A stereoselective synthesis of squalamine

Article abstract of DOI:10.1016/S0040-4020(02)01413-8

Squalamine (1) was synthesized stereoselectively in 14 steps and 19% overall yield from 3-keto-5α-chenodeoxycholanate (2) by using a modified Sharpless asymmetric dihydroxylation as the key step.

Full text of DOI:10.1016/S0040-4020(02)01413-8

Tetrahedron 58 (2002) 10293–10299
A stereoselective synthesis of squalamine
†
Xiang-Dong Zhou, Feng Cai and Wei-Shan Zhou
*
Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 354 Fenglin Lu, Shanghai 200032, People’s Republic of China
Received 30 August 2002; revised 7 October 2002; accepted 31 October 2002
Abstract—Squalamine (1) was synthesized stereoselectively in 14 steps and 19% overall yield from 3-keto-5a-chenodeoxycholanate (2) by
using a modified Sharpless asymmetric dihydroxylation as the key step. q 2002 Published by Elsevier Science Ltd.
1. Introduction
and asthma.4c Coupled with this promising biological
activity, the recent discovery of several analogues of
squalamine from the stomach of the dogfish shark
S. acanthias,5 has ushered in a new era of research on
squalamine.
Squalamine, 3b-N-1-{N-[3-(4-aminobutyl)]-1,3-diamino-
propane}-7a, 24R-dihydroxy-5a-cholestane-24-sulfate (1)
(Fig. 1), the first example of an aminosterol, was isolated by
Zasloff1 in minute quantities from the stomach of the
dogfish shark Squalus acanthias in 1993.2a Squalamine,
whose structure was determined by 1H NMR and 13C NMR
spectroscopy and FAB mass spectrometry,2,3a possesses
both amphipathic and zwitterionic properties, due to the
lipophilic steroidal skeleton, the hydophilic 3b-spermine
and 24R-sulfate, and the cationic spermidine and anionic
24-sulfate moieties.
This compound cannot be obtained in a large amount from
natural sources. Therefore, much effort has been expended
in synthesizing it and its analogues. Moriarty’s3b and
Pechulis’6 groups have synthesized squalamine in racemic
form, and the former group also synthesized the 24-R-
hydroxy side chain.3a Kinney7 and his co-workers also have
obtained good results. Although, this group obtained the
24-R-hydroxy compounds in 91% d.e., the undesired 24-S-
isomer had to be removed in the last step by HPLC. Herein,
we report a highly stereoselective new synthetic route to
squalamine by using an improved Sharpless catalytic
asymmetric dihydroxylation8as a key step. Thus, the 24-R-
hydroxy group was introduced9 in 100% d.e. and the pure
squalamine was obtained in an overall yield of 19%.
Squalamine displays potent bactericidal activities against
both Gram-negative and Gram-positive bacteria.2 However,
the most significant property is its anti-angiogenic
activity,4a which first led to the development of squalamine
for cancer chemotherapy.4b In particular, the combination of
squalamine and cisplatin was found to be highly active
against human cancer cells.4d Squalamine also has activity
against age-related macular degeneration, malaria, obesity
2. Results and discussion
The starting material, methyl 3-keto-5a-chenodeoxy-
cholanate 2, was prepared from methyl chenodeoxy-
cholanate according to the literature.10 In addition, our
group also developed a method11 to prepare it from methyl
hyodeoxycholanate (Me-HDCA), which is readily available
in China. Our synthesis of squalamine was divided into
three stages. First, compound 7 with the 24-R-hydroxyl
group was prepared. Then, the side chain on ring D was
constructed through the synthesis of compound 11. Finally,
the spermidine side chain was connected to ring A,
providing the target compound.
Figure 1. Squalamine (1).
Keywords: squalamine; improved Sharpless AD; aminosterol;
stereoselective.
As depicted in Scheme 1, the 7a-hydroxyl group of 2 was
converted by treatment with dimethoxymethane in the
presence of P2O5 in chloroform into the 7a-methoxymethyl
ether 3 (94%). Then the 3-keto group was protected with
*
Corresponding author. Tel.: þ86-21-6416-3300; fax: þ86-21-6416-6128;
e-mail: zhws@pub.sioc.ac.cn
Present address: Department of Chemistry, The Third Millitary Medical
University, Chongqing 400038, People’s Republic of China.
†
0040–4020/02/$ - see front matter q 2002 Published by Elsevier Science Ltd.
PII: S0040-4020(02)01413-8

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X.-D. Zhou et al. / Tetrahedron 58 (2002) 10293–10299
Scheme 1. (a) CH3OCH2OCH3, P2O5, CHCl3, room temperature, 94%; (b) ethylene glycol, PTSA, benzene, K 96%; (c) LiAlH4, THF, room temperature 94%;
(d) (1), (COCl)2, DMSO, Et3N, CH2Cl2, 2788C, (2) BuLi, Ph3PþCH(CH3)2I2, THF, room temperature, 91% for two steps; (e) (DHQD)2PHAL,
K2OsO2(OH)4, K3Fe(CN)6, K2CO3, CH3SO2NH2, t-butanol–methyl t-butyl ether–H2O (2.5:3:2.5), room temperature, 97%, 100% d.e.
ethylene glycol to give the ethylene acetal 4 (96%). The side
chain ester in 4 was reduced with LiAlH4 to the 24-alcohol 5
(94%), Swern oxidation of which followed by Wittig
olefination with isopropyltriphenylphosphonium iodide
and BuLi in THF at room temperature gave the desmoster-
oid derivative 6 in 91% overall yield. Compound 6 could
be used as a key intermediate for transformation into
squalamine and its cognates,5 for example, 24-one-26-
cysteine squalamine, D25-24-one squalamine, etc. In the
course of our synthesis of squalamine, the key step is the
stereoselective introduction of the 24R, 25-dihydroxy group
into the side chain of 6 via an improved Sharpless catalytic
asymmetric dihydroxylation.8 Dihydroxylation of 6 with
(DHQD)2PHAL and K2OsO2(OH)4 in tert-BuOH-methyl
tert-butyl ether–H2O (2.5:3:2.5) solvent system gave
24R,25-dihydroxy compound 7 in 6 h in 97% yield and
with 100% d.e. This solvent system has the advantages over
the usual solvent system (1:1 t-BuOH–H2O12 or 1.5:1
t-BuOH–H2O13) in the AD reaction in that it can greatly
shorten the reaction time and increase the diastereo-
selectivity due to the larger solubility of steroids therein.
Thereby 24-R-hydroxyl group was successfully introduced
with complete stereoselectivity.
In the second stage (Scheme 2), compound 7 was acetylated
by using acetic anhydride and pyridine to give the acetate 8
(92%). Dehydration of the 25-tert-hydroxy group in 8
with methanesulfonyl chloride and triethylamine gave the
D25-24R-acetoxyl-compound 9 (91%). This result is better
than that obtained from the reaction in which DMAP is used
as a catalyst, as described in our preliminary communi-
cation.9 Diimide reduction14 of 9 followed by hydrolysis
with potassium hydroxide in methanol afforded com-
pound 10 (86%). Removal of the 7a-MOM and 3-acetal
Scheme 2.

X.-D. Zhou et al. / Tetrahedron 58 (2002) 10293–10299
10295
Scheme 3.
protecting groups of 10 with PPTS in tert-BuOH gave 11 in
92% yield.
Chemical shifts are reported in ppm relative to TMS as
internal standard. Mass spectra were recorded by EI or ESI
methods. Flash column chromatography was carried out
with silica gel (300–400 mesh). THF was distilled over
sodium and dichloromethane was distilled over CaH2. The
d.e. value was determined by HPLC analysis on Inersil
ODS-3 column with CH3CN–H2O as an eluent.
With the steroidal skeleton in hand, our attention was turned
to the introduction of the spermidine side chain into ring A
(Scheme 3). This was achieved via a reductive amination of
compound 11 with the protected spermidine 1215 utilizing
sodium borohydride as the reducing agent, and gave a
mixture of the aminosterols 13 and 14. These were separated
with flash chromatography with silica gel to give the
3b-aminosterol 13 (66%) and the 3a-aminosterol 14 (10%).
Removal of the Boc protecting groups of 13 by treatment
with a solution of HCl in MeOH gave 15 as the hydrochloric
acid salt (91%), which without further purification was
treated with sulfur trioxide–pyridine complex in pyridine to
give squalamine in 55% yield. This synthetic squalamine
obtained from 3b-aminosterol 13 was identical to natural
squalamine5 according to 1H NMR and 13C NMR
spectroscopy, and HRMS (ESI).
4.1.1. Methyl 7a-methoxymethyl-5a-cholanate-3-one
(3). To a solution of 3-keto-5a-chenodeoxycholanate 2
(50 mg, 0.12 mmol) in dry chloroform (1 mL) and
dimethoxymethane (0.23 mL, 2.6 mmol) was added phos-
phorus pentoxide (100 mg, 0.7 mmol) with stirring at room
temperature. After being stirred for 1 h, the mixture was
filtered through a pad of silica gel and washed with
chloroform (3£30 mL). Removal of the solvent in vacuo
afforded a light yellow oil (79 mg), which was purified by
flash chromatography (pet. ether/ethyl acetate 8:l) to afford
pure 3 (52 mg, 94%). Recrystallization from methanol
gave colorless needles; mp 147–1488C; [a]2D0¼23 (c
0.15, CHCl3); MS-EI (m/z): 416 (Mþ2CH3OH), 386
(Mþ2HOCH2OCH3); IR (cm21): 1741 (COOCH3); 1H
NMR d 0.67 (3H, s, 18-CH3), 0.92 (3H, d, J¼6.3 Hz,
21-CH3), 1.01 (3H, s, 19-CH3), 1.10–1.20 (3H, m), 1.22–
1.45 (11H, m), 1.52–1.68 (5H, m) 1.72–2.20 (5H, m), 2.30–
2.50 (3H, m), 3.35 (3H, s, OCH3), 3.69 (3H, s, COOCH3),
4.60 (1H, d, A of AB, J¼6.9 Hz, –OCH2O–), 4.67 (1H, d, B
of AB, J¼6.9 Hz, –OCH2O–). Anal. calcd for C27H44O5: C,
72.28%; H, 9.89%. Found C, 72.22%; H, 9.81%.
3. Conclusion
There are two key elements in our synthesis of squalamine.
The first one is the highly stereoselective introduction of the
24R-hydroxy group into the key intermediate, desmosteroid
derivative 6, via an improved Sharpless asymmetric
dihydroxylation in 100% d.e. The second is the reductive
replacement of the 3-keto group in ring A by the spermidine
side chain to give a mixture of 3b-aminosterol 13 and
3a-aminosterol 14 in a ratio of 6:1.
4.1.2. Methyl 7a-methoxymethyl-3-dioxolane-5a-chola-
nate (4). To a solution of 3 (183 mg, 0.41 mmol) in 20 mL
benzene was added ethylene glycol (25 mL) and p-toluene-
sulfonic acid (500 mg). The resulting solution was refluxed
for 3 h with use of a Dean–Stark trap to remove water, and
then cooled to room temperature. The solvent was removed
in vacuo then extracted with ethyl acetate (3£30 mL). The
organic layer was washed with sodium bicarbonate solution
(2£10 mL), distilled water (10 mL) and brine (10 mL),
respectively. The organic layer was dried over Na2SO4, and
the solvent was removed in vacuo, and the residue was
purified by flash chromatography (pet. ether/ethyl acetate
10:1) to afford pure compound 4 (193 mg, 96%, white
Another novel synthetic route to squalamine and its
analogues is in progress.
4. Experimental
4.1. General
All melting points are uncorrected. IR spectra were recorded
with FT-IR apparatus. 1H NMR and 13C NMR spectra were
recorded at 300 and 75 MHz in CDCl3 except those noted.

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X.-D. Zhou et al. / Tetrahedron 58 (2002) 10293–10299
1
solid); mp 100–1028C; [a]2D0¼29.6 (c 0.65, CHCl3); MS-EI
(m/z): 492 (Mþ), 460 (Mþ2CH3OH), 432 (Mþ2HOCH2-
OCH3); IR (cm21): 1734 (COOCH3); 1H NMR d 0.64
(3H, s, 18-CH3), 0.80 (3H, s, 19-CH3), 0.92 (3H, d, J¼
6.3 Hz, 21-CH3), 1.02–1.70 (11H, m), 1.80–2.10 (3H, m),
2.20–2.40 (2H, m), 3.36 (3H, s, OCH3), 3.64 (1H, m, 7b-H),
3.66 (3H, s, COOCH3), 3.92 (4H, s, OCH2CH2O), 4.60 (1H,
d, A of AB, J¼6.9 Hz, –OCH2O–), 4.69 (1H, d, B of AB,
J¼6.9 Hz, –OCH2O–); HRMS calcd for C29H48O6:
492.3451. Found 492.3454.
(Mþ2HOCH2OCH3), 426 (Mþ2HOCH2OCH3); H NMR
d 0.64 (3H, s, 18-CH3), 0.81 (3H, s, 19-CH3), 0.93 (3H, d,
J¼6.5 Hz, 21-CH3), 1.02–1.40 (11H, m), 1.52–1.70 (18H,
m), 1.82–2.10 (4H, m), 3.38 (3H, s, OCH3), 3.61 (1H, m,
7b-H), 3.92 (4H, s, –OCH2CH2O–), 4.61 (1H, d, A of AB,
J¼6.9 Hz, –OCH2O–), 4.69 (1H, d, B of AB, J¼6.9 Hz,
–OCH2O–), 5.07 (1H, t, 24-H); 13C NMR d 130.96, 125.35,
109.41, 96.18, 75.36, 64.18, 64.15, 56.11, 55.69, 50.11,
45.94, 42.64, 39.90, 39.51, 37.68, 36.50, 36.20, 35.88,
35.75, 35.59, 33.57, 31.29, 28.26, 25.78, 24.86, 23.84,
21.11, 18.72, 17.71, 11.90, 10.60. Anal. calcd for C31H52O4:
C, 76.18%; H, 10.72%. Found C, 76.30%; H, 11.01%.
4.1.3. 7a-Methoxymethyl-3-dioxolane-5a-cholane-24-ol
(5). A solution of 4 (1.2 g, 2.44 mmol) in dry tetrahydro-
furan (20 mL) was added to a suspension of lithium
aluminum hydride (114 mg, 3.0 mmol) in dry tetrahydro-
furan (30 mL) under argon over 3 h. The reaction mixture
was stirred for an additional hour before being quenched
with Na2SO4·10H2O, filtered through a pad of celite, and
washed with ethyl acetate. Removal of the solvent in vacuo
and purification by flash chromatography (pet. ether/ethyl
acetate 6:1) afforded pure compound 5 (1.09 g, 94%, white
solid); mp 133–1358C; [a]2D0¼211.7 (c 0.4, CHCl3); MS-EI
(m/z): 464 (Mþ), 420 (Mþ2CO2), 403 (Mþþ1–HOCH2-
4.1.5. 7a-Methoxymethyl-3-dioxolane-5a-24R,25-dihy-
droxy-cholestane (7). A solution of K3Fe(CN)6 (820 mg,
2.48 mmol), K2CO3 (350 mg, 2.54 mmol), CH3SO2NH2
(80 mg, 0.84 mmol), (DHQD)2PHAL (40 mg, 9 mol%), and
K2OsO2(OH)4 (10 mg, 5 mol%) in t-butanol–water (1:1,
40 mL) was cooled to 08C. A solution of 6 (268 mg,
0.55 mmol) in methyl t-butyl ether (30 mL) was added
dropwise. The resulting mixture was stirred vigorously for
6 h. Then the reaction was quenched at 08C with sodium
sulfite (1.0 g). Stirring was continued for an additional hour.
The aqueous layer was extracted with ethyl acetate
(3£3 mL). The combined organic layer was washed with
2 M KOH solution (25 mL), 10% HCl solution (10 mL), sat.
NaHCO3 solution (10 mL), brine (10 mL), dried over
sodium sulfate and evaporated to give the crude product,
which was purified by flash chromatography (pet. ether/
acetone 2:1) to afford pure 9 (279 mg, 100% d.e., 97%,
white solid); mp 167,1698C, [a]2D3¼þ0.68 (c 4.1, CHCl3);
MS-EI (m/z): 522 (Mþ), 443 (Mþþ1–HOCH2OCH3–
H2O); IR (cm21): 3443 (OH); 1H NMR d 0.65 (3H, s,
18-CH3), 0.88 (3H, s, 19-CH3), 0.92 (3H, d, J¼6.2 Hz,
21-CH3), 0.90–1.05 (8H, m), 1.07 (3H, s, 26-CH3), 1.13
(3H, s, 27-CH3), 1.30–1.7 (16H, m), 1.80–2.10 (2H, m)
3.25 (1H, m, 24-H), 3.39 (3H, s, CH3O), 3.59 (H, m, 3-H),
3.92 (4H, s, –OCH2CH2O–), 4.61 (1H, d, A of AB,
J¼6.9 Hz, –OCH2O–), 4.69 (1H, d, B of AB, J¼6.9 Hz,
–OCH2O–); 13C NMR d 109.31, 96.08, 78.78, 75.26,
64.08, 64.05, 55.98, 55.62, 50.00, 45.81, 39.77, 37.55,
36.40, 35.76, 35.64, 34.47, 33.45, 32.86, 31.17, 30.32,
29.69, 28.26, 28.13, 26.55, 23.72, 23.27, 21.00, 18.55,
11.83, 10.51. Anal. calcd for C31H54O6: C, 71.23%; H,
10.41%. Found C, 71.33%; H, 10.21%.
1
OCH3); IR (cm21): 3317 (OH); H NMR d 0.65 (3H, s,
18-CH3), 0.81 (3H, s, 19-CH3) 0.91 (3H, d, J¼6.4 Hz,
21-CH3), 1.02–1.20 (4H, m), 1.22–1.60 (10H, m), 1.62–
1.80 (10H, m), 1.80–2.10 (3,H, m), 3.37 (3H, s, OCH3),
3.60 (3H, m, 7b-H and 24-H), 3.92 (4H, s, OCH2CH2O),
4.60 (1H, d, A of AB, J¼6.9 Hz, –OCH2O–), 4.69 (1H, d,
B of AB, J¼6.9 Hz, –OCH2O–). Anal. calcd for C28H48O5:
C, 72.37%; H, 10.41%. Found C, 72.55%; H, 10.52%.
4.1.4. D24,7a-Methoxymethyl-3-dioxolane-5a-cholestene
(6). Dimethyl sulfoxide (0.42 mL, 5 mmol) was added
dropwise to a solution of oxalyl chloride (0.23 mL,
2.5 mmol) in dry dichloromethane (2 mL) at 2788C. The
solution was stirred in a dry ice–acetone bath under argon
over 35 min. A solution of 5 (525 mg, 1.13 mmol) in
dichloromethane (3 mL) was added dropwise over 2 min.
The stirring was continued for an additional 1 h. When
triethylamine (1.6 mL) was added dropwise to the reaction
mixture, a large amount of white solid appeared. After being
stirred for 10 min, the reaction mixture was allowed to
warm to room temperature and washed in turn with sat.
NH4Cl solution (2£5 mL), sat. NaHCO3 solution (5 mL),
brine (10 mL) and dried over sodium sulfate. Removal of
the solvent in vacuo afforded yellow solid (570 mg).
Without chromatography, it was directly used for the next
step. Thus, n-butyl lithium (1.6 M in hexane, 2.2 mL) was
added to a suspension of isopropyltriphenylphosphonium
iodide (1.25 g, 2.9 mmol) in dry tetrahydrofuran (6 mL) at
room temperature, giving a deep red solution, to which the
compound (570 mg) from last step in dry tetrahydrofuran
(6 mL) was added to it. After being stirred for 2 h, the
reaction was quenched with ethyl acetate (20 mL), and a
large amount of white solid appeared. The mixture was
filtered through a pad of celite and washed with ethyl acetate
(20 mL). The filtrate was then washed with sat. NH4Cl
(2£5 mL) and brine (10 mL), respectively, and dried over
sodium sulfate. Removal of the solvent in vacuo and
purification by flash chromatography (pet. ether/ethyl
acetate 10:1) afforded 6 (504 mg, 91.3%, white solid); mp
111–1138C; [a]2D0¼219 (c 0.12, CHCl3); MS-EI (m/z): 456
4.1.6. 7a-Methoxymethyl-3-dioxolane-5a-24R,25-dihy-
droxy-cholestan-24-acetate (8). A solution of 7 (118 mg,
0.226 mmol) in acetic anhydride (15 mL) and pyridine
(1.5 mL) was stirred at room temperature for 9 h. The
reaction mixture was partitioned between ethyl acetate
(20 mL) and water. The organic layer was washed with 5%
HCl (5 mL), sat. NaHCO3 (5 mL), and brine (2£5 mL),
respectively, and dried over sodium sulfate, evaporated in
vacuo and purified by flash chromatography (pet. ether/
acetone 4:1) to afford pure compound 8 (117 mg, 92%,
white solid); mp 76–788C; [a]2D2¼25.6 (c 1.6, CHCl3);
MS-EI (m/z): 564 (Mþ), 519 (Mþ2CH3OCH2), 502 (Mþ2
CH3OCH2OH); IR (cm21): 3450 (OH), 1736 (CH3CO2); 1H
NMR d 0.64 (3H, s, 18-CH3), 0.81 (3H, s, 19-CH3), 0.91
(3H, d, J¼6.4 Hz, 21-CH3), 0.90–1.05 (3H, m), 1.20 (3H,
s, 26-CH3), 1.20 (3H, s, 27-CH3), 1.25–1.45 (12H, m),
1.50–1.85 (9H, m), 1.90–2.00 (3H, m), 2.11 (3H, s,

X.-D. Zhou et al. / Tetrahedron 58 (2002) 10293–10299
10297
CH3CO2–), 3.38 (3H, s, –OCH3), 3.59 (1H, s, 7b-H), 3.92
(4H, s, –OCH2CH2O–), 4.72 (1H, d, A of AB, J¼6.9 Hz,
–OCH2O–), 4.76 (1H, d, B of AB, J¼6.9 Hz, –OCH2O–),
4.78 (1H, dd, J¼10.2, 2.4 Hz, 24-H); 13C NMR d 171.26,
109.27, 96.01, 80.03, 75.17, 72.49, 64.06, 64.02, 55.70,
55.61, 49.96, 45.77, 42.47, 39.70, 39.35, 37.51, 36.35,
35.71, 35.44, 35.32, 33.40, 32.04, 31.13, 28.03, 26.82,
25.68, 24.85, 23.64, 21.06, 20.95, 18.42, 11.76, 10.49;
HRMS calcd for C33H56O7: 564.4026. Found 564.4004.
white solid.); mp 110–1128C; [a]2D4¼20.3 (c 0.91, CHCl3);
MS-EI (m/z): 506 (Mþ), 461 (Mþ2CH2OCH3), 444 (Mþ2
1
HOCH2OCH3); IR (cm21): 3487 (OH); H NMR d 0.65
(3H, s, 18-CH3), 0.68 (3H, d, J¼6.8 Hz, 21-CH3), 0.81 (3H,
s, 19-CH3), 1.01 (3H, s, 26-CH3), 1.10–1.80 (22H, m),
1.90–2.10 (6H, m), 2.20–2.40 (3H, m), 3.33 (1H, t, J¼
4.5 Hz, 24-H), 3.35 (3H, s, OCH3), 3.59 (1H, s, 7b-H), 3.92
(4H, s, –OCH2CH2O–), 4.61 (1H, d, A of AB, J¼6.9 Hz,
–OCH2O–), 4.69 (1H, d, B of AB, J¼6.9 Hz, –OCH2O–);
13C NMR d 109.40, 96.14, 75.32, 64.18, 64.14, 56.01,
55.71, 50.09, 45.90, 42.61, 39.86, 39.48, 37.65, 36.48,
35.85, 35.83, 35.57, 33.63, 32.13, 31.26, 30.69, 29.78,
28.33, 23.80, 21.09, 19.00, 18.73, 17.32, 11.91, 10.60, 1.10;
HRMS calcd for C31H54O5: 506.3971. Found 506.3922.
4.1.7. D25,7a-Methoxymethyl-3-dioxolane-5a-24R-
hydroxy-cholesten-24-acetate (9). To a solution of
8 (908 mg, 1.61 mmol) and triethylamine (1.92 mL,
138.7 mmol) in dichloromethane (70 mL) stirring at 08C
under argon was added dropwise methanesulfonyl chloride
(5.6 mL, 72.2 mmol). After 20 h, water (30 mL) was
dropped in. The reaction mixture was extracted with
dichloromethane (30£3 mL). The organic lay was washed
with brine (20 mL), dried over sodium sulfate and
concentrated in vacuo. The residue was purified by flash
chromatography (pet. ether/acetone 4:1) to afford pure 9
(658 mg, 75%, white solid, 162 mg 8 was recovered); mp
104–1088C; [a]2D0¼213 (c 0.23, CHCl3); MS-EI (m/z): 546
(Mþ), 484 (Mþ2HOCH2OCH3); IR (cm21): 1737
(CH3CO2), 1655 (CvCH2), 902 (CvCH2); 1H NMR d
0.63 (3H, s, 18-CH3), 0.91 (3H, d, J¼6.5 Hz, 21-CH3), 0.81
(3H, s, 19-CH3), 1.02–1.10 (4H, m), 1.20–1.60 (12H, m)
1.75–1.90 (8H, m), 1.92–2.00 (2H, m), 1.71 (3H, s,
27-CH3), 2.06 (3H, s, 24-CH3CO2), 3.38 (3H, s, –OCH3),
3.58 (1H, s, 7b-H), 3.92 (4H, s, –OCH2CH2O–), 4.64 (1H,
d, A of AB, J¼7.0 Hz, –OCH2O–), 4.64 (1H, d, B of AB,
J¼7.0 Hz, –OCH2O–), 4.90 (2H, d, J¼7.0 Hz, 26-H), 5.11
(1H, t, J¼6.5 Hz, 24-H); 13C NMR d 170.48, 143.51,
112.56, 109.34, 96.15, 77.71, 75.31, 64.18, 64.14, 55.78,
55.72, 50.09, 45.90, 42.60, 39.85, 39.45, 37.64, 36.48,
35.85, 35.56, 35.53, 33.53, 31.40, 31.26, 29.19, 28.19,
23.78, 21.31, 21.08, 18.71, 18.26, 11.87, 10.60; HRMS
(ESI) calcd for C33H54O6: 546.3920. Found 546.3955.
4.1.9. 7a,24R-Dihydroxy-5a-cholestane-3-one (11). A
mixture of 10 (300 mg, 0.593 mmol) and PPTS (400 mg,
1.6 mmol) in t-butanol (30 mL) was refluxed for 10 h.
Removal of the solvent in vacuo and purification by flash
chromatography (pet. ether/acetone 4:1) afforded pure 11
(white prism crystal, 228 mg, 92%); mp 149–1518C (lit.7e
151–1538C), [a]D20¼þ22.6 (c 0.32, CHCl3); IR (cm21):
3447 (–OH), 1707 (3-one); 1H NMR d 0.70 (3H, s,
18-CH3), 0.91 (9H, m, 19-CH3), 1.01 (3H, s), 1.10–1.70
(18H, m), 1.90–2.10 (7H, m), 2.20–2.30 (2H, m), 2.30–
2.40 (2H, m), 3.32 (1H, m, 24-H) 3.86 (1H, m, 7b-H);
MS-EI (m/z): 419 (4.8%, [Mþþ1]), 400 (24.2%, [Mþ2
H2O]), 382 (33.4%, [Mþ22H2O]); 13C NMR d 211.57,
77.97, 67.55, 56.05, 50.47, 45.24, 44.14, 42.68, 39.51, 39.44,
39.08, 38.16, 38.12, 36.57, 35.69, 33.61, 32.00, 30.92, 30.55,
28.24, 23.67, 21.22, 18.90, 18.60, 17.25, 11.88, 10.46;
HRMS calcd for C27H46O3: 418.3447. Found 418.3419.
4.1.10. 3b-[5,10Bis(tert-butoxycarbonyl)-1,5,10-triaza-
decyl]-5a-7a,24R-dihydroxy-cholestane (13) and
3a-[5,10-bis(tert-butoxycarbonyl)-1,5,10-triazadecyl]-
5a-7a,24R-dihydroxy-cholestane (14). A mixture of
compound 11 (32 mg, 0.076 mmol), amino compound 12
˚
(95 mg, 0.28 mmol) and 3 A molecular sieves (0.35 g) in
4.1.8. 7a-Methoxymethyl-3-dioxolane-5a-24R-hydroxy-
cholestane (10). To a suspension of NH2OH·HCl (3.59 g,
51.7 mmol) in DMF (10 mL) with stirring at 08C was added
KOH (85%, 3.41 g, 51.7 mmol). After being stirred for
30 min, the mixture was filtered and the solid was washed
with DMF (ca. 2 mL). The combined filtrate was cooled to
08C, and then ethyl acetate (2.22 mL, 22.6 mmol) was added
dropwise. After being stirred for an additional 30 min, the
solution was added dropwise to another flask containing 9
(413 mg, 0.756 mmol) with stirring at 90–958C. After being
stirred for 10 h, the mixture was cooled to room tempera-
ture, and water (15 mL) was added. The mixture was then
extracted with ethyl acetate (3£30 mL). The combined
organic layers were washed with water (15 mL), 5% HCl
(15 mL), sat. NaHCO3 (20 mL), and brine (20 mL),
respectively, and then was dried over sodium sulfate and
evaporated to afford the crude product. It was dissolved in
methanol (20 mL) containing water (1 mL) and KOH
(400 mg) and refluxed for 5 h. After removal of solvents
in vacuo, the residue was dissolved in ethyl acetate (50 mL),
washed with water (2£10 mL) and brine (2£10 mL),
respectively, and dried over sodium sulfate, evaporated
and purified by flash chromatography (pet. ether/ethyl
acetate 6:1) to afford pure 10 (329 mg, 86%, 100% d.e.,
absolute methanol (3 mL) was stirred for 18 h at room
temperature under argon. NaBH4 (40 mg) was added and
the solution stirred for 4 h at 2788C. Then acetic acid
(20 mL) was dropped until pH¼7.0 to quench the reaction.
The mixture was filtered through celite, the cake was
washed well with MeOH and CH2Cl2. Removal of the
solvent in vacuo and purification by flash chromatography
(pet. ether/ethyl acetate/triethyl amine 6:4:1) afforded
3b-compound 13 (38 mg, 66%, colorless oil) and 3a-com-
pound 14 (6 mg, 10%, colorless oil).
Compound 13. 1H NMR d 0.66 (3H, s, 18-CH3), 0.79 (3H, s,
19-CH3), 0.91 (9H, m, 21-, 26-, 27-CH3), 1.02–1.80 (50H,
m, including 18H of BOC), 1.90–2.00 (3H, m), 3.1–3.4
(7H, m, NCH and 3a-H), 3.48 (1H, t, J¼6.0 Hz), 3.54 (1H,
t, J¼4.5 Hz), 3.73 (1H, t, J¼4.4 Hz), 3.82 (1H, br); 1H NMR
(CD3OD) d 0.74 (3H, s, 18-CH3), 0.87 (3H, s, 19-CH3), 0.93
(3H, s), 0.95 (3H, s), 0.98 (3H, d, J¼6 Hz), 1.12–1.60 (44H,
m, including 18H of BOC), 1.72–1.90 (7H, m), 1.92–2.10
(2H, m), 3.06–3.26 (9H, m, NCH), 3.82 (1H, br, 7b-H); 13C
NMR d 156.00, 79.34, 76.82, 76.68, 71.83, 71.03, 67.70,
61.59, 57.23, 56.03, 50.55, 46.55, 45.84, 42.60, 40.15,
39.54, 39.48, 37.45, 37.06, 36.55, 36.00, 35.69, 34.36,
33.56, 32.04, 30.50, 29.61, 28.38, 28.20, 27.36, 23.57,

10298
X.-D. Zhou et al. / Tetrahedron 58 (2002) 10293–10299
20.86, 19.24, 18.88, 18.57, 17.29, 11.82, 11.20. MS (ESI):
770.8 [MþNaþ], 748.8 [MþHþ], 747.8 [Mþ]; HRMS (ESI)
calcd for C44H82N3O6: 748.6203. Found 748.6187.
analysis. We thank Mr Ji-Ming Sheng and Ms Qun-Yan Pan
for preparation of the starting material, and Mr Wei Gong
for performing the 1H 1H COSY and 13C NMR
spectroscopy.
Compound 14. 1H NMR d 0.66 (3H, s, 18-CH3), 0.80 (3H, s,
19-CH3), 0.92 (9H, m, 21-, 26-, 27-CH3), 1.12–1.80 (44H,
m, including 18H of BOC), 1.92–2.10 (3H, m), 2.62–2.70
(3H, m), 3.05–3.35 (9H, m, NCH and 3b-H), 3.49 (1H, t,
J¼6.2 Hz), 3.54 (1H, t, J¼4.5 Hz), 3.75 (1H, t, J¼4.6 Hz),
3.81 (1H, br); 13C NMR d 125.51, 67.79, 61.89, 56.07,
50.63, 46.60, 42.57, 39.65, 39.50, 35.76, 33.57, 32.05,
31.93, 30.61, 30.33, 29.70, 29.66, 28.42, 28.21, 27.38,
23.61, 18.90, 18.62, 17.26, 14.86, 11.89, 10.51; MS (ESI):
770.8 [MþNaþ], 748.8 [MþHþ], 747.8 [Mþ]; HRMS (ESI)
calcd for C44H82N3O6: 748.6203. Found 748.6172.
References
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4.1.11. 3b-(1,5,10-Triazadecyl)-5a-7a,24R-dihydroxy-
cholestane trihydrochloride (15). Compound 13 (29 mg,
0.04 mmol) was dissolved in a solution of HCl in MeOH
(dry HCl gas was dissolved in 6 mL MeOH until pH ,1).
The solution was stirred about 29 h at room temperature.
The solvent was removed in vacuo. The desired product 15
as the salt of HCl (white solid, 23 mg, 90.7%) was obtained,
it was pure from TLC detection and could be used without
1
further purification. H NMR (CD3OD) d 0. 74 (3H, s, 18-
CH3), 0.91 (3H, s, 19-CH3), 0.92 (3H, s), 0.95 (3H, s), 0.98
(3H, d, J¼6 Hz), 0.99–1.11 (3H, m), 1.15–1.20 (3H, m),
1.20–1.50 (13H, m), 1.60–1.80 (8H, m), 1.80–1.90 (3H,
m), 2.02–2.10 (12H, m), 3.03–3.25 (9H, m, NCH), 3.83
(1H, br, 7b-H); 13C NMR (CD3OD) d 77.79, 68.34, 58.94,
57.64, 51.70, 46.75, 46.09, 43.67, 42.96, 40.92, 40.12,
38.52, 37.62, 37.08, 36.78, 34.91, 33.38, 32.02, 31.49,
30.78, 29.32, 25.94, 25.64, 24.43, 24.47, 24.32, 22.05,
19.48, 19.21, 18.03, 12.31, 11.54; HRMS (ESI) calcd for
C34H66N3O2: 548.5155. Found 548.5150.
5. Rao, M. N.; Shinnar, A. E.; Noecker, L. A.; Chao, T. L.;
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5121–5126.
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SO3–pyridine complex (11 mg, 0.07 mmol, 2 equiv.) was
added to a flask, flushed with argon. Dry pyridine (1 mL)
was added, and the solution was warmed to 408C in an oil
bath and stirred 2 h. MeOH (1 mL) was added to quench the
reaction. The flask was removed from the oil bath, and the
mixture was stirred for 15 min. The solution was concen-
trated in vacuo, and the residue was resuspended in MeOH
and filtered through a pad of celite. Flash chromatography
(CH2Cl2/MeOH/NH4OH 14:4:1) gave the desired product 1
7. (a) Rao, M. N.; McGuigan, M. A.; Zhang, X. H.; Ze’ev, S.;
Kinney, W. A.; Bulliard, M.; Laboue, B.; Lee, N. E. J. Org.
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1
(10.0 mg, 55%, white solid). MS/ESI: 628.6 [Mþ1]þ; H
NMR (CD3OD) d 0.75 (3H, s, 18-CH3), 0.92 (3H, s,
19-CH3), 0.96 (3H, s), 0.99 (3H, s), 1.00 (3H, s), 1.10–1.3
(8H, m), 1.40–1.70 (10H, m), 1.80–2.00 (8H, m), 2.10–
2.30 (5H, m), 3.04 (2H, t, J¼7.4 Hz, NCH), 3.11–3.25 (7H,
m, NCH), 3.84 (1H, s, 7b-H), 4.13 (1H, dd, J¼10.0 Hz,
24H); 13C NMR (CD3OD) d 86.60, 68.52, 59.20, 57.57,
51.93, 48.50, 46.86, 46.13, 43.88, 43.11, 41.00, 40.24,
38.70, 37.92, 37.59, 37.38, 36.99, 32.70, 32.17, 32.09,
29.28, 28.02, 26.01, 25.74, 24.61, 24.50, 24.39, 22.16,
19.56, 18.58, 18.44, 12.58, 11.79; HRMS (ESI) calcd for
C34H66N3O5S: 628.4723. Found 628.4718.
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12. Sharpless dihydroxylation of (22E)-3a,5-cyclo-5a-ergost-22-
en-6-one with DHQD-PHN in 1:1 t-BuOH–H2O proceeded in
4–6 days to give a mixture of (22R,23R,24R)-2a,3a,22,23-
tetrahydroxy and (22S,23S,24R)-2a,3a,22, 23-tetrahydroxy
Acknowledgements
We thank Professor Li-Jun Xia for performing the HPLC

X.-D. Zhou et al. / Tetrahedron 58 (2002) 10293–10299
10299
compounds in 78% yield and with 93:7 diastereo-selectivity
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13. Sharpless dihydroxylation of desmosterol with (DHQD)2-
PYDZ in 1.5:1 t-BuOH–H2O proceeded in 52 h to give 24(R),
25-dihydroxycholesterol in 83% yield and with 96:4 dia-