Stereoselective synthesis and moulting activity of 2,3-diepi-20- hydroxyecdysone and 2,3-diepi-5α-20-hydroxyecdysone

Article abstract of DOI:10.1016/j.tet.2004.02.042

The ecdysteroid analogues 2,3-diepi-20-hydroxyecdysone and 2,3-diepi-5α-20-hydroxyecdysone have been synthesized from the readily available ecdysteroid, 20-hydroxyecdysone, and moulting activity has been determined using the Musca bioassay. As expected, the 2,3-diepi-analogue was less active than the parent ecdysteroid, 20-hydroxyecdysone. However, the 2,3-diepi-5α-analogue, which was expected to be inactive in the assay, exhibited moulting activity though it was approximately 1.5-fold less active than its 5β-analogue. The activity of the 5α-analogue could possibly result from the ability of this compound to bind to the ecdysteroid receptor. Alternatively, a possible in vivo C-5 epimerization of the 2,3-diepi-5α- analogue to the corresponding 5β-analogue could account for its activity.

Full text of DOI:10.1016/j.tet.2004.02.042

Tetrahedron 60 (2004) 3433–3438
Stereoselective synthesis and moulting activity of
2,3-diepi-20-hydroxyecdysone
and 2,3-diepi-5a-20-hydroxyecdysone
Sureeporn Homvisasevongsa,a Aporn Chuaynugul,a Nitirat Chimnoib and Apichart Suksamrarna
,
*
aDepartment of Chemistry, Faculty of Science, Ramkhamhaeng University, Huamark, Bangkapi, Bangkok 10240, Thailand
bChulabhorn Research Institute, Vipavadee-Rangsit Highway, Bangkok 10210, Thailand
Received 14 November 2003; revised 27 January 2004; accepted 19 February 2004
Abstract—The ecdysteroid analogues 2,3-diepi-20-hydroxyecdysone and 2,3-diepi-5a-20-hydroxyecdysone have been synthesized from
the readily available ecdysteroid, 20-hydroxyecdysone, and moulting activity has been determined using the Musca bioassay. As expected,
the 2,3-diepi-analogue was less active than the parent ecdysteroid, 20-hydroxyecdysone. However, the 2,3-diepi-5a-analogue, which was
expected to be inactive in the assay, exhibited moulting activity though it was approximately 1.5-fold less active than its 5b-analogue. The
activity of the 5a-analogue could possibly result from the ability of this compound to bind to the ecdysteroid receptor. Alternatively, a
possible in vivo C-5 epimerization of the 2,3-diepi-5a-analogue to the corresponding 5b-analogue could account for its activity.
q 2004 Elsevier Ltd. All rights reserved.
1. Introduction
Ecdysteroids are arthropod moulting hormones found in
invertebrates and plant species and 20-hydroxyecdysone (1)
is a representative of this class of compounds.1–3 The
physiological functions in invertebrates including insects
are to control moulting and metamorphosis processes and
are involved in the control of reproduction. Essential
features contributing to high moulting activity include a
cis-A/B ring junction, a 6-keto-7-ene system, a full sterol
side chain and a free 14a-hydroxyl group.4 The number,
location and stereochemistry of the hydroxyl groups in the
molecule are also responsible for the high activity of
ecdysteroids. Many works indicated that the 3b-hydroxyl
group is required for high activity of ecdysteroids and that
the 2-hydroxyl group is not essential to such activity.4–6
Previous works have shown that moulting activity of
ecdysteroids decreased in going from the 3b-hydroxyl to
the corresponding 3a-hydroxyl analogues.4 All active
ecdysteroids have a cis-fused A/B ring junction, whereas
the 5a-epimers are inactive.4,7 5a-Ecdysteroid analogues,
which have a trans-A/B ring junction, have an approxi-
mately planar ring structure while ecdysteroids (5b) are
non-planar in the region of the A ring with C-2, C-3 and C-4
lying below the plane of the other rings.
It was found that the brassinosteroid castasterone (2), the
7,8-dihydro analogue of ecdysteroid with the 2a,3a-
dihydroxyl groups and 5a-orientation, inhibited ecdysteroid
activity and the effects could be explained by competitive
displacement of ecdysteroids, for example, compound 1.8
By comparing molecular models of 1 and 2 it was found that
the 3b-hydroxyl group of 1 and the 3a-hydroxyl group of 2
Keywords: Ecdysteroid; 2,3-diepi-20-Hydroxyecdysone; 2,3-diepi-5a-20-
Hydroxyecdysone; Synthesis; Moulting activity.
*
Corresponding author. Tel.: þ662-3191900; fax: þ662-3108381;
e-mail address: apichart@ram1.ru.ac.th
0040–4020/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2004.02.042

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S. Homvisasevongsa et al. / Tetrahedron 60 (2004) 3433–3438
product. The presence of the mesylate groups at the 2- and
3-positions in 7 was evident from a 1.13 and 1.11 ppm
downfield shifts of H-2 and H-3 as compared with that of the
starting acetonide 5 and the presence of two mesylate
methyl signals at d 3.10 and 3.11 in the 1H NMR spectrum
of 7. Reaction of 7 with NaI and Zn in DMF at 80 8C
afforded the olefin acetonide 8 in 75% yield. The ESMS
exhibited a sodiated molecular ion [MþNa]þ at m/z 509 and
a pseudo-molecular ion [MþH]þ at m/z 487, corresponding
to a molecular formula of C30H46O5. This was confirmed by
positive-ion HRFABMS which exhibited a pseudo-molecu-
lar ion [MþH]þat m/z 487.3428. The 1H NMR spectrum of
8 indicated the presence of two olefinic (H-2 and H-3)
signals at d 5.52 and 5.69. The rest of the 1H NMR spectro-
scopic data were consistent with structure (Scheme 1).
Figure 1. The A-ring geometry of the ecdysteroid 1 (solid line) and
brassinosteroid 2 (broken line) and the near spatial coincidence of the 3b-
hydroxyl group in 1 with the 3a-hydroxyl group in 2. Some hydrogens are
omitted in the structure for clarity.
occupied the same space (Fig. 1) and this might lead to
effective competition in binding the ecdysteroid receptor of
2. It was therefore of interest to study moulting activity of a
2,3-diepi-5a-ecdysteroid and, for comparison, the activity
of a 2,3-diepi-ecdysteroid should also be evaluated.
The present work deals with the stereoselective synthesis
of 2,3-diepi-20-hydroxyecdysone (3) and 2,3-diepi-5a-20-
hydroxyecdysone (4) from the readily available ecdysteroid
1 and evaluation of moulting activity of these two
ecdysteroid analogues. Compound 4 possesses a structural
framework in the A-ring region comparable to that of the
brassinosteroid 2.
Dihydroxylation of the olefin acetonide 8 with OsO4 in
pyridine, followed by treatment with 5% aq. NaHSO3
afforded two products in high yield. The more polar product
(58% yield) was identified as 20-hydroxyecdysone 20,22-
acetonide (5) by comparison of spectroscopic (IR, 1H NMR
and mass spectral) data with the reported value.9 The less
polar product (32% yield) was compound 9, the C-2 and C-3
epimer of 5. That the ecdysteroid 5 was the major product
was expected, since dihydroxylation of 8 took place on the
1
less-hindered b-face. The H and 13C NMR spectroscopic
signals of 9 were assigned from 2D (COSY, HMQC,
HMBC) and DEPT techniques and were found to be much
different from those of 5. A striking difference was the
unusual downfield H-9 signal of 9 (in C5D5N) at d 4.70,
whereas that of 5 appeared at d 3.54. A close proximity of
H-9 and the 2a-hydroxyl group resulted in a large downfield
shift of the former signal. A marked upfield shift of the H-5
signal of 9 at d 2.37, as compared with that of 5 at d 2.99,
was also noted. The strong steric interaction between the
2a-hydroxyl group and H-9 might lead to facile C-5
epimerization of 9. The structure of this minor dihydroxyl-
ation product could possibly, therefore, be 9 or its C-5
epimer of 9. To prove whether the stereochemistry at C-5
was in the b-orientation, a NOE experiment was performed.
The key experiment was to irradiate the 19-Me signal to see
if enhancement of the H-5 signal would occur.11 The result,
however, was not conclusive since the H-5 signal was
obscured by other signals. Compound 9 was therefore
subjected to acetylation to the corresponding 2,3,25-
triacetate acetonide 10. Assignments of the NMR spectro-
scopic signals of 10 were achieved by 2D NMR techniques.
Irradiation at the 19-Me frequency of 10 resulted in NOE
enhancement of the H-5 signal at d 2.20, whereas irradiation
at the H-9 frequency caused enhancement of the 2a-
acetoxyl signal at d 1.98. To confirm that the position of the
H-5 resonance was at d 2.20, the H-3 resonance was
irradiated and enhancement of this proton signal was also
observed. The results indicated that H-5 of compound 10
was in the b-orientation. It was thus concluded that
compound 9 was a 5b-ecdysteroid as shown. Acetonide
deprotection of 9 with 70% AcOH in the presence of the
phase transfer catalyst benzyltrimethylammonium chloride
afforded 2,3-diepi-20-hydroxyecdysone (3) in 81% yield.
2. Results and discussion
The first ecdysteroid analogue we planned to synthesize was
the 2,3-diepi-ecdysteroid 3. It was then expected that this
compound would be transformed into the corresponding
C-5 epimer, 2,3-diepi-5a-ecdysteroid 4, by base-catalyzed
epimerization. Starting from 1, the 20,22-acetonide 5 was
prepared by the literature procedure.9 Mesylation of 5 with
mesyl chloride in pyridine yielded the corresponding
2-mesylate 610 and 2,3-dimesylate 7 in 30 and 66% yields,
respectively. The former could be recycled in the mesyla-
tion step. Prolonged reaction time gave only the dimesylate
7, but it was usually accompanied by a less polar side
In order to increase the ratio of the product 9:5, asymmetric
dihydroxylation12 was investigated. The chiral ligands used
were those of the dihydroquinidine (DHQD) series, that is,

S. Homvisasevongsa et al. / Tetrahedron 60 (2004) 3433–3438
3435
Scheme 1. Synthesis of ecdysteroid analogues 3 and 4. Reagents and conditions: (a) CH3COCH3, p-TsOH; (b) MsCl, pyridine; (c) NaI, Zn, DMF, 80 8C;
(d) OsO4, ligand, solvent (see text); (e) Ac2O, pyridine; (f) 70% AcOH, PhCH2NMeþ3 Cl2; (g) 2% Na2CO3, MeOH; (h) 70% AcOH, PhCH2NMe3þCl2.
dihydroquinidine 4-methyl-2-quinolyl ether (DHQD-
MQE), dihydroquinidine 9-phenanthryl ether (DHQD-PE)
and dihydroquinidine 1,4-phthalazinediyl diether
(DHQD)2-PHAL), and the dihydroquinine (DHQ) series,
that is, DHQ-MQE, DHQ-PE and (DHQ)2-PHAL (Table 1).
The best 9:5 product ratio was 4:1, the chiral ligand of
which was (DHQD)2-PHAL. In this case the yield of 9 was
raised to 68%. The overall yield of 3 from the starting
ecdysteroid 1 was 25%, based on the utilization of the chiral
ligand (DHQD)2-PHAL in the dihydroxylation step.
11 indicated the proximity of this proton and the 3a-
hydroxyl group. The H-5 proton of 11 was further confirmed
to be in the a-orientation by NOE experiments. Thus
irradiation at the H-5 frequency did not cause enhancement
of the 19-Me signal at d 0.96, whereas irradiation at the H-9
frequency resulted in enhancement of the H-5 signal at d
3.02. Compound 11 was subjected to deacetonation to give
2,3-diepi-5a-20-hydroxyecdysone (4) in 72% yield, the
spectroscopic data of which were consistent with the
structure. The overall yields of 4 from the starting
ecdysteroid 1 was 18%, or 58% from compound 9.
To effect C-5 epimerization, compound 9 was treated with
2% Na2CO3 in MeOH and, as expected, epimerization
occurred more readily than the 2b,3b-dihydroxyl analogue7
(e.g., compound 5) to give the corresponding 5a-analogue
11 in 80% yield. The trans-A/B ring fusion of 11 was
evident from a large upfield shift (1.47 ppm) of H-9 in going
from 9 to 11. A downfield shift (0.65 ppm) of H-5 signal of
2.1. Biological activity
The Musca bioassay13 has been used to evaluate moulting
activity of ecdysteroid and their analogues in our study. As
expected for a 3a-hydroxy ecdysteroid, compound 3 which
is the 2a,3a-analogue of the parent ecdysteroid 1 was much
less active; it was 30-fold less active than compound 1.
Surprisingly, compound 4, the 5a-analogue of compound 3
and was expected to be inactive,4 was active in the assay. It
was 42-fold less active than compound 1. The activity of 4,
though it was approximately 1.5-fold less active than that of
3, deserved special attention. One possible explanation for
the activity of 4 was that it could bind to the ecdysteroid
receptor by analogy to that which occurred in castasterone
(2). Unlike 2, compound 4 possesses a 6-keto-7-ene system,
the essential structural requirement for moulting activity.
The a-nature of the 2- and 3-hydroxyl groups resulted in
effective binding to the receptor and this could possibly
Table 1. Asymmetric dihydroxylation of compound 8
Entry
Ligand
Ratio of products 5:9
Yield (%)
1
2
3
4
5
6
DHQD-MQE
DHQD-PE
(DHQD)2-PHAL
DHQ-MQE
DHQ-PE
5:6
7:3
1:4
7:3
3:1
5:4
77
83
85
76
80
84
(DHQ)2-PHAL
The ratio of the ligand:OsO4: compound 8 was 3:3:1. tert-BuOH–THF–
H2O (7:4:1) was used as a solvent.

3436
S. Homvisasevongsa et al. / Tetrahedron 60 (2004) 3433–3438
compensate the trans-A/B ring junction of 4. An alternative
explanation was that compound 4 could undergo in vivo C-5
epimerization to compound 3. The unfavorable steric
interaction of the 2a-hydroxyl group and H-9 in 3 could,
in part, be compensated for by the absence of 1,3-diaxial
interaction between 19-Me and the 2b- and 4b-H (see
Fig. 1). One supported example was the activity of
compound 12, a 2-deoxy-5a-ecdysteroid analogue, which
exhibited low moulting activity in the Musca assay.14
Whether the first or second hypothesis was more likely
could not be judged from the existing data.
and zinc dust (196 mg, 3 mmol) was added. The reaction
mixture was left to stir at 80 8C for 3 days. Water was added
and the mixture was extracted with CHCl3. The combined
CHCl3 layer was washed with water, dried over anhydrous
Na2SO4 and evaporated under reduced pressure to dryness.
The product was purified by column chromatography (silica
gel 30 g) using CHCl3–MeOH with gradually increasing
concentration of MeOH to give compound 8 (520 mg, 75%),
eluted by CHCl3–MeOH (99:1).
Compound 8. Colorless needles (from CHCl3–MeOH), mp
117–119 8C; nmax 3426, 2971, 1659, 1443, 1376, 1213,
1
1200 cm21; H NMR (400 MHz, CDCl3) d 0.75 (s, 3H,
18-Me), 1.02 (s, 3H, 19-Me), 1.12 (s, 3H, 21-Me), 1.20 (s,
3H, 26-Me), 1.21 (s, 3H, 27-Me), 1.29, 1.38 (each s, 2£3H,
acetonide Me), 2.22 (t, J¼ca 9 Hz, 1H, H-17), 2.85 (br m,
1H, H-9), 3.63 (br d, J¼7.8 Hz, 1H, H-22), 5.50 and 5.67
(each br d, J¼10 Hz, 2£1H, H-2 and H-3), 5.76 (d,
J¼1.3 Hz, 1H, H-7); ESMS (positive ion mode) m/z (%
rel. intensity) 995 [2MþNa]þ (25), 509 [MþNa]þ (100),
487 [MþH]þ (17); HRFABMS (positive ion mode) m/z
487.3428 [MþH]þ (calcd for C30H46O5þH, 487.3423).
3. Experimental
3.1. General experimental procedures
Melting points were determined on an Electrothermal
1
melting point apparatus and were uncorrected. H and 13C
NMR spectra were recorded on a Bruker AVANCE 400
spectrometer. FAB and ES mass spectra were measured on a
Finnigan MAT 90 and a Bruker Esquire-LC instruments.
Column chromatography and TLC were carried out using
Merck silica gel 60 (,0.063 mm) and precoated silica gel
60 F254 plates, respectively. Spots on TLC were visualized
under UV light and by spraying with anisaldehyde–H2SO4
reagent followed by heating.
3.1.3. Reaction of compound 8 with osmium tetroxide.
Synthesis of 2,3-diepi-20-hydroxyecdysone 20,22-aceto-
nide (9) and 20-hydroxyecdysone 20,22-acetonide (5). To
a solution of compound 8 (134 mg, 0.275 mmol) in pyridine
(0.8 mL) was added OsO4 in pyridine (0.55 mL, prepared by
dissolving 500 mg of OsO4 in 3 mL of pyridine and the
amount used was equivalent to 92 mg or 0.36 mmol of
OsO4). The mixture was stirred for 5 min and 5% NaHSO3
(2 mL) was added. Stirring was continued for 15 min and
the mixture was extracted with EtOAc (3£15 mL). The
combined organic layer was washed with water, dried over
anhydrous Na2SO4 and evaporated to dryness. The crude
mixture (150 mg) was subjected to column chromatography
(silica gel 25 g), using CHCl3–MeOH as eluting solvent,
with increasing amount of MeOH. Fractions eluted by
CHCl3–MeOH (95:5) afforded 46 mg (32%) of 2,3-diepi-
20-hydroxyecdysone 20,22-acetonide (9). The second frac-
tion (83 mg, 58%) eluted by CHCl3–MeOH (94:6), was
identified as 20-hydroxyecdysone 20,22-acetonide (5).
3.1.1. 20-Hydroxyecdysone 20,22-acetonide 2,3-dimesyl-
ate (7). Compound 59 (1.4 g, 2.69 mmol) was dissolved in
pyridine (4 mL) and the mixture stirred at 0–5 8C for
10 min, then mesyl chloride (1.5 mL, 19.30 mmol) was
added. The reaction mixture was left to stir at 0–5 8C for 1 h
and at ambient temperature for another 1 h. Water was
added and the mixture extracted with CHCl3 (4£25 mL).
The combined chloroform extract was washed with water,
dried over anhydrous Na2SO4 and evaporated under reduced
pressure. The residue was subjected to column chromato-
graphy (silica gel 50 g) using CHCl3–MeOH, with
gradually increasing concentration of MeOH, to give
20-hydroxyecdysone 20,22-acetonide 2,3-dimesylate (7)
(1.2 g, 66%) eluted by CHCl3–MeOH (98.5:1.5) and
20-hydroxyecdysone 20,22-acetonide 2-mesylate (6)
(490 mg, 30%), eluted by CHCl3–MeOH (96:4).
Compound 5. Colorless needles (from acetone–hexane), mp
221–223 8C (lit.9 222–224 8C); nmax 3423, 2974, 1649,
1
1454, 1377, 1216, 1170, 1103, 1057, 1001, 877 cm21; H
Compound 6. Spectroscopic (1H NMR and mass spectral)
data were identical to those reported in literature.10
NMR (400 MHz, C5D5N) d 1.00, 1.03 (each s, 2£3H,
18-Me, 19-Me), 1.32 (s, 3H, acetonide Me),a 1.34 (s, 3H,
26-Me), 1.35 (s, 3H, 27-Me), 1.44 (s, 3H, acetonide Me),
1.53 (s, 3H, 21-Me),a 2.75 (t, J¼8.6 Hz, 1H, H-17), 2.99
(dd, J¼13.1, 3.5 Hz, 1H, H-5), 3.54 (m, 1H, H-9), 3.93 (dd,
J¼9.6, 2.4 Hz, 1H, H-22), 4.16 (m, 1H, H-2), 4.22 (br s, 1H,
H-3), 6.24 (d, J¼2.1 Hz, 1H, H-7), (‘a’ stands for the
assignments may be reversed for signals with the same
Compound 7. Amorphous; nmax 3436, 2971, 1660, 1449,
1
1354, 1176, 1106, 1024, 909 cm21; H NMR (400 MHz,
CDCl3) d 0.77 (s, 3H, 18-Me), 1.03 (br s, 3H, 19-Me), 1.14
(s, 3H, 21-Me), 1.22 (s, 3H, 26-Me), 1.23 (s, 3H, 27-Me),
1.31, 1.39 (each s, 2£3H, acetonide Me), 2.22 (dd, J¼9.1,
7.9 Hz, 1H, H-17), 2.98 (m, 1H, H-9), 3.10, 3.11 (each s,
2£3H, 2-OMs, 3-OMs), 3.63 (dd, J¼9.4, 2.4 Hz, 1H, H-22),
4.94 (m, 1H, H-2), 5.12 (br s, 1H, H-3), 5.87 (d, J¼2.1 Hz,
1H, H-7); ESMS m/z (% rel. intensity) 699 [MþNa]þ(100)];
HRFABMS (positive ion mode) m/z 677.3045 [MþH]þ
(calcd for C32H52O11S22H, 677.3029).
1
superscript); H NMR (400 MHz, CDCl3) d 0.76 (s, 3H,
18-Me), 0.93 (s, 3H, 19-Me), 1.12 (s, 3H, 21-Me), 1.18 (s,
3H, 26-Me),b 1.26 (s, 3H, 27-Me),b 1.29,b 1.38 (each s,
2£3H, acetonide Me), 2.38 (dd, J¼12.9, 4.4 Hz, 1H, H-5),
2.95 (m, 1H, H-9), 3.59 (m, 1H, H-22), 3.81 (m, 1H, H-2),
4.01 (br s, 1H, H-3), 5.81 (d, J¼1.8 Hz, 1H, H-7), (‘b’ stands
for the assignments may be reversed for signals with the
same superscript); ESMS (positive ion mode) m/z (% rel.
intensity) 543 [MþNa]þ (100); HRFABMS (negative ion
3.1.2. 2,3-Didehydro-2,3-dideoxy-20-hydroxyecdysone
20,22-acetonide (8). Compound 7 (960 mg, 1.42 mmol)
was dissolved in DMF (5 mL) and NaI (820 mg, 5.47 mmol)

S. Homvisasevongsa et al. / Tetrahedron 60 (2004) 3433–3438
3437
mode) m/z 519.3322 [M2H]2. (calcd for C30H48O72H,
519.3321).
30.9, 31.9 (C-12, C-15), 35.0 (C-9), 36.1 (C-10), 36.7 (C-1),
38.5 (C-24), 47.2 (C-13), 49.0 (C-17), 55.8 (C-5), 69.3, 71.0
(C-2, C-3), 81.4 (C-22), 82.0 (C-25), 84.0 (C-20), 84.9
(C-14), 106.8 (acetonide C), 120.9 (C-7), 165.8 (C-8), 170.0
(2£C, acetate CO), 170.5 (acetate CO), 200.6 (C-6);
HRFABMS (positive ion mode) m/z 647.3787 [MþH]þ.
(calcd for C36H54O10þH, 647.3787).
Compound 9. Amorphous; nmax 3418, 2970, 1654, 1458,
1
1384, 1258, 1200, 1173, 1075, 1002, 927, 867 cm21; H
NMR (400 MHz, C5D5N) d 0.99 (br s, 3H, 19-Me), 1.03 (s,
3H, 18-Me), 1.30 (s, 3H, 21-Me), 1.37 (s, 2£3H, 26-Me,
27-Me), 1.45, 1.53 (each s, 2£3H, acetonide Me), 2.37
(obscured signal, 1H, H-5), 2.77 (t, J¼8.3 Hz, 1H, H-17),
3.92 (obscured signal, 1H, H-3), 3.94 (dd, J¼9.3, 2.8 Hz,
1H, H-22), 4.33 (br s, W1/2¼12 Hz, 1H, H-2), 4.70 (br, 1H,
H-9), 6.22 (d, J¼2.3 Hz, 1H, H-7); 13C NMR (100 MHz,
C5D5N) d 17.3 (C-18), 21.3 (C-16), 22.1 (C-11), 22.4
(C-21), 24.2 (C-19), 24.4 (C-23), 27.2 (acetonide Me), 29.4
(acetonide Me), 29.7 (C-4), 29.8 (C-26), 30.1 (C-27), 31.6
(C-12), 31.8 (C-15), 36.2 (C-9), 36.6 (C-10), 39.9 (C-1),
42.1 (C-24), 47.9 (C-13), 49.9 (C-17), 57.7 (C-5), 69.2
(C-25), 70.4 (C-2), 71.5 (C-3), 82.5 (C-22), 84.0 (C-14),
85.1 (C-20), 106.9 (acetonide C), 121.1 (C-7), 167.8 (C-8),
202.1 (C-6); HRFABMS (negative ion mode) m/z 519.3324
[M2H]2. (calcd for C30H48O72H, 519.3321).
3.2.2. Acetonide deprotection of compound 9. Compound
9 (36 mg, 0.069 mmol) was dissolved in 70% AcOH
(0.7 mL, excess) and benzyltrimethylammonium chloride
(25 mg, 0.135 mmol) was added. The reaction mixture was
left to stir for 4 h; water was then added and the mixture
extracted with n-BuOH (3£15 mL). The combined organic
layer was washed with water; the solvent was removed by
co-evaporation with water under reduced pressure. The
crude product was purified by column chromatography
using CHCl3–MeOH as eluting solvent to afford 2,3-diepi-
20-hydroxyecdysone (3) (27 mg, 81%).
Compound 3. Colorless needles (from MeOH–EtOAc), mp
204–206 8C; nmax 3420, 2965, 1654, 1383, 1065, 929 cm21
;
3.2. Asymmetric dihydroxylation of 8 with OsO4 and
chiral ligands
1H NMR (400 MHz, C5D5N) d 1.00 (s, 3H, 19-Me), 1.22 (s,
3H, 18-Me), 1.38 (s, 2£3H, 26-Me, 27-Me), 1.54 (s, 3H,
21-Me), 2.40 (obscured signal, 1H, H-5), 3.00 (t, J¼8.9 Hz,
1H, H-17), 3.87 (br d, J¼8.7 Hz, 1H, H-22), 3.90 (obscured
signal, 1H, H-3), 4.34 (br s, W1/2¼8.5 Hz, 1H, H-2), 4.76 (br
m, 1H, H-9), 6.22 (d, J¼2.6 Hz, 1H, H-7); 13C NMR
(100 MHz, C5D5N) d 18.0 (C-18), 21.4 (C-16), 21.5 (C-11),
21.6 (C-21), 24.2 (C-19), 27.5 (C-23), 29.8 (C-4), 29.9
(C-26), 30.3 (C-27), 31.8 (C-12), 32.1 (C-15), 36.3 (C-9),
36.7 (C-10), 39.9 (C-1), 42.7 (C-24), 48.2 (C-13), 50.1
(C-17), 57.8 (C-5), 69.6 (C-25), 70.5 (C-2), 71.6 (C-3), 76.9
(C-20), 77.5 (C-22), 84.1 (C-14), 121.0 (C-7), 168.4 (C-8),
202.2 (C-6); HRFABMS (negative ion mode) m/z 479.3001
[M2H]2. (calcd for C27H44O72H, 479.3008).
General procedure. To a solution of 0.03 mmol of a chiral
ligand in tert-BuOH–THF–H2O (7:4:1, 0.6 mL) was added
a THF solution of OsO4 (14 mL, 0.03 mmol. The solution
was prepared by dissolving 500 mg of OsO4 in 9 mL of
THF.) and the mixture stirred for 3 min. A solution of the
olefin acetonide 8 (5 mg, 0.01 mmol) in tert-BuOH–THF–
H2O (7:4:1, 0.5 mL) was then added and stirring continued
for 5 min. The ratio of the ligand, OsO4 and olefin acetonide
was 3:3:1. A 5% solution of NaHSO3 (10 mL) was added
and stirring continued for another 10 min. The mixture was
extracted with EtOAc (4£20 mL); the combined organic
phase was evaporated and the residue was chromatographed
to separate compounds 5 and 9 from the ligand. Since the
two products could easily be separated from each other by
column chromatography, the 5:9 ratio for each ligand was
determined from the isolated products 5 and 9. The results
are shown in Table 1.
3.2.3. Epimerization of compound 9. A mixture of
compound 9 (35 mg, 0.067 mmol) in MeOH (0.8 mL) and
2% Na2CO3 (0.2 mL, 0.038 mmol) was stirred at ambient
temperature for 5 h and water was then added. The solution
was extracted with n-BuOH (3£10 mL); the combined
butanol layer was washed with water and the solvent
removed by co-evaporation with water. The product was
purified by column chromatography to afford 2,3-diepi-5a-
20-hydroxyecdysone 20,22-acetonide (11) (28 mg, 80%).
3.2.1. Acetylation of compound 9. A mixture of compound
9 (9 mg, 0.017 mmol), Ac2O (0.1 mL, 1.05 mmol) and
pyridine (0.7 mL) was stirred for 6 h. The reaction mixture
was worked up in the usual manner and the product purified
by column chromatography to give 2,3-diepi-20-hydroxy-
ecdysone 2,3,25-triacetate (10) (8 mg, 72%).
Compound 11. Amorphous; nmax 3422, 2971, 2942, 1664,
1458, 1375, 1219, 1175, 1105, 1054, 1001, 905, 868 cm21
;
1H NMR (400 MHz, C5D5N) d 0.95 (s, 3H, 19-Me), 0.99 (s,
3H, 18-Me), 1.30 (s, 3H, 21-Me), 1.35 (s, 2£3H, 26-Me,
27-Me), 1.43, 1.52 (each s, 2£3H, acetonide Me), 2.74 (t,
J¼ca 8 Hz, 1H, H-17), 3.02 (br d, J¼11.1 Hz, 1H, H-5),
3.23 (m, 1H, H-9), 3.93 (br d, J¼8.5 Hz, 1H, H-22), 4.02 (m,
W1/2¼21 Hz, 1H, H-2), 4.42 (br s, W1/2¼8 Hz, 1H, H-3),
6.16 (br s, 1H, H-7); 13C NMR (100 MHz, C5D5N) d 13.6
(C-19), 17.3 (C-18), 20.8 (C-16), 22.0 (C-11), 22.4 (C-21),
24.4 (C-23), 27.2 (acetonide Me), 28.2 (C-4), 29.5
(acetonide Me), 29.9 (C-26), 30.1 (C-27), 31.5 (C-12),
31.6 (C-15), 40.3 (C-10), 41.2 (C-1), 42.2 (C-24), 46.7
(C-9), 47.6 (C-13), 48.7 (C-5), 49.9 (C-17), 68.3 (C-2), 69.1
(C-3), 69.3 (C-25), 82.5 (C-22), 83.9 (C-14), 85.1 (C-20),
106.9 (acetonide C), 123.3 (C-7), 164.4 (C-8), 201.7 (C-6);
Compound 10. Amorphous; nmax 3482, 2977, 1744, 1666,
1
1458, 1370, 1246, 1168, 1137, 1106, 928, 874 cm21; H
NMR (400 MHz, CDCl3) d 0.76 (s, 3H, 18-Me), 0.94 (br s,
3H, 19-Me), 1.12 (s, 3H, 21-Me), 1.29, 1.38 (each s, 2£3H,
acetonide Me), 1.43 (s, 3H, 26-Me), 1.45 (s, 3H, 27-Me),
1.97 (s, 2£3H, 3-OAc, 25-OAc), 1.98 (s, 3H, 2-OAc), 2.20
(partially obscured signal, 1H, H-5), 3.59 (dd, J¼ca 9,
2.5 Hz, 1H, H-22), 3.75 (br, W1/2¼21 Hz, 1H, H-9), 4.88
(br, W1/2¼20 Hz, 1H, H-3), 5.26 (br s, W1/2¼13 Hz 1H,
H-2), 5.86 (d, J¼1.9 Hz, 1H, H-7); 13C NMR (100 MHz,
C5D5N) d 17.1 (C-18), 20.9 (acetate Me), 21.1 (2£C, acetate
Me), 21.9 (C-21), 23.2 (C-16), 23.6 (C-19), 25.7 (C-26),
26.1 (C-27), 26.8 (acetonide Me), 28.9 (acetonide Me),

3438
S. Homvisasevongsa et al. / Tetrahedron 60 (2004) 3433–3438
HRFABMS (negative ion mode) m/z 519.3313 [M2H]2.
(calcd for C30H48O72H, 519.3322).
(TRF). One of us (S.H.) acknowledges financial support
from the Royal Golden Jubilee Program of TRF.
3.2.4. Acetonide deprotection of compound 11. Com-
pound 11 (15 mg, 0.029 mmol) was subjected to acetonide
deprotection in the same manner as described for the
preparation of 3 from 9. The product was purified by column
chromatography to afford 2,3-diepi-5a-20-hydroxy-
ecdysone (4) (10 mg, 72%).
References and notes
1. Rees, H. H. Phytoecdysteroids: structures and occurrence. In
Ecdysone-from chemistry to mode of action; Koolman, J., Ed.;
Georg Thieme: Stuttgart, 1989; pp 28–38.
2. Lafont, R.; Horn, D. H. S. Zooecdysteroids: structures and
occurrence. In Ecdysone-from chemistry to mode of action;
Koolman, J., Ed.; Georg Thieme: Stuttgart, 1989; pp 39–64.
3. Camps, F. Plant ecdysteroids and their interaction with insects.
In Ecological chemistry and biochemistry of plant terpenoids;
Harborne, J. B., Tomas-Barberan, F. A., Eds.; Claredon:
Oxford, 1991; pp 331–376.
Compound 4. Amorphous; nmax 3415, 2925, 1660, 1384,
1
1062 cm21; H NMR (400 MHz, C5D5N) d 0.84 (s, 3H,
19-Me), 1.21 (s, 3H, 18-Me), 1.38 (s, 2£3H, 26-Me,
27-Me), 1.58 (s, 3H, 21-Me), 2.99 (t, J¼9.2 Hz, 1H, H-17),
3.03 (dd, J¼12.4, 3.6 Hz, 1H, H-5), 3.28 (m, 1H, H-9), 3.89
(br d, J¼8.9 Hz, 1H, H-22), 4.04 (m, W1/2¼20 Hz, 1H, H-2),
4.44 (br s, W1/2¼9 Hz, 1H, H-3), 6.18 (d, J¼2.4 Hz, 1H,
H-7); 13C NMR (100 MHz, C5D5N) d 13.6 (C-19), 17.9
(C-18), 20.9 (C-16),a 21.4 (C-11),a 21.7 (C-21), 27.4
(C-23),b 28.3 (C-4),b 29.9 (C-26), 30.2 (C-27), 31.8 (C-12,
C-15), 40.3 (C-10), 41.3 (C-1), 42.7 (C-24), 46.7 (C-9), 47.9
(C-13), 48.7 (C-5), 50.1 (C-17), 68.4 (C-2), 69.1 (C-3), 69.6
(C-25), 76.9 (C-20), 79.8 (C-22), 84.0 (C-14), 123.3 (C-7),
164.9 (C-8), 201.8 (C-6), (‘a and b’ stand for assignments
may be reversed for signals with the same superscript);
HRFABMS (negative ion mode) m/z 479.3018 [M2H]2.
(calcd for C27H44O72H, 479.3008).
4. Bergamasco, R.; Horn, D. H. S. The biological activities of
ecdysteroids and analogue. In Progress in ecdysone research;
Hoffmann, J. A., Ed.; Elsevier/North Holland Biomedical:
Amsterdam, 1980; pp 299–324 and references cited therein.
5. Horn, D. H. S.; Bergamasco, R. Chemistry of ecdysteroids. In
Comparative insect physiology, biochemistry and pharma-
cology; Kerkut, G. A., Gilbert, L. I., Eds.; Pergamon: Oxford,
1985; Vol. 7, pp 185–248.
6. Robbins, W. E.; Kaplanis, J. N.; Thompson, M. J.; Shortino,
T. J.; Joyner, S. C. Steroids 1970, 16, 105–125.
7. Ogawa, S.; Nishimoto, N.; Okamoto, N.; Takemoto, T.
Yakugaku Zasshi 1971, 91, 916–920.
3.5. Moulting bioassay
8. Luu, B.; Werner, F. Pestic. Sci. 1996, 46, 49–53.
9. Suksamrarn, A.; Pattanaprateep, P. Tetrahedron 1995, 51,
10633–10650.
Compounds 3 and 4 were subjected to the Musca bioassay,
using Musca domestica larvae.13 The purity of the
ecdysteroid and their analogues was checked by reversed-
phase HPLC. The bioassay results were scored15 and EC50,
the molar concentration of each steroid required to effect
puparium formation of 50% effectiveness, of each com-
pound was determined by plotting concentrations against %
effectiveness of puparium formation.16 The EC50 values of
compounds 3 and 4 were 5.0£1024 and 7.0£1024 M,
respectively, whereas that of the reference compound 1 was
1.65£1025 M.
10. Suksamrarn, A.; Yingyongnarongkul, B. Tetrahedron 1996,
52, 12623–12630.
11. Suksamrarn, A.; Jankam, A.; Tarnchompoo, B.; Putchakarn, S.
J. Nat. Prod. 2002, 65, 1194–1197.
12. Kolb, H. C.; VanNieuwenhze, M. S.; Sharpless, K. B. Chem.
Rev. 1994, 94, 2483–2547.
13. Kaplanis, J. N.; Tabor, L. A.; Thompson, M. J.; Robbins,
W. E.; Shortino, T. J. Steroids 1966, 8, 625–631.
14. Suksamrarn, A.; Yingyongnarongkul, B. Tetrahedron 1997,
53, 3145–3154.
15. Ohtaki, T.; Milkman, R. D.; Williams, C. M. Proc. Natl Acad.
Sci. USA 1967, 58, 981–984.
Acknowledgements
16. Suksamrarn, A.; Pattanaprateep, P.; Tanachatchairatana, T.;
Haritakun, W.; Yingyongnarongkul, B.; Chimnoi, N. Insect
Biochem. Mol. Biol. 2002, 32, 193–197.
This work was supported by the Thailand Research Fund