79 (1960) RECUEIL 771

577.161.2:542.952 1
INVESTIGATIONS ON STEROLS XV *
The syntheses and properties of 9P,lOa-progesterone and
6-dehydro-9/3,lOa-progesterone
BY
P. U'ESTERHOF and E. H. REERINK
(Central Laboratory, N.V. Philips-Duphar, Weesp)

A description is given of the syntheses of 98, 10a-progesterone

and 6-dehydro-48.1 Om-progesterone using lumisterolz as the starting
material.

Introduction
In the last decmnium the investigations in the field of steroid
hormones have been directed particularly to the preparation of
compounds with stronger or more dissociated physiological activities.
Most attention has been paid to introduction of substituents into the
steroid molecule. On the other hand, the preparation of analogues with
abnormal configuration a t the centres of asymmetry of the steroid
skeleton, from which the influence of such stereochemical changes on
the physiological properties can be evaluated, has received relatively
little consideration.
I n this laboratory Bots 1 according to this train of thought prepared
13a-steroids, using the Butenandt 2 9 3 technique. These compounds
showed no androgenic or anabolic activity 1.
The present publ-ication deals with the first part of the investigations
in preparing steroid hormone analogues inverted a t other centres of
asymmetry i.e. 98,lOa steroids (as in lumisterolz). ** Up to now no
steroid hormone analogues with this configuration have been described
in the literature. The transformation of the normal steroid configuration

Previous communication: M . P. Rappoldt, Rec. trav. chim. 79, 392 (1960).

** The trivial name proposed for this series of steriods is vetro-steroids, e.g.
9p, 1Ox-progesterone = velvo-progesterone.
1 J P. L . Hots, Rec. trav. chim. 77, 1010 (1958).
2 A . Butenandt, W . Fviedvich and L. Poschman, Ber. 75, 1931 (1942).
3 A . Butenandt and L. Poschman, Ber. 77, 394 (1944).
772 P. Weslerhof and E. H . Reerink,

into the 9p, 10a configuration is found to have a remarkable effect on the
physiological properties of the compounds in comparison with those of
the analogues in the normal 9a,lOb series *. This prompted us to syn-
thesize a large number of representative structures in the 9/l, I Oa series
and to investigate the physiological properties of the compounds
prepared. The present paper describes the syntheses of 9b, 1Oa-proges-
terone (9p,10a-pregn-4-ene73,20-dione)and 6-dehydro-9p, 1Oa-pro-
gesterone (9p,1Oa-pregna-4,6-diene-3,20-dione)from lumisterolz.
Recently Castells et al. 5 established that lumisterol2 has the 9P,1 Oa
49

configuration, which fact was derived from rotation data and degra-
dation of the four C-9, C-10 stereoisomers, involving the elimination of
the stereochemical differences at position 10.
For the preparation of the title compounds from lumisterole we
made use of the route employed by Shepherd et al. ti for the synthesis of
progesterone from ergosterol.

Chemistry **
The Oppenauer oxidation of lumisterole (I) to lumisterone (11)
(lumista-4,7,22-trien-3-one) 7 was carried out according to the method
of Shefherd et a,?. 6 .The yield was somewhat lower (55-60%) than in
the ergosterol series.
At the time of our investigations the isomerisatiom from lumisterone
to isolumisterone (111) (lumista-4,6,22-trien-3-one) had not yet been
described in the literature.
We found that this isomerisation can be carried out with good
results if the reaction takes place under substantially waterfree and
acidic conditions. When using hydrohalogenic acids as the isomerisa-
tion agents and a solvent in which the acids are ionised to a great
extent particularly fair results were obtained with lower aliphatic
alcohols. The most satisfying results were achieved while using hydro-
chloric acid. Thus, waterfree lower aliphatic alcohols saturated for 60
t o 100% with dry hydrochloric acid appeared to give high yields.
When lumisterone was reacted with a saturated solution of hydro-
* Preliminary communication: Nature 186, 168 (1960).
** I n this paper by lumistane is meant 9/?, l0a-ergostane.
4 J . Castells, E . R . H . Jones, R , W . J . Williams and G . D . Meakins. Proc.
Chem. SOC.1958, 7.
5 J . Castells, E . R . H . Jones, G . D. Meakins and R . W . J . Williams, J. Chem.
SOC.1959, 1 159.
6 D , A . Shepherd, R . A . Donia, J . A . Campbell, B . A . Johnson, R . P . Holysz,
G . Slomp J r . , J . E . Stafford, R.L . Pederson and A . C . Ott, J. Am. Chem. SOC.
77, 1212 (1955).
7 I . M . Heilbron, T . Kennedy, F . S . Spying and G . Swain, J. Chem. SOC.1938,
869.
 Investimtions on sterols, X V etc. 79 (1960) RECUEIL 773

Chart. I

C H,O
IX

chloric acid in isopropanol for 30 minutes a t 10" a crude isomerisation

product was obtained which contained 70-90% of 4,6,22-trien-3-one.
A single recrystallisation from petroleum ether afforded nearly pure
isolumisterone in a yield of 60-70%. The compound appeared to be
polymorphous, because in the early experiments crystals with a
melting point of 88-89" were obtained. After allowing to stand for
some months the crystals melted a t 100". We did not succeed in
N

obtaining the lower melting modification afterwards.

Other methods based on known technics for the normal series were
investigated too, but all of them failed to give satisfying results.
Application of the method employed by Shepherd et al. 6 in the
774 P . Westerhof and E . H . Reerink,

ergosterol series, consisting in the isomerisation of the 4,7,22-trien-3-

one in a boiling mixture of methanol and concentrated hydrochloric
acid, only resulted in the formation of the 3-en01 ether (3-methoxy-
lumista-3,5,7,22-tetraene) (IV) in a moderate yield. The rotations of
chloroform solutions of this enol ether showed a shift to the left when
allowed to stand a t room temperature, while on the contrary Shepherd
et al. 6 observed for the corresponding compound in the ergosterol
series a shift to the right.
Isomerisation of the 4,7,22-trien-3-one with concentrated hydro-
chloric acid in boiling acetic acid according to the method of Antonucci
et al. 8 and hydrolysis of the 3-en01 acetate under acidic 7 and alkaline 9
conditions gave bad results. Reaction of the starting material with
a saturated solution of dry hydrochloric acid in chloroform at 0" 109 l1
provided a crude isomerisation product, which contained only small
amounts of the desired isomer. Under optimal reaction conditions for
this solvent, i.e. by reacting the 4,7,22-trien-3-one for 74 hours a t 45"
in chloroform saturated with dry hydrochloric acid, the content of
4,6,22-trien-3-one in the crude reaction product could be raised to
40-50%.
After completion of our investigations on the isomerisation reaction
Castells et al. 5 described the conversion of lumisterone into isolumis-
terone by a reaction with hydrogen bromide in chloroform-acetic acid.
By adsorbing the crude reaction product on alumina unchanged
lumisterone was transformed into non-elutable material. Elution with

yield of -
petroleum ether-benzene and crystallisation gave isolumisterone in a
40%. Elution with the same mixture of solvents also gave
a not yet identified unconj ugated ketone.
I n our experiments this compound was only obtained when the
isomerisation was camed out in solvents which gave isolumisterone
in low yields (ethyl bromide, chloroform). The ultraviolet and infrared
absorption spectra indicated the probable presence of a 6,8(9) or a
6,8(14) diene system 1 2 (V), as already suggested by Castells et al. 5.
Catalytic hydrogenation of the 4,6-dien-3-one system of isolumiste-
rone in weakly alkaline methanol 49 5,6913 in the presence of pre-
reduced palladium-norite gave dihydroisolumisterone (VII) (lumista-

* R . Antonucci, S . Bernstein, D. Giancola a n d K . J . Sax, J . Org. Chcm. 16,

1453 (1951).
8 A . F . Daglish, J. Green a n d V . D. Poole, J . Chem. SOC. 1954, 2627.
10 F. Johnson, G . T . Newbold a n d F . S . S@ing, J . Chem. SOC. 1954, 1302.
11 D . H . R . Barton, J . D. Cox and N . J . Holness, J . Chem. SOC. 1949, 1771.
12 F. Reindel, E . Walter and H . Rauch, Ann. 452, 34 (1927).
1s E . R . Garret, R . A . Donia, B . A . Johnson a n d L . Scholten, J. Am. Chem.
SOC. 78, 3340 (1956).
 Investigations
-~
on sterols, X V etc. 79 (1960) RECUEIL 775

4,22-dien-3-one) in a rather good yield. A much better result, however,

was obtained when the reduction was carried out with lithium in
liquid ammonia according to a modification of the method of Green
et al. 1 4 and Johnson et al. 10. As an excess of lithium metal caused over
reduction in the lumisterol series, the reaction was carried out by
adding a solution of the metal in liquid ammonia to a well stirred
solution of isolumisterone in a mixture of diethyl ether and ammonia
until the spontaneous decolourisation of the added solution ceased and
the reaction mixture remained blue for a short time. Decomposition
with dry ethanol and working up by dilution with water resulted in a
crude reaction product which showed a low absorption maximum at
240 mp, Ei& N 100-200. Crystallisation from a mixture of acetone
and methanol gave a compound which melted at 88-90' and did not
show an ultraviolet absorption a t wavelengths > 220 mp. The in-
frared absorption spectrum showed a strong band at 1720 cm-1
indicating the presence of an unconjugated keto group. Boiling this
compound with alkali in ethanol 15 for a short time caused isomerisation
to the conjugated 4,22-dien-3-one in a high yield. Apparently 1,4-
addition at the system of double bonds had initially formed lumista-
5,22-dien-3-one (VI), which by working up was only partly isomerised
to lumista-4,22-dien-3-one (VII).
The low yield of ergosta-4,22-dien-3-one obtained by Green et al. 14
by reducing ergosta-4,6,22-trien-3-one with an excess of lithium in
ammonia and deca3mposing the reaction mixture with ammonium
chloride instead of ethanol, is probably caused by the neutralizing
action of the inorganic salt on the reaction medium, which causes the
5,22-dien-3-one to be only partly isomerised t o the desired end
product.
Ozonisation of lumista-4,22-dien-3-one (VII) in methylene chloride
in the presence of pyridine a t -80" according to Shepherd et al. 6 and
Slomp and Johnson 16 was followed by reductive decomposition with
zinc dust and glacial acetic acid. The aldehyde obtained (9p,10a-
bisnorchol-4-en-3-01i-22-al) (VIII) could readily be recrystallised from
diethyl ether. The ultraviolet absorption maximum a t 242.5 mp and
the bands at 1610 cm-1 and 1662 cm-l in the infrared spectrum
confirmed the presence of the 4-en-3-one system. Decomposition of
the ozonisation-mix ture with chromic acid formed the corresponding
3-oxo-9b, 1Oa-bisnorchol-4-enic 22-acid (X). This compound could also
be obtained by oxidation of the aldehyde (VIII) with the same reagent.
l4 J . Green, A . F . Daglish and V . D . Poole (Vitamins Limited), British Patent
755, 103 (1956).
l5 A . Butenandt and J . Schmidt-Thomd, Ber. 69, 882 (1936).
l6 G . Slomp Jr. and J . D . Johnson, J. Am. Chem. SOC.80, 915 (1958).
776 P. Westerhof and E. H . Reerink,

Chart. 2
c H3
I
H-C-C
eo

__L

The enamine [22-(1'-piperidyl)-9@,1Oa-bisnorchola-4,20(22)-dien-3-

one] (IX) was prepared by reacting the aldehyde with a nearly equi-
valent amount of piperidine in boiling benzene in the presence of p-
toluenesulfonic acid 17. The oxidation of the enamine to 9,9,10a-
progesterone (9p,lOa-pregn-4-ene-3,20-dione) (XI) was effected by a
solution of sodium dichromate in a mixture of benzene and acetic
acid. The infrared absorption spectrum showed a strong band at
1690cm-1 which may be attributed to the 20-keto group and bands
a t 1662 cm-1 and 1615 cm-1 indicating the presence of the conjugated
4-en-3-one system.
The 9p, 1Oa-pregn-4-ene-3,20-dionecould also be obtained by
ozonising the 20(22)-unsaturated 22-en01 acetate according to the
method of Heyl and Herr 18. The latter compound had been prepared
by reacting 9@,1Oa-bisnorchol-4-en-3-on-22-al with acetic anhydride
and sodium acetate.
17 M . E . Herr and F . W . Heyl, J. Am. Chem. SOC.74, 3627 (1952).
18 F . W . Heyl and M . E . Herr, J. Am. Chem. SOC. 72, 2617 (1950).
 InvestiEaticm on sterols, X V etc. 79 (1960) RECUEIL 777

The introduction of a double bond in order to prepare 6-dehydro-

9/?,1Oa-progesterone (9/?,1Oa-pregna-4,6-diene-3,20-dione)(XII) was
performed by the dehydrogenating action of chloranil (tetrachloro-p-
benzoquinone) in refluxing tert. butanol according to the method of
Agnello and Laubach 19.
The compound obtained showed a large negative rotation ([a12 =
- 484.5"), as already observed for other 4,6-dien-3-ones in the 9/?,lOa
series 20.

Discussion
The microanalytical data, the ultraviolet and the infrared spectra
of the compounds prepared confirm the presence of the expected
groups and chemical systems.
In the reaction sequence from lumista-4,22-dien-3-one (VII) to
the title compounds no reactions are used which attack the centres of
asymmetry a t C-8, C-9 and C-10. Castells et al. 435 have proved the
8/?,9/?,1Oa structure of lumista-4,22-dien-3-one (VII). We therefore
assume that the title compounds also possess the 8/?,9/?,10a configu-
ration and consequently differ from the naturally occurring steroid
hormones a t the C-9 and C-10 positions. This assumption is supported
by comparison of the differences of the molecular rotations of some
compounds in the normal series (8B,9cr,lOB) with those of the corre-
sponding compounds in our series (table I).

Bio-assays
In contrast to the analogues in the normal series (9a,lO/?)the title
compounds 9B, 1Oa-pregn-4-ene-3,20-dione(XI) and 9/?,1Oa-pregna-
Table I
I Compounds 1 M D (CHC13) I ML8*lor- MOD*''
lumista-4,22-dien-3-one (VII)
ergosta-4,22-dien-3-one 6
1
1
- 608
+ 178
1 - 786

bisnorchol-4-en-3-on-22-al
6
(VIII)
9/?,10~~-bisnorchol-4-c:n-3-on-22-a1
I - 817

98,l Oa-22-piperidinoenamine (IX)

9a, 1O/?-ZZ-piperidinoenamine 6 I + I - 485
340 - 825
98, ION-progesterone (XI)
progesterone (pregn-4-ene-3.20-dione)
21
I J ::: 1 - 830

19 E. J. Agnello and G . D . Lnubach, J . Am. Chem. SOC.79, 1258 (1957).

20 P. Bladon, J. Chem. SOC.1955, 2176.
21 J. P. Mafhieu and A . Petit, Tables de constantes et donnCes numeriques.
Pouvoir rotatoire naturel, I Steroides ( 1956).
778 P . Westevhof and E . H . Reerink,

4,6-diene-3,20-dione (XII) both show a pronounced progestational

activity on oral administration.
Whereas 6-dehydro-progesterone (pregna-4,6-diene-3,20-dione) has
about one third the activity of progesterone (pregn-4-ene-3,20-
dione) 22 the introduction of a double bond between carbon atoms 6
and 7 in 9@,10a-pregn-4-ene-3,20-dione(XI) in order to form 9@,10a-
pregna-4,6-diene-3,2O-dione(XII) considerably increases the pro-
gestational activity.
The animal 23 and clinical 24 experiments will be described in detail
elsewhere.

Experimental Part *
(With the collaboration of Mr. J. Hartog)

a. Lumista-4,7,22-trien-3-one(11)
This reaction was carried out according to the method of Shepherd et al. 6 by
oxidation of lumisteroln (I) with aluminum isopropoxide in toluene and cyclo-
hexanone. The crude oxidation product obtained could readily be purified by
recrystallisation from a mixture of acetone and diethyl ether ( 1 +
l), from
methanol or from ethyl acetate. Melting point 141-142", [a]: = +
39" (CHClS)
and &(ilmaxlmum = 240 mp) = 14 450.
The infrared absorption spectrum showed characteristic bands a t 964 cm-1
and 1675 cm-1.

b. Lumista-4,6,22-trien-3-one (111)
By stirring vigorously, 125 g of lumista-4,7,22-trien-3-one(11) were dissolved
at 10" in 2.2 1 of dry isopropanol, which had previously been saturated with dry
hydrochloric acid gas. After further passing a slow stream of hydrochloric acid
for half an hour the red-coloured solution was poured out as rapidly as possible
into a well-stirred suspension of 4 kg of sodium bicarbonate in 8 1 of water.
After standing for a while, the yellow layer of isopropanol was separated and
the salt layer extracted twice with 1 1 of petroleum ether. The collected extracts

* Melting points were corrected for extending stem and were determined at
atmospheric pressure. Ultraviolet absorption measurements were made in
methanol, unless stated otherwise, by the Analytical Department of N.V.
Philips-Duphar (Dr. K . J . Keuning) under the supervision of Mr. H . Keuker.
The infrared spectra were obtained with a Hilger H 800 spectrometer in
KBr tablets.
Microanalyses were performed by the Laboratory of Organic Chemistry,
Analytical Department (head Mr. W . M . Hazenberg), Groningen, and the
Laboratory of Organic Chemistry, Micro-Analytical Department (head Mr.
P . J . Hubers), University of Amsterdam.
22 A . Butenandt and J . Schmidt-Thomt, Ber. 72, 182 (1939).
R . E . Marker, T . Tsukamoto and D . L . Tuvner, J. Am. Chem. Soc. 62, 2525
( 1940).
23 H . F . L . Scholer, et al., Acta Endocrinologica (to be published).
24 K . C. Tillinger and E . Diczfalusy, ibid. (to be published).
 Investipations on sterols, X V etc. 79 (1960) RECUEIL 779

were subsequently washed three times with a sodium bicarbonate solution and
water, dried over sodium sulphate and evaporated to dryness. The ultraviolet
absorption spectrum of the residue obtained showed a maximum at 287mp,
Ei:L = 562.
This resin was dissolved in 250 ml of boiling petroleum ether (40-60"), crys-
tallised for several hours at - 5" and finally overnight at - 25". After filtering
with suction and washing twice with 50 ml of petroleum ether at - 25" 80.5 g
of lumista-4,6,22-triert-3-one (111) were obtained having a melting point of
99-100.5" (yield 64%). After some recrystallisations the pure compound had a
melting point of 101-102".
= - 632" (CHC13) &(jlmasimum = 287 mp) = 26 200
Found : C 85.7, 85.7; H 10.7, 10.8
Calc. for C28H420 (394.65): ,, 85.22 ; ,, 10.73.
The infrared spectrum showed characteristic bands at 984, 1586, 1622 and
1661 cm-l.
By chromatography in petroleum ether on alumina a further fraction could
be obtained from the mother liquor by elution with a mixture of petroleum ether
and diethyl ether (19 -- 1). This increased the yield of lumista-4.6.22-trien-3-one
(111) by another 5%.

c. 3-Methoxy-lumista-3,5,7,22-tetraene(IV)
To a boiling solution of 2.5 g of lumista-4,7.22-trien-3-one (11) in 150 ml of
methanol were added some drops of concentrated hydrochloric acid, turning the
colour of the solution into orange-red. Then 6.2 ml of concentrated hydrochloric
acid were added to the boiling solution.
After refluxing had been continued for an additional 1.5 hours the solution
was cooled to room temperature and cautiously neutralised with 6.2 g of sodium
bicarbonate. Working up was performed by adding water and extracting with
diethyl ether. The collected extracts were thoroughly washed with water, dried,

+
-
filtered and evaporated to dryness. The residue showed an ultraviolet absorption
maximum at 295-305 m p , Ei& 300. Four recrystallisations from a mixture
of acetone and methanol (3 20) afforded 200mg of needles. Melting point
89.5-90.5".
[a]: = + 230" (CHUB) c(jlmaxlmum = 320 mp) = 20 200
The absorption spectrum showed shoulders at 310 and 325 mp.
After the solution ha.d been kept at room temperature for 20 minutes, a shift
to the left could already be observed in the optical activity.

d. Lumista-5,22-dien-3-one(VI)
Under vigorous stirring a solution of 1 g of lumista-4,6,22-trien-3-one (111) in
50 ml of dry diethyl ether was added in 10 minutes to a solution of 100 mg of
lithium in 200 ml of 1i.quid ammonia. During the addition a precipitate was
formed. The excess of lithium was decomposed by dripping anhydrous ethanol
into the reaction mixture and after stirring for some minutes the solution was
diluted with water and extracted with diethyl ether. The collected extracts
were adequately washed with water, dried over sodium sulphate and, after
filtering, evaporated to dryness. The residue was dissolved in dry benzene,
filtered through a column of alumina and eluted with 60 ml of benzene. The
resin obtained after evaporation in vacuo (906 mg) exhibited an ultraviolet
absorption maximum a t 242 mp, Ef:k = 209.
780 P. Westerhof and E . H . Reerink,

Crystallisation from acetone-methanol ( 1 + 1) at - 25" yielded 510 mg of

crystals, melting at from 70 to 100". Recrystallisation from ethyl acetate at 20"
gave a small quantity of crystals melting at 110-114".
Upon crystallisation a t - 25" the filtrate gave 100 mg of needles, which when
recrystallised from acetone-methanol ( 1 + 4) at - 5". finally yielded 80 mg of
lumista-5,22-dien-3-one (VI) with a melting point of 88-90",
No absorption was detected in the ultraviolet region (A > 220 mp). The infra-
red spectrum exhibited a strong band at 1720cm-1 and a weaker band at
962cm-1. [a]: = - 61" (CHC13).
Found : C 84.3, 84.3; H 10.9, 11.0
Calc. for C28H440 (396.66): ,, 84.79 ; ,, 11.17.
The yield of lumista-5,22-dien-3-one (VI) could be considerably improved
(up to 70%) by using the reduction method described under e) and by working
up as quickly as possible.

e. Lumista-4,22-dien-3-one(VII)
Whilst stirring vigorously, a solution of 3.0 g of lumista-4,6,22-trien-3-one
(111) in 300ml of dry diethyl ether was added to 450ml of liquid ammonia.
Then, again under vigorous stirring, a solution of 420 mg of lithium in 100 ml of
ammonia was carefully added in drops, until the spontaneous decolourisation
ceased. After dripping in 90ml of dry ethanol, stirring was continued for 30
minutes, the mixture was diluted with water and extracted with diethyl ether.
The ethereal extracts were thoroughly washed with water, dried, filtered and
evaporated t o dryness. The resin obtained (El:'& 242 mp = 196) was dissolved
in 100 ml of boiling ethanol, 6 ml of 2 N sodium hydroxide were added t o the
solution and it was boiled for five minutes. Then cooling was performed rapidly,
the reaction mixture diluted with water and extracted with diethyl ether. The
collected extracts were subsequently washed with water, dried, filtered and
evaporated to dryness.
The solid residue finally obtained exhibited an ultraviolet absorption maxi-
mum at 242mp, E!i& = 420. The substance dissolved in 24ml of benzene
was filtered through 30 g of alumina and eluted with 75 ml of the same solvent,
a dark brown ring remaining at the upper end of the column. The dry eluate was
crystallised from 45 ml of methanol at 5", yielding 2.43 g of needles with melting
point 122-124". Two recrystallisations of 500 mg of the compound finally gave
313 mg of colourless crystals of lumista-4,22-dien-3-one (VII) with melting
point 122-124'. ~ (= 242 , mp)l= 16~ ~
800 [a]"," ~ (CHC13)
= - 153.5" ~ ~ ~
Found : C 85.0, 84.9; H 11.0, 11.0
Calc. for C28H440 (396.66): ,, 84.79 ; ,, 11.17.
In the infrared spectrum bands were exhibited at 962, 978, 1620 and 1665
cm-1.
Lumista-4.22-dien-3-one (VII) could also be obtained in a high yield by
isomerisation of the purified lumista-5,22-dien-3-one (VI) in the same way with
sodium hydroxide in methanol.

f . 9/3,lOa-Bisnorchol-4-en-3-on-22-al (VIII)
20 g of lumista-4,22-dien-3-one (VII) were dissolved in a mixture of 750 ml of
freshly distilled dry methylene chloride and 5.75 ml of dry pyridine.
Whilst stirring at - 80" the solution was ozonised for 4) hours. The speed of
 Investigations on sterols, X V etc. 79 (1960) RECC'EIL 781

the passing oxygen was 9-10 Ilhour, while 0.205 mmole of ozone/minute were
absorbed.
The reaction complex was then decomposed by adding 20 g of zinc dust and
105 ml of glacial acetic acid to the reaction mixture, taking away the cooling
bath and stirring subsequently one hour at 0" and 5 minutes at 35". The zinc
was removed by filtration. After cooling to 5" the filtrate was washed with
75 ml and 50 ml of cooled 10% sodium carbonate solution, 50 ml of 10% sodium
hydroxide solution a t 0" and four times with 300 ml of cold water. The methyl-
ene chloride extract was dried on sodium sulphate, filtered and the filtrate
evaporated to dryness. The final part of the evaporation was carried out in
vacuo. The colourless, almost completely crystalline residue was recrystallised
from 50 ml of diethyl ether at - 25" yielding 10.1 g of 9p, lO~bisnorchol-4-en-
3-on-22-al (VIIl) with a melting point of 119-121". Recrystallisations from
petroleum ether (40-60°), containing a few drops of ethanol, and from diethyl
ether raised the melting point to 122-123'.
E(Amaximum = 242.5 mp) = 16 700 = - 165' (CHC13)
Found : C 79.9, 80.1; H 9.7, 9.8
Calc. for C22H3202 (328.50): ,, 80.43 ; ,, 9.82.
The infrared spectrum exhibited characteristic bands a t 1610, 1642 and
1712 cm-l.
The yield of aldehyde could readily be improved to 70-75% by using a larger
excess of ozone in the: ozonisation reaction (- 1.4 mmole of ozone/mmole of
starting material).

g. 3-0xo-9/3,10a-bisnorchol-4-enic 22-acid (X)

A solution of 450 mg of the aldehyde, obtained by method f ) , in 15 ml of
chloroform was oxidised by stirring with a solution of 200 mg of chromic acid
and 0.2 ml of water in 25 ml of acetic acid at about 30" for 16 hours.
After the excess of chromic acid had been decomposed by stirring for 30
minutes with 1.5 ml o [ methanol, the mixture was diluted with water and ex-
tracted with benzene. 'The collected extracts were washed with water to neutral
reaction, dried, filtercd and evaporated to dryness. Recrystallisation from
diethyl ether yielded 340 mg of 3-ox0-9p. 1O~bisnorchol-4-enic22-acid (X)
with melting point 194-198'.
A few recrystallisations from methanol (90%) raised the melting point to
202-204".
E(Amaxfmum = 242 mp) = 16 800
Found : C 76.9, 76.9; H 9.4, 9.5
Calc. for C22H320:) (344.50): ,, 76.70 ; ,, 9.36.
The infrared spectrum exhibited bands at 868, 1039, 1611, 1649 and
1729 crn-'.

h. 2 2 4 1'-Piperidyl) -9/3,1 Oa-bisnorchola-4,20(22) -dien-3-one (IX)

A solution of 10 g of the aldehyde, prepared by method f), in 180 ml of dry
benzene was refluxed in a nitrogen atmosphere in the presence of 3.8 ml of
piperidine and 30mg of p-toluenesulfonic acid for 3 hours. The condensed
benzene was dried by passing through powdered barium oxide.
The reaction mixture was then distilled in vacuo to complete dryness. A
crystalline residue was obtained, which, after recrystallisation from 10 ml of
782 P . Westerhof and E . H . Reerink,

methanol, yielded 9.3 g of enamine (IX) with melting point 98 (sintering)-

107-111". For analytical purposes a portion of this fraction, which was ade-
quately pure for further processing, was recrystallised several times from
acetone until a constant melting point of 114-1 15" was attained.
E (Amsximum = 241.5 mp) = 22 000 [a]: = - 123" (CHCl3)
Found : C 81.9, 90.0; H 10.5, 10.5; N 3.4, 3.6
Calc. for C27H41NO (395.64): ,, 81.97 ; ,, 10.45 ; ,, 3.54.
The infrared spectrum exhibited bands at 874 and 1610 cm-1.
An intensive band at 1660cm-1 distinctly overlapped a band with lower
extinction at 1650 cm-1.

i. 9/3,lOa-Pregn-4-ene-3,2O-dione (XI)
1. A solution of 300 mg of the enamine, prepared by method h), in 4.5 ml
of dry thiophene-free benzene, was added dropwise (in 45 minutes) at 0-5" to a
well-stirred solution of 452 mg of sodium dichromate (2 aq) in 4.5 ml of acetic
acid and 3 ml of benzene. After additional stirring for 2 hours at O", 0.75 ml of
methanol was added and stirring continued for 30 minutes at the same temper-
ature in order to decompose the excess of dichromate. The reaction mixture was
then poured out into 25 ml of water and extracted with benzene. The benzene
extracts were washed consecutively with water, twice with 3 ml of cold 10%
sodium hydroxide solution, with water, with 3 ml of cold 10% hydrochloric
acid solution and finally again four times with water. The solution was dried,
filtered and evaporated t o dryness affording a completely crystalline residue.
Recrystallisation from a mixture of 1 ml of methylene chloride and 4 ml of
petroleum ether at - 25" yielded 150 mg of crystals of 9b. l0a-pregn-4-ene-
3.20-dione (XI) with melting point 161- 163". Repeated crystallisation raised
the melting point to 163-164'.
E (Amaximum = 241.5 mp) = 16 800 [a]: = - 62" (CHC13)
Found : C 79.9, 79.9; H 9.7, 9.8
Calc. for CzlH3oOz (314.47): ,, 80.20 ; ,, 9.62.
The infrared spectrum exhibited strong bands at 1690 and 1662 cm-1 and a
weaker band at 1615 cm-1.
2. To a solution of 1 g of aldehyde, obtained by method f) in 10 ml of freshly
distilled acetic anhydride was added 0.5 g of freshly melted sodium acetate and
the mixture was refluxed in a nitrogen atmosphere for 16 hours.
Then, under reduced pressure, the solvent was distilled off as far as possible
and the residue obtained was dissolved in 25 ml of chloroform. After the sodium
acetate had been filtered off, the filtrate was filled to 150 ml with chloroform.
Under cooling with ice-water this solution was ozonised for 14 minutes,
10.5 mg of ozone being absorbed per minute. Then, after the addition of 15 ml
of acetic acid and 2 g of zinc dust, the mixture was shaken for 10 minutes. The
filtered solution was washed with 10% sodium hydroxide solution and water to
neutral reaction. The dried and filtered solution was evaporated to dryness.
In order to hydrolyse any 3-en01 acetate which might have been formed, the
residue obtained was refluxed in 45 ml of methanol and 25 ml of 2 N sulphuric
acid for 45minutes. After concentration and dilution with water the crude
product was extracted with diethyl ether. The extracts were washed with 10%
sodium hydroxide solution and water to neutral reaction, dried, evaporated to
dryness and crystallised from 3 ml of diethyl ether. Recrystallisation from a
 Investigatioizs on sterols. X V etc. 79 (1960)RECUEIL 783

mixture of methylene chloride and acetone gave 113 mg with a melting point of
160-163°C.No depression of the melting point was observed when mixed with
9p,lOa-pregn-4-ene-3.2O-dione (XI) obtained by method il).

j. 9P,lO~-Pregna-4,6-diene-3,20-dione (XII)
A solution of 7.5 g of 9p.lOa-pregn-4-ene-3,2O-dione (XI)in 500 ml of freshly
distilled tert. butanol was refluxed in the presence of 12.75g of finaly powdered
chloranil whilst stimng for 5 hours in a nitrogen atmosphere.
After cooling, 2 1 of water were added and the mixture was extracted with
three 200 ml portions of methylene chloride. After dilution with 1 1 of petroleum
ether (40-60") the combined extracts were washed successively with a diluted
sodium hydrosulphite solution (100ml), four times with 1 N sodium hydroxide
and with water to neutral reaction. The dried organic layer was filtered and
evaporated to dryness yielding 3.7 g of a crystalline residue. The crude product
was filtered in benzene solution through a column containing 35 g of alumina.
Elution with benzene and evaporation of the filtrate gave 3.11 g of residue,
which was recrystallised successively from acetone, ethanol, a mixture of ethanol
and n-hexane and a mixture of acetone and n-hexane, finally giving 1.0g of
9p,lOa-pregna-4.6-diene-3,20-dione (XII) with melting point 169-170".
From the mother liquors a second crop of 0.44g with melting point 168-169"
could be obtained.
E (Amaximum = 286.5mp) = 26 400 [a]: = - 484.5"(CHC13)
Found : C 81.1, 81.3; H 9.2,9.1
Calc. for C2lH2802 (312.45):,, 80.73 ; ,, 9.03.
The infrared spectrum exhibited inter alia bands at 888, 1574, 1617, 1656 and
1695 cm-1.

Acknowledgment
The authors wish to thank Dr. J . Bakker of the Philips' Research
Laboratory for the measurement and discussion of the infrared spectra.

(Received February 6th 1960).