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.

Synthesis of pyrogallolestrogens (2,3,4-trihydroxy-1,3,5(10)-estratrienes) and their methyl ethers.

849 SYNTHESIS OF PYROGALLOLESTROGENS estratrienes) AND THEIR G. Stubenrauch, Abteilung 0. Haupt fUr Biochemische Hochschule LUbeck, Received...
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