Synthesis, antitumor activity, and mechanism of action of benzo[a]pyrano[3,2-h]acridin-7-one analogues of acronycine

Article abstract of DOI:10.1021/jm0602007

Twenty-two derivatives belonging to the cis-1,2-diacyloxy-6-methoxy-3,3,14- trimethyl-1,2,3,14-tetrahydro-7H-benzo[a]pyrano[3,2-h]acridin-7-one series were synthesized in nine steps starting from 3,5-dimethoxy-acetanilide (5) and 2-methoxy-1-naphthalenecarboxylic acid (7). Most of them exhibited submicromolar cytotoxicity when tested against murine leukemia (L1210) and human epidermoid carcinoma (KB-3-1) cell lines. The cytotoxic activity correlated strongly with the ability of the compounds to form covalent adducts with purified DNA. Among the most active compounds, 25, with IC₅₀ values of 0.7 and 0.15 μM against L1210 and KB-3-1, respectively, was selected for evaluation in vivo against Colon 38 adenocarcinoma implanted in mice. This compound was active at 3 mg/kg iv (day 12 and 24) with 3/7 tumor free mice by day 80.

Full text of DOI:10.1021/jm0602007

J. Med. Chem. 2006, 49, 3383-3394
3383
Synthesis, Antitumor Activity, and Mechanism of Action of Benzo[a]pyrano[3,2-h]acridin-7-one
Analogues of Acronycine
Tuan Minh Nguyen,† Chavalit Sittisombut,† Sabrina Boutefnouchet,† Marie-Christine Lallemand,† Sylvie Michel,† Michel Koch,†
Franc¸ois Tillequin,*,† Romain Mazinghien,‡ Ame´lie Lansiaux,‡ Marie-He´le`ne David-Cordonnier,‡ Bruno Pfeiffer,§
Laurence Kraus-Berthier,§ Ste´phane Le´once,§ and Alain Pierre´§
Laboratoire de Pharmacognosie de l’UniVersite´ Rene´ Descartes, U.M.R./C.N.R.S. No. 8638, Faculte´ des Sciences Pharmaceutiques et
Biologiques, 4 AVenue de l’ObserVatoire, 75006 Paris, France, INSERM U-814, IRCL, UniVersite´ de Lille II, Place de Verdun,
59045 Lille Cedex, France, and Institut de Recherches SerVier, DiVision Recherche Cance´rologie,
125 Chemin de Ronde, 78290 Croissy sur Seine, France
ReceiVed February 21, 2006
Twenty-two derivatives belonging to the cis-1,2-diacyloxy-6-methoxy-3,3,14-trimethyl-1,2,3,14-tetrahydro-
7H-benzo[a]pyrano[3,2-h]acridin-7-one series were synthesized in nine steps starting from 3,5-dimethoxy-
acetanilide (5) and 2-methoxy-1-naphthalenecarboxylic acid (7). Most of them exhibited submicromolar
cytotoxicity when tested against murine leukemia (L1210) and human epidermoid carcinoma (KB-3-1) cell
lines. The cytotoxic activity correlated strongly with the ability of the compounds to form covalent adducts
with purified DNA. Among the most active compounds, 25, with IC50 values of 0.7 and 0.15 µM against
L1210 and KB-3-1, respectively, was selected for evaluation in vivo against Colon 38 adenocarcinoma
implanted in mice. This compound was active at 3 mg/kg iv (day 12 and 24) with 3/7 tumor free mice by
day 80.
Chart 1. Acronycine (1), Acronycine Epoxide (2), and
(()-cis-1,2-Diacetoxy-1,2-dihydrobenzo[b]acronycine (3)
Introduction
The natural pyranoacridone acronycine (1, Chart 1), first
isolated from Acronychia baueri Schott (Rutaceae) in 1948,1,2
was subsequently shown to exhibit a broad spectrum of activity
against numerous experimental tumor models, including sar-
coma, myeloma, carcinoma, and melanoma.2,3 Nevertheless, the
moderate potency and very low solubility in aqueous solvents
of this alkaloid severely hampered the subsequent clinical trials,
which gave only poor results.4
Following the isolation of the unstable acronycine epoxide
(2) from several New-Caledonian Sarcomelicope species, efforts
toward the design of more potent derivatives were guided by a
hypothesis of bioactivation of the 1,2-double bond of acronycine
into the corresponding oxirane in vivo.2,5 Significant improve-
ments in terms of potency were obtained with derivatives
modified in the pyran ring, which had a similar reactivity toward
nucleophilic agents as acronycine epoxide but an improved
chemical stability. Such compounds are exemplified by diesters
of cis-and trans-1,2-dihydroxy-1,2-dihydroacronycine, which
exhibited marked antitumor properties, with a broadened
spectrum and increased potency when compared to acronycine.6
Further on, structural analogues in the related benzo[b]acrony-
cine series, including an additional aromatic ring linearly fused
on the natural alkaloid skeleton, were developed, and several
cis-1,2-dihydroxy-6-methoxy-3,3,14-trimethyl-1,2,3,14-tetrahy-
dro-7H-benzo[b]pyrano[3,2-h]acridin-7-one esters and diesters
proved even more potent.7,8 A representative of this latter series,
diacetate 3, currently under phase I clinical trials under the code
S23906-1, displayed a particularly impressive broad preclinical
antitumor spectrum. Indeed, when evaluated against aggressive
orthotopic models of human ovarian, lung, and colon cancers,
compound 3 demonstrated comparable and/or better activity than
paclitaxel, vinorelbine, and irinotecan, respectively.9 The mech-
anism of its action was shown to imply alkylation of the 2-amino
group of DNA guanine residues by the carbocation resulting
from the elimination of the ester leaving group at position 1 of
the drug.8,10
In a continuation of our studies on the structure-activity
relationships in the acronycine series,11 we describe here the syn-
thesis and the biological properties of 6-methoxy-3,3,14-tri-
methyl-3,14-dihydro-7H-benzo[a]pyrano[3,2-h]acridin-7-one (4)
and of related cis-1,2-dihydro-1,2-diol esters and diesters. The
aim of the present work is to determine the influence of the
mode of fusion of the additional aromatic ring onto the natural
acronycine tetracyclic core on DNA alkylation and cytotoxic
and antitumor properties.
Chemistry
* To whom correspondence should be addressed. Tel: + 33-1-53-73-
98-10. Fax: + 33-1-40-46-96-58. E-mail: francois.tillequin@univ-paris5.fr.
† Laboratoire de Pharmacognosie de l’Universite´ Rene´ Descartes.
‡ INSERM U-524 et Centre Oscar Lambret.
The well-documented facile decarboxylation of 2-amino-1-
naphthalenecarboxylic acid derivatives12 and the toxicity of
2-naphthylamine, which had to be avoided as starting material
§ Institut de Recherches Servier.
10.1021/jm0602007 CCC: $33.50 © 2006 American Chemical Society
Published on Web 05/09/2006

3384 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 11
Nguyen et al.
Scheme 1. Synthesis of 6-Methoxy-3,3,14-trimethyl-3,14-dihydro-7H-benzo[a]pyrano[3,2-h]acridin-7-one (4)a
a Reagents and conditions: (i) SOCl2, 60 °C; (ii) AlCl3, CH2Cl2, 3 h at 0 °C, and 12 h at rt, (55%); (iii) NaH, DMF, rt (95%); (iv) HBr/H2O/CH3COOH,
reflux (94%); (v) K2CO3/KI, DMF, 65 °C (40 °C); (vi) DMF, 130 °C (95%); (vii) K2CO3/MeI, Me2CO, reflux (90%); (viii), NaH/MeI, Me2CO, reflux
(75%); (ix) NaH/(CH3)2SO4, DMF, rt (65%).
in the case of a large-scale synthesis, did not permit us to build
up the pentacyclic basic benzo[a]pyrano[3,2-h]acridin-7-one
core using a strategy similar to those previously developed in
the isomeric benzo[b]pyrano[3,2-h]acridin-7-one series.7,8 Con-
sequently, the general approach was inspired by the synthesis
of acronycine developed by Lewis et al.,13 which involved base-
catalyzed cyclization of an intermediate diphenyl ketone to
construct the acridone skeleton (Scheme 1).14
rolo[1,2,3-fg]acridin-15-one (16). In addition, a fifth compound
was obtained in mixture with 16, and the two compounds could
only be separated as their methylated derivatives 17 and 18.
All these secondary products resulted from the C-alkylation of
10 or 13 by 3-chloro-3-methylbut-1-yne, eventually followed
by subsequent cyclization in alkaline medium.19 As expected,
Claisen rearrangement of 13 by heating at 130 °C in dimeth-
ylformamide gave the required 6-hydroxy-3,3-dimethyl-3,14-
dihydro-7H-benzo[a]pyrano[3,2-h]acridin-7-one (19) in 95%
yield.
Methylation of 19 in acetone, using potassium carbonate as
base and methyl iodide as alkylating agent, gave 6-hydroxy-
3,3,14-trimethyl-3,14-dihydro-7H-benzo[a]pyrano[3,2-h]acridin-
7-one (20) in almost quantitative yield. In contrast, when the
reaction was carried out with dimethyl sulfate in dimethylfor-
mamide, in the presence of an excess of sodium hydride, the
desired 6-methoxy-3,3,14-trimethyl-3,14-dihydro-7H-benzo[a]-
pyrano[3,2-h]acridin-7-one (4) was obtained in 65% yield,
together with smaller amounts of 6,7-dimethoxy-3,3-dimethyl-
3H-benzo[a]pyrano[3,2-h]acridin (21, Chart 2) and 6-methoxy-
3,3-dimethyl-3,14-dihydro-7H-benzo[a]pyrano[3,2-h]acridin-7-
one (22). In a similar way, the unresolved mixture obtained in
the course of the synthesis of 13 was converted into the
corresponding O-methyl derivatives 17 and 18. A phase-
sensitive NOESY experiment performed on 18 permitted us to
ascribe unambiguously the linear fusion of the dihydrofuran to
the benzo[a]acridinone core.
Friedel-Crafts reaction of 3,5-dimethoxyacetanilide (5)15 with
2-methoxy-1-naphthoyl chloride (6), prepared extemporaneously
by treatment of 2-methoxy-1-naphthalenecarboxylic acid (7)16
with thionyl chloride, afforded 2-methoxy-1-naphthyl (6-aceta-
mido-2,4-dimethoxy)phenyl ketone (8) in 55% yield. Cyclization
of 8 to 9,11-dimethoxybenzo[a]acridin-12(7H)-one (9) was
achieved in 95% yield by the use of sodium hydride in
dimethylformamide. Treatment of 9 with hydrogen bromide in
acetic acid gave the required 9,11-dihydroxybenzo[a]acridin-
12(7H)-one (10) in 94% yield, accompanied by minute amounts
of 11-hydroxy-9-methoxybenzo[a]acridin-12(7H)-one (11, Chart
2). Construction of the dimethylpyran ring onto the phenol at
9-position of 10 was performed by a Claisen rearrangement of
the corresponding dimethylpropargyl ether.7a,17 Treatment of 10
with 3-chloro-3-methylbut-1-yne (12)18 at 65 °C in dimethyl-
formamide, in the presence of potassium carbonate and potas-
sium iodide, gave the desired 11-hydroxy-9-(1,1-dimethylpropyn-
1-oxy)benzo[a]acridin-12(7H)-one (13) isolated in 40% yield
after purification by column chromatography. This compound
was accompanied by 3% of 11-hydroxy-9-(6-hydroxy-1,1,6-
trimethylhepta-2,4-diynyloxy)-12H-benzo[a]acridin-12(7H)-
one (14, Chart 2), 2% of 10,12-dihydroxy-9,9-dimethyl-8-
methylidene-8,9-dihydro-15H-benzo[a]pyrrolo[1, 2,3-fg]acridin-
13-one (15), and 9% of 12-hydroxy-9,9-dimethyl-8-methylidene-
8,9-dihydro-10-(1,1-dimethylpropyn-1-oxy)-15H-benzo[a]pyr-
The (()-cis-diol 23, accompanied by small amounts of the
corresponding keto alcohol 24 (Chart 3),8 was conveniently
obtained by catalytic osmium tetroxide oxidation of 4 using
N-methylmorpholine N-oxide to regenerate the oxidizing agent
(Scheme 2).7a,20 Treatment of diol 23 with an excess of acylating
reagent, acyl anhydride, or acyl chloride afforded the corre-

Antitumor Benzo[a]pyrano[3,2-h]acridin-7-ones
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 11 3385
Scheme 2. Synthesis of
Chart 2. Compounds 11, 14-18, and 20-22
(()-cis-1,2-Dihydroxy-6-methoxy-3,3,14-trimethyl-
1,2,3,14-tetrahydro-7H-benzo[a]pyrano[3,2-h]acridin-7-one
Esters and Diesters
Chart 3. Compounds 24 and 44
sponding diesters exemplified by diacetate 25, dipropionate 26,
diisovalerate 27, and dipentenoate 28. Under controlled condi-
tions, monoesters at the less hindered 2-position, 29-33, were
obtained. Treatment of monovalerate 31, monopentenoate 32,
and monobenzoate 33 with excess acetic anhydride led to the
mixed esters 34, 35, and 36, respectively. The reaction of diol
23 with N,N′-carbonyldiimidazole in 2-butanone under reflux
afforded the cyclic carbonate 37. Finally, di-N,N-diethylcar-
bamate 38 and mono-N,N-dimethylcarbamate 39, whose coun-
terparts in the benzo[b]pyrano[3,2-h]acridin-7-one series had
been recently prepared,7b were obtained upon treatment of diol
23 with N,N-diethylcarbamoyl and N,N-dimethylcarbamoyl
chloride, respectively, in tetrahydrofuran, in the presence of
potassium hydride (Scheme 3).
To better investigate the structure-activity relationships, the
diacetate analogous to 25 was prepared in the isomeric benzo-
[a]pyrano[3,2-h]acridin series (Scheme 4). For this purpose, 6,7-
dimethoxy-3,3-dimethyl-3H-benzo[a]pyrano[3,2-h]acridin (21)
was submitted to catalytic osmium tetroxide oxidation. The
expected cis-diol 40 was obtained in a moderate 15% yield under
those conditions, accompanied by the keto alcohol 41 and the
diol 42 (Chart 4), both isolated in 16% yield from the reaction
mixture. Acetylation of 40 with acetic anhydride afforded the
desired (()-cis-1,2-Diacetoxy-6,7-dimethoxy-3,3-dimethyl-3H-
benzo[a]pyrano[3,2-h]acridine (43). Similarly, acetylation of 24
gave 2-acetoxy-6-methoxy-3,3,14-trimethyl-1,2,3,14-tetrahydro-
7H-benzo[a]pyrano[3,2-h]acridin-1,7-dione (44, Chart 3).
Results and Discussion
All the new benzo[a]acronycine derivatives were first evalu-
ated in vitro for their cytotoxicity against two tumor cell lines,
a murine leukemia cell line (L1210) and a human epidermoid
carcinoma cell line (KB-3-1). The results (IC50) are reported in
Table 1. As expected, all (()-cis-1,2-dihydroxy-6-methoxy-3,3,-
14-trimethyl-1,2,3,14-tetrahydro-7H-benzo[a]pyrano[3,2-h]acri-
din-7-one esters and diesters 25-35, as well as the cyclic
carbonate 37 and the carbamates 38 and 39, exhibited cytotoxic
properties, with submicromolar IC50 (between 0.06 and 1 µM)
against L1210 cells. It is worth noticing that these compounds
were significantly more potent on the solid tumor KB-3-1 cell
line than on the L1210 leukemia (8-fold increase for compound
34, 0.1 versus 0.8µM), as previously observed for their

3386 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 11
Nguyen et al.
Scheme 3. Synthesis of
Chart 4. Compounds 41 and 42
(()-cis-1,2-Dihydroxy-6-methoxy-3,3,14-trimethyl-
1,2,3,14-tetrahydro-7H-benzo[a]pyrano[3,2-h]acridin-7-one
Carbamates
Table 1. Cytotoxicity of Compounds 4, 21, 23, 25-41, 43, and 44 in
Comparison with Acronycine (1) and S 23906-1 (3)
cytotoxicity, IC50, µMa
% of L1210
cells in S phase
[concn, µM]b
in vitro
DNA
L1210
cells
KB-3-1
cells
compound
alkylationc
1 (acronycine) 23
3 (S 23906-1) 0.7
3.7
0.1
8.6
33
nt
73 [5]
na
nt
++e
nt
4
2.5
28
21
23
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
43
44
nt
49
32
0.15
0.1
0.15
0.13
0.2
0.1
0.2
0.25
0.3
0.1
nt
0
++
+
++
++
++
++
+
0
+
++
++
nt
0
+
++
0
0.7
0.5
0.7
0.5
0.7
0.5
1.0
0.8
0.8
0.8
0.7
insoluble
0.06
0.9
0.5
1.4
0.5
8.0
2.0
68 [5]
70 [5]
60 [10]
66 [10]
76 [5]
69 [5]
73 [5]
70 [5]
73 [2.5]
76 [5]
78 [5]
insoluble
64 [0.25]
43 [10]
73 [10]
nt
Scheme 4. Synthesis of
(()-cis-1,2-Diacetoxy-6,7-dimethoxy-3,3-dimethyl-
3H-benzo[a]pyrano[3,2-h]acridine 43
0.1
insoluble
0.015
0.8
0.1
6.3
2.0
7.2
6.3
na
nt
nt
0
0
0
a Inhibition of cell proliferation measured by the MTT assay (mean of
at least three values obtained in separate experiments). b Highest percentage
of L1210 cells arrested in S phase after a 21 h exposure to the indicated
concentration. Untreated control: 32% on average; na: inactive; nt: not
tested. c The capacity of the tested compounds to form complexes with
purified DNA was investigated by a gel shift assay. Symbols ++ and +
refer to strong and weak alkylation, respectively, whereas 0 means no DNA
alkylation at all.
Figure 1A) or a longer delay (24 h, Figure 1B).8 This result
clearly identifies some compounds as efficient DNA binding
agents with the most efficient one at 2 h being the diacetate
derivative 25. Inactive DNA binding molecules were also
identified (see compounds 23, 44, 41, 40, and 37). Indeed,
compounds 44 and 41 are not able to react with DNA, in full
agreement with the data previously published for their benzo-
[b]acronycine counterparts.8 Similarly, the diols 23 and 40 failed
to react with DNA, since they do not bear any reactive group
on the pyran ring, which is a major requisite element for the
alkylating reaction.10 Surprisingly, the benzo[a]acronycine
monoacetate 29 binds DNA less efficiently than the benzo[a]-
acronycine diacetate 25, by contrast with the higher reactivity
of benzo[b]acronycine monoacetate previously shown to be
more reactive than compound 3.8 In the same manner, the
monopentenoate 32 totally failed to supershift DNA, whereas
the corresponding diesters bearing an additional acetate (35) or
a second pentenoate group (28) efficiently delayed the DNA
migration as a marker of DNA binding. The same observation
was done using the isovalerate derivatives 27, 34, and 31. In
term of kinetic of reaction, some compounds achieve their
maximum of reactivity after 2 h, and further incubation for 24
h did not allow additional binding/bonding of the compounds
counterparts in the isomeric benzo[b]acronycine series.8 In
contrast, 6-methoxy-3,3,14-trimethyl-3,14-dihydro-7H-benzo-
[a]pyrano[3,2-h]acridin-7-one (benzo[a]acronycine) (4), diol 23,
keto alcohol 44, and compounds 21, 40, and 43, with an
aromatized C ring, only displayed marginal cytotoxic activity
or were found to be inactive. Therefore, the structure-
cytotoxicity relationships observed within the benzo[a]acrony-
cine series are comparable to those previously established in
the benzo[b]acronycine series.7a,8 The presence of an ester
leaving group on the pyran ring appears as an important
structural requirement to observe significant cytotoxic activity
in both series.
The perturbation of the cell cycle induced by the new benzo-
[a]acronycine derivatives was studied on L1210 cell line. As
previously observed in the isomeric benzo[b]acronycine series,
all active compounds induced accumulation in the S phase.
The compounds were also evaluated for their ability to form
covalent complexes with DNA using gel shift assay.8 As shown
in Figure 1, the various benzo[a]acronycine derivatives were
used at a fixed concentration (50 µM) and incubated with the
radiolabeled 117-bp DNA fragment for a fixed short time (2 h,

Antitumor Benzo[a]pyrano[3,2-h]acridin-7-ones
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 11 3387
observed after 2 to 3 h, but a small decrease in the alkylation
efficiency from 3 to 24 h suggested the release of the adduct
from DNA.
Finally, compound 25 was selected for an in vivo evaluation,
comparatively with compound 3, on an established C38 colon
adenocarcima (sc implantation) in mice. Although less potent
than compound 3 (Figure 3), compound 25 administered twice
(day 12, day 24) by the iv route at the optimal dose of 4 mg/kg
proved to be significantly active, inhibiting tumor growth by
more than 80% and with three of seven mice tumor-free on
day 80.
Conclusion
In summary, 1,2-dihydroxy-3,3,14-trimethyl-1,2,3,14-tetrahy-
dro-7H-benzo[a]pyrano[3,2-h]acridin-7-one esters and diesters
were markedly more potent than acronycine in terms of
cytotoxicity, when tested against L1210 and KB-3-1 cell lines.
As previously observed in the isomeric benzo[b]acronycine
series, the cytotoxic activity appeared to be strongly correlated
with the ability of the compounds to give covalent adducts with
DNA. Compound 25, although less potent than its benzo[b]-
pyrano[3,2-h]acridin-7-one counterpart 3, proved to be signifi-
cantly active in vivo on the murine C38 colon adenocarcinoma
model. The new benzo[a]acronycine series appears very promis-
ing, and various pharmacomodulations are currently being
carried out.
Figure 1. DNA bonding analysis by benzo[a]acronycine derivatives
using gel shift experiments. The various compounds (50 µM) were
incubated for 2 h (panel A) or 24 h (panel B) with the 117-bp
radiolabeled DNA substrate in 1 mM Na cacodylate buffer prior to be
subjected to electrophoresis on a 10% native polyacrylamide gel. The
lane “0” refers to the control DNA fragment alone. Bound and free
DNA fragments are referred as “b” and “f”, respectively.
Experimental Section
Chemistry. Melting points were determined on a hot stage
Reichert microscope and are uncorrected. Mass spectra were
recorded with ZQ 2000 Waters and Q-Tof1 Micromass spectrom-
eters using electrospray ionization (ESI-MS; Vc ) 30 V) or with
a Nermag R-10-10C spectrometer using desorption-chemical ion-
ization (DCI-MS; reagent gas: NH3). UV spectra (λmax in nm) were
recorded in spectroscopic grade MeOH on a Beckman Model 34
spectrophotometer. IR spectra (νmax in cm-1) were obtained from
potassium bromide pellets or sodium chloride films on a Perkin-
1
Elmer 257 instrument. H NMR [δ (ppm), J (Hz)] spectra were
run at 400 MHz and 13C NMR spectra at 75 MHz, using Bruker
AVANCE-400 and AC-300 spectrometers, respectively. When
necessary, the structures of the novel compounds were ensured and
the signals unambiguously assigned by 2D NMR techniques: 1H-
1H COSY, 1H-1H NOESY, 13C-1H HMQC, and 13C-1H HMBC.
These experiments were performed using standard Bruker micro-
programs. Column chromatographies were carried out with silica
gel 20-45 µm. Flash column chromatographies were conducted
using silica gel 60 Merck (35-70 µm) with an overpressure of
300 mbars. Microanalyses were in agreement with calculated values
(0.4%.
Cell Culture and Cytotoxicity. Cell Culture and Cytotoxicity.
L1210 and KB-3-1 cells were cultivated in RPMI 1640 or DMEM
medium, respectively (Gibco) supplemented with 10% fetal calf
serum, 2 mM L-glutamine, 100 units/mL penicillin, 100 µg/mL
streptomycin, and 10 mM HEPES buffer (pH 7.4). Cytotoxicity
was measured by the microculture tetrazolium assay (MTA) as
described.21 Cells were exposed to graded concentrations of drug
(nine serial dilutions in triplicate) for four doubling times (48 h
for L1210 cells and 96 h for KB-3-1 cells). Results are expressed
as IC50, the concentration that reduced by 50% the optical density
of treated cells with respect to the optical density of untreated
controls.
Figure 2. Kinetics of DNA bonding using electromobility shift assay.
The radiolabeled 117-bp DNA fragment was incubated with 50 µM of
27, 33, or 39 derivatives for the appropriate time indicated on the top
of the lanes (min). Bound and free DNA fragments are referred as “b”
and “f”, respectively.
to the DNA fragment (see the diacetate 25 and monoacetate
29, the di- and monopropionate 26 and 30, the acetate-
isovalerate 34 and the acetate-pentenoate 35), whereas other
derivatives presented delayed reaction, as shown by comparing
the 2 h (Figure 1, panel A) with the overnight (Figure 1, panel
B) incubation time. This is notably the case for compounds 27
and 39, the latter one clearly presenting a slow and progressive
alkylation process as revealed by kinetics measurements (Figure
2B). Kinetics of alkylation reaction using the diisovalerate 27
(Figure 2A) reveals a slow reactivity with nearly no gel shift
after 1 h of incubation. However, this compound appears to be
a very efficient DNA alkylating agent after a 24-h incubation,
suggesting a very slow isovalerate release and/or transesterifi-
cation process. By contrast, the monobenzoate derivative 33
quickly reacted with DNA with a maximum of alkylation
For the cell cycle analysis, L1210 cells (5 × 105 cells/mL) were
incubated for 21 h with various concentrations of drugs. Cells were
then fixed by 70% ethanol (v/v), washed, and incubated in PBS
containing 100 µg/mL RNAse and 50 µg/mL propidium iodide for
30 min at 20 °C. For each sample, 10 000 cells were analyzed on
an XLMCL flow cytometer (Beckman Coulter, France). Results
are expressed as the percentage of cells in the S phase of the cell
cycle.

3388 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 11
Nguyen et al.
Figure 3. In vivo evaluation of compound 25 in comparison with compound 3, on an established C38 colon adenocarcima (sc implantation) in
mice.
Antitumor Activity. The antitumor activity of the compounds
3 and 25 was evaluated on the murine colon 38 adenocarcinoma
implanted in B6D2F1 (C57B1/6 x DBA2) mice. The colon
adenocarcinoma C38 (NCI, Frederick) was introduced by sc
implantation of a tumor fragment into the dorsal flank. The
compounds were solubilized in a preparation of cremophor ELP/
ethanol (10% each in physiological saline) and administered by iv
injection 12 and 22 days after the tumor graft. Compounds were
administered at their maximal tolerated dose (MTD) of 3 mg/kg,
which was the highest nontoxic dose, and at one-third of the MTD,
1 mg/kg. A dose is considered as toxic when the weight loss is
higher than 20% or when it induces toxic deaths. Tumors were
measured twice a week and tumor volumes (Vt) were calculated
using the following formula: length (mm) × width2 (mm2)/2. At
the end of the experiment, on day 80, mice were palpated and the
number of tumor-free mice was recorded.
DNA Restriction Fragments. The 117-bp DNA fragment was
obtained from the pBS plasmid digestion using Eco RI and PVu II
restriction enzymes in their respective digestion buffers and was
then labeled at the Eco RI site using R-[32P]dATP (Amersham)
and AMV reverse transcriptase (Ozyme). The radiolabeled DNA
was then purified by electrophoresis on a nondenaturing 10% (w/
v) polyacrylamide gel with the desired 3′-end-labeled product being
cut out of the gel and eluted overnight in 500 mM ammonium
acetate, 10 mM magnesium acetate.
Gel Shift Studies. A typical cross-linking reaction consisted of
incubating 50 µM of the drug with the radiolabeled DNA in 1 mM
Na cacodylate, pH 7.0 (Tris buffer must be avoided due to the
presence of reactive amine functions) and incubated in the dark at
room temperature during the period specified in the legend. After
the desired incubation time, 5 µL of a 50% glycerol containing
tracking dyes solution was added to each DNA sample, which were
then resolved by electrophoresis under nondenaturing conditions
in 6% polyacrylamide gels for about 5 h at 300 V at room
temperature in TBE buffer (89 mM boric acid, 2.5 mM Na2EDTA,
pH 8.3). Gels were transferred to Whatman 3MM paper, dried under
vacuum at 80 °C, and then analyzed on a phosphorimager
(Molecular Dynamics 445SI).
2-Methoxy-1-naphthyl (2-Acetamido-4,6-dimethoxy)phenyl
Ketone (8). Thionyl chloride (60 mL, 405 mmol) was added
dropwise to 2-methoxynaphthalene-1-carboxylic acid (7) (30.3 g,
150 mmol). The mixture was heated at 60 °C for 3 h, and the excess
of thionyl chloride was evaporated under reduced pressure. The
acyl chloride 6 obtained was immediately dissolved in dry CH2Cl2
(100 mL) and added to an iced-cooled suspension of 3,5-
dimethoxyacetanilide (5) (25.4 g, 120 mmol) and anhydrous AlCl3
(25 g, 186.6 mmol) in dry CH2Cl2 (100 mL). The mixture was
stirred under argon at 0 °C for 3 h and then at room temperature
for 12 h. The cooled reaction mixture was poured onto ice-cooled
1 N aqueous HCl (300 mL) and extracted with CH2Cl2 (5 × 250
mL). The combined organic layers were washed with 1 M NaHCO3
solution (3 × 100 mL) and water (5 × 100 mL), dried over NaSO4,
filtered, and evaporated under reduced pressure. Purification by flash
chromatography (solvent CH2Cl2 and then CH2Cl2/MeOH 99.5:
0.5 to 95:5) gave 8 (25.1 g, 55%) as white crystals: mp 150 °C
1
(crystallized from Me2CO); H NMR (400 MHz, CDCl3) δ 2.27
(s, 3H, NHCOCH3), 3.02 (s, 3H, C6′-OCH3), 3.81 (s, 3H, C2-OCH3),
3.88 (s, 3H, C4′-OCH3), 5.99 (d, J ) 2 Hz, 1H, H-5′), 7.27 (d, J )
9 Hz, 1H, H-8), 7.32 (td, J ) 8, 1 Hz, 1H, H-7), 7.36 (td, J ) 8,
1 Hz, 1H, H-6), 7.53 (dd, J ) 8, 1 Hz, 1H, H-5), 7.79 (dd, J ) 8,
1 Hz, 1H, H-8), 7.83 (d, J ) 9 Hz, 1H, H-4), 8.17 (d, J ) 2 Hz,
1H, H-3′), 12.27 (s, 1H, NH); 13C NMR (100 MHz, CDCl3) δ 26.0
(NHCOCH3), 55.7 (C5′-OCH3 + C3′-OCH3), 57.0 (C2-OCH3), 94.6
(C-5′), 96.9 (C-3′), 109.8 (C-1′), 113.7 (C-3), 123.8 (C-7 + C-5),
126.9 (C-6), 128.0 (C-8), 128.9 (C-4a + C-1), 129.8 (C-4), 130.8
(C-8a), 144.7 (C-2′), 152.8 (C-2), 163.6 (C-4′), 165.9 (C-6′), 170.1
(NHCOCH3), 198.3 (CO); DCI-MS m/z 380 [MH]+; IR (KBr) ν
3196, 3138, 3006, 2944, 2839, 1693, 1612, 1507, 1448, 1297, 1200,
1107, 889, 823, 757 cm-1; UV λ (MeOH) (log ꢀ) 230 (4.90), 250
(sh) (4.48), 306 nm (4.24). Anal. (C22H21NO5) C, H, N.
9,11-Dimethoxybenzo[a]acridin-12(7H)-one (9). Sodium hy-
dride (4.95 g of 80% oil dispersion, 165 mmol) was added to an
ice-cooled solution of 8 (12.50 g, 33 mmol) in dry N,N-dimethyl-
formamide (150 mL). The mixture was stirred under nitrogen for
30 min at 0 °C and then for 15 h at room temperature and poured
onto ice water (1 L). The precipitate was filtered, washed with water
(4 × 200 mL), and dried in a vacuum over P2O5 to afford 9 (9.6 g,
95%) as a white amorphous solid: 1H NMR (400 MHz, CDCl3) δ
3.87 (s, 6H, 2 × OCH3), 6.34 (d, J ) 2 Hz, 1H, H-10), 6.50 (d, J
) 2 Hz, 1H, H-8), 7.48 (td, J ) 8, 1 Hz, 1H, H-3), 7.51 (d, J ) 9
Hz, 1H, H-6), 7,62 (td, J ) 8, 1 Hz, 1H, H-2), 7.90 (dd, J ) 8, 1
Hz, 1H, H-4), 8.08 (d, J ) 9 Hz, 1H, H-5), 10.06 (dd, J ) 8, 1 Hz,
1H, H-1), 11.68 (s, 1H, NH); 13C NMR (100 MHz, CDCl3) δ 56.6
(OCH3), 56.8 (OCH3), 91.4 (C-8), 94.7 (C-10), 95.4 (C-11a), 110.0
(C-12a), 118.6 (C-6), 125.5 (C-3), 126.7 (C-1), 129.2 (C-2), 129.4
(C-4), 129.9 (C-4a), 132.6 (C-12b), 135.2 (C-5), 141.7 (C-7a), 144.0
(C-6a), 162.8 (C-9), 163.6 (C-11), 178.3 (C-12); DCI-MS m/z 306
[MH]+; IR (KBr) ν 3421, 3283, 2994, 2955, 1639, 1584, 1452,
1410, 1161, 1130, 823, 749 cm-1; UV λ (MeOH) (log ꢀ) 233 (4.07),
288 (4.67), 368 (3.69), 386 nm (3.66). Anal. (C19H15NO3) C, H,
N.
9,11-Dihydroxybenzo[a]acridin-12(7H)-one (10) and 11-Hy-
droxy-9-methoxybenzo[a]acridin-12(7H)-one (11). To a solution
of 9 (2.5 g, 8.2 mmol) in acetic acid (90 mL) was added 48%
hydrogen bromide aqueous solution (90 mL). The reaction mixture
was stirred and refluxed for 4 days. The cooled mixture was poured
onto ice water (500 mL). The brown precipitate was filtered, washed
with water (4 × 100 mL), and dried in a vacuum over P2O5. Column
chromatography on silica gel (solvent, CH2Cl2 and then CH2Cl2/
MeOH, 99:1 to 90:10) gave 10 (2.14 g, 94%) as yellow sheets and
11 (0.071 g, 3%) as a yellow amorphous solid.
Compound 10: mp 312 °C (crystallized from acetone/ethyl
acetate, 1:1); 1H NMR (400 MHz, DMSO-d6) δ 6.05 (s, 1H, H-10),
6.39 (s, 1H, H-8), 7.53 (td, J ) 8, 1 Hz, 1H, H-3), 7.57 (d, J ) 9

Antitumor Benzo[a]pyrano[3,2-h]acridin-7-ones
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 11 3389
Hz, 1H, H-6), 7.70 (td, J ) 8, 1 Hz, 1H, H-2), 7.96 (dd, J ) 8, 1
Hz, 1H, H-4), 8.16 (d, J ) 9 Hz, 1H, H-5), 10.02 (dd, J ) 8, 1 Hz,
1H, H-1), 10.47 (s, 1H, C9-OH), 12.21 (s, 1H, NH), 15.00 (s, 1H,
C11-OH); 13C NMR (75 MHz, DMSO-d6) δ 91.2 (C-8), 97.2 (C-
10), 106.0 (C-11a), 110.8 (C-12a), 118.3 (C-6), 125.5 (C-3), 126.4
(C-1), 129.3 (C-2 + C-4), 129.4 (C-4a), 131.6 (C-12b), 136.1 (C-
5), 142.0 (C-7a), 142.7 (C-6a), 163.9 (C-9), 164.2 (C-11), 182.5
(CO); DCI-MS m/z 278 [MH]+; IR (KBr) ν 3344, 3048, 1639, 1584,
1506, 1460, 1421, 1270, 1208, 827 cm-1; UV λ (MeOH) (log ꢀ)
238 (5.22), 287 (5.54), 381 nm (5.44). Anal. (C17H11NO3) C, H,
N.
Compound 11: 1H NMR (400 MHz, DMSO-d6) δ 3.86 (s, 3H,
C9-OCH3), 6.23 (d, J ) 2, 1H, H-10), 6.47 (d, J ) 2, 1H, H-8),
7.56 (td, J ) 8, 1 Hz, 1H, H-3), 7.58 (d, J ) 9 Hz, 1H, H-6), 7.73
(td, J ) 8, 1 Hz, 1H, H-2), 7.99 (dd, J ) 8, 1 Hz, 1H, H-4), 8.20
(d, J ) 9 Hz, 1H, H-5), 10.02 (dd, J ) 8, 1 Hz, 1H, H-1), 12.39
(s, 1H, NH), 14.98 (s, 1H, C11-OH); 13C NMR (75 MHz, DMSO-
d6) δ 56.4 (OCH3), 89.4 (C-8), 95.2 (C-10), 104.2 (C-11a), 114.4
(C-12a), 118.6 (C-6), 126.7 (C-1), 127.0 (C-1), 129.8 (C-2), 130.0
(C-4), 131.0 (C-4a), 131.7 (C-12b), 137.1 (C-5), 154.2 (C-7a), 157.1
(C-6a), 158.4 (C-9), 165.3 (C-11), 181.0 (CO); DCI-MS m/z 292
[MH]+; IR (KBr) ν 3382, 3274, 2998, 2936, 1643, 1577, 1511,
1411, 1239, 1153, 823 cm-1; UV λ (MeOH) (log ꢀ) 213 (3.56),
251 (3.95), 285 (4.40), 299 (sh), 324 (sh), 378 nm (3.81). Anal.
(C18H13NO3) C, H, N.
Reaction of 10 with 3-Chloro-1-methylbut-1-yne (12). A
solution of 10 (3 g, 10.8 mmol) in dry N,N-dimethylformamide
(100 mL) was stirred and heated to 65 °C for 15 min, under
nitrogen, in the presence of anhydrous potassium carbonate (4.47
g, 32.4 mmol). Then, dry potassium iodide (5.38 g, 32.4 mmol)
and excess 3-chloro-3-methylbut-1-yne (6.64 g, 64.8 mmol) were
added, and the mixture was stirred and heated at 65 °C for 24 h.
After addition of cold water (200 mL), the reaction mixture was
extracted with CH2Cl2 (4 × 250 mL). The combined organic layers
were washed with water, dried over anhydrous MgSO4, filtered,
and evaporated under reduced pressure. Purification by flash
chromatography (solvent, cyclohexane and then cyclohexane/
acetone, 98:2 to 90:10) afforded successively 16 (0.4 g, 9%); an
inseparable mixture, which was further methylated to 17 and 18
(0.2 g); 13 (1.5 g, 40%), 15 (0.07 g, 2%), and 14 (0.15 g, 3%) as
yellow solid products.
11), 183.4 (CO); ESI-MS m/z 426 [MH]+, 448 [MNa]+, 464 [MK]+;
IR (NaCl) ν 3428, 3204, 3078, 3055, 2921, 2849, 1630, 1600, 1583,
1492, 1361, 1155, 1024, 752 cm-1; UV λ (MeOH) (log ꢀ) 210
(4.41), 247 (4.15), 293 (4.45), 308 (4.44), 400 nm (3.52). Anal.
(C27H23NO4) C, H, N.
10,12-Dihydroxy-9,9-dimethyl-8-methylidene-8,9-dihydro-
15H-benzo[a]pyrrolo[1,2,3-fg]acridin-13-one (15): yellow amor-
1
phous solid; H NMR (400 MHz, DMSO-d6) δ 1.56 (s, 6H, C9-
(CH3)2), 5.17 (d, J ) 3 Hz, 1H, H-1′a), 5.76 (d, J ) 3 Hz, 1H,
H-1′b), 6.23 (s, 1H, H-11), 7.63 (td, J ) 8, 1 Hz, 1H, H-3), 7.73
(td, J ) 8, 1 Hz, 1H, H-2), 7.87 (dd, J ) 8, 1 Hz, 1H, H-4), 8.31
(d, J ) 9 Hz, 1H, H-5), 8.40 (d, J ) 9 Hz, 1H, H-6), 10.16 (dd, J
) 8, 1 Hz, 1H, H-1), 10.72 (s, 1H, C10-OH), 12.94 (s, 1H, C12-
OH); 13C NMR (75 MHz, DMSO-d6) δ 27.4 [C9-(CH3)2], 46.5 (C-
9), 92.6 (C-1′), 98.4 (C-11), 104.9 (C-12a), 110.3 (C-9a), 113.7
(C-13a), 115.9 (C-6), 126.1 (C-3), 127.4 (C-1), 128.6 (C-4a), 129.7
(C-4), 129.8 (C-2), 132.9 (C-13b), 137.0 (C-5), 141.1 (C-9b), 143.6
(C-6a), 158.9 (C-8), 160.0 (C-10), 161.8 (C-12), 182.9 (CO); ESI-
MS m/z 344 [MH]+, 366 [MNa]+; IR (KBr) ν 3432, 2976, 2930,
2851, 1650, 1600, 1598, 1558, 1523, 1453, 1357, 1279, 1137, 1086,
1061, 825, 757 cm-1; UV λ (MeOH) (log ꢀ) 203 (4.34), 243 (4.31),
299 (4.50), 325 (4.21), 389 nm (3.77). Anal. (C22H17NO3) C, H,
N.
12-Hydroxy-9,9-dimethyl-8-methylidene-8,9-dihydro-10-(1,1-
dimethylpropyn-1-oxy)-15H-benzo[a]pyrrolo[1,2,3-fg]acridin-
15-one (16): yellow amorphous solid; 1H NMR (400 MHz, CDCl3)
δ 1.61 (s, 6H, C9-(CH3)2), 1.81 (s, 6H, C1′′-(CH3)2), 2.74 (s, 1H,
H-3′′), 4.99 (d, J ) 3 Hz, 1H, H-1′a), 5.60 (d, J ) 3 Hz, 1H, H-1′b),
7.14 (s, 1H, H-11), 7.58 (td, J ) 8, 1 Hz, 1H, H-3), 7.74 (td, J )
8, 1 Hz, 1H, H-2), 7.87 (dd, J ) 8, 1 Hz, 1H, H-4), 8.01 (d, J )
9 Hz, 1H, H-5), 8.25 (d, J ) 9 Hz, 1H, H-6), 10.26 (dd, J ) 8, 1
Hz, 1H, H-1), 13.05 (s, 1H, C12-OH); 13C NMR (100 MHz, CDCl3)
δ 27.7 [C9-(CH3)2], 29.8 [C1′′-(CH3)2], 46.9 (C-9), 72.3 (C-1′′), 75.2
(C-3′′), 85.2 (C-2′′), 92.2 (C-1′), 99.1 (C-11), 104.6 (C-12a), 112.8
(C-9a), 115.4 (C-13a), 115.6 (C-6), 125.7 (C-3), 126.9 (C-1), 128.3
(C-4), 129.4 (C-2), 132.5 (C-13b + C-4a), 135.4 (C-5), 140.8 (C-
9b), 142.5 (C-6a), 156.4 (C-10), 158.6 (C-8), 160.8 (C-12), 182.8
(C-13); ESI-MS m/z 410 [MH]+, 432 [MNa]+, 448 [MK]+; IR
(KBr) ν 3244, 2975, 2926, 2861, 1670, 1623, 1608, 1558, 1512,
1343, 1297, 1179, 1136, 1086, 1061, 825, 664 cm-1; UV λ (MeOH)
(log ꢀ) 246 (4.32), 299 (4.53), 417 nm (3.61). Anal. (C27H23NO3)
C, H, N.
6-Hydroxy-3,3-dimethyl-3,14-dihydro-7H-benzo[a]pyrano-
[3,2-h]acridin-7-one (19). A solution of 13 (3 g, 8.75 mmol) in
dry N,N-dimethylformamide (100 mL) was heated at 130 °C for 3
h. The solvent was removed by evaporation under reduced pressure.
Flash chromatography (solvent, cyclohexane and then cyclohexane/
acetone, 98:2 to 90:10) gave 19 (2.85 g, 95%) as a yellow
amorphous solid: 1H NMR (400 MHz, DMSO-d6) δ 1.47 (s, 6H,
2 × CH3), 5.74 (d, J ) 10 Hz, 1H, H-2), 6.12 (s, 1H, H-5), 7.17
(d, J ) 10 Hz, 1H, H-1), 7.56 (td, J ) 8, 1 Hz, 1H, H-10), 7.71
(td, J ) 8, 1 Hz, 1H, H-9), 7.93 (d, J ) 9 Hz, 1H, H-13), 7.97 (dd,
J ) 8, 1 Hz, 1H, H-11), 8.19 (d, J ) 9 Hz, 1H, H-12), 10.19 (dd,
J ) 8, 1 Hz, 1H, H-8), 11.46 (s, 1H, NH), 15.39 (s, 1H, C6-OH);
13C NMR (100 MHz, DMSO-d6) δ 28.0 (2 × CH3), 77.6 (C-3),
97.8 (C-5), 98.6 (C-14b), 106.7 (C-6a), 111.2 (C-7a), 116.7 (C-1),
118.7 (C-13), 125.7 (C-10), 126.4 (C-2), 126.6 (C-8), 129.2 (C-
11), 129.4 (C-9), 129.7 (C-11a), 131.2 (C-8a), 136.2 (C-12), 136.5
(C-14a), 143.1 (C-13a), 158.8 (C-4a), 164.4 (C-6), 183.0 (C-7);
DCI-MS m/z 344 [MH]+; IR (KBr) ν 3340, 3048, 2971, 2924, 1639,
1588, 1550, 1499, 1359, 1173, 1142, 1126, 823, 746 cm-1; UV λ
(MeOH) (log ꢀ) 245 (4.38), 295 (4.86), 331 (4.10), 398 nm (3.68).
Anal. (C22H17NO3) C, H, N.
11-Hydroxy-9-(1,1-dimethylpropyn-1-oxy)benzo[a]acridin-12-
(7H)-one (13): yellow sheets; mp 212 °C (crystallized from CH2-
1
Cl2/acetone, 1:1); H NMR (400 MHz, DMSO-d6) δ 1.72 (s, 6H,
2 × CH3), 3.89 (s, 1H, H-3′), 6.34 (d, J ) 2 Hz, 1H, H-10), 6.92
(d, J ) 2 Hz, 1H, H-8), 7.58 (td, J ) 8, 1 Hz, 1H, H-3), 7.61 (d,
J ) 9 Hz, 1H, H-6), 7.73 (td, J ) 8, 1 Hz, 1H, H-2), 8.00 (dd, J
) 8, 1 Hz, 1H, H-4), 8.21 (d, J ) 9 Hz, 1H, H-5), 10.02 (dd, J )
8, 1 Hz, 1H, H-1), 12.46 (s, 1H, NH), 14.90 (s, 1H, C11-OH); 13C
NMR (75 MHz, DMSO-d6) δ 30.4 (2 × CH3), 73.2 (C-1′), 78.9
(C-3′), 86.1 (C-2′), 95,2 (C-8), 100.3 (C-10), 107.6 (C-11a), 111.6
(C-12a), 118.9 (C-6), 126.2 (C-3), 127.0 (C-1), 129.8 (C-4), 129.9
(C-2), 130.0 (C-4a), 131.9 (C-12b), 137.0 (C-5), 141.6 (C-7a), 143.4
(C-6a), 161.6 (C-9), 164.0 (C-11), 183.2 (CO); DCI-MS m/z 344
[MH]+; IR (KBr) ν 3340, 3048, 2971, 2924, 1639, 1588, 1550,
1499, 1359, 1173, 1142, 1126, 823, 746 cm-1; UV λ (MeOH) (log
ꢀ) 221 (sh), 242 (3.42), 296 (3.82), 332 (sh), 399 nm (2.70). Anal.
(C22H17NO3) C, H, N.
11-Hydroxy-9-(6-hydroxy-1,1,6-trimethylhepta-2,4-diynyloxy)-
12H-benzo[a]acridin-12(7H)-one (14): yellow amorphous solid;
1H NMR (400 MHz, CDCl3) δ 1.47 (s, 6H, C6′-(CH3)2), 1.51 (s,
6H, C1′-(CH3)2), 5.47 (s, 1H, C6′-OH), 6.16 (s, 1H, H-10), 7.52 (s,
1H, H-8), 7.59 (td, J ) 8, 1 Hz, 1H, H-3), 7.73 (td, J ) 8, 1 Hz,
1H, H-2), 7.95 (d, J ) 9 Hz, 1H, H-6), 7.99 (dd, J ) 8, 1 Hz, 1H,
H-4), 8.23 (d, J ) 9 Hz, 1H, H-5), 10.25 (dd, J ) 8, 1 Hz, 1H,
H-1), 11.62 (br.s, 1H, NH), 15.58 (s, 1H, C11-OH); 13C NMR (75
MHz, CDCl3) δ 27.3 [C1′-(CH3)2], 32.7 [C6′-(CH3)2], 64.4 (C-5′),
78.8 (C-1′), 78.9 (C-2′ + C-3′), 79.2 (C-4′), 98.2 (C-10), 99.3 (C-
8), 99.9 (C-6′), 106.9 (C-11a), 111.4 (C-12a), 118.8 (C-6), 126.0
(C-3), 126.6 (C-1), 129.3 (C-4), 129.5 (C-2), 129.8 (C-4), 131.1
(C-12b), 137.0 (C-5), 143.7 (C-6a + C-7a), 158.7 (C-9), 165.8 (C-
6-Hydroxy-3,3,14-trimethyl-3,14-dihydro-7H-benzo[a]pyrano-
[3,2-h]acridin-7-one (20). Dry sodium carbonate (1.324 g, 9.6
mmol) was added to a solution of 19 (0.412 g, 1.2 mmol) in dry
acetone (50 mL) and the mixture was stirred under argon at 0 °C
for 30 min. Methyl iodide (0.852 g, 6 mmol) was added and the
reaction mixture was refluxed for 2 h. Methanol (40 mL) and water
(50 mL) were added to the cooled reaction mixture, and the solvents
were removed under reduced pressure. After extraction with CH2-

3390 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 11
Nguyen et al.
Cl2 (4 × 50 mL), the combined organic layers were washed with
1 M NaOH aqueous solution (3 × 30 mL) and water (5 × 50 mL),
dried over NaSO4, and evaporated under reduced pressure. Silica
gel column chromatography (solvent, cyclohexane and then cyclo-
hexane/acetone, 99.5:0.5 to 95.5) gave 20 (0.412 g, 96%) as a
yellow amorphous solid: 1H NMR (400 MHz, CDCl3) δ 1.57 (s,
6H, 2 × CH3), 4.03 (s, 3H, NCH3), 5.53 (d, J ) 10 Hz, 1H, H-2),
6.35 (s, 1H, H-5), 6.59 (d, J ) 10 Hz, 1H, H-1), 7.56 (td, J ) 8,
1 Hz, 1H, H-10), 7.60 (d, J ) 9 Hz, 1H, H-13), 7.76 (td, J ) 8, 1
Hz, 1H, H-9), 7.88 (dd, J ) 8, 1 Hz, 1H, H-11), 8.11 (d, J ) 9 Hz,
1H, H-12), 10.09 (dd, J ) 8, 1 Hz, 1H, H-8), 15.37 (s, 1H, C6-
OH); 13C NMR (75 MHz, CDCl3) δ 26.9 (2 × CH3), 44.7 (NCH3),
76.2 (C-3), 90.5 (C-5), 100.7 (C-14b), 109.1 (C-6a), 114.6 (C-7a),
115.9 (C-13), 121.3 (C-1), 123.0 (C-2), 125.5 (C-10), 126.9 (C-8),
128.2 (C-11), 128.9 (C-11a), 129.2 (C-9), 131.1 (C-8a), 135.7 (C-
12), 145.9 (C-13a + C-14a), 160.4 (C-4a), 164.8 (C-6), 182.7 (C-
7); DCI-MS m/z 358 [MH]+; IR (KBr) ν 3433, 3052, 2971, 2920,
) 8, 1 Hz, 1H, H-10), 7.63 (td, J ) 8, 1 Hz, 1H, H-9), 7.88 (d, J
) 9 Hz, 1H, H-13), 7.91 (dd, J ) 8, 1 Hz, 1H, H-11), 8.08 (d, J
) 9 Hz, 1H, H-12), 9.99 (dd, J ) 8, 1 Hz, 1H, H-8), 10.75 (s, 1H,
NH); 13C NMR (100 MHz, CDCl3) δ 28.0 (2 × CH3), 56.4 (OCH3),
77.3 (C-3), 94.9 (C-5), 100.1 (C-14b), 109.6 (C-6a), 114.2 (C-7a),
116.9 (C-1), 118.6 (C-13), 125.1 (C-10), 126.3 (C-8), 126.8 (C-2),
128.7 (C-9), 128.9 (C-11), 129.6 (C-11a), 131.7 (C-8a), 134.5 (C-
12), 138.1 (C-13a), 141.4 (C-14a), 156.9 (C-4a), 162.3 (C-6), 178.0
(C-7); ESI-MS m/z 358 [MH]+, 380 [MNa]+; IR (KBr) ν 3429,
2962, 2934, 1664, 1592, 1564, 1434, 1364, 1136, 1059, 820, 754
cm-1; UV λ (MeOH) (log ꢀ) 242 (4.39), 292 (4.82), 384 nm (3.77).
Anal. (C23H19NO3) C, H, N.
9,9-Dimethyl-12-methoxy-8-methylidene-8,9-dihydro-10-(1,1-
dimethylpropyn-1-oxy)benzo[a]pyrrolo[1,2,3-fg]acridin-13-
one (17) and 4-Methoxy-3,3,12-trimethyl-2-methylidene-2,3-
dihydrobenzo[a]furo[3,2-i]acridin-5(13H)-one (18). Compounds
17 and 18 were synthesized from the unresolved mixture obtained
in the course of the synthesis of 13, according to the procedure
described for the preparation of 4. To a solution of the mixture
(0.1 g) in dry N,N-dimethylformamide (15 mL) were added sodium
hydride (50 mg of 80% oil dispersion, 1.65 mmol) and dimethyl
sulfate (0.16 mL, 1.65 mmol). After the usual workup, the crude
reaction mixture was purified by silica gel column chromatography
(solvent, CH2Cl2 and then CH2Cl2/MeOH, 99.9:0.1 to 98:2) to give
17 (0.05 g) and 18 (0.025 g) as yellow amorphous products.
Compound 17: 1H NMR (400 MHz, CDCl3) δ 1.61 (s, 6H, C9-
(CH3)2), 1.81 (s, 6H, C1′′-(CH3)2), 2.78 (s, 1H, H-3′′), 4.04 (OCH3),
4.92 (d, J ) 3 Hz, 1H, H-1′a), 5.50 (d, J ) 3 Hz, 1H, H-1′b), 7.27
(s, 1H, H-11), 7.54 (td, J ) 8, 1 Hz, 1H, H-3), 7.71 (td, J ) 8, 1
Hz, 1H, H-2), 7.85 (dd, J ) 8, 1 Hz, 1H, H-4), 8.03 (d, J ) 9 Hz,
1H, H-5), 8.25 (d, J ) 9 Hz, 1H, H-6), 10.38 (dd, J ) 8, 1 Hz, 1H,
H-1); 13C NMR (75 MHz, CDCl3) δ 27.5 [C9-(CH3)2], 29.9 [C1′′-
(CH3)2], 45.2 (C-9), 56.5 (OCH3), 72.2 (C-1′′), 74.7 (C-3′′), 86.1
(C-2′′), 90.9 (C-1′), 96.4 (C-11), 107.6 (C-12a), 115.3 (C-9a), 115.6
(C-6), 118.2 (C-13a), 125.4 (C-3), 127.4 (C-1), 128.1 (C-4), 129.0
(C-2), 129.6 (C-4a), 132.6 (C-13b), 134.2 (C-5), 139.4 (C-9b), 144.6
(C-6a), 154.7 (C-10), 157.9 (C-8), 159.8 (C-12), 178.9 (C-13); ESI-
MS m/z 424 [MH]+, 446 [MNa]+; IR (KBr) ν 3054, 2980, 2956,
2863, 1655, 1621, 1600, 1554, 1507, 1299, 1257, 1180, 1116, 1086,
1065, 825, 723 cm-1; UV λ (MeOH) (log ꢀ) 245 (4.51), 294 (4.71),
397 nm (3.86). Anal. (C28H25NO3) C, H, N. Compound 18: 1H
NMR (400 MHz, CDCl3) δ 1.65 (s, 6H, 2 × CH3), 3.92 (s, 3H,
NCH3), 4.07 (s, 3H, OCH3), 4.32 (d, J ) 3 Hz, 1H, H-1′a), 4.73
(d, J ) 3 Hz, 1H, H-1′b), 6.77 (s, 1H, H-14), 7.53 (td, J ) 8, 1 Hz,
1H, H-9), 7.59 (d, J ) 9 Hz, 1H, H-12), 7.73 (td, J ) 8, 1 Hz, 1H,
H-8), 7.85 (dd, J ) 8, 1 Hz, 1H, H-10), 8.03 (d, J ) 9 Hz, 1H,
H-11), 10.07 (dd, J ) 8, 1 Hz, 1H, H-7); 13C NMR (75 MHz,
CDCl3) δ 29.3 (2 × CH3), 36.7 (NCH3), 44.2 (C-3), 62.8 (OCH3),
83.3 (C-1′), 90.7 (C-14), 114.9 (C-12 + C-4a), 117.4 (C-5a), 120.3
(C-3a), 125.3 (C-9), 126.6 (C-1), 128.1 (C-10), 129.0 (C-10a), 129.2
(C-8), 131.6 (C-6a), 134.5 (C-11), 142.5 (C-12a), 145.2 (C-13a),
158.0 (C-14a), 160.3 (C-4), 172.3 (C-2), 179.0 (C-5); ESI-MS m/z
372 [MH]+, 394 [MNa]+; IR (KBr) ν 2957, 2920, 2848, 1694, 1622,
1594, 1520, 1482, 1436, 1206, 1094, 1025, 951, 923 cm-1; UV λ
(MeOH) (log ꢀ) 203 (4.48), 245 (4.36), 295 (4.57), 342 (3.90), 399
nm (3.70). Anal. (C24H21NO3) C, H, N.
1627, 1580, 1542, 1456, 1344, 1262, 1172, 1138, 819, 753 cm-1
;
UV λ (MeOH) (log ꢀ) 247 (4.23), 280 (sh), 303 (4.69), 411 nm
(3.62). Anal. (C23H19NO3) C, H, N.
Methylation of 19 with Dimethyl Sulfate. Sodium hydride (1.05
g of 80% oil dispersion, 35 mmol) was added to an iced-cooled
solution of 19 (2 g, 5.83 mmol) in dry N,N-dimethylformamide
(100 mL) and the mixture was stirred under argon for 30 min at 0
°C. After addition of dimethyl sulfate (3.3 mL, 35 mmol), the
reaction mixture was stirred at room temperature for 4 h, poured
carefully onto ice water, and extracted with ethyl acetate (5 × 100
mL). The combined organic layers were washed with 1 M NaOH
aqueous solution (3 × 150 mL) and water (3 × 250 mL), dried
over NaSO4, and evaporated under reduced pressure. Flash chro-
matography (solvent, CH2Cl2 and then CH2Cl2/MeOH, 99.5:0.5 to
90:10) gave successively 21 as an amorphous orange solid (216.3
mg, 10%) and 4 (1.41 g, 65%) and 22 (416 mg, 20%) as yellow
amorphous solids.
6-Methoxy-3,3,14-trimethyl-3,14-dihydro-7H-benzo[a]pyrano-
[3,2-h]acridin-7-one (4): 1H NMR (400 MHz, CDCl3) δ 1.56 (s,
6H, 2 × CH3), 3.92 (s, 3H, NCH3), 4.02 (s, 3H, OCH3), 5.54 (d, J
) 10 Hz, 1H, H-2), 6.39 (s, 1H, H-5), 6.60 (d, J ) 10 Hz, 1H,
H-1), 7.50 (td, J ) 8, 1 Hz, 1H, H-10), 7.53 (d, J ) 9 Hz, 1H,
H-13), 7.66 (td, J ) 8, 1 Hz, 1H, H-9), 7.82 (dd, J ) 8, 1 Hz, 1H,
H-11), 8.00 (d, J ) 9 Hz, 1H, H-12), 9.90 (dd, J ) 8, 1 Hz, 1H,
H-8); 13C NMR (75 MHz, CDCl3) δ 27.0 (2 × CH3), 45.1 (NCH3),
56.5 (OCH3), 76.4 (C-3), 95.2 (C-5), 103.0 (C-14b), 113.5 (C-6a),
116.2 (C-13), 119.1 (C-7a), 121.6 (C-1), 123.6 (C-2), 125.3 (C-
10), 127.0 (C-8), 128.0 (C-11), 128.7 (C-9), 129.4 (C-11a), 131.2
(C-8a), 134.2 (C-12), 144.9 (C-13a + C-14a), 158.4 (C-4a), 162.2
(C-6), 179.4 (C-7); DCI-MS m/z 372 [MH]+; IR (KBr) ν 3433,
3048, 2967, 2920, 2850, 1623, 1592, 1511, 1460, 1340, 1204, 1134,
819, 753 cm-1; UV λ (MeOH) (log ꢀ) 245 (4.38), 291 (sh), 298
(4.67), 390 nm (3.79). Anal. (C24H21NO3) C, H, N.
6,7-Dimethoxy-3,3-dimethyl-3H-benzo[a]pyrano[3,2-h]acri-
din (21): 1H NMR (400 MHz, DMSO-d6) δ 1.49 (s, 6H, 2 × CH3),
3.89 (s, 3H, C7-OCH3), 4.04 (s, 3H, C6-OCH3), 5.68 (d, J ) 10
Hz, 1H, H-2), 6.69 (s, 1H, H-5), 7.45 (d, J ) 10 Hz, 1H, H-1),
7.66 (td, J ) 8, 1 Hz, 1H, H-10), 7.71 (td, J ) 8, 1 Hz, 1H, H-9),
7.79 (d, J ) 9 Hz, 1H, H-13), 7.98 (dd, J ) 8, 1 Hz, 1H, H-11),
8.03 (d, J ) 9 Hz, 1H, H-12), 9.45 (dd, J ) 8, 1 Hz, 1H, H-8); 13
C
Catalytic Osmium Tetroxide Oxidation of 4. Compound 4
(2.43 g, 6.55 mmol) was added to a solution of osmium tetroxide
(2.5% in 2-methyl-2-propanol) (5.5 mL) and N-methylmorpholine
N-oxide dihydrate (0.97 g, 10.32 mmol) in t-BuOH/THF/H2O (10:
3:1, v/v/v, 100 mL). The reaction mixture was stirred at room
temperature for 4 days. After addition of saturated aqueous
NaHSO3, the mixture was stirred for 1 h and then extracted with
CH2Cl2 (6 × 150 mL). The combined organic layers were dried
over Na2SO4 and evaporated under reduced pressure. Flash chro-
matography (solvent, CH2Cl2 then CH2Cl2/MeOH, 99.5:0.5 to 90:
10) gave successively 24 (0.264 g, 10%) as bright yellow crystals
and 23 (1.89 g, 71%) as a white amorphous solid.
NMR (100 MHz, DMSO-d6) δ 28.4 (2 × CH3), 57.1 (C6-OCH3),
62.2 (C7-OCH3), 78.2 (C-3), 99.0 (C-5), 107.5 (C-14b), 110.6 (C-
6a), 115.3 (C-7a), 118.6 (C-1), 126.2 (C-2), 127.4 (C-10), 128.0
(C-8), 128.8 (C-9), 128.9 (C-13), 129.6 (C-11), 131.8 (C-8a +
C-11a), 133.9 (C-12), 147.0 (C-14a), 151.5 (C-13a), 155.4 (C-4a),
157.4 (C-4a), 165.4 (C-6); DCI-MS m/z 372 [MH]+; IR (NaCl) ν
2974, 2928, 2849, 1612, 1592, 1557, 1469, 1435, 1350, 1307, 1197,
1130, 1030, 856, 759 cm-1; UV λ (MeOH) (log ꢀ) 205 (4.40), 224
(4.48), 262 (4.37), 292 (4.58), 360 nm (3.77). Anal. (C24H21NO3)
C, H, N.
6-Methoxy-3,3-dimethyl-3,14-dihydro-7H-benzo[a]pyrano-
[3,2-h]acridin-7-one (22): 1H NMR (400 MHz, CDCl3) δ 1.47
(s, 6H, 2 × CH3), 4.03 (s, 3H, OCH3), 5.75 (d, J ) 10 Hz, 1H,
H-2), 6.33 (s, 1H, H-5), 7.20 (d, J ) 10 Hz, 1H, H-1), 7.49 (td, J
(()-cis-1,2-Dihydroxy-6-methoxy-3,3,14-trimethyl-1,2,3,14-
1
tetrahydro-7H-benzo[a]pyrano[3,2-h]acridin-7-one (23): H NMR
(400 MHz, DMSO-d6) δ 1.38 (s, 3H, CH3), 1.42 (s, 3H, CH3), 3.66

Antitumor Benzo[a]pyrano[3,2-h]acridin-7-ones
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 11 3391
(t, J ) 4.5 Hz, 1H, H-2), 3.83 (s, 3H, OCH3), 3.94 (s, 3H, NCH3),
4.63 (d, J ) 9 Hz, 1H, C1-OH), 5.05 (d, J ) 4.5 Hz, 1H, C2-OH),
5.09 (dd, J ) 9, 4.5 Hz, 1H, H-1), 6.26 (s, 1H, H-5), 7.48 (td, J )
8, 1 Hz, 1H, H-10), 7.60 (td, J ) 8, 1 Hz, 1H, H-9), 7.81 (d, J )
9 Hz, 1H, H-13), 8.01 (dd, J ) 8, 1 Hz, 1H, H-11), 8.13 (d, J )
9 Hz, 1H, H-12), 9.78 (dd, J ) 8, 1 Hz, 1H, H-8); 13C NMR (100
MHz, DMSO-d6) δ 23.4 (CH3), 26.0 (CH3), 42.9 (NCH3), 56.9
(OCH3), 64.7 (C-1), 71.2 (C-2), 78.4 (C-3), 95.9 (C-5), 104.2 (C-
14b), 114.8 (C-6a), 118.3 (C-13), 118.5 (C-7a), 125.7 (C-10), 126.5
(C-8), 128.0 (C-11), 129.1 (C-9), 129.6 (C-11a), 131.3 (C-8a), 134.4
(C-12), 145.5 (C-13a), 147.7 (C-14a), 159.0 (C-4a), 161.0 (C-6),
178.6 (C-7); DCI-MS m/z 406 [MH]+; IR (KBr) ν 3402, 3045,
H-1), 7.37 (d, J ) 9 Hz, 1H, H-13), 7.50 (td, J ) 8, 1 Hz, 1H,
H-10), 7.67 (td, J ) 8, 1 Hz, 1H, H-9), 7.82 (dd, J ) 8, 1 Hz, 1H,
H-11), 8.00 (d, J ) 9 Hz, 1H, H-12), 9.78 (dd, J ) 8, 1 Hz, 1H,
H-8). Anal. (C30H31NO7) C, H, N.
(()-cis-1,2-Diisovaleroyloxy-6-methoxy-3,3,14-trimethyl-1,2,3,-
14-tetrahydro-7H-benzo[a]pyrano[3,2-h]acridin-7-one (27): amor-
phous solid; 1H NMR (400 MHz, CDCl3) δ 0.88-0.93 (m, 12H, 2
× (CH3)2), 1.46 (s, 3H, CH3), 1.55 (s, 3H, CH3), 2.01-2.06 (m,
4H, 2 × CH, CH2), 2.17 (d, J ) 7 Hz, 2H, CH2), 3.75 (s, 3H,
NCH3), 4.05 (s, 3H, OCH3), 5.48 (d, J ) 5 Hz, 1H, H-2), 6.38 (s,
1H, H-5), 6.62 (d, J ) 5 Hz, 1H, H-1), 7.39 (d, J ) 9 Hz, 1H,
H-13), 7.52 (td, J ) 8, 1 Hz, 1H, H-10), 7.65 (td, J ) 8, 1 Hz, 1H,
H-9), 7.82 (dd, J ) 8, 1 Hz, 1H, H-11), 8.00 (d, J ) 9 Hz, 1H,
H-12), 9.79 (dd, J ) 8, 1 Hz, 1H, H-8). Anal. (C34H39NO7) C, H,
N.
2971, 2928, 1623, 1592, 1514, 1456, 1382, 1208, 1153, 819 cm-1
;
UV λ (MeOH) (log ꢀ) 249 (4.54), 294 (4.78), 310 (sh), 342 (sh),
378 (3.92), 398 nm (sh). Anal. (C24H23NO5) C, H, N.
2-Hydroxy-6-methoxy-3,3,14-trimethyl-1,2,3,14-tetrahydro-
7H-benzo[a]pyrano[3,2-h]acridin-1,7-dione (24): mp 150 °C
(crystallized in CH2Cl2/acetone, 1:1); 1H NMR (400 MHz, CDCl3)
δ 1.32 (s, 3H, CH3), 1.67 (s, 3H, CH3), 3.80 (s, 3H, NCH3), 4.04
(d, J ) 2 Hz, 1H, C2-OH), 4.11 (s, 3H, OCH3), 4.39 (d, J ) 2 Hz,
1H, H-2), 6.34 (s, 1H, H-5), 7.61 (td, J ) 8, 1 Hz, 1H, H-10), 7.66
(d, J ) 9 Hz, 1H, H-13), 7.69 (td, J ) 8, 1 Hz, 1H, H-9), 7.86 (dd,
J ) 8, 1 Hz, 1H, H-11), 8.07 (d, J ) 9 Hz, 1H, H-12), 9.91 (dd,
J ) 8, 1 Hz, 1H, H-8); 13C NMR (100 MHz, CDCl3) δ 17.9 (CH3),
27.1 (CH3), 46.0 (NCH3), 57.0 (OCH3), 76.4 (C-3), 84.7 (C-2),
95.5 (C-5), 102.1 (C-14b), 113.6 (C-6a), 116.8 (C-13), 119.7 (C-
7a), 125.9 (C-10), 127.0 (C-8), 128.1 (C-11), 129.0 (C-9), 129.8
(C-11a), 130.9 (C-8a), 134.7 (C-12), 144.3 (C-13a), 146.6 (C-14a),
166.2 (C-4a), 167.8 (C-6), 178.2 (C-7), 189.1 (C-1); ESI-MS m/z
404 [MH]+, 426 [MNa]+; IR (NaCl) ν 3434, 3057, 3022, 2974,
2920, 2844, 1669, 1628, 1602, 1580, 1564, 1517, 1406, 1209, 1108,
1070, 1029, 823, 750 cm-1; UV λ (MeOH) (log ꢀ) 214 (4.53), 241
(4.34), 296 (4.49), 330 (4.16), 391 nm (3.92). Anal. (C24H21NO5)
C, H, N.
General Procedure for the Preparation of (()-cis-1,2-Diacy-
loxy-6-methoxy-3,3,14-trimethyl-1,2,3,14-tetrahydro-7H-benzo-
[b]pyrano[3,2-h]acridin-7-ones 25-28. An ice-cooled mixture of
the appropriate acid anhydride (25, 26) or acyl chloride (27, 28)
(52.5 mmol) and dry pyridine (20 mL) was added to 23 (610 mg,
1.5 mmol) and 4-(dimethylamino)pyridine (0.005 g). After stirring
at room temperature for 2 days the mixture was evaporated under
reduced pressure (t < 40 °C) or poured on cold water (20 mL) to
give a precipitate, which was isolated by filtration. The crude
product was purified by silica gel column chromatography (solvent,
CH2Cl2/MeOH) (26-28) or by crystallization (25).
(()-cis-1,2-Bis(4-pentenoyloxy)-6-methoxy-3,3,14-trimethyl-
1,2,3,14-tetrahydro-7H-benzo[a]pyrano[3,2-h]acridin-7-one (28):
Amorphous solid; 1H NMR (400 MHz, CDCl3) δ 1.47 (s, 3H, CH3),
1.56 (CH3), 2.27 (m, 4H, 2 × OCOCH2CH2CHCH2), 2.38 (m, 4H,
2 × OCOCH2CH2CHCH2), 3.70 (s, 3H, NCH3), 4.05 (s, 3H, OCH3),
4.96 (m, 4H, 2 × OCOCH2CH2CHCH2), 5.49 (d, J ) 5 Hz, 1H,
H-2), 5.73 (m, 2H, 2 × OCOCH2CH2CHCH2), 6.38 (s, 1H, H-5),
6.60 (d, J ) 5 Hz, 1H, H-1), 7.39 (d, J ) 9 Hz, 1H, H-13), 7.50
(td, J ) 8, 1 Hz, 1H, H-10), 7.67 (td, J ) 8, 1 Hz, 1H, H-9), 7.82
(dd, J ) 8, 1 Hz, 1H, H-11), 8.00 (d, J ) 9 Hz, 1H, H-12), 9.79
(dd, J ) 8, 1 Hz, 1H, H-8). Anal. (C34H35NO7) C, H, N.
General Procedure for the Preparation of (()-cis-1-Hydroxy-
2-acyloxy-6-methoxy-3,3,14-trimethyl-1,2,3,14-tetrahydro-7H-
benzo[b]pyrano[3,2-h]acridin-7-ones 29-33. To an iced-cooled
solution of 23 (0.05 g, 0.124 mmol) in dry pyridine (5 mL) was
added the appropriate acid anhydride (29, 30, 33) or acyl chloride
(31, 32) (0.372 mmol). After stirring at room temperature for 2
days, the reaction mixture was evaporated under reduced pressure
(t < 40 °C). The crude product was purified by flash chromatog-
raphy (solvent, CH2Cl2, then CH2Cl2/MeOH).
(()-cis-1-Hydroxy-2-acetoxy-6-methoxy-3,3,14-trimethyl-1,2,3,-
14-tetrahydro-7H-benzo[a]pyrano[3,2-h]acridin-7-one (29): amor-
1
phous solid; H NMR (400 MHz, CDCl3) δ 1.44 (s, 3H, CH3),
1.56 (s, 3H, CH3), 1.93 (d, J ) 9 Hz, 1H, C1-OH), 2.01 (s, 3H,
CH3CO), 3.95 (s, 3H, NCH3), 4.01 (s, 3H, OCH3), 5.35 (dd, J )
9, 5 Hz, 1H, H-1), 5.38 (d, J ) 5 Hz, 1H, H-2), 6.34 (s, 1H, H-5),
7.49 (td, J ) 8, 1 Hz, 1H, H-10), 7.52 (d, J ) 9 Hz, 1H, H-13),
7.65 (td, J ) 8, 1 Hz, 1H, H-9), 7.82 (dd, J ) 8, 1 Hz, 1H, H-11),
8.00 (d, J ) 9 Hz, 1H, H-12), 9.83 (dd, J ) 8, 1 Hz, 1H, H-8); 13
C
NMR (100 MHz, CDCl3) δ 21.0 (CH3CO), 22.5 (CH3), 25.4 (CH3),
42.2 (NCH3), 56.4 (OCH3), 63.8 (C-1), 72.2 (C-2), 76.5 (C-3), 95.1
(C-5), 101.4 (C-14b), 114.8 (C-6a), 116.3 (C-13), 119.2 (C-7a),
125.3 (C-10), 126.8 (C-8), 128.0 (C-11), 128.7 (C-9), 129.3 (C-
11a), 130.9 (C-8a), 134.3 (C-12), 145.0 (C-13a), 146.6 (C-14a),
157.9 (C-4a), 161.7 (C-6), 171.0 (C2OCO), 179.4 (C-7); ESI-MS
m/z 448 [MH]+, 470 [MNa]+; IR (KBr) ν 3430, 2975, 2925, 1741,
1662, 1591, 1153, 820 cm-1; UV λ (MeOH) (log ꢀ) 239 (4.51),
246 (4.51), 294 (4.70), 374 (3.87), 391 nm (3.89). Anal. (C26H25-
NO6) C, H, N.
(()-cis-1,2-Diacetoxy-6-methoxy-3,3,14-trimethyl-1,2,3,14-tet-
rahydro-7H-benzo[a]pyrano[3,2-h]acridin-7-one (25): white
1
needles; mp 162 °C; H NMR (400 MHz, CDCl3) δ 1.48 (s, 3H,
CH3), 1.56 (s, 3H, CH3), 1.98 (s, 3H, CH3CO), 2.03 (s, 3H, CH3-
CO), 3.71 (s, 3H, NCH3), 4.05 (s, 3H, OCH3), 5.67 (d, J ) 5 Hz,
1H, H-2), 6.38 (s, 1H, H-5), 6.55 (d, J ) 5 Hz, 1H, H-1), 7.39 (d,
J ) 9 Hz, 1H, H-13), 7.50 (td, J ) 8, 1 Hz, 1H, H-10), 7.67 (td,
J ) 8, 1 Hz, 1H, H-9), 7.82 (dd, J ) 8, 1 Hz, 1H, H-11), 8.01 (d,
J ) 9 Hz, 1H, H-12), 9.78 (dd, J ) 8, 1 Hz, 1H, H-8); 13C NMR
(100 MHz, CDCl3) δ 20.8 (CH3CO), 21.0 (CH3CO), 23.6 (CH3),
24.5 (CH3), 42.9 (NCH3), 56.5 (OCH3), 65.6 (C-1), 69.6 (C-2),
76.3 (C-3), 95.7 (C-5), 97.7 (C-14b), 115.1 (C-6a), 115.9 (C-13),
119.5 (C-7a), 125.4 (C-10), 126.7 (C-8), 128.0 (C-11), 128.8 (C-
9), 129.4 (C-11a), 131.0 (C-8a), 134.5 (C-12), 145.3 (C-13a), 147.4
(C-14a), 158.9 (C-4a), 162.0 (C-6), 170.6 (CH3CO), 171.1 (CH3CO),
179.4 (C-7); ESI-MS m/z 490 [MH]+, 512 [MNa]+; IR (KBr) ν
3048, 2975, 2936, 1740, 1627, 1592, 1514, 1371, 1235, 1157, 1025,
904, 815, 753 cm-1; UV λ (MeOH) (log ꢀ) 247 (4.37), 292 (4.58),
311 (sh), 336 (sh), 374 (sh), 391 nm (3.78). Anal. (C28H27NO7) C,
H, N.
(()-cis-1-Hydroxy-2-propioxy-6-methoxy-3,3,14-trimethyl-
1,2,3,14-tetrahydro-7H-benzo[a]pyrano[3,2-h]acridin-7-one (30):
1
amorphous solid; H NMR (400 MHz, CDCl3) δ 1.09 (t, J ) 7.5
Hz, 3H, CH3CH2CO), 1.44 (s, 3H, CH3), 1.57 (s, 3H, CH3), 1.92
(d, J ) 9 Hz, 1H, C1-OH), 2.32 (q, J ) 7.5 Hz, 2H, CH3CH2CO),
3.95 (s, 3H, NCH3), 4.04 (s, 3H, OCH3), 5.35 (dd, J ) 9, 5 Hz,
1H, H-1), 5.39 (d, J ) 5 Hz, 1H, H-2), 6.34 (s, 1H, H-5), 7.49 (td,
J ) 8, 1 Hz, 1H, H-10), 7.52 (d, J ) 9 Hz, 1H, H-13), 7.67 (td, J
) 8, 1 Hz, 1H, H-9), 7.82 (dd, J ) 8, 1 Hz, 1H, H-11), 8.00 (d, J
) 9 Hz, 1H, H-12), 9.83 (dd, J ) 8, 1 Hz, 1H, H-8). Anal. (C27H27-
NO6) C, H, N.
(()-cis-1,2-Dipropanoyloxy-6-methoxy-3,3,14-trimethyl-1,2,3,-
14-tetrahydro-7H-benzo[a]pyrano[3,2-h]acridin-7-one (26): amor-
phous solid; 1H NMR (400 MHz, CDCl3) δ 1.06 (m, 6H, 2 × CH3),
1.47 (s, 3H, CH3), 1.56 (s, 3H, CH3), 2.18 (q, J ) 8 Hz, 2H, CH2),
2.31 (m, 2H, CH2), 3.70 (s, 3H, NCH3), 4.05 (s, 3H, OCH3), 5.48
(d, J ) 5 Hz, 1H, H-2), 6.38 (s, 1H, H-5), 6.58 (d, J ) 5 Hz, 1H,
(()-cis-1-Hydroxy-2-isovaleroyloxy-6-methoxy-3,3,14-trimethyl-
1,2,3,14-tetrahydro-7H-benzo[a]pyrano[3,2-h]acridin-7-one (31):
1
amorphous solid; H NMR (400 MHz, CDCl3) δ 0.87-0.90 (m,
6H, (CH3)2CHCH2CO), 1.43 (s, 3H, CH3), 1.52 (s, 3H, CH3), 2.01-
2.05 (m, 2H, C1-OH, (CH3)2CHCH2CO), 2.20 (d, J ) 7 Hz, 2H,
(CH3)2CHCH2CO), 3.94 (s, 3H, NCH3), 4.04 (s, 3H, OCH3), 5.33

3392 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 11
Nguyen et al.
(d, J ) 5 Hz, 1H, H-1), 5.39 (d, J ) 5 Hz, 1H, H-1), 6.33 (s, 1H,
H-5), 7.49 (td, J ) 8, 1 Hz, 1H, H-10), 7.52 (d, J ) 9 Hz, 1H,
H-13), 7.66 (td, J ) 8, 1 Hz, 1H, H-9), 7.82 (dd, J ) 8, 1 Hz, 1H,
H-11), 8.00 (d, J ) 9 Hz, 1H, H-12), 9.83 (dd, J ) 8, 1 Hz, 1H,
H-8). Anal. (C29H31NO6) C, H, N.
(()-cis-1-Hydroxy-2-(4-pentenoyloxy)-6-methoxy-3,3,14-tri-
methyl-1,2,3,14-tetrahydro-7H-benzo[a]pyrano[3,2-h]acridin-7-
one (32): amorphous solid; 1H NMR (400 MHz, CDCl3) δ 1.43 (s,
3H, CH3), 1.53 (s, 3H, CH3), 1.93 (d, J ) 9 Hz, 1H, C1-OH), 2.31
(m, 2H, OCOCH2CH2CHCH2), 2.49 (m, 2H, OCOCH2CH2CHCH2),
3.95 (s, 3H, NCH3), 4.03 (s, 3H, OCH3), 4.80 (m, 2H, OCOCH2-
CH2CHCH2), 5.32 (dd, J ) 9, 5 Hz, 1H, H-1), 5.40 (d, J ) 5 Hz,
1H, H-2), 5.66 (m, 1H, OCOCH2CH2CHCH2), 6.34 (s, 1H, H-5),
7.48 (td, J ) 8, 1 Hz, 1H, H-10), 7.52 (d, J ) 9 Hz, 1H, H-13),
7.66 (td, J ) 8, 1 Hz, 1H, H-9), 7.82 (dd, J ) 8, 1 Hz, 1H, H-11),
8.01 (d, J ) 9 Hz, 1H, H-12), 9.83 (dd, J ) 8, 1 Hz, 1H, H-8).
Anal. (C29H29NO6) C, H, N.
(()-cis-1-Hydroxy-2-benzoyloxy-6-methoxy-3,3,14-trimethyl-
1,2,3,14-tetrahydro-7H-benzo[a]pyrano[3,2-h]acridin-7-one (33):
amorphous solid; 1H NMR (400 MHz, CDCl3) δ 1.50 (s, 3H, CH3),
1.60 (s, 3H, CH3), 3.00 (br s, 1H, C1-OH), 3.98 (s, 3H, NCH3),
4.08 (s, 3H, OCH3), 5.44 (d, J ) 5 Hz, 1H, H-1), 5.64 (d, J ) 5
Hz, 1H, H-2), 6.43 (s, 1H, H-5), 7.36 (m, 1H, H-5′), 7.46-7.52
(m, 3H, H-10, H-13, H-3′), 7.61-7.66 (m, 2H, H-9, H-4′), 7.80
(dd, J ) 8, 1 Hz, 1H, H-11), 7.86 (td, J ) 8, 1.5 Hz, 1H, H-6′),
7.98 (d, J ) 9 Hz, 1H, H-12), 8.11 (td, J ) 8, 1.5 Hz, 1H, H-2′),
9.85 (dd, J ) 8, 1 Hz, 1H, H-8). Anal. (C31H27NO6) C, H, N.
General Procedure for the Preparation of (()-cis-1-Acetoxy-
2-acyloxy-6-methoxy-3,3,14-trimethyl-1,2,3,14-tetrahydro-7H-
benzo[b]pyrano[3,2-h]acridin-7-ones 34-36. Acetic anhydride
(0.098 mL, 1.03 mmol) was added to an iced-cooled solution (0
°C) of the appropriate (()-cis-1-hydroxy-2-acyloxy-6-methoxy-3,3,-
14-trimethyl-1,2,3,14-tetrahydro-7H-benzo[b]pyrano[3,2-h]acridin-
7-one (31, 32, or 33) (0.103 mmol) and 4-(dimethylamino)pyridine
(0.005 g) in dry pyridine (3 mL). After stirring at room temperature
during 15 h, the reaction mixture was evaporated under reduced
pressure (t < 40 °C) and the crude product was purified by flash
chromatography (solvent, CH2Cl2 and then CH2Cl2/MeOH).
(dd, J ) 8, 1 Hz, 1H, H-11), 8.01 (d, J ) 9 Hz, 1H, H-12), 9.80
(dd, J ) 8, 1 Hz, 1H, H-8). Anal. (C31H31NO7) C, H, N.
(()-cis-1-Acetoxy-2-benzoyloxy-6-methoxy-3,3,14-trimethyl-
1,2,3,14-tetrahydro-7H-benzo[a]pyrano[3,2-h]acridin-7-one (36):
amorphous solid; 1H NMR (400 MHz, CDCl3) δ 1.54 (s, 3H, CH3),
1.65 (s, 3H, CH3), 1.88 (s, 3H, CH3CO), 3.73 (s, 3H, NCH3), 4.09
(s, 3H, OCH3), 5.74 (d, J ) 5 Hz, 1H, H-2), 6.47 (s, 1H, H-5),
6.64 (d, J ) 5 Hz, 1H, H-1), 7.37 (m, 3H, H-13, H-3′, H-5′), 7.46-
7.50 (m, 2H, H-10, H-4′), 7.65 (td, J ) 8, 1 Hz, 1H, H-9), 7.80
(dd, J ) 8, 1 Hz, 1H, H-11), 7.85 (m, 2H, H-2′, H-6′), 7.98 (d, J
) 9 Hz, 1H, H-12), 9.80 (dd, J ) 8, 1 Hz, 1H, H-8). Anal. (C33H29-
NO7) C, H, N.
(()-cis-7-Methoxy-4,4,15-trimethyl-15,15c-dihydro-4H-benzo-
[a][1,3]dioxolo[4′,5′:4,5]pyrano[3,2-h]acridin-2,8[3aH]-dione (37).
N,N′-Carbonyldiimidazole (0.231 g, 1.35 mmol) was added to a
solution of 23 (0.109 g, 0.27 mmol) in 2-butanone (5 mL). The
reaction mixture was refluxed for 2 h under argon and after cooling,
5% aqueous NaHCO3 (7 mL) was added. The solution was extracted
with ethyl acetate (3 × 15 mL), and the combined organic layers
were dried over anhydrous MgSO4, filtered, and evaporated under
reduced pressure. Flash chromatography (solvent, cyclohexane and
then cyclohexane/acetone, 98:2 to 95:5) afforded 37 (0.066 g, 56%)
as a white amorphous solid: 1H NMR (400 MHz, CDCl3) δ 1.42
(s, 3H, CH3), 1.60 (s, 3H, CH3), 3.92 (s, 3H, NCH3), 4.02 (s, 3H,
OCH3), 4.79 (d, J ) 8 Hz, 1H, H-2), 6.27 (d, J ) 8 Hz, 1H, H-1),
6.36 (s, 1H, H-5), 7.48 (d, J ) 9 Hz, 1H, H-13), 7.51 (td, J ) 8,
1 Hz, 1H, H-10), 7.67 (td, J ) 8, 1 Hz, 1H, H-9), 7.83 (dd, J ) 8,
1 Hz, 1H, H-11), 8.00 (d, J ) 9 Hz, 1H, H-12), 9.70 (dd, J ) 8,
1 Hz, 1H, H-8); 13C NMR (100 MHz, CDCl3) δ 22.0 (CH3), 24.3
(CH3), 43.9 (NCH3), 56.6 (OCH3), 71.0 (C-1), 74.1 (C-2), 78.9
(C-3), 96.2 (C-5), 97.2 (C-14b), 115.1 (C-6a), 116.4 (C-13), 119.9
(C-7a), 125.7 (C-10), 126.5 (C-8), 128.1 (C-11), 128.9 (C-9), 129.5
(C-11a), 130.4 (C-8a), 134.4 (C-12), 145.4 (C-13a), 147.2 (C-14a),
153.7 (CO), 158.3 (C-4a), 162.8 (C-6), 179.5 (C-7); DCI-MS m/z
432 [MH]+; IR (KBr) ν 3441, 3080, 2979, 2932, 1794, 1631, 1588,
1511, 1456, 1406, 1173, 1138, 1033, 815, 749 cm-1; UV λ (MeOH)
(log ꢀ) 244 (4.54), 290 (4.78), 309 (sh), 332 (sh), 369 (sh), 388 nm
(3.96). Anal. (C25H21NO6) C, H, N.
(()-cis-1,2-Bis(N,N-diethylcarbamoyloxy)-6-methoxy-3,3,14-
trimethyl-1,2,3,14-tetrahydro-7H-benzo[a]pyrano[3,2-h]acridin-
7-one (38). Potassium hydride (0.141 g of 35% oil dispersion, 1.24
mmol) was added to a solution of 23 (0.1 g, 0.25 mmol) in dry
tetrahydrofuran (10 mL) at -10 °C. The mixture was stirred under
argon for 15 min at -10 °C and the N,N-diethylcarbamoyl chloride
(0.078 mL, 0.62 mmol) was added. After stirring at room temper-
ature for 15 h, the reaction mixture was poured carefully onto ethyl
acetate (50 mL) and saturated aqueous NaHCO3 (10 mL) (pH 8).
The organic layer was washed with water (3 × 10 mL), dried over
anhydrous MgSO4, and evaporated under reduced pressure (t < 40
°C). Flash chromatography (solvent, CH2Cl2 and then CH2Cl2/
MeOH, 99.5:0.5 to 98:2) gave 38 (0.045 g, 30%) as a white
amorphous solid: 1H NMR (400 MHz, CDCl3) δ 0.57 (t, J ) 7
Hz, 3H, CH3CH2), 0.79 (t, J ) 7 Hz, 3H, CH3CH2), 1.03 (t, J ) 7
Hz, 3H, CH3CH2), 1.20 (t, J ) 7 Hz, 3H, CH3CH2), 1.47 (s, 3H,
CH3), 1.58 (s, 3H, CH3), 2.77-2.82 (m, 2H, CH2), 2.93-2.95 (m,
1H, CH), 3.00-3.04 (m, 1H, CH), 3.23-3.27 (m, 1H, CH), 3.32-
3.36 (m, 2H, CH2), 3.77 (s, 3H, NCH3), 4.03 (s, 3H, OCH3), 5.48
(d, J ) 5 Hz, 1H, H-2), 6.37 (s, 1H, H-5), 6.48 (d, J ) 5 Hz, 1H,
H-1), 7.39 (d, J ) 9 Hz, 1H, H-13), 7.48 (td, J ) 8, 1 Hz, 1H,
H-10), 7.51 (td, J ) 8, 1 Hz, 1H, H-9), 7.81 (dd, J ) 8, 1 Hz, 1H,
H-11), 7.99 (d, J ) 9 Hz, 1H, H-12), 9.91 (dd, J ) 8, 1 Hz, 1H,
H-8); 13C NMR (100 MHz, CDCl3) δ 13.4 (2 × CH3CH2), 13.7
(CH3CH2), 13.8 (CH3CH2), 23.0 (CH3), 24.9 (CH3), 41.0 (CH2),
41.5 (CH2), 42.0 (CH2), 42.1 (CH2), 42.9 (NCH3), 56.5 (OCH3),
67.1 (C-1), 70.1 (C-2), 77.0 (C-3), 95.3 (C-5), 98.7 (C-14b), 114.7
(C-6a), 116.3 (C-13), 118.9 (C-7a), 125.3 (C-10), 127.0 (C-8), 128.0
(C-11), 128.7 (C-9), 129.3 (C-11a), 131.2 (C-8a), 134.1 (C-12),
145.1 (C-13a), 147.2 (C-14a), 154.8 (C-4a), 155.0 (C-6), 159.3
(C1OCO), 161.9 (C2OCO), 179.3 (C-7); ESI-MS m/z 604 [MH]+,
626 [MNa]+; 642 [MK]+; IR (KBr) ν 2974, 2068, 2926, 2845, 1711,
1700, 1630, 1591, 1509, 1133, 1070, 817 cm-1; UV λ (MeOH)
(()-cis-1-Acetoxy-2-isovaleroyloxy-6-methoxy-3,3,14-trimeth-
yl-1,2,3,14-tetrahydro-7H-benzo[a]pyrano[3,2-h]acridin-7-one
(34): Amorphous solid; 1H NMR (400 MHz, CDCl3) δ 0.86-0.88
(m, 6H, (CH3)2CHCH2CO), 1.47 (s, 3H, CH3), 1.57 (s, 3H, CH3),
1.99 (s, 3H, CH3CO), 2.01-2.03 (m, 1H, (CH3)2CHCH2CO), 2.16
(d, J ) 7 Hz, 2H, (CH3)2CHCH2CO), 3.71 (s, 3H, NCH3), 4.05 (s,
3H, OCH3), 5.48 (d, J ) 5 Hz, 1H, H-1), 6.38 (s, 1H, H-5), 6.55
(d, J ) 5 Hz, 1H, H-1), 7.39 (d, J ) 9 Hz, 1H, H-13), 7.50 (td, J
) 8, 1 Hz, 1H, H-10), 7.66 (td, J ) 8, 1 Hz, 1H, H-9), 7.82 (dd,
J ) 8, 1 Hz, 1H, H-11), 8.01 (d, J ) 9 Hz, 1H, H-12), 9.80 (dd,
J ) 8, 1 Hz, 1H, H-8); 13C NMR (100 MHz, CDCl3) δ 21.1 (CH3-
CO), 22.4 ((CH3)2CHCH2CO), 23.6 (CH3), 24.6 (CH3), 25.5
((CH3)2CHCH2CO), 42.9 ((CH3)2CHCH2O), 43.1 (NCH3), 56.5
(OCH3), 65.8 (C-1), 69.2 (C-2), 76.4 (C-3), 95.6 (C-5), 97.8 (C-
14b), 115.0 (C-6a), 115.9 (C-13), 119.5 (C-7a), 125.4 (C-10), 126.7
(C-8), 128.0 (C-11), 128.8 (C-9), 129.4 (C-11a), 131.0 (C-8a), 134.5
(C-12), 145.3 (C-13a), 147.4 (C-14a), 159.0 (C-4a), 162.1 (C-6),
171.0 (C1OCO), 172.6 (C2OCO), 179.3 (C-7); ESI-MS m/z 532
[MH]+, 554 [MNa]+, 570 [MK]+; IR (NaCl) ν 3053, 3016, 2983,
2963, 2920, 2868, 2851, 1744, 1738, 1635, 1592, 1573, 1513, 1490,
1460, 1430, 1368, 1219, 1150, 1090, 1070, 909, 823, 753 cm-1
;
UV λ (MeOH) (log ꢀ) 201 (4.77), 246 (4.41), 290 (4.63), 375 (3.70),
389 nm (3.73). Anal. (C31H33NO7) C, H, N.
(()-cis-1-Acetoxy-2-(4-pentenoyloxy)-6-methoxy-3,3,14-tri-
methyl-1,2,3,14-tetrahydro-7H-benzo[a]pyrano[3,2-h]acridin-7-
one (35): amorphous solid; 1H NMR (400 MHz, CDCl3) δ 1.49 (s,
3H, CH3), 1.56 (s, 3H, CH3), 1.96 (s, 3H, CH3CO), 2.30-2.41 (m,
4H, OCO(CH2)2CHCH2), 3.71 (s, 3H, NCH3), 4.05 (s, 3H, OCH3),
4.93-4.98 (m, 2H, OCOCH2CH2CHCH2), 5.48 (d, J ) 5 Hz, 1H,
H-2), 5.70-5.72 (m, 1H, OCOCH2CH2CHCH2), 6.38 (s, 1H, H-5),
6.58 (d, J ) 5 Hz, 1H, H-1), 7.39 (d, J ) 9 Hz, 1H, H-13), 7.48
(td, J ) 8, 1 Hz, 1H, H-10), 7.66 (td, J ) 8, 1 Hz, 1H, H-9), 7.82

Antitumor Benzo[a]pyrano[3,2-h]acridin-7-ones
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 11 3393
1617, 1600, 1496, 1450, 1436, 1380, 1213, 1133, 1103, 756 cm-1
(log ꢀ) 204 (4.71), 247 (4.47), 291 (4.69), 374 (3.80), 390 nm (3.82).
Anal. (C34H41N3O7) C, H, N.
;
UV λ (MeOH) (log ꢀ) 203 (4.34), 214 (4.38), 235 (4.21), 281 (4.32),
(()-cis-1-Hydroxy-2-(N,N-dimethylcarbamoyloxy)-6-methoxy-
3,3,14-trimethyl-1,2,3,14-tetrahydro-7H-benzo[a]pyrano[3,2-h]-
acridin-7-one (39). Compound 39 was obtained from 23 (0.050 g,
0.124 mmol) according to the procedure described for the prepara-
tion of 38, using N,N-dimethylcarbamoyl chloride (0.046 mL, 0.494
mmol), potassium hydride (0.085 g of 35% oil dispersion, 0.741
mmol), and dry tetrahydrofuran (8 mL). Flash chromatography
(solvent, CH2Cl2 and then CH2Cl2/MeOH, 99.5:0.5 to 98:2) gave
39 (0.019 g, 32%) as a yellow amorphous solid: 1H NMR (400
MHz, CDCl3) δ 1.48 (s, 3H, CH3), 1.52 (s, 3H, CH3), 1.65 (br.s,
1H, C1-OH), 2.73 (s, 3H, CH3N), 2.86 (s, 3H, CH3N), 3.97 (s, 3H,
NCH3), 4.02 (s, 3H, OCH3), 5.20 (d, J ) 5 Hz, H-1), 5.36 (d, J )
5 Hz, 1H, H-2), 6.33 (s, 1H, H-5), 7.50 (td, J ) 8, 1 Hz, 1H, H-10),
7.54 (d, J ) 9 Hz, 1H, H-13), 7.66 (td, J ) 8, 1 Hz, 1H, H-9),
7.81 (dd, J ) 8, 1 Hz, 1H, H-11), 8.00 (d, J ) 9 Hz, 1H, H-12),
9.86 (dd, J ) 8, 1 Hz, 1H, H-8); 13C NMR (100 MHz, CDCl3) δ
22.6 (CH3), 25.3 (CH3), 35.8 (CH3N), 36.8 (CH3N), 42.0 (NCH3),
56.3 (OCH3), 64.2 (C-1), 73.4 (C-2), 76.6 (C-3), 94.7 (C-5), 104.7
(C-14b), 114.7 (C-6a), 116.2 (C-13), 118.9 (C-7a), 125.1 (C-10),
126.7 (C-8), 127.8 (C-11), 128.5 (C-9), 129.2 (C-8a), 130.9 (C-
11a), 134.1 (C-12), 144.9 (C-13a), 146.6 (C-14a), 156.5 (C-4a),
157.8 (C-6), 161.5 (C2OCO), 179.1 (C-7); ESI-MS m/z 477 [MH]+,
499 [MNa]+; 515 [MK]+; IR (KBr) ν 3429, 2935, 2880, 1701, 1633,
1623, 1598, 1520, 1399, 1145, 1056, 823 cm-1; UV λ (MeOH)
(log ꢀ) 233 (4.28), 247 (4.42), 295 (4.62), 375 (3.75), 392 nm (3.77).
Anal. (C27H28N2O6) C, H, N.
Catalytic Osmium Tetroxide Oxidation of 21. Compound 21
(0.243 g, 0.65 mmol) was added to a solution of osmium tetroxide
(2.5% in 2-methyl-2-propanol) (0.53 mL) and N-methylmorpholine
N-oxide dihydrate (0.097 g, 1.31 mmol) in t-BuOH/THF/H2O (10:
3:1, v/v/v, 10 mL). The reaction mixture was stirred at room
temperature for 2 days. After addition of saturated aqueous
NaHSO3, the mixture was stirred for 1 h and extracted with CH2-
Cl2 (6 × 25 mL). The combined organic layers were dried over
Na2SO4 and evaporated under reduced pressure. Flash chromatog-
raphy (solvent, CH2Cl2 and then CH2Cl2/MeOH, 99.5:0.5 to 90:
10) gave successively 41 (0.04 g, 16%), 40 (0.04 g, 15%), and 42
(0.04 g, 16%) as amorphous solids.
(()-cis-1,2-Dihydroxy-6,7-dimethoxy-3,3-dimethyl-3H-benzo-
[a]pyrano[3,2-h]acridine (40): 1H NMR (400 MHz, CDCl3) δ
1.26 (s, 3H, CH3), 1.37 (s, 3H, CH3), 3.97 (s, 3H, C7-OCH3), 4.01
(d, J ) 4.5 Hz, 1H, H-2), 4.10 (s, 3H, C6-OCH3), 5.53 (d, J ) 4.5
Hz, 1H, H-1), 6.56 (s, 1H, H-5), 7.63 (td, J ) 8, 1 Hz, 1H, H-10),
7.72 (td, J ) 8, 1 Hz, 1H, H-9), 7.82 (d, J ) 9 Hz, 1H, H-13),
7.87 (dd, J ) 8, 1 Hz, 1H, H-11), 7.93 (d, J ) 9 Hz, 1H, H-12),
9.57 (dd, J ) 8, 1 Hz, 1H, H-8); 13C NMR (75 MHz, CDCl3) δ
24.8 (CH3), 29.3 (CH3), 56.5 (C6-OCH3), 62.2 (C7-OCH3), 64.6
(C-1), 70.9 (C-2), 78.2 (C-3), 98.7 (C-5), 105.0 (C-14b), 111.3 (C-
6a), 115.3 (C-7a), 126.9 (C-10), 127.6 (C-8), 128.1 (C-9), 128.3
(C-13), 129.0 (C-11), 129.6 (C-11a), 131.6 (C-8a), 134.0 (C-12),
147.3 (C-14a), 152.0 (C-13a), 155.1 (C-4a), 156.8 (C-7), 165.9 (C-
6); ESI-MS m/z 406 [MH]+, 428 [MNa]+, IR (NaCl) ν 3390, 2920,
2854, 1610, 1581, 1555, 1448, 1435, 1407, 1363, 1302, 1201, 1137
cm-1; UV λ (MeOH) (log ꢀ) 225 (4.38), 289 (4.56), 332 (3.80),
347 (3.73), 367 nm (3.71). Anal. (C24H23NO5) C, H, N.
2-Hydroxy-6-methoxy-3,3-dimethyl-1,2,3,14-7H-benzo[a]py-
rano[3,2-h]acridin-1,7-dione (41): 1H NMR (400 MHz, DMSO-
d6) δ 1.39 (s, 3H, CH3), 1.51 (s, 3H, CH3), 3.97 (s, 3H, OCH3),
4.30 (d, J ) 5 Hz, 1H, H-2), 6.24 (d, J ) 5 Hz, 1H, C2-OH), 6.40
(s, 1H, H-5), 7.56 (td, J ) 8, 1 Hz, 1H, H-10), 7.67 (td, J ) 8, 1
Hz, 1H, H-9), 7.69 (d, J ) 9 Hz, 1H, H-13), 7.99 (dd, J ) 8, 1 Hz,
1H, H-11), 8.18 (d, J ) 9 Hz, 1H, H-12), 10.00 (dd, J ) 8, 1 Hz,
1H, H-8), 12.80 (s, 1H, NH); 13C NMR (75 MHz, DMSO-d6) δ
20.5 (CH3), 26.5 (CH3), 57.5 (OCH3), 76.4 (C-3), 83.6 (C-2), 95.5
(C-5), 104.7 (C-14b), 114.0 (C-6a), 116.9 (C-13), 119.6 (C-7a),
125.7 (C-10), 127.0 (C-8), 128.4 (C-11), 129.3 (C-9), 130.0 (C-
11a), 131.1 (C-8a), 135.2 (C-12), 144.3 (C-13a), 147.1 (C-14a),
166.5 (C-4a), 169.0 (C-6), 177.4 (C-7), 195.1 (C-1); DCI-MS m/z
390 [MH]+; IR (NaCl) ν 3380, 3059, 3025, 2919, 2845, 1654, 1633,
293 (4.34), 333 (3.86), 388 nm (3.85). Anal. (C23H19NO5) C, H,
N.
(()-cis-1,2-Dihydroxy-6-methoxy-3,3-dimethyl-1,2,3,14-tet-
rahydro-7H-benzo[a]pyrano[3,2-h]acridin-7-one (42): 1H NMR
(400 MHz, DMSO-d6) δ 1.31 (s, 3H, CH3), 1.52 (s, 3H, CH3), 3.70
(dd, J ) 6, 5 Hz, 1H, H-2), 3.82 (s, 3H, OCH3), 5.11 (d, J ) 7, 6,
1H, H-1), 5.34 (d, J ) 7 Hz, 1H, C1-OH), 5.47 (d, J ) 5 Hz, 1H,
C2-OH), 6.20 (s, 1H, H-5), 7.49 (td, J ) 8, 1 Hz, 1H, H-10), 7.66
(td, J ) 8, 1 Hz, 1H, H-9), 7.70 (d, J ) 9 Hz, 1H, H-13), 7.92 (dd,
J ) 8, 1 Hz, 1H, H-11), 8.09 (d, J ) 9 Hz, 1H, H-12), 10.03 (dd,
J ) 8, 1 Hz, 1H, H-8), 10.57 (s, 1H, NH); 13C NMR (75 MHz,
DMSO-d6) δ 23.2 (CH3), 25.5 (CH3), 56.2 (OCH3), 62.8 (C-1),
71.1 (C-2), 79.2 (C-3), 94.7 (C-5), 100.3 (C-14b), 109.7 (C-6a),
114.0 (C-7a), 118.9 (C-13), 125.1 (C-10), 126.4 (C-8), 128.7 (C-
11a), 128.9 (C-9), 129.6 (C-11), 131.8 (C-8a), 134.6 (C-12), 140.6
(C-13a), 142.4 (C-14a), 156.6 (C-4a), 161.5 (C-6), 177.9 (C-7);
DCI-MS m/z 392 [MH]+; IR (NaCl) ν 3400, 3056, 3025, 2921,
2847, 1633, 1623, 1451, 1154, 1030 cm-1; UV λ (MeOH) (log ꢀ)
203 (4.56), 213 (4.35), 243 (4.30), 288 (4.58), 348 (3.64), 367
(3.67), 386 nm (3.66). Anal. (C23H21NO5) C, H, N.
(()-cis-1,2-Diacetoxy-6,7-dimethoxy-3,3-dimethyl-3H-benzo-
[a]pyrano[3,2-h]acridine (43). Compound 43 was obtained from
40 (0.02 g, 0.049 mmol) according to the procedure described for
the preparation of 25 from 23, using excess acetic anhydride (0.1
mL, 1.04 mmol) and 4-(dimethylamino)pyridine (0.002 g). Flash
chromatography (solvent, CH2Cl2 and then CH2Cl2/MeOH, 99.5:
0.5 to 98:2) gave 43 (0.02 g, 83%) as a yellow amorphous solid:
1H NMR (400 MHz, CDCl3) δ 1.34 (s, 3H, CH3), 1.37 (s, 3H,
CH3), 2.07 (s, 3H, CH3CO), 2.15 (s, 3H, CH3CO), 3.96 (s, 3H,
C7-OCH3), 4.10 (s, 3H, C6-OCH3), 5.42 (d, J ) 5 Hz, 1H, H-2),
6.50 (s, 1H, H-5), 7.03 (d, J ) 5 Hz, 1H, H-1), 7.61 (td, J ) 8, 1
Hz, 1H, H-10), 7.66 (td, J ) 8, 1 Hz, 1H, H-9), 7.76 (d, J ) 9 Hz,
1H, H-13), 7.86 (dd, J ) 8, 1 Hz, 1H, H-11), 7.88 (d, J ) 9 Hz,
1H, H-12), 9.56 (dd, J ) 8, 1 Hz, 1H, H-8); 13C NMR (75 MHz,
CDCl3) δ 20.6 (CH3CO), 20.8 (CH3CO), 22.4 (CH3), 25.9 (CH3),
56.8 (C6-OCH3), 61.6 (C7-OCH3), 62.4 (C-1), 71.7 (C-2), 77.0 (C-
3), 97.6 (C-5), 105.0 (C-14b), 111.3 (C-6a), 115.3 (C-7a), 126.7
(C-10), 127.4 (C-8), 127.8 (C-9), 128.6 (C-13), 128.9 (C-11), 129.6
(C-11a), 131.6 (C-8a), 133.0 (C-12), 147.3 (C-14a), 152.0 (C-13a),
154.7 (C-4a), 157.2 (C-7), 166.3 (C-6); 169.9 (OCOCH3), 170.5
(OCOCH3); DCI-MS m/z 490 [MH]+; IR (NaCl) ν 2920, 2847,
1742, 1722, 1610, 1584, 1462, 1432, 1153 cm-1; UV λ (MeOH)
(log ꢀ) 224 (4.28), 260 (4.06), 285 (4.46), 329 (3.42), 362 nm (3.43).
Anal. (C28H27NO7) C, H, N.
2-Acetoxy-6-methoxy-3,3,14-trimethyl-1,2,3,14-tetrahydro-
7H-benzo[a]pyrano[3,2-h]acridin-1,7-dione (44). Compound 44
was synthesized from 41 (0.050 g, 0.124 mmol) according to the
procedure described for the preparation of 25 from 23, using excess
acetic anhydride (0.2 mL, 2.08 mmol) and 4-(dimethylamino)-
pyridine (0.002 g). Flash chromatography (solvent, CH2Cl2 and then
CH2Cl2/MeOH, 99.5:0.5 to 98:2) gave 44 (0.054 g, 98%) as a
yellow amorphous solid: 1H NMR (400 MHz, CDCl3) δ 1.46 (s,
3H, CH3), 1.62 (s, 3H, CH3), 2.26 (CH3CO), 3.75 (s, 3H, NCH3),
4.09 (s, 1H, H-2), 6.36 (s, 1H, H-5), 7.54 (td, J ) 8, 1 Hz, 1H,
H-10), 7.61 (d, J ) 9 Hz, 1H, H-13), 7.68 (td, J ) 8, 1 Hz, 1H,
H-9), 7.83 (dd, J ) 8, 1 Hz, 1H, H-11), 8.03 (d, J ) 9 Hz, 1H,
H-12), 9.91 (dd, J ) 8, 1 Hz, 1H, H-8); 13C NMR (100 MHz,
CDCl3) δ 20.3 (CH3), 20.8 (CH3CO), 26.1 (CH3), 45.9 (NCH3),
57.0 (OCH3), 76.3 (C-3), 82.0 (C-2), 95.0 (C-5), 103.2 (C-14b),
113.6 (C-6a), 116.7 (C-13), 119.5 (C-7a), 125.8 (C-10), 127.0 (C-
8), 128.1 (C-11), 128.9 (C-9), 129.8 (C-11a), 130.8 (C-8a), 134.7
(C-12), 144.2 (C-13a), 146.4 (C-14a), 165.2 (C-4a), 167.4 (C-6),
170,0 (C2OCO), 178.2 (C-7), 183.7 (C-1); ESI-MS m/z 446 [MH]+,
468 [MNa]+; IR (NaCl) ν 2927, 1747, 1654, 1630, 1581, 1404,
1206, 1067, 1026 cm-1; UV λ (MeOH) (log ꢀ) 224 (4.28), 260
(4.06), 285 (4.46), 329 (3.42), 362 nm (3.43). Anal. (C26H23NO6)
C, H, N.

3394 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 11
Nguyen et al.
tumors. Clin. Cancer Res. 2001, 7, 2573-2580. (b) Guilbaud, N.;
Le´once, S.; Tillequin, F.; Koch, M.; Hickman, J.; Pierre´, A.
Acronycine derivatives as promising antitumor agents. Anti-Cancer
Drugs 2002, 13, 445-449.
Acknowledgment. The authors thank Prof. John A. Hickman
(Institut de Recherches Servier, Croissy sur Seine, France) for
his kind interest in this work.
(10) (a) David-Cordonnier, M.-H.; Laine, W.; Lansiaux, A.; Kouach, M.;
Briand, G.; Pierre´, A.; Hickman, J. A.; Bailly, C. Alkylation of
guanine by S-23906-1, a novel potent antitumor compound derived
from the plant alkaloid acronycine. Biochemistry 2002, 41, 9911-
9920. (b) David-Cordonnier, M.-H.; Laine, W.; Kouach, M.; Briand,
G.; Vezin, H.; Gaslonde, T.; Michel, S.; Doan Thi Mai, H.; Tillequin,
F.; Koch, M.; Le´once, S.; Pierre´, A.; Bailly, C. A transesterification
reaction is implicated in the covalent binding of benzo[b]acronycine
anticancer agents with DNA and glutathion. Bioorg. Med. Chem.
2004, 12, 23-29. (c) David-Cordonnier, M.-H.; Laine, W.; Gaslonde,
T.; Michel, S.; Tillequin, F.; Koch, M.; Le´once, S.; Pierre´, A.; Bailly,
C. Design of novel antitumor DNA alkylating agents: The benza-
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Med. Chem. 2004, 39, 649-655.
Supporting Information Available: Experimental procedures
for the preparation of compounds 25-36; 13C NMR, MS, IR, and
UV spectral data for compounds 26-28, 30-33, 35, and 36;
elemental analysis data of compounds 4 and 8-44. This material
is available free of charge via the Internet at http://pubs.acs.org.
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