Dimeric ecdysteroid analogues and their interaction with the Drosophila ecdysteroid receptor

Article abstract of DOI:10.1135/cccc20061229

Three structurally related specific ecdysteroid derivatives, 7,7′-dimers of 14-deoxy-8(14)-ene transformed 20-hydroxyecdysone, ponasterone A and ajugasterone C, were obtained by photochemical transformation. The structures of the dimeric ecdysteroids were identified mainly by NMR spectroscopy supported by MS and IR spectroscopy. Yields of the dimerisation products were dependent on the reactant concentrations and photoreaction conditions. Inert gas atmosphere supported high yields, whereas oxygen atmosphere fully prevented the dimer formation. All the three dimers retained a rather high agonistic activity at the ecdysteroid receptor in the Drosophila B_II bioassay when compared with the relevant original ecdysteroids.

Full text of DOI:10.1135/cccc20061229

Dimeric Ecdysteroid Analogues
1229
DIMERIC ECDYSTEROID ANALOGUES AND THEIR INTERACTION
WITH THE Drosophila ECDYSTEROID RECEPTOR
a1,
a2
a3
b
Juraj HARMATHA *, Miloš BUDĚŠÍNSKÝ , Karel VOKÁČ , Laurence DINAN and
c
René LAFONT
a
Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic,
Flemingovo nám. 2, 166 10 Prague 6, Czech Republic; e-mail: harmatha@uochb.cas.cz,
budesinsky@uochb.cas.cz, vokac@uochb.cas.cz
Department of Biological Sciences, University of Exeter,
Prince of Wales Road, Exeter, EX4 4PS, U.K; e-mail: laurie@dinan.wanadoo.co.uk
Laboratoire Protéines – Biochimie structurale et fonctionnelle, Université P. et M. Curie,
1
2
3
b
c
7
Quai Saint-Bernard, 75252 Paris 05, France; e-mail: rene.lafont@snv.jussieu.fr
Received Jun e 6, 2006
Accepted July 6, 2006
Dedicated to Professor Antonín Holý on the occasion of his 70th birthday.
Th ree structurally related specific ecdysteroid derivatives, 7,7′-dim ers of 14-deoxy-8(14)-en e
tran sform ed 20-h ydroxyecdyson e, pon asteron e A an d ajugasteron e C, were obtain ed by ph o-
toch em ical tran sform ation . Th e structures of th e dim eric ecdysteroids were iden tified m ain ly
by NMR spectroscopy supported by MS an d IR spectroscopy. Yields of th e dim erisation prod-
ucts were depen den t on th e reactan t con cen tration s an d ph otoreaction con dition s. In ert gas
atm osph ere supported h igh yields, wh ereas oxygen atm osph ere fully preven ted th e dim er
form ation . All th e th ree dim ers retain ed a rath er h igh agon istic activity at th e ecdysteroid
receptor in th e Drosophila BII bioassay wh en com pared with th e relevan t origin al ecdy-
steroids.
Keyw ord s: Steroids; Ecdysteroids; 20-Hydroxyecdyson e; Pon asteron e A; Ajugasteron e C; Ph o-
toch em ical tran sform ation s; Drosophila melanogaster BII bioassay; Ecdysteroid receptor
agon ists; NMR spectroscopy.
Ecdysteroids in teract with th e ligan d bin din g site of th e in sect ecdysteroid
1
receptor m ostly as agon ists . On ly on e an alogue, a side-ch ain -m odified
lacton e derivative, h as been so far foun d to dem on strate an tagon istic activ-
2
ity . Th ese activities were detected an d quan tified usin g th e Drosophila
3
melanogaster B cell bioassay . Th e activities of a series of n atural an d ch em -
II
ically m odified ecdysteroids prepared in our laboratory h ave already been
4
com pared in th is bioassay . Th e n atural ecdysteroids were isolated from
h igh er plan ts, m ain ly from Leuzea carthamoides5 , or from fun gi . Th e
,6
7,8
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 8, pp. 1229–1238
©
2006 Institute of Organic Chemistry and Biochemistry
doi:10.1135/cccc20061229

1
230
Harmatha, Buděšínský, Vokáč, Dinan, Lafont:
m ajority of structural an alogues were prepared by targeted ch em ical tran s-
9
,10
form ation s . Later, a ph otoch em ically in duced tran sform ation was in tro-
1
1
duced as an altern ative approach to ecdysteroid structure m odification s .
Th is m eth od was origin ally used for preparation of stan dard 20-h ydroxy-
1
2
ecdyson e derivatives , in order to m on itor wh eth er such structural an a-
logues could stem from th e n ative com poun d as UV ligh t-in duced artefacts.
Ph ototran sform ation of 20-h ydroxyecdyson e, as described by Can on ica et
1
2,13
al.
, yielded a series of ecdysteroid deoxy-, h ydroperoxy- an d C/D-rin g-
1
1
rearran ged derivatives. Wh en th e m eth od was used in our laboratory , var-
ious addition al an alogues were obtain ed. Th e m ost surprisin g ph oto-
tran sform ation product obtain ed from our experim en t was a dim eric
ecdysteroid , wh ich was n ot described in th e papers of Can on ica et al.12,13.
11
Th e biological activity data of th e ph otoch em ically tran sform ed ecdy-
1
4
steroids were used to in vestigate structure–activity relation sh ips , an d for
1
5
design in g furth er targeted structure m odification s . Th e activities in th e
in vitro bioassay gen erally con firm ed th ose previously obtain ed by in vivo
tests, with exception of th e dim eric ecdysteroids, wh ich h ave n ot yet been
assessed in vivo. In order to exten d th is study, we prepared an d tested n ew
specific an alogues an d ecdysteroid dim ers obtain ed by ph ototran sform -
ation of 20-h ydroxyecdyson e (1) ponasterone A (2) and ajugasterone C (3), re-
spectively.
RESULTS AND DISCUSSION
Ph otoch em ical tran sform ation s were perform ed in a stan dard ph oto-
reactor un der con trolled reaction con dition s, as described for 20-h ydroxy-
1
1
ecdyson e (1) in our previous paper . Th e resultin g referen ce dim er 4, as
well as th e experien ce gain ed durin g its ph otoproduction , isolation an d
iden tification , con tributed to th e ph ototran sform ation of two oth er related
ecdysteroids, th e 25-deoxy an alogue pon asteron e A (2) an d its 11-h ydroxy
an alogue ajugasteron e C (3). Both com poun ds yielded dim eric derivatives 5
an d 6, respectively (Sch em e 1). Th e ph otoreaction m ixtures were an alyzed,
an d in dividual com poun ds separated by a com bin ation of reversed-ph ase
an d n orm al-ph ase HPLC. Th e reten tion tim es of th e targeted dim ers 4, 5
an d 6 are com pared with th ose of th e startin g ecdysteroids 1, 2 an d 3 in
Table I. Th e structures of th e dim ers 4–6 were determ in ed m ain ly by NMR
spectroscopy, supported by IR an d MS, utilizin g results of th e exten ded an d
com plete structure an alysis of th e dim eric 20-h ydroxyecdyson e derivative 4,
1
1
described in our previous paper .
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 8, pp. 1229–1238

Dimeric Ecdysteroid Analogues
1231
SCHEME 1
TABLE I
HPLC reten tion tim es an d biological activities of ecdysteroids 1–3 an d th eir dim ers 4–6
Reten tion tim e, m in
Agon ist activity
(EC50 value)
Com poun d
a
b
c
M
System 1
System 2
System 3
–
9
2
0-Hydroxyecdyson e (1)
34.2
47.8
39.2
51.8
74.2
67.2
50.7
19.2
32.3
65.9
15.5
25.2
–
117.4
–
7.5 × 10
3.1 × 10
3.0 × 10
2.1 × 10
4.6 × 10
8.2 × 10
–
10
Pon asteron e A (2)
Ajugasteron e C (3)
–
–
–
–
8
7
8
7
7
7
7
-7′-bis-14-Deoxy-8(14)-en -20-h ydroxyecdyson e (4)
-7′-bis-14-Deoxy-8(14)-en -pon asteron e A (5)
-7′-bis-14-Deoxy-8(14)-en -ajagasteron e C (6)
–
72.4
–
a
System 1: Separon SGX C-18 colum n (7 µm , 250 × 4 m m i.d.), elution with a lin ear gra-
dien t of 10–70% m eth an ol in water over 50 m in , followed by isocratic elution for 30 m in
b
at a flow-rate of 0.6 m l/m in . System 2: Silasorb 600 colum n (5 µm , 250 × 4 m m i.d.),
c
elution with n -h exan e/eth an ol/water (810:180:0.8, v/v/v), flow-rate 0.8 m l/m in . System 3:
Silasorb 600 colum n (5 µm , 250 × 4 m m i.d.), elution with n -h exan e/eth an ol/water
(
900:99:0.5, v/v/v), flow-rate 0.8 m l/m in .
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 8, pp. 1229–1238

1
232
Harmatha, Buděšínský, Vokáč, Dinan, Lafont:
Th e n um bers of sign als in both H an d 13C NMR spectra of com poun ds 5
1
an d 6 (Table II), correspon din g to th e em pirical com position s C H O an d
2
7
43
5
C27H43O , respectively, in dicate th e sym m etry of th e m olecules. Th is
6
m ean s, th at com poun ds 5 an d 6 are h om o-dim eric, com posed of two iden -
tical parts lin ked at th e sam e position s. Th e absen ce of ch aracteristic sign als
for th e olefin ic proton H(7) an d carbon C(14)–OH, th e observed upfield
sh ift of th e C=O carbon at C(6) (δ 215.89 an d 215.54 in dim ers 5 an d 6, re-
spectively, versus δ 206.50 an d 206.66 in th e startin g com poun ds 2 an d 3),
togeth er with sign als for a tetrasubstituted double bon d (olefin ic carbon s at
δ 124.51/153.00 an d 123.68/152.63, respectively) an d th e n ewly appeared
sin glet for h ydrogen H(7) at δ 3.54 in dicated th at th e m on om eric un its in
dim ers 5 an d 6 con tain a C=C double bon d between C(8) an d C(14) an d are
1
13
lin ked at position s C(7). Th e H an d C NMR data of dim ers 5 an d 6 (see
1
1
Table II) are very sim ilar to th ose we described previously for th e dim er of
0-h ydroxyecdyson e 4, with ch aracteristic differen ces on ly for carbon atom s
2
an d proton s, wh ich are close to th e differin g structure m oieties (th e ab-
sen ce of a 25-OH group in both 5 an d 6, an d th e presen ce of a 11α-OH
group in 6), already presen t in th e correspon din g startin g com poun ds 2
an d 3. Th e determ in ation of th e con figuration at carbon atom s C(7)/C(7′)
by th e an alysis of a 2D-ROESY spectrum an d m olecular m odellin g is de-
1
1
scribed in detail in our previous paper .
Differen ces in th e water solubility of th e ecdysteroids 1–3 led us to per-
form in g th e ph otoreaction at rath er low ecdysteroid con cen tration s
1
1
(
0.6–0.8 m m ol/l). We already kn ew from our previous observation th at
th e reaction yielded on ly 3% of dim er 4, wh en th e 20-h ydroxyecdyson e
was added at a low con cen tration (0.83 m m ol/l). Ajugasteron e C (3) did n ot
dissolve com pletely at th is con cen tration , but after dissolvin g durin g th e
reaction , it yielded 4.5% of dim er 5, besides 12% of un reacted ajugasteron e
C (3). Pon asteron e A (2) is even less soluble in water. Th erefore, th e ph oto-
reaction was perform ed at a con cen tration of 0.65 m m ol/l (wh ere a part of
th e reactan t still rem ain ed in suspen sion ). All th e reactan t did n ot dissolve
com pletely, even in a prolon ged reaction tim e. Th us, pon asteron e A (2)
yielded on ly 2.5% of dim er 5, with up to 32.5% of un reacted 2 rem ain in g
in th e reaction m ixture. A h igh er solubility, but a differen t reaction product
profile, was obtain ed by addin g organ ic solven t to water (tetrah ydrofuran /
water, 1:1 v/v). Th ese results, as well as effects of th e presen ce of oxygen in
th e reaction m ixture in stead of usin g in ert argon protection , will be pub-
lish ed later in an oth er con text. Th e presen ce of oxygen h ad a greater in flu-
en ce on th e reaction product com position th an th e reactan t con cen tration .
However, in con n ection with th is report, it is relevan t on ly th at th e dim er
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 8, pp. 1229–1238

Dimeric Ecdysteroid Analogues
1233
TABLE II
1H an d C NMR ch em ical sh ifts of com poun ds 4–6 in CD OD
13
3
Proton
4a
5
6
Carbon
4a
5
6
1
1
2
3
4
4
5
7
9
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
α
β
α
α
α
β
1.75
1.47
3.81
4.04
2.24
2.13
2.42
3.53
2.99
1.87
1.60
1.92
1.59
2.38
2.31
1.92
1.85
2.37
0.90
0.70
1.13
3.48
1.60
1.68
1.46
1.75
–
1.75
1.46
3.80
4.03
2.24
2.12
2.42
3.54
3.00
1.87
1.60
1.95
1.53
2.37
2.32
1.89
1.89
2.24
0.94
0.71
1.12
3.45
1.50
1.60
1.30
1.45
1.57
0.91
0.93
2.02
1.48
4.12
4.03
2.20
2.13
2.43
3.54
2.83
–
1
2
36.88
68.37
69.05
33.88
56.03
215.99
59.07
124.54
33.49
41.39
21.42
36.70
43.95
152.97
26.54
23.04
57.79
19.68
25.33
78.33
21.24
77.52
27.36
41.25
71.58
28.95
29.93
36.69
68.41
69.05
33.97
56.03
215.89
59.09
124.51
33.52
41.30
21.43
37.32
44.27
153.00
26.71
22.94
57.81
19.69
25.36
78.31
21.26
77.30
37.32
30.53
29.35
22.81
23.54
38.15
68.74
69.19
33.95
56.18
215.54
58.77
123.68
43.30
41.02
67.50
48.14
45.54
152.63
26.70
23.19
57.60
19.88
27.10
78.18
21.39
77.46
36.96
30.54
29.23
22.88
23.66
3
4
5
6
7
8
9
1α
1β
2α
2β
5α
5β
6α
6β
7
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
4.03
2.18
1.57
2.43
2.32
1.99
1.89
2.18
0.96
0.75
1.15
3.46
1.49
1.58
1.28
1.43
1.58
0.92
0.93
8
9
1
2
3a
3b
4a
4b
5
6
1.19
1.21
7
a
Data from lit.11
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 8, pp. 1229–1238

1
234
Harmatha, Buděšínský, Vokáč, Dinan, Lafont:
form ation was n ot detected in th e presen ce of oxygen durin g th e ph oto-
reaction . Th is correspon ds with th e observation th at dim eric steroids with
C–C lin ked B rin gs, wh en subjected to ph otooxidation un der fluorescen t
1
6
ligh t, were tran sform ed to th eir m on om eric un its .
Th e biological activities of th e relatively large dim eric ecdysteroid an a-
logues 4–6 were com pared with th ose of th eir n ative precursors 1–3 in th e
BII bioassay, in wh ich poten cy reflects th e affin ity of bin din g to th e
1
,3
ligan d-bin din g site of th e Drosophilla melanogaster ecdysteroid receptor
.
We h ave already com pared12 th e activity of th e dim eric 20-h ydroxy-
ecdyson e derivative 4 with its structurally related, but con siderably sm aller,
m on om eric an alogues. Th e results led to th e correction of som e form er gen -
eral presum ption s con cern in g ecdysteroid structure–activity relation sh ips,
con cern in g in particular th e in fluen ce of th e C(14)–OH con figuration an d
th e correspon din g C/D-rin g an elation , as well as un usual double bon d loca-
tion . We can n ow com pare addition al data obtain ed from th e B bioassay
II
for th e structurally tran sform ed dim eric ecdysteroids 5 an d 6 in relation to
dim er 4 an d to th eir n ative, structurally rath er differen t precursors (Table I).
All th e com pared com poun ds possessed agon istic activity; n on e of th em
sh owed an y an tagon istic activity. Th e relative poten cies of th e agon ist
activity of dim eric ecdysteroids 4–6 (with EC
values in th e ran ge
0
5
–
8
–7
1
0 –10 M) are un expectedly h igh . Th ey are on ly two orders of m agn itude
lower th an th e activities of th e very active paren t ecdysteroids 1–3, an d are
sign ifican tly h igh er th an th e activities of som e structurally-related m on o-
m ers, e.g. podecdyson e B (EC50 = 1.2 × 10–5 M) . Th is fin din g sh ould be
con sidered in th e followin g con text: (i) Dim ers are con siderably bulkier
th an m on om eric ecdysteroids an d on e m igh t expect th is to preven t in terac-
tion with th e receptor bin din g site. However, oth er large m olecules, such as
11
1
7,18
oligostilben es, can also in teract with th is bin din g site
. (ii) If th e dim ers
can accom m odate th em selves at th e bin din g site, th en dim erisation effec-
tively doubles th e con cen tration of ecdysteroid m oieties an d m ay facilitate
th e reloadin g of th e bin din g site after dissociation of th e ligan d–receptor
com plex. If th e association rate con stan t (k ) is en h an ced wh ile th e dissoci-
a
ation rate con stan t (k ) is n ot sign ifican tly affected, th en th e dissociation
d
con stan t (K = k /k ) is lower (i.e. th e affin ity in creases). (iii) Th e dim er
d
d
a
m igh t in teract sim ultan eously with th e two receptor–protein s (EcR/USP
com plex), both of wh ich are m em bers of th e n uclear receptor fam ily. Si-
m ultan eous ecdysteroid in teraction with EcR an d USP (th e n atural ligan d
for USP is curren tly un kn own ) m igh t accoun t for th e h igh activity of th e
1
1
dim ers. Th e rath er h igh stability of th e dim ers an d low activity of struc-
1
4
turally related m on om ers , expected as products of possible degradation ,
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 8, pp. 1229–1238

Dimeric Ecdysteroid Analogues
1235
do n ot support th e opin ion th at th e activity could depen d on th e reverse
m on om er form ation at th e ligan d bin din g site.
9
4
Th e less bulky 20-h ydroxyecdyson e glucosides h ave lower activity by
–
6
–5
two orders of m agn itude (EC50 = 10 –10 M) th an th e dim ers (Table I). Th e
glucosides are substituted in position s 2, 3, 22 or 25, wh ich are critical for
th e ecdysteroid activity accordin g to th e ph arm acoph ore h ypoth esis ,
wh ereas dim erisation , creatin g lin ks th rough C(7), leaves th ese position s
free, but affects th e region between rin gs B, C an d D, wh ere m olecular m od-
ellin g does n ot in dicate essen tial roles for th e C(6)=O an d 14α-OH in recep-
1
5
1
9
tor in teraction s .
Our experim en ts h ave proven th at th e specific dim erisation of ecdy-
steroids in duced by UV ligh t (un der in ert gas con dition s) is n ot an exclu-
sive ph otoreaction occurrin g on ly with 20-h ydroxyecdyson e; it is m ore
gen eral, in depen den t of th e n um ber an d location of h ydroxyl groups in th e
ecdysteroid m olecule. Th e un expectedly h igh biological activity in term s of
th e in teraction with th e ecdysteroid receptor is eviden tly also m ore gen eral.
EXPERIMENTAL
1
NMR spectra were m easured on a Varian Un ity-500 an d Bruker Avan ce-500 in strum en t ( H
1
3
at 500 MHz, C at 125.7 MHz) in CD OD. Ch em ical sh ifts in CD OD were referen ced to th e
3
3
1
13
solven t sign al at 3.31 ppm ( H) an d 49.00 ppm ( C). Hom on uclear 2D-COSY an d 2D-
1
3
ROESY spectra were used for structure assign m en t of proton s. C APT spectra an d h etero-
correlated 2D-HSQC an d 2D-HMBC spectra were com bin ed to assign sign als of all carbon
atom s. Mass spectra were recorded on a ZAB-EQ spectrom eter with fast atom bom bardm en t
(
FAB) ion ization usin g a glycerol–th ioglycerol m ixture as m atrix. In frared spectra (ν
in
m ax
–
1
cm ) were recorded on a Bruker IPS-88 in strum en t usin g KBr pellets. Meltin g poin ts were
determ in ed on a Boetius apparatus an d are un corrected.
Ph ototran sform ation s were perform ed in a 500-m l origin al Han au Ph otoreactor, equipped
with a m agn etic turbin e pum p for forced circulation , with a jacket for water stream coolin g,
an d with an im m ersed Pyrex-filtered m edium -pressure broad-ban d (250–600 n m ) Hg lam p.
Th e reaction was perform ed in water solution (500 m l) circulatin g in th e reactor un der in ert
gas (argon ) an d at con trolled tem perature (25 °C). Th e reaction tim e was adjusted accordin g
to th e results of RP-HPLC m on itorin g of sam ples taken an d an alyzed every 30 m in un til th e
con version of th e reactan t reach ed a steady state. Th e reaction m ixture was tran sferred to a
flask, an d water was evaporated to give a dry residue.
Th e residue was an alyzed by RP-HPLC (reversed-ph ase) usin g a 4 × 250 m m colum n
packed with 7 µm size Separon SGX C-18 (Tessek, Prague). Elution was perform ed with sol-
ven t System 1 (as in dicated in Table I). Th e residue was later fraction ated by preparative RP-
HPLC usin g a 26 × 600 m m colum n packed with th e sam e station ary ph ase an d eluted with
th e sam e m obile ph ase at th e sam e gradien t over 300 m in an d at a flow rate of 5 m l/m in .
Collected fraction s were m on itored by NP-HPLC (n orm al-ph ase) usin g a 4 × 250 m m col-
um n packed with 5 µm size Silasorb 600 (Lach em a, Brn o) an d a m obile ph ase (System 2 in
Table I). Fraction s con tain in g dim eric ecdysteroid an alogues were furth er separated an d puri-
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 8, pp. 1229–1238

1
236
Harmatha, Buděšínský, Vokáč, Dinan, Lafont:
fied by NP-HPLC usin g a 12 × 500 m m colum n packed with th e sam e station ary ph ase an d
eluted with th e sam e m obile ph ase at a flow rate of 3 m l/m in . For an alysis of th e dim eric
pon asteron e A derivative 5 System 3 (Table I) was m ore effective. Ch aracteristic HPLC data
recorded un der an alytical con dition s are sum m arized in Table I.
2
0-Hydroxyecdyson e (1) an d ajugasteron e C (3) were obtain ed in sufficien t quan tity an d
6
h igh quality (over 98% purity) from roots of Leuzea carthamoides . Pon asteron e A (2) was
prepared from 20-h ydroxyecdyson e (1) by th e followin g four-step procedure: 1. Con version
of 20-h ydroxyecdyson e in to its 2,3,22-triacetate an d purification of th e product by LC.
2
. Deh ydration of 20-h ydroxyecdyson e-2,3,22-triacetate with POCl in pyridin e, wh ich pro-
3
2
4
25
duced a m ixture of ∆ an d ∆ derivatives. Th e reaction m ixture was purified by LC with out
separation of th e two derivatives. 3. Deprotection with potassium carbon ate in aqueous
m eth an ol givin g a m ixture of ∆
tion of th e m ixture from step 3 with h ydrogen usin g 10% Pd/C catalyst. Th is procedure,
2
4/25
derivatives (stach ysteron e C an d its isom er). 4. Reduc-
2
0–22
based on previously kn own origin al tran sform ation s
, represen ts upgraded straigh t-
forward m odification suitable for large-scale preparation . Th e resultin g pon asteron e A was
1
13
purified by preparative RP-HPLC an d iden tified by H an d C NMR an d by com parison with
an auth en tic stan dard by NP-HPLC (System 3, Table I).
Gen eral Procedure for Ph ototran sform ation of Ecdysteroids 1–3
Th e ph otoreactor (see above) was filled with water (500 m l), an d th e reactan t was added in
on e portion . Th e m ixture was stirred by forced circulation un der a con tin uous flow of
argon . After dissolvin g th e reactan t (in th e case of 1) an d furth er argon rin sin g (1 h ), th e UV
lam p was switch ed on , an d th e reaction was perform ed. Th e reaction m ixture was tran s-
ferred to a flask, an d water was evaporated. Th e residue was fraction ated by preparative
RP-HPLC (usin g System 1 adapted for preparative separation s). Th e m ain con stituen t of th e
ph otoreaction product (dim eric ecdysteroid) was separated from th e m ajor fraction as de-
scribed above.
Th e ph ototran sform ation reaction was perform ed in th e sam e way with pon asteron e A (2)
an d ajugasteron e C (3), with th e on ly differen ce th at both reactan ts at th e sam e con cen tra-
tion did n ot dissolve com pletely. Th us, th ese reaction s were perform ed partly in suspen sion .
Th e dim eric com poun ds were obtain ed from reaction m ixtures in various yields (varied from
2
.5 to 4.5%).
(
20R,20′R,22R,22′R)-2β,2′β,3β,3′β,20,20′,22,22′,25,25′-Decah ydroxy-[7αH,7′αH-bi(5β-
ch olest-8(14)-en e)]-6,6′-dion e, i.e. 7-7′-bis-14-Deoxy-8(14)-en e-20-h ydroxyecdyson e (4)
2
0-Hydroxyecdyson e (1; 500 m g) was added to th e ph otoreactor filled with water (500 m l).
After dissolvin g th e reactan t (10 m in ), th e ph otoreaction was perform ed for 4 h . Th e reac-
tion m ixture, after evaporation of water, was fraction ated by RP-HPLC (usin g m odified
System 1) yieldin g 13 fraction s. Com poun d 4 (87 m g, 17%) was obtain ed directly from frac-
tion 11 after solven t evaporation an d crystallization from m eth an ol–water. M.p. 171–173 °C.
IR: 3485, 3290 (O–H), 1695 (C=O), 1059 (C–O). UV (n m , MeOH): 210 (4.06), 242 (3.69), 310
(
[
2.78). CD (MeOH): ∆ε209 +55.0, ∆ε237 +4.5, ∆ε259 –12.2, ∆ε299 –1.8. FAB-MS, m/z (%): 949
M + Na] (11), 927 [M + H] (6), 891 (8), 873 (10), 464 [1/2 M + 1] (100), 429 (55). Com posi-
tion C54H86O12 (M = 926) determ in ed by HR FAB-MS: 949.6111 [M + Na], for C54H86O12Na
1
13
calculated 949.6017. For H an d C NMR data, see Table II.
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 8, pp. 1229–1238

Dimeric Ecdysteroid Analogues
1237
(
8
20R,20′R,22R,22′R)-2β,2′β,3β,3′β,20,20′,22,22′-Octoh ydroxy-[7αH,7′αH-bi(5β-ch olest-
(14)-en e)]-6,6′-dion e, i.e. 7-7′-bis-14-Deoxy-8(14)-en e-pon asteron e A (5)
Pon asteron e A (2; 150 m g) was added to th e ph otoreactor filled with water (500 m l). Th e
m ixture was stirred un der a con tin uous flow of argon for m ore th an 1 h . Th e reactan t did
n ot dissolve com pletely. Th e ph otoreaction was perform ed in suspen sion for 4 h . Owin g to
sm all con version of pon asteron e A an d low yield of th e dim er, th e reaction was repeated
3
tim es. Th e com bin ed reaction m ixtures (450 m g), after evaporation of water, were frac-
tion ated by RP-HPLC (usin g adapted System 1). Com poun d 5 (11 m g, 2.5%) was obtain ed
in addition to un ch an ged pon asteron e A (145 m g, 32.5%) from th e relevan t fraction s. Com -
poun d 5 was purified by NP-HPLC (usin g adapted System 3) givin g a wh ite solid. IR: 3440
(
O–H), 1698 (C=O), 1060 (C–O). FAB-MS, m/z (%): 893 [M – H] (57), 447 [1/2 M] (27), 255
(
8
34), 153 (100), 113 (33). Com position C54H86O10 (M = 894) determ in ed by HR FAB-MS:
95.632213 [M + H], for C54H87O10 calculated 895.629925. For H an d C NMR data, see
1
13
Table II.
(
20R,20′R,22R,22′R)-2β,2′β,3β,3′β,11α,11′α,20,20′,22,22′-Decah ydroxy-[7αH,7′αH-bi(5β-
ch olest-8(14)-en e)]-6,6′-dion e, i.e. 7-7′-bis-14-Deoxy-8(14)-en -ajugasteron e C (6)
Ajugasteron e C (3; 200 m g) was added to th e ph otoreactor filled with water (500 m l). Th e
m ixture was stirred un der a con tin uous flow of argon for m ore th an 1 h . Th e reactan t did
n ot dissolve com pletely. Th e ph otoreaction was in itiated in suspen sion . Th e reactan t gradu-
ally dissolved durin g th e reaction over 8 h . Th e reaction m ixture, after evaporation of water,
was fraction ated by RP-HPLC (usin g adapted System 1). Com poun d 6 (9 m g, 4.5%) was ob-
tain ed in addition to un ch an ged ajugasteron e C (25 m g, 12.5%) from th e relevan t fraction s.
Com poun d 6 was purified by NP-HPLC (usin g adapted System 2) givin g a wh ite solid. IR:
3
[
418 (O–H), 1698 (C=O), 1054 an d 1023 (C–O). FAB-MS, m/z (%): 949 [M + Na] (22), 926
M] (2), 486 (57), 464 [1/2 M + 1] (31), 413 (41), 231 (29), 149 (82), 57 (100). Com position
C54H86O12 (M = 926) determ in ed by HR FAB-MS: 949.599062 [M + Na], for C54H86O12Na cal-
1
13
culated 949.601699. For H an d C NMR data, see Table II.
The research was supported by the Grant Agency of Czech Republic, grant No. 203/04/0298, and in
part from research project Z40550506. We thank Dr J. Kohoutová and Ms P. Loudová for recording
MS and Dr S. Vašíčková for recording IR and CD spectra.
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