), 7.36 (t, J ) 7.1
Hz, 1H), 7.10 (s, 1H), 5.63 (s, 2H), 1.60 (s, 9H); 13C NMR
(CDCl3, 100 MHz) δ 160.4, 152.0, 144.9, 140.3, 137.1, 132.3,
130.5, 128.7 (2C), 128.6 (2C), 128.2, 126.3, 124.8, 118.6, 81.1,
D
D
0.047, CH2Cl2).
3-N-(ter t-Bu t yloxyca r b on yl)-1-(ch lor om et h yl)-1,2-d i-
h yd r op yr r olo[2,3-c]isoq u in olin -5-on e (15, seco-N-Boc-
CBA). A solution of 14 (22.8 mg, 0.0538 mmol) in THF (0.4
mL) was treated with a catalytic amount of 10% Pd/C (5 mg)
and placed under 1 atm H2. The mixture was stirred for 2 h
at 25 °C, filtered through a Celite plug, and concentrated in
vacuo. The residue was dissolved in a minimal amount of
CHCl3 and then precipitated by the slow addition of hexanes
to afford 15 (18.0 mg, 100%) as a white solid: mp 134-136 °C
1
(dec); Rf ) 0.38 (25% EtOAc-hexanes); H NMR (CDCl3, 500
MHz) δ 10.61 (br s, 1H), 8.35 (d, J ) 8.1 Hz, 1H), 7.62 (t, J )
7.7 Hz, 1H), 7.30 (m, 2H), 4.09 (m, 2H), 3.91 (m, 1H) 3.79 (m,
1H), 3.49 (t, J ) 10.0 Hz, 1H) 1.57 (s, 9H); 13C NMR (CDCl3,
150 MHz) δ 162.0, 152.6, 140.4, 135.8, 133.5, 129.4, 124.3,
122.7, 120.9, 96.3, 83.6, 52.2, 46.4, 40.4, 28.5 (3C); IR (film)
νmax 3323, 1697, 1636, 1410, 1144 cm-1; MALDIFT-HRMS
(DHB) m/z 357.0975 (M + Na+, C17H19ClN2O3 requires
357.0976). (1S)-15: [R]23 -58 (c 0.1, CH2Cl2). (1R)-15: [R]23
77.1, 68.8, 28.6 (3C); IR (film) νmax 3279, 1721, 1574, 1162 cm-1
;
MALDIFT-HRMS (DHB) m/z 499.0483 (M + Na+, C21H21IN2O3
D
D
requires 499.0489).
+56 (c 0.1, CH2Cl2).
3-[N-(ter t-Bu tyloxyca r bon yl)-N-(3-ch lor op r op -2-en -1-
yl)a m in o]-1-ben zyloxy-4-iod oisoqu in olin e (13). A solution
of 12 (0.264 g, 0.554 mmol) in DMF (2.5 mL) cooled to 0 °C
was treated with NaH (60% suspension in mineral oil, 33.2
mg, 0.831 mmol) in portions and stirred for 0.5 h. A catalytic
amount of Bu4NI (10 mg) and 1,3-dichloropropene (0.154 mL,
1.66 mmol) were added sequentially, and the solution was
warmed to 25 °C and stirred for 3 h before being treated with
saturated NaHCO3 (5 mL). The aqueous layer was extracted
with Et2O (3 × 25 mL), and the combined organic layers were
washed with H2O (3 × 20 mL), dried (Na2SO4), and concen-
trated in vacuo. Flash chromatography (SiO2, 10% EtOAc-
hexanes) afforded 13 (0.306 g, 99%) as a tan oil as a mixture
of E- and Z-olefin isomers, amide rotamers: Rf ) 0.65 (10%
EtOAc-hexanes); 1H NMR (CDCl3, 600 MHz) δ 8.28 (br s, 1H),
8.09 (d, J ) 8.5 Hz, 1H), 7.75 (br s, 1H), 7.59 (m, 1H), 7.51 (d,
J ) 7.0 Hz, 2H), 7.41 (t, J ) 7.0 Hz, 2H), 7.35 (t, J ) 7.0 Hz,
1H), 5.91-6.10 (m, 2H), 5.57 (s, 2H), 4.66 and 4.46 (two d, J
) 12.7 and 9.6 Hz, 1H), 4.13-4.33 (two m, 1H), 1.60, 1.39 and
1.37 (three s, 9H); 13C NMR (CDCl3, 150 MHz) δ 140.6, 137.2
and 137.1 (C), 132.4 and 132.3 (C), 131.9 and 131.8 (C), 129.3,
128.7 (2C), 128.6, 128.23 and 128.20 (C), 128.17 (2C), 127.9,
127.8, 124.7, 121.0, 119.8, 81.1, 77.4, 68.6, 48.1 and 45.4 (C),
3-N-(ter t-Bu tyloxyca r bon yl)-1,2,9,9a -tetr a h yd r ocyclo-
p r op a [c]b en z[e]-3-a za in d ol-4-on e (16, N-Boc-CBA). A
sample of 15 (2.5 mg, 7.5 µmol) was dissolved in freshly
distilled CH3CN (100 µL) under Ar. Anhydrous DBU (5.7 µL,
37 µmol) was added at 23 °C, and the mixture was stirred for
1 h. The reaction mixture was then concentrated under a
stream of N2 and applied directly to PTLC (SiO2, 10 × 20 cm,
EtOAc) to afford N-Boc-CBA (16, 2.1 mg, 95%) as a white
solid: mp 157-160 °C (dec); Rf ) 0.38 (EtOAc); 1H NMR
(CDCl3, 600 MHz) δ 8.23 (d, J ) 7.9 Hz, 1H), 7.52 (t, J ) 7.7
Hz, 1H), 7.42 (t, J ) 7.7 Hz, 1H), 6.83 (d, J ) 7.4 Hz, 1H),
4.05 (m, 2H), 2.79 (m, 1H), 1.68 (app dd, J ) 4.9, 2.2 Hz, 1H),
1.62 (app t, J ) 4.9 Hz, 1H), 1.57 (s, 9H); 13C NMR (CDCl3,
150 MHz) δ 176.1, 174.2, 150.2, 138.6, 133.4, 129.9, 129.5,
128.1, 121.1, 84.8, 51.8, 31.4, 28.5 (3C), 23.5, 1.9; IR (film) νmax
2915, 1748, 1601, 1160 cm-1; UV (MeOH) 270 nm (ꢀ 11000),
202 nm (ꢀ 21000); MALDIFT-HRMS (DHB) m/z 321.1211 (M
+ Na+, C17H18N2O3 requires 321.1210). Natural-(8bA,9aS)-
16: [R]23 +30 (c 0.06, CH2Cl2). ent-(8bS,9aR)-16: [R]23 -32
D
D
(c 0.06, CH2Cl2). The sample for X-ray analysis was obtained
by recrystallization from 10% CH2Cl2-hexanes, which pro-
vided colorless needles.
1,2,9,9a -Tetr a h yd r ocyclop r op a [c]ben z[e]-3-a za in d ol-4-
J . Org. Chem, Vol. 68, No. 23, 2003 8989

Parrish et al.
on e (17, CBA). A sample of 15 (3.3 mg, 9.9 µmol) was treated
with 4 N HCl/EtOAc (500 µL) and stirred at 23 °C for 1 h.
The solvent was removed under a steam of N2 and dried under
high vacuum for 30 min. The gray residue was dissolved in
THF (500 µL) and treated with saturated aqueous NaHCO3
(500 µL), and the mixture was stirred for 3 h at 23 °C. The
mixture was extracted with EtOAc (3 × 10 mL), and the
organic layers were combined, dried over Na2SO4, and con-
centrated. The solid was dissolved in THF and purified by
PTLC (SiO2, 10 × 20 cm, 50% EtOAc-EtOH) to afford CBA
(17, 1.7 mg, 89%) as a pale yellow solid: Rf ) 0.15 (50%
EtOAc-EtOH); 1H NMR (acetone-d6, 500 MHz) δ 8.07 (d, J )
8.1 Hz, 1H), 7.53 (t, J ) 7.7 Hz, 1H), 7.37 (t, J ) 7.7 Hz, 1H),
7.04 (d, J ) 7.7 Hz, 1H), 3.86 (dd, J ) 8.1, 5.5 Hz, 1H), 3.72
(d, J ) 13.6 Hz, 1H), 3.10 (m, 1H), 1.62 (dd, J ) 8.5, 4.1 Hz,
1H), 1.28 (t, J ) 3.7 Hz, 1H); IR (film) νmax 2923, 1739, 1595,
1550 cm-1; MALDIFT-HRMS (DHB) m/z 199.0868 (M + H+,
) 9.5 Hz, 1H), 3.72 (m, 2H), 3.45 (app t, J ) 8.0 Hz, 1H), 3.33
(s, 3H), 1.57 (s, 9H); IR (film) νmax 3333, 1692, 1667, 1333 cm-1
;
MALDIFT-HRMS (DHB) m/z 353.1473 (M + Na+, C18H22N2O4
requires 353.1472).
Ad d ition of HCl to N-Boc-CBA. A solution of 16 (1.5 mg,
5.0 µmol) in THF (50 µL) was cooled to -78 °C and treated
with 4 N HCl-EtOAc (50 µL). The mixture was stirred for 2
min before the solvent was removed in vacuo. PTLC (SiO2, 10
× 10 cm, 25% EtOAc-hexanes) afforded 15 (1.7 mg, 99%).
Hyd r olysis of N-Boc-CBA w ith LiOH. A solution of 16
(800 µg, 2.7 µmol) in 3:1:1 (v/v/v) THF-CH3OH-H2O (30 µL)
was treated with LiOH (310 µg, 7.4 µmol). The mixture was
stirred for 5 min before the solvent was removed with a stream
of N2. PTLC (SiO2, 10 × 10 cm, 10% EtOH-EtOAc) of the
residue afforded 17 (520 µg, 98%).
Aqu eou s Solvolysis Rea ctivity, p H 3. Compounds 16 and
17 (50 µg) were dissolved in CH3OH (1.5 mL) and mixed with
pH 3 aqueous buffer (1.5 mL). The buffer contained 4:1:20 (v/
v/v) 0.1 M citric acid, 0.2 M Na2HPO4, and deionized H2O,
respectively. Immediately after mixing, the UV spectra of the
solution was measured against a reference solution containing
CH3OH (1.5 mL) and the aqueous buffer (1.5 mL), and this
reading was used as the initial absorbance value. The solution
was stoppered, protected from light, and allowed to stand at
25 °C. The UV spectra were recorded at regular intervals until
a constant value was obtained for the long-wavelength absor-
bance. The increase of the absorbance at 300 nm was moni-
tored. The solvolysis rate constant for 17 was determined from
the slope of the line obtained from the least-squares treatment
(r2 ) 0.959) of the plot of ln[(Af - Ai)/(Af - A)] versus time; k
) 6.44 × 10-5 s-1, t1/2 ) 3.0 h (Table 1).
C12H10N2O requires 199.0866). Natural-(8bR,9aS)-17: [R]23
D
+165 (c 0.03, CH2Cl2). ent-(8bS,9aR)-17: [R]23 -167 (c 0.03,
D
CH2Cl2).
seco-CBA-TMI (18). A sample of 15 (9.7 mg, 29.0 µmol)
was treated with 4 N HCl/EtOAc (300 µL) and stirred at 23
°C for 1 h. The solvent was removed under a stream of N2 and
dried under high vacuum for 30 min. The gray residue was
dissolved in DMF (300 µL), and 5,6,7-trimethoxyindole-2-
carboxylic acid64 (7.3 mg, 29.0 µmol) and EDCI (16.8 mg, 87.0
µmol) were added. The mixture was stirred under Ar at 23 °C
for 18 h in the absence of light before the solvent was removed
under a stream of N2. Flash chromatography (SiO2, 3 × 17
cm, 10-50% EtOAc-hexanes gradient) afforded seco-CBA-TMI
(18, 6.7 mg, 50%) as a yellow solid: Rf ) 0.51 (EtOAc); 1H
NMR (CDCl3, 500 MHz) δ 9.89 (br s, 1H), 8.44 (d, J ) 8.1 Hz,
1H), 7.67 (t, J ) 8.1 Hz, 1H), 7.40 (m, 2H), 7.03 (d, J ) 2.2
Hz, 1H), 6.84 (br s, 1H), 4.67 (m, 2H), 4.12 (s, 3H), 4.00 (m,
2H), 3.97 (s, 3H), 3.92 (s, 3H), 3.56 (app t, J ) 10.7 Hz, 1H);
13C NMR (CDCl3, 125 MHz) δ 190.9, 164.0, 161.9, 144.7, 142.3,
135.7, 134.1, 131.3, 130.2, 127.2, 125.8, 124.4, 122.1, 108.4,
98.3, 86.5, 62.4, 62.1, 57.1, 55.0, 46.9, 42.3; IR (film) νmax 3238,
1646, 1312 cm-1; MALDIFT-HRMS (DHB) m/z 468.1324 (M
DNA Alk yla tion Stu d ies. Selectivity a n d Efficien cy.
General procedures, the preparation of singly 32P 5′ end-labeled
double-stranded DNA, the compound binding studies, gel
electrophoresis, and autoradiography were conducted accord-
ing to procedures described elsewhere.66 Eppendorf tubes
containing the 5′ end-labeled DNA (w794, 9 µL) in TE buffer
(10 mM Tris, 1 mM EDTA, pH 7.5) were treated with the agent
in DMSO (1 µL at the specified concentration). The solution
was mixed by vortexing and brief centrifugation and subse-
quently incubated at 4-37 °C for 24-120 h. The covalently
modified DNA was separated from unbound agent by EtOH
precipitation and resuspended in TE buffer (10 µL). The
solution of DNA in an Eppendorf tube sealed with parafilm
was warmed to 100 °C for 30 min to induce cleavage at the
alkylation sites, allowed to cool to 25 °C, and centrifuged.
Formamide dye (0.03% xylene cyanol FF, 0.03% bromophenol
blue, 8.7% Na2EDTA 250 mM) was added (5 µL) to the
supernatant. Prior to electrophoresis, the sample was dena-
tured by warming at 100 °C for 5 min, placed in an ice bath,
and centrifuged, and the supernatant was loaded directly onto
the gel. Sanger dideoxynucleotide sequencing reactions were
run as standards adjacent to the reaction samples. Polyacry-
lamide gel electrophoresis (PAGE) was run on an 8% sequenc-
ing gel under denaturing conditions (8 M urea) in TBE buffer
(100 mM Tris base, 100 mM boric acid, 0.2 mM Na2EDTA)
followed by autoradiography.
+ H+, C24H22ClN3O5 requires 468.1321). (1S)-18: [R]23 -14
D
(c 0.09, CH2Cl2). (1R)-18: [R]23 +16 (c 0.09, CH2Cl2).
D
CBA-TMI (19). A sample of 18 (2.1 mg, 4.5 µmol) was
dissolved in freshly distilled CH3CN (50 µL) under Ar. Anhy-
drous DBU (3.4 µL, 22.4 µmol) was added at 23 °C, and the
mixture was stirred for 1 h. The reaction mixture was then
concentrated under a stream of N2. PTLC (SiO2, 20 × 20 cm,
EtOAc) afforded CBA-TMI (19, 1.5 mg, 71%) as a white solid:
1
Rf ) 0.49 (EtOAc); H NMR (CDCl3, 600 MHz) δ 8.33 (d, J )
7.9 Hz, 1H), 7.54 (t, J ) 7.9 Hz, 1H), 7.46 (t, J ) 7.9 Hz, 1H),
7.01 (d, J ) 7.9 Hz, 1H), 6.85 (s, 1H), 6.80 (s, 1H), 6.31 (s,
1H), 4.16 (d, J ) 11.8 Hz, 1H), 3.87 (s, 3H), 3.71 (s, 3H), 3.87
(d, J ) 11.8 Hz, 1H), 2.98 (s, 3H), 2.00 (t, J ) 7.9 Hz, 1H),
1.59 (br s, 1H), 0.99 (t, J ) 6.1 Hz, 1H); 13C NMR (CDCl3, 150
MHz) δ 163.4, 151.8, 140.9, 140.6, 138.2, 133.9, 133.0, 129.6,
129.5, 129.3, 128.9, 127.5, 123.6, 122.4, 101.6, 98.8, 95.3, 62.0,
60.4, 57.0, 44.4, 37.7, 31.4; IR (film) νmax 3257, 1713, 1683 cm-1
;
MALDIFT-HRMS (DHB) m/z 432.1558 (M + H+, C24H21N3O5
requires 432.1554). Natural-(8bR,9aS)-19: [R]23D +51 (c 0.05,
CH2Cl2). ent-(8bS,9aR)-19: [R]23 -49 (c 0.04, CH2Cl2).
D
Acid -Ca ta lyzed Ad d ition of CH3OH to N-Boc-CBA. 3-N-
(ter t-Bu tyloxyca r bon yl)-1-m eth oxym eth yl-4H-5-oxo-1,2-
d ih yd r op yr r olo[2,3-c]isoqu in olin e (20). A solution of 16
(1.0 mg, 3.4 µmol) in CH3OH (70 µL) was treated with CF3-
SO3H-CH3OH (17 µL, 0.02 M, 0.34 µmol) at 0 °C. The ice bath
was removed, and the reaction mixture was stirred for 30 min
at 23 °C before being quenched by the addition of NaHCO3
(2.0 mg), filtered through Celite, and concentrated in vacuo.
PTLC (SiO2, 10 × 20 cm, 50% EtOAc-hexanes) afforded 20
(1.0 mg, 90%) as a white solid: Rf ) 0.71 (50% EtOAc-
Ack n ow led gm en t. We gratefully acknowledge the
financial support of NIH (CA41986) and the Skaggs
Institute for Chemical Biology. We especially thank Dr.
R. Chadha for the X-ray structure determination of 16
and H. Schmitt for initial studies. D.B.K. is a Skaggs
Fellow.
Su p p or tin g In for m a tion Ava ila ble: 1H NMR spectra of
10-20 are provided. This material is available free of charge
via the Internet at http://pubs.acs.org.
1
hexanes); H NMR (acetone-d6, 500 MHz) δ 10.43 (br s, 1H),
8.20 (d, J ) 7.0 Hz, 1H), 7.64 (t, J ) 6.0 Hz, 1H), 7.56 (d, J )
7.0 Hz, 1H), 7.29 (t, J ) 6.2 Hz, 1H), 4.12 (m, 1H), 4.00 (d, J
J O035119F
8990 J . Org. Chem., Vol. 68, No. 23, 2003