Synthesis, cytotoxic activity, and DNA binding properties of antitumor cis-1,2-dihydroxy-1,2-dihydrobenzo[b]acronycine cinnamoyl esters

Article abstract of DOI:10.1016/j.bmc.2009.01.062

Monocinnamoyl esters at position 2 of (±)-cis-1,2-dihydroxy-6-methoxy-3,3,14-trimethyl-1,2,3,14-tetrahydro-7H-benzo[b]pyrano[3,2-h]acridin-7-one and their acetyl derivatives at position 1 were prepared as stabilized analogues of the anticancer alkylating

Full text of DOI:10.1016/j.bmc.2009.01.062

Bioorganic & Medicinal Chemistry 17 (2009± 1918–1927
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, cytotoxic activity, and DNA binding properties of antitumor
cis-1,2-dihydroxy-1,2-dihydrobenzo[b]acronycine cinnamoyl esters
a
a
a
a
b
b
Quyên Do , Wen Tian , Rodrigue Yougnia , Thomas Gaslonde , Bruno Pfeiffer , Alain Pierré ,
b
b
c
c
Stéphane Léonce , Laurence Kraus-Berthier , Marie-Hélène David-Cordonnier , Sabine Depauw ,
Amélie Lansiaux , Romain Mazinghien , Michel Koch , François Tillequin , Sylvie Michel ,
Hanh Dufat
c,d
c,d
a
a,
a
*
a
a
Laboratoire de Pharmacognosie de l’Université Paris Descartes, U.M.R./C.N.R.S. No. 8638, Faculté des Sciences Pharmaceutiques et Biologiques, 4 Avenue de l’Observatoire,
7
5006 Paris, France
b
Institut de Recherches Servier, Division Recherche Cancérologie, 125 Chemin de Ronde, 78290 Croissy sur Seine, France
INSERM U-837-Centre de Recherches Jean-Pierre Aubert (Team 4 Molecular and Cellular Targeting for Cancer Treatment) and IMPRT-IFR114, IRCL, 59045 Lille Cedex, France
Laboratoire de Pharmacologie Antitumorale du Centre Oscar Lambret, IRCL, 59045 Lille Cedex, France
c
d
a r t i c l e i n f o
a b s t r a c t
Article history:
Monocinnamoyl esters at position 2 of (± ±-cis-1,2-dihydroxy-6-methoxy-3,3,14-trimethyl-1,2,3,14-tetra-
hydro-7H-benzo[b]pyrano[3,2-h]acridin-7-one and their acetyl derivatives at position 1 were prepared as
stabilized analogues of the anticancer alkylating agent S23906-1. Monocinnamoyl esters at position 2
were slower DNA alkylators than the reference 2-monoacetate. Mixed esters bearing an acetyl ester
group at position 1 and a cinnamoyl ester group at position 2 alkylated DNA slower than S23906-1. A
strong correlation was observed between cytotoxicity and DNA alkylation kinetics, with slower alkylators
displaying more potent antiproliferative activities. The most cytotoxic compounds proved to be signifi-
cantly active in vivo against murine C-38 adenocarcinoma implanted in mice, but less potent than
S23906-1.
Received 19 December 2008
Revised 15 January 2009
Accepted 20 January 2009
Available online 31 January 2009
Keywords:
Acronycine
S-23906-1
DNA alkylator
Cinnamoyl esters
Ó 2009 Elsevier Ltd. All rights reserved.
7
1
. Introduction
There is an urgent need for the discovery and development of
double helix, with the formation of single-stranded DNA, which fi-
nally leads to cell apoptosis.
8
Surprisingly, monoesters at position 2 of cis-1,2-dihydroxy-6-
methoxy-3,3,14-trimethyl-1,2,3,14-tetrahydro-7H-benzo[b]pyrano
[3,2-h]acridin-7-one, exemplified by acetate 2, which is the major
metabolite of 1 in vivo, also exhibit both potent cytotoxic activities
in vitro and marked antitumor activities in vivo, despite the lack of
leaving group at the benzylic position 1. These latter results can
be rationalized on the basis of a spontaneous transesterification
process, leading to the more reactive but unstable isomeric cis-
monoesters at position 1 in aqueous media.
new anticancer agents. Much effort is directed toward the identifi-
cation of cytostatic agents targeting the cell cycle pathway, angio-
genesis or cell differentiation, but conventional cytotoxic agents
interfering with DNA remain actively searched as well. Indeed,
the current development of small molecules that bind directly to
DNA, such as ecteinascidin 743 (ET-743, Trabectedin±, which in-
duces stabilization of the DNA double helix after alkylation of the
2
1
5
,6
2
-amino groups of guanine residues, clearly indicate the resur-
gence of interest in DNA alkylating agents. The mechanism of
action of the acronycine derivative (± ±-cis-1,2-diacetoxy-6-meth-
oxy-3,3,14-trimethyl-1,2,3,14-tetrahydro-7H-benzo[b]pyrano[3,2-h]
O
6OCH3
5
9
8
7
1
0
3
acridin-7-one (1±, which recently underwent phase I clinical trials
4
1
1
O
under the code S23906-1, also implies 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
12
13
14N
CH3
1
2
3
R1O
OR2
4
–6
drug.
However, in strong contrast with ecteinascidin 743, DNA
1 R = R = COCH
1 2
3
alkylation by S23906-1, induces a marked destabilization of the
2 R1 = H R2 = COCH3
The ultimate metabolite of both S23906-1 (1± and (± ±-cis-
-acetoxy-1-hydroxy-6-methoxy-3,3,14-trimethyl-1,2,3,14-tetra-
*
Corresponding author. Tel.: +33 1 53 73 98 10; fax: +33 1 40 46 96 58.
E-mail addresses: francois.tillequin@parisdescartes.fr, tillequi@pharmacie.
2
univ-par (F. Tillequin±.
hydro-7H-benzo[b]pyrano[3,2-h]acridin-7-one (2± is the diol
0
968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.01.062

Q. Do et al. / Bioorg. Med. Chem. 17 (2009) 1918–1927
Table 1
1919
(
± ±-cis-1,2-dihydroxy-6-methoxy-3,3,14-trimethyl-1,2,3,14-tetra-
Cytotoxic activity of monocinnamoyl esters 4–16 against murine leukemia cell line
L1210 and human epidermoid carcinoma cell line KB-3-1, in comparison with
S23906-1 (1± and (± ±-cis-2-acetoxy-1-hydroxy-6-methoxy-3,3,14-trimethyl-1,2,3,14-
tetrahydro-7H-benzo[b]pyrano[3,2-h]acridin-7-one (2±
hydro-7H-benzo[b]pyrano[3,2-h]acridin-7-one (3±, which is devoid
of any ester leaving group and consequently biologically inactive.
In this context, stabilized esters presenting a better stability to-
ward hydrolytic processes appear as possible new drug candidates,
provided they are still able to undergo additions onto intracellular
nuclophilic targets. Recent results obtained in the field of prodrugs
of anticancer agents underline the stabilizing effect toward hydro-
lysis of the ‘acrylic’ double bond conjugated with the aromatic ring
Compound
Cytotoxicity L1210
Cytotoxicity KB-3-1
IC50
(lM±
IC50 (lM±
1
2
4
0.79
0.62
0.12
0.04
0.05
0.06
0.035
0.035
0.05
0.08
0.09
0.10
0.24
0.13
1.1
0.10
0.16
0.03
0.008
0.02
9
in various cinnamoic esters. In a continuation of our studies on the
structure activity relationships in the benzoacronycine series,
we describe here the synthesis and biological activities of mono-
cinnamoyl esters at position 2 of cis-1,2-dihydroxy-6-methoxy-
5
6
7
8
1
0–16
0.02
0.006
0.006
0.006
0.02
0.02
0.015
0.03
9
3
,3,14-trimethyl-1,2,3,14-tetrahydro-7H-benzo[b]pyrano[3,2-h]
10
11
acridin-7-one, and their acetyl derivatives at position 1. Several
types of substituents on the cinnamoic aromatic ring have been
envisaged, including halogens (Cl or Br±, electron withdrawing
1
1
1
1
1
2
3
4
5
6
2 3 3
groups (NO or CF ± and electron donating groups (OCH ±.
0.03
0.08
2
2
. Results
trimethyl-1,2,3,14-tetrahydro-7H-benzo[b]pyrano[3,2-h]acridin-
.1. Chemistry
7
-one (2±. The results (IC50± are reported in Tables 1 and 2.
As expected, the cinnamoyl esters 4–16 as well as their acetyl
Treatment
of
(± ±-cis-1,2-dihydroxy-6-methoxy-3,3,14-tri-
counterparts 17–29 displayed significant cytotoxic properties,
most of them with submicromolar IC50 against both cell lines. With
a few exceptions (compounds 16, 17, 25, and 28±, all of them were
found more potent than the reference compounds in the series,
S23906-1 (1± and 2. As a general rule, monocinnamoyl esters 4–
methyl-1,2,3,14-tetrahydro-7H-benzo[b]pyrano[3,2-h]acridin-7-
one (3± with 1 equiv of the appropriate cinnamoyl chloride in dry
pyridine afforded the corresponding monoesters at the less hin-
3
dered 2-position (4–16± with an excellent regioselectivity, as pre-
viously observed for their benzoic conterparts.5
,17
Further
1
6 were found more potent against both cell lines than the corre-
treatment of monoesters 4–16 with excess acetic anhydride gave
the desired mixed esters 17–29 in almost quantitative yield.
sponding acetates 17–29. The IC50 values of the esters unsubstitut-
ed on the cinnamic aromatic ring (4 and 17± were within the same
range of magnitude as the parent compounds. Substitution by elec-
tron withdrawing groups (NO or CF ± or electron donating groups
2
.2. Cytotoxic activity
2
3
(
3
OCH ± did not result in a significant increase of the antiprolifera-
Monocinnamoyl esters 4–16 and the corresponding mixed es-
tive activity against KB-3-1 cell line, except in the case of the acet-
ylated trifluoromethylcinnamate 26. Excellent results in terms of
cytotoxicity against both cell lines were obtained with compounds
bearing halo substituents at position 4 on the cinnamic aromatic
ring. Indeed, the most active monocinnamate derivative, (± ±-cis-
ters 17–29 were evaluated in vitro for their cytotoxicity against
two tumor cell lines, a murine leukemia cell line (L1210± and a hu-
man epidermoid carcinoma cell line (KB-3-1±, in comparison with
S23906-1 (1± and (± ±-cis-2-acetoxy-1-hydroxy-6-methoxy-3,3,14-
COCl
O
OCH3
O
OCH3
O
OCH3
R1,2,3
Ac2O, 4-DMAP
pyridine
N
CH3
O
N
CH3
O
pyridine
N
CH3
O
5'
R3
6'
1'
4'
R3
HO
HO
O
OH
O
O
R2
3' R2
R1
2'
O
3
O
R1
O
Monocinnamoyl esters
R = H R = R = R = H
Mixed acetyl-cinnamoyl esters
17 R = COCH3 R = R = R = H
4
5
6
7
8
9
1
1
1
1
1
1
1
1
2
3
1
2
3
R = H R = R = H R = Cl
18 R = COCH3 R = R = H R = Cl
1
2
3
1 2 3
R = H R = R = H R = Cl
19 R = COCH3 R = R = H R = Cl
2 3 1
2
3
1
R = H R = R = H R = Cl
20 R = COCH3 R = R = H R = Cl
1
3
2
1 3 2
R = H R = H R = R = Cl
21 R = COCH3 R = H R = R = Cl
1 2 3
1
2
3
R = H R = H R = R = Cl
22 R = COCH3 R = H R = R = Cl
2
1
3
2 1 3
0 R = H R = R = H R = Br
23 R = COCH3 R = R = H R = Br
1 2 3
1
2
3
1 R = H R = R = H R = Br
24 R = COCH3 R = R = H R = Br
1
3
2
1 3 2
2 R = H R = R = H R = CF3
25 R = COCH3 R = R = H R = CF
1 2 3
1
2
3
3
3 R = H R = R = H R = CF3
26 R = COCH3 R = R = H R = CF
1
3
2
1 3 2
3
4 R = H R = R = H R = NO2
27 R = COCH3 R = R = H R = NO2
1 2 3
1
2
3
5 R = H R = R = H R = OCH3
28 R = COCH3 R = R = H R = OCH3
1
2
3
1 2 3
6 R = H R = H R = R = OCH
29 R = COCH3 R = H R = R = OCH
1 2 3 3
1
2
3
3

1
920
Q. Do et al. / Bioorg. Med. Chem. 17 (2009) 1918–1927
Table 2
Kinetics of DNA alkylation reactions were determined in both
series for the most active halogeno derivatives 5, 8, and 10 on
the one hand, and 18, 21, and 23 on, the other hand, in comparison
with unsubstituted compounds 4 and 17, and reference com-
pounds 1 and 2. The results are summarized on Tables 3 and 4
and a representative experiment is presented in Figure 1. As a gen-
eral rule, monocinnamoyl esters alkylate DNA faster than the cor-
responding mixed esters. In both series, a strong correlation
between antiproliferative activity and DNA alkylation kinetics
can be observed. Indeed, the most cytotoxic compounds, monocin-
namate 8 and mixed ester 21, appear as the slowest DNA alkylators
in their respective series.
Cytotoxic activity of mixed esters 17–29 against murine leukemia cell line L1210 and
human epidermoid carcinoma cell line KB-3-1, in comparison with S23906-1 (1± and
(± ±-cis-2-acetoxy-1-hydroxy-6-methoxy-3,3,14-trimethyl-1,2,3,14-tetrahydro-7H-
benzo[b]pyrano[3,2-h]acridin-7-one (2±
Compound
Cytotoxicity L1210
Cytotoxicity KB-3-1
IC50 (lM±
IC50
(l
M±
1
2
0.79
0.62
1.1
0.5
0.6
Insoluble
0.4
0.6
0.7
0.6
0.10
0.16
0.15
0.07
0.09
Insoluble
0.05
0.09
0.08
0.12
0.3
1
1
1
2
2
2
2
2
2
2
2
2
2
7
8
9
0
1
2
3
4
5
6
7
8
9
2.4. Antitumor in vivo evaluation
1.5
0.7
0.5
1.2
The most cytotoxic compounds in both series, 8 and 21, were
selected for an in vivo evaluation, comparatively with compound
0.06
0.14
0.24
0.15
1
(S23906-1±, on the murine C38 colon adenocarcinoma model im-
0.9
planted sc. Although less active and less potent than compound 1,
compound 8 administered twice by iv route at the optimal dose of
2
mg/kg proved to be significantly active, inhibiting tumor growth
by 90% and with one out of seven mice tumor-free on day 90
Fig. 2±. Compound 8 was toxic when administered at 4 mg/kg
2
-(3,4-dichlorocinnamoyloxy±-1-hydroxy-6-methoxy-3,3,14-tri-
(
methyl-1,2,3,14-tetrahydro-7H-benzo[b]pyrano[3,2-h]acridin-7-
one (8± was about 20–30-fold more potent than S-23906-1 (1± and
its monoacetyl counterpart 2. Similarly, (± ±-cis-1-acetoxy-(3,4-
dichlorocinnamoyloxy±-6-methoxy-3,3,14-trimethyl-1,2,3,14-tetra-
hydro-7H-benzo[b]pyrano[3,2-h]acridin-7-one (21± was the most
interesting compound in the mixed ester series, but was only two-
fold more potent than the reference compound S-23906-1 (1±.
since 6 mice out of seven died after the treatment.
In the same experiment, compound 21 administered at the opti-
mal dose of 4 mg/kg induced 84% of tumor growth inhibition with-
out tumor-free mice on day 90 (Fig. 3±. Compound 1 (S 23906-1±,
administered at its optimal dose of 4 mg/kg induced 99% of tumor
growth inhibition with five mice out of seven tumor-free on day
9
0.
2
.3. DNA alkylation
21
8
The compounds were also evaluated for their ability to form
covalent complexes with DNA using gel shift assay. S23906-1 (1±
and its mono-acetylated derivative 2 were previously analysed
4
for DNA alkylation and revealed in kinetics reaction that half of
the DNA was alkylated (t1/2± after 30 and 5 min, respectively
(Tables 3 and 4±.
b
Table 3
Kinetics of DNA alkylation by halogenated monocinnamoyl esters 4, 5, 8, and 10, in
comparison with S23906-1 (1± and (± ±-cis-2-acetoxy-1-hydroxy-6-methoxy-3,3,14-
trimethyl-1,2,3,14-tetrahydro-7H-benzo[b]pyrano[3,2-h]acridin-7-one (2±
f
17
4
Compound
DNA alkylation
1/2 (min±
t
1
2
4
5
8
30
5
20
60
60
60
1
0
b
Table 4
f
Kinetics of DNA alkylation by halogenated mixed esters 17, 18, 21, and 23, in
comparison with S23906-1 (1± and (± ±-cis-2-acetoxy-1-hydroxy-6-methoxy-3,3,14-
trimethyl-1,2,3,14-tetrahydro-7H-benzo[b]pyrano[3,2-h]acridin-7-one (2±
Compound
DNA alkylation
1/2 (min±
Figure 1. Kinetics for DNA alkylation measured using electromobility shift assay—
the radiolabelled 117 bp DNA fragment was incubated with 50 M of the indicated
compound at room temperature for the time specified on the top of the lanes (min±.
b’ and ‘f’ correspond to bound or free DNA, respectively. Open arrow refers to the
reached plateau for the alkylation of the DNA by compound 8. The t1/2 referred in
Tables 3 and 4 corresponds to the incubation time requires to obtain one half of the
migration retardation obtained at the plateau. When the plateau is far to be reached
as for compound 21 the t1/2 is referred as > of the maximum of experiment time
t
l
1
2
30
5
90
360
>1440
360
‘
1
1
2
2
7
8
1
3
(
24 h, 1440 min±.

Q. Do et al. / Bioorg. Med. Chem. 17 (2009) 1918–1927
1921
5
4
3
2
1
000
000
000
000
000
0
400 MHz and 100 MHz, respectively, using a Bruker AVANCE-400
spectrometer. When necessary, the signals were unambiguously
Control
1
1
1
1
assigned by 2D NMR techniques: H– H COSY, H– H NOESY,
1
3
1
13
1
C– H HMQC, and C– H HMBC. These experiments were per-
formed using standard Bruker microprograms. Flash column chro-
matographies were conducted using silica gel 60 Merck (35–
70 lm± with an overpressure of 300 mbar. Microanalyses were in
cpd8 1mg/kg
agreement with calculated values ± 0.4%. The starting (± ±-cis-1,2-
dihydroxy-6-methoxy-3,3,14-trimethyl-1,2,3,14-tetrahydro-7H-
benzo[b]pyrano[3,2-h]acridin-7-one (3± was prepared essentially
cpd8 2 mg/kg
cpd1 2 mg/kg
cpd1 4 mg/kg
60
3
as previously described.
10
20
30
40
50
Days
4
.2. General procedure for the synthesis of monocinnamoyl
Figure 2. In vivo evaluation of compound 8 (S72419-1± administered by the iv
route, in comparison with S23906-1 (1±, on an established C38 colon adenocarci-
noma (sc implantation± in mice.
esters 4–16
Thionyl chloride (7.30 mL, 100 mmol±, was added dropwise to a
suspension of the appropriate cinnamoic acid (10 mmol± in anhy-
drous CH Cl (40 mL±. The reaction mixture was stirred under reflux
for 3 h, filtered, and the solvent and excess thionyl chloride were re-
moved under reduced pressure to give the crude cinnamoyl chloride
as a whitish solid, which was immediately used without further
purification in the following step. The crude cinnamoyl chloride
5
4
3
2
1
000
000
000
000
000
0
2
2
cpd21 1mg/kg
Control
cpd21 2 mg/kg
(
1.15 mmol± was added to a solution of (± ±-cis-1,2-dihydroxy-6-
methoxy-3,3,14-trimethyl-1,2,3,14-tetrahydro-7H-benzo[b]pyrano-
3,2-h]acridin-7-one (419 mg, 1.03 mmol± in dry pyridine (10 mL±.
cpd21 4 mg/kg
cpd1 2 mg/kg
[
The reaction mixture was stirred at room temperature for 48 h and
then evaporated under reduced pressure. Flash chromatography
cpd1 4 mg/kg
60
(
2 2 2 2
solvent: CH Cl , then CH Cl /MeOH, 99:1± afforded the correspond-
10
20
30
40
50
ing (± ±-cis-2-cinnamoyloxy-1-hydroxy-6-methoxy-3,3,14-tri-
methyl-1,2,3,14-tetrahydro-7H-benzo[b]pyrano[3,2-h]acridin-7-
one 4–16 as a yellow amorphous solid in 23–42% yield.
Days
Figure 3. In vivo evaluation of compound 21 (S72422-1± administered by the iv
route, in comparison with S23906-1 (1±, on an established C38 colon adenocarci-
noma (sc implantation± in mice.
4
.3. (± ±-cis-2-Cinnamoyloxy-1-hydroxy-6-methoxy-3,3,14-
trimethyl-1,2,3,14-tetrahydro-7H-benzo[b]pyrano[3,2-
h]acridin-7-one (4±
3
. Discussion and conclusion
Introduction of a cinnamoyl ester group at position 2 of (± ±-cis-
,2-dihydroxy-6-methoxy-3,3,14-trimethyl-1,2,3,14-tetrahydro-
H-benzo[b]pyrano[3,2-h]acridin-7-one resulted, as expected, in
Yield 34%, UV k nm (log
2.63±, 340 (2.83±, 436 (2.53±; IR (KBr±
637, 1610, 1585, 1469, 1467, 1395, 1205, 1167, 1148, 1086 cm
e
± (MeOH± 236 (3.23±, 283 (3.74±, 330
1
7
(
m
3415, 2982, 2932, 1707,
1
the obtainment of slower DNA alkylators than the reference mono-
acetyl ester 2. Similarly, mixed esters bearing an acetyl ester group
at position 1 and a cinnamoyl ester group at position 2 alkylated
DNA slower than S23906-1 (1±. A strong correlation between cyto-
toxicity in vitro and DNA alkylation kinetics was observed. Indeed,
slower alkylators displayed more potent antiproliferative activities
in both series. When tested in vivo against the murine C-38 adeno-
carcinoma model implanted in mice, the most cytotoxic com-
pounds, 8 and 21, proved to be significantly active, but less
potent than the reference compound S23906-1 (1±. The lack of di-
rect correlation between cytotoxic properties in vitro and antitu-
mor activity in vivo has been previously observed in the
benzoacronycine series.3 It should be most probably related to
the fact that several other factors (i.e., distribution, metabolism,
and general toxicity± modulate the biological activity in vivo.
ꢀ1
1
;
3
H NMR (400 MHz, CDCl ± d 8.86 (1H, s, H-8±, 8.00 (1H, dd,
J = 8, 1.5, H-9±, 7.83 (1H, dd, J = 8, 1.5, H-12±, 7.67 (1H, d, J = 16,
OCOCH@CH±, 7.65 (1H, s, H-13±, 7.51 (1H, td, J = 8, 1.5, H-11±,
0
0
7
7
.44–7.42 (2H, m, H-2 , H-6 ±, 7.40 (1H, td, J = 8, 1.5, H-10±, 7.34–
0
0
0
.30 (3H, m, H3 , H4 , H5 ±, 6.42 (1H, d, J = 16, OCOCH@CH±, 6.29
(
1H, s, H-5±, 5.55 (1H, d, J = 5.5, H-2±, 5.42 (1H, m ? d, J = 5.5 upon
O addn., H-1±, 4.02 (3H, s, OCH ±, 3.96 (3H, s, NCH ±, 2.13 (1H, br
s, D O exch., OH-1±, 1.59 (3H, s, CH -C-3±, 1.49 (3H, s, CH
NMR (100 MHz, CDCl ± d 178.4 (C-7±, 166.8 (CO-O-C-2±, 162.6 (C-
±, 159.5 (C-4a±, 149.8 (C-14a±, 142.1 (OCOCH@CH±, 142.1 (C-
D
2
3
3
13
2
3
3
-C-3±;
C
3
6
1
0
0
3a±, 135.8 (C-1 ±, 135.3 (C-12a±, 130.6 (C-4 ±, 129.5 (C-9±, 128.9
0
0
0
0
(
2C, C-3 , C-5 ±, 128.6 (C-8a±, 128.2 (2C, C-2 , C-6 ±, 128.1 (C-11±,
27.8 (C-8±, 126.8 (C-12±, 125.5 (C-7a±, 124.4 (C-10±, 116.8
OCOCH@CH±, 112.0 (C-13±, 111.1 (C-6a±, 102.0 (C-14b±, 93.8 (C-
1
(
5
2
±, 76.7 (C-3±, 72.1 (C-2±, 64.1 (C-1±, 56.3 (OCH
3 3
±, 42.4 (NCH ±,
+
5.5 (CH -C-3±, 22.6 (CH -C-3±; ESI-MS m/z 536 [MH] . Anal. Calcd
3
3
4
4
. Experimental
for (C33H29NO ±: C, 74.00; H, 5.46; N, 2.62. Found: C, 73.82; H, 5.48;
N, 2.57.
6
.1. General
4
.4. (± ±-cis-2-(4-Chlorocinnamoyloxy±-1-hydroxy-6-methoxy-
Mass spectra were recorded with ZQ 2000 Waters and Q-Tof1
3,3,14-trimethyl-1,2,3,14-tetrahydro-7H-benzo[b]pyrano[3,2-
h]acridin-7-one (5±
Micromass spectrometers using electrospray ionization (ESI-MS;
Vc = 30 V±. UV spectra (kmax in nm± were recorded in spectroscopic
grade MeOH on a Beckman Model 34 spectrophotometer. IR spec-
Yield 40%, UV k nm (loge± (MeOH± 226 (3.37±, 234 (3.35±, 284
ꢀ1
tra (
or sodium chloride films on a Perkin–Elmer 257 instrument.
NMR (d [ppm], J [Hz]± spectra and 13C NMR spectra were run at
m
max in cm ± were obtained from potassium bromide pellets
(3.78±, 341 (2.87±, 382 (2.06±, 437 (2.55±, 496 (1.34±; IR (KBr±
3425, 2974, 2928, 1718, 1640, 1613, 1589, 1492, 1393, 1202,
1151, 1088, 1033, 1009, 819
1
H
ꢀ1
1
3
m cm ; H NMR (400 MHz, CDCl ± d

1
922
Q. Do et al. / Bioorg. Med. Chem. 17 (2009) 1918–1927
8
.82 (1H, s, H-8±, 7.98 (1H, dd, J = 8, 1.5, H-9±, 7.78 (1H, dd, J = 8, 1.5,
(C-7a±, 124.5 (C-10±, 118.6 (OCOCH@CH±, 111.9 (C-13±, 110.9 (C-
H-12±, 7.64 (1H, d, J = 16, OCOCH@CH±, 7.61 (1H, s, H-13±, 7.49 (1H,
td, J = 8, 1.5, H-11±, 7.39 (1H, td, J = 8, 1.5, H-10±, 7.35 (2H, d, J = 8.5,
H-2 , H-6 ±, 7.27 (2H, d, J = 8.5, H-3 , H-5 ±, 6.43 (1H, d, J = 16,
OCOCH@CH±, 6.25 (1H, s, H-5±, 5.55 (1H, d, J = 5, H-2±, 5.42 (1H,
6a±, 101.9 (C-14b±, 93.8 (C-5±, 76.8 (C-3±, 72.4 (C-2±, 64.1 (C-1±,
56.2 (OCH
m/z 570 and 572 [MH] , 592 and 594 [MNa] . Anal. Calcd for
28ClNO ±: C, 69.53; H, 4.95; N, 2.46. Found: C, 69.52; H,
4.91; N, 2.41.
3 3 3 3
±, 42.4 (NCH ±, 25.5 (CH -C-3±, 22.6 (CH -C-3±; ESI-MS
0
0
0
0
+
+
(C33H
6
m ? d, J = 5 upon D
2
O addn., H-1±, 3.99 (3H, s, OCH
3
±, 3.95 (3H, s,
-C-3±,
± d 178.4 (C-7±,
NCH ±, 2.35 (1H, br s, D
3
2
O exch., OH-1±, 1.58 (3H, s, CH
3
1
3
1
.49 (3H, s, CH
3
-C-3±; C NMR (100 MHz, CDCl
3
4.7. (± ±-cis-2-(3,4-Dichlorocinnamoyloxy±-1-hydroxy-6-
methoxy-3,3,14-trimethyl-1,2,3,14-tetrahydro-7H-
benzo[b]pyrano[3,2-h]acridin-7-one (8±
1
66.6 (CO-O-C-2±, 162.6 (C-6±, 159.4 (C-4a±, 149.2 (C-14a±, 145.1
0
(
OCOCH@CH±, 142.0 (C-13a±, 136.6 (C-4 ±, 135.7 (C-12a±, 132.5
0
0
0
0
0
(
C-1 ±, 129.5 (C-9±, 129.4 (2C, C-2 , C-6 ±, 129.2 (2C, C-3 , C-5 ±,
128.6 (C-8a±, 128.2 (C-11±, 127.9 (C-8±, 126.8 (C-12±, 125.5 (C-
7a±; 124.4 (C-10±, 117.5 (OCOCH@CH±, 111.9 (C-13±, 111.7 (C-6a±,
101.9 (C-14b±, 93.8 (C-5±, 76.8 (C-3±, 72.2 (C-2±, 64.1 (C-1±, 56.1
Yield 31%, UV k nm (log
(2.89±, 437 (2.90±; IR (KBr±
1588, 1491, 1394, 1200, 1149, 1087 cm
e
m
± (MeOH± 283 (4.09±, 338 (3.23±, 436
3351, 2982, 2928, 1724, 1642, 1615,
ꢀ1
1
; H NMR (400 MHz,
(
5
OCH
3
±, 42.3 (NCH
70 and 572 [MH] , 592 and 594 [MNa] . Anal. Calcd for
28ClNO ±: C, 69.53; H, 4.95; N, 2.46. Found: C, 69.72; H,
.02; N, 2.47.
3
±, 25.4 (CH
3
-C-3±, 22,6 (CH
3
-C-3±; ESI-MS m/z
CDCl ± d 8.78 (1H, s, H-8±, 7.97 (1H, dd, J = 8, 1.5, H-9±, 7.74 (1H,
3
+
+
dd, J = 8, 1.5, H-12±, 7.61 (1H, d, J = 16, OCOCH@CH±, 7.56 (1H, s,
0
(
C
33
H
6
H-13±, 7.50 (1H, d, J = 2, H-2 ±, 7.47 (1H, td, J = 8, 1.5, H-11±, 7.38
0
5
(1H, td, J = 8, 1.5, H-10±, 7.36 (1H, d, J = 8.5, H-5 ±, 7.23 (1H, dd,
0
J = 8.5, 2, H-6 ±, 6.48 (1H, d, J = 16, OCOCH@CH±, 6.22 (1H, s, H-5±,
4
3
.5. (± ±-cis-2-(2-Chlorocinnamoyloxy±-1-hydroxy-6-methoxy-
,3,14-trimethyl-1,2,3,14-tetrahydro-7H-benzo[b]pyrano[3,2-
5.56 (1H, d, J = 5.5, H-2±, 5.43 (1H, m ? d, J = 5.5 upon D
H-1±, 3.97 (3H, s, OCH ±, 3.94 (3H, s, NCH ±, 2.56 (1H, br s, D
exch., OH-1±, 1.58 (3H, s, CH -C-3±, 1.49 (3H, s, CH
NMR (100 MHz, CDCl ± d 178.2 (C-7±, 166.6 (CO-O-C-2±, 162.5 (C-
6±, 159.5 (C-4a±, 149.8 (C-14a±, 143.6 (OCOCH@CH±, 141.9 (C-
2
O addn.,
3
3
2
O
C
-C-3±; 1
3
h]acridin-7-one (6±
3
3
3
Yield 40%, UV k nm (log
e± (MeOH± 206 (3.54±, 223 (3.42±, 229
0
0
0
(
3.41±, 284 (3.87±, 325 (2.93±; IR (KBr±
m
3343, 3056, 2982, 2932,
13a±, 135.7 (C-12a±, 134.5 (C-4 ±, 134.3 (C-1 ±, 133.2 (C-3 ±, 130.9
ꢀ1
1
0
0
2
846, 1720, 1642, 1615, 1588, 1495, 1390, 1153, 1087 cm
;
H
(C-5 ±, 129.9 (C-2 ±, 129.5 (C-9±, 128.5 (C-8a±, 128.3 (C-11±, 127.9
0
NMR (400 MHz, CDCl
3
± d 8.81 (1H, s, H-8±, 8.09 (1H, d, J = 16,
(C-8±, 127.1 (C-6 ±, 126.8 (C-12±, 125.3 (C-7a±, 124.4 (C-10±, 119.3
OCOCH@CH±, 7.97 (1H, dd, J = 8, 1.5, H-9±, 7.76 (1H, dd, J = 8, 1.5,
H-12±, 7.59 (1H, s, H-13±, 7.49 (1H, dd, J = 7.5, 1.5, H-6 ±, 7.48
(OCOCH@CH±, 111.9 (C-13±, 110.8 (C-6a±, 102.0 (C-14b±, 93.7 (C-
0
5±, 76.8 (C-3±, 72.5 (C-2±, 64.1 (C-1±, 56.0 (OCH
25.6 (CH -C-3±, 22.6 (CH -C-3±; ESI-MS m/z 604, 606 et 608
[MH] . Anal. Calcd for (C33 NO ±: C, 65.57; H, 4.50; N, 2.32.
Found: C, 65.43; H, 4.64; N, 2.27.
3 3
±, 42.4 (NCH ±,
(
1H, td, J = 8, 1.5, H-11±, 7.38 (1H, td, J = 8, 1.5, H-10±, 7.34 (1H,
3
3
0
0
+
dd, J = 7.5, 1.5, H-3 ±, 7.24 (1H, td, J = 7.5, 1.5, H-5 ±, 7.15 (1H, td,
J = 7.5, 1.5, H-4 ±, 6.48 (1H, d, J = 16, OCOCH@CH±, 6.21 (1H, s, H-
H
27Cl
2
6
0
5
±, 5.54 (1H, d, J = 5.5, H-2±, 5.42 (1H, m ? d, J = 5.5 upon D
addn., H-1±, 3.95 (3H, s, OCH ±, 3.94 (3H, s, NCH ±, 2.55 (1H, br s,
O exch., OH-1±, 1.58 (3H, s, CH -C-3±, 1.50 (3H, s, CH
NMR (100 MHz, CDCl ± d 178.4 (C-7±, 166.5 (CO-O-C-2±, 162.6 (C-
±, 159.5 (C-4a±, 149.7 (C-14a±, 142.3 (OCOCH@CH±, 142.0 (C-
2
O
3
3
4.8. (± ±-cis-2-(2,4-Dichlorocinnamoyloxy±-1-hydroxy-6-
methoxy-3,3,14-trimethyl-1,2,3,14-tetrahydro-7H-
benzo[b]pyrano[3,2-h]acridin-7-one (9±
1
3
D
2
3
3
-C-3±;
C
3
6
1
0
0
0
3a±, 135.8 (C-2 ±, 135.2 (C-12a±, 132.3 (C-1 ±, 131.4 (C-5 ±, 130.2
Yield 30%, UV k nm (log
e± (MeOH± 203 (3.61±, 206 (3.61±, 227
0
(
(
C-3 ±, 129.6 (C-9±, 128.6 (C-8a±, 128.3 (C-11±, 127.9 (C-8±, 127.7
C-6 ±, 127.1 (C-4 ±, 126.9 (C-12±, 125.6 (C-7a±, 124.4 (C-10±,
(3.49±, 283 (3.90±, 331 (2.93±, 341 (2.95±; IR (KBr±
m
3394, 2982,
0
0
ꢀ1
2920, 2846, 1716, 1638, 1584, 1495, 1394, 1153, 1091, 815 cm
;
1
1
9
19.6 (OCOCH@CH±, 111.9 (C-13±, 110.9 (C-6a±, 101.9 (C-14b±,
3.8 (C-5±, 76.8 (C-3±, 72.4 (C-2±, 64.2 (C-1±, 56.2 (OCH ±, 42.4
-C-3±; ESI-MS m/z 570 and 572
3
H NMR (400 MHz, CDCl ± d 8.75 (1H, s, H-8±, 8.01 (1H, d, J = 16,
3
OCOCH@CH±, 7.95 (1H, dd, J = 8, 1.5, H-9±, 7.70 (1H, dd, J = 8, 1.5,
H-12±, 7.53 (1H, s, H-13±, 7.44 (1H, td, J = 8, 1.5, H-11±, 7.40 (1H,
(
[
(
NCH
3
±, 25.5 (CH
+
3
-C-3±, 22.6 (CH
3
+
+
0
MH] , 592 and 594 [MNa] , 608 and 610 [MK] . Anal. Calcd for
C
d, J = 8.5, H-6 ±, 7.36 (1H, td, J = 8, 1.5, H-10±, 7.36 (1H, d, J = 2, H-
0
0
33
H
28ClNO
6
±: C, 74.00; H, 5.46; N, 2.62. Found: C, 74.11; H,
3 ±, 7.11 (1H, dd, J = 8.5, 2, H-5 ±, 6.44 (1H, d, J = 16, OCOCH@CH±,
6.17 (1H, s, H-5±, 5.56 (1H, d, J = 5.5, H-2±, 5.43 (1H, m ? d,
5
.51; N, 2.54.
J = 5.5 upon D
OCH ±, 2.80 (1H, br s, D
1.51 (3H, s, CH
2
O addn., H-1±, 3.92 (3H, s, NCH
3
±, 3.91 (3H, s,
-C-3±,
3
-C-3±; C NMR (100 MHz, CDCl ± d 178.3 (C-7±,
4
3
.6. (± ±-cis-2-(3-Chlorocinnamoyloxy±-1-hydroxy-6-methoxy-
,3,14-trimethyl-1,2,3,14-tetrahydro-7H-benzo[b]pyrano[3,2-
3
2
O exch., OH-1±, 1.59 (3H, s, CH
3
1
3
3
h]acridin-7-one (7±
166.5 (CO-O-C-2±, 162.6 (C-6±, 159.5 (C-4a±, 149.8 (C-14a±, 141.9
0
0
(
C-13a±, 140.9 (OCOCH@CH±, 136.7 (C-4 ±, 135.7 (2C, C-12a
,
C-2 ±,
0
0
0
Yield 31%, UV k nm (log
e
± (MeOH± 219 (3.49±, 283 (3.85±, 328
3378, 2920, 2846, 1712,
642, 1588, 1203, 1149, 1083 cm ; H NMR (400 MHz, CDCl ± d
.82 (1H, s, H-8±, 7.98 (1H, dd, J = 8, 1.5, H-9±, 7.78 (1H, dd, J = 8,
.5, H-12±, 7.62 (1H, d, J = 16, OCOCH@CH±, 7.60 (1H, s, H-13±,
130.9 (C-1 ±, 130.0 (C-3 ±, 129.5 (C-9±, 128.5 (C-8a±, 128.4 (C-6 ±,
128.3 (C-11±, 127.9 (C-8±, 127.6 (C-5 ±, 126.8 (C-12±, 125.5 (C-7a±,
0
(
2.94±, 340 (2.95±, 437 (2.62±; IR (KBr±
m
ꢀ1
1
1
8
1
7
7
6
3
124.4 (C-10±, 120.2 (OCOCH@CH±, 111.9 (C-13±, 110.8 (C-6a±,
101.9 (C-14b±, 93.6 (C-5±, 76.8 (C-3±, 72.6 (C-2±, 64.2 (C-1±, 56.1
(OCH
604, 606, and 608 [MH] , 626, 628, and 630 [MNa] . Anal. Calcd
for (C33 NO ±: C, 65.57; H, 4.50; N, 2.32. Found: C, 65.71; H,
4.53; N, 2.39.
3 3 3 3
±, 42.4 (NCH ±, 25.6 (CH -C-3±, 22.6 (CH -C-3±; ESI-MS m/z
0
+
+
.49 (1H, td, J = 8, 1.5, H-11±, 7.41–7.37 (2H, m, H-2 , H-10±,
0
0
0
.30–7.22 (3H, m, H4 ,
.25 (1H, s, H-5±, 5.55 (1H, d, J = 5.5, H-2±, 5.42 (1H, m ? d,
O addn., H-1±, 3.99 (3H, s, OCH ±, 3.94 (3H, s,
±, 2.35 (1H, br s, D O exch., OH-1±, 1.59 (3H, s, CH -C-3±,
± d 178.4 (C-7±,
,
H5 , H6 ±, 6.46 (1H, d, J = 16, OCOCH@CH±,
H27Cl
2
6
J = 5.5 upon D
NCH
2
3
3
2
3
4.9. (± ±-cis-2-(4-Bromocinnamoyloxy±-1-hydroxy-6-methoxy-
3,3,14-trimethyl-1,2,3,14-tetrahydro-7H-benzo[b]pyrano[3,2-
h]acridin-7-one (10±
1
3
1
3 3
.49 (3H, s, CH -C-3±; C NMR (100 MHz, CDCl
1
66.8 (CO-O-C-2±, 162.6 (C-6±, 159.4 (C-4a±, 149.8 (C-14a±, 144.9
0
(
(
(
OCOCH@CH±, 142.0 (C-13a±, 135.9 (C-1 ±, 135.7 (C-12a±, 134.9
0
0
0
C-3 ±, 130.5, 130.2 (C-4 , C-5 ±, 129.6 (C-9±, 128.6 (C-8a±, 128.3
Yield 32%, UV k nm (log
e± (MeOH± 284 (3.84±, 340 (2.87±, 438
0
0
C-11±, 128.0 (C-2 ±, 127.9 (C-8±, 126.8 (C-12±, 126.4 (C-6 ±, 125.5
(2.51±; IR (KBr± 3335, 2986, 2928, 2846, 1712, 1642, 1615,
m

Q. Do et al. / Bioorg. Med. Chem. 17 (2009) 1918–1927
1923
1
1
583, 1495, 1394, 1269, 1087, 815, 737 cmꢀ1; H NMR (400 MHz,
CDCl ± d 8.79 (1H, s, H-8±, 7.97 (1H, dd, J = 8, 1.5, H-9±, 7.75 (1H,
dd, J = 8, 1.5, H-12±, 7.64 (1H, d, J = 16, OCOCH@CH±, 7.57 (1H, s,
(OCH
604 [MH] . Anal. Calcd for (C34
2.32. Found: C, 67.50; H, 4.65; N, 2.37.
3
±, 42.2 (NCH
3
±, 25.6 (CH
3
-C-3±, 22.5 (CH
3
-C-3±; ESI-MS m/z
+
3
H
28
F
3
NO ±.: C, 67.66; H, 4.68; N,
6
0
0
H-13±, 7.47 (1H, td, J = 8, 1.5, H-11±, 7.44 (2H, d, J = 8.5, H3 , H5 ±,
0
0
7
(
2
.37 (1H, td, J = 8, 1.5, H-10±, 7.27 (2H, d, J = 8.5, H-2 , H-6 ±, 6.48
1H, d, J = 16, OCOCH@CH±, 6.22 (1H, s, H-5±, 5.55 (1H, d, J = 5, H-
±, 5.42 (1H, m ? d, J = 5 upon D O addn., H-1±, 3.98 (3H, s,
OCH ±, 3.94 (3H, s, NCH ±, 2.55 (1H, br s, D O exch., OH-1±, 1.53
3H, s, CH -C-3±, 1.49 (3H, s, CH
-C-3±; 13C NMR (100 MHz, CDCl
d 178.4 (C-7±, 166.8 (CO-O-C-2±, 162.6 (C-6±, 159.5 (C-4a±, 149.8 (C-
4a±, 145.2 (OCOCH@CH±, 142,0 (C-13a±, 135.8 (C-12a±, 133.1 (C-
±, 132.3 (2C, C-3 , C-5 ±, 129.6 (2C, C-2 , C-6 ±, 129.6 (C-9±, 128.6
C-8a±, 128.3 (C-11±, 127.9 (C-8±, 126.8 (C-12±, 125.5 (C-7a±,
25.0 (C-4 ±, 124.5 (C-10±, 117.8 (OCOCH@CH±, 112.0 (C-13±,
4.12. (± ±-cis-2-(3-Trifluoromethylcinnamoyloxy±-1-hydroxy-6-
methoxy-3,3,14-trimethyl-1,2,3,14-tetrahydro-7H-
benzo[b]pyrano[3,2-h]acridin-7-one (13±
2
3
3
2
(
3
3
3
±
Yield 23%, IR (KBr±
m 3336, 2982, 2926, 2849, 1713, 1641, 1591,
1488, 1396, 1333, 1270, 1027, 1164, 1144, 1121, 1084, 816, 740,
ꢀ1
1
1
1
3
687, 657 cm ; H NMR (400 MHz, CDCl ± d 8.70 (1H, s, H-8±,
0
0
0
0
0
7.94 (1H, dd, J = 8, 1.5, H-9±, 7.77 (1H, d, J = 16, OCOCH@CH±, 7.66
0
(
(1H, dd, J = 8, 1.5, H-12±, 7.64 (1H, m, H-2 ±, 7.56–7.52 (2H, m, H-
0
0
0
0
1
1
6
3
4 , H-6 ±, 7.50 (1H, s, H-13±, 7.43–7.33 (3H, m, H-11, H-5 , H-10±,
11.0 (C-6a±, 102.0 (C-14b±, 93.8 (C-5±, 76.8 (C-3±, 72.3 (C-2±,
6.64 (1H, d, J = 16, OCOCH@CH±, 6.15 (1H, s, H-5±, 5.59 (1H, d,
4.1 (C-1±, 56.2 (OCH
±; ESI-MS m/z 614 and 616 [MH] , 636 and 638 [MNa] . Anal.
28BrNO ±: C, 64.50; H, 4.59; N, 2.28. Found: C,
4.38; H, 4.69; N, 2.34.
3
±, 42.4 (NCH
3
±, 25.5 (CH
3
-C-3±, 22.6 (CH
3
-C-
J = 5.5, H-2±, 5.44 (1H, m ? d, J = 5.5 upon D
(3H, s, NCH ±, 3.89 (3H, s, OCH ±, 3.52 (1H, br s, D
1±, 1.60 (3H, s, CH -C-3±, 1.52 (3H, s, CH
(100 MHz, CDCl
59.5 (C-4a±, 149.8 (C-14a±, 144.6 (OCOCH@CH±, 141.9 (C-13a±,
2
O addn., H-1±, 3.93
+
+
3
3
2
O exch., OH-
13
Calcd for (C33
6
H
6
3
3
-C-3±;
C NMR
3
± d 178.2 (C-7±, 166.7 (CO-O-C-2±, 162.5 (C-6±,
1
0
0
0
4
3
.10. (± ±-cis-2-(3-Bromocinnamoyloxy±-1-hydroxy-6-methoxy-
,3,14-trimethyl-1,2,3,14-tetrahydro-7H-benzo[b]pyrano[3,2-
135.7 (C-12a±, 135.1 (C-1 ±, 131.6 (q, J = 32, C-3 ±, 131.2 (C-6 ±,
129.5 (2C, C-9, C-5 ±, 128.5 (C-8a±, 128.2 (C-11±, 127.9 (C-8±,
0
0
0
h]acridin-7-one (11±
126.9 (2C, C-12, C-4 ±, 125.2 (C-7a±, 124.8 (C-2 ±, 124.4 (C-10±,
19.4 (OCOCH@CH±, 119.2 (q, J = 270, CF ±, 111.9 (C-13±, 111.8
(C-6a±, 102.0 (C-14b±, 93.6 (C-5±, 76.8 (C-3±, 72.5 (C-2±, 64.2 (C-
1±, 56.0 (OCH ±, 42.4 (NCH ±, 25.6 (CH -C-3±, 22.6 (CH -C-3±; ESI-
MS m/z 604 [MH] . Anal. Calcd for (C34 NO ±: C, 67.66; H,
4.68; N, 2.32. Found: C, 67.72; H, 4.74; N, 2.28.
1
3
Yield 30%, IR (KBr±
m 3354, 2977, 2912, 2847, 1716, 1645, 1612,
584, 1490, 1458, 1271, 1207, 1153, 1088, 754 cm
400 MHz, CDCl ± d 8.78 (1H, s, H-8±, 7.97 (1H, dd, J = 8, 1.5, H-9±,
.76 (1H, dd, J = 8, 1.5, H-12±, 7.62 (1H, d, J = 16, OCOCH@CH±, 7.58
1H, s, H-13±, 7.56 (1H, t, J = 1.5, H-2 ±, 7.48 (1H, td, J = 8, 1.5, H-11±,
.43 (1H, dt, J = 8, 1.5, H-4 ±, 7.38 (1H, td, J = 8, 1.5, H-10±, 7.32 (1H,
dt, J = 8, 1.5, H-6 ±; 7.16 (1H, t, J = 8, H-5 ±, 6.48 (1H, d, J = 16,
OCOCH@CH±, 6.21 (1H, s, H-5±, 5.56 (1H, d, J = 5, H-2±, 5.42 (1H,
ꢀ1
1
1
(
7
(
;
H NMR
3
3
3
3
+
3
28
H F
3
6
0
0
7
4.13. (± ±-cis-1-Hydroxy-6-methoxy-3,3,14-trimethyl-2-(4-
nitrocinnamoyloxy±-1,2,3,14-tetrahydro-7H-
benzo[b]pyrano[3,2-h]acridin-7-one (14±
0
0
m ? d, J = 5 upon D
2
O addn., H-1±, 3.95 (3H, s, OCH
O exch., OH-1±, 1.59 (3H, s, CH
-C-3±; C NMR (100 MHz, CDCl ± d 178.4 (C-7±, 166.6
3
±, 3.94 (3H, s,
NCH ±, 3.58 (1H, br s, D
3
2
3
-C-3±, 1.50
Yield 37%, UV k nm (log
(2.17±, 418 (2.51±, 425 (2.53±; IR (KBr±
1640, 1617, 1589, 1570, 1518, 1492, 1396, 1343, 1024, 1165,
e
± (MeOH± 233 (3.35±, 287 (3.74±, 379
1
3
(
3H, s, CH
3
3
m
3348, 2982, 2932, 1718,
(
(
4
1
1
CO-O-C-2±, 162.6 (C-6±, 159.5 (C-4a±, 149.8 (C-14a±, 144.8
0
ꢀ1
1
OCOCH@CH±, 142.0 (C-13a±, 136.2 (C-1 ±, 135.8 (C-12a±, 133.4 (C-
3
1151, 1087, 1029 cm ; H NMR (400 MHz, CDCl ± d 8.72 (1H, s,
0
0
0
0
0
±, 130.9 (C-2 ±, 130.4 (C-5 ±, 129.6 (C-9±, 128.6 (C-8a±, 128.3 (C-
H-8±, 8.13 (2H, d, J = 8.5, H-3 , H-5 ±, 7.94 (1H, dd, J = 8, 1.5, H-9±,
7.77 (1H, d, J = 16, OCOCH@CH±, 7,69 (1H, dd, J = 8, 1.5, H-12±,
0
1±, 127.9 (C-8±, 126.9 (2C, C-12, C-6 ±, 125.5 (C-7a±, 124.5 (C-10±,
23.1 (C-3 ±, 118.6 (OCOCH@CH±, 111.9 (C-13±, 111.0 (C-6a±, 101.9
0
0
0
7.55 (2H, d, J = 8.5, H-2 , H-6 ±, 7.51 (1H, s, H-13±, 7.43 (1H, td,
J = 8, 1.5, H-11±, 7.38 (1H, td, J = 8, 1.5, H-10±, 6.68 (1H, d, J = 16,
OCOCH@CH±, 6.21 (1H, s, H-5±, 5.59 (1H, d, J = 5, H-2±, 5,44 (1H,
(
C-14b±, 93.8 (C-5±, 76.8 (C-3±, 72.4 (C-2±, 64.1 (C-1±, 56.2 (OCH
3
±,
-C-3±; ESI-MS m/z 614 and
16 [MH] , 636 and 638 [MNa] . Anal. Calcd for (C33 28BrNO ±: C,
4.50; H, 4.59; N, 2.28. Found: C, 64.59; H, 4.53; N, 2.22.
4
6
6
3 3 3
2.4 (NCH ±, 25.5 (CH -C-3±, 22.6 (CH
+
+
H
6
m ? d, J = 5 upon D ±, 3.93 (3H, s,
NCH ±, 2.35 (1H, br s, D -C-3±,
3 3
.51 (3H, s, CH -C-3±; C NMR (100 MHz, CDCl ± d 177.9 (C-7±,
2
O addn., H-1±, 3.95 (3H, s, OCH
3
3
2
O exch., OH-1±, 1.60 (3H, s, CH
3
1
3
1
4
.11. (± ±-cis-2-(4-Trifluoromethylcinnamoyloxy±-1-hydroxy-6-
167.7 (CO-O-C-2±, 162.6 (C-6±, 159.3 (C-4a±, 149.2 (C-14a±, 147.3
0
methoxy-3,3,14-trimethyl-1,2,3,14-tetrahydro-7H-
benzo[b]pyrano[3,2-h]acridin-7-one (12±
(C-4 ±, 143.1 (OCOCH@CH±, 142.9 (C-13a±, 135.5 (C-12a±, 132.4
0
0
0
(C-1 ±, 129.5 (C-9±, 128.8 (C-8a±, 128.7 (2C, C-2 , C-6 ±, 128.2 (C-
1±, 127.9 (C-8±, 126.9 (C-12±, 126.7 (C-7a±, 124.4 (C-10±, 124.1
1
0
0
Yield 24%, IR (KBr±
m
3327, 2950, 2922, 2848, 1721, 1648, 1613,
(2C, C-3 , C-5 ±, 117.1 (OCOCH@CH±, 111.3 (C-13±, 104.6 (C-6a±,
1
6
588, 1498, 1459, 1393, 1320, 1281, 1166, 1009, 974, 834, 737,
101.9 (C-14b±, 93.6 (C-5±, 76.6 (C-3±, 72.2 (C-2±, 64.1 (C-1±, 56.1
ꢀ1
1
98 cm
;
3
H NMR (400 MHz, CDCl ± d 8.82 (1H, s, H-8±, 7.99
(OCH
3
±, 42.3 (NCH
3
±, 25.4 (CH
3
-C-3±, 22.6 (CH
3
-C-3±; ESI-MS m/z
±:
+
+
+
(
1H, dd, J = 8, 1.5, H-9±, 7.77 (1H, dd, J = 8, 1.5, H-12±, 7.73 (1H, d,
581 [MH] , 603 [MNa] , 619 [MK] . Anal. Calcd for (C33H N O
28 2 8
0
J = 16, OCOCH@CH±, 7.68 (1H, s, H-13±, 7.61–7.52 (4H, m, H-2 , H-
C, 68.27; H, 4.86; N, 4.83. Found: C, 68.11; H, 4.91; N, 4.91.
0
0
0
6
, H-3 , H-5 ±, 7.49 (1H, td, J = 8, 1.5, H-11±, 7.39 (1H, td, J = 8,
.5, H-10±, 6.56 (1H, d, J = 16, OCOCH@CH±, 6.26 (1H, s, H-5±, 5.59
O addn., H-
±, 2.42 (1H, br s, D O exch.,
1
4.14. (± ±-cis-1-Hydroxy-6-methoxy-2-(4-
methoxycinnamoyloxy±-3,3,14-trimethyl-1,2,3,14-tetrahydro-
7H-benzo[b]pyrano[3,2-h]acridin-7-one (15±
(
1H, d, J = 5.5, H-2±, 5.45 (1H, m ? d, J = 5.5 upon D
2
1
±, 4.00 (3H, s, OCH
3
±, 3.96 (3H, s, NCH
-C-3±; 1.52 (3H, s, CH
± d 177.8 (C-7±, 167.1 (CO-O-C-2±, 162.3 (C-6±,
59.6 (C-4a±, 149.9 (C-14a±, 144.2 (OCOCH@CH±, 141.5 (C-13a±,
3
2
1
3
OH-1±, 1.58 (3H, s, CH
100 MHz, CDCl
3
3
-C-3±; C NMR
(
3
Yield 42%, UV k nm (log
e
± (MeOH± 232 (3.46±, 286 (3.88±, 379
3351, 2928, 1712, 1638, 1592, 1506,
3
1394, 1153, 1079 cm ; H NMR (400 MHz, CDCl ± d 8.85 (1H, s,
1
1
1
(2.08±, 437 (2.66±; IR (KBr± m
0
0
ꢀ1
1
37.9 (C-12a±, 135.4 (C-1 ±, 131.8 (q, J = 35, C-4 ±, 129.2 (C-8±,
28.3 (3C, C-11, C-2 , C-6 ±, 127.8 (C-8a±, 126.7 (C-9±, 125.8 (2C,
0
0
H-8±, 7.99 (1H, dd, J = 8, 1.5, H-9±, 7.82 (1H, dd, J = 8, 1.5, H-12±,
7.64 (1H, s, H-13±, 7.62 (1H, d, J = 16, OCOCH@CH±, 7.51 (1H, td,
J = 8, 1.5, H-11±, 7.39 (1H, td, J = 8, 1.5, H-10±, 7.37 (2H, d, J = 8.5,
0
0
C-3 , C-5 ±, 124.4 (C-7a±, 124.2 (C-12±, 122.4 (C-10±, 120.5
OCOCH@CH±, 120.1 (q, J = 270, CF ±, 111.6 (C-13±, 110.3 (C-6a±,
02.5 (C-14b±, 93.3 (C-5±, 76.9 (C-3±, 72.6 (C-2±, 64.1 (C-1±, 55.6
(
1
3
0
0
0
0
H-2 , H-6 ±, 6.82 (2H, d, J = 8.5, H-3 , H-5 ±, 6.28 (1H, d, J = 16,

1
924
Q. Do et al. / Bioorg. Med. Chem. 17 (2009) 1918–1927
OCOCH@CH±, 6.26 (1H, s, H-5±, 5.54 (1H, d, J = 5, H-2±, 5.42 (1H,
(1H, d, J = 5, H-1±, 6.37 (1H, d, J = 16, OCOCH@CH±, 6.35 (1H, s, H-
5±, 5.67 (1H, d, J = 5, H-2±, 4.05 (3H, s, OCH ±, 3.73 (3H, s, NCH ±,
1.95 (3H, s, OCOCH ±, 1.64 (3H, s, CH -C-3±, 1.48 (3H, s, CH -C-3±;
C NMR (100 MHz, CDCl ± d 178.5 (C-7±, 171.1 (CO-O-C-1±, 166.5
m ? d, J = 5 upon D
3H, s, NCH
OH-1±, 1.58 (3H, s, CH
2
O addn., H-1±, 4.00 (3H, s, CH
3
O-C-6±, 3.95
O exch.,
3
3
0
(
3
±, 3.94 (3H, s, CH
3
O-C-4 ±, 2.18 (1H, br s, D
2
3
3
3
-C-3±; 1 C NMR
3
13
3
-C-3±, 1.49 (3H, s, CH
3
3
(
100 MHz, CDCl
3
± d 178.4 (C-7±, 167.3 (CO-O-C-2±, 162.6 (C-6±,
(CO-O-C-2±, 163.1 (C-6±, 160.4 (C-4a±, 150.4 (C-14a±, 146.5
0
0
1
1
1
1
1
7
61.4 (C-4 ±, 159.5 (C-4a±, 149.8 (C-14a±, 146.5 (OCOCH@CH±,
42.1 (C-13a±, 135.8 (C-12a±, 130.1 (2C, C-2 , C-6 ±, 129.6 (C-9±,
28.6 (C-8a±, 128.3 (C-11±, 127.9 (C-8±, 126.9 (C-12±, 126.8 (C-1 ±,
25.8 (C-7a±, 124.5 (C-10±, 114.4 (2C, C3 , C5 ±, 114.2 (OCOCH@CH±,
(OCOCH@CH±, 142.5 (C-13a±, 136.0 (C-12a±, 135.3 (C-1 ±, 130.7
0
0
0
0
0
(C-4 ±, 129.8 (C-9±, 128.9 (C-3 , C-5 ±, 128.8 (C-8a±, 128.4 (3C, C11,
0
0
0
C2 , C6 ±, 128.2 (C-8±, 126.8 (C-12±, 126.0 (C-7a±, 124.6 (C-10±,
0
0
116.9 (OCOCH@CH±, 111.7 (C-13±, 111.4 (C-6a±, 97.9 (C-14b±,
12.0 (C-13±, 111.1 (C-6a±, 102.1 (C-14b±, 93.9 (C-5±, 76.8 (C-3±,
94.4 (C-5±, 76.7 (C-3±, 69.2 (C-2±, 66.1 (C-1±, 56.5 (OCH
(NCH ±, 24.9 (CH -C-3±, 23.1 (CH -C-3±, 20.4 (OCOCH ±; ESI-MS
m/z 578 [MH] . Anal. Calcd for (C35 ±: C, 72.78; H, 5.41; N,
2.42. Found: C, 72.90; H, 5.37; N, 2.32.
3
±, 43.0
0
1.9 (C-2±, 64.1 (C-1±, 56.3 (CH
±, 25.5 (CH -C-3±, 22.7 (CH
Anal. Calcd for (C34 ±: C, 72.20; H, 5.52; N, 2.48. Found: C,
3
O-C-6±, 55.4 (CH
3
O-C-4 ±, 42.4
3
3
3
3
+
+
(
NCH
3
3
3
-C-3±; ESI-MS m/z 566 [MH] .
H31NO
7
H31NO
7
7
2.09; H, 5.39; N, 2.57.
4
.18. (± ±-cis-1-Acetoxy-2-(4-chlorocinnamoyloxy±-6-methoxy-
4
.15. (± ±-cis-1-Hydroxy-6-methoxy-2-(3,4-
3,3,14-trimethyl-1,2,3,14-tetrahydro-7H-benzo[b]pyrano[3,2-
h]acridin-7-one (18±
dimethoxycinnamoyloxy±-3,3,14-trimethyl-1,2,3,14-
tetrahydro-7H-benzo[b]pyrano[3,2-h]acridin-7-one (16±
Yield 98%, UV k nm (loge± (MeOH± 226 (3.44±, 286 (3.74±, 375
Yield 25%, IR (KBr±
m
3356, 2937, 1716, 1700, 1643, 1616, 1538,
(2.02±, 383 (2.06±, 431 (2.46±; IR (KBr± 2982, 2928, 1735, 1720,
1
8
513, 1494, 1459, 1394, 1335, 1262, 1203, 1149, 1132, 1089, 1027,
1648, 1591, 1491, 1400, 1226, 1206, 1153, 1086, 1033, 1009, 819
m cm ; H NMR (400 MHz, CDCl ± d 8.87 (1H, s, H-8±, 8.01 (1H,
3
ꢀ1
1
ꢀ1
1
3
14, 744 cm ; H NMR (400 MHz, CDCl ± d 8.77 (1H, s, H-8±, 7.98
(
1H, dd, J = 8, 1.5, H-9±, 7.74 (1H, dd, J = 8, 1.5, H-12±, 7.66 (1H, d,
dd, J = 8, 1.5, H-9±, 7.84 (1H, dd, J = 8, 1.5, H-12±, 7.55 (1H, d,
J = 16, OCOCH@CH±, 7.57 (1H, s, H-13±, 7.45 (1H, td, J = 8, 1.5, H-
J = 16, OCOCH@CH±, 7.53 (1H, td, J = 8, 1.5, H-11±, 7.52 (1H, s, H-
0
0
0
1
6
1±, 7.36 (1H, td, J = 8, 1.5, H-10±, 6.97 (1H, dd, J = 8.5, 1.5, H-6 ±,
.87 (1H, d, J = 1.5, H-2 ±, 6.74 (1H, d, J = 8.5, H-5 ±, 6.38 (1H, d,
13±, 7.41 (1H, td, J = 8, 1.5, H-10±, 7.38 (2H, d, J = 8.5, H-2 , H-6 ±,
0
0
0
0
7.30 (2H, d, J = 8.5, H-3 , H-5 ±, 6.60 (1H, d, J = 5, H-1±, 6.35 (1H, s,
H-5±, 6.34 (1H, d, J = 16, OCOCH@CH±, 5.66 (1H, d, J = 5, H-2±,
J = 16, OCOCH@CH±, 6.11 (1H, s, H-5±, 5.57 (1H, d, J = 5.5, H-2±,
5
3
.43 (1H, m ? d, J = 5.5 upon D
.85 (6H, s, 2 ꢁ OCH ±, 3.66 (3H, s, OCH
-C-3±, 1.52 (3H, s, CH
± d 178.2 (C-7±, 167.3 (CO-O-C-2±, 162.6 (C-6±,
2
O addn., H-1±, 3.97 (3H, s, NCH
3
±,
4.05 (3H, s, OCH
3
±, 3.72 (3H, s, NCH
3 3
±, 1.94 (3H, s, OCOCH ±, 1.63
1
3
3
3
±, 2.82 (1H, br s, D O exch.,
2
(3H, s, CH -C-3±, 1.51 (3H, s CH
3
3
-C-3±; C NMR (100 MHz, CDCl ±
3
-C-3±; 1 C NMR
3
d 178.4 (C-7±, 171.1 (CO-O-C-1±, 166.3 (CO-O-C-2±, 163.1 (C-6±,
160.4 (C-4a±, 150.4 (C-14a±, 144.9 (OCOCH@CH±, 142.4 (C-13a±,
OH-1±, 1.59 (3H, s, CH
100 MHz, CDCl
59.5 (C-4a±, 151.4 (C-4 ±, 149.9 (C-14a±, 149.2 (C-3 ±, 146.5
3
3
(
1
3
0
0
0
0
0
136.7 (C-4 ±, 135.9 (C-12a±, 132.5 (C-1 ±, 129.8 (C-9±, 129.5 (C-2 ,
0
0
0
(
OCOCH@CH±, 141.9 (C-13a±, 135.8 (C-12a±, 129.5 (C-9±, 128.5 (C-
C6 ±, 129.3 (C-3 , C-5 ±, 128.8 (C-8a±, 128.4 (C-11±, 128.2 (C-8±,
126.8 (C-12±, 125.9 (C-7a±, 124.7 (C-10±, 117.5 (OCOCH@CH±,
111.6 (C-13±, 111.4 (C-6a±, 97.8 (C-14b±, 94.4 (C-5±, 76.6 (C-3±,
0
8
a±, 128.2 (C-11±, 127.9 (C-8±, 127.2 (C-1 ±, 126.8 (C-12±, 125.3
0
(
C-7a±, 124.4 (C-10±, 123.2 (C-6 ±, 114.7 (OCOCH@CH±, 111.8 (C-
0
0
1
3±, 110.9 (C-5 ±, 110.8 (C-6a±, 109.4 (C-2 ±, 102.2 (C-14b±, 93.7
±,
-C-3±; ESI-MS m/z 596
±: C, 70.58; H, 5.58; N, 2.35.
69.3 (C-2±, 66.1 (C-1±, 56.5 (OCH
23.1 (CH -C-3±, 21.2 (OCOCH ±; ESI-MS m/z 612 and 614 [MH] ,
634 and 636 [MNa] . Anal. Calcd for (C35 30ClNO ±: C, 68.68; H,
4.94; N, 2.29. Found: C, 68.57; H, 5.01; N, 2.17.
3 3 3
±, 42.9 (NCH ±, 24.9 (CH -C-3±,
+
(
4
C-5±, 76.8 (C-3±, 71.9 (C-2±, 64.2 (C-1±, 56.0, 55.9, 55.7 (3 ꢁ OCH
3
3
3
+
2.3 (NCH
3
±, 25.5 (CH
3
-C-3±, 22.6 (CH
3
H
7
+
[
MH] . Anal. Calcd for (C35
H33NO
8
Found: C, 70.50; H, 5.42; N, 2.41.
4
.19. (± ±-cis-1-Acetoxy-2-(2-chlorocinnamoyloxy±-6-methoxy-
4
.16. General procedure for the synthesis of mixed esters 17–29
3,3,14-trimethyl-1,2,3,14-tetrahydro-7H-benzo[b]pyrano[3,2-
h]acridin-7-one (19±
Acetic anhydride (0.15 mL, 1.57 mmol± was added to an iced-
cooled solution (0 °C± of the appropriate (± ±-cis-2-cinnamoyloxy-
-hydroxy-6-methoxy-3,3,14-trimethyl-1,2,3,14-tetrahydro-7H-
benzo[b]pyrano[3,2-h]acridin-7-one 4–16 (0.15 mmol± and 4-
dimethylaminopyridine (0.01 g± in dry pyridine (5 mL±. After stir-
ring at 25 °C for 5 h, the reaction mixture was poured on cold water
Yield 94%, UV k nm (log
(3.79±, 338 (2.87±, 434 (2.49±; IR (KBr±
1720, 1646, 1588, 1495, 1398, 1157, 1087, 1029, 737 cm
NMR (400 MHz, CDCl ± d 8.87 (1H, s, H-8±, 8.04 (1H, d, J = 16,
e
± (MeOH± 224 (3.31±, 230 (3.31±, 286
1
m
3056, 2978, 2939, 1747,
ꢀ1
1
; H
3
OCOCH@CH±, 8.01 (1H, dd, J = 8, 1.5, H-9±, 7.84 (1H, dd, J = 8, 1.5,
0
(
20 mL±. The precipitate was filtered, washed with water
3 ꢁ 15 mL±, and dried in vacuum over P , to afford the corre-
(± ±-cis-1-acetoxy-2-cinnamoyloxy-6-methoxy-3,3,14-
trimethyl-1,2,3,14-tetrahydro-7H-benzo[b]pyrano[3,2-h]acridin-
H-12±, 7.55 (1H, dd, J = 7.5, 1.5, H-3 ±, 7.53 (1H, td, J = 8, 1.5, H-
(
2
O
5
11±, 7.52 (1H, s, H-13±, 7.41 (1H, td, J = 8, 1.5, H-10±, 7,36 (1H, dd,
0
0
sponding
J = 7.5, 1 Hz, H-6 ±, 7.26 (1H, td, J = 7.5, 1.5, H-5 ±, 7.21 (1H, td,
0
J = 7.5, 1, H-4 ±, 6.60 (1H, d, J = 5, H-1±, 6.36 (1H, d, J = 16,
7
-one 17–29 as a yellow amorphous solid in 70–100% yield.
OCOCH@CH±, 6.35 (1H, s, H-5±, 5.66 (1H, d, J = 5, H-2±, 4.03 (3H,
s, OCH
3
±, 3.72 (3H, s, NCH
3
±, 1.96 (3H, s, OCOCH
-C-3±; C NMR (100 MHz, CDCl ± d
3
±, 1.64 (3H, s,
1
3
4
.17. (± ±-cis-1-Acetoxy-2-cinnamoyloxy-6-methoxy-3,3,14-
CH -C-3±, 1,53 (3H, s, CH
3
3
3
trimethyl-1,2,3,14-tetrahydro-7H-benzo[b]pyrano[3,2-
h]acridin-7-one (17±
178,4 (C-7±, 171.2 (CO-O-C-1±, 165.9 (CO-O-C-2±, 163.1 (C-6±,
160.4 (C-4a±, 150.4 (C-14a±, 142.4 (C-13a±, 142.1 (OCOCH@CH±,
0
0
0
0
1
35.9 (C-12a±, 135.2 (C-2 ±, 132.2 (C-1 ±, 131.5 (C-5 ±, 130.3 (C-6 ±,
0
Yield 71%, UV k nm (log
3.82±, 337 (2.93±, 431 (2.57±; IR (KBr±
618, 1577, 1429, 1328, 1205, 1155, 1086, 1029 cm
400 MHz, CDCl ± d 8.87 (1H, s, H-8±, 8.01 (1H, dd, J = 8, 1.5, H-9±,
.84 (1H, dd, J = 8, 1.5, H-12±, 7.62 (1H, d, J = 16, OCOCH@CH±,
e
± (MeOH± 222 (3.40±, 236 (3.35±, 286
129.7 (C-9±, 128.8 (C-8a±, 128,4 (C-11±, 128.2 (C-8±, 127.7 (C-3 ±,
127.1 (C-4 ±, 126.8 (C-12±, 126.0 (C-7a±, 124.6 (C-10±, 119.3
0
(
1
(
7
7
m
2974, 2935, 1735, 1638,
ꢀ1
1
;
H NMR
(OCOCH@CH±, 111.7 (C-13±, 111.4 (C-6a±, 97.8 (C-14b±, 94.4 (C-
3
5±, 76.6 (C-3±, 69.5 (C-2±, 66.2 (C-1±, 56.5 (OCH
24.9 (CH -C-3±, 23.2 (CH -C-3±, 21.2 (OCOCH ±; ESI-MS m/z 612
and 614 [MH] . Anal. Calcd for (C35 30ClNO ±: C, 68.68; H, 4.94;
N, 2.29. Found: C, 68.74; H, 5.03; N, 2.27.
3 3
±, 42.9 (NCH ±,
3
3
3
0
0
+
.57–7.52 (2H, m, H-11, H-13±, 7.46–7.44 (2H, m, H2 , H6 ±, 7.41
H
7
0
0
0
(
1H, td, J = 8, 1.5, H-10±, 7.36–7.32 (3H, m, H-3 , H-4 , H-5 ±, 6.62

Q. Do et al. / Bioorg. Med. Chem. 17 (2009) 1918–1927
1925
0
0
0
4
3
.20. (± ±-cis-1-Acetoxy-2-(3-chlorocinnamoyloxy±-6-methoxy-
,3,14-trimethyl-1,2,3,14-tetrahydro-7H-benzo[b]pyrano[3,2-
136.0 (C-12a±, 135.8 (C-2 ±, 130.8 (C-1 ±, 130.1 (C-3 ±, 129.7 (C-9±,
0
0
128.8 (C-8a±, 128.5 (C-6 ±, 128.4 (C-11±, 128.2 (C-8±, 127.6 (C-5 ±,
h]acridin-7-one (20±
126.8 (C-12±, 125.9 (C-7a±, 124.7 (C-10±, 119.8 (OCOCH@CH±,
1
11.7 (C-13±, 111.4 (C-6a±, 97.7 (C-14b±, 94.4 (C-5±, 76.8 (C-3±,
69.6 (C-2±, 66.2 (C-1±, 56.4 (OCH ±, 42.9 (NCH ±, 24.9 (CH -C-3±,
23.1 (CH -C-3±, 21.2 (OCOCH ±; ESI-MS m/z 646, 648, and 650
[MH] . Anal. Calcd for (C35 NO ±: C, 65.02; H, 4.52; N, 2.17.
Found: C, 65.13; H, 4.57; N, 2.19.
Yield 99%, UV k nm (log
e± (MeOH± 206 (3.60±, 220 (3.53±, 226
3
3
3
(
1
3.51±, 286 (3.88±, 327 (2.99±; IR (KBr±
724, 1642, 1592, 1401, 1157, 1087, 1033 cm
m
3056, 2982, 2016, 2846,
3
3
ꢀ1
1
+
;
H NMR
H
29Cl
2
7
(
400 MHz, CDCl
3
± d 8.87 (1H, s, H-8±, 8.01 (1H, dd, J = 8, 1.5, H-9±,
7
7
.84 (1H, dd, J = 8, 1.5, H-12±, 7.55 (1H, d, J = 16, OCOCH@CH±,
.54 (1H, td, J = 8, 1.5, H-11±, 7.52 (1H, s, H-13±, 7.45 (1H, t,
4.23. (± ±-cis-1-Acetoxy-2-(4-bromocinnamoyloxy±-6-methoxy-
3,3,14-trimethyl-1,2,3,14-tetrahydro-7H-benzo[b]pyrano[3,2-
h]acridin-7-one (23±
0
J = 1.5, H-2 ±, 7.41 (1H, td, J = 8, 1.5, H-10±, 7.34–7.25 (3H, m, H-
0
0
0
4
, H-6 , H-5 ±, 6.60 (1H, d, J = 5, H-1±, 6.37 (1H, d, J = 16,
OCOCH@CH±, 6.35 (1H, s, H-5±, 5.66 (1H, d, J = 5, H-2±, 4.05 (3H,
s, OCH ±, 3.72 (3H, s, NCH ±, 1.94 (3H, s, OCOCH ±, 1.63 (3H, s,
CH -C-3±, 1.51 (3H, s, CH ± d
-C-3±; 13C NMR (100 MHz, CDCl
3
3
3
Yield 96%, UV k nm (log
e
± (MeOH± 205 (3.61±, 286 (3.87±, 335
3
3
3
(2.94±, 434 (2.61±; IR (KBr± m 3048, 2978, 2932, 2850, 1743, 1646,
ꢀ1
1
1
1
1
1
1
78.4 (C-7±, 171.1 (CO-O-C-1±, 166.1 (CO-O-C-2±, 163.1 (C-6±,
60.4 (C-4a±, 150.4 (C-14a±, 144.8 (OCOCH@CH±, 142.4 (C-13a±,
1588, 1405, 1087, 1005, 916, 819, 730, 699 cm
;
H NMR
(400 MHz, CDCl ± d 8.90 (1H, s, H-8±, 8.01 (1H, dd, J = 8, 1.5, H-9±,
3
0
0
0
0
35.9 (C-12a±, 135.8 (C-1 ±, 135.0 (C-3 ±, 130.6 (C-5 ±, 130.2 (C-4 ±,
7.84 (1H, dd, J = 8, 1.5, H-12±, 7.54 (1H, d, J = 16, OCOCH@CH±, 7.53
0
0
29.8 (C-9±, 128.8 (C-8a±, 128.4 (C-8±, 128.2 (C-11±, 128.0 (C-2 ±,
26.8 (C-12±, 126.6 (C-6 ±, 126.0 (C-7a±, 124.7 (C-10±, 118.4
(1H, td, J = 8, 1.5, H-11±, 7.52 (1H, s, H-13±, 7.46 (2H, d, J = 8.5, H-3 ,
0
0
0
0
H-5 ±, 7.41 (1H, td, J = 8, 1.5, H-10±, 7.30 (2H, d, J = 8.5, H-2 , H-6 ±,
6.60 (1H, d, J = 5, H-1±, 6.36 (1H, d, J = 16, OCOCH@CH±, 6.35 (1H, s,
(
OCOCH@CH±, 111.7 (C-13±, 111.4 (C-6a±, 97.8 (C-14b±, 94.4 (C-
5
2
±, 76.6 (C-3±, 69.4 (C-2±, 66.1 (C-1±, 56.5 (OCH
4.9 (CH -C-3±, 23.1 (CH -C-3±, 21.2 (OCOCH ±; ESI-MS m/z 612
30ClNO ±: C, 68.68; H, 4.94;
N, 2.29. Found: C, 68.79; H, 4.89; N, 2.33.
3
±, 42.9 (NCH
3
±,
H-5±, 5.67 (1H, d, J = 5, H-2±, 4.02 (3H, s, OCH
1.94 (3H, s, OCOCH ±, 1.63 (3H, s, CH -C-3±, 1.51 (3H, s, CH
C NMR (100 MHz, CDCl ± d 178.4 (C-7±, 171.1 (CO-O-C-1±, 166.2
(CO-O-C-2±, 163.1 (C-6±, 160.4 (C-4a±, 150.4 (C-14a±, 145.0
3
±, 3.72 (3H, s, NCH
3
±,
3
3
3
3
3
3
-C-3±;
+
13
and 614 [MH] . Anal. Calcd for (C35
H
7
3
0
0
(
OCOCH@CH±, 142.4 (C-13a±, 136.0 (C-12a±, 132.9 (C1 ±, 132.2 (C3 ,
0
0
0
4
.21. (± ±-cis-1-Acetoxy-(3,4-dichlorocinnamoyloxy±-6-
C5 ±, 129.8 (C-9±, 129.7 (C-2 , C-6 ±, 128.8 (C-8a±, 128.4 (C-11±,
128.2 (C-8±, 126.8 (C-12±, 125,9 (C-7a±, 125.1 (C-4 ±, 124.7 (C-10±,
0
methoxy-3,3,14-trimethyl-1,2,3,14-tetrahydro-7H-
benzo[b]pyrano[3,2-h]acridin-7-one (21±
117.6 (OCOCH@CH±, 111.7 (C-13±, 111.4 (C-6a±, 97.8 (C-14b±, 94.4
(
C-5±, 76.6 (C-3±, 69.3 (C-2±, 66.1 (C-1±, 56.5 (OCH
24.9 (CH -C-3±, 23.1 (CH -C-3±, 21.2 (OCOCH ±; ESI-MS m/z 656
and 658 [MH] . Anal. Calcd for (C35 30BrNO ±: C, 64.03; H, 4.61; N,
2.13. Found: C, 63.92; H, 4.62; N, 2.05.
3 3
±, 42.9 (NCH ±,
Yield 84%, UV k nm (log
3.10±, 433 (2.75±; IR (KBr±
576, 1398, 1157, 1087, 1033, 815, 737 cm ; H NMR (400 MHz,
e
m
± (MeOH± 230 (3.61±, 286 (3.97±, 337
3056, 2978, 2935, 2854, 1747, 1646,
3
3
3
+
(
1
H
7
ꢀ1 1
CDCl
3
± d 8.87 (1H, s, H-8±, 8.01 (1H, dd, J = 8, 1.5, H-9±, 7.84 (1H, dd,
0
J = 8, 1.5, H-12±, 7.56–7.48 (4H, m, H-11, H-2 , H-13, OCOCH@CH±,
4.24. (± ±-cis-1-Acetoxy-2-(3-bromocinnamoyloxy±-6-methoxy-
3,3,14-trimethyl-1,2,3,14-tetrahydro-7H-benzo[b]pyrano[3,2-
h]acridin-7-one (24±
0
0
7
.43–7.39 (2H, m, H-10, H-5 ±, 7.27 (1H, dd, J = 8.5, 2, H-6 ±, 6.60
(
1H, d, J = 5, H-1±, 6.35 (1H, d, J = 16, OCOCH@CH±, 6.34 (1H, s, H-5±,
5
(
.66 (1H, d, J = 5, H-2±, 4.05 (3H, s, OCH
3H, s, OCOCH ±, 1.63 (3H, s, CH -C-3±, 1.51 (3H, s, CH
NMR (100 MHz, CDCl
O-C-2±, 163.1 (C-6±, 160.3 (C-4a±, 150.4 (C-14a±, 143.6 (OCOCH@CH±,
3
±, 3.71 (3H, s, NCH
3
±, 1.94
1
3
3
3
3
-C-3±;
C
Yield 90%, IR (KBr±
1494, 1464, 1399, 1227, 1205, 1153, 1083, 1028, 747 cm ; H
m 2916, 2846, 1731, 1651, 1621, 1586, 1569,
ꢀ1
1
3
± d 178.4 (C-7±, 171.1 (CO-O-C-1±, 165.9 (CO-
NMR (400 MHz, CDCl
H-9±, 7.84 (1H, dd, J = 8, 1.5, H-12±, 7.61–7.52 (4H, m, H-2 , H-11,
H-13, OCOCH@CH±, 7.47 (1H, dt, J = 8, 1.5, H-4 ±, 7.41 (1H, td, J = 8,
1.5, H-10±, 7.37 (1H, dt, J = 8, 1.5, H-6 ±, 7.22 (1H, t, J = 8, H-5 ±, 6.61
(1H, d, J = 5, H-1±, 6.37 (1H, d, J = 16, OCOCH@CH±, 6.36 (1H, s, H-5±,
3
± d 8.88 (1H, s, H-8±, 8.01 (1H, dd, J = 8, 1.5,
0
0
0
0
1
1
42.4 (C-13a±, 135.9 (C-12a±, 134.7 (C-1 ±, 134.0 (C-3 ±, 133.4 (C-4 ±,
0
0
0
30.9(C-5 ±, 129.9(C-2 ±, 129.8 (C-9±, 128.8 (C-8a±, 128.4(C-8±, 128.2
0
0
0
(
(
C-11±, 127.3 (C-6 ±, 126.8 (C-12±, 126.0 (C-7a±, 124.7 (C-10±, 118.8
OCOCH@CH±, 111.6 (C-13±, 111.5 (C-6a±, 97.7 (C-14b±, 94.4 (C-5±,
7
(
6.6 (C-3±, 69.5 (C-2±, 66.1 (C-1±, 56.5 (OCH
CH -C-3±, 23.1 (CH -C-3±, 21,2 (OCOCH ±; ESI-MS m/z 646, 648,
and 650 [MH] . Anal. Calcd for (C35 NO ±: C, 65.02; H, 4.52; N,
3
±, 42.9 (NCH
3
± ; 24.9
5.66 (1H, d, J = 5, H-2±, 4.05 (3H, s, OCH
(3H, s, OCOCH ±, 1.64 (3H, s, CH -C-3±, 1.52 (3H, s, CH
NMR (100 MHz, CDCl
O-C-2±, 163.1 (C-6±, 160.4 (C-4a±, 150.4 (C-14a±, 144.7 (OCOCH@CH±,
3
±, 3.73 (3H, s, NCH
3
±, 1.94
13
3
3
3
3
3
3
-C-3±;
C
+
H29Cl
2
7
3
± d 178.4 (C-7±, 171.1 (CO-O-C-1±, 166.1 (CO-
2
.17. Found: C, 64.90; H, 4.42; N, 2.09.
0
0
0
1
42.4 (C-13a±, 136.1 (C-1 ±, 136.0 (C-12a±, 133.5 (C-4 ±, 131.0 (C-2 ±,
0
4
.22. (± ±-cis-1-Acetoxy-(2,4-dichlorocinnamoyloxy±-6-
130.5 (C-5 ±, 129.8 (C-9±, 128.8 (C-8a±, 128.4 (C-11±, 128.2 (C-8±,
127.0 (C-6 ±, 126.8 (C-12±, 126.0 (C-7a±, 124.7 (C-10±, 123.1 (C-3 ±,
0
0
methoxy-3,3,14-trimethyl-1,2,3,14-tetrahydro-7H-
benzo[b]pyrano[3,2-h]acridin-7-one (22±
118.5 (OCOCH@CH±, 111.7 (C-13±, 111.4 (C-6a±, 97.8 (C-14b±, 94.4
(
C-5±, 76.6 (C-3±, 69.4 (C-2±, 66.1 (C-1±, 56.5 (OCH
24.9 (CH -C-3±, 23.1 (CH -C-3±, 21.2 (OCOCH ±; ESI-MS m/z 656
and 658 [MH] . Anal. Calcd for (C35 30BrNO ±: C, 64.03; H, 4.61; N,
2.13. Found: C, 64.20; H, 4.53; N, 2.21.
3 3
±, 42.9 (NCH ±,
Yield 100%, UV k nm (log
3.05±, 434 (2.66±; IR (KBr±
e
± (MeOH± 233 (3.54±, 286 (3.99±, 337
2924, 2850, 1728, 1638, 1623, 1588,
; H NMR (400 MHz, CDCl ± d 8.87
3
3
3
3
+
(
m
H
7
ꢀ
1
1
1
398, 1207, 1149, 1087 cm
1H, s, H-8±, 8.01 (1H, dd, J = 8, 1.5, H-9±, 7.95 (1H, d, J = 16,
OCOCH@CH±, 7.84 (1H, dd, J = 8, 1.5, H-12±, 7.56–7.48 (3H, m, H-
(
4.25. (± ±-cis-1-Acetoxy-2-(4-trifluoromethylcinnamoyloxy±-1-
hydroxy-6-methoxy-3,3,14-trimethyl-1,2,3,14-tetrahydro-7H-
benzo[b]pyrano[3,2-h]acridin-7-one (25±
0
0
1
1, H-13, H-6 ±, 7.43–7.39 (2H, m, H-10, H-3 ±, 7.20 (1H, dd, J = 8,
0
1
.5, H-5 ±, 6.60 (1H, d, J = 5, H-1±, 6.34 (1H, s, H-5±, 6.33 (1H, d,
J = 16, OCOCH@CH±, 5.66 (1H, d, J = 5, H-2±, 4.03 (3H, s, OCH
3
±,
-C-3±,
3
± d 178.4 (C-7±,
3
1
1
1
.72 (3H, s, NCH
3
±, 1.95 (3H, s, OCOCH
3
±, 1.64 (3H, s, CH
3
Yield 88%, IR (KBr±
m 2981, 2930, 2849, 1723, 1642, 1615, 1592,
13
.52 (3H, s, CH -C-3±; C NMR (100 MHz, CDCl
3
1490, 1460, 1392, 1372, 1352, 1281, 1206, 1159, 1122, 1027, 1014,
ꢀ
1 1
71.2 (CO-O-C-1±, 165.8 (CO-O-C-2±, 163.1 (C-6±, 160.4 (C-4a±,
50.4 (C-14a±, 142.4 (C-13a±, 140.9 (OCOCH@CH±, 136.9 (C-4 ±,
831 cm ; H NMR (400 MHz, CDCl
3
± d 8.88 (1H, s, H-8±, 8.01 (1H,
0
dd, J = 8, 1.5, H-9±, 7.84 (1H, dd, J = 8, 1.5, H-12±, 7.66–7.53 (7H, m,

1
926
Q. Do et al. / Bioorg. Med. Chem. 17 (2009) 1918–1927
0
0
0
0
H-2 , H-6 , H-3 , H-5 , H-11, H-13, OCOCH@CH±, 7.41 (1H, td, J = 8,
4.28. (± ±-cis-1-Acetoxy-6-methoxy-2-(4-
1
6
.5, H-10±, 6.61 (1H, d, J = 5, H-1±, 6.45 (1H, d, J = 16, OCOCH@CH±,
.36 (1H, s, H-5±, 5.68 (1H, d, J = 5, H-2±, 4.05 (3H, s, OCH ±, 3.73
±, 1.94 (3H, s, OCOCH ±, 1.65 (3H, s, CH -C-3±, 1.53
-C-3±; C NMR (100 MHz, CDCl ± d 178.3 (C-7±, 171.0
CO-O-C-1±, 165.9 (CO-O-C-2±, 163.0 (C-6±, 160.3 (C-4a±, 150.3 (C-
methoxycinnamoyloxy±-3,3,14-trimethyl-1,2,3,14-tetrahydro-
7H-benzo[b]pyrano[3,2-h]acridin-7-one (28±
3
(
(
(
3H, s, NCH
3H, s, CH
3
3
3
1
3
3
3
Yield 90%, UV k nm (log
(3.28±, 373 (1.99±, 418 (2.44±, 425 (2.45±; IR (KBr±
2924, 2850, 1735, 1650, 1588, 1510, 1153, 1083, 1029, 737 cm ;
e± (MeOH± 230 (3.34±, 286 (3.68±, 308
m
3052, 2955,
ꢀ
1
1
1
2
4a±, 144.4 (OCOCH@CH±, 142.3 (C-13a±, 137.3 (C-12a±, 135.9 (C-
0
0
0
1
±, 131.8 (q, J = 35, C-4 ±, 129.7 (C-8±, 128.7 (C-11±, 128.4 (2C, C-
3
H NMR (400 MHz, CDCl ± d 8.87 (1H, s, H-8±, 8.01 (1H, dd, J = 8,
0
0
, C-6 ±, 128.3 (C-8a±, 128.1 (C-9±, 126.7 (C-12±, 125.8 (2C, C-3 ,
1.5, H-9±, 7.84 (1H, dd, J = J = 8, 1.5, H-12±, 7.57 (1H, d, J = 16,
OCOCH@CH±, 7.54 (1H, td, J = 8, 1.5, H-11±, 7.53 (1H, s, H-13±,
0
C-5 ±, 125.5 (C-7a±, 124.6, (C-10±, 119.4 (OCOCH@CH±, 118.4 (q,
0
0
J = 270, CF
7.2 (C-3±, 69.4 (C-2±, 66.0 (C-1±, 55.4 (OCH
CH -C-3±, 23.0 (CH -C-3±, 21.1 (OCOCH ±; ESI-MS m/z 646 [MH] .
Anal. Calcd for (C36 NO ±: C, 66.97; H, 4.68; N, 2.17. Found:
C, 67.08; H, 4.53; N, 2.24.
3
±, 111.6 (C-13±, 111.3 (C-6a±, 97.6 (C-14b±, 94.3 (C-5±,
7.41 (1H, td, J = 8, 1.5, H-10±, 7.40 (2H, d, J = 8.5, H-2 , H-6 ±, 6.84
0
0
7
(
3
±, 42.9 (NCH ±, 24.8
3
(2H, d, J = 8.5, H-3 , H-5 ±, 6.60 (1H, d, J = 5, H-1±, 6.36 (1H, s, H-
+
3
3
3
5±, 6.23 (1H, d, J = 16, OCOCH@CH±, 5.66 (1H, d, J = 5, H-2±, 4.05
0
H
30
F
3
7
(3H, s, CH
3
O-C-6±, 3.81 (3H, s, NCH
±, 1.63 (3H, s, CH
3
±, 3.73 (3H, s, CH
-C-3±, 1.59 (3H, s, CH
3
O-C-4 ±,
1.94 (3H, s, OCOCH
3
3
3
-C-3±;
1
3
C NMR (100 MHz, CDCl
3
± d 178.4 (C-7±, 171.1 (CO-O-C-1±, 166.8
0
4
.26. (± ±-cis-1-Acetoxy-2-(3-Trifluoromethylcinnamoyloxy±-6-
(CO-O-C-2±, 163.1 (C-6±, 161.7 (C-4 ±, 160.5 (C-4a±, 150.4 (C-14a±,
0
methoxy-3,3,14-trimethyl-1,2,3,14-tetrahydro-7H-
benzo[b]pyrano[3,2-h]acridin-7-one (26±
146.1 (OCOCH@CH±, 142.5 (C-13a±, 135.9 (C-12a±, 130.1 (2C, C-2 ,
0
C-6 ±, 129.7 (C-9±, 128.8 (C-8a±, 128.4 (C-11±, 128.2 (C-8±, 126.8
0
0
0
(
2C, C-12, C-1 ±, 126.0 (C-7a±, 124.6 (C-10±, 114.4 (2C, C-3 , C-5 ±,
Yield 98%, IR (KBr±
m 2982, 2926, 2835, 1729, 1646, 1621, 1589,
114.4 (OCOCH@CH±, 111.7 (C-13±, 111.4 (C-6a±, 97.9 (C-14b±,
1
1
8
573, 1494, 1462, 1400, 1373, 1332, 1270, 1227, 1205, 1159, 1127,
94.4 (C-5±, 76.8 (C-3±, 68.9 (C-2±, 66.2 (C-1±, 56.5 (CH
3
O-C-6±,
-C-3±,
ꢀ1
1
0
089, 1033, 914, 868, 806, 746 cm ; H NMR (400 MHz, CDCl
3
± d
55.5 (CH
21.2 (OCOCH
for (C36
5.54; N, 2.34.
3
O-C-4 ±, 42.9 (NCH
3
±, 24.9 (CH
3
-C-3±, 23.1 (CH
3
+
+
.87 (1H, s, H-8±, 8.01 (1H, dd, J = 8, 1.5, H-9±, 7.84 (1H, dd, J = 8, 1.5,
3
±; ESI-MS m/z 608 [MH] ; 630 [MNa] . Anal. Calcd
8
H33NO ±: C, 71.16; H, 5.47; N, 2.31. Found: C, 71.01; H,
0
0
0
H-12±, 7.70–7.59 (4H, m, H-2 , H-4 , H-6 , OCOCH@CH±, 7.54 (1H, td,
J = 8, 1.5, H-11±, 7.53 (1H, s, H-13±, 7.45 (1H, t, J = 8, H-5 ±, 7.41 (1H,
0
td, J = 8, 1.5, H-10±, 6.62 (1H, d, J = 5, H-1±, 6.44 (1H, d, J = 16,
OCOCH@CH±, 6.36 (1H, s, H-5±, 5.67 (1H, d, J = 5, H-2±, 4.05 (3H,
4.29. (± ±-cis-1-Acetoxy-6-methoxy-2-(3,4-
s, OCH
3
±, 3.72 (3H, s, NCH
3
±, 1.95 (3H, s, OCOCH
–C-3±; 13C NMR (100 MHz, CDCl
3
3
±, 1.64 (3H, s,
dimethoxycinnamoyloxy±-3,3,14-trimethyl-1,2,3,14-
tetrahydro-7H-benzo[b]pyrano[3,2-h]acridin-7-one (29±
CH –C-3±, 1.52 (3H, s, CH
3
3
± d
1
1
1
1
1
1
1
78.4 (C-7±, 171.1 (CO-O-C-1±, 166.0 (CO–O–C-2±, 163.1 (C-6±,
60.4 (C-4a±, 150.4 (C-14a±, 144.5 (OCOCH@CH±, 142.4 (C-13a±,
Yield 98%, IR (KBr±
m 2982, 2932, 2833, 1740, 1717, 1651, 1624,
0
0
0
36.0 (C-12a±, 134.8 (C-1 ±, 131.7 (q, J = 30, C-3 ±, 131.4 (C-6 ±,
1588, 1508, 1462, 1389, 1263, 1230, 1027, 1141, 1028, 905, 813,
0
ꢀ1
1
29.8 (C-5 ±, 129.5 (C-9±, 128.8 (C-8a±, 128.4 (C-11±, 128.2 (C-8±,
3
733 cm ; H NMR (400 MHz, CDCl ± d 8.88 (1H, s, H-8±, 8.01
0
0
27.1 (C-4 ±, 126.8 (C-12±, 126.0 (C-7a±, 124.8 (C-2 ±, 124.7 (C-
0±, 119.2 (q, J = 270, CF ±, 118.9 (OCOCH@CH±, 111.7 (C-13±,
11.4 (C-6a±, 97.8 (C-14b±, 94.4 (C-5±, 76.6 (C-3±, 69.5 (C-2±, 66.1
C-1±, 56.5 (OCH ±, 42.9 (NCH ±, 24.9 (CH –C-3±, 23.1 (CH -C-3±,
±; ESI-MS m/z 646 [MH] . Anal. Calcd for
±: C, 66.97; H, 4.68; N, 2.17. Found: C, 66.88; H,
(1H, dd, J = 8, 1.5, H-9±, 7.90 (1H, dd, J = 8, 1.5, H-12±, 7.58–7.53
(3H, m, H-11, H-13, OCOCH@CH±, 7.41 (1H, td, J = 8, 1.5, H-10± ;
3
0
0
7.01 (1H, dd, J = 8.5, 1.5, H-6 ±, 6,98 (1H, d, J = 1.5, H-2 ±, 6.80 (1H,
0
(
3
3
3
3
d, J = 8.5, H-5 ±, 6.61 (1H, d, J = 5, H-1±, 6.36 (1H, s, H-5±, 6.19 (1H,
+
2
1.2 (OCOCH
NO
.74; N, 2.20.
3
d, J = 16, OCOCH@CH±, 5.68 (1H, d, J = 5, H-2±, 4.05 (3H, s, CH
3
O-
0
0
(
C
36
H
30
F
3
7
C-6±, 3.88 (6H, s, CH
(3H, s, OCOCH ±, 1.64 (3H, s, CH
NMR (100 MHz, CDCl ± d 178.4 (C-7±, 171.1 (CO-O-C-1±, 166.8
3
O-C-3 , CH
3
O-C-4 ±, 3.73 (3H, s, NCH
3
±, 1.94
13
4
3
3
-C-3±, 1.52 (3H, s, CH -C-3±;
3
C
3
0
0
4
.27. (± ±-cis-1-Acetoxy-6-methoxy-3,3,14-trimethyl-2-(4-
(CO-O-C-2±, 163.1 (C-6±, 160,4 (C-4a±, 151.5 (C-4 ±, 149.3 (C-3 ±,
150.4 (C-14a±, 146.4 (OCOCH@CH±, 142.4 (C-13a±, 135.9 (C-12a±,
129.8 (C-9±, 128.8 (C-8a±, 128.4 (C-8±, 128.2 (C-11±, 127.0 (C-1 ±,
26.8 (C-12±, 126.0 (C-7a±, 124.7 (C-10±, 123.4 (C-6 ±, 114.4
(OCOCH@CH±, 111.6 (2C, C-13, C-6a±, 111.0 (C-5 ±, 109.5 (C-2 ±,
nitrocinnamoyloxy±-1,2,3,14-tetrahydro-7H-
benzo[b]pyrano[3,2-h]acridin-7-one (27±
0
0
1
0
0
Yield 84%, UV k nm (log
2.65±; IR (KBr± 3056, 2961, 2928, 2851, 1729, 1641, 1590,
080, 1025, 740 cm
H-8±, 8.19 (2H, d, J = 8.5, H-3 , H-5 ±, 8.01 (1H, dd, J = 8, 1.5, H-9±,
e± (MeOH± 233 (3.47±, 287 (3.85±, 431
(
1
m
97.9 (C-14b±, 94.4 (C-5±, 76.8 (C-3±, 68.9 (C-2±, 66.2 (C-1±, 56.5,
ꢀ1
1
;
H NMR (400 MHz, CDCl
3
± d 8.87 (1H, s,
56.1, 56.0 (3 ꢁ OCH
3±, 21.2 (OCOCH ±; ESI-MS m/z 638 [MH] . Anal. Calcd for
9
(C37H35NO ±: C, 69.69; H, 5.53; N, 2.20. Found: C, 69.82; H, 5.67;
3 3 3 3
±, 42.9 (NCH ±, 24.9 (CH -C-3±, 23.0 (CH -C-
0
0
+
3
7
7
.84 (1H, dd, J = 8, 1.5, H-12±, 7.63 (1H, d, J = 16, OCOCH@CH±,
.60 (2H, d, J = 8.5, H-2 , H-6 ±, 7.54 (1H, td, J = 8, 1.5, H-11±, 7.52
0
0
N, 2.29.
(
1H, s, H-13±, 7.41 (1H, td, J = 8, 1.5, H-10±, 6.61 (1H, d, J = 5, H-
±, 6.49 (1H, d, J = 16, OCOCH@CH±, 6.35 (1H, s, H-5±, 5.67 (1H, d,
J = 5, H-2±, 4.05 (3H, s, OCH ±, 3.72 (3H, s, NCH ±, 1.95 (3H, s,
OCOCH ±, 1.64 (3H, s, CH -C-3±, 1.55 (3H, s, CH -C-3±; C NMR
100 MHz, CDCl
1
4.30. DNA alkylation
3
3
1
3
Kinetics for DNA alkylation were performed using a 117-bp
DNA fragment obtained from EcoRI and PvuII restriction enzymes
3
3
3
(
3
± d 178.4 (C-7±, 171.1 (CO-O-C-1±, 165.6 (CO-O-
0
C-2±, 163.1 (C-6±, 160.3 (C-4a±, 150.4 (C-14a±, 148.7 (C-4 ±, 143.1
digestion of the pBS plasmid which was then labeled at the EcoRI
0
32
(
(
8
1
9
OCOCH@CH±, 142.4 (C-13a±, 140.0 (C-1 ±, 135.9 (C-12a±, 129.8
site using a-[ P]-dATP (Amersham± and AMV reverse transcrip-
0
0
C-9±, 128.9 (2C, C-2 , C-6 ±, 128.8 (C-8a±, 128.4 (C-11±, 128.2 (C-
±, 126.8 (C-12±, 126.1 (C-7a±, 124.7 (C-10±, 124.2 (2C, C-3 , C-5 ±,
21.3 (OCOCH@CH±, 111.6 (C-13±, 111.4 (C-6a±, 97.6 (C-14b±,
tase (Ozyme± and purified as previously described (David-Cordon-
nier et al., 2002±. The radiolabelled DNA fragment was then
incubated with a fixed concentration of drug (50 lM± in 1 mM
Na cacodylate, pH 7.0 in the dark and room temperature during
5, 10, 20, 30, 45 min, 1, 2, 3, 4, 6, 8, 16 and 24 h. At the desired incu-
bation time, the reaction was stopped by adding 5
erol containing tracking dyes solution and subsequent freezing of
0
0
4.3 (C-5±, 76.5 (C-3±, 69.7 (C-2±, 66.1 (C-1±, 56.5 (OCH
±, 24.9 (CH -C-3±, 23.0 (CH -C-3±, 21.2 (OCOCH ±; ESI-MS
m/z 623 [MH] . Anal. Calcd for (C35 ±: C, 67.52; H, 4.86; N,
.50. Found: C, 67.39; H, 4.71; N, 4.61.
3
±, 42.9
(
NCH
3
3
3
3
+
H
30
N
2
O
9
lL of a 50% glyc-
4

Q. Do et al. / Bioorg. Med. Chem. 17 (2009) 1918–1927
1927
the reaction samples at ꢀ80 °C. The DNA samples were then re-
solved by electrophoresis under non-denaturing conditions in 6%
polyacrylamide gels for around 5 h at 300 V at room temperature
Compounds were administered at their maximal tolerated dose
(MTD± which was the highest non-toxic dose (4 mg/kg±; MTD/2
(2 mg/kg± and MTD/4 (1 mg/kg±. A dose is considered as toxic
when it induces toxic deaths or when the weight loss is higher than
20%. Tumor were measured twice a wekk and tumor volume (Vt±
were calculated using the following formula: length
2
in TBE buffer (89 mM boric acid, 2.5 mM Na EDTA, pH 8.3±. Gels
were transferred to Whatman 3MM paper, dried under vacuum
at 80 °C, and finally analyzed on a phosphorimager (Molecular
Dynamics 445SI±. The t1/2 corresponds to the incubation time re-
quires to obtain one half of the maximum of alkylation retardation
as a plateau (tmax±. When the plateau is not reached the t1/2 is re-
ferred as > of the half of the maximum of experiment time if the
plateau is nearly reached or > of the maximum of experiment time
2
2
(mm± ꢁ width (mm ±/2. At the end of the experiment, on day
80, mice were palpated and tumor-free mice were recorded.
Acknowledgment
(
24 h± if the plateau is far to be reached (Fig. 1±.
M.-H.D.-C thanks the Ligue Nationale contre le Cancer (Comité
du Nord± for a grant.
4
.31. Cytotoxicity
References and notes
L1210 and KB-3-1 cells were cultivated in RPMI 1640 or DMEM
1
2
.
.
Hotta, K.; Ueoka, H. Crit. Rev. Oncol. Hematol. 2005, 55, 45.
Van Kesteren, Ch.; De Vooght, M. M. M.; Lopez-Lazaro, L.; Mahot, R. R. A.;
Schellens, J. H. M.; Jimeno, J. M.; Beijnen, J. H. Anti-Cancer Drugs 2003, 14, 487.
medium, respectively (Gibco± supplemented with 10% fetal calf
serum, 2 mM -glutamine, 100 units/mL penicillin, 100 g/mL
L
l
streptomycin, and 10 mM HEPES buffer (pH 7.4±. Cytotoxicity
3. Costes, N.; Le Deit, H.; Michel, S.; Tillequin, F.; Koch, M.; Pfeiffer, B.; Renard, P.;
Léonce, S.; Guilbaud, N.; Kraus-Berthier, L.; Pierré, A.; Atassi, Gh. J. Med. Chem.
2000, 43, 2395.
was measured by the microculture tetrazolium assay (MTA± as de-
1
7
scribed. Cells were exposed to graded concentrations of drug
nine serial dilutions in triplicate± for 4 doubling times (48 h for
4.
David-Cordonnier, M.-H.; Laine, W.; Lansiaux, A.; Kouach, M.; Briand, G.; Pierré,
A.; Hickman, J. A.; Bailly, C. Biochemistry 2002, 41, 9911.
(
5.
Doan Thi Mai, H.; Gaslonde, T.; Michel, S.; Tillequin, F.; Koch, M.; Bongui, J.-B.;
Elomri, A.; Seguin, E.; Pfeiffer, B.; Renard, P.; David-Cordonnier, M.-H.; Tardy,
C.; Laine, W.; Bailly, C.; Kraus-Berthier, L.; Léonce, S.; Hickman, J. A.; Pierré, A. J.
Med. Chem. 2003, 46, 3072.
L1210 cells and 96 h for KB-3-1 cells±. Results are expressed as
IC50, the concentration which reduced by 50% the optical density
of treated cells with respect to the optical density of untreated
controls.
6. David-Cordonnier, M.-H.; Laine, W.; Kouach, M.; Briand, G.; Vézin, H.;
Gaslonde, T.; Michel, S.; Doan Thi Mai, H.; Tillequin, F.; Koch, M.; Léonce, S.;
Pierré, A.; Bailly, C. Bioorg. Med. Chem. 2004, 12, 23.
4
.32. Cell cycle analysis
7.
8.
9.
David-Cordonnier, M.-H.; Laine, W.; Lansiaux, A.; Rosu, F.; Colson, P.; De Pauw,
E.; Tillequin, F.; Michel, S.; Koch, M.; Pierré, A.; Hickman, J. A.; Bailly, C. Mol.
Cancer Ther. 2005, 4, 71.
Léonce, S.; Perez, V.; Lambel, S.; Peyroulan, D.; Tillequin, F.; Michel, S.; Koch,
M.; Pfeiffer, B.; Atassi, Gh.; Hickman, J. A.; Pierré, A. Mol. Pharmacol. 2001, 60,
1383.
5
For the cell cycle analysis, L1210 cells (5 ꢁ 10 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 con-
Damen, E. W. P.; Nevalainen, T. J.; van den Bergh, T. J. M.; de Groot, F. M. H.;
Scheeren, H. W. Bioorg. Med. Chem. 2002, 10, 71–77.
taining 100
lg/mL RNAse and 50 lg/mL propidium iodide for
3
0 min at 20 °C. For each sample, 10,000 cells were analyzed on
10. Doan Thi Mai, H.; Gaslonde, T.; Michel, S.; Koch, M.; Tillequin, F.; Bailly, C.;
an XLMCL flow cytometer (Beckman Coulter, France±. Results are
expressed as the% of cells in the S phase of the cell cycle.
David-Cordonnier, M.-H.; Pfeiffer, B.; Léonce, S.; Pierré, A. Chem. Pharm. Bull.
2005, 53, 919–922.
1
1
1
1. Bongui, J.-B.; Elomri, A.; Cahard, D.; Tillequin, F.; Pfeiffer, B.; Pierré, A.; Seguin,
E. Chem. Pharm. Bull. 2005, 53, 1540.
2. Sittisombut, C.; Boutefnouchet, S.; Trinh Van-Dufat, H.; Tian, W.; Michel, S.;
Koch, M.; Tillequin, F.; Pfeiffer, B.; Pierré, A. Chem. Pharm. Bull. 2006, 54, 1113.
3. Nguyen, T. M.; Sittisombut, C.; Boutefnouchet, S.; Lallemand, M.-C.; Michel, S.;
Koch, M.; Tillequin, F.; Mazinghien, R.; Lansiaux, A.; David-Cordonnier, M.-H.;
Pfeiffer, B.; Kraus-Berthier, L.; Léonce, S.; Pierré, A. J. Med. Chem. 2006, 49, 3383.
4. Gaslonde, T.; Michel, S.; Koch, M.; Pfeiffer, B.; Léonce, S.; Pierré, A.; Tillequin, F.
Chem. Pharm. Bull. 2007, 55, 734.
4
.33. Antitumor activity
The antitumor activity of the compounds 1, 8 and 21 was eval-
uated on the murine colon 38 adenocarcinoma implanted in
1
1
B6D2F1 (C57B1/6 ꢁ DBA2± mice. The colon adenocarcinoma C38
(
NCI, Frederick± was introduced by sc implantation of a tumor frag-
5. Tillequin, F. Phytochem. Rev. 2007, 6, 65.
ment 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.
[16]. Do, Q.; Doan Thi Mai, H.; Gaslonde, T.; Pfeiffer, B.; Léonce, S.; Pierré, A.;
Michel, S.; Tillequin, F.; Dufat, H. Eur. J. Med. Chem. 2008, 43, 2677.
1
7. Elomri, A.; Mitaku, S.; Michel, S.; Skaltsounis, A.-L.; Tillequin, F.; Koch, M.;
Pierré, A.; Guilbaud, N.; Léonce, S.; Kraus-Berthier, L.; Rolland, Y.; Atassi, Gh. J.
Med. Chem. 1996, 39, 4762.