Biosynthesis of 20-hydroxyecdysone in Ajuga hairy roots: Fate of 6α- and 6β-hydrogens of lathosterol

Article abstract of DOI:10.1016/S0968-0896(99)00243-6

The fate of 6α- and 6β-hydrogens of lathosterol during the transformation into 20-hydroxyecdysone was chased by feeding [3α,6β-²H₂]- and [3α,6α-²H₂]-lathosterols to hairy roots of Ajuga reptans var. atropurpurea. The behavior of 6β-hydrogen, which mostly migrated to the C-5 position of 20-hydroxyecdysone, was in agreement with that of C-6 hydrogen of cholesterol. The results strongly supported the view that cholesterol and lathosterol are first metabolized into 7-dehydrocholesterol, which is then converted into 20-hydroxyecdysone via 7-dehydrocholesterol 5α,6α-epoxide in the hairy roots. Copyright (C) 1999 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(99)00243-6

Bioorganic & Medicinal Chemistry 7 (1999) 2925±2930
Biosynthesis of 20-Hydroxyecdysone in Ajuga Hairy Roots:
Fate of 6ꢀ- and 6ꢁ-Hydrogens of Lathosterol
Kiyoshi Ohyama, Tetsuo Kushiro, Kinya Nakamura and Yoshinori Fujimoto*
Department of Chemistry and Materials Science, Tokyo Institute of Technology, Meguro, Tokyo 152-8551, Japan
Received 3 June 1999; accepted 23 July 1999
AbstractÐThe fate of 6a- and 6b-hydrogens of lathosterol during the transformation into 20-hydroxyecdysone was chased by
feeding [3a,6b-2H2]- and [3a,6a-2H2]-lathosterols to hairy roots of Ajuga reptans var. atropurpurea. The behavior of 6b-hydrogen,
which mostly migrated to the C-5 position of 20-hydroxyecdysone, was in agreement with that of C-6 hydrogen of cholesterol. The
results strongly supported the view that cholesterol and lathosterol are ®rst metabolized into 7-dehydrocholesterol, which is then
converted into 20-hydroxyecdysone via 7-dehydrocholesterol 5a,6a-epoxide in the hairy roots. # 1999 Elsevier Science Ltd. All
rights reserved.
Introduction
roots, the insects Schistocerica gregaria11 and the fern
Polypodium vulgare.12 This suggests that three distinct
mechanisms are operating in the construction of the 5b-
H-7-en-6-one structure of 20-hydroxyecdysone. Further
studies on the mechanism of the early stages of ecdy-
steroid biosynthesis are thus required.
20-Hydroxyecdysone (1) is a molting hormone of
most arthropods and it is also distributed in the plant
kingdom. The structure of the steroidal hormone is
characterized by polyhydroxyl groups, a 7-en-6-one
conjugated system and a cis-A/B-ring junction. It is well
documented that cholesterol (2) serves as a biosynthetic
precursor of the hormone in both insects and plants.1
However, the mechanism of the early stages in 20-
hydroxyecdysone biosynthesis (i.e. the mechanism of
the formation of the 5b-H-7-en-6-one structure) has
remained uncertain.1±3
Adler and his co-workers have demonstrated that
lathosterol (6) is incorporated into 1 in spinach leaves
without information of the intermediary role of 7-dehy-
drocholesterol.13 We expected that feeding studies of
labeled lathosterols would provide a clue to the
mechanism of the early stages of 20-hydroxyecdysone
biosynthesis. The present paper describes the fate of 6a-
and 6b-hydrogens of 6 during the conversion into 1. In
addition, a feeding study of a postulated intermediate,
7-dehydrocholesterol, is described.
In previous papers, we reported that 3a, 4a, and 4b-
hydrogens of 2 are retained at the original positions
after the conversion into 1,4 and that 3b-hydroxy-5b-
cholest-7-en-6-one (5b-ketol) (3) is converted into 14 in
hairy roots of Ajuga reptans var. atropurpurea.5,6
Recently, we have found that most of C-6 hydrogen of
cholesterol migrates to the C-5 position of 1.7 On the
basis of these ®ndings, together with the obligatory
intermediacy of 7-dehydrocholesterol 4 in insects,1±3 we
proposed a pathway, 2!4!5!3!1 (Scheme 1), in
Ajuga hairy roots. An intermediary role of 7-dehy-
drocholesterol 5a,6a-epoxide 5 has been previously
postulated by our8 and other9,10 groups.
Results and Discussion
A preliminary feeding of [3a-2H]lathosterol14 to Ajuga
hairy roots showed that 6 is incorporated into 1 as
e€ectively as [3a-2H]cholesterol. Then, [6b-2H]lathos-
terol (6a) (for the synthesis, see Scheme 2) was fed to see
2
the fate of 6b-hydrogen. The H NMR spectrum of the
biosynthesized 1 exhibited a signal at d 2.95 (data not
shown), which corresponds to the 5-H signal of 1. The
results indicated that 6b-hydrogen of 6 migrates to the
C-5 position of 1 during the conversion. The fate of
6b-hydrogen of 6 coincides with that reported for C-6
hydrogen of 2.7 The observed 1,2-hydrogen migration
ruled out a direct C-6 oxidation pathway of 6 leading to
Interestingly, the metabolic fates of 3a-, 4a-, 4b- and
6-hydrogens of 2 are di€erent between Ajuga hairy
* Corresponding author. Tel.: +81-3-5734-2241; fax: +81-3-5734-
7655; e-mail: fujimoto@cms.titech.ac.jp
0968-0896/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(99)00243-6

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K. Ohyama et al. / Bioorg. Med. Chem. 7 (1999) 2925±2930
Scheme 1. Conversion of cholesterol (2) and lathosterol (3) into 20-hydroxyecdysone (1) via 7-dehydrocholesterol in Ajuga hairy roots with 1,2-shift
of hydrogen (Hb) from C-6 to C-5.
Scheme 2. Synthesis of [3a,6b-2H2]lathosterol. Reagents and conditions: (i) NBS; Bu4NBr, Bu4NF; KOH-MeOH (32%), (ii) Raney Ni (91%), (iii)
PCC (91%), (iv) LiAlD4 (71%).
3, since the route cannot account for the behavior of 6b-
hydrogen of 6. This implies that 6 has to be converted
into 4, which would serve as a substrate for the sub-
sequent oxidation leading to the epoxide 5.
[3a,6a-2H2]Lathosterol (6c) was synthesized from the
known bromoketone (10)16 according to Scheme 3. Thus,
compound 10 was converted into [6a-2H]-3b,7a-diol
(14) via bromohydorin 11, 6-ene 12 and 6a,7a-epoxide
13, according to a slight modi®cation of the published
method.18,19 Mono-benzoylation of 14 gave 15 which,
upon dehydration, a€orded [6a-2H]lathosterol 16. Com-
pound 6c was obtained from 16 via 3-ketone 17 in the
same manner as described for the conversion of 6a to 6b.
We have recently reported that ca 70% of C-5 hydrogen
of 1 originates from C-6 hydrogen of 2, whereas the
remaining (30%) comes from another source, pre-
sumably from water.7 If the fate of 6b-hydrogen of 6 is
the same as that of C-6 hydrogen of 2, it would further
substantiate the obligatory intermediacy of 4. To
obtain such a quantitative information, [3a,6b-2H2]-
and [3a,6a-2H2]-lathosterols (6b and 6c ) were synthe-
sized and subjected to the feeding. The doubly deuter-
ium-labeled substrates were used to eliminate a possible
deuterium isotope e€ect.
The two labeled sterols, 6b and 6c, were separately fed
to Ajuga hairy roots. The 2HNMR spectra of the
resulting 20-hydroxyecdysone (1) are shown in Figure 1.
20-Hydroxyecdyone derived from 6b exhibited two
peaks at d 2.95 and 4.18 in the ratio of ca 7:10, which
correspond to 5b-H and 3a-H of 1, respectively. In
contrast, 1 derived from 6c showed a single peak at d
4.18. The results unambigously established the loss of
6a-hydrogen of 6 and the migration of 6b-hydrogen of 6
to the C-5 position of 1. This is in accord with the
known steric course, i.e. cis-elimination in the forma-
tion of Á5-sterol from Á7-sterol in plants.20±22 More
importantly, the behavior of 6b-hydrogen of 6 (ca 70%
shifted to C-5 of 1) is in excellent agreement with that
of C-6 hydrogen of 2. The whole data described above
further supported that 2 and 6 once lead to 4 which, in
turn, is transformed into 1 in Ajuga hairy roots and
presumably in spinach leaves.
The synthesis of 6b is outlined in Scheme 2. [6-2H]Cho-
lesterol acetate (7) was converted into [6-2H]-7-dehy-
drocholesterol (8) according to the method of Rappoldt
et al.15 Hydrogenation of 8 with Raney nickel a€orded
[6b-2H]lathosterol (6a).16,17 This was converted into
[3a,6b-2H2]lathosterol (6b) via 3-ketone (9).
The sterol fraction was separated from the hairy roots
in the above experiments and cholesterol was isolated

K. Ohyama et al. / Bioorg. Med. Chem. 7 (1999) 2925±2930
2927
Scheme 3. Synthesis of [3a,6a-2H2]lathosterol. Reagents and conditions: (i) LiAlD4 (34%), (ii) Zn, AcOH; Ac2O/Py (81%), (iii) mCPBA (99%), (iv)
LiAlH4(100%), (v) BzCl/Py (73%), (vi) POCl3/Py; KOH, MeOH (38%), (vii) PCC (71%), (viii) LiAlD4 (86%).
Figure 1. The 2H-NMR spectra (77 MHz, pyridine) of 20-hydroxyecdysone (1). Top: derived from [3a,6b-2H2]lathosterol (6b); Bottom: derived from
[3a,6a-2H2]lathosterol (6c). The three intense signals at d 7.19, 7.56 and 8.69 are those of the solvent.
by HPLC. The 2HNMR spectrum of cholesterol derived
from 6b showed two signals at d 3.51 and 5.35 due to
3-2H and 6-2H, respectively, in equal intensity, whereas
that from 6c showed a single signal at d 3.51. This ®rmly
established that the elimination of 6a-hydrogen is highly
stereospeci®c and no loss of 6b-hydrogen of 6 takes
place during the conversion of 6 into 2 via 4. Moreover,
we recently proved that C-5 hydrogen of 5b-ketol 3 is
retained completely during the subsequent conversion
into 1.23 Thus, 30% of C-5 hydrogen of 1, not derived
from 6b-H of 6, should be introduced in the step of cis-
A,B-ring formation, i.e. 5!3.
of 20-hydroxyecdysone, whereas the remaining (ca
30%) of C-5 hydrogen of 20-hydroxyecdysone origi-
nated from another hydrogen source. This observation
is very similar to that found in C-6 hydrogen of cho-
lesterol. (3) 7-Dehydrocholesterol was shown to be
converted into 20-hydroxycdysone in plants for the ®rst
time.
The present ®ndings strongly suggested that the inter-
mediacy of 7-dehydrocholesterol as well as 7-dehydro-
cholesterol 5a,6a-epoxide (Scheme 1). The partial (ca.
30%) loss of Hb (see Scheme 1) appears to be due to the
instability of the epoxide, but a precise mechanism is an
open question.
Although 7-dehydrocholesterol (4) is accepted as an
obligatory intermediate in ecdysteroid biosynthesis in
insects, no feeding studies of 4 have been reported in
plants. Thus, [4-13C]-7-dehydrocholesterol24 was fed to
Ajuga hairy roots to see whether it is metabolized into 1
or not. Figure 2 shows the 13C-NMR spectrum of the
resulting 1, indicating incorporation of 4 into 1. Rather
poor incorporation of 4 into 1, compared to that found
in cholesterol, may be attributed to the instability of 4 in
the incubation medium.
In conclusion, the present studies have provided several
lines of evidence on the early steps of ecdysteroid bio-
synthesis in Ajuga hairy roots: (1) lathosterol was
incorporated into 20-hydroxyecdysone. (2) Ca 70% of
6b-hydrogen of lathosterol migrated to the C-5 position
Figure 2. The 13C-NMR spectrum (100 MHz, pyridine-d5) of 20-
hydroxyecdysone (1) derived from [4-13C]-7-dehydrocholesterol.

2928
K. Ohyama et al. / Bioorg. Med. Chem. 7 (1999) 2925±2930
Experimental
122±122.5ꢀC). 1H NMR d: 0.53 (3H, s, 18-H3), 0.79
(3H, s, 19-H3), 0.86 (6H, d, J=6.8 Hz, 26, 27-H3), 0.92
1H NMR spectra were obtained on a JEOL EX-400
(400 MHz) or JEOL GSX-500 (500 MHz) spectrometer
in CDCl3 solutions and chemical shifts (d) are reported
in ppm down®eld from tetramethylsilane (used as inter-
nal reference). 13C-NMR spectra were recorded on the
same spectrometer (100 or 125 MHz) and chemical
shifts are referenced to the CDCl3 solvent (d 77.0) or
(3H, d, J=6.8 Hz, 21-H3), 3.59 (1H, m, 3-H), 5.14 (1H,
2
brd, J=4.0 Hz, 7-H). H NMR d: 1.74 (6b-2H). 13C-
NMR d: 29.24 (t, J=73 Hz, C-6). Anal. Calcd. for
C27H45DO: C, 83.65%; H+D, 11.96%. Found: C,
83.60%; H+D, 12.26%.
[6ꢁ-2H]-5ꢀ-Cholest-7-en-3-one (9). Molecular seives 4A
(powder, 340 mg) and PCC (340 mg, 1.55 mM) were
added to a solution of 6a (300 mg, 0.775 mM) in dry
CH2Cl2 (10 mL) and the mixture was stirred at room
temperature for 4 h. The mixture was diluted with dry
ether and ®ltered through a Florisil pad. Concentration
of the ®ltrate gave a crude ketone which was chroma-
tographed over silica gel with hexane:AcOEt (30:1) to
a€ord 14 (273 mg, 91%) as white crystals, mp 142±
143ꢀC (from acetone) (lit.28 143±145ꢀC for unlabeled
2
2,5-C of pyridine-d5 (d 149.8). H NMR spectra were
obtained on a JEOL GSX-500 (77 MHz) spectrometer
in a CHCl3 or pyridine solution and chemical shifts (d)
are referenced to d 7.26 for CHCl3 or d 7.19 for 2,5-H2
of pyridine. HPLC were performed on Shimadzu LC-
6A with SPD-6A UV detector using an ODS column.
[6-2H]Cholesterol acetate (7). This compound, mp 114±
116ꢀC (lit.25 114±115ꢀC), was prepared from 3b-tetra-
hydropyranyloxy-5a-cholestan-6-one26 in four steps
(reduction with LiAlD4, dehydration with POCl3/Py,
deprotection of the THP group under an acidic condi-
tion, and acetylation with Ac2O/Py).
1
compound). H NMR d: 0.56 (3H, s, 18-H3), 0.87 (6H,
d, J=6.5 Hz, 26, 27-H3), 0.93 (3H, d, J=6.2 Hz, 21-
H3), 1.01 (3H, s, 19-H3), 5.18 (1H, brs, 7-H). Anal.
Calcd. for C27H43DO: C, 84.09%; H+D, 11.50%.
Found: C, 84.38%; H+D, 11.79%.
[6-2H]Cholesta-5,7-dien-3ꢁ-ol (8). N-bromosuccinimide
(1.00 g, 5.62 mM) was added to a stirred solution of 7
(2.80 g, 6.53 mM) in dry CCl4 (100 ml) under N2 and
the mixture was heated at re¯ux for 10 min and then
cooled to 0ꢀC. The resulting precipitate was ®ltered o€
and the ®ltrate was concentrated to dryness. The resi-
due, dissolved in dry THF (15 ml), was treated with
tetra-N-butylammonium bromide (29 mg, 0.091 mM).
The mixture was stirred at room temperature for 45 min
in the dark. Tetra-N-butyl-ammonium ¯uoride in THF
(1 M solution, 20 ml) was added and the mixture was
stirred for 15 min in the dark. AcOEt and water were
added to the mixture and the separated organic layer
was washed with brine, dried over Na2SO4 and con-
centrated. The residue was chromatographed over silica
gel with hexane:AcOEt (50:1) to give a crude diene (2.3
g). This was further puri®ed on an AgNO3 (20 g)
impregnated silica gel (100 g) column hexane:CH2Cl2
(2:1) to give the diene acetate (1.3 g) as a yellow solid.
This was dissolved in KOH/THF±MeOH (KOH 1.0 g,
THF 25 ml, MeOH 75 ml) and stirred at room tem-
perature for 1 h. Extractive (ether) workup and recrys-
tallization of the residue from acetone a€orded 8 (812
mg, 32%) as white crystals, mp 132±133ꢀC (lit.27 143ꢀC
[3ꢀ,6ꢁ-2H2]Lathosterol (6b). LiAlD4 (14 mg, 0.333
mM) was added to a stirred solution of 9 (254 mg, 0.660
mM) in dry ether (10 mL) at room temperature under
N2. The mixture was stirred for 30 min and then diluted
with moist ether and dil. HCl. Extractive (AcOEt) work
up and puri®cation of the residue on a silica gel Lobar
column with hexane:AcOEt (6:1) gave 6b (196 mg,
71%) as a white solid, mp 124±125ꢀC (from MeOH)
(lit.18 120±123ꢀC for unlabeled compound). 1H NMR d:
0.53 (3H, s, 18-H3), 0.79 (3H, s, 19-H3), 0.86 (6H, d,
J=6.4 Hz, 26, 27-H3), 0.92 (3H, d, J=6.4 Hz, 21-H3),
2
5.16 (1H, brd, J=5.8 Hz, 7-H). H NMR d: 1.74 (6b-
2H), 3.58 (3-2H). Anal. calcd for C27H44D2O: C,
83.44%; H+D, 11.93%. Found: C, 83.53%; H+D,
12.04%.
[6ꢀ - 2H] - 7ꢀ - Bromo - 5ꢀ - cholestane - 3ꢁ,6ꢁ - diol (11).
LiAlD4 (1.00 g, 9.07 mM) was added to a stirred solu-
tion of 3b-acetoxy-7a-bromo-5a-cholestan-6-one 1018
(2.80 g, 5.32 mM) in dry ether (30 mL) at 0ꢀC under N2
and the mixture was stirred for 1.5 h. The mixture was
worked up in a manner similar to that described for 6b
[Lobar column was eluted with CHCl3:AcOEt (10:1)] to
a€ord 11 (894 mg, 34%), mp 95±97ꢀC. 1H NMR d: 0.71
(3H, s, 18-H3), 0.87 (6H, d, J=5.9 Hz, 26, 27-H3), 0.91
(3H, d, J=6.8 Hz, 21-H3), 1.03 (3H, s, 19-H3), 3.71 (1H,
m, 3-H), 3.92 (1H, brs, 6-H), 4.24 (1H, brs, 7-H). Anal.
Calcd. for C27H46DBrO2: C, 66.53%; H+D, 10.81%.
Found: C, 66.92%; H+D, 9.78%.
1
for unlabeled compound). H NMR d: 0.62 (3H, s, 18-
H3), 0.86 (6H, d, J=6.8 Hz, 26, 27-H3), 0.94 (3H, d,
J=6.2 Hz, 21-H3), 0.94 (3H, s, 19-H3), 3.63 (1H, m, 3-
H), 5.38 (1H, brs, 7-H). Anal. Calcd. for C27H43DO: C,
84.10%; H+D, 11.50%. Found: C, 83.82%; H+D,
11.83%.
[6ꢁ-2H]Lathosterol (6a). Raney nickel (W-2) (40 mg)
was added to a solution of 8 (812 mg, 2.11 mM) in dry
dioxane (11 ml) and the mixture was stirred for 2 days
at room temperature under H2. The catalyst was
removed by ®ltration through a pad of Celite. The ®l-
trate was washed with brine and the organic layer was
dried over Na2SO4 and concentrated. The residue
was recrystallized from 90% aqueous acetone to give 6a
(744 mg, 91%) as white crystals, mp 123±124ꢀC (lit.18
[6-2H]-3ꢁ-Acetoxy-5ꢀ-cholest-6-ene (12). Zn powder
(1.80 g, 334 mM) was added to a solution of 11 (894 mg,
1.85 mM) in acetic acid (20 mL) and the mixture was
heated at re¯ux for 1.5 h under N2. The mixture was
cooled to room temperature and diluted with water and
AcOEt. Extractive (AcOEt) work up gave 6-en-3b-ol
(696 mg) as a white solid. This was treated with pyridine
and acetic anhydride to give a crude acetate which was
chromatographed over silica gel hexane:AcOEt (50:1).

K. Ohyama et al. / Bioorg. Med. Chem. 7 (1999) 2925±2930
2929
1
Recrystallization of the residue from MeOH gave 12
(644 mg, 81%) as white crystals, mp 108±109ꢀC (lit.19
MeOH) (lit.18 120±123ꢀC for unlabeled compound). H
NMR d: 0.53 (3H, s, 18-H3), 0.86 (6H, d, J=6.6 Hz, 26,
27-H3), 0.92 (3H, d, J=6.6 Hz, 21-H3), 0.80 (3H, s, 19-
H3), 3.59 (1H, m, 3-H), 5.15 (1H, brs, 7-H). 2H NMR d:
1.77 (6a-2H). Anal. Calcd. for C27H45DO: C, 83.65%;
H+D, 11.50%. Found: C, 83.50%; H+D, 12.24%.
ꢀ
1
105.8±106.6 C for unlabeled compound), H NMR d:
0.69 (3H, s, 18-H3), 0.86 (6H, d, J=6.5 Hz, 26, 27-H3),
0.91 (3H, d, J=6.5 Hz, 21-H3), 0.79 (3H, s, 19-H3), 2.03
(3H, s, Ac), 4.73 (1H, m, 3-H), 5.48 (1H, brs, 7-H).
Anal. Calcd. for C29H47DO2: C, 81.06%; H+D,
11.26%. Found: C, 80.93%; H+D, 11.54%.
[6ꢀ-2H]-5ꢀ-Cholest-7-en-3-one (17). Compound 16 (336
mg, 0.87 mM) was oxidized in a manner similar to that
described for 9 to a€ord 17 (238 mg, 71%) as a white
solid, mp 141±142ꢀC (from acetone). (lit.27 143±145ꢀC
[6ꢁ-2H]-3ꢁ-Acetoxy-6ꢀ,7ꢀ-epoxy-5ꢀ-cholestane (13).
A solution of 12 (612 mg, 1.43 mM) in dry CH2Cl2 (10
mL) was treated with mCPBA (492 mg, 2.85 mM) and
the mixture was stirred at room temperature for 1 h
under N2. Addition of sat. aq NaHCO3 and extractive
(AcOEt) workup gave a crude product which was chro-
matographed over silica gel column hexane:AcOEt
(10:1) to give 13 (629 mg, 99%) as a white solid, mp
180±181ꢀC (lit.18 176.5±177.8ꢀC for unlabeled com-
1
for unlabeled compound). H NMR d: 0.56 (3H, s, 18-
H3), 0.87 (6H, d, J=6.6 Hz, 26, 27-H3), 0.93 (3H, d,
J=6.5 Hz, 21-H3), 1.02 (3H, s, 19-H3), 5.18 (1H, brs, 7-
H). Anal. Calcd. for C27H43DO: C, 84.09%; H+D,
11.50%. Found: C, 84.33%; H+D, 11.73%.
[3ꢀ,6ꢀ-2H2]Lathosterol (6c). Compound 17 (227 mg,
0.59 mM) was reduced with LiAlD4 (12 mg, 0.29 mM)
in a manner similar to that described for 11 to give 6c
(206 mg, 86%) as a white solid, mp 124±125ꢀC (from
1
pound). H NMR d: 0.71 (3H, s, 18-H3), 0.87 (6H, d,
J=6.5 Hz, 26, 27-H3), 0.90 (3H, d, J=6.8 Hz, 21-H3),
0.79 (3H, s, 19-H3), 2.04 (3H, s, Ac), 3.02 (1H, d, J=2.2
Hz, 7-H), 4.74 (1H, m, 3-H). Anal. Calcd. for C29H47
DO3: C, 78.15%; H+D, 10.85%. Found: C, 78.27%;
H+D, 11.14%.
1
MeOH). H NMR d 0.53 (3H, s, 18-H3), 0.86 (6H, d,
J=6.8 Hz, 26, 27-H3), 0.92 (3H, d, J=6.5 Hz, 21-H3),
0.79 (3H, s, 19-H3), 5.15 (1H, brs, 7-H). Anal. calcd for
C27H44D2O: C, 83.44%; H+D, 11.93%. Found: C,
83.39%; H, 12.23%.
[6ꢀ-2H]-5ꢀ-Cholestane-3ꢁ,7ꢀ-diol (14). Reduction of
13 (597 mg, 1.34 mM) with LiAlH4 (357 mg, 9.39 mM)
and workup in a manner similar to that described for 6b
gave, after concentration of the extract, 14 (600 mg,
quant.) as a white solid, mp 156±157ꢀC (from MeOH)
(lit.29 151±152ꢀC for unlabeled compound). 1H NMR d:
0.66 (3H, s, 18-H3), 0.86 (6H, d, J=6.5 Hz, 26, 27-H3),
0.91 (3H, d, J=6.2 Hz, 21-H3), 0.81 (3H, s, 19-H3), 3.63
(1H, m, 3-H), 3.83 (1H, brs, 7-H). Anal. Calcd. for
C27H45DO2: C, 79.94%; H+D, 11.93%. Found: C,
80.18%; H+D, 12.23%.
Incubation with Ajuga hairy roots. Ajuga hairy roots
were maintained as described previously.6 To pre-incu-
bated cultures (four 500-mL ¯asks, each containing 250
mL of MS medium) were added the labeled sterols (100
mg each), 6a, 6b or 6c, dissolved in acetone (4 mL) and
Tween 80 (2 mL) as described previously.5 The roots,
weighing ca 110 g (wet wt), were extracted and sepa-
rated to furnish 4.2, 2.1 and 2.8 mg of 1 from 6a, 6b and
6c, respectively, as described previously.5 The sterol
fraction was obtained from the fractions eluted with
CHCl3:MeOH (10:1±7:1) and chromatographed again
over silica gel with hexane-AcOEt (3:1) to yield a pur-
i®ed sterol fraction. A portion of the sterol fraction was
further puri®ed by HPLC to furnish cholesterol (col-
umn, Shimadzu Shimpack CLC-ODS; 15Â6 mm i.d.;
solvent, MeOH; ¯ow rate, 1.0 ml/min; detection at 210
nm, retention time 16.6 min).
[6ꢀ-2H]-5ꢀ-Cholestane-3ꢁ,7ꢀ-diol 3-benzoate (15).
BzCl (332 mL, 2.83 mM) was added to a stirred solu-
tion of 14 (574 mg, 1.42 mM) in pyridine (5 mL) at
room temperature under N2. The mixture was reacted at
room temperature for 2 h. Ice chips were added and the
mixture was stirred for 10 min. Extractive (AcOEt)
workup gave a crude product which was chromato-
graphed over silica gel with hexane:AcOEt (10:1) to
a€ord 15 (526 mg, 73%) as a white solid, mp 163±165ꢀC
[4-13C]-7-Dehydrocholesterol (41 mg) was fed to the
hairy roots in a similar manner to give 1.1 mg of 20-
hydroxyecdysone.
1
(from MeOH). H NMR d: 0.67 (3H, s, 18-H3), 0.87
(6H, d, J=6.5 Hz, 26, 27±H3), 0.91 (3H, d, J=6.5 Hz,
21-H3), 0.88 (3H, s, 19-H3), 3.84 (1H, brs, 7-H), 4.97
(1H, m, 3-H), 7.4±8.1 (5H, m, aromatic). Anal. Calcd.
for C34H51DO3: C, 80.11%; H+D, 10.28%. Found: C,
80.39%; H+D, 10.27%.
Acknowledgements
We thank Drs. T. Matsumoto and N. Tanaka, Bioassay
Laboratory, Research Center, Daicel Chemical Indus-
tries Ltd., for providing Ajuga hairy root culture.
[6ꢀ-2H]Lathosterol (16). POCl3 (174 mL, 1.87 mM) was
added to a solution of 15 (640 mg, 1.26 mM) in pyridine
(6.0 mL) at room temperature under N2 and the mixture
was stirred overnight. Water and AcOEt were added to
the mixture and extractive (AcOEt) work up gave 7-ene
(573 mg). This was treated with 10% KOH±MeOH
(3 mL) and THF (6 mL) at room temperature for 1 h.
Extractive (AcOEt) work up and puri®cation of the
residue over silica gel with hexane:AcOEt (9:1) a€orded
16 (183 mg, 38%) as a white solid, mp 123±125ꢀC (from
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