Synthesis and cytotoxic activity of pyranocarbazole analogues of ellipticine and acronycine

Article abstract of DOI:10.1248/cpb.52.540

Various 2,2,5,11-tetramethyl- and 2,2,5,6,11-pentamethyl-2,6- dihydropyrano[3,2-b]carbazole derivatives were synthesized by condensation of 3-methylbut-2-enal or 3-chloro-3-methylbut-1-yne with an appropriate hydroxycarbazole. These compounds associate th

Full text of DOI:10.1248/cpb.52.540

540
Chem. Pharm. Bull. 52(5) 540—545 (2004)
Vol. 52, No. 5
Synthesis and Cytotoxic Activity of Pyranocarbazole Analogues of
Ellipticine and Acronycine
Hong Anh TRAN-THI,a Thuan NGUYEN-THI,a Sylvie MICHEL,a Francois TILLEQUIN,a Michel KOCH,a
b
c
,a
*
Bruno PFEIFFER, Alain PIERRÉ, and Hanh TRINH-VAN-DUFAT
a Laboratoire de Pharmacognosie de l’Université René Descartes, U.M.R./C.N.R.S. N° 8638, Faculté des Sciences
Pharmaceutiques et Biologiques; 4 Avenue de l’Observatoire, 75006 Paris, France: b Les Laboratoires Servier, and Institut
de Recherches Internationales Servier; 1 rue Carle Hébert, 92415 Courbevoie Cedex, France: and c Division Recherche
Cancérologie, 125 Chemin de Ronde; 78290 Croissy sur Seine, France.
Received November 21, 2003; accepted February 5, 2004
Various 2,2,5,11-tetramethyl- and 2,2,5,6,11-pentamethyl-2,6-dihydropyrano[3,2-b]carbazole derivatives
were synthesized by condensation of 3-methylbut-2-enal or 3-chloro-3-methylbut-1-yne with an appropriate hy-
droxycarbazole. These compounds associate the tricyclic system responsible for the intercalating properties of el-
lipticine related drugs, with the dimethylpyran pharmacophore of acronycine derivatives. The study of the bio-
logical properties of the new pyrano[3,2-b]carbazole derivatives was carried out in vitro on L1210 murine
leukaemia cell line. The three (ꢀ)-cis-diol diesters 15, 16, and 18 were the most active compounds.
Key words ellipticine; acronycine; 2,6-dihydropyrano[3,2-b]carbazole; cytotoxicity
Natural products including a carbazole basic skeleton responsible for the intercalating properties of ellipticine
fused with another heterocyclic system currently receive a related drugs, with the dimethylpyran pharmacophore of
strong attention, due to the promising antitumor properties of acronycine derivatives.
several of their representatives.1) Of particular interest in this
field are the pyrido[4,3-b]carbazole alkaloids and the in- Chemistry
dolo[2,3-a]carbazole antibiotics. The first series, illustrated
For the synthesis of 2,2,5,11-tetramethyl-2,6-dihydropy-
by ellipticine (1) and 9-methoxyellipticine (2), both isolated rano[3,2-b]carbazole (7), the readily available 1,4-dimethyl-
from Ochrosia species (Apocynaceae) include compounds 9H-carbazole-3-carbaldehyde (8), previously used as key-in-
which act by intercalation within the DNA-double strand, fol- termediate in several ellipticine syntheses,15) was chosen as
lowed by inhibition of topoisomerase II.2) A derivative of el- starting material. It was first oxidized by use hydrogen perox-
lipticine, elliptinium (3), has been commercialized for the ide in acidic medium, into the corresponding 1,4-dimethyl-
treatment of metastatic breast cancer.3) Rebeccamycine (4), 9H-carbazol-3-ol (9), according to the Matsumoto’s proce-
isolated from Saccharothrix aerocolonigenes, is a typical dure which has been applied by Langendoen et al. to the syn-
representative of the second series currently under clinical thesis of related hydroxycarbazoles.16) Construction of the
trials. It induces topoisomerase I mediated DNA-cleavage.4) fused dimethylpyran ring was ensured by condensation of 9
The antitumor activity of several related indolocarbazoles
has been recently discussed.5)
The structurally related pyrano[2,3-c]acridone alkaloid
acronycine (5) isolated from Achronycia baueri SCHOTT. (Ru-
taceae) was shown to exhibit antitumor properties against a
broad spectrum of solid tumor models.6—8) Nevertheless, its
moderate potency and low water solubility rapidly appeared
as severe drawbacks for a possible introduction into the
clinic. A hypothesis of bioactivation of the 1,2-double bond
of the chromene ring system of acronycine into the corre-
sponding epoxide9) led to the development of several series
of more active structural analogues. The most significant im-
provements were obtained with diesters of 1,2-dihydroxy-
1,2-dihydroacronycine10) and, more recently, of 1,2-dihy-
droxy-1,2-dihydrobenzo[b]acronycine.11) Representatives of
this latter series, such as diacetate 6, are considered as valu-
able candidates for clinical studies.12) The mechanism of
their action implies alkylation of the 2-amino group of DNA
guanine residues by the ester leaving group at position 1 of
the drug.13,14)
We describe here the synthesis and cytotoxic properties of
various 2,2,5,11-tetramethyl- and 2,2,5,6,11-pentamethyl-
2,6-dihydropyrano[3,2-b]carbazole derivatives. Such com-
pounds associate the 1,4-dimethylcarbazole tricyclic system
∗ To whom correspondence should be addressed. e-mail: dufat@pharmacie.univ-paris5.fr
© 2004 Pharmaceutical Society of Japan

May 2004
541
Chart 1
Chart 2
with 3-methylbut-2-enal (senecioaldehyde) in the presence of sioned to create the dimethylpyran ring of 2,2,5,6,11-pen-
phenylboronic acid as Lewis acid catalyst17) which gave the tamethyl-2,6-dihydropyrano[3,2-b]carbazole (19).19) The
target compound 7. The same reaction sequence, applied to starting 1,4,9-trimethyl-9H-carbazol-3-ol (20) was first con-
6-methoxy-1,4-dimethyl-9H-carbazole-3-carbaldehyde veniently prepared by hydrogen peroxide oxidation of the
(10),15) furnished successively 6-methoxy-1,4-dimethyl-9H- known 1,4,9-trimethyl-9H-carbazole-3-carbaldehyde (21).15)
carbazol-3-ol (11) and 9-methoxy-2,2,5,11-tetramethyl-2,6- Further treatment of 20 with 3-chloro-3-methylbut-1-yne20)
dihydropyrano[3,2-b]carbazole (12) (Chart 1).
in the presence of sodium hydride in refluxing toluene af-
Functionalization of the pyran 3,4-double bond of 7 and forded the desired 19 (Chart 2). Nevertheless, the difficulties
12 in order to prepare corresponding (ꢀ)-cis-dihydrodiol di- encountered in both terms of yield and separation of isomers
esters was further envisaged, since this pharmacomodulation in the course of the preparation of 21 from 1,4,9-trimethyl-
had previously given outstanding results in terms of antitu- carbazole severely limited the practical scope of syntheses
mor potency in the acronycine series.10)
using N-methycarbazoles as starting materials. In contrast,
Accordingly, the racemic cis-diols 13 and 14 were conve- N-methylation of a preformed pyranocarbazole gave excel-
niently obtained by catalytic osmium tetroxide oxidation of 7 lent results, illustrated by the obtainment in 89% yield of 9-
and 12, using N-methylmorpholine N-oxide to regenerate the methoxy-2,2,5,6,11-pentamethyl-2,6-dihydropyrano[3,2-
oxidizing agent.18) Treatment of diols 13 and 14 with excess b]carbazole (22), when 12 was treated by methyl iodide and
acetic anhydride afforded the desired diacetates 15 and 16, sodium hydride in dimethylformamide (Chart 3).
respectively. Reaction of the same diols with N,Nꢁ-carbony-
diimidazole in 2-butanone under reflux afforded the corre- Pharmacology
sponding cyclic carbamates 17 and 18.
The study of the biological properties of the new
An alternative approach involving the Claisen rearrange- pyrano[3,2-b]carbazole derivatives was carried out in vitro
ment of an intermediate dimethylpropargylic ether was envi- on L1210 murine leukaemia cell line. The results (IC50) are

542
Vol. 52, No. 5
Chart 3
Table 1. Inhibition of L1210 Cell Proliferation
Compound
R1
R2
R3
R4
IC50 mM
1
5
0.1
10.4
0.6
6
7
H
OCH3
H
H
H
CH3
CH3
H
H
H
H
H
H
H
H
19.9
19.2
31.2
ꢂ50
12
19
22
OCH3
(ꢀ)-13
(ꢀ)-15
(ꢀ)-17
(ꢀ)-14
(ꢀ)-16
(ꢀ)-18
H
H
H
OCH3
OCH3
OCH3
H
H
H
H
H
H
OH
OH
25.3
16.8
25.2
20.0
13.6
16.6
OCOCH3
OCOO
OH
OCOCH3
OCOO
OCOCH3
OCOO
OH
OCOCH3
OCOO
reported in Table 1.
presence of a Lewis acid catalyst described here, appears as
The three (ꢀ)-cis-diol diesters 15, 16, and 18 were the the first efficient method giving a facile access to the
most active compounds.
pyrano[3,2-b]carbazole series.
Considering the structure–activity relationships, it appears
that in both 2,2,5,11-tetramethyl- and 6-methoxy-2,2,5,11-
Results and Discussion
From a chemical point of view, no synthetic method giving tetramethyl-2,6-dihydropyrano[3,2-b]carbazole series, func-
a general entry to the pyrano[3,2-b]carbazole system had tionalization of the pyran double bond to give (ꢀ)-cis-dihy-
been developed up to now. Consequently, only two com- dro-diol diesters results in an increase of the cytotoxic activ-
pounds deriving from this basic skeleton have been described ity. Nevertheless, none of these esters exhibit an antiprolifer-
in the literature to our knowledge. Pyrayaquinone-A is the ative activity within the same range of magnitude as the cor-
only natural product belonging to this series. It was first iso- responding acronycine derivatives.
lated from Murraya euchrestifolia HAYATA (Rutaceae) and
In contrast, N-methylation reduces significantly the biolog-
subsequently synthesized, although in low yield, by palla- ical activity in both chemical series. Indeed, 19 only exhibits
dium-assisted intramolecular ring closure of a suitable a marginal activity, whereas 22 was devoid significant cyto-
chromenylamino-1,4-benzoquinone.21) The other pyrano[3,2- toxic activity.
b]carbazole previously described, isoglycomaurin, was ob-
The effect of the substitution by a methoxy group at posi-
tained as a minor product in the course of the synthesis of tion 9 seems to be more discrete.
glycomaurin, through Claisen rearrangement of the corre-
However, methoxy compounds appear, as a whole, slightly
sponding dimethylpropargylic ether.22) Therefore, the con- more cytotoxic than their unsubstituted counterparts.
densation of a hydroxycarbazole with 3-methylbut-2-enal in

May 2004
543
(CDCl3) d: 1.48 (6H, s, (CH3)2-2), 2.45 (3H, s, CH3-5), 2.72 (3H, s, CH3-
11), 3.95 (3H, s, OCH3), 5.81 (1H, d, Jꢄ10 Hz, C3-H), 6.73 (1H, d,
Jꢄ10 Hz, C4-H), 7.03 (1H, dd, Jꢄ9, 2.5 Hz, C8-H), 7.32 (1H, d, Jꢄ9 Hz,
C7-H), 7.64 (1H, D2O exch, s, NH), 7.68 (1H, d, Jꢄ2.5 Hz, C10-H). 13C-
NMR (CDCl3) d: 12.0 (CH3-11), 12.1 (CH3-5), 27.2 (2C, (CH3)2-2), 56.2
(OCH3), 74.4 (C-2), 106.3 (C-10), 110.9 (C-7), 112.1 (C-4a), 113.6 (C-8),
117.0 (C-10b), 118.8 (C-5), 120.3 (C-4), 121.8 (C-11), 125.3 (C-10a), 131.9
Experimental
The melting points were determined on a Leica VM apparatus and are not
corrected. IR spectra (nmax in cmꢃ1) with KBr pellets were obtained on a
Nicolet FT-IR instrument. UV spectra (lmax in nm) were determined in
ꢂ99.5% (v/v) EtOH on a Beckman DU® 600 spectrophotometer. H-NMR
1
(d in ppm, J in Hz) and 13C-NMR spectra were recorded at 400 MHz and
100 MHz respectively, using a Bruker Avance 400 spectrometer. When nec-
essary, the signals were unambiguously assigned by 2D NMR techniques:
COSY, NOESY, HMQC, and HMBC. These experiments were performed
using standard Bruker microprograms. Mass spectra were recorded with a
Nermag R-10-10C spectrometer using chemical ionization technique
(reagent gas: NH3) (CI-MS) and with a ZQ 2000 Waters using a Zspray
(ESI-MS). Column chromatographies were conducted using silica gel 60
Merck (35—70 mm) with an overpressure of 200 mbars.
(C-3), 134.9 (C-5a), 135.5 (C-6a), 144.0 (C-11a), 153.4 (C-9). IR nmax cmꢃ1
:
3431, 3356, 3045, 2961, 2922, 1482, 1314, 1214, 1135. UV lmax nm (log e):
210 (4.15), 257.5 (4.52), 282 (3.84), 321.5 (4.31), 334 (4.44). CI-MS m/z:
307 ([MH]ꢅ). Anal. Calcd for C20H21NO2: C, 78.15; H, 6.89; N, 4.56.
Found: C, 78.02; H, 6.88; N, 4.57.
(3RS,4RS)-2,2,5,11-Tetramethyl-2,3,4,6-tetrahydropyrano[3,2-b]car-
bazole-3,4-diol (13) Compound 7 (135 mg, 0.49 mmol) was added to a so-
lution of osmium tetroxide (2.5% in 2-methyl-2-propanol) (0.31 ml) and N-
methylmorpholine N-oxide dihydrate (65 mg, 0.54 mmol) in a mixture of t-
BuOH/THF/H2O: 10/3/1, v/v/v (3 ml). The reaction mixture was stirred at
25 °C for 48 h. After addition of aqueous saturated NaHSO3 and stirring for
1 h, the mixture was extracted with CH2Cl2 (3ꢆ50 ml). The combined or-
ganic layers were dried over Na2SO4, filtered, and evaporated under reduced
pressure. Column chromatography of the crude product on silica gel
(CH2Cl2/MeOH: 99/1, v/v) gave 13 as a whitish amorphous product
(141 mg, 93%). 1H-NMR (CD3OD) d: 1.38 (3H, s, CH3-2), 1.45 (3H, s,
CH3-2), 2.58 (3H, s, CH3-5), 2.65 (3H, s, CH3-11), 3.75 (1H, d, Jꢄ5 Hz, C3-
H), 4.97 (1H, d, Jꢄ5 Hz, C4-H), 7.06 (1H, td, Jꢄ8, 1 Hz, C9-H), 7.30 (1H,
td, Jꢄ8, 1 Hz, C8-H), 7.42 (1H, dd, Jꢄ8, 1 Hz, C7-H), 8.10 (1H, dd, Jꢄ8,
1 Hz, C10-H). 13C-NMR (CD3OD) d: 12.7 (CH3-5), 13.4 (CH3-11), 21.4
(CH3-2), 27.2 (CH3-2), 66.1 (C-4), 74.6 (C-3), 77.0 (C-2), 111.6 (2C, C-7
and C-4a), 117.5 (C-10b), 118.6 (C-5), 119.0 (C-9), 121.3 (C-11), 123.5 (C-
10), 125.3 (C-10a), 125.9 (C-8), 135.8 (C-5a), 142.6 (C-6a), 144.8 (C-11a).
IR nmax cmꢃ1: 3523, 3450, 3362, 3282, 3050, 2971, 2918, 1504, 1458, 1392,
1301, 1149, 1102, 1007, 746. UV lmax nm (log e): 224 (4.42), 240 (4.57),
257.5 (4.31), 271 (4.09), 290 (4.08), 299 (4.32). CI-MS m/z: 312 ([MH]ꢅ).
Anal. Calcd for C19H21NO3: C, 73.29; H, 6.80; N, 4.50. Found: C, 73.12; H,
6.82; N, 4.51.
1,4-Dimethyl-9H-carbazol-3-ol (9) To an ice-cooled solution of 1,4-di-
methyl-9H-carbazole-3-carbaldehyde15) (7.8 g, 35.0 mmol) in methanol
(400 ml) were added 33% aqueous hydrogen peroxide (5 ml) and 95% sulfu-
ric acid (2.5 ml). After stirring for 15 min at 0 °C, the reaction mixture was
diluted with a mixture of crushed ice and water and neutralized with aque-
ous 1 M NaOH to give a whitish precipitate which was filtered off and
washed with water. Crystallization from MeOH gave 1,4-dimethyl-9H-car-
bazol-3-ol as whitish needles (6.5 g, 88%), mp 246—247 °C. 1H-NMR
(DMSO-d6) d: 2.45 (3H, s, CH3-1), 2.60 (3H, s, CH3-4), 6.77 (1H, s, C2-H),
7.07 (1H, t, Jꢄ8 Hz, C6-H), 7.30 (1H, t, Jꢄ8 Hz, C7-H), 7.44 (1H, d,
Jꢄ8 Hz, C8-H), 8.08 (1H, d, Jꢄ8 Hz, C5-H), 8.65 (1H, D2O exch, s, OH),
10.80 (1H, D2O exch, s, NH). 13C-NMR (DMSO-d6) d: 13.7 (CH3-4), 18.1
(CH3-1), 112.0 (C-8), 115.1 (C-4a), 116.8 (C-2), 118.3 (C-4), 119.0 (C-6),
122.8 (C-1), 123.3 (C-5), 124.9 (C-4b), 125.6 (C-7), 134.4 (C-9a), 141.7 (C-
8a), 148.7 (C-3). IR nmax cmꢃ1: 3493, 3319, 3056, 2959, 2933, 2917, 1457,
1306, 1090, 756, 735. UV lmax nm (log e): 222 (4.38), 238 (4.45), 252.5
(4.23), 267.5 (4.23), 295 (4.07). CI-MS m/z: 212 ([MH]ꢅ). Anal. Calcd for
C14H13NO: C, 79.59; H, 6.20; N, 6.63. Found: C, 79.31; H, 6.18; N, 6.61.
2,2,5,11-Tetramethyl-2,6-dihydropyrano[3,2-b]carbazole (7) A solu-
tion of 9 (5.0 g, 23.7 mmol), phenylboronic acid (4.3 g, 35.5 mmol), 3-
methylbut-2-enal (3.0 g, 35.5 mmol), glacial acetic acid (90 ml) in anhydrous
toluene (300 ml) was refluxed for 22 h under N2 in an apparatus fitted with a
Dean-Stark trap. After cooling, the mixture was concentrated in vacuo and
the residue was extracted with CH2Cl2 (4ꢆ100 ml). The combined organic
phase was washed successively with H2O (150 ml), 5% NaHCO3 solution
(200 ml), and brine (150 ml), dried over Na2SO4, filtered, and the solvent was
evaporated under reduced pressure. Column chromatography of the crude
product on silica gel (CH2Cl2/cyclohexane: 7/3, v/v) gave 7 as a pale yellow
crystalline product (1.76 g, 27%), mp 139—140 °C. 1H-NMR (CDCl3) d:
1.45 (3H, s, CH3-2), 1.48 (3H, s, CH3-2), 2.50 (3H, s, CH3-5), 2.75 (3H, s,
CH3-11), 5.81 (1H, d , Jꢄ10 Hz, C3-H), 6.74 (1H, d, Jꢄ10 Hz, C4-H), 7.18
(1H, ddd, Jꢄ8, 7, 1.5 Hz, C9-H), 7.37 (1H, td, Jꢄ7, 1 Hz, C8-H), 7.43 (1H,
dd, Jꢄ7, 1.5 Hz, C7-H), 7.78 (1H, D2O exch, s, NH), 8.17 (1H, dd, Jꢄ8,
1 Hz, C10-H). 13C-NMR (CDCl3) d: 12.0 (CH3-5), 12.2 (CH3-11), 27.2 (2C,
(CH3)2-2), 74.5 (C-2), 110.4 (C-7), 112.0 (C-4a), 117.2 (C-10b), 118.8 (C-
5), 118.9 (C-9), 120.3 (C-10), 121.7 (C-11), 122.6 (C-4), 124.7 (C-10a),
124.9 (C-8), 131.8 (C-3), 133.8 (C-5a), 140.2 (C-6a), 144.2 (C-11a). IR nmax
cmꢃ1: 3412, 3050, 2973, 2915, 1301, 1147, 1135, 746, 725. UV lmax nm
(log e): 258 (4.58), 313 (4.34), 325 (4.44). CI-MS m/z: 278 ([MH]ꢅ). Anal.
Calcd for C19H19NO: C, 82.28; H, 6.90; N, 5.05. Found: C, 82.28; H, 6.88;
N, 5.03.
(3RS,4RS)-9-Methoxy-2,2,5,11-tetramethyl-2,3,4,6-tetrahydropy-
rano[3,2-b]carbazole-3,4-diol (14) Oxidation of 12 (50 mg, 0.16 mmol)
according to the same procedure as that described for the preparation of 13,
1
afforded diol 14 as a whitish amorphous product (38 mg, 68%). H-NMR
(DMSO-d6) d: 1.30 (3H, s, CH3-2), 1.41 (3H, s, CH3-2), 2.52 (3H, s, CH3-
5), 2.60 (3H, s, CH3-11), 3.65 (1H, dd , Jꢄ7, 5 Hz, C3-H), 3.83 (3H, s,
OCH3), 4.78 (1H, D2O exch, d, Jꢄ5 Hz, C4-OH), 4.85 (1H, t, Jꢄ5 Hz, C4-
H), 5.00 (1H, D2O exch, d, Jꢄ5 Hz, C3-OH), 7.01 (1H, dd, Jꢄ9, 2 Hz, C8-
H), 7.37 (1H, d, Jꢄ9 Hz, C7-H), 7.59 (1H, d, Jꢄ2 Hz, C10-H), 10.60 (1H,
D2O exch, s, NH). 13C-NMR (DMSO-d6) d: 12.8 (CH3-11), 13.7 (CH3-5),
21.5 (CH3-2), 27.8 (CH3-2), 56.2 (OCH3), 64.5 (C-4), 73.2 (C-3), 76.3 (C-
2), 106.1 (C-10), 111.8 (C-7), 114.2 (C-8), 115.4 (C-10b), 118.2 (C-4a),
121.7 (C-11), 121.9 (C-5), 124.2 (C-10a), 135.5 (C-5a), 136.4 (C-6a), 143.4
(C-11a), 152.9 (C-9). IR nmax cmꢃ1: 3557, 3431, 3271, 2990, 2916, 2870,
1480, 1218, 1145, 1095. UV lmax nm (log e): 238 (4.52), 250 (4.35), 260.5
(4.08), 274.5 (3.68), 299 (4.09), 308.5 (4.34). ESI-MS m/z: 364 ([MꢅNa]ꢅ).
Anal. Calcd for C20H23NO4: C, 70.36; H, 6.79; N, 4.10. Found: C, 70.35; H,
6.80; N, 4.11.
(3RS,4RS)-2,2,5,11-Tetramethyl-2,3,4,6-tetrahydropyrano[3,2-b]car-
bazole-3,4-diyle Diacetate (15) Diol 13 (305 mg, 0.98 mmol) was acety-
lated with acetic anhydride in anhydrous pyridine under Ar at 25 °C for 18 h.
The mixture was evaporated under reduced pressure and the residue gave 15
6-Methoxy-1,4-dimethyl-9H-carbazol-3-ol (11) Oxidation of 6-
methoxy-1,4-dimethyl-9H-carbazole-3-carbaldehyde (10)15) (4.0 g, 15.8
mmol) under conditions identical with those described for the preparation of
9, afforded 11 as pale green yellow needles (3.5 g, 91%), mp 185—186 °C.
1H-NMR (DMSO-d6) d: 2.50 (3H, s, CH3-1), 2.70 (3H, s, CH3-4), 3.90 (3H,
s, OCH3), 6.85 (1H, s, C2-H), 7.04 (1H, dd, Jꢄ8, 1 Hz, C7-H), 7.43 (1H, d,
Jꢄ8 Hz, C8-H), 7.67 (1H, d, Jꢄ1 Hz, C5-H), 8.60 (1H, D2O exch, s, OH),
10.68 (1H, D2O exch, s, NH). 13C-NMR (CDCl3–CD3OD) d: 12.1 (CH3-4),
16.4 (CH3-1), 56.2 (OCH3), 106.4 (C-5), 110.9 (C-8), 113.5 (C-7), 115.1 (C-
4a), 115.7 (C-2), 117.3 (C-4), 122.3 (C-1), 124.9 (C-4b), 134.6 (C-9a),
135.5 (C-8a), 146.6 (C-3), 153.0 (C-6). IR nmax cmꢃ1: 3452, 3421, 3348,
2967, 2916, 1482, 1215, 1091, 1020. UV lmax nm (log e): 231 (4.47), 258
(3.05), 271.5 (2.84), 305.5 (3.18), 357 (2.57). CI-MS m/z: 242 ([MH]ꢅ).
Anal. Calcd for C15H15NO2: C, 74.67; H, 6.27; N, 5.81. Found: C, 74.48; H,
6.26; N, 5.79.
1
as a pale yellow amorphous product (349 mg, 90%). H-NMR (DMSO-d6)
d: 1.36 (3H, s, CH3-2), 1.37 (3H, s, CH3-2), 2.05 (3H, s, OCOCH3) 2.07
(3H, s, OCOCH3), 2.32 (3H, s, CH3-5), 2.63 (3H, s, CH3-11), 5.17 (1H, d,
Jꢄ5 Hz, C3-H), 6.33 (1H, d, Jꢄ5 Hz, C4-H), 7.12 (1H, t, Jꢄ8 Hz, C9-H),
7.37 (1H, t, Jꢄ8 Hz, C8-H), 7.48 (1H, d, Jꢄ8 Hz, C7-H), 8.12 (1H, d,
Jꢄ8 Hz, C10-H), 11.00 (1H, D2O exch, s, NH). 13C-NMR (DMSO-d6) d:
12.8 (CH3-11), 13.4 (CH3-5), 20.9 (OCOCH3), 21.1 (OCOCH3), 22.1 (CH3-
2), 26.4 (CH3-2), 64.6 (C-4), 72.2 (C-3), 74.7 (C-2), 111.5 (C-7), 115.9 (C-
4a), 116.7 (C-10a), 117.6 (C-5), 118.8 (C-9), 122.8 (C-11), 123.1 (C-10),
123.5 (C-10a), 126.0 (C-8), 134.7 (C-5a), 141.6 (C-6a), 144.0 (C-11a),
170.2 (COCH3), 170,6 (COCH3). IR nmax cmꢃ1: 3406, 3049, 2990, 2940,
1747, 1728, 1369, 1242, 1016, 741. UV lmax nm (log e): 240 (4.28), 247.5
(4.22), 258.5 (3.99), 270 (3.71), 290.5 (3.73), 299.5 (4.05). ESI-MS m/z:
418 ([MꢅNa]ꢅ), 336 ([MꢃOAc]ꢅ). Anal. Calcd for C23H25NO5: C, 69.86;
H, 6.37; N, 3.54. Found: C, 69.90; H, 6.40; N, 4.53.
9-Methoxy-2,2,5,11-tetramethyl-2,6-dihydropyrano[3,2-b]carbazole
(12) Synthesis of 12 from 11 (3.46 g, 14.4 mmol) carried out under the
same conditions as those applied for the preparation of compound 7, af-
forded 12 as a pale yellow amorphous product (1.27 g, 29%). 1H-NMR
(3RS,4RS)-9-Methoxy-2,2,5,11-tetramethyl-2,3,4,6-tetrahydropy-
rano[3,2-b]carbazole-3,4-diyle Diacetate (16) Diol 14 (80 mg, 0.23

544
Vol. 52, No. 5
mmol) was acetylated according to the same procedure as that used for the
dium hydride (50% dispersion in oil, 160 mg, 6.66 mmol) was added to a
preparation of compound 15, to afford the diacetate 16 as a pale yellow stirred solution of 20 (750 mg, 3.33 mmol) in dry toluene (20 ml) under Ar.
1
amorphous product (79.5 mg, 79%). H-NMR (DMSO-d6) d: 1.35 (3H, s, 3-Chloro-3-methylbut-1-yne20) (1.70 g, 16.6 mmol) and potassium iodide
CH3-2), 1.37 (3H, s, CH3-2), 2.05 (3H, s, OCOCH3), 2.07 (3H, s, OCOCH3),
(2.75 g, 16.6 mmol) were then added. The reaction mixture was refluxed for
2.30 (3H, s, CH3-5), 2.64 (3H, s, CH3-11), 3.84 (3H, s, OCH3), 5.16 (1H, d, 17 h, cooled, washed with water, dried over Na2SO4, filtered, and evaporated
Jꢄ5 Hz, C3-H), 6.32 (1H, d, Jꢄ5 Hz, C4-H), 7.05 (1H, dd, Jꢄ9, 2 Hz, C8-
H), 7.38 (1H, d, Jꢄ9 Hz, C7-H), 7.60 (1H, d, Jꢄ2 Hz, C10-H), 10.80 (1H,
D2O exch, s, NH). 13C-NMR (DMSO-d6) d: 12.7 (CH3-11), 13.3 (CH3-5),
20.9 (OCOCH3), 21.1 (OCOCH3), 22.1 (CH3-2), 26.4 (CH3-2), 56.2
(OCH3), 64.6 (C-4), 72.2 (C-3), 74.7 (C-2), 106.1 (C-10), 112.0 (C-7), 115.0
(C-8), 116.0 (C-4a), 116.6 (C-10b), 117.7 (C-5), 122.8 (C-11), 123.8 (C-
10a), 135.5 (C-5a), 136.6 (C-6a), 143.6 (C-11a), 153.1 (C-9), 170.2
under reduced pressure. Column chromatography of the crude product on
silica gel (cyclohexane/CH2Cl2: 6/4, v/v) gave 19 which crystallized from
the eluent as pale yellow needles (243 mg, 25%), mp 180—181 °C. 1H-NMR
(CDCl3) d: 1.48 (6H, s, (CH3)2-2), 2.74 (3H, s, CH3-5), 2.78 (3H, s, CH3-
11), 4.08 (3H, s, NCH3), 5.82 (1H, d, Jꢄ12 Hz, C3-H), 6.81 (1H, d,
Jꢄ12 Hz, C4-H), 7.18 (1H, dd, Jꢄ8, 7 Hz, C9-H), 7.35 (1H, d, Jꢄ8 Hz, C7-
H), 7.44 (1H, dd, Jꢄ8, 7 Hz, C8-H), 8.20 (1H, d, Jꢄ8 Hz, C10-H). 13C-
(COCH3), 170,6 (COCH3). IR nmax cmꢃ1: 3424, 2979, 2936, 2831, 1734, NMR (CDCl3) d: 12.2 (CH3-5), 14.3 (CH3-11), 27.1 (2C, (CH3)2-2), 33.4
1250, 1220, 1018. UV lmax nm (log e): 239.5 (4.68), 250.5 (4.52), 262.5 (NCH3), 74.1 (C-2), 108.4 (2C, C-7 and C-4a), 113.2 (C-10b), 117.4 (C-5),
(4.24), 273.5 (3.89), 298.5 (4.24), 308.5 (4.48). ESI-MS m/z: 425 118.4 (C-9), 119.6 (C-11), 120.6 (C-10), 122.5 (C-4), 123.9 (C-10a), 124.9
([MꢅNa]ꢅ). Anal. Calcd for C24H27NO6: C, 67.75; H, 6.40; N, 3.29. Found: (C-8), 131.8 (C-3), 135.5 (C-5a), 143.2 (C-6a), 144.1 (C-11a). IR nmax cmꢃ1
C, 67.73; H, 6.38; N, 3.29.
:
3433, 3048, 2968, 2920, 2862, 1467, 1300, 1133, 745, 715. UV lmax nm
(3aRS,12bRS)-4,4,6,12-Tetramethyl-3a,4,11,12b-tetrahydro-1,3-diox- (log e): 260.5 (4.53), 302 (3.94), 314 (4.77), 326.5 (4.37). CI-MS m/z: 292
olo[4ꢁ,5ꢁ:4,5]pyrano[3,2-b]carbazol-2-one (17) To a solution of 13 (103 ([MH]ꢅ). Anal. Calcd for C20H21NO: C, 82.44; H, 7.26; N, 4.81. Found: C,
mg, 0.33 mmol) in 2-butanone (5 ml) was added N,Nꢁ-carbonyldiimidazole 82.50; H, 7.27; N, 4.82.
(261 mg, 1.61 mmol). The reaction mixture was refluxed under Ar for 1.5 h
and after cooling, aqueous 5% NaHCO3 solution (8 ml) was added. The so-
9-Methoxy-2,2,5,6,11-pentamethyl-2,6-dihydropyrano[3,2-b]carbazole
(22) Sodium hydride (50% dispersion in oil, 10 mg, 0.4 mmol) was added
lution was extracted with CH2Cl2 (3ꢆ20 ml) and the combined organic lay- to an ice-cooled solution of 12 (25.0 mg, 0.08 mmol) in dry N,N-dimethyl-
ers were dried over Na2SO4, filtered, and evaporated under reduced pressure.
Column chromatography of the crude product on silica gel (cyclohexane/
formamide (5 ml). After stirring for 10 min at 0 °C, methyl iodide (17.0 mg,
0.12 mmol) was added and the reaction mixture was stirred for 5 min at 0 °C.
EtOAc: 5/5, v/v) gave 17 as a whitish amorphous product (48 mg, 43%). 1H- The reaction mixture was diluted with ice water, extracted with CH2Cl2
NMR (DMSO-d6) d: 1.13 (3H, s, CH3-2), 1.51 (3H, s, CH3-2), 2.56 (3H, s,
CH3-5), 2.63 (3H, s, CH3-11), 5.07 (1H, d, Jꢄ8 Hz, C3-H), 6.36 (1H, d,
Jꢄ8 Hz, C4-H), 7.13 (1H, t, Jꢄ8 Hz, C9-H), 7.39 (1H, t, Jꢄ8 Hz, C8-H),
(3ꢆ10 ml), and the combined organic layers were dried over Na2SO4, fil-
tered, and evaporated under reduced pressure. Column chromatography of
the crude product on silica gel (cyclohexane/EtOAc: 99/1, v/v) gave 22
7.52 (1H, d, Jꢄ8 Hz, C7-H), 8.13 (1H, d, Jꢄ8 Hz, C10-H), 10.14 (1H, D2O which crystallized from the eluent as pale yellow needles (23 mg, 89%), mp
exch, s, NH). 13C-NMR (DMSO-d6) d: 12.6 (CH3-11), 13.5 (CH3-5), 22.4
(CH3-2), 24.6 (CH3-2), 71.9 (C-4), 75.3 (C-2), 79.4 (C-3), 111.6 (C-7),
115.2 (C-4a), 117.8 (C-10b), 118.5 (C-5), 119.0 (C-9), 123.1 (C-10 and C-
11), 123.5 (C-10a), 126.2 (C-8), 134.9 (C-5a), 141.5 (C-6a), 143.7 (C-11a),
154.7 (CꢄO). IR nmax cmꢃ1: 3365, 3050, 2979, 2970, 1782, 1182, 1006,
166—167 °C. 1H-NMR (CDCl3) d: 1.48 (6H, s, (CH3)2-2), 2.70 (3H, s, CH3-
5), 2.75 (3H, s, CH3-11), 3.94 (3H, s, OCH3), 4.02 (3H, s, NCH3), 5.82 (1H,
d, Jꢄ10 Hz, C3-H), 6.79 (1H, d, Jꢄ10 Hz, C4-H), 7.08 (1H, dd, Jꢄ9,
2.5 Hz, C8-H), 7.24 (1H, d, Jꢄ9 Hz, C7-H), 7.71 (1H, d, Jꢄ2.5 Hz, C10-H).
13C-NMR (CDCl3) d: 12.1 (CH3-11), 14.3 (CH3-5), 27.1 (2C, (CH3)2-2),
744. UV lmax nm (log e): 241.5 (4.56), 248.5 (4.51), 258 (4.23), 270 (3.84), 33.7 (NCH3), 56.0 (OCH3), 74.1 (C-2), 106.4 (C-10), 108.9 (C-7), 113.3 (C-
290.5 (3.99), 300.5 (4.28). ESI-MS m/z: 360 ([MꢅNa]ꢅ). Anal. Calcd for 8), 113.5 (C-4a), 117.2 (C-10b), 119.8 (C-5), 120.5 (C-4), 122.5 (C-11),
C20H19NO4: C, 71.20; H, 5.68; N, 4.15. Found: C, 71.16; H, 5.65; N, 4.16.
124.2 (C-10a), 128.8 (C-5a), 130.8 (C-6a), 131.9 (C-3), 144.4 (C-11a),
(3aRS,12bRS)-8-methoxy-4,4,6,12-tetramethyl-3a,4,11,12b-tetrahy- 153.0 (C-9). IR nmax cmꢃ1: 3448, 3045, 2967, 2932, 2827, 1484, 1222, 1156.
dro-1,3-dioxolo[4ꢁ,5ꢁ:4,5]pyrano[3,2-b]carbazol-2-one (18) Compound UV lmax nm (e): 259.5 (4.53), 322 (4.33), 334.5 (4.44). CI-MS m/z: 322
18 was obtained from diol 14 (100 mg, 0.29 mmol) according to the same
procedure as that described for the preparation of 17, as a whitish amor- 78.52; H, 7.22; N, 4.35.
([MH]ꢅ). Anal. Calcd for C21H23NO2: C, 78.47; H, 7.21; N, 4.36. Found: C,
1
phous product (57 mg, 53%). H-NMR (DMSO-d6) d: 1.28 (3H, s, CH3-2),
1.51 (3H, s, CH3-2), 2.53 (3H, s, CH3-5), 2.63 (3H, s, CH3-11), 3.84 (3H, s,
OCH3), 5.06 (1H, d, Jꢄ8 Hz, C3-H), 6.35 (1H, d, Jꢄ8 Hz, C4-H), 7.07 (1H,
dd, Jꢄ9, 2 Hz, C8-H), 7.42 (1H, d, Jꢄ9 Hz, C7-H), 7.60 (1H, d, Jꢄ2 Hz,
Cytotoxicity Murine leukaemia L1210 cells from the American Type
Culture Collection (Rockville Pike, MD) were grown in RPMI medium
1640 supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 U/ml
penicillin, 100 mg/ml streptomycin and 10 mM HEPES buffer (pH 7.4). The
C10-H), 10.93 (1H, D2O exch, s, NH). 13C-NMR (DMSO-d6) d: 12.5 (CH3- cytotoxicity was measured by microculture tetrazolium assay essentially as
11), 13.4 (CH3-5), 22.5 (CH3-2), 24.6 (CH3-2), 56.2 (OCH3), 71.9 (C-4),
75.3 (C-2), 79.4 (C-3), 106.0 (C-10), 112.2 (C-7), 115.2 (C-4a), 115.4 (C-8),
117.7 (C-10b), 118.6 (C-5), 122.9 (C-11), 123.8 (C-10a), 135.7 (C-5a),
described.23) Cells were exposed for 48 h to nine graded concentrations in
triplicate of the test drug. Results are expressed as IC50 (mean, nꢄ3), which
is defined as the drug concentration inhibiting the absorbance by 50% with
respect to that of untreated cells.
136.5 (C-6a), 143.2 (C-11a), 153.2 (C-9), 154.7 (CꢄO). IR nmax cmꢃ1
:
3381, 2982, 2932, 2823, 1784, 1225, 1181. UV lmax nm (log e): 242.5
(4.71), 251.5 (4.58), 263.5 (4.24), 301 (4.26), 309.5 (4.47). ESI-MS m/z: References
390 ([MꢅNa]ꢅ). Anal. Calcd for C21H21NO5: C, 68.65; H, 5.76; N, 3.81.
Found: C, 68.64; H, 5.77; N, 3.81.
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were added 33% aqueous hydrogen peroxide (5 ml) and 95% sulfuric acid
(2.5 ml). After stirring for 15 min at 0 °C, the reaction mixture was diluted
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carbazol-3-ol as white needles (6.5 g, 88%), mp 208—209 °C. 1H-NMR
(DMSO-d6) d: 2.61 (3H, s, CH3-1), 2.72 (3H, s, CH3-4), 4.00 (3H, s, NCH3),
6.79 (1H, s, C2-H), 7.12 (1H, td, Jꢄ8, 1 Hz, C6-H), 7.39 (1H, td, Jꢄ8, 1 Hz,
C7-H), 7.48 (1H, dd, Jꢄ8, 1 Hz, C8-H), 8.11 (1H, dd, Jꢄ8, 1 Hz, C5-H),
8.73 (1H, D2O exch, s, OH). 13C-NMR (DMSO-d6) d: 13.5 (CH3-1), 20.8
(CH3-4), 33.1 (NCH3), 109.8 (C-8), 115.5 (C-4a), 118.8 (C-4), 119.0 (2C,
C-2 and C-6), 123.2 (C-5), 123.5 (C-1), 124.1 (C-4b), 125.8 (C-7), 134.5
(C-9a), 142.9 (C-8a), 148.6 (C-3). IR nmax cmꢃ1: 3445, 1473, 1394, 1299,
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