Syntheses of dexamethasone conjugates of the phytohormones gibberellin A3 and 24-epicastasterone

Article abstract of DOI:10.1135/cccc20020103

The syntheses of N-[10-(9α-fluoro-11β,17α-dihydroxy-16α-methyl-3- oxoandrosta-1,4-diene-17β-carboxamido)decyl]gibberellamide (7) and 6-[({N-[10-(9α-fluoro-11β,17α-dihydroxy-16α-methyl-3- oxoandrosta-1,4-diene-17β-carboxamido)decyl]carbamoyl}methoxy)imino] -24-epicastasterone (10) are described. [(Benzotriazol-1-yl)oxy]bis(pyrrolidin-1-yl)methylium hexafluorophosphate (HBPyU) was used as the coupling agent for the reaction of gibberellic acid as well as of 24-epicastasterone-O-(carboxymethyl)oxime with N-(10-aminodecyl)-9αfluoro-11β,17α-hydroxy-16α- methyl-3-oxoandrosta-1,4-diene-17β-carboxamide (4). The gibberellic acid conjugate 7 was also synthesised by the coupling of succinimidyl gibberellate 6 with amine 4.

Full text of DOI:10.1135/cccc20020103

Syntheses of Phytohormones
103
SYNTHESES OF DEXAMETHASONE CONJUGATES OF THE
PHYTOHORMONES GIBBERELLIN A3 AND 24-EPICASTASTERONE
1
2
3
4
Adelheid KOLBE , Robert KRAMELL , Andrea PORZEL , Jürgen SCHMIDT ,
5
6,
Gernot SCHNEIDER and Günter ADAM *
Institut für Pflanzenbiochemie, Weinberg 3, D-06120 Halle/S., Germany;
1
2
3
e-mail: adelheid@Kolbe-net.de, rkramell@ipb-halle.de, aporzel@ipb-halle.de,
4
5
6
jschmidt@ipb-halle.de, gschneid@ipb-halle.de, gadam@ipb-halle.de
Received October 5, 2001
Accepted Jan uary 21, 2001
Dedicated to the memory of Dr Václav Černý.
Th e syn th eses of N-[10-(9α-fluoro-11β,17α-dih ydroxy-16α-m eth yl-3-oxoan drosta-1,4-dien e-
17β-carboxam ido)decyl]gibberellam ide (7) an d 6-[({N-[10-(9α-fluoro-11β,17α-dih ydroxy-
16α-m eth yl-3-oxoan drosta-1,4-dien e-17β-carboxam ido)decyl]carbam oyl}m eth oxy)im in o]-24-
epicastasteron e (10) are described. [(Ben zotriazol-1-yl)oxy]bis(pyrrolidin -1-yl)m eth ylium
h exafluoroph osph ate (HBPyU) was used as th e couplin g agen t for th e reaction of gibberellic
acid as well as of 24-epicastasteron e-O-(carboxym eth yl)oxim e with N-(10-am in odecyl)-
9α-fluoro-11β,17α-dih ydroxy-16α-m eth yl-3-oxoan drosta-1,4-dien e-17β-carboxam ide (4). Th e
gibberellic acid con jugate
7 was also syn th esised by th e couplin g of succin im idyl
gibberellate 6 with am in e 4.
Keyw ord s: Ph ytoh orm on es; Gibberellin s; Steroids; Brassin osteroids; Con jugation ; Dexam eth -
ason e; Gibberellin A3; 24-Epicastasteron e.
Heterodim er h ybrid m olecules derived from several n atural products are
useful tools in m olecular biology to study th e regulation of protein –protein
in teraction s, to trigger sign al tran sduction path ways an d for th e detection
of ligan d–protein receptor in teraction s1–3. As first m odels in such direction
in th e field of ph ytoh orm on es, we describe h erein th e syn th eses of
h eterodim ers of gibberellin A3 (ref.4) an d th e brassin osteroid 24-epicasta-
steron e5 h avin g dexam eth ason e as th e con jugated m oiety.
RESULTS AND DISCUSSION
For th e syn th eses of ph ytoh orm on e con jugates coupled with dexam eth a-
son e (9α-fluoro-16α-m eth ylpredn isolon e) via th e decan e-1,10-diam in e
Collect. Czech. Chem. Commun. (Vol. 67) (2002)
doi:10.1135/cccc20020103

104
Kolbe et al.:
spacer, dexam eth ason e (1) was oxidised in th e first step with periodic acid.
Th e obtain ed 9α-fluoro-11β,17α-dih ydroxy-16α-m eth yl-3-oxoan drosta-
1,4-dien e-17β-carboxylic acid6,7 (2) was con verted in to th e succin im idyl es-
ter 3 by th e reaction with N-h ydroxysuccin im ide usin g dicycloh exyl-
carbodiim ide as th e couplin g agen t. Th e active ester 3 was treated with
decan e-1,10-diam in e to afford N-(10-am in odecyl)-9α-fluoro-11β,17α-di-
hydroxy-16α-methyl-3-oxoandrosta-1,4-diene-17β-carboxamide2 (4) (Scheme 1),
a suitable com pon en t for th e couplin g with th e ph ytoh orm on e gibberellin
A3 (5) as well as 24-epicastasteron e (8). Com poun ds 2, 3, an d 4 h ave been
described2,6,7, but with out detailed spectroscopic data.
Startin g from GA3 (5), th e correspon din g succin im idyl ester was prepared
by th e reaction with N-h ydroxysuccin im ide usin g dicycloh exylcarbodi-
im ide as th e couplin g agen t. Th e obtain ed active ester 6 was treated with
am in e 4 to give, after repeated colum n ch rom atograph y an d in a poor yield
(16%), N-[10-(9α-fluoro-11β,17α-dih ydroxy-16α-m eth yl-3-oxoan drosta-
1,4-dien e-17β-carboxam ido)decyl]gibberellam ide (7). In a m ore efficien t
procedure, com poun d 7 was obtain ed in 57% yield by th e couplin g of 5
HOH2C
HO
20'
C O
OH
C O
18'
12'
OH
11'
HO
HO
21'
17'
19'
13'
16'
(i)
(ii)
1'
9'
2'
14'
15'
8'
7'
10'
F
F
3'
5'
O
O
4'
6'
1
2
O
O
O
N
O
H
N
1"-10"
20'
O
[CH2]10
NH2
18'
12'
OH
OH
16'
11'
HO
HO
21'
17'
14'
19'
13'
(iii)
1'
9'
2'
15'
8'
7'
10'
F
F
3'
5'
O
O
4'
6'
3
4
(i) EtOH, H5IO6,H2SO4; (ii) THF, dioxane, DCC, N-hydroxysuccinimide;
(iii) CH2Cl2, decane-1,10-diamine
SCHEME 1
Syn th esis of N-(10-am in odecyl)-9α-fluoro-11β,17α-dih ydroxy-16α-m eth yl-3-oxoan drosta-
1,4-dien e-17β-carboxam ide (4)
Collect. Czech. Chem. Commun. (Vol. 67) (2002)

Syntheses of Phytohormones
105
with 4 usin g th e excellen t peptide couplin g agen t [(ben zotriazol-1-yl)-
oxy]bis(pyrrolidin-1-yl)methylium hexafluorophosphate8,9 (HBPyU) (Scheme 2).
Because of th e absen ce of a carboxylic fun ction in th e brassin osteroid
24-epicastasteron e (8), a differen t couplin g procedure via th e 6-oxo fun c-
tion was used in th is case. Th us, 24-epicastasteron e-O-(carboxy-
m eth yl)oxim e (9) was syn th esised by th e reaction of 24-epicastasteron e (8)
with O-(carboxym eth yl)h ydroxylam in e h em ih ydroch loride in 0.1
M
eth an olic NaOH in a procedure sim ilar to th at described in th e ecdyson e se-
ries10. In th e n ext step, oxim e 9 was coupled with am in e 4 usin g HBPyU
(refs7,8). After purification by colum n ch rom atograph y, 58% of am orph ous
am ide 10 was obtain ed (Sch em e 3).
Th e spectral data of th e n ew com poun ds are in agreem en t with th e given
structures. 13C an d relevan t 1H NMR data of 2, 7, 9 an d 10 from on e- an d
1
two-dim en sion al NMR experim en ts in cludin g H-13C sh ift correlated spec-
tra (HSQC, HMBC) are sh own in Tables I–IV. Th e un equivocal assign m en t
of 1H an d 13C NMR sign als in th e dexam eth ason e part an d th e GA3 an d
O
O
1
11
10
19
CO
13
CO
3
8
(i)
14
15
OH
OH
6
HO
HO
H
H
16
COOH
CO
O
CH2
CH2
18
7
17
N
O
O
6
5
4
4
(iii)
(ii)
O
1
11
10
9
8
12
13
2
19
CO
3
5
14
15
OH
6
7
HO
4
H
16
CH2
18
O
H
N
17
1"-10"
[CH2]10
20'
O
N
18'
H
12'
11'
7
OH
16'
HO
21'
17'
14'
19'
13'
1'
9'
2'
15'
8'
7'
10'
(i) THF, dioxane, DCC, N-hydroxysuccinimide;
(ii) EtOAc, Et3N, EtOH, 72 h RT;
(iii) THF, Et3N, HBPyU, 45 min RT
F
3'
5'
O
4'
6'
SCHEME 2
Syn th esis of N-[10-(9α-fluoro-11β,17α-dih ydroxy-16α-m eth yl-3-oxoan drosta-1,4-dien e-
17β-carboxam ido)decyl]gibberellam ide (7)
Collect. Czech. Chem. Commun. (Vol. 67) (2002)

106
Kolbe et al.:
24-epicastasteron e m oieties in 7 an d 10 was m ade by m ean s of on e- an d
two-dim en sion al NMR experim en ts in cludin g COSY, NOESY, HSQC, an d
HMBC tech n iques. Th e NMR sign als of th e m eth ylen e ch ain could n ot be
assign ed. Th e 13C sign al at δ 175.4 (7) an d δ 173.3 (10), respectively, was
un am biguously assign ed to C-20′ by th e HMBC correlation with H-16′β.
Th e HMBC correlation between th e m eth ylen e proton s at δ 3.2 an d C-20′
proved th e couplin g of decan e-1,10-diam in e with C-20′. In a sim ilar m an -
n er, th e HMBC correlation s H-5/C-7, H-6/C-7, an d CH2/C-7 con firm ed th e
couplin g of th e gibberellin m oiety with th e decan e-1,10-diam in e spacer. In
th e case of 10, th e couplin g of th e spacer with O-(carboxym eth yl)oxim e 9
was verified by HMBC correlation s between H-1a/C-1b an d CH2/C-1b.
28
OH
OH
21
18
20
17
24
22
25
OH
11
OH
19
13
8
H2NOCH2COOH
HO
HO
HO
HO
15
1
10
3
6
1a
1b
H
H
O
N O CH2 COOH
8
9
28
24
OH
21
27
20
23
22
18
25
26
12
11
OH
17 16
15
19
THF
Et3N
HBPyU
13
1
4
9
HO
HO
14
2
3
10
8
7
4
5
6
H
N O
1a
CH2
O
H
N
1b
H
N
1"-10"
[CH2]10
20'
O
18'
12'
OH
16'
11'
HO
21'
17'
14'
19'
13'
1'
9'
2'
15'
8'
7'
10'
10
F
3'
5'
O
4'
6'
SCHEME 3
Syn th esis of 6-[({N-[10-(9α-fluoro-11β,17α-dih ydroxy-16α-m eth yl-3-oxoan drosta-1,4-dien e-
17β-carboxam ido)decyl]carbam oyl}m eth oxy)im in o]-24-epicastasteron e (10)
Collect. Czech. Chem. Commun. (Vol. 67) (2002)

Syntheses of Phytohormones
107
TABLE I
1H NMR data (δ, m ultiplicity, couplin g con stan ts [Hz]) for proton s of com poun ds 2 an d 7
(th e values with out m ultiplicities are ch em ical sh ifts of HSQC correlation peaks)
Posi-
tion
Posi-
tion
Posi-
tion
2
7
7
1′ 7.415 d [10.1]
1
2
6.371 dd
[9.4/0.6]
1′ 7.418 d [10.1]
2′ 6.282 dd [10.1/1.9]
5.866 dd
[9.3/3.5]
2′ 6.279 dd
[10.2/2.0]
3′
4′ 6.077 dd [1.9/1.5]
5′
–
3
4
5
6
3.980 d [3.7]
–
3′
–
–
4′ 6.074 br s
5′
–
3.256 d [10.1]
2.608 d [10.4]
6′ α: 2.390 ddd [13.6/5.1/1.7]
β: 2.718 tdd [13.7/6.2/1.6]
6′ α: 2.38
β: 2.719 td
[13.1/6.1]
7′ α: 1.517 m
7
–
7′ α: 1.51 m
β: 1.88 m
β: 1.885 dt [13.0/5.3]
8′ 2.444 dtd [29.0/12.0/4.9]
8
9
–
1.96
8′ 2.44
9′
–
–
9′
–
10′
10
–
10′
–
11′ 4.242 ddd [10.9/4.0/2.1]
12′ α: 2.12; β: 1.592 m
11
12
13
14
15
1.70/1.83
1.76/1.97
–
11′ 4.230 m
12′ α: 2.19; β: 1.43
13′
–
13′
–
14′ 2.14
1.64/1.90
2.21/2.21
14′ 2.18
15′ α: 1.201 ddd [12.2/8.3/4.2]
β: 1.743 dddd
15′ α: 1.19
β:1.74 m
[14.2/12.2/11.0/1.5]
16′ 2.996 dqd [11.2/7.2/4.2]
16
17
–
16′ 3.10
17′
17′
–
5.177/4.91
5
–
–
18′ 1.137
19′ 1.591
20′
18
19
1.203 s
–
18′ 1.089 s
19′ 1.589 s
20′
–
1′′
ca 3.2
21′ 0.940 d [7.2]
2′′–9′′ 1.30–1.50
10′′ ca 3.2
21′ 0.885 d [7.3]
Collect. Czech. Chem. Commun. (Vol. 67) (2002)

108
Kolbe et al.:
TABLE II
1H NMR data (δ, m ultiplicity, couplin g con stan ts [Hz]) for proton s of com poun ds 9 an d 10
(th e values with out m ultiplicities are ch em ical sh ifts of HSQC correlation peaks)
Posi-
tion
Posi-
tion
Posi-
tion
9
10
10
1
2
α: 1.45; β: 1.66
1
2
3
4
5
6
7
8
9
α: 1.43; β: 1.74
1′
2′
3′
4′
5′
6′
7′
8′
9′
7.301 d [10.0]
3.659 d [1.5]
3.70
6.321 dd [10.1/1.8]
3
3.928 d [2.4]
3.98
–
4
α: 1.797; β: 1.709
α: 1.85; β: 1.68
6.112
5
α: 2.401 dd [12.3/3.4]
α: 2.41
–
6
–
–
α: 2.36; β: 2.62
7
α: 1.29; β: 3.32
α: 1.30; β: 3.21
α: 1.53; β: 1.85
8
1.59
1.50
2.38
9
1.10
1.09
–
10
11
12
13
14
15
16
17
18
19
20
21
22
23
–
10
–
10′
–
α: 1.66; β: 1.33
α: 1.25; β: 2.03
–
11
12
13
14
15
16
17
18
19
20
21
22
23
α: 1.61; α: 1.28
α: 1.24; β: 2.01
–
11′
12′
13′
14′
15′
16′
17′
18′
19′
20′
21′
1′′
4.27
α: 2.18; β: 1.39
–
1.24
1.22
2.11
1.19/1.67
1.30/2.02
1.57
1.15/1.64
1.30/2.00
1.56
α: 1.20; β: 1.75
3.10
–
0.713
0.677
1.098
0.768
0.736
1.556
1.49
1.45
–
0.972 d [6.7]
3.65
0.965 d [6.7]
3.67
0.918 d [5.3]
3.27/3.16
3.30
3.36
2′′–9′′ ca 1.5 (8 H)
ca 1.3 (8 H)
24
25
26
27
28
1.48
24
25
26
27
28
1.48
10′′
3.28
1.94
1.92
0.913 d [7.0]
0.856 d [6.7]
0.832 d [7.0]
0.862 d [6.7]
0.915 d [7.0]
0.836 d [6.7]
4.42
1a 4.500/4.503
1a
1b
1b
–
–
Collect. Czech. Chem. Commun. (Vol. 67) (2002)

Syntheses of Phytohormones
109
TABLE III
13C NMR ch em ical sh ifts an d couplin g con stan ts JCF [Hz] for carbon s of com poun ds 2 an d 7
Position
2
Position
7
Position
7
1′
2′
156.2
1
2
3
4
5
6
7
8
9
133.2
134.1
70.7
55.1
54.3
53.6
173.5
51.7
52.1
92.8
18.1
39.8
78.8
46.1
44.6
158.2
107.2
14.9
181.3
40.4
1′
2′
156.1
129.8
129.7
3′
189.2
3′
189.0
4′
125.1
4′
125.0
5′
171.3
5′
171.2
6′
32.2
6′
32.3
7′
28.8
7′
28.8
8′
35.7 [19.0]
102.5 [175.7]
8′
35.8 [20.1]
102.6 [175.9]
50.3 [22.6]
73.1 [37.7]
36.9
9′
9′
10′
11′
12′
13′
14′
15′
16′
17′
18′
19′
20′
21′
50.3 [22.5] 10
73.1 [37.4] 11
10′
11′
12′
13′
14′
15′
16′
17′
18′
19′
20′
36.9
49.2
12
13
14
15
16
17
18
19
49.4
44.3
44.8
33.5
33.4
37.2
36.3
87.8
88.1
17.7
17.8
23.6 [5.8]
177.0
15.3
23.6 [6.3]
175.4
1′′
2′′–9′′
28.0/28.1/30.3/30.4 21′
15.2
30.5/30.6/30.6/30.8
10′′
40.4
Collect. Czech. Chem. Commun. (Vol. 67) (2002)

110
Kolbe et al.:
TABLE IV
13C NMR ch em ical sh ifts an d couplin g con stan ts JCF [Hz] for carbon s of com poun ds 9 an d
10
Position
9
Position
10
Position
10
1
2
40.7
69.4
69.9
29.2
44.6
174.4
31.7
36.8
55.6
41.4
22.3
41.1
43.9
57.9
25.0
28.9
54.2
12.3
13.3
41.7
13.1
73.5
77.4
42.7
28.0
22.6
17.6
11.2
70.8
162.9
1
2
39.2
68.1
68.3
27.3
43.3
162.6
30.4
35.6
53.9
39.5
21.0
39.4
42.6
56.2
23.9
27.5
52.5
11.8
12.8
40.2
12.2
72.3
75.8
41.4
26.8
17.0
22.0
10.6
72.1
171.0
1′
153.5
129.2
187.3
124.6
167.8
31.0
2′
3
3
3′
4
4
4′
5
5
5′
6
6
6′
7
7
7′
27.4
8
8
8′
34.2 [19.6]
100.9 [175.9]
49.9 [21.1]
71.7 [37.7]
35.7
9
9
9′
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
1a
1b
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
1a
1b
10′
11′
12′
13′
49.2
14′
43.6
15′
32.1
16′
35.1
17′
86.7
18′
17.0
19′
22.7 [5.3]
173.3
14.3
20′
21′
1′′
39.19
29.29
29.24
29.22
29.16
29.00
29.00
26.68
26.55
38.62
2′′–9′′
2′′–9′′
2′′–9′′
2′′–9′′
2′′–9′′
2′′–9′′
2′′–9′′
2′′–9′′
10′′
Collect. Czech. Chem. Commun. (Vol. 67) (2002)

Syntheses of Phytohormones
111
In con clusion , we h ave prepared th e first h eterodim er h ybrid m olecules
of ph ytoh orm on es gibberellin A3 an d 24-epicastasteron e with dexam eth a-
son e as th e con jugated m oiety, wh ich could be used as probes for m olecular
biological studies of protein –protein in teraction s, sign al tran sduction path -
ways an d ligan d–protein receptor in teraction s.
EXPERIMENTAL
Materials an d Meth ods
Merck silica gel 60 (particle size 0.040–0.063 m m , 230–400 m esh ASTM) was used for col-
um n ch rom atograph y. Meltin g poin ts (un corrected) were determ in ed on a Boetius h eatin g
table. High -resolution MS were m easured on an API Qstar/pulsar h ybrid quadrupole TOF MS
in strum en t (Applied Biosystem s). EIMS (DIS): 70 eV, AMD 402 (AMD In tectra). Th e positive-
ion electrospray (ESI) m ass spectra were obtain ed on a Fin n igan MAT TSQ 7000 in strum en t
(electrospray voltage 4.5 kV, h eated capillary tem perature 220 °C, sh eath gas n itrogen ) cou -
p led with a Micro-Tech Ultra-Plu s MicroLC system eq u ip p ed with an RP 18-colu m n
(5 µm , 1 × 100 m m , Ultrasep). For HPLC, a gradien t system was used startin g from H2O :
CH3CN 1 : 1 (each con tain in g 0.2% HOAc) to 1 : 9 with in 15 m in followed by a 5-m in
isocratic period; flow rate 70 µl m in –1. IR spectra (νm ax, cm –1) were obtain ed on a Bruker IFS 25.
UV spectra were m easured with a Perkin –Elm er Lam bda 16. NMR spectra 1D: Varian Gem in i
300 spectrom eter, 300.24 an d 75.5 MHz, solven ts CD3OD an d CDCl3; 2D: (1H-1H-COSY,
NOESY, GHMBC, an d GHSQC) Varian Un ity 500 spectrom eter, 499.83 MHz, solven ts
CD3OD an d CDCl3, TMS in tern al stan dard. Ch em ical sh ifts are given in ppm (δ-scale), cou-
plin g con stan ts (J) in Hz.
9α-Fluoro-11β,17α-dih ydroxy-16α-m eth yl-3-oxoan drosta-1,4-dien e-17β-carboxylic Acid (2)
9α-Fluoro-16α-m eth ylpredn isolon e (dexam eth ason e) (1; 2 g, 5.1 m m ol) was dissolved in eth -
an ol (400 m l). To th e stirred solution , periodic acid (1.37 g, 6 m m ol) in water (240 m l) an d
sulfuric acid (1.53 m l of con cen trated H2SO4 in 156 m l of distilled water) were added. After
stirrin g for 24 h at room tem perature, eth an ol was rem oved un der reduced pressure. Th e
crude product was collected by filtration , wash ed with water an d dried in a desiccator with
ph osph orus pen toxide. Th e crude acid was recrystallized to yield th e product 2 (1.82 g,
94.3%), m .p. 287 °C decom p. (MeOH–H2O). EIMS, m/z (% rel. in t.): 378 (M+, 2), 358 ([M – HF]+,
26), 160 (14), 147 (15), 135 (18), 122 (100), 120 (56), 107 (10), 91 (17). For 1H and 13C NMR data
see Tables I an d III.
Succin im idyl-9α-fluoro-11β,17α-dih ydroxy-16α-m eth yl-3-oxoan drosta-1,4-dien e-
17β-carboxylate (3)
Carboxylic acid 2 (1.7 g, 4.5 m m ol) an d N-h ydroxysuccin im ide (517 m g, 4.5 m m ol) were
dissolved in
a m ixture of dry THF (36 m l) an d dry dioxan e (36 m l). A solution of
dicycloh exylcarbodiim ide (927 m g, 4.5 m m ol) in dry THF (9 m l) an d dry dioxan e (9 m l) was
added, an d th e reaction m ixture was stirred at room tem perature overn igh t. Dicyclo-
h exylurea was rem oved by filtration , an d th e filtrate was con cen trated un der reduced pres-
sure to yield th e title com poun d (2.1 g, 98.3%), m .p. 254–256 °C decom p. (EtOAc), TLC
Collect. Czech. Chem. Commun. (Vol. 67) (2002)

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Kolbe et al.:
(silufol) RF 0.44 (CHCl3–MeOH 9 : 1, v/v). EIMS, m/z (% rel. in t.): 475 (M+, 0.2), 455 ([M –
HF]+, 15), 340 (6), 312 (12), 237 (8), 223 (8), 160 (15), 147 (17), 135 (15), 122 (100), 115
(15), 107 (19), 91 (17), 87 (24). LC-ESIMS: reten tion tim e 3.30 m in ; m/z 476 ([M + H]+). An a-
lytical HPLC: Hewlett–Packard 1100, LiCh rosph er 100 RP 18, 5 µm , 125 × 4 m m ,
MeCN–H2O (35 : 65), 1 m l m in –1, 254 n m , reten tion tim e 16.83 m in .
N-(10-Am in odecyl)-9α-fluoro-11β,17α-dih ydroxy-16α-m eth yl-3-oxoan drosta-1,4-dien e-
17β-carboxam ide (4)
To a stirred solution of decan e-1,10-diam in e (1.58 g, 9.2 m m ol) in EtOAc (500 m l),
succin im idyl ester 3 (1.45 g, 3.05 m m ol) in EtOAc (75 m l) was added un der n itrogen atm o-
sph ere an d th e reaction m ixture was stirred at room tem perature for 20 h . Th e solven t was
evaporated un der reduced pressure. Th e residue was purified by colum n ch rom atograph y
(CHCl3–MeOH–Et3N 3 : 1 : 0.04, v/v/v) to yield am orph ous product 4 (982 m g, 60.5%).
LC-ESIMS: reten tion tim e 2.82 m in ; m/z 533 ([M + H]+). An alytical HPLC: Hewlett–Packard
1100, LiCh rocart 100 RP 18, 5 µm , 125 × 4 m m , MeCN–0.1% Et3N (7 : 3), 1 m l m in –1, 254 n m ,
reten tion tim e 12.46 m in .
Succin im idyl Gibberellate (6)
GA3 (5; 1.03 g, 3 m m ol) an d N-h ydroxysuccin im ide (347 m g, 3 m m ol) were dissolved in a
m ixture of dry THF (15 m l) an d dry dioxan e (15 m l). Th en , at –10 °C, a solution of DCC
(638 m g, 3.1 m m ol) in dry THF (5 m l) an d dry dioxan e (5 m l) was added. Th e reaction m ix-
ture was stirred at –10 °C for 2 h , at –5 °C for 16 h , an d at room tem perature for 5 h . After
filtration of precipitated dicycloh exylurea, th e solven t was evaporated to yield 1.3 g of prod-
uct 6 (98.5%), m .p. 205–210 °C (EtOAc), RF 0.22 (CHCl3–MeOH 9 : 1, v/v). EIMS, m/z (% rel.
in t.): 443 (M+, 5), 425 (8), 329 (25), 310 (9), 301 (11), 283 (10), 267 (23), 255 (15), 238 (43),
209 (21), 195 (12), 136 (36), 121 (34), 107 (19), 91 (24), 88 (96), 77 (16), 69 (15), 58 (100).
N-[10-(9α-Fluoro-11β,17α-dih ydroxy-16α-m eth yl-3-oxoan drosta-1,4-dien e-
17β-carboxam ido)decyl]gibberellam ide (7)
Method A. To a stirred solution of com poun d 4 (402 m g, 0.76 m m ol) in EtOAc (70 m l)
an d trieth ylam in e (2 m l), succin im idyl ester 6 (356 m g, 0.8 m m ol) dissolved in EtOAc
(30 m l) was added. Th e reaction m ixture was stirred at room tem perature for 24 h . After ad-
dition of eth an ol (15 m l) to dissolve th e precipitate, th e reaction m ixture was stirred at
room tem perature for an oth er 48 h . Th e organ ic solven t was evaporated an d th e residue was
purified by colum n ch rom atograph y (CHCl3–MeOH 95 : 5, v/v) to yield th e crude product
(184 m g), wh ich was rech rom atograph ed (EtOAc–CHCl3 4 : 1, v/v) to yield 111 m g (16%) of
am orph ous con jugate 7. IR (KBr): 3 421 (NH); 2 929 (CH); 2 855 (CH); 1 760 (CO); 1 661
(CO); 1 525, 1 455, 891. UV (MeOH), λm ax, nm (ε, dm 3 m ol–1 cm –1): 206 (18 000), 239 (15 800).
EIMS, m/z (% rel. in t.): 798 ([M – CO2 – H2O]+, 2), 778 (9), 760 (19), 523 (71), 466 (35), 439
(55), 277 (73), 248 (100), 239 (70), 222 (75), 199 (49), 156 (61), 121 (72), 69 (45), 55 (78).
LC-ESIMS: reten tion tim e 3.87 m in ; m/z 861 ([M + H]+). ESI-TOF MS, m/z: 861.4870, calcu-
lated for C50H70FN2O9 861.5060. An alytical HPLC: Hewlett–Packard 1100, LiCh rosph er 100
RP 18, 5 µm , 250 × 4 m m , MeOH–0.2% HOAc (7 : 3), 1 m l m in –1, 254 n m , reten tion tim e
11.93 m in . For 1H an d 13C NMR data, see Tables I an d III.
Collect. Czech. Chem. Commun. (Vol. 67) (2002)

Syntheses of Phytohormones
113
Method B. To a stirred solution of GA3 (5; 34.2 m g, 0.1 m m ol) an d com poun d 4 (53.2 m g,
0.1 m m ol) in THF (3 m l), HBPyU (43.13 m g, 0.1 m m ol) an d Et3N (27.8 µl) was added. Th e
reaction m ixture was stirred at room tem perature for 45 m in an d th e solven t was evapo-
rated. Th e residue was purified by colum n ch rom atograph y (CHCl3–MeOH 95 : 5, v/v) to
give a wh ite product, wh ich was rech rom atograph ed (EtOAc–CHCl3 4 : 1, v/v) to yield 48.4 m g
(56.9%) of am orph ous con jugate 7. Th e spectral data were con sisten t with th e data for th is
com poun d obtain ed by method A.
24-Epicastasteron e-O-(carboxym eth yl)oxim e (9)
O-(Carboxym eth yl)h ydroxylam in e h em ih ydroch loride (275.3 m g, 1.26 m m ol) was added to
a stirred solution of 24-epicastasteron e (8; 464 m g, 1 m m ol) in 0.1 M eth an olic NaOH (17 m l).
Th e solution was stirred at 90 °C for 3 h an d th en at room tem perature for 16 h . Eth an ol
was rem oved un der reduced pressure an d th e residue was dissolved in water. Th e solution
was acidified with h ydroch loric acid an d extracted with EtOAc. Th e organ ic ph ase was
wash ed several tim es with water, dried over an h ydrous Na2SO4 an d th e solven t was rem oved
un der reduced pressure. Th e residue was purified by colum n ch rom atograph y
(CHCl3–MeOH–HOAc 9 : 1 : 0.1, v/v/v) an d recrystallized from EtOAc to give 527 m g of 9
(com poun d 9 was prepared via m odified procedure by B. Voigt (person al com m un ication )),
m .p. 173–174 °C. EIMS, m/z (% rel. in t.): 537 (M+, 0.2), 463 (7), 448 (20), 364 (100), 345
(54), 327 (37), 287 (20), 263 (13), 245 (14), 189 (12), 173 (20), 151 (27), 135 (24), 120 (58),
107 (60), 95 (50), 81 (63), 71 (55). For 1H an d 13C NMR data see Tables II an d IV.
6-[({N-[10-(9α-Fluoro-11β,17α-dih ydroxy-16α-m eth yl-3-oxoan drosta-1,4-dien e-
17β-carboxam ido)decyl]carbam oyl}m eth oxy)im in o]-24-epicastasteron e (10)
To a stirred solution of com poun d 9 (489 m g, 0.91 m m ol) an d carboxam ide 4 (480 m g,
0.9 m m ol) in THF (5 m l), HBPyU (392 m g, 0.91 m m ol) an d Et3N (276 µl) were added. Th e
reaction m ixture was stirred at room tem perature for 1 h , an d THF was evaporated. Th e resi-
due was purified by colum n ch rom atograph y (CHCl3–MeOH 95 : 5, v/v) to give (555 m g,
58%) of am orph ous con jugate 10. IR (KBr): 3 426 (NH); 2 932 (CH); 2 868 (CH); 1 664 with
lon g-wave sh oulder (CO); 1 531, 1 455, 1 066, 754. UV (MeOH), λm ax, n m (ε, dm 3 m ol–1 cm –1):
206 (18 000), 239 (18 200). LC-ESIMS: reten tion tim e 14.92 m in ; m/z 1 052 ([M + 1]+).
ESI-TOF MS, m/z: 1 052.7103, calculated for C61H99FN3O10 1 052.7309. An alytical HPLC:
Hewlett–Packard 1100, LiCh rosph er 100 RP 18, 5 µm , 250 × 4 m m , MeCN–0.2% HOAc (8 : 2),
1 m l m in –1, 254 n m , reten tion tim e 25.21 m in . For 1H an d 13C NMR data see Tables II
an d IV.
The authors are indebted to the Fonds der chemischen Industrie and to the Schering AG for
financial support. Further we are indebted to Prof. C. Gatz, Göttingen, for stimulating discussions and
Dr T. Degenkolb for the ESI-TOF measurements.
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