Inhibition of DNA topoisomerase II by azaelliptitoxins functionalized in the variable substituent domain

Article abstract of DOI:10.1021/jm9508806

A series of novel C₁₁-substituted derivatives of azaelliptitoxin (azatoxin) have been synthesized and tested for their inhibitory activity against human DNA topoisomerase II. Incorporation of a C₁₁ polyamine or amine resulted in an i

Full text of DOI:10.1021/jm9508806

2188
J . Med. Chem. 1996, 39, 2188-2196
In h ibition of DNA Top oisom er a se II by Aza ellip titoxin s F u n ction a lized in th e
Va r ia ble Su bstitu en t Dom a in
J etze J . Tepe, J ose S. Madalengoitia, Kelli M. Slunt, Karl W. Werbovetz, P. Grant Spoors, and
Timothy L. Macdonald*
Department of Chemistry, University of Virginia, Charlottesville, Virginia 22901
Received December 4, 1995X
A series of novel C11-substituted derivatives of azaelliptitoxin (azatoxin) have been synthesized
and tested for their inhibitory activity against human DNA topoisomerase II. Incorporation
of a C11 polyamine or amine resulted in an increase in the intercalation properties of the drug
and a decrease of topoisomerase II activity. The structure-activity relationship (SAR) profile
of the nonintercalating C11 anilino azatoxin class follows the SAR of the (anilino)acridine family.
11-(4-Cyanoanilino)azatoxin (14) was found to be the most active analog in this series, exhibiting
∼10-fold higher activity than azatoxin 12 and etoposide.
In tr od u ction
azatoxin analogs represent two classes of substituents
in the variable substituent domain. The synthesis of
one class, the C11 alkoxy and aminoalkyl analogs, is
depicted in Scheme 1. The synthesis of the N-aryl
derivatives is outlined in Schemes 2 and 3.
Azaelliptitoxin (referred to as azatoxin) is a potent
inhibitor of DNA topoisomerase II (topo II) and tubulin
polymerization. This agent was rationally designed as
a hybrid of etoposide and ellipticine.1,2 As part of our
continuing efforts aimed at the design of novel DNA topo
II inhibitors, we have been investigating the individual
contributions to activity from each of the three domains,
hypothesized in a model pharmacophore of the azatoxin
class.1 Azatoxin and its analogs have been established
to stabilize the ternary complex of drug, DNA, and DNA
topo II, termed the “cleavable complex”.3,4
We have previously reported the structure-activity
relationship (SAR) of pendant group and indole-substi-
tuted azatoxin analogs.5 The pendant group domain in
the azatoxin and epipodophyllotoxin series has strict
structural requirements for activity.5,6 The variable
substituent domain can be substituted by a variety of
structurally diverse groups, with little SAR data avail-
able.7,8 Since a significant increase in potency and
selectivity, with regard to topo II versus tubulin,9,10 has
been observed in some 4-epi-functionalized podophyllo-
toxins, the incorporation of a substituent in the variable
substituent domain of the azatoxin class was under-
taken.
The structural requirements of the variable substitu-
ent domain in 4-epi-odophyllotoxin derivatives indicates
that O-alkyl and N-alkyl chains of two or carbons with
a terminal polar functionality enhances topo II activ-
ity.11 In addition, N-aryl-substituted epipodophyllotox-
ins have been found to be potent topo II inhibitors.12
As such, a representative group of substituted azatoxin
analogs that fit the aforementioned structural require-
ments was synthesized and evaluated for their ability
to promote protein-associated DNA strand breaks, a
measure of cleavable complex formation. In addition,
in an attempt to further define the structural demands
for activity of the variable substituent domain, several
C11 (anilino)azatoxin analogs were synthesized and
tested for topo II inhibition.
Oxazolidinone 1, generated from L-tryptophanol and
diethyl carbonate with sodium ethoxide (98% yield), is
the precursor for both classes (Scheme 1). Treatment
of 1 with syringaldehyde dimethyl acetal and catalytic
trifluoroacetic acid (15 mol %) gave azatoxin (91% yield).
Functionalization of 1 at C11 to yield 2, as a 2:3 mixture
of diastereomers, was accomplished by DDQ oxidation13
of oxazolidinone 1 in THF/acetic acid at -78 °C (98%).
Acetate 2 was unstable and was used as quickly as
possible. Cyclization of 2 with syringaldehyde dimethyl
acetal and catalytic p-TsOH (10 mol %) provided the 11-
methoxyazatoxin 3 as a single diastereomer in 47%
yield. Due to the insolubility of 3 in many solvent
systems, the phenol was protected as the methyl car-
bonate, yielding 4, which had a wider spectrum of
solubility.
Acid-catalyzed exchange of the C11 methoxy with
water (5) or ethylene glycol (7) could be effected with
catalytic p-TsOH in either a dioxane/water or dioxane/
ethylene glycol mixture, respectively (Scheme 1). Depro-
tection of 5 was then accomplished with sodium meth-
oxide in methanol. In order to functionalize the C11
position with an amine, a variation of the literature
procedure for the elaboration of the C4 position of
podophyllotoxin was developed (Scheme 1). The “stan-
dard method” of treatment of methoxyazatoxin 4 with
HBr resulted in decomposition. A milder procedure
involved treatment of alcohol 5 with acetyl chloride and
TEA, followed by N,N-dimethylethlenediamine to yield
9 (54% yield). However, when anilines were used as
nucleophiles, the yields were rather low. An alternative
procedure utilizing BF3‚OEt2-catalyzed addition of the
desired aniline was employed for synthesis of the
anilino-substituted azatoxins. Treatment of 4 with
4-fluoroaniline, 4-nitroaniline, or 4-cyanoaniline and
BF3‚OEt2 at 0 °C in CH2Cl2 afforded compounds 10a -c
in 50-60% yield (Scheme 2). Deprotection of 10a -c
proceeded smoothly with sodium methoxide in methanol
to yield the arylamines 11, 13 and 14 (78%).
Ch em istr y
Thirteen derivatives were synthesized and function-
alized at the C11 position of the azatoxin nucleus. These
The methyl chloroformate protection group was re-
placed with the carbobenzyloxy group in the synthesis
X Abstract published in Advance ACS Abstracts, May 1, 1996.
S0022-2623(95)00880-6 CCC: $12.00 © 1996 American Chemical Society

Inhibition of DNA Topoisomerase II
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 11 2189
Sch em e 1
of 4′-hydroxy-substituted anilines, since basic or acidic
deprotection often resulted in elimination of the aniline.
The aniline precursors were synthesized through well-
established procedures. Subsequent treatment of 15
with the aforementioned anilines and BF3‚OEt2 at 0 °C
followed by hydrogenation over 10% Pd/C provided the
compounds 26-31 (Scheme 3). However, oxidation of
30 occurred rapidly, resulting in the elimination of the
aniline group.
Table 1 provides a summary of the SAR data, and a
representative cleavage gel is shown in Figure 1. The
SAR data indicate that incorporation of a para-substi-
tuted anilino group in the variable substituent domain
of azatoxin significantly increases the overall activity
of the drug. Interestingly, an electron-withdrawing
functionality in the paraposition of the aniline appears
to show the highest increase in the potency of the agent.
As illustrated by the di- and trisubstituted anilines, the
SAR of the variable substituent domain of azatoxin
follows the SAR of the (anilino)acridine family. The
effects on in vitro topo II-mediated DNA cleavage
activity of anilino-substituted in the (anilino)acridine
series has demonstrated that (i) a para H-bonding
substituent is not essential for activity and (ii) in
Resu lts a n d Discu ssion
Compounds 3, 7-9, 11-14, and 26-31 were assayed
for cleavable complex formation in the presence of
linearized pUC18 DNA and topo II purified from human
placenta, according to previously described techniques.14

2190 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 11
Tepe et al.
Sch em e 2
Sch em e 3
p-hydroxyanilines, ortho substitution depresses activity.
Thus, (p-hydroxyanilino)acridine > (o-methoxyhydroxy-
anilino)acridine > (o,o′-dimethoxyhydroxyanilino)acri-
dine. In addition, the results are consistent with
previous studies which found that analogously substi-
tuted podophyllotoxins exhibited a closely related SAR
for aniline substitution at the C4 position of podophyl-
lotoxins. These results support our hypothesis that the

Inhibition of DNA Topoisomerase II
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 11 2191
Ta ble 1. Biological Evaluation of Azatoxin Substituted in the
Variable Substituent Domain
F igu r e 1. DNA cleavable complex assay with azatoxin
analogs: lane A, DNA only; lane B, DNA +topo II; lane C, 1%
DMSO; lanes D-F, 5, 10, 50 µM azatoxin; lanes G-I, 5, 10,
50 µM (4-fluoroanilino)azatoxin (11); lanes J -L, 5, 10, 50 µM
anilinoazatoxin (26); lanes M-O, 5, 10, 50 µM (4-hydroxy-
anilino)azatoxin (27); lanes P-R, 5, 10, 50 µM (4-(carboxy-
methyl)anilino)azatoxin (28).
inhibitor of tubulin polymerization and topo II.10 Based
on the similarity in structure and the protein-associated
DNA cleavage patterns of 4′-demethyl-4-deoxypodophyl-
lotoxin (DDP) and azatoxin 12, it has been postulated
that tubulin might have a comparable drug binding
site.3 The association with the tubulin binding site can
be eliminated when the drug is substituted in the
variable substituent domain of our model pharmaco-
phore. The enhancement of topo II selectivity has been
previously illustrated by the transformation of the dual
inhibitor, DDP, into the selective topo II inhibitor,
etoposide, by substitution in the variable substituent
region. In the case of azatoxin, the incorporation of a
substituent in the variable substituent domain similarly
eliminates the inhibition of tubulin polymerization and
results in selective topo II inhibition.3 Our data indicate
that the variable substituent domain contributes to both
drug selectivity, by eliminating the association with
tubulin binding sites, and drug potency at the topo II
target site.
In contrast to the increase in potency and selectivity
resulting from the substitution of para-substituted
anilines in the variable substituent domain, the other
substituents depressed topo II activity. Although 4′-
demethyl-epipodophyllotoxin has been reported to be
equipotent to etoposide, the analogously substituted
azatoxin 8 is considerably less active than 12. However,
the reduced activity of the glycol ether 7 is comparable
to that observed in the epipodophyllotoxin series.16
The difference in cleavage patterns, between azatoxin
and the anilino-substituted azatoxins, observed in Fig-
ure 1, is thought to be the result of the subtly different
orientations of the drug within the enzyme-DNA
complex. Azatoxin 12 and the anilino-substituted aza-
toxin analogs (11, 13, 14, 26-28) are nonintercalative
topo II inhibitors which exert their cytotoxicity through
the stabilization of the cleavable complex in the catalytic
cycle of topo II. The association of the drug with the
DNA-enzyme complex can occur through several
“modes” of interaction, illustrated by the diversity of
DNA cleavage patterns.17, 18 Each cleavage site presum-
* Relative DNA cleavage was determined by densitometric
analysis of the protein-associated DNA cleavage assays and
quantified through comparison with the previously reported drug-
induced cleavage of several of the azatoxin derivatives.3 The
cleavage activities are expressed relative to azatoxin and were
reproducible within 30% of the reported activities.
anilino group of the aminoacridine family resides in the
variable substituent domain of the model pharmaco-
phore.
Of all the compounds tested, compound 14 is the most
active analog in this series exhibiting ∼10-fold higher
activity than azatoxin 12 and etoposide. The (nitro-
anilino)azatoxin 13 was shown to be sensitive to P-
glycoprotein-mediated cell efflux as observed in multiple
drug resistance (MDR).15 The (fluoroanilino)azatoxin
derivative 11 was not found to be sensitive to the
multidrug transporter and was selected for preclinical
evaluation of its antitumor activity.
In addition to the potential increase in potency of
these functionalized azatoxin analogs, the C11 aniline-
substituted azatoxins exhibit a profound increase in topo
II selectivity. Azatoxin 12 was designed to be a dual

2192 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 11
Tepe et al.
(m, 2H), 3.04 (m, 2H); [R]24 +8.4° (c 9.4, MeOH). Anal.
ably reflects on destinct drug-DNA-enzyme complex
with a distinct, but similar, drug-induced DNA confor-
mation. As illustrated by our model of the pharma-
cophore for topo II active agents, we postulate that the
variable substituent domain of azatoxin 12 is located
in the minor groove of the DNA. The additional binding
energy derived from placement of a group in this
structurally accesible locus contributes to the stability
of the drug in the DNA-enzyme complex. We hypoth-
esize that the aniline in the variable substituent domain
of azatoxin enables a drug-induced DNA conformation
with which the enzyme binds more tightly, thereby
increasing the stabilization of the DNA-enzyme cleav-
able complex and resulting in a profound increase of
potency.
It should be notes that structual restrictions on the
C11 substituent are apparent. For example, incorpora-
tion of a polyamine or amine into the nucleus of a topo
II inhibitor structure normally results in an analog with
increased activity, although the azatoxin polyamine
derivative 9 is less active than azatoxin 12. Interest-
ingly, the cleavage intensity of 9 does not increase from
50 to 100 mM, but appears to decrease slightly. This
result suggests the same autoinhibitory phenomenon
that is observed with intercalators. Compound 9 ex-
hibits limited intercalative ability in an ethidum bro-
mide displacement assay.19 The resulting concentration-
response curve reveals that at 50 mM of 9, 15%
displacement occurs, while displacement does not sig-
nificantly increase at 100 mM. This asymptotic behav-
ior is typical of weaker intercalative agents. Further-
more, 9 is the first epipodophyllotoxin-like topo II
inhibitor to exhibit any “classical” intercalative behav-
ior. This result represents a significant observation in
that it exemplifies the continuous spectrum of intercala-
tion and topo II activity suggested by the composite
pharmacophore model.
D
(C12H12N2O2) C, H, N.
P r ep a r a tion of Syr in ga ld eh yd e Dim eth yl Aceta l. Sy-
ringaldehyde (1 g) was dissolved in trimethyl orthoformate (7
mL), and a catalytic amount of p-TsOH (40 mg) was added.
The reaction was followed to completion by TLC. The solvent
was removed under reduced pressure. The remaining oil was
dissolved in CHCl3 and filtered through a plug of silica. The
solvent was again removed under reduced pressure. The
remaining oil was stored in a desicator until use.
(5R,11a S)-4,5,11,11a -Tet r a h yd r o-5-(3,5-d im et h oxy-4-
h yd r oxyp h en yl)-1H,6H-oxa zolo[3′,4′:1,6]p yr id o[3,4-b]in -
d ol-3-on e (12). TFA (0.15 equiv) was added to a solution of
oxazolidinone 1 (0.522 mmol) in 3 mL of THF and syringal-
dehyde dimethyl acetal (1.5 equiv). The solution was refluxed
for 5 h, cooled to room temperature, quenched with saturated
aqueous NaHCO3, and extracted with ethyl acetate. The
organic layer was separated, dried over Na2SO4, and filtered.
The product was purified by flash chromatography (12%
acetone in CHCl3, Rf ) 0.28), producing a white solid in 91%
1
yield: mp dec slow 175 °C; [R]22 -139.6° (c 1.0, CHCl3); H
D
NMR (300 MHz, CD3CN) δ 7.94 (sb, 1H), 7.51 (d, J ) 7.91 Hz,
1H), 7.30 (d, J ) 7.57 Hz, 1H), 7.09 (m, 2H), 6.59 (s, 2H), 6.27
(s, 1H), 5.88 (d, J ) 1.7 Hz, 1H), 4.54 (dd app t, J ) 8.3 Hz,
1H), 4.33 (m, 1H), 4.21 (dd, J ) 8.5, 4.7 Hz, 1H), 3.75 (s, 6H),
3.16, (dd, J ) 15, 4.6 Hz, 1H), 2.76 (ddd, J ) 15, 10.38 Hz,
1.73 Hz, J ) 1.73 Hz, 1H). Anal. (C21H20N2O5) C, H, N.
(5R,11R,11a S)-4,5,11,11a -Tetr a h yd r o-5-(3,5-d im eth oxy-
4-h yd r oxyp h en yl)-11-m et h oxy-1H ,6H -oxa zolo[3′,4′:1,6]-
p yr id o[3,4-b]in d ol-3-on e (3). Glacial acetic acid (30 mL) was
added dropwise to a solution of oxazolidinone 1 (1.02 g, 4.72
mmol) in THF (30 mL) at -78 °C. The cooling bath was
removed. When dissolution of the acetic acid occurred, the
reaction flask was reemerged in the cooling bath, and im-
mediately a solution of DDQ (1.17 g, 5.2 mmol) in THF (30
mL) was added over 5 min. The solution was allowed to stir
at -78 °C for 30 min. The cooling bath was removed, and the
reaction mixture was allowed to stir at room temperature for
24 h. Toluene (60 mL) was added, and the solvent was
removed under reduced pressure. The remaining brown oil
was redissolved in EtOAc (200 mL) and washed with saturated
aqueous NaHCO3 (5 × 75 mL). The organic layer was dried
over Na2SO4 and filtered quickly through a plug of florisil. The
solvent was removed under reduced pressure to yield the
acetate 2 (1.28 g, 98%) as a mixture of diastereomers.
The incorporation of several other substituents in the
variable substituent domain of azatoxin, such as several
glycosyl units and additional N-arylamino units, are
under investigation, and their biological results will be
published in the near future.
p-TsOH (0.80 mg, 0.47 mmol) was added to a solution of
syringaldehyde dimethyl acetal (1.60 g, 7.01 mmol) in anhy-
drous CH2Cl2/MeOH (9:1 mixture, 8 mL) and was allowed to
stir for 5 min. The solution was cooled to 0 °C, and a solution
of acetate 2 (1.28 g, 4.67 mmol) in 9:1 CH2Cl2/MeOH (8 mL)
was added in small portions. The precipitate which was
formed after 4 h was collected by filtration in a scintered glass
funnel and washed with cold CH2Cl2. The remaining solid was
Exp er im en ta l Section
All melting points were taken on a Thomas-Hoover UNI-
MELT melting point apparatus and are uncorrected. 1H NMR
spectra were obtained using a General Electric QE300 spec-
trometer at 300 MHz. All chemical shifts are recorded in ppm.
Elemental analyses were performed by Atlantic Microlab Inc.,
Norcross, GA, and in house on a Perkin-Elmer PE 2400 C, H,
N analyzer. Analytical thin-layer chromatography (TLC) was
carried out on Merck precoated silica gel 60 F-254 plates and
were visualized with a phosphomolybdic acid/ethanol solution.
dried in a vacuum desicator to yield the methoxyazatoxin
1
analog 3 (0.91g, 47%); [R]22 ) -134 (c 0.9, DMSO); H NMR
D
(300 MHz, DMSO-d6) δ 11.50 (sb, 1H), 8.42 (sb, 1H), 7.65 (d,
J ) 7.28 Hz, 1H), 7.31 (d, J ) 7.71 Hz, 1H), 7.06 (m, 2H), 6.48
(s, 2H), 5.85 (s, 1H), 4.63 (d, J ) 1.8 Hz, 1H), 4.43 (m, 3H),
3.64 (s, 6H), 3.28 (s, 3H); 13C NMR (DMSO-d6) 157.5, 148.9,
137.3, 136.8, 135.0, 130.7, 127.6, 122.5, 120.4, 119.2, 112.5,
108.5, 106.6, 68.0, 65.1, 57.2, 57.0, 55.4, 54.2. Anal.
(C22H22N2O6) C, H, N.
4(S)-(1H-In d ol-3-ylm eth yl)-2-oxa zolid in on e (1). Diethyl
carbonate (8.83 g, 74.8 mmol) and L-tryptophanol (12.94 g,
68.02 mmol) in ethanol (100 mL) was added to a solution of
sodium (1.56 g, 68.1 mmol) in absolute ethanol (150 mL). The
solution was heated at reflux for 5 h and cooled to room
temperature, and the solvent was removed under reduced
pressure. Saturated aqueous NH3Cl (100 mL) and CH2Cl2 (200
mL) were added, and the organic layer was separated. The
aqueous layer was washed with CH2Cl2 (2 × 100 mL), and
the organic fractions were combined and dried over Na2SO4.
The solvent was removed under reduced pressure. Recrys-
tallization from MeOH/H2O yielded a white solid (11.66 g,
Gen er a l P r oced u r e A for Meth yl F or m a te P r otection
of P h en ols. Methyl chloroformate (3.69 mL, 47.7 mmol) was
added dropwise to a suspension of methoxy azatoxin 3 (1.96
g, 4.77 mmol) in CH2Cl2 (10 mL) and TEA (6.7 mL, 47.7 mmol)
at 0 °C. The solution was diluted with CH2Cl2 after 2 h and
washed with water. The organic layer was dried over Na2-
SO4 and filtered, and the solvent was removed under reduced
pressure. The product was purified by flash chromatography
(eluting with acetone-chloroform).
(5R,11R,11a S)-4,5,11,11a -Tetr a h yd r o-5-(3,5-d im eth oxy-
4-(ca r bom eth oxyoxyp h en yl)-11-m eth oxy-1H,6H-oxa zolo-
[3′,4′:1,6]pyr ido[3,4-b]in dol-3-on e (4). Methoxyazatoxin ana-
log 4 was prepared as described in procedure A. The product
was purified by flash chromatography eluting with 8% acetone
1
79%): mp 155 °C; H NMR (300 MHz, CDCl3) δ 8.17 (sb,1H),
7.57 (d, J ) 7.94 Hz, 1H), 7.40 (d, J ) 8.06 Hz, 1H), 7.19 (m,
2H), 7.08 (d, J ) 2.2 Hz, 1H), 5.22 (sb, 1H), 4.50 (m, 1H), 4.21

Inhibition of DNA Topoisomerase II
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 11 2193
in CHCl3 to yield the product as a white solid (2.09 g, 94%).
An analytical sample was obtained by recrystallization from
reaction mixture was heated at 50 °C, and the reaction was
followed to completion by TLC. The solvent was removed
under reduced pressure, and the remaining solid was purified
by flash chromatography, eluting with 8% TEA in CH2Cl2 (Rf
ethyl acetate: mp dec 219-220 °C; [R]22 ) -83.5 (c 1.25,
D
CHCl3); 1H NMR (300 MHz, CDCl3) δ 8.26 (sb, 1H), 7.71 (d, J
) 6.72 Hz, 1H), 7.37 (d, J ) 6.37 Hz, 1H), 7.28-7.21 (m, 2H),
6.51 (s, 2H), 6.08 (s, 1H), 4.75 (dd, J ) 8.32 Hz, J ) 3.82 Hz,
1H), 4.67 (d, J ) 2.29 Hz, 1H), 4.45 (t, J ) 8.71 Hz, 1H), 4.20
(dt, J ) 8.83 Hz, 1H), 3.89 (s, 3H), 3.72 (s, 6H), 3.4 (s, 3H).
Anal. (C24H24N2O8) C, H.
) 0.35) to afford the product 9 as a white solid (54 mg, 54%):
1
[R]22 -145° (c 0.5, DMSO); H NMR (300 MHz, DMSO-d6) δ
D
8.41 (sb, 1H), 7.59 (d, J ) 7.34 Hz, 1H), 7.27 (d, J ) 7.77 Hz,
1H), 7.07-6.97 (m, 2H), 6.47 (s, 2H), 5.78 (s, 1H), 4.63 (dd, J
) 7.31 Hz, J ) 4.23 Hz, 1H), 4.42-4.31 (m, 2H), 3.97 (d, J )
2.11 Hz, 1H), 3.65 (s, 6H), 2.74-2.7 (m, 1H), 2.63-2.60 (m,
1H), 2.26-2.17 (m, 2H), 1.96 (s, 6H). Anal. (C25H30N4O5) C,
H, N.
(5R,11R,11a S)-4,5,11,11a -Tetr a h yd r o-5-(3,5-d im eth oxy-
4-(ca r bom eth oxyoxyp h en yl)-11-h yd r oxy-1H,6H-oxa zolo-
[3′,4′:1,6]p yr id o[3,4-b]in d ol-3-on e (5). p-TsOH (130 mg,
0.70 mmol) was added to a solution of the methoxyazatoxin
analog 4 (1.88 g, 7.00 mmol) in a dioxane/water solution (9:1
mixture, 30 mL). The solution was followed to completion by
TLC and diluted with CHCl3. The resulting mixture was
washed with saturated aqueous NaHCO3, dried over Na2SO4
and filtered. The solvent was removed under reduced pres-
sure. The product was purified by flash chromatography
eluting with 20% acetone in CHCl3, yielding the hydroxyaza-
toxin analog 5 as white solid (1.21 g, 66%) and the epimer as
the minor product (0.16 g, 13%). An analytical sample was
obtained by recrystallization from toluene: mp dec 205-207
°C; 1H NMR (300 MHz, CDCl3) 8.09 (sb, 1H), 7.75 (d, J ) 7.17
Hz, 1H), 7.38 (d, J ) 7.29 Hz, 1H), 7.32-7.22 (m, 2H), 6.53 (s,
2H), 6.14 (s, 1H), 5.01 (sb, 1H), 4.84 (dd, J ) 8.39 Hz, J )
4.56 Hz, 1H), 4.49 (t, J ) 8.8 Hz, 1H), 4.22-4.16 (m, 1H), 3.91
(s, 3H), 3.37 (s, 6H). Anal. (C23H22N2O8) C, H.
(5R,11R,11a S)-4,5,11,11a -Tetr a h yd r o-5-(3,5-d im eth oxy-
4-h yd r oxyp h en yl)-11-h yd r oxy-1H ,6H -oxa zolo[3′,4′:1,6]-
p yr id o[3,4-b]in d ol-3-on e (8). A solution of the hydroxyaza-
toxin analog 5 (64.5 mg, 141 mmol) in absolute ethanol was
added to a solution of sodium methoxide (0.51 mmol) in
absolute methanol (1 mL). The solution was allowed to stir
at room temperature for 3 h. The reaction mixture was
carefully acidified to pH 7 by the addition of 1% HCl, and
stirring was continued for an additional 20 min. A precipitate
formed, which was collected by filtration in a scintered glass
funnel and dried in a vacuum desiccator to yield the product
8 as a white solid (41 mg, 73%): [R]22D -126.7° (c 0.3, DMSO);
1H NMR (300 MHz, DMSO-d6) δ 10.9 (sb, 1H), 8.42 (sb, 1H),
7.54 (d, J ) 7.29 Hz, 1H), 7.28 (d, J ) 7.78 Hz, 1H), 7.08-
7.00 (m, 2H), 6.47 (s, 2H), 5.80 (s, 1H), 5.26 (d, J ) 6.7 Hz,
1H), 4.79 (dd, J ) 6.43 Hz, J ) 2.25 Hz, 1H), 4.55-4.51 (m,
1H), 4.37 (t, J ) 8.37 Hz, 1H), 4.29-4.27 (m, 1H), 3.65 (s, 6H).
Anal. (C21H20N2O6) C, H, N.
(5R,11R,11a S)-4,5,11,11a -Tetr a h yd r o-5-(3,5-d im eth oxy-
4-h yd r oxyp h en yl)-11-h yd r oxy-1H ,6H -oxa zolo[3′,4′:1,6]-
p yr id o[3,4-b]in d ol-3-on e (7). p-TsOH (10 mol %) was added
to a solution of the hydroxyazatoxin analog 5 (0.2 g, mmol) in
dioxane/ethylene glycol (9:1 mixture, 6 mL), and the reaction
mixture was followed to completion by TLC. The reaction was
quenched by the addition of saturated aqueous NaHCO3 and
CH2Cl2. The organic layer was separated, dried over Na2SO4,
and filtered. The solvent was removed under reduced pres-
sure. Purification by flash chromatography yielded the prod-
uct 7 as a white solid (60%): 1H NMR (300 MHz, DMSO-d6) δ
11.55 (sb, 1H), 7.66 (d, J ) 7.19 Hz, 1H), 7.32 (d, J ) 8.00 Hz,
1H), 7.11-7.02 (m, 2H), 6.63 (s, 2H), 5.94 (s, 1H), 4.81 (s, 1H),
4.64 (d, J ) 4.78 Hz, 1H), 4.58 (t, J ) 3.64 Hz, 1H), 4.55-4.48
(m, 2H), 3.76 (s, 3H), 3.70 (s, 6H), 3.66-3.59 (m, 1H), 3.51-
3.43 (m, 3H).
3,4,5-Tr im eth oxyn itr oben zen e (16). Fuming nitric acid
(3.5 mL) was added to a solution of trimethoxybenzene (60
mmol) in 20 mL of glacial acetic acid at 0 °C. The solution
was stirred for 1 h, after which it was poured over ice. The
beige/brown precipitate was filtered off by suction, yielding a
orange/yellow solid. The product was purified by flash chro-
matography eluting with 5% acetone in chloroform (Rf ) 0.8),
yielding trimethoxynitrobenzene as a beige/yellow solid (39%
yield): 1H NMR (300 MHz, CDCl3) δ 7.52 (s, 2H), 3.88 (s, 9H).
3,5-Dim eth oxy-4-h yd r oxyn itr oben zen e (17). A solution
of 3,4,5-trimethoxynitrobenzene (0.034 mol) in 20% KOH/H2O
(100 mL) was refluxed for 2 days. The pH was adjusted to
pH ∼3 with a 10% HCl solution. The solution was extracted
with CHCl3 (3 × 25 mL), and the organic layers were
combined, dried over Na2SO4, and filtered. The solvent was
removed under reduced pressure. The product was purified
by flash chromatography eluting with 5% acetone in chloro-
form (Rf ) 0.5), yielding the product 17 as a yellow solid (600
mg, 9% yield). The remaining 91% was starting material
which was demethylated by repeating the procedure (∼10%
yield per repeat): 1H NMR (300 MHz, CDCl3) δ 7.57 (s, 2H),
6.12 (sb, 1H), 4.01 (s, 6H).
Gen er a l P r oced u r e B for Ca r boben zoxy P r otection of
Nitr op h en ols. CBZCl (19.6 mmol) was added to a solution
of the nitrophenol (7.18 mmol) and TEA (20.0 mmol) in 22 mL
CH2Cl2 at 0 °C. The solution was stirred for 2 h. The solution
was diluted with of CH2Cl2 and extracted with H2O (3 × 10
mL). The organic layer was separated, dried over Na2SO4, and
filtered. The solvent was removed under reduced pressure.
The product was purified by flash chromatography (acetone-
chloroform).
4-(Ca r boben zoxyoxy)ben zen e. This nitrophenol analog
was prepared as described in procedure B (95% yield) and
purified by flash chromatography eluting with 10% acetone
in chloroform (Rf ) 0.45): 1H NMR (300 MHz, CDCl3) δ 8.27
(d, J ) 9.3 Hz, 2H), 7.24 (m, 7H), 5.38 (s, 2H).
3-Meth oxy-4-(ca r boben zoxyoxy)n itr oben zen e. This ni-
trophenol analog was prepared as described in procedure B
(96%) and purified by flash chromatography eluting with 5%
acetone in chloroform (Rf ) 0.58): 1H NMR (300 MHz, CDCl3)
δ 7.85 (m, 2H), 7.42 (m, 5H), 7.27 (d, J ) 13.9 Hz, 1H), 5.29
(s, 2H), 3.87 (s, 3H).
3,5-Dim eth oxy-4-(ca r boben zoxyoxy)n itr oben zen e (18).
Nitrophenol analog 18 was prepared as described in procedure
B (89%) and purified by flash chromatography eluting with
5% acetone in chloroform (Rf ) 0.7): 1H NMR (300 MHz,
CDCl3) δ 7.52 (s, 2H), 7.39 (m, 5H), 5.31 (s, 2H), 3.84 (s, 6H).
Gen er a l P r oced u r e
C for P r ep a r a t ion of 4-(Ca r -
boben zoxyoxy)a n ilin es. SnCl2‚H2O and H2O (0.6 mL) were
added to a solution of the 4-(carbobenzoxyoxy)nitrobenzene
(3.34 mmol) in ethanol (30 mL). The solution was refluxed
until completed by TLC (∼30 min). The solution was cooled
to room temperature and poured onto ice. The pH was
neutralized to pH 7-8 with NaHCO3 powder, and the aqueous
layer was extracted with ethyl acetate (3 × 10 mL). The
organic layers were combined and washed with a brine solution
(3 × 25 mL). The organic layer was separated, dried over Na2-
SO4, and filtered. The solvent as removed under reduced
pressure. The product was purified by flash chromatography
(acetone-chloroform).
Deprotected as in 8 (72%): 1H NMR (300 MHz, DMSO-d6)
δ 11.02 (sb, 1H), 8.43 (sb, 1H), 7.65 (d, J ) 7.26 Hz, 1H), 7.30
(d, J ) 7.9 Hz, 1H), 7.12-7.03 (m, 2H), 6.46 (s, 2H), 5.83 (s,
1H), 4.78 (s, 1H), 4.61 (d, J ) 3.84 Hz, 1H), 4.56 (t, J ) 5.15
Hz, 1H), 4.49-4.42 (m, 2H), 3.65 (s, 6H), 3.64-3.59 (m, 1H),
4.57-4.42 (m, 3H).
(5R,11R,11a S)-4,5,11,11a -Tetr a h yd r o-5-(3,5-d im eth oxy-
4-h yd r oxyp h e n yl)-11-((2-N ,N -d im e t h yla m in o)e t h yl)-
am in o)-1H,6H-oxazolo[3′4′:1,6]pyr ido[3,4-b]in dol-3-on e (9).
TEA (640 mmol) and acetyl chloride (535 mmol) were added
to a solution of 5 (97 mg, 214 mmol) in anhydrous dioxane (2
mL) at 0 °C. After 15 min of stirring, the solvent was removed
under reduced pressure, and BaCO3 (1.1 mmol), N,N-dimeth-
ylethylenediamine (1.1 mmol), dioxane (3 mL) were added. The
4-(Ca r boben zoxyoxy)a n ilin e (22). Aniline analog 22 was
prepared as described in procedure C (93%) and purified by
flash chromatography eluting with 5% acetone in chloroform

2194 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 11
Tepe et al.
(Rf ) 0.48): 1H NMR (300 MHz, CDCl3) δ 7.48 (m, 7H), 6.96
(d, J ) 8.8 Hz, 2H), 6.63 (d, J ) 8.8 Hz, 2H), 5.22 (s, 2H), 3.63
(sb, 2H).
3-Met h oxy-4-(ca r b ob en zoxyoxy)a n ilin e (21). Aniline
analog 21 was prepared as described in procedure C (97%) and
purified by flash chromatography eluting with 5% acetone in
chloroform (Rf ) 0.40): 1H NMR (300 MHz, CDCl3) δ 7.40 (m,
5H), 6.88 (d, J ) 8.4 Hz, 1H), 6.29 (d, J ) 2.4 Hz, 1H), 6.20
(dd, J ) 8.5 Hz, 2.59 Hz, 1H), 5.23 (s, 2H), 3.75 (s, 3H), 3.62
(sb, 2H).
form (Rf ) 0.6): 1H NMR (300 MHz, DMSO-d6) δ 8.42 (sb, 1H),
7.41-7.18 (m, 7H), 7.05 (t, J ) 7.5 Hz, 1H), 6.90 (t, J ) 7.3
Hz, 1H), 6.48 (s, 2H), 6.07 (s, 2H), 5.82 (s, 1H), 5.21 (s, 2H),
4.97 (dd, J ) 7.7, 2.69 Hz, 1H), 4.52 (m, 1H), 4.45 (t, J ) 8.5
Hz, 1H), 4.18 (dd, J ) 6.4, 4.87 Hz, 1H), 3.66 (s, 6H), 3.63 (s,
6H), 3.52 (s, 3H).
(5R,11R,11a S)-4,5,11,11a -Tetr a h yd r o-5-(3,5-d im eth oxy-
4-(car boben zoxyoxy)ph en yl)-11-(4-am in oben zoxy)-1H,6H-
oxa zolo[3′,4′:1,6]p yr id o[3,4-b]in d ol-3-on e. The protected
(aminobenzoxy)azatoxin was prepared as described in proce-
dure D (55%) and purified by flash chromatography eluting
with 10% acetone in chloroform (Rf ) 0.55): 1H NMR (300
MHz, DMSO-d6) δ 8.49 (sb, 1H), 7.69 (d, J ) 8.43 Hz, 2H),
7.38-7.20 (m, 6H), 7.15 (d, J ) 7.82 Hz, 1H), 7.04 (t, J ) 7.5
Hz, 1H), 6.87 (m, 2H), 6.68 (d, J ) 8.82 Hz, 1H), 6.49 (s, 2H),
5.87 (s, 1H), 5.16 (s, 2H), 5.12 (dd, J ) 8.7, 2.74 Hz, 1H), 4.55
(m, 1H), 4.45 (t, J ) 8.7 Hz, 1H), 4.03 (dd, J ) 8.2 Hz, 5.42
Hz, 1H), 3.72 (s, 3H), 3.66 (s, 6H).
3,5-Meth oxy-4-(ca r boben zoxyoxy)a n ilin e (19). Aniline
analog 19 was prepared as described in procedure C (93%) and
purified by flash chromatography eluting with 5% acetone in
chloroform (Rf ) 0.30): 1H NMR (300 MHz, CDCl3) δ 7.40 (m,
5H), 5.92 (s, 2H), 5.25 (s, 2H), 3.78 (s, 6H), 3.63 (sb, 2H).
Gen er a l P r oced u r e D for th e Su bstitu tion of An ilin es
in th e Va r ia ble Su bstitu en t Dom a in . 4-Carbobenzoxyoxy-
protected aniline (0.366 mmol) was added to a solution of 25
(0.183 mmol) in anhydrous CH2Cl2 (3 mL) over 4A molecular
sieves in an argon-charged flame-dried 10 mL two-neck round
bottom flask. The solution was stirred at room temperature,
and BF3‚OEt2 (5-10 mol % BF3‚OEt2, 1-2 µL) was added. The
reaction was judged to completion by TLC after 4 h. The
reaction was quenched with saturated aqueous NaHCO3 (5
mL) and extracted with CH2Cl2 (3 × 10 mL). The organic
layers were combined, dried over Na2SO4, and filtered. The
solvent was removed under reduced pressure. The product
was purified by flash chromatography (acetone-chloroform).
(5R,11R,11a S)-4,5,11,11a -Tetr a h yd r o-5-(3,5-d im eth oxy-
4-(ca r b ob en zoxyoxy)p h en yl)-11-(4-(ca r b ob en zoxyoxy)-
a n ilin o)-1H ,6H -oxa zolo[3′,4′:1,6]p yr id o[3,4-b]in d ol-3-
on e. The protected ((carbobenzoxyoxy)anilino)azatoxin was
prepared as described in procedure D (23%) and purified by
flash chromatography eluting 5% acetone in chloroform (Rf )
0.32); 1H NMR (300 MHz, CDCl3) δ 8.20 (s, 1H), 7.47-7.20
(m, 13H), 7.11 (t, J ) 7.61 Hz, 1H), 7.07 (d, J ) 8.80 Hz, 2H),
6.66 (d, J ) 8.89 Hz, 2H), 6.54 (s, 2H), 6.06 (s, 1H), 5.27 (s,
4H), 4.86 (dd, J ) 7.80, 2.74 Hz, 1H), 4.48 (m, 3H), 3.82 (db,
1H), 3.69 (s, 6H).
(5R,11R,11a S)-4,5,11,11a -Tetr a h yd r o-5-(3,5-d im eth oxy-
4-(ca r b ob e n zoxyoxy)p h e n yl)-11-(3-m e t h oxy-4-(ca r b o-
ben zoxyoxy)a n ilin o)-1H,6H-oxa zolo[3′,4′:1,6]p yr id o[3,4-
b]in d ol-3-on e. The protected (methoxy(carbobenzoxyoxy)-
anilino)azatoxin was prepared as described in procedure D
(33%) and purified by flash chromatography eluting with 20%
acetone in chloroform (Rf ) 0.55): 1H NMR (300 MHz, CDCl3)
δ 8.38 (s, 1H), 7.46-7.22 (m, 14H), 7.10 (t, J ) 7.55 Hz, 1H),
7.01 (d, J ) 8.23 Hz, 1H), 6.52 (s, 2H), 6.22 (s, 1H), 6.08 (s,
1H), 5.28 (s, 2H), 5.26 (s, 2H), 4.86 (dd, J ) 7.8, 2.74 Hz, 1H),
4.49-4.44 (m, 3H), 3.85 (db, 1H), 3.73 (s, 3H), 3.66 (s, 6H).
(5R,11R,11a S)-4,5,11,11a -Tetr a h yd r o-5-(3,5-d im eth oxy-
4-(ca r b ob en zoxyoxy)p h en yl)-11-(3,5-d im et h oxy-4-(ca r -
b ob en zoxyoxy)a n ilin o)-1H ,6H -oxa zolo[3′,4′:1,6]p yr id o-
[3,4-b]in d ol-3-on e. The protected (dimethoxy(carbobenzoxy-
oxy)anilino)azatoxin was prepared as described in procedure
D (23%) and purified by flash chromatography eluting with
15% acetone in chloroform (Rf ) 0.65): 1H NMR (300 MHz,
CDCl3) δ 8.27 (s, 1H), 7.46-7.26 (m, 13H), 7.13 (t, J ) 7.54
Hz, 1H), 6.53 (s, 2H), 6.11 (s, 1H), 5.90 (s, 2H), 5.29 (s, 2H),
5.27 (s, 2H), 4.87 (dd, J ) 7.8, 2.74 Hz, 1H), 4.58-4.43 (m,
3H), 3.85 (db, 1H), 3.77 (s, 6H), 3.67 (s, 6H).
Gen er a l P r oced u r e E for th e Dep r otection of 4-(Ca r -
boben zoxyoxy)ben zen es. A suspension of the 4-carboben-
zoxyoxy-protected phenol (0.013 mmol) in ethanol (5 mL)
containing 10% Pd/C (20 mg) was stirred under a H2
atmo-
sphere (balloon pressure) at room temperature overnight. The
solution was filtered through a plug of Celite. The solvent was
evaporated off under reduced pressure, and the product was
purified by flash chromatography (acetone-chloroform).
(5R,11R,11a S)-4,5,11,11a -Tetr a h yd r o-5-(3,5-d im eth oxy-
4-h yd r oxyp h en yl)-11-(4-h yd r oxya n ilin o)-1H,6H-oxa zolo-
[3′,4′:1,6]pyr ido[3,4-b]in dol-3-on e (27). The (hydroxyanilino)-
azatoxin analog 27 was prepared as described in procedure E
(80%) and purified by flash chromatography eluting with 15%
acetone in chloroform (Rf ) 0.42): 1H NMR (300 MHz, CDCl3)
δ 8.02 (sb, 1H), 7.40-7.18 (m, 4H), 7.09 (t, J ) 7.4 Hz, 1H),
6.78 (d, J ) 8.82 Hz, 2H), 6.59 (d, J ) 8.87 Hz, 2H), 6.54 (s,
2H), 6.61 (s, 1H), 5.56 (s, 1H), 4.74 (dd, J ) 7.8, 2.72 Hz, 1H),
4.58 (m, 1H), 4.42 (m, 2H), 3.80 (s, 6H), 3.44 (db, 1H). Anal.
(C26H27N3O6) C, H, N.
(5R,11R,11a S)-4,5,11,11a -Tetr a h yd r o-5-(3,5-d im eth oxy-
4-h yd r oxyp h e n yl)-11-(3-m e t h oxy-4-h yd r oxya n ilin o)-
1H,6H-oxa zolo[3′,4′:1,6]p yr id o[3,4-b]in d ol-3-on e (29). The
(methoxyhydroxyanilino)azatoxin analog 29 was prepared as
described in procedure E (81%) and purified by flash chroma-
tography eluting with 20% acetone in chloroform (Rf ) 0.32):
1H NMR (300 MHz, CDCl3) δ 8.06 (sb, 1H), 7.35-7.20 (m, 3H),
7.11 (t, J ) 7.4 Hz, 1H), 6.85 (d, J ) 8.36 Hz, 1H), 6.53 (s,
2H), 6.25 (dd, J ) 8.4, 2.45 Hz, 1H), 6.20 (d, J ) 2.41 Hz, 1H),
6.11 (s, 1H), 5.56 (sb, 1H), 5.13 (sb, 1H), 4.80 (dd, J ) 7.5,
2.74 Hz, 1H), 4.59 (m, 1H), 4.43 (m, 2H), 3.81 (s, 3H), 3.79 (s,
6H), 3.49 (db, 1H). Anal. (C27H29N3O7) C, H, N.
(5R,11R,11a S)-4,5,11,11a -Tetr a h yd r o-5-(3,5-d im eth oxy-
4-h yd r oxyp h en yl)-11-(3,5-d im eth oxy-4-h yd r oxya n ilin o)-
1H,6H-oxazolo[3′,4′:1,6]pyr ido[3,4-b]in dole-3-on e (30). The
(dimethoxyhydroxyanilino)azatoxin analog 30 was prepared
as described in procedure E and purified by flash chromatog-
raphy eluting with 20% acetone in chloroform (Rf ) 0.23): 1H
NMR (300 MHz, CDCl3) δ 7.99 (sb, 1H), 7.36-7.15 (m, 3H),
6.52 (s, 2H), 6.11 (s, 2H), 6.07 (s, 1H), 5.54 (sb, 1H), 5.23 (sb,
1H), 4.71 (dd, J ) 7.5, 2.73 Hz, 1H), 4.67 (d, J ) 1.7 Hz, 1H),
4.43 (t, J ) 8.4 Hz, 1H), 4.22 (m, 1H), 3.66 (s, 6H), 3.58 (s,
6H), 3.52 (db, J ) 7.2 Hz, 1H).
(5R,11R,11a S)-4,5,11,11a -Tetr a h yd r o-5-(3,5-d im eth oxy-
4-p h yd r oxyh en yl)-11-(3,4,5-t r im et h oxya n ilin o)-1H ,6H -
oxa zolo[3′,4′:1,6]p yr id o[3,4-b]in d ol-3-on e (31). The (tri-
methoxyanilino)azatoxin analog 31 was prepared as described
in procedure E (78%) and purified by flash chromatography
eluting with 20% acetone in chloroform (Rf ) 0.42): 1H NMR
(300 MHz, DMSO-d6) δ 8.42 (sb, 1H), 7.28 (d, J ) 8.0 Hz, 1H),
7.19 (d, J ) 7.6 Hz, 1H), 7.05 (t, J ) 7.2 Hz, 1H), 6.90 (t, J )
7.4 Hz, 1H), 6.48 (s, 2H), 6.07 (s, 2H), 5.82 (s, 1H), 5.50 (d, J
) 8.41 Hz, 1H), 4.97 (dd, J ) 7.7, 2.69 Hz, 1H), 4.52 (m, 1H),
4.45 (t, J ) 8.6 Hz, 1H), 4.18 (dd, J ) 7.5, 4.87 Hz, 1H), 3.66
(s, 6H), 3.64 (s, 6H), 3.52 (s, 3H). Anal. (C30H33N3O8) C, H,
N.
(5R,11R,11a S)-4,5,11,11a -Tetr a h yd r o-5-(3,5-d im eth oxy-
4-(ca r boben zoxyoxy)p h en yl)-11-a n ilin o-1H,6H-oxa zolo-
[3′,4′:1,6]p yr id o[3,4-b]in d ol-3-on e. The protected (anilino)-
azatoxin was prepared as described in procedure D (75%) and
purified by flash chromatography eluting with 5% acetone in
chloroform (Rf ) 0.35): 1H NMR (300 MHz, CDCl3) δ 8.27 (s,
1H), 7.43-7.19 (m, 10H), 7.07 (t, J ) 7.41 Hz, 1H), 6.81 (t, J
) 7.18 Hz, 1H), 6.67 (d, J ) 8.23 Hz, 2H), 6.55 (s, 2H), 6.13 (s,
1H), 5.27 (s, 2H), 4.91 (dd, J ) 7.8, 2.75 Hz, 1H), 4.81 (db,
1H), 4.52-4.43 (m, 3H), 3.69 (s, 6H).
(5R,11R,11a S)-4,5,11,11a -Tetr a h yd r o-5-(3,5-d im eth oxy-
4-(car boben zoxyoxy)ph en yl)-11-(3,4,5-tr im eth oxyan ilin o)-
1H,6H-oxa zolo[3′,4′:1,6]p yr id o[3,4-b]in d ol-3-on e. The pro-
tected (trimethoxyanilino)azatoxin was prepared as described
in procedure D (60%) and purified by 15% acetone in chloro-
(5R,11R,11a S)-4,5,11,11a -Tetr a h yd r o-5-(3,5-d im eth oxy-
4-h yd r oxyp h e n yl)-11-a n ilin o-1H ,6H -oxa zolo[3′,4′:1,6]-
p yr id o[3,4-b]in d ole-3-on e (26). The anilinoazatoxin analog

Inhibition of DNA Topoisomerase II
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 11 2195
1
26 was prepared as described in procedure E (63%) and
215 °C; H NMR (300 MHz, DMSO-d6) δ 11.06 (s, 1H), 7.49
purified by flash chromatography eluting with 5% acetone in
(d, J ) 8.47 Hz, 1H), 7.31 (d, J ) 8.06, 1H), 7.15 (d, J ) 7.86
Hz, 1H), 7.07 (dd apparent t, J ) 7.32 Hz, 1H), 6.95-6.86 (m,
3H), 6.65 (s, 2H), 5.96 (s, 1H), 5.15 (dd, J ) 8.98 Hz, 2.92 Hz,
1H), 4.68-4.64 (m, 1H), 4.50 (t, J ) 8.68, 1H), 4.02 (dd, J )
8.52, 5.15 Hz, 1H), 3.71 (s, 6H). Anal. (C26H26N4O7) C, H, N.
1
chloroform (Rf ) 0.19): H NMR (300 MHz, CDCl3) δ 8.27 (s,
1H), 7.34-7.19 (m, 4H), 7.10 (t, J ) 7.3 Hz, 1H), 6.81 (t, J )
7.2 Hz, 1H), 6.68 (d, J ) 8.66 Hz, 2H), 6.52 (s, 2H), 6.11 (s,
1H), 5.57 (sb, 1H), 4.92 (dd, J ) 7.6, 2.52 Hz, 1H), 4.52-4.42
(m, 3H), 3.81 (db, 1H), 3.77 (s, 6H). Anal. (C26H27N3O5) C, H,
N.
(5R,11R,11a S)-4,5,11,11a -Tetr a h yd r o-5-(3,5-d im eth oxy-
4-h yd r oxyp h en yl)-11-(4-cya n oa n ilin o)-1H ,6H -oxa zolo-
[3′,4′:1,6]p yr id o[3,4-b]in d ol-3-on e (14). The (cyanoanilino)-
azatoxin was deprotected as in 11. Purification by flash
chromatography eluting with 15% acetone in CH2Cl2 (Rf )
0.29) yielded the product 14 as a white solid (71%): mp dec
(5R,11R,11a S)-4,5,11,11a -Tetr a h yd r o-5-(3,5-d im eth oxy-
4-h yd r oxyp h en yl)-11-(4-a m in oben zoxy)-1H,6H-oxa zolo-
[3′,4′:1,6]pyr ido[3,4-b]in dol-3-on e (33). The (aminobenzoxy)-
azatoxin analog 33 was prepared as described in procedure E
(82%) and purified by flash chromatography eluting with 5%
acetone in chloroform (Rf ) 0.19): 1H NMR (300 MHz, DMSO-
d6) δ 8.49 (s, 1H), 7.69 (d, J ) 8.43 Hz, 2H), 7.30 (d, J ) 8.11
Hz, 1H), 7.15 (d, J ) 7.82 Hz, 1H), 7.04 (t, J ) 7.3 Hz, 1H),
6.87 (m, 2H), 6.68 (d, J ) 8.82 Hz, 1H), 6.49 (s, 2H), 5, 87 (s,
1H), 5.12 (dd, J ) 8.74, 2.74 Hz, 1H), 4.55 (m, 1H), 4.45 (t, J
) 8.7 Hz, 1H), 4.03 (dd, J ) 8.2, 5.42 Hz, 1H), 3.72 (s, 3H),
3.66 (s, 6H). Anal. (C28H29N3O7) C, H, N.
(5R,11R,11a S)-4,5,11,11a -Tetr a h yd r o-5-(3,5-d im eth oxy-
4-(car bom eth oxyoxy))-11-(4-flu or oan ilin o)-1H,6H-oxazolo-
[3′,4′:1,6]p yr id o[3,4-b]in d ol-3-on e (10a ). The protected (flu-
oroanilino)azatoxin analog 10a was prepared as described in
procedure A and purified by flash chromatography eluting with
6% acetone in chloroform (Rf ) 0.30): 1H NMR (300 MHz,
DMSO-d6) δ 7.31 (d, J ) 8.05 Hz, 1H), 7.08 (m, 2H), 6.96 (m,
3H), 6.81 (m, 2H), 6.65 (s, 2H), 5.94 (s, 1H), 5.66 (d, J ) 8.31
Hz, 1H), 4.87 (dd, J ) 2.81, J ) 8.04 Hz, 1H), 4.62 (m, 1H),
4.49 (t, J ) 8.5 Hz, 1H), 4,18 (dd, J ) 5.27, J ) 8.04 Hz, 1H),
3.77 (s, 3H), 3.71 (s, 6H).
(5R,11R,11a S)-4,5,11,11a -Tetr a h yd r o-5-(3,5-d im eth oxy-
4-(car bom eth oxyoxy))-11-(4-n itr oan ilin o)-1H,6H-oxazolo-
[3′,4′:1,6]p yr id o[3,4-b]in d ol-3-on e (10b). The protected (ni-
troanilino)azatoxin analog 10b was prepared as described in
procedure A and purified by flash chromatography eluting with
10% acetone in chloroform (Rf ) 0.42): 1H NMR (300 MHz,
CDCl3) δ 8.76 (s, 1H), 8.14 (d, J ) 9.17 Hz, 2H), 7.35 (m, 2H),
7.23 (t, J ) 7.78 Hz, 1H), 7.14 (t, J ) 7.27 Hz, 1H), 6.76 (d, J
) 9.11 Hz, 2H), 6.46 (s, 2H), 6.06 (s, 1H), 5.17 (m, 2H), 4.47
(m, 2H), 4.34 (m, 1H), 3.86 (s, 3H), 3.63 (s, 6H).
(5R,11R,11a S)-4,5,11,11a -Tetr a h yd r o-5-(3,5-d im eth oxy-
4-(car bom eth oxyoxy)-11-(4-cyan oan ilin o)-1H,6H-oxazolo-
[3′,4′:1,6]p yr id o [3,4-b]in d ol-3-on e (10c). The protected
(cyanoanilino)azatoxin analog 10c was prepared as described
in procedure A and purified by flash chromatography eluting
with 6% acetone in CH2Cl2 (Rf ) 0.35): 1H NMR (300 MHz,
CDCl3) δ 8.39 (s, 1H), 7.52 (d, J ) 8.59 Hz, 2H), 7.35-7.26
(m, 3H), 7.14 (t, J ) 7.58 Hz, 1H), 6.73 (d J ) 8.73 Hz, 2H),
6.51 (s, 2H), 6.11 (s, 1H), 5.02 (dd, J ) 8.50, 2.03 Hz, 1H),
4.46-4.36 (m, 3H), 3.89 (s, 3H), 3.70 (s, 6H).
1
190-194 °C; H NMR (300 MHz, DMSO-d6) δ 11.10 (s, 1H),
8.49 (s, 1H),7.48 (d, J ) 8.32 Hz, 2H), 7.30 (d, J ) 8.03 Hz,
1H), 7.14 (d, J ) 7.84 Hz, 1H), 7.05 (dd apparent t, J ) 7.33
Hz, 1H), 6.94-6.83 (m, 3H), 6.49 (s, 2H), 5.88 (s, 1H), 5.15
(dd, J ) 8.59, 2.40 Hz, 1H), 4.56-4.52 (m, 1H), 3.98 (dd, J )
7.99, 5.70 Hz, 1H), 3.66 (s, 6H). Anal. (C27H27N4O5) C, H, N.
Ack n ow led gm en t. We would like to thank Michael
L. Spera for his help in obtaining the elemental analysis.
This research was supported by the National Institutes
of Health (CA 54347).
Refer en ces
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(5R,11R,11a S)-4,5,11,11a -Tetr a h yd r o-5-(3,5-d im eth oxy-
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(10.8 mg, 73%) as a white solid: mp slow dec 150 °C; [R]22
D
1
-139° (c 0.3, DMSO); H NMR (300 MHz, DMSO-d6) δ 8.48
(s, 1H), 7.28 (d, J ) 8.0 Hz, 1H), 7.09-7.04 (m, 2H), 7.01-6.86
(m, 2H), 6.78 (dd, J ) 8.92 Hz, J ) 4.55 Hz, 2H), 6.49 (s, 2H),
5.86 (s, 1H), 5.65 (d, J ) 8.26 Hz, 2H), 4.85 (dd, J ) 8.14 Hz,
J ) 2.95 Hz, 1H), 4.53-4.49 (m, 1H), 4.41 (t, J ) 4.95 Hz,
1H), 4.12 (dd, J ) 8.07 Hz, J ) 5.41 Hz, 1H), 3.66 (s, 6H).
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