A simple chemical method for synthesizing malonyl hemiesters of 21-hydroxypregnanes, potential intermediates in cardenolide biosynthesis

Article abstract of DOI:10.1016/j.steroids.2007.12.012

A simple and versatile method for the chemical synthesis of 21-hydroxypregnane 21-O-malonyl hemiesters which may be important intermediates of cardenolide biosynthesis is described. Starting from commercial β-methyldigitoxin, acid hydrolysis followed by 3β-O-acetylation and ozonolysis with reductive cleavage of the ozonides afforded 3β-acetoxy-5β-pregnane-14β,21-diol-20-one which was finally converted into the target compound by treatment with malonyl chloride. The malonylation protocol was optimized using deoxycorticosterone (DOC) as the pregnane educt.

Full text of DOI:10.1016/j.steroids.2007.12.012

steroids 7 3 ( 2 0 0 8 ) 458–465
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journal homepage: www.elsevier.com/locate/steroids
A simple chemical method for synthesizing malonyl
hemiesters of 21-hydroxypregnanes, potential
intermediates in cardenolide biosynthesis
Rodrigo M. Pa´duaa, Reiner Waibelb, Serge Philibert Kuatea, Pia K. Schebitza,
Stefanie Hahna, Peter Gmeinerb, Wolfgang Kreisa,∗
a
¨
¨
¨
¨
Friedrich-Alexander-Universitat, Lehrstuhl fur Pharmazeutische Biologie, Staudtstr. 5, D-91058 Erlangen, Germany
b
Friedrich-Alexander-Universitat, Lehrstuhl fur Pharmazeutische Chemie, Schuhstr. 19, D-91052 Erlangen, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
A simple and versatile method for the chemical synthesis of 21-hydroxypregnane
Received 19 July 2007
21-O-malonyl hemiesters which may be important intermediates of cardenolide biosyn-
thesis is described. Starting from commercial ␤-methyldigitoxin, acid hydrolysis followed
by 3␤-O-acetylation and ozonolysis with reductive cleavage of the ozonides afforded
3␤-acetoxy-5␤-pregnane-14␤,21-diol-20-one which was finally converted into the target
compound by treatment with malonyl chloride. The malonylation protocol was optimized
using deoxycorticosterone (DOC) as the pregnane educt.
Received in revised form
7 December 2007
Accepted 13 December 2007
Published on line 23 December 2007
© 2007 Elsevier Inc. All rights reserved.
Keywords:
Malonylation
21-Hydroxypregnane
Malonyltransferase
Butenolide ring
Cardiac glycoside
Digitalis
1.
Introduction
to the 3␤-OH group, comprising glucose and rare 6-deoxy, 2,6-
dideoxy or 6-deoxy-3-methoxy sugars [8].
Cardiac glycosides are valuable drugs in the medication of
patients suffering from cardiac insufficiency due to their abil-
ity to bind to and to inhibit Na+–K+-ATPase. Additionally, these
steroids exhibit a wide spectrum of biological activities, and
have, for instance, anti-carcinogenic, acaricidal, antifilarial,
molluscicidal and antibacterial properties [1–7].
The cardenolide genins are composed of a steroid nucleus
with its four rings connected cis-trans-cis and ␤-hydroxyl
groups at carbons C-3 and C-14 and an unsaturated five-
membered lactone ring (butenolide ring) at C-17␤. Their
glycosides have a sugar side chain of varying length attached
The production of important Digitalis cardenolides, such as
digitoxin, digoxin and lanatoside C is dependent on plant cul-
tivation as these products have a high structural complexity
which makes their chemical synthesis economically imprac-
ticable [9,10]. Therefore, a more detailed knowledge of the
compounds, enzymes and genes involved in cardenolide for-
mation in plants is necessary with a view to studying the
regulation and engineering of the cardenolide biosynthetic
pathway.
Although features of the biosynthesis pathway of car-
denolides have been elucidated with the use of labeled
∗
Corresponding author. Tel.: +49 9131 8528241; fax: +49 9131 8528243.
E-mail address: wkreis@biologie.uni-erlangen.de (W. Kreis).
0039-128X/$ – see front matter © 2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2007.12.012

steroids 7 3 ( 2 0 0 8 ) 458–465
459
and unlabeled precursors, it is still not fully understood
2.2.
Spectroscopic techniques
[8,11–13]. It was shown by feeding experiments that the
carbon atoms C-20 and C-21 of the butenolide ring are
derived from the side chain of pregnanes, whereas the
atoms C-22 and C-23 have origin from malonate [14,15]. The
incubation of a 21-hydroxy-20-oxo-pregnane precursor, for
example 3␤-O-acetoxy-5␤-pregnane-14␤,21-diol-20-one with
malonyl-coenzyme A in the presence of cell-free extracts
from cardenolide-producing plants leads to the formation of
its 21-O-malonyl hemiester [16]. The enzyme catalyzing the
formation of the malonyl hemiester was termed malonyl-
coenzyme A: 21-hydroxypregane 21-O-malonyltransferase
(MHPMT) and is supposed to play an important role in carde-
nolide biosynthesis. The chemical structure of the enzymatic
product, namely the malonyl hemiester of 3␤-O-acetoxy-5␤-
pregnane-14␤,21-diol-20-one was originally only proposed on
basis of the GC–MS analysis of its decarboxylated product
[16].
While investigating the biosynthesis of cardenolides, one
of our aims was the development of a chemical method for
the synthesis of the malonyl hemiesters of pregnanes in order
to use these compounds as substrates to investigate the next
step in cardenolide biosynthesis, namely the butenolide ring
formation. This may be achieved by intramolecular nucle-
ophilic attack of methylene-deprotonated malonyl at the C-20
carbonyl under decarboxylation and dehydration.
2.2.1. NMR
1H NMR, 13C NMR, DEPT-135, 1H–1H COSY, HSQC, HMBC and
NOESY spectra were recorded on Bruker Avance 600 NMR spec-
trometer (1H 600 MHz and 13C 150 MHz) in CDCl3 at 300 K with
tetramethylsilane (TMS) as internal standard.
2.2.2. GC–MS
Analyses were performed on a GC Hewlett-Packard HP 5890A,
MSD type HP 5971A. An Optima®
5 column coated 5%
phenyl/95% dimethyl-poly-siloxane (0.25 ␮m) (30 m × 250 ␮m
i.d.) was used as the stationary phase. Helium served as mobile
phase with a constant pressure of 8.0 psi. Injection of the sam-
ples (1 ␮L of the standard solution, concentration 0.5 mg/mL)
was performed in a splitless mode with an inlet temperature
of 220 ◦C, according to the literature [17]. The applied temper-
ature programme included an initial step for 4 min at 200 ◦C,
temperature shifted up to 300 ◦C with 5 ◦C/min, followed by
40 min at 300 ◦C.
The MS-conditions were scan-modus, transfer line tem-
perature 280 ◦C, MS-temperature 162 ◦C and ionization energy
−70 eV.
2.2.3. LC–APCI-MS
Mass spectrum (MS) of the malonyl hemiester 4 was acquired
using atmospheric pressure chemical ionization (APCI) in the
negative ion mode with a Bruker Daltonics Esquire 2000 cou-
pled to Agilent 1100 LC—dry temp (Set) 260 ◦C; APCI temp (Set)
250 ◦C; nebulizer (Set) 13.00 psi; dry gas (Set) 4.5 L min−1. An
ODS column (Symmetry®—150 mm × 4.6 mm i.d., 5 ␮m) was
employed with a flow rate of 1.0 mL min−1 and a wavelength
of 205 nm. A gradient elution of CH3CN (A) and water pH 2.5
(adjusted with H3PO4) (B) was performed; 0 min 35% A, 65%
B; 4 min 55% A, 45% B; 12 min 66% A, 34% B; 13 min 100%
A; 17 min 100% A; 18 min 35% A, 65% B. The negative ion-
ization was obtained with a solution of 20% NH3 in MeOH
(3 mL h−1/syringe pump).
In this paper we report
a simple, fast and efficient
method for the chemical synthesis of 21-hydroxypregnane-
21-O-malonyl hemiesters as well as the complete assignment
of 1H and 13C data of these compounds by 1D and 2D NMR
experiments.
2.
Experimental
2.1.
General
Reagents and solvents were obtained from commercial
sources and were used as delivered unless stated other-
wise. The ketol 3 was isolated by thin layer chromatography
(TLC) on silica gel 60, 1.0 mm, 10 cm × 20 cm, F254 plates
(Merck, Darmstadt, Germany). The plates were developed four
times with a mixture of ethyl acetate-CH2Cl2 (20–80%, v/v). 3
(Rf = 0.60) was eluted from the silica gel with ethyl acetate.
The malonyl hemiesters 4 and 5 were separated on RP-18
preparative TLC plates, 1.0 mm, 10 cm × 20 cm, F254 (Macherey
Nagel, Du¨ ren, Germany). The plates were developed three
times with a mixture of CHCl3-acetone (90–10%, v/v), tetra-
zolium blue or UV were used for detection. 4 (Rf = 0.65) and
5 (Rf = 0.70) were eluted from the silica gel with acetone.
The compounds were dissolved in a small volume of CH2Cl2
and after addition of n-pentane, the solvent was evaporated.
The purity of the compounds was checked by GC–MS and
TLC on aluminum sheets silica gel 60 F254, precoated plates
(Merck, Darmstadt, Germany) in 7–93% (v/v) MeOH–CH2Cl2
(two times developed), 80–18–2% (v/v/v) CH2Cl2–MeOH–H2O
and 60–40% (v/v) ethyl acetate–CH2Cl2 (UV, anisaldehyde
and/or tetrazolium blue detection). The melting points were
determined by Bu¨ chi 535 equipment using capillary tubes
(80 mm × 1.0 mm).
Mass spectrum of the malonyl hemiester 5 was acquired
using APCI in the negative and positive ion modes with
the same conditions as 4, with exception of the wavelength
(240 nm) and the mobile phase of the analysis. A gradient
elution of CH3CN (A) and a solution of 0.1 mol L−1 HCOOH
(B); 0 min 20% A, 80% B; 10 min 60% A, 40% B; 15 min 60%
A, 40% B; 17 min 100% A; 20 min 100% A; 22 min 20% A, 80%
B.
ESI–MS were recorded on a Finnigan LCQ (Thermo Fisher
Scientific, Waltham, MA, U.S.A.) with quaternary pump Rheos
4000 (Ercatech, Bern, Switzerland).
2.3.
Synthesis
2.3.1. Digitoxigenin (1)
Digitoxigenin 1 was obtained by the acid hydrolysis of 4.0 g
of ␤-methyl-digitoxin, using published protocols (Scheme 1)
[18]. After extraction the residue (3.424 g) was recrystallized
(acetone/n-pentane) (1.148 g, 60%, m.p. 242–244 ◦C). GC–MS
analysis: m/z (relative abundance), 374 ([M+] < 1%), 356 (7%),
246 (11%), 203 (100%), and 111 (69%). The retention time of
digitoxigenin 1 was 36.3 min.

460
steroids 7 3 ( 2 0 0 8 ) 458–465
Scheme 1 – Synthesis of 3␤-acetoxy-5␤-pregnane-14␤-ol-21-O-malonyl-20-one 4 from ␤-methyl-digitoxin: (i) HCl/CH3OH,
35 min, 55 ◦C; (ii) AC2O-pyridine, 28 h, room temperature (24 ◦C); (iii) O3/EtOAc/pyridine, −70 ◦C, 3.6 h; (iv) Zn–HOAc, 12 h, 8 ◦C;
(v) (ClCO)2CH2, 1 min, −70 ◦C.
paper no longer turned violet. After 12 h at 8 ◦C the exces-
2.3.2. 3ˇ-Acetoxy-digitoxigenin (2)
sive zinc was filtered out of the solution. After adding 30 mL
of ice water, the reaction solution was extracted three times
with 30 mL of ice-cold dichloromethane. The organic phase
was washed twice with 30 mL saturated sodium hydrogen
carbonate solution and twice with 30 mL deionized water to
remove the excess of acetic acid. The yield was 50% (0.471 g,
m.p. 147–150 ◦C). GC–MS analysis: m/z (relative abundance),
392 ([M+] < 1%), 374 (<1%), 301 (100%), 283 (9%) and 255 (9%).
The retention time of the ketol 3 was 25.6 min.
1 g of digitoxigenin 1 was dissolved in 4 mL of dry pyridine.
Then 6 mL acetic anhydride were added and the solution was
stirred for 28 h at room temperature (24 ◦C) under CaCl2 pro-
tection using a published method with slight modifications
only [11]. The reaction was stopped by adding ice, so that the
product precipitated. The filtered precipitate was dissolved
in 100 mL dichloromethane, washed twice with 60 mL deion-
ized water and finally the organic solvent was evaporated.
The yield was 98% (1.090 g, m.p. 222–224 ◦C). GC–MS analy-
sis: m/z (relative abundance), 416 ([M+], < 1%), 356 (15%), 246
(34%), 231 (9%), 203 (100%) and 111 (35%). The retention time
of 3␤-acetoxydigitoxigenin 2 was 42.9 min.
2.3.4. 3ˇ-Acetoxy-5ˇ-pregnane-14ˇ-ol-21-O-malonyl-20-
one (4)
2 mL of malonyl dichloride (Fluka, Buchs, Switzerland) was
cooled with an acetone/dry ice mixture (−70 ◦C) for 5 min, then
100 mg of cold (−70 ◦C) ketol 3 was added. The reaction solu-
tion was shaken at −70 ◦C and CaCl2 protection for 1 min. Ice
was then added until the product precipitated. The precip-
itate was quickly filtered, then dissolved in chloroform and
the solution washed five times with water until the solution
reached pH 6.0. The organic phase was dried over Na2SO4 and
then evaporated at 20 ◦C. The malonyl hemiester 4 was iso-
lated from the residue (140 mg) as described in Section 2.1. The
yield was 45% (54.8 mg, m.p. 140 ◦C; decomp.). GC–MS analy-
sis: m/z (relative abundance), 434 ([M+ − CO2] < 1%), 416 (<1%),
406 (3%), 392 (3%), 301 (85%), 283 (6%), 273 (12%), 255 (8%) and
43 (100%). The retention time of 4 was 27.5 min. LC–APCI-
MS analysis: negative ionization (relative abundance)— 575
2.3.3. 3ˇ-Acetoxy-5ˇ-pregnane-14ˇ,21-diol-20-one (3)
1 g of 3␤-acetoxydigitoxigenin 2 was dissolved in 60 mL of
dry ethyl acetate and 3.6 mL of dry pyridine. The solution
was cooled with an acetone/dry ice mixture to −70 ◦C. The
ozonolysis was carried out basically as described with minor
modifications only [11,19,20]. First, a stream of a synthetic
air/ozone mixture was led in the reaction solution. This mix-
ture was initially added twice over 15 min and then six times
for 10 min with breaks of 10 min each between the additions.
After 50 min at −70 ◦C the solution was allowed to warm to
0 ◦C. The reductive cleavage of the ozonide was achieved by
adding 10 mL acetic acid, 6 mL deionized water and zinc dust
under shaking while keeping the temperature below 30 ◦C.
The zinc dust was added until moistened potassium iodide

steroids 7 3 ( 2 0 0 8 ) 458–465
461
in GC–MS (m/z = 374 for acetoxylated 6 and 7) or its spectrum
in high resolution electrospray ionization mass spectrometry
(m/z = 563.26130 [M + Na]+, 523 [M − H2O + H+] (calcd. 563.26154
for C31H40O8Na) for 8. The yields were found similar to those
of the malonyl hemiesters 4 and 5, ranging from 45 to 55%.
Table 1 – 13C chemical shifts (ppm) for
3␤-acetoxy-5␤-pregnane-14␤-ol-21-O-malonyl-20-one 4
and 21-O-malonyl deoxycorticosterone 5 obtained in
CDCl3 solution
Chemical shift (ppm)
Carbon
4
5
3.
Results and discussion
1
2
3
4
5
6
7
8
30.5
25.5
70.5
30.5
36.9
26.3
21.4
40.0
35.2
35.2
20.8
39.2
49.9
85.1
34.0
24.9
57.8
15.3
23.8
210.4
70.0
166.2
40.0
168.3
170.8
21.5
35.7
33.8
199.9
123.9
171.4
32.8
31.9
35.6
53.6
38.6
21.0
38.3
44.8
56.2
24.5
22.9
59.1
13.2
17.4
202.9
69.9
166.3
40.3
169.2
–
Malonyl hemiesters of 21-hydroxypregnanes may be the direct
precursors of butenolide ring formation in cardenolide biosyn-
thesis [8,16]. To test this assumption on the enzyme level, it
is necessary to have at least mg quantities of the respective
substrates for a putative ring-forming enzyme (cardeno-
lide synthase). 14␤-Hydroxylated 21-malonyloxypregnanes
such as the target malonyl hemiester 4 may be the ideal
candidate substrates for this reaction. Readily available ␤-
methyldigitoxin was used as the starting material for the
synthesis of the malonyl hemiester 4 via degradation of the
butenolide ring and subsequent 21-O-malonylation of the
intermediary pregnane-14␤,21-diol.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
Digitoxigenin 1, 3␤-acetoxydigitoxigenin 2 and the ketol 3
were characterized by GC–MS according to their molecular ion
peaks; M+, 374, 416 and 392, respectively, and compared with
authentic samples.
Malonyl dichloride reacts by cyclocondensation with di-
nucleophiles giving rise to a variety of so-called “malonyl
heterocycles”. In our study, we exploited the high reactivity
of malonyl dichloride as electrophile at low temperatures [21].
An excess of malonyl dichloride was used to avoid the forma-
tion of a polymer with two pregnane derivatives, as reported
for a method using methyl thiocarbamate [22]. When ketol 3
has reacted with malonyl dichloride at room temperature, the
main product was a non-polar compound with the MS spec-
trum of malonyl hemiester 4 but minus a water molecule (M+,
416), thus demonstrating the considerable influence of tem-
perature on the malonylation reaction. Increasing the reaction
time from 1 to 5 or 10 min did not improve yield.
C
O(3-OAc)
Me(3-OAc)
–
[M − H + H3PO4]− (100%), 559 [M − H + 2CH3CN]− (83%), 557
[M − H − H2O + H3PO4]− (39%), 433 [M − H − CO2]− (3%), 415
[M − H − H2O − CO2]− (34%), 397 [M − H − 2H2O − CO2]− (20%),
319 [M − 2H − H2O + 2CH3CN + H3PO4]2− (60%). The retention
time of 4 was 8.9 min. 13C and 1H NMR data (Tables 1 and 2).
2.3.5. 21-O-Malonyl deoxycorticosterone (5)
The target compound 3␤-acetoxy-5␤-pregnane-14␤-ol-21-
O-malonyl-20-one 4 was isolated as a white amorphous solid.
The atmospheric pressure chemical ionization mass spectrum
(APCI-MS) of 4 showed adduct ions [M − H + H3PO4]− at m/z 575,
that represent an interference on the fragmentation of 4 by
the mobile phase (CH3CN and water pH 2.5— adjusted with
H3PO4) used in the LC. The fragmentation ions at m/z 433 and
the adduct ion m/z 557 were due to the loss of a CO2 group
of the malonyl moiety and the loss of a water molecule of
the adduct ion at m/z 575, respectively. Malonyl hemiester 4
loses water easily because of its tertiary hydroxyl group at C-
14. Another adduct ion at m/z 559 was due to fragmentation
with two molecules of CH3CN of the mobile phase. The appli-
cation of APCI as an ionization method of sterols present some
limitations, because these compounds are generally highly
lipophilic and have few polar functional groups leading to a
small sensitivity and explaining the failure in obtaining the
MS spectrum of malonyl hemiester 4 by positive ionization
[23,24]. The GC–MS spectrum exhibited a molecular ion peak
at m/z 434 of decarboxylated 4.
21-O-Malonyl deoxycorticosterone
5 was obtained in an
analogous way as the malonyl hemiester 4 using deoxy-
corticosterone (DOC) as the pregnane educt (Scheme 2).
After isolation (Section 2.1), the yield was 50% (63.0 mg,
m.p. 146–148 ◦C). GC–MS spectra analysis: m/z (relative abun-
dance), 372 ([M+ − CO2], 4%), 354 (3%), 312 (21%), 299 (91%), 271
(40%), 253 (40%) and 43 (100%). The retention time of 5 was
26.1 min. LC–APCI-MS analysis: negative ionization (relative
abundance)— 415 [M − H]− (7%), 371 [M − H − CO2]− (100%), 353
[M − H − CO2 − H2O]− (83%), 329 [M − H − C3H2O3]− (7%); posi-
tive ionization (relative abundance)— 417 [M + H]+ (100%), 373
[M + H − CO2]+ (37%). The retention time of 5 was 12.1 min. 13
C
and 1H NMR data are shown in Tables 1 and 2.
2.3.6. Other 21-O-malonylated pregnanes
The 21-O-malonyl hemiesters of 5␤-pregnan-21-ol-3,20-dione
(6), 5␣-pregnan-21-ol-3,20-dione (7) and 3␤-benzoyloxy-5␤-
pregnane-14␤,21-diol-20-one (8) were synthesized using the
method described above (Scheme 2). The chemical structures
of the malonylated products were proposed on the basis of
the fragmentation pattern of their decarboxylated products
The 1H NMR spectrum of 4 showed the 3H singlets of the
methyl groups at C-18/C-19 and of the acetoxy moiety at ı

462
steroids 7 3 ( 2 0 0 8 ) 458–465
Table 2 – 1H chemical shifts (ppm) and coupling constants (Hz) for 3␤-acetoxy-5␤-pregnane-14␤-ol-21-O-malonyl-20-one
4 and 21-O-malonyl deoxycorticosterone 5 obtained in CDCl3 solution
Hydrogen
4
5
ı (ppm)
Coupling constants (Hz)
ı (ppm)
Coupling constants (Hz)
1␣
1␤
2␣
2␤
3␣
4
4
5␤
6␣
6␤
7␣
7␤
8␤
9␣
11␣
11␤
12␣
12␤
14␣
15␣
15␤
16␣
16␤
17␣
18
1.34
1.53
1.53
1.61
5.08
1.89 (␣)
1.44 (␤)
1.65
1.23
1.89
1.88
1.17
1.64
1.56
1.39
1.23
1.34
1.53
–
2.18
1.84
2.06
1.93
2.83
0.96
0.96
4.77
4.84
3.57
2.05
1.69
2.02
2.35
2.43
–
5.74
–
–
J1␣,1␤ = 13.5 Hz
J1␤, 2␣ = 3.9 Hz; J1␤, 2␤ = 3.9 Hz
J2␣,2 ␤ = 17.4 Hz; J2␣,1␣ = 3.9 Hz
J2␤,1␣ = 13.5 Hz
–
J4␣,3␣ = 3.0 Hz
J4␤,4␣ = 15.0 Hz; J4␤,3␣ = 3.0 Hz
J5␤,4␤ = 6.6 Hz
–
–
J6␣,7␤ = 11.7 Hz
2.27
2.40
1.05
1.85
1.56
0.96
1.62
1.45
1.38
2.05
1.18
1.73
1.31
1.71
2.20
2.51
0.69
1.17
4.59
4.80
3.55
–
J6␣,7␤ = 3.6 Hz; J6␣,7␣ = 3.6 Hz
J6␤,6␣ = 14.4 Hz; J7␣,6␤ = 12.8 Hz
J7␣,7␤ = 12.8 Hz
J7␤,8␤ = 3.6 Hz; J7␤,6␤ = 3.6 Hz
J8␤,7␣ = 12.8 Hz; J8␤,9␣ = 12.8 Hz
J9␣,11␣ = 3.8 Hz J9␣,11␤ = 12.8 Hz
J11␣,12␤ = 3.8 Hz; J11␣,12␣ = 3.8 Hz
J11␤,11␣ = 12.8 Hz, J11␤,12␣ = 12.8 Hz
J12␣,12␤ = 12.8 Hz; J12␣,11␤ = 12.8 Hz
J12␤,11␤ = 3.8 Hz
J7␣,7␤ = 11.7 Hz
J7␤,8␤ = 11.7 Hz; J7␤,6␤ = 3.3 Hz
J8␤,9␣ = 11.7 Hz
J9␣,11␣ = 3.3 Hz; J9␣,11␤ = 11.7 Hz
J11␣,12␤ = 3.3 Hz; J11␣,12␣ = 3.3 Hz
J11␤,11␣ = 13.2 Hz
–
J14␣,8␤ = 12.8 Hz
J15␣,15␤ = 18.8 Hz; J15␣,16␤ = 10.2 Hz
J15␤,16␣ = 3.1 Hz;
J16␣,16␤ = 10.2 Hz; J16␣,15␣ = 10.2 Hz
J16␤,15␤ = 10.2 Hz
J16␣,16␤ = 10.0 Hz
J16␤,15␤ = 2.6 Hz; J16␤,15␣ = 10.0 Hz
J17␣,16␤= 10.0 Hz; J17␣,16␣ = 10.0 Hz
J17␣,16␣ = 10.2 Hz; J17␣,16␤ = 4.2 Hz
19
21a
21b
23
J21a, 21b = 17.5 Hz
J21a, 21b = 17.5 Hz
Me(3-OAc)
–
atoms in compound 4. Complete 1H/13C one-bond shift cor-
relations of all protonated carbon atoms are presented in
Table 2. NOESY data, coupling constants and comparison with
the literature data [25] were used to distinguish diastereotopic
protons within methylene groups which exhibited character-
istic patterns for axially or equatorially located hydrogens
(Tables 2 and 3). The HMBC spectrum of 4 was also recorded
to assign the 13C NMR chemical shift assignments of qua-
ternary atoms and to confirm the substitution of functional
groups in compound 4. The C-21 methylene proton (ı 4.84
and 4.77) exhibited HMBC interactions with C-20 (ı 210.4) and
C-22 (ı 166.2). These HMBC observations indicated that C-21
was bonded to C-22 through an ester linkage. The presence
of an ester bond in compound 4 was also supported by 1H,
13C and mass spectral data. The C-23 methylene proton (ı
3.57) showed cross-peaks with the C-22 (ı 166.2) and C-24 (ı
168.3). The long-range hetero-nuclear multiple bond couplings
of C-17 methine proton (ı 2.83) with the C-20 (ı 210.4), C-14
(ı 85.1), C-21(ı 70.0), C-13 (ı 49.9), C-12 (ı 39.2), C-15 (ı 34.0)
and C-16 (24.9) were also observed in the HMBC spectrum
(Table 3).
0.96 and 2.05, respectively. This observation was confirmed
by the HSQC spectrum. Two doublets at ı 4.84 and 4.77 (d,
J21a,21b = 17.5 Hz) were ascribed to the geminal coupling methy-
lene protons at C-21. The methylene protons of the malonyl
side chain at C-21 resonated as a two-proton singlet at ı 3.57.
The C-3 methine proton, geminal to an acetoxy moiety, res-
onated at ı 5.08 and C-17 methine proton appeared as a double
doublet at ı 2.83 (J17␣,16␣ = 10.2 Hz and J17␣,16␤ = 4.2 Hz).
The 1H–1H COSY spectrum was helpful in the 1H NMR
chemical shift assignments of malonyl hemiester 4. The C-3
methine proton (ı 5.08) showed cross-peaks with C-2 methy-
lene (ı 1.53 and 1.61) and C-4 methylene (ı 1.44 and 1.89)
protons. The C-17 methine proton (ı 2.83) exhibited cross-
peaks with C-16 methylene (ı 2.06 and 1.93) protons.
The 13C NMR spectrum of compound 4 showed the reso-
nances of all 26 carbon atoms (Table 1). The DEPT experiment
indicated the presence of 3 methyl, 11 methylene and 5
methine carbon atoms in compound 4 and by comparison
with broadband 13C NMR spectrum revealed the presence
of seven quaternary carbon atoms in malonyl hemiester 4.
Four downfield resonances at ı 210.4, 170.8, 168.3 and 166.2
were due to the C-20, C O (acetoxy moiety), C-24 and C-
22 carbon atoms, respectively. The downfield chemical shift
values of C-14, C-3 and C-21 were due to the presence of
geminal oxygen functionalities on these carbon atoms. The
NOESY cross-peaks H12␣/H15␣, H12␣/H16␣, H12␣/H17␣
and H9␣/H15␣ indicate the presence of C/D cis ring junction
in 4. Additionally, evidence for the type of the ring junctions
of 4 was provided by the lower ı-values of C-5, C-7 and C-
9, characteristic of 5␤-pregnanes when compared with their
5␣-derivatives [25].
HSQC spectrum was very helpful to determine the 1H/13
one-bond shift correlations of all hydrogen-bearing carbon
C

steroids 7 3 ( 2 0 0 8 ) 458–465
463
Scheme 2 – Synthesis of 21-O-malonyl hemiesters 5, 6, 7, 8 from deoxycorticosterone, 5␤-pregnane-21-ol-3,20-dione,
5␣-pregnane-21-ol-3,20-dione and 3␤-benzoyloxy-5␤-pregnane-14␤,21-diol-20-one, respectively: (i) (ClCO)2CH2, 1 min,
−70 ◦C.
These spectroscopic studies led us to propose structure 4
for this putative intermediate of butenolide-ring formation in
cardenolide biosynthesis.
C-21 methylene protons at ı 4.59 and 4.80 at downfield as com-
pared with the literature data of DOC [27]. In contrast, the
signal of C-20 at ı 202.9 showed diamagnetic shift in compar-
ison with that of the DOC, as consequence of the proximity of
the malonyl side chain. The 1H and 13C of 5 were assigned with
help of HSQC, HMBC and NOESY spectra (Tables 1–3). Addition-
ally, we used the application of templates for the recognition
of signals due to particular protons from their profiles as sug-
gested in the literature [28].
The method was successfully applied for the malony-
lation of three other 21-hydroxy-pregnanes, namely 5␤-
pregnan-21-ol-3,20-dione, 5␣-pregnan-21-ol-3,20-dione and
3␤-benzoyloxy-5␤-pregnane-14␤,21-diol-20-one. The prod-
ucts 6 and 7 were identified by GC–MS and their spectra
compared to those of pregnane acetyl esters as the malonyl
hemiesters were decarboxylated to the corresponding acetyl
esters at the high temperatures used in GC analysis [16]. The
structure of compound 8, which could not be analysed by
GC–MS, was elucidated by HRESI–MS.
The malonylation protocol was optimized with the sur-
rogate compound DOC which is a commercially available
21-O-pregnane sharing some structure similarities with the
target substrate, 3␤-acetoxy-5␤-pregnane-14␤,21-diol-20-one.
The corresponding 21-O-pregnane malonyl hemiester, namely
21-O-malonyl deoxycorticosterone 5 was also isolated as
a white amorphous solid. The APCI–MS spectrum showed
[M + H]+ ion peak m/z 417 and [M − H]− ion peak m/z 415,
as well as their decarboxylated ions, as reported in the lit-
erature with carboxylated compound [26]. GC–MS spectrum
exhibited molecular ion peak at m/z 372 of 5 decarboxy-
lated.
Finally, the 1H and 13C NMR spectra of compound 5 were
nearly identical to those of DOC with the exception of some
resonances due to the 21-OH bound malonyl moiety. The 1H
NMR spectrum of malonyl hemiester 5 showed the resonance

464
steroids 7 3 ( 2 0 0 8 ) 458–465
Table 3 – Characteristic 1H–1H proximities (NOESY) and 13C–1H long-range correlations (HMBC) of
3␤-acetoxy-5␤-pregnane-14␤-ol-21-O-malonyl-20-one (4) and 21-O-malonyl deoxycorticosterone (5) obtained in CDCl3
solution
1H
4
5
NOESY, 1
1␤, 2␣, 2␤, 19
1␣, 19
1␣, 2␤, 3␣
1␣, 2␣, 3␣
2␣, 2␤, 4␤, 4␣, Me (3-OAc)
3␣, 4␤
3␣, 4␣, 5␤, 6␣
4␤, 6␤, 19
4␤, 6␤, 7␣
5␤, 6␣, 7␤, 8␤, 19
6␣, 7␤, 9␣
6␤, 7␣, 8␤, 15␣
6␤, 7␤, 11␤, 18, 19
7␣, 11␣, 15␣, 19
9␣, 11␤
8␤, 11␣, 12␤, 18
12␤, 15␣, 16␣, 17␣
11␤, 12␣, 17␣, 18
–
7␤, 9␣, 12␣, 15␤
15␣, 16␣
12␣, 15␤, 16␤, 17␣
16␣, 17␣, 21a, 21b
12␤, 12␣, 16␣, 16␤, 18, 21a, 21b
8␤, 11␤, 12␤, 17␣, 21a, 21b
1␣, 1␤, 5␤, 6␤, 8␤, 9␣, 11␤
16␤, 17␣, 18
H
HMBC, 13
3, 9, 10,19
C
NOESY, 13
1␤, 2␣, 9␣
1␣, 2␣, 2␤, 19
1␣, 1␤, 2␤
1␤, 2␣, 19
–
6␣
–
C
HMBC, 13
C
1␣
1␤
2␣
2␤
3␣
4(␣)
4␤
5␤
6␣
6␤
7␣
7␤
8␤
2, 3, 5, 9, 10, 19
2, 3, 5, 10, 19
1, 3, 4, 10
1, 3, 4, 10
–
5, 9, 10
3
a
a
a
a
a
a
2, 10
–
–
–
6␤, 7␣, 7␤
6␣, 7␤, 8␤, 19
6␣, 7␤
4, 5, 7, 8, 10
4, 5, 7, 8, 10
5, 6, 8, 14
5, 9
5, 7
14
a
6␣, 6␤, 7␣, 8␤
9, 10, 14
8, 14
12
9
13,18
9, 14
–
6␤, 7␤, 15␤, 18, 19
1␣, 11␣, 12␣, 14␣
9␣, 12␣, 12␤
11␣, 12␤, 18, 19
9␣, 12␤, 11␣, 14␣, 17␣
11␣, 11␤, 12␣, 18, 21a
9␣, 12␣, 16␣, 17␣
15␤
7, 9, 11, 14
9␣
1, 5, 7, 8, 10, 11, 14, 19
8, 9, 12, 13
9, 12
11, 13, 17, 18
9, 11, 13, 14, 18
9, 13, 15, 18
13
11␣
11␤
12␣
12␤
14␣
15␣
15␤
16␣
16␤
17␣
18
16
13, 14, 16, 17
15, 20
15, 17
12, 13, 14, 15, 16, 20, 21
12, 13, 14, 17
1, 5, 9, 10
20, 22
20, 22
22, 24
8␤, 15␣, 16␤, 18
16␤, 17␣
15␤, 16␣, 18
8, 14, 16
15, 17
14, 15, 17, 20
12, 13, 16, 18, 20
12, 13, 14, 17
1, 5, 9, 10
20, 22
12␣, 14␣, 16␣
8␤, 11␤, 12␤, 15␤, 16␤
1␤, 2␤, 6␤, 8␤, 11␤
17␣, 21b
19
21a
21b
23
16␤, 17␣, 18
a
12␤, 17␣, 21a
a
20, 22
22, 24
Me (3-OAc)
3␣
C
O (acetoxy group), 3
–
–
a
Non-observed cross-peaks.
r e f e r e n c e s
The compounds synthesized here may help to elucidate
butenolide ring formation in cardenolide biosynthesis fur-
ther. Actually, the 21-O-malonylated compounds synthesized
and characterized here have already been used to unambigu-
ously identify the reaction product of a 21-hydroxypregnane
21-O-malonyltransferase as being compound 4 [29]. In addi-
tion, we believe that the malonylation method described here
for the efficient production of malonyl hemiesters of ketol
structures may also find application in other synthetic pro-
tocols.
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