Biochemical Pharmacology, Vol. 19, pp. 2979-2993. Pergamon Press, 1970. Printed in Great Britain

METABOLISM OF ACETANILIDES AND ANISOLES WITH RAT LIVER MICROSOMES

JOHN DALY

Laboratory of Chemistry, National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, Public Health Service, U.S. Department of Health, Education and Welfare, Bethesda, Md.

20014, U.S.A.

(Received 3 December 1969; accepted 20 April 1970)

Abstract-The metabolism of various substituted (F, Cl, Br, I, CF3, NOZ) acetanitides and anisoles have been studied with 3-methylcholanthrene-induced rat liver micro- somes. Metabolism of acetanilides and anisoles, which occurs primarily at the position paw to the acetamido or methoxy function, is markedly influenced by the presence of other substituents in the o&o-, meta- or para-positions. A novel loss of an adjacent substituent during hydroxylation o&o- to an iodo group was observed in the conversion of 4-iodoanisole to 3-hydroxyanisoie. Migration of substituents (the NIH Shift) was not a significant meta~lism with any of these substrates.

DURING the course of studies on the migration of ring substituents during hydroxyla- tion of aromatic substrates,‘* z the metabolism of a variety of substituted anisoles and acetanilides have been investigated with preparations of liver microsomes. The metabolism of these substrates by liver microsomes from 3-methylcholanthrene- treated rats is reported in this paper.

MATERIALS AND METHODS

The substrates were from commercial sources or were prepared by standard proce- dures. Reference phenolic products were from commercial sources or were prepared by standard procedures including hydroxylation with Fenton’s reagent.* Livers from Sprague-Dawley male rats pretreated with 3-methylcholanthrene4 were homogenized with 3 vol. (w/v) of isotonic KCl, centrifuged at 20,000 g for 20 min to afford a crude microsomal suspension. Incubations were carried out as described in the footnotes to the tables, and the products identified by comparison to reference standards. Products were arbitrarily classified as major, minor and trace metabolites, as described in the tables. The remainder of the substrate (> 90 per cent) was recovered unchanged.

Typical identifications for products obtained after incubations of 2-, 3- and 4-fluoro- acetanilide and of 2-, 3- and 4-fluoroanisole are given as examples.

2-Fluoroacetanilide. Two trace metabolites were detected by paper chromatography (R,s 0.15 and 0.62). Insufficient matter (~0.1 pmole) was obtained for accurate quantitation by ultraviolet spectroscopy. Both compounds gave color reactions typical of phenols with Folin’s reagent and diazotized~-nitroaniline. The product with &O-62 gave a color reaction with Gibbs’ reagent indicative of a phenol unsubstituted in the para-position. The high Rf and color reactions of this product are compatible only

* Extensive loss of fluorine to form 4-hydroxyanisole was noted during hydroxylation of 4-thtoro- anisole with Fenton’s reagent (Table 1, footnoteI’; cf. ref. 3).

2979

TABLE ~.METABOL~MOFA~~LEANDHAL~S~S~~D

ANISOLESWITX RAT LIVER MICRO~~MES*

Subs

trat

es

Ani

sole

S

2-Fl

uoro

anis

ole

3-Fl

uoro

anis

ole

4-Fl

uoro

anis

olel

~

2-C

hlor

oani

sole

3-C

hlor

oani

sole

a

4-C

hlor

oani

sole

~

2-B

rom

oani

sole

3-B

rom

oani

sole

~~

4-~r

omoa

niso

le~

p-H

ydro

xyla

tion

m-H

ydro

xyla

tion

o-H

ydro

xyla

tion

Dem

ethy

latio

n --

1___

R

eten

tion

Ret

entio

n R

eten

tion

Ret

entio

n Pr

oduc

t C

onve

r-

time

Prod

uct

Con

ver-

tim

e Pr

oduc

t C

onve

r-

time

Con

ver-

T

ime

sion

t (T

emp.

):

sion

t (T

emp.

)S

sion

t (T

emnX

si

ont

(Tem

p.)f

9 4-

OH

4-O

H

4-O

H

4-O

H

4-O

H

4.O

H

4-O

H

Maj

or

(4)

Maj

or

(2)

Maj

or

(3)

125

min

(1

38”)

16

.3 m

in

(130

”)

4-8

min

(1

30”)

3-

OH

T

race

Maj

or

(2)

Tra

ce

30.5

min

(1

38”)

6.

7 m

in

(138

”)

4.7

min

(1

30”)

2-O

H

6-O

H

6-O

H

2-O

H

6-O

H

2-O

H

2-O

H

6-O

H

Min

or

(0.8

) M

inor

(O

-4)

Min

or

(0‘5

) M

ajor

(4

) M

ajor

(1

) M

ajor

(4

) M

ajor

(4

) M

ajor

(2

)

2.5

min

(1

38”)

2.

1 m

in

(130

”)

2.6

min

(1

30”)

2.

3 m

in

(130

”)

5.1

min

(1

38”)

6.

7 m

in

(138

0)

7.2

min

(1

38”)

4.

4 m

in

(148

”)

Min

or

(0.2

)

Trace

Major

(3)

2.8

mitt

(1

38”)

9 U

3 5

3.8

min

(1

300)

3.

5 m

in

(130

”)

Tra

ce

3-O

H

Maj

or

(2)

Min

or

(0.5

)

Bro

ad

41 m

in

(148

”)

5.1

min

(1

48”)

Min

or

(@4)

6.

7 m

in

(138

”)

Maj

or

(3)

10.6

min

(1

38”)

10

.4 m

in

(138

”)

2-O

H

Tra

ce

3-O

H

Min

or

(0*3

) 4.

8 m

in

(148

”)

2-O

H

Maj

or

(6)

Maj

or

(3)

5.1

min

(1

48”)

58

m

in

(148

”)

Maj

or

(3)

9.6

min

(1

48O

) 9.

7 m

in

(148

”)

ZIo

doan

isoi

e 4-

OH

M

ajor

B

road

6-

OH

M

ajor

4.

3 m

in

(2)

37 m

in

(3)

1148

”)

(162

”)

3.Io

doan

isol

e 4-

OH

M

ajor

12

.6 m

in

6-O

H

Tra

ce

10.9

min

T

race

19

.2 m

in

(4)

(140

(1

48”)

(1

48”)

4-

Iodo

an~s

o~e

4-O

H

Maj

or

7*6 m

in

3-O

H

Maj

or

8.1

min

(L

oss

I)

(2)

048”

) ‘L

7;$

(2)

(148

”)

Maj

or

12.2

min

2-

OH

M

ajor

13

.0 m

in

Maj

or

196

min

2,ld

Dic

hlor

oani

sole

(1

1 (1

48”)

(3

) ( 1

4S0)

(3

) (1

48”)

S-

OH

M

inor

2.

3 m

in

2-O

H

Min

or

5.3

min

(0

.31

(148

”)

(0.3

) (1

48”)

* In

cuba

tions

w

ere

carr

ied

out

at 3

7” fo

r 15

min

with

10

ml

of m

icro

som

al p

repa

ratio

n,

3.5

ml

of

0-J

M T

ris

buff

er,

pH 8

.0,

15 p

mol

es A

TP,

50 c

trno

les

E

gluc

ose-

6-ph

osph

ate,

10

units

glu

cose

d-ph

osph

ate

dehy

drog

enas

e, 2

mg

Tw

een

80 a

nd 5

0 pm

oles

of

subs

trat

e ad

ded

in 0

.1 m

l ac

eton

e in

a f

inal

vol

ume

of 1

5 $,

m

l. So

lutio

n w

as e

xtra

cted

with

equ

al v

olum

e of

eth

yl a

ceta

te.

The

ext

ract

was

dri

ed N

a2S0

4, c

once

ntra

ted

in u

ucuo

and

the

phe

nolic

pro

duct

s is

olat

ed b

y g

thin

-lay

er c

hrom

atog

raph

y, s

ilica

gel

GF,

chl

orof

orm

, be

nzen

e, e

thyl

ace

tate

(65

: 15:

1.5

). R

etec

tion

was

with

Gib

b’s

or

Foiin

’s r

eage

nt.

Phen

ols

wer

e ex

trac

ted

$’

from

sili

ca g

el s

crap

ings

int

o et

hyl

acet

ate,

con

cent

rate

d an

d su

bjec

ted

to c

ombi

ned

gas

chro

mat

ogra

phy-

mas

s sp

ectr

omet

ry w

ith a

LK

B-9

000

com

bina

tion

gas

~ ch

rom

atog

raph

-mas

s sp

ectr

omet

er.

t C

onve

rsio

ns f

or m

ajor

and

min

or p

rodu

cts

@m

oles

in p

aren

thes

es)

are

estim

ates

bas

ed o

n ga

s ch

rom

atog

raph

y.

Tra

ce p

rodu

cts

wer

e fo

rmed

in

quan

titie

s 5

of l

ess

than

0.2

pm

ole.

$

Ret

entio

n tim

es a

re o

n a

6-ft

col

umn

with

3 p

er c

ent

EC

NSS

-M o

n G

as C

hrom

Q a

t th

e te

mpe

ratu

re i

ndic

ated

. B

!?

?.

§ Pr

etre

atm

ent

of r

ats

with

3-m

ethy

lcho

lant

hren

e ap

pear

ed t

o en

hanc

e 2-

and

Chy

drox

ylat

ion

of a

niso

le b

ut n

ot d

emet

hyla

tion

to p

heno

l. 0

II 4-

Fluo

roan

isol

e w

as c

onve

rted

with

Fen

ton’

s re

agen

t to

a m

ixtu

re o

f 4-

hydr

oxya

niso

le

(los

s fl

uori

ne)

and

2-hy

drox

y-4-

fluo

roan

isol

e.

A s

mal

l am

ount

of

s

4-fl

uoro

phen

o1 a

nd a

tra

ce o

f 3-

hydr

oxy~

-flu

oroa

niso

l~

wer

e al

so f

orm

ed.

ll T

he p

heno

lic p

rodu

cts

wer

e al

so c

onve

rted

top

heno

land

hy

drox

yani

sole

s by

red

uctiv

e de

halo

gena

tion

usin

g pa

lladi

um o

n ch

arco

al c

atal

yst,

hydr

ogen

gas

8 2

and

a tr

ace

of t~

ethy

iam

~e

in t

etra

hydr

ofur

an

for

16 h

r. T

he a

mou

nts

of p

heno

l an

d hy

drox

y~is

o1~

wer

e th

en a

naly

zed

by g

as c

hrom

ato~

aphy

-mas

s sp

ectr

a-

VJ

met

ry.

2982 JOHN DALY

with the ortho-hy-droxylated acetanilide, 6-hydroxy-2-fluoroacetanilide. This product, after isolation by paper chromatography, gave the expected mass spectrum (parent ion, m/e 169; base peak, m/e 127). The identity of this compound was confirmed by comparison with synthetic material. The other product appeared on the basis of R, value, color reactions and mass spectrum (parent ion, m/e 169; base peak, m/e 127) to be 4-hydroxy-2-fluoroacetanilide. Comparison of R, values, color reactions and mass spectrum with those of synthetic 4-hydroxy-2-fluoroacetanilide confirmed its identity.

3-Fluoroacetanilide. One major metabolite (R, 0.23) and a trace metabolite (R, 0.45) were detected by paper chromatography. The major product gave R, values, color reactions, ultraviolet spectrum (h,,, 250 rnp, fmax 8800) and mass spectrum (parent ion, m/e 169; base peak, m/e 127) identical to those of synthetic 4-hydroxy-3-fluoroacetani- lide. Based on ultraviolet spectroscopy, 1.4 f 0.1 pmoles of this product were formed in 15 min from 25 pmoles of substrate. The trace metabolite (< 0.1 pmole) had an R, value and gave color reactions with Folin’s reagent, diazotized sulfanilic acid and Gibbs’ reagent typical of an ortho-hydroxyacetanilide. It was, therefore, either 6-hydroxy-3-fluoroacetanilide or 2-hydroxy-3-fluoroacetanilide. The mass spectrum

exhibited the required parent ion, m/e 169 and base peak, m/e 127. 4-Fluoroacetanilide. Three metabolites (R,s 0.10, 0.18 and 0.48) were detected by

paper chromatography. These were identified by comparison to synthetic compounds as, respectively, 4-hydroxyacetanilide (mass spectrum; parent ion, m/e 151; base peak, m/e 109), 3-hydroxy-4-fluoroacetanilide (mass spectrum; parent ion, m/e 169; base peak, m/e 127) and 2-hydroxy-4-fluoroacetanilide (mass spectrum: parent ion, m/e 169; base peak, m/e 127).

2-Fluoroanisole. Phenolic metabolites were isolated by thin-layer chromatography and subjected to combined gas chromatography-mass spectrometry (Table 1). Two products were detected with retention times of 2.1 and 16.3 min. The mass spectra of these products (parent ion, m/e 142; base peak, m/e 127) identified them as ortho- orpara-hydroxyanisoles since meta-hydroxyanisoles exhibit, in addition to those peaks, a significant parent-30 peak in their mass spectra. The low retention time (2.1 min) of the minor component is typical of ortho-hydroxyanisoles, indicating that this product is 6-hydroxy-2-fluoroanisole. The major product with retention time of 16.3 min must be 4-hydroxy-2-fluoroanisole. This was confirmed by comparison to syn- thetic material. Approximate conversions were estimated as 0.2 and 4 pmoles, respec- tively, by gas chromatographic comparison with standard solutions of hydroxyanisoles or hydroxyfluoroanisoles.

3-Fluoroanisole. Phenolic products were isolated by thin-layer chromatography and analyzed by combination gas chromatography-mass spectrometry. Three metabolites were detected with retention times of 2.6, 3.8 and 4.8 min. The mass spectrum (parent ion, m/e 142; base peak, m/e 127) of the minor metabolite in conjunction with its low retention time of 2.6 min indicated it was one of the two possible fluorinated ortho- hydroxyanisoles. Its identity as 6-hydroxy-3-fluoroanisole was confirmed by compari- son (retention time, mass spectrum) with synthetic material. The trace metabolite with retention time of 3.8 min was identified as 3-fluorophenol (mass spectrum: parent ion and base peak, m/e 112) by comparison with synthetic material. The major metabolite with retention time of 4.8 min was identified as 4-hydroxy-3-fluoroanisole by compari- son with synthetic material.

Metabolism of aromatic substrates 2983

4-Fluoroanisole. Phenolic products were isolated by thin-layer chromatography and analyzed by combination gas chromatography-mass spectrometry. Three metabolites were detected with retention times of 2.3, 3.5 and 4.7 min. These were identified as 2-hydroxy+fluoroanisole (major), 4-fluorophenol (major), and 3-hydroxy-4-fluoro- anisole (trace) by comparison with synthetic compounds. With the latter compound, the mass spectrum with peaks at m/e 142, 127 and 112 had provided initial evidence for a mela-hydroxyanisole since the parent ion rather than the parent-15 peak was the base peak and a parent-30 peak, typical of meta-hydroxyanisoles, was present. This identification was confirmed by comparison with synthetic material.

RESULTS AND DISCUSSION

The metabolism in vitro of various acetanilides and anisoles with 3-methylcholan- threne-induced rat liver microsomes are presented in Tables 1 to 6. The semiquantita- tive results presented in the tables are in substantial agreement with earlier findings’ that the extent and position of enzymatic aryl hydroxylation with microsomal prepara- tions is correlated with the reactivity of the aromatic ring to electrophilic attack and that attack at a preferred ring position may be blocked by a halo or alkyl substituent.5 Steric factors cause the microsomal metabolism of aromatic rings to favor para- over ortho-hydroxylation. It is apparent in the present study that steric factors are indeed very important in determining the extent and direction of metabolism of aromatic substrates.

Acetanilides.” For example, the effect of ortho-substituents (F, Cl, Br, I, CH,, CF,) in blocking the normal para-hydroxylation of acetanilides was particularly striking (Tables 2 and 3). The extent of this effect appears to be much greater than would have been predicted from consideration of ring deactivation because of steric effects of the o&o-substituent on electron donation by the acetamido group. The effect of ortho-F, Cl, Br or CH, substituents on para-hydroxylation of acetanilides was similar. With 2-iodo or 2-trifluoromethylacetanilide, the blocking effect was greater and no para- hydroxylated or other metabolites could be detected. In the case of 2-methoxyacetani- lide, the ortho-substituent effect on para-hydroxylation was less pronounced.

No hydroxylation at the 5-position (para- to methoxy) was observed. The same ortho-substituent effect was also noted in acetanilides where the para-directed meta- bolism was by either alkyl hydroxylation (4-methylacetanilides) or by dealkylation (4-methoxyacetanilides). Thus, presence of a 2-methyl or 2-chloro substituent markedly reduced the hydroxylation of the 4-methyl group (Table 3) or the dealkylation of the 4-methoxy substituent in acetanilides. In both types of metabolisms, the 2-methyl group was more effective than the 2-chlorogroup in blocking attack of the para- substituent. It appears likely that steric effects of the ortho-substituent, which may involve the relative configuration of the acetamido group and the aromatic ring, prevent proper binding of acetanilides to the enzyme(s) and thus prevent metabolism by para-attack.

Meta-substituents either had no effect (CH,) on the para-hydroxylation of acetani- lides or caused only a slight reduction (F, Cl, Br, I, CF,, OCH,) in this metabolism. With 3_methoxyacetanilide, hydroxylation para- to the methoxy group yielded the 6-hydroxy derivative as a minor metabolite. With a disubstituted compound, such as

* For acetanilides, the terms orfho, metu and paru are referenced to the acetamido group, while for anisoles they are referenced to the methoxyl group.

TABLE

2. METABOLISM OF ACETANILIDE AND HALO-SUBSTITUTED ACETANILIDES WITH RAT LIVERMICROXIMES*

-

p-H

ydro

xyla

tion

m-H

ydro

xyla

tion

o-H

ydro

xyla

tion

%::

-

e_I_

__p

Subs

trat

e Pr

oduc

t C

onve

rsio

nt

Prod

uct

Con

vers

iont

R

fS

Prod

uct

Con

vers

iont

&

$ %

00

A

ceta

nilid

e J

2-Fl

uoro

acet

anili

de

3-Fl

uoro

acet

anili

de~

4-Fl

uoro

acet

anili

de

4-O

H

Maj

or

(2.6

1 T

race

M

ajor

(1

.41

Tra

ce

0.10

0.15

@

23

@IO

0.28

0.

23

0.11

0.23

0.33

0.

27

0.11

2-O

H

4-08

4-

OH

6-

OH

4(

2)-O

HII

4-O

H

(Los

s F)

4-

OH

4-

OH

3-O

H

Tra

ce

0.18

Z

-OH

Tra

ce

Tra

ce

Tra

ce

Tra

ce

0.38

a

0.62

0.

45

0.48

2-C

hlor

oace

tani

lide

3-C

hlor

oace

tani

lide§

4Chl

oroa

ceta

nilid

e$

2-B

rom

oace

tani

lide

3-B

rom

oace

tani

hde

4-B

rom

oace

tani

lide

4-Io

doac

etan

ilide

3,

5-D

ichl

oroa

ceta

nilid

e

4-O

H

“Los

‘osD

3-C

l -

4-O

H

4-O

H

4-O

H

(Los

s B

r)

4-O

H

4-O

H

Tra

ce

Tra

ce

Maj

or

(2.0

) T

race

(F

riz1

(Min

or)

Tra

ce

Maj

or

(2.0

) T

rm

Maj

or

(1%

0.

27

_**

6CW

OH

lI T

race

05

6

3-O

H

Tra

ce

0.22

2-

OH

T

race

04

4

W-O

Hll

Tra

ce

3-01

1 T

race

0.

32

2-O

H

Tra

ce

6-O

H

Tra

Ce

0.62

b

@62

B

0.63

3-O

H

Tra

ce

0.28

6-

OH

T

race

060

* In

cuba

tions

w

ere

carr

ied

out

at 3

7” f

or

15 m

in

with

10

ml

of m

icro

som

al

prep

arat

ion,

3.

5 m

l O

-5 M

Tri

s bu

ffer

, pH

8.

0, 1

5 pm

oles

A

TP,

50

/*m

oles

glu

- co

se &

phos

phat

e,

10 u

nits

glu

cose

6-

phos

phat

e de

hydr

ogen

ase,

2

mg

Tw

een

80 a

nd

25 p

mol

es

of s

ubst

rate

ad

ded

in O

-2 m

l ac

eton

e in

a f

inal

vo

lum

e of

15

ml.

Solu

tion

was

sat

urat

ed

with

NaC

l an

d ex

trac

ted

two

times

w

ith

equa

l vo

lum

e of

eth

yl

acet

ate.

T

he e

xtra

ct

was

dri

ed

Na,

SO,,

conc

entr

ated

in

uam

o an

d th

e pr

oduc

ts

isol

ated

by

pap

er

c~om

atog

raph

y an

d id

entif

ied

by m

ass

spec

trom

etry

us

ing

a H

it~bi

-Per

kin-

Elm

er

RM

UdD

m

ass

spec

trom

eter

. t

Con

vers

ions

in

pm

oles

pr

oduc

t ha

ve b

een

estim

ated

fr

om

uhra

viol

et

spec

tra

of t

he

maj

or

prod

ucts

&

mol

es

in p

aren

thes

is).

T

race

pr

oduc

ts

(0.2

@m

ole

or

less

) ha

ve b

een,

in

add

ition

, de

sign

ated

as

min

or

whe

n th

e am

ount

is

bar

ely

dete

ctab

le

(< 0

.1 t

imol

e).

$ &

valu

es,

Wha

tman

N

o.

1, b

enze

ne-a

cetic

ac

id-w

ater

(2

: 2:

1).

Man

y pr

oduc

ts

wer

e th

en

subs

eque

ntly

ch

rom

atog

raph

ed

on S

i02-

GF

thin

-lay

er

chro

mat

o-

plat

es

with

ben

zene

-met

hano

l-ac

etic

ac

id (

90:

16: 8

). 5

. 6 I

n m

ost

case

s R

~vaI

ues

wer

e co

mpa

red

with

au

then

tic

refe

renc

e co

mpo

unds

. G

ibbs

’ re

agen

t, Fo

hn’s

(p

heno

l)

reag

ent

and

diaz

otiz

ed

p-ni

troa

nifi

ne

wer

e us

ed t

o de

tect

ph

enol

s an

d in

the

cas

e of

the

Gib

bs’

reag

ent

to d

istin

guis

h be

twee

n or

rho-

an

d m

eta-

subs

titu-

te

d,

whi

ch

give

a p

ositi

ve

colo

r re

actio

n an

d pa

ra-s

ubst

itute

d ph

enol

s,

whi

ch

do n

ot.

§ Pr

evio

usly

re

port

ed

hydr

oxyl

atio

n?

with

ra

bbit

live

r m

icro

som

es

gave

sim

ilar

resu

hs

exce

pt

that

~c

hlor

oace

~nili

de

did

not

form

de

tect

able

am

ount

s of

4c

hlor

o-3-

hydr

oxya

ceta

nilid

e or

3-c

hlor

o-4-

hydr

oxya

ceta

nilid

e.

(See

tex

t.)

11 Iden

tific

atio

n of

thi

s ph

enol

ic

prod

uct

is t

enta

tive.

O

ther

po

ssib

le

isom

ers

in p

aren

thes

es.

7 N

o m

etab

olite

s de

tect

ed.

** C

hrom

atog

raph

y ca

rrie

d ou

t on

SiO

z-G

F th

in-l

ayer

ch

rom

atop

late

s,

benz

enee

thyl

ac

etat

e (4

9,

R,fo

r 3,

5-di

chlo

ro-4

_hyd

roxy

acet

anili

de,

0.54

. Id

entil

i-

catio

n by

red

uctiv

e de

halo

gena

tion

to 4

-hyd

roxy

acet

anili

de

usin

g pa

lladi

um

on c

harc

oal,

hydr

ogen

ga

s an

d a

trac

e of

tri

ethy

lam

ine

in t

etra

hydr

ofur

an

for

16 h

r.

TA

BLE

3. METABOLISMOF

AC

ET

AN

ILID

E

AND METHYL- ANDTRIFLUOKOMETHYL-SUBSTITUTED

ACETANILIDES W

ITH

R

AT

LIVER MICROSOMES*

p_H

ydro

xyla

tion

m-H

ydro

xyla

tion

o-H

ydro

xyla

tion

Subs

trat

e _.

.___

__.

___

- __

__

.~.

- --

_.

Prod

uct

Con

vers

ion?

R

fZ

Prod

uct

Con

vers

iont

R

,::

Prod

uct

Con

vers

iont

R

,::

F

Ace

tani

lideS

4-

OH

M

ajor

O

*lO

2-

OH

T

race

0.

38

(2.6

) H

2-M

ethy

lace

tani

iide

4-O

H

Tra

ce

0.25

6-

OH

T

race

05

.5

B

3-M

ethy

lace

tani

i~de

§ 4-

OH

M

ajor

o-

19

6(2)

-OH

i/

Tra

ce

o-41

%

(2

5)

g:

4-M

ethy

lace

tani

lide§

Ib

CH

;?O

H

Maj

or

0.16

3-

OH

T

race

0.

28

2-O

H

Tra

ce

0.45

g

(1.6

) (M

inor

) 2,

4-D

imet

hyla

ceta

nilid

e 4-

CH

zOH

T

race

0.

19

g.

2-C

hlor

o-4-

met

hyla

ceta

nilid

e 4-

CH

zOH

M

ajor

0.

38

05)

2-T

rifl

uoro

met

hyla

ceta

nilid

e’lf

k

3-T

rifl

uoro

met

hyla

ceta

nilid

e 4-

OH

M

ajor

0.

23

i8

(1.9

) 4-

Tri

fluo

rom

ethy

lace

tani

lide~

*,t,$

,§,

I/, q

See

foo

tnot

es

to T

able

2.

2986 JOHN DALY

3,5dichloroacetanilide, steric effects may well have been responsible for the marked reduction in the extent of para-hydroxylation.

Pura-substituents either reduced the extent of para-hydroxylation of the acetanilides to a trace metabolism (F, Cl, Br) or completely blocked this route of metabolism (I, CH,, CFB, OCH,). With certain par&substituted acetanilides, hydroxylation meta-

to the acetamido group was now noted either as a trace (F, Cl, Br, CHJ, I) or minor (OCH,) metabolic route. With 4-methyl- or 4-methoxyacetanilides, oxidative attack on the para-substituent was the major metabolism.J Pura-hydroxylation of 4-halo- acetanilides was accompanied by loss of halogen (F, Cl, Br) or by migration of halogen (Cl), but in all cases these were quantitatively insignificant metabolisms. The migration of halogen during hydroxylation of 4-chloroacetanilide and 4-bromoacetanilide with benzypyrene-induced rat liver microsomes has been reported6 and the conversions reported were much higher than those obtained in the present study. Migration of halogen, however, in both the present study and the earlier report was found to be the least significant metabolic route. Meta- and ortho-hydroxylation and loss of halogen during paru-hydroxylation were the major pathways. The results with 4-chloroacetani- lide in both studies are in at least qualitative agreement as to the ratio of metabolites which are formed. In another study5 using normal rabbit liver microsomes and 4-chloroacetanilide, only para-hydroxylation with loss of halogen could be detected. The mechanism of oxidative dehalogenation with para-haloacetanilides presumably involves formation of an oxidized intermediate which undergoes reductive loss of halogen. Such a mechanism has been proposed for the conversion ofpara-halopheny- lalanines to tyrosine.7

orfho

meia

, I. CF3

NHAc

NHAc NHAc

CH3

t

OCH,

t

FIG. 1. Effect of substituents on hydroxylation of acetanilides with rat liver microsomes. (bold arrow), major metabolism; +, minor metabolism; and ----+, trace or no metabolism.

TA

BL

E 4. M

ET

AB

OL

EM

O

F A

CE

TA

NIL

WE

A

ND

M

ET

HO

XY

-SU

BST

ITU

TE

D

AC

ET

AN

ILW

ES

W

ITH

R

AT

LIW

R M

KR

~SO

ME

S~

Subs

trat

e

Ace

tani

lides

p-H

ydro

xyla

tion

m-H

ydro

xyla

tion

o-H

ydro

xyla

tion

- .~

Pr

oduc

t C

onve

rsio

nt

RfS

Pr

oduc

t C

onve

rsio

nt

R,S

Pr

oduc

t C

onve

rsio

n?

RJS

Z=

4-O

H

2-M

etho

xyac

etan

ilide

4-

OH

/(

Maj

or

(2.6

) M

inor

(@

3)

2-O

H

Tra

ce

O-3

8 6 s

2-O

H

Tra

ce

0.36

g

(des

met

hyl)

F

6-O

H

Maj

or

0.50

3

(0.8

) s

3-O

H

Tra

ce

0.14

&

OH

M

inor

0.

37

g (~

~hY

1)

(O-2

) 8

Min

or

Oal

O

2-O

H

Tra

ce

0.30

B

0

(0.2

) 2 a :: D

1 H

3-M

etho

xya~

tani

lide

4-M

etho

xyac

etan

ilide

§

2-M

ethy

l-Q

-met

hoxy

acet

anili

de

2-C

hlor

o4m

etho

xyac

etan

ilide

4-O

H

4-O

H

(Des

met

hyl)

4-

OH

(D

esm

ethy

l)

4-O

H

(Des

met

hyl)

Maj

or

t0.g

) M

ajor

(0

%

Tra

ce

Min

or

W2)

0.22

q

@II

0.29

O-2

6

** f*

z*

f Se

e fo

otno

tes

to T

able

2.

II T

his

prod

uct

was

ide

ntif

ied

by c

onve

rsio

n to

2,4

-dim

etho

xyac

etan

ilide

w

ith d

iazo

met

hane

. A

naly

sis

by T

LC

(si

lica

gel,

benz

ene,

eth

yl a

ceta

te,

CH

C&

, 2:

1: 1

) dis

tingu

ishe

d 2,

4_di

met

hoxy

acet

anili

de (

R,0

*36)

fro

m 2

,5-d

imet

hoxy

acet

anili

de

(R,0

*45)

. 7

The

sepa

ra-s

ubst

itute

d ph

enol

s ga

ve a

pos

itive

Gib

b’s

reac

tion.

2988 JOHN DALY

Ortho-hydroxylation of acetanilides was observed in the present study as a signifi- cant metabolism only with 2- and 3-methoxyacetanilide. The results of these studies on metabolism of substituted acetanilides are presented in Fig. 1 and Tables 24.

Anisoles. The pathways of metabolism of anisole with microsomal preparations from rats that have been induced with 3-methylcholanthrene are by para-hydroxyla- tion (major), ortho-hydroxylation (minor) and by 0-demethylation (minor). With normal rat or rabbit’ microsomes, the major metabolisms are paru-hydroxylation and 0-demethylation.

Most ortho-substituents (F, Cl, Br, I, CH,) greatly reduced metabolism by O-de- methylation but had little effect on para-hydroxylation. The ratio of ortho- to para-

hydroxylation, however, tended to increase from 0.2 in anisole and 0.2 in 2-fluoroani- sole to 0.25 in 2-methylanisole, 0.5 in 2-chloroanisole, 1.0 in 2-bromoanisole, and 1.5 in 3-iodoanisole, in spite of the fact that one less ortho-position was available for hydroxylation in these ortho-substituted anisoles. If this is because of the same steric effect which was noted in the ortho-substituted acetanilides, it is much less effective with the ortho-substituted anisoles in blocking metabolism by paru-hydroxylation.

Me&z-substitution with F, CH3, or I substituents had little effect on ortho- or para-

ring hydroxylation of anisoles. However, with Cl or Br substituents in the meta-

position, ortho-hydroxylation (para- to the halogen) was greatly enhanced with a con- comitant decrease in paru-hydroxylation. This is in contrast to acetanilide metabolism where F, Cl, Br, I, CH,, and 0CH3 substituents in the meta-position had little effect on para-hydroxylation. The explanation of this rather remarkable effect of meta-Cl

or Br substituents in the anisole series might involve steric interactions which result

para

ortho

meta

R= t I, Br,I

,*’ I

t R = Cl, Br

OCH,

R = F. Cl. Br,

,’

t t

CH3

t

t R =F,1

FIG. 2. Effect of substituents on the hydroxylation of anisoles with rat liver microsomes. (bold arrow), major metabolism; +, minor metabolism; and ----3, trace or no metabolism.

TABLE ~.M~ABOLISMOF

ANISOLE AND METHYL-SUBS~TUTED ANISOLESWITSI RAT LIVER MICR~S~MES*

Subs

trat

es

p-H

ydro

xyla

tion

~-H

ydro

xy I

at~o

n o-

Hyd

roxy

latio

n D

emet

hyla

tion

_ -~

~-,_

~I-_

C

omer

- R

eten

tion

Con

ver-

R

eten

tion

Con

ver-

R

eten

tion

Con

vet-

R

eten

tion

Prod

uct

sion

t tim

e Pr

oduc

t si

ont

time

Prod

uct

sion

t tim

e si

ont

time

(148

”):

(148

”)t:

(148

”)$

(148

”)::

Ani

sob

$

2-M

ethy

lani

sole

3-M

ethy

lani

sole

4-M

ethy

lani

sote

2,3-

Dim

ethy

lani

sole

2,4-

Dim

ethy

lani

sole

2,5-

Dim

ethy

lani

sole

2,6-

Dim

ethy

Iani

sole

3,4-

Dim

ethy

lani

sole

3,5-

Dim

ethy

lani

sole

3-M

ethy

l-4-

chlo

roan

isol

e

4-O

H

4-O

H

4-O

H

Maj

or

(4)

Maj

or

(4)

Maj

or

(5)

4.8

min

6.2

min

5.6

min

4-O

H

Min

or

76 m

in

(@8)

4-O

H

4-O

H

Maj

or

(3)

Tra

ce

6.3

min

7.0

min

3-O

H

Min

or

(0.2

) 5.

9 m

in

5-O

H

Maj

or

7.7

min

(f

J.8)

3-O

H

Tra

ce

5.3

min

4-O

H

Maj

or

(1.5

) 5.

6 m

in

S-O

H

Tra

ce

~7

min

Z-O

H

2-O

H

6-O

H

2-O

H

&O

H

6-O

H

6-O

H

&O

H

2-O

H

6-O

H

Min

or

(o-8

) M

inor

(0

.8)

Min

or

(1)

Min

or

(0.6

) M

inor

(0

.2)

Min

or

04)

Tra

ce

Maj

or

(2)

Tra

ce

Min

or

(1)

I.2

min

1.3

min

I.6

min

1.4

min

25 m

in

26 m

in

25 m

in

3.2

min

2.9

min

5.1

min

Min

or

(0.2

) T

race

Tra

ce

Maj

or

(3)

Tra

ce

Tra

ce

Tra

ce

Min

or

(0.2

) T

race

(0*2

>

Maj

or

(4)

1.5

min

1.3

min

E

%

1.

5 m

in

&

B

1.8

min

3

2.8

min

f?

k!

2.7

min

R

3.4

min

3.0

min

6.8

min

** t*

$*

§ Se

e fo

otno

tes

to T

able

I.

~onp

heno

lic

met

abot

ites

wer

e fo

rmed

by

hydr

oxyl

atio

n of

rin

g-m

ethy

l su

bstit

uent

s in

ail

case

s bu

t th

ese

mct

abol

ites

wer

e no

t ex

amin

ed a

fter

sep

arat

ion

by t

he t

hin-

laye

r ch

rom

atog

raph

y.

2990 JOHN DALY

in differing modes of binding only with Cl and Br. Anisoles containing meta-substitu- ents (F, Cl, Br, I, CHJ) exhibited greatly decreased metabolism by 0-demethylation.

In 3,5_dimethylanisole, para-hydroxylation between the methyl groups was still a major metabolism. In other dimethylanisoles, para-hydroxylation was blocked in 2,4-, and 2,6-dimethylanisoles, reduced in 2,3_dimethylanisole and unaffected in 2,5- dimethylanisole.

Para-substitution (F, Cl, Br, CH,) in the anisole series blocked para-hydroxylation of the ring and markedly stimulated 0-demethylation. This enhanced 0-demethylation was blocked by a second substitutent ortho- to the methoxy group (2,4-dichloroanisole, 2,4_dimethylanisole) and, in one case, by a second substituent meta- to the methoxy group (3,4-dimethylanisole). In another case of 3,4+ubstitution, 3-methyl-4-chloro- anisole, 0-demethylation was still a major metabolism. Ortho-hydroxylation of anisole appeared to be stimulated by the presence of a paru-halogen substituent (F, Cl, Br, I) but not by a paru-methyl group. No migration or loss of F, Cl or Br substituents from the para-position was observed in the anisoles in distinction to the metabolism of 4-haloacetanilides (see above). Me&z-hydroxylation was observed in anisoles which contained Cl, Br, I, or CH, substituents in the para-position. The metabolism of halo- and methyl-substituted anisoles is summarised in Fig. 2 and Tables 1, 4 and 5. With many of the isomeric halogenated hydroxyanisoles, separation by GLC or TLC was difficult and final identification often necessitated conversion of the products to ortho-,

mefa- and para-hydroxyanisoles by reductive dehalogenation (Table 1, footnote I). The mass spectra of hydroxyanisoles were also useful in identification of the isomeric hydroxyanisoles, since the ortho- and para-isomers tend to lose methyl to form the most intense peak in the mass spectrum. This loss is not as prominent with meta-

hydroxyanisoles and an alternate fragmentation involving loss of OCH, is present. The results obtained with microsomal metabolism of 4-iodoanisole were unexpected.

In contrast to the other 4-haloanisoles, formation of 4-hydroxyanisole with loss of iodide was observed. In addition, 3-hydroxyanisole was a major metabolite as were 2-hydroxy-4-iodoanisole and 3-hydroxy-4-iodoanisole (Fig. 3). Incubation of iodin- ated hydroxyanisoles with microsomes did not result in deiodination which demon- strates that the 3-hydroxy-4-iodoanisole was not an intermediate in the formation of

I)CH3 OCH,

Q - Q”

I I

+ OH

I

+

OH

FIG. 3. Metabolism of iodoanisole with rat liver microsomes. The products are formed in a ratio of 3 : 1 : 3 : 2 : 2 respectively.

Subs

trat

e Pr

oduc

t C

onve

rsio

nt

W

Col

or$

Gib

bs3

2-N

itroa

niso

le

2-N

itro+

hydr

oxya

niso

le

Tra

ce

0.26

Fa

int

gree

n 2-

Nitr

o-6-

hydr

oxya

niso

le

Tra

ce

035

Fain

t bl

ue

5

2-A

min

oani

sole

M

inor

0.

50

Lig

ht

purp

le

2-N

itrop

heno

l M

ajor

08

6 Y

ello

w

i 3 3-

Nitr

oani

sole

3-

Nitr

o-6-

hydr

oxya

niso

le

Min

or

0.22

Y

ello

w

B?

3-A

min

oani

sole

M

ajor

0.

32

Blu

e-pu

rple

e

3-N

itrop

heno

i M

inor

04

5 L

ight

ye

llow

L

ight

gr

een

i;

cl-N

itroa

niso

le

~Nitr

o-2-

hydr

oxya

niso

le

Maj

or

0.26

L

ight

ye

llow

L

ight

gr

een

B

c)

~~itr

ophe

noi

Maj

or

0.35

Y

eilo

w

4-A

min

oani

sole

T

race

04

6 B

fue-

purp

le

$ %

* In

cuba

tions

ca

rrie

d ou

t as

des

crib

ed

in T

able

1.

Eth

yl

acet

ate

extr

acts

ch

rom

atag

raph

ed

on t

hin-

laye

r ch

rom

atop

late

s,

SiO

a-G

F,

benz

enee

thyl

ac

etat

e,

2

95:s

. D

etec

tion

with

Gib

bs’

or F

olin

’s

reag

ent.

Prod

ucts

w

ere

extr

acte

d fr

om

plat

e in

to

ethy

l ac

etat

e fo

r es

timat

ion

by u

ltrav

iole

t sp

ectr

osco

py

and

iden

tifi-

ca

tion

by m

ass

spec

trom

etry

. t

Maj

or

prod

uct,

> 1

pm

ole;

tr

ace

prod

uct,

<: 0

.1 p

mol

e.

z O

rtho

- an

d pa

ra-n

itrop

heno

ls

are

yello

w

com

poun

ds,

whi

ch

give

no

colo

r w

ith

Gib

bs’

reag

ent.

2992 JOHN DALY

3-hydroxyanisole. At the present time, the mechanism of the novel formation of 3- hydroxyanisole from 4-iodoanisole is unknown. This type of reaction was not observed with 4-iodoacetanilide. The only report of such a reaction is the conversion of 3-fluoroaniline to 4-hydroxyaniline with microsomes.8

Nitroanisoles. The metabolism of the three nitroanisoles was examined (Table 6). Ring hydroxylation, reduction of the nitro group to an amine and dealkylation were observed and the fractionation between these pathways varied with the substitution pattern. The major metabolism with 2-nitroanisole was dealkylation. The principal metabolism of 3nitroanisole was reduction to 3-aminoanisoles, but ring hydroxylation para- to the nitro group and dealkylation were also observed as minor pathways. 4- Nitroanisole underwent both dealkylation and ring hydroxylation, ortho to the methoxy group, as major metabolisms (see Fig. 4).

OCH,

ON” - tiNoz + GNHz + T”AC,‘_N”~~“o~NfROXY-

MAJOR MINOR

0CH3

ONO,-- 6N0, + GNH, + HoaNo2

MINOR MAJOR MINOR

OCH,

(p-$+6+@

NOo NOz NHz NO2

MAJOR TRACE MAJOR

FIG. 4. Metabolism of nitroanisoles with rat liver microsomes.

The results presented in this paper further delineate the complex of reactions cata- lyzed by drug-metabolising enzymes and indicate the importance of both electronic and steric factors in determining the metabolism of aromatic substrates. It is note- worthy that methyl or halogen migration has not been observed as a major metabolic route with any of these substrates. This is in contrast to the results obtained with 4- methyLg and 4-chlorophenylalanine” with the highly specific enzyme, phenylalanine hydroxylase, where migration of the substituent is the major metabolic pathway. As yet there is only one report” of the “NIH Shift” of halogen as a relatively major metabolism during oxidation of an aromatic substrate by nonspecific microsomal enzymes. In all other cases4’ 5, 12* l3 the migration of halogen during metabolism by nonspecific enzymes has been a minor route or has not been observed. The significance of these observations and their relation to the role of arene oxides in the hydroxylation of aromatic substrates are under investigation.14-l6

Acknowledgement-The author wishes to acknowledge the excellent technical assistance of Mrs. Marci a Phyillaier.

Metabolism of aromatic substrates 2993

REFERENCES

1. J. DALY, D. JERINA and B. WITKOP, Archs Biochem. Biophys. 128, 517 (1968). 2. J. DALY and D. JERINA, Archs Biochem. Biopbys. 134, 226 (1969). 3. G. M. K. HUGHES and B. C. SAUNDERS, .I. ihem. Sot. 4360 (1954). 4. J. DALY. D. JERINA. J. FARNSWORTH and G. GIJROFF. Archs. Biochem. Biodws. 131,238 (1969). 5. J. W. DALY, G. G&ROFF, S. UDENFRIEND and B. WIT~OP, Biochem. Pharmk: 17,3i (1968). 6. U. ULLRICH, J. WOLF, E. AMADORI and H. STAUDINGER, Hoppe-Seyler’s Z. physiol. Chem. 349,85

(1968). 7. S. KAUFMAN, Biochim. biophys. Acta 51,619 (1961). 8. J. RENSON and U. BOIJRDON, Archs int. Pharmacodyn. Ther. 171,240 (1968). 9. J. DALY and G. GUROFF, Archs. Biochem. Biophys. 125, 136 (1968).

10. G. GUROFF, K. KONDO and J. DALY, Biochem. biophys. Res. Commun. 25, 622 (1966). 11. D. M. FOULKES, Nature, Land. 221, 582 (1969). 12. J. K. FAULKNER and D. WOODCOCK, J. them. Sot. 1187 (1965). 13. E. W. THOMAS, B. C. LOUGHMAN and R. G. POWELL, Nature, Lond. 204,884 (1964). 14. D. JEIUNA, J. DALY, B. WITKOP, P. ZALTZMAN-NIRENBERG and S. UDENFRIEND, Archs Biochem.

Biophys. 128, 176 (1968). 15. D. JERINA, J. DALY and B. WITKOP, J. Am. them. Sot. 90, 6523 (1968). 16. D. JERINA, J. DALY, B. WITKOP, P. ZALTZMAN-NIRENBERG and S. UDENFRIEND. .I. Am. them. Sot.

90,6525 (1968).