Electrooxidative simulation of stereoselectivity in microsomal allylic hydroxylation

Tetrahedron Printed in

Letters,Vol.25,No.l,pp Great Britain

91

ELECTROOXIDATIVE

-

SIMULATION

IN MICROSOMAL

of Synthetic

Kyoto

University, Toshiki

HYDROXYLATION’

Shone*

Chemistry, Yoshida,

Toda

Research

II,

Faculty

Sakyo,

606, Japan

Oshino

Schering

Y odogawa,

of Engineering,

Kyoto

and Nozomu Nihon

2- 6- 64, Nishimiyahara,

.OO Ltd.

OF STEREOSELECTIVITY

ALLYLIC Tatsuya

Department

0040-4039/84 $3.00 + 01984 Pergamon Press

94,1984

K .K.,

Osaka

532, Japan

A comparison of the stereochemistry of liver microsomal y-hydroxylation of some cyclic ~1,R-unsaturated ketones with that of electrochemical y-acetoxylation of the corresponding dienol esters and with that of peracid oxidation of the dienol esters has been carried out. One of the important monooxygenase ated

processes

in metabolism

is oxidation

catalyzed

by the cytochrome

P-450

system.

Secondary and tertiary amines and amides are oxidized to give N-dealkyl2 while the oxidation at saturated aliphatic carbon atoms leads to 3 of hydroxylated metabolites.

amines and amides,

the formation We have microsomal tools

suggested

to simulate

that an electron transfer process of amines and amides 4,5 and electrooxidative

the microsomal

On the other proceed

previously

N-dealkylation

hand,

with homolytic

microsomal

hydroxylation

abstraction

at aliphatic

by an iron -oxygen

on the stereochemistry of enzymatic hydroxylation at primary, 10 the configuration of hydroxylated products has been

atoms,

controlling

stereoselectivity

in the liver

method

is one of the best

4,6

N-dealkylation.

hydrogen

is involved

of hydroxylation

still remains

carbons

has been

suggested

to

species.

7 In a number of studies 8 9 secondary, and tertiary carbon

discussed, 11 obscure.

whereas

the mechanism

Previously, we have shown that anodic allylic acetoxylation proceeds with a remarkable stereo12 selectivity. Since both the electrode reactions and the enzymatic reactions have similar heterogeneous

characters,

y-hydroxylation

we compared

of cyclic

tion of the corresponding of the dienol

esters

~1,S-unsaturated

get a stereochemical

dienol

(eq.3)

ketones

in the present

CI,R-unsaturated esters

to find catalyzed

insight

‘I Microsomes

(eq.

study

ketones

(eq.

the stereoselectivity

2) and with that of peracid

an efficient

method

by the cytochrome

to simulate

P-450 catalyzed

b

aq,-

(‘) OH

0

91

oxidation

the metabolism

P- 450 monooxygenase

into the cytochrome

&@

of liver

1) with that of electrochemical

microsomal y-acetoxyla-

(y-hydroxylation) of steroidal

system,

and also to

hydroxylation.

CH$OH@

o@

(2) OCOCH3

92

The were

substrates

used in the present study 13 (I-7) and their analogs

A 4- 3-ketosteroids

shown in Table

1.

The microsomal

hydroxylation

of the cyclic

~1,B-unsaturated

ketones

was carried

(l-3)14

(I-7)

under

glucose ase,

conventional

6-phosphate,

0.1 M Tris

protein/ml,

0.5mM NADPH,

and substrate 7).

room temperature 2 Flmol

potassium

istry

solution

y-acetoxylated

compounds21 The

results

to simulate

gave

hydroxylation complex

acid,

the reaction

extracted

with ether.

oxidation

acetate

mixture

electrode.

and 10 mM

was poured

The enol acetates

into were

B-isomers

produced

by microsomal

oxidation,

and y-hydroxylated products obtained 19 with the reference and HPLC”

TLC

of y-substituted

with peracid

products

were

determined

by

by HPLC.

1.

examined,

microsomal

y-hydroxylation

and anodic

almost exclusively. between

microsomal

y-hydroxylation

and anodic

oxidation,

a much greater

stereoselectivity

was observed

in

oxidation. suggest

that the present

of some steroidal

electrooxidative

a,6-unsaturated

method

ketones

is the most effective

catalyzed

by liver

cytochrome

system.

of studies, with

step

viously

that the initial

formed

from one-electron

ation known

by anodic

the microsomal hydroxylation at C-6 position of steroids has been 22 as same as the present study. The stereoselectivity of

B-orientation

may be determined

or at some

of 0.5 mM dienol

metabolites

of the stereoselectivity

strongly

to proceed

at 2.0 V vs. SCE with a platinum

was observed.

the metabolism

In a number reported

in acetic

by comparative

ratios

the electrochemical

P-450 monooxygenase

potential

are shown in Table

y-acetoxylation

results

10 min at 37OC, the reaction was termi17 of dienol acetates was carried out at

the solution

were

4, 5, 6,

for

oxidation

through

to all the compounds

Good accordance

These

anodic

y-Hydroxylated

detected

and the isomer

Comparing

dehydrogen-

3. Omg of

acid in refluxing ether for 16h to compare the stereochem18 oxidations. The ethereal solution was washed with sodium bi-

obtained

were

y-acetoxylation

(3)

incubated

electrolyte

and anodic

With respect

(2)

B-phosphate

containing

4 mM

with perbenzoic

products

following

(1)

of glucose

suspension

contained

n

and the products

and evaporated.

oxidation

0.5 unit/ml

mixture

of 50 UM (1, 2, and 3) or 1 FM (JH-labeled

of controlled

was passed

The incubation

microsomal

were

The

as a supporting

with microsomal

chemical

the mixtures

conditions

chemically

dried

(pH 7.4))

of methanol.

under

acetate

carbonate,

(3)

o ha

16

conditions.

in the concentration

of electricity

sodium bicarbonate also oxidized

C6H5COOOH

c ti H3

OH

5mM MgC12,

buffer

After

by the addition

After

centrifugal

hydrochloride

and 14C-labeled nated

c

R>

out with incubation 15 with rat liver microsomes

of the substrates prepared

“\

after active

either

species

oxidation

in the anodic

explained

by a selective

allylic

in the anodic

acetoxylation

adsorption

is formed.

reaction

of cytochrome

On the other allylic

of the substrate.

as the Kharasch-Sosnovsky

observed

at the step of formation

the complex

l2

substitution Compared

(cis/trans=O.

of methylcyclohexenes

of methylcyclohexenes

hand,

P-450-substrate

we have

reaction

with chemical

proposed

is a cation allylic

2-O. 3)) a remarkable

pre-

radical

benzoyloxyl-

cis-selectivity

(cis I trans=2.8- 3.4) has been

to the anode

from the less

sterically

93

Table

Stereoselectivity in Microsomal Hydroxylation, Acetoxylation and Chemical Oxidation ----__

1.

Product Substrate

Electrooxidative -

Ratio (B -isomer/n-isomer)a _--.--

Chemical Method

Electrooxidative Method

Microsomal Method

0 co

1

14.1 +_ 1.2

13.9 +_0.9

3.0 2 1.6

0

2

7.3 2 1.6

7.2 5 0.4

3.2 k 0.8

3

13.5 2 2.1

13.0 + 0.8

2.9 2 0.6

4

11.8 + 2,l

12.3 i: 0.9

2.8 + 1.1

5

12.1 2 2.6

13.3 2 1.7

3.9 ir 0.7

6

8.4 ?r 2.7

10.3 2 1.1

2.9 2 1.0

10.8 + 1.9

9.0 + 1.8

3.5 t 0.5

JP

0cb

“p

aAnodic oxidation, peracid oxidation and the incubation of substrates with liver microsomes of a rat were carried out three times for each substrates. The isomer ratios (B-isomer/~-isomer) of v-substituted products were given as means f SD.

hindered

side,

namely

tion of an acetoxyl methyl

group

the reverse

group

to give

to the allylic

predominantly

Since

the anodic y-acetoxylation 23 lar mechanism, the S-oriented lar steric

interaction

selectivity

between

side

position

acetates

stereoselectivity

to the methyl

of allylically has been observed

substrate-nucleophile.

y-hydroxylation

group,

followed

from the side of solution,

the cis-isomer

of dienol

of electrodemicrosomal

with respect

and anodic

proposed

the good

y-acetoxylation

the side of the

methylcyclohexenes.

to proceed

in the present Thus,

that is,

acetoxylated

by the introduc-

study

through

the simi-

suggests

the simi-

accordance

of the stereo-

shown in Table

1 may

94

suggest

that the similar

P-450 enzyme

is involved

tion of steroidal in the chemical

stereoselective

of the substrates

in the stereoselectivity

~1,B-unsaturated oxidation

approach

ketones,

in a homogeneous

while

determining such a control

solution

using

to the surface

step of the microsomal of stereochemistry

References

4. 5.

6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.

19.

20.

21.

22.

23.

y-hydroxylais not expected

peracid.

Acknowledgment. One of the authors (T.S.) wishes to thank Science, and Culture, Japan, for the Grant-in-Aid for Special Project 56109011, and 57102010).

1. 2. 3.

of cytochrome

the Ministry of Education, Research (1) (No. 521313,

and Notes

Electroorganic Chemistry. 78. R.E. McMahon, H. W. Culp, and J.C. Occolowitz, J. Am. Chem. Sot., 91. 3389 (1969). I. B joerkhem , “Cytochrome P-450,” ed by R. Sato, T. Omura, Academic Press, New York (1978), p. 645. T. Shono, T. Toda, and N. Oshino, J. Am. Chem. Sot., l&4, 2639 (1982). As for the electron transfer mechanism of the cytochrome P-450 catalyzed oxidation of amines, see also: (a) R.B. Silverman, S.J. Hoffman, and W .B. Catus, III, J. Am. Chem. Sot., 102, 7126 (1980); (b) P. Shannon and T.C. Bruice, ibid., 103, 4580 (1981). T. Shono, T. Toda, and N. Oshino, Drug Metab. Dispos., 9, 481 (1981). R.C. Blake, II and M.J. Coon, J. Biol. Chem., 256, 12127 (1981). S. Shapiro, J.U. Piper, and E. Caspi, J. Am. Chem. Sot., 104, 2301 (1982). I. Bjoerkhem, Eur. J. Biochem., 51, 137 (19’75). S. Burstein and M. Gut, Adv. Lipid Res., 9, 291 (1971). C .E. Castro, ed by R . Sato, T . Omura, Academic Press, New York “Cytochrome P-450,” (1978), p. 547. T. Shono and A. Ikeda, J. Am. Chem. Sot., 9rl, 7892 (1972). 3H- And “C-labeled and unlabeled substances were supplied from Schering AG (Berlin, GFR). The analogs of A4-3-ketosteroids were prepared from the reaction of 2--substituted cyclohexanone with corresponding vinyl ketone. Liver microsomal fractions were prepared from the liver homogenates of male Sprague-Dawley rats. Microsomal protein was determined by the method of Lowry. P. Mazel, “Fundamentals of Drug Metabolism and Drug Disposition ,” ed by B .N. La Du, H.G. Mandel, E .L. !ITay, Williams & Wilkins, Baltimore (1971)) p. 527. Dienol acetates were prepared from the corresponding a, P-unsaturated ketones. The peracid oxidation of dienol ether prepared from a cyclic a,R-unsaturated ketone has been described to give y-hydroxylated products: H .L. Holland and B . L. Auret , Can. J. Chem. , 53, 2041 (1975). TLC was carried out on silica gel plates with a solvent system of ethyl acetate-hexane and visualized under UV (254 nm) . Rs values varied with the substrates are as follows: dienol acetates; a,~‘!-unsaturated ketones; 1.0; a-isomer of y-acetoxy derivative; 1.1-O. 9, a-isomer of y-acetoxy derivative; 0.9-O. 8, a-isomer of y-hydroxy derivative: 0.5-O. 4, cl-isomer of y-hydroxy derivative; 0.5-0.3. HPLC was monitored with UV absorbance at 235nm or with measurements of radioactivity of effluents collected at 0.5 min intervals, with a mobile phase containing 0.5- 1% methanol in dichloromethane by using a Zorbax SIL column. The retention volumes of B-isomers were always smaller than those of a-isomers. The reference compounds for microsomal y-hydroxylation were prepared in large scale by Using isolated perfused rat livers: T. Toda and N. Oshino, Drug Metab. Dispos. , 9, 108 (1981). The allylic hydroxylated metabolites in the perfusate were separated by TLC. NMR (CDCl3) for y-hydroxylated derivatives of 1-T (6 -values varied somewhat with the substrates) : B-isomer, 6 5.80-5.87 (s, 1, cl-CH), 4.32-4.39 (t, 1, y-CH) and a-isomer, 6 6.18-6.20 (d, 1, a-CH), 4.10-4.40 (m, 1, y-CH). The reference compounds for anodic y-acetoxylation were obtained by preparative electrolysis of dienol acetates. NMR (CDC13) for y-acetoxylated derivatives of l-7; 6 5.90-5.97 (s, 1, a-CH), 5.40-5.45 (t, 1, y-CH), 2.04-2.06 (s, 3, acetate). M. J. Coon, S.D. Black, D.R. Koop, E.T. Morgan, and G.E. Tarr, “Microsomes, Drug Oxidaed by R. Sato, R. Kato, Japan Scientific Societies Press, Tokyo tions and Drug Toxicity,” (1982), p. 13 and p. 35. The initiation process of this anodic oxidation has been proposed to be the electron transfer from dienol esters to anode yielding cationic species: T. Shono, I. Nishiguchi, S. Kashimura, and M. Okawa, Bull. Chem. Sot. Jpn., 51, 2181 (1978). (Received

in

Japan

29

September

1983)