Synthesis of "glycospirostanes" via ring-closing metathesis

Article abstract of DOI:10.1016/j.steroids.2009.09.001

Syntheses of "glycospirostanes" from 3β-hydroxy-23,24-dinor-5α-cholano-22,16-lactone and 3α-hydroxy-23,24-dinor-5β-cholano-22,16-lactone were performed. The key step of these syntheses was ring-closing metathesis of the corresponding C,O-diallyl derivativ

Full text of DOI:10.1016/j.steroids.2009.09.001

Steroids 74 (2009) 1073–1079
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Steroids
journal homepage: www.elsevier.com/locate/steroids
Synthesis of “glycospirostanes” via ring-closing metathesis
a,∗
a,∗∗
b
a
Dorota Czajkowska , Jacek W. Morzycki
, Rosa Santillan , Leszek Siergiejczyk
a
Institute of Chemistry, University of Białystok, Piłsudskiego 11/4, 15-443 Białystok, Poland
Departamento de Química, Centro de Investigación y de Estudios Avanzados del IPN, Apdo. Postal 14-740, 07000 México, D.F., Mexico
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 6 July 2009
Received in revised form 2 September 2009
Accepted 2 September 2009
Available online 11 September 2009
Syntheses of “glycospirostanes” from 3␤-hydroxy-23,24-dinor-5␣-cholano-22,16-lactone and 3␣-
hydroxy-23,24-dinor-5␤-cholano-22,16-lactone were performed. The key step of these syntheses was
ring-closing metathesis of the corresponding C,O-diallyl derivative. Further elaboration of the six-
membered ring F consisted of allylic hydroxylation with SeO2 followed by OsO4 dihydroxylation of
the C24–C25 double bond. The obtained final products proved to be simultaneously O- and C-l-
arabinopyranosides.
Keywords:
Glycospirostanes
©
2009 Elsevier Inc. All rights reserved.
Ring-closing metathesis
Polyhydroxylated compounds
1
. Introduction
announced the first synthesis of “glycospirostanes” [9] by step-
by-step transformations of steroidal sapogenins. In the present
communication a different strategy to synthesize such compounds
is demonstrated. The idea consists of removal of the entire ring F
of sapogenin and its reconstruction using a ring-closing metathe-
sis (RCM) as the key step. Such approach allows to get rid of the
superfluous 27-methyl group, which was retained in the previous
strategy.
Spirostane saponins constitute a large group of glycosides,
widely occurring in higher plants. They have found application in
pharmaceutical industry mainly as precursors of corticosteroids [1]
and other steroidal hormones due to the simple way of degra-
dation of the spiroketal moiety elaborated by Marker et al. [2].
The most common steroidal sapogenins used as raw materials for
industry are diosgenin from Dioscorea tokoro and hecogenin from
Agave sisalana. Many other saponins demonstrate significant role in
phytotherapy, especially those present in the roots of the ginseng
The starting compounds for the syntheses of glycospirostanes
were commercially available tigogenin (1a) and epismilagenin (1b)
(Scheme 1).
(
Panax ginseng) or soybeans (Glycine max) [3]. The other activities
of saponins, e.g. antiviral [4], antibacterial [5], antifungal [6], anti-
inflammatory [7], and antitumor [8], have also been reported in the
literature.
2. Experimental
2.1. General remarks
Biological activity and bioavailability of saponins depend both
on the structure of the aglycone and the sugar part. Introduc-
tion of a sugar moiety containing many hydroxyl groups into the
molecule decreases hydrophobic character of the steroidal agly-
cone and makes it more available for biochemical processes in the
organism. Therefore it could be expected that introduction of addi-
tional hydroxyl groups into the ring F, rendering it similar to sugar
pyranose rings, may also substantially change biological properties
of the sapogenin. Of course, molecules obtained in this way will be
O-glycosides and C-glycosides at the same time. We have recently
Melting points were determined on a Kofler apparatus of the
Boetius type. NMR spectra were recorded with a Bruker Avance
II 400 MHz spectrometer with TMS or residual solvent as internal
1
standard (only selected signals in the H NMR spectra are reported).
Infrared spectra were recorded on a Nicolet series II Magna-IR 550
FT-IR spectrometer in chloroform solutions or KBr pellets. Mass
spectra were obtained at 70 eV with an AMD-604 spectrometer.
The reaction products were isolated by column chromatography
performed on 70–230 or 230–400 mesh silica gel (J.T. Baker).
2
3
3
.2. Synthesis of
ˇ-hydroxy-23,24-dinor-5˛-cholano-22,16-lactone (2a) and
˛-hydroxy-23,24-dinor-5ˇ-cholano-22,16-lactone (2b)
∗
Corresponding author. Tel.: +48 85 7457604; fax: +48 85 7457581.
Corresponding author. Tel.: +48 85 7457585; fax: +48 85 7457581.
E-mail addresses: dorotac@uwb.edu.pl (D. Czajkowska), morzycki@uwb.edu.pl
∗
∗
To a mixture of AcOH (22 ml) and concentrated sulfuric
(
J.W. Morzycki).
acid (0.4 ml), iodine (220 mg, 0.87 mmol) was added and it was
0
039-128X/$ – see front matter © 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2009.09.001

1
074
D. Czajkowska et al. / Steroids 74 (2009) 1073–1079
Scheme 1.
stirred for 30 min at room temperature. Then 3 g (7.2 mmol)
of compound 1b was added and the mixture was stirred for
further 10 min. To the reaction mixture 8 ml (75 mmol) of per-
2.4. Synthesis of 3ˇ-t-butyldimethylsilyloxy-23,24-dinor-5˛-
cholano-22,16-lactone (4a) or
3˛-t-butyldimethylsilyloxy-23,24-dinor-5ˇ-cholano-22,16-
oxyacetic acid in CH COOH (39%) was added dropwise. After
lactone
3
1
4 h, the reaction was saponified with 5N NaOH to pH = 13 at
(4b)
room temperature and left overnight. The crude product was
filtered off and dried in vacuo. Flash chromatography (ethyl
acetate/hexane 2:8) provided 2b as white crystalline compound
To a solution of compound 2b (1.05 g; 3.03 mmol) in 20 ml of
dry DMF, 413 mg (6.07 mmol) of imidazole and 685 mg (4.54 mmol)
of t-butyldimethylsilyl chloride were added. The reaction mixture
was stirred at room temperature for 3 h, poured into saturated
aqueous solution of NaCl and extracted with CH Cl . The organic
(
2071 mg, 83%).
˛-Hydroxy-23,24-dinor-5ˇ-cholano-22,16-lactone (2b), yield:
3
◦
−1
8
3
4
2
3%; m.p. 212–214 C (ethyl acetate/hexane); IR, ꢀmax (cm ): 3605,
2
2
1
459, 2930, 2859, 1758, 1452, 1189, 1033; H NMR (CDCl ) ı (ppm):
layers were combined, dried over Na SO , and evaporated in
3
2 4
.98–4.93 (m, 1H), 3.68–3.63 (m, 1H), 2.58 (dq, J = 7.6, 0.8 Hz, 1H),
vacuo. The crude product was purified over silica gel with ethyl
acetate/hexane (7:93) elution to afford compound 4b (1255 mg,
90%).
.31–2.24 (m, 1H), 1.87 (d, J = 8.4 Hz, 2H), 1.32 (d, J = 7.6 Hz, 3H), 0.95
+
(
s, 3H), 0.74 (s, 3H); HRMS (EI) calcd. for C22H34O Na 369.2406,
3
found 369.2401.
ˇ-Hydroxy-23,24-dinor-5˛-cholano-22,16-lactone (2a) was
3˛-t-Butyldimethylsilyloxy-23,24-dinor-5ˇ-cholano-22,16-
◦
3
lactone (4b), yield: 90%; m.p. 127–129 C (ethyl acetate/hexane);
◦
IR, ꢀmax (cm ): 2932, 2858, 1758, 1182, 1092, 1068, 837; 1H NMR
−1
analogously obtained in 85% yield; m.p. 237–239 C [12] (ethyl
−1
acetate/hexane); IR, ꢀmax (cm ): 3605, 3484, 2934, 1757, 1452,
(CDCl ) ı (ppm): 4.97–4.92 (m, 1H), 3.63–3.57 (m, 1H), 2.58 (dq,
3
1
1
1
0
3
189, 1034, 1016, 960; 1H NMR (CDCl ) ı (ppm): 4.96–4.91 (m,
H), 3.61–3.56 (m, 1H), 2.57 (dq, J = 7.6, 0.9 Hz, 1H), 2.29–2.22 (m,
J = 7.7, 0.8 Hz, 1H), 2.30–2.23 (m, 1H), 1.86 (d, J = 7.0 Hz, 2H), 1.32
3
(d, J = 7.6 Hz, 3H), 0.93 (s, 3H), 0.90 (s, 9H), 0.73 (s, 3H), 0.07 (s, 6H);
+
H), 1.86 (d, J = 7.6 Hz, 1H), 1.31 (d, J = 7.6 Hz, 3H), 0.82 (s, 3H),
HRMS (EI) calcd. for C28H48O SiNa 483.3270, found 483.3291.
3
+
.74 (s, 3H); HRMS (EI) calcd. for C22H34O Na 369.2406, found
C28H48O Si: calcd. C, 72.99; H, 10.50. Found. C, 73.10; H, 10.49.
3
3
69.2421.
3ˇ-t-Butyldimethylsilyloxy-23,24-dinor-5˛-cholano-22,16-
lactone (4a) was obtained from 2a in an analogous way, yield: 69%;
◦
−1
m.p. 116–118 C (ethyl acetate/hexane); IR, ꢀmax (cm ): 2931,
2
4
2
855, 1757, 1183, 1092, 1059, 837; 1H NMR (CDCl ) ı (ppm):
.96–4.91 (m, 1H), 3.58–3.52 (m, 1H), 2.58 (dq, J = 7.6, 1.0 Hz, 1H),
.30–2.23 (m, 1H), 1.86 (d, J = 7.7 Hz, 1H), 1.32 (d, J = 7.6 Hz, 3H),
3
2.3. Synthesis of 27-nor-5˛-furost-25-en-3ˇ,22-diol (3a)
To a stirred solution of 2a (407 mg, 1.17 mmol) in dry THF
◦
0.89 (s, 9H), 0.82 (s, 3H), 0.75 (s, 3H), 0.06 (s, 6H); HRMS (EI) calcd.
(
10 ml) cooled to −23 C (CCl /acetone bath) 1.23 ml of vinyl-
4
+
for C28H48O SiNa 483.3270, found 483.3283. C28H48O Si: calcd.
3
3
magnesium bromide solution (1.23 mmol; 1 M in THF) was
added dropwise. Two further portions (1.23 ml each) of the
Grignard reagent were added after 3 h and 6 h. The reaction
C, 72.99; H, 10.50. Found. C, 73.12; H, 10.52.
mixture was stirred for another 1 h, quenched with saturated
◦
2.5. Synthesis of (20S,22S)-3ˇ-t-butyldimethylsilyloxy-26,27-
dinor-5˛-furost-24-en-22-ol (5a) and
aqueous solution of NH Cl at −23 C, and allowed to stand
4
at room temperature. The product was extracted three times
with diethyl ether. The combined organic extracts were washed
with saturated aqueous NaCl solution, dried over Na SO , and
(
20S,22S)-3˛-t-butyldimethylsilyloxy-26,27-dinor-5ˇ-furost-24-
en-22-ol
5b)
2
4
(
concentrated under reduced pressure. The residue was puri-
fied by flash column chromatography on silica gel with ethyl
acetate/hexane (25:75) elution to afford lactol 3a (255 mg,
To a solution of 4b (1255 mg, 2.72 mmol) in dry THF (15 ml)
◦
cooled to −78 C (dry ice/acetone bath) 2.9 ml of allylmagnesium
5
4%).
7-Nor-5˛-furost-25-en-3ˇ,22-diol (3a), yield 54%; m.p.
bromide solution (2.90 mmol; 1 M in diethyl ether) was added
2
◦
−1
dropwise. The reaction mixture was stirred for 15 min under argon,
1
3
05–107 C (ethyl acetate/hexane); IR, ꢀmax (cm ): 3598,
◦
1
quenched with saturated aqueous solution of NH Cl at −78 C and
445, 2932, 2850, 1641, 1452, 1381, 1279, 1033, 918;
H
4
allowed to stand at room temperature. The product was extracted
three times with diethyl ether. The combined organic extracts
were washed with saturated aqueous solution of NaCl, dried over
NMR (CDCl ) ı (ppm): 5.93–5.81 (m, 1H), 5.07 (ddt, J = 17.1,
3
1
.7, 1.7 Hz, 1H), 4.97 (ddt, J = 10.2, 1.5, 1.5 Hz, 1H), 4.63–4.56
(
m, 1H), 3.64–3.54 (m, 1H), 2.27–2.20 (m, 2H), 1.03 (d,
Na SO , and concentrated under reduced pressure. The crude prod-
J = 7.0 Hz, 3H), 0.83 (s, 3H), 0.79 (s, 3H), 0.69–0.62 (m, 1H);
2
4
+
uct was purified by flash column chromatography on silica gel
with ethyl acetate/hexane (6:94) elution to afford compound 5b
HRMS (EI) calcd. for C26H42O Na 425.3032, found 425.3026.
3
C26H42O : calcd. C, 77.56; H, 10.51. Found. C, 77.42; H,
3
(1151 mg, 84%).
9
.53.

D. Czajkowska et al. / Steroids 74 (2009) 1073–1079
1075
3
˛-t-Butyldimethylsilyloxy-26,27-dinor-5ˇ-furost-24-en-22-ol
Evaporation of the solvent afforded crude product, which was puri-
fied by flash column chromatography on a silica gel using ethyl
acetate/hexane (4:96) to afford spiroketal 7b (369 mg, 91%).
27-Nor-5ˇ-spirost-24-en-3˛-ol t-butyldimethylsilyl ether (7b),
◦
(
(
8
2
5b), yield 84%; m.p. 121–123 C (ethyl acetate/hexane); IR, ꢀmax
−
1
cm ): 3581, 3561, 2932, 2857, 1452, 1254, 1183, 1068, 869,
37; 1H NMR (CDCl ) ı (ppm): 5.98–5.87 (m, 1H), 5.21–5.16 (m,
3
◦
−1
H), 4.61–4.56 (dq, J = 7.4, 2.2 Hz, 1H), 3.63–3.54 (m, 1H), 2.47 (dd,
J = 13.6, 6.4 Hz, 1H), 2.39 (dd, J = 13.6, 8.4 Hz, 1H), 2.19 (s, 1H), 1.03
yield 91%; m.p. 164–166 C (ethyl acetate/hexane); IR, ꢀmax (cm ):
2931, 2857, 1451, 1373, 1254, 1077, 869, 837; 1H NMR (CDCl ) ı
3
(
d, J = 7.0 Hz, 3H), 0.92 (s, 3H), 0.90 (s, 9H), 0.77 (s, 3H), 0.06 (s, 6H);
(ppm): 5.76–5.75 (m, 2H), 4.54–4.47 (m, 1H), 4.25 (ddd, J = 15.8, 3.3,
3.3 Hz, 1H), 4.10–4.03 (m, 1H), 3.63–3.55 (m, 1H), 1.03 (d, J = 7.0 Hz,
3H), 0.93 (s, 3H), 0.90 (s, 9H), 0.78 (s, 3H), 0.07 (s, 6H); HRMS (EI)
+
HRMS (EI) calcd. for C31H54O SiNa 525.3740, found 525.3761.
C31H54O Si: calcd. C, 74.05; H, 10.82. Found. C, 74.09; H, 10.80.
3
3
+
3
ˇ-t-Butyldimethylsilyloxy-26,27-dinor-5˛-furost-24-en-22-
calcd. for C32H54O SiNa 537.3740, found 537.3761. C32H54O Si:
3
3
ol (5a) was analogously obtained from 4a in 82% yield; m.p.
calcd. C, 74.65; H, 10.57. Found. C, 74.55; H, 10.58.
◦
−1
1
2
17–120 C (ethyl acetate/hexane); IR, ꢀmax (cm ): 3582, 3559,
27-Nor-5˛-spirost-24-en-3ˇ-ol t-butyldimethylsilyl ether (7a)
was analogously obtained from ketal 6a in 91% yield; m.p.
930, 2855, 1382, 1253, 1093, 1059, 869, 837; 1H NMR (CDCl ) ı
3
◦
−1
(
ppm): 5.98–5.87 (m, 1H), 5.21–5.15 (m, 2H), 4.61–4.55 (m, 1H),
189–191 C (ethyl acetate/hexane); IR, ꢀmax (cm ): 2931, 2854,
1
3
8
3
5
.57–3.52 (m, 1H), 2.47 (dd, J = 13.6, 6.4 Hz, 1H), 2.39 (dd, J = 13.6,
1096, 1083, 1068, 869, 836; H NMR (CDCl ) ı (ppm): 5.75–5.75 (m,
3
.4 Hz, 1H), 2.19 (s, 1H), 1.02 (d, J = 7.0 Hz, 3H), 0.89 (s, 9H), 0.82 (s,
2H), 4.50 (m, 1H), 4.24 (ddd, J = 14.7, 3.9, 3.9 Hz, 1H), 4.09–4.02 (m,
1H), 3.56–3.53 (m, 1H), 1.02 (d, J = 7.0 Hz, 3H), 0.89 (s, 9H), 0.82 (s,
3H), 0.79 (s, 3H), 0.05 (s, 6H); HRMS (EI) calcd. for C32H54O SiNa
+
H), 0.78 (s, 3H), 0.05 (s, 6H); HRMS (EI) calcd. for C31H54O SiNa
3
+
25.3740, found 525.3757. C31H54O Si: calcd. C, 74.05; H, 10.82.
3
3
Found. C, 74.13; H, 10.84.
537.3740, found 537.3751. C32H54O Si: calcd. C, 74.65; H, 10.57.
3
Found. C, 74.50; H, 10.59.
2
2
.6. Synthesis of (22S)-3ˇ-t-butyldimethylsilyloxy-22-allyloxy-
6,27-dinor-5˛-furost-24-en-22-ol (6a) and
2.8. Synthesis of
(
22S)-3˛-t-butyldimethylsilyloxy-22-allyloxy-26,27-dinor-5ˇ-
furost-24-en-22-ol
6b)
(23R)-3ˇ-t-butyldimethylsilyloxy-27-nor-5˛-spirost-24-en-23-ol
(8a) and
(23R)-3˛-t-butyldimethylsilyloxy-27-nor-5ˇ-spirost-24-en-23-ol
(
(
8b)
To
a solution of 5b (1100 mg, 2.19 mmol) in anhydrous
CH Cl (15 ml), Tf NH (646 mg, 2.30 mmol), 2-propen-1-ol (1.5 ml,
To a solution of 7b (360 mg, 0.70 mmol) in dry dioxane (12 ml)
2
2
2
◦
2
1.95 mmol), and molecular sieves 4 Å were added at −78 C. The
39 mg of SeO2 (0.35 mmol) was added. The reaction mixture was
refluxed for 15 h, poured into saturated aqueous NaCl solution and
extracted with ethyl acetate. The organic layer was dried over
solution was stirred for 10 min before quenching with 0.5 ml Et N at
3
the same temperature. The mixture was warmed to room temper-
ature and filtered through Celite. After evaporation of the solvent
under reduced pressure, the resulting crude product was puri-
fied by flash column chromatography on silica gel with ethyl
acetate/hexane (1.5:98.5) elution to afford cyclic ketal 6b (831 mg,
Na SO4 and evaporated in vacuo. The crude product was purified
2
over silica gel with ethyl acetate/hexane (15:85) elution to afford
pure product 8b (104 mg, 28%).
(23R)-3˛-t-Butyldimethylsilyloxy-27-nor-5ˇ-spirost-24-en-23-ol
◦
7
0%).
22S)-3˛-t-Butyldimethylsilyloxy-22-allyloxy-26,27-dinor-
(8b), yield 28%; m.p. 209–211 C (ethyl acetate/hexane); IR, ꢀmax
−
1
(
(cm ): 3587, 3481, 2931, 2857, 1254, 1085, 1058, 1022, 868, 837;
◦
1
5
ˇ-furost-24-en-22-ol (6b), yield 70%; m.p. 123–125 C (ethyl
H NMR (CDCl ) ı (ppm): 6.01–5.93 (m, 2H), 4.57–4.50 (m, 1H),
3
−1
acetate/hexane); IR, ꢀmax (cm ): 2931, 2857, 1686, 1450, 1374,
4.56–4.50 (m, 1H), 4.13–4.07 (m, 1H), 3.75–3.70 (m, 1H), 3.63–3.54
1
1
254, 1070, 837; H NMR (CDCl ) ı (ppm): 5.98–5.86 (m, 1H),
(m, 1H), 1.17 (d, J = 7.0 Hz, 3H), 0.92 (s, 3H), 0.90 (s, 9H), 0.81 (s,
3
+
5
.84–5.77 (m, 1H), 5.35–5.26 (m, 1H), 5.13–5.04 (m, 3H), 4.43–4.37
3H), 0.06 (s, 6H); HRMS (EI) calcd. for C32H54O SiNa 553.3689,
4
(
5
m, 1H), 4.06 (dddd, J = 13.4, 4.8, 1.8, 1.8 Hz, 1H), 3.95 (dddd, J = 13.4,
.0, 1.7, 1.7 Hz, 1H), 3.63–3.52 (m, 1H), 2.52–2.48 (m, 2H), 1.03
found 553.3699. C32H54O Si: calcd. C, 72.40; H, 10.25. Found. C,
72.59; H, 10.27.
4
(
d, J = 6.9 Hz, 3H), 0.92 (s, 3H), 0.90 (s, 9H), 0.76 (s, 3H), 0.07 (6H);
(23R)-3ˇ-t-Butyldimethylsilyloxy-27-nor-5˛-spirost-24-en-23-ol
(8a) was obtained from 7a in an analogous way (yield 32%); m.p.
+
HRMS (EI) calcd. for C34H58O SiNa 565.4053, found 565.4062.
C34H58O Si: calcd. C, 75.22; H, 10.77. Found. C, 75.35; H, 10.80.
3
◦
−1
176–180 C (ethyl acetate/hexane); IR, ꢀmax (cm ): 3588, 2931,
3
(
22S)-3ˇ-t-Butyldimethylsilyloxy-22-allyloxy-26,27-dinor-5˛-
2854, 1093, 1060, 1022, 909, 836; 1H NMR (CDCl ) ı (ppm):
3
furost-24-en-22-ol (6a) was obtained in a similarly way from 5a
6.01–5.93 (m, 2H), 4.56–4.50 (m, 1H), 4.28–4.21 (m, 1H), 4.14–4.07
(m, 1H), 3.74–3.71 (m, 1H), 3.59–3.51 (m, 1H), 1.17 (d, J = 7.0 Hz,
3H), 0.89 (s, 9H), 0.82 (s, 6H), 0.06 (s, 6H); HRMS (EI) calcd. for
◦
in 75% yield; m.p. 140–142 C (ethyl acetate/hexane); IR, ꢀmax
−
1
(
(
cm ): 2930, 2856, 1097, 1058, 869, 836; 1H NMR (CDCl ) ı
3
+
ppm): 5.95–5.88 (m, 1H), 5.85–5.78 (m, 1H), 5.32–25 (m, 1H),
C32H54O SiNa 553.3689, found 553.3702. C32H54O Si: calcd. C,
4
4
5
1
1
3
5
.12–5.05 (m, 3H), 4.42–4.35 (m, 1H), 4.06 (dddd, J = 13.4, 4.6, 1.8,
.8 Hz, 1H), 3.94 (dddd, J = 13.4, 4.9, 1.7, 1.7 Hz, 1H), 3.58–3.52 (m,
72.40; H, 10.25. Found. C, 72.63; H, 10.29.
H), 2.52–2.47 (m, 2H), 1.03 (d, J = 7.0 Hz, 3H), 0.89 (s, 9H), 0.82 (s,
2.9. Synthesis of (23R,24S,25S)-3ˇ-t-butyldimethylsilyloxy-27-
nor-5˛-spirost-23,24,25-triol (9a) and
(23R,24S,25S)-3˛-t-butyldimethylsilyloxy-27-nor-5ˇ-spirost-
23,24,25-triol
+
H), 0.77 (s, 3H), 0.05 (6H); HRMS (EI) calcd. for C34H58O SiNa
3
65.4053, found 565.4074. C34H58O Si: calcd. C, 75.22; H, 10.77.
3
Found. C, 75.33; H, 10.81.
(
9b)
2.7. Synthesis of 27-nor-5˛-spirost-24-en-3ˇ-ol
t-butyldimethylsilyl ether (7a) and
To a solution of 8b (80 mg, 0.15 mmol) in benzene (20 ml)
and pyridine (0.3 ml), 1 M solution of OsO4 in benzene (0.16 ml;
.16 mmol) was added. The reaction mixture was stirred at room
27-nor-5ˇ-spirost-24-en-3˛-ol t-butyldimethylsilyl (7b)
0
To a stirred solution of ketal 6b (428 mg, 0.79 mmol) in dry
toluene (20 ml) Grubbs’ first generation catalyst (Fluka; 65 mg;
0 mol%) was added at room temperature. Reaction was stirred for
5 min under argon and quenched with 0.3 ml of ethylvinyl ether.
temperature for 20 h, quenched with 40% aqueous solution of
NaHSO3 and stirred for another 2 h. The solution was poured into
water and product was extracted with diethyl ether. The extract
1
1
was washed with water, dried over Na SO4 and evaporated in
2

1
076
D. Czajkowska et al. / Steroids 74 (2009) 1073–1079
vacuo. The crude product was purified on silica gel by flash col-
umn chromatography with ethyl acetate/hexane (50:50) elution to
afford triol 9b (75 mg, 88%).
473.2895. C26H42O : calcd. C, 69.30; H, 9.39. Found. C, 69.26; H,
9.41.
6
(
23R,24S,25S)-3˛-t-Butyldimethylsilyloxy-27-nor-5ˇ-spirost-
2
.11. Crystal structure determination of compound 10a
◦
2
3,24,25-triol (9b), yield 88%; m.p. 234–236 C (ethyl
acetate/hexane); IR, ꢀmax (cm ): 3566, 2930, 2857, 1472,
259, 1093, 1058, 868, 837; H NMR (CDCl ) ı (ppm): 4.58–4.50
m, 1H), 3.96–3.81 (m, 3H), 3.69 (dd, J = 10.6, 6.6 Hz, 1H), 3.56 (dd,
J = 11.2, 11.2 Hz, 1H), 3.63–3.54 (m, 1H), 2.37–2.30 (m, 1H), 1.11 (d,
J = 6.9 Hz, 3H), 0.93 (s, 3H), 0.90 (s, 9H), 0.79 (s, 3H), 0.06 (s, 6H);
HRMS (EI) calcd. for C32H56O SiNa 587.3744, found 587.3770.
C32H56O Si: calcd. C, 68.04; H, 9.99. Found. C, 68.21; H, 10.01.
−1
Crystals of compound 10a suitable for X-ray analysis were
1
1
(
3
obtained by slow evaporation of its CH Cl /MeOH solution at room
2 2
temperature. The X-ray measurements were performed at 293 K
on an Enraf–Nonius–Kappa CCD diffractometer with Mo K␣ radia-
tion, ꢁ = 0.71073 A. The structure was solved using Sir-92 program
[10] and refined using SHELXL-97 [11]. All non-hydrogen atoms
were refined anisotropically, the H atoms were placed at calculated
positions using a riding model.
+
6
6
(
23R,24S,25S)-3ˇ-t-Butyldimethylsilyloxy-27-nor-5˛-spirost-
2
9
3
3,24,25-triol (9a) was obtained from 8a in an analogous way, yield
(23R,24S,25S)-27-nor-5␣-spirost-3␤,23,24,25-tetraol
(10a)
◦
−1
3%; m.p. 239–243 C (ethyl acetate/hexane); IR, ꢀmax (cm ):
crystallized in a monoclinic system with space group P21 and Z = 4
and 2 independent molecules in the unit cell. The X-ray structure
proved the configuration of the newly formed stereogenic centers,
i.e. the hydroxyl groups at the 23 and 24 positions are axial and
483, 2931, 2855, 1097, 1071, 870, 836, 774, 669; 1H NMR (CDCl3)
ı (ppm): 4.57–4.49 (m, 1H), 3.96–3.80 (m, 3H), 3.67 (dd, J = 11.2,
.9 Hz, 1H), 3.55 (dd, J = 11.2, 11.2 Hz, 1H), 3.60–3.49 (m, 1H),
.37–2.30 (m, 1H), 1.10 (d, J = 6.9 Hz, 3H), 0.88 (s, 9H), 0.81 (s,
H), 0.79 (s, 3H), 0.05 (s, 6H); HRMS (EI) calcd. for C32H56O SiNa
87.3744, found 587.3759. C32H56O Si: calcd. C, 68.04; H, 9.99; Si,
.97. Found. C, 68.25; H, 10.02; Si, 4.98.
5
2
3
5
4
2
5 is equatorial. The A–C and F rings assume a chair conformation,
+
6
the five-membered rings D and E are envelopes.
6
Crystallographic data have been deposited at the Cambridge
Crystallographic Center as supplementary material No. 736578.
Copy of the data can be obtained free of charge to CCDC, 12 Union
Road, Cambridge CB2 1EZ, UK. E-mail: deposit@ccdc.cam.ac.uk.
2
.10. Synthesis of
(
(
23R,24S,25S)-27-nor-5˛-spirost-3ˇ,23,24,25-tetraol (10a) and
23R,24S,25S)-27-nor-5ˇ-spirost-3˛,23,24,25-tetraol (10b)
3. Results and discussion
A solution of triol 9b (75 mg, 0.13 mmol) in THF (10 ml) was
The starting sapogenins 1a and 1b were subjected to par-
tial degradation to lactones 2a and 2b according to the recently
described procedure [12]. Thus, sapogenins were treated with per-
treated with 0.9 ml of tetrabutylammonium fluoride solution (1 M
in THF, 0.9 mmol) and stirred for 4 h at room temperature. Then, the
mixture was poured into water and extracted with ethyl acetate
oxyacetic acid in the presence of catalytic amounts of H SO4 and
2
The extract was dried over Na SO4 and evaporated in vacuo. The
crude product was purified by flash column chromatography on
silica gel with ethyl acetate/hexane (90:10) elution to afford tetraol
iodine (Scheme 2). This procedure proved superior to other meth-
ods of degradation and afforded the corresponding lactones in
83–85% yields.
2
1
0b (57 mg, 94%).
23R,24S,25S)-27-Nor-5ˇ-spirost-3˛,23,24,25-tetraol (10b), yield
Two alternative routes were designed (Scheme 3) to rebuilt the
pyranose ring F devoid of the methyl group at C25 and with the
double bond enabling introduction of hydroxyl groups to the ring.
The first option (route A) was to attach the vinyl group to the lac-
tone carbonyl group (C22) followed by reaction with 3-buten-1-ol
(
◦ −1
4%; m.p. 281–283 C (CH Cl /MeOH); IR, ꢀmax (cm ): 3422, 2929,
2 2
065, 1042, 966, 866; H NMR (CD OD) ı (ppm): 4.57–4.50 (m, 1H),
.92 (ddd, J = 11.1, 5.6, 3.4 Hz, 1H), 3.76 (ddd, J = 3.4, 3.4, 1.1 Hz, 1H),
.68 (dd, J = 11.1, 10.8 Hz, 1H), 3.67 (d, J = 3.4 Hz, 1H), 3.59–3.50 (m,
H), 3.47 (ddd, J = 10.8, 5.6, 1.1 Hz, 1H), 2.38–2.30 (m, 1H), 2.07–1.99
9
1
3
3
1
1
3
promoted by Tf NH as a Brønsted acid [13], and the second one
2
(route B)—addition of the three-carbon atom fragment (allyl group)
and ketalization of resulting lactol with 2-propen-1-ol under the
same reaction conditions. The following RCM reactions would form
dihydropyran rings with the double bonds C23–C24 or C24–C25,
respectively.
(
m, 1H), 1.09 (d, J = 7.0 Hz, 3H), 0.97 (s, 3H), 0.83 (s, 3H); HRMS (EI)
+
calcd. for C26H42O Na 473.2879, found 473.2891. C26H42O : calcd.
C, 69.30; H, 9.39. Found. C, 69.41; H, 9.41.
6
6
(
23R,24S,25S)-27-Nor-5˛-spirost-3ˇ,23,24,25-tetraol (10a) was
◦
analogously obtained from triol 9a in 93% yield; m.p. 298–302 C
Unfortunately, the route A did not provide the desired prod-
uct. The reaction of the lactone 2a with vinylmagnesium bromide
CH Cl /MeOH); IR, ꢀmax (cm ): 3438, 2926, 1065, 963, 867; 1
−1
H
(
2 2
◦
NMR (CD OD) ı (ppm): 4.56–4.49 (m, 1H), 3.92 (ddd, J = 11.2, 5.6,
in THF at −78 C led to the double addition of the vinyl group.
3
3
1
.4 Hz, 1H), 3.76 (ddd, J = 3.4, 3.4, 1.1 Hz, 1H), 3.68 (dd, J = 11.2,
0.8 Hz, 1H), 3.67 (d, J = 3.4 Hz, 1H), 3.55–3.43 (m, 1H), 3.47
Presumably, the initially formed intermediate I (Scheme 4) prefer-
entially exists in an open-chain form II. The 1,4-addition of another
molecule of the Grignard reagent to this form afforded intermediate
III, which in turn underwent cyclization to the final product—lactol
3a (a single stereoisomer at C22 was formed but configuration at
(
ddd, J = 10.8, 5.6, 1.1 Hz, 1H), 2.37–2.31 (m, 1H), 2.05–1.99 (m,
1
H), 1.08 (d, J = 7.0 Hz, 3H), 0.85 (s, 3H), 0.82 (s, 3H), 0.72–0.65
+
(
m, 1H); HRMS (EI) calcd. for C26H42O Na 473.2879, found
6
Scheme 2.

D. Czajkowska et al. / Steroids 74 (2009) 1073–1079
1077
Scheme 3.
Scheme 4.
this chiral center was not established). In this case, the cyclic lac-
tol form is apparently more thermodynamically stable than the
hydroxy-ketone form, which is not even detectable by IR (no car-
bonyl band). In spite of numerous attempts to stop the reaction
after addition of only one molecule of Grignard reagent (slow addi-
tion of the reagent, low temperature, reaction in the presence of
trimethylsilyl chloride), the desired product could not be obtained.
Also protection of a 3␤-hydroxyl group as a t-butyldimethylsilyl
ether (compound 4a) did not change the reaction course. Therefore
an alternative route B was subsequently explored.
The 3-OTBDMS-lactones (4a or 4b) were treated with allylmag-
◦
nesium bromide in THF at −78 C. The reactions were completed
Scheme 5.

1
078
D. Czajkowska et al. / Steroids 74 (2009) 1073–1079
Fig. 1. X-ray structure of (23R,24S,25S)-27-nor-5␣-spirost-3␤,23,24,25-tetraol (10a).
within 15 min and the corresponding lactols (5a or 5b) were
obtained in 82% or 84% yield, respectively (Scheme 5). Subse-
revealed by X-ray analysis. Typical diequatorial coupling constant
(J23,24 = 3.4 Hz) confirms axial arrangement of both 23-OH and 24-
OH, whereas the 25-OH group occupies the equatorial position as
it follows from the diaxial coupling constant J25,26ax = 11.1–11.2 Hz.
Moreover, long range coupling (J24,26eq = 1.1 Hz) indicates W-like
shape of the 24H–24C–25C–26C–26Heq moiety. The structures of
10a and 10b are also consistent with their C NMR spectra. The
full assignment of signals in the 13C NMR spectra for all compounds
described throughout the paper is given in Table 1 [14,15].
Using carbohydrate nomenclature compounds 10a and 10b are
l-arabinopyranosides.
quent reactions with 2-propen-1-ol catalyzed by Tf NH yielded
2
the expected ketals (6a or 6b), which were subjected to the RCM
reactions with Grubbs’ first generation catalyst. The reactions pro-
ceeded fast affording dihydropyrans (7a or 7b) in excellent yields
13
(
91%).
The next step—allylic hydroxylation with SeO2 proved to be
more demanding. The reaction was optimized in terms of amount
of reagent, solvent (dioxane or t-butanol) used, temperature, and
time. The best results were achieved in refluxing dioxane with
0
.5 equiv. of SeO ; 15 h reaction afforded 8a (or 8b) in about 30%
The glycospirostane 10a was tested on various malignant cancer
cell lines. However, it appeared to be not active at concentra-
tion below 50 ␮mol/L against tumor cells of MCF7 and HELA,
only against CEM compound 10a was slightly cytotoxic (IC50
36.9 ␮mol/L). Further biological studies are in progress.
2
yield with recovery of 40% of the starting material. The reaction was
highly regio- and stereoselective—the hydroxyl group was intro-
duced at C23 from the less hindered ␤-side (23R configuration
was found). However, reactions with SeO2 became less clean on
prolonging the reaction time.
Then, the allylic alcohols (8a or 8b) were subjected to dihy-
droxylation with OsO4 in pyridine/benzene mixture at room
temperature. The reaction afforded products 9a or 9b with hydroxyl
groups on the opposite side to the 23-OH group already present
in ring F of the starting compound 8a or 8b. Final cleavage of
the TBDMS group yielded crystalline (23R,24S,25S)-5␣-spirostane-
3
␤,23,24,25-tetraol (10a). The configurations at all newly formed
stereogenic centers in 10a was unequivocally proved by an X-ray
analysis (Fig. 1). The configurations of the ring F hydroxyl groups in
compounds 9b and 10b belonging to the 3␣-OH-5␤-series, which
do not form crystals suitable for X-ray measurements, were found
1
13
by H and C NMR analysis to be the same as those in the 3␤-OH-
␣-series. The chemical shift data and coupling constants patterns
5
for the ring F protons of 10a and 10b proved to be almost iden-
Fig. 2. Comparison of 1H NMR chemical shifts and coupling constants of the F ring
tical (Fig. 2). The NMR spectral data reflect structural features
protons in the spectra of 10a and 10b.

D. Czajkowska et al. / Steroids 74 (2009) 1073–1079
1079
Table 1
1
3
a
C
N
M
R
c
h
e
m
i
c
a
l
s
h
i
f
t
s
(
ı
;
p
p
m
)
i
n
C
D
C
l
3
f
o
r
a
l
l
c
o
m
p
o
u
n
d
s
d
e
s
c
r
i
b
e
d
t
h
r
o
u
g
h
o
u
t
t
h
e
p
a
p
e
r
.
C no.
2a
36.9
2b
30.4
3a
37.0
4a
37.1
4b
31.0
5a
37.2
5b
31.0
6a
37.2
6b
31.0
7a
37.2
7b
31.0
8a
37.2
8b
31.0
9a
37.2
9b
31.0
10ab
10bb
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
38.2
32.1
71.8
38.9
46.2
29.9
33.4
36.6
55.8
36.7
22.1
40.8
42.1
57.7
32.9
84.1
64.5
16.8
12.8
41.9
16.9
110.8
74.2
73.5
64.5
60.1
31.1
36.5
72.4
37.2
43.5
28.2
27.8
37.0
41.9
35.8
21.6
40.9
42.2
57.8
32.9
84.2
64.5
16.7
23.1
41.9
16.8
110.8
74.2
73.5
64.6
60.1
31.4
71.1
38.1
44.8
28.4
32.1
34.9
54.4
35.6
20.5
38.3
41.7
54.6
33.0
82.8
59.0
13.8
12.3
36.0
17.9
181.3
35.3
71.6
36.3
41.8
26.9
26.6
35.2
40.6
34.7
20.1
38.5
41.8
54.6
33.0
82.8
59.1
13.8
23.3
36.1
18.0
181.3
31.5
71.2
38.2
44.8
28.6
32.2
35.1
54.3
35.6
21.0
39.9
40.9
56.2
31.7
81.4
62.7
16.4
12.3
39.9
15.4
31.9
72.0
38.4
45.0
28.5
32.2
34.9
54.5
35.6
20.5
38.6
41.7
54.7
33.0
82.8
59.1
13.9
12.4
36.1
18.0
35.5
72.6
36.9
42.1
27.0
26.6
35.2
40.4
34.7
20.1
38.5
41.8
54.6
33.0
82.9
59.1
13.8
23.3
36.1
18.0
31.9
72.1
38.7
45.0
28.7
32.3
35.1
54.5
35.6
21.0
40.0
40.9
56.3
31.7
81.6
62.6
16.5
12.4
40.2
15.3
35.6
72.7
36.9
42.2
27.2
26.6
35.5
40.3
34.7
20.5
40.0
41.0
56.2
31.8
81.6
62.6
16.4
23.4
40.1
15.2
31.9
72.1
38.7
45.0
28.7
32.3
35.1
54.5
35.6
21.0
40.0
41.0
56.3
31.6
81.5
63.0
16.5
12.4
40.0
15.1
35.6
72.7
36.9
42.2
27.2
26.6
35.4
40.3
34.7
20.5
40.0
41.1
56.2
31.6
81.5
63.1
16.4
23.4
40.0
15.1
31.8
72.1
38.7
45.0
28.6
32.3
35.1
54.4
35.6
21.0
40.0
40.7
56.4
31.9
81.8
62.0
16.4
12.4
41.6
14.3
35.6
72.7
36.9
42.2
27.2
26.6
35.5
40.3
34.7
20.6
40.1
40.7
56.3
31.8
81.8
62.0
16.4
23.4
41.6
14.3
31.9
72.1
38.7
45.0
28.7
32.3
35.2
54.5
35.6
20.9
39.7
41.1
56.5
32.1
83.1
64.1
16.2
12.4
40.1
16.5
35.6
72.7
36.9
42.2
27.2
26.6
35.6
40.3
34.7
20.5
39.8
41.1
56.4
32.1
83.2
64.1
16.5
23.4
40.1
16.2
31.9
72.1
38.6
45.0
28.6
32.2
35.2
54.4
35.6
20.9
39.5
41.0
56.5
31.8
83.3
62.9
16.2
12.4
40.8
16.3
35.6
72.7
36.9
42.1
27.1
26.6
35.6
40.3
34.7
20.4
39.6
41.1
56.4
31.8
83.4
62.8
16.3
23.4
40.8
14.1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
110.3 181.3 181.4 108.9 108.9 111.7 111.6 108.4 108.4 109.2 109.2 109.0 109.0
28.2
37.9
138.6
114.7
43.4
43.4
38.4
38.4
31.2
31.2
67.2
67.2
72.7
71.5
62.9
72.7
71.5
62.9
132.9 133.0 133.8 133.8 122.4 122.5 125.5 125.5
119.6 119.5 117.4 117.4 125.5 125.5 129.7 129.8
60.0
60.0
60.4
60.3
60.1
59.7
c
c
c
c
c,d
c,d
c
c
c
c
c
c
Other C
a
Some signals may be interchanged.
Spectra taken in CD3OD.
Carbon atom signals of TBDMS at ı: 26.0 (3 × CH3), 18.3 (C), and −4.6 (2 × CH3).
Carbon atom signals of O-allyl at ı: 135.8 (CH), 114.6 (CH2), and 60.6 (CH2).
b
c
d
In conclusion, the two new glycospirostanes 10a and 10b were
in vitro properties for their in vivo impact. J Agric Food Chem 1998;46:
178–86.
6] Sindambiwe JB, Calomme M, Geerts S, Pieters L, Vlietinck AJ, Vanden Berghe DA.
Evaluation of biological activities of triterpenoid saponins from Maesa lanceo-
lata. J Nat Prod 1998;61:585–90.
7] Li DW, Lee EB, Kang SS, Hyun JE, Whang WK. Activity-guided isolation of
saponins from Kalopanax pictus with anti-inflammatory activity. Chem Pharm
Bull 2002;50:900–3.
3
synthesized using ring-closing metathesis as a key step. These
crystalline compounds can be considered as O-glycosides and C-
glycosides at the same time. Unfortunately, the biological tests
proved their marginal cytotoxic activity.
[
[
Acknowledgements
[8] Itabashi M, Segawa K, Ikeda Y, Kondo S, Naganawa H, Koyano T, et al. A new
bioactive steroidal saponin, furcreastatin, from the plant Furcraea foetida. Car-
bohydr Res 1999;323:57–62.
Financial support from the Polish Ministry of Science and Higher
Education (Grant nos. PBZ-KBN-126/T09/2004 and N N204 240334)
is gratefully acknowledged. We thank Dr. Miroslav Strnad and Dr.
Lucie Hoffmannova from the Laboratory of Growth Regulators,
Palacky University & Institute of Experimental Botany ASCR, Olo-
mouc, Czech Republic for performing cytotoxicity tests.
[
9] López Y, Jastrz e˛ bska I, Santillan R, Morzycki JW. Synthesis of
“glycospirostanes”—steroid sapogenins with
2008;73:449–57.
a sugar-like ring F. Steroids
[
10] Altomare A, Cascarano G, Giacovazzo C, Guagliardi A. Completion and
refinement of crystal structures with SIR92.
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J Appl Crystallogr 1993;26:
[11] Sheldrick GM. SHELXL-97. A program for the refinement of crystal structure.
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[
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