Synthesis and cytotoxic activity of pyranocoumarins of the seselin and xanthyletin series

Article abstract of DOI:10.1021/np9800295

The synthesis of known (3-6) and new (7-10 and 14-22) coumarins in the seselin and xanthyletin series is described. The cytotoxic activity of compounds 3-22 was carried out in vitro on L-1210 cells. The most active compounds were 9, 16, 18, and 20 in the seselin series and 10, 17, and 19 in the xanthyletin series. Structure-activity relationships are discussed.

Full text of DOI:10.1021/np9800295

982
J . Nat. Prod. 1998, 61, 982-986
Syn th esis a n d Cytotoxic Activity of P yr a n ocou m a r in s of th e Seselin a n d
Xa n th yletin Ser ies
Prokopios Magiatis,† Eleni Melliou,† Alexios-Leandros Skaltsounis,† Sofia Mitaku,*,† Ste´phane Le´once,‡
Pierre Renard,‡ Alain Pierre´,‡ and Ghanem Atassi‡
Laboratory of Pharmacognosy, University of Athens, Panepistimiopolis, Zografou, GR-15771 Athens, Greece, and
Institut de Recherche SERVIER, 11 rue des Moulineaux, 92150 Suresnes, France
Received J anuary 29, 1998
The synthesis of known (3-6) and new (7-10 and 14-22) coumarins in the seselin and
xanthyletin series is described. The cytotoxic activity of compounds 3-22 was carried out in
vitro on L-1210 cells. The most active compounds were 9, 16, 18, and 20 in the seselin series
and 10, 17, and 19 in the xanthyletin series. Structure-activity relationships are discussed.
Coumarins constitute a major class of widely distrib-
uted O-heterocyclic natural products exhibiting a broad
pharmacological profile,1 including anticancer activity.2
Naturally occurring pyranocoumarins show interesting
cytotoxic3-5 and antitumor-promoting activity.5 More-
over, seselin (1) and xanthyletin (2) have been reported
to act as DNA-damaging agents,6 and 3′,4′-dihydroxy-
3′,4′-dihydroseselin derivatives were found to display
significant cytotoxic activity against P-388 lymphocytic
leukemia.3,4 As part of our research program on anti-
tumor drugs from natural products, we synthesized
known and new derivatives in the seselin and xanth-
yletin series.
δ ) 1.93 (1H, dd, J ) 14.6, 4.8 Hz), which are in
agreement with those described for other natural and
hemisynthetic products containing the dimethylpyrano
ring system11,13 of 12 and 13. The 13C NMR spectrum
of 9 also showed a signal pattern of the dimethylpyrano
ring similar to that of 12. On the basis of these results,
the structure of praeroside V should be revised.
Treatment of bromohydrins 7 and 8 with excess Ac2O
or benzoic anhydride in pyridine afforded the corre-
sponding esters 14-17. In a similar way, alcohols 9 and
10 afforded the corresponding esters 18-21. Finally,
carbamate 22 was obtained by treatment of 7 with
excess chloroethyl isocyanate in pyridine.
The cytotoxic activity of compounds 3-22 was mea-
sured in vitro using L-1210 leukemia.14,15 The results
(IC50) are reported in Table 3. The most active com-
pounds were 9, 16, 18, and 20 in the seselin series and
10, 17, and 19 in the xanthyletin series. The perturba-
tion of the cell cycle induced by the most active
compounds was studied using the same cell line. Com-
pounds 16 and 18 induced a partial accumulation of
cells in the G1 phase of the cell cycle. In contrast, the
cell cycle was not modified by the most potent com-
pounds 9, 10, and 19. Considering the structure-
cytotoxic activity relationships, it appears (with one
exception) that compounds of the seselin series exhibit
significantly more potent activity than compounds of the
xanthyletin series. It appears also that compounds
bearing a hydroxy or acetoxy group at the 4′-position
and compounds bearing a bromo group at the 3′-position
and a benzoxy group at the 4′-position are the most
potent in inhibiting cell proliferation.
Resu lts a n d Discu ssion
The previously described7-9 diols 3 and 4 have been
obtained by catalytic osmium oxidation of 1 and 2,
respectively, using N-methylmorpholine N-oxide to re-
generate the oxidizing agent (Scheme 1). Treatment of
cis diols 3 and 4 with excess Ac2O in pyridine afforded
the corresponding diesters8-10 5 and 6.
(()-trans-4′-Hydroxy-3′-bromo-3′,4′-dihydroseselin (7)
and (()-trans-4′-hydroxy-3′-bromo-3′,4′-dihydroxanth-
yletin (8) were obtained by treatment of 1 and 2,
respectively, with NBS in aqueous THF solution.11 The
bromohydrins 7 and 8 were smoothly debrominated with
tributyltin hydride,11 to 9 and 10, respectively. It is
interesting to point out that 4′-hydroxy-3′,4′-dihydrose-
selin (9) has recently been described as praeroside V
aglycon,12 the enzymatic hydrolysis product of praero-
side V (11), a new natural coumarin glycoside isolated
from the roots of Peucedanum praeruptorum. However,
careful examination of 1H (Table 1) and 13C NMR (Table
2) spectra of compound 9, praeroside V (11), and
praeroside V aglycon showed significant differences,
particularly with respect to the signals of dihydropyran
ring.
Exp er im en ta l Section
Gen er a l Exp er im en ta l P r oced u r es. Spectra were
recorded on the following apparatus: MS, Nermag R10-
10C in desorption-chemical ionization, using NH3 as
reagent gas; NMR, Bruker AC200, 1H NMR (200
MHz),13C NMR (50 MHz) and a Bruker DRX400, 1H
NMR (400 MHz). Chemical shifts are given in δ with
TMS as an internal standard. Coupling constants (J )
are given in Hz. The signals of 1H and 13C spectra were
unambiguously assigned by using 2D NMR tech-
niques: 1H-1H-COSY, 13C-1H HETCOR, and HMBC.
These 2D experiments were performed using standard
The 1H NMR spectrum of 9 showed characteristic
signals at δ ) 5.02 (1H, ddd, J ) 4.8, 4.4, 3.0 Hz, dd, J
) 4.8, 3.0 Hz after addition of D2O), 5.30 (1H, d, J ) 4
Hz, exch with D2O), 2.05 (1H, dd, J ) 14.6, 3.0 Hz) and
* To whom correspondence should be addressed. Tel.: (301) 7284594.
Fax: (301) 7238297. E-mail: mls@galenos.dpharm.uoa.gr.
† University of Athens.
‡ Institut de Recherche SERVIER.
S0163-3864(98)00029-9 CCC: $15.00
© 1998 American Chemical Society and American Society of Pharmacognosy
Published on Web 07/11/1998

Synthesis and Cytotoxic Activity of Pyranocoumarins
J ournal of Natural Products, 1998, Vol. 61, No. 8 983
Sch em e 1a
a
Key: (i) OsO4, N-methylmorpholine N-oxide, t-BuOH, THF, H2O, rt; (ii) Ac2O, Py, rt; (iii) NBS, THF, H2O, 0 °C; (iv) (PhCO)2O, Py,
rt; (v) Bu3SnH, AIBN, toluene, 110 °C; (vi) ClCH2CH2CNO, Py, Me2CO, rt.
Ta ble 1. 1H NMR (DMSO/TMS, 200 MHz, δ (ppm), J (Hz)
atom
9
11 (praeroside V)a
praeroside V aglycona
3
4
5
6
6.26, d, J ) 9.5
7.97, d, J ) 9.5
7.51, d, J ) 8.5
6.77, d, J ) 8.5
2.05, dd, J ) 3.0, 14.6
6.23, d, J ) 9.5
7.94, d, J ) 9.5
7.43, d, J ) 8.5
6.88, d, J ) 8.5
3.01, d, J ) 6.1
(2H: 3′a, 3′b)
6.14, d, J ) 9.5
7.89, d, J ) 9.5
7.35, d, J ) 8.5
6.79, d, J ) 8.5
2.93, dd, J ) 2.4, 13.6
(2H: 3′a, 3′b)
3′a
3′b
4′
Me
Me
4′-OH
1.93, dd, J ) 4.8, 14.6
5.02, ddd, J ) 4.8, 4.4, 3.0
1.44 s
1.37 s
5.30, d, J ) 4.4
3.92, t, J ) 6.1
1.22 s
3.52, dd, J ) 2.4, 10.1
1.13, s (6H: Me1, Me2)
1.17 s
G-1: 4.09, d, J ) 7.6
a
From ref 12.
Ta ble 2. 13C NMR (DMSO/TMS, 50 MHz, δ ppm)
Ta ble 3. Cytotoxic Activitya
11a,b
(praeroside V)
praeroside V
compd
IC50, µM
atom
9
aglycona
3
4
5
6
7
>100
>100
>100
>100
>100
>100
0.9
2
3
4
5
6
7
8
9
10
2′
3′
4′
Me
Me
160.95
111.89
144.23
128.18
114.91
157.01
112.06
154.19
114.91
76.24
160.55
112.36
144.90
127.01
113.07
159.19
111.10
153.92
111.58
71.58
160.68
112.72
144.91
126.64
114.25
159.22
110.13
153.79
110.89
71.77
8
9
10
14
15
16
17
18
19
20
21
22
6.5
60.3
>100
10.3
36.5
24.7
5.0
40.18
59.02
26.45
27.95
24.84
85.04
25.44
25.94
24.96
76.94
25.16
25.65
30.9
90.1
67.2
a
b
From ref 12. Carbons of the glucose moiety are not men-
tioned.
a
Inhibition of L-1210 cell proliferation measured by the MMT
assay (mean of 2 values obtained in independent experiments).
Bruker microprograms. Column chromatography was
conducted using flash Si gel 60 Merck (40-63 µm), with
an overpressure of 300 mbar. Medium-pressure liquid
chromatography (MPLC) was performed with a Bu¨chi
model 688 apparatus on columns containing Si gel 60

984 J ournal of Natural Products, 1998, Vol. 61, No. 8
Magiatis et al.
Merck (20-40 µm). All new compounds gave satisfac-
tory combustion analyses (C, H, N, within (0.4% of
calculated values). The murine leukemia L-1210 cell
line was from the American Type Culture Collection
(Rockville Pike, MD).
J ) 5 Hz, OH-4′), 4.30 (1H, d, J ) 5 Hz, H-3′), 5.41 (1H,
t, J ) 5 Hz, H-4′), 6.29 (1H, d, J ) 9.5 Hz, H-3), 6.82
(1H, d, J ) 8.5 Hz, H-6), 7.36 (1H, d, J ) 8.5 Hz, H-5),
7.67 (1H, d, J ) 9.5 Hz, H-4);13C NMR (CDCl3, 50 MHz)
δ 24.27 (Me), 26.25 (Me), 57.53 (C-3′), 67.12 (C-4′), 78.85
(C-2′), 110.45 (C-8), 112.66 (C-3), 113.43 (C-10), 114.72
(C-6), 128.76 (C-5), 144.01 (C-4), 153.93 (C-9), 155.66
(C-7), 160.59 (C-2); MS-DCI m/z 344 (M + NH4)+, 342
(M + NH4)+.
(()-tr a n s-3′-Br om o-4′-h yd r oxy-3′,4′-d ih yd r oxa n -
th yletin (8). Treatment of 2 (145 mg, 0.64 mmol) under
conditions essentially the same as those described for
the preparation of 7 afforded compound 8 (160 mg,
83%): 1H NMR (DMSO, 200 MHz) δ 1.45 (3H, s, Me),
1.60 (3H, s, Me), 4.30 (1H, d, J ) 8 Hz, H-3′), 4.80 (1H,
t, J ) 8 Hz, H-4′), 6.30 (1H, d, J ) 9.5 Hz, H-3), 6.78
(1H, s, H-8), 7.75 (1H, s, H-5), 8.04 (1H, d, J ) 9.5 Hz,
H-4);13C NMR (DMSO, 50 MHz) δ 21.87 (Me), 27.59
(Me), 60.99 (C-3′), 68.05 (C-4′), 79.71 (C-2′), 103.22 (C-
8), 113.04 (C-3,7), 121.91 (C-6), 129.04 (C-5), 144.25 (C-
4), 154.31 (C-9), 154.90 (C-10), 160.11 (C-2); MS-DCI
m/z 344 (M + NH4)+, 342 (M + NH4)+.
(()-cis-3′,4′-Dih yd r oxy-3′,4′-d ih yd r oseselin (3).
Compound 3 was synthesized according to the published
procedure.10 1H and 13C NMR.10,16
(()-cis-3′,4′-Dih yd r oxy-3′,4′-d ih yd r oxa n t h ylet in
(4). Treatment of 2 under conditions essentially the
same as those described for the preparation of 3 afforded
4: 1H NMR (DMSO, 200 MHz) δ 1.25 (3H, s, Me), 1.40
(3H, s, Me), 3.63 (1H, t, J ) 4.2 Hz, H-3′), 4.75 (1H, dd,
J ) 7.9, 4.2 Hz, H-4′), 5.09 (1H, d, J ) 4.2 Hz, OH-3′),
5.34 (1H, d, J ) 7.9 Hz, OH-4′), 6.22 (1H, d, J ) 9.5 Hz,
H-3), 6.60 (1H, s, H-8), 7.75 (1H, s, H-5), 8.00 (1H, d, J
) 9.5 Hz, H-4); 13C NMR (DMSO, 50 MHz) δ 24.45 (Me),
24.88 (Me), 63.62 (C-4′), 69.86 (C-3′), 79.81 (C-2′), 102.41
(C-8), 112.08 (C-3,7), 122.06 (C-6), 128.51 (C-5), 144.60
(C-4), 154.17 (C-9), 156.37 (C-10), 160.49 (C-2); MS-DCI
m/z 280 (M + NH4)+.
(()-cis-3′,4′-Dia cetoxy-3′,4′-d ih yd r oseselin (5). To
a solution of 3 (20 mg, 0.08 mmol) in dry pyridine (1.5
mL) was added Ac2O (1.5 mL, 15 mmol). The reaction
mixture was stirred for 24 h at room temperature, and
then the reagents were removed under reduced pressure
(using a high-vacuum pump). The residue was com-
pound 5 (24 mg, 92%): 1H NMR (CDCl3, 200 MHz) δ
1.33 (3H, s, Me), 1.37 (3H, s, Me), 2.05 (3H, s, CH3CO),
2.08 (3H, s, CH3CO), 5.22 (1H, d, J ) 5 Hz, H-3′), 6.16
(1H, d, J ) 9.5 Hz, H-3), 6.45 (1H, d, J ) 5 Hz, H-4′),
6.73 (1H, d, J ) 8.5 Hz, H-6), 7.32 (1H, d, J ) 8.5 Hz,
H-5), 7.57 (1H, d, J ) 9.5 Hz, H-4); 13C NMR (CDCl3,
50 MHz) δ 20.46 (1-CH3CO, 2-CH3CO), 22.42 (Me),
24.57 (Me), 60.68 (C-4′), 69.95 (C-3′), 77.27 (C-2′), 106.51
(C-8), 112.39 (C-10), 112.86 (C-3), 114.25 (C-6), 129.17
(C-5), 143.31 (C-4), 153.67 (C-9), 156.37 (C-7), 159.79
(C-2), 169.83 (1-CH3CO, 2-CH3CO); MS-DCI m/z 364 (M
+ NH4)+.
(()-cis-3′,4′-Diacetoxy-3′,4′-dih ydr oxan th yletin (6).
Treatment of 4 (20 mg, 0.08 mmol) under conditions
essentially similar to those described for the preparation
of 5 afforded compound 6 (24 mg, 92%): 1H NMR
(CDCl3, 200 MHz) δ 1.41 (6H, s, Me), 2.04 (3H, s,
CH3CO), 2.13 (3H, s, CH3CO), 5.32 (1H, d, J ) 4 Hz,
H-3′), 6.16 (1H, d, J ) 4 Hz, H-4′), 6.24 (1H, d, J ) 9.5
Hz, H-3), 6.78 (1H,s, H-8), 7.28 (1H, s, H-5), 7.59 (1H,
d, J ) 9.5 Hz, H-4);13C NMR (CDCl3, 50 MHz) δ 20.60
(CH3CO), 20.86 (CH3CO), 24.37 (Me), 24.61 (Me), 64.83
(C-4′), 68.99 (C-3′), 77.99 (C-2′), 104.67 (C-8), 113.05 (C-
7), 113.73 (C-3), 115.31 (C-6), 127.26 (C-5), 143.06 (C-
4), 155.21 (C-9), 156.24 (C-10), 160.87 (C-2), 170.41
(CH3CO), 170.61 (CH3CO); MS-DCI m/z 364 (M +
NH4)+.
(()-tr a n s-3′-Br om o-4′-a cetoxy-3′,4′-d ih yd r osese-
lin (14). Treatment of 7 (31 mg, 0.09 mmol) under
conditions essentially the same as those described for
the preparation of 5 afforded 14 (33 mg, 95%): 1H NMR
(CDCl3, 200 MHz) δ 1.57 (6H, s, Me), 2.13 (3H, s,
CH3CO), 4.31 (1H, d, J ) 4.5 Hz, H-3′), 6.23 (1H, d, J )
9.5 Hz, H-3), 6.49 (1H, d, J ) 4.5 Hz, H-4′), 6.80 (1H, d,
J ) 8.5 Hz, H-6), 7.37 (1H, d, J ) 8.5 Hz, H-5), 7.60
(1H, d, J ) 9.5 Hz, H-4);13C NMR (CDCl3, 50 MHz) δ
20.79 (CH3CO), 25.36 (Me), 26.13 (Me), 53.96 (C-3′),
66.82 (C-4′), 77.93 (C-2′), 105.94 (C-8), 112.63 (C-10),
113.27 (C-3), 114.48 (C-6), 129.35 (C-5), 143.34 (C-4),
153.87 (C-9), 156.22 (C-7), 159.88 (C-2), 169.82 (CH3CO);
MS-DCI m/z 386 (M + NH4)+, 384 (M + NH4)+.
(()-tr a n s-3′-Br om o-4′-a cet oxy-3′,4′-d ih yd r oxa n -
th yletin (15). Treatment of 8 (20 mg, 0.06 mmol)
under conditions essentially the same as those described
for the preparation of 5 afforded 15 (21 mg, 95%): 1H
NMR (CDCl3, 200 MHz) δ 1.50 (3H, s, Me), 1.62 (3H, s,
Me), 2.21 (3H, s, CH3CO), 4.22 (1H, d, J ) 7.8 Hz, H-3′),
6.23 (1H, d, J ) 9.5 Hz, H-3), 6.26 (1H, d, J ) 7.8 Hz,
H-4′), 6.76 (1H, s, H-8), 7.23 (1H, s, H-5), 7.57 (1H, d, J
) 9.5 Hz, H-4); 13C NMR (CDCl3, 50 MHz) δ 21.06 (CH3-
CO), 22.26 (Me), 27.46 (Me), 54.87 (C-3′), 70.38 (C-4′),
79.65 (C-2′), 105.00 (C-8), 113.45 (C-7), 114.12 (C-3),
116.91 (C-6), 128.11 (C-5), 142.93 (C-4), 155.36 (C-9),
155.80 (C-10), 160.58 (C-2), 170.87 (CH3CO); MS-DCI
m/z 386 (M + NH4)+, 384 (M + NH4)+.
(()-tr a n s-3′-Br om o-4′-ben zoyloxy-3′,4′-dih ydr ose-
selin (16). To a solution of 7 (31 mg, 0.09 mmol) in
dry pyridine (1.5 mL) was added benzoic anhydride (62
mg, 0.29 mmol). The reaction mixture was stirred for
48 h at room temperature, and then the reagents were
removed under reduced pressure (using a high-vacuum
pump). The residue was extracted with EtOAc-
NaHCO3 (saturated), and the organic layer was col-
lected. The solvent was removed under reduced pres-
sure, and the remaining residue was purified by flash
chromatography on Si gel with cyclohexane-EtOAc (90:
10) to give compound 16 (31 mg, 76%): 1H NMR (CDCl3,
200 MHz) δ 1.67 (6H, s, Me), 4.52 (1H, d, J ) 3 Hz,
H-3′), 6.23 (1H, d, J ) 9.5 Hz, H-3), 6.78 (1H, d, J ) 3,
(()-tr a n s-3′-Br om o-4′-h yd r oxy-3′,4′-d ih yd r osese-
lin (7). To a solution of 1 (145 mg, 0.64 mmol) in THF
(5 mL) and H2O (5 mL) was added N-bromosuccinimide
(117 mg, 0.66 mmol). The reaction mixture was stirred
for 4 h at 0 °C, the reaction mixture was extracted with
NaCl (saturated)-Et2O, and the organic layer was
collected. The solvent was removed under reduced
pressure, and compound 7 was purified by crystalliza-
tion with Et2O (170 mg, 87%): 1H NMR (CDCl3, 200
MHz) δ 1.52 (3H, s, Me), 1.64 (3H, s, Me), 4.02 (1H, d,

Synthesis and Cytotoxic Activity of Pyranocoumarins
J ournal of Natural Products, 1998, Vol. 61, No. 8 985
H-4′), 6.90 (1H, d, J ) 8.5 Hz, H-6), 7.41 (2H, tt, J ) 7,
1.5 Hz, H-3′′, 5′′) 7.44 (1H, d, J ) 8.5 Hz, H-5), 7.55
(1H, tt, J ) 7, 1.5 Hz, H-4′′), 7.63 (1H, d, J ) 9.5 Hz,
H-4), 8.02 (2H, dt, J ) 7, 1.5 Hz, H-2′′, 6′′); 13C NMR
(CDCl3, 50 MHz) δ 24.76 (Me), 28.03 (Me), 53.48 (C-3′),
67.17 (C-4′), 77.00 (C-2′), 106.00 (C-8), 112.65 (C-10),
113.48 (C-3), 114.58 (C-6), 128.48 (C-3′′, 5′′), 129.50 (C-
1′′), 129.55 (C-2′′, 6′′), 129.85 (C-5), 133.36 (C-4′′), 143.18
(C-4), 154.37 (C-9), 156.38 (C-7), 159.77 (C-2), 165.05
(OCO); MS-DCI m/z 448 (M + NH4)+, 446 (M + NH4)+.
(()-tr a n s-3′-Br om o-4′-(ben zoyloxy)-3′,4′-dih ydr ox-
a n th yletin (17). Treatment of compound 8 (31 mg,
0.09 mmol) under conditions essentially the same as
those described for the preparation of 16 afforded
compound 17 (29 mg, 71%): 1H NMR (CDCl3, 200 MHz)
δ 1.58 (3H, s, Me), 1.70 (3H, s, Me), 4.41 (1H, d, J ) 7.5
Hz, H-3′), 6.23 (1H, d, J ) 9.5, H-3), 6.52 (1H, d, J )
7.5 Hz, H-4′), 6.82 (1H, s, H-8), 7.35 (1H, s, H-5), 7.46
(2H, tt, J ) 7, 1.5 Hz, H-3′′, 5′′), 7.54 (1H, d, J ) 9.5
Hz, H-4), 7.61 (1H, tt, J ) 7, 1.5 Hz, H-4′′), 8.07 (2H,
dt, J ) 7, 1.5 Hz, H-2′′, 6′′); 13C NMR (CDCl3, 50 MHz)
δ 22.99 (Me), 27.27 (Me), 54.73 (C-3′), 70.83 (C-4′), 79.43
(C-2′), 105.07 (C-8), 113.43 (C-7), 114.10 (C-3), 116.66
(C-6), 128.64 (C-5, 5′′, 3′′), 129.00 (C-1′′), 130.00 (C-2′′,
6′′), 133.82 (C-4′′), 143.00 (C-4), 155.43 (C-9), 155.95 (C-
10), 160.64 (C-2), 166.24 (OCO); MS-DCI m/z 448 (M +
NH4)+, 446 (M + NH4)+.
(()-4′-Hydr oxy-3′,4′-dih ydr oseselin (9). Compound
7 (90 mg,0.28 mmol) was dissolved in anhydrous toluene
(10 mL), and the solution was refluxed for 15 min under
argon. Then AIBN (azo-bis-2,2′(methyl-2-propionitrile))
(9 mg) was added, and after 5 min a solution of
tributyltin hydride (0.5 mL in 4 mL of toluene) was
added for 40 min. The reaction mixture was refluxed
for 1 h. Then the solvent was evaporated and the
residue was purified by flash chromatography on Si gel
with cyclohexane-EtOAc (70:30) to give compound 6 (55
mg, 78%): 1H NMR (DMSO, 200 MHz) Table 1; 13C
NMR (DMSO, 50 MHz) Table 2; MS-DCI m/z 264 (M +
NH4)+.
(C-4′), 75.78 (C-2′), 107.56 (C-8), 112.07 (C-10), 112.77
(C-3), 114.75 (C-6), 128.98 (C-5), 143.54 (C-4), 153.98
(C-9), 157.71 (C-7), 160.42 (C-2), 170.36 (CH3CO); MS-
DCI m/z 306 (M + NH4)+.
(()-4′-Acet oxy-3′,4′-d ih yd r oxa n t h ylet in
(19).
Treatment of 10 (14 mg, 0.06 mmol) under conditions
essentially the same as those described for the prepara-
tion of 5 afforded compound 19 (16 mg, 95%). 1H NMR
(CDCl3, 200 MHz) δ 1.40 (3H, s, Me), 1.45 (3H, s, Me),
1.99 (1H, dd, J ) 14, 6 Hz, H-3′a), 2.13 (3H, s, CH3CO),
2.23 (1H, dd, J ) 14, 6 Hz, H-3′b), 6.00 (1H, t, J ) 6
Hz, H-4′), 6.21 (1H, d, J ) 9.5 Hz, H-3), 6.73 (1H, s,
H-8), 7.35 (1H, s, H-5), 7.58 (1H, d, J ) 9.5 Hz, H-4);
13C NMR (CDCl3, 50 MHz) δ 21.33 (CH3CO), 27.15 (Me),
27.61 (Me), 38.34 (C-3′), 64.99 (C-4′), 77.20 (C-2′), 104.87
(C-8), 112.69 (C-7), 113.42 (C-3), 117.43 (C-6), 128.53
(C-5), 143.24 (C-4), 155.28 (C-9), 157.37 (C-10), 161.02
(C-2), 170.87 (CH3CO); MS-DCI m/z 306 (M + NH4)+.
(()-4′-Ben zoyloxy-3′,4′-dih ydr oseselin (20). Treat-
ment of 9 (31 mg, 0.13 mmol) under conditions es-
sentially the same as those described for the preparation
of 16 afforded compound 20 (31 mg, 68%): 1H NMR
(CDCl3, 200 MHz) δ 1.67 (6H, s, Me), 4.52 (2H, d, J ) 3
Hz, H-3′a, 3′b), 6.23 (1H, d, J ) 9.5 Hz, H-3), 6.78 (1H,
d, J ) 3 Hz, H-4′), 6.90 (1H, d, J ) 8.5 Hz, H-6), 7.41
(2H, tt, J ) 7, 1.5 Hz, H-3′′, 5′′) 7.44 (1H, d, J ) 8.5 Hz,
H-5), 7.55 (1H, tt, J ) 7, 1.5 Hz, H-4′′), 7.63 (1H, d, J )
9.5 Hz, H-4), 8.02 (2H,dt, J ) 7, 1.5 Hz, H-2′′, 6′′); 13C
NMR (CDCl3, 50 MHz) δ 25.60 (Me), 29.20 (Me), 38.35
(C-3′), 61.50 (C-4′), 75.57 (C-2′), 107.44 (C-8), 111.97 (C-
10), 112.93 (C-3), 114.75 (C-6), 128.33 (C-3′′, 5′′), 129.10
(C-5), 129.70 (C-2′′, 6′′), 130.09 (C-1′′), 132.95 (C-4′′),
143.32 (C-4), 154.44 (C-9), 157.79 (C-7), 160.21 (C-2),
165.59 (OCO); MS-DCI m/z 368 (M + NH4)+.
(()-4′-Ben zoyloxy-3′,4′-d ih yd r oxa n th yletin (21).
Treatment of 10 (31 mg, 0.13 mmol) under conditions
essentially the same as those described for the prepara-
tion of 16 afforded compound 21 (31 mg, 68%): 1H NMR
(CDCl3, 200 MHz) δ 1.48 (3H, s, Me), 1.58 (3H, s, Me),
2.15 (1H, dd, J ) 14, 6 Hz, H-3′a), 2.28 (1H, dd, J ) 14,
6 Hz, H-3′), 6.21 (1H, d, J ) 9.5 Hz, H-3), 6.29 (1H, t, J
) 6 Hz, H-4′), 6.78 (1H, s, H-8), 7.44 (1H, s, H-5), 7.46
(2H, tt, J ) 7, 1.5 Hz, H-3′′, 5′′), 7.56 (1H, tt, J ) 7, 1.5
Hz, H-4′′), 7.56 (1H, d, J ) 9.5 Hz, H-4), 8.03 (2H, dt, J
) 7, 1.5 Hz, H-2′′, 6′′); 13C NMR (CDCl3, 50 MHz) δ 27.49
(Me, Me), 38.55 (C-3′), 65.39 (C-4′), 77.00 (C-2′), 105.00
(C-8), 112.81 (C-7), 113.46 (C-3), 117.44 (C-6), 128.50
(C-1′′), 128.53 (C-5′′, 3′′), 128.96 (C-5), 129.70 (C-2′′, 6′′),
133.43 (C-4′′), 143.30 (C-4), 155.36 (C-9), 157.48 (C-10),
161.02 (C-2), 166.31 (OCO); MS-DCI m/z 368 (M +
NH4)+.
(()-4′-H yd r oxy-3′,4′-d ih yd r oxa n t h ylet in (10).
Treatment of compound 8 (90 mg, 0.28 mmol) under
conditions essentially the same as those described for
the preparation of 9 afforded 10 (50 mg, 71%): 1H NMR
(CDCl3, 200 MHz) δ 1.30 (3H, s, Me), 1.47 (3H, s, Me),
1.87 (1H, dd, J ) 14, 8 Hz, H-3′a), 2.20 (1H, dd, J ) 14,
6 Hz, H-3′b), 2.74 (1H, d, J ) 8 Hz,OH-4′), 4.85 (1H,
dd, J ) 14, 8 Hz, H-4′), 6.08 (1H, d, J ) 9.5 Hz, H-3),
6.60 (1H, s, H-8), 7.53 (1H, d, J ) 9.5 Hz, H-4), 7.57
(1H, s, H-5); 13C NMR (CDCl3, 50 MHz) δ 25.67 (Me),
29.21 (Me), 42.24 (C-3′), 62.81 (C-4′), 77.28 (C-2′), 104.24
(C-8), 112.40 (C-7), 112.64 (C-3), 122.59 (C-6), 127.35
(C-5), 143.67 (C-4), 154.69 (C-9), 156.97 (C-10), 161.68
(C-2); MS-DCI m/z 264 (M + NH4)+.
(()-4′-Acetoxy-3′,4′-d ih yd r oseselin (18). Treat-
ment of compound 9 (31 mg, 0.13 mmol) under condi-
tions essentially the same as those described for the
preparation of 5 afforded 18 (34 mg, 95%): 1H NMR
(CDCl3, 200 MHz) δ 1.40 (6H, s, Me), 2.05 (3H, s,
CH3CO), 2.15 (2H, d, J ) 5 Hz, H-3′a, 3′b), 6.20 (1H, d,
J ) 9.5 Hz, H-3), 6.25 (1H, d, J ) 5 Hz, H-4′), 6.75 (1H,
d, J ) 8.5 Hz, H-6), 7.31 (1H, d, J ) 8.5 Hz, H-5), 7.58
(1H, d, J ) 9.5 Hz, H-4);13C NMR (CDCl3, 50 MHz) δ
21.09 (CH3CO), 25.9 (Me), 28.48 (Me), 38.33 (C-3′), 61.21
(()-4′-(2-Ch lor oeth ylca r ba m a te)-3′,4′-d ih yd r ose-
selin (22). To a solution of 9 (30 mg, 0.12 mmol) in
dry pyridine (1 mL) and dry Me2CO was added 2-chlo-
roethyl isocyanate (0.5 mL, 5.8 mmol). The reaction
mixture was stirred for 24 h at room temperature, and
then the reagents were removed under reduced pressure
(using a high-vacuum pump). The residue was submit-
ted to flash chromatography on Si with cyclohexane-
EtOAc (95:5-50:50) to give compound 22 (18 mg,
50%): 1H NMR (CDCl3, 200 MHz) δ 1.42 (6H, s, Me),
2.14 (1H, dd, J ) 15, 5 Hz, H-3′b), 2.26 (1H, dd, J ) 15,
4 Hz, H-3′a), 3.60 (4H, m, H-6′,7′), 5.10 (1H, br, N-H),
6.20 (1H, d, J ) 9.5 Hz, H-3), 6.20 (1H, dd, J ) 5, 4 Hz,

986 J ournal of Natural Products, 1998, Vol. 61, No. 8
Magiatis et al.
H-4′), 6.74 (1H, d, J ) 8.5 Hz, H-6), 7.30 (1H, d, J ) 8.5
Hz, H-5), 7.57 (1H, d, J ) 9.5 Hz, H-4); 13C NMR (CDCl3,
50 MHz) δ 25.79 (Me), 28.57 (Me), 38.68 (C-3′), 42.88
(C-7′), 44.41 (C-6′), 61.94 (C-4′), 75.79 (C-2′), 107.85 (C-
8), 111.96 (C-10), 112.79 (C-3), 114.73 (C-6), 128.90 (C-
5), 143.44 (C-4), 153.50 (C-9), 155.68 (OCO), 157.60 (C-
7), 160.32 (C-2); MS-DCI m/z 369 (M + NH4)+.
Cell Cu ltu r e a n d Cytotoxicity. L-1210 cells were
cultivated in RPMI 1640 medium (Gibco) supplemented
with 10% fetal calf serum, 2 mM L-glutamine, 100 units/
mL penicillin, 100 µg/mL streptomycin, and 10 mM
HEPES buffer (pH ) 7.4). Cytotoxicity was measured
by the microculture tetrazolium assay.14 Cells were
exposed to graded concentrations of drug (nine serial
dilutions in triplicate) for 48 h. Results are expressed
as IC50, the concentration needed to reduce by 50% the
optical density of treated cells with respect to the optical
density of untreated controls.
Refer en ces a n d Notes
(1) Murray, R. D. H. In Progress in the Chemistry of Organic Natural
Products; Herz, W., Kirby, G. W., Steglich, W., Tamm, C., Eds.;
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(2) Rosskopf, F.; Kraus, J .; Franz, G. Pharmazie 1992, 47, 139-
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(3) Duh, C.-Y.; Wang, S.-K.; Wu, Y.-C. Phytochemistry 1991, 30,
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(4) Duh, C.-Y.; Wang, S.-K.; Wu, Y.-C. Phytochemistry 1992, 31,
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(5) Nishino, H.; Nishino, A.; Okuyama, T.; Shibata, S.; Kyoto, J .
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(6) Gunatilaka, L.; Kingston, D.; Wijeratne, K.; Bandara, R.; Hof-
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(7) Lemmich, J .; Lemmich, E.; Nielse, B. E. Acta Chem. Scand. 1966,
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(8) El-Antably, S. M.; Soine, T. O. J . Pharm. Sci. 1973, 62, 1643-
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(9) Gonzalez, A. G.; Barroso, J . T.; Lopez-Dorta, H.; Luis, J .;
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(10) Tembe, S. B.; Bhedi, D. N.; de Souza, N. J .; Rupp, R. H.
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(11) Mitaku, S.; Skaltsounis, A. L.; Tillequin, F.; Koch, M. Planta
Med. 1989, 55, 24-27.
(12) Takata, M.; Okuyama, T.; Shibata, S. Planta Med. 1988, 54,
323-327.
For the cell-cycle analysis, L-1210 cells (5 × 105 cells/
mL) were incubated for 21 h with various concentrations
of drugs. Cells were then fixed by 70% EtOH (v/v),
washed, and incubated with PBS containing 100 µg/mL
RNAse and 25 µg/mL propidium iodide for 30 min at
20 °C. For each sample, 10 000 cells were analyzed on
an ATC3000 flow cytometer (Bruker, Wissenbourg,
France).
(13) Mitaku, S.; Skaltsounis, A. L.; Tillequin, F.; Koch, M.; Pusset,
J .; Chauviere, G. Heterocycles 1987, 26, 2057-2063.
(14) Pierre, A.; Dunn, T. A.; Kraus-Berthier, L.; Leonce, S.; Saint-
Dizier, D.; Regnier, G.; Dhainaut, A.; Berlion, M.; Bizzari, J . P.;
Atassi, G. Invest. New Drug 1992, 10, 137-148.
(15) Leonce, S.; Perez, V.; Casabianca-Pignede, M. R.; Anstett, M.;
Bisagni, E.; Pierre, A.; Atassi, G. Invest. New Drug 1996, 14,
169-180.
(16) Mac´ıas, F. A.; Massanet, G. M.; Rodr´ıguez-Luis, F.; Salva´, J .;
Fronczek, F. R. Magn. Reson. Chem. 1989, 27, 653-658.
Ack n ow led gm en t. We are grateful to Dr. F. Libot
for recording the mass spectra.
NP9800295