Synthesis of 5α,8α-ethanoergosta-2,6,22-triene-2'β-hydroxymethylene-1'β-carboxylic acid lactone, a key intermediate for the synthesis of a brassinolide analogue

Article abstract of DOI:10.1016/S0009-3084(99)00100-0

Metal hydride reduction of the 5α,8α-ethanoergosta-2,6,22-triene-1'β,2'β-dicarboxylic acid anhydride prepared by a new route afforded in prevalence the corresponding 4'-lactone, a known steroidal intermediate affording a brassinolide congener by hydroxyla

Full text of DOI:10.1016/S0009-3084(99)00100-0

Chemistry and Physics of Lipids
103 (1999) 117–123
www.elsevier.com/locate/chemphyslip
Synthesis of
5a,8a-ethanoergosta-2,6,22-triene-2%b-hydroxymethylene-1%b-ca
rboxylic acid lactone, a key intermediate for the synthesis of
a brassinolide analogue
Pietro Allevi *, Alessandra Longo, Mario Anastasia
Dipartimento di Chimica e Biochimica Medica, Uni6ersita` degli Studi di Milano, Via Saldini 50, 20133 Milan, Italy
Received 3 February 1999; received in revised form 19 July 1999; accepted 19 July 1999
Abstract
Metal hydride reduction of the 5a,8a-ethanoergosta-2,6,22-triene-1%b,2%b-dicarboxylic acid anhydride prepared by a
new route afforded in prevalence the corresponding 4%-lactone, a known steroidal intermediate affording a brassino-
lide congener by hydroxylation with osmium tetraoxide. © 1998 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Brassinosteroids; Steroidal lactones; Inhoffen adducts; Hydride reduction
lactone group of 3 is obtained, in a regioselective
way, by reduction with metal hydrides of the
Inhoffen adducts 5a or b, probably since the
3b-substituents direct the reductions to the nearest
carbonyl (Allevi et al., 1997; Rickard and Le
Quesne, 1998). On the contrary, the lactone group
of the compound 4 is obtainable only by sodium
borohydride reduction of the 3b-thexyldimethylsi-
lyloxy anhydride 5c (Allevi et al., 1999) in a
reaction in which an equal amount of the re-
gioisomer lactone parent of 3 is also formed.
Here we report a more satisfactory synthesis of
the lactone 4 and consequently the brassinosteroid
2. This steroid is useful for continue some prelim-
inary studies in which it has shown (unpublished
results) some ability to stimulate the lamina incli-
nation of rice (Maeda, 1965; Wada et al., 1981).
1. Introduction
In a recent paper from our laboratory (Allevi et
al., 1999) we reported a simple synthesis of the
two steroidic lactones 1 and 2 (Fig. 1) having
some structural features of brassinosteroids, plant
growth hormones, and of ecdysteroids, the moult-
ing hormones of insects (Cutler et al., 1991;
Yokota, 1997). Compounds 1 and 2 are prepared
(Allevi et al., 1999) starting from the unsaturated
lactones 3 and 4 which derive from the anhydrides
5a–c (Inhoffen adducts of ergosterol) (Inhoffen,
1934; Birckelbaw et al., 1970). In particular, the
* Corresponding author. Tel.: +39-2-70645231; fax: +39-
2-2361407.
E-mail address: pietro.allevi@unimi.it (P. Allevi)
0009-3084/99/$ - see front matter © 1998 Elsevier Science Ireland Ltd. All rights reserved.
PII: S0009-3084(99)00100-0

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P. Alle6i et al. / Chemistry and Physics of Lipids 103 (1998) 117–123
2. Materials and methods
Usual work-up refers to washing the organic
layer with water, drying over anhydrous Na2SO4
and evaporating the solvent under reduced
pressure.
2.1. General
All chemical materials were purchased from
Aldrich Chemical Co. (Milwaukee, WI, USA).
NaBH4 was crystallised from 2-methoxyethyl
ether and dried under vacuum.
Proton nuclear magnetic resonance spectra
were recorded as CDCl3 solution at 303 K on
Bruker AM-500 spectrometer operating at 500.13
MHz. All chemical shifts are reported in ppm
relative to CHCl3 fixed at 7.24 ppm. Signal multi-
plicity was designated according to the following
abbreviations: s=singlet, d=doublet, t=triplet,
q=quartet, m=multiplet.
All reactions and chromatographic separations
were monitored by thin-layer chromatography
(TLC) carried out on 0.25 mm E. Merck silica gel
plates (60 F254) using UV light or 50% sulphuric
acid and heat as developing agent. E Merck 230-
400 mesh silica gel was used for flash column
chromatography (Still et al., 1978).
2.2. Dehydration of the hydroxyanhydride 5a
2.2.1. Tosylation of the hydroxyanhydride 5a
The hydroxyanhydride 5a (Inhoffen, 1934; Bir-
ckelbaw et al., 1970) (0.9 g; 1.8 mmol), dissolved
in pyridine, (10 ml) was treated with p-toluenesul-
fonyl chloride (1.03 g; 5.4 mmol) at 0°C. The
mixture was then allowed to warm to 23°C and
was stirred for a further 20 h. The mixture was
diluted with an ice cold aqueous solution of HCl
(5 M; 30 ml) and filtered. The crude solid ob-
tained was dissolved in ethyl acetate (30 ml) and
worked-up to afford the crude p-toluenesulfony-
loxyanhydride 5d (0.870 g; Y 74%): m.p. 181–
182°C (from diisopropyl ether); TLC Rf 0.26
(eluting with hexane–ethyl acetate; 70:30, v/v);
1
[h]2D0 +9.3 (CHCl3, c 1); H NMR l 7.32 (2H, d,
J 8.0 Hz, aromatics), 7.83 (2H, d, J 8.0 Hz,
aromatics), 6.21 (1H, d, J 9.1 Hz, H-7), 5.70 (1H,
Fig. 1. Brassinolide analogues and their synthetic precursors.

P. Alle6i et al. / Chemistry and Physics of Lipids 103 (1998) 117–123
119
d, J 9.1 Hz, H-6), 5.22-5.13 (2H, overlapping,
H-22 and H-23), 4.88 (1H, dddd, J 10.5 Hz, 10.5,
6.1 and 5.0 Hz, H-3a), 3.26 (1H, d, J 8.5 Hz,
H-2%), 2.82 (1H, d, J 8.5 Hz, H-1%), 2.53 (1H, ddd,
J 13.0 Hz, 5.0 and 1.5 Hz; H-4a), 2.42 (3H, s,
CH3-Ar), 2.40 (1H, dddd, J 13.0, 10,0, 7.2 and 3.5
Hz, H-15a), 0.99 (3H, d, J 6.5 Hz, 21-CH3), 0.90
(3H, s, 19-CH3), 0.89 (3H, d, J 5.0 Hz, 28-CH3),
0.81 (3H, d, J 7.0 Hz, 26-CH3 or 27-CH3), 0.79
(3H, d, J 7.0 Hz, 27-CH3 or 26-CH3), 0.69 (3H, s,
18-CH3). Anal. Calcd. for C39H52O6S: C 72.19, H
8.08. Found: C 73.7, H, 8.2.
CH3), 0.90 (3H, d, J 7.0 Hz, 28-CH3), 0.83 (3H, s,
19-CH3), 0.82 (3H, d, J 6.8 Hz, 26-CH3 or 27-
CH3), 0.81 (3H, d, J 6.8 Hz, 27-CH3 or 26-CH3),
0.74 (3H, s, 18-CH3). Anal. Calcd. for C32H44O3:
C, 80.63; H, 9.30. Found: C, 80.7, H, 9.3.
2.3. Synthesis of 2,5,7,22-ergostatetraene 10 from
the urazole adduct 9
2.3.1. Synthesis by lithium aluminum hydride
reduction
To a solution of 3%,5%-dioxo-4%-phenyl-5,8[1%,2%]-
1%,2%,4%-triazolidino-5a,8a-ergosta-2,6,22-triene
9
2.2.2. Dehydrotosylation of
(Anastasia et al., 1985) (500 mg; 0.9 mmol) in dry
tetrahydrofuran (25 ml), lithium aluminum hy-
dride (340 mg) was added with stirring and the
mixture was refluxed for 3 h under argon (Barton
et al., 1971). Ethyl acetate (0.2 ml) was then
added to the mixture, followed by water (7.0 ml)
at 0°C. The mixture was then decanted and the
solid residue dissolved into a saturated solution of
sodium and potassium tartrate (10 ml) and ex-
tracted with ethyl acetate (20 ml). The organic
layer collected were worked up to afford a residue
which was crystallised from methanol to give the
tetraene 10 (277 mg; 81%), homogeneous on TLC
(plates containing 10% AgNO3; eluent hexane–
ethyl acetate; 90:10, v/v): m.p. 71–73°C (from
methanol); [h]2D0 −3.4 (c 1, CHCl3); UV umax 276
(m 9400), 286 (m 10 400), 298 nm (m 6700); 1H NMR
l 5.65–5.60 (2H, AB system, H-2 and H-3), 5.56
(1H, ddd, J 5.6, B1 and B1 Hz, H-6), 5.36 (1H,
ddd, J 5.6, 2.8 and 2.8 Hz, H-7), 5.23–5.14 (2H,
overlapping, H-22 and H-23), 3.00 (1H, ddd, J
22.0, B1 and B1 Hz, H-4a), 2.77 (1H, ddd, J
22.0, B1 and B1 Hz, H-4b), 1.02 (3H, d, J 6.6
Hz, 21-CH3), 1.00 (3H, s, 19-CH3), 0.90 (3H, d, J
6.8 Hz, 28-CH3), 0.82 (3H, d, J 7.0 Hz, 26-CH3 or
27-CH3), 0.80 (3H, d, J 7.0 Hz, 27-CH3 or 26-
CH3), 0.61 (3H, s, 18-CH3). Anal. Calcd. for
C28H42: C, 88.82; H, 11.18. Found: C, 89.0, H,
11.2.
p-toluenesulfonyloxyanhydride 5d
The p-toluenesulfonyloxyanhydride 5d (800 mg;
1.2 mmol) dissolved in sym-collidine (1.5 ml) was
refluxed for 8 h under argon. At this time the
mixture was diluted with an ice cold aqueous
solution of HCl (2 M; 10 ml), extracted with ethyl
acetate (15 ml) and worked-up to afford, after
purification by column chromatography (eluting
with hexane–ethyl acetate; 90:10, v/v), the 5a,8a-
ethanoergosta-2,6,22-triene-1%b,2%b-dicarboxylic
acid anhydride 6 (340 mg; Y 59%): m.p. 118–
1
119°C; [h]2D0 +0.10 (CHCl3, c 1); H NMR l 6.22
(1H, d, J 9.0 Hz, H-7), 5.94 (1H, d, J 9.0 Hz,
H-6), 5.74–5.66 (2H, overlapping, H-2 and H-3),
5.23–5.14 (2H, overlapping, H-22 and H-23), 3.27
(1H, d, J 8.5 Hz, H-2%), 3.25 (1H, ddd, J 13.0 Hz,
5.5 and 2.0 Hz; H-4a), 2.85 (1H, d, J 8.5 Hz,
H-1%), 1.00 (3H, d, J 6.7 Hz, 21-CH3), 0.90 (3H, d,
J 7.0 Hz, 28-CH3), 0.82 (3H, d, J 7.0 Hz, 26-CH3
or 27-CH3), 0.81 (3H, d, J 7.0 Hz, 27-CH3 or
26-CH3), 0.80 (3H, s, 19-CH3), 0.77 (3H, s, 18-
CH3). Anal. Calcd. for C32H44O3: C, 80.63; H,
9.30. Found: C, 80.5, H, 9.4.
Further elution yielded the 5a,8a-ethanoer-
gosta-3,6,22-triene-1%b,2%b-dicarboxylic acid anhy-
dride 7 (183 mg; Y 32%): m.p. 150–152°C (from
1
diisopropyl ether); [h]2D0 −13.8 (CHCl3, c 1); H
NMR l 6.28 (1H, d, J 9.0 Hz, H-7), 6.07 (1H,
ddd, J 10.5, 2.5 and 2.5 Hz, H-4), 6.00 (1H, d, J
9.0 Hz, H-6), 5.84 (1H, ddd, J 10.5, 3.5 and 3.5
Hz, H-3), 5.21–5.13 (2H, overlapping, H-22 and
H-23), 3.29 (1H, d, J 8.5 Hz, H-2%), 2.84 (1H, d, J
8.5 Hz, H-1%), 2.43 (1H, dddd, J 13.0, 10.0, 7.2
and 3.5 Hz, H-15a), 1.01 (3H, d, J 6.5 Hz, 21-
2.3.2. Synthesis by reflux with sym-collidine
A solution of the adduct 9 (500 mg; 0.9 mmol)
in sym-collidine (5 ml), was heated at reflux for 15
min under argon (Anastasia and Derossi, 1979).
The solution was then cooled at room tempera-

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P. Alle6i et al. / Chemistry and Physics of Lipids 103 (1998) 117–123
ture and diluted with an ice cold aqueous solution
of HCl (2 M; 20 ml). The resulting suspension was
then extracted with ethyl acetate and the organic
layer was worked up to afford a residue which
was crystallised from methanol to give the te-
traene 10 (236 mg; 69%), homogeneous on TLC
(plates coated with 10% AgNO3; eluent hexane–
ethyl acetate; 90:10, v/v): m.p. 71–73°C (from
methanol); [h]2D0 −4.1 (c 1, CHCl3); with spectro-
scopic properties identical with those of the com-
pound described above.
and 6.0, H-4%a), 3.04 (1H, ddd, J 9.7, 9.2 and 6.0,
H-1%), 2.53 (1H, dddd, J 12.5, 9.8, 7.9 and 3.5,
H-15a), 2.42 (1H, d, J 9.7, H-2%), 1.00 (3H, d, J
6.8 Hz, 21-CH3), 0.90 (3H, d, J 6.8 Hz, 28-CH3),
0.83 (3H, d, J 7.0 Hz, 26-CH3 or 27-CH3), 0.81
(3H, d, J 7.0 Hz, 27-CH3 or 26-CH3), 0.79 (3H, s,
19-CH3), 0.77 (3H, s, 18-CH3).
Further elution yielded 5a,8a-ethanoergosta-
2,6,22-triene-2%b-hydroxymethylene-1%b-carboxylic
acid lactone 4 (Allevi et al., 1999) (178 mg; Y
61%) as a white solid: m.p. 120–122°C; [h]D20
1
+58.3; H NMR l 6.19 (1H, d, J 9.1, H-7), 5.89
(1H, d, J 9.1, H-6), 5.72 (1H, m, H-3), 5.63 (1H,
m, H-2), 5.21 (1H, dd, J 15.0 and 7.5, H-22 or
H-23), 5.14 (1H, dd, J 15.0 and 8.0, H-23 or
H-22), 4.12 (1H, dd, J 9.1 and 9.1, H-3%b), 3.76
(1H, dd, J 9.1 and 5.8, H-3%a), 3.21 (1H, dd, J
18.0 and 6.1, H-4a), 2.77 (1H, d, J 9.5, H-1%), 2.52
(1H, ddd, J 9.5, 9.1 and 5.8, H-2%), 0.99 (3H, d, J
6.8 Hz, 21-CH3), 0.90 (3H, d, J 6.8 Hz, 28-CH3),
0.82 (3H, d, J 7.0 Hz, 26-CH3 or 27-CH3), 0.80
(3H, d, J 7.0 Hz, 27-CH3 or 26-CH3), 0.79 (3H, s,
19-CH3), 0.77 (3H, s, 18-CH3).
2.4. Synthesis of 5h,8h-ethanoergosta-2,6,22-
triene-1%i,2%i-dicarboxylic acid anhydride 6
Ergosta-2,5,7,22-tetraene 10 (5.0 g; 13.2 mmol)
was dissolved in xylene (50 ml) and treated with
maleic anhydride (1.35 g; 13.8 mmol) under argon
at 135°C for 6 h. Then the mixture was cooled,
poured into ice cold water and extracted. After
usual work-up, the crude residue obtained was
chromatographed (eluting with hexane–ethyl ac-
etate; 95:5, v/v) to afford the anhydride 6 (4.20 g;
Y 67%) m.p. 118–119°C; [h]2D0 +0.18 (CHCl3,
c
1); with spectroscopic properties identical with
those of the compound described above.
2.5.2. Reduction with lithium borohydride
Treatment of anhydride 6 (200 mg; 0.42 mmol)
with LiBH4 in the conditions described above for
the reduction with NaBH4, afforded the lactone 3
(50 mg; Y 26%) and lactone 4 (109 mg; Y 56%).
2.5. Reduction of 5h,8h-ethanoergosta-2,6,22-
triene-1%i,2%i-dicarboxylic acid anhydride 6
2.5.1. Reduction with sodium borohydride
2.5.3. Reduction with L-selectride
The anhydride 6 (300 mg; 0.63 mmol), dis-
solved in a solution of tetrahydrofuran–methanol
(8 ml; 1:1, v/v), was treated with NaBH4 (23.3 mg;
0.62 mmol) under stirring at room temperature
for 12 h. At this time, the mixture was treated
with water (5 ml) and extracted with ethyl acetate
(15 ml). Usual work-up and rapid chromatogra-
phy (eluting with hexane-dichloromethane; 50:50,
v/v) afforded the 5a,8a-ethanoergosta-2,6,22-
triene-1%b-hydroxymethylene-2%b-carboxylic acid
lactone 3 (Allevi et al., 1999) (57 mg; Y 20%) as a
white solid: m.p. 132–133°C; [h]2D0 −131.7; 1H
NMR l 6.26 (1H, d, J 9.1, H-7), 5.82 (1H, d, J
9.1, 6-H), 5.68 (1H, m, H-3), 5.57 (1H, m, H-2),
5.22-5.14 (2H, overlapping, H-22 and H-23), 4.05
(1H, dd, J 9.2 and 9.2, H-4%b), 3.78 (1H, dd, J 9.2
The anhydride 6 (225 mg; 0.47 mmol) was
dissolved in dry, freshly distilled THF (15 ml) at
25°C under argon. L-Selectride (1.4 ml of a 1 M
solution in THF; 1.4 mmol) was then injected
slowly and the reaction mixture was stirred for 5
h at 25°C. At this time NaOH (0.7 ml of an
aqueous 4 M solution) and H2O2 (1 ml of a 30%
solution) were added and the stirring was contin-
ued overnight. The reaction was then acidified
with an ice cold aqueous solution of HCl (2 M)
and extracted with ethyl acetate. Usual work-up
and rapid chromatography (eluting with hexane–
dichloromethane; 50:50, v/v) afforded lactone 3
(116 mg; Y 53%) and lactone 4 (60 mg; Y 28%)
identical in all aspects with the compounds above
described.

P. Alle6i et al. / Chemistry and Physics of Lipids 103 (1998) 117–123
121
Fig. 2. Schematic pathway of the synthesis of D2-unsaturated anhydride 6 via dehydration of Inhoffen adduct. Reagents and reaction
conditions: (i) p-toluenesulfonyl chloride, pyridine, 23°C, 20 h; (ii) sym-collidine, reflux, 8 h.
2.5.4. Reduction with K-selectride
regioselectivity of the hydride reduction and could
decrease the steric hindrance at the 4%-carbonyl
which is believed one of the factors which favour
its reduction (Soucy et al., 1987 and references
therein).
Treatment of anhydride 6 (225 mg; 0.47 mmol)
with K-selectride in the conditions described
above for the reduction with L-selectride, af-
forded the lactone 3 (111 mg; Y 51%) and lactone
4 (54 mg; Y 25%).
Unvaried results were obtained performing the
reductions with NaBH4 or LiBH4 at 0°C and at
−10°C and the reductions with L- and K-selec-
tride at −40°C.
With this in mind we decided to prepare the
anhydride 6 starting from the Inhoffen adduct 5a
(Fig. 2) which was transformed into the corre-
sponding D2-unsaturated compound 6 via dehy-
drotosylation
of
the
corresponding
3b-p-toluensulfonate 5d, by treatment with sym-
collidine at reflux. These conditions preserved the
anhydride group but promote a non regiospecific
dehydrotosylation which affords the compound 6
(in 59% yield; 7% total yield from ergosterol)
always accompanied by considerable amounts of
the D3-isomer 7 (32%) unsuitable for the synthesis.
The formation of the undesired D3-anhydride
was quite unexpected considering our previous
result in the dehydration of the Diels–Alder ad-
duct of ergosterol 8 which was transformed exclu-
sively into the D2-compound 9 by a similar
reaction sequence (Anastasia et al., 1985). On the
other hand, the obtaining of the D2-compound 9
from ergosterol (in 65% yield) suggested that the
anhydride 6 could be prepared from ergosterol via
the adduct 9 (Fig. 3). In fact this compound
contains a masked D5,7 system which could be
regenerated (Barton et al., 1971; Anastasia and
Derossi, 1979; Anastasia et al., 1985) in a reaction
giving the triene 10 able to react with maleic
anhydride to afford the anhydride 6. Thus, the
treatment of the adduct 9 with LiAlH4 (Barton et
al., 1971) or sym-collidine (Anastasia and
3. Results and discussion
Earlier work (Allevi et al., 1997; Rickard and
Le Quesne, 1998) on the metal hydride reduction
of the cyclic anhydrides 5a and b suggested that
the 3b-substituents influence the regiochemistry of
the reductions of the anhydride group. In fact, the
3b-hydroxy and the 3b-acetoxy substituents direct
the reduction completely to the carbonyl nearest
to the ring A (Allevi et al., 1997; Rickard and Le
Quesne, 1998) while the 3b-methyl (Burke and Le
Quesne, 1971) and 3b-thexyldimethylsilyloxy sub-
stituents (Allevi et al., 1999) show lower directing
effect and permit the obtaining of both possible
lactones in roughly equal amounts, almost using
sodium borohydride. Because of these results we
considered that the D2 anhydride 6 (Fig. 2), lack-
ing of any substituent at the 3b-position of the
steroid nucleus, could be a suitable precursor of
the unsaturated lactone 4. In fact we were confi-
dent that the absence of any 3b-substituent elimi-
nates its unfavourable influence on the

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P. Alle6i et al. / Chemistry and Physics of Lipids 103 (1998) 117–123
Fig. 3. Schematic pathway of the synthesis of D2-unsaturated anhydride 6 via tetraene 10. Reagents and reaction conditions: (i)
p-toluenesulfonyl chloride, pyridine, 23°C, 21 h; (ii) basic Al2O3, toluene, stirring at r.t. for 10 h; (iii) LiAlH4, THF, reflux, 3 h; (iv)
maleic anhydride, xylene, 135°C, 6 h.
Derossi, 1979) regenerated the D5,7 dienic system
typical of ergosterol affording the crystalline te-
traene 10 uncontaminated by the D3-isomer. This
tetraene was then reacted with maleic anhydride
in a Diels–Alder reaction affording the anhydride
6 in satisfactory yields (67% yield).
The anhydride 6 was then subjected to reduc-
tion with different hydride (sodium and lithium
borohydride, L- and K-selectride (Makhlouf and
Rickborn, 1981) at different temperatures. The
obtained results showed that, in all cases, an
incomplete regioselectivity of the reduction oc-
curred since both possible lactones 3 and 4 were
formed. However while L- and K-selectride af-
forded as major compound the undesired lactone
3, NaBH4 and LiBH4 afforded the lactone 4 as
major component (56–61%) which contained the
lactone 3 in minor ratio (20–26%), independently
from the temperature.
1999; Rickard and Le Quesne, 1998) show that
the presence and the nature of a 3b-substituent in
the Inhoffen anhydrides are dominant factors in
influencing the course of the metal hydride reduc-
tions of Inhoffen adducts. In their absence steric
effects prevail and favour the reduction of the
more hindered carbonyl (Bloomfield and Lee,
1967). In this case, the nucleophile’s reactivity and
the effect of the counterions of the hydride
reagents appear on the contrary negligible
(Bloomfield and Lee, 1967).
Acknowledgements
This work was supported by Ministero dell’
Universita` e della Ricerca Scientifica e Tecnolog-
ica (MURST; fondi 60%).
Therefore, the lactone 4 was prepared using
NaBH4 and transformed into the brassinolide
analogue 2 according to the previously reported
procedure (Allevi et al., 1999). Thus, the improve-
ment of its preparation by a different route repre-
sents an improvement of the synthesis of lactone
2.
References
Allevi, P., Ciuffreda, P., Anastasia, M., 1997. Revised struc-
ture for the lactones obtained by reduction of the adduct
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Allevi, P., Longo, A., Anastasia, M., 1999. Synthesis of two
analogues of brassinolide, possible plant growth promoting
steroids. Tetrahedron 55, 4167–4176.
In conclusion, we report here an improved
method for converting inexpensive ergosterol to
lactone 4 (in 21% total yield) and consequently to
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obtainable in amounts suitable for biological ex-
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