The synthesis and biological activity of potent, selective, luteolytic prostaglandins

THE SYNTHESIS AND BIOLOGICAL ACTIVITY OF POTENT, SELECTIVE, LUTEOLYTIC PROSTAGLANDINS

N.S.

Crossley

ICI Pharmaceuticals Division Alderley Park, Macclesfield, Cheshire, England.

The regulation of luteal function as a means of controlling human and animal fertility has been of academic interest for many years but, since the discovery that prostaglandins (PGs) have a luteolytic action in a variety of laboratory and farm animals, it is now of considerable practical importance. In particular lute01 tic prostaglandins are of value for the control of oestrus in cattle, P for the induction of parturition in pigs2 and for the treatment of infertility due to persistent luteal function in horses.3 It remains to be seen whether it will be possible to use these compounds as contraceptives in women. The work described here is concerned with our attempts to find PG analogues which are selective in the sense that they retain the luteolytic properties of the natural PGs but have fewer undesirable effects. In the laboratory the luteolytic activity of PGs can be measured using a termination of pregnancy test in hamsters. In our routine screening procedure the compounds are dosed subcutaneously or orally to hamsters on days 4,5 and 6 of pregnancy (day 1 being the day that sperm are found in the vaginal smear and day 8 being the day of autopsy). We have also measured the ability of compounds to stimulate isotonic contractions of the isolated uterus of dioestrus guinea-pigs and the value of this test is that it seems to give some indication of the severity of the side effects which occur in vivo. The synthesis of PG analogues to put into these tests can be achieved in many ways but one of the most convenient is that due to Professor Corey. 4 A key step in his synthesis is reaction of the aldehyde (Fig. 1) with a phosphonate reagent to give an enone intermediate. The group R attached to the carbon atom which will become C-15 in the final PG is derived from an ester or acid chloride and therefore can be varied very easily. For example an homologous series where R is an alkyl group is readily made using this procedure and the activity of four of these PGF2o analogues in the hamster test is shown in Fig. 2. Surprisingly two of these compounds are more potent than PGF2, as luteolytic agents in hamsters and the oral activity of 20-ethylPGF2, (ICI 74,205) is particularly striking.5

PROSTAGLANDINS JULY 1975

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5

PROSTAGLANDINS

On a speculative basis we looked next at the effect of having an ether oxygen link in the alkyl chain and, because of the potent of we chose to make oxa analogues of this compound. % The 20-ethylPGF2,, biological activity of these compounds when dosed subcutaneously to The most interesting of the five pregnant hamsters is shown in Fig. 3. isomers is the 17-oxa compound, which is five times more potent than In view of this result we looked at the effect of varying the chain PGF2c. but none of these analogues was more potent length in the 17-oxa series, 17-oxaPGF2, (bottom of Fig. 3) for than the pentyloxy compound itself. In the in vitro smooth example, is very similar in potency to PGF2,. muscle test (Fig. 4) the pentyloxy compound 20-ethyl-17-oxaPGF2o, is only one-tenth as potent as PGF2a and therefore in this compound there is an encouraging separation between the luteolytic and smooth muscle effects. A logical extension of this work was to replace the alkyl group attached to the ether oxygen with a phenyl ring and this compound was easily made starting with commercially available phenoxyacetyl chloride. In the hamster test it is an extremely potent luteolytic agent (Fig. 5), being at least one hundred times more potent than PGF2c. Many related analogues with substituents in the phenyl ring were found to be equally (ICI 79,939), m-chloro (ICI 80,996) and potent and the p-fluoro 7 m-trifluoromethyl (ICI 81,008) compounds were selected for further study. For comparison results are given for the so- alled metabolically-blocked PGs, 16,16-dimethylPGF2c and 15-methylPGF2,. s When these compounds were tested in the smooth muscle assay very 6), the largest being between 79,939 large differences were found (Fig. 79,939 is in fact a very potent smooth muscle stimulant, whereas and 81,008. Very similar results were obtained using 81,008 is on1 weakly effective. gerbil colon. 4; These smooth muscle tests can be used as a rough guide to the toxicity if the ability of the compounds to cause of the compounds , particularly diarrhoea in young male rats is used as a measure of in vivo toxicity In these animals, by comparison with PGF2c, 79,939 is a very (Fig. 7). toxic compound, but very large doses of 81,008 can be given without any sign of toxicity. Both ICI 80,996 and ICI 81,008 are as effective at terminating pregnancy in hamsters when dosed subcutaneously on day 4 only as on each of days 4 to 6 (Fig. 8) and when dosed by the intraperitoneal or intravaginal route. When given by mouth they compare even more favourably In the pregnant rat Professor Csapo has shownro that 81,008 with PGF2c. is very effective when dosed intravaginally and in particular on day 8 pregnancy can be terminated with a single dose of 10 a. Details of the biological activity of these compounds in large animals has been published elsewhere, but briefly ICI 81,008 (fluprostenol) can be used to treat persistent luteal function in horses and the recommended intramuscular dose is 250 pg. ICI 80,996 (cloprostenol) at

JULY 1975

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PROSTAGLANDINS

an intramuscular cycle in cows.

dose

of

500 ug is

preferred

for

controlling

the oestrous

Both of these compounds are racemic but in the case of 81,008 we have shown that the enantiomer having the same stereochemistry as the natural PGs is twice as active as the racemate, whereas the other enantiomer has no luteolytic activity. At this stage it is not known why these compounds are so potent as luteolytic agents and apparently selective but it is of interest that they have no significantly greater affinity than PGF2, for the PGF2, receptor In vitro they are not substrates for the in bovine corpora lutea. 15-dehydrogenase enzyme which so readily deactivates the natural compounds. although they are rapidly P-oxidised,they may have a longer Therefore, biological half-life than PGF2, in viva. Structure-activity relationships are of particular interest to the medicinal chemist and we have made several attempts to rationalise our In general it seems results with aryl PGs in terms of a physical parameter. that electron-withdrawing groups such as chlorine and trifluoromethyl In agreement with this the analogue in the aromatic ring are preferred. with an electron-releasing p-methoxy group (Fig. 9) is only one-tenth as potent as the unsubstituted compound. With these results in mind the p-nitro and p-methylsulphinyl analogues were synthesised but neither is of any interest. A series of compounds has also been made with various groups linking the aromatic ring to C-15. As far as luteolytic potency in hamsters is concerned the oxygen atom in the linking group is important since the or with CH2 instead of oxygen, are less analogous compounds without it, As shown (Fig. potent. 10) the oxygen atom cannot be replaced by other hetero atoms nor by a linking group containing two oxygen atoms without a considerable drop in potency. If there is a relationship between structure and activity these and other results point to it being a complex one. The differences in properties between PGs of the F and E series are well known and as far as luteolysis in hamsters is concerned PGE2 is at least four times less potent than PGF2,. It was no surprise to find therefore that the E analogues of the aryloxy compounds are not as potent as the corresponding F analogues although the p-chloro compound ICI 80,205 (Fig. 11) is still much more potent than PGF2u. However, ICI 80,205 is one of the most potent stimulators of guinea-pig uterus muscle in vitro that we have tested, being something like six times more potent than PGE2 in this test. Turning now to chemistry and first of all the problems connected with the large scale preparation of PGs. Although the Corey synthesis is a very suitable procedure for the preparation of PG analogues in the laboratory, it is not an easy recess to operate on a larger scale. In 72 particular the preferred route involves the alkylation of the thallium salt of cyclopentadiene with chloromethyl ether and the hazards associated with the use of thallium and any chloromethyl ether are well known.

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7

PROSTAGLANDINS

A modification which avoids this problem uses the literature compound acetoxyfulvene13 as the diene in the Diels-Alder reaction with Details of this route were published recently14 chloroacrylonitrile. and the advantages are listed (Fig. 12).

(Fig. steps

The enol acetate group in the Diels-Alder adduct can be hydrolysed 13) and the aldehyde protected as a dimethyl acetal. Subsequent are then very similar to those in the Corey procedure.

the intermediate aldehyde can be reacted with the Alternatively, appropriate phosphonate reagent to give the bicyclic enone shown and then the Corey procedure can be used again. This route avoids the use of protecting groups and is one of the shortest that has been described.15 Another major problem which had to be solved concerned the reduction of the intermediate enones. This can be done with sodium or zinc borohydride but in the case of the aryloxy enones unacceptably large amounts of dihydro compounds are formed by reduction of the double bond A very satisfactory solution was found in the use of aluminium (Fig. 14). isopropoxide and this reagent is particularly suitable for large scale preparations.‘6 Finally, a refinement to our synthesis has enabled us to make PGFl,, PGF2a, PGEl and PGE2 analogues from a common intermediate (Fig. 15). In this sequence the top side chain is attached before the bottom one using the standard Wittig procedure. The double bond in the Wittig product can then be hydrogenated to give an intermediate suitable for conversion to the PGl series and, if the h drogenation step is omitted) compounds of the PG2 series are obtained. TJ I would like to thank Dr Arthur Walpole, Dr Mike Dukes,and their colleagues for the biological results. I would also like to acknowledge the work of my chemical colleagues - their names are mentioned in the appropriate references. REFERENCES 1.

Tervit,

2.

Ash,

3.

Allen,

4.

Corey, E.J., Weinshenker, J. Amer. Chem. Sot a:5675,

5.

Labhsetwar,

A.P.,

6.

Bowler, J., 391, 1975.

Crossley,

7.

Binder, D., Bowler, J., Brown, E.D., Crossley, N.S., Hutton, Senior, M., Slater, L., Wilkinson, P. and Wright, N.C.A., Prostaglandins h:87, 1974.

8

H.R.

and Rowson,

R.W. and Heap,

L.E.A.,

R.B.,

.I. Agri.

W.R. and Rossdale,

Nature

J.

P.D.,

Reprod. Sci.

Equine

N.M., Schaaf, 1969. 238:400,

N.S.

Fert.

g:365,

1973.

1973.

Veterinary T.F.

34:179,

Journali:137,

and Huber,

1973.

W.,

1972.

and Dowell,

R.I.,

Prostaglandins

JULY 1975

2:

J.,

VOL. 10 NO. 1

PROSTAGLANDINS

8.

Magerlein, B.J., DuCharme, D.W., Magee, W.E., Miller, W.L., Robert, A. and Weeks, J.R., Prostaglandins &:143, 1973.

9.

Dukes, M., Russell, W. and Walpole,

10. Csapo, A.I., Prostaglandins

1974.

7_:141, 1974.

11. Cooper, M., Vet. Rec. 95:200, 12. Corey, E.J., Koelliker, 93:1489, 1971.

A.L., Nature 250:330,

1974.

U. and Neuffer,

J., J. Amer. Chem. Sot.

13. Hafner, K., Schulz, G. and Wagner, K., Annalen 678:39, 14. Brown, E.D., Clarkson, R., Leeney, T.J. and Robinson, J.C.S. Chem. Corma., 642, 1974.

1964. G.E.,

15. Brown, E.D. and Lilley, T.J., J.C.S. Chem. Comm., 39, 1975. 16. Bowler, J., Mallion, K.B. and Raphael, R.A., Synthetic Communications 4:211, 1974. 17. Mallion, K.B. and Walker, E.R.H., Synthetic Communications, in the press.

JULY 1975

VOL. 10 NO. 1

9

PROSTAGLANDINS

0

9’5

(MeO12POCH2!I!R

1

-(FL

/ AC0

R 0

!JhPh I (Me012POCH’2

TABLE

H? c

0

R

1 HO

Minimum effective dose to terminate pregnancy

C02H

/

1

given on each of days 4-6 vg I hamster S.C.

Hb R

=

C5Hll

25

Am

‘gH13

25

-

C7H15

10

100

‘gH17

10

500

s50

C9H19

TABLE

10

p.0. - PGFp

- ICI 74,205

SHW)

2

JULY 1975

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PROSTAGLANDINS

H?

Minimum effectivedose -CO2H

&A

toterminatepregnancy givens.c. on eachof days 4-6 vglhamster

'R

Hb R

=w

25 Hb 7

10

HO TOW I

5

100

;oI HO 70-

,100

I &

00

10

Hb 70'

20

HO TO-

20

HO

TABLE 3

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VOL. 10 NO. 1

11

PROSTAGLANDINS

R

Lowest daily dose, s.c., given on days 4- 6. needed to terminate pregnancy.pg/ hamster

Contraction of guinea-pig uterus in vitro Relative potency ---

25

10

10

4

5

1

=V Hb

HO

\/‘0Hb

TABLE

4

HO

I

Lowest daily dose, s.c., given on Days 4-6, needed to terminate pregnancy. vgl hamster

d

R = ‘f-”

25.0

PGFp

10.0

ICI 74,205

Hb

’

7

&&I I

1.0

l

0.125

ICI SO.996 *

0.25

ICI 81,wB

*

Dimethyl

PGFp

“” > 5.0

1.0

TABLE

12

5

l

16,16-

I)-Methyl

PGFp

(a and 8)

Racemic cormuwnd

JULY 1975

VOl,.

JO NO. 1

PRO’STAGLANDINS

Relative Potencies

Termination of pregnant Y in hamster 1 S.C.

dosing,

1

Cont f-action of (dioestrous) guineapig uterus in vitro

PGF2, ICI 79,939

1

50

200

500

ICI 80,996

200

50

ICI 81,008

100

1

I

i

Doses of PGF2, and Analogues

Compound

which Cause Diarrhoea

Dose (pgglkg)

Incidence

in Young Male Rats.

of diarrhoea

120-240 1,000

50% of rats lOOTI II

I 79,939

100

acutely lethal

I C I 80,996

1,000 2,400

50% of rats 100% ”

ICI 81,003

12,000

PGF2,

IC

TABLE

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VOL. 10 NO. 1

no side-effects

seen

7

13

PROSTAGLANDINS

Oral and Vaginal Dosing,

pregnancy given on each of days 4-6

Lp. or S.C. F2tl

p.0.

25

1,000

terminate

pregnancy given on day 10

p.v. -

1,000 -

80,996

0.125 (day 4 only)

2.5

0.125

81,008

0.25

5.0

0.25

40

20 (day 8 = 10)

TABLE 8

Minimum effective dose to terminate pregnancy given on each of days 4 - 6 ug I hamster

R

=

H

0.25

=

F

0.125

=

OMe

2.5

=

NO2

2.5

=

ICH3 0

5.0

TABLE 9

14

JULY 1975

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PROSTAGLANDINS

Minimum effective dose to terminate pregnancy given on each of days 4- 6 ug I hamster

R

=

00

-0

u -s u -s u d

-0

0

0.25

0.125

Cl

0

Cl

0

Cl

1.0

>50

-00 file

-N

2.5

x”D

5.0

0

0

TABLE

JULY 1975

VOL. 10 NO. 1

10

15

PROSTAGLANDINS

IC I 80,205

Relative Potencies. Termination of pregnancy in hamster kc. dosing) PGF2cl ICI 81,008 PGE;, ICI 80,205

TABLE

11

Acetoxyfulvene

Contraction of guinea-pig uterus in vitro

4

50

400

1

1

1,800

100

10,800

Route. O”

HCOZEt

Na@

OAc

v

A /

CN

Cl CN

1. Sodium salt of cyclopentadiene. 2. Innocuous

reagents.

3. No isomers. TABLE

16

12

A

Protected

aldehyde

not methyl ether.

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PRO’STAGLANDINS

OMe

CN

\

qx 0

0

0

-

CF3

/

Cl

dN

TABLE 13

L Rb

,ac,

)

0

komc,+

dihydro

Rd

HO

i!n(BH4)p

> 2041

Al(OiPd3

0%

TABLE 14

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VOL. 10 NO. 1

17

PROSTAGLANDINS

koMe _fJ-$-co2H_7$---.-~co PCFla

THPb

OMe

THPO

OMe

HA

TABLE

18

OMe

15

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