A low-toxicity method for the separation of lanosterol and dihydrolanosterol from commercial mixtures

Article abstract of DOI:10.1016/j.steroids.2004.07.003

We describe an inexpensive, low-toxicity and high-yielding method for the production of pure lanosterol and dihydrolanosterol from the commercially available mixture. Optimum conditions are presented for the one-pot production of the intermediate 24,25 vicinal diol of lanosterol acetate (via either epoxidation or hydroxyhalogenation) which is readily separated from the unreacted dihydrolanosterol acetate. The lanosterol diol can then be converted to pure (>97%) lanosterol. Hypophosphorous acid was used for both the conversion of the epoxide to the diol, and as a catalyst for the hydroxyhalogenation by N-halosuccinimides of the olefinic bond.

Full text of DOI:10.1016/j.steroids.2004.07.003

Steroids 69 (2004) 697–700
A low-toxicity method for the separation of lanosterol and
dihydrolanosterol from commercial mixtures
Levan K. Kavtaradze, Merilyn Manley-Harris∗, Brian K. Nicholson
Chemistry Department, University of Waikato, Private Bag 3105, Hamilton, New Zealand
Received 2 February 2004; received in revised form 3 May 2004; accepted 28 July 2004
Available online 17 September 2004
Abstract
We describe an inexpensive, low-toxicity and high-yielding method for the production of pure lanosterol and dihydrolanosterol from the
commercially available mixture. Optimum conditions are presented for the one-pot production of the intermediate 24,25 vicinal diol of
lanosterol acetate (via either epoxidation or hydroxyhalogenation) which is readily separated from the unreacted dihydrolanosterol acetate.
The lanosterol diol can then be converted to pure (>97%) lanosterol. Hypophosphorous acid was used for both the conversion of the epoxide
to the diol, and as a catalyst for the hydroxyhalogenation by N-halosuccinimides of the olefinic bond.
© 2004 Elsevier Inc. All rights reserved.
Keywords: Lanosterol; Dihydrolanosterol; Hydroxyhalogenation; Dihydroxylation; Hypophosphorous acid
1. Introduction
We here report a technically simple method for the pro-
duction of pure lanosterol in high yield without recourse to
highly toxic reagents. Pure dihydrolanosterol, which is also
of interest as a starting material for synthesis [9], is also iso-
lated. The method has evolved from our earlier development
of techniques for efficient synthesis of epimerically pure side-
chain derivatives of lanosterol [8].
Side chain derivatives of lanosterol (lanosta-8,24-dien-
3␤-ol, 1a) have been shown to act as inhibitors of ꢀ24(25)
sterol methyl transferase, which is an essential enzyme in
the sterol biosynthesis pathway of protozoa, fungi and plants
[1]. Because of this activity these compounds have potential
therapeutic applications. Significant chemopreventive activ-
ity of lanosterol against colon carcinogenesis in rat has been
reported [2].
2. Experimental
Pure lanosterol for bioassay or as a starting material for
synthesis is expensive and the usual starting material for syn-
thesis is a commercial “lanosterol” containing approximately
40–50% of dihydrolanosterol 2a, as the major impurity as
well as other steroids as minor impurities. Various methods
of obtaining pure lanosterol have been reported in the liter-
ature but most are limited in their suitability for scaling-up
either by their low yield [3–6] or the toxic or hazardous nature
of the reagents involved [7].
2.1. General
Lanosterol 50–60% was purchased from Sigma and used
without any further purification. Hypophosphorous acid
(50 wt.%inwater)andallotherreagentswerepurchasedfrom
Aldrich and used as such. Column chromatography was car-
ried out on Merck silica gel (70–250 mesh) using the eluents
indicated. TLC was carried out using Alugram® Sil G/UV254
plates. All melting points were obtained on a Reichert-Jung
micro-melting point determination apparatus and are uncor-
rected. Optical rotations were measured on an Optical Activ-
ity, Ltd. automatic polarimeter.
∗
Corresponding author. Tel.: +64 7 838 4384; fax: +64 7 838 4219.
E-mail address: manleyha@waikato.ac.nz (M. Manley-Harris).
0039-128X/$ – see front matter © 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2004.07.003

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L.K. Kavtaradze et al. / Steroids 69 (2004) 697–700
Gas chromatography with flame ionisation detection (GC-
spectrum of the mixture was assigned by comparison with
the spectra in reference [8]; carbons 20–24 were sufficiently
different in chemical shift to be distinguished by the 2D
HSQC experiment. In the case of 22 and 23 the different
shifts of the pairs of diastereotopic protons on these two
atoms were clearly observable in the HSQC. The side-chain
carbons had the following shifts (R,S ppm): 36.34, 36.78 (C-
20); 18.66, 18.86 (C-21); 33.18, 33.59 (C-22); 28.32, 28.73
(C-23); 78.77, 79.63 (C-24). The H-24 signals (3.31 and
3.26 ppm R and S, respectively) could be resolved to baseline
FID) was carried out using a Hewlett-Packard Ultra 2 (25 m
× 0.33 mm) crosslinked phenyl methyl silicone fused sil-
ica capillary column. Conditions used were 55 ◦C (1 min),
30 ◦C min−1 to 200 ◦C, 4 ◦C min−1 to 320 ◦C.
Gas chromatography with mass spectrometry was carried
out using a Hewlett-Packard HP 1 cross-linked methyl sili-
cone fused silica capillary column interfaced with a Hewlett-
Packard 5970 mass spectrometer (70 eV). Conditions used
were 200 ◦C (1 min), 30 ◦C min−1 to 260 ◦C, 3 ◦C min−1 to
295 ◦C.
1
in the 1D H spectrum using resolution enhancement and
upon integration gave the ratio R:S of 1.00:0.84.
1H and 13C NMR and HSQC spectra were obtainedusinga
Bruker Avance DRX 400 MHz spectrometer, in chloroform-d
and referenced to chloroform.
2.3.2. Bromohydroxy route to 4
To a solution of lanosterol acetates 1b and 2b (10 g)
(1b = 6.15 g, 2b = 3.85 g by GC) in acetone (900 mL),
water (15 mL) and hypophosphorous acid [2.7 mL (50%
in water)] was added N-bromosuccinimide (NBS, 3.04 g,
0.017 mol). The reaction mixture was stirred at room tem-
perature for 5 min, NaHCO3 (3 g) was added and the mix-
ture concentrated under vacuum. The residue was dissolved
in 2-propanol (300 mL), water (100 mL) and hypophospho-
rous acid (7.2 mL, 50% in water). More NaHCO3 (4.5 g) was
added, and the reaction mixture was refluxed (4 h), diluted
with water, filtered and washed until neutral. Separation by
flash column chromatography (dichloromethane) yielded di-
hydrolanosterol acetate, 2b (3.3 g, 85%), m.p. 119–120 ◦C
(recrystallized twice from acetone; Ref. [7] 120–122 ◦C).
NMR spectra were as reported [12]. Further elution with
ethyl acetate afforded a stereoisomeric mixture of the diol,
24(R,S)-3␤-acetoxy-24,25-dihydroxy-5␣-lanost-8-ene, 4, as
colourless needles (5.8 g, 88%), m.p. 172–175 ◦C (Ref. [10]
166–169 ◦Cor[11]183–185 ◦C, dependingonepimericcom-
position).
2.2. Acetylation of mixed lanosterols (1a + 2a)
This was carried out using commercial lanosterol (80 g,
0187 mol, 50–60% pure) as previously described [8] to
give 3␤-acetoxy-5␣-lanost-8,24-diene (1b) and 3␤-acetoxy-
5␣-lanost-8-ene (2b) as a colourless solid (72 g), m.p.
125–129 ◦C.
2.3. 24(R,S)-3␤-acetoxy-24,25-dihydroxy-5␣-
lanost-8-ene (4)
Compound
4
was prepared by two different
routes—epoxidation and hydroxyhalogenation (hydroxy-
bromide or hydroxy-iodide).
2.3.1. Epoxide route to 4
To a solution of the mixed lanosterol acetates 1b
and 2b (20 g) (1b = 12.3 g, 2b = 7.7 g by GC-FID) in
dichloromethane (600 mL) a mixture of m-chloroperbenzoic
acid (70%) (5.5 g) and sodium hydrogen carbonate (2.2 g,
0.05 mol) was added in the following manner: half added
at room temperature over 3 h and the remainder at 0 ◦C (ice
bath), also over 3 h. The mixture was stirred vigorously (1 h)
and left in a refrigerator overnight. The reaction mixture was
diluted with 2-propanol (500 mL), hypophosphorous acid
(20 mL) and water (200 mL). Dichloromethane was removed
by distillation at atmospheric pressure, an additional amount
of hypophosphorous acid (10 mL) added and the mixture
heated under reflux (2.5 h). The reaction mixture was poured
into water (1.2 L) and the solid filtered and washed with wa-
ter. Purification by short column chromatography (CH2Cl2)
yielded dihydrolanosterol acetate, 2b (7.3 g, 95% recovery),
m.p. 120–121 ◦C (acetone–methanol, Ref. [7] 120–122 ◦C).
NMR spectra were identical with the literature [12]. Fur-
ther elution with ethyl acetate afforded a stereoisomeric
mixture of the diol, 24(R,S)-3␤-acetoxy-24,25-dihydroxy-
5␣-lanost-8-ene, 4, as colourless needles (10.7 g, 82%), m.p.
173–176 ◦C (Ref. [10] 166–169 ◦C or [11] 183–185 ◦C; the
melting point depends on proportions of R and S epimers [8]).
Analytical calculation for C32H54O4: C, 76.44; H, 10.83.
Found: C, 76.52; H, 10.84. NMR spectra have previously
NMR was identical with that described above for the epox-
ide route. The ratio R:S from the 1H spectrum was 1.00:1.20.
2.3.3. Iodohydroxy route to 4
The reaction was carried out as described in Section
2.3.2 with N-iodosuccinimide (NIS) in place of N-
bromosuccinimide to give 2b (3.3 g, 81%), m.p. 119–120 ◦C
(recrystallized twice from acetone) and 4 (5.6 g, 86%) m.p.
170–174 ◦C. NMR was identical with that described above
1
for the epoxide route. The ratio R:S from the H spectrum
was 1.00:1.14.
2.4. Conversion of
24(R,S)-3␤-acetoxy-24,25-dihydroxy-5␣-lanost-8-ene, 4,
to lanosterol acetate, 1b
To a solution of 24(R,S)-3␤-acetoxy-24,25-dihydroxy-
5␣-lanost-8-ene (4, 10 g, 0.0199 mol) in dichloromethane
(150 mL) was added N,N-dimethylformamide dimethylacetal
(13.2 mL, 0.597 mol) and the mixture was refluxed (2.5 h).
The reaction mixture was cooled, acetic anhydride (20 mL)
was added and the dichloromethane was distilled under re-
been assigned for the individual R and S epimers [8] the 13
C

L.K. Kavtaradze et al. / Steroids 69 (2004) 697–700
699
duced pressure. An additional amount of acetic anhydride
(100 mL) was added and the mixture containing acetal 6
was refluxed at 130 ◦C (3.5 h), cooled, poured into ice wa-
ter and the solid filtered and washed until neutral to yield
a light brown powder. The crude product after chromatog-
raphy through silica gel (eluent dichloromethane) afforded
lanosterol acetate, 1b (8.3 g, 90%), as a white powder (97%
purity by GC), m.p. 129.5–131.5 ◦C [␣]D = +58.6◦ (c 1.16,
CHCl3). [Ref. [6,7] m.p. = 129–130 ◦C, [␣]D = +57.1◦ (c 1.1,
CHCl3) or m.p. = 129–130 ◦C, [␣]D = +59.0◦ (c 1.0 CHCl3)].
NMR spectra matched those in the literature [12]. Purifica-
tion by recrystallization at this stage resulted in poor recovery
of material, so the crude product was used for the next stage.
The resulting mixture was poured into ice water and after
standing 6–7 h was collected by filtration, dried and recrys-
tallized from methanol–benzene to yield lanosta-8,24-dien-
3␤-ol (1a, 4.2 g, 92%), m.p. 139–140 ◦C (Ref. [6] 140 ◦C).
NMR was identical with literature [12]. GC-FID analysis in-
dicated a purity of 98% lanosterol with the major impurity
being agnosterol (2%).
Similar treatment of dihydrolanosterol acetate (2b)
afforded lanosta-8-en-3␤-ol (2a, 4.25 g, 93%), m.p.
144–145 ◦C (Ref. [9] 144–144.5 ◦C). NMR was identical
with literature [12]. GC-FID indicated 98.6% dihydrolanos-
terol and 1.4% of dihydroagnosterol.
3. Results and discussion
2.5. Conversion of lanosterol and dihydrolanosterol
acetates (1b and 2b) to the corresponding hydroxy
derivatives (1a and 1b)
Lanosterol has been purified by three different routes,
all of which have in common the formation of 24(R,S)-3␤-
acetoxy-24,25-dihydroxy-5␣-lanost-8-ene, 4, Scheme 1. In
the first stage, Scheme 1, the epimeric 24,25-epoxylanosterol
Lanosterol acetate (1b, 5 g) was hydrolysed with 10%
potassium hydroxide in ethanol (150 mL) at 60 ◦C for 2 h.
Scheme 1. Separation of lanosterol from dihydrolanosterol.

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L.K. Kavtaradze et al. / Steroids 69 (2004) 697–700
acetate 3, or 24-halo-25-hydroxylanosterol acetates, 5a, 5b,
were formed as a precursor to the diols, 4. Separation from
2b was effected by short column chromatography at the diol
stage (a significant difference between the solubility of 2b
and 4 in organic solvents makes this process very easy for
large scale production). The mixture of epimeric diols was
then converted via the N,N-dimethylacetal, 6, to lanosterol
acetate, 1a.
In the first method, epoxidation [7,8] of acetylated com-
mercial lanosterol (mixture of 1b and 2b in the ratio
61.5:38.5) with m-chloroperbenzoic acid afforded a mix-
ture of the epimeric 24,25-epoxylanosterol acetates, 3, which
were not isolated. Upon refluxing in 2-propanol and aqueous
hypophosphorous acid the epoxide, 3, afforded the mixed
epimers of 24,25-dihydroxylanosterol acetate, 4. 2-Propanol
was chosen as the organic solvent to afford miscibility with
water without competing as a nucleophile and thereby form-
ing unwanted ethers.
Alternatively, treatment of acetylated commercial lanos-
terol (1b and 2b) with NBS or NIS in aqueous acetone in
the presence of a catalytic amount of hypophosphorous acid
afforded a mixture of the corresponding 24(R,S)-halo-25-
hydroxylanosterol acetates 5a and 5b (cf. [8]). These were
converted directly to the mixed epimeric diol, 4, by reflux-
ing in 2-propanol with aqueous hypophosphorous acid and
its sodium salt, which was generated in situ by addition of
NaHCO3 to the reaction mixture.
The epoxidation route to purification, when scaled-up, is
complicated by the possible formation of the 8,9 epoxide
due to “over-oxidation”, which, under these conditions, can
give rise to undesirable agnosterol and dihydroagnosterol by-
products. The hydroxyhalogenation routes are free from this
problem.
4. Conclusion
A simple, inexpensive and low-toxicity route is de-
scribed for the purification of lanosterol from mixtures of
lanosterol and dihydrolanosterol. The method makes use
of two different routes to a common intermediate 24,25-
dihydroxylanosterol acetate, which is then readily converted
to lanosterol. High yields are made possible by the use of
hypophosphorous acid as a reagent for the ring opening of
the intermediate epoxides, or as a catalyst for the hydroxy-
halogenation.
Acknowledgements
We thank Dr Stephen Parker of the Horticultural and Food
Research Institute of New Zealand, Ltd. for helpful discus-
sion. This work was supported by a grant from the Vice-
Chancellor and from the School of Science and Technology
of the University of Waikato.
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than 90% yields at each stage. This new method of separation
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We believe that one-pot dihydroxylation reaction of the
24,25-double bond of lanosterol through hydroxyhalides
could be successfully applied to other olefins as well.