849
SYNTHESIS
OF PYROGALLOLESTROGENS estratrienes)
AND THEIR
G. Stubenrauch,
Abteilung
0. Haupt
fUr Biochemische
Hochschule
LUbeck,
Received:
4-4-77
(2,3,4-trihydroxy-1,3,5(10)METHYL
ETHERS
and R. Knuppen
Endokrinologie
Germany,
D-24
der Medizinischen
LUbeck,
Ratzeburger Allee 160
ABSTRACT The preparation of 2,4_dihydroxyestrone, 2,4_dihydroxyestradiol-178 and their methyl ethers (14 compounds) is described. The structures were established by nuclear magnetic resonance, infrared and mass spectra as well as by elemental analyses, alternative synthetic routes and by microchemical reactions, their chromatographic properties. INTRODUCTION Pyrogallolestrogen derivatives incubation liver
have
recently
Cl]. For these
has been worked
out
ethers
nitrosodisulfonate conditions.
(Fig.
were
investigations
(Fremy's
salt)
The o-alkoxy
to 2,4_dihydroxyestrone by selective
and KI catechols
TTEIPCOfDl6
after
with
rat
an efficient methyl
ethers
catecholestrogen with
potassium
under
carefully
formed
were
and the corresponding
alkylationldealkylation
S
ethers
and their
1). Appropriate
ethers
29, Number 6
as metabolites
ortho-hydroxylated
verted
Vohze
isolated monomethyl
of 2,4_dihydroxyestrogens
monomethyl defined
been
of catecholestrogen
tissue
synthesis
(2,3,4-trihydroxy-1,3,5(10)-estratrienes)
con-
methyl
steps.
June,
2977
m
850
2 o: R1*OCH3;
TIIEOTD-
Rz=H
b:R’-H; R2-0CH3 c: R 1 l OCH2C@5;
&o:R3=OCH3;
Rt=OH
b:R3=0H;
RL=OCH3
R -H
..,.:,
2s
‘:~~~~‘;
RC=oH
r’ CH30 CH30
/ w
CH30
I
I
’
HO OH 3, &i
0CH3 &
’ 1. CH2N2 “2’Pdl------
w
I
’
2. (C li3O)2 SO2 _ 3. H2 /Pd
I 1. CH2N2 2. H2/Pd r
I
:I&
Fig.1
Ct+a”@
1
C;;@
OH
OH
OCH3
5
P
I
: Synthetic
routes
to
pyrogollolestrogens
S
TIIROIDI)
RESULTS The
syntheses
diol-17R ready
corresponding
described
by other
the tedious
procedures
dered
synthetic
these
DISCUSSION
of 2,4_dihydroxyestrone,
and their
been
AND
851
of the desired
trimethyl
workers
and/or routes
2,4_dihydroxyestraethers
C2a-dl.
However,
the poor overall unsuitable
pyrogallolestrogen
have al-
yields
ren-
for the preparation
mono-
and dimethyl
ethers. Gelbke
C31 observed
the formation
estradiene-3,4,17-trione ne (la) with aqueous mined been
potassium
acetone,
mixture after
that
graphy
of the quinone acid
yield
followed
by column
droxyestrone high
reactivity
for a directed
methyl
ether, sulfate.
zyl group tained
separation 12%)
with then
product
yielded
group
chromato-
C41 pure with
a
diazomethane
2-methoxy-4-hy2,4-di-
The obvious
12:l.
of 2a was
chloride/K1
mainly
directly
Subsequent
of the
by column
benzyl
containing
selectively
as pure
ethereal
65%);
utilized
of 4. 2-Methoxy-4-hydroxyestrone
synthesis
was
gel
be isolated
of approx.
of the 3-hydroxy
the mixture,
3-benzyl
and column
(3) and the isomeric
(4) in a ratio
was monobenzylated and
ether
(approx.
silica
(2a) could
chromatography
3-methyl
methoxyestrone
in situ
of 2a with
it has
KHZPOL, in the reaction
in yield
impregnated
of 50%. Methylation
10% as deter-
investigation
of solid
increase
2-methoxy-4-hydroxyestrone
in KH2P04-buffered
did not exceed
In the present
the presence
on ascorbic
of 2-methoxyestro-
nitrosodisulfonate
led to a marked
reduction
treatment
but the yield
by titration. found
after
of 2-methoxy-1,5(10)-
under
the desired methylated
hydrogenolysis
in approx.
isomeric
30% yield
by-product
chromatography.
with
removed
and 2,4-dimethoxyestrone
nitrogen
2-methyldithe ben-
(4) was (from
(3) (yield
ob-
2a) after approx.
Z-~ydroxy-4-methoxyestrone
(2b) was
estrone
(lb) which
estrone
T61 by the sequence
hydrogenolysis tography merit
exclusively
formed
with
after
when
time
2-hydroxy-4-methoxyestrone
was
synthesized
treated
with
with
from
benzyl
monobenzyl
ethers.
that
the 2-benzyl
ether
fore,
salt while
the mixture
without
prior
obtained
yielded
of 2c with
afforded after
diazomethane
hydroxyl
161, which
yielding
was used
After
a mixture
3-methyl
and then with
and column ether
ration
4-methoxyestrone
with
inert.
with
iso-
indicated Therestep
KI and column (2~) was
crystallisation.
diazomethane
first
of the
in the oxidation
chromatography
3-methyl
at C2,
reacted
completely
for
was
experiments
reduction
resisted
4-methoxyestrone of 7 from
key intermediate
2,4-dihydroxyestrone
hydrogenolysis
in 35% yield
2-benzyloxy-4-hydroxyestrone
2,4_dihydroxyestrone
of 2c with
to 10 min,
consumed).
directly
ethereal
above
in 10% acetic
isolated
Preliminary
of 2c and column
conditions
out
of 2-hydroxyestrone
as an oil which
genolysis ment
pure
showed
as described
an unsubstituted
separation.
chromatography
of li-hydroxy-
(2b) was
the 3-isomer was
almost
for 4-methoxyestrone.
carried
(2c),
chloride
was
experiments
2-hydroxyestrone
merit
Fremy's
methylation
limited
2-Benzyloxy-4-hydroxyestrone pyrogallolestrogens
chroma-
be isolated.
ether
strictly
on 4-methoxyestrone
-
15% of the iso-
procedure
was
column
could
not applicable
the oxidation
and the reaction
(55% based
ether
Preliminary
hydroxylation was
Following
3-methyl
direct
diazomethane.
for E-methoxyestrone However,
3-methyl
rl-methoxy-
- methylation
(lb) and
4-hydroxyestrone
an analogous
acid
above.
from
now from 4-hydroxy-
benzylation
35% of 4-methoxyestrone
4-hydroxyestrone
estrone
is available
as described
Interestingly,
that
easily
prepared
under
Hydro-
reducing
(5) [Za,bl.
Treat-
and hydrogenolysis ether
(6). Methylatior
dimethyl
sulfate
chromatography,
2-hydroxy-
(7). The attempted 3-methyl
ether
gave,
prepaby direct
S benzoyloxylation
or an acetylation/Baeyer-Villiger-oxidation
of 4-hydroxyestrone Treatment
of the
TIIZROIDS
dimethyl 17-0~0
steroids
to the
17B-hydroxy
analogues.
pounds
were
by their
troscopical reactions
proved data
(IR,
and their
ether
lH-NMR
proved
with
unsuccessful.
sodium
The structures synthetic
routes,
and MS [71),
chromatographic
borohydride
led
of the comtheir
spec-
microchemical
properties
181.
EXPERIMENTAL Estrone and estradiol were gifts from Schering AG, Berlin. P-Methoxyestrone was prepared according to Fishman 191, 2- and 4-hydroxyestrone according to Stubenrauch and Knuppen 161. Infrared spectra, using pressed KBr-discs, were recorded with a Beckman Acculab 4 spectrophotometer. Ultraviolet spectra of ethanolic solutions were run with a Shimadzu double beam spectrophotometer UV-200. Nuclear magnetic resonance spectra were recorded with a Varian HA-100 spectrometer with tetramethylsilane as internal standard in CDCls solution. Mass spectra were obtained by using a gas chromatography-mass spectrometer LKB 9000. Melting points were determined with a microscope hot-stage and are uncorrected. Analyses were performed by Bistrich und Jeschke, Mikroanalytisches Labor, Bottrop, Germany. For column chromatography, silica gel refers to Kieselgel 60 (0.063-0.2; E. Merck, Germany) and alumina to Aluminiumoxid neutral Woelm (Woelm, Germany). Chromatography on ascorbic acid impregnated stationary phases was carried out as described elsewhere [41. (2a). To a solution of 500 mg of 2-methoxyestrone (la) in 150 ml of acetone a solution of 3 g potassiumnitrosodisulfonate in 200 ml of 0,l M KHzP04buffer was added at once. After the addition of 160 ml of acetone the mixture was vigorously shaken for 25 min. 200 ml of water were then added and the solution extracted twice with chloroform. The combined extracts were washed once with 0.1 N HCl and twice with water. 100 ml of glacial acetic acid and 2.5 g of potassium iodide were then added and the mixture was shaken for 2 min. The iodine formed was removed with approx. 80 ml of 5% aqueous hydrogen sulfite solution, the chloroform extracts washed with water, and the combined aqueous layers reextracted once with chloroform. After short drying over sodium sulfate 2 ml of acetic acid and 2 ml of a solution of ascorbic acid in methanol (2 g/100 ml) were added and the solvents were evaporated. The residual oil was chromatographed on 150 g of silica gel impregnated with ascorbic acid. Elution with cyclohexane/ chloroform/acetic acid (40:40:3) afforded 255 mg (49%) of 2-Methoxy-4-hydroxyestrone
2-methoxy-4_hydroxyestrone, m.p. 199-201°C (cyclohexanel benzenelaletic acid). i.r.: 3550 cm-l, 3420 (vO-H), 1720 (vC=O). M peak at m/e = 316. Anal. Calcd. C 72.13; H 7.64; found C 72.4; H 7.6. 2-Methoxy-4-hydroxyestrone a-methyl ether (31. A solution of 110 mg of 2-methoxy-4-hydroxyestrone (2a) in 60 ml of ethano was stored for 5h at -15OC with an excess of diazomethane in ether. After evaporation of the solvents and excess diazomethane the oily residue was chromatographed on 140 g of silica gel. Elution with cyclohexane/chloroform/acetic acid (20:20:1) gave 70 mg (60%) of 2-methoxy-4-hydroxyestrone 3-methyl ether, m.p. 146-7OC (pethanol/water); i.r.: 3395 cm-l (vO-H), 1730 (vC=O), M peak at m/e = 330. Further elution with the same solvent system afforded 10 mg of 2,4_dimethoxyestrone. 2,4-DCmethoxyestrone (4). To a solution of 322 mg of 'L-methoxy-4-hydroxyestrone in 25 ml of dry acetone, 150 mg of potassium iodide, 120 ~1 of benzyl chloride and 600 mg of dry potassium carbonate were added with stirring. After refluxing and stirring for 24h under nitrogen the mixture was filtered and the solvent removed i. vat. The oily residue was dissolved in 25 ml of ethanol and 5 ml of 40% (w/v) sodium hydroxide solution. 5 ml of dimethyl sulfate were added dropwise with stirring at a rate that the temperature did not rise above 55OC. Additional dimethyl sulfate (2 portions of 3 ml each) and sodium hydroxide solution were then added alternately, keeping the mixture alkaline at all times. After 45 min, the mixture was poured into water and extracted 3times with ether. The combined ethereal extracts were washed with water and dried over sodium sulfate. After evaporation of the solvent the residue was dissolved in approx. 30 ml of ethanol. 500 mg of 10% palladium on charcoal were then added and the suspension was stirred overnight under a stream of hydrogen. After filtration the solvent was evaporated and the residue chromatographed on 150 g of silica gel impregnated with ascorbic acid. Elution with cyclohexane/chloroform/acetic acid (20:20:1) gave 40 mg of 2-methoxy-4-hydroxyestrone 3-methyl ether and 100 mg (30%) of the desired 2,4dimethoxyestrone, m.p. 149-150°C (methanol/water); i.r.: 3400 cm-l (vO-H), 1720 (vC=O); M* peak at m/e = 330. Further elution with the same solvent system afforded starting material. 2-Hydroxy-4-methoxyestrone (2bl. To a solution of 1 g of 4-hydroxyestrone in 60 ml of dry acetone, 300 mg of potassium iodide, 390 ~1 of benzyl chloride and 2.2 g of potassium carbonate were added with stirring. After refluxing and stirring for 24h under nitrogen, the mixture was filtered and the solvent evaporated. The residue was suspended in 30 ml of ethanol, methylated with dimethyl sulfate and subsequently hydrogenolysed as described above. After evaporation of the solvent the residual oil was chromatographed on 150 g
S
TEIROID~
855
of silica gel. Elution with cyclohexane/chloroform/acetic acid (20:20:1) gave 180 mg (21%) of 4-hydroxyestrone 3-methy1 ether and 365 mg (35%) of 4-methoxyestrone, m.p. undepressed on admixture of an authentic sample. 4-Methoxyestrone (500 mg) was dissolved in 150 ml of acetone and a solution of 3 g of potassiumnitrosodisulfonate in 220 ml of 10% aqueous acetic acid was added at once. The mixture was vigorously shaken for 10 min and the reaction product extracted with chloroform. The extract was processed as described above. Chromatography on 150 g of sjlica gel impregnated with ascorbic acid and elution with cyclohexane/ chloroform/acetic acid (5:5:1) gave 180 mg (34%; 55% based on 4-methoxyestrone consumed) of 2-hydroxy-4-methoxyestrone, rn.p-;185-6OC (cyclohexane/b$nzene/acetic acid); i.r.: 3400 (vO-H), 1730 (vC=O); M peak at m/e = 316. Anal.: E!lcd. C 72.13; H 7.6$; found C 72.3; H 7.6. Further elution with the same solvent system afforded 185 mg of unreacted 4-methoxyestrone. 4.3 g of 2-hydroxyestrone 2-BenzyZoxy-4-hydroxyestrone (Be). dissolved in 300 ml of dry acetone was refluxed with 600 mg of potassium iodide, 1.8 ml of benzyl chloride and 9 g of dry potassium carbonate for 24h with stirring under nitrogen. After filtration, most of the solvent was evaporated and the residue partitioned between ether and water. The aqueous layer was reextracted with ether and the combined organic extracts were dried over sodium sulfate. After evaporation of the solvent the residue was chromatographed on alumina (200 g, activity II). Elution with benzene afforded a small amount of dibenzyl ether. With benzene/l% ethanol a mixture of the monobenzyl ethers (4 g, containing approx. 2 g of the desired ?-benzyl ether) was eluted. 1 g of the mixture was dissolved in 210 ml of acetone and a solution of 3 g of potassiumnitrosodisulfonate in 200 ml of 0.1 M KHzPO,-buffer was added at once. Acetone (approx. 100 ml) was then added until a white precipitate appeared and the mixture was vigorously shaken for 25 min. The reaction product was then isolated as described above. After column chromatography on impregnated silica gel and elution with cyclohexane/chloroform/acetic acid (40:40:1) 200 mg of 2-benzyloxy-4-hydroxyestronq were obtained as an oil which resisted crystallisation. M peak at m/e = 392. 2,4-Dihydroxyestrone (5). To a solution of 200 mg of 2benzyloxy-4-hydroxyestrone in 30 ml of ethanol, 500 mg of 10% palladium on charcoal and 2 ml of a methanolic solution of ascorbic acid (2g/lOO ml) and 1 ml of acetic acid were added. The mixture was stirred overnight under a stream of hydrogen. Another 2 ml portion of ascorbic acid solution was added, the mixture filtered and after evaporation of the solvent the residue was chromatographed on 150 g of silica gel impregnated with ascorbic acid. Elution with cyclohexane/chloroform/acetic acid (3:3:lh gave 110 mg (70%) of 2,4_dihydroxyestrone, m.p. 231-2 C (lit. L2a]
856
S
TDPBOIDI)
2$8-30°C); i.r.: 3550 cm-l, M peak at m/e = 302.
3505,
3450
(uO-H),
1720
(vC=O);
2,4-Dihydroxyestrone S-methyl ether (6). A solution of 200 mg of 2-benzyloxy-4-hydroxyestrone in 50 ml of ethanol was stored for 6h at -15OC with an excess of diazomethane in ether. After evaporation of the solvents and excess diazomethane the residue was hydrogenolysed as described above. Column chromatography on 140 g of silica gel impregnated with ascorbic acid and elution with cyclohexane/chloroform/ acetic acid (8:8:1) gave 70 m (43%) of 2,4-dihydroxyestrone 3-methyl ether, m.p. 234-7 ? C (dec.; metha?ol-water); 3460 cm-l, 3310 (vO-H), 1740 (vC=O); M peak at m/e = ’
Xl
2-Hydroxy-4-methoxyestrone 3-methy ether (71. A solution of 200 mg of 2-benzyloxy-4-hydroxyestrone in 20 ml of ethanol was stored for lh at room temperature with an excess of diazomethane in ether. After the usual work-up the residue was methylated with dimethyl sulfate and hydrogenolysed as described above. The oily reaction product was chromatographed on 120 g of silica gel. Elution with cyclohexane/ chloroform/acetic acid (20:20:1) gave 90 mg (53%) of 2-hydroxy-4-methoxyestrone 3-methyl ether, m.p. 199-202°C idec.; methanol-water; i.r.: 3450 cm-l (vO-H), 1730 (vC=O); M peak at m/e = 330. 2,4-Dimethoxyestrone a-me-thy2 ether. 2-Methoxy-4-hydroxyestrone or 2-hydroxy-4-methoxyestrone were methylated with etheral diazomethane in ethanol for 1.5h at room temperature. After removing of the solvent in vat. the residue was treated with dimethyl sulfate in alkaline ethanol in the usual manner. Short column chromatography on alumina (activity II) and elution with benzene afforded 2,4-dimethoxyestrone 3-methyl ether in 50% yield, m.p. 120-2OC (lit. [2c,dl 132-3OC); i&r.: 1730 cm-l (vC=O), no absorption in the OH-region. M peak at m/e = 344. IHnmr: S 6.65 (sill, C-lH), 3.86 and 3.88 (sC91, 3 0CH3), 0.85 (s[31, C-18H,); resonances of the remaining protons between 3.11.1. Anal.: Calcd. C 73.23; H 8.19; found C 73.2, 73.3; H 8.2, 8.1. 2,4-DihydroxyestradioZ-17k3 and methyl ethers. Reduction of the 17-0~0 steroids with sodiumborohydride was carried out in the usual manner. The physical data of the compounds are given in Table 1.
164-6 156-7 125-6, 180-l:: 211-2 263-6 Idec.1 145-7 129-30
2-methoxy-4-hydroxyestradiol 3-methyl ether
2,4-dimethoxyestradiol
2-hydroxy-4-methoxyestradiol
2,4-dihydroxyestradiol
2,4-dihydroxyestradiol 3-methyl ether;:::
2-hydroxy-4-methoxyestradiol 3-methyl ether
2,4-dimethoxyestradiol 3-methyl ether
i.r. vO-H
ether/petroleum ether
ether/petroleum ether
methanol/water
methanol/water
ether/petroleum ether
ether/petroleum ether
methanol/water
3540
3350
3260,348O
::::Purification of the reaction mixture by chromatography on silica gel, elution with cyclohexane/ chloroform/aceticacid (3:3:1).
346
332
318
304
318
3420
3420
332
332
318
M+ at m/e
3250,346O
3485,355O
cvclohexane/benzene/acetic 3400,3515 acid
tryst. from
::Aftermelting at 125-6'C the substance resolidified (needles) and melted again at 180-1'C.
205-6
m.p. [‘Cl
2-methoxy-4-hydroxyestradiol
Compound
Table 1. Physical data of 2,4-dihydroxyestradiol-178and its methyl ether derivatives.
; u
0
:
d
w
858
ACKNOWLEDGEMENT This investigation was supported by the Deutsche Forschungsgemeinschaft. REFERENCES 1. Stubenrauch, G., Gelbke, H.P., and Knuppen, R., HOPPE-SEYLER'S Z. PHYSIOL. CHEM. 357, 75 (1976). 2a. Kovacs, K., Rakonczay, Z., and Matkovics, B., ACTA PHYS. CHEM. (SZEGED) 19, 287 (1973). b. Matkovics, B., STEROIDS LIPID RESEARCH 4, 153 (1973). c. Axelrod, L.R., Rao, P.N., and Baeder, D.H., J. AM. CHEM. SOC. 88, 856 (1966). d. Rao, P.N., Jacob, E.J., and Axelrod, L.R., J. CHEM. SOC., C, 1971, 2855. 3.
Gelbke, H.P., DISSERTATION (Heidelberg), 1973.
4.
Gelbke, H.P., and Knuppen, R., J. CHROMATOGR. 71, 465 (1972).
5.
Kraychy, S., J. AM. CHEM. SOC. 81, 1702 (1959).
6.
Stubenrauch, G., and Knuppen, R., STEROIDS 28, 733 (1976).
7.
Spiegelhalder, B., and Stubenrauch, G., to be published.
8.
Gelbke, H.P., and Stubenrauch, G., J. CHROMATOGR. 120, 239 (1976).
9.
Fishman, J., J. AM. CHEM. SOC. 80, 1213 (1958).
S
TPROIPS
859
Nomenclature list Estrone = 3-hydroxy-1,3,5(10)-estratrien-17-one; estradiol-17R = estra-1,3,5(10)-triene-3,17l3-diol; Z-hydroxyestrone = 2,3-dihydroxy-1,3,5(10)-estratrien-17-one; 4-hydroxyestrone = 3,4-dihydroxy-1,3,5(10)-estratrien-17-one; 2-methoxyest~ne = 2,3-dihydroxy-1,3,5(10)-estratrien-17-one 'L-methyl ether; It-methyl 4-methoxyestrone = 3,4-dihydroxy-1,3,5(10)-estratrien-17-one ether; 2,4_dihydroxyestrone= 2,3,4-trihydro~-l,3,5(lO)-estratrien-l7-one~ 2,4-dihydroxyestradiol-178= estra-1,3,5(10)-triene-2,3,4,17&tetrol; Z-methoxy-4-hydroxyestrone= 2,3,4-trihydroxy-1,3,5(10)-estratrien17-one Z-methyl ether; 2-methoxy-4-hydroxyestrone3-methyl ether = 2,3,4-trihydroxy-1,3,5(10)estratrien-17-one 2,3-dimethyl ether; 2,4-dimethoxyestrone= 2,3,4-trihydroxy-1,3,5(10)-estratrien-17-one 2,4-dimethyl ether; 2-hydroxy-4-methoxyestrone= 2,3,4-trihydroxy-1,3,5(10)-estratrien17-one 4-methyl ether; 2,4_dihydroxyestrone3-methyl ether = 2,3,4-trihydroxy-1,3,5(10)estratrien-17-one 3-methyl ether; 2-benzyloxy-4-hydroxyestrone= 2,3,4-trihydroxy-1,3,5(10)-estratrien17-one 2-benzyl ether; 2-hydroxy-4-methoxyestrone3-methyl ether = 2,3,4-trihydroxy-1,3,5(10)estratrien-17-one 3,4-dimethyl ether; 2,4-dimethoxyestrone3-methyl ether = 2,3,4-trihydroxy-X,3,5(10)estratrien-17-one 2,3,4-trimethyl ether; 2-methoxy-4-hydroxyestradiol-17~= estra-1,3,5(10)-triene-2,3,4,17l3tetrol 2-methyl ether; 2-methoxy-4-hydroxyestradiol-1783-methyl ether = estra-1,3,5(10)triene-2,3,4,17&tetrol 2,3-dimethyl ether; 2,4-dihydroxyestradiol-1783-methyl ether = estra-1,3,5(10)-triene2,3,4,17B-tetrol 3-methyl ether; 2,4-dimethoxyestradiol-17B= estra-1,3,5(10)-triene-2,3,4,17& tetrol 2,4-dimethyl ether; 2-hydroxy-4-methoxyestradiol-17R= estra-1,3,5(10)-triene-2,3,4,17& tetrol 4-methyl ether; Z-hydroxy-4-methoxyestradiol-1703-methyl ether = estra-1,3,5(10)triene-2,3,4,17&tetrol 3,4-dimethyl ether; 2,4-dimethoxyestradiol-17R3-methyl ether = estra-1,3,5(10)-triene2,3,4,17Ptetrol 2,3,4-trimethyl ether.