Synthesis and antiestrogenic activity of 2,4-bis(bromomethyl)-estrone methyl ether∗: A bifunctional alkylating steroid

63 SYNTHESISANDANTIESTROGENIC ACTIVITY OF 2,4_BIS(BROMOMETHYL)ALKYLATING STEROID. ESTRONEMETHYL ETHER*: A BIFDNCTIONAL

Narasimha rao Kanamarlapudi,

Frederick

Sweet and James C. Warren

Departments of Obstetrics-Gynecology and Biological Chemistry Washington University School of Medicine St. Louis, Missouri 63110 Received:

3/U/74

ABSTRACT The synthesis of 2,4-bis(bromomethyl)estrone methyl ether was accomplished by reacting estrone methyl ether with formaldehyde and hydrogen bromide in s-tetrachloroethane. The compound readily reacts with cysteine, tryptophan and histidine in 0.05 E phosphate It also reacts with compounds containing sulfhydryl buffer pH 7.0. and the reduced form of 5,5’groups, such as 2-mercaptoethanol Structural analysis and stoichiodithiobis (2-nitrobenzoic acid). metry of the reaction of the bromomethylsteroid with cysteine indicates that the title compound contains two reactive bromoThe biological activity of the bromomethylsteroid methyl groups. was evaluated by analysis of its effect on uterine glucose-6phosphate dehydrogenase activity after administration into the While it lacked estrogenic uterine lumen of ovariectomized rats. activity, the compound did display an antiestrogenic effect that was both greater and more persistent than that of an equimolar quantity of estrone methyl ether.

*IUPAC nomenclature for the compound: methoxy-1,3,5(10)-estratrien-17-one.

Volwne 24, Number 1

s

WD1LOXDrn

2,4-bis

(bromomethyl) -3-

July,

1974

S

64

TmEOXDI INTRODUCTION

Previous

reports

from this

of 4-mercuri-17B-estradiol the estrogen

receptor

protein

labeling

mercury coordinated

capable

acid

at the steroid

is highly binding

desirable. sites

limited

binding

because

of estrone

silica

resonance tryptophan,

spectral

bond formation,

iodo

and bromo derivatives

respectively

methyl ether methyl ether

histidine,

*Abbreviations used: DBX, m-dibromoxylene;

of

(2-6). procedure,

(BBEIM*) was prepared

with aqueous formaldehyde at room temperature.

by chromatography

of elemental

data.

protein

via true covalent

The pure crystalline

as BBEIMon the basis

Therefore,

of progesterone

was purified

gel plates.

forms only

of the receptor

and hydrogen bromide in s-tetrachloroethane The crude product

activity,

bond with amino

of a known halomethylation

2,4-bis(bromomethyl)estrone

Although

labeling

reactive

and progesterone

of

(1).

it

residues.

site

sites

estrogenic

of forming a true covalent

in proteins

By modification

by reaction

persistent

For affinity

we have synthesized cortisone

is

the synthesis

binding labeling

bonds with cysteine

an “estrogen” residues

by affinity

possesses

use in affinity

described

to study steroid

4-mercuri-17B-estradiol its

laboratory

product

analysis

was identified

and nuclear

This compound reacts 2-mercaptoethanol

on preparative

magnetic

with cysteine,

and reduced

ElM, estrone methyl ether; BB, benzyl bromide.

E2, 17B-estradiol;

5,5’-dithiobis(2-nitrobenzoic alkylation

confirms

The biological

17g-estradiol activity

Antiestrogenic

estrogenic

induced elevation

in the uterus activity

activity

steroid.

in ovariectomized

and measurement of 24 hours later.

activity,

acts

of the bromosteroid

The

but inhibits

of glucose-6-phosphate

and therefore

of cysteine

alkylating

compound was tested

administration

dehydrogenase

does not posses

The stoichiometry

is a bifunctional

of this

intraluminal

glucose-6-phosphate bromosteroid

that it

activity

rats by uterine

acid).

dehydrogenase

as an antiestrogen. persists

for over

48 hours. EXPERIMENTAL Reagent grade solvents and formaldehyde were obtained Materials. Co. Hydrogen bromide gas was purchased from Fisher Scientific Steroids and other reagents were from Matheson Gas Products. supplied by Sigma Chemical Co. L-Cysteine- 14C(U)hydrochloride (32 mCi/mmole) was purchased from Amersham/Searle Co. This preparation was diluted with nonradioactive cysteine hydrochloride to obtain the desired specific activity. Preparative chromatographic silica gel plates containing fluorescent indicator DBX and BB were supplied by were obtained from Analtech Inc. Aldrich Chemical Company Inc. of a procedure by Hoehn and Preparation of BBElM. Modification Johns (7) was used to prepare BBElM. Hydrogen bromide gas was continuously dispersed through a stirred solution of 1.5 g of estrone methyl ether in 15 ml of s-tetrachloroethane and 1.0 ml After 2 hours, of 40% aqueous formaldehyde at room temperature. 1.0 ml of 40% aqueous formaldehyde was added to the reaction After mixture and dispersion of hydrogen bromide continued. a total of 8 hours the contents of the reaction flask were diluted with 75 ml of a 1 to 1 mixture of saturated aqueous NaHCO3and 10% NaOH. The resulting mixture was extracted with chloroform and the chloroform oxtract washed thorougly with distilled water, The dry chloroform extract was then dried (MgSO4) overnight. concentrated and chromatographed on preparative silica gel (1 mmthick) plates containing a fluorescent indicator and

developed with benzene:ethyl acetate (19:l). The forward migrating product (Rf value 0.66 on silica gel plates, Eastman No. 6060) was eluted with chloroform, evaporated to dryness and the residue crystallized from ether-hexane to give 0.4 g of the bromosteroid (16% yield). Recrystallization from ether yielded pure compound, m.p. 128-130'. Calc. for C21H2sBr20,: C 53.64, H 5.57, Br 33.98 Found: C 53.58, H 5.64, Br 33.72 The structural assignment for the title compound was obtained by comparing the nuclear magnetic resonance spectra of BBElM with known m-dibromoxylene. Signals due to m-dibromoxylene (CDC13 solution, tetramethyl silane internal standard) were observed at 6 4.45 (S, 4 protons) produced by the CHzBr groups, a broad singlet at 6 7.42 (1 proton, H-2) and a narrow sharp multiplet centered at 6 7.30 (3 protons, H-4, H-5, H-6) produced by the aromatic ring protons. Corresponding resonance signals from BBElM (CDC13solution)were observed at 6 4.52 (S, 2 protons) and 6 4.60 (S, 2 protons) due to the two CHzBr groups and 6 7.36 (S, 1 proton) due to H-l. Additional signals observed at 6 0.88 (S) (18-CH3) and 6 4.00 (S) (3-OCH3) are characteristic of estrone 3-methyl ether analogs (7). Reaction of BBElM with reduced 5,5'dithiobis(2-nitrobenzoicacid). The yellow colored anion of 5-thio-2-nitrobenzoicacid (0.4 pmole) was produced by reducing 0.4 umole of 5,5'-diothiobis(2-nitrobenzoic acid) with 0.4 pmole of reduced glutathione (8) in 2.8 ml of 0.05 E potassium phosphate buffer, pH 7.0. To this solution was added 0.04 umole of BBElM in 0.2 ml of ethanol. Reduction in absorption at 412 nm due to alkylation of the anion was monitored in a Cary-14 spectrophotometer. Reaction of BBElM with cysteine and its stoichiometry. A solution of 6.59 umole of BBElM in 1.5 ml of ethanol was mixed with 18.75 pmole of L-cysteine-I4C hydrochloride (40 uCi/mmole) in 1.5 ml of 0.05 M potassium phosphate buffer, pH 7.0 and incubated at room temperature for 2 hours. Aliquots of the reaction mixture (0.5 ml per plate) were applied to 20 x 20 cm size, silica gel (0.1 mm thickness) plate. Cysteine, cystine and BBElM were also applied to the same plates to serve as standards. The plates were developed with n-butanol:acetic acid:water (12:3:5), dried and visualized under ultraviolet light. A portion of the plate containing the standards and a small part of the reaction mixture was cut off and sprayed with ninhydrin reagent. Spots on the chromatograms which absorbed ultraviolet light and reacted with

S

?FB=OLDI

67

ninhydrin reagent were assumed to be conjugates of the steroid and the amino acid. These conjugates were extracted with appropriate solvents (spots B and C with methanol and spot A with water) at room temperature followed by heating at 60' for 10 minutes. Clear supernatants were obtained by centrifuging the extracts. The steroid content of the extracts was estimated by a modified Zimmerman reaction (9) using estrone methyl ether as the standard. The cysteine content was quantitiated by counting radioactivity in the extracts in a Packard Trio-Carb liquid scintillation spectrometer (86% efficiency). Reactions of BBElM with other amino acids and 2-mercaptoethanol. Solutions containing 2.5 umoles of BBE,M in 0.6 ml of ethanol were incubated with-S.0 umole of histidine, tryptophan, methionine or 2-mercaptoethanolin 0.6 ml of 0.05 E potassium phosphate buffer, pH 7.0 at room temperature. At regular time intervals during 24 hours of incubation 0.03 ml aliquots of the reaction mixture were applied to 20 x 20 cm silica gel (0.1 mm thickness) plates. Appropriate amino acids and BBElM were applied to the same plates to serve as standards. The chromatograms were developed and visualized as described in the preceeding experiment. Animal studies. Female Holtzman rats (250-280 g) were bilaterally ovariectomized and used four weeks later. Steroids and non-steroidal compounds to be tested for biological activity were dissolved in ethanol and added to a solution of 1.8% NaCl in a proportion to give a final ethanol concentration of 1%. The total volume injected into one horn was 0.025 ml. The molar quantities of BBElM, ElM and DBX used in these studies were 23 times greater than 178-estradiolwhile BB was 46 times greater. Assay of enzyme activity. Determination of total activity of glucose-6-phosphatedehydrogenase (D-glucose-6-P:NADP+oxidoreductase, EC 1.1.1.49) in uterus following estrogen administration had previously shown to be a valid assessment of estrogenicity (10). The animals were injected with appropriate compounds and sacrificed 24 hours following last injection. The uteri were removed and homogenized in 0.04 E Tris; 0.0015 E EDTA buffer, pH 7.5. The homogenates were centrifuged for 15 min at 30,000 x g. The supernatants were assayed for glucose-6-phosphatedehydrogenase activity. The enzyme activities are expressed as milliunits/horn,where a unit is defined as that amount of enzyme capable of producing 1 pmole of NADPH per min under the conditions of assay. Data are reported as a mean f standard error of the mean. Statistical significance was analyzed by Student's ttest for non paired variates.

S

68

-JYDEOIDII

RESULTS

Reaction of BBE,M with reduced 5,5'-dithiobis(Z-nitrobenzoic acid. The reaction of BBE,M with the colored anion of S-thio2-nitrobenzoic acid was continuously monitored at 412 nm in a Cary-14 spectrophotometer. Addition of BBEIM to a cuvette containing the aromatic thiol resulted in rapid decrease in the absorbance at 412 nm which indicates S-alkylation of S-thio2-nitrobenzoic acid by BBEIM to a colorless product.

Alkylation of cysteine with BBElM and stoichiometry of the reaction. A solution containing 1 part of the bromosteroid to 2.85 parts of L-cysteine-14C was incubated for 2 hours at room temperature. The incubation mixture and appropriate standards were chromatographed on silica gel plates with n-butanol:acetic acid:water (12:3:5). Examination of the chromatogram under UV light revealed the presence of a small amount of unreacted BBEIM (Rf value 0.77), a major spot with Rf value 0.42 (spot A) and two minor spots with Rf values 0.52 and 0.57 (spots B and C respectively). Treatment of the same chromatogram with ninhydrin reagent then heating the plates at 110' for 1 minute produces a blue color in spots A, B and C.

Unreacted cysteine (Rf 0.35)

and a portion of cysteine oxidized to cystine (Rf 0.2) formed a purple colored product with ninhydrin. Since spots A, B and C absorbed W

light, reacted with ninhydrin reagent and possesed

s Rf values

intermediate

were considered criterion

between those

to be steroid-amino

for detecting

established

to determine

These results

two cysteine

of bromoprogesterones

residues

the basis

of stoichiometric

and in spots

of steroid

per molecule

conjugate

the 2- and 4- isomers

of monocysteinyl-steroid

methionine

of BBElMwith other

amino acids

Tryptophan and histidine acid conjugates.

acids

incubation

Reaction

longer

between methionine

the same period

of incubation.

periods

B and On

spot A contains

B and C contain conjugates.

and 2-mercaptoethanol.

were analyzed

to form steroid-amino required

and spots

between BBE,M and tryptophan,

or 2-mercaptoethanol

chromatography.

spots

is

in spot A contains

of steroid.

a dicysteinyl-steroid

of the reaction

B and C it

data and Rf values, while

to the

of the steroid

that the conjugate

residue

solvents

of the steroid

The molar ratio

per molecule

one cysteine

with appropriate

the molar ratio

indicate

C contain

Products

had been

(4,s).

to the amino acid in spot A is 1:2,

Reactions

This

acid conjugates

work on reactions

amino acid in the conjugates.

1:l.

they

acid conjugates.

Spot A, B and C were extracted and analyzed

of the reactants,

steroid-amino

in previous

with cysteine

69

TllltOXDrn

by silica

reacted

gel

with BBElM

However these (over

histidine,

amino

12 hours).

and BBE,M could not be detected

within

Chromatography of the incubation

S mixture spot of

containing

which

gation

BBE,M and 2-mercaptoethanol

absorbed

BBEIM.

UV light

The relative

are

TIIEOXD6

in the

and had a lower

rates

order:

of

the

cysteine

tryptophan

> histidine.

Biological

activity

of

estrogenic

activity

by intraluminal

to ovariectomized phosphate

of

6-phosphate

greater

bromosteroid

intraluminal

application

administered induction

molar

2 hours

later

(Table

II).

glucose-6-phosphate estrone

induced

rise

These

activity resides

experimental

molecule

ether

conditions

specific.

that

the

of

were

methyl to

antiestrogenicity

exert

ether

17B-estradiol suppressed

while

about

an equi-

52% of

dehydrogenase

by the bromosteroid

did not

23

experiments.

BBEIM completely

reactive

of

was studied.

uterus

portion

steroid

glucose-

ether

similar

suppressed

for

quantities

methyl

the

steroid

a chemically

demostrate

and is

activity

under

>

Intraluminal

dehydrogenase,

exhibited

in the

conju-

glucose-6-

later.

in glucose-6-phosphate

m-dibromoxylene,

data

methyl

uterine

BBEIM or estrone of

and specificity

similar

of

of

bromosteroid

response

The antiestrogenic

since

the

the

amount of

estradiol

used of

that

was tested

The molar

blocked

of

of

increase I).

17B-estradiol

than

acid

administration

and estrone

activity

significantly

the

(Table

Antiesirogenic Prior

to

a new

(and 2-mercaptoethanol)

24 hours

BBElM failed

than

steroid-amino

measurement

activity

dehydrogenase

administered

times

with

Rf value

The bromosteroid

BBElM.

dehydrogenase

administration

the

rats

produced

of

the

analog

activity. is

specific

molecule,

of

BBElM, under

antiestrogenic resides

17B-

in the

activity. steroid

TABLE I Estrogenic

activity

Group

Treatment

1

vehicle

2

E2

3 4

of 2,4-bis

(bromomethyl) estrone

Dose of steroid (r&horn ) ---

methyl ether.

G-6-PD activity (mu/horn) 65.0f5.1

5

101.2k4.1

BBElM

200

74.1k3.5

EIM

121

66.524.1

Animals (250-280 g) used in these experiments were ovariectoThe steroids or vehicle alone mized four weeks prior to use. were administered intraluminally and animals killed 24 hours later. Each value reported is mean of five animals per group. Group 2 value is significant (Pc.01) from the control. No significant variation from control was observed in group 3 and 4.

TABLE II Antiestrogenic activity of 2,4-bis(bromomethyl)estronemethyl ether.

Group 1st admin.

Treatment Time 2nd admin. (hr)

G-6-PD activity (mu/horn)

Experiment I 1

vehicle

2

vehicle

2

vehicle

2

E2

3

BBE,M

2

4

EIM

2

5

BBElM

24

E,

6

EIM

24

E2

7

BBEIM

48

8

EIM

48

E2 E2

E2 E2

74.27k3.43 141.32k3.38 77.17k2.26 106.11+6.40 77.97k1.67 110.91+6.62 90.8354.46 131.83i2.93

Experiment II 1

vehicle

2

vehicle

2

vehicle

2

E2

131.18+5.85

3

DBX

2

E2

134.37k3.71

4

BB

2

E2

138.90?7.39

71.85k4.84

Animals (250-280 g) were ovariectomized four weeks prior to use. First administration was vehicle, BBElM (200 ng/horn), EIM (121 ng/horn), DBX (110 ng/horn) or BB (144 ng/horn). After the times shown vehicle or E2 (5 ng/horn) were applied to the same uterine horn. Enzyme activity in the uterine horns was evaluated 24 hours after second administration. In experiment I the activity in group 2 is significantly (Pc.01) greater than groups 3, 4, 5, 6, and 7, but not 8. Further activities of BBElM groups (3, 5, and 7) are significantly (Pc.01) lower than activities of E,M groups (4, 6, and 8). In experiment II the activity in group 2 did not vary significantly from groups 3 and 4.

SC Thus modification alkylating its

was administered

and 4

of antiestrogenic

does not appear to effect

of alkylating

methyl ether.

administration

Glucose-6-phosphate

in uterus was measured 24 hours after

The antiestrogenic

activity

24 hours then gradually

estrogenic

activity

of estrone

to the extent

induced glucose-6-phosphate

last

dehydrogenase injection

methyl ether was evident

diminshed within

of BBElMpersisted

48 hours suppression

groups results

178-estradiol

activity.

24 hours or 48 hours following

of BBEIMor estrone

for

2

methyl ether by introducing

The presence

activity.

in the persistence

activity

of estrone

groups at positions

biological

73

TBEOIDI

48 hours.

The anti-

beyond 24 hours and after

of about 70% of the 176-estradiol

dehydrogenase

activity

was observed.

DISCUSSION The 2,4_bis(bromomethyl) was obtained

by the classical

ethers

according

(ll),

by Hoehn and Johns (7). formaldehyde

crude product resulting

to a modification

esters

2-hydroxymethyl-

of phenolic

of a procedure

reportedly estrone

followed

which produced the isolated

described

methyl ether with gave a mixture of

methyl ether.

were based on subsequent

with sodium acetate

methyl ether

reaction

Treatment of estrone

and 4-chloromethyl

assignments

of estrone

halomethylation

and hydrogen chloride

2-chloromethylstructural

derivative

These

reaction

of the

by hydrolysis

of the

and characterized

and 4-hydroxymethylestrone-3-methyl

ether

(7).

S

TDEOIDI

We repeated the halomethylation with hydrogen bromide and the crude reaction mixture was purified by chromatography with preparative layer silica gel plates. Purification of the isolated halogen containing component by recrystallization gave a crystalline product found to be homogeneous on the basis of thin layer chromatographic analysis. Elemental analysis indicated the compound to have the empirical formula CZ1H26Br202, which is clearly consistent with a bis(bromomethy1) derivative of estrone methyl ether. The nuclear magnetic resonance spectrum of this compound when compared with that from m-dibromoxylene is consistent with the structure of 2,4-bis(bromomethyl)estronemethyl ether. Moreover, this is the anticipated product of bromomethylation of estrone

methyl ether since the strongly ortho directing J-methyl

ether group is expected to favor electrophilic substitution at positions 2

and 4

similar to the transient 2- and 4- mono-

halomethylation found by Hoehn and Johns (7). BBEIM is capable of reacting with compounds containing a sulfhydryl group such as 2-mercaptoethanol and reduced 5,5'-dithiobis(2-nitrobenzoicacid). Progress of the reaction between the bromosteroid and the latter compound can be monitored by decrease in the absorption at 412 nm due to alkylation of the -So auxochrome. The capacity of the bromosteroid to alkylate amino acids was shown by the reaction of the bromosteroid with cysteine. The reaction products have Rf values intermediate between those

indicating

of the reactants,

that they are steroid-amino

Stoichiometric

conjugates.

analysis

the ratio

of steroid

products,

and 1:2 in the third.

tory

(4,s)

Previous

that alkylation

bromide produces

acid and steroid.

Therefore,

2,4-bis(S-s-

a covalent

spots

form steroid

Stoichiometric

tive

alkylating

in the biological

the target

These implications

that uptake and retention binding

proteins

sulfhydryl (12,13). are either

of estradiol

significantly

slower

These observations

indicate

at the steroid

in some way in the biological

that this

and spot

methyl ether.

rate than cysteine. extent.

estrogen

derivagroups

of estrogens

on

were made on the basis by mouse uterus following

and estrogen

blocking

of

or p-chloromercuribenzoate that the sulfhydryl

binding

action

of cysteine

Sulfhydryl

action

decreased

groups by N-ethylmaleimide

present

isomers

amino acid conjugates

steroid.

have been implicated tissues.

estrone

from the reaction

study demonstrate

is a bifunctional

between amino

with BBE,M to a detectable

data obtained

with BBElM in this

linkage

methyl ether

with BBElM at pH 7.0 at a significantly does not react

laboraamino acids

B and C constitute

cysteinylmethyl)

Tryptophan and histidine

Methionine

work in this

of nucleophilic

of 2- (or 4-)S-L-cysteinylmethylestrone A constitutes

showed

to amino acid to be 1: 1 in two of the

demonstrated

by a steroidal

of the conjugates

acid

site

groups

or are involved

of the hormone.

BBElM was tested for biological activity by administering solutions of the steroid intraluminally to ovariectomized rats and measuring glucose-6-phosphatedehydrogenase activity 24 hours later. Neither BBEIM nor estrone methyl ether possessed estrogenie activity, however, they both inhibit 17$-estradiol induced glucose-6-phosphate dehydrogenase activity in the uterus. Antiestrogenic activity exhibited by BBElM is greater than that produced by an equivalent amount of estrone methyl ether and the effect persists even after 48 hours.

Antiestrogenicity

exhibited by BBEIM is specific since a bifunctional analog, m-dibromoxylene administered under similar conditions fails to show antiestrogenicity. These data are consistent with a mechanism whereby the affinity-labeling steroid occupies the estrogen binding site then forms a covalent bond with an amino acid residue at the site.

This prevents 178-estradiol from

binding.

ACKNOWLEDGEMENTS

This research was supported by NIH grant AM 15708. N. Kanamarlapudi is a Ford Foundation Postdoctoral Fellow. We thank Drs. C. David Gutsche and T.C. Kung of the Chemistry Department of Washington University for NMB Spectra.

77

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