Dipeptide glycols: A new class of renin inhibitors

Vol.

132,

October

No.

1, 1985

15,

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS Pages

1985

DIPEPTIDE

GLYCOLS:

155-l

61

A NEW CLASS OF RENIN INHIBITORS

Gunnar J. Hanson: John S. Baran, Thomas Lindberg, Gerald M. Walsh, S.E. Papaioannou, Maribeth Babler, Stephen E. Bittner, Po-Chang Yang and Mary Dal Corobbo G.D. Searle and Co. Parkway, Skokie, Ill.

4901 Searle

60077

Received August 26, 1985 SUMMARY: The discovery of a new class of novel renin inhibitors consisting of protected dipeptide amides derived from aminoglycols (Formula I) prompted a study of structure-activity in vitro and efficacy in vivo. Thus, Boc-&-Phe-N-[(lS,2R)-1-benzyl-(2,3-dihydroxy)propyl]-L-leucinamide (1) and the corresponding histidinamide (2) inhibit human renin in vitro (IC50: 8.7 x 10-6 M and 2.6 x lo-6 Ij, respectively). Compound 1 has a slight inhibitory effect on pepsin and compound 2 does not inhibit pepsin at all (at lo-4q; these compounds are inactive against rat renin. Compound 1 is efficacious in lowering plasma renin activity in the Rhesus monkey (i.v.). Results indicate that this new class of low molecular weight inhibitors is specific for human renin and thus constitutes a new source of drug candidates. 0 1985 Academic Press, Inc.

The search 3.4.99.19)

for

greatly

in

clinical

remains

(EC 3.4.15.1)

catalyzes

N-terminus

inhibitors

(1),

but

are

should

the

hydrolytic

Design candidates

potentially

clinical

of trial,

whom correspondence

the

inhibition

release

of

angiotensin

this

process

is

substance

high

I from

the

rate-limiting II.

Renin

of plasma

pressure.

In seeking

we focused

enzyme

successful.

modulators

Inhibitors.

should

the

and

as an alter-

angiotensin

blood

such

commercial

be equally

important

(EC

interest,

I converting

renin

in principle

and pathologically

for to

in

of pressor

and Selection

tremendous

of angiotensin

interest

that

thus

despite

of human renin has intensified

Nevertheless,

inhibitors

fueled

inhibitor

of hypertension

elusive.

formation

activity

* author

active

of angiotensinogen;

in the

renin

of

has

approach

Renin

treatment years

success

native

an orally

the

recent

an inhibitor

step

for

on compounds

new chemical that

be addressed 0006-291X/85 $1.50

155

Copyright 0 1985 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol.

132,

No.

1, 1985

satisfied

BIOCHEMICAL

the

exhibit

a high

degree

that

an oral

insuring pepsin,

following

(~600)

2) the to

of

dose

3) it

should

peptide

the

to or greater

lower

plasma

should

we wish

satisfy

the

amides

derived

that

be of

must

of other

low molecular

weight

a primate

a new class

one func-

potency

should

5) the

substance

model.

In this

renin

inhibitors

of

N-terminal

aminoglycols

at most

electrophilic

in vitro

(Formula

acid

absorption;

possessing

of reactive

requirements:

from

inhibition

of pepstatin; in

to report

substance

of gastrointestinal

4) the

activity

above

of

COMMUNICATIONS

--for renin vs pepsin, bound to gastric

become

stable,

be free

than

renin

not

likelihood

as aldehyde;

equal

specificity

probability

and should

such

nication

the

be metabolically

bond

tionality

will

RESEARCH

1) the

in vitro

inhibitor

increase

BIOPHYSICAL

requirements:

and to decrease

proteases;

AND

protected

be must

Commuthat

dipeptide

I).

METHODS AND MATERIALS: In vitro inhibition assays for human renin, rat renin, and porcine pepsin were performed using known methods (2,3,4) as A monkey model for assaying the effect of modified recently (5). test compound on plasma renin activity (PRA) was developed: Rhesus monkeys (6/group) weighing 6.3-9.9 kg were treated with Lasix (furosemide) at 2 mg/kg, both in the a.m. and p.m. on Day 1. intravenousOn Day 2, test compound at 10 mg/kg was administered Plasma samples (K-EDTA) were taken at intervals as indicated ly. in Table 2; PRA was then determined by a known method (6). Details of the dipeptide glycol synthesis will be published elsewhere. Compounds 1 through 9 were prepared by amidation of a protected dipeptide or amino acid with an appropriate aminoglycol (see Table 1 and Figure 1 for structures). All amino acids are of the & configuration.

RESULTS: Table

1 summarizes

dipeptide

glycols

inhibitors

have

the

in vitro

and related the

general

inhibition

data

for

the

The new glycol

compounds. formula:

N-protected-Dipeptide-NHCH(CHZPh)&i([CHz],-0H)OH To demonstrate i.v.

to Rhesus

pepstatin

treated

in vivo

monkeys.

efficacy,

Table

and compound

compound

2 shows the 1 groups. 156

(1) 1 was administered

PRA for For both

both groups,

the PRA is

Vol.

132,

No.

BIOCHEMICAL

1, 1985

Table

AND

BIOPHYSICAL

In Vitro

1.

COMMUNICATIONS

Inhibitiona

Human Renin no.

RESEARCH

Pepsin

Rat Renin

structure

%

IC50

%

IC50

1

Boc-Phe-Leu-V

87

8.7

67

50

0

2

Boc-Phe-His-V

90

2.6

4

>lOOO

0

84

15.4

63

0

59

0

3

Boc-Nal(l)-Leu-V

%

4

tBuAc-Phe-Leu-V

84

14.0

5

Boc-Phe-Lys(Cbz)-V

70

24.0

Boc-Phe-Leu-W

25

>lOO

7

Boc-Phe-Leu-X

70

63

59

8

Boc-Phe-Phe-Y

34

>lOO

4

9

Boc-Phe-Leu-2

8

>lOO

10 Boc-Phe-Phe-Phe-ol

22

>lOO

11 Boc-Phe-Leu-Leu-al

98

2.2

46

100

50

86

13.6

100

0.34

60.3

6

Pepstatin

0 0

apercent (%) figures indicate percent inhibition at low4 M. Is are in micromolar units. Compounds l-9 are the formal 24 It of linking a protected dipeptide acid via an amide bond to an appropriate aminoglycol. V= (lS,2R)-1-benzyl-(2,3-dihydroxypropyl)amino; W= (lS,2S)-1-benzyl-(2,3-dihydroxypropyl)amino; x= (lS,2S)-1-benzyl-(2,4-dihydroxybutyl)amino: Y= (lS,ZS)-2-hydroxy1-hydroxymethyl-(2-phenylethyl)amino; Z= (lS,2R)-l-benzyl-(2,4dihydroxybutyl)amino. Nal(1) denotes 3-(ll-naphthyl)-&-alaninyl.

2. The Effect of Dipeptide Plasma Renin Activity of Lasix

Table

Glycol 1 and Pepstatin on Treated Rhesus Monkeys

Time

Glycol (n=6)

Pre-Lasix -24 hr

12.2 -+ 2.9

13.9 + 2.5b

Post-Lasix 0 hr

47.2 + 7.9

53.1 t 6.1

2 min

13.0 + 0.2

5 min

8.4 + - 2.6 15.1 t 3.5

15 min

23.5 -+ 4.6

44.7 + 5.4

1

b mean + S.E. 157

Pepstatin (n=6)

22.7 + 9.2

Vol.

132,

No.

1, 1985

increased after

BIOCHEMICAL

by Lasix

and is

treatment.

diminished of the

AND

returned

The PRA for

to

50% of the

pepstatin

group

BIOPHYSICAL

to pre-Lasix

the

initial

RESEARCH

group

had returned

values

treated

value

after

COMMUNICATIONS

at 2 min

with

1 was still

15 min,

to post-Lasix

while

the

PRA

levels

within

15

(8)

postulated

min.

DISCUSSION: Recent that

work

a C-terminal

be the

on aldehydic

geminal

pharmacophore,

Because

the

inhibitory

glycols:

1,2

respectively,

aldehyde

in

Formula

As can be seen

Compounds respectively pepstatin;

I;

from

renin

compounds

to explore more

would

our

1, this

approach

inhibitor

leads,

IC50,s

inhibitors

I

R= i-h

2

R=CH2

Most

vitro

and

n= 1,2 hypothesis

that

formyl. was successful especially

of 8.7 than

Potent

158

and 2.6

the

when n=l: micromolar

standard,

respectively,

The one-carbon

Dipeptide

in

naphthylalaninyl,

substitutions

to pepstatin.

1.

in

under

homologous

this

test

might

mimic.

the

stable

3, 4, and 5 possessing

in potency

hydrate)

to be unstable

where

and Cbz-lysinyl

Figure

likely

substances

1) with

more potent

aldehyde

i.e.

Table

1 and 2 (Figure are

is

of the

with

new primate

tert-butylacetyl, comparable

moiety

diols,

the

as a transition-state

properties and 1,3

inhibitors

(i.e.

we decided

may be bioisosteric

generating

glycol

conditions,

in vivo

renin

functioning

parent

physiological

diol

(7)

Glycols.

homologue

are 7

Vol.

132,

No.

(n=2)

BIOCHEMICAL

1, 1985

was also

than

active

pepstatin.

important weakly clear

(IC50

activity

active.

That

because

the

the

diol

example,

and the

configuration

epimer

of I (n=l),

of 7 (n=2),

i.e.

at this

compounds

derived

(S,S)-statine

center

from

(9),

possibly,

1 and 2 occupy

inhibitor

pepstatin

potency there

is a closer

enzymatic

reaction

active-site.

It

with

transition

state

A remarkable

illustrates

the

specificity

of 1 and 2 for

385:l

glycol these

substances inhibit

also rat

homologue

dipeptides render over

for

such them

pepsin

containing

during

renin,

whereas

Table

versus

has not

over

Compounds

human renin; even

(11). I59

pepsin:

specificity: the

I)

been obtained

pepsin

Interestingly,

11,

such for

specificity a large

3

a greater

1 and 2

postulated

It is remarkable -is active. as 1 and 2 contain sufficient information for

their

preference.

aldehyde

as

(10). is

2 exhibits

at the acting

or pepsin. renin

that

the

10,Valll,,,

human renin

6:l

fact

2 and the

diols

renin

Compound

show species

(n=O in

specific statine

rat

inhibiting

1 shows a corresponding

do not lower

inhibitors.

preference

the

inhibitor

inhibitory

versus

than

to

inhibitors

human renin

other

competitive

and collected-substrate

for

with

Quite

1 and 2 are

of these

those

at C-3 in

glycols

feature

The

The superior

---His'-Leu

specificity

compared

inactive.

as the

is positioned

that

is

the

(i.e.

be linked

between

sequence

is possible

series

space

2 could

the

is also

of renin.

angiotensinogen

substrate:

9,

of pepstatin.

same

analogy

activity

bearing

same as that

active-site

sequence

only

inactive.

active

principle

of histidine-containing

natural

both

in the

for

carbon

compound

much the

8 is

for activity: -is critical i.e. compound 6, is inactive,

in the

active

is probably

necessary

10 is

V and X) is the

the

potent

derivative

is

I)

less

hydroxyls

asymmetric

(C* in Formula

the

epimer

the

COMMUNICATIONS

although

phenylserinol

alcohol

at

RESEARCH

x 10m5 M),

moiety

monohydric

hydroxyl

BIOPHYSICAL

of the

because

The stereochemistry

for

6.3

The orientation

for

secondary

AND

(7)

that

mere

to for

heptapeptide

renin

Vol.

132,

No.

BIOCHEMICAL

1, 1985

Table

3.

AND

BIOPHYSICAL

Specificity

RESEARCH

of Renin

COMMUNICATIONS

Inhibitors

SPECIFICITY (IC50 for pepsin) Renin

Inhibitor

(IC50 for 0.025 23 6

Pepstatin Iva-His-Pro-Phe-His-Sta-Ile-Phe-NH2 1 Boc-Phe-Leu-V 2 Boc-Phe-His-V

As part

of

trial,

we tested

(Table

2).

the

Compound

pepstatin,

exhibiting

consistent

with

prospective

for

1 for 1 proved

an agent activity

duration that

than

of action. a minimum

is desirable

for

for in

more efficacious

hypothesis

inhibitors

selecting

PRA lowering

greater

our

11

>385

protocol

diol

Reference

renin)

the the

This

of peptide

in vivo

clinical monkey standard is bonds

in

utility.

CONCLUSION: The novel 2,

represent

compounds

are

attractively

Because

subsites for

1,2-diol

lowest possess

stable

function

theme

the

known,

cophore.

glycols

an important

inhibitors

could

dipeptide

new class molecular only

of their

as probes their

the

design

constituting

Formula

I,

of renin weight

non-aldehydic

one peptide

demonstated for

in vivo

SAR studies activity

bond, 1,2

an addition

the

These renin

and contain diol

an

pharma-

activity, they 12 of S ,S , and S3 renin

makes them

to be bioisosteric to

by 1 and

in vitro

of new therapeutics.

was shown

typified inhibitors.

and convenient-to-handle

(7);

unit

of

armamentarium

an attractive

In addition,

the

with

thus

formyl, of the

medicinal

chemist. REFERENCES: 1.

2. 3.

Haber, E. (1983) Clin. and Exper. Hyper. A5(7&8), 1193. Burton, J., Poulsen, K., and Haber, E. (1975) Biochemistry 14, 3892. Haber, E., Koerner, T., Page, L.B., Kliman, B., and Purnode, A. (1969) J. Clin. Endocr., 29, 1349. 160

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4.

5. 6. 7. 8. 9. 10. 11.

132,

No.

1, 1985

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Anson, M.L. (1938) J. Gen. Physiol. 22, 79. Papaioannou, S., Hansen, D., Babler, M. P.-C. Yang, Bittner, S ., Miller, A., and Clare, M. (1985) Clin. and Exper. Hyper., in press. Sealey, J.E. and Laragh J.H. (1977) Cardiovascular Medicine 2,

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Fehrentz, J.-A., Heitz, A., Castro, B., Cazaubon, C., and Nisato, D. (1984) FEBS Letters 167, 273. Kokubu,T., Hiwada, K., Sato, Y., Iwata, T., Imamura! Y., Matsueda, R., Yabe, Y., Kogen, H., Yamazaki, M., Ii]ima, Y and Baba, Y. (1984) BBRC 118, 929. Um&zawa, H., Aoyagi, T., Morishima, H., Matsuzaki, M., Hamada, M., and Takeuchi, T. (1970) J. Antibiot. Tokyo 23, 259. Rich, D.H. (1985) J. Med. Chem. 28, 263. Boger, J., Lohr, N-S., Ulm, E.H, Poe, M., Blaine, E.H., Fanelli, G.M., Lin, T.-Y., Payne, L.S., Schorn, T.W., LaMont, B.I., Vassil, T.C., Stabilito, I.I., and Veber, D.F. (1983) Nature 303, 81.

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