Biochemical Pharmacology, Vol. 25, pp. 993

996. Pergamon Press, 1976. Printed in Great Britain

OBSERVATIONS AND

ON

BIOLOGICAL

THE

DIFFERENTIAL

ACTIVITY

OF

THE

METABOLISM

OPTICAL

ISOMERS

OF

CYCLOPHOSPHAMIDE

P. J.Cox, Chester Royal

P. B. Farmer,

Beatty

Cancer

Research

Hospital,

M. Jarman* Institute

Institute,

Fulham

and M. Jones

Road,

of Cancer

London

SW3

Research:

6JB,

England

and W. J.Stec Centre

for Molecular

and Macromolecular 90-362

Cyclaphosphamide chiral

phosphorus

(.I ), a widely

atom and optical

CH2

--

NH

Lodz,

used anticancer isomers

‘P

drug,

are therefore

\

0”

recent

synthesis

stereoselective antitumour Total

was laevorotatary,

(5) of the optical metabolism

effects 24-hour

of racemic

samples

(1 g).

cyclophosphamide

was recovered

(Merck,

Kieselgel

Kieselgel

G) using

in Table

I show that

metabolism

*

Biology

in animal

were collected

60) with

sample

two of the sumples

of the dextrorotatary

Unit,

molecule

because

of metabolism

(1,2)

use of the drun have hitherto

racemic

(?I)

that

(4) that,

of synthesis

following

cyclophosphamide

stereoselectjve has allowed

produced (3).

the by

The

the administration

to patients,

metabolism

in man and a demonstration

of

the dtug

had occurred.

an assessment

a

and the

involved

form of cyclophosphamide method

it contains

The

of the degree of

of markedly

different

tests. from 3 putients was extracted

from the extracted chloroform

of Sciences,

clinical

of cyclophosphamide

chloroform-methanol

of Human Cancer

Studies

possible.

cyclophosphamide

Each urine

cyclophosphamide

gel

isomrs

of the enantiomers urine

is a dissymmetric

racemic indicated

Academy

Poland

observation

1

from the urine

5,

Polish

the conventional

N(CH2CH2C1)2 ‘i

recovered

Studies,

Boczna

40

CH2’ ‘CH2-

and R. Kinas

followed (19:l).

each treated with

material

chloroform

by elution

by thin-layer The products

were markedly

i.v.

from a column

chromatography

thereby

racemic

(3 x 500 ml) and

were crystallized

laevorotatory

with

(25 x 2 cm) of silica

on silica from water

demonstrating

gel

(Merck,

and the data stereoselective

form.

Ludwig

Institute

for Cancer

993

Research

at the Institute

of Cancer

Research

994

Preliminary Communications

Table Patient

I

Cyclophosphamide

recovered

(24-hour (ref.4)

1

mg

-1.5’

2 0. lo (c 0.95,

2

16.9

mg

-0.3O

f O.1°

3

17.5

mg

-1.9’2

of cyclophosphamide

(-)-N-(3-hydroxypropyl)-1 and fractionation

purity

of each resulting

in MeOH,

of these enantiomers

cyclophosphamide

respectively,

of the (-)-isomer.

tumour

to yield

(6)

(+)-

and -2.3+0.1’

samples

(Table

N, N-bis(2-chloroethyl)phosphoroamidic

(c 4.6),

respectively.

in Table

were tested

PC6

Further

I are calculated

(two-fold

Tumour

Test

Therapeutic

LD5O

ID90

(mg/Kg)

(mg/Kg)

index

68.9

(-)-cyclophosphamide

365

2.85

128.1

Racemic

335

3.6

93.0

cyclophosphamide

the (-)-isomer cells

with

is considerably

is the (+)-isomer. had greater

toxicity

of the inhibition

the drugs (7) showed the metabolic

that

products

more effective Preliminary

the drug in the presence

Measurement

[a],

on the optical

values,

against

kO.1’

the two most

83 and 91%,

the ADJ/PC6

plasma

Results

5.3

than

studies

+2.3

2

365

system

[a12:

to contain

dose spacing)

(+)-cyclophosphamide

(-)-isomer

having

2)

Table

The

dichloride

of 3N-(1-phenylethyl)cyclophosphamide,

on the basis of the foregoing

reported

(c 0.88)

of (+)- or

and (-)-cyclophosphamides

but,

MeOH)

(c 0.84)

0.1’

(5) by condensation

of diastereoisomers

of cyclophosphamide

in mice

with

are in progress,

iaevorotatory

cell

pair

hydrogenation 1 dm cell)

The enantiomers

were synthesized

-phenylethylamine

bycatalytic

(c 12.2

(1 dm cell)

9.5

The enantiomers

followed

[a]2,8-29

urine)

(lower

experiments

towards

tumour

of a microsomal

of growth

when metabolism

were of equal

lD90)

of Walker

cells

(8).

the tumour

using

a bioossay

in tests

involving

activating ascites

of the racemate

toxicity

in killing

cells

(LD5dIDgO)

cells

technique in vitro

in culture

after

(2) showed

incubation

system and subsequent

and the (+)- and (-)-

in this

that

of TLX

injection

microsomal

test

into mice.

activation

forms was 98%

5

of

complete,

Preliminary

The metabolism quantitating

of the racemic,

the unchanged

(+)-

and (-)-

drug by stable

cyclophosphamide-4,5,6-d6

OS internal

identical,

of metabolism

The

as was the extent rates and patterns

investigated

in relation

correlation (10) that activity

between the

Assessment of optical investigating utilising

enantiomers

of the differential

an alternative

deuterated

approach

This

(Institute

of Cancer The A.E.

(G973/786/K). from the Medical

Research

and encouragement,

involving in which

Research:RoyaI MS-12

quantities

WCIS supported

(4,

9,12,

on the basis

We are presently by mass spectrometry

deuterium.

to the Chester

from the

A.B.Foster

for expert

in biological

A variety

of

13).

Medical

WCIS purchased

Professor

cyclophosphamide

with

known

(11).

and analysis

by grants

differences

material.

is labelled

Hospital)

and M.H.Baker

purified

and the

It has long been

animals.

in racemic

further

metabolism

transformations

of carefully

is now avoilable

are being

may show marked

metabolic

one enantiomer

We thank

and L.J.G:iggs

structure

microgram

Cancer

racemic

forms were virtually

of differential

in experimental

mass spectrometer

Council.

of cyclophosphamide

implications

different

quantities

investigation

I.

rates for the three

of the enantiomers

of cyclophosphomide

Acknowledgments: Institute

milligram

(9) using

by

15 minutes.

activity

undergo

cyclophosphamide

derivatives

after

of the clinical

metabolism

data requires

rocemic

(45%)

was monitored

spectrometry

initial

of drugs having a dissymmetric

certain

rotation

dilution-mass The

rates and anti-tumour

enantiomers

and that

isotope

of the enantiomers

to an assessment

99s

forms of cyclophosphamide

standard.

of metabolism

these

Communications

Beatty

Research

on CI special

grant

and Dr T.A.Connors

technical

Research Council (G969/189/C) for their

interest

assistance.

References 1.

USA 2.

A Review

D.L.HILL,

pp 25-59,

Charles

C.Thomos,

Springfield,

III.

(1975)

T.A.CONNORS, 3,

of Cyclophosphamide,

P. J.COX,

P.B.

FARMER,

A. B. FOSTER

and

M. JARMAN,

Biochem.

Pharmac.

115 (1974)

3.

H.ARNOLD

4.

P.J.COX,

and F. BOURSEAUX, P.B.FARMER,

Angew

A.B.FOSTER,

. Chem. E.D.GILBY

70,

539

(1958)

and M.JARMAN,

Cancer

Chemother.Rep.

in press. 5.

W.J.STEC,

R.KINAS

6.

T.A.CONNORS,

and K.PANKIEWICZ, M.JONES,

Chem.-Biol.lnteractions 7.

P.J.COX,

8.

P.J.COX

B.J.PHILLIPS and

B.J.PHILLIPS,

W.C.J.ROSS, J,

415

and

submitted

for publication

P.D.BRADDOCK,

A.R.KHOKHARandM.L.TOBE,

(1972)

P.THOMAS, unpublished

Cancer work

Res.

35,

3755

(1975)

Preliminary Communications

996

9.

M.JARMAN,

E.D.GILBY,

10.

C.C.STOCK,

H.C.REILLY,

A.B.FOSTERand

P.K.BONDY,

Clin.Chim.Acta

S.M.BUCKLEY,

D.A.CLARKE

and C.P.RHOADS,

g,

61 (1975) Nature%

71 (1954) 11.

H.KEBERLE,

K.HOFFMANN

12.

T.A.CONNORS,

P.J. COX,

and K.BERNHARD, P.B.FARMER,

Experientia18,

A.B.FOSTER,

M.JARMAN and J.K.MACLEOD,

Biomed. Mass Spectrom. l_, 130 (1974) 13.

L.J.GRIGGS

and M.JARMAN,

105 (1962)

J.Med.Chem. 18, 1102 (1975)