Synthesis, antitumor activity, and mechanism of action of benzo[b]chromeno[6,5-g][1,8]naphthyridin-7-one analogs of acronycine

Article abstract of DOI:10.1021/jm500927d

A series of 6-methoxy-3,3,14-trimethyl-3,14-dihydro-7H-benzo[b]chromeno[6,5-g][1,8]naphthyridin-7-one (4), 13-aza derivatives of benzo[b]acronycine, the isomeric 5-methoxy-2,2,13-trimethyl-2,13-dihydro-6H-benzo[b]chromeno[7,6-g][1,8]naphthyridin-6-one (5)

Full text of DOI:10.1021/jm500927d

Article
pubs.acs.org/jmc
Synthesis, Antitumor Activity, and Mechanism of Action of
Benzo[b]chromeno[6,5‑g][1,8]naphthyridin-7-one Analogs of
Acronycine
Wen Tian,† Rodrigue Yougnia,† Sabine Depauw,‡ Amelie Lansiaux,‡ Marie-Helene David-Cordonnier,‡
́
́
̀
Bruno Pfeiffer,§ Laurence Kraus-Berthier,§ Stephane Leonce,§ Alain Pierre,§ Hanh Dufat,†
́
́
́
and Sylvie Michel*,†,∥
†Laboratoire de Pharmacognosie, Universite
́ ́
Paris Descartes, U.M.R./C.N.R.S. n° 8638, Faculte des Sciences Pharmaceutiques
et Biologiques, 4 Avenue de l’Observatoire, 75006 Paris, France
‡INSERM U837-JPARC (Jean-Pierre Aubert Research Center), Team “Molecular and Cellular Targeting for Cancer Treatment”,
Universite
France
́
Lille Nord de France, IMPRT-Institut Fed
́
er
́
atif de Recherche 114, IRCL, Place de Verdun, 59045 Lille Cedex,
§Institut de Recherches Servier, Division Recherche Cancer
́
ologie, 125 Chemin de Ronde, 78290 Croissy sur Seine, France
S
* Supporting Information
ABSTRACT: A series of 6-methoxy-3,3,14-trimethyl-3,14-dihydro-
7H-benzo[b]chromeno[6,5-g][1,8]naphthyridin-7-one (4), 13-aza
derivatives of benzo[b]acronycine, the isomeric 5-methoxy-2,2,13-
trimethyl-2,13-dihydro-6H-benzo[b]chromeno[7,6-g][1,8]-
naphthyridin-6-one (5), and related cis-diols mono- and
diesters were designed and synthesized. Their in vitro and
in vivo biological activities were evaluated. As previously
observed in the acronycine series, esters were the most potent
derivatives exhibiting submicromolar activities; among them
monoesters are particularly active. Racemic diacetate 21
showed a strong activity against KB-3-1 cell lines and was
selected for in vivo evaluation and proved to be active,
inhibiting tumor growth by more than 80%. After separation of the two enantiomers, compounds 21a and 21b were also
evaluated against C38 colon adenocarcinoma; their activities were found to be significantly different.
which underwent phase I clinical trials under the code
S23906-1 (3).6b
Similar activity observed with cis-1,2-dihydroxy-1,2-dihydrobenzo-
[b]acronycine monoesters at position 2 could be rationalized
on the basis of spontaneous transesterification into the isomeric
cis-monoesters at position 1.7
The mechanism of action of 3 implies an unusual alkylation of
the 2-amino group of DNA guanine residues, in the minor groove,
by a carbocation. The latter results from the elimination of
the ester leaving group at position 1 of the drug.8 A marked
destabilization of the double helix, with the formation of single-
stranded DNA during the S-phase of the cell cycle, causes cell
death by apoptosis.9a,b This atypical alkylating agent inhibits
DNA synthesis, increases cyclin E1 and B1 protein levels,
associated with CDk1 kinase activity, and suggests the induction
of a mitotic catastrophe.9c Furthermore, Nucleotide Excision
Repair (NER) proteins were shown to regulate sensitivity of the
cells to 3.9d,e
INTRODUCTION
■
After the isolation of the pyranoacridone alkaloid acronycine
(1) (Chart 1) from Acronychia baueri Schott (Rutaceae),1 the
interest in this compound was underlined by Svoboda et al.,2
highlighting antitumor properties against a large panel of murine
solid tumor models. However, subsequent clinical trials were
hampered by the moderate potency and the very low solubility
of 1 in aqueous solvents.3 Consequently, the development of
structural analogs with increased potency and/or better solubility
in biocompatible solvents was highly desirable.
The isolation from New-Caledonian Sarcomelicope species of
a natural unstable acronycine epoxide 2 permitted establishment
of an hypothesis of bioactivation, in vivo, of the pyran 1,2-double
bond into the corresponding oxirane. This led us to design structural
analogs4 with a double bond of the pyran ring functionalized.
New compounds, with similar reactivity toward nucleophilic agents
such as acronycine epoxide but with an improved chemical stability
were obtained. Thus, diesters of cis- and trans-1,2-dihydroxy-1,2-
dihydroacronycine5 and cis-1,2-dihydroxy-1,2-dihydrobenzo[b]-
acronycine6 were shown to be very potent and are exempli-
fied by ( )-cis-1,2-diacetoxy-1,2-dihydrobenzo[b]acronycine,
Received: June 18, 2014
© XXXX American Chemical Society
A
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Treatment of 11 with hydrogen bromide in acetic acid under reflux
gave the required 8,10-dihydroxy-6-methyldibenzo[b,g][1,8]-
naphthyridin-11(6H)-one (12) in 86% yield, accompanied by smaller
amounts of 10-hydroxy-8-methoxy-6-methyldibenzo[b,g][1,8]-
naphthyridin-11(6H)-one (13). Construction of the dimethylpyran
ring onto the phenol at the 8-position of 12 was performed by a
thermic rearrangement of the corresponding dimethylpropargyl
ether.6a,13 Treatment of 12 with 3-chloro-3-methylbut-1-yne (14)14
at 65 °C in dimethylformamide in the presence of potassium carbonate
and potassium iodide gave the desired 10-hydroxy-8-(1,1-dimethyl-
propyn-1-oxy)-6-methyldibenzo[b,g][1,8]naphthyridin-11(6H)-one
(15) isolated in 39% yield after purification by column chromatog-
raphy, accompanied by 6% of the linearly fused 5-hydroxy-2,2,13-
trimethyl-2,13-dihydro-6H-benzo[b]chromeno[7,6-g][1,8]-
naphthyridin-6-one (16). When Claisen rearrangement was per-
formed on compound 15, by heating at 130°C in dimethylformamide,
the desired angular 6-hydroxy-3,3,14-trimethyl-3,14-dihydro-7H-
benzo[b]chromeno[6,5-g][1,8]naphthyridin-7-one (17) was ob-
tained in a moderate 32% yield, accompanied by its linear isomer 16,
isolated in 50% yield after column chromatography. Better results
in terms of angular regioselectivity were obtained by thermal
rearrangement of 10-methoxy-8-(1,1-dimethylpropyn-1-oxy)-6-
methyldibenzo[b,g][1,8]naphthyridin-11(6H)-one (18), prepared
in 79% yield by methylation of 15 with methyl iodide, in the
presence of sodium hydride in dimethylformamide. Indeed, heating
18 in dimethylformamide at 130 °C gave the desired angular 6-
methoxy-3,3,14-trimethyl-3,14-dihydro-7H-benzo[b]chromeno-
[6,5-g][1,8]naphthyridin-7-one (4) in a satisfactory 93% yield,
accompanied by only 6% of the linear 5-methoxy-2,2,13-trimethyl-
2,13-dihydro-6H-benzo[b]chromeno[7,6-g][1,8]naphthyridin-6-
one (5) (Scheme 1). A series of phase sensitive NOESY experi-
ments permitted us to ascribe unambiguously the angular structures
of 17 and 4, and the linear structures of 16 and 5.
Conversion of 4 and 5 into the corresponding ( )-cis-diols 19
and 20, respectively, was conveniently obtained by catalytic
osmium tetroxide oxidation, using N-methylmorpholine N-oxide
to regenerate the oxidizing agent (Scheme 2).6a,15 Treatment
of diol 19 (Chart 3) with an excess of acylating reagent, acyl
anhydride or acyl chloride, afforded the corresponding diesters
exemplified by diacetate 21, dicinnamate 22, di-4-methoxycin-
namate 23, and di-4-trifluoromethylcinnamate 24. Under
controlled conditions, monoesters at the less hindered
2-position, 25−27, were obtained. Treatment of monocinnamate
25, mono-4-methoxycinnamate 26, and mono-4-trifluoro-
methylcinnamate 27 with excess acetic anhydride led to the mixed
esters 28, 29, and 30, respectively. The reaction of diol 19
with N,N′-carbonyldiimidazole in 2-butanone under reflux
afforded the cyclic carbonate 31. In the linear 6H-benzo[b]-
chromeno[7,6-g][1,8]naphthyridin-6-one series, diacetate 32 (Chart 4)
and cyclic carbonate 33 were prepared similarly from diol 5.
Finally, in order to better investigate the structure−activity relation-
ships, the two enantiomeric (+)-(1R,2R)-1,2-diacetoxy-6-methoxy-
3,3,14-trimethyl-1,2,3,14-tetrahydro-7H-benzo[b]chromeno[6,5-g]-
[1,8]naphthyridin-7-one (21a) and (−)-(1S,2S)-1,2-diacetoxy-6-
methoxy-3,3,14-trimethyl-1,2,3,14-tetrahydro-7H-benzo[b]-
chromeno[6,5-g][1,8]naphthyridin-7-one (21b) were separated by
preparative HPLC on a ChiralPak AS-H column, starting from racemic
( )-cis-diacetate 21,sincedifferences in terms of cytotoxicity have been
recently observed between the two enantiomeric forms of the related
cis-1,2-diacetoxy-6-methoxy-3,3,14-trimethyl-1,2,3,14-tetrahydro-7H-
benzo[b]pyrano[3,2-h]acridin-7-one (3).16 The absolute config-
urations of 21a and 21b were deduced from their CD curves, in
comparison with those obtained for the enantiomers of 3.16
Chart 1. Acronycine (1), Acronycine Epoxide (2), and
( )-cis-1,2-Diacetoxy-1,2-dihydrobenzo[b]acronycine (3)
Chart 2. 6-Methoxy-3,3,14-trimethyl-3,14-dihydro-7H-
benzo[b]chromeno[6,5-g][1,8]naphthyridin-7-one (4) and
5-Methoxy-2,2,13-trimethyl-2,13-dihydro-6H-
benzo[b]chromeno[7,6-g][1,8]naphthyridin-6-one (5)
In a continuation of our studies on the structure−activity
relationships in the acronycine series,10 we describe here the
synthesis and the biological properties of 6-methoxy-3,3,14-
trimethyl-3,14-dihydro-7H-benzo[b]chromeno[6,5-g][1,8]-
naphthyridin-7-one (4) (Chart 2), the isomeric 5-methoxy-
2,2,13-trimethyl-2,13-dihydro-6H-benzo[b]chromeno[7,6-g]-
[1,8]naphthyridin-6-one (5), and related cis-diols mono- and
diesters. The aim of the present work is to determine the influence of
the introduction of a nitrogen atom in the polyaromatic benzacridone
core, while conserving unchanged the methoxydimethylchromene
pharmacophore, which was previously shown to be indispensable
to observe significant biological activity.8b Indeed, such derivatives
should possess different pharmacokinetic and distribution properties
together with a better solubility in biocompatible solvents than the
parent compounds, in relation with the presence of the additional
basic aromatic nitrogen atom.
CHEMISTRY
■
The construction of the required dibenzo[b,g][1,8]naphthyridin-
11(6H)-one core was envisioned through Ullmann condensation11
of a suitable halo-quinoline carboxylic acid with 3,5-dimethoxyaniline
(6), followed by cyclization under acidic conditions (Scheme 1).
Thus, reaction of 6 with 2-chloro-3-quinolinecarboxylic acid (7),
prepared by oxidation of commercially available 2-chloro-3-
quinolinecarbaldehyde (8), afforded the corresponding carboxylic
diarylamine 9 in 48% yield. Cyclization of 9 to 8,10-
dimethoxydibenzo[b,g][1,8]naphthyridin-11(6H)-one (10) was
achieved in 91% yield by the use of polyphosphoric acid.12
Subsequent methylation of 10 in acetone, using potassium hydroxide
as base and methyl iodide as alkylating agent, gave 8,10-dimethoxy-6-
methyldibenzo[b,g][1,8]naphthyridin-11(6H)-one (11) in 65% yield.
B
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Scheme 1. Synthesis of 6-Methoxy-3,3,14-trimethyl-3,14-dihydro-7H-benzo[b]chromeno[6,5-g][1,8]naphthyridin-7-one (4), the
a
Isomeric 5-Methoxy-2,2,13-trimethyl-2,13-dihydro-6H-benzo[b]chromeno[7,6-g][1,8]naphthyridin-6-one (5)
a
Reagents and conditions: (i) Ag2O, H2O, 0 °C, 3 h (86%); (ii) CH3COOK/(CH3COO)2Cu.H2O/Et3N, (CH3)2CHOH, 100 °C, 72 h (48%); (iii)
PPA, 100 °C, 2 h (91%); (iv) CH3I, KOH, (CH3)2CO, reflux, 24h (65%); (v) HBr/H2O/CH3COOH, reflux, 72 h; (vi) K2CO3/KI, DMF, 65 °C,
96 h; (vii) DMF, 130 °C, 24−48 h; (viii), NaH/MeI, DMF, 50 °C, 2 h (79%).
anticipated from results previously obtained in the isoacronycine
series,17 linear 5-methoxy-2,2,13-trimethyl-2,13-dihydro-6H-benzo-
[b]chromeno[7,6-g][1,8]naphthyridin-6-one derivatives only dis-
played marginal cytotoxic activities against both cell lines. In contrast,
the angular esters and diesters of cis-1,2-dihydroxy-6-methoxy-3,3,
14-trimethyl-1,2,3,14-tetrahydro-7H-benzo[b]chromeno[6,5-g]-
RESULTS AND DISCUSSION
■
All the new compounds were first evaluated 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 Tables 1 and 2. As could be
C
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Scheme 2. Synthesis of ( )-cis-1,2-Dihydroxy-6-methoxy-3,3,14-trimethyl-1,2,3,14-tetrahydro-7H-benzo[b]chromeno[6,5-g]-
[1,8]naphthyridin-7-one (19) and ( )-cis-3,4-Dihydroxy-5-methoxy-2,2,13-trimethly-2,3,4,13-tetrahydro-6H-
benzo[b]chromeno[7,6-g][1,8]naphthyridin-6-one (20)
Chart 3. ( )-cis-1,2-Dihydroxy-6-methoxy-3,3,14-trimenthly-1,2,3,14-tetrahydro-7H-benzo[b]chromeno[6,5-g]-
[1,8]naphthyridin-7-one Esters and Diesters
[1,8]naphthyridin-7-one exhibited antiproliferative activities (be-
tween 5 μM to 0.01 μM) within the same order of magnitude as cis-
1,2-dihydroxy-1,2-dihydrobenzoacronycine diesters.
All compounds were founded significantly more potent on the
solid tumor KB-3-1 cell line than on the L1210 leukemia (6- to
10-fold increase).
D
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The cytotoxic activities of the two enantiomers of compound
21 were evaluated: the (+)-(1R,2R)-1,2-diacetoxy-6-methoxy-
3,3,14-trimethyl-1,2,3,14-tetrahydro-7H-benzo[b]chromeno-
[6,5-g][1,8]naphthyridin-7-one (21a) (0.11 μM) was found less
active than the corresponding (−)-(1S,2S)-1,2-diacetoxy-6-
methoxy-3,3,14-trimethyl-1,2,3,14-tetrahydro-7H-benzo[b]-
chromeno[6,5-g][1,8]naphthyridin-7-one (21b) (0.035 μM)
against the KB-3-1 cell line.
Chart 4. Compounds 32 and 33
In the case of cinnamate esters, there is no influence of the
substitution of the aromatic ring on the cinnamate moiety (no
substitution, p-OCH3 or p-CF3). In contrast, some differences
appeared between mono- and diesters. Diesters (22, 23, 24 and
28, 29, 30) are about 10-fold less active than the corresponding
monoesters (25, 26, 27) with IC50 0.4 versus 0.04 μM on average.
The perturbation of the cell cycle induced by the new deriv-
atives was studied on the L1210 cell line. As previously observed
in the benzo[a]acronycine6 and benzo[b]acronycine series,6 all
active compounds induced accumulation in the S phase.
The compounds were also evaluated for their ability to form
covalent complexes with DNA using the gel shift assay.7 As
shown in Figure 1, the various 13-aza derivatives were incubated
at increasing concentrations (up to 50 μM) with the 117bp radio-
labeled DNA fragment for 2 or 24 h (Figures 1A and 2B,
respectively). A fixed concentration of 3 (50 μM) was used as a
control. This result clearly identifies some compounds as efficient
DNA binding agents. Monoesters 25 and 27 are strongly efficient
even after a 2 h incubation period; as previously observed in the
benzo[b]acronycine series, monocinnamoyl esters alkylate DNA
faster than the corresponding diesters.6d Surprisingly, the cis-
racemate diacetylated compound 21 was less potent than its pure
cis-isomers 21a and 21b after either a 2 h or a 24 h reaction (parts
A and B, respectively, of Figure 1).
Table 1. Cytotoxicity of 5-Methoxy-2,2,13-trimethyl-2,13-
dihydro-6H-benzo[b]chromeno[7,6-g][1,8]naphthyridin-6-
one derivatives 5, 20, 32, and 33 in Comparison with 1 and 3
Cytotoxicity
Cytotoxicity
L1210 cells
% of cells in
In vitro
(L1210 cells,
(KB-3-1 cells,
DNA
a
a
b
c
Compound
IC50, μM)
IC50, μM)
S phase (μM) alkylation
1
23
3.7
0.1
14
n.t.
c
3
0.7
24
73% 5 μM
n.a.
++
n.t.
n.t.
0
5
20
32
33
12.5
10.3
20
7
n.t.
8.8
12
n.t.
68% 5 μM
++
a
Inhibition of cell proliferation measured by the MTT assay (mean of
b
at least 3 values obtained in separate experiments). Highest per-
centage of L1210 cells arrested in the S phase after a 21 h exposure to
the indicated concentration. Untreated control: 32% on average; n.a.:
c
inactive; n.t.: not tested. 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.
Table 2. Cytotoxicity of 6-Methoxy-3,3,14-trimethyl-3,14-
dihydro-7H-benzo[b]chromeno[6,5-g][1,8]naphthyridin-7-
one Derivatives 4, 19, and 21−31, in Comparison with 1 and 3
Kinetic studies of the alkylation reaction were performed
(Figure 2A), and the percent of maximum of band shifting was
quantified for each compound relative to 3 (Figure 2B). The T1/2
values corresponding to the time for which half of the maximum
of the covalent reaction (gel shift of the radiolabeled DNA) was
reached are deduced for Figure 2B and presented in Table 1.
From comparison to 3, the aza-derivative 21 presents huge
differences in the alkylation kinetics since no plateau (maximum
values) could be reached, even after a 24 h incubation time.
Similar difference (but to a much lesser extent) are obtained
from comparison of the cinnamate derivative 27 in the 13-aza-
series (120 min) to the corresponding compound in the
benzo[b]acronycine series6d presenting a T1/2 value of 60 min
(personal data).
L1210 cells
Cytotoxicity
Cytotoxicity
% of cells in
In vitro
DNA
(L1210 cells,
(KB-3-1 cells,
S phase
a
a
b
c
Compound
IC50, μM)
IC50, μM)
(μM)
alkylation
1
23
3.7
n.t.
++
n.t.
n.t.
++
++
++
n.t.
+
3
0.7
0.1
73% 5 μM
n.a.
4
11
3.7
19
21
21a
21b
22
23
24
25
26
27
28
29
30
31
5.1
2
n.t.
0.20
0.19
0.21
5
0.037
0.11
0.035
0.54
0.37
0.42
0.03
0.023
0.013
0.32
0.47
0.39
0.041
68% 5 μM
9.6
70% 5 μM
60% 10 μM
66% 10 μM
76% 5 μM
69% 5 μM
73% 5 μM
70% 5 μM
73% 2.5 μM
76% 5 μM
9.1
++
++
++
++
+
0.11
0.29
0.09
2.1
Replacement of the cinnamate 25 by a 4-trifluoromethylcinna-
mate 27 reduces the alkylation efficiencies, as evidenced from
dose effects (Figure 1) and kinetic measurements (Figure 2 and
Table 1). By using the two cis-pure enantiomers rather than the
cis-racemate 21, it is evidenced that the pure enantiomers are
more reactive (intensity of the shift and speed of reaction) than
the racemate. This could be attributed to molecular stacking of
the two enantiomers present in the racemate aza-molecule in a
head to tail orientation, affecting both compound solubility and
alkylation potency, at least in the in vitro experiments. The more
soluble pure enantiomers present much higher DNA alkylation
potencies with T1/2 values of 60 and 90 min for 21a and 21b,
respectively (Table 3). Those T1/2 values are lower than that
obtained using the two pure enantiomers of the reference drug 3
(20−30 min).16
2.9
0
3.8
+
0.12
++
a
Inhibition of cell proliferation measured by the MTT assay (mean of
b
at least 3 values obtained in separate experiments). Highest percent-
age of L1210 cells arrested in the S phase after a 21 h exposure to the
indicated concentration. Untreated control: 32% on average; n.a.:
c
inactive; n.t.: not tested. Relative quantification of bound/free ratio
from EMSA. ++ means strong bonding, equivalent to that of 3; +
corresponds to much lower bonding propensity that failed to reach a
plateau even at 24 h; 0 relates to no bonding; n.t.: not tested.
Diacetate 21 and cyclic carbonate 31 displayed similar good
cytotoxic activity (respectively 0.037 μM and 0.041 μM).
Finally, diacetate 21 and the two enantiomers 21a and 21 b
were selected for an in vivo evaluation, comparatively with
E
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Figure 1. DNA bonding analysis using gel shift experiments. Increasing of fixed concentrations of the various compounds (μM) upon incubation for 2 h (panel
A) or 24 h (panel B) with the 117-bp radiolabeled DNA fragment in 1 mM Na cacodylate buffer prior to being 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 to as “b” and “f ”, respectively.
Figure 2. Kinetic measurements of DNA bonding using electrophoretic mobility shift assay. (A) Gel shift assays for kinetic measurements. The
radiolabeled 117bp DNA fragment was incubated with 50 μM of the various indicated compounds for the appropriate time indicated on the top of the
lanes (min). Bound and free DNA fragments are referred as “b” and “f ”, respectively. (B) Quantification of the retarded migration expressed as the
percentage of the maximum of electrophoretic migration of the DNA alone (lanes “0” in panel A).
compound 3, on an established C38 colon adenocarcinoma
(sc implantation) in mice. Although less potent than com-
pound 3 (Figure 3), compound 21 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
95% and with four of seven tumor free mice at the end of the
experiment.
At the dose of 4 mg/kg, the enantiomer 21b was slightly less
active than the racemate on the tumor growth, inducing two of
seven cured mice (tumor free mice) at the end of the experiment.
F
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Table 3. Half Time and Plateau Values for Covalent Complex
Formation
CONCLUSION
■
a
In conclusion, introduction of a basic nitrogen on the B ring of
the benzo[b]acronycine led to the development of a new series of
derivatives. As previously observed in the acronycine series,
esters of the pyranediol were the most potent derivatives
exhibiting submicromolar activities; among them monoesters are
particularly active. Compound 21, the corresponding aza
derivative of 3, was shown to be the most promising compound.
Both enantiomeric compounds 21a and 21b were evaluated after
separation on chiral chromatography. 21a and 21b presented
some different properties, where 21b was shown to be more
cytotoxic on the KB-3-1 cell line but less effective in alkylating
DNA. The good activity of compound 21b was confirmed by
in vivo evaluation.
Compounds T1/2 (min) Tmax (H) Compounds T1/2 (min) Tmax (H)
3
30
45
2
3
8
21
nd
60
90
nd
3
25
27
21a
21b
120
3
a
The T1/2 values (expressed in minutes) correspond to the incubation
time required to obtain half of the maximum of the covalent drug/
DNA complex that could be obtained. The values (expressed in
minutes) were deduced from the quantification plots (Figure 2B) of
the kinetic gels (Figure 2A). Values could not be determined (nd)
when a Tmax is not reached.
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-Tof1Micromass spectrometers using electro-
spray ionization (ESI-MS; Vc = 30 V), or with a Nermag R-10-10C
spectrometer using desorption−chemical ionization (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
1
chloride films on a PerkinElmer 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 micropro-
grams. Circular dichroism (CD) spectra were recorded with a Jasco
J-810 apparatus between 300 and 400 nm, at room temperature.
The samples were prepared in methanol at 10−4 g/mL. Column
chromatographies were carried out with silica gel 20−45 mm.
Microanalyses were in agreement with calculated values 0.4%,
confirming a purity ≥95%.
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 mg/mL streptomycin, and 10 mM HEPES buffer
(pH = 7.4). Cytotoxicity was measured by the microculture tetrazolium
assay (MTA) as described.18 Cells were exposed to graded concentrations
of drug (nine serial dilutions in triplicate) for 4 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.
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 mg/mL RNase and 50 mg/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 % of
cells in the S phase of the cell cycle.
Antitumor Activity. The antitumor activity of the diacetate 21 and
the two enantiomers 21a and 21b was evaluated on murine colon C38
adenocarcinoma implanted in B6D2F1 (C57B1/6 x DBA2) mice. A C38
tumor fragment of approximately 50 mg was subcutaneously implanted into
the dorsal flank of seven mice. The evaluated compounds were prepared in a
mixture of 10% cremophor ELP, 10% ethanol and administrated intravenous
at indicated doses in two administrations 10 days apart.
Mice bearing s.c. implanted tumors were treated when the tumors
reached a volume about 150 to 220 mm3. 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 each experi-
ment, tumor-free animals were defined as cured animals.
Figure 3. In vivo evaluation of 21a and 21b comparatively with
compound 3 on C38 adenocarcinoma in mice.
In the same experiment, the enantiomer 21a was inactive and did
not induce any tumor growth inhibition (Figure 3B).
G
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DNA Restriction Fragments. The 117-bp DNA fragment was
obtained as previously described7 from the pBS plasmid digestion using
EcoRI and PvuII restriction enzymes in their respective digestion buffers.
The generated 117 bp DNA was then labeled at the EcoRI restriction site
by incorporation of a-[32P]-dATP (GEHealthcare) using AMV reverse
transcriptase (Ozyme). The generated 117bp radiolabeled DNA
fragment was purified by electrophoresis on a nondenaturing 10%
(w/v) polyacrylamide gel. The portion of gel containing the 117bp DNA
was cut out of the gel, crushed, and eluted overnight in 500 mM
ammonium acetate, 10 mM magnesium acetate. After filtration to
remove the polyacrylamide gel, the radiolabeled DNA was precipitated
using cold ethanol, dried, and dissolved in MQ water.
Gel Shift Studies. The cross-linking reaction was conducted as
previously described.16 Briefly, it consists of incubating the drug at
appropriate concentrations (increasing concentration or a fixed 50 μM
concentration for kinetic studies) with the radiolabeled DNA in 1 mM
Na cacodylate, pH 7.0, and incubation in the dark at room tempera-
ture during the period specified in the legend. After various incubation
times from 5 min to 24 h, 5 mL of a 50% glycerol containing tracking
dyes solution was added to each DNA sample. The covalent complex
was evidenced as a shifted band resolved after electrophoresis on a
nondenaturing 6% polyacrylamide gel in TBE buffer (89 mM boric acid,
2.5 mM Na2EDTA, pH 8.3) for about 5 h at 300 V and room tem-
perature. Gels were transferred to Whatman 3MM paper, dried under
vacuum at 80 °C, and then analyzed on a Storm phosphorimaging
system to evidence the shifted complex.
was heated at 100 °C for 2 h. The reaction mixture was slowly poured
onto ice water (300 mL) and neutralized to pH = 7 by addition of a 30%
aqueous NaOH solution. The obtained precipitate was collected,
washed with water, and dried in vacuum over P2O5. Silica gel column
chromatography (solvent: CH2Cl2, then CH2Cl2/MeOH, 99.9:0.1 to
98:2) gave 10 (4.84 g, 91%) as pale yellow crystals: mp 309 °C
(crystallized from CH2Cl2/MeOH 1/1). 1H NMR (400 MHz, CDCl3) δ
3.77 (s, 3H, OCH3), 4.00 (s, 3H, OCH3), 6.18 (s, 1H, H-7), 6.25 (s, 1H,
H-9), 7.49 (td, J = 8, 1 Hz, 1H, H-2), 7.81 (td, J = 8, 1 Hz, 1H, H-3), 7.97
(dd, J = 8, 1 Hz, 1H, H-4), 8.05 (dd, J = 8, 1 Hz, 1H, H-1), 9.26 (s, 1H,
H-12), 9.93 (s, 1H, D2O exch., NH); 13C NMR (75 MHz, DMSO-d6)
δ 55.5 (C8−OCH3), 56.2 (C10-OCH3), 91.3 (C-7), 93.3 (C-9), 106.3
(C-10a), 118.9 (C-11a), 124.4 (C-2), 125.1 (C-12a), 126.2 (C-1), 130.0
(C-4), 132.5 (C-3), 139.2 (C-12), 145.5 (C-6a), 149.0 (C-4a), 149.5
(C-5a), 163.4 (C-10), 165.2 (C-8), 177.2 (C-11); DCI-MS m/z 307
[MH]+; IR (KBr) ν cm−1: 3448 (br.), 1624, 1618, 1271, 1170, 1098; UV
λ nm (MeOH) (log ε): 224 (3.77), 278 (4.16), 338 (3.47).
8,10-Dimethoxy-6-methyldibenzo[b,g][1,8]naphthyridin-11(6H)-
one (11). Potassium hydroxide pellets (3.9 g) were added to a solution
of 10 (3.5 g, 15 mmol) in dry acetone (250 mL). The mixture refluxed
for 15 min under argon and methyl iodide (30 mL, 480 mmol) was
added. After 24 h of heating under reflux, the reaction mixture was
evaporated under reduced pressure. Silica gel column chromatography
(solvent: CH2Cl2, then CH2Cl2/MeOH, 99.9:0.1 to 99.5:0.5) afforded
11 (3.1 g, 65%) as yellow crystals: mp 335 °C (crystallized from
1
CH2Cl2/MeOH 1/1). H NMR (400 MHz, CDCl3) δ 3.94 (s, 3H,
2-Chloro-3-quinolinecarboxylic Acid (7). A solution of sodium
hydroxyde (16.8 g/L, 420 mmol) in water (80 mL) was added to a
solution of silver nitrate (36.1 g, 200 mmol) in water (80 mL). 2-Chloro-
3-quinolinecarbaldehyde (8) (20.0 g, 120 mmol) was added to the
resulting silver hydroxide suspension, and the reaction mixture was
stirred at 0 °C for 3 h and filtered. The filtrate was acidified to pH = 6
with conc. HCl aqueous solution, and the obtained precipitate was
collected, washed with water, and dried. Purification by silica gel column
chromatography (solvent: CH2Cl2, then CH2Cl2/MeOH 99.9:0.1 to
95:5) gave 7 (18.7 g, 86%) as pale crystals: mp 263 °C (crystallized from
OCH3), 4.02 (s, 3H, OCH3), 4.11 (s, 3H, NCH3), 6.25 (s, 1H, H-9),
6.49 (s, 1H, H-7), 7.44 (ddd, J = 9, 7, 1 Hz, 1H, H-2), 7.75 (ddd, J = 9, 7,
1 Hz, 1H, H-3), 7.95 (dd, J = 9, 1 Hz, 1H, H-1), 7.98 (dd, J = 9, 1 Hz, 1H,
H-4), 9.21 (s, 1H, H-12), 10.96 (br. s, 1H, D2O exch., C8−OH), 14.23
(s, 1H, D2O exch., C10−OH); 13C NMR (75 MHz, DMSO-d6) δ 31.5
(NCH3), 55.4 (C8−OCH3), 56.3 (C10-OCH3), 91.4 (C-7), 92.0 (C-9),
107.1 (C-10a), 119.3 (C-11a), 124.3 (C-3), 124.5 (C-12a), 127.4 (C-1),
129.4 (C-4), 131.9 (C-2), 138.5 (C-12), 147.6 (C-6a), 149.1 (C-4a),
149.7 (C-5a), 163.8 (C-10), 164.9 (C-8), 177.2 (C-11); DCI-MS m/z
321 [MH]+; IR (KBr) ν cm−1: 1630, 1583, 1514, 1425, 1257, 1202,
1159, 1145; UV λ nm (MeOH) (log ε): 227 (4.36), 245 (4.24), 281
(4.59), 340 (4.16).
1
MeOH/Me2CO 1/1). H NMR (400 MHz, DMSO-d6) δ 7.72 (dd,
J = 8, 7 Hz, 1H, H-6), 7.91 (dd, J = 8, 7 Hz, 1H, H-7), 7.99 (d, J = 8 Hz,
1H, H-5), 8.16 (d, J = 8 Hz, 1H, H-8), 8.92 (s, 1H, H-4), 13.81 (br. s, 1H,
D2O exch., COOH); 13C NMR (75 MHz, DMSO-d6) δ 126.6 (C-3),
126.9(C-4a), 128.7(C-5), 129.0(C-6), 130.0(C-8), 133.7(C-7),
142.2(C-4), 147.5(C-2), 148.4(C-8a), 166.7(COOH); DCI-MS m/z
208, 210 [MH]+; IR (KBr) ν cm−1: 3423 (br.), 3073, 2924, 2568, 1731,
1618, 1567, 1489, 1449, 1251, 1234, 1191, 1127, 1023, 899, 789, 767,
748; UV λ nm (MeOH) (log ε): 238 (4.48), 280 (3.52), 309 (3.32), 323
(3.34).
2-(3,5-Dimethoxyphenylamino)-3-quinolinecarboxylic Acid (9). A
mixture of 3,5-dimethoxyaniline (6) (20.5 g, 130 mmol), 7 (21.3 g,
100 mmol), potassium acetate (23.2 g), and cupric acetate monohydrate
(1.3 g) in triethylamine (17.1 mL) and 2-propanol (370 mL) was heated
at 100 °C for 72 h. The reaction mixture was evaporated under reduced
pressure, and the residue was partitioned between CH2Cl2 and
1 N aqueous HCl. The aqueous phase was extracted with CH2Cl2
(4 × 200 mL). The combined organic layers were dried over anhydrous
Na2SO4, filtered, and evaporated under reduced pressure. Silica gel
column chromatography (solvent: CH2Cl2, then CH2Cl2/MeOH,
99.9:0.1 to 98:2) gave 9 (16 g, 48%) as an amorphous yellowish solid.
1H NMR (400 MHz, DMSO-d6) δ 3.78 (s, 6H, 2 OCH3), 6.20 (t, J =
2 Hz, 1H, H-4′), 7.25 (m, 2H, H-2′, H-6′), 7.36 (ddd, J = 8, 7, 2 Hz, 1H,
H-6), 7.69 (dd, J = 8, 2 Hz, 1H, H-5), 7.72 (ddd, J = 8, 7, 2 Hz, 1H, H-7),
7.93 (dd, J = 8, 2 Hz, 1H, H-8), 8.92 (s, 1H, H-4), 10.60 (s, 1H, D2O exch.,
NH), 13.81 (br. s, 1H, D2O exch., COOH); 13C NMR (75 MHz, DMSO-
d6) δ 56.1 (2C, 2 OCH3), 95.6 (C-4′), 98.7(2C, C-2′, C-6′), 112.3(C-3),
123.3 (C-4a), 124.6 (C-6), 127.1 (C-5), 130.4 (C-8), 133.7 (C-7), 142.7
(C-1′), 143.7 (C-4), 149.6 (C-8a), 153.4 (C-2), 161.7 (2C, C-3′, C-5′),
169.8(COOH); DCI-MS m/z 325 [MH]+, 347 [MNa]+; IR (KBr) ν cm−1:
3448 (br.), 1655, 1649, 1605, 1544, 1477, 1459, 1425, 1204, 1156, 1066; UV
λ nm (MeOH) (log ε): 216 (4.61), 280 (4.44), 370 (3.84).
8,10-Dihydroxy-6-methyldibenzo[b,g][1,8]naphthyridin-11(6H)-
one (12) and 10-Hydroxy-8-methoxy-6-methyldibenzo[b,g][1,8]-
naphthyridin-11(6H)-one (13). Acetic acid (50 mL) was added to a
solution of 11 (3.0 g, 9.4 mmol) in 48% hydrogen bromide aqueous
solution (150 mL). The reaction mixture was stirred and refluxed for
3 days. The cooled mixture was poured onto ice water (500 mL). The
precipitate was filtered, washed with water (4 × 50 mL), and dried
in vacuum over P2O5. Column chromatography on silica gel (solvent:
CH2Cl2, then CH2Cl2/MeOH, 99.9:0.1 to 98:2) gave successively 12
(2.35 g, 86%) and 13 (0.37 g, 13%) as yellow amorphous solids.
Compound 12: 1H NMR (400 MHz, DMSO-d6) δ 4.03 (s, 3H, NCH3),
6.10 (d, J = 2 Hz, 1H, H-9), 6.40 (d, J = 2 Hz, 1H, H-7), 7.51 (ddd, J = 8,
7, 1 Hz, 1H, H-2), 7.85 (ddd, J = 8, 7, 1 Hz, 1H, H-3), 7.92 (dd, J = 8, 1
Hz, 1H, H-1), 8.17 (dd, J = 8, 1 Hz, 1H, H-4), 9.21 (s, 1H, H-12), 10.96
(br. s, 1H, D2O exch., C8−OH), 14.23 (s, 1H, D2O exch., C10−OH); 13
C
NMR (75 MHz, DMSO-d6) δ 31.9 (N-CH3), 93.5 (C-7), 97.0 (C-9),
103.6 (C-10a), 116.9 (C-11a), 124.8 (C-12a), 125.8 (C-2), 128.1(C-4),
130.7 (C-1), 134.2 (C-3), 138.7(C-12), 146.5 (C-6a), 149.8 (C-4a),
150.1 (C-12a), 166.3 (C-10), 167.1 (C-8), 181.4 (C-11); DCI-MS m/z
293 [MH]+; IR (KBr) ν cm−1: 3448 (br.), 3138 (br.), 1638, 1627, 1611,
1571, 1551, 1518, 1477, 1429, 1346, 1304, 1274, 1186, 1170, 1150, 809,
756, 727; UV λ nm (MeOH) (log ε): 225 (4.28), 281 (4.70), 355 (4.04).
Compound 13: 1H NMR (400 MHz, CDCl3) δ 3.93 (s, 3H, OCH3),
4.06 (s, 3H, NCH3), 6.29 (d, J = 1.5 Hz, 1H, H-9), 6.38 (d, J = 1.5 Hz,
1H, H-7), 7.46 (ddd, J = 9, 7, 1 Hz, 1H, H-2), 7.79 (ddd, J = 9, 7, 1 Hz,
1H, H-3), 7.95 (dd, J = 9, 1 Hz, 1H, H-1), 7.99 (dd, J = 9, 1 Hz, 1H, H-4),
9.19 (s, 1H, H-12), 14.25 (s, 1H, D2O exch., C10−OH); 13C NMR (75
MHz, CDCl3) δ 31.2 (N-CH3), 55.7 (O-CH3), 91.6 (C-7), 94.0 (C-9),
104.1 (C-10a), 116.6 (C-11a), 124.2 (C-12a), 124.8 (C-3), 127.7 (C-4),
129.4 (C-1), 132.8 (C-2), 138.0 (C-12), 145.5 (C-6a), 149.2 (C-4a),
149.9 (C-5a), 166.5 (C-10), 167.1 (C-8), 181.7 (C-11); DCI-MS m/z
307 [MH]+; IR (KBr) ν cm−1: 3448 (br.), 3138 (br.), 1638, 1605, 1585,
8,10-Dimethoxydibenzo[b,g][1,8]naphthyridin-11(6H)-one (10).
A suspension of 9 (5.6 g, 17 mmol) in polyphosphoric acid (122 g)
H
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1508, 1477, 1458, 1425, 1402, 1349, 1251, 1211, 1164, 1100, 1062, 813,
753; UV λ nm (MeOH) (log ε): 282 (4.70), 353 (4.13).
0.28 mmol) in dry N,N-dimethylformamide (20 mL), and the mixture
was stirred under argon for 15 min. After addition of methyl iodide
(1 mL, 16 mmol), the reaction mixture was stirred at 50 °C for 2 h and
then poured carefully onto ice water. The precipitate was filtered and
dried in vacuum over P2O5. Column chromatography on silica gel
(solvent: CH2Cl2 then CH2Cl2/MeOH, 99.9:0.1 to 99:1) gave 18
(0.082 g, 79%) as yellow crystals: mp 223 °C (crystallized from
CH2Cl2). 1H NMR (400 MHz, CDCl3) δ 1.82 (s, 6H, C(CH3)2), 2.78
(s, 1H, H-3′), 4.07 (s, 3H, OCH3), 4.12 (s, 3H, NCH3), 6.64 (d, J = 2
Hz, 1H, H-9), 7.05 (d, J = 2 Hz, 1H, H-7), 7.42 (ddd, J = 9, 7, 1 Hz, 1H,
H-2), 7.74 (ddd, J = 9, 7, 1 Hz, 1H, H-3), 7.96 (dd, J = 9, 1 Hz, 1H, H-1),
7.98 (dd, J = 9, 1 Hz, 1H, H-4), 9.22 (s, 1H, H-12); 13C NMR (75 MHz,
CDCl3) δ 29.7 (2C, C(CH3)2), 31.6 (N-CH3), 56.3 (O-CH3), 72.5
(C-1′), 75.0 (C-3′), 85.4 (C-2′), 96.2 (C-9), 97.1 (C-7), 107.6 (C-10a),
119.3 (C-11a), 124.3 (C-3), 124.5 (C-12a), 127.4 (C-1), 129.4 (C-4),
131.9 (C-2), 138.6 (C-12), 147.0 (C-6a), 149.2 (C-4a), 149.7 (C-5a),
161.5 (C-8), 163.3 (C-10), 177.4 (C-11); DCI-MS m/z 373 [MH]+; IR
(KBr) ν cm−1: 1648, 1618, 1604, 1586, 1508, 1474, 1421, 1350, 1257,
1137, 1096, 1031; UV λ nm (MeOH) (log ε): 228 (4.36), 279 (4.74),
340 (4.11).
Thermal Rearrangement of 10-Methoxy-8-(1,1-dimethylpropyn-
1-oxy)-6-methyldibenzo[b,g][1,8]naphthyridin-11(6H)-one (18). A
solution of 15 (0.1 g, 0.27 mmol) in N,N-dimethylformamide
(50 mL) was heated under nitrogen at 130 °C for 24 h. The reaction
mixture was poured carefully onto ice water (100 mL) and extracted
with CH2Cl2 (4 × 50 mL). The combined organic layers were washed
with water, dried over anhydrous Na2SO4, filtered, and evaporated under
reduced pressure. Purification by silica gel column chromatography
(solvent: CH2Cl2, then CH2Cl2/MeOH, 99.9:0.1 to 99.5:0.5) gave
successively 4 (0.93 g, 93%) and 5 (0.06 g, 6%).
Reaction of 12 with 3-Chloro-1-methylbut-1-yne (14). A solution
of 12 (2 g, 6.8 mmol) and 3-chloro-3-methylbut-1-yne (14) (5.0 g,
48 mmol) in dry N,N-dimethylformamide (200 mL) was stirred and
heated at 65 °C for 4 days, under nitrogen, in the presence of anhydrous
potassium carbonate (3.0 g, 23 mmol) and potassium iodide (3.0 g, 8
mmol). After addition of ice water (200 mL), the reaction mixture was
extracted with CH2Cl2 (4 × 100 mL). The combined organic layers were
washed with water, dried over anhydrous Na2SO4, filtered, and
evaporated under reduced pressure. Purification by silica gel column
chromatography (solvent: CH2Cl2, then CH2Cl2/MeOH, 99.9:0.1 to
99.6:0.4) successively afforded 15 (0.97 g, 39%) and 16 (0.15 g, 6%).
10-Hydroxy-8-(1,1-dimethylpropyn-1-oxy)-6-methyldibenzo[b,g]-
[1,8]naphthyridin-11(6H)-one (15). Reddish crystals: mp 240 °C
(crystallized from CH2Cl2/MeOH 1/1); 1H NMR (400 MHz, CDCl3)
δ 1.80 (s, 6H, C(CH3)2), 2.75 (s, 1H, H-3′), 4.12 (s, 3H, NCH3), 6.73
(d, J = 1.5 Hz, 1H, H-7), 6.79 (d, J = 1.5 Hz, 1H, H-9), 7.49 (ddd, J = 9, 7,
1 Hz, 1H, H-2), 7.83 (ddd, J = 9, 7, 1 Hz, 1H, H-3), 7.92 (dd, J = 9, 1 Hz,
1H, H-1), 7.99 (dd, J = 9, 1 Hz, 1H, H-4), 9.26 (s, 1H, H-12), 14.15 (s,
1H, D2O exch., C10−OH); 13C NMR (75 MHz, CDCl3) δ 29.7 (2C,
C(CH3)2), 31.1 (N-CH3), 72.7 (C-1′), 75.2 (C-3′), 85.0 (C-2′), 95.5
(C-7), 99.0 (C-9), 104.7 (C-10a), 115.6 (C-11a), 124.2 (C-12a), 124.8
(C-3), 127.7 (C-4), 129.4 (C-1), 132.8 (C-2), 138.0 (C-12), 145.1
(C-6a), 149.4(C-5a), 149.9 (C-4a), 163.7 (C-8), 165.5 (C-10), 181.9
(C-11); DCI-MS m/z 359 [MH]+; IR (KBr) ν cm−1: 3448 (br.), 3231,
1638, 1585, 1516, 1492, 1423, 1382, 1342, 1304, 1257, 1214, 1164,
1133, 1029, 824, 747, 727; UV λ nm (MeOH) (log ε): 283 (4.64), 345
(3.93).
5-Hydroxy-2,2,13-trimethyl-2,13-dihydro-6H-benzo[b]chromeno-
[7,6-g][1,8]naphthyridin-6-one (16). Bright yellow crystals: mp 250 °C
(crystallized from CH2Cl2); 1H NMR (400 MHz, CDCl3) δ 1.46 (s, 6H,
C(CH3)2), 4.03 (s, 3H, NCH3), 5.51 (d, J = 10 Hz, 1H, H-3), 6.31 (s,
1H, H-14), 6.70 (d, J = 10 Hz, 1H, H-4), 7.39 (ddd, J = 9, 7, 1 Hz, 1H,
H-9), 7.73 (ddd, J = 9, 7, 1 Hz, 1H, H-10), 7.88 (dd, J = 9, 1 Hz, 1H,
H-8), 7.91 (dd, J = 9, 1 Hz, 1H, H-11), 9.14 (s, 1H, H-7), 14.49 (s, 1H,
D2O exch., C5−OH); 13C NMR (75 MHz, CDCl3) δ 28.6 (2C,
C(CH3)2), 31.1 (N-CH3), 78.4 (C-2), 92.7 (C-14), 102.5 (C-4a), 103.9
(C-5a), 115.6 (C-4), 116.5 (C-6a), 124.2(C-7a), 124.8 (C-10), 126.6
(C-3), 127.7 (C-11), 129.3 (C-8), 132.7 (C-9), 137.8 (C-7), 145.1
(C-13a), 149.1 (C-12a), 149.8 (C-11a), 160.2(C-5), 161.2 (C-14a),
181.5 C-6); DCI-MS m/z 359 [MH]+; IR (KBr) ν cm−1: 3448 (br.),
1637, 1586, 1559, 1491, 1430, 1342, 1312, 1202, 1160, 1129, 811, 780,
763; UV λ nm (MeOH) (log ε): 250 (4.39), 312 (4.80), 364 (4.17).
Thermal Rearrangement of 10-Hydroxy-8-(1,1-dimethylpropyn-
1-oxy)-6-methyldibenzo[b,g][1,8]naphthyridin-11(6H)-one (15). A solu-
tion of 15 (0.5 g, 1.4 mmol) in N,N-dimethylformamide (170 mL) was
heated under nitrogen at 130 °C for 2 days. The solvent was evaporated
under reduced pressure. Silica gel column chromatography (solvent:
CH2Cl2, then CH2Cl2/MeOH, 99.9:0.1 to 99.6:0.4) gave successively
17 (0.16 g, 32%) and 16 (0.25 g, 50%). 6-Hydroxy-3,3,14-trimethyl-
3,14-dihydro-7H-benzo[b]chromeno[6,5-g][1,8]naphthyridin-7-
one (17): bright yellow crystals: mp 322 °C (crystallized from CH2Cl2/
MeOH 1/1); 1H NMR (400 MHz, CDCl3) δ 1.56 (s, 6H, C(CH3)2),
4.10 (s, 3H, NCH3), 5.59 (d, J = 10 Hz, 1H, H-2), 6.29 (s, 1H, H-5), 6.70
(d, J = 10 Hz, 1H, H-1), 7.50 (ddd, J = 9, 7, 1 Hz, 1H, H-10), 7.83 (ddd,
J = 9, 7, 1 Hz, 1H, H-11), 8.00 (dd, J = 9, 1 Hz, 1H, H-9), 8.04 (dd, J = 9,
1 Hz, 1H, H-12), 9.18 (s, 1H, H-8), 14.32 (s, 1H, D2O exch., C6−OH);
13C NMR (75 MHz, CDCl3) δ 27.0 (2C, C(CH3)2), 41.7 (N-CH3), 76.6
6-Methoxy-3,3,14-trimethyl-3,14-dihydro-7H-benzo[b]-
chromeno[6,5-g][1,8]naphthyridin-7-one (4). Yellow crystals: mp
275 °C (crystallized from CH2Cl2/MeOH 1/1); 1H NMR (400 MHz,
CDCl3) δ 1.50 (s, 6H, C(CH3)2), 3.94 (s, 3H, OCH3), 3.98 (s, 3H,
NCH3), 5.50 (d, J = 10 Hz, 1H, H-2), 6.27 (s, 1H, H-5), 6.60 (d, J =
10 Hz, 1H, H-1), 7.39 (ddd, J = 9, 7, 1 Hz, 1H, H-10), 7.71 (ddd, J = 9, 7,
1 Hz, 1H, H-11), 7.92 (dd, J = 9, 1 Hz, 1H, H-9), 7.95 (dd, J = 9, 1 Hz,
1H, H-12), 9.08 (s, 1H, H-8); 13C NMR (75 MHz, CDCl3) δ 26.9 (2C,
C(CH3)2), 42.4 (N-CH3), 56.3 (O-CH3), 76.6 (C-3), 94.4 (C-5), 103.6
(C-14b), 109.4 (C-6a), 120.0 (C-7a), 121.9 (C-1), 123.3 (C-2), 124.6
(C-11), 124.9 (C-8a), 127.5 (C-12), 129.4 (C-9), 131.9 (C-10), 137.9
(C-8), 147.1 (C-14a), 149.4 (C-12a), 152.4 (C-13a), 160.2 (C-4a),
163.3 (C-6), 177.8 (C-7); HRMS (ESI) calcd for C23H20N2O3 ([MH]+)
m/z: 373.1547, found: 373.1548; IR (KBr) ν cm−1: 1645, 1621, 1587,
1561, 1493, 1395, 1344, 1203, 1134, 1120, 1093, 1032, 812, 758; UV λ
nm (MeOH) (log ε): 236 (4.35), 276 (4.62), 299 (4.61), 368 (3.84);
Anal. Calcd for C23H20N2O3: C, 74.18; H, 5.41; N, 7.52. Found: C,
74.05; H, 5.40; N, 7.50.
5-Methoxy-2,2,13-trimethyl-2,13-dihydro-6H-benzo[b]-
chromeno[7,6-g][1,8]naphthyridin-6-one (5). Yellow crystals: mp
239 °C (crystallized from CH2Cl2/MeOH 1/1); 1H NMR (400 MHz,
CDCl3) δ 1.45 (s, 6H, C(CH3)2), 3.97 (s, 3H, OCH3), 4.05 (s, 3H,
NCH3), 5.62 (d, J = 10 Hz, 1H, H-3), 6.68 (s, 1H, H-14), 6.73 (d, J =
10 Hz, 1H, H-4), 7.36 (ddd, J = 9, 7, 1 Hz, 1H, H-9), 7.69 (ddd, J = 9, 7,
1 Hz, 1H, H-10), 7.91 (m, 2H, H-8, H-11), 9.16 (s, 1H, H-7); 13C NMR
(75 MHz, CDCl3) δ 28.5 (2C, C(CH3)2), 31.5 (N-CH3), 62.4 (O-CH3),
77.9 (C-2), 98.1 (C-14), 109.9 (C-4a), 110.3 (C-5a), 116.2 (C-4), 118.8
(C-6a), 124.4 (2C, C-10, C-7a), 127.5 (C-11), 129.2 (C-3),129.3 (C-8),
132.1 (C-9), 138.5 (C-7), 146.6 (C-13a), 149.2 (C-11a), 149.5 (C-12a),
158.1 (C-5), 159.3 (C-14a), 177.0 (C-6); HRMS (ESI) calcd for
C23H20N2O3 ([MH]+) m/z: 373.1547, found: 373.1550; IR (KBr) ν
cm−1: 3052, 2968, 2920, 1650, 1631, 1608, 1589, 1551, 1486, 1425,
1343, 1121, 1091, 1021, 958, 821, 784, 754; UV λ nm (MeOH) (log ε):
242 (4.44), 301 (4.85), 356 (4.13); Anal. Calcd for C23H20N2O3: C,
74.18; H, 5.41; N, 7.52. Found: C, 73.95; H, 5.39; N, 7.49.
(C-3), 98.1 (C-5), 101.9 (C-14b), 105.9 (C-6a), 117.1 (C-7a), 121.6
(C-1), 123.5 (C-2), 124.5 (C-8a), 125.0 (C-11), 127.8 (C-12), 129.4
(C-9), 132.7 (C-10), 137.4 (C-8), 144.7 (C-14a), 150.0 (C-12a), 151.8
(C-13a), 162.6 (C-4a), 165.7 (C-6), 181.7 (C-7); DCI-MS m/z 359
[MH]+; IR (KBr) ν cm−1: 3448 (br.), 1625, 1578, 1560, 1501, 1405,
1375, 1338, 1313, 1277, 1174, 1156, 1137, 1092, 1025, 850, 825, 750;
UV λ nm (MeOH) (log ε): 234 (4.12), 277 (4.40), 305 (4.43), 376
(3.70).
( )-cis-1,2-Dihydroxy-6-methoxy-3,3,14-trimethyl-1,2,3,14-tetra-
hydro-7H-benzo[b]chromeno[6,5-g][1,8]naphthyridin-7-one (19).
Compound 4 (0.500 g, 1.35 mmol) was added to a solution of osmium
tetroxide (2.5% in 2-methyl-2-propanol) (3.9 mL) and N-methyl-
morpholine N-oxide dihydrate (0.63 g, 4.6 mmol) in t-BuOH/THF/H2O
10-Methoxy-8-(1,1-dimethylpropyn-1-oxy)-6-methyldibenzo-
[b,g][1,8]naphthyridin-11(6H)-one (18). Sodium hydride (0.41 g
of 50% oil dispersion, 8.5 mmol) was added to a solution of 15 (0.1 g,
I
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(10:3:1, v/v/v, 55 mL). The reaction mixture was stirred at room
temperature for 3 days. After addition of saturated aqueous NaHSO3
(55 mL), the mixture was stirred for 1 h and then extracted with CH2Cl2
(5 × 100 mL). The combined organic layers were dried over Na2SO4
and evaporated under reduced pressure. Silica gel column chromatog-
raphy (solvent: CH2Cl2 then CH2Cl2/MeOH, 99.9:0.1 to 98:2) gave 19
(0.315 g, 58%) as pale yellow crystals crystals: mp 302 °C (crystallized
from Me2CO/MeOH 1/1). 1H NMR (400 MHz, DMSO-d6) δ 1.40 (s,
3H, CCH3), 1.43 (s, 3H, CCH3), 3.68 (m, 1H, H-2), 3.83 (s, 3H,
OCH3), 3.99 (s, 3H, NCH3), 4.68 (br. s., 1H, D2O exch., OH), 5.10 (m,
2H, H-1, OH), 6.25 (s, 1H, H-5), 7.50 (t, J = 8 Hz, 1H, H-10), 7.83 (t, J =
8 Hz, 1H, H-11), 7.95 (d, J = 8 Hz, 1H, H-9), 8.18 (dd, J = 8 Hz, 1H,
H-12), 9.01 (s, 1H, H-8); 13C NMR (75 MHz, DMSO-d6) δ 23.2
(C-CH3), 26.1 (C-CH3), 40.0 (N-CH3), 56.9 (O-CH3), 64.9 (C-1), 71.1
(C-2), 78.8 (C-3), 95.2 (C-5), 105.3 (C-14b), 110.7 (C-6a), 120.4
(C-7a), 125.1 (C-8a), 125.4 (C-11), 128.0 (C-9), 130.6 (C-12), 133.1
(C-10), 137.8 (C-8), 149.7 (C-12a), 150.8 (C-14a), 152.7 (C-13a),
161.1 (C-4a), 162.4 (C-6), 177.1 (C-7); DCI-MS m/z 407 [MH]+; IR
(KBr) ν cm−1: 3422 (br.), 1635, 1618, 1585, 1505, 1397, 1351, 1212,
1140, 1117, 1097, 1037, 815; UV λ nm (MeOH) (log ε): 232 (4.35),
288 (4.73), 350 (4.06); Anal. Calcd for C23H22N2O5: C, 67.97; H, 5.46;
N, 6.89. Found: C, 67.81; H, 5.45; N, 6.87.
( )-cis-1,2-Dicinnamoyloxy-6-methoxy-3,3,14-trimethyl-1,2,3,14-
tetrahydro-7H-benzo[b]chromeno[6,5-g][1,8]naphthyridin-7-one
(22). Thionyl chloride (7.30 mL, 100 mmol), was added dropwise to a
suspension of cinnamoic acid (1.48 g, 10 mmol) in anhydrous CH2Cl2
(40 mL). The reaction mixture was stirred under reflux for 3 h and
filtered, and the solvent and excess thionyl chloride were removed 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 (0.98 g, 5.9 mmol) was
added to a solution of 19 (0.239 g, 0.59 mmol) in dry pyridine (10 mL)
containing 4-dimethylaminopyridine (0.01 g). The reaction mixture was
stirred at room temperature for 48 h and then evaporated under reduced
pressure. Silica gel column chromatography (solvent: CH2Cl2, then
CH2Cl2/MeOH 99.9:0.1 to 99:1) afforded 22 as a yellow amorphous
1
solid (0.133 g, 34%). H NMR (400 MHz, CDCl3) δ 1.51 (s, 3H,
CCH3), 1.66 (s, 3H, CCH3), 3.82 (s, 3H, OCH3), 4.08 (s, 3H, NCH3),
5.88 (d, J = 5 Hz, 1H, H-2), 6.26 (d, 1H, J = 16 Hz, 2−OCOCHCH),
6.34 (d, 1H, J = 16 Hz, 1−OCOCHCH), 6.41 (s, 1H, H-5), 6.67 (d,
J = 5 Hz, 1H, H-1), 7.07 (m, 3H, H-3″, H-4″, H-5″), 7.24 (m, 2H, H-2″,
H-6″), 7.31 (m, 3H, H-3′, H-4′, H-5′), 7.36 (m, 2H, H-2′, H-6′), 7.42 (t,
J = 8 Hz, 1H, H-10), 7.45 (d, 1H, J = 16 Hz, 1−OCOCHCH), 7.53 (d,
1H, J = 16 Hz, 2−OCOCHCH), 7.63 (t, J = 8 Hz, 1H, H-11), 7.78 (d,
1H, J = 8 Hz, H-9), 7.97 (d, 1H, J = 8 Hz, H-12), 9.11 (s, 1H, H-8); 13C
NMR (75 MHz, CDCl3) δ 23.1 (C-CH3), 25.0 (C-CH3), 39.7 (N-CH3),
56.4 (O-CH3), 66.7 (C-1), 69.1 (C-2), 77.2 (C-3), 94.7 (C-5), 98.5
(C-14b), 110.9 (C-6a), 116.9 (2−OCOCHCH), 117.0 (1−
OCOCHCH), 119.9 (C-7a), 124.6 (C-11), 124.9 (C-8a), 127.9
(C-9), 128.1 (2C, C3′, C5′), 128.2 (2C, C3″, C5″), 128.5 (2C, C2″,
C6″), 128.7 (2C, C2′, C6′), 129.2 (C-12), 130.2 (C-4″), 130.4 (C-4′),
131.7 (C-10), 133.9 (C-1′), 134.0 (C-1″), 137.6 (C-8), 146.1 (1−
OCOCHCH), 146.2 (2−OCOCHCH), 149.6 (C-12a), 150.0
(C-14a), 152.1 (C-13a), 160.8 (C-4a), 163.1 (C-6), 166.1 (2-OCOCH
CH), 166.7 (1-OCOCHCH), 178.1 (C-7); HRMS (ESI) calcd for
C41H34N2O7 ([MH]+) m/z: 667.2439, found: 667.2440; IR (KBr)
ν cm−1: 2967, 2360, 1733, 1644, 1590, 1575, 1450, 1399, 1204, 1172,
1150, 1091, 979, 766; UV λ nm (MeOH) (log ε): 283 (4.95), 344
(4.14), 428 (3.89); Anal. Calcd for C41H34N2O7: C, 73.86; H, 5.14; N,
4.20. Found: C, 73.72; H, 5.13; N, 4.19.
( )-cis-3,4-Dihydroxy-5-methoxy-2,2,13-trimethyl-2,3,4,13-tetra-
hydro-6H-benzo[b]chromeno- [7,6-g][1,8]naphthyridin-6-one (20).
Compound 20 was synthesized from 5 (0.500 g, 1.35 mmol) according
to the procedure described for the preparation of 19 from 4, using
osmium tetroxide and N-methylmorpholine N-oxide dihydrate in
t-BuOH/THF/H2O. Purification by silica gel column chromatography
(solvent: CH2Cl2, then CH2Cl2/MeOH, 99.9:0.1 to 98:2) gave 20
1
(0.388 g, 71%) as a pale yellow amorphous product. H NMR (400
MHz, DMSO-d6) δ 1.41 (s, 6H, C(CH3)2), 3.63 (dd, J = 6, 5 Hz, 1H,
H-3), 3.93 (s, 3H, OCH3), 4.02 (s, 3H, NCH3), 4.89 (dd, J = 5, 4 Hz, 1H,
H-4), 5.05 (m, 2H, D2O exch., OH-3, OH-4), 6.77 (s, 1H, H-14), 7.51
(td, J = 8, 1 Hz, 1H, H-9), 7.85 (td, J = 8, 1 Hz, 1H, H-10), 7.94 (dd, J = 8,
1 Hz, 1H, H-8), 8.17 (dd, J = 8, 1 Hz, 1H, H-11), 9.19 (s, 1H, H-7); 13C
NMR (75 MHz, DMSO-d6) δ 22.6 (C-CH3), 28.2 (C-CH3), 32.3 (N-
CH3), 61.9 (C-4), 63.5 (O-CH3), 72.7 (C-3), 80.2 (C-2), 99.0 (C-14),
110.5 (C-5a), 114.4 (C-4a), 119.3 (C-6a), 124.9 (C-7a), 125.5 (C-10),
127.9 (C-8), 130.6 (C-11), 133.6 (C-9), 139.2 (C-7), 147.0 (C-13a),
149.6 (C-11a), 150.3 (C-12a), 159.7 (C-14a), 163.9 (C-5), 176.6 (C-6);
DCI-MS m/z 407 [MH]+; IR (KBr) ν cm−1: 3438, 2936, 1650, 1602,
1589, 1553, 1491, 1423, 1399, 1344, 1224, 1193, 1128, 1104, 1025, 953,
823, 758; UV λ nm (MeOH) (log ε): 228 (4.27), 289 (4.51), 340
(4.11); Anal. Calcd for C23H22N2O5: C, 67.97; H, 5.46; N, 6.89. Found:
C, 67.72; H, 5.44; N, 6.86.
( )-cis-1,2-Diacetoxy-6-methoxy-3,3,14-trimethyl-1,2,3,14-tetra-
hydro-7H-benzo[b]chromeno[6,5-g][1,8]naphthyridin-7-one (21).
An ice-cooled mixture of acetic anhydride (10 mL, 105 mmol) and
dry pyridine (45 mL) was added to 19 (0.200 g, 0.5 mmol) and
4-dimethylaminopyridine (0.01 g). After stirring at room temperature
for 24 h, the mixture was poured on cold water (30 mL). The precipitate
was filtered, washed with water (3 × 15 mL), dried in vacuum over P2O5,
and crystallized from CH2Cl2/cyclohexane (1/1) to afford 21 (0.223 g,
92%) as pale yellow needles: mp 267 °C. 1H NMR (400 MHz, CDCl3) δ
1.48 (s, 3H, CCH3), 1.60 (s, 3H, CCH3), 1.95 (s, 3H, CH3CO), 2.01 (s,
3H, CH3CO), 3.80 (s, 3H, OCH3), 4.02 (s, 3H, NCH3), 5.54 (d, J = 5
Hz, 1H, H-2), 6.33 (s, 1H, H-5), 6.55 (d, J = 5 Hz, 1H, H-1), 7.46 (t, J = 8
Hz, 1H, H-10), 7.76 (t, J = 8 Hz, 1H, H-11), 7.99 (m, 2H, H-9, H-12),
9.11 (s, 1H, H-8); 13C NMR (75 MHz, CDCl3) δ 20.6 (CH3CO), 21.0
(CH3CO), 23.1 (C-CH3), 24.8 (C-CH3), 39.8 (N-CH3), 56.3 (O-CH3),
66.0 (C-1), 69.1 (C-2), 76.5 (C-3), 94.7 (C-5), 98.3 (C-14b), 110.9
(C-6a), 120.0 (C-7a), 124.6 (C-11), 124.9 (C-8a), 127.8 (C-12), 129.4
(C-9), 131.9 (C-10), 137.6 (C-8), 149.6 (C-12a), 149.9 (C-14a), 152.3
(C-13a), 160.6 (C-4a), 160.3 (C-6), 170.4 (CH3CO), 170.9 (CH3CO),
178.0 (C-7); HRMS (ESI) calcd for C27H26N2O7 ([MH]+) m/z
491.1813, found: 491.1814; IR (KBr) ν cm−1: 2985, 2361, 2344, 1752,
1645, 1619, 1590, 1502, 1459, 1397, 1374, 1350, 1235, 1214, 1159,
1090, 1030, 813, 759; UV λ nm (MeOH) (log ε): 231 (4.36), 286
(4.72), 343 (4.08); Anal. Calcd for C27H26N2O7: C, 66.11; H, 5.34; N,
5.71. Found: C, 65.95; H, 5.34; N, 5.71.
( )-cis-6-Methoxy-1,2-di-(4-methoxycinnamoyloxy)-3,3,14-tri-
methyl-1,2,3,14-tetrahydro-7H-benzo[b]chromeno[6,5-g][1,8]-
naphthyridin-7-one (23). Compound 23 was synthesized from 19
(0.239 g, 0.59 mmol) according to the procedure described for the
preparation of 22 from 19, using 4-methoxycinnamoyl chloride (1.15 g,
5.9 mmol) prepared extemporaneously from 4-methoxycinnamoic
acid. Silica gel column chromatography (solvent: CH2Cl2, then
CH2Cl2/MeOH 99.9:0.1 to 99:1) afforded 23 as a yellow amorphous
1
solid (0.098 g, 22%). H NMR (400 MHz, CDCl3) δ 1.53 (s, 3H,
CCH3), 1.69 (s, 3H, CCH3), 3.73 (s, 3H, 4″-OCH3), 3.78 (s, 3H, 4′-
OCH3), 3.86 (s, 3H, 6-OCH3), 4.08 (s, 3H, NCH3), 5.87 (d, J = 5 Hz,
1H, H-2), 6.14 (d, 1H, J = 16 Hz, 1−OCOCHCH), 6.20 (d, 1H, J =
16 Hz, 2−OCOCHCH), 6.41 (s, 1H, H-5), 6.58 (d, J = 9 Hz, 2H,
H-3″, H-5″), 6.64(d, J = 5 Hz, 1H, H-1), 6.80 (d, J = 9 Hz, 2H, H-3′, H-5′),
7.01 (d, J = 9 Hz, 2H, H-2″, H-6″), 7.33 (d, J = 9 Hz, 2H, H-2′, H-6′),
7.38 (d, 1H, J = 16 Hz, 1−OCOCHCH), 7.44 (t, J = 8 Hz, 1H, H-10),
7.47 (d, 1H, J = 16 Hz, 2−OCOCHCH), 7.62 (t, J = 8 Hz, 1H, H-11),
7.77 (d, 1H, J = 8 Hz, H-9), 7.97 (d, 1H, J = 8 Hz, H-12), 9.13 (s, 1H,
H-8); 13C NMR (75 MHz, CDCl3) δ 23.1 (C-CH3), 25.0 (C-CH3), 39.7
(N-CH3), 55.3 (2C, 4′-O-CH3, 4″-O-CH3), 56.4 (6-O-CH3), 66.6
(C-1), 68.8 (C-2), 77.5 (C-3), 94.7 (C-5), 98.7 (C-14b), 110.9 (C-6a),
113.9 (2C, C3″, C5″), 114.2 (2C, C3′, C5′), 114.5 (2C, 1−OCOCH
CH, 2−OCOCHCH), 119.8 (C-7a), 124.5 (C-11), 124.8 (C-8a),
126.8 (2C, C-1′, C-1″), 127.9 (C-9), 129.2 (C-12), 129.9 (2C, C2″,
C6″), 129.9 (2C, C2′, C6′), 131.7 (C-10), 137.6 (C-8), 145.8 (2C,
1−OCOCHCH, 2−OCOCHCH), 149.6 (C-12a), 150.0 (C-14a),
152.0 (C-13a), 160.8 (C-4a), 161.3 (C-4″), 161.5 (C-4′), 163.0 (C-6),
166.4 (1-OCOCHCH), 167.0 (2-OCOCHCH), 178.2 (C-7);
HRMS (ESI) calcd for C43H38N2O9 ([MH]+) m/z: 727.2650, found:
727.2650; IR (KBr) νcm−1: 2979, 2935, 2836, 2363, 1719, 1647, 1591, 1512,
1397, 1255, 1156, 1092, 1032, 827; UV λ nm (MeOH) (log ε): 288 (4.85),
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312 (4.65), 429 (3.87); Anal. Calcd for C43H38N2O9: C, 71.06; H, 5.27;
N, 3.85. Found: C, 70.82; H, 5.26; N, 3.84.
(0.194 g, 45%). H NMR (400 MHz, CDCl3) δ 1.53 (s, 3H, CCH3),
1.58 (s, 3H, CCH3), 3.38 (br. d, J = 9 Hz, 1H, D2O exch., OH-1), 3.77 (s,
3H, 4′-OCH3), 3.89 (s, 3H, 6-OCH3), 4.05 (s, 3H, NCH3), 5.49 (dd, J =
9, 5 Hz, 1H, H-1), 5.62 (d, J = 5 Hz, 1H, H-2), 6.18 (s, 1H, H-5), 6.40 (d,
1H, J = 16 Hz, OCOCHCH), 6.76 (d, J = 9 Hz, 2H, H-3′, H-5′), 7.32
(d, J = 9 Hz, 2H, H-2′, H-6′), 7.38 (t, J = 8 Hz, 1H, H-10), 7.63 (t, J = 8
Hz, 1H, H-11), 7.69 (d, 1H, J = 16 Hz, OCOCHCH), 7.84 (d, 1H, J =
8 Hz, H-9), 7.91 (d, 1H, J = 8 Hz, H-12), 8.90 (s, 1H, H-8); 13C NMR
(75 MHz, CDCl3) δ 22.6 (C-CH3), 25.5 (C-CH3), 39.4 (N-CH3), 55.3
(4′-O-CH3), 56,0 (6-O-CH3), 63.8 (C-1), 72.1 (C-2), 77.1 (C-3), 94.2
(C-5), 103.1 (C-14b), 110.3 (C-6a), 114.3 (2C, C3′, C5′), 114.4
(OCOCHCH), 119.4 (C-7a), 124.3 (C-11), 124.6 (C-8a), 126.8 (C-
1′), 127.2 (C-9), 129.3 (C-12), 129,9 (2C, C2′, C6′), 131.7 (C-10),
137.7 (C-8), 146.1 (OCOCHCH), 149.2 (C-12a), 149.7 (C-14a),
151.8 (C-13a), 159.8 (C-4a), 161.5 (C-4′), 162.5 (C-6), 167.1
(OCOCHCH), 177.5 (C-7); HRMS (ESI) calcd for C33H30N2O7
([MH]+) m/z: 567.2126, found: 567.2127; IR (KBr) ν cm−1: 3398 (br.),
2974, 2931, 2834, 1720, 1641, 1588, 1503, 1394, 1254, 1209, 1146,
1095, 1032, 819; UV λ nm (MeOH) (log ε): 288 (4.84), 434 (3.88);
Anal. Calcd for C33H30N2O7: C, 69.95; H, 5.34; N, 4.94. Found: C,
69.80; H, 5.33; N, 4.93.
( )-cis-1-Hydroxy-6-methoxy-2-(4-trifluoromethylcinnamoyl-
oxy)-3,3,14-trimethyl-1,2,3,14-tetrahydro-7H-benzo[b]chromeno-
[6,5-g][1,8]naphthyridin-7-one (27). Compound 27 was synthesized
from 19 (0.431 g, 1.06 mmol) according to the procedure described for
the preparation of 25 from 19, using 4-trifluoromethylcinnamoyl
chloride (0.471 g, 2.0 mmol) prepared extemporaneously from
4-trifluoromethylcinnamoic acid. Silica gel column chromatography
(solvent: CH2Cl2, then CH2Cl2/MeOH 99.9:0.1 to 99:1) afforded 27 as
a yellow amorphous solid (0.243 g, 38%). 1H NMR (400 MHz, CDCl3)
δ 1.54 (s, 3H, CCH3), 1.61 (s, 3H, CCH3), 3.63 (br. d, J = 9 Hz, 1H, D2O
exch., OH-1), 3.89 (s, 3H, OCH3), 4.09 (s, 3H, NCH3), 5.51 (dd, J = 9, 5
Hz, 1H, H-1), 5.63 (d, J = 5 Hz, 1H, H-2), 6.19 (s, 1H, H-5), 6.72 (d, 1H,
J = 16 Hz, OCOCHCH), 7.35 (t, J = 8 Hz, 1H, H-10), 7.46 (d, J = 8
Hz, 2H, H-3′, H-5′), 7.50 (d, J = 8 Hz, 2H, H-2′, H-6′), 7.56 (t, J = 8 Hz,
1H, H-11), 7.74 (d, 1H, J = 8 Hz, H-9), 7.82 (d, 1H, J = 16 Hz,
OCOCHCH), 7.85 (d, 1H, J = 8 Hz, H-12), 8.82 (s, 1H, H-8); 13C
NMR (75 MHz, CDCl3) δ 22.4 (C-CH3), 25.6 (C-CH3), 39.2 (N-CH3),
55.6 (O-CH3), 63.8 (C-1), 72.6 (C-2), 77.2 (C-3), 93.8 (C-5), 103.0
(C-14b), 110.0 (C-6a), 118.8 (C-7a), 120.2 (OCOCHCH), 124.2
(C-8a), 124.3 (C-11), 125.7 (4′-CF3), 125.8 (2C, C3′, C5′), 127.1
(C-9), 128.1 (2C, C2′, C6′), 129.2 (C-12), 131.4 (C-4′), 131.8 (C-10),
131.9 (C-1′), 137.7 (C-8), 144.2 (OCOCHCH), 148.9 (C-12a),
149.8 (C-14a), 151.3 (C-13a), 159.9 (C-4a), 162.3 (C-6), 166.8
(OCOCHCH), 177.2 (C-7); HRMS (ESI) calcd for C33H27F3N2O6
([MH]+) m/z: 605.1894, found: 605.1894; IR (KBr) ν cm−1: 3372 (br.),
2970, 2931, 2363, 1727, 1639, 1588, 1503, 1394, 1326, 1210, 1159,
1124, 1067, 1035, 834, 812, 747; UV λ nm (MeOH) (log ε): 288 (4.83),
348 (4.13), 432 (3.90); Anal. Calcd for C33H27F3N2O6: C, 65.56; H,
4.50; N, 4.63. Found: C, 65.42; H, 4.50; N, 4.62.
( )-cis-1-Acetoxy-2-cinnamoyloxy-6-methoxy-3,3,14-trimethyl-1,2,3,14-
tetrahydro-7H-benzo[b]chromeno[6,5-g][1,8]naphthyridin-7-one (28).
Acetic anhydride (1.5 mL, 15.7 mmol) was added to an iced-cooled
solution (0 °C) of monoester 25 (0.080 g, 0,15 mmol) and
4-dimethylaminopyridine (0.01 g) in dry pyridine (5 mL). After
stirring at 25 °C for 5 h, the reaction mixture was poured on cold
water (20 mL). The precipitate was filtered, washed with water
(3 × 15 mL), and dried in vacuum over P2O5, to afford 28 as a yellow
amorphous solid (0.072 g, 83%). 1H NMR (400 MHz, CDCl3) δ 1.51
(s, 3H, CCH3), 1.65 (s, 3H, CCH3), 1.92 (s, 3H, CH3CO), 3.81 (s,
3H, OCH3), 4.06 (s, 3H, NCH3), 5.73 (d, J = 5 Hz, 1H, H-2), 6.35 (d,
1H, J = 16 Hz, OCOCHCH), 6.38 (s, 1H, H-5), 6.58 (d, J = 5 Hz,
1H, H-1), 7.34 (m, 3H, H-3′, H-4′, H-5′), 7.45 (m, 3H, H-2′, H-6′,
H-10), 7.60 (d, 1H, J = 16 Hz, OCOCHCH), 7.76 (t, J = 8 Hz, 1H,
H-11), 7.99 (m, 2H, H-9, H-12), 9.11 (s, 1H, H-8); 13C NMR (75
MHz, CDCl3) δ 21.0 (CH3CO), 22.7 (C-CH3), 25.0 (C-CH3), 39.7
(N-CH3), 56.4 (O-CH3), 66.3 (C-1), 68.8 (C-2), 77.2 (C-3), 94.5
(C-5), 98.2 (C-14b), 110.9 (C-6a), 116.8 (OCOCHCH), 120.0
(C-7a), 124.6 (C-11), 124.9 (C-8a), 127.8 (C-12), 128.3 (2C, C3′,
C5′), 128.8 (2C, C2′, C6′), 129.4 (C-9), 130.6 (C-4′), 131,9 (C-10),
( )-cis-6-Methoxy-1,2-di-(4-trifluoromethylcinnamoyloxy)-
3,3,14-trimethyl-1,2,3,14-tetrahydro-7H-benzo[b]chromeno[6,5-g]-
[1,8]naphthyridin-7-one (24). Compound 24 was synthesized from 19
(239 mg, 0.59 mmol) according to the procedure described for the
preparation of 22 from 19, using 4-trifluoromethylcinnamoyl chloride
(1.38 g, 5.9 mmol) prepared extemporaneously from 4-trifluoro-
methylcinnamoic acid. Silica gel column chromatography (solvent:
CH2Cl2, then CH2Cl2/MeOH 99.9:0.1 to 99:1) afforded 24 as a yellow
amorphous solid (0.175 g, 37%). 1H NMR (400 MHz, CDCl3) δ 1.53 (s,
3H, CCH3), 1.69 (s, 3H, CCH3), 3.81 (s, 3H, OCH3), 4.08 (s, 3H,
NCH3), 5.91 (d, J = 5 Hz, 1H, H-2), 6.34 (d, 1H, J = 16 Hz,
1−OCOCHCH), 6.41 (d, 1H, J = 16 Hz, 2−OCOCHCH), 6.43
(s, 1H, H-5), 6.64 (d, J = 5 Hz, 1H, H-1), 7.06 (d, J = 9 Hz, 2H, H-3″,
H-5″), 7.28 (d, J = 9 Hz, 2H, H-2″, H-6″), 7.36 (d, 1H, J = 16 Hz,
1−OCOCHCH), 7.48 (m, 6H, H-2′, H-3′, H-5′, H-6′. H-10,
2−OCOCHCH), 7.59 (t, J = 8 Hz, 1H, H-11), 7.68 (d, 1H, J = 8 Hz,
H-9), 7.98 (d, 1H, J = 8 Hz, H-12), 9.11 (s, 1H, H-8); 13C NMR (75
MHz, CDCl3) δ 22.9 (C-CH3), 25.0 (C-CH3), 39.7 (N-CH3), 56.4
(O-CH3), 67.1 (C-1), 69.2 (C-2), 77.1 (C-3), 94.7 (C-5), 98.1 (C-14b),
110.8 (C-6a), 119.4 (C-7a), 119.8 (1−OCOCHCH), 120.4 (2−
OCOCHCH), 124.8 (C-11), 125.0 (C-8a), 125.5 (2C, C3″, C5″),
125.8 (2C, C3′, C5′), 127.7 (C-9), 128.1 (2C, C2″, C6″), 128.3 (2C,
C2′, C6′), 129.3 (4″-CF3), 129.4 (C-12), 129.8 (4′-CF3), 131.9 (C-10),
132.0 (C-4″), 132.3 (C-4′), 137.0 (C-1″), 137.2 (C-1′), 137.7 (C-8),
144.2 (1−OCOCHCH), 144.4 (2−OCOCHCH), 149.5 (C-12a),
150.0 (C-14a), 152.1 (C-13a), 160.7 (C-4a), 163.2 (C-6), 165.6
(1-OCOCHCH), 166.1 (2-OCOCHCH), 178.1 (C-7); HRMS (ESI)
calcd for C43H32F6N2O7 ([MH]+) m/z: 803.2186, found: 803.2186; IR
(KBr) ν cm−1: 2974, 2934, 2855, 2367, 1728, 1648, 1592, 1501, 1397,
1326, 1206, 1161, 1127, 1067, 1017, 832, 758; UV λ nm (MeOH) (log
ε): 288 (4.63), 343 (4.05), 423 (3.80); Anal. Calcd for C43H32F6N2O7:
C, 64.34; H, 4.02; N, 3.49. Found: C, 64.12; H, 4.01; N, 3.49.
( )-cis-2-Cinnamoyloxy-1-hydroxy-6-methoxy-3,3,14-trimethyl-
1,2,3,14-tetrahydro-7H-benzo[b]chromeno[6,5-g][1,8]-
naphthyridin-7-one (25). Cinnamoyl chloride (0.186 g, 1.12 mmol),
prepared extemporaneously from 4-methoxycinnamoic acid, was added
to a solution of 19 (0.239 g, 0.59 mmol) in dry pyridine (10 mL). The
reaction mixture was stirred at room temperature for 48 h and then
evaporated under reduced pressure. Silica gel column chromatography
(solvent: CH2Cl2, then CH2Cl2/MeOH, 99:1) afforded 25 as a yellow
amorphous solid (0.196 g, 62%). 1H NMR (400 MHz, CDCl3) δ 1.55 (s,
3H, CCH3), 1.61 (s, 3H, CCH3), 3.67 (br. d, J = 9 Hz, 1H, D2O exch.,
OH-1), 3.88 (s, 3H, OCH3), 4.04 (s, 3H, NCH3), 5.50 (dd, J = 9, 5 Hz,
1H, H-1), 5.64 (d, J = 5 Hz, 1H, H-2), 6.17 (s, 1H, H-5), 6.58 (d, 1H, J =
16 Hz, OCOCHCH), 7.25 (m, 3H, H-3′, H-4′, H-5′), 7.36 (m, 3H,
H-2′, H-6′, H-10), 7.58 (t, J = 8 Hz, 1H, H-11), 7.77 (d, 1H, J = 16 Hz,
OCOCHCH), 7.78 (d, 1H, J = 8 Hz, H-9), 7.88 (d, 1H, J = 8 Hz,
H-12), 8.85 (s, 1H, H-8); 13C NMR (75 MHz, CDCl3) δ 22.6 (C-CH3),
25.5 (C-CH3), 39.3 (N-CH3), 55.9 (O-CH3), 63.8 (C-1), 72.3 (C-2),
77.2 (C-3), 94.1 (C-5), 103.1 (C-14b), 110.3 (C-6a), 117.1
(OCOCHCH), 119.3 (C-7a), 124.3 (C-11), 124.5 (C-8a), 127.1
(C-9), 128.1 (2C, C3′, C5′), 128.8 (2C, C2′, C6′), 129.3 (C-12), 130.4
(C-4′), 131.7 (C-10), 134.1 (C-1′), 137.7 (C-8), 146.4 (OCOCH
CH), 149.1 (C-12a), 149.7 (C-14a), 151.7 (C-13a), 159.8 (C-4a), 162.5
(C-6), 167.1 (OCOCHCH), 177.4 (C-7); HRMS (ESI) calcd for
C32H28N2O6 ([MH]+) m/z: 537.2020, found: 537.2021; IR (KBr) ν
cm−1: 3406 (br.), 2975, 2933, 2359, 1719, 1638, 1589, 1503, 1394, 1159,
1095, 1033, 818, 769; UV λ nm (MeOH) (log ε): 288 (4.76), 348
(4.03), 433 (3.79); Anal. Calcd for C32H28N2O6: C, 71.63; H, 5.26; N,
5.22. Found: C, 71.42; H, 5.25 N, 5.21.
( )-cis-1-Hydroxy-6-methoxy-2-(4-methoxycinnamoyloxy)-
3,3,14-trimethyl-1,2,3,14-tetrahydro-7H-benzo[b]chromeno[6,5-g]-
[1,8]naphthyridin-7-one (26). Compound 26 was synthesized from 19
(0.307 g, 0.76 mmol) according to the procedure described for the
preparation of 25 from 19, using 4-methoxycinnamoyl chloride (0.282 g,
1.44 mmol) prepared extemporaneously from 4-methoxycinnamoic
acid. Silica gel column chromatography (solvent: CH2Cl2, then CH2Cl2/
MeOH 99.9:0.1 to 99:1) afforded 26 as a yellow amorphous solid
K
dx.doi.org/10.1021/jm500927d | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
133.9 (C-1′), 137.6 (C-8), 146,3 (OCOCHCH), 149.6 (C-12a),
149.9 (C-14a), 152.2 (C-13a), 160.6 (C-4a), 163.0 (C-6), 166.3
(OCOCHCH), 171.1 (CH3CO), 178.1 (C-7); HRMS (ESI) calcd
for C34H30N2O7 ([MH]+) m/z: 579.2126, found: 579.2126; IR
(KBr) ν cm−1: 2976, 2360, 1746, 1717, 1641, 1589, 1500, 1399, 1217,
1153, 1092, 1035, 815, 768; UV λ nm (MeOH) (log ε): 288 (4.45),
343 (3.85), 427 (3.55); Anal. Calcd for C34H30N2O7: C, 70.58; H,
5.23; N, 4.84. Found: C, 70.43; H, 5.22; N, 4.83.
CH2Cl2 then CH2Cl2/MeOH, 99.9:0.1 to 98:2) gave 31 (0.078 g, 37%)
as a yellowish amorphous solid. 1H NMR (400 MHz, DMSO) δ 1.45 (s,
3H, CCH3), 1.56 (s, 3H, CCH3), 3.89 (s, 3H, OCH3), 4.02 (s, 3H,
NCH3), 5.24 (d, J = 8 Hz, 1H, H-2), 6.46 (s, 1H, H-5), 6.78 (d, J = 8 Hz,
1H, H-1), 7.56 (ddd, J = 8, 7, 1 Hz, 1H, H-10), 7.88 (ddd, J = 8, 7, 1 Hz,
1H, H-11), 8.00 (dd, J = 8, 1 Hz, 1H, H-9), 8.22 (dd, J = 8, 1 Hz, 1H,
H-12), 9.05 (s, 1H, H-8); 13C NMR (75 MHz, DMSO) δ 22.0 (C-CH3),
25.3 (C-CH3), 42.0 (N-CH3), 57.3 (O-CH3), 71.7 (C-1), 76.4 (C-3),
79.4 (C-2), 96.5 (C-5), 99.8 (C-14b), 111.3 (C-6a), 120.6 (C-7a), 125.5
(C-8a), 126.0 (C-11), 128.2 (C-9), 130.7 (C-12), 133.6 (C-10), 138.2
(C-8), 149.6 (C-12), 150.2 (C-14a), 152.9 (C-13a), 154.5 (CO), 160.6
(C-4a), 164.0 (C-6), 177.3 (C-7); DCI-MS m/z 433 [MH]+, 431, 389,
373; IR (KBr) ν cm−1: 1810, 1640, 1619, 1588, 1572, 1501, 1459, 1399,
1345, 1213, 1199, 1172, 1158, 1097, 1032, 981, 814, 754; UV λ nm
(MeOH) (log ε): 286 (4.56), 343 (3.98); Anal. Calcd for C24H20N2O6:
C, 66.66; H, 4.66; N, 6.48. Found: C, 66.49; H, 4.65; N, 6.47.
( )-cis-1-Acetoxy-6-methoxy-2-(4-methoxycinnamoyloxy)-
3,3,14-trimethyl-1,2,3,14-tetrahydro-7H-benzo[b]chromeno[6,5-g]-
[1,8]naphthyridin-7-one (29). Acetic anhydride (1.5 mL, 15.7 mmol)
was added to an iced-cooled solution (0 °C) of monoester 26 (0.131 g,
0,23 mmol) and 4-dimethylaminopyridine (0.01 g) in dry pyridine
(5 mL). Following the procedure described for the preparation of 28
from 25, the mixed ester 29 was obtained as a yellow amorphous solid
1
(0.129 g, 92%). H NMR (400 MHz, CDCl3) δ 1.50 (s, 3H, CCH3),
1.64 (s, 3H, CCH3), 1.91 (s, 3H, CH3CO), 3.79 (s, 6H, 6-OCH3,
4′-OCH3), 4.05 (s, 3H, NCH3), 5.72 (d, J = 5 Hz, 1H, H-2), 6.22 (d, 1H,
J = 16 Hz, OCOCHCH), 6.38 (s, 1H, H-5), 6.57 (d, J = 5 Hz, 1H,
H-1), 6.84 (d, J = 8 Hz, 2H, H-3′, H-5′), 7.39 (d, J = 8 Hz, 2H, H-2′,
H-6′), 7.45 (t, J = 8 Hz, 1H, H-10), 7.55 (d, 1H, J = 16 Hz, OCOCH
CH), 7.76 (t, J = 8 Hz, 1H, H-11), 7.98 (m, 2H, H-9, H-12), 9.11 (s, 1H,
H-8); 13C NMR (75 MHz, CDCl3) δ 21.0 (CH3CO), 22.7 (C-CH3),
25.0 (C-CH3a), 39.7 (N-CH3), 55.4 (4′-O-CH3), 56.4 (6-O-CH3), 66.3
(C-1), 68.5 (C-2), 77.8 (C-3), 94.6 (C-5), 98.2 (C-14b), 110.9 (C-6a),
113.4 (OCOCHCH), 114.3 (2C, C3′, C5′), 120.0 (C-7a), 124.6
(C-11), 124.9 (C-8a), 126.7 (C-1′), 127.8 (C-9), 129.4 (C-12), 130.0
(2C, C2′, C6′), 131.9 (C-10), 137.6 (C-8), 146.0 (OCOCHCH),
149.6 (C-12a), 149.9 (C-14a), 152.2 (C-13a), 160.1 (C-4a), 161.6
(C-4′), 163.0 (C-6), 166.6 (OCOCHCH), 171.1 (CH3CO), 178.1
(C-7); HRMS (ESI) calcd for C35H32N2O8 ([MH]+) m/z: 609.2231,
found: 609.2232; IR (KBr) ν cm−1: 2974, 1745, 1718, 1648, 1592, 1501,
1397, 1346, 1254, 1214, 1151, 1092, 1031, 815; UV λ nm (MeOH) (log
ε): 288 (4.82), 314 (4.55), 427 (3.86); Anal. Calcd for C35H32N2O8: C,
69.07; H, 5.30; N, 4.60. Found: C, 68.98; H, 5.29; N, 4.59.
( )-cis-3,4-Diacetoxy-5-methoxy-2,2,13-trimethyl-2,3,4,13-tetra-
hydro-6H-benzo[b]chromeno[7,6-g][1,8]naphthyridin-6-one (32).
Compound 32 was synthesized from 20 (0.200 g, 0.49 mmol) according
to the procedure described for the preparation of 21 from 19, using
acetic anhydride (10 mL, 105 mmol) and 4-dimethylaminopyridine
(0.01 g) in dry pyridine (40 mL). Crystallization from CH2Cl2 gave 32
(0.213 g, 88%) as pale yellow needles: mp 313 °C. 1H NMR (400 MHz,
CDCl3) δ 1.47 (s, 3H, CCH3), 1.51 (s, 3H, CCH3), 2.09 (s, 3H,
CH3CO), 2.11 (s, 3H, CH3CO), 3.98 (s, 3H, OCH3), 4.12 (s, 3H,
NCH3), 5.27 (d, J = 5 Hz, 1H, H-3), 6.48 (d, J = 5 Hz, 1H, H-4), 6.78 (s,
1H, H-14), 7.46 (ddd, J = 8, 7, 1 Hz, 1H, H-9), 7.77 (ddd, J = 8, 7, 1 Hz,
1H, H-10), 7.97 (m, 2H, H-8, H-11), 9.23 (s, 1H, H-7); 13C NMR (75
MHz, CDCl3) δ 20.7 (COCH3), 20.9 (COCH3), 21.9 (C-CH3), 26.3
(C-CH3), 31.6 (N-CH3), 61.0 (C-4), 62.4 (O-CH3), 71.5 (C-3), 77.3
(C-2), 98.4 (C-14), 107.8 (C-4a), 110.4 (C-5a), 118.7 (C-6a), 124.5
(C-7a), 124.6 (C-10), 127.5 (C-11), 129.4 (C-8), 132.4 (C-9), 138.7
(C-7), 147.4 (C-13a), 149.4 (C-11a), 158.9 (C-14a), 163.5 (C-5), 169.4
(COCH3), 170.0 (COCH3), 177.4 (C-6); HRMS (ESI) calcd for
C27H26N2O7 ([MH]+) m/z: 491.1813, found: 491.1814; IR (KBr)
ν cm−1: 2936, 1754, 1741, 1648, 1618, 1608, 1592, 1577, 1560, 1501,
1425, 1373,1344, 1241, 1196, 1153, 1127, 1100, 1049, 1026, 824, 760;
UV λ nm (MeOH) (log ε): 228 (4.39), 289(4.83), 337 (4.18); Anal.
Calcd for C27H26N2O7: C, 66.11; H, 5.34; N, 5.71. Found: C, 65.98; H,
5.33; N, 5.70.
( )-cis-3,4-Di-O-carbonyl-3,4-dihydroxy-5-methoxy-2,2,13-tri-
methyl-2,3,4,13-tetrahydro-6H-benzo[b]chromeno[7,6-g][1,8]-
naphthyridin-6-one (33). Compound 33 was synthesized from 20
(0.200 g, 0.49 mmol) according to the procedure described for the pre-
paration of 31 from 19, using carbonyldiimidazole (0.64 g, 3.92 mmol)
in 2-butanone (80 mL). Purification by silica gel column chromatog-
raphy (solvent: CH2Cl2, then CH2Cl2/MeOH, 99.9:0.1 to 99.2:0.8)
gave 33 (0.143 g, 67%) as pale yellow crystals: mp 272 °C (crystallized
from CH2Cl2/MeOH 1/1). 1H NMR (400 MHz, CDCl3) δ 1.26 (s, 3H,
CCH3), 1.59 (s, 3H, CCH3), 4.05 (s, 3H, OCH3), 4.10 (s, 3H, NCH3),
4.73 (dd, J = 8 Hz, 1H, H-3), 6.04 (d, J = 8 Hz, 1H, H-4), 6.78 (s, 1H,
H-14), 7.44 (ddd, J = 8, 7, 1 Hz, 1H, H-9), 7.75 (ddd, J = 8, 7, 1 Hz, 1H,
H-10), 8.15 (m, 2H, H-8, H-11), 9.20 (s, 1H, H-7); 13C NMR (75 MHz,
CDCl3) δ 22.5 (C-CH3), 24.3 (C-CH3), 31.7 (N-CH3), 63.9 (O-CH3),
68.6 (C-4), 76.2 (C-2), 78.1 (C-3), 99.4 (C-14), 106.0 (C-4a), 110.9
(C-5a), 118.7 (C-6a), 124.6 (C-7a), 124.9 (C-10), 127.6 (C-11), 129.4
(C-8), 132.6 (C-9), 138.8 (C-7), 148.1 (C-13a), 149.5 (2C, C-11a,
C-12a), 153.9 (CO), 158.3 (C-14a), 164.1 (C-5), 177.0 (C-6); HRMS
(ESI) calcd for C24H20N2O6 ([MH]+) m/z: 433.1394, found: 433.1394;
IR (KBr) ν cm−1: 2940, 2363, 1794, 1649, 1619, 1607, 1577, 1498, 1425,
1345, 1206, 1177, 1158, 1100, 1080, 1029, 750; UV λ nm (MeOH)
(log ε): 228 (4.39), 288 (4.78), 335 (4.20); Anal. Calcd for C24H20N2O6: C,
66.66; H, 4.66; N, 6.48. Found: C, 66.50; H, 4.66; N, 6.47.
( )-cis-1-Acetoxy-6-methoxy-2-(4-trifluoromethylcinnamoyloxy)-
3,3,14-trimethyl-1,2,3,14-tetrahydro-7H-benzo[b]chromeno[6,5-g]-
[1,8]naphthyridin-7-one (30). Acetic anhydride (1.5 mL, 15.7 mmol)
was added to an iced-cooled solution (0 °C) of monoester 27 (0.072 g,
0.12 mmol) and 4-dimethylaminopyridine (0.01 g) in dry pyridine
(5 mL). Following the procedure described for the preparation of 28
from 25, the mixed ester 30 was obtained as a yellow amorphous solid
1
(0.054 g, 70%). H NMR (400 MHz, CDCl3) δ 1.51 (s, 3H, CCH3),
1.64 (s, 3H, CCH3), 1.92 (s, 3H, CH3CO), 3.80 (s, 3H, OCH3), 4.04
(s, 3H, NCH3), 5.73 (d, J = 5 Hz, 1H, H-2), 6.39 (s, 1H, H-5), 6.43 (d,
1H, J = 16 Hz, OCOCHCH), 6.58 (d, J = 5 Hz, 1H, H-1), 7.46 (t, J = 8
Hz, 1H, H-10), 7.56 (m, 5H, H-2′, H-3′, H-5′, H-6′, OCOCHCH),
7.77 (t, J = 8 Hz, 1H, H-11), 7.98 (m, 2H, H-9, H-12), 9.11 (s, 1H, H-8);
13C NMR (75 MHz, CDCl3) δ 21.0 (CH3CO), 22.7 (C-CH3), 25.0
(C-CH3a), 39.7 (N-CH3), 56.3 (O-CH3), 66.2 (C-1), 69.2 (C-2), 77.2
(C-3), 94.5 (C-5), 98.1 (C-14b), 110.9 (C-6a), 119.5 (C-7a), 120.0
(OCOCHCH), 124.7 (C-11), 124.9 (C-8a), 125.8 (4′-CF3), 125.8
(2C, C3′, C5′), 127.8 (C-9), 128.4 (2C, C2′, C6′), 129.4 (C-12), 130.0
(C-4′), 131.8 (C-11), 131.9 (C-10), 137.6 (C-8), 144.3 (OCOCH
CH), 149.6 (C-12a), 150.0 (C-14a), 152.2 (C-13a), 160.6 (C-4a), 163.1
(C-6), 165.7 (OCOCHCH), 171.0 (CH3CO), 178.0 (C-7); HRMS
(ESI) calcd for C35H29F3N2O7 ([MH]+) m/z: 647.2000, found:
647.1999; IR (KBr) ν cm−1: 2971, 2931, 2362, 1751, 1736, 1647,
1592, 1502, 1399, 1324, 1214, 1161, 1094, 1067, 1033, 835; UV λ nm
(MeOH) (log ε): 288 (4.63), 343 (4.03), 427 (3.77); Anal. Calcd for
C35H29F3N2O7: C, 65.01; H, 4.52; N, 4.33. Found: C, 64.82; H, 4.51; N, 4.32.
( )-cis-1,2-Di-O-carbonyl-1,2-dihydroxy-6-methoxy-3,3,14-tri-
methyl-1,2,3,14-tetrahydro-7H-benzo[b]chromeno[6,5-g][1,8]-
naphthyridin-7-one (31). N,N′-Carbonyldiimidazole (0.64 g, 3.92
mmol) was added to a solution of 19 (0.20 g, 0.49 mmol) in 2-butanone
(90 mL). The reaction mixture was refluxed for 4 days under argon, and
after cooling, 5% aqueous NaHCO3 (15 mL) was added. The solution
was extracted with ethyl acetate (3 × 30 mL), and the combined organic
layers were dried over anhydrous Na2SO4, filtered, and evaporated
under reduced pressure. Silica gel column chromatography (solvent:
(+)-(1R,2R)-1,2-Diacetoxy-6-methoxy-3,3,14-trimethyl-1,2,3,14-
tetrahydro-7H-benzo[b]chromeno[6,5-g][1,8]naphthyridin-7-one
(21a) and (−)-(1S,2S)-1,2-Diacetoxy-6-methoxy-3,3,14-trimethyl-
1,2,3,14-tetrahydro-7H-benzo[b]chromeno[6,5-g][1,8]-
naphthyridin-7-one (21b). Chiral separation of the isomers was per-
formed on Preparative pump K-1800 KNAUER using a Chiralpak AS-V
(600 mm × 60 mm, 20 μm) column with CH3OH−CH3CN−DEA
L
dx.doi.org/10.1021/jm500927d | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(diethylamine) 900-100-1 as the mobile phase and a flow rate of
50 mL/min at room temperature from the racemic compound 21
(269 mg). The desired compounds 21a and 21 b were isolated in
quantitative yields. The absolute stereochemistry was deduced from the
optical rotation sign and by observation of the curves of the Cotton
effect by comparison with those of 3 enantiomers. For 3 enantiomers the
absolute configuration of one of them was previously determined by RX
diffraction of a brominated compound.16
activity of esters in the trans-1,2-dihydroxy-1,2-dihydroacronycine
series. J. Nat. Prod. 1998, 61, 198−201.
(6) (a) Costes, N.; Le Deit, H.; Michel, S.; Tillequin, F.; Koch, M.;
Pfeiffer, B.; Renard, P.; Leo
́
nce, S.; Guilbaud, N.; Kraus-Berthier, L.;
Pierre, A.; Atassi, Gh. Synthesis and cytotoxic and antitumor activity of
́
benzo[b]pyrano[3,2-h]acridin-7-one analogues of acronycine. J. Med.
Chem. 2000, 43, 2395−2402. (b) Guilbaud, N.; Kraus-Berthier, L.;
Meyer-Losic, F.; Malivet, V.; Chacun, C.; Jan, J.; Tillequin, F.; Michel, S.;
21a[α]: D = +0.028° (CH2Cl2), negative Cotton effect R,R
21b[α]: D = −0.019° (CH2Cl2), positive Cotton effect S,S
́
Koch, M.; Pfeiffer, B.; Atassi, Gh.; Hickman, J.; Pierre, A. Marked
antitumor activity of a new potent acronycine derivative in orthotopic
models of human solid tumors. Clin. Cancer Res. 2001, 7, 2573−2580.
(c) Doan Thi Mai, H.; Gaslonde, T.; Michel, S.; Koch, M.; Tillequin, F.;
ASSOCIATED CONTENT
■
́ ́
Pfeiffer, B.; Renard, P.; Kraus-Berthier, L.; Leonce, S.; Pierre, A.
S
* Supporting Information
Synthesis and cytotoxic and antitumor activity of 1,2-dihydroxy-1,2-
dihydrobenzo[b]acronycine diacid hemiesters and carbamates. Chem.
Pharm. Bull. 2004, 52, 293−297. (d) Do, Q.; Tian, W.; Yougnia, R.;
1H and 13C NMR spectra. This material is available free of charge
via the Internet at http://pubs.acs.org.
́ ́
Gaslonde, T.; Pfeiffer, B.; Pierre, A.; Leonce, S.; Kraus-Berthier, L.;
AUTHOR INFORMATION
Corresponding Author
*Telephone number: +331 53 73 98 03. E-mail address: sylvie.
michel@parisdescartes.fr.
■
David-Cordonnier, M.-H.; Depauw, S.; Lansiaux, A.; Mazinghien, R.;
Koch, M.; Tillequin, F.; Michel, S.; Dufat, H. Synthesis, cytotoxic
activity, and DNA binding properties of antitumor cis-1,2-dihydroxy-1,2-
dihydrobenzo[b]acronycine cinnamoyl esters. Bioorg. Med. Chem. 2009,
17, 1918−1927.
Notes
(7) David-Cordonnier, M.-H.; Laine, W.; Kouach, M.; Briand, G.;
Vezin, H.; Gaslonde, T.; Michel, S.; Doan Thi Mai, H.; Tillequin, F.;
The authors declare no competing financial interest.
́ ́
Koch, M.; Leonce, S.; Pierre, A.; Bailly, C. A transesterification reaction
ACKNOWLEDGMENTS
M.-H.D.-C. thanks the IRCL and the Ligue Nationale contre le
is implicated in the covalent binding of benzo[b]acronycine anticancer
agents with DNA and glutathion. Bioorg. Med. Chem. 2004, 12, 23−29.
(8) (a) David-Cordonnier, M.-H.; Laine, W.; Lansiaux, A.; Kouach, M.;
■
Cancer (Comite
for access to the Storm facilities.
́
du Nord) for grants, and the IFR114-IMPRT
́
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) 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.;
DEDICATION
∥Dedicated to Professor Franco
■
̧
is Tillequin.
Laine, W.; Bailly, C.; Kraus-Berthier, L.; Leo
́
nce, S.; Hickman, J. A.;
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