Oxidation of oxyphenbutazone by sheep vesicular gland microsomes and lipoxygenase

VoI. 63, No. 3, 1975

BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS

OXIDATION SHEEP V E S I C U L A R

Philip

S. P o r t o g h e s e ,

Department

GLAND MICROSOMES

Kerstin

January

and Bengt

Karolinska

01 S t o c k h o l m

60,

BY

AND L I P O X Y G E N A S E

Svanborg,

of Chemistry,

S-I04 Received

OF O X Y P H E N B U T A Z O N E

Samuelsson

Institutet

Sweden

30,1975

SUMMARY O x y p h e n b u t a z o n e was o x i d i z e d w h e n i n c u b a t e d w i t h a c e t o n e powder p r e p a r e d from sheep v e s i c u l a r g l a n d m i c r o s o m e s or w i t h l i p o x y g e n a s e at pH 4 or 5. O x i d a t i o n also o c c u r r e d at pH 8 or 9, if a r a c h i d o n a t e or l i n o l e a t e was a d d e d to either of the incub a t i o n m i x t u r e s . The o x y g e n a t e d p r o d u c t was found to be i d e n t i c a l w i t h 4 - h y d r o x y o x y p h e n b u t a z o n e , w h i c h was s y n t h e s i z e d and a n a l y z e d by gas l i q u i d c h r o m a t o g r a p h y and mass s p e c t r o m e t r y . The o x y g e n a t e d c o m p o u n d was not an i n h i b i t o r of p r o s t a g l a n d i n b i o s y n t h e s i s .

INTRODUCTION gated

Although

extensively

turated

prostaglandin

concerning

fatty acids

(i),

its a b i l i t y

there

of other c l a s s e s

of p r o s t a g l a n d i n

synthetase.

involving agent.

oxyphenbutazone

Since

there

m o d e of a c t i o n hibition gated

of n o n - s t e r o i d a l

the c o n s t i t u t i o n

against prostaglandin glands.

In addition,

prostaglandin to d e t e r m i n e

synthetase

whether

and

derived

Copyright©19~ ~ AcademicPress,~ AH~gh~ ~ r e p r o ~ c ~ n m a ~ r m r e s e ~ e d .

748

the

agents with

in-

investiit

f r o m sheep v e s i c u l a r features

s h a r e d by

(6) have p r o m p t e d

enzyme c a t a l y z e s

tion of o x y p h e n b u t a z o n e .

linking

p r o d u c t and tested

lipoxygenase

the latter

such e x a m p l e

(4, 5), we h a v e

the c o m m o n m e c h a n i s t i c

synthetase

the a c t i o n

used a n t i i n f l a m m a t o r y

antiinflammatory

of the o x i d a t i o n

unsa-

(2) p e r t a i n i n g

body of e v i d e n c e

biosynthesis

investi-

various

during

to r e p o r t one

a clinically

is a s u b s t a n t i a l

of p r o s t a g l a n d i n

to o x y g e n a t e

of c o m p o u n d s

we w i s h (3),

has b e e n

is o n l y one r e p o r t

to the o x i d a t i o n

In this c o m m u n i c a t i o n

synthetase

a similar

us oxida-

Vol. 63, No. 3, 1975

MATERIALS

BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS

AND METHODS

Materials:

~,5-14C~-Oxyphenbutazone

Ciba-Geigy,

Basel,

Switzerland.

diluted with unlabeled M~indal,

Sweden)

Arachidonic

to a specific

acid was p r e p a r e d

to a s p e c i f i c

obtained

somes:

Frozen

grinder

glands

and 1 0 - 3 M was

powder

blended

directly

for one m i n u t e

in a P o t t e r - E l v e h j e m

carried

o u t at 4°C.

The cell debris,

centrifuged microsomes buffer was

for

further were

1,000 ml c o l d stirring. mixture

a B~chner portions washing

Minn.,

to -23°C.

at i0,000 g for

g. The

USA.

gland micro-

continued

until

to a w a t e r

1,000 ml d i e t h y l

(-20°C) ether

749

containing

and homowere

were removed supernatant The

The s u s p e n s i o n dropwise

into

strong m a g n e t i c of the through

p u m p and w a s h e d w i t h

at -20°C,

was

50 ml of s u c r o s e

then f i l t e r e d

butanol.

by

sedimented

the t e m p e r a t u r e

The s u s p e n s i o n was

of cold

a meat-

mlxer

and a d d e d

constant,

lipoxy-

. The s u s p e n s i o n

and h o m o g e n i z e d .

with

was

, 0.05 M Tris,

75 m i n u t e s .

funnel

Minn.,

All o p e r a t i o n s

w i t h an a d d i t i o n a l

acetone,

connected

form

mitochondria

into a s e p a r a t o r y

(50 ml each) with

and

homogenizer

(-40°C)

funnel

(reduced

and

and s o y b e a n

into 500 ml of b u f f e r

homogenizer.

at i00,000

S t i r r i n g was

rose

aspirin

2 N HCl to pH 7.4

12 m i n u t e s

transferred

into a glass

transferred

nuclei

(7

Indomethacin

in an U l t r a - T u r r a x

genized

1-14CI -

Austin,

from sheep v e s i c u l a r

(adjusted w i t h

centrifugation

by S t o f f e l

St. Louis,

10-4M g l u t a t h i o n e

EDTA

of 0.03 Ci/mol.

(about 150 g) w e r e m i n c e d w i t h

and t r a n s f e r r e d

0.25 M sucrose,

AB,

0.36 Ci/mol.

f r o m Sigma,

of a c e t o n e

(H~ssle-Ciba-Geigy

Institute,

Sharp and Dohme;

g e n a s e was p u r c h a s e d

was

as d e s c r i b e d

of

by

material

activity

(Hormel

activity

from Merck,

Preparation

acid

provided

The r a d i o l a b e l e d

oxyphenbutazone

diluted with unlabeled USA)

was k i n d l y

After

3

exhaustive

the p o w d e r w a s

Vol. 63, No. 3, 1975

collected, hour

and

dried

trode

in a 1 ml

acetone

powder,

a sufficient tion of

3 minutes

or

terminated

CH3OH

termination

Extraction tained

buffer

were

scraped

was

run

a final

was

of

added

concentra(20 ~g

added

after

oxyphenbutazone

labeled

The

incuba-

acetate.

arachidonate.

above

by a d d i n g

but

in the

7 ml of CHCl 3-

D~nnschicht

liquid

buffered (8:2,

and

phase

in b e n z e n e - d i o x a n e - a c e t i c arachidonate

v/v).

and c o u n t e d

adding was

was

con-

was

6 with McIlvaine After

detection

in a P a c k a r d

to s i l i c a

zones

Tri-carb

If l a b e l e d

continued

(80:20:2,

from products. 750

material

arachi-

by r e m o v i n g

3 ml CHCI 3 a n d

applied

acid

incubation

the r a d i o a c t i v e

spectrometer.

the e x t r a c t i o n

protein

to pH

II scanner,

scintillation

utilized,

If the

the e x t r a c t e d

in C H C I 3 - H O A c

The c h l o r o f o r m

uncoverted

or

or 4 - h y d r o x y o x y p h e n b u t a z o n e

Chromatography:

f r o m the p l a t e s

the p r e c i p i t a t e d HCOOH.

t h e r e was

i0 ml of e t h y l

as d e s c r i b e d

accomplished

gel p l a t e s

(8) a n d r u n

3375

was

elec-

otherwise)

3 minutes.

out with

oxyphenbutazone

oxyphenbutazone,

to s i l i c a

a Berthold

donate

performed

and T h i n L a y e r

with

model

carried

con-

oxygen

substrate

radioactive

with

were

v/v).

labeled

applied

were

to m a k e

for an a d d i t i o n a l

also

of u n l a b e l e d

(i:i,

to 1

of 0.i M T r i s - H C l

respectively)

by e x t r a c t i n g

were

a Clark

unlabeled

containing

continued

incubations

the

1/2

lipoxygenase,

experiments,

i00 ]~g l i n o l e a t e ,

Experiments

and

for

(when not s p e c i f i e d

of o x y p h e n b u t a z o n e

In some

of

4 mg

with

to 1 ml

20 ~g of s o y b e a n

to the m i x t u r e

t i o n was

presence

as f o l l o w s :

quantity

incubation

These

(37 ° ) f i t t e d

containing

10-3M.

arachidonate

vacuum

of I 3 , 5 - 1 4 C l - o x y p h e n b u t a z o n e

chamber

and accomplished buffer

under

at -20°C.

Incubations

acetate

and

in a d e s s i c c a t o r

stored

Incubations: ducted

BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS

2 ml

1%

gel p l a t e s

V/v/v)

and

to s e p a r a t e

Vol. 63, No. 3, 1975

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Synthesis

of

zone

(0.342

-H20

4-Hydroxyoxyphenbutazone:

and C H 3 0 H ( 3 . 5 After

g,

ml)

1 mmole), 30% H202

was

that p e r i o d

allowed

and

acetate

phase was

removed

in vacuo w i t h o u t elution

ting w i t h

99:1

acetate.

with

ethyl

separated,

column

eluted

to 98:2.

: 0.47 on silica elemental

a solvent

point was

(SilicAR

177.5-179.5°C

The ethyl

CC-4,

agreed

with

calculated

C,

67.1;

H,

was

taken

using

values:

the formula 5.9;

was m a i n t a i n e d

34 g)

to star-

The product

in c o m p o s i t i o n from ethyl

and it had an Rf

run in C H C I 3 - H O A c

N, 8.3; w h i c h well

energy

5% HCI

subjected

was r e c r y s t a l l i z e d

following

electron

was

which varied

showed

spectrum

into

with CHCI3-HOAc(96:4).

analysis

Mass

13 hours.

(2x20 ml).

The r e s i d u e

chromatography

gel plates,

values:

(0.i ml)

dried over Na3CO 3, and the solvent

The m a t e r i a l

Its m e l t i n g

1 N NaOH

was p o u r e d

acetate,

heating.

using

of o x y p h e n b u t a -

at 22°C for

mixture

CHCL 3 and t e r m i n a t i n g

(94 mg) was from

extracted

(0.7 ml),

to stand

the r e a c t i o n

(20 ml)

gradient

A mixture

C,

(8:2,v/v). 66.8;

The

H, 5.9;

CI9H20N204

and its

N, 8.3.

a LKB

9000

at 70 eV and

instrument, the trap

the

current

was

60 ~A. The 55(11);

spectrum 57(94);

107(25);

m/e

65(15);

119(31);

ed a m o l e c u l a K

(% intensity): 77(40);

120(36);

ion at m / e

79(19);

135(72);

ion from o x y p h e n b u t a z o n e

the butyl

group

rings w e r e This

identical

indicates

attached

at m / e

(9).

119,

to the butyl

group

92(21);

199 w i t h

93(55);

340(6)

higher

Ions at m / e

135,

than

showthe

57 c o n t a i n i n g the a r o m a t i c

from o x y p h e n b u t a z o n e .

introduced

or located

52(32);

205(48);

is 16 units

to those o b t a i n e d

that the newly

51(18);

85(100);

199(16);

340, w h i c h

molecular

and ions

41(30);

OH group c a n n o t

in either

be

of the aromatic

rings. The

synthesized

compound

was

also

751

converted

into

the trimethyl-

Vol. 63, No. 3, 1975

BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS

100

4,84

0 ,d"~,/Osi (CH3)3 (CH3)3Si O ~ J , ~ j

80-

HgC4"

73

60-

271

403

-

2o-

&28

207

.J.1,..<

..... J

50

100

200

150

'

250

•

i

300

.

.

.

.

i

350

.

.

.

.

i

.

400

.

.

.

i

~50

.

.

500

rnle

Fig.

silyl

i.

The mass s p e c t r u m of 4 - h y d r o x y o x y p h e n b u t a z o n e t r i m e t h y l s i l y l ether.

ether

and a n a l y z e d

a gas c h r o m a t o g r a p h

by the mass

spectrometer

equipped with a 1%

SE-30

tion time of the c o m p o u n d was

converted

using methyl

fatty acids

mass

esters

spectrum

(Fig.

of normal

i) was o b t a i n e d

at 22.5 eV and the trap c u r r e n t at m / e

484, w h i c h

presence

60 pA.

units

aromatic

rings w e r e

that the i n t r o d u c e d

the free r a d i c a l

both carbonyl

of

tion M i x t u r e s :

After

was

[3,5-14C3

scraped

A n a l i q u o t was analyzed

the e l e c t r o n

23.6

(i0). The energy

then 340,

due

compared

The f r a g m e n t a t i o n

susceptible

to the

of ions

upward,

is l o c a t e d

ion

pattern

in the

4 posi-

site for o x i d a t i o n

w o u l d be s t a b i l i z e d

by

groups.

Identification

Rf=0.47

the C-value,

72 units

OH g r o u p s

intermediate

The r e t e n -

It had a m o l e c u l a r

shifted

This w o u l d be a p a r t i c u l a r l y

because

column.

as s t a n d a r d s

higher

the s p e c t r u m of o x y p h e n b u t a z o n e .

indicates tion.

with

combined with

of two t r i m e t h y l s i l y l groups. The m / e v a l u e s

containing with

is 2x72=144

into

as

4-Hydroxyoxyphenbutazone

separation

on TLC,

from the p l a t e

converted

into

by gas c h r o m a t o g r a p h y

the r a d i o a c t i v e

and e l u t e d w i t h

the t r i m e t h y l s i l y l u s i n g a Barber

752

in Incubazone at

ethyl

ether

acetate.

and

Colman model

Vol. 63, No. 3, 1975

BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS

5000 i n s t r u m e n t radioactivity.

with

The m a s s

the t r i m e t h y l s i l y l

RESULTS

AND

derivative

within

five minutes.

above.

In the a b s e n c e

no o x y g e n

uptake.

c a n t l y by a s p i r i n of e f f e c t i n g Because acetone

responsible

as to w h i c h for o x y g e n

soybean

It was

found

of o x y g e n

experiments

to air o x i d a t i o n be i n h i b i t e d

lipoxygenase component

found that

application

incubation

with

~,5-14C~-oxyphenbutazone

whose Rf=0.47 unbuffered

is i d e n t i c a l

silica

at pH 6 u n d e r 3% c o n v e r s i o n .

gel G.

otherwise

agents

whether

similar

experi-

it bears in p r o s t a -

u p t a k e was

oxygen

During

were

the c o u r s e

of

to TLC plates,

but this could

powder

to pH 6. A f t e r at pH 5, 15 %

to a s i n g l e product, by air o x i d a t i o n

on

of[3,5-14C]-oxyphenbutazone

conditions

therefore

753

to a

is s u c c e p t i b l e

converted

Incubation

consump-

or m e r e l y

experiments

to that p r o d u c e d

These results

in the

this c o m p o u n d

the a c e t o n e

identical

capable

5.

enzyme

gel G b u f f e r e d

was

signifi-

since

of o x y p h e n b u t a z o n e

silica

there was

at pH 9 this did not occur.

to d e t e r m i n e

by u t i l i z i n g

three m i n u t e s of

after

these

uptake,

u p t a k e by the enzyme,

it was

enzyme

component

carried out with~3,5-14C~-oxyphenbutazone. these

at pH 6 or

that at pH 5 o x y g e n

while

to o x i d a t i o n

in the c h a m b e r

not i n h i b i t e d

nor w e r e

at

sheep v e s i c u l a r

occurred

(5) to the d i o x y g e n a s e

it was of i n t e r e s t

triggering

from

on their o w n at pH 4 or

c a r r i e d out w i t h

tion was r e l a t e d

incubated

consumption

U p t a k e was

of the u n c e r t a i n t y

synthetase.

was

or w i t h b o i l e d

for

compound.

oxygen

of e n z y m e

i n d u c e d by o x y p h e n b u t a z o n e , As

prepared

and

as d e s c r i b e d

(185nmoles)of

No o x y g e n

oxygen uptake

a resemblance glandin

uptake

or i n d o m e t h a c i n ,

p o w d e r was

ments were

powder

The o x y g e n

of m a s s

of the s y n t h e s i z e d

When oxyphenbutazone

25 mg a c e t o n e

there was full

registration

s p e c t r u m was o b t a i n e d

DISCUSSION:

pH 4 or 5 w i t h glands,

simultaneous

resulted

indicate

only

that o x y g e n

in a up-

Vol. 63, No. 3, 1975

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

take at pH 5 is r e l a t e d zone.

Similarly,

at pH 5 w h e n

the same o x i d a t i o n

the drug was

Of p a r t i c u l a r conversion

(15%)

tion p r o d u c t powder

of

oxidation

was

mixture.

of this type c o u l d

pared

metry which

showed

oxyphenbutazone oxygen

subjected

of the a c e t o n e

it is p o s s i b l e in vivo as well,

and p r o p e r t i e s

that it was

of the

and the s y n t h e t i c a l l y

into t r i m e t h y l s i l y l

C-values

and mass

with acetone

and this fact also

is r e s i s t e n t

Oxyphenhutazone

b o t h the o x i d a t i o n

to gas c h r o m a t o g r a p h y -

identical

incubated

uptake

the m o l e c u l e

acetone

to the same oxida-

by c o n v e r t i n g

mixtures

4-hydroxyoxyphenbutazone

(ii,

substantial

or l i n o l e a t e , r e s p e c t i v e l y ,

Since

the s t r u c t u r e

from the i n c u b a t i o n

were

sis

that

product.

derivatives

mote

lipoxygenase.

take p l a c e

Its i d e n t i t y was d e t e r m i n e d products

(13%) was p r o d u c e d

the finding

when arachidonate

to i n v e s t i g a t e

of o x y p h e n b u t a -

at pH 8 in the p r e s e n c e

to the i n c u b a t i o n

of i n t e r e s t

product

[3,5-14C]-oxyphenbutazone

also o c c u r r e d

co-oxidation

oxidation

incubated with

significance

or l i p o x y g e n a s e

were added

to e n z y m a t i c

12).

When arachidonate

powder

in the p r e s e n c e

was

under

mass

The

spectro-

spectra.

4-Hydroxy-

at pH 5 did not pro-

suggests

to o x i d a t i o n

is an i n h i b i t o r

powder

ethers.

pre-

that the rest of

these

conditions.

of p r o s t a g l a n d i n incubated

biosynthe-

at pH 8 w i t h

of o x y p h e n b u t a z o n e ,

the

there was an i n -

(CH2)3 CH3

R=H, Oxyphenbutazone R= OH, 4 - Hyd roxyoxyphenbutazone R=OOH, 4-Hydroperoxyoxyphenbutazone Fig.

2.

The s t r u c t u r e derivatives.

of o x y p h e n b u t a z o n e

754

and its o x y g e n a t e d

Vol. 63, No. 3, 1975

hibition

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of the o x y g e n

uptake.

by 4 - h y d r o x y o x y p h e n b u t a z o n e ,

When oxyphenbutazone

the o x y g e n

transformation

of

also

by o x y p h e n b u t a z o n e

inhibited

butazone. future

The o x y g e n a t i o n s

work

tase and

a Gustavus

Council

2. Marnett, Biochem.

A.

powder

was

of action

of p r o s t a g l a n d i n

for

synthe-

(P.S.P.)

Pfeiffer

Memorial

by a grant

wishes

to thank

Education

Research

for support

Fellowship.

from the S w e d i s h

Medical

(03X-217).

B.

(1972)

Fed.

Proc.

31,

1442-1450.

L.J., Wlodawer, P., and Samuelsson, B. Biophys. Res. Commun. 6_O0, 1286-1294.

(1974)

"The P h a r m a c o l o g i c a l Basis of T h e r a p e u t i c s " , fourth ed., Goodman, L.S., and Gilman, A., editors. The M a c m i l l a n Co., N.Y., 1970, p. 337.

4. Ferreira, S.H., i_44, 57-73. 5. Flower,

R.J.

7. Stoffel,

W.

and Vane,

(1974)

6. Hamberg, M., 5329-5335.

9. Unterhalt,

J.R.

and Samuelsson,

(1964)

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12.

The

to be of i m p o r t a n c e

for P h a r m a c e u t i c a l

supported

i. Samuelsson,

i0,

by the a c e t o n e

seem

One of the authors

Foundation

This w o r k was

3.

enhanced.

its inhibitiors.

the A m e r i c a n

Research

was

but not by 4 - h y d r o x y o x y p h e n -

observed

on the m e c h a n i s m s

ACKNOWLEDGEMENT:

through

[l-14C]-arachidonate

uptake

was r e p l a c e d

J. Pharm.

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L.H.

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